mRNA folding facilitates efficient and spleen-targeted delivery by highly branched poly(β-amino ester)s

Messenger RNA (mRNA)-based therapeutics hold great promise in preventing and treating various diseases. However, the instability and high immunogenicity of raw mRNA molecules, along with the inefficient delivery vectors, strictly limited the proteins translation and therapeutic applications. In this work, we successfully engineered a folded structural mRNA for enhancing its stability by leveraging a magnesium ion (Mg²⁺)-mediated charge neutralization and structural compression strategy. Further, a functionalized highly branched poly(β-amino ester)s (HPAEs) was utilized to efficiently deliver the folded mRNA, achieving specifically targeting to the spleen. Notably, compared with polyplexes containing non-folded mRNA, the optimal folded mRNA-based polyplexes exhibited stronger mRNA encapsulation, smaller size, and a more homogeneous morphology; ultimately, demonstrated higher mRNA transfection efficiency up to 85.4% across five different cell types, maintaining high cell viability. Mechanistic studies demonstrated that Mg²⁺-mediated mRNA folding strategy dramatically enhanced the folded mRNA-formulated polyplexes cellular uptake and endosomal escape. Encouragingly, when administered systemically, the representative folded mRNA-based polyplexes demonstrated robust protein expression, yielding 3-fold enhancement in spleen, compared to the unfolded mRNA-based nanoparticles, showing superior biosafety in vivo. Collectively, the work establishes a new class of the folded structure mRNA polymer delivery system, and provides a foundation for the next generation of RNA-based therapeutic drugs for spleen-related diseases.