Beyond the genetic code: orchestrating epigenetic and immune landscapes with multivalent mRNA-exosome vaccines

Huiqin Bian; William Tse; Gang Huang; Shujun Liu

doi:10.1093/pcmedi/pbag019

. 2026 Jul 7;9(3):pbag019. doi: 10.1093/pcmedi/pbag019

Beyond the genetic code: orchestrating epigenetic and immune landscapes with multivalent mRNA-exosome vaccines

Huiqin Bian ^1,^✉, William Tse ^2,³, Gang Huang ⁴, Shujun Liu ^5,^6,^✉

PMCID: PMC13445983 PMID: 42565117

Abstract

mRNA therapeutics are transitioning from transient anti-viral vaccines into precise cancer immunotherapies capable of orchestrating potent antigen-specific T-cell and humoral responses. However, therapeutic resistance within immunologically “cold” tumors remains a formidable barrier, necessitating multiaxial optimization across transcript architecture, neoantigen selection, delivery vector engineering, and tumor microenvironment (TME) reprogramming. This review synthesizes critical breakthroughs in mRNA biochemistry—including chemical nucleotide modifications, optimized untranslated regions, structural codon adjustments, and stringent purification methodologies—that extend transcript longevity while limiting off-target reactogenicity to maximize functional antigen expression. We evaluate multiomic neoantigen discovery workflows leveraging genomics, transcriptomics, immunoproteomics, and computational HLA-binding algorithms to refine patient-specific target selection. Next, we dissect advanced lipid nanoparticles, surface-functionalized biomaterials, and engineered extracellular vesicles optimized to enhance antigen-presenting cell tropism and lymphoid homing. We further detail how vaccine-induced cytokine fluxes actively remodel the TME, successfully reversing local immune tolerance and driving robust effector leukocyte infiltration into the tumor stroma. Specifically, we highlight the convergence of mRNA-mediated cytokine signaling and epigenetic imprinting, which cooperatively induce trained immunity for durable preventive surveillance. Finally, we delineate rational combinations with immune checkpoint blockades while addressing translational challenges: identifying predictive biomarkers, mapping presentation kinetics, and structuring adaptive clinical trial frameworks.

Keywords: multivalent mRNA vaccines, exosome-mediated delivery, epigenetic reprogramming, tumor microenvironment plasticity, trained immunity, precision immunotherapy

Graphical Abstract

For image description, please refer to the figure legend and surrounding text. — The journey from code to cure. A schematic overview mapping the design, delivery, and immunological mechanisms of next-generation mRNA vaccines.

Introduction

Conventional cytotoxic chemotherapy and many targeted agents yield meaningful clinical responses but are constrained by systemic toxicity and the near–universal emergence of drug resistance [1–6]. Cancer vaccines aim to harness adaptive immunity to produce antigen–specific cytotoxic T lymphocytes (CTLs) and durable memory that can eliminate minimal residual disease while minimizing off–target toxicity [7–11]. Neoantigen vaccines—focused on tumor–restricted somatic mutations—are especially promising because they present truly non–self epitopes that escape central tolerance, offering a pathway to highly specific, long–lasting anti–tumor immunity [8, 10, 12–14]. To generate robust immunity, researchers employ diverse platforms—including nucleic acids, viral vectors, and peptides—alongside personalized dendritic cell (DCs) therapies that prime a patient’s own immune cells ex vivo. Yet, despite such innovation, few therapeutic candidates reach regulatory approval. While preventive vaccines for HPV and HBV are global standards, therapeutic successes remain limited to agents like Sipuleucel-T and intravesical Bacillus Calmette-Guerin (BCG). However, the field has reached a clinical inflection point. Recent landmark data for personalized neoantigen vaccines—demonstrating significant reductions in recurrence for melanoma and pancreatic ductal adenocarcinoma—signal a definitive shift toward precision immuno-oncology [15, 16]. Accelerated by the clinical success of COVID-19 therapeutics [17–22], mRNA vaccines are now a leading modality due to their inherent modularity and rapid manufacturing timelines [23–30].

Translation of cancer vaccines has been uneven. Mechanistic barriers include incomplete identification of immunogenic neoepitopes, insufficient antigen processing and cross–presentation by DCs, limited duration or magnitude of antigen expression, and the dominant influence of an immunosuppressive tumor microenvironment (TME) that blunts effector responses [7, 31–38]. These constraints help explain why many peptide–, protein–, and viral–vector approaches have achieved limited durable efficacy despite strong preclinical rationale [39–46]. To circumvent these specific bottlenecks, mRNA platforms offer distinct mechanistic advantages. Delivered mRNA is translated in the cytosol, enabling endogenous antigen synthesis and efficient major histocompatibility complex (MHC) class I presentation to CD8⁺ T cells; antigen sequence and dose are readily tuned; and innate RNA sensing provides an adjustable adjuvant signal that can be modulated through nucleotide chemistry and purification [34, 47–51]. Importantly, mRNA does not require nuclear entry for expression, avoiding mechanisms linked to genomic integration; advances in untranslated region design, codon optimization, and nucleotide modifications have increased transcript stability and controlled innate activation, improving both potency and tolerability [9, 23–30, 52].

Effective delivery remains pivotal. Lipid nanoparticles (LNPs) have enabled lymphoid targeting and clinical translation but also engage hepatic clearance pathways and innate components that can limit bioavailability [53–56]. Concurrently, engineered extracellular vesicles (EVs/exosomes) are under active preclinical investigation because they can display altered biodistribution and may reduce complement activation relative to some synthetic formulations; however, comparative data in humans remain preliminary, and categorical claims of superiority over established LNPs are premature [53–56]. Multivalent vaccine designs—defined here as the genetic combining of multiple neoantigens and selected tumor–associated antigens to counteract intratumoral (IT) heterogeneity—are actively being pursued alongside strategies to leverage the multifunctional characteristics of delivery systems, which encompass engineered, payload-independent trafficking across complex biological barriers, while strategies to reprogram the TME seek to sustain effector function and memory formation [57–61].

In this review, we synthesize mechanistic and translational advances across four integrated domains—mRNA engineering, multi–omic neoantigen discovery, delivery technologies (including EV strategies), and TME modulation by epigenetic dynamics—and discuss how combining these approaches can overcome resistance and enhance durable clinical benefit. We emphasize mechanistic principles that link molecular design to immune outcomes and highlight experimental and clinical evidence supporting rational combination strategies.

Mapping the antigenic landscape: from discovery to validation

The multiomics pipeline: integrating genomics, proteomics, and immunopeptidomics for target identification

The identification of high–quality neoantigens requires a coordinated multi–omics workflow applied to defined sample inputs: tumor tissue and matched normal tissue (DNA and RNA), tumor RNA–seq, tumor–enriched immunopeptidomics material (MHC eluates), and—when available—plasma–derived material such as circulating tumor DNA/RNA for longitudinal monitoring (Fig. 1A) [62, 63]. Initial genomic profiling uses whole exome sequencing (WES) for coding SNVs/indels and whole genome sequencing (WGS) to capture noncoding variants, structural variants (SVs), and complex gene fusions. In our pipeline, SVs and fusion events detected by WGS are translated into neoantigen candidates via fusion callers and junction reconstruction, in silico translation to candidate junctional peptides, subsequent HLA-binding prediction, and RNA–level confirmation (RNA–seq) followed by proteogenomic or immunopeptidomic validation when material permits [64, 65].

We define a neoepitope as a peptide derived from a somatic or tumor–specific event (mutation, splice variant, retained intron, fusion, or noncoding translation) that can be presented by MHC and recognized by T cells [66]; a “non–self neoepitope” denotes sequences absent from the normal human proteome and thus minimally subject to central tolerance. DNA–level fusion detection (WGS/WES) provides genomic breakpoint certainty and can reveal structural events not well captured by short–read RNA–seq; RNA–level detection confirms transcription and splicing but may miss lowly expressed or unstable transcripts and can be biased by expression noise. Where complex isoforms are suspected, long–read sequencing and careful transcript annotation improve resolution of splice and fusion isoforms; ultimately, proteogenomic confirmation (MS–based or targeted proteomics) is required to demonstrate peptide generation [67–70].

To distinguish functional variants from background transcriptional variation, transcriptomic validation via RNA-seq is utilized as a critical biological filter. This integration confirms that somatic mutations are not merely present at the DNA level but are actively transcribed into mRNA. By excluding transcriptionally silent “passenger” mutations, RNA-seq ensures that vaccine candidates are restricted to variants capable of being translated and subsequently processed for MHC presentation. Furthermore, transcriptomic quantification allows for the assessment of variant expression levels, which serves as a primary determinant in the hierarchy of neoantigenic potency.

Contemporary discovery also extends into non–canonical sequence space—alternative splicing, intron retention, and translation of noncoding regions—identified through appropriate sequencing platforms, calling pipelines, and orthogonal validation (long reads, targeted proteomics) [71, 72]. Deep sequencing per se is not the sole determinant; rather, the sequencing platform, variant calling pipeline, transcript annotation, long–read capabilities, and downstream validation determine whether abnormal RNA events produce translated peptides [73, 74]. Integrating genomics, transcriptomics, and immunopeptidomics through proteogenomic pipelines increases confidence in truly foreign epitopes by confirming transcription, translation, processing, and MHC presentation, thereby reducing false–positive candidates [62, 73, 75].

Prioritizing targets: predicting MHC-binding affinity and TCR cross-reactivity

Accurate target prioritization combines high–resolution HLA typing with computational models (Fig. 1A). Representative tools include NetMHCpan and MHCflurry for peptide–MHC binding affinity and stability prediction; NetChop and ProteaSMM for proteasomal processing likelihood; and immunogenicity or presentation models such as DeepImmuno, MARIA, MHCnuggets, and IEDB–derived predictors for T–cell recognition probability. Binding predictors generally achieve high sensitivity for candidate elimination but do not guarantee immunogenicity [62, 76]. To address this, pipelines compute “agretopicity” (the ratio of mutant to wild–type binding affinity) and “foreignness” (sequence divergence from the self–proteome and predicted exposure of TCR–facing residues); these metrics are calculated by comparing predicted binding scores between mutant and wild–type sequences and by assessing sequence similarity against reference proteomes and TCR–facing residue conservation. While these parameters improve prioritization in practice, their positive predictive value remains limited, and they are best used to rank candidates for experimental benchmarking [76, 77].

Functional benchmarking: bridging the gap between in silico predictions and biological reality

Immunopeptidomics by LC–MS provides the most direct evidence of presentation but has limitations: limited sensitivity for low–abundance peptides, substantial input requirements, HLA allele coverage bias, peptide detectability biases, cost, and restricted routine availability [78]. These constraints must be acknowledged when interpreting negative MS results. Therefore, MS should be complemented by orthogonal approaches. Functional validation should extend beyond single cytokine readouts (IFN-γ) to include cytotoxicity assays, multi–cytokine profiling, proliferation and activation marker assays, single–cell TCR sequencing to link specificity and clonotype, and assays using patient–derived immune cells or engineered APC/target systems. The paragraph addressing T–cell response validation has been separated from the antigen–presentation discussion to reflect this conceptual distinction [79–81].

Finally, selection filters should integrate clonality and genetic context. Prioritizing truncal or “trunk” mutations—those present in the common ancestor of all malignant cells—can increase coverage of the tumor mass, but clonality alone is insufficient: whether a mutation occurs in a driver, passenger, or resistance–associated gene influences therapeutic relevance. The potential emergence of resistant subclones after therapy means that the most clonal mutation is not always the optimal target; therefore, clonality assessment should be combined with functional relevance and evolutionary context when filtering candidates [35, 40, 82, 83].

Optimizing the payload: engineering mRNA sequences for maximum translational efficiency

mRNA vaccine design must balance multi–epitope breadth with processing fidelity. Two principal strategies exist to deliver these polyvalent payloads: (i) a single synthetic transcript encoding concatenated epitopes, and (ii) a formulation composed of a cocktail of multiple individual mRNAs, each encoding one or a few discrete epitopes. Single–transcript constructs simplify manufacturing chemistry, scaling, and dosing parameters, but concurrently increase the risk of creating unintended junctional epitopes; conversely, multi–mRNA formats reduce junctional risk and bypass long-transcript translation bottlenecks, but exponentially increase formulation complexity, analytical characterization, and manufacturing burdens. In clinical practice, practical epitope loads are generally restricted to ~20–30 epitopes per vaccine dose. This clinical ceiling reflects critical biological and technical trade-offs among manufacturing fidelity (which declines with extreme transcript length), intracellular antigen expression thresholds, host APC processing capacity saturation, systemic immune competition—where dominant peptides can suppress responses to subdominant neoantigens—and strict regulatory dosing and toxicity considerations [62, 71, 84].

This polyvalent approach mimics the multifaceted nature of natural anti-tumor immunity, thereby reducing the probability of immune escape. However, the concatenation of heterologous sequences introduces the risk of creating “junctional epitopes”—unintended, non-native sequences at the boundaries of neoantigenic units that may divert the immune response away from the target antigens. To mitigate this risk, sophisticated sequence optimization and linker engineering are employed via advanced computational pipelines. Utilizing bioinformatic tools such as NeoDesign, researchers leverage combinatorial optimization algorithms to evaluate millions of potential epitope permutations, systematically ordering the sequences to minimize unintended junctional binders while optimizing flanking cleavage motifs [85]. These pipelines incorporate structural or antigen-processing spacers, deploying rigid linkers (such as alanine-alanine-tyrosine (AAY)) or flexible linkers (such as glycine-glycine-serine (GGS) pairs) [85]. These engineered spacers facilitate autonomous proteasomal processing of each discrete neoantigen while minimizing junctional interference with the surrounding cassette.

Furthermore, the structural integrity of the mRNA serves as a primary determinant of its intracellular half-life. By utilizing thermodynamic modeling tools like RNAfold, the secondary structure and stability of the transcript can be refined [86, 87]. This process involves optimizing GC content and codon usage to eliminate inhibitory secondary configurations, such as tight hairpins, that could impede ribosomal translocation [88]. By harmonizing thermodynamic stability with translational throughput, these strategies ensure resilience against ribonuclease-mediated degradation while maximizing the magnitude of neoantigen synthesis within host APCs [89].

From bench to bedside: critical bottlenecks in clinical antigen selection

Translating personalized mRNA vaccines from design to clinical reality requires overcoming three distinct translational bottlenecks. The first is the strict selection bottleneck; despite bioinformatic filtering, empirical data indicates only ~1%–6% of predicted neoantigens elicit a functional T-cell response [71, 90]. This low conversion rate underscores algorithmic limitations in modeling stochastic intracellular processes like proteasomal cleavage and endogenous T-cell repertoire nuances [78]. To mitigate this, clinical workflows are shifting toward refined artificial intelligence (AI) and integrative proteogenomics to enhance true-positive predictive accuracy [78].

The second major barrier is the highly immunosuppressive TME, which frequently attenuates vaccine efficacy. Malignancies actively deploy evasion mechanisms, including inhibitory ligand upregulation (e.g. PD-L1) and regulatory T-cell (Treg) recruitment, causing profound T-cell exhaustion [62]. To circumvent this and revitalize effector cells, contemporary strategies utilize combinatorial regimens integrating neoantigen vaccines with ICIs, such as anti-PD-1 or anti-CTLA-4 antibodies [91]. This synergy primes the immune system with tumor-specific antigens while simultaneously lowering localized activation thresholds [92].

Finally, the transition to the bedside is hampered by the logistical complexity of personalized GMP manufacturing [93]. Unlike “off-the-shelf” therapeutics, neoantigen vaccines require rapid, patient-specific production cycles tailored to an individual’s unique mutational profile, where manufacturing delays risk clinical deterioration [93]. Transitioning to automated, modular manufacturing platforms is essential to compress the vein-to-vein timeline, ensuring personalized neoantigen therapy becomes a scalable, timely, and clinically viable reality [69].

The frontiers of mRNA synthesis and quality control

Architecture of the transcript: engineering UTRs and poly(A) tails for sustained expression

Efficient and reproducible in vitro transcription (IVT) production begins with a rigorously defined DNA template and a streamlined, QC–driven workflow (Fig. 1B, Table 1). Plasmid DNA (pDNA) design or alternative linear templates (PCR–amplified templates, dbDNA™, and linear duplexes) must integrate high–efficiency bacteriophage promoters (T7, SP6, and T3) together with engineered cis–elements—5′ and 3′ untranslated regions (UTRs) and a defined poly(A) tract—optimized to maximize mRNA stability and translational capacity while avoiding inhibitory secondary structures that induce polymerase stalling [94–100]. Template linearization downstream of the poly(A) sequence is required to prevent run–around transcription by processive bacteriophage polymerases and to yield uniform product length; residual circular DNA markedly lowers batch purity and must be minimized [101, 102].

Table 1.

Optimization strategies for mRNA vaccine development.

Strategy category	Specific techniques	Achieved outcomes and benefits	Potential pitfalls
IVT synthesis efficacy	Engineered polymerases (e.g. T7 RNAP mutants like K389A, G753A); High-yield buffers (optimized Mg²⁺)	Increases mRNA integrity to ~85%+; reduces truncated transcripts; lowers production costs	Mutants may reduce overall yield while fixing quality; buffer sensitivity can cause hydrolysis
Stability and translation	Nucleoside modification (ψ, m1ψ); UTR Engineering (Globin UTRs); Poly(A) tail lengthening	Enhances translation efficiency by up to 7-fold; extends protein expression duration	Optimal UTRs vary by cell type; long Poly(A) tails can be difficult to synthesize and verify
Reducing adverse immunity	Cap 0/1/CleanCap® technology (Cap 0 (m7GpppN; Cap 1, m7GpppNm; Cap 2, m7GpppNmNm); Cellulose-based purification to remove dsRNA	Blocks RIG-I/MDA5 sensing; prevents “cytokine storms” and premature mRNA degradation	Purification adds manufacturing complexity; incomplete capping still triggers innate immunity
Future modalities	Circular RNA (circRNA); AI-driven codon optimization (GC content balance)	Dramatically increases half-life (days vs. hours); avoids exonuclease degradation	Complex circularization processes; AI models require vast datasets for high accuracy

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Core IVT reaction parameters—enzyme (polymerase), DNA template concentration, nucleotide triphosphates (NTPs), and Mg²⁺—are tuned to balance yield, fidelity, and byproduct formation. The Mg²⁺ to NTPs ratio is a pivotal determinant of reaction kinetics and the propensity for immunogenic dsRNA byproducts [103–106]. Nucleoside modification, most commonly N1–methylpseudouridine (ψ), is widely used to decrease innate immune sensing (toll-like receptor 7/8 (TLR7/8), RIG–I/MDA5), suppress type I interferon induction, and thereby enable higher and more sustained protein expression—an essential feature for both vaccine and protein–replacement applications [105, 107]. For scale–up, fed–batch additions of NTPs and Mg²⁺, and thermostable polymerase variants, address inorganic pyrophosphate accumulation and maintain T7 activity, enabling reproducible kilogram–scale synthesis required for population–level deployment [106, 108, 109].

Capping and poly(A) strategies: practical considerations and GMP usage

Terminal modifications dictate translation initiation kinetics and transcript stability (Fig. 1C, Table 1). These structural features facilitate direct recognition by eukaryotic initiation factor 4E (eIF4E) to initiate translation while shielding mRNA from exonuclease-mediated decay [110–112]. Specifically, the 5′ cap serves as a molecular “self” marker, whereas the poly(A) tail modulates intracellular half-life and ribosome loading [113]. Co–transcriptional capping with anti–reverse cap analogs (ARCA) or advanced reagents like CleanCap® ensures correct chemical orientation; CleanCap® consistently yields a natural Cap–1 structure (typically >95%) (Table 2) that evades innate immune sensors and streamlines GMP workflows for clinical lots. Conversely, post–transcriptional capping with vaccinia-derived enzymes remains a robust alternative for discovery research and specific GMP settings requiring unique cap chemistries; both modalities achieve near–quantitative Cap–0/Cap–1 formation but differ markedly in unit operation complexity and raw material economics [114–116]. For the 3′ terminus, poly(A) tails can be template–encoded to ensure defined, homogeneous lengths—an advantage for regulatory potency assays—or appended postsynthetically via recombinant poly(A) polymerase. While template–encoded tails simplify downstream quality control (QC), enzymatic tailing permits broad length optimization, meaning each approach introduces distinct trade-offs regarding critical quality attributes and batch-to-batch homogeneity [117, 118].

Table 2.

Characterization of various caps used in mRNA vaccine development.

Cap type/tech	Structure and key modifications	Advantages	Disadvantages	FDA status	Translation efficacy
Cap 0	m7GpppN	Simple to produce; binds eIF4E to initiate translation	Highly immunogenic; recognized as “foreign” by RIG-I	Not used in approved vaccines	Baseline efficiency
Cap 1	m7GpppNm (2’-o-methylation)	Escapes innate immune sensing (RIG-I/IFIT1); high stability	Requires specific enzymatic or trinucleotide reagents	Approved (Standard for Pfizer and Moderna)	High; superior to Cap 0
Cap 2	m7GpppNmNm (two methylation)	3–5x higher protein yield than Cap 1; even lower inflammatory response	Technically difficult to synthesize with high purity	Preclinical/in development	Highest recorded
CleanCap	Trinucleotide analog (m7GpppApG)	Yields > 95% Cap 1 co-transcriptionally; “one-pot” manufacturing	High proprietary costs; requires specific start sequences (e.g. AG)	Approved (used in Pfizer-BioNTech)	Very high; consistent results
ARCA	Anti-reverse Cap analog	Prevents “reverse” cap orientation; doubles translation vs. standard Cap 0	Only produces Cap 0; still induces significant innate immunity	Primarily research use	2 × better than Cap 0

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Purification and QC: removing DNA and dsRNA to mitigate off–target inflammation

Downstream purification converts a crude IVT mixture into a therapeutic–grade drug substance, ensuring clinical safety and biological potency. Removal of template DNA by high–efficiency DNase I digestion (and orthogonal clearance) is essential to mitigate integration risk and meet residual DNA regulatory thresholds [119, 120]. The principal immunogenic contaminant—dsRNA—arises from antisense transcription, abortive initiation, or 3′ extension and potently activates MDA5 and RIG–I, triggering type I interferon responses that cause translational arrest and mRNA degradation [86, 121–123]. Thus, scalable purification strategies are selected to reduce dsRNA while maximizing recovery.

Typical GMP purification workflows pair tangential flow filtration (TFF) for diafiltration and concentration with affinity chromatography (oligo–dT capture) to enrich full–length polyadenylated transcripts and remove truncated species, residual NTPs, and enzymatic proteins. For stringent dsRNA clearance, reversed-phase high-performance liquid chromatography (RP–HPLC) or hydrophobic interaction chromatography (HIC) is employed where throughput and recovery economics permit; the choice reflects a tradeoff between purity and yield and the need for high–resolution impurity removal in clinical products [124, 125]. Comprehensive QC encompasses residual DNA, dsRNA quantitation, cap and tail integrity, sequence identity, endotoxin, bioburden, and potency assays to confirm translational competence and absence of innate activation.

Technical hurdles: identifying the persistent “blind spots” in large-scale mRNA production

Despite rapid clinical progress, several biochemical and engineering bottlenecks impede the scalable manufacturing of mRNA therapeutics. A primary hurdle is maintaining structural integrity; premature transcriptional termination generates truncated fragments that are immunologically inert and complicate QC metrics. Furthermore, persistent dsRNA generation by T7 RNA polymerase triggers unwanted cellular responses [126–129]. Resolving this requires a distinct purification trade-off: high-resolution methodologies like RP-HPLC deliver superior purity but suffer from limited scalability and low recovery yields, constraining manufacturing throughput. IVT is further complicated by a lack of universal reaction parameters. Sequence-specific tuning of reaction temperature, magnesium (Mg²⁺) concentrations, and NTP ratios is routinely required—particularly for complex architectures like multivalent cancer neoantigens—to stabilize yield profiles and suppress truncation or dsRNA formation. From a commercial perspective, high manufacturing costs for cGMP-grade enzymes and synthetic cap analogs restrict personalized medicine workflows [130]. Mechanistically, incomplete capping poses a clear safety risk, as residual 5′-triphosphate moieties trigger RIG-I-mediated immune sensing and downstream inflammatory signaling [131]. Transcript architecture decisions also directly impact downstream processing and QC. While maximizing “optimal” codons increases translation rates, overly aggressive codon optimization alters ribosome transit speed and disrupts the kinetics of co-translational folding [132]. This accelerated ribosome velocity can prevent nascent polypeptides from achieving native conformations, causing protein misfolding that potentially alters therapeutic functionality or antigenicity [132]. Ultimately, mitigating these bioprocessing blind spots requires the integration of real-time process analytics, sequence-aware reaction design, scalable high-resolution purification alternatives, and cost-reduction strategies to make patient-specific mRNA therapies broadly viable.

Navigating the body: delivery and in vivo efficacy

Vehicle design: evolving delivery platforms for enhanced stability and uptake

The transition of mRNA therapeutics from conceptual frameworks to clinical realities has been driven by iterative innovations in nanocarrier engineering [133] (Fig. 1D, Tables 3 and 4). At present, LNPs represent the absolute clinical gold standard and the most widely used delivery platform, uniquely demonstrated by their monumental success in global vaccination campaigns [53, 134]. Their efficacy is predicated on a synergistic multicomponent architecture: ionizable lipids facilitate high encapsulation efficiency and pH-dependent endosomal escape, while PEGylated lipids provide steric stabilization, and cholesterol preserves bilayer structural integrity.

Table 3.

Comparisons of mRNA delivery vehicles.

Vehicle type	Key characteristics and outcomes	Benefits	Pitfalls	Clinical/FDA status
LNPs	Sphere-shaped vesicles with ionizable lipids	High encapsulation efficiency; excellent endosomal escape; proven scalability	Requires cold chain (−20°C to −80°C); potential for PEG-related allergic reactions	Approved for human use (e.g. Comirnaty, Spikevax)
Polymers and polyplexes	Cationic or biodegradable polymers (e.g. PLGA, PBAE)	High stability; customizable for mucosal or localized delivery	Potential for cytotoxicity and particle aggregation; lower transfection than LNPs	Preclinical; some in clinical trials for cancer
Cationic nanoemulsions	O/W droplets stabilized by cationic lipids	Highly biocompatible; can incorporate various adjuvants (e.g. MF59)	Complex manufacturing; potential for instability	Preclinical and early Phase I trials (e.g. Rabies)
Exosomes (EVs)	Natural 30–150 nm vesicles secreted by living cells	Natural targeting; crosses biological barriers (e.g. blood–brain barrier); no immunogenicity	High production costs; difficult to purify and scale; low loading efficiency	Preclinical; No FDA-approved products to date.
Peptides and CPPs	Short amino acid sequences that penetrate membranes	Simple to synthesize; target-specific	Often requires complexation with other carriers; rapid clearance	Preclinical

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Table 4.

Comparative analysis of nanoparticle carrier platforms for vaccine delivery.

Carrier platform	Key physicochemical features	Major strengths	Principal bottlenecks	Optimal vaccine context	Regulatory/manufacturing precedent
LNPs	Ionizable lipids, helper lipids, PEGylated; fluid bilayer	High transfection; proven mRNA delivery; rapid scalable synthesis	Cold-chain dependence; potential transient pro-inflammatory profile	Systemic mRNA delivery; rapid-response viral vaccines	High (FDA-approved COVID-19 mRNA platforms)
Polymer-based carriers	PLA/PLGA, chitosan, dendrimers; rigid matrix	High thermal stability; highly tunable controlled sustained release	Risk of cargo degradation during organic solvent formulation	Localized delivery; single-dose depot vaccines; protein antigens	Moderate (PLGA approved for drug delivery; fewer vaccine precedents)
Hybrid carrier systems	Lipid-polymer cores; biomimetic membranes	Dual benefits: polymer stability with lipid biocompatibility	Multistep synthesis; batch-to-batch variability challenges	Complex multiantigen co-delivery; mucosal immunization.	Low (predominantly preclinical; entering early phase trials)
“Smart” delivery systems	pH, thermal, or enzyme-responsive linkages	Targeted intracellular release; minimal off-target exposure	High chemical complexity; strict regulatory hurdles for multicomponent systems	Adjuvant-targeted delivery to lymph nodes; therapeutic cancer vaccines	Low (primarily advanced preclinical development)

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Despite these successes, conventional LNPs face a major translational bottleneck due to their inherent, rapid sequestration by the liver, which causes off-target accumulation and limits extrahepatic applications [135]. To mitigate this, the field has diversified into alternative synthetic and bio-inspired materials, though each carries its own set of technical challenges. Polymer-based carriers, such as polyethylenimine (PEI), chitosan, and poly(β-amino esters) (PBAE) [136], offer excellent structural modularity for calibrating the balance between transfection efficiency and cytotoxicity via side-chain modification; however, high-molecular-weight polymers frequently suffer from high polydispersity and slow in vivo clearance, leading to long-term cumulative toxicity. Stimulus-responsive “smart” delivery systems offer precise spatiotemporal control by triggering mRNA release only upon encountering specific physiological cues—such as an acidic TME, ultrasound, or thermal induction [137–141]. Yet, these smart platforms are held back by highly complex, hard-to-scale chemical manufacturing and unpredictable triggering thresholds in living tissues. Hybrid vector systems—incorporating viral capsids or dendrimers—are evaluated for cargo protection [142], but they often trigger unintended host immune responses against the vehicle itself. These persistent synthetic limitations explain why researchers are pivoting toward endogenous exosome-based platforms, because they are derived from native biological cells, exosomes possess inherently superior biocompatibility, low immunogenicity, and natural biochemical tropisms that synthetic platforms simply cannot replicate. Concurrently, innovative modifications to synthetic baselines continue to advance; a major breakthrough in bypassing the “hepatic bottleneck” involves the development of selective organ targeting (SORT), where the addition of a fifth lipid component shifts biodistribution from the liver to the lungs or spleen [143], while the integration of biodegradable components ensures long-term biocompatibility [144].

Deployment strategies: how routes of administration dictate immunological outcomes

The therapeutic profile of mRNA interventions is inextricably linked to the administration route, which dictates systemic biodistribution and the qualitative nature of the immune response (Table 5). Traditionally, local depot administration via IM [145], and subcutaneous (SC) injections [146] serves as the cornerstone for prophylaxis. These routes establish localized microenvironments that recruit antigen-presenting cells (APCs), fostering sustained immune priming with a manageable reactogenicity profile. Building upon these traditional techniques, precision oncology has increasingly adopted IT delivery [147–149]. By bypassing high interstitial fluid pressures and systemic barriers, IT injection allows for the localized expression of immunomodulators (e.g. cytokines), converting “immunologically cold” tumors into “hot” microenvironments while avoiding systemic cytokine storms. Furthermore, intranodal (IN) administration [150, 151] targets lymph-node-resident DCs directly to accelerate T-cell priming, while mucosal routes—such as intranasal and inhalation methods—are optimized for respiratory pathologies by inducing localized mucosal-associated lymphoid tissue (MALT) responses [152–156]. Conversely, systemic administration (intravenous) remains reserved for disseminated pathologies [79, 157], though it requires robust engineering to evade rapid clearance by the mononuclear phagocyte system.

Table 5.

Summary of mRNA vaccine delivery routes.

Delivery route	Developed/tested stages	Achieved outcomes and benefits	Potential pitfalls	FDA approved status
Intramuscular (IM)	Preclinical and clinical	Efficiently recruits APCs; induces robust systemic immunity	Local pain or swelling; potential for rare myocarditis	Approved (e.g. COVID-19)
Intradermal (ID)	Preclinical and clinical	Higher density of dermal DCs; can induce stronger initial immune responses	High risk of local adverse effects (erythema, pruritus); limited injection volume	Preclinical/clinical trials
Subcutaneous (SC)	Preclinical and clinical	Permits larger volumes than ID; less painful than ID	Slower absorption; higher risk of mRNA degradation by local enzymes	Preclinical/clinical trials
Intravenous (IV)	Preclinical and clinical	Fastest systemic distribution; ideal for therapeutic antibodies or targeting the liver/spleen	High risk of systemic side effects (cytokine storms, spleen injury)	Preclinical/cancer trials
Intranasal (IN)	Preclinical and in vitro	Induces mucosal immunity; needle-free and potentially self-administered	Complex formulation needed for mucosal penetration; risk of rapid clearance	Preclinical
Intranodal (INo)	Preclinical	Direct delivery to lymph nodes; highly efficient APC activation	Requires ultrasound-guided injection; highly invasive and technically complex	Preclinical

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Crucially, the chosen route of administration behaves differently depending on the specific structural platform used. While highly stable endogenous exosomes can navigate mucosal and systemic barriers with minimal structural degradation, synthetic polymeric and hybrid systems are far more prone to aggregate and shear under the mechanical forces of direct local injections. Furthermore, a critical pharmacokinetic distinction must be made regarding the gold-standard route: although IM injection is highly optimized to anchor the initial dose within skeletal muscle tissue, it is a common misconception that local delivery entirely prevents systemic escape. In reality, IM injection is not sufficient to alter the inherent liver-homing tendency of standard LNPs; a significant fraction of locally injected LNPs inevitably enters local capillaries, drains into the systemic bloodstream, and binds to circulating apolipoprotein E (ApoE), resulting in subsequent off-target accumulation in hepatic tissues.

Among these diversified strategies, IM injection remains the standardized route for prophylactic mRNA delivery, utilizing skeletal muscle as a transient “immunological factory” and biochemical depot. The dense vascularization of muscle tissue facilitates the rapid recruitment of professional APCs, such as DCs, while its anatomical proximity to major axillary lymph node clusters ensures efficient lymphatic transport for robust B and T cell activation [158]. Furthermore, the interstitial architecture of the muscle acts as a reservoir that sustains the presence of LNPs for ~24–28 hours. This localized retention provides an extended “immunological training session” that enhances potency while mitigating the risks of systemic inflammatory cascades or organ-specific toxicities associated with intravenous administration. Compared to the restricted volumes of intradermal or SC routes, the high volumetric capacity of the deltoid allows for consistent dosing with significantly reduced risks of local tissue necrosis or severe granulomatous reactions. Ultimately, the IM route offers an optimal nexus of pharmacokinetic stability, superior safety, and clinical scalability essential for global immunization efforts.

Overcoming biological barriers: defining the metrics for successful systemic delivery

Systemic mRNA efficacy requires navigating a series of extracellular and intracellular physiological barriers. Protective nanocarriers provide the first line of defense, shielding the transcript against degradation by extracellular ribonucleases (RNases) [159]. Upon reaching target tissues, crossing the anionic cell membrane presents a distinct kinetic bottleneck. Incorporating pH-responsive ionizable lipids resolves this by facilitating endosomal escape, preventing the diversion of entrapped mRNA to lysosomal degradation pathways [160, 161]. Once inside the cytoplasm, mRNA translation duration dictates therapeutic efficacy, contrasting sharply with the pharmacokinetics of protein-based biologics. Advanced bioluminescence tracking reveals that vehicle composition—specifically the molecular weight of polymer backbones—can modulate these expression kinetics and systemic biodistribution [148], shifting output from a transient protein “burst” to a sustained-release profile [162]. Beyond nucleic acid delivery, modern vehicles are increasingly engineered as “self-adjuvants” that provide intrinsic TLR stimulation to amplify host immunogenicity [163]. This adjuvant activity can be leveraged in combination regimens; co-administering mRNA-encoded neoantigens with ICIs yields a synergistic therapeutic effect capable of reversing T-cell exhaustion and extending survival in treatment-resistant tumor models [71].

The rise of exosomes as natural delivery vehicles

Nature vs. synthetic: the inherent advantages of exosomes over LNPs

As endogenolong-termus EVs, exosomes represent a profound biological paradigm shift over synthetic LNPs [107], offering superior biocompatibility and minimal immunogenicity (Fig. 2) [164]. While conventional LNPs rely heavily on ionizable lipids—which are known to trigger pro–inflammatory cascades or cause the accelerated blood clearance (ABC) phenomenon upon repeated clinical dosing—exosomes possess an inherent “biological passport”. Their complex lipid bilayer is naturally enriched with cholesterol, sphingomyelin, and saturated phospholipids, providing a robust, native shield that sequesters mRNA from RNase degradation while circumventing the mononuclear phagocyte system (MPS) [165, 166].

A defining mechanistic advantage of this natural vehicle is the surface display of CD47, a potent “don’t eat me” signal. By interacting directly with SIRPα on macrophages to inhibit phagocytosis, preclinical studies have shown that CD47 grants exosomes an extended circulation half–life that consistently outperforms traditional PEGylated synthetic carriers [167]. Crucially, this extended circulation is vital for directing mRNA delivery to the correct target cells. For prophylactic vaccines and cancer immunotherapies to achieve optimal clinical efficacy, the mRNA cargo must be selectively delivered into APCs—specifically DCs and splenic macrophages—which are equipped to process antigens and orchestrate downstream adaptive T–cell and B–cell immunity.

This absolute immunological requirement highlights a severe limitation of conventional synthetic LNPs; because synthetic lipids naturally bind to blood–borne ApoE, standard LNPs are heavily sequestered by hepatocytes in the liver rather than preferentially targeting professional APCs. Crucially, data obtained from mouse models demonstrate that exosomes naturally bypass this hepatic bottleneck, thereby successfully overcoming the rigid liver-targeting restrictions inherent to conventional synthetic matrices.

Furthermore, in initial preclinical testing, exosomes excel at multifunctional trafficking, a concept distinct from the multivalent multiantigen targeting (TSA/TAA combinations). Here, multifunctional trafficking refers to a vehicle’s simultaneous ability to cross formidable physical boundaries and deeply infiltrate dense tissues. Unlike conventional LNPs, which require extensive, multi–step surface chemical functionalization to avoid liver capture, preclinical evaluations suggest that exosomes leverage endogenous transcytosis pathways to traverse the blood–brain barrier (BBB) and penetrate the high interstitial fluid pressure of the blood–tumor barrier (BTB) [168].

The integration of tetraspanins (CD9, CD63, and CD81) and cell–specific integrins directly into the exosomal membrane facilitates precise cellular tropism and docking. Upon reaching the target, exosomes facilitate mRNA delivery via direct membrane fusion or specialized endocytosis. By “hijacking” the recipient cell’s internal trafficking machinery, it has been observed in vitro and in vivo that they achieve highly efficient cytosolic release, completely bypassing the endosomal entrapment and lysosomal degradation that frequently limit the potency of synthetic delivery systems [169]. Consequently, preclinical models indicate that exosomes provide a “stealth” delivery architecture that marries structural stability with sophisticated cellular signaling, offering a level of biological integration that synthetic LNPs have yet to fully replicate.

These mechanistic advantages, however, should be interpreted with appropriate caution. Practical challenges for exosomal therapeutics include manufacturing scale–up, donor–cell–dependent vesicle heterogeneity, variable cargo–loading efficiencies, and current gaps in precise biodistribution control; importantly, robust clinical efficacy data remain limited at present. By contrast, LNPs provide well–established scalable manufacturing and tunable chemistry that favor reproducible dosing and regulatory pathfinding. Mechanistic claims such as consistent BBB crossing or uniform integrin–mediated homing are supported in select preclinical models but require further validation in large–animal and clinical contexts. Thus, exosomes and LNPs represent complementary platforms: exosomes offer native tropism and reduced systemic reactogenicity in specific settings, whereas LNPs retain advantages in scalability and predictable systemic delivery. Later sections compare these platforms in the context of immune training and TME remodeling, emphasizing that platform choice should be guided by therapeutic context and translational feasibility.

Loading the cargo: strategies for endogenous and exogenous mRNA packaging

To address the challenge of low natural mRNA encapsulation, researchers have developed robust engineering protocols. Endogenous loading exploits the cellular secretory pathway by modifying donor cells to express RNA–binding fusion proteins (e.g. L7Ae, HuR, or MS2). These proteins recognize specific motifs on the target mRNA to actively shuttle it into nascent vesicles [170, 171]. A prominent example is the exosomal transfer into cells (EXOtic) system, which utilizes an L7Ae–functionalized CD63 scaffold to achieve precise mRNA sequestration, significantly enhancing delivery in neurodegenerative models. Conversely, exogenous loading involves the post–isolation manipulation of the exosomal bilayer. While electroporation and sonication are standard, they often suffer from mRNA aggregation or compromised membrane integrity; electroporation, in particular, may induce RNA artifacts that skew efficiency data [172]. Cellular nanoporation (CNP) represents a paradigm shift in this space; by applying focal electrical pulses to donor cells via nano–engineered silicon chips, CNP stimulates a 1000–fold increase in mRNA encapsulation compared to bulk methods [173]. This high–throughput approach enables the delivery of large transcripts, such as PTEN mRNA, to dense solid tumors, bridging the gap between bench–scale isolation and clinical–grade production [174, 175].

Surface customization: modular engineering and the development of chimeric hybrids

Targeting specificity is typically achieved through the genetic engineering of donor cells, where ligands are fused to exosomal “anchors” like Lamp2b, tetraspanins (CD63 and CD81), or the C1C2 domain of lactadherin. For instance, fusing the RVG peptide to Lamp2b enables neurotropism [168], while the iRGD peptide enhances tumor penetration via αv integrin recognition [176].

Complementing genetic methods, bio-orthogonal “click chemistry” (e.g. CuAAC or the copper-free SPAAC) allows for the covalent attachment of complex moieties—such as monoclonal antibodies or aptamers—directly to the membrane [177]. This post-isolation modification avoids the complexities of cellular synthesis and prevents metal-induced toxicity. Furthermore, exosome-liposome hybrids have emerged as “engineered chimeras”, integrating the high loading capacity of synthetic liposomes with the “self” signaling (e.g. CD47) of natural exosomes. These hybrids can incorporate ionizable lipids to combine the pH-responsive endosomal escape of LNPs with superior biological stealth.

Intracellular logic: mastering biodistribution and the kinetics of endosomal escape

The therapeutic potential of exosomes is anchored in their intrinsic homing capabilities and specialized surface topographies. Dendritic cell-derived exosomes (Dex) naturally display MHC-I/II complexes and costimulatory molecules (CD80/CD86), facilitating targeted trafficking to draining lymph nodes for optimal antigen presentation [178]. This makes Dex an ideal scaffold for mRNA vaccines designed to elicit robust anti-tumor immunity. Similarly, mesenchymal stem cell (MSC)-derived exosomes exhibit innate biochemical tropism for inflamed or infarcted tissues, particularly in myocardial and ischemic stroke models [179].

A burgeoning frontier in EV therapeutics utilizes red blood cell-derived exosomes (RBC-Exos). These vesicles offer unique advantages: they lack nuclear and mitochondrial DNA, minimizing horizontal gene transfer risks, and can be harvested in vast quantities from clinical-grade blood units. RBC-Exos naturally possess glycophorin A and high levels of CD47, which drastically reduces splenic sequestration and extends circulation half-life, making them highly efficient “stealth” vehicles for systemic mRNA delivery.

Despite these advantages, endosomal sequestration remains a primary kinetic bottleneck. To circumvent this, “smart” exosomes have been engineered to incorporate pH-sensitive pore-forming proteins, such as Listeriolysin O (LLO). LLO-mediated membrane destabilization occurs rapidly upon endosomal acidification, ensuring prompt cytosolic release of mRNA. Alternative strategies utilize pH-responsive fusogenic peptides (e.g. GALA), which undergo a conformational shift at low pH to disrupt the endosomal bilayer and enhance translational output [180–182].

These advancements have yielded significant preclinical outcomes in oncology. In glioblastoma models, exosomal vehicles—particularly those derived from RBCs or DCs—have successfully traversed the BBB to deliver tumor-suppressor transcripts. Furthermore, exosome-mediated delivery of PTEN mRNA has been shown to restore phosphatase activity and suppress PI3K/Akt signaling in PTEN-deficient prostate cancer models [183]. These studies document substantial tumor regression and improved survival, highlighting the superior precision and kinetic efficiency of exosomal delivery over conventional synthetic systems.

Scaling for the clinic: addressing the hurdles of exosome standardization and yield

The clinical translation of exosome-based mRNA therapeutics is primarily obstructed by the standardization of isolation and purification. Traditional differential ultracentrifugation is insufficient for cGMP manufacturing due to its inability to resolve homogeneous populations from large-scale media. Nonexosomal contaminants, such as protein aggregates and lipoproteins, compromise biological activity and safety [184]. Consequently, the field is shifting toward TFF and size-exclusion chromatography (SEC). Coupled together, these methods provide an optimal balance of yield and purity, removing over 95% of soluble protein contaminants [185].

Passive loading methods (e.g. simple incubation) yield notoriously low drug-loading efficiency (often <10%), while active methods (e.g. electroporation, sonication, or transient membrane permeabilization) risk destabilizing the exosome’s delicate lipid bilayer and promoting structural aggregation. This fundamental loading limitation directly compounds the issue of batch-to-batch variability. Because exosome cargo profiles and surface proteins are highly sensitive to subtle alterations in donor cell states and 3D culture microenvironments, achieving consistent molecular identity across industrial batches remains a formidable hurdle. This variability is further exacerbated by a lack of universal QC standards. Current regulatory pathways are restricted by the absence of consensus metrics defining acceptable particle-to-protein ratios, precise single-vesicle cargo quantification, and standardized identity markers to assure cGMP-grade purity and safety prior to clinical release.

While RBC-Exos offer a scalable, DNA-free alternative, they introduce unique pitfalls. The high concentration of hemoglobin in RBC-Exos can interfere with downstream analytical assays and potentially trigger oxidative stress if not meticulously removed. Furthermore, the lack of a nucleus precludes the use of endogenous genetic engineering for surface targeting, forcing reliance on complex, often inefficient post-isolation chemical conjugation or hydrophobic insertion of ligands. Industrial scalability remains a significant barrier. Moving beyond 2D culture requires 3D bioreactor systems, such as hollow-fiber bioreactors, which offer superior surface-area-to-volume ratios and nutrient exchange [186]. These systems can achieve up to a 40-fold increase in EV concentration while preserving cargo potency [187]. However, ensuring batch-to-batch consistency and uniform mRNA loading at this scale remains a complex engineering challenge. Finally, storage stability imposes logistical constraints. Current formulations necessitate −80°C storage to prevent membrane degradation. To achieve “off-the-shelf” utility, research into lyophilization is intensifying. Incorporating cryoprotectants like trehalose has been shown to maintain vesicle morphology and mRNA integrity for up to six months at 4°C [188]. Despite this, the precise impact of lyophilization on the cellular uptake kinetics of these biologics requires further longitudinal study to ensure that long-term storage does not diminish therapeutic efficacy.

Decoding the multivalent immune response

Bridging the divide: PAMP recognition and the activation of DC maturation

The efficacy of mRNA-LNP vaccines on immune responses (Fig. 3A and B, Tables 6 and 7) is predicated on their “autoadjuvant” properties, where the mRNA cargo and lipid scaffold function as potent pathogen-associated molecular patterns (PAMPs). Upon endosomal uptake, mRNA is recognized by TLR7/8, while LNP components can activate the cGAS-STING pathway. This dual recognition triggers a transcriptional program resulting in Type I interferons (IFN-α/β) and pro-inflammatory cytokines like IL-6 and TNF-α [62, 84, 189]. This signaling milieu transforms the injection site into an active immunological hub. Within hours, this gradient recruits neutrophils and inflammatory monocytes (Ly6C⁺ in mice; CD14⁺ in humans) [190] to initiate the primary inflammatory hub. These cells differentiate into monocyte-derived DCs (moDCs), which, alongside conventional DCs (cDCs), serve as translational engines for mRNA translation and upregulate costimulatory ligands CD80 and CD86 [191, 192]. Adaptive immunity is governed by specialized subsets: cDC1s (CD8α⁺/CD141⁺) drive CD8⁺ T-cell responses via cross-presentation—facilitated by the LNP’s ability to provide cytosolic access for MHC-I loading [62, 79, 84], while cDC2s (CD11b⁺/CD1c⁺) orchestrate CD4⁺ helper T-cell differentiation [193].

Table 6.

Immune dynamics of mRNA vaccine delivery.

Category	Immune pathways/mechanisms	Key immune cell types
Innate sensing and activation	TLR7/8 and cGAS-STING: recognition of mRNA/LNP as PAMPs Type I IFN signaling: production of IFN-α/β, IL-6, and TNF-α Pro-presentation: exosomes carrying preformed MHC-peptide complexes	Neutrophils, inflammatory monocytes (Ly6C⁺/CD14⁺), NK cells
Antigen presentation	Cross-presentation: cytosolic access for MHC-I loading Noncanonical autophagy: shunting antigens for MHC-II processing CCR7-dependent trafficking: migration of APCs to draining lymph nodes (dLNs)	cDC1 (CD8α⁺/CD141⁺), cDC2 (CD11b⁺/CD1c⁺), monocyte-derived DCs (moDCs)
Adaptive effector response	Th1 polarization: IFN-γ and IL-2 rich environment Targeted lysis: Perforin/granzyme-mediated cell death ADCC: antibody-mediated cytotoxicity via Fc receptor engagement	CD8⁺ cytotoxic T lymphocytes (CTLs), CD4⁺ Th1 cells, NK cells, B cells
TME reprogramming	M2-to-M1 switch: converting TAMs to a tumoricidal phenotype Hot tumor conversion: increasing TIL infiltration and reducing exhaustion markers (PD-1, TIM-3) Stromal targeting: cxosomal delivery of IL-12/costimulatory ligands	Tumor-associated macrophages (TAMs), tumor-infiltrating lymphocytes (TILs), stromal cells
Immunological memory	Signal 3 modulation: cytokine-driven differentiation of long-lived memory Systemic surveillance: interception of micrometastases	Central memory (TCM), effector memory (TEM), tissue-resident memory (TRM)

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Table 7.

Role of immune responses in vaccine efficacy.

Category	Component/pathway	Role in vaccine efficacy
Immune pathways	PAMP-mediated signaling	Recognition of mRNA via TLR7/8 and LNP via cGAS-STING; triggers type I IFNs (IFN-α/β) and cytokines (IL-6, TNF-α)
	Antigen presentation	Simultaneous MHC-I (cytosolic access) and MHC-II (via noncanonical autophagy) loading for T-cell priming
	Th1 polarization	Creation of an IFN-γ/IL-2 rich milieu to favor CD8⁺ CTLs and bypass Treg suppression
	TME reprogramming	Reversal of immunosuppression; converting M2 (pro-tumor) macrophages to M1 (tumoricidal) phenotypes
Innate immune cells	Dendritic cells (cDCs and moDCs)	cDC1 drive CD8⁺ responses; cDC2 orchestrate CD4⁺ helper T-cells; moDCs serve as translation engines
	Neutrophils and monocytes	Recruited to the injection site to initiate the inflammatory hub
	Natural killer (NK) cells	Rapid effector function via granzyme B; mediated by IL-12 and IFN-γ gradients
	TAMs	Tumor-associated macrophages are reprogrammed to M1 to secrete TNF-α and promote antigen presentation
Adaptive immune cells	CD8⁺ CTLs	Primary “strike force” executioners using perforin and granzymes for targeted lysis
	CD4⁺ Th1 clls	Provide essential “help” for durable memory and coordinate the multipronged response
	Memory T-cells	TCM, TEM, and TRM subsets provide long-term surveillance and intercept metastatic seeding
	B-cells	Produce antibodies that enable persistent protection through ADCC

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Exosome-delivered mRNA vaccines offer a distinct “innate-adaptive bridge”. Unlike the often harsh, reactive inflammatory profile of synthetic LNPs, exosomes modulate the innate response through their host-derived membrane proteins, frequently outperforming synthetic systems without the strict requirement for additional, exogenous chemical adjuvants [193]. For instance, exosome-encapsulated mRNA can still trigger necessary TLR signaling, but the delivery kinetics are stealthier, reducing systemic reactogenicity while preserving local potency.

Furthermore, when harvested from matured or antigen-pulsed donor antigen-presenting cells, these exosomes carry preformed, antigen-specific MHC-peptide complexes embedded directly within their lipid bilayers. This architecture enables a specialized phenomenon termed “pro-presentation”. Here, the vehicle itself functions as a direct antigen presentation platform immediately upon reaching recipient T-cells, serving as an instantaneous “Signal 1” activation wave. This cell-derived presentation operates in tandem with the encapsulated mRNA cargo, which enters the host cell cytoplasm to initiate a delayed, secondary wave of sustained endogenous antigen translation [194]. mRNA-LNP and exosomal technologies bypass traditional antigen compartmentalization, ensuring simultaneous MHC-I and MHC-II loading. While endogenous synthesis feeds the MHC-I pathway, the induction of noncanonical autophagy by delivery components shunts intracellular antigens into the lysosomal compartment for MHC-II processing, ensuring the CD4⁺ T-cell help required for durable memory [195–199].

Orchestrating the attack: the spatiotemporal mobilization of CTL and NK cells

The protective efficacy of mRNA vaccines relies on coordinated adaptive and innate immune responses. CD8⁺ CTLs serve as primary effectors, executing targeted lysis of malignant or infected cells via perforin and granzymes [62, 84]. The activation of these CTLs is driven by a Th1–dominant polarization inherent to mRNA-LNP formulations, which counteracts the immunosuppressive influence of regulatory T cells (Tregs) by creating a cytokine milieu rich in IFN–γ and IL–2 [62, 84, 200]. Concurrently, vaccine–induced B–cell responses provide humoral synergy through antibody–dependent cellular cytotoxicity (ADCC). This cellular arm is augmented by natural killer (NK) cell activation; driven by dose-dependent IL-12 and IFN-γ gradients, NK cells upregulate CD69 and granzyme B to provide a rapid effector wave [193, 201]. Mechanistically, antibody Fc fragments engage receptors on NK cells and macrophages to destroy targets expressing vaccine–derived surface antigens [191]. This ADCC activity remains detectable for over a year, providing persistent protection against variants that might evade direct neutralization [193].

Delivery platform choice significantly modulates these downstream dynamics. LNPs reliably induce robust systemic Th1 polarization via innate sensors (TLR7/8, cGAS–STING) and reproducibly generate potent CTL responses across multiple clinical models. Exosome–delivered mRNA vaccines offer distinct immunomodulatory features alongside notable processing limitations. Because exosomes can harbor membrane–bound costimulatory molecules (e.g. CD80 and CD86) and pre–formed MHC–peptide complexes from parent cells, they deliver coordinated Signal 1 and Signal 2 to cognate T cells prior to de novo antigen expression, favoring metabolic fitness and high–avidity memory formation in preclinical models. However, the prevalence and functional consistency of such surface displays remain donor– and preparation–dependent, and comparative clinical data demonstrating superior long–term memory over conventional synthetic systems remain limited.

Flipping the switch: reprogramming the immunosuppressive TME

The immunosuppressive TME remains a formidable barrier to immunotherapy, characterized by dense extracellular matrix, hypoxia, and a wealth of inhibitory signaling. However, mRNA–based strategies offer a robust mechanism for phenotypic reprogramming (Fig. 3C and D). A central pillar of this remodeling is the conversion of tumor-associated macrophages (TAMs) from a pro–tumor, angiogenic M2–like state toward a tumoricidal M1 phenotype [202, 203]. This polarization reverses local immunosuppression, as M1 macrophages actively secrete IFN–γ and TNF–α to inhibit tumor progression and promote antigen presentation.

Exosome–delivered mRNA vaccines introduce a sophisticated layer to this remodeling. Unlike synthetic LNPs, which can sometimes trigger generalized inflammation, exosomes possess innate biochemical tropism for the TME, leveraging surface integrins—such as αvβ3 and αvβ5—to home into malignant niches. This targeted homing remains highly relevant even in the context of conventional IM injection; following local depot administration, a defined fraction of integrin–bearing exosomes or the DCs that internalize them enter the systemic circulation. These vesicles naturally dock at distant tumor sites because their surface integrins bind tightly to vitronectin and fibronectin overexpressed on tumor vascular endothelial cells and stromal elements. Once internalized within the malignant niche, exosomal mRNA specifically reprograms the TME by delivering transcripts encoding cytokines (e.g. IL–12) or costimulatory ligands directly to stromal cells and TAMs, minimizing systemic cytokine storms while maximizing local “hot” tumor conversion.

Crucially, this exosome–mediated mechanism remains effective even in advanced tumors, where systemic immunity and the TME are substantially altered compared to healthy tissue or early–stage disease. Advanced tumors present a hostile architecture with dense physical collagen barriers, extreme hypoxia, and deep systemic immune tolerance. While standard LNPs often fail to penetrate these dense, high–pressure environments and risk causing systemic toxicities, the natural transcytosis capacity of exosomes allows them to penetrate deeply into advanced tumor cores. The targeted delivery of IL–12 or costimulatory transcripts directly to advanced TAMs effectively circumvents systemic tolerance, locally forcing the downregulation of exhaustion markers such as PD–1, TIM–3, and LAG–3, particularly when integrated with immune checkpoint inhibitors (ICIs), thereby restoring the proliferative capacity and effector function of tumor–infiltrating lymphocytes (TILs) [204–206].

It should be noted that integrin–mediated homing and deep tumor penetration reported for exosomes show variability across models and preparations; reproducibility in large–animal and clinical settings remains to be established. Engineered LNPs, especially with IT delivery or tailored size/charge, can also achieve meaningful IT distribution and TME reprogramming. Consequently, choice of platform for TME remodeling should be guided by the specific therapeutic context—targeted, low–reactogenicity modulation may favor exosomes in select preclinical scenarios, whereas LNPs offer advantages in scalability and dose control for broader clinical application.

Lasting protection: establishing the foundations of immunological memory and surveillance

The long–term efficacy of mRNA vaccines in preventing infectious relapse and tumor recurrence is anchored in the robust generation of long–lived memory T–cell subsets, specifically central memory central memory T-cell (TCM), effector memory T-cell (TEM), and tissue-resident memory T-cell (TRM) cells. Conventional mRNA–LNP formulations, typically delivered via IM injection, induce potent TCM and CD8⁺ T–cell responses that form the basis for durable protective immunity [62, 207]. While TCM cells primarily reside in secondary lymphoid organs to maintain high proliferative potential, cells circulate through peripheral tissues to provide immediate effector recall. This diverse memory pool is essential for systemic surveillance, executing continuous host protection by intercepting metastatic seeding and eliminating dormant micrometastases before they progress to clinical recurrence [71].

A critical frontier in enhancing this memory architecture involves the use of exosomes as mRNA delivery vehicles. Direct comparative preclinical studies demonstrate that while conventional LNPs induce sharp, ntier in enhancing this memory architecturet ransient central memory bursts, exosome–based platforms exhibit a distinct capacity in rodent models for generating sustained, high–avidity memory T–cell responses due to their specialized tissue–homing properties and non–exhausting priming kinetics. In these animal models, experiments suggest that exosomal delivery can modulate the Signal 3 cytokine environment during priming, driving cell differentiation toward long-lived memory precursors rather than short-lived effectors.

Furthermore, while intramuscular (IM) administration of standard LNPs is highly effective for systemic priming, it exhibits reduced efficiency in eliciting lung–resident memory (TRM) compared to natural infection. However, preclinical evaluations show that the modular nature of exosomes allows for surface engineering to target specific distal mucosal sites even after IM injection [208–210]. These CD103⁺ and CD69⁺ cells function as tissue-resident memory populations within the tissue architecture, providing a site–specific response that bypasses the need for leukocyte recruitment from peripheral blood [81, 211]. Recent longitudinal preclinical studies demonstrate that mRNA–LNP and exosomal platforms promote the expansion of these persistent subsets, ensuring CD8⁺ T–cell durability for several months, even as neutralizing antibody titers naturally decline [207, 212, 213]. By maintaining this diverse memory landscape—integrating systemic circulation with localized resident populations—next–generation mRNA vaccines provide a continuous immunological shield against both evolving viral variants and emergent tumor neoantigens.

In summary, preclinical observations confirm that both mRNA–LNP and exosomal vaccines activate innate and adaptive immunity via TLR7/8 and cGAS–STING pathways, fostering a Th1–polarized milieu essential for DC maturation. While intramuscularly delivered LNPs act as potent “auto–adjuvants”—triggering rapid systemic inflammation and effector responses—preclinical models indicate that exosome platforms offer superior biochemical tropism for TME via integrin–mediated delivery. By providing “dual–signals” (mRNA and pre–formed MHC), it has been observed in mouse models that exosomes facilitate more durable memory T–cell formation with reduced systemic reactogenicity. Consequently, based on current preclinical metrics, while LNPs provide a robust initial burst, exosomal systems achieve a more balanced, long–lasting strike force mobilization for sustained anti–tumor efficacy (Tables 6 and 7).

The multidimensional landscape of immune reprogramming

The therapeutic efficacy of cancer mRNA vaccines is governed by a highly synchronized, multitiered reprogramming of the host immune system. This complex phenomenon operates across two distinct but deeply interconnected dimensions: upstream extracellular cytokine dynamics that orchestrate immediate tissue plasticity and downstream intranuclear epigenetic modifications that permanently hardwire long-term immune vigilance. Together, these complementary axes convert local, transient vaccine sensing into a durable, systemic anti-tumor response.

Extracellular signaling: reshaping the TME through cytokine orchestration

The therapeutic efficacy of mRNA vaccines is predicated on a coordinated, cytokine-driven metamorphosis of the immune landscape, transitioning from innate sensing to adaptive execution (Table 8). While conventional synthetic LNPs activate an intense, systemic pro-inflammatory cascade driven by endosomal TLR7/8 sensing, next-generation exosomal vehicles utilize their native host-derived endogenous surface marker profiles (e.g. CD47 expression) to elicit a highly localized, nontoxic cytokine profile. Preclinical studies demonstrate that exosome-mediated delivery induces a tightly synchronized, Th1-skewed cytokine microenvironment characterized by high local concentrations of IL-12 and IFN-γ, without inducing the off-target systemic reactogenicity or Th2-associated IL-4 spikes often seen with high-dose synthetic lipid matrices [71].

Table 8.

Cytokine dynamics in mRNA vaccine efficacy.

Cytokine type	Key cytokines	Role	Target immune cells/pathways	Effect on vaccine efficacy
Type I interferons	IFN-α, IFN-β	Good	Activates cDC1s and NK cells; promotes MHC-I expression via JAK-STAT pathway	Promotes: essential for the “autoadjuvant” effect of LNPs; initiates T-cell priming
Th1/pro-inflammatory	IFN-γ, IL-12, IL-2	Good	Drives CD8⁺ CTL activation and Th1 differentiation; activates M1 macrophages	Promotes: creates a “hot” TME; ensures durable memory (TCM) and cytotoxic killing
Effector/pyrogenic	TNF-α, IL-6	Mixed	Recruits neutrophils and monocytes to the injection site (IM route)	Promotes: local recruitment; Suppresses: High systemic levels (LNP “burst”) cause reactogenicity
Immunosuppressive	IL-10, TGF-β	Bad	Activates tregs and MDSCs; induces M2 macrophage polarization	Suppresses: shuts down T-cell proliferation; reinforces the TME’s “cold” architecture
Checkpoint-related	IL-35	Bad	Promotes T-cell exhaustion; upregulates PD-1/LAG-3 expression	Suppresses: limits the persistence and “strike force” of vaccine-induced CTLs

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This balanced cytokine orchestration is essential for “licensing” DCs within the TME. The localized accumulation of IL-12 and IFN-γ lowers the activation threshold for naïve T-cells and provides critical paracrine cytokine support, specifically IL-2, which is required to sustain robust cytotoxic CD8⁺ T-cell proliferation and function [71]. Consequently, this precise signaling control dictates overall vaccine efficacy by shifting the quality of the adaptive response. Clinical observations confirm that mRNA-induced cytokine signaling significantly amplifies the expansion of long-lived effector memory (TEM) cells and stem cell-like memory cells [207, 212]. By utilizing exosomal platforms to sustain these antigen-specific CD4⁺ and CD8⁺ T-cell reactivities locally for months—even as neutralizing antibody titers naturally decline—the immune setpoint is effectively shifted from local tolerance to long-term active surveillance, providing a durable defense mechanism against tumor evolution and metastatic spread.

Breaking resistance: turning “cold” tumors “hot” to enhance ICI sensitivity

The clinical utility of mRNA vaccines extends beyond antigen delivery; they function as potent immunomodulators capable of converting “immunologically cold” tumors into “hot”, inflamed environments (Fig. 3C and D). A primary mechanism for this transformation is the cytokine-mediated repolarization of myeloid cells, where the localized production of IFN-γ and TNF-α drives the phenotypic shift of pro-tumorigenic M2 macrophages into tumoricidal M1 phenotypes [71]. These reprogrammed M1 macrophages secrete high levels of IL-12, further amplifying the Th1 response and effectively heating up resistant tumors by disrupting the immunosuppressive stroma.

The emergence of exosome-delivered mRNA vaccines adds a highly sophisticated layer to this molecular remodeling. Direct preclinical evidence demonstrates that engineered exosomes achieve exceptionally deep tissue infiltration in poorly vascularized tumors, triggering a dense, localized IFN surge that enhances CD8⁺ T-cell priming directly within the malignant core [212]. Importantly, advanced modified LNPs—such as SORT or ligand-functionalized synthetic nanoparticles—have been engineered to achieve improved organ-specific trafficking and tumor localized delivery. However, even these advanced synthetic variants frequently face limitations in dense solid tumors; they remain susceptible to the high interstitial fluid pressures of the tumor stroma and risk triggering ABC upon repeated dosing due to their synthetic components.

Exosomes possess specific, native structural features that allow them to overcome these exact translational bottlenecks. Because exosomes inherit an endogenous nanoscale lipid asymmetry and superior membrane lipid deformability from their parent cells, they can mechanically navigate tight extracellular matrix gaps that trap rigid synthetic nanoparticles. Furthermore, their surface display of native tetraspanins and cell-specific integrins promotes active, receptor-mediated transcytosis through the tumor endothelium, while surface CD47 protects them from macrophage clearance within the stroma. This deep penetration allows exosome-delivered mRNA to establish a highly uniform cytokine surge. This localized induction of Type I IFNs and IFN-γ leads to a compensatory upregulation of PD-L1 on tumor cells and infiltrating immune cells. While this is a classical defensive mechanism by cancer to evade T-cell attack, it effectively sensitizes the tumor to immune ICIs. Previous research [62] suggests that this mRNA-induced “inflamed” state makes the TME highly responsive to anti-PD-1/PD-L1 therapies, providing the cellular fuel for checkpoint blockade and significantly improving overall survival across diverse histologies.

Global effects: leveraging systemic reprogramming for long-term preventive surveillance

The long-term preventive efficacy of mRNA vaccines lies in their ability to establish “high-alert” memory pools through specialized cytokine-driven maturation. The induction of IL-15 and Type I Interferons is critical for the survival and homeostatic proliferation of TCM and stem cell-like memory T-cell (TSCM). As previous studies [212] suggest, these cytokine profiles facilitate epigenetic remodeling that ensures cellular persistence for years, maintaining a “ready-to-act” state where memory B-cells and T-cells undergo rapid clonal expansion upon detecting nascent neoantigens.

Exosome-delivered mRNA vaccines offer a distinct advantage in executing this systemic macro-environmental remodeling and distal lymphatic niche cross-priming. Unlike traditional IM injections of LNPs, which primarily drive localized inflammation or un-targeted systemic drainage via TLR7/8 [62], engineered exosomes exploit natural bio-tropisms to achieve targeted homing directly to distant secondary lymphoid organs, such as the spleen and remote lymph nodes. Once localized in these distal niches, exosomes deliver their mRNA cargo to resident antigen-presenting cells, favoring the differentiation of high-avidity memory precursors over short-lived, exhausted effectors.

Furthermore, evidence indicates that these exosomal vehicles significantly enhance trained immunity—a broad-scale epigenetic and metabolic reprogramming of the innate immune system. By shifting distant myeloid cell baseline profiles from passive tolerance to active vigilance, mRNA-LNP and exosomal platforms prime the entire systemic immune architecture to eliminate malignant clones through enhanced immunosurveillance. This proactive defense is reinforced by the poly-specific nature of mRNA-induced immunity, providing a safety net against tumor heterogeneity [71]. The resulting global memory landscape—characterized by CD103⁺ TRM tissue-resident memory populations and circulating TEM populations—acts as a proactive shield capable of eradicating dormant micrometastases before they achieve clinical escape, ensuring sustained protection against tumor recurrence.

Intranuclear hardwiring: epigenetic priming and chromatin readiness

While cytokine networks initiate immediate immune remodeling, the ultimate sustainability of this cellular state is governed by “epigenetic priming”—the intranuclear molecular hardwiring that dictates host immune responsiveness [214–216]. This baseline readiness is established through two primary molecular axes: DNA methylation landscapes in lineage commitment and histone acetylation for rapid transcriptional induction (Fig. 4A, Table 9). Baseline DNA methylation patterns within hematopoietic stem cells (HSCs) serve as a critical determinant of the readiness to respond to mRNA-encoded antigens. Research indicates that DNA methyltransferase 3a (DNMT3a) is essential for maintaining the homeostatic balance between HSC self-renewal and differentiation [217]. Furthermore, IM injection of mRNA-LNPs can trigger “trained immunity”, involving the epigenetic reprogramming of bone marrow progenitors where specific methylation marks dictate the lineage bias and functional responsiveness of innate immune cells—particularly monocytes and neutrophils—to subsequent vaccine stimuli [218, 219].

Table 9.

Epigenetic regulation of vaccine-induced cytokines and immune cell function.

Epigenetic factor	Target machinery	Regulated cytokines	Affected immune cells/pathways	Impact on vaccine effect
DNA methylation	DNMT1, DNMT3a/b	IFN-γ, IL-2, TNF-α	CD8⁺ CTLs and Th1: promotes demethylation of effector gene promoters (e.g. Ifng)	Good: essential for the rapid “recall” of memory T-cells upon tumor challenge
Histone acetylation	HATs (p300/CBP)	IL-12, IFN-β	cDC1 and moDCs: increases H3K27ac at enhancer regions for antigen presentation	Good: enhances DC maturation and TLR7/8 signaling efficiency in LNP-IM delivery
Histone deacetylation	HDACs (HDAC1/2)	IL-10, TGF-β	Tregs and MDSCs: removes acetyl groups from pro-inflammatory loci, silencing them	Bad: suppresses the “strike force”; often active in “cold” TMEs to block mRNA vaccine effects
Histone methylation	H3K4me3 (Trithorax)	TNF-α, IL-6	Trained innate immunity: epigenetic “memory” in monocytes/macrophages	Good: leads to a faster, more robust innate response upon secondary vaccine doses
Histone methylation	H3K27me3 (EZH2)	IFN-γ, Granzyme B	T-cell exhaustion: silences effector genes via the Polycomb Repressive Complex	Bad: drives T-cell “sidedness” toward exhaustion; must be reversed via exosome/LNP TME reprogramming
Chromatin remodeling	SWI/SNF complex	Type I IFNs (IFN-α/β)	cGAS-STING pathway: opens chromatin to allow IRF3/IRF7 binding	Good: critical for the initial “autoadjuvant” burst required for LNP-based mRNA translation

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A significant advancement in this field is the use of exosome-delivered mRNA vaccines. Unlike synthetic LNPs, which can induce pronounced cellular toxicity, exosomes utilize native endogenous surface marker profiles (e.g. CD47) to navigate the body with superior biocompatibility. Emerging evidence suggests that exosomal vehicles may modulate the epigenetic landscape of cDCs and resident macrophages more precisely, reducing systemic inflammatory spikes while maintaining robust “open” chromatin states (euchromatin) at key pro-inflammatory gene loci [167].

Recent longitudinal studies reveal that mRNA vaccines—particularly after two consecutive doses—induce significant H3K27ac at the promoters of monocyte-derived macrophages [220]. This modification, often linked to G-quadruplex DNA structures, creates a transcriptionally “ready” state. This allows for the immediate and robust transcription of pro-inflammatory cytokines—such as IL-1β, IL-6, and TNF-α—upon the sensing of delivered mRNA. By bridging innate recognition within myeloid lineages with long-term adaptive memory in T-cell compartments, these persistent epigenetic marks ensure a sustained and rapid immune response against evolving tumor neoantigens [220].

However, the therapeutic exploitation of trained immunity poses critical challenges regarding long-term predictability and systemic safety. Because epigenetic memory in myeloid progenitors alters the long-term baseline reactivity of the innate immune system, the absolute duration and final predictability of these changes in the highly variable human physiological landscape remain uncertain. Maintaining chromatin in a permanently “poised” or hyperaccessible state presents a profound double-edged sword [221]. If these epigenetic modifications lack absolute temporal control, they run the risk of inciting chronic, low-grade systemic inflammation or precipitating severe autoimmune activation. Prolonged or uncontrolled hyperresponsiveness of monocyte-derived macrophages can break central immunotolerance, turning a targeted anti-tumor vaccine response into a destructive, autoreactive pathology against healthy somatic tissues. Consequently, fine-tuning the temporal resolution of these exosome-mediated chromatin alterations is paramount to ensuring that transient epigenetic priming does not devolve into persistent autoinflammatory disease.

Signaling plasticity: JAK-STAT-mediated remodeling of the immune landscape

The efficacy of mRNA vaccines relies on systemic cytokine surges that reconfigure the host’s epigenetic landscape through interferon-mediated chromatin remodeling. IFNs, triggered by the endosomal sensing of LNP-encapsulated mRNA, serve as master regulators of innate memory. Utilizing ATAC-seq, researchers have demonstrated that IM administration of mRNA vaccines induces significant increases in chromatin accessibility at the loci of interferon-stimulated genes (ISGs) [212]. These modifications—enriched with motifs for interferon regulatory factors (IRFs)—effectively prime monocytes, establishing a state of “long-term immune alertness”.

A critical frontier involves exosome-delivered mRNA, which can refine this “epigenetic reset”. Unlike synthetic LNPs that often trigger a broad, nonspecific inflammatory spike, engineered exosomes leverage surface proteins like integrins 7o deliver cargo to specific cellular niches. This targeted delivery may modulate the JAK-STAT signaling axis more precisely, recruiting histone acetyltransferases (HATs) such as CBP/p300 to specific transactivation domains. This recruitment facilitates localized histone acetylation, promoting an “open” chromatin configuration that favors the expression of anti-tumor genes while potentially minimizing the exhaustion-related epigenetic marks associated with chronic systemic inflammation.

Furthermore, the transition from transient cytokine sensing to stable epigenetic change ensures that immune effectors remain transcriptionally poised. This “transcriptional readiness” is maintained through persistent STAT-mediated remodeling, which keeps cytotoxic program genes accessible even during periods of low antigen exposure. By integrating systemic IM priming with the nuanced delivery capabilities of exosomal platforms, mRNA vaccines create a plastic yet persistent immune architecture. This ensures that CD8⁺ T-cells and NK cells are functionally equipped to execute rapid cytotoxic programs upon the detection of evolving tumor neoantigens or viral variants.

Epigenetic memory: reversing T-cell exhaustion

The downstream structural consolidation of the immune response relies on stable, heritable epigenetic modifications across both innate and adaptive compartments (Fig. 4C and D). Central to this process is the induction of trained immunity in the myeloid lineage. Following IM injection, mRNA-LNP systems act as potent stimuli that drive significant increases in histone H3 lysine 4 trimethylation (H3K4me3) at the promoters of pro-inflammatory genes in circulating monocytes. This epigenetic mark identifies active promoters and enhances the production of IL-6 and TNF-α upon subsequent tumor challenge, establishing a heightened state of innate vigilance. Emerging research into exosome-delivered mRNA vaccines suggests that these natural vesicles may further refine this myeloid shift. By leveraging endogenous surface marker profiles like CD47 and specific integrins, exosomes achieve superior biocompatibility and targeted delivery to bone marrow niches, inducing highly durable epigenetic reprogramming of progenitor cells compared to synthetic LNPs [215].

In the adaptive compartment, the transition of naïve T-cells into functional effectors is governed by the selective removal of repressive DNA methylation marks. This lineage commitment is accompanied by the DNA demethylation of the IFNG and GZMB (Granzyme B) promoters, ensuring rapid cytotoxic secretion upon antigen encounter [222, 223]. mRNA platforms provide the necessary costimulatory milieu to maintain these loci in an accessible state. However, chronic antigen exposure within the TME typically leads to T-cell exhaustion, a dysfunctional state characterized by a stable epigenetic “lock-in” maintained by high-mobility group box proteins like TOX that permanently limits effector function. A critical hallmark of this terminal differentiation is the progressive demethylation of the PDCD1 (PD-1) promoter [224].

Modern mRNA strategies, particularly those utilizing exosomal delivery for enhanced TME infiltration, aim to circumvent this terminal lock-in by delivering potent stimulatory signals that recruit de novo epigenetic remodelers, such as ten-eleven translocation (TET) methylcytosine dioxygenases. These enzymes facilitate DNA hydroxymethylation to restore a youthful transcriptional profile and reopen closed chromatin states at effector loci, effectively reversing exhaustion in TILs. By integrating systemic IM priming with localized exosomal delivery, next-generation mRNA vaccines can maintain a “transcriptionally poised” immune state, providing a persistent shield against tumor evolution and metastatic escape while synergizing with ICIs [225].

Targeting the “epigenetic TME”: overcoming resistance at the molecular level

The final step in this global reprogramming framework is the direct modification of the TME chromatin landscape to benefit vaccine efficacy (Fig. 4B–D, Table 9). This transformation is driven by two critical axes: macrophage repolarization via histone deacetylase (HDAC) modulation and cytokine-induced alterations in tumor DNA methylation. TAMs frequently adopt a pro-tumor M2-like phenotype, a state maintained by specific HDACs that silence pro-inflammatory gene expression. Research indicates that inhibiting class IIa HDACs can epigenetically “reprogram” myeloid cells. mRNA vaccine-delivered signals, particularly those involving TLR ligands, can suppress the activity of HDAC6 and HDAC10. This leads to increased histone acetylation at the promoters of M1-associated genes, such as Nos2 and Il12, ensuring that the TME facilitates, rather than hinders, the vaccine-induced T-cell response.

The mode of vaccine administration—IM injection versus exosome-delivered mRNA—significantly dictates the spatial and temporal dynamics of this epigenetic remodeling. Traditional IM injections primarily activate localized immune responses in draining lymph nodes, inducing a systemic cytokine surge—specifically Type I and Type II Interferons (IFN-α/β and IFN-γ)—that indirectly alters the tumor’s epigenetic signature. Prolonged cytokine exposure recruits TET DNA hydroxylases, resulting in the demethylation of promoters for MHC class I molecules and tumor-suppressor genes like PTEN.

In contrast, exosome-delivered mRNA vaccines offer superior biocompatibility and cell-specific tropism, enabling the direct delivery of mRNA to the TME or distal tissues with reduced immunogenicity. Direct comparative evidence demonstrates that while advanced modified LNPs (such as ligand-functionalized or SORT platforms) can alter gross organ-level biodistribution, they remain highly susceptible to the elevated interstitial fluid pressures of the tumor stroma and risk causing ABC upon repeated clinical dosing. Exosomes inherently overcome these synthetic barriers due to their natural cellular lineage; their highly deformable lipid bilayers and native surface protein architectures (including tetraspanins and CD47) allow them to penetrate deep into dense tumor cores where rigid synthetic nanoparticles become physically trapped. This permits exosomes to directly enter myeloid-derived suppressor cell (MDSCs) and cancer-associated fibroblasts (CAFs) at the tumor site to deliver specific epigenetic factors, including miRNAs and DNA methyltransferases. This targeted approach bypasses the limitations of systemic administration, reversing “epigenetic silencing” within the TME more efficiently than conventional or modified synthetic methods. By synergizing direct epigenetic reprogramming via exosomes with the systemic inflammatory profile of IM vaccines, mRNA-based therapies can effectively transform the TME from a suppressive niche into an immunostimulatory environment, thereby enhancing overall tumor recognition and therapeutic durability.

Future outlook: sustaining preventive immunity through epigenetic “imprinting”

The clinical longevity of cancer mRNA vaccines is anchored in their ability to instill “epigenetic memory” (Fig. 4D), ensuring the immune system remains vigilant against recurrence long after the initial series of injections. This surveillance is facilitated by epigenetic imprinting of memory T-cells and stable cytokine regulation. mRNA vaccines drive the differentiation of naïve T-cells into long-lived memory subsets, a process defined by stable histone modifications at memory-specific loci. The acquisition of an “effector-poised” state involves the enrichment of activating marks—specifically H3K4me3 and histone H3 lysine 27 acetylation (H3K27ac)—at the promoters of genes required for rapid recall [226]. In neoantigen-based platforms, these marks ensure that the immune system “remembers” specific mutations for years. Research confirms that mRNA platforms effectively expand these polyfunctional memory populations, maintaining chromatin accessibility even in the absence of chronic antigen stimulation [207].

The maintenance of a “poised” transcriptional state through DNA methylation further mediates the prevention of cancer recurrence. DNA methylation serves as a critical regulatory gatekeeper for cytokine production [227, 228]. mRNA vaccination promotes a specialized profile where the promoters of key anti-tumor cytokines, such as IFN-γ and TNF-α, remain in a partially demethylated state. This allows for an almost instantaneous transcriptional surge upon re-exposure, bypassing the need for de novo chromatin remodeling. This mechanism was clinically reflected in the KEYNOTE-942 trial, where a personalized mRNA vaccine combined with pembrolizumab significantly reduced recurrence or death risk by 44% in high-risk melanoma patients by fostering a durable, transcriptionally ready immune environment.

The delivery method—IM injection versus exosome-delivered mRNA—critically influences the breadth of this memory. While IM injections primarily drive systemic adaptive memory through lymph node activation, exosome-mediated delivery can induce potent “trained innate immunity”. Recent studies indicate that mRNA-loaded exosomes can establish persistent H3K27ac marks in monocyte-derived macrophages, preserving an inflammatory “memory” for at least six months. Furthermore, exosome-targeted vaccines have demonstrated a four-fold increase in long-term memory CD8⁺ T-cell survival compared to conventional DC-based vaccines. By integrating these targeted exosomal pathways with the systemic priming of IM injections, mRNA platforms can achieve a dual-layered epigenetic defense, providing both rapid innate recall and high-fidelity adaptive surveillance against metastatic relapse.

Maintaining chromatin in a permanently “poised” or hyperaccessible state presents a profound double-edged sword. If these epigenetic modifications lack absolute temporal control, they run the risk of inciting chronic, low-grade systemic inflammation or precipitating severe autoimmune activation. Prolonged or uncontrolled hyperresponsiveness of monocyte-derived macrophages can break central immunotolerance, turning a targeted anti-tumor vaccine response into a destructive, autoreactive pathology against healthy somatic tissues. Consequently, fine-tuning the temporal resolution of these exosome-mediated chromatin alterations is paramount to ensuring that transient epigenetic priming does not devolve into persistent autoinflammatory disease [229–231].

Concluding remarks and future perspectives

Integrating exosome bioengineering with mRNA technology marks a paradigmatic shift toward highly adaptive, multivalent therapeutics. By combining the inherent structural integrity of cell-derived vesicles with the programmable precision of synthetic mRNA transcripts, this approach directly overcomes the strict pharmacological constraints of first-generation lipid carriers. Exosomes possess a distinct biomimetic signature—a specialized lipid bilayer enriched with native self-signaling molecules like CD47. This natural shield protects delicate mRNA strands from ribonuclease degradation and allows the vehicle to cross formidable biological barriers, such as the BBB, with minimal reactogenicity. This highly modular architecture enables the simultaneous delivery of a complex multiplex of tumor neoantigens and immunomodulatory cytokines. Doing so drives immediate antigen presentation while concurrently initiating the deep epigenetic priming of memory T-cell compartments. By leveraging the body’s endogenous communication networks to deliver these therapeutic instructions, the field is moving away from transient vaccination toward a durable framework of long-term, systemic immune reprogramming.

However, translating these platforms successfully from bench to bedside requires resolving major manufacturing and regulatory bottlenecks. Standardizing exosome isolation remains a primary challenge for the field. Legacy techniques like differential ultracentrifugation are no longer viable and must be replaced by scalable, high-resolution technologies—specifically tangential flow filtration and SEC—to guarantee GMP-compliant purity and reliable batch-to-batch consistency. Furthermore, current regulatory frameworks under the FDA and EMA categorize engineered exosomes as advanced therapy medicinal products (ATMPs). This designation mandates a rigorous, highly detailed characterization of their exact mechanisms of action and precise spatiotemporal biodistribution in vivo. Establishing validated potency assays and completely xeno-free production pipelines is absolutely essential for gaining clinical approval. As these technical hurdles are cleared, adopting advanced process analytical technologies alongside thermostable lyophilization will be paramount for securing reliable global cold-chain distribution for these next-generation biologics.

The true future of precision oncology lies in developing off-the-shelf personalized platforms that effectively harmonize patient-specific care with industrial-scale manufacturing efficiency. This hybrid paradigm relies on premanufactured, modular exosome chassis lines that can be rapidly loaded and functionalized with patient-specific mRNA libraries. This approach can compress the prolonged turnaround time for personalized neoantigen vaccines from months down to under a week. Additionally, sophisticated computational deep learning architectures will increasingly evaluate a patient’s multiomic profile in real-time—integrating TCR sequencing data and specific HLA-binding affinities—to optimize mRNA ratios for maximal immune cross-reactivity. Ultimately, these advanced vaccines will function as dynamic orchestrators of the TME, leveraging trained immunity and epigenetic plasticity to permanently block malignant recurrence. By opening up widespread access to elite precision medicine, this unified platform is poised to completely redefine the boundaries of modern immunotherapy, ensuring durable, programmable protection for patient populations worldwide.

Acknowledgments

This work was supported in part by the MetroHealth Foundation (start-up fund, S.L.) through the Case Western Reserve University, and the National Cancer Institute (Bethesda, MD) (grant Nos. R01CA248019 (S.L.) and R01CA266256 (S.L.)).

Contributor Information

Huiqin Bian, Department of Medicine, The MetroHealth System, Case Western Reserve University, Cleveland, OH 44109, USA.

William Tse, Department of Medicine, The MetroHealth System, Case Western Reserve University, Cleveland, OH 44109, USA; Cell and Gene Therapy Institute, The MetroHealth System, Case Western Reserve University, Cleveland, OH 44109, USA.

Gang Huang, Department of Cell Systems & Anatomy Department of Pathology & Laboratory Medicine UT Health San Antonio, Joe R. and Teresa Lozano Long School of Medicine Mays Cancer Center at UT Health San Antonio, San Antonio, TX 78229, USA.

Shujun Liu, Department of Medicine, The MetroHealth System, Case Western Reserve University, Cleveland, OH 44109, USA; Cell and Gene Therapy Institute, The MetroHealth System, Case Western Reserve University, Cleveland, OH 44109, USA.

Author contributions

Huiqin Bian (Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing – original draft, Writing – review & editing), William Tse (Writing – review & editing), Gang Huang (Writing – review & editing), and Shujun Liu (Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Visualization, Writing – original draft, Writing – review & editing).

Conflicts of interest

The authors declare no competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

No new datasets were generated during this study. All discussed data are derived from cited literature, and the schematic figures were originally created by the authors.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

No new datasets were generated during this study. All discussed data are derived from cited literature, and the schematic figures were originally created by the authors.

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PERMALINK

Beyond the genetic code: orchestrating epigenetic and immune landscapes with multivalent mRNA-exosome vaccines

Huiqin Bian

William Tse

Gang Huang

Shujun Liu

Roles

Abstract

Graphical Abstract

Graphical Abstract.

Introduction

Mapping the antigenic landscape: from discovery to validation

The multiomics pipeline: integrating genomics, proteomics, and immunopeptidomics for target identification

Figure 1.

Prioritizing targets: predicting MHC-binding affinity and TCR cross-reactivity

Functional benchmarking: bridging the gap between in silico predictions and biological reality

Optimizing the payload: engineering mRNA sequences for maximum translational efficiency

From bench to bedside: critical bottlenecks in clinical antigen selection

The frontiers of mRNA synthesis and quality control

Architecture of the transcript: engineering UTRs and poly(A) tails for sustained expression

Table 1.

Capping and poly(A) strategies: practical considerations and GMP usage

Table 2.

Purification and QC: removing DNA and dsRNA to mitigate off–target inflammation

Technical hurdles: identifying the persistent “blind spots” in large-scale mRNA production

Navigating the body: delivery and in vivo efficacy

Vehicle design: evolving delivery platforms for enhanced stability and uptake

Table 3.

Table 4.

Deployment strategies: how routes of administration dictate immunological outcomes

Table 5.

Overcoming biological barriers: defining the metrics for successful systemic delivery

The rise of exosomes as natural delivery vehicles

Nature vs. synthetic: the inherent advantages of exosomes over LNPs

Figure 2.

Loading the cargo: strategies for endogenous and exogenous mRNA packaging

Surface customization: modular engineering and the development of chimeric hybrids

Intracellular logic: mastering biodistribution and the kinetics of endosomal escape

Scaling for the clinic: addressing the hurdles of exosome standardization and yield

Decoding the multivalent immune response

Bridging the divide: PAMP recognition and the activation of DC maturation

Figure 3.

Table 6.

Table 7.

Orchestrating the attack: the spatiotemporal mobilization of CTL and NK cells

Flipping the switch: reprogramming the immunosuppressive TME

Lasting protection: establishing the foundations of immunological memory and surveillance

The multidimensional landscape of immune reprogramming

Extracellular signaling: reshaping the TME through cytokine orchestration

Table 8.

Breaking resistance: turning “cold” tumors “hot” to enhance ICI sensitivity

Global effects: leveraging systemic reprogramming for long-term preventive surveillance

Intranuclear hardwiring: epigenetic priming and chromatin readiness

Figure 4.

Table 9.

Signaling plasticity: JAK-STAT-mediated remodeling of the immune landscape

Epigenetic memory: reversing T-cell exhaustion

Targeting the “epigenetic TME”: overcoming resistance at the molecular level

Future outlook: sustaining preventive immunity through epigenetic “imprinting”

Concluding remarks and future perspectives

Acknowledgments

Contributor Information

Author contributions

Conflicts of interest

Data availability

References

Associated Data

Data Availability Statement

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