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Cell-Penetrating Peptides as Vectors for Delivery of Nucleic Acids 351
penetratin by disulphide bridge. This construct blocked the expression of galanin
receptors in Bowes cells. The intrathecal administration of the peptide–PNA construct
resulted in a decrease in galanin binding in the dorsal horn and inability of
galanin to inhibit the C fibers stimulation-induced facilitation of flexor reflex in rat.
Oligonucleotides are efficiently taken up by retro-inverso penetratin as demonstrated
by Aldrian–Herrada (Table 16.1). 45 PNA antisense sequence for the AUG
translation initiation region of prepro-oxytocin mRNA was covalently coupled to
retro-inverso penetratin. This conjugate was internalized by cultured cerebral cortex
neurons within minutes, while the uptake of naked PNA was slow. The internalization
and functional activity also occurred after maintaining cells at 4°C for 5 min and
then incubating them at the same temperature for 10 min with the penetratin–PNA;
PNA alone was internalized too slowly to be studied by this method without causing
cell damage. The result is consistent with the hypothesis of direct penetration across
the cell membrane. They also showed that PNA and vector peptide–PNA decreased
the prepro-oxitocin mRNA level in a dose- and time-dependent manner in cultured
magnocellular neurons from the hypothalamic supraoptic nucleus as shown by RT- PCR.
Penetratin has been used to deliver other modified oligonucleotide analogs, most
commonly, phosphorothioates used in antisense experiments. Astriab–Fisher et al. 46
demonstrated by fluorescence microscopy that application of penetratin and penetratin–ON
conjugate resulted in accumulation in the cell nucleus. Cells treated with
naked ON showed no intracellular localization. They also attained significant inhibition
of P-glycoprotein, a multidrug-resistance gene1 (MDR1) expression product,
with submicromolar amounts of phosphorothioate oligonucleotide in NIH 3T3 cells.
The presence of 10% serum enhanced the antisense effects, a surprising finding
anomalous to other similar studies. Unfortunately, this study does not describe the
degree of CPP–ON conjugates needed to affect drug resistance and is restricted to
estimating the potential of these constructs in vivo. This study uses, in parallel,
another type of CPP, a Tat protein-derived peptide.
Villa et al. 47 have used penetratin to target PNA and phosphorothioate ODNs to
human melanoma cell line JR8 (Table 16.1). Biotinylated PNA was utilized to detect
internalization, which occurred only when penetratin was coupled to PNA, whereas
naked oligomers did not enter the cells. Moreover, these constructs were able to
inhibit telomerase activity in these cells, suggesting such constructs have high
potential in cancer treatment.
Very recently, Kokunai et al. 48 demonstrated use of Antennapedia homeodomain
in antisense ODN delivery to malignant glioma cell lines. They showed that these
ODNs are capable of enhancing γ irradiation-induced apoptosis, probably by p21
antisense.
16.4.2 TAT
Astriab–Fisher et al. 46 demonstrated similar results with Tat (see Chapter 1) coupled
to phosphorothioates as previously described with penetratin (Table 16.1). These
results further strengthen the assumptions that the uptake mechanism for Tat and
penetratin peptides, could be similar and they could form a subclass of nonamphiphilic
CPPs.