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Cell-Penetrating Peptides as Vectors for Delivery of Nucleic Acids 355 hydrophilic part of Simian virus SV40 large tumor antigen) have been shown to act as carriers of nucleic acids at physiological and decreased temperatures (Table 16.1). 55 The ability of this peptide to act as carrier for nucleic acids was further investigated using antisense technology. Antisense phosphorothioate ODN covalently attached to the formerly described peptide was able to inhibit L-type Ca 2+ channel β subunit synthesis in cardiac cell line H9C2. Another report showed the use of MTS (MPM) derived from the hydrophobic region of signal peptide sequence of the Kaposi fibroblast growth factor, or of the above-mentioned MTS in complex with NLS of transcription nuclear factor NFκB. 56 The phosphorothioate ODNs were cross-linked to the carrier peptides by disulphide bond. These constructs translocated membranes and localized in the nucleus. Unfortunately, the constructs did not show any antisense effect in vitro. Experiments with a cell-free luciferase reporter assay were successful as all antisense sequences decreased luciferase expression by 60 to 80% of basal level at 2 µM. The inhibition of luciferase expression by control sequences was observed by 10 to 20%. We explain it by the fact that the authors of this study used high concentrations of construct. Morris et al. 57 developed a 27-amino acid-long peptide called MPG that contains a hydrophobic domain derived from the fusion sequence of HIV gp41 and a hydrophilic domain derived from the nuclear localization sequence of SV40 T-antigen (Table 16.1). Binding of both single- and double-stranded phosphorothioate ODNs to MPG occurs through electrostatic interactions, which involve basic residues of the peptide vector. MPG peptide translocates fluorescein-labeled phosphorothioate ODN conjugates into cultured mammalian cells in less than 1 h with high efficiency (90%, as measured by confocal microscopy). The speed of the uptake indicates that the mechanism does not follow the endosomal pathway. Experiments with MPGmediated ON uptake on HS68 fibroblasts at low temperature to block the endosomal pathway confirm the assumption. At 4°C the oligonucleotides were also rapidly internalized into cells in the previously described time frame and localized to the nucleus. Vidal et al. 58 used MPG peptide for transmembrane delivery of fluorescentlabeled single-stranded mRNA encoding the p66 subunit of the HIV-1 reverse transcriptase. In the presence of the peptide vector, the mRNA was delivered into the cytoplasm of HS68 human fibroblasts. The same authors used a similar strategy for gene delivery into cultured cells. 59 MPG promoted delivery and expression of the pRL-SV40 plasmid encoding the reporter R. reniformis luciferase in HS-68 and NIH 3T3 fibroblasts, C2C12 myoblasts and COS-7 cells, as well as human CEM-SS lymphoblasts (Table 16.1). The degree of transfection using MPG was at least twoand sevenfold higher than that obtained with Lipofectamine ® for fibroblasts or COS- 7 and C2C12 myoblasts, respectively. They also demonstrated that MPG-mediated delivery of a plasmid carrying full-length antisense cdc25C cDNA into mammalian fibroblasts promotes cell cycle arrest at the G2/M transition. Zanta et al. 60 demonstrated that NLS peptide (PKKKRKVEDPYC) alone was sufficient to deliver 3.3 kbp CMV luciferase gene. The efficiency of transfection was enhanced 10- to 1000-fold as a result of addition of NLS. Whether the enhancement of transfection is caused by improved membrane penetration or just by
356 Cell-Penetrating Peptides: Processes and Applications increased nuclear localization remains unclear as transfections were performed by standard transfection protocol by Transfectam ® or branched polyethylenimine (PEI). The same NLS has been demonstrated to be involved in antigene PNA delivery to intact cells in culture. 61 The cell lines derived from Burkitt’s lymphomas presenting translocated and hyperexpressed c-myc oncogene. An NLS coupled to 17-mer anti-myc PNA, complementary to myc sequence of the second exon of the oncogene, caused rapid down-regulation of c-myc expression. PNA-myc-NLS construct was shown to be localized predominantly in the cell nuclei. The data shown in this paper do not explain the type of uptake; similarities to previously used constructs cause one to think about penetration. The putative roles of NLS in membrane penetration is further confirmed by a recent study that utilizes a similar transfection strategy. 62 Brandén et al. coupled SV40 NLS directly to PNA/Cy-3 oligonucleotide complex. These complexes were injected into various mouse tissues. The complex transfected cells in subcutaneous, muscular, or hepatic tissue, as measured by fluorescense microscopy. Comparative transfections with PEI/Cy-3 complexes uncover differences in transfection; PEIcondensed ODNs are detected in the cell as large complexes with nonhomologous distribution, while NLS-PNA/ODN complexes show less aggregated intracellular dispersion. When using a PNA/Cy-3 complex without NLSs, no detectable transfection took place, indicating the crucial role of NLSs in this experimental system. 16.4.5 PVEC A recent article by Elmquist et al. 63 reported on novel CPP, an 18-amino acid-long peptide derived from the murine sequence of the cell adhesion molecule vascular endothelial cadherin, amino acids 615–632. They show that human aortic endothelial cells, brain capillary endothelial cells, Bowes melanoma cells, and murine brain endothelial cells efficiently take up this peptide. pVEC is also shown to transport hexameric PNA molecule at physiological and decreased (4°C) temperatures. 16.4.6 OTHER PEPTIDE VECTORS There are several examples of peptide-mediated transmembrane delivery of ON or dsDNA when utilized peptides are not strictly CPPs by definition. Whether this is a general virtue of these peptides or whether the delivery is dependent on cell type or experimental conditions remains to be discovered. Recently, novel gene transfer technique, in which an amphiphilic α-helical peptide containing cationic amino acids is used as a gene carrier into cells, has been reported. Wyman et al. 64 studied a peptide, KALA (WEAK-LAKA-LAKA-LAKH- LAKA-LAKA-LKAC-EA), derived from the sequence of the amino-terminal segment of the HA-2 subunit of the influenza virus hemagglutinin involved in fusion of the viral envelope with the endosomal membrane. The peptide displays functions necessary in transfection process, e.g., condensing DNA and causing an endosome–membrane perturbation and transfection of cells. At physiological pH, the KALA peptide exists in the α-helical conformation in spite of the number of positive charges from the protonated Lys side chains. The peptide undergoes an α-helical structure to mixed random coil if pH is decreased
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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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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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116 Cell-Penetrating Peptides: Proc
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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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Page 418 and 419: Index A Abaecin, 129 Abz radiolabel
Page 420 and 421: Index 399 Diffraction, 168 Disulphi
Page 422 and 423: Index 401 structure prediction, 187
Page 424 and 425: Index 403 pRB proteins, Tat-E1A bin
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Index 405 lipid perturbation (secon
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