Virus-Like Particles of mRNA with Artificial Minimal Coat Proteins: Particle Formation, Stability, and Transfection Efficiency

Abstract

RNA has enormous potential as a therapeutic, yet, the successful application depends on efficient delivery strategies. In this study, we demonstrate that a designed artificial viral coat protein, which self-assembles with DNA to form rod-shaped virus-like particles (VLPs), also encapsulates and protects mRNA encoding enhanced green fluorescent protein (EGFP) and luciferase, and yields cellular expression of these proteins. The artificial viral coat protein consists of an oligolysine (K) for binding to the oligonucleotide, a silk protein-like midblock S = (GAGAGAGQ) that self-assembles into stiff rods, and a long hydrophilic random coil block C that shields the nucleic acid cargo from its environment. With mRNA, the C-S-K protein coassembles to form rod-shaped VLPs each encapsulating about one to five mRNA molecules. Inside the rod-shaped VLPs, the mRNAs are protected against degradation by RNAses, and VLPs also maintain their shape following incubation with serum. Despite the lack of cationic surface charge, the mRNA VLPs transfect cells with both EGFP and luciferase, although with a much lower efficiency than obtained by a lipoplex transfection reagent. The VLPs have a negligible toxicity and minimal hemolytic activity. Our results demonstrate that VLPs yield efficient packaging and shielding of mRNA and create the basis for implementation of additional virus-like functionalities to improve transfection and cell specificity, such as targeting functionalities.

Nanomedicines to Deliver mRNA: State of the Art and Future Perspectives

“…In another work, artificial viral coat protein consisting of an oligolysine (K12), a silk protein-like midblock S10, and a long hydrophilic random coil block C was generated and complexed with mRNA to form rod-shaped VLPs. This system transfected cells with both EGFP and luciferase, but the efficacy was low compared to that obtained with a lipoplex transfection reagent [146]. More recently, VLPs prepared by fusing protein G of Vesicular stomatitis virus (VSV-G) with a ribosomal protein L7Ae of Archeoglobus fulgidus, resulted in the efficient delivery of EGFP in human induced pluripotent stem cells (iPSCs) and monocytes [147].…”

Nanoscale platforms for messenger RNA delivery

“…Coat proteins derived from viruses were also used for mRNA delivery (Jekhmane et al, 2017;Li et al, 2014;Sun, Sun, Zhao, & Gao, 2016). Li et al (2014) assembled the recombinant coat protein of bacteriophage MS2 with mRNA encoding granulocyte macrophage colony-stimulating factor.…”

Nanomaterials for Cancer Precision Medicine

“…As is well known, virus RNA carrier exhibits high transfection efficiency, but at the cost of strong immunogenicity. Virus‐like nanoparticles have therefore been developed as gene and drug carriers from different plant protein or Hepatitis B core protein …”