Cell-penetrating peptides in protein mimicry and oligonucleotide delivery Applications and mechanisms

The plasma membrane separates the interior and exterior of the cell by functioning as a barrier, which regulates and restricts entry of molecules into the cell. Consequently, the plasma membrane imposes a major hurdle for passage of many hydrophilic pharmaceutical agents into the cell. A group of peptides, denoted cell-penetrating peptides (CPPs) was found about a decade ago to be able to transport bioactive cargos into the cell. Since then they have been extensively investigated for their mechanism of entry and capability to deliver various cargos spanning from peptides to plasmids. The main aim of this thesis was to investigate the mechanism and capability of some of these CPPs to deliver mainly oligonucleotides and peptides into the cell. Oligonucleotides have shown great promise in modifying gene expression, but are normally excluded from cellular entry. Oligonucleotides in the form of double-stranded DNA decoy for sequestering of transcription factors or peptide nucleic acids for blockade and redirection of splicing were explored in the thesis. In addition, peptides derived from the interaction interface of a tumor suppressor protein, were investigated for their potential to combine cell-penetrating and pro-apoptotic properties. The results show that all peptides with or without any cargo are predominantly dependent on some form of endocytic mechanism for internalization. The involvement of endocytosis was substantiated by using a functional assay based on splice correction, where all tested CPPs were associated with endocytosis for delivery of splice correcting peptide nucleic acids. A new CPP denoted M918 proved most efficient in promoting splice correction and was found to internalize mainly via macropinocytosis. In addition, the CPP TP10 efficiently delivered dsDNA decoy oligonucleotides for sequestering of the transcription factor Myc with a concomitant biological response, i.e. reduced proliferation. Finally, for the first time, to our knowledge, a novel pro-apoptotic peptide with cell-penetrating properties was designed from the tumor suppressor p14ARF, which decreased proliferation and induced apoptosis in cancer cell-lines, potentially mimicking the full-length protein. Altogether, this thesis highlights the functionality of CPPs and the possibility to develop new CPPs with improved or new properties, having the potential to advance delivery of therapeutic compounds.