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William Hardy Biomembranes: Structure and Biophysical Properties University of Florida Spring 2009 Introduction Cell Penetrating Principles Mechanisms Specific Pathways Specific Applications Conclusion Questions References Short polycationic or amphiphilic peptides which facilitate cellular uptake of various molecular cargo linked to them either in a covalent or non-covalent fashion. The first CPP was discovered independently by two laboratories in 1988 It was found that the trans-activating transcriptional activator (Tat) from Human Immunodeficiency Virus 1 (HIV-1) could be efficiently taken up from the surrounding media by numerous cell types in culture. Most peptide- and nucleic acid-based drugs are poorly taken up in cells, and this is considered a major limitation in their development as therapeutic agents Conjugation of therapeutic agents to CPPs could thus become a strategy of choice to improve their pharmacological properties. The mechanism of internalization of CPPs and their cargo is not well understood and has recently been the subject of controversies Kelly M. Stewart, Kristin L. Horton and Shana O. Kelley Org. Biomol. Chem., 2008, 6, 2242 CPPs are of various different types and sizes The functionally significant part of the peptides seems to be a sequence of amino acids about 10 residues Called protein transduction domains (PTDs) A part of protein sequence and structure that can evolve, function, and exist independently of the rest of the protein chain They drive the uptake of cargo through various pathways. Endocytosis- Pinocytosis, Macropinocytosis ??? Directly Penetrating Cell ??? The mechanism of internalization of CPPs and their cargo is not well understood and has recently been the subject of controversies Veldhoen, S., Laufer, S.D., Trampe, A. and Restle, T., (NAR, 34 (22), 6561–6573 Int. J. Mol. Sci. 2008, 9 ttp://www.youtube.com/watch?v=4gLtk8Yc1Zc Int. J. Mol. Sci. 2008, 9 Two Complex delivery systems 1. A branched polymer consisting of alternating histidines and lysines. HK peptides was the starting material. This polymer showed high serum stability an efficiently delivered plasmids not only into cultured cells but also tumor mouse models 2. Tat-grafted PEGylated nanocarrier, these carriers have been successfully applied for nucleic acid or drug delivery in severl cell types and mouse models Fluorescence microscopy on fixed cells Fluorescence-activated cell sorter (FACS) analysis These flurescense based spectroscopies examine intracelluar localization of fluorescently labelled peptides in the absence or presense of cargo Current reports provide increasing evidence that peptides represent a promising alternative to viral and lipid-based nucleic acid delivery systems. After two decades of intensive research, we now can chose from a constantly growing arsenal of different peptide-based transfection systems each suitable for a particular application. 1. Opalinska, J. B.; Gewirtz, A. M. Nucleic-acid therapeutics: basic principles and recent applications. Nat. Rev. Drug Discov. 2002, 1, 503-514. 2. Eckstein, F. The versatility of oligonucleotides as potential therapeutics. Expert. Opin. Biol. Ther. 2007, 7, 1021-1034. 3. Kootstra, N. A.; Verma, I. M. Gene therapy with viral vectors. Annu. Rev. Pharmacol. Toxicol. 2003, 43, 413-439. 4. Verma, I. M.; Weitzman, M. D. Gene therapy: twenty-first century medicine. Annu. Rev. Biochem. 2005, 74, 711-738. 5. Raper, S. E.; Yudkoff, M.; Chirmule, N.; Gao, G. P.; Nunes, F.; Haskal, Z. J.; Furth, E. E.; Propert, K. J.; Robinson, M. B.; Magosin, S.; Simoes, H.; Speicher, L.; Hughes, J.; Tazelaar, J.; Wivel, N. A.; Wilson, J. M.; Batshaw, M. L. A pilot study of in vivo liver-directed gene transfer with an adenoviral vector in partial ornithine transcarbamylase deficiency. Hum. Gene Ther. 2002, 13, 163-175. 6. Hacein-Bey-Abina, S.; Von Kalle, C.; Schmidt, M.; McCormack, M. P.; Wulffraat, N.; Leboulch, P.; Lim, A.; Osborne, C. S.; Pawliuk, R.; Morillon, E.; Sorensen, R.; Forster, A.; Fraser, P.; Cohen, J. I.; de Saint, B. G.; Alexander, I.; Wintergerst, U.; Frebourg, T.; Aurias, A.; Stoppa-Lyonnet, D.; Romana, S.; Radford-Weiss, I.; Gross, F.; Valensi, F.; Delabesse, E.; Macintyre, E.; Sigaux, F.; Soulier, J.; Leiva, L. E.; Wissler, M.; Prinz, C.; Rabbitts, T. H.; Le Deist, F.; Fischer, A.; Cavazzana-Calvo, M. LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science 2003, 302, 415-419. 7. Raper, S. E.; Chirmule, N.; Lee, F. S.; Wivel, N. A.; Bagg, A.; Gao, G. P.; Wilson, J. M.; Batshaw, M. L. Fatal systemic inflammatory response syndrome in a ornithine transcarbamylase deficient patient following adenoviral gene transfer. Mol. Genet. Metab 2003, 80, 148-158. 8. Check, E. Gene therapy put on hold as third child develops cancer. Nature 2005, 433, 561. 9. Luo, D.; Saltzman, W. M. Synthetic DNA delivery systems. Nat. Biotechnol. 2000, 18, 33-37. 10. Frankel, A. D.; Pabo, C. O. Cellular uptake of the tat protein from human immunodeficiency virus. Cell 1988, 55, 1189-1193.