Application of PepFect peptides for the delivery of splice-correcting oligonucleotides

One oligonucleotide-based approach that appear very promising for the treatment of different genetic disorders are based on so-called splice-correcting oligonucleotides (SCOs) that are exploited to manipulate splicing patterns. In order to increase the bioavailability, cell-penetrating peptides (CPPs) have readily been covalently conjugated to SCOs to facilitate cellular internalization. While being a successful strategy for the delivery of uncharged oligonucleotides (ONs), it is extremely difficult to generate covalent conjugates between commonly used negatively charged ON analogs and cationic CPPs. Furthermore, high concentrations of ONs in the micromolar range are often needed to obtain biological responses, most likely as a result of endosomal entrapment of material. Therefore, exploring other vectorization methods using CPPs with endosomolytic properties are highly desired.A method of using stearyl modified CPP (i.e., TP10) analogs, named PepFect3 and PepFect4, are being described for the transfection of antisense SCOs using a simple one-step co-incubation procedure. These peptides form complexes with SCOs and efficiently promote cellular uptake by facilitating endosomal escape. This chapter describes the methods of how to form and characterize these nanoparticles and the cellular assay used to address the delivery.