Transcript Cell Penetrating Peptides

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.