Engineering filamentous potato virus X as a platform nanotechnology for nucleic acid gene delivery

Abstract

Abstract Nucleic acid therapeutics and gene delivery platforms have the potential to change the way we treat disease, enabling precision medicine. Advances in biotechnology have allowed for production of virtually any functional protein or peptide in the human body through the introduction of various classes of RNA, including regulatory and coding RNAs (e.g., mRNA) in the form of linear or circular RNA (circRNA). To be effective, nucleic acid therapeutics require safe, stable, and efficient delivery vehicles that protect the otherwise labile cargo from degradation, promote cellular uptake, and facilitate payload release. Lipid nanoparticles (LNPs) have enjoyed recent clinical success for the delivery of mRNA to combat disease – especially against coronavirus 2019 (SARS CoV-2) and, more recently, against high-grade ductal breast carcinoma (clinical trial) – marking significant milestones for RNA nanomedicines. As an alternative to LNPs and mammalian viral vectors, we report the use of nucleoprotein components from a filamentous plant virus, potato virus X (PVX) as an RNA platform delivery technology. Designer nucleoprotein complexes of filamentous and halo-shaped nanoparticles were obtained through self-assembled templating on custom mRNAs and circRNAs. The flexuous nature of the PVX platform enabled the packing of circRNA yielding a unique nanoparticle platform. We explored assembly from coat protein (CP) obtained from PVX particles produced in planta but also extended the design space by producing recombinant CP by bacterial expression. Controlled mixed assemblies were achieved allowing the programming of mosaic particles with integrated functionality; length control was also demonstrated, resulting in bespoke nucleic acid delivery vehicles for tunable pharmacology. Using reporter genes, we demonstrate successful transfection of eukaryotic cells and target protein expression.