Vectorization of oligonucleotides with cell-penetrating peptides Characterization of uptake mechanisms and cytotoxicity

The hydrophobic plasma membrane constitutes an indispensable barrier for cells in living animals, allowing the constitutive and regulated influx of essential molecules while preventing access to the interior of cells of other macromolecules. Albeit being pivotal for the maintenance of cells, the inability to cross the plasma membrane is still one of the major obstacles to overcome in order to progress current drug development. A group of substances that has shown great promise for future clinical use is oligonucleotides that are exploited to interfere with gene expression. Short interfering RNAs that are utilized to confer gene silencing and splice correcting oligonucleotides, applied for the manipulation of splicing patterns, are two classes of oligonucleotides that have been explored in this thesis. Despite being efficient compounds for regulating gene expression, their hydrophilic nature prohibits cellular internalization. Cell-penetrating peptides (CPPs) are a class of peptides that has gained increasing focus in last years. This ensues as a result of their remarkable ability to convey various, otherwise impermeable, macromolecules across the plasma membrane of cells in a relatively non-toxic fashion. Since the initial discovery over a decade ago, their uptake mechanism has been under intense investigation. Although results from earlier studies favored a direct translocation of peptides independent of receptors, an increasing number of studies is now emphasizing the importance of endocytosis in this process. This thesis aims at further characterizing well-established, and newly designed, CPPs in terms of toxicity, delivery efficacy, and internalization mechanism. Furthermore, we employ various CPPs for the delivery of the abovementioned bioactive cargos and analyze the impact of endosomolysis on the bioavailabilty of the cargos. Our results demonstrate that different CPPs display different toxic profiles and that cargo conjugation alters the toxicity and uptake conferred by CPPs. Furthermore, we confirm the involvement of endocytosis in translocation of CPPs using a functional assay based on splice correction. All tested peptides facilitate the delivery of splice correcting oligonucleotides with varying efficacy, the newly designed CPP, M918, being the most potent. Further characterization of this peptide signified the importance of macropinocytosis in cellular uptake which is in concurrence with another CPP, penetratin. Finally we conclude that by promoting endosomolysis, using lysosomotrophic agents, or by exploring new CPPs with improved endosomolytic properties, the biological response increases significantly. In conclusion, we believe that these results will facilitate the development of new CPPs with improved delivery properties that could be used for transportation of oligonucleotides in clinical settings.