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Cell-Penetrating Peptides as Vectors for Delivery of Nucleic Acids 351 penetratin by disulphide bridge. This construct blocked the ex<strong>press</strong>ion 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) ex<strong>press</strong>ion 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.
TABLE 16.1 Examples of Transport of Nucleic Acids by CPP Cargo type Cargo function Target cell/tissue Applied CPP Conjugation method Ref. deoxyoligonucleotides antisense 21-mer PC-12 cells pAntp(43 to 58) RQIKIWFQNRRMKWKK peptide nucleic acids antisense 11-mer DU145 cells pAntp analog, GGRQIKIWFQNRRMKWKK antisense 21-mer Bowes cells; Penetratin rat spinal cord RQIKIWFQNRRMKWKK transportan GWTLNSAGYLLGKINLKALAALAKKIL antisense 16-mer Cultured cerebral retro-inverso-penetratin cortex neurons KKWKMRRNQFWVKVQR antisense 13-mer JR8 cells pAntp(44 to 58) 19 25-mers X-chromosome silencing, interference mapping C57BL/6 dermal fibroplasts QIKIWFQNRRMKWKK a transportan GWTLNSAGYLLGKINLKALAALAKKIL 6 mer bEnd cells pVEC LLIILRRRIRKQAHAHSK phosphorothioates antisense 20-mer NIH 3T3 cells pAntp analog, Ant-20 RQIKIWFQNRRMKWKKGGC Tat analog, Tat-20 RKKRRQRRRPPQC S–S 41 peptide bond 42 S–S 44 attachment via βAla-LysβAla linker 45 S–S 47 S–S 53 peptide bond 63 S–S 46 352 Cell-Penetrating Peptides: Processes and Applications
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CELL- PENETRATING PEPTIDES Processe
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Pharmacology and Toxicology: Basic
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Library of Congress Cataloging-in-P
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to the handbook are prominent resea
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REFERENCES 1. Green, M. and Loewens
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Contributors Mats Andersson Microbi
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Erin T. Pelkey Department of Chemis
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Contents Section I Classes of Cell-
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Chapter 16 Cell-Penetrating Peptide
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1 CONTENTS 0-8493-1141-1/02/$0.00+$
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The Tat-Derived Cell-Penetrating Pe
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24 Cell-Penetrating Peptides: Proce
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3 Transportans Margus Pooga, Mattia
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Transportans 55 Indeed, galparan is
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Transportans 57 especially the endo
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Transportans 59 In the penetration
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Transportans 61 A 21-mer antisense
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Transportans 63 Commonly, the prote
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Transportans 65 antibiotin antibodi
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Transportans 67 For cross-linking o
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Transportans 69 and still retain ef
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Model Amphipathic Peptides 73 its D
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Model Amphipathic Peptides 75 pmol/
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TABLE 4.1 Internalization of Peptid
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TABLE 4.2 Internalization of Peptid
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Model Amphipathic Peptides 81 relat
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Model Amphipathic Peptides 87 A cat
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Model Amphipathic Peptides 89 At th
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Model Amphipathic Peptides 91 16. H
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Signal Sequence-Based Cell-Penetrat
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Interactions of Cell-Penetrating Pe
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Structure Prediction of CPPs and It
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FIGURE 9.7 (Color Figure 9.7 follow
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FIGURE 9.8 The center of the figure
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Biophysical Studies of Cell-Penetra
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Toxicity and Side Effects of Cell-P
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Kinetics of Uptake of Cell-Penetrat
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Page 350 and 351: Cell-Penetrating Peptide Conjugatio
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Index 401 structure prediction, 187
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Index 403 pRB proteins, Tat-E1A bin
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Index 405 lipid perturbation (secon
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