Development of mRNA-Based Therapeutics

Abstract

Messenger RNA (mRNA) therapeutics have undergone a paradigm shift from experimental construct to licensed pharmaceutical product, catalysed by the unprecedented clinical success of COVID-19 mRNA vaccines and sustained by expanding applications in oncology, rare metabolic disease, and protein-replacement therapy. This study systematically evaluates the formulation, immunogenicity, and translational efficiency of four mRNA therapeutic platforms -- unmodified mRNA, N1-methylpseudouridine (m1psi) modified mRNA, self-amplifying RNA (saRNA), and circular RNA (circRNA) - delivered by four lipid nanoparticle (LNP) compositions across five cell types in vitro and two murine in vivo models. Protein expression was quantified by luciferase reporter and ELISA (n = 480 experimental conditions); immunogenicity was assessed by innate immune activation assays (IFN-alpha, TNF-alpha, IL-6) and adaptive antibody titres (IgG ELISA) at days 14 and 28. m1psi-modified mRNA formulated in ionisable LNP-C achieved the highest hepatocyte protein expression (mean 4.82 x 10^6 RLU/mg, 6.4-fold above unmodified) with the lowest innate immune activation (IFN-alpha induction 0.9-fold vs. baseline). saRNA produced sustained antigen expression for 28 days at one-tenth the mRNA dose and elicited IgG titres 3.8-fold higher than equimolar conventional mRNA at day 28. circRNA demonstrated superior nuclease resistance (t1/2 = 72.4 h vs. 4.8 h for linear unmodified mRNA) and acceptable immunogenicity in innate immune-competent cells. These findings delineate the translational efficiency-immunogenicity trade-off landscape across mRNA platforms and provide evidence-based formulation guidance for therapeutic application selection.