Encapsulin-Protected Immunotherapeutic Complexes: Bacteria-Derived Nanoparticles for mRNA Delivery to Eukaryotic Cells.

Encapsulins are protein nanocompartments that self-assemble into icosahedral structures that capture cargo proteins through a cargo-loading peptide (CLP). Here, we aimed to develop an mRNA delivery vector for eukaryotic cells by fusing the CLP from Myxococcus xanthus encapsulin cargoes with a short peptide that binds to a complementary RNA aptamer, creating an encapsulin-RNA adaptor peptide (ERAP). Additionally, we designed a chimeric mRNA (CmRNA) molecule for translation in eukaryotic cells, featuring an internal ribosome entry site (IRES) in the 5'-UTR to enable cap-independent translation. Downstream of the IRES, genes coding for model proteins were included. Finally, an aptamer that binds the Rev-derived peptide was added to the 3'-UTR of the RNA molecule. The CmRNA, ERAP, and encapsulin were coexpressed in Escherichia coli, and it was demonstrated that the produced nanocompartments contained the ERAP and bacterial RNA. The RNA was reverse-transcribed and identified by nested PCR, confirming that the encapsulin captured the CmRNA. qPCR analysis of encapsulin-protected immunotherapeutic complexes (EPICs) demonstrated the loading of 2.9 CmRNA molecules per particle. These RNA-loaded encapsulins were taken up by macrophage cells, and the expression of model proteins was confirmed. Finally, immunization of mice with these particles induced IgG antibodies against the model proteins.