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350 Cell-Penetrating Peptides: Processes and Applications
16.4 PEPTIDE VECTORS FOR OLIGONUCLEOTIDE DELIVERY
In accordance with the definition, cell membrane penetration of CPPs takes place
at 4°C and under conditions that prevent normal endocytosis. 37 Cellular penetration
is not receptor-mediated; thus it is cell type-unspecific. This virtue allows using
these delivery vehicles as universal transporters of conjugated hydrophilic macromolecules,
including nucleic acids. Recent reports suggest that CPPs are able to
transport these macromolecules across the blood–brain barrier, 38 enabling use of
these delivery vectors in gene therapy applications. Here we summarize recent
applications of CPPs in ON delivery (cf. Figure 16.1). In addition, peptides that do
not strictly fulfill the definition of CPPs but have been used in nucleic acid delivery
are briefly described (Sections 16.4.6 and 16.4.7).
16.4.1 PENETRATIN
The use of penetration in transmembrane delivery of nucleic acids was first demonstrated
by Allinquant et al. in 1995. 39 Their study demonstrated that amyloid precursor
protein (APP) antisense ODN was capable of transiently inhibiting neurite
outgrowth and decreasing APP levels in embryonic cortical neurons in vitro. Oligonucleotide
internalization was achieved by linking ODN to a polypeptide derived
from Drosophila melanogaster Antennapedia gene homeobox. The encoded protein
is a 60 amino acid-long highly conserved sequence responsible for membrane translocation.
The whole Antennapedia homeodomain is not necessary for translocation
through membrane; earlier site-directed mutagenesis studies demonstrated that the
third helix of Antennapedia (amino acids 43–58) is the shortest fragment of the
homeodomain capable of membrane penetration. 40
Troy et al. demonstrated down-regulation of Cu 2+ /Zn 2+ superoxide dismutase
(SOD1) activity and following apoptotic death in PC12 cells using penetratinmodified
antisense ON (Table 16.1). This showed efficient cellular uptake in the
presence of serum and also 100-fold increased effect compared to that with naked
ONs. 41
Simmons et al. 42 demonstrated that peptides derived from the third helix of the
homeodomain of Antennapedia mediate the uptake of a novel type of DNA analog
with backbone modification, a peptide nucleic acid (PNA; for review, see
Reference 43) to human prostate tumor-derived cells DU145 (Table 16.1). These
results were obtained by using fluorescence microscopy and FACS analysis; the
latter showed that about 99% of DU145 cells were transfected by fluorescein- or
rhodamine-labeled PNA oligomers at concentrations as low as 500 nM — efficiency
rarely observed by other transfection techniques. Naked PNA did not internalize at
that concentration. The authors did not observe any toxicity at conjugate concentrations
up to 500 nM and showed that PNA–peptide conjugates in vitro, as well as
unmodified PNAs, inhibit human telomerase, thus demonstrating that the peptide
moiety does not interfere with hybridization.
Pooga et al. (Table 16.1) further proved the potential of this delivery vector in
transmembrane delivery of PNA. 44 A 21-mer antisense PNA complementary to the
human neuropeptide galanin receptor type 1 mRNA was used after coupling to the