Engineered VPg saRNA achieves cap-independent, low-immunogenic and precise encoding of therapeutic proteins in vivo

Domain: neuroscience
Published: 2026-01-27
Editorial outcome: minor_revision

Abstract
Self-amplifying mRNA (saRNA) vectors hold promise for the sustained expression of mRNA vaccines in vivo. However, their inherently high immunogenicity and low-fidelity replication—stemming from the RNA viral genome’s replication mechanisms—limit their efficacy as replacements or adjuncts to protein therapies. Here we report an engineered viral protein genome-linked (VPg) saRNA vector derived from a Norovirus replicon, designed for rapid loading of therapeutic protein mRNAs in vitro. The engineered VPg saRNA is adapted for a range of therapeutic scenarios, including treatment of tumor-associated cachexia under conditions of translational restriction in cap-dependent metabolism, precise encoding of oncolytic mRNAs in vivo to achieve complex functionality, and therapy for graft-versus-host disease in highly auto-immune environments. VPg saRNA addresses key limitations of linear mRNA and conventional saRNA therapies, broadening the potential applications of mRNA-based treatments.

Introduction
The demonstrated efficacy of mRNA vaccines against rapidly mutating epidemic RNA virus infections1,2 has established cell-free in vitro transcribed (IVT) mRNA technology as a cornerstone in the development of nucleic acid-based therapeutics3. This technology has expanded into applications for the treatment, replacement, or supplementation of protein therapies4. Notably, mRNA avoids nuclear entry, thereby mitigating the risk of altering the host cell’s genetic material5. However, exogenous mRNA is vulnerable to recognition by cytoplasmic nucleic acid receptors, which can trigger the host’s antiviral response, leading to mRNA degradation and significantly diminishing protein expression6. This vulnerability poses minimal challenges for mRNA vaccines, as even small quantities can elicit robust immune responses, generating sufficient antigen levels7. In contrast, preclinical research focused on encoding active enzymes, cytokines, and therapeutic proteins often necessitates up to a 1000-fold increase in active protein levels to achieve therapeutic thresholds8. This demand has spurred the development of various strategies to enhance protein expression from equivalent mRNA doses, including self-amplifying mRNA (saRNA)9, circular mRNA (circRNA)10, and trans-amplifying mRNA11 constructs.

SaRNA is an engineered mRNA structure that incorporates single-stranded RNA (ssRNA) viral replicon elements, enabling it to replicate itself and achieve long-term, efficient expression of the encoded open reading frame (ORF) sequence. The most extensively studied and utilized saRNA derives from the genomic RNA (gRNA) replicon elements of the alpha-virus (αV) family, including the Venezuelan equine encephalitis virus (VEEV), Sindbis virus (SINV), and Semliki Forest virus (SFV)12. In αV saRNA, the replicase expressed in the nonstructural ORF1 region facilitates self-cloning, while the structural gene region in its subgenomic (sgRNA) is replaced by a gene of interest (GOI), driven by the subgenomic promoter located upstream. This replication and translation mechanism significantly reduces the dosage required for saRNA vaccines compared to conventional linear mRNA vaccines, which require 30–100 μg per dose, saRNA only requires 10 ng–5 μg13. The performance of the gRNA replicon is critical for the saRNA architecture, serving as the foundation for designing effective protein-encoding vectors.

Despite their potential, αV self-amplifying mRNA (αV saRNA) vectors face major engineering challenges in the development of mRNA-encoded therapeutics. First, the lengthy sequence of αV saRNA replicon elements—approximately 10 kb—complicates their separation and purification, affects the performance of lipid delivery carriers, and raises concerns regarding structural stability7. Second, the double-stranded RNA (dsRNA) replicative form (RF) generated during αV saRNA replication can activate cytoplasmic nucleic acid receptors PKR, leading to inhibition of host cell translation by phosphorylated eIF2α14,15, while inducing a severe type I interferon response16,17, triggering programmed apoptosis through antiviral response mechanisms18,19. This strong immune activation poses risks in treating certain self-limiting diseases. Third, the replication of positive-strand viral gRNA relies on RNA-dependent RNA polymerase (RdRp), which typically lacks a proofreading mechanism20 and operates with low fidelity21. This leads to the incorporation of incorrect bases, potentially resulting in reduced long-term titers of the encoded protein and inducing cytotoxicity. These limitations hinder the research, development, and application of αV saRNA in nucleic acid-based therapeutics.

In this study, we demonstrate that human norovirus gRNA (huNoV gRNA) replicates in human cells with relatively high fidelity. This fidelity is attributed to the interplay between the viral RdRp and the virion gene-binding protein (VPg), which regulate the incorporation of bases during replication. Building on our understanding of the replication mechanism of huNoV-gRNA, we engineer VPg saRNA to achieve high-fidelity replication and low immunogenicity in vivo, while retaining the characteristics of long-term and cap-independent expression. This approach provides a versatile framework with broad potential for the development of mRNA-encoded protein therapeutics.

Results
Characterization of functional elements in the huNoV replicon
Human norovirus (huNoV) is a small positive-sense RNA virus of the Caliciviridae family, widely recognized as a leading cause of acute gastroenteritis. To date, huNoV remains challenging to culture in mammalian cells22. We constructed huNoV gRNA saRNA using in vitro recombination and IVT technologies (Fig. 1A) to observe its replication in human cell lines and determine whether its replicon could serve as an engineered element for encoding exogenous protein genes in cells. As shown in Fig. 1B, the replicase RdRp and helicase 2C-L from the huNoV genome were expressed in transfected cells. Additionally, huNoV genome (ORF1, VP1, and VP2) mRNAs could be amplified from cells (Fig. 1C). We also detected substantial amounts of dsRNA RF generated by huNoV gRNA saRNA amplification (Fig. 1D). These observations indicate that huNoV genome replication occurs in human cells using IVT technology and lipid nanoparticles (LNPs) delivery systems.

In host cells, huNoV gRNA saRNA also expressed the N-terminal protein (p48) and 3A-like protein (p22) (Fig. 1E). Deletion of p22 (Δp22) severely impaired the replication of huNoV gRNA saRNA and its ability to drive ORF2 protein expression in host cells (Fig. 1F, G). Confocal microscopy showed that the p22 element of huNoV gRNA saRNA could form vesicle-like structures in the host cell endoplasmic reticulum, recruiting viral genome-linked protein VPg (Fig. 1H), consistent with membrane-bound vesicles formed by positive-strand RNA viruses that facilitate RNA synthesis and protect nascent RNA from nucleases23,24. Endolysosomal TLR3 and TLR7 respond to dsRNA and ssRNA and ssRNA, respectively25,26. The presence of p22 prevented activation of TLR7 and TLR3 in host cell endolysosomes (Fig. 1I, J), although it did not affect TLR9 activation (activated by unmethylated CpG DNA) (Supplementary Fig. 1A). These findings suggest that p22-associated vesicles help protecting nascent ssRNA from recognition and degradation.

The VPg is a 133-amino-acid peptide covalently attached to the 5′ end of huNoV genome27. VPg plays a role in the initiation and elongation phases of picornavirus replication and is proposed to function as a cap analog in the genomes of Caliciviridae and Picornaviridae, distinct from the 5′ m7G methylated cap of linear mRNAs28. VPg was also expressed in huNoV gRNA saRNA-transfected host cells (Fig. 1K). However, the precise contribution of huNoV VPg to gRNA replication remains unclear22. Using a biotin-labeled probe to capture the “daughter” replicative chains of huNoV gRNA saRNA (Supplementary Fig. 1B) and analyzing by capillary electrophoresis, we observed that approximately 20% of the negative-sense progeny strands carried VPg at the 5′ end, while 100% of the positive-sense progeny strands were VPg-linked (Fig. 1L, M). The cap-independent translation mechanism of huNoV gRNA saRNA may mitigate limitations traditional linear m7G-capped mRNAs in translation-suppressive disease settings.

We employed huNoV gRNA saRNA to load GOI for replication and expression in eukaryotic cells. In the huNoV gRNA saRNA, we replaced VP1/VP2 with four distinct GOI ORFs of varying lengths: ghrelin (84 nt), Egfp (717 nt), proc1 (1452 nt), and GSDMDENG (1535 nt), each intended for therapeutic application in specific disease contexts in further studies. Four separate IVT plasmids were generated using a two-step Golden Gate reaction, each encoding a distinct huNoV (GOI)-saRNA construct. These plasmids were then transcribed into linear IVT mRNA using T7 polymerase. Each of the four huNoV (GOI)-saRNA constructs was individually encapsulated into LNPs (Supplementary Fig. 1C), yielding uniform and well-defined particle size and morphology across all constructs (Supplementary Fig. 1D). Upon transfecting huNoV (GOI)-saRNA-LNPs into 293T cells, VPg-saRNA concomitantly expressed VPg, RdRp, and 2C-L with endonuclease/helicase activities (Supplementary Fig. 1E), followed by detectable expression of the four respective GOI proteins (Supplementary Fig. 1F).

In brief, we established a huNoV replicon-based self-amplifying RNA system based on the huNoV replicon to drive therapeutic protein expression in eukaryotic cells.

Self-amplifying huNoV saRNA shows relatively high fidelity
The precision of saRNA in encoding is crucial for the execution of protein function, minimizing cytotoxicity and cellular defensive immune responses. Next, we examined the replication fidelity of huNoV gRNA saRNA in vitro. Studies have shown that RdRp acts as the primary executor of RNA viral genome replication29,30. In Coxsackievirus B3 (Picornaviridae), VPg has been observed to bind to the base of the thumb subdomain of RdRp; however, whether huNoV VPg affects RdRp function to participate in or regulate the RNA replication process remains unknown.

HuNoV RdRp comprises seven conserved motifs that form a palm-shaped RNA synthesis center (Fig. 2A). Molecular docking predictions indicated that VPg residues Lys40 and Gly99 form hydrogen bonds with Lys371 and Asp376 in Motif D at the NTP entry port (Fig. 2B). Biolayer interferometry (BLI) experiments revealed that the binding affinity of recombinant VPg to RdRp lacking Motif D was reduced (Supplementary Fig. 2A). Local rearrangements within Motif D are critical for fidelity control in small RNA viruses31. Using the T32/P2 construct32, we evaluated the effect of VPg on the base incorporation error rate of RdRp in vitro. An artificial RNA template-primer complex (T30/P2) was used to observe the extension of the RNA template catalyzed by RdRp. Samples were collected at different timepoints (time-course) to monitor the conversion rate from 9-mer to 42-mer, which reflects the G:U mismatch incorporation rate (Rmis), in order to analyze the error-incorporation capacity of different RdRp constructs (Fig. 2C).

Our findings showed that huNoV RdRp generated up to 54.5% G:U mismatch events within 3 h, while the inclusion of recombinant VPg reduced the mismatch rate to 16.9% (Fig. 2D). Based on the molecular docking predictions results, we engineered VPg K40N and VPg G99V mutant huNoV-gRNAs, as shown in Fig. 2D, disruption of the binding interface with Motif D in VPg largely attenuated the reduction of the mismatch rate of RdRp.

The mismatch rate constant (Kmis) and Michaelis constant (KMapp) were determined using supersaturated UTP substrates (Supplementary Fig. 2B, C). The Kmis value for RdRp alone was approximately 3.3 times greater than that of RdRp/VPg (1.806 h−1 vs. 0.555 h−1), while KMapp values remained consistent (451.9 μM vs. 463.3 μM) (Fig. 2E). These data suggest that VPg regulates RdRp fidelity by modulating the catalytic rate, rather than by altering the affinity for the NTP substrate, consistent with its action on Motif D at the active site. PCR products were digested with T7 endonuclease I (T7EI) enzyme to quantify mutations in the daughter chains (Fig. 2F). Briefly, huNoV gRNA saRNA served as a template for in vitro self-replication using recombinant RdRp/2C-L and a His-VPg-pU primer. The resulting mRNA was reverse transcribed and amplified by RT-PCR with VP1/VP2 primers. Replication errors led to mismatched base pairs and heteroduplex formation between wild-type and mutant cDNA strands, which were recognized and cleaved by T7EI. The extent of digestion indirectly reflected the mutation frequency in the amplified products. The replication products of huNoV gRNA saRNA with VPg K40N and VPg G99V mutations exhibited an increased in mutation frequency in cells (Fig. 2G). Compared to egfp sequences loaded by three types of Alphavirus-based saRNA, progeny strands from the huNoV egfp-saRNA platform show virtually no detectable mutation sites (Fig. 2H).

In brief, VPg-dependent initiation represents a high-fidelity replication mechanism, and there exists potential to develop high-fidelity replication saRNA vectors.

Engineering realization of low immunogenicity of huNoV gRNA saRNA
The p22 element ensures that nascent single-stranded huNoV mRNA is not recognized by antiviral responses. However, this does not prevent the activation of double-stranded DNA cytoplasmic receptors by the dsRNA RF. Therefore, we next focused on eliminating the dsRNA immunogenicity of huNoV gRNA saRNA.

The replication of the positive strand of huNoV is not sequence-dependent33, but requires guanylylated VPg (VPg-pG) as a peptide primer for initiation34. In contrast, the negative strand requires either de novo initiation or uridylylated VPg (VPg-pU) for replication33. By engineering a system to generate solely VPg-pU primers within the cell, we aim to reduce the VPg-pG-dependent positive strand to inhibit the formation of paired dsRNA intermediates, thereby reducing immunogenicity. To accomplish this, we first examined the mechanism of VPg nucleosidyl acylation. Recombinant huNoV RdRp35 was employed to synthesize VPg-pU products in vitro (Supplementary Fig. 3A, B). We then constructed an in vitro replication system comprising huNoV gRNA mRNA template, VPg-pU primer, recombinant RdRp, and helicase 2C-L (Supplementary Fig. 3C). The replication system containing only the VPg-pU primer effectively inhibited the generation of VPg-independent de novo 5′ gRNA (−) and markedly reduced the synthesis of 5′ VPg gRNA (+) (Supplementary Fig. 3D, E).

To explore methods for spontaneously generating single VPg-pU in cells, we assessed the selectivity of RdRp for VPg nucleotide acylation using mixed NTPs. The results revealed that the covalent attachment of VPg to NTPs did not preferentially favor UTP (accounting for only 10.04%) but rather predominantly produced VPg-pA and VPg-pC (Supplementary Fig. 3F). RdRp consists of a 19.2 kDa NT segment (Pro) and a 57.5 kDa CT segment (pol) connected by a flexible linker36 (Supplementary Fig. 3G). Deletion of the CT region abolished VPg nucleotidylation, while deletion of the NT segment compromised selectivity, resulting in equal ratios of all four nucleosidyl VPg products (Supplementary Fig. 3H). This confirms that RdRp (pro) directly participates in the differential nucleotidylation of huNoV VPg.

Molecular docking predictions indicated that the NT of RdRp contains an NTP binding pocket formed by two large β-folds, displaying distinct preferred binding free energies for different NTPs, consistent with our experimental findings regarding RdRp (pro) NT’s nucleotidylation selectivity (Fig. 3A). Within this pocket, the residues Thr84, Lys146, and Tyr151 of RdRp (pro) tightly interact with the phosphate group of NTP (Fig. 3B). Deleting any of these residues abolished the nucleotidylation function of RdRp (pro) NT (Fig. 3C). Additionally, residues Ala1, Pro2, and Val82 are crucial for base recognition (Fig. 3B). Mutations of Ala1 and Pro2 to polar amino acids Gly (c.3031C>G) and Gln (c.3034C>A), respectively, reduced affinity for A, C, and G, while Val82 was substituted with polar Gly (c.3274T>G) to enhance affinity for polar purine groups. The triple mutant RdRp (pro) nearly completely inhibited the generation of VPg-pA, -pG, and -pC, while increasing VPg-pU production to 93.7 ± 1.79% (Fig. 3D).

Next, we generated a HuNoV gRNA saRNA triple mutant carrying the c.3031C>G, c.3034C>A, and c.3274T>G substitutions, which we designated as HuNoV gRNA tirM-saRNA (Supplementary Fig. 3I). Transfecting the triple mutant huNoV gRNA tirM-saRNA into Huh-7 cells eliminated to the limit of detection positive-sense 5′ VPg gRNA (+) and 5′ sgRNA (+) (Supplementary Fig. 3J). The triple mutant RdRp reduced the abundance of dsRNA RFs in the cell lysate supernatant, as well as ISG expression of in cells, and the release of IFN-related cytokines, thereby enhancing the translation of VP1/VP2 proteins (Fig. 3E–H).

An antisense sequence of huNoV saRNA based on a triple mutant RdRp (designated huNoV GOI triM-saRNA) was constructed. This structural rearrangement strategy compensates for the loss of the positive-sense progeny strand by generating 5′ VPg GOI mRNA progeny coding strands within the cell (Fig. 3I). To assess the immunogenicity of huNoV triM-saRNA, traditional VEEV-based saRNA was used as a control. At equivalent doses, the amount of paired dsRNA RF from the daughter mRNA of huNoV triM-saRNA was negligible (Fig. 3J). Although huNoV triM-saRNA did not amplify exponentially within cells, it evaded host immune responses, yielding 2.68 times more protein than VEEV saRNA during the same phase (Fig. 3K, L). The low levels of dsRNA produced by huNoV triM-saRNA avoided the activation of the RNA sensor PKR and reduced eIF2α phosphorylation, thereby prevents translation repression. Furthermore, huNoV triM-saRNA exhibited weaker activation of RIG-I and MDA5 (Fig. 3M), as well as lower ISGs expression and IFN-related cytokines release, compared to VEEV egfp saRNA (Fig. 3N, O).

In brief, these RdRp (pro) mutation strategies facilitate the production of supersaturated VPg-pU in cells, reducing dsRNA-induced immunogenicity by simultaneously suppressing positive-sense chain production, without affecting protein coding capacity.

Optimizing 3′ UTR to enhance translation efficiency
We employed HuNoV GOI triM-saRNA as a template to assess the integrity of VPg-mediated saRNA replication progeny in vitro. As shown in Fig. 4A, in vitro replication of VPg saRNA produced multiple incompletely amplified VPg ssRNAs, especially during reactions involving longer mRNA templates. These truncated 5′ Vpg-GOI mRNAs contain potential start codons in the +1 or +2 reading frames, which may lead to the translation of truncated frameshift peptides. The potential impact on biosafety and expression efficiency warrants further investigation.

The HuNoV 3′ UTR typically functions as a recruitment sequence for VPg-pU primers, driving the replication of the negative-sense strand. However, the synthetic ghrelin(-) mRNA (Kd=4.6×102 nM) forms a secondary structure with slightly weaker affinity for VPg compared to the HuNoV 3′ UTR (Kd=2.1×102 nM) (Fig. 4B). We analyzed the RNA secondary structures of sequences near the breakpoints of the progeny VPg ssRNAs. As depicted in Fig. 4B and Supplementary Fig. 4A–C, the AXXU sequence in interior-loops, stem-loops, or bulge-loops appears to serve as a nonspecific recruitment site for VPg-pU primers to initiate replication. This suggests that VPg recruitment is structure-dependent rather than sequence-dependent. The presence of AXXC-containing neck-loops may result in nonspecific de novo replication events. As shown in Supplementary Fig. 4D, removal of the AC site (Δ33A and Δ30U) or disruption of the lock structure (T29A) significantly reduced the synthetic ghrelin mRNA’s affinity for recombinant VPg. Conversely, mutating site 41 from C to A, thereby generating two consecutive AXXU sequences, increased ghrelin mRNA affinity for VPg.

Based on these insights, we designed and screened modified huNoV 3′ UTRs with randomly inserted AXXU sequences as VPg-affinity RNA aptamers (apt-VPg) to inhibit the generation of truncated VPg ssRNAs (Supplementary Fig. 4E). A 42-nt high-affinity apt-VPg proved effective in eliminating truncated VPg ghrelin ssRNA production (Supplementary Fig. 4F), even in the context of long-chain template replication (Supplementary Fig. 4G). The apt-VPg-modified huNoV 3′ UTR competitively binds to the VPg-pU peptide, thereby suppressing nonspecific short ssRNA amplification (Fig. 4C). Transfecting cells with huNoV saRNA containing the 42-nt apt-VPg-modified 3′ UTR reduced the production of nontheoretical length progeny 5′ VPg-GOI mRNA (Fig. 4D). Notably, the inhibition of truncated 5′ VPg-GOI mRNA production was associated with increased GOI protein expression by minimizing waste in mRNA templates and worker proteins (Fig. 4E). In conclusion, optimizing the VPg saRNA 3′ UTR reduces nonspecific short-chain amplification and enhances protein production. We simplified the name of the triM-saRNA construct containing the 42-nt apt-VPg-modified 3′ UTR to VPg GOI saRNA for the purpose of establishing an in vivo dosing regimen. The four types of GOI saRNA exhibited minimal activation of lysosomal and cytosolic RNA sensors, indicating reduced innate immune stimulation compared to the original huNoV gRNA saRNA sequence (Supplementary Fig. 4H, I). This suggests that the VPg-GOI saRNA self-replicating vector is less prone to innate immune activation within cells.

The studies outlined highlight three key features of VPg-saRNA: cap-independent translation, high-fidelity replication, and low immunogenicity, which collectively support the design of therapeutic applications of huNoV-saRNA. Leveraging these attributes, we designed therapeutic scenarios to illustrate the advantages of huNoV-saRNA.

Cap-independent translation VPg saRNA circumvents metabolic translational repression
The VPg can serve as a substitute for the cap structure, facilitating the efficient assembly of the 43S pre-initiation complex on its mRNA. This mechanism is therefore termed cap-independent translation37. HuNoV VPg hijacks host cell machinery for translation through eIF4G, distinguishing it from the linear mRNA Cap1-m7G-dependent initiation translation mode that relies on eIF4E (Supplementary Fig. 5A). eIF4E is regulated by mTORC1 signaling, which releases it from the phosphorylated substrate release of the competitive inhibitor 4E-BP1, thereby mediating Cap1-m7G-dependent translation38. In mTOR-deficient fibroblasts (MEF mTOR−/−), the phosphorylation levels of 4E-BP1 and eIF4E were suppressed (Supplementary Fig. 5B). This suppression led to a reduced in the translation of m7G-cap1-driven linear mRNA in MEF mTOR−/− cells, while VPg saRNA effectively bypassed the translation block in these cells (Supplementary Fig. 5C).

The suppression of mRNA cap-dependent translation due to mTOR inactivation is reminiscent of cancer-associated cachexia syndrome (CACs), an energy-wasting syndrome characterized by protein synthesis repression resulting from mTORC1 impairment39. We observed mTORC1 inactivation and dephosphorylation of 4E-BP1/eIF4E in several postmortem muscle fibers from cancer patients who succumbed to wasting, while the phosphorylation level of eIF4G remained relatively unaffected (Fig. 5A). Inactivation of mTORC1/4E-BP1/eIF4E signaling lead to disrupted skeletal muscle protein fiber and fat synthesis (Fig. 5B), resulting in significant weight loss among CAC patients, which severely impacts the quality of life of approximately 50–80% of cancer patients40. Ghrelin41 and selective androgens42 are considered to counteract the anabolic deficiencies associated with CACs. VPg saRNA is expected to overcome the limitations of these short-chain recombinant peptides, which include short half-life, inconvenient administration, and high cost.

Approximately 80% of pancreatic cancer patients develop CACs40. We employed KPC (Pdx1-cre/LSL-KrasG12D/P53R172H) mice43, a transgenic model that spontaneously develops pancreatic ductal adenocarcinoma (PDAC), to evaluate VPg Ghrelin saRNA. Mouse ghrelin is a 28-amino-acid polypeptide (GSSFLSPEHQKAQQRKESKKPPAKLQPR) with 92.9% homology to human ghrelin44. KPC mice begin to exhibit progressive weight loss between 15 and 17 weeks of age, and by over 25 weeks, all surviving mice experience nearly 20% body weight loss (Supplementary Fig. 5D). We administered intraperitoneal injections of VPg Ghrelin saRNA-LNP to KPC mice that had lost more than 5% of their body weight, followed by tissue dissection at the terminal point or upon humane euthanasia (weight loss exceeding 20%) (Fig. 5C). The phosphorylation level of 4E-BP1/eIF4E was inhibited in the muscle tissue of KPC mice after more than 5% body weight loss, while phosphorylated eIF4G only began to decrease when body weight loss exceeded 20% (Fig. 5D).

Due to the cascade inhibition of eIF4E, the serum levels of ghrelin driven by linear cap1-m7G mRNA in KPC mice were lower than those driven by VPg Ghrelin saRNA-LNP (Fig. 5E). Additionally, VPg Ghrelin saRNA did not induce weight loss or organ damage in BALB/c background mice (Supplementary Fig. 5E, F). Repeated administration of VPg Ghrelin saRNA does not affect plasma levels of Ghrelin or antiviral IFN-α in mice (Supplementary Fig. 5G, H). KPC mice progressively exhibited severe loss of subcutaneous white adipose tissue (WAT), muscle fiber atrophy (Fig. 5F, G), feeding disorders, and weight loss (Fig. 5H, I). Monthly intraperitoneal injections of VPg Ghrelin saRNA-LNP alleviated the CAC symptoms in KPC mice (Fig. 5F–H) and improved their metabolic impairments, including blocked liver glycogensynthesis, fatty acid metabolism in WAT, and disrupted myofibril protein synthesis (Supplementary Fig. 5I, J). Moreover, ribosomes, endoplasmic reticulum, and mitochondria in the skeletal muscle of mice treated with VPg Ghrelin saRNA-LNP were found to be active (Supplementary Fig. 5K–N). Notably, while VPg Ghrelin saRNA improved the quality of life and metabolic status of the mice, it also extended the survival rate of KPC mice to a certain extent (median 149 days vs. 214 days) (Fig. 5J).

In brief, the cap-independent and long-lasting expression of VPg saRNA encoding ghrelin can enhance the quality of life and moderately prolong survival in cachectic KPC mice.

High-fidelity VPg saRNA ensures the precise realization of complex protein functions
In previous studies, we constructed an oncolytic mRNA vaccine called GSDMDENG, featuring a precise mRNA structure with long hGSDMD c.825T>A; c.884A>G mutation sequences that encode mitochondrial cardiolipin inner membrane-targeting toxicity. This oncolytic mRNA vaccine can induce mitochondrial autophagy and present tumor antigens in KrasG12D mutant tumor cells45 (Supplementary Fig. 6A). GSDMDENG consists of three key structural elements: a 614 nt HRV2 IRES, a 1455 nt GSDMD ORF, and an 84 nt F1L CT, each serving distinct functions in tumor conditional expression, mitochondrial inner membrane pore toxicity, and mitochondrial signal peptide activity (Fig. 6A). This complex and delicate functional structure necessitates high fidelity in saRNA vector performance.

After transfecting AsPC1 cells with VEEV saRNA and VPg saRNA loaded with GSDMDENG, we performed 15 subcultures. T7EI digestion revealed multiple mutations in the daughter mRNA (Fig. 6B). Sanger sequencing46 of the reverse-transcribed mRNA identified a total of 22 random mutation sites in the daughter mRNA, with 7 located in HRV2 IRES, 12 in the ORF region, and 2 in the F1L CT region (Supplementary Fig. 6B). This is because AsPC1 cancer cells are killed by GSDMDNT, while sequence mutations that impair GSDMDENG function tend to be retained. We constructed various mutants based on these random mutations to evaluate their effects on linear Cap1-m7G GSDMDENG mRNA function. Three categories of mutations diminished the functionality of linear GSDMDENG mRNA to varying degrees, including specific expression and driving intensity in tumor cells (Supplementary Fig. 6C), GSDMDNT mitochondrial import (Supplementary Fig. 6D), and cancer cell toxicity (Supplementary Fig. 6E). Since mutation points in the daughter mRNA cannot be exponentially amplified, we found no mutation sites in the high-fidelity VPg GSDMDENG saRNA-transfected cells (Fig. 6B). Therefore, high-fidelity VPg saRNA vectors are essential for realizing the functional potential of long-chain mRNAs with complex roles.

Next, we tested the ablation effect of GSDMDENG integrated into the VPg saRNA vector on tumor tissue (Fig. 6A), using VEEV GSDMDENG saRNA as a control. VPg GSDMDENG saRNA, containing the non-mutated adenocarcinoma-targeting HRV2 IRES sequence, effectively drove GSDMD expression specifically in AsPC-1 cells (Fig. 6C). The F1L CT signal peptide facilitated its transport to mitochondria, activating GSDMDNT with pore-forming activity (Fig. 6D). In contrast, VEEV saRNA loaded with GSDMDENG was less stable and showed reduced conditional selectivity for cancer cells in this experiment. (Fig. 6C, D). Exogenously active GSDMDNT induced mitochondrial damage, leading to mtROS leakage and mtDNA release (Fig. 6E, F). In a mouse model bearing AsPCs-1 with a humanized immune system, peritumoral injection of 1 μg/kg VPg GSDMDENG saRNA increased tumor tissue ablation compared to the same dose of VEEV GSDMDENG saRNA (Fig. 6G). VPg GSDMDENG saRNA prompted the uptake and maturation of the mitochondrial antigen mtOVA by antigen-presenting cells (APCs) (Fig. 6H), resulting in sustained proliferation of OT-I and OT-II cells and the production of IFN-γ in AsPCs-mtOVA tumor-bearing mice (Supplementary Fig. 6F–H). The AsPC-1 model, linked to mitochondrial autophagy and presentation, indicated that VPg GSDMDENG saRNA triggered PINK1- and MHCI-mediated mitophagy and antigen presentation, whereas VEEV GSDMDENG saRNA did not induce mitophagy or antigen presentation (Supplementary Fig. 6I).

KPC mice were utilized to observe specific cytotoxic T responses elicited by mitophagy. This model, characterized by constitutive KrasG12D expression and p53 deletion, spontaneously develops PDAC in adulthood. We intraperitoneally injected VPg GSDMDENG saRNA into newborn mice as a prophylactic measure (Fig. 6I). One week later, VPg saRNA successfully drove GSDMDNT expression in KrasG12D-positive pancreatic cancer cells (Fig. 6J), correlating with the abnormal activation of Raf-MEK-MAPK signaling induced by KrasG12D47, which activated EIF4G2/PTBP1 to mediate HRV2 IRES-driven mRNA expression45. Four weeks later, mice treated with VPg GSDMDENG saRNA exhibited IFN responses, activation of antigen-presenting dendritic cells (DCs), and sustained cytotoxic T cell immunity (Fig. 6K, L). Cytotoxic T lymphocytes sorted from the spleen demonstrated a robust in vitro killing effect on KrasG12D AsPC-1 cells but were unresponsive to other Kras mutant cell lines (Fig. 6M). Ultimately, a single intraperitoneal injection of VPg GSDMDENG saRNA increased the 24-week survival rate of KPC mice from 20 to 62.5% (Fig. 6N). These findings indicate that high-fidelity VPg GSDMDENG saRNA supports sustained specific cytotoxic T cell responses, resulting in lasting tumor protection.

Low immunogenic VPg saRNA protects against autoimmune organ injury
The long-term expression, low dosage, and low immunogenicity of VPg saRNA are maybe advantageous for treating chronic autoimmune-related diseases. Activated protein C (APC, also known as Proc1) is a plasma serine protease, as depicted in Fig. 7A. It cleaves protease-activated receptor 1 at the classical Arg41 or nonclassical Arg46 sites48. The latter site is located on the surface of allogeneic T cells49. Its activation promotes follicular TFH cell differentiation, germinal center (GC) reactions, and GC B cell maturation, leading to the deposition of nonspecific immunoglobulin (Ig) G in the lungs, liver, and colon, ultimately causing chronic fibrosis. Pulmonary fibrosis is the most serious complication of cGVHD, and utilizing Proc1 to protect alveolar cells from autologous IgG attacks represents a potential strategy for mitigating cGVHD50.

We used Proc1 mRNA loaded with Alphavirus saRNA as a control to evaluate the therapeutic effect of VPg saRNA encoding Proc1 on cGVHD. Both VPg Proc1 saRNA and VEEV Proc1 saRNA effectively drive PROC1 expression in mouse airway epithelial cells (Supplementary Fig. 7A) and enable its secretion into the cell culture supernatant for detection (Supplementary Fig. 7B, C). Compared to an equivalent amount of Cap1-m7G linear mRNA transfected, the half-life of VEEV Proc1 saRNA and VPg Proc1 saRNA increased by 3–5 times, respectively (Fig. 7B). VPg Proc1 saRNA exhibited lower immunogenicity in vivo. Intravenous injection of VEEV Proc1 saRNA induced systemic innate immune responses in C57BL/6 (B6) background mice, including activation of PKR, eIF2α, MDA5, RIG-I, and expression of ISG in the lungs, liver, and intestines (Supplementary Fig. 7D, E). In contrast, VPg Proc1 saRNA did not detectably activate dsRNA receptors or induce an IFN-I response. In an acute GVHD model using MHC-incompatible allo-HSCT mice (C57BL/6 (B6) donor mice, B6D2F1 recipient mice) (Supplementary Fig. 7F), pre-injection of VPg Proc1 saRNA improved survival rates and reduced aGvHD scores, while VEEV Proc1 saRNA worsened mortality and aGvHD scores (Fig. 7C, D).

To assess the protective effects of VPg Proc1 saRNA on the organs of chronic GVHD mice, we used an MHC-matched allo-HSCT mouse model (C57BL/6 (B6) as donor mice and B10.BR as recipient mice). A dose of 0.2 μg/kg VPg Proc1 saRNA was administered via inhalation three days prior to observation of protection against chronic pulmonary fibrosis induced by cGVHD (Fig. 7E). VPg Proc1 saRNA was able to drive the replication and expression of 5′ VPg mRNA in the airway cells (VPg+ cells), with secreted Proc1 (Proc1+ cells) distributed throughout the airway bronchioles and terminal alveoli (Fig. 7F). Immunological examinations showed that VPg Proc1 saRNA reduced the proportion of allogeneic GC B cells and Tfh in the mouse lung lymph nodes while increasing the proportion of Tfr cells (Fig. 7G, H). Additionally, it reduced the abundance of nonspecific IgG antibodies against various self-antigens in the alveolar lavage fluid (Fig. 7I). In functional tests, VPg Proc1 saRNA decreased lung resistance and improved lung compliance in mice (Supplementary Fig. 7G). Pathological assessments revealed that VPg Proc1 saRNA reduced lung damage, collagen deposition, and accumulation of IgG immunoglobulins in the lungs (Fig. 7J).

In conclusion, VPg saRNA functions as a low-immunogenicity carrier suitable for mRNA therapy targeting active autoimmune diseases.

Methods
Cell culture
AsPC-1 human pancreatic adenocarcinoma cells (ATCC CRL-1682), hTERT-immortalized human pancreatic ductal epithelial cells (hTRET-hPNE; ATCC CRL-4023), HEK293T cells (ATCC CRL-3216), and HuH-7 human hepatocellular carcinoma cells (Procell CL-0120) were obtained from the indicated repositories and authenticated by the suppliers. AsPC-1, hTRET-hPNE, and HuH-7 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; high glucose) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin (P/S). HEK293T cells were cultured under the same conditions unless otherwise specified. All cells were incubated at 37 °C in a humidified atmosphere containing 5% CO2 and were routinely tested to confirm the absence of mycoplasma contamination.

Acquisition of norovirus genomic cDNA
Fecal samples from patients suspected of norovirus infection were tested using multiplex real-time quantitative PCR (qRT-PCR) to confirm positivity. Subsequently, the genogroup and genotype of the norovirus were determined by Sanger sequencing. A BLAST search against the NCBI nucleotide database confirmed that the obtained strain was Hu/GII.P4 New Orleans 2009_GII.4 Sydney 2012/NSW789Z/2016/AU (99.71% match, GenBank: KY905331.1). Additionally, blood samples were collected from the patients, and total RNA was extracted for reverse transcription to synthesize first-strand cDNA. The norovirus gRNA region, encompassing the 5′ UTR, ORF1, VP1, VP2, and 3′ UTR, was amplified using the following primers: forward primer: 5′-GACTCAGGCGCCctagaaagcacttggcctctaa-3′ (the underlined sequence indicates the KasI restriction site); reverse primer: 5′-CAGTCACCTG CAGGtaaagaaaaagaaagataatcaattttgtcttttca-3′ (the underlined sequence indicates the SbfI restriction site). The amplified product was digested with KasI and SbfI and ligated into a similarly digested pUC19 cloning vector (New England Biolabs) using T4 DNA ligase. The resulting construct, designated pUC19-hNoV-gRNA, was further verified by Sanger sequencing to confirm the correct insertion of the target DNA fragment.

Generation of huNoV gRNA mutations and deletions
Site-directed mutagenesis or gene deletion was performed on the pUC19-hNoV gRNA plasmid template using the Q5 site-directed mutagenesis kit (New England Biolabs). The following primers were designed to construct various mutants: RdRp Mutants, RdRp ΔT84-his forward: 5′-actactcatcaaaaggtccac-3′, reverse: 5′-taaccacggtgccctc-3′; RdRp ΔK146-his: forward: 5′-aaggggtaatgactatgtg-3′, reverse: 5′-tgta gatgtaggggc-3′; RdRp ΔY151-his: forward: 5′-tgtggtcattggagtc-3′, reverse: 5′-agtcattaccccttttgtag-3′; RdRp A1Q/P2Q-his: forward: 5′-cat catcatcatgcaacagcaagcatttg-3′, reverse: 5′-atgatgatgatgcatgaattc-3′; RdRp V82G-his: forward: 5′-gagggcaccgatattacactactcatcaaaag-3′, reverse: 5′-aggtgcgccttcttc-3′; HuNoV gRNA Deletions, HuNoV gRNA Δp22: forward: 5′-ggcaagaaggggaag-3′, reverse: 5′-ctgcagttcaaattcatc-3′; HuNoV gRNA Δp48: forward: 5′-gctgcaagacctgag-3′, reverse: 5′-gtaaagaaaaagaaagataatcaattttgtc-3′; VPg Mutants, VPg K40N (K915N in full-length sequence): forward: 5′-aaggaatggcaactactctatagaag-3′, reverse: 5′-aaggaatggcaactactctatagaag-3′; VPg G99V (G974V in full-length sequence): forward: 5′-tttggtcacagtgtctgaaattaggaaaag-3′, reverse: 5′-ccgagagagactctttc-3′; All constructs were verified by Sanger sequencing to confirm the desired mutations or deletions.

In vitro transcription (IVT)
The PUC19-hNoV gRNA plasmid and its mutant derivatives were digested with KasI/SbfI, followed by PCR amplification of the gRNA region using: forward primer: 5′-GACTCAGAATTCctagaaagcacttggcctctaa-3′ (EcoRI site underlined, start codon in lowercase), reverse primer: 5′- GTCACTGCGGCCGCtcaGTGGTGGTGGTGGTGGTGCTCtaaagaaaaa gaaagataatcaattttgtcttttca-3′ (NotI site underlined, stop codon in lowercase, His-tag in bold). The amplified product was digested with EcoRI/NotI and ligated into the similarly digested pIVTRup-T7 expression vector (Biovector) using T4 DNA ligase, generating the construct pIVTRup-his-HuNoV-gRNA. The plasmid pIVTRup-his-HuNoV-gRNA was linearized by PCR using primers containing the T7 promoter and terminator sequences: forward primer (T7 promoter): 5′-taatacgactcacta taggggaagcacacggcc-3′, reverse primer (terminator): 5′-ctagcataaccccttggggcctctaaacgggtcttgaggggttttttaaagaaaaagaaaga taatcaattttgtctttt-3′. After 30 PCR cycles, the linearized DNA was column-purified and used as a template for IVT with T7 RNA polymerase (NEB). Each 20 µL IVT reaction contained 1 µg DNA template and was incubated at 37 °C for 2 h with shaking at 1000 xg. 4 mM hm5CTP (5-hydroxymethylcytidine triphosphate) was used to replace CTP to incorporate methylation modifications into IVT saRNA. Post-IVT, residual DNA was degraded using 2 µL DNase I (37 °C, 20 min, 1000 xg). The unmodified linear mRNA was purified using the MEGAclear transcription clean-up kit (Ambion). Capillary electrophoresis was used to assess the integrity of IVT products, as shown in Supplementary Information Table 1 and Supplementary Fig. 8.

For the three linear mRNAs, a co-transcriptional capping strategy was employed using the HiScribe™ T7 ARCA mRNA Kit (NEB #E2065). After purification, the transcripts were converted from Cap0 to Cap1 using the mRNA Cap 2′-O-Methyltransferase (NEB #M0366), following the manufacturer’s protocol. In addition, 4 mM N1Ψ (N1-Pseudouridine) substituted for UTP to enhance mRNA stability. For all self-amplifying RNAs (saRNAs), an enzymatic capping approach was adopted. The Cap1 structure was introduced using the Vaccinia capping system (NEB #M2080) in combination with the mRNA Cap 2′-O-Methyltransferase (NEB #M0366), according to the technical guidelines provided by the manufacturer. In addition, 4 mM hm5CTP (5-hydroxymethylcytidine triphosphate) was used to replace CTP to incorporate methylation modifications into IVT saRNA. The final processed saRNA (HuNoV gRNA saRNA) and mRNA (Cap1-m7G mRNA) were repurified and quantified.

LNP encapsulation and characterization
Self-assembly of saRNA-LNPs: a lipid mixture containing ionizable lipid, DSPC, cholesterol, and PEG-lipid (50:10:38.5:1.5 molar ratio) was combined with HuNoV gRNA saRNA in citrate buffer (1:2 ratio) using a microfluidic mixing system. The mixture was filtered (0.22 µm) and stored at 4 °C. Particle size, concentration, PDI, and zeta potential were measured using NanoSight NS300 (Malvern Instruments, UK). Encapsulation efficiency (EE%) was determined via ultrafiltration centrifugation. TEM imaging (JEM-1230, JEOL, JPN) was performed after staining with 2% uranyl acetate. RNA quantification was conducted using a GeneQuant pro RNA/DNA analyzer (Cytiva). All HuNoV gRNA saRNA doses refer to the mass of the GOI. For example, 1 µg HuNoV gRNA saRNA corresponds to 7.53×1011 copies of VP1/VP2 mRNA. Details of all saRNA variants, including sequence modifications and physical characteristics, are summarized in Supplementary Information Table 1.

Detection of progeny 5′ VPg mRNA
To determine the proportion of progeny strands carrying 5′ VPg in the HuNoV gRNA saRNA replicons, 1 μg of huNoV gRNA saRNA-LNP was transfected into 107 Huh-7 cells. After 72 h, cells were harvested by scraping into 10 mL of ice-cold phosphate-buffered saline (PBS) and pelleted by centrifugation at 1500 xg for 5 min at 4 °C. The cell pellet was resuspended in 5 mL of cytoplasmic lysis buffer containing: 25 mM HEPES (pH 7.9), 5 mM KCl, 0.5 mM MgCl2, 0.5% (v/v) NP-40, protease inhibitors (Halt Protease and Phosphatase Inhibitor Cocktail, Thermo Fisher Scientific), RNase inhibitor (RiboLock RNase Inhibitor, Thermo Fisher Scientific). After 5 min of incubation on ice, cell lysates were clarified by centrifugation at 14,000 xg for 10 min at 4 °C in a microcentrifuge. For RNA capture, 100 nM of biotinylated VP1 sense or antisense primer was added to the lysate and hybridized using a thermal cycler with the following program: 85 °C for 15 min, 9–65 °C at 2 °C/s, 65–25 °C at 0.1 °C/s. Subsequently, 500 μL of pre-equilibrated Pierce™ Streptavidin Magnetic Beads (Thermo Fisher Scientific) were added and incubated at room temperature (RT) for 30 min with gentle mixing. The beads were then washed three times with cytoplasmic lysis buffer and resuspended in 300 μL of resuspension buffer from the Thermo Scientific GeneJet RNA Cleanup and Concentration Kit for RNA purification according to the manufacturer’s protocol.

Progeny mRNA analysis was performed by capillary gel electrophoresis as described by Denise A. Warzak60. Briefly: mRNA samples were diluted to 100 ng/μL in nuclease-free water. 1 μL of intercalating dye was added per 10 mL of RNA separation gel to prepare the gel/dye mixture. Appropriate markers and 2 μL of sample (or Agilent RNA Ladder, 200–6000 nt) were loaded into an Eppendorf 96-well Twin.Tec PCR plate. Samples were analyzed using an Agilent 2100 Bioanalyzer system with a 12-capillary short array.

Predicted structure of VPg/RdRp and autodocking analysis
The sequences of huNoV RdRp and huNoV VPg were retrieved from the GenBank database (GenBank: KY905331.1). Structural predictions for huNoV RdRp and VPg were generated using AlphaFold2 (https://colab.research.google.com/drive/1uAJDgAu-ejsG70h6cG-glm8YYU3-GHGt#scrollTo=kOblAo-xetgx). The resulting PDB files were visualized using PyMOL software. To assess the interaction between VPg and RdRp, the PDB files were submitted to the HDOCK online server (http://hdock.phys.hust.edu.cn/data) for docking analysis. The 376–384 region, corresponding to the A motif D, was designated as the RdRp binding site for VPg. Docking predictions were ranked by docking scores, calculated using the knowledge-based ITScorePP or ITScorePR scoring functions. Based on docking results, 10 VPg mutants were selected for further experimental analysis. Mutations at the binding sites K40 and G99 significantly affected the interaction with motif D in RdRp.

T32/P2 construct and in vitro misbase incorporation assay
The T32 template strand was chemically synthesized and purified using 10% polyacrylamide gel electrophoresis, followed by electro-elution with an Elu-Trap device (GE Healthcare). After annealing at 95 °C for 3 min, the template was stored in RNA storage buffer containing 50 mM NaCl, 5 mM Tris (pH 7.5), and 5 mM MgCl2. The T32/P2 construct was formed by annealing the T32 strand with UU dinucleotide primers at a molar ratio of 1:1.25, incubated at 45 °C for 3 min, then cooled to 4 °C for storage.

In vitro RNA replication reactions were performed in 20 µl reactions at 30 °C for 3 h, containing 4 µM T32/P2 construct, 50 µM synthetic VPg, 1 µM purified RdRp, 300 µM ATP, 300 µM UTP, 20 mM NaCl, 50 mM Tris (pH 7.0), 5 mM MgCl2, and 2.5 mM DTT. Following the reaction, RNA was analyzed by high-resolution capillary gel electrophoresis, and the misincorporation fraction (rmis) of mismatch products P9 and P42 was calculated. The rmis values were fitted to a Michaelis–Menten model: Rmis = k mis × [S] / (KMapp + [S]), where k mis is the rate constant, and KMapp is the Michaelis constant for UTP.

Biolayer interferometry (BLI) experiments
BLI experiments were conducted using an Octet 8-channel system (Sartorius) in HBS buffer supplemented with 0.05% (w/v) Tween-20 as the blocking buffer. VPg (30 nM) was immobilized onto Octet protein A biosensors. Subsequently, the biosensors were immersed in wells containing purified RdRp proteins, lacking specific motifs, at concentrations of 320, 160, 80, 40, 20, and 10 nM for 200 s. The biosensors were then transferred to wells containing HBS buffer supplemented with 0.05% (w/v) Tween-20 for 300 s to measure dissociation. The data were reference-subtracted and curve-fitted using the “association-dissociation” model in GraphPad Prism.

Determination of progeny mRNA mutation frequency by T7 endonuclease I cleavage assay
Huh-7 human hepatoma cells were maintained in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin at 37 °C under 5% CO2. For transfection, 1×107 cells were seeded in 6-well plates and transfected at 70–80% confluency with 1 μg of HuNoV gRNA saRNA-LNP and the corresponding VPg mutant strains. Untreated cells and empty LNP-transfected controls were included. Cells were harvested 72 h post-transfection for RNA extraction. Total RNA was isolated using TRIzol Reagent (Invitrogen). Briefly, cells were lysed in TRIzol, mixed with chloroform, and centrifuged at 12,000 xg for 15 min at 4 °C. The aqueous phase was precipitated with isopropanol, washed with 75% ethanol, and dissolved in RNase-free water. RNA concentration and purity (A260/A280 ≥ 1.8) were verified by NanoDrop™ spectrophotometry. Reverse transcription was performed with 1 μg RNA using a PrimeScript™ RT Reagent Kit (Takara) with oligo(dT)18 and random hexamer primers in a 20 μL reaction (25 °C for 5 min, 42 °C for 60 min, 70 °C for 5 min). VP1 and VP2 genes were amplified using gene-specific primers (VP1-F: 5′-GGGACCTGGGTGGCGACTA-3′, VP1-R: 5′-CAGCCACACCTTCGGCATA-3′; VP2-F: 5′-TGGGTGCTGGAGTTGATGG-3′, VP2-R: 5′-CCTCGGTGGCAATCTCTTC-3′). GAPDH (F: 5′-GAAGGT GAAGGTCGGAGTC-3′, R: 5′-GAAGATGGTGATGGGATTTC-3′) served as the endogenous control. PCR reactions (25 μL) contained 2 μL cDNA, 10 μM primers, and 2× Taq PCR Master Mix (Thermo Fisher), with cycling conditions: 95 °C for 5 min; 35 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 45 s; final extension at 72 °C for 5 min. The reverse transcription product was subjected to denaturation and hybridization in a PCR thermocycler (95 °C for 5 min, followed by temperature reduction from 95 to 85 °C at 2 °C/s, then from 85 to 25 °C at 0.1 °C/s) to ensure generation of mutant chimeras. Subsequently, 10 U of T7 Endonuclease I (Beyotime) was added to the hybridization product and incubated at 37 °C for 30 min for enzymatic digestion. Finally, the digestion results were analyzed by capillary gel electrophoresis (Agilent 2100 Bioanalyzer system), and the number of resulting cleavage fragments was quantified.

RdRp and VPg recombinant proteins
To obtain recombinant RdRp and its mutant variants for in vitro experiments, the PUC19-hNoV gRNA plasmid (digested with KasI/SbfI) and corresponding RdRp mutant constructs were used as templates for PCR amplification. The RdRp region (nt 3025-5097) was amplified using the following primers: forward primer: 5′-GACTCA GAATTCatgGCCCCGCCAAGCATTTGGT-3′ (EcoRI site underlined, start codon in lowercase), reverse primer: 5′-GTCACTGCGGCCGCt caGTGGTGGTGGTGGTGGTGCTCTCACTCGACGCCATCTTCATTTCA-3′ (NotI site underlined, stop codon in lowercase, His-tag in bold). The PCR product was digested with EcoRI/NotI and ligated into the similarly digested pET-28b(+) expression vector (Novagen) using T4 DNA ligase, generating the construct pET-his-RdRp. For VPg protein expression, the VPg region (nt 2626–3024) was amplified from KasI/SbfI-digested PUC19-hNoV gRNA using: forward primer: 5′-CAGT CAGGTACCatgGGCAAGAAGGGGAAGAACA-3′ (Acc65I site underlined, start codon in lowercase), reverse primer: 5′-CAGTCACTCGAGt caAAAACTGAGTTTCACATT-3′ (PaeR7I site underlined, stop codon in lowercase). The amplified product was digested with Acc65I/PaeR7I and cloned into the pE-SUMO vector (LifeSensors) via T4 DNA ligase, yielding pE-His-SUMO-VPg.

The constructed pET- and pSUMO-based plasmids were transformed into E. coli BL21 (DE3) cells (Novagen). Protein expression was induced with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG), and recombinant proteins were successfully expressed in the E. coli lysate. All recombinant proteins were purified using Ni-NTA affinity chromatography. Elution was performed using a buffer containing 300 mM NaCl, 50 mM NaHPO4, 10% glycerol, and 60 mM imidazole (pH 8.0).

The recombinant his-tagged proteins—his-RdRp ΔT84, his-RdRp ΔK146, his-RdRp ΔY151, his-RdRp A1Q/P2Q, and his-RdRp V82I—were dialyzed overnight at 4 °C in a buffer containing 150 mM KCl, 50 mM Tris (pH 8.0), 2 mM DTT, and 20% glycerol. The dialyzed proteins were aliquoted and stored at −80 °C.

For His-SUMO-VPg, the proteins were dialyzed overnight in PBS (pH 7.4) with 10% glycerol and 2 mM DTT at 4 °C. SUMO tags were removed by incubating the purified fusion proteins at 1 mg/ml with His-tagged SUMO protease (1 U/20 μg) (LifeSensors, Inc.) overnight at 4 °C. Following tag removal, the protein mixtures were diluted to 2 ml and incubated with 250 μl of a 50% Ni-NTA resin suspension (Qiagen) for 1 h at 4 °C. After incubation, the supernatant containing untagged VPg K40N or G99V was collected by centrifugation at 10,000 xg for 10 min.

The supernatant was concentrated using a 10 kDa cutoff Centricon column (Millipore) by centrifugation at 3000 xg for 45 min, reducing the final volume to 1 ml. The concentrated proteins were then dialyzed overnight at 4 °C in a buffer containing 150 mM KCl, 50 mM Tris-HCl (pH 8.0), 2 mM DTT, and 20% glycerol. This was followed by a second round of concentration using a 10 kDa cutoff Microcon column (Millipore) by centrifugation at 10,000 xg for 30 min. Protein concentrations were determined using the Bradford assay, and samples were stored at −80 °C.

In vitro generation of nucleosidylated VPg
Purified HuNoV RdRp and VPg peptides (>99% purity, confirmed by Coomassie staining) were used to nucleosidylate VPg in a 20 µl reaction mixture containing 50 mM HEPES buffer (pH 7.0–7.5), 3.5 mM magnesium acetate, 6–8% glycerol, 1 µl NTP mix (10 mM), 50 µM recombinant VPg, and 1 µM recombinant RdRp. The reaction was incubated at 33 °C for 60 min. The formation of VPg-pU was assessed by liquid chromatography–Tandem mass spectrometry (LC–MS/MS)46, and the selectivity of VPg nucleosidylation was evaluated using high-performance liquid chromatography (HPLC).

HPLC separation and LC–MS/MS analysis of nucleosidylated VPg
LC–MS/MS analysis of nucleosidylated VPg was performed on a total of 2 samples, including unmodified VPg (n=1) and nucleosidylated VPg (n=1) species with 3 technical replicates. Samples were prepared by diluting purified VPg or in vitro nucleotidylation reaction mixtures in LC–MS-grade water, centrifuging at 12,000 xg to remove particulates, and injecting 1 μl of each preparation onto a CAPCELL CORE AQS 2.7 column (2.1 × 150 mm). HPLC separation was carried out on a Waters Alliance 2795 system equipped with a 2996 PDA detector using 10 mmol/L ammonium phosphate as the mobile phase at a flow rate of 400 μl/min, allowing up to 200 μg of VPg per injection without loss of resolution. Fractions corresponding to individual nucleosidylated VPg species were manually collected and stored at −80 °C for subsequent LC–MS analysis. Mass spectrometry was performed on a Waters Q-TOF Micro instrument equipped with a dual electrospray ionization lockspray source, acquiring data in positive-ion mode under identical conditions for sample eluates and a VPg reference infused at 2 μl/min. Source parameters included a source temperature of 120 °C, desolvation temperature of 300 °C, and cone voltages of 25 V (reference) and 20 V (sample). Raw spectra were processed and deconvoluted using MassLynx 4.1 with the MaxEnt 1 Transform algorithm to obtain zero-charge intact molecular masses. Because nucleotidylated VPg yields intact mass shifts rather than proteolytic peptides, peptide-database searches, missed-cleavage criteria, FDR filtering and peptide-scoring parameters were not applicable. Instead, VPg nucleosidylation states were identified by theoretical-to-observed mass matching, with VPg-pU confirmed by an intact mass increase of 303.17 Da corresponding to uridylylation at Tyr30 (Y30). All LC–MS analyses were repeated independently at least 3 times to ensure reproducibility.

In vitro extension reaction system for HuNoV gRNA mRNA
The purity of HuNoV 2C-L helicase and RdRp proteins (>99%, confirmed by Coomassie staining) was assessed prior to their use in in vitro extension reactions. Reactions were carried out in a 25 µl system containing 50 mM HEPES buffer (pH 7.0–7.5), 3.5 mM magnesium acetate, 6–8% glycerol, 50 µM VPg-pU peptide primer or 80 nM pU22 primer, 0.5–16 µM HuNoV gRNA saRNA template, and 1 µM each of purified 2C-L helicase and RdRp. After incubation at 37 °C for 60 min, the reaction mixture was applied to a Ni-NTA column and eluted with 60 mM imidazole buffer. The production of progeny his-5′ VPg mRNA was finally assessed by capillary gel electrophoresis (Agilent 2100 Bioanalyzer system).

Structural rearrangement of huNoV gRNA saRNA and insertion of GOI sequences
To construct the huNoV gRNA saRNA backbone, the ORF1 (nt 1–5105) regions was amplified from the PUC19-hNoV gRNA template using BsmBI-restricted primers, followed by Golden Gate assembly into the pRC0569 vector. Concurrently, synthetic DNA fragments containing: Rts+polyT, GFP1(-), Kozak(-), HuNoV 3′ UTR were similarly assembled into pRC0569 via BsmBI-mediated Golden Gate cloning (all sequence details in Supplementary Information Table 2). The reaction mixture (2 µL) was transformed into Turbo Competent Cells (NEB), plated on carbenicillin agar, and nongreen colonies were selected for miniprep and sequencing. For secondary assembly, the above fragments were cloned into the pIVTRup-T7 vector using a BsaI-based Golden Gate reaction, generating pIVTRup-T7 VPg saRNA. Transformed cells (2 µL plasmid product) were plated on kanamycin agar, and nongreen colonies were verified by sequencing. The Ghrelin, proc1, and GSDMDENG sequences were inserted into pIVTRup-T7 VPg saRNA via BamHI/MfeI restriction sites. The plasmid was PCR-amplified using restriction-site-flanked primers, followed by column purification. 1 µg linearized template per 20 µL reaction with T7 RNA polymerase (NEB).

Optimization of the 3′ UTR sequence
To enhance the translation efficiency of huNoV gRNA, the 3′ UTR was optimized by designing randomized insertions of the AXXU sequence into a VPg-binding RNA aptamer (apt-VPg). Among seven different-length apt-VPg candidates theoretically predicted to exhibit high VPg-binding affinity, in vitro experiments involving 2C-L RdRp and VPg-pU elongation on a ghrelin mRNA template confirmed that the 42-nt apt-VPg (tgtgaaaagacaaaattgattatctattcatttattcattta) demonstrated superior secondary structure stability and higher VPg-binding activity. For the construction of huNoV gRNA saRNA with an optimized 3′ UTR sequence, the 42-nt apt-VPg was used to replace the original 3′ UTR in the triple-mutant PUC19-hNoV triM-gRNA template plasmid using the Q5 Site-Directed Mutagenesis Kit (New England Biolabs). To generate replace, the following primers were employed: forward primer: 5′-atctattcatttattcatttactagaaagcacacgg-3′, reverse primer: 5′-aatcaattttgtcttttcacattaagcccgtgactc-3′. Subsequently, standard procedures were carried out, including IVT, methylation modification, tailing, capping, and LNP encapsulation.

Fully automated Western blot analysis
Total protein concentration in the cell lysates was determined using a Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Briefly, a standard curve was generated using bovine serum albumin (BSA) 0–2000 μg/mL, and sample absorbance was measured at 562 nm using a microplate reader (BioTek Synergy H1). For Western blot analysis, 5 μg of total protein per sample was mixed with 1× fluorescent master mix (ProteinSimple) containing a fluorescent molecular weight marker and reducing agent (DTT). The samples were heated at 95 °C for 5 min to ensure complete denaturation before loading. Protein separation and immunodetection were performed using the Jess™ Fully Automated Western blot system (Bio-Techne) with the following parameters: separation module: 12–230 kDa (SM-W008, 25-capillary cartridge), separation time: 30 min (optimized for high-resolution separation). Automated data collection: the Jess system performed capillary-based size separation, immunoprobing, washing, and chemiluminescent detection without manual intervention. Raw data were processed using Compass for Simple Western™ (ProteinSimple, v6.1.0). Peak area quantification was used to determine relative protein expression levels.

Dot blot analysis of viral replication intermediate dsRNA
To detect dsRNA replication intermediates generated during viral infection, a dot blot assay was performed as follows: cells were lysed in TRIzol reagent (Invitrogen) for total RNA extraction, followed by phase separation with chloroform. The aqueous phase containing RNA was mixed with an equal volume of 2× RNA loading buffer (95% formamide, 0.025% SDS, 0.5 mM EDTA, 0.02% bromophenol blue) and heated at 65 °C for 5 min to denature secondary structures. Ten microliters of the RNA-containing supernatant was spotted directly onto a nitrocellulose membrane (0.45 μm pore size, Millipore) using a Bio-Dot microfiltration apparatus (Bio-Rad). The membrane was air-dried for 15 min to allow RNA binding. UV cross-linking (254 nm, 120 mJ/cm², Stratagene Stratalinker) was performed to immobilize RNA onto the membrane. To confirm equal sample loading, the membrane was stained with Ponceau S (Beyotime) for 5 min, followed by destaining in PBS-T (0.1% Tween-20) until protein spots were clearly visible. The membrane was blocked in 5% nonfat milk in PBS-T for 1 h at RT to prevent nonspecific antibody binding. Anti-dsRNA monoclonal antibody (Scicons, 1:1000 dilution in blocking buffer) was applied overnight at 4 °C with gentle agitation. HRP-conjugated anti-mouse IgG (Cell Signaling, 1:5000 dilution) was applied for 1 h at RT. Enhanced chemiluminescence (ECL, Thermo Fisher) was used for visualization. Images were acquired using a ChemiDoc MP Imaging System (Bio-Rad) with Image Lab software.

Confocal microscopy
293T cells were seeded at low density in 35 mm glass-bottom dishes (MatTek) and cultured overnight at 37 °C in 5% CO2. Non-adherent cells were washed with DMEM (HyClone) supplemented with 10 mM HEPES, 100 μg/ml penicillin, 100 μg/ml streptomycin, and 10% heat-inactivated FBS. Following transfection with huNoV gRNA saRNA-LNPs, cells were fixed with paraformaldehyde. After fixation, primary antibodies (rabbit anti-his-VPg, mouse anti-p22, goat anti-erp72) were applied in the dark, followed by fluorescent secondary antibodies. Imaging was performed using a Leica STELLARIS DIVE confocal system (Wetzlar) according to the excitation wavelength specified by the fluorescent antibody instructions.

qPCR
Gene-specific TaqMan primers were used for qPCR, and reactions were carried out using LightCycler 480 Probe Master Mix and a LightCycler 480 instrument (Roche). Each sample was analyzed in technical replicates for data reliability. Gene expression levels were normalized to GAPDH for accurate comparisons using the comparative Ct method. The required primer sequences are as follows: VP1 primer 5′-atgaa gatggcgtcgagtgacg-3′, 5′-ttataccacacgtctacgcc-3′; VP2 primer 5′-atggctggagctttctttgctg-3′, 5′-ttaagcccgtgactcccctc-3′; ORF1 primer 5′-cacctccgagtggaagtggt-3′, 5′-tcgacctcgacagccgagtc-3′; ifit1 primer 5′-cagctgcagaggtgtgaaga-3′, 5′-tggtgcagattccaggtgat-3′; mx2 primer 5′-gctgggacttcttcgacctg-3′, 5′-cagccacattcggttcctta-3′; infa1 primer 5′-atggctgtccttgctcagac-3′, 5′-ggcagacaggaggtccaatc-3′; infb1 primer 5′-aaactcatgagcagtctgca-3′, 5′-aggagatcttcagtttcggagg-3′.

ELISA assay to detect cytokine release
Huh-7 cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin–streptomycin at 37 °C under 5% CO2. Cells were seeded in 6-well plates (1×106 cells/well) and treated according to experimental conditions. Supernatants were collected at specified time points, centrifuged (300 xg, 5 min, 4 °C) to remove debris, and stored at −80 °C until analysis. Cytokine levels were quantified using commercial ELISA kits (IFN-α: Invitrogen; IFN-β: R&D Systems; CXCL10: Abcam) following manufacturers’ protocols. Briefly, standards and samples were loaded in duplicate onto pre-coated plates, incubated with detection antibodies, and developed using TMB substrate. Absorbance was measured at 450 nm (reference 570 nm) using a microplate reader (BioTek Synergy H1). Standard curves were generated using a 4-parameter logistic model, and sample concentrations were interpolated from the standard curve. Assay validity was confirmed by R2 > 0.99 for standard curves, intra-assay CV < 10%, and inter-assay CV < 15%. For normalization, total protein concentration was determined by BCA assay when required.

Posthumous muscle fiber analysis from cancer patients with cachexia
The CRISPR/Cas9 system was delivered into target cells using third-generation lentiviral vectors (pLenti-CRISPRv2, Addgene). The lentiviral transfer plasmid was co-transfected with packaging plasmids (psPAX2 and pMD2.G) into HEK293T cells using Lipofectamine 3000 (Thermo Fisher). Viral supernatants were collected at 48 and 72 h post-transfection, concentrated via ultracentrifugation (25,000 xg, 2 h, 4 °C), and titered using a qPCR-based lentivirus titration kit (Takara). Single-guide RNAs (sgRNAs) targeting key GOI were designed using the CRISPR Design Tool (Broad Institute) and CHOPCHOP v3, with selection based on high on-target efficiency and minimal off-target effects. The following sgRNA sequences were cloned into the BsmBI-digested pLenti-CRISPRv2 vector via Golden Gate Assembly (NEB): target gene sgRNA sequence PAM mTOR: attagttcctggccgggttt; eif4e: agaaatctag tagactccta; Eif4g1: cttgccctgttgattactca; pink1: actggcggaagaagcggaga; MHC-I (HLA-A): acggccatcctcggcgtctg. Cloning success was confirmed by Sanger sequencing (Tsingke Biotechnology) using the U6-F primer (5′-gagggcctatttcccatgatt-3′). Target cells were transduced with lentiviral particles at an MOI of 5–10 in the presence of 8 µg/mL polybrene (Sigma). After 48 h, stable integrants were selected using 2 µg/mL puromycin (Thermo Fisher) for 7 days. GFP-positive cells were further enriched via fluorescence-activated cell sorting (FACS, BD FACSAria III) to generate a polyclonal knockout pool.

Human skeletal muscle tissue collection
Postmortem skeletal muscle tissue samples were retrospectively collected from the biobank of pathology at the Red Cross Body Donation Center, Wannan Medical College (Wuhu, China). The tissues were resected, formalin-fixed, and paraffin-embedded (FFPE). Samples were obtained from 14 body donors aged 35–58 years, of whom 42.8% were female. These donors exhibited significant weight loss and were pathologically diagnosed with cancer between 2015 and 2021, forming an independent cohort that included 4 cases of lung adenocarcinoma (LUAD), 4 cases of pancreatic adenocarcinoma (PAAD), and 3 cases of colon adenocarcinoma, as well as 3 healthy donors who died from accidental trauma.

Posthumous muscle fiber analysis from cancer patients with cachexia
Muscle tissues were fixed in 10% neutral buffered formalin for 24–48 h, followed by standard paraffin embedding. Serial sections (4 μm thickness) were prepared using a rotary microtome (Leica RM2235). Slides were subjected to heat-induced epitope retrieval in citrate buffer (pH 6.0) at 95 °C for 20 min. Primary antibodies (all diluted in PBS with 1% BSA): phosphorylated 4E-BP1 (Thr37/46); phospho-mTOR (Ser2448); phospho-eIF4E (Ser209); phospho-eIF4G (Ser1108). HRP-conjugated secondary antibodies (Dako EnVision+ System), DAB chromogen and Hematoxylin counterstaining. Images of sections were collected using an (Olympus IX73) microscope and quantified using ImageJ.

KPC mouse model for cachexia, weight loss, and organ damage
KPC mice (Trp53LSL-R172H; Kras+/LSL-G12D; Pdx1-Cre) were used as a spontaneous pancreatic cancer model to monitor cachexia. Body weight, food intake, and survival were recorded weekly starting at 8 weeks of age. When KPC mice lost >5% body weight, they were administered VPg Ghrelin saRNA-LNP intraperitoneally. Mice were euthanized with pentobarbital (200 mg/kg, intraperitoneally) once body weight loss reached 20%. Muscle, subcutaneous fat, liver, and blood samples were collected post-mortem.

Blood was analyzed for liver and kidney damage (AST/ALT, creatinine, and urea nitrogen levels) and platelet counts to assess hematopoietic damage. Muscle and subcutaneous white adipose tissue were fixed in formaldehyde for 48 h, embedded in paraffin, and sectioned for H&E staining. Immunohistochemistry was performed to evaluate phosphorylated 4E-BP1 Thr37/46, mTOR Ser2448, eIF4E Ser209, and eIF4G Ser1108 expression. Images were captured with an Olympus BX51 microscope equipped with a DP72 camera. RNA was extracted from muscle, fat, and liver tissue for qPCR analysis of ribosome, endoplasmic reticulum, and mitochondria-associated gene expression.

Construction of linear mRNA and VEEV saRNA controls
The construction of linear Cap1-m7G GSDMDENG mRNA-LNP was performed as previously described45. Briefly, the coding sequences of hGSDMD FL and hGSDMD NT were amplified from human fibroblasts. The IRES and F1L CT sequences were chemically synthesized by GenScript (New Jersey, USA). Gibson assembly was performed using the NEBuilder HiFi DNA Assembly Kit (NEB) on a linearized pUC19 vector containing the T7 promoter. The ATG start codon and hGSDMD mutations (c.884A>G/c.825T>A) were introduced using the Q5 site-directed mutagenesis kit (NEB). The assembled product was transformed into 5-α competent E. coli (NEB), and single clones were selected after carbenicillin screening and verified by Sanger sequencing. IVT was performed using the HiScribe T7 high-yield RNA synthesis kit (NEB), with 4 mM hm5CTP substituted for CTP to enhance mRNA stability. The IVT product was digested with DNase I to remove the DNA template, and unmodified linear mRNA was purified using the MEGAclear transcription clean-up kit (Ambion). Capping with m7G-cap and poly(A) tailing were conducted at 70 °C (5 min) and 4 °C (3 min), respectively. Further purification by column chromatography yielded high-purity Cap1-m7G-modified GSDMDENG mRNA for subsequent LNP encapsulation.

The VEEV, VEEV-SINV, and SFV backbone saRNA T7-IVT vectors were all provided by VectorBuilder. Using VEEV GSDMDENG saRNA as an example, the full-length GSDMDENG oncolytic sequence was amplified from the linear GSDMDENG mRNA vector using restriction enzyme digestion primers: forward primer 5′-GGACA TATGgggtcggcctttgagcggg-3′ (the underlined sequence indicates the NdeI site) and reverse primer 5′-CTCCTCGAGttatcctatcatgtatttga gagtttt-3′ (the underlined sequence indicates the XhoI site). The amplified product was digested with NdeI/XhoI restriction enzymes and ligated into the NdeI/XhoI-digested VEEV-saRNA T7-IVT plasmid (VectorBuilder, Guangzhou, CHN) using T4 DNA ligase. Subsequently, standard procedures were performed, including IVT, methylation modification, tailing, capping, and LNP encapsulation.

Mitochondrial analysis
Mitochondrial and cytosolic fractions were separated using the mitochondria/cytosol isolation kit (Biovision, CA, USA) following the manufacturer’s protocol. Approximately 106 cells were resuspended in 2 ml cytosol extraction buffer and disrupted using a Potter-Elvehjem PTFE grinder (Sigma). The homogenate was centrifuged at 1200 xg for 10 min at 4 °C, and the supernatant was further centrifuged at 12,000 xg for 10 min at 4 °C. The supernatant served as the cytosolic fraction, while the pellet (mitochondrial fraction) was washed and collected. GSDMD expression in both fractions was analyzed by immunoblotting, with TOM20 serving as a mitochondrial marker. Total DNA was extracted from 300 µl of cytosol using the QIAquick Nucleotide Removal Kit (Qiagen) and eluted in 30 µl. Mitochondrial DNA (mtDNA) was quantified using the QX200 Droplet Digital PCR System (Bio-Rad) with ND1 as a marker and β-actin as an internal control.

Animal and animal ethics
All animal procedures were approved by the Animal Ethics Committee of Wenzhou Medical University (approval no. XMSQ2024-0471) and conducted in accordance with institutional and national guidelines. All mice were obtained from Cyagen Biosciences or Jackson lab and housed in a specific pathogen-free facility under controlled temperature (20–24 °C), humidity (40–60%) and a 12-h light/dark cycle, with ad libitum access to sterilized water and a standard rodent chow diet (LabDiet 5001, catalog no. 0007688; protein 23.9%, fat 5.0%, carbohydrate 55.2%, fiber 5.0%; 3.36 kcal/g). Animals were maintained in individually ventilated cages with autoclaved corncob bedding and provided with environmental enrichment. NSG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl) were used for immunodeficient studies. KPC mice (LSL-KrasG12D; LSL-Trp53R172H; Pdx1-Cre) were used as a spontaneous PDAC model. Wild-type C57BL/6 mice were used as background controls. Age- and sex-matched mice were used for all in vivo experiments, and humane endpoints were implemented according to the approved protocol to minimize animal distress.

Tumor-bearing mouse model with humanized immune system
CD34+ cells were isolated using the direct CD34+ precursor cell isolation kit (Miltenyi Biotec). Newborn NSG mice were sublethally irradiated with 100× cGy and subsequently received CD34+ stem cells injected into the liver. Human CD45+ cells in peripheral blood were assessed by flow cytometry at 8 and 12 weeks post-transplantation to confirm engraftment. Beginning on day 8 post-transplant, mice were administered 30 µg of recombinant human FLT3-L protein intraperitoneally each day. At 13 weeks post-transplantation, 5×105 AsPC-1 tumor cells were implanted subcutaneously in humanized mice. VPg GSDMDENG saRNA or VEEV GSDMDENG saRNA was injected peritumoral around the tumor on day 5 post-implantation. Tumor size was measured every 2–4 days with an electronic digital caliper, and tumor volume was calculated as length×width×height (mm3). Mice were euthanized when tumor volume exceeded 1000 mm³.

Construction and application of AsPC1-mtOVA-mCherry cells
To generate AsPC1 cells expressing mitochondrially-targeted ovalbumin (OVA257-264) fused with mCherry, we first cloned the OVA257-264 sequence into the PHR-SIN-mCherry vector to create an N-terminal mCherry fusion construct. Lentiviral particles were then produced using this plasmid and subsequently transduced into AsPC1 human pancreatic cancer cells. For in vivo studies, humanized immune system mice bearing AsPC1-mtOVA-mCherry tumors received weekly VPg GSDMDENG saRNA or VEEV GSDMDENG saRNA peritumoral injections (1 μg/kg). On day 42 post-implantation (with analysis performed at day 14), mice were sacrificed and draining lymph node cells were harvested. These cells were then stained and analyzed for CD80, CD86, and CD40 expression by flow cytometry to assess antigen-presenting cell (APC) (CD45+ CD103+ CD11c+ CD11b−) uptake of the mCherry-tagged antigen.

IFN-γ ELISPOT analysis
Fourteen days after peritumoral administration of either VPg GSDMDENG saRNA or VEEV GSDMDENG saRNA, tumor-bearing mice were euthanized, and spleens were aseptically harvested. Single-cell suspensions were prepared by mechanically dissociating splenic tissues through a 70-µm nylon cell strainer (BD Biosciences). Erythrocytes were subsequently lysed using ACK lysis buffer (BioWhittaker), and the remaining leukocytes were washed and resuspended in complete RPMI-1640 medium supplemented with 10% FBS, 1% penicillin-streptomycin, and 50 µM β-mercaptoethanol. For IFN-γ detection, 96-well ELISPOT plates (pre-coated with PVDF membranes containing anti-IFN-γ capture antibody, Biosciences) were seeded with 2× 105 splenocytes per well and incubated for 24 h at 37 °C in a 5% CO2 humidified incubator. Following stimulation, cells were removed, and plates were washed extensively before incubation with a biotinylated anti-IFN-γ detection antibody (Biosciences). After additional washing, spots were developed using streptavidin-conjugated fluorescent secondary antibody and visualized under a fluorescence microscope (Olympus IX73). Spot enumeration was performed using an automated ELISPOT reader (AID iSpot) and analyzed with AID ELISPOT software.

In vivo T cell proliferation assay
OT-I cells originated from transgenic mice expressing CD8+ T cell receptors specific for MHC-I-restricted OVA peptide (257–264), while OT-II cells originated from mice with CD4+ T cell receptors specific for MHC-II-restricted OVA peptide (323–339). Two days before VPg GSDMDENG saRNA treatment, 2× 106 OT-I cells or OT-II cells were purified and transferred intravenously into tumor-bearing mice. Four days later, draining lymph nodes were isolated, and OT-I or OT-II cell proliferation was analyzed by flow cytometry. Cells were stained with anti-CD16/CD32 antibodies to block Fc receptors, followed by labeling with anti-CD4, CD8, CD3, CD19, and MHC-I dextramer H-2 Kb/SINFEKL. After permeabilization, Ki67 expression levels were measured to determine proliferation activity.

In vitro CLT cell killing assay
Tumor-draining lymph nodes were harvested from KPC mice treated with VPg-GSDMDENG or VEEV-GSDMDENG saRNA-LNP. Antigen-specific CD8+ T cells were isolated via H-2Db KrasG12D tetramer sorting or IFN-γ capture after AsPC-1 lysate stimulation, then co-cultured with IFN-γ-primed KRAS-mutant lines (AsPC-1, MDA-MB-231, HT-29, NCI-H358, SW480) in real-time impedance (xCELLigence), flow cytometric (CFSE/7-AAD), and MHC-I-blockade-controlled cytotoxicity assays at E:T ratios of 20:1-2.5:1.

Establishment of mouse GvHD model
Pan T cells were isolated from mouse spleens by depleting non-T cells using the Mouse Pan T Cell Isolation Kit II (Miltenyi Biotec) according to manufacturer guidelines. The purity of T cells (95–98%) was confirmed by flow cytometry. Bone marrow cells were flushed from the tibiae and femurs, with RBCs lysed using ACK buffer (BioWhittaker). For MHC-incompatible GvHD induction, C57BL/6-derived bone marrow and splenic T cells were transplanted into B6D2F1 recipients. For MHC-matched GvHD induction, C57BL/6-derived bone marrow and T cells were transplanted into B10.BR recipients. Recipient mice (8–10 weeks old) underwent total body irradiation with a 12.5 Gy single dose using a BioBeam 8000 (Gamma Service Medical).

Mice were anesthetized with 100 µl of a solution containing Ketavet (20 mg/ml) and Rompun (1 mg/ml) and immobilized in an irradiation chamber. Four hours post-irradiation, recipients were injected intravenously with 4× 106 mismatched bone marrow cells and 4× 106 purified mismatched Pan T cells. Body weight was monitored on day 1 and weekly thereafter. Survival was assessed daily. GvHD clinical scoring followed an established system61, including weight loss, posture, activity, hair texture, and skin integrity, with weekly evaluations and a cumulative score based on the sum of the five parameters.

Flow cytometric analysis of GCB and T follicular cells
Single-cell suspensions from lymphoid tissues were prepared by mechanical dissociation, followed by RBC lysis (ACK buffer) and viability staining (Ghost Violet™ 510). Cells were Fc-blocked (anti-CD16/32) and stained for surface markers: GCB cells (CD19+ CD95hi GL7+), TFH cells (CD4+ PD-1+ CXCR5+ FoxP3−), and TFR cells (CD4+ PD-1+ CXCR5+ FoxP3+). Intracellular FoxP3 staining was performed using a fixation/permeabilization kit. Samples were acquired on a BD LSRFortessa™, with doublets excluded (FSC-H vs. FSC-W) and live cells gated (Ghost Violet™ 510).

Detection of autoantibodies in mice
Serum levels of IgM, IgG, IgA, and IgE were quantified using commercial ELISA kits (eBioscience) with absorbance measurements performed on a SpectraMax M5 microplate reader (Molecular Devices). For autoantibody detection, ELISA plates were pre-coated with either 2.5 μg/ml calf thymus double-stranded DNA (Sigma) or recombinant/isolated autoantigens, including La protein, Ro52, and Ro60 (10 μg/ml for recombinant proteins; 5 μg/ml for native Ro60; Arotec Diagnostics). Serially diluted serum samples were incubated with the coated antigens, followed by detection using alkaline phosphatase (AP)-conjugated goat anti-mouse secondary antibodies specific for IgM, IgG, IgA, or IgE (Southern Biotech).

3D immunofluorescence staining of VPg and PROC1 in mouse lung tissue
Mouse lung tissues were fixed in 4% paraformaldehyde at 4 °C for 24 h, followed by three washes in PBS (pH 7.4). Tissues were permeabilized with 0.5% Triton X-100 in PBS for 1 h at RT and blocked with 5% BSA containing 0.1% Triton X-100 for 2 h at RT. For immunostaining, tissues were incubated with primary antibodies against VPg (1:200) and PROC1 (1:200) diluted in blocking buffer at 4 °C for 48 h with gentle agitation. After washing with PBST (PBS + 0.1% Tween-20) three times (1 h each), samples were incubated with fluorophore-conjugated secondary antibodies (Alexa Fluor 594 for VPg, Alexa Fluor 488 for PROC1; 1:500 and DAPI for nuclear counterstaining at RT for 4 h. For 3D visualization, tissues were cleared using the CUBIC method: Dehydration in CUBIC-1 solution (25% urea, 25% N,N,N′,N′-tetrakis(2-hydroxypropyl)ethylenediamine) for 3 days at 37 °C. Refractive index matching in CUBIC-2 solution (50% sucrose, 25% urea) for 1 day. Cleared tissues were imaged using a light-sheet fluorescence microscope (e.g., Zeiss Lightsheet Z.1) with a Z-step size of 2 μm. 3D reconstruction and colocalization analysis were performed using Imaris 9.0 (Bitplane).

Statistics and reproducibility
No statistical method was used to predetermine sample size in all the highly controlled in vitro or in vivo experiments, but our sample sizes are similar to those reported in previous publications. For each experiment, we aimed for at least three samples or animals per group to allow basic statistical significance. The exact sample sizes used for each experiment are provided in the corresponding figure legends. No data were excluded from the analyses.

All experiments were reliably reproduced, and data are presented as mean ± s.d. unless otherwise stated. All experiments were repeated with at least three independent biological donors or performed independently three times. Statistical significance between experimental groups was assessed using two-tailed Student’s t tests or one-way or two-way analysis of variance (ANOVA) followed by the appropriate post hoc test or other specified statistical methods, as indicated in the respective figure legends. A P value of 0.05 or less was considered statistically significant.

For in vitro experiments, cells were randomly allocated into control and experimental groups. For in vivo experiments, age- and sex-matched mice were randomized into control and experimental groups. For therapeutic animal experiments, investigators responsible for data collection were blinded to treatment allocation. No blinding was used for in vitro experiments. The majority of data collection involved quantifiable endpoints for which blinding would not influence measurement bias.

Ethical statement
All cadaveric specimens were obtained through the Wannan Medical College Red Cross Body Donation Center with full ethical compliance. Each donation was accompanied by properly executed informed consent documents, signed by the donors during their lifetime, explicitly permitting the use of their remains for scientific research. The study protocol was approved by the Medical Ethics Committee of the First Affiliated Hospital of Wannan Medical College (approval number: 2022-LSX-12). In accordance with the body donation agreement, all tissue samples were destroyed after analysis, as they are permitted for research use for only 2 years.

All protocols involving animals in this study were approved by the Institutional Animal Care and Use Committee of Wenzhou Medical University and were carried out in accordance with the approved guidelines (approval no. XMSQ2024-0471).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript by Feng et al, describes the design of a new self-amplifying RNA candidate, aimed at facilitating protein\nreplacement therapy. To achieve this, they engineered a Vpg saRNA vector from human Norovirus isolates, as the Vpg will\nhelp facilitate high-fidelity replication. The Norovirus isolate replicon was initially characterised in vitro, leading to further\nsequence modifications and later, adjustments to the GOI orientation. The cap-independent translation of the Vpg saRNA\nwas confirmed in mTOR knockout cells, prior to testing the vector in a CAC model. Further engineering of the Vpg was then\napplied to increase fidelity of RdRp replication, which was confirmed using the GSDMDENG model. Other adjustments to\nthe vector, including mutations within the RdRp, UTR and arrangement of the construct were later applied. Overall, the\nmanuscript shows an interesting approach to the rational design of a Vpg saRNA vector\nspecifically for gene therapy/protein replacement approaches.\nWhile the topic is interesting and relevant, the layout of the manuscript is confusing and often important concepts and\nachievements (particularly with regards to the engineering of the vector) get lost in background literature and proof of\nconcept studies. It is also unclear why proof of concept studies was performed after each iteration, and not at the end of the\ndesign process. Lack of appropriate controls and incomplete methods add to the confusion.\nGeneral Major comments:\n1. The statement ‘The engineered Vpg saRNA is adapted for a range of therapeutic scenarios, including treatment of tumor-\nassociated cachexia under conditions of translational restriction in cap-dependent metabolism, precise encoding of oncolytic\nmRNAs in vivo to achieve complex functionality, and therapy for graft-versus-host disease in highly auto-immune\nenvironments’ is not accurate.\nThere are several iterations of the engineered Vpg saRNA described in this manuscript and it is not clear why each iteration\nwas tested in a different disease model. A diagram to show the different iterations and their purpose, as well as a complete\ncomplementary methods sections are needed.\n2. The methods section appears to be written by several individuals and is lacking coherence and appropriate details. In\naddition, certain methods do not describe the procedures in their titles (particularly the immunogenicity studies). Most of the\nmethods need more detail for experiments to be reproducible. Some results don’t have associated methods, which need to\nbe included\n3. Clear distinctions between mRNA, saRNA, capped mRNA, capped saRNA and uncapped saRNA need to be maintained\nthroughout the text to avoid confusion.\n4. Information relating to the IVT, capping, polyadenylation and formulation (both in the methods and results sections) are\nlacking. As noted by the authors, IVT of longer saRNA is complicated. It is important to show that the transcripts produced\nare of a high quality (size, integrity, purity) and that formulation was successful (size of nanoparticle, PDI and encapsulation\nefficiency). No information about polyA tail length is included, which is important for functioning of the mRNA transcripts\nwhich have been included as controls throughout the study.\n5. Appropriate controls need to be used throughout the study. Comparing the efficacy of mRNA to saRNA without copy\nnumber comparisons (important when looking at transcripts of vastly different lengths), and comparing three other alphavirus\nsaRNA constructs without any information on their RdRp design, sequence or transcript length makes it hard to determine if\nthe engineered Vpg saRNA is in fact superior to current vectors. We assume from the figures that the VEEV, VEEV-SINV\nand SFV saRNA vectors make up the three alphaviruses tested, yet there is no information about these sequences, which\nderivatives they are, and how they compare (structurally and functionally) to the Vpg saRNA. In addition, later studies simply\ndesignate an αV saRNA as a comparator without specific identification of which alphavirus has been used.\n6. Figures – in general the figures are very small and difficult to read. Many of the figures have sections of the figure,\nincluding axis and labels, cropped off, and typos. This needs to be addressed throughout the manuscript.\nSpecific major comments:\n7. Importance of TLR3 needs to be addressed. Later in the manuscript there is a focus on ablating TLR3 activation,\npresumably based on the results shown in Fig S1A. However, TLR3 seems barely activated in this study, and could be a\nresult of residual dsRNA from the IVT process (which was not confirmed). It is also unclear how this activation might occur\nduring replication given the conclusion by the authors that ‘These findings suggest that p22-induced vesicles are crucial\nstructures protecting nascent single-stranded RNA from recognition and degradation’ (line 146 – 147). If replication, and thus\nproduction of dsRNA also occurs in these vesicles, why does TLR3 get activated even in the presence of p22?\n8. Particles in Fig. S1D do not look uniform. Confirm size and PDI of LNPs with zeta sizing (as described in the methods but\nno results provided). Encapsulation efficiency is also required to confirm dose for in vivo studies – doses are given but no\nEE is described in the methods or results. How did the authors determine dose of their vaccines?\n9. Line 167 – ‘Vpg-saRNA sequentially expressed Vpg’. Please can the authors confirm how they came to the conclusion\nthat these were sequentially expressed as a single timepoint (72 hours)?\n10. Line 202 – 203: The Vpg Ghrelin saRNA constructs have an IRES. Where is this IRES derived from and how does it\nimpact VPg-mediated translation?\n11. In figure 2, the results of the KPC mice show those treated with mRNA, no untreated controls are shown (as described).\n12. Line 237 – more information on the principle of the T32/P2 construct need to be included.\n13. Line 248-249. More information needs to be included about the mismatch cleavage assay. what was done first. Chance\nof PCR introducing mutations? PCR was also done to produce the template.\nCould this also not introduce mutations? The figure shown in 3F is not correct. It is an RT-PCR so the T7E1 cleavage is on\nthe cDNA, not on mRNA. The fragmented DNA is run on a gel.\n14. Line 252 – 253. Example of where there is little to no information on the three additional alphaviruses tested. Were these\nsaRNAs also produced from a PCR amplified template? If so, there is a higher chance of mutations since alphavirus\nsequences are significantly longer.\n15. Lines 274 – 275: It appears the VPg construct used here was only described in the following paragraph. This is thus not\na comparison of just Vpg fidelity but also sequence optimisation and engineered Vpg to reduce dsRNA. See S4B.\nThe rationale and sequence elements behind this construct should be discussed. Also, these modifications to the sequence\nis only described in the next section. The order of sequence elements in alphavirus and Vpg constructs are not the same\nFig4A and S4B\n16. Line 282 – ‘The F1LCT signal peptide facilitated its transport to mitochondria, activating GSDMDNT with pore-forming\nactivity (Fig. S4D).’\nThis shows alphavirus GSDMD expression in hTERT mito, but this is not the case in Fig4E? Why is this seen in one figure\nbut not the next?\n17. Please confirm that the labels are correct in Fig 4G – 4H. Are labels correct for Vpg saRNA? Wouldn’t one expect that\nthe Vpg saRNA induces higher mtROS in ASPC1 cells compared to hTERT?\n18. Line 286 – ‘injection of 1 μg/kg Vpg GSDMDENG saRNA significantly enhanced tumor tissue ablation compared to the\nsame dose of αV GSDMDENG saRNA (Fig. 4I)’.\nWhile dose may be the same, information relating to copy number should be included. If the Vpg GSDMDENG saRNA is a\nmuch shorter transcript than the αV GSDMDENG saRNA, it is not surprising that there would be a difference in therapeutic\neffect since more copies of the Vpg saRNA were initially administered.\n19. Line 307 ‘These findings indicate that high-fidelity Vpg GSDMDENG saRNA continuously activates specific cytotoxic T\ncell responses, resulting in lasting tumor protection’.\nIs the Vpg or any of the Norovirus sequences used in the engineered saRNA designs immunogenic? Are T cells targeting\nVpg or RdRp generated? This could impact repeat administration of therapy.\n20. Lines 349 – 360. This section should be presented in the main figures, and not just in the supplementary. Further\nquestions related to this section include:\na. The authors didn’t compare WT with TriM VPG - did these changes result in significant improvements?\nb. How is Vpg saRNA affected by PKR or IFIT? Translation is cap independent, so please explain the importance of\nmeasuring the activation of PKR in the context of Vpg?\nc. Even though there is less dsRNA, is there a significant difference in protein expression compared to WT?\nd. TLR3 activation was not examined even though this was the reason for optimising the sequences? This relates to\ncomment 6.\ne. PKR, eIF and IFIT 1 were examined but alphaviruses are resistant to these so why is there less expression from the\nalphaviruses?\n21. Lines 361 – 363 – ‘In brief, these RdRp (pro) mutation strategies facilitate the efficient production of supersaturated VPg-\npU in cells, reducing dsRNA-induced immunogenicity by simultaneously suppressing positive-sense chain production,\nwithout affecting protein coding capacity.’\nProtein coding capacity between WT and Trim Vpg was not show. Please can the authors comment on how they arrived as\nthis conclusion?\n22. Line 376 – ‘effect of Vpg saRNA encoding Proc1 on cGVHD’. Is this the TriM-Vpg?\n23. Line 379 – why was Cap 1 nucleoside modified mRNA not used as a comparator? In the current form, this Cap 0 mRNA\nis not suitable for in vivo studies, nor does it serve as an appropriate comparison for therapeutic applications. By using an\ninferior mRNA design, it is not surprising that the saRNA outperforms the mRNA.\n24. Line 382 – 383. ‘saRNA induced systemic autoimmune responses in C57BL/6 (B6) background mice, including\nactivation of PKR, eIF2α, MDA5, RIG-I, and expression of ISG in the lungs, liver’\nThese are innate immune responses (not autoimmune). Also, in the supplementary information it mentions B6D2F1 mice,\nnot C57BL/6.\n25. Line 387-388. What are the possible reasons for such serious adverse effects in the αV Proc1 saRNA?\n26. Line 406 – is this the WT or engineered Vpg?\n27. Line 409 – 410 ‘While these truncated 5' Vpg-Goi mRNAs lack a start codon and thus cannot be translated into truncated\npeptides’\nWere the sequences evaluated to see if there are additional downstream start codons within the sequence (or in +1, +2\nsequence)?\n28. Lines 431-433: Transfecting cells with Vpg saRNA containing the 42-nt apt-Vpg-modified 3'UTR effectively inhibited the\nproduction of non-theoretical length progeny 5' Vpg-Goi mRNA (Fig. 7D).\nSome non-specific products can still be seen. Was this effect tested using different GOI’s? The results may be specific to the\nGOI.\nMinor comments:\n29. General throughout text - consistent use of VPg – in manuscript it referred to VPg or Vpg or vpg. Also spell out\nabbreviation in abstract.\n30. In vitro transcribed does not need to be italicised (line 73)\n31. Line 93 - 95 – sentence needs rephrasing. It is not an engineered sequence. The structural genes are replaced with a\nGOI.\n32. Line 96 – 97 – need reference for statement ‘decreasing it by 30-1000 times compared to conventional linear mRNA\nvaccines’\n33. Line 104 – 107 – reference 14 does not mention the following ‘Second, the double-stranded RNA (dsRNA) replicative\nform (RF) generated during αV saRNA replication can activate cytoplasmic nucleic acid receptors, leading to inhibition of\nhost cell translation, including translation of the saRNA itself’. Please cite appropriate reference.\n34. Line 108 – reference 15 discusses the ability of alphaviruses to avoid translation initiation by PKR, not ‘triggering\nprogrammed apoptosis through antiviral response mechanisms’\n35. Line 110 – ‘which typically lacks a proofreading mechanism [17]’ – needs to be cited correctly. Ref 17 is specifically\nabout coronaviruses.\n36. Line 131 – ‘mRNAs could be self-amplified from cells’ is this refereeing to PCR amplification? If so, this should be\nrephrased.\n37. Line 148 need to be referenced accurately (not 24).\n38. Line 149 – 151 requires citation.\n39. Line 161 – 162 – rephrase sentence so that it is clear that four separate constructs were made.\n40. Line 175 – typo ‘mmNoV’\n41. Line 297 – ‘newborn mice’ stated in text but in the figure the mice are injected at 4-weeks old. Please correct.\n42. Line 377 - Supp info says TECs instead of AECs\n43. Line 401 – aGVHD mentioned in the figure legend. cGVHD mentioned in the text. Please confirm which it is.\n44. In methods – identification of the restriction sites used for cloning (in multiple methods) is not accurate. The underlined\nRE site is longer than the actual RE site.\n45. Line 860 – this is not a method for an ELISPOT\n46. Line 869 – this is not a method for T-cell proliferation\n47. Line 1094 – triggered is not the correct word\n48. Line 1100 – the control is 5’m7G Ghrelin mRNA not saRNA\n49. Line 1147 – ‘showed a reduction’ is not the correct word choice\n50. Line 1160 – figure 4L – check the labelling consistence of kras/anti-kras\n51. Supp. Figure S5H – Y-axis cut off\n52. Supp. Figure S4 – typos for hTERT-hPEN\n53. Supp. Figure S4G – label under bar graph is GSDMD circRNA?", "reviewer_2": "In the current manuscript, the authors explore the use of a norovirus derived saRNA for delivery of gene therapy and cancer\ntherapy.\nMajor comments (please address 1 and 2).\n1. In Figure 3 where you explore fidelity, can you also look at impact on RNA sensing of the different fragment lengths?\n2. Likewise in Figure 5 – you mention low immunogenicity – but there is only a readout of ISG expression, but more is\nneeded – e.g. IFN or IP10. Might be better to rename the figure about incorporation rather than immunogenicity.\nantigen. (nice to add, but not vital)\nMinor comments\n1. Fig 1C – why is the VP1 bar a different colour, it is a bit confusing\n2. What was the primary antibody in 1B (add to legend)\n3. N number of repeats for 1F and 1G\n4. Where’s the weight loss data for 2D in the wild type mice?\n5. On panel 2C – might be helpful to indicate the monthly injections\n6. The histology needs scale bars\n7. Figure 2H – was there a no injection control?\n8. Does Figure 4 need tumour size?", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts."}, "author_response": "Thank you very much for your careful review of our manuscript. We agree that the layout of\nthe original version was indeed confusing. In fact, all Vpg-saRNAs with in vivo therapeutic\nfunctions were developed based on the final version of the huNoV saRNA, which incorporated\nstructural rearrangements, a triple mutation in the RdRp, and 3′UTR optimization to enhance Vpg\naffinity.\nIn the revised manuscript, we have significantly reorganized the overall structure to improve\nclarity and logical flow. Specifically, we moved the sections describing the therapeutic\napplications of the three types of Vpg-saRNA to follow the vector engineering and\nproof-of-concept sections. The revised manuscript now follows a seven-part narrative structure:\ni.) Initial construction of the huNoV replicon vector and its ability to drive gene-of-interest (Goi)\nexpression;\nii.) Investigation of the Vpg-dependent, high-fidelity self-replication mechanism;\niii) Structural rearrangement and RdRp triple mutation to achieve reduced immunogenicity;\niv.) 3′UTR optimization to minimize non-specific replication initiation and enhance translation\nefficiency;\nv.) Application of the cap-independent translation feature for treating cancer-associated cachexia\n(CAC) using Vpg-Ghrelin saRNA;\nvi.) Use of high-fidelity replication to improve the performance of a GSDMD^ENG-based oncolytic\nmRNA tumor vaccine;\nvii.) Exploitation of low immunogenicity to treat graft-versus-host disease (GvHD).\nAdditionally, we have included a graphical abstract (now presented as Figure 8) to illustrate\nthe overall workflow, and we have added comprehensive Methods section detailing the iterative\ndevelopment and validation of each saRNA variant.\nWe sincerely appreciate your insightful and constructive suggestions.\n2. The methods section appears to be written by several individuals and is lacking coherence and\nappropriate details. In addition, certain methods do not describe the procedures in their titles\n(particularly the immunogenicity studies). Most of the methods need more detail for\nexperiments to be reproducible. Some results don’t have associated methods, which need to be\nincluded\nIn the revised manuscript, we have made substantial revisions to the Materials and\nMethods section to improve its coherence and level of detail. We have also included several\nmethodological experiments that were previously omitted. We sincerely thank you for your\nthorough review of our manuscript.\n3. Clear distinctions between mRNA, saRNA, capped mRNA, capped saRNA and uncapped saRNA\nneed to be maintained throughout the text to avoid confusion.\nThank you for your careful review. To avoid confusion, we have standardized the full names\nof all saRNAs and mRNAs throughout the manuscript. Detailed information for each individual\nsaRNA construct is now provided in Supplementary Table 1. We greatly appreciate your thorough\nevaluation.\n4. Information relating to the IVT, capping, polyadenylation and formulation (both in the\nmethods and results sections) are lacking. As noted by the authors, IVT of longer saRNA is\ncomplicated. It is important to show that the transcripts produced are of a high quality (size,\nintegrity, purity) and that formulation was successful (size of nanoparticle, PDI and encapsulation\nefficiency). No information about polyA tail length is included, which is important for functioning\nof the mRNA transcripts which have been included as controls throughout the study.\nThank you for your review. We have added Supplementary Table 1, which includes the\nsequences and production details for all IVT mRNAs and saRNAs. We sincerely appreciate your\nthoughtful evaluation.\n5. Appropriate controls need to be used throughout the study. Comparing the efficacy of mRNA\nto saRNA without copy number comparisons (important when looking at transcripts of vastly\ndifferent lengths), and comparing three other alphavirus saRNA constructs without any\ninformation on their RdRp design, sequence or transcript length makes it hard to determine if\nthe engineered Vpg saRNA is in fact superior to current vectors. We assume from the figures that\nthe VEEV, VEEV-SINV and SFV saRNA vectors make up the three alphaviruses tested, yet there is\nno information about these sequences, which derivatives they are, and how they compare\n(structurally and functionally) to the Vpg saRNA. In addition, later studies simply designate an αV\nsaRNA as a comparator without specific identification of which alphavirus has been used.\nAll mRNA doses mentioned in the manuscript (e.g., 1 μg, 0.2 μg) refer specifically to the\nmass of the gene-of-interest (Goi) sequence, i.e., the microgram (μg) amount of the mRNA\nmolecules encoding the target protein (such as EGFP). For example, 1 μg of EGFP mRNA\ncorresponds to approximately 2.55 × 10¹² copies of the Goi sequence, while 1 μg of ghrelin mRNA\nrepresents approximately 2.17 × 10¹³ copies. Regardless of the vector used—whether αV saRNA\nor Vpg saRNA—these constructs have the same mass, which effectively represents the same\nnumber of transcript copies. This clarification is important because, in therapeutic applications of\nmRNA-based drugs, dosing is typically based on mass rather than transcript copy number.\nTo avoid potential confusion for readers, we have added this explanation to the figure\nlegends and the Methods section of the revised manuscript. We sincerely appreciate your\nattention to this detail.\nAdditionally, we have included Supplementary Tables 1 and 2, which provide detailed\nsequence information for all mRNA and saRNA constructs, including the full genomic RNA\nsequences of the three alphavirus-based vectors: VEEV, VEEV-SINV, and SFV. These alphavirus\nvectors are commercially available plasmid backbones, and we have now included this\ninformation in the Methods section.\nAs alphaviruses and noroviruses belong to different families and genera, their structural and\nfunctional differences are not directly relevant for our study. Alphaviruses were used as a\ncomparison group because they represent the most widely used commercial saRNA vectors\ncurrently available. Our aim was to evaluate the replication fidelity of the αV saRNA vector by\nfocusing on the EGFP mRNA segment (717 nt), where we observed multiple erroneous base\nincorporations. In contrast, our engineered Vpg saRNA vector maintained high fidelity, and this\ndifference was statistically significant.\nIn our follow-up studies, all αV saRNA constructs were based on the VEEV backbone, and we\nhave clarified this point in the revised manuscript.\n6. Figures – in general the figures are very small and difficult to read. Many of the figures have\nsections of the figure, including axis and labels, cropped off, and typos. This needs to be\naddressed throughout the manuscript.\nWe have uploaded higher-resolution images and carefully reviewed all figures to ensure\nthat none are obscured or improperly cropped. We also checked all figure labels for spelling and\ngrammatical errors. We sincerely appreciate your thorough review.\nSpecific major comments:\n7. Importance of TLR3 needs to be addressed. Later in the manuscript there is a focus on ablating\nTLR3 activation, presumably based on the results shown in Fig S1A. However, TLR3 seems barely\nactivated in this study, and could be a result of residual dsRNA from the IVT process (which was\nnot confirmed). It is also unclear how this activation might occur during replication given the\nconclusion by the authors that ‘These findings suggest that p22-induced vesicles are crucial\nstructures protecting nascent single-stranded RNA from recognition and degradation’ (line 146 –\n147). If replication, and thus production of dsRNA also occurs in these vesicles, why does TLR3\nget activated even in the presence of p22?\nThere was a labeling error. In the original manuscript, Fig. 1I and Fig. 1J should have been\nlabeled as TLR7 and TLR3, respectively, and Fig. S1A should have indicated TLR9. We have\ncorrected these mistakes in the revised version. Due to the extended duration of the\nexperimental timeline, we unfortunately failed to identify this issue prior to submission, which\nled to the inaccurate description at the beginning of part 3.\nAs background, endolysosomal TLR7 and TLR3 recognize ssRNA and dsRNA, respectively.\nDuring Vpg saRNA replication, both long dsRNA and ssRNA intermediates are produced. In\ncontrast, TLR9 is not activated by RNA; it primarily responds to unmethylated CpG DNA, such as\nCpG ODN, which are not generated during Vpg saRNA replication.\nWhile p22 can block the activation of endolysosomal RNA sensors, it cannot inhibit cytosolic\nnucleic acid sensors. Therefore, our subsequent modifications to Vpg saRNA focused on\neliminating the activation of cytosolic RNA receptors such as PKR, RIG-I, and IFIH1 by dsRNA\nintermediates, thereby aiming to suppress the type I interferon (IFN-I) response.\nWe sincerely apologize for the previous mislabeling and incorrect interpretation, and we are\nvery grateful for your careful and insightful review.\n8. Particles in Fig. S1D do not look uniform. Confirm size and PDI of LNPs with zeta sizing (as\ndescribed in the methods but no results provided). Encapsulation efficiency is also required to\nconfirm dose for in vivo studies – doses are given but no EE is described in the methods or results.\nHow did the authors determine dose of their vaccines?\nWe have added Supplementary Table 1, which includes detailed production information for\nall saRNA-LNP and mRNA-LNP formulations. As for the in vivo dosing of saRNAs, it is quantified\nbased on the microgram (μg) amount of mRNA molecules encoding the target protein, as\naddressed in our response to Comment No. 5. We sincerely appreciate your thoughtful review.\n9. Line 167 – ‘Vpg-saRNA sequentially expressed Vpg’. Please can the authors confirm how they\ncame to the conclusion that these were sequentially expressed as a single timepoint (72 hours)?\nThis was an inaccurate description. These proteins are expressed simultaneously, not\nsequentially. We have corrected this in the revised manuscript. Thank you very much for pointing\nit out.\n10. Line 202 – 203: The Vpg Ghrelin saRNA constructs have an IRES. Where is this IRES derived\nfrom and how does it impact VPg-mediated translation?\nThe schematic diagrams contained incorrect information. Specifically, we mistakenly labeled\nthe huNoV 5′UTR as \"huNoV 5′IRES.\" In reality, huNoV translation is not IRES-dependent but\ninstead relies on the Vpg protein to initiate translation.\nAfter the initial (parental) strand is synthesized via in vitro transcription (IVT), it is capped\nwith an m⁷GpppNm structure. This capping ensures immunological stability and allows for the\nfirst round of translation of the parental mRNA strand, including ORF1 (encoding Vpg/RdRp/2C-L).\nDuring subsequent rounds of replication, the newly synthesized progeny strands lose the 5′ cap\nstructure and are instead primed with Vpg at the 5′ end through Vpg-dependent replication. This\n5′ Vpg modification facilitates more efficient translation.\nIn this context, Vpg plays a dual role in both replication and translation initiation (as\ndiscussed in the relevant part of the Discussion section), whereas the 5′ cap (cap1) is primarily\ninvolved in the first round of translation after saRNA enters the cell—a step that is nevertheless\nessential for ensuring the initial stability of the saRNA.\nWe sincerely apologize for presenting an inaccurate schematic and are grateful for your\ncareful and constructive review.\n11. In figure 2, the results of the KPC mice show those treated with mRNA, no untreated controls\nare shown (as described).\nThe \"0 days\" labels in Fig. 2H and 2I (renumbered as Fig. 5H and 5I in the revised manuscript)\nrepresent the baseline body weight and food intake of the mice and can be considered as\ninternal pre-treatment controls. This is because body weight loss is evaluated relative to each\nmouse’s own baseline.\nAs the study progressed, the number of mice decreased over time due to\nmalnutrition-induced mortality. Therefore, Fig. 2F and 2G (now Fig. 5F and 5G in the revised\nversion) present representative images of mice that died from cachexia.\nIncluding untreated control mice at each time point would not be meaningful in this context,\nas it would not reflect the progressive nature of cachexia in the same individuals.\nWe sincerely thank you for your thoughtful review.\n12. Line 237 – more information on the principle of the T32/P2 construct need to be included.\nWe have added a detailed explanation of the design rationale for the T32/P2 constructs in\nthe Results section of the revised manuscript. We sincerely appreciate your valuable suggestion.\n13. Line 248-249. More information needs to be included about the mismatch cleavage assay.\nwhat was done first. Chance of PCR introducing mutations? PCR was also done to produce the\ntemplate.\nCould this also not introduce mutations? The figure shown in 3F is not correct. It is an RT-PCR so\nthe T7E1 cleavage is on the cDNA, not on mRNA. The fragmented DNA is run on a gel.\nIn brief, huNoV-gRNA saRNA was used as a template for in vitro self-replication using\nrecombinant RdRp and 2C-L proteins, along with a His-Vpg-pU primer. The resulting\nself-replicated mRNA products were then subjected to RT-PCR using VP1-VP2 primers (for\ncomparison with the αV system, EGFP primers were used instead).\nIf base misincorporations occur during self-replication, they may result in mismatched base\npairs (i.e., mutations) between otherwise identical cDNA molecules. These mismatches form\nheteroduplex double-stranded DNA, which can be specifically recognized and cleaved by T7\nEndonuclease I (T7EI), thereby indirectly indicating the presence and frequency of\nreplication-induced mutations.\nRT-PCR itself does not introduce additional mutations, as high-fidelity reverse transcriptase\nenzymes are used. This assay was designed to evaluate the mutational load introduced during\nhuNoV-gRNA saRNA self-replication.\nWe have added an explanation of the T7EI assay principle to the Results section of the\nrevised manuscript (Fig. 2F, schematic), and provided detailed experimental procedures in the\nMaterials and Methods section. We sincerely appreciate your insightful suggestion.\n14. Line 252 – 253. Example of where there is little to no information on the three additional\nalphaviruses tested. Were these saRNAs also produced from a PCR amplified template? If so,\nthere is a higher chance of mutations since alphavirus sequences are significantly longer.\nThe three alphavirus (αV) saRNAs used in this study are widely adopted self-replicating\nmRNA vectors. Their sequences are primarily derived from synthetic constructs and\ncommercially available backbones. We have added the relevant sequence and construct\ninformation in Supplementary Tables 1 and 2, and included a detailed description in the Methods\nsection.\nTo compare replication fidelity, we focused specifically on the EGFP mRNA region (717 nt)\nthat was encoded by both the αV saRNA and huNoV saRNA vectors. The EGFP sequence was\nloaded at equal copy numbers in both vectors, as referenced in our responses to Comments No.\n5 and No. 8. Our results show that, compared to the huNoV saRNA vector carrying the same\nnumber of EGFP mRNA transcripts, the αV saRNA vector introduced significantly more mutations\nin this region, thereby demonstrating a statistically significant difference in replication fidelity\nbetween the two systems.\nWe sincerely thank you for your thorough and constructive review.\n15. Lines 274 – 275: It appears the VPg construct used here was only described in the following\nparagraph. This is thus not a comparison of just Vpg fidelity but also sequence optimisation and\nengineered Vpg to reduce dsRNA. See S4B.\nThe rationale and sequence elements behind this construct should be discussed. Also, these\nmodifications to the sequence is only described in the next section. The order of sequence\nelements in alphavirus and Vpg constructs are not the same Fig4A and S4B\nThe structural rearrangement of the huNoV gRNA mRNA, as well as the incorporation of\nTriM-huNoV and 3′UTR Vpg-affinity aptamers, were applied consistently across all in\nvivo-delivered Vpg saRNA constructs. The confusion was due to the previously unclear\nmanuscript layout and narrative structure.\nIn the revised version, we have comprehensively clarified the iterative development of all\nhuNoV gRNA mRNA constructs. Specifically, Fig. 3I in the revised manuscript illustrates the design\nrationale behind the structural rearrangement of the huNoV gRNA mRNA. This design was based\non its self-replicating properties and was aimed at suppressing the formation of dsRNA\nreplication intermediates.\nWe sincerely appreciate your careful review of our manuscript.\n16. Line 282 – ‘The F1LCT signal peptide facilitated its transport to mitochondria, activating\nGSDMDNT with pore-forming activity (Fig. S4D).’\nThis shows alphavirus GSDMD expression in hTERT mito, but this is not the case in Fig4E? Why is\nthis seen in one figure but not the next?\nThank you very much for your thorough review.\nIn the revised manuscript, we considered it more important to highlight the differences\nbetween VEEV GSDMDENG saRNA and Vpg GSDMDENG saRNA. Therefore, we moved the\ndescription of the effects of random mutations on the linear GSDMD^ENG mRNA vaccine to the\nsupplementary figures. Specifically, the original Fig. 4C, 4D, 4E, and 4F have been moved to\nSupplementary Fig. S6B, S6C, S6D, and S6E, while Fig. S4B, S4C, and S4D have been incorporated\ninto the main figures as Fig. 6A, 6C, and 6D in the revised version.\nFig. 4E (now Fig. S6D) illustrates the functional impact of random mutations on the linear\nGSDMDENG mRNA vaccine. Since the HRV2 IRES sequence was not mutated, the GSDMDENG mRNA\nselectively expressed full-length GSDMD in AsPC-1 cells. In contrast, in hTERT-hPNE cells, the\nF1LCT sequence carried two engineered mutations (2090A>T and 2103C>G), which were\nidentified through low-fidelity VEEV saRNA screening (cells without effective mutations would be\neliminated, and functional-impacting mutations would be retained). These mutations interfered\nwith GSDMD’s mitochondrial localization in AsPC-1 cells.\nFig. S4D (now Fig. 6D) compares the VEEV and Vpg saRNA vectors. Due to the low fidelity of\nthe VEEV saRNA vector, the HRV2 IRES sequence accumulated mutations, resulting in loss of\nexpression selectivity of GSDMD between AsPC-1 and hTERT-hPNE cells; GSDMD was expressed\nin both. Additionally, because the F1LCT sequence remained unmutated in this case, GSDMD\ncould localize to mitochondria in hTERT-hPNE cells as well. In contrast, the Vpg saRNA vector\ndisplayed much higher stability, maintaining both translational selectivity and mitochondrial\ntargeting specificity.\nWe have added detailed explanations of these points in the revised manuscript.\n17. Please confirm that the labels are correct in Fig 4G – 4H. Are labels correct for Vpg saRNA?\nWouldn’t one expect that the Vpg saRNA induces higher mtROS in ASPC1 cells compared to\nhTERT?\nThis was a labeling error, and we have corrected it in the revised manuscript. We sincerely\nappreciate your excellent attention to detail.\n18. Line 286 – ‘injection of 1 μg/kg Vpg GSDMDENG saRNA significantly enhanced tumor tissue\nablation compared to the same dose of αV GSDMDENG saRNA (Fig. 4I)’.\nWhile dose may be the same, information relating to copy number should be included. If the Vpg\nGSDMDENG saRNA is a much shorter transcript than the αV GSDMDENG saRNA, it is not surprising\nthat there would be a difference in therapeutic effect since more copies of the Vpg saRNA were\ninitially administered.\nThe information regarding transcript copy numbers has been added to the revised\nmanuscript. This issue is related to our responses to Comments 5, 8, and 14. In the animal\nexperiments, the dosage of 1 μg/kg for both Vpg GSDMDENG saRNA and αV GSDMDENG saRNA\nrefers specifically to the mass of the GOI-encoding RNA sequence itself, which corresponds to\napproximately 1.19 × 10¹² copies of saRNA.\nWe sincerely appreciate your review.\n19. Line 307 ‘These findings indicate that high-fidelity Vpg GSDMDENG saRNA continuously\nactivates specific cytotoxic T cell responses, resulting in lasting tumor protection’.\nIs the Vpg or any of the Norovirus sequences used in the engineered saRNA designs\nimmunogenic? Are T cells targeting Vpg or RdRp generated? This could impact repeat\nadministration of therapy.\nNon-structural proteins encoded by viral vector-based saRNAs typically function within the\nhost cell cytoplasm and are not directly exposed to the extracellular environment, nor are they\nprimary targets of humoral immune responses. Compared to viral structural proteins (e.g., spike\nor capsid proteins), non-structural proteins generally induce weaker antibody responses.\nPrevious studies have shown that self-amplifying RNAs based on viral RdRp (such as those using\nalphavirus replicon systems) can be administered multiple times in animal models without\nsignificantly impairing their expression function (Erasmus JH et al., Sci Transl Med, 2020;\nDominguez F et al., J Virol, 2023; Leitner WW et al., Nat Med, 2003). These findings suggest that\nthe viral RdRp itself is not the primary limiting factor in repeat dosing.\nEven if a certain degree of RdRp-specific immune response occurs, it is more likely to involve\ncytotoxic T cell responses rather than neutralizing antibodies, and therefore does not lead to\ndirect clearance of subsequently administered saRNA-LNPs. As such, the impact of this response\non saRNA expression and function remains limited, especially in non-viral infection contexts.\nIn our revised manuscript, we have included additional in vivo repeat-dosing experiments\n(Supplementary Fig. S5F and S5G). Multiple administrations of Vpg saRNA did not result in a\nsignificant decrease in ghrelin expression (as assessed by plasma ghrelin levels), and systemic\nantiviral inflammatory responses remained low (plasma IFN-α < 100 pg/mL). These findings\nfurther support that Vpg saRNA does not robustly activate immune responses in vivo, and that its\nnon-structural protein immunogenicity does not significantly compromise the efficacy of repeat\ndosing.\nWe sincerely thank you for raising this important concern, which prompted us to strengthen\nour manuscript with additional validation.\n20. Lines 349 – 360. This section should be presented in the main figures, and not just in the\nsupplementary. Further questions related to this section include:\nThank you very much for your thoughtful suggestion. In the revised manuscript, we have\nmoved the phenotypic data related to the low immunogenicity of the huNoV gRNA TriM-saRNA\nconstruct—originally shown in Fig. S5E, Fig. S5G, Fig. S5H, Fig. S5I, and Fig. S5J—into the main\nfigures (now presented as Fig. 3I, Fig. 3J, Fig. 3K, Fig. 3L, and Fig. 3M), in order to better highlight\nthe functional importance of these findings.\nTo avoid overcrowding the main figures, we have correspondingly moved some of the data\nvalidating the positive-strand replication mechanism of huNoV gRNA saRNA—originally shown in\nFig. 5A, Fig. 5B, Fig. 5C, Fig. 5E, and Fig. 5F—into the supplementary figures. These are now\nshown as Fig. S3A, Fig. S3B, Fig. S3C, Fig. S3D, Fig. S3E, and Fig. S3F in the revised version.\nWe believe this restructuring improves the clarity and focus of the main results, and we\nsincerely appreciate your input, which helped enhance the overall presentation of our data.\na. The authors didn’t compare WT with TriM VPG - did these changes result in significant\nimprovements?\nThe abundance of dsRNA replication intermediates (RFs) generated by WT and TriM-saRNA\nconstructs in cells is shown in the revised Fig. 3E. Additionally, to assess downstream effects,\nwe\nhave included new data comparing the induction of interferon-stimulated genes (ISGs) and type I\ninterferon release between the huNoV gRNA WT-saRNA and TriM-saRNA constructs, as\npresented in Fig. 3G and Fig. 3H. These results demonstrate that the triple mutations significantly\nlimit the type I interferon response elicited by the huNoV gRNA saRNA.\nWe sincerely thank you for your insightful review and suggestions.\nb. How is Vpg saRNA affected by PKR or IFIT? Translation is cap independent, so please explain\nthe importance of measuring the activation of PKR in the context of Vpg?\nOn one hand, the activation of PKR, RIG-I, and MDA5 was assessed to confirm that the\nstructural rearrangement and triple mutations introduced into the huNoV gRNA saRNA reduced\nthe generation of dsRNA replication intermediates (RFs), thereby significantly attenuating type I\ninterferon activation in host cells and contributing to the observed low immunogenicity. On the\nother hand, Vpg serves two key functions: it acts as a primer to mediate saRNA replication and\nenables 5′Vpg-dependent protein translation. In contrast, alphavirus-derived saRNA relies on\ncap-dependent translation, which is susceptible to inhibition by phosphorylated eIF2α. This\ntranslational blockade reduces the expression of viral proteins despite high levels of replication.\nIn contrast, the 5′Vpg-dependent translation of huNoV gRNA saRNA is resistant to\neIF2α phosphorylation, allowing continued protein synthesis. This mechanistic difference explains why\nalphavirus-based saRNA exhibits higher replication activity (revised Fig. 3K) but lower protein\nexpression (revised Fig. 3L), whereas huNoV-based saRNA maintains efficient translation under\nthe same conditions.\nWe sincerely thank you for your careful review and insightful comments.\nc. Even though there is less dsRNA, is there a significant difference in protein expression\ncompared to WT?\nWe have added data comparing the expression of interferon-stimulated genes (ISGs)\nbetween the huNoV gRNA WT saRNA and huNoV gRNA TriM-saRNA constructs and their impact\non VP1/VP2 protein expression, as shown in the revised Fig. 3F. Compared to the WT, the\nTriM-saRNA construct did not show a significant reduction in protein expression levels. This\nsuggests that despite the attenuated innate immune activation, the translation efficiency of the\ntarget protein remains unaffected.\nWe sincerely appreciate your thoughtful review.\nd. TLR3 activation was not examined even though this was the reason for optimising the\nsequences? This relates to comment 6.\nThe original huNoV gRNA does not activate TLR3 or TLR7, likely due to vesicle shielding\nmediated by the p22 non-structural protein, as demonstrated in the first part of our results.\nHowever, replication products of huNoV gRNA still trigger a subsequent type I interferon (IFN-I)\nresponse. In this section, our goal was to eliminate this residual immune activation by\nengineering a structurally rearranged and triple-mutant RdRp variant.\nWe sincerely appreciate your thoughtful review.\ne. PKR, eIF and IFIT 1 were examined but alphaviruses are resistant to these so why is there less\nexpression from the alphaviruses?\nThank you for your thorough and insightful review. Regarding the seemingly paradoxical\nphenomenon of alphavirus replicons exhibiting high replication activity but low protein\nexpression, we believe this is closely related to the dynamic host response to translation\nsuppression mechanisms, such as eIF2α phosphorylation.\nTranslation of alphavirus genomic RNA initially relies on a 5′ cap structure (methylated by\nthe viral non-structural protein nsP1), following a typical cap-dependent mechanism. Upon\nactivation of host antiviral sensors—such as PKR—eIF2α becomes phosphorylated, leading to\nglobal suppression of cap-dependent translation. Consequently, the expression of viral proteins\nis significantly reduced, as shown in Fig. 3L. This observation is consistent with previous reports\n(e.g., Ventoso et al., J Virol, 2006).\nDespite this translational blockade, alphaviruses can sustain high levels of RNA replication\n(Fig. 3K), likely due to several factors: (1) in the early phase of infection, before translation is shut\ndown, sufficient quantities of viral replicase complexes (e.g., nsP1–nsP4) are synthesized,\nallowing replication to proceed even under translational inhibition (Ventoso et al., 2006); and (2)\nreplication often occurs within membrane-associated compartments (e.g., endosomal or\nvesicular structures), which may provide partial shielding from host antiviral factors such as PKR\n(Fros et al., PLoS Pathog, 2012).\nThus, this “high replication–low translation” balance may represent an immune evasion\nstrategy employed by alphaviruses—minimizing overt protein production to reduce\nimmunogenicity, while maintaining sufficient genome replication to ensure progeny virion\nproduction.\nHowever, this feature poses a significant limitation when repurposing alphavirus replicons\nas vectors for mRNA-based therapeutics. Excessive replication can trigger strong host antiviral\nresponses, while inefficient protein translation compromises therapeutic efficacy. Therefore,\naddressing this imbalance is critical for optimizing such replicon systems for clinical applications.\n21. Lines 361 – 363 – ‘In brief, these RdRp (pro) mutation strategies facilitate the efficient\nproduction of supersaturated VPg-pU in cells, reducing dsRNA-induced immunogenicity by\nsimultaneously suppressing positive-sense chain production, without affecting protein coding\ncapacity.’\nProtein coding capacity between WT and Trim Vpg was not show. Please can the authors\ncomment on how they arrived as this conclusion?\nThank you very much for your thoughtful review. This issue is consistent with the concern\nraised in point 20.c. We have addressed this by providing comparative data on VP1/VP2\nexpression between the WT and TriM-saRNA constructs (see revised Fig. 3F). Notably, the\nTriM-saRNA construct does not impair protein expression capacity when compared to the WT. It\nis important to clarify that the role of the TriM-saRNA is to maintain protein expression, rather\nthan to enhance it per se. It is only after structural reorganization—implemented in the\nTriM-saRNA construct—that the expression efficiency of the GOI-encoded protein surpasses that\nof conventional alphavirus-based saRNA vectors. This improvement is due to the TriM-saRNA's\ncorrection of the previously discussed “high replication–low translation” imbalance, a limitation\ninherent to alphavirus replicons.\nFurthermore, in the fourth part of our study, we demonstrate that the addition of the\naptamer-linked VPg (apt-VPg) to the 3′UTR further enhances the expression efficiency of the\nhuNoV-based saRNA platform.\n22. Line 376 – ‘effect of Vpg saRNA encoding Proc1 on cGVHD’. Is this the TriM-Vpg?\nAll in vivo-delivered Vpg saRNAs used in this study contain the structurally rearranged\nhuNoV gRNA mRNA sequence, the TriM-huNoV mutations, and the 3′UTR Vpg-affinity aptamers.\nWe sincerely apologize for the confusion caused by the poor layout and lack of clarity in the\noriginal manuscript. This clarification was also stated in our response to point 15.\n23. Line 379 – why was Cap 1 nucleoside modified mRNA not used as a comparator? In the\ncurrent form, this Cap 0 mRNA is not suitable for in vivo studies, nor does it serve as an\nappropriate comparison for therapeutic applications. By using an inferior mRNA design, it is not\nsurprising that the saRNA outperforms the mRNA.\nThank you very much for your careful review. In the original Materials and Methods section\n(\"Construction of 5' m⁷G Linear mRNA\"), we stated that 3'-O-Me-m⁷GpppG (NEB) was used for\ncapping linear mRNA. 3'-O-Me-m⁷GpppG is a commonly used Cap 1 structure that enhances\nmRNA stability, translational efficiency, and modulates innate immune activation. We agree that\nthe previous description of “5' m⁷G mRNA” was inaccurate. In the revised manuscript, we have\nreplaced all instances of “5' m⁷G mRNA” with “Cap1-m⁷G mRNA” to avoid confusion.\nAdditionally, saRNAs are also capped after in vitro transcription (typically with an\nm⁷GpppN₁m cap). Essentially, saRNAs remain linear mRNAs, and the first strand entering the cell\nstill requires a 5′ cap for stability and for the translation of nonstructural proteins. We have\nadded this capping information to Supplementary Table 1.\n24. Line 382 – 383. ‘saRNA induced systemic autoimmune responses in C57BL/6 (B6) background\nmice, including activation of PKR, eIF2α, MDA5, RIG-I, and expression of ISG in the lungs, liver’\nThese are innate immune responses (not autoimmune). Also, in the supplementary information\nit mentions B6D2F1 mice, not C57BL/6.\nThank you very much for your thorough review. We have revised the inappropriate and\nincorrect text accordingly.\n25. Line 387-388. What are the possible reasons for such serious adverse effects in the αV Proc1\nsaRNA?\nThank you for your careful review. GVHD patients already suffer from severe autoimmune\nattacks in vivo. The purpose of Proc1 is to eliminate the humoral immunity mediated by\nTfh-driven germinal center (GC) B cells. It is clear that due to the replication characteristics of the\nαV saRNA, a strong type I interferon (IFN-I) response is induced, which, especially in the acute\nphase of GVHD (aGVHD), directly promotes the formation and function of GC B cells. We\nsincerely appreciate your review and valuable suggestions.\n26. Line 406 – is this the WT or engineered Vpg?\nAs previously clarified in our responses to Questions 15 and 22, we apologize again for the\nconfusion arising from the suboptimal layout of the original manuscript.\n27. Line 409 – 410 ‘While these truncated 5' Vpg-Goi mRNAs lack a start codon and thus\ncannot be translated into truncated peptides’\nWere the sequences evaluated to see if there are additional downstream start codons within the\nsequence (or in +1, +2 sequence)?\nThank you very much for your insightful suggestion. We have screened the target sequences\nfor potential open reading frames (ORFs), as summarized in the table below. Except for ghrelin,\nthe other three GOIs theoretically contain in-frame start codons that may lead to the translation\nof truncated frameshift peptides. This highlights the necessity of optimizing the 3′UTR to\nenhance Vpg recruitment and translation specificity.\nseq +1 reading frames +2 reading frames\nghrelin 0 0\neGFP 2 1\nGSDMD-ENG 7 6\nPROC1 1 8\nAccordingly, we have revised the original inaccurate description to: “These truncated 5′\nVpg-GOI mRNAs contain potential start codons in the +1 or +2 reading frames, which may lead to\nthe translation of truncated frameshift peptides. The potential impact on biosafety and\nexpression efficiency warrants further investigation.”\n28. Lines 431-433: Transfecting cells with Vpg saRNA containing the 42-nt apt-Vpg-modified\n3'UTR effectively inhibited the production of non-theoretical length progeny 5' Vpg-Goi mRNA\n(Fig. 7D).\nSome non-specific products can still be seen. Was this effect tested using different GOI’s? The\nresults may be specific to the GOI.\nThank you very much for your thoughtful review. The 42-nt apt-Vpg functions primarily by\nenhancing the recruitment of Vpg-pU primers through high-affinity interactions, thereby\ncompetitively reducing non-specific initiation events. We acknowledge that this strategy does\nnot completely eliminate the generation of non-specific products. However, it effectively reduces\nsuch products by over 95%, which significantly improves the translational efficiency of progeny\nmRNAs (as shown in Fig. 4E of the revised manuscript).\nMoreover, the occurrence of non-specific products is closely related to the secondary\nstructure of cryptic initiation sites within the GOI sequence. The stronger the unintended affinity\nto Vpg, the more abundant these non-specific products tend to be.\nMinor comments:\n29. General throughout text - consistent use of VPg – in manuscript it referred to VPg or Vpg or\nvpg. Also spell out abbreviation in abstract.\nWe have standardized the notation of \"VPg\" throughout the revised manuscript. Thank you\nvery much for your careful suggestion.\n30. In vitro transcribed does not need to be italicised (line 73)\nThe changes have been made accordingly. Thank you very much for your suggestion.\n31. Line 93 - 95 – sentence needs rephrasing. It is not an engineered sequence. The structural\ngenes are replaced with a GOI.\nWe have rephrased the relevant statements. Thank you again for your valuable feedback.\n32. Line 96 – 97 – need reference for statement ‘decreasing it by 30-1000 times compared to\nconventional linear mRNA vaccines’\nAccording to ref13 in the original manuscript, \"Current COVID-19 vaccines contain 30–100\nμg mRNA per dose. Self-amplifying (sa) RNA vaccines require a reduced initial amount of RNA,\nbecause the mRNA is expanded intracellularly. For example, a dose of only 10 ng saRNA was able\nto induce a SARS-CoV-2 specific immune response in mice and 5 µg saRNA were successfully used\nin a clinical trial. \"\nWe changed it to a more rigorous statement: \"This replication and translation mechanism\nsignificantly reduces the required dose of saRNA vaccines. Compared to conventional linear\nmRNA vaccines, which require 30 to 100μg per dose, saRNA only requires 10ng to 5μg [13].\"\n33. Line 104 – 107 – reference 14 does not mention the following ‘Second, the double-stranded\nRNA (dsRNA) replicative form (RF) generated during αV saRNA replication can activate\ncytoplasmic nucleic acid receptors, leading to inhibition of host cell translation, including\ntranslation of the saRNA itself’. Please cite appropriate reference.\nWe mistakenly reversed the order of references 14 and 15 in the original manuscript.\nReference 15 describes the role of host PKR-eIF2α signaling in suppressing alphavirus mRNA\ntranslation, while reference 14 pertains to antiviral IFN-induced apoptosis. We have corrected\nthese citation errors in the revised manuscript. Thank you very much for your careful review.\n34. Line 108 – reference 15 discusses the ability of alphaviruses to avoid translation initiation by\nPKR, not ‘triggering programmed apoptosis through antiviral response mechanisms’\nThis issue is the same as the previous one — we mistakenly reversed the order of references\n14 and 15. We have corrected these errors in the revised manuscript. Thank you very much for\nyour thorough review.\n35. Line 110 – ‘which typically lacks a proofreading mechanism [17]’ – needs to be cited correctly.\nRef 17 is specifically about coronaviruses.\nThank you. We have replaced the reference as suggested. We greatly appreciate your\nreview.\n36. Line 131 – ‘mRNAs could be self-amplified from cells’ is this refereeing to PCR amplification?\nIf so, this should be rephrased.\nYes, this was a misstatement and has now been corrected. Thank you very much for your\ncareful examination.\n37. Line 148 need to be referenced accurately (not 24).\nA more accurate reference has been cited in the revised manuscript. Thank you for your\nvaluable suggestion.\n38. Line 149 – 151 requires citation.\nThe appropriate citation has been added. We sincerely thank you for your insightful\ncomments.\n39. Line 161 – 162 – rephrase sentence so that it is clear that four separate constructs were\nmade.\nWe have revised the text to more clearly convey that “a total of four different saRNA\nconstructs were generated and each was individually encapsulated into LNPs.” Thank you very\nmuch for your careful review.\n40. Line 175 – typo ‘mmNoV’\nThe issue has been corrected. Thank you very much for your review.\n41. Line 297 – ‘newborn mice’ stated in text but in the figure the mice are injected at 4-weeks old.\nPlease correct.\nThe term has been corrected to “newborn mice.”\n42. Line 377 - Supp info says TECs instead of AECs\nThank you for your careful review. The errors in the supplementary figures have been\ncorrected accordingly.\n43. Line 401 – aGVHD mentioned in the figure legend. cGVHD mentioned in the text. Please\nconfirm which it is.\nWe would like to clarify that two distinct GVHD models were employed in this study: the\nMHC-mismatched allo-HSCT mouse model (C57BL/6 (B6) donors and B6D2F1 recipients), which\nserves as an acute GVHD (aGVHD) model, and the MHC-matched allo-HSCT mouse model\n(C57BL/6 (B6) donors and B10.BR recipients), representing chronic GVHD (cGVHD). The\nMHC-mismatched model was primarily utilized to investigate the advantages of the low\nimmunogenicity profile of Vpg saRNA, whereas the MHC-matched model was used to evaluate\nthe organ-protective effects mediated by Proc1. We sincerely appreciate your thorough and\ninsightful review.\n44. In methods – identification of the restriction sites used for cloning (in multiple methods) is\nnot accurate. The underlined RE site is longer than the actual RE site.\nWe have revised the manuscript accordingly. In the original version, the underlined\nrestriction sites included protective bases. We sincerely appreciate the reviewer's meticulous\nattention to this detail.\n45. Line 860 – this is not a method for an ELISPOT\nWe sincerely appreciate the reviewer's careful reading. We have corrected the mislabeled\nsection titles between 'IFN-γ ELISPOT Assay' and 'In Vivo T Cell Proliferation Assay' in the revised\nmanuscript.\n46. Line 869 – this is not a method for T-cell proliferation\nWe sincerely appreciate the reviewer's careful reading. As noted, the section titles for 'IFN-γ\nELISPOT Assay' and 'In Vivo T Cell Proliferation Assay' were inadvertently reversed and have now\nbeen corrected in the revised manuscript.\n47. Line 1094 – triggered is not the correct word\nWe have adopted the new expression as suggested and appreciate the reviewer's valuable\ncomment.\n48. Line 1100 – the control is 5’m7G Ghrelin mRNA not saRNA\nWe have corrected this error and thank the reviewer for identifying it.\n49. Line 1147 – ‘showed a reduction’ is not the correct word choice\nThe imprecise expression has been revised accordingly. We appreciate the reviewer's\nattention to this detail.\n50. Line 1160 – figure 4L – check the labelling consistence of kras/anti-kras\nThe text has been modified to maintain consistency throughout. We thank the reviewer for\nthis observation.\n51. Supp. Figure S5H – Y-axis cut off\nThis has been corrected as requested (Fig. 3J has been moved to the main figures following\ncomment #20). We are grateful for the reviewer's suggestion.\n52. Supp. Figure S4 – typos for hTERT-hPEN\nThe suggested modification has been implemented. We appreciate the reviewer's input.\n53. Supp. Figure S4G – label under bar graph is GSDMD circRNA?\nWe have corrected the misquoted statistical analysis. We sincerely thank the reviewer for\ncatching this oversight."}, {"round": 2, "reviewer_comments": {"reviewer_1": "Thank you to the authors for making the extensive changes to the manuscript. Some of the changes still require further\nclarification and are listed below, suing the original numbering:\n3. Clear distinctions between mRNA, saRNA, capped mRNA, capped saRNA and uncapped saRNA need to be maintained\nthroughout the text to avoid confusion.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts."}, "author_response": "Thank you for your careful review. We appreciate the reviewer’s comment regarding the labeling in Figure 2F. The PCR\nproducts were mistakenly labeled as “mRNA.” We have corrected the labels to “DNA” in the revised figure, and the figure\nlegend has been updated accordingly."}]

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