Introducing a Single-Point Modification to the Cell-Penetrating Peptide PepFect14 Analogue Sequence for Optimising Oligonucleotide Delivery

Extracellular synthetic nucleic acids (NA), including various types of RNAs, encounter challenges in effectively reaching their intended targets. To improve the efficacy of RNA as a therapeutic agent, delivery methods such as cell-penetrating peptides (CPP) are being explored to enhance their levels inside cells and aid their escape from endosomes, resulting in productive delivery. One such CPP, PepFect14 (PF14), has been recognised as an efficient peptide-based delivery vector used to deliver RNA into cells in vitro and in vivo. To further optimise nanoparticle physicochemical characteristics, as well as intracellular delivery and bioactivity of CPP/RNA nanoparticles, we previously modified PF14 by replacing ornithine residues in the sequence with lysine, resulting in a CPP we named PF14-Lys, which demonstrated delivery efficacy on a similar level as original PF14 but has several advantages in synthesis and modelling. In this project, we went a step further by creating a comprehensive single-mutation library of PF14-Lys to explore how the introduced modifications impact the characteristics of the analogues. These modifications were implemented at various positions in the peptide sequence and focused on the cationic or hydrophobic face modulation of the projected α-helix. We evaluated the peptide sequences using predictions and calculations, and a small subset of analogues underwent further characterisation and experimental testing. Following analysis of secondary structure, nanoparticle formation, nanoparticle stability, transfection efficacy and cell viability post-transfection, we identified several CPPs with high efficacy. Furthermore, we gained insights into potential directions for peptide characteristics to enhance further design approaches for creating efficient delivery peptides for specific cargoes and applications.