Abstract
The development of therapeutic small interfering RNAs (siRNAs) has lately gained significant momentum due to their ability to silence genes in a highly specific manner. The main obstacle withholding the wider translation of siRNA-based drug modalities is their limited half-life and poor bioavailability, especially in extra-hepatic tissues. Consequently, various drug delivery systems (DDSs) have been developed to improve the delivery of siRNAs, including short delivery peptides called cell-penetrating peptides (CPPs). In this study, we explore the potential of using alkenyl-alanine modifications to enhance the siRNA delivery efficacy with CPPs. We demonstrate on hPep peptides that incorporation of alkenyl-alanines enhances the encapsulation of siRNAs into stable nanoparticles and contributes to increased cellular uptake. Furthermore, we demonstrate that the lead peptide, hPep3, induces effective RNAi-mediated gene silencing in a reporter cell model as well as on the disease-implicated endogenous CD45 gene target. The biodistribution studies in mice show that the alkenyl-alanines are systemically well tolerated, and employing such modifications in the peptide backbone improves siRNA delivery in several tissues, including extra-hepatic sites. As demonstrated on hPep peptides, alkenyl-alanines offer a simple yet robust way to enhance the delivery efficacy of CPPs and have the potential to advance siRNA therapeutics beyond the liver targets.