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A peptide carrier for the delivery of biologically active proteins into mammalian cells

Abstract

The development of peptide drugs and therapeutic proteins is limited by the poor permeability and the selectivity of the cell membrane. There is a growing effort to circumvent these problems by designing strategies to deliver full-length proteins into a large number of cells (Schwarze and Dowdy in Trends Pharmacol. Sci. 21:45, 2000, Ford et al. in Gene Ther. 8:1, 2001, Prochiantz in Curr. Opin. Cell Biol. 12:400, 2000). A series of small protein domains, termed protein transduction domains (PTDs), have been shown to cross biological membranes efficiently and independently of transporters or specific receptors and to promote the delivery of peptides and proteins into cells. TAT protein from human immunodeficiency virus (HIV-1) is able to deliver biologically active proteins in vivo and has been shown to be of considerable interest for protein therapeutics (Frankel and Pabo in Cell 55:1189, 1998, Mann and Frankel in EMBO J. 10:1733, 1991, Fawell in Proc. Natl. Acad. Sci. USA 91:664, 1994, Vives et al. in J. Biol. Chem. 272:16010, 1997, Schwarze et al. in Science 285:1569, 1999, Nagahara in Nat. Med. 4:1449, 1998). Similarly, the third α-helix of Antennapedia homeodomain (Derossi et al. in J. Biol. Chem. 269:10444, 1994, Lindgren et al. in Trends Pharmacol. Sci. 21:99, 2000, Chen in Proc. Natl. Acad. Sci USA 96:4325, 1999) and VP22 protein from herpes simplex virus (Elliott and O'Hare in Cell 88:223, 1997, Phelan et al. in Nat. Biotechnol. 16:440, 1998) promote the delivery of covalently linked peptides or proteins into cells. However, these PTD vectors display a certain number of limitations in that they all require crosslinking to the target peptide or protein. Moreover, protein transduction using PTD–TAT fusion protein systems may require denaturation of the protein before delivery to increase the accessibility of the TAT–PTD domain. This requirement introduces an additional delay between the time of delivery and intracellular activation of the protein (Schwarze and Dowdy in Trends Pharmacol. Sci. 21:45, 2000). In this report, we propose a new strategy for protein delivery based on a short amphipathic peptide carrier, Pep-1. This peptide carrier is able to efficiently deliver a variety of peptides and proteins into several cell lines in a fully biologically active form, without the need for prior chemical covalent coupling or denaturation steps. In addition, this peptide carrier presents several advantages for protein therapy, including stability in physiological buffer, lack of toxicity and lack of sensitivity to serum. Pep-1 technology should be extremely useful for targeting specific protein–protein interactions in living cells and for screening novel therapeutic proteins.

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Acknowledgements

This work was supported in part by the Centre National de la Recherche Scientifique (CNRS) and by grants from the Association pour la Recherche sur le Cancer (ARC-5271) and the Agence Nationale de Recherche sur le Sida (ANRS). The Pep-1/Chariot project was supported by Active Motif (Carlsbad, CA). We thank K. Hondorp, J. Archdeacon and L. Chaloin for constructive discussions and advice. We also thank M. Dorée for continuous support and P. Travo, head of IFR 24 Integrated Imaging Facility, for technical advice on microscopy.

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Morris, M., Depollier, J., Mery, J. et al. A peptide carrier for the delivery of biologically active proteins into mammalian cells. Nat Biotechnol 19, 1173–1176 (2001). https://doi.org/10.1038/nbt1201-1173

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