Noncovalent peptide-mediated delivery of chemically modified steric block oligonucleotides promotes splice correction: quantitative analysis of uptake and biological effect.
Despite numerous encouraging reports in the literature, the efficiency of cell penetrating peptides (CPPs) in promoting cellular delivery of bioactive cargos is still limited. To extend our current understanding of the underlying limitations of such approaches, we performed quantitative uptake studies of different chemically modified (2'-O-methyl, LNA and PNA) steric block oligonucleotides, targeted against a mutated splice site inserted in a firefly luciferase reporter gene construct, applying the peptide carrier MPGalpha as a model system. The peptide formed stable noncovalent complexes with phosphorothioate oligonucleotide (PTO) and locked nucleic acid (LNA) modified oligonucleotides, whereas the neutral peptide nucleic acid (PNA) had to be hybridized to an unmodified DNA to allow for complex formation. Detailed quantitative uptake studies revealed comparable numbers of intracellular PTO and LNA oligonucleotides after peptide-mediated delivery. Surprisingly, the PTO derivative showed the strongest upregulation of reporter gene activity of about 100-fold followed by the PNA (40-fold) and LNA (10-fold). Electroporation and microinjection studies proved that delivery itself was not the limiting factor for the low activity of the LNA derivative. Maximal achievable reporter gene activity could be observed only after addition of chloroquine (CQ), indicative of an endosomal pathway involved. This is in line with nuclear microinjection experiments, which show that the minimal number of steric block molecules needed to trigger the observed reporter upregulation is about two orders of magnitude lower than determined after peptide or cationic lipid delivery.
Laufer
Sandra D
SD
Institut für Molekulare Medizin, Universitätsklinikum Schleswig-Holstein, Universität Lübeck, Lübeck, Germany.
Recke
Anna Lena
AL
Veldhoen
Sandra
S
Trampe
Alexander
A
Restle
Tobias
T
D000595:Amino Acid Sequence; D024202:Electrophoretic Mobility Shift Assay; D018274:Electroporation; D006367:HeLa Cells; D006801:Humans; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000008:administration & dosage*; D010455:Peptides / Q000032:analysis* / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction
D009841:Oligonucleotides; D010455:Peptides