MULTI-FUNCTIONAL SELF-ASSEMBLING PEPTIDE VECTORS FOR GENE DELIVERY

Messenger RNA (mRNA) as a therapeutic agent has recently gained significant attention, in particular after the introduction of COVID-19 pandemic vaccines. However, mRNA delivery presents several drawbacks, because it is prone to degradation by nucleases and its polyanionic nature obstacles its entrance into the apolar cellular and tissue barriers. For these reasons, a suitable delivery system is essential to provide protection and stability to the mRNA, improving cellular uptake while preserving its biological function. To date, lipid and polymer nanoparticles (NPs) are the most commonly used delivery materials, although they show problems related to biocompatibility and possible toxicity of the biodegradation products. Self-assembling peptides (SAPs) can offer a promising alternative as delivery vectors thanks to their biocompatibility, biodegradability and ease of synthesis. Currently, nanoparticles achieve mRNA binding through electrostatic interactions between the negatively charged nucleic acid and a positively charged layer added to the NP. Our project introduced a different methodology where a peptide nucleic acid (PNA), acting as an RNA mimetic, was conjugated to a self-assembling peptide (SAP) core, forming a novel delivery vector that can be used also in combination with other SAP NPs. The PNA sequence, consisting of polyT bases, was designed to exploit the polyA tail found in all synthetic mRNA strands, anchoring the cargo via formation of hydrogen bonds between complementary bases. This method offers two distinct advantages: first, it does not need a cationic element for mRNA anchoring; second, the polyT-polyA interaction allows for the efficient binding of any mRNA type without requiring the customized synthesis of a PNA sequence complementary to the mRNA coding strand. Using an mRNA encoding for the firefly luciferase, or for the green fluorescent protein, we were able to demonstrate that these nanoparticles could bind the mRNA and deliver it into the cell cytoplasm, thereby allowing the production of the protein of interest with different efficiencies mediated by NPs size and composition.