Noncovalent Peptide-Mediated Delivery of Chemically Modified Steric Block Oligonucleotides Promotes Splice Correction: Quantitative Analysis of Uptake and Biological Effect

Abstract

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.

Cell-Penetrating Peptides—Mechanisms of Cellular Uptake and Generation of Delivery Systems

“…Several CPPs have been used to mediate the delivery of PNAs and PMOs through covalent linkage of both entities [111,113,114,116,117,118,119]. The formation of efficient non-covalent complexes comprising CPPs and both charged and uncharged steric block oligonucleotides, namely 2'-O-methyl, LNA, PNA and charged PNA derivatives, has also been described [120,121,122,123,124]. …”

“…Importantly, the stearic acid modification of these peptides was associated with increased endosomal escape. MPGα has also been used to mediate the uptake of different chemically modified (2'-O-methyl, LNA and PNA) steric block oligonucleotides [120]. …”

Cell Penetrating Peptides in the Delivery of Biopharmaceuticals

“…They have been used to mediate the delivery of PNAs and PMOs through covalent linkage [33]. The formation of efficient non-covalent complexes comprising CPPs and both charged and uncharged steric block oligonucleotides like 2'- O -methyl, LNA, PNA and charged PNA derivatives, has also been described [34].…”

Peptide-mediated Cell and In Vivo Delivery of Antisense Oligonucleotides and siRNA

“…7,59,60 The uptake mechanisms of peptide-complexed oligonucleotides appear to be more diverse. Some peptide complexing agents such as MPG appear to utilize endocytosis, 61 whereas for other similar peptides a non-energy-dependent membrane-crossing pathway is proposed. 62 To obtain splicing redirection, the oligonucleotide must be delivered into the cell nucleus.…”

“…Debate continues as to whether endocytosis or energy-independent mechanisms of delivery are optimal in such applications in terms of bioavailability, which is known to be poorly correlated with cell uptake. 42,61 In recent studies of conjugates of charge-neutral antisense PNA and PMO, earlier Penetratin and Tat transduction domains have now been replaced by Arg-rich CPPs ( Table 1). In the paradigm peptides, Arg residues are spaced by aminohexanoyl (Ahx or X) and/or -alanyl ( ) moieties to enhance hydrophobicity and proteolytic resistance in serum, such as (R-Ahx-R) 4 -Ahx-Ala and (R-Ahx-R-R-Ala-R) 2 -Ahx-Ala called RXR4 and B-peptide respectively.…”