Modified cell-penetrating peptides for delivery of splice-correction oligonucleotides and small interfering RNA

Abstract

The therapeutic potential of nucleic acids (NA) is often constrained by their poor intracellular uptake and subsequent loss of activity. Cell-penetrating peptides (CPPs) offer a promising strategy for enhancing the delivery and resulting effect of splice-correcting oligonucleotides (SCO) and small interfering RNA (siRNA) into cells. This study aims to evaluate the delivery efficiency of novel CPPs designed for short nucleic acid delivery. The characterization of the nanoparticles formed between these CPPs, and their nucleic acid cargo was performed using dynamic light scattering analysis. Moreover, the functional delivery was evaluated using reporter cell lines and CPP/siRNA and CPP/SCO nanoparticles. The results demonstrated that several peptide-NA formulations mediated efficient intracellular delivery of siRNA and SCO, leading to significant levels of reporter rescue through splice correction in HeLa pLuc705 cells and efficient gene silencing in U87 MG-Luc2 cells. Notably, one of the CPPs, RDP18, formed nanoparticles with the hydrodynamic diameter of ~70 nm and induced ~100-fold reporter rescue in the cells, when nanoparticles were formed at a CPP/SCO molar ratio 10:1, while maintaining cell viability. Additionally, RDP7, regardless of larger particles with hydrodynamic diameter around 205 nm, also led to similar levels of reporter rescue. The observed variations in delivery efficacy were attributed to the differences in CPP sequence and used formulation conditions. These findings highlight the importance of optimization of peptide characteristics, and formulation conditions to achieve suitable nanoparticle for specific applications.