William Hardy Biomembranes: Structure and Biophysical Properties University of Florida Spring 2009.

Cell Penetrating Peptides

William HardyBiomembranes: Structure and Biophysical Properties

University of FloridaSpring 2009

Introduction Cell Penetrating Principles Mechanisms Specific Pathways Specific Applications Conclusion Questions References

Outline

Short polycationic or amphiphilic peptides which facilitate cellular uptake of various molecular cargo linked to them either in a covalent or non-covalent fashion.

What are Cell Penetrating Peptides ?

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.

History

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.

Why do CPP Research

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

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)

Mechanism

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.

Protein Transduction Domains

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

Which Pathways ??? Who Knows

Problem With Mechanism

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

Endocytosis

Int. J. Mol. Sci. 2008, 9

Endocytosis

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

Examples of CPP

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

Recent Advancements in Vivo Delivery

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

Characterization

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.

Conclusion

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.

References

Questions