mRNA-based therapeutics: powerful and versatile tools to combat diseases

Purpose: mRNA therapeutics have fueled hope to combat incurable diseases, but insufficient understanding of mRNA instability, immunogenicity, and delivery has impeded progress. The review argues that mRNA-based drugs—especially after the COVID-19 vaccine success—can become powerful, versatile tools, and that mRNA optimization and delivery systems are the key bottlenecks to solve.

Hypothesis: No formal experimental hypothesis. Central thesis: with appropriate structural optimization (5′ cap, UTRs, poly(A) tail, ORF, nucleoside modification) and engineered delivery systems (LNPs, polymers, CNEs, etc.), mRNA can serve as a versatile platform for vaccines, protein replacement, gene editing, and cancer immunotherapy.

Aims: Comprehensively describe mRNA-based therapeutics: principles, manufacture, application, effects, and shortcomings. - Highlight the importance of mRNA optimization and delivery systems in successful mRNA therapeutics. - Discuss key challenges and opportunities for developing mRNA into powerful and versatile tools against genetic, infectious, cancer, and other refractory diseases.

Delivery system: mRNA formats: conventional IVT mRNA; self-amplifying RNA (saRNA, alphavirus-derived nsP1–4 replicase); circular RNA; noncoding RNA/ceRNA concepts. - Structural elements: 5′ cap analogs (ARCA, m⁷Gppp(m²′-O)ApG, phosphorothioate, dithiodiphosphate), 5′/3′ UTRs (α/β-globin, CYBA, VEE), ORF codon optimization, poly(A) tail (100–150 nt optimal; UGC linker strategy). - Lipid nanoparticles: cationic lipids (DOTMA, DOTAP, DSTAP, DMTAP, DDA, DOBAQ, DC-Chol) and ionizable lipids (DLin-MC3-DMA, SM-102, ALC-0315, A6, Lipid 5, C12-200, 5A2-SC8, cKK-E12, G0-C14, OF-02, 306Oi10, 93-O17S, A18-Iso5-2DC18, TT3, BAMEA-O16B, FTTS, Vc-Lipid, C14-4, Lipid 14, 4A3-Cit, ssPalmO-Phe). - LNP components: cholesterol or variants (β-sitosterol, 20α-hydroxycholesterol), helper lipids (DSPC, DOPE), PEGylated lipids (PEG-DMG, ALC-0159). - Polymeric nanoparticles: PEI, PEI-gPEG, PBAE, hPBAE, OMPBAE, PEG-PAsp(DET), PAsp(DET/R), CARTs, PCL-based PBAE, PEG[Glu(DET)]2. - Cationic nanoemulsions: squalene core, DOTAP, Tween 80, Span 85. - Other: protamine-condensed mRNA (RNActive), exosomes/EVs, mesoporous silica, CaP, nucleoside lipids, polypex micelles, coordination polymer NPs. - Payloads: antigens (SARS-CoV-2 S/RBD, influenza HA, Zika prM-E, RSV F, HIV Gag/Pol/Nef, Ebola/rabies glycoproteins, VZV gE, HCMV gB, PMIF, PfGARP), monoclonal antibodies (VRC01, ZIKV-117, CHKV-24, trastuzumab, bispecific T cell engagers), protein replacement (CFTR, FVIII, FIX, FAH, PBGD, MMUT, OTC, α-galactosidase A, AAT, VEGF-A, PTEN, p53, IL-22BP), and gene-editing nucleases (ZFN, TALEN, CRISPR/Cas9). - Routes: intravenous, intramuscular, intradermal, subcutaneous, intranasal, intratracheal/inhaled, intracerebroventricular, intrathecal, intraperitoneal.

Approach: Review of preclinical and clinical literature spanning mRNA design, manufacture, delivery, and applications. Preclinical models include mice, rats, ferrets, rabbits, pigs, guinea pigs, Syrian hamsters, cynomolgus macaques, rhesus macaques, and Aotus monkeys. Clinical trials cover infectious disease vaccines (SARS-CoV-2, influenza, Zika, RSV, rabies, HIV, CMV, chikungunya), cancer vaccines (melanoma, glioblastoma, AML, RCC, NSCLC, prostate), protein replacement (CFTR, VEGF-A), and gene editing (CCR5, TRAC, PD-1, HLA class I). Doses range from nanograms (saRNA) to 1,000 µg; controls include placebo, convalescent serum, and licensed vaccines.

Key methods: mRNA characterization: capping efficiency, poly(A) tail length, HPLC/cellulose chromatography purification, dsRNA removal, mass spectrometry, NMR. - Nanoparticle characterization: particle size, zeta potential, encapsulation efficiency, micromorphology, isoelectric point, lipid identity/ratio. - Immunogenicity: neutralizing antibody titres, anti-RBD IgG, CD4⁺/CD8⁺ T cell responses, germinal centre reactions, mucosal IgA. - Functional readouts: luciferase/EGFP reporter expression, CFTR chloride efflux, FVIII/FIX activity, plasma methylmalonic acid, ammonia levels, tumor growth inhibition, survival. - Safety: adverse events, anti-PEG antibodies, ADE risk assessment. - Gene editing: indel frequency, off-target analysis, CAR expression, TCR knockout rate.

Key results: COVID-19 mRNA vaccines: ~90% effectiveness for full vaccination, 80% for partial; BNT162b2 95% efficacy in phase III; mRNA-1273 94.1% efficacy; Omicron-neutralizing antibodies largely undetectable in most recipients, improved by booster. - Influenza: 50 ng nucleoside-modified mRNA vaccine encoding HA stalk + neuraminidase + nucleoprotein + M2 protected mice against 500× LD50; H10N8 and H7N9 candidates showed seroconversion and seroprotection in phase I. - Zika: single 30 µg or 50 µg dose protected mice and macaques; neutralizing titres 50–100× higher than inactivated virus or DNA vaccines; mRNA-1893 induced 94–100% seroconversion in phase I. - HIV: single 0.7 mg/kg IV dose of VRC01 mRNA produced antibody concentrations comparable to 10–20 mg/kg mAb protein and protected humanized mice from HIV-1 challenge. - Rabies: CV7202 two 1 µg doses yielded high neutralizing titres and strong adaptive responses in phase I. - Cancer: personalized melanoma mRNA vaccine (NCT02035956) remained recurrence-free at 23 months; DC-based glioblastoma vaccine extended progression-free survival 2.9×; WT1 mRNA DC vaccine improved relapse-free survival in AML responders; mRNA-based CAR T cells achieved 94% CAR expression in >80% viable T cells. - Protein replacement: CFTR mRNA restored up to 55% net chloride efflux in mice; FAH mRNA normalized liver function and extended survival in HT-1 mice; methylmalonic acid reduced 75–85%. - Gene editing: TALEN mRNA + 5 gRNAs achieved up to 81% TCR knockout in primary T cells; ZFN mRNA edited CD34⁺ HSPCs with multilineage engraftment; RNase III purification reduced immunogenicity and improved CAR T cytotoxicity. - Purification: HPLC purification increased protein expression 1,000-fold and eliminated immune response of modified mRNA.

Interpretation: mRNA-based therapeutics have achieved remarkable improvement in stability, function, and production over 30 years and are now powerful, versatile tools. The authors are confident of accelerated development in the next decade, potentially providing solutions for currently incurable diseases. They emphasize that mRNA optimization and engineering precision nanoparticles are crucial to realizing this potential.

Limitations: Review article; no primary data. - mRNA instability (~7 h median intracellular half-life) and susceptibility to RNases remain core challenges. - Only <1/10,000 input mRNAs reach cytoplasm and translate protein. - Delivery barriers: cell membrane, endosomal escape, lysosomal degradation, tissue targeting, and inconsistent in vitro/in vivo transfection. - Long saRNA sequences (9–12 kb) complicate manufacturing and delivery. - Codon optimization may alter protein folding and create novel peptides with unknown activity. - Anti-PEG antibodies (~40% of population) may accelerate clearance and reduce efficacy. - Reactogenicity: CV7201 caused grade 2 Bell’s palsy and other severe AEs; CV7202 5 µg dose was unacceptable; Moderna H10N8 400 µg dose stopped. - Most protein replacement and gene-editing applications remain preclinical; long-term safety and duration data are limited. - Mucosal immunity, cellular immunity, and durability of response require further study.