Characterization of mRNA formulation and delivery by histidine-modified peptides

Abstract

The growing interest in gene therapy has resulted in the development and application of many novel therapeutic approaches, which can offer potential cure or treatment to previously incurable diseases, by the delivery of genetic materials inside the cells. Consequently restoring, replacing, or inhibiting gene expression.The effector molecules deployed in gene therapy are based on nucleic acids. However, physicochemical properties of these molecules, such as high negative charge and large size, limit their effective uptake by target cells and this has been identified as the key obstacle impeding the wider use of gene therapy. To improve the bioavailability, various drug delivery systems (DDSs) have been developed. One group of such DDSs are thecell penetrating peptides (CPPs). CPPs are short peptides (consisting of 3-40 amino acids) usually with a positive charge and possess high transport capability for biomolecules across the cell membrane. The working mechanism of CPPs is based on the formation of non covalent complexes/nanoparticles through electrostatic interactions with the negatively charged nucleic acids, such as mRNA. In this thesis, a number of histidine-modified PepFect14 peptide CPP analogs were studiedwith the aim of tailoring pH-sensitivity with varying degrees of ionizability and cationic charge. It was found that replacing up to 3 out of 5 cationic amino acids in the PF14 peptide with histidines didn’t affect its ability to form nanoparticles or reduce its mRNA delivery efficacy. Peptides with only 1 or no cationic charge were able to form complexes with mRNA but with sizes in the low micrometer range. Their inability to form nanoparticles together with their low cationic charge correlated with their low mRNA delivery efficacy in cell culture. Conclusively, these findings show that PepFect14 requires at the least 2 positive charges or more to be able to encapsulate mRNA into stable nanoparticles without losing efficacy.