Cell Penetrating Peptides

1. Cell Penetrating Peptides William Hardy Biomembranes: Structure and Biophysical Properties University of Florida Spring 2009

2. Outline • Introduction • Cell Penetrating Principles • Mechanisms • Specific Pathways • Specific Applications • Conclusion • Questions • References

3. What are Cell Penetrating Peptides ? • Short polycationic or amphiphilicpeptides which facilitate cellular uptake of various molecular cargo linked to them either in a covalent or non-covalent fashion.

4. History • 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.

5. Why do CPP Research • Most peptide-and nucleic acid-based drugs are poorly taken up in cells, andthis is considered a major limitation in their development astherapeutic agents • Conjugationof therapeutic agents to CPPs could thus become a strategy ofchoice to improve their pharmacologicalproperties.

6. Schematic Diagram • 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

7. Mechanism • 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)

8. Protein Transduction Domains • 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.

9. Which Pathways ??? Who Knows • 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

10. Problem With Mechanism Veldhoen, S., Laufer, S.D., Trampe, A. and Restle, T., (NAR, 34 (22), 6561–6573

11. Endocytosis Int. J. Mol. Sci. 2008, 9

12. Endocytosis ttp://www.youtube.com/watch?v=4gLtk8Yc1Zc

13. Examples of CPP Int. J. Mol. Sci. 2008, 9

14. Recent Advancements in Vivo Delivery • Two Complex delivery systems • 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 • Tat-grafted PEGylatednanocarrier, these carriers have been successfully applied for nucleic acid or drug delivery in severl cell types and mouse models

17. Characterization • Fluorescence microscopy on fixedcells • Fluorescence-activated cell sorter (FACS) analysis • These flurescense based spectroscopies examine intracelluar localization of fluorescently labelled peptides in the absence or presense of cargo

18. Conclusion • 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.

19. References • 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 ornithinetranscarbamylase 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 ornithinetranscarbamylase • deficient patient following adenoviral gene transfer. Mol. Genet. Metab2003, 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. Cell1988, 55, 1189-1193.

20. Questions