Peptide-Based Delivery Vectors
Lab Members: Elenora Bolewska-Pedyczak, Molly Stief

Jean Gariépy Research Laboratory has been working for many years on the design of peptide-based delivery vehicles.^1-13 Drug delivery often remains the key step in turning new drugs (small molecules, peptides, proteins or DNA) into useful medicines. Delivery systems commonly used in the pharmaceutical industry can be grouped into either physical approaches (needles, gene gun, etc.) or chemical formulations (liposomes, polymer/nanoparticles and more recently cell-penetrating peptides). Viral vectors, although attractive as a strategy for gene delivery, present major challenges (immune response, large-scale production, toxicity) that can be more effectively answered with future advances in chemical, non-viral formulations.¹⁴

Currently some of the most interesting new vehicles for delivering drug cargos are cationic polymers. Cationic lipids and polymers (such as polyethylenimine, PEI) enter cells efficiently and are widely used as transfection agents in delivering plasmids and oligonucleotides into eukaryotic cells.^15,16 These agents titrate the negative charges on nucleic acids resulting in condensed, cationic complexes that are readily internalized by cells. Short cationic peptides can also act as protein transduction domains (PTDs, also termed cell-penetrating peptides, or CPPs) and shuttle drugs, peptides, proteins, oligonucleotides as well as large macromolecules (up to several hundreds of nanometers in size) into cells.^17-19 PTDs can also be introduced directly into recombinant proteins or synthetic peptides, generating biomolecular conjugates with a built-in ability to be internalized by cells. Typically, PTDs are sequences rich in lysine or arginine (R_n, where n ≥ 8) residues, with some sequences originally derived from basic domains of proteins, such as the Tat peptide from the HIV-1 Tat transcriptional transactivator and the Antennapedia homeodomain.^20-29

Our laboratory has recently established the importance of multivalency in the endocytosis of PTDs into eukaryotic cells: when presented on a defined scaffold as a multivalent ligand, the overall affinity, or avidity, for its receptor is greatly enhanced over the affinity of a monovalent counterpart.^1,2 Our laboratory has demonstrated that peptide dendrimers act as efficient delivery vehicles for drugs, biogenic peptides and nucleic acids in vitro^3,5-7 and in vivo.⁴ A rich source of peptide dendrimers can be found in the quaternary structure of natural proteins, and peptides derived from their oligomerization domains represent excellent candidates for engineering multivalent scaffolds. With a targeting ligand, such as a peptide hormone analogue, incorporated directly in the primary sequence, these oligomeric peptides self-assemble in a Lego-like fashion into compact, defined multivalent ligands.⁸ We have used, for example, the 30-residue tetramerization domain of the human p53 tumour suppressor, p53tet, as a scaffold to construct highly avid, tetravalent PTDs. These constructs exhibit greatly enhanced cell entry by receptor-mediated endocytosis relative to monovalent PTDs (Figure 1 = movie).^1,2

An important design aspect with respect to noncovalently linked oligomers is the stability of their quaternary structure. Specifically, these oligomeric scaffolds must remain stable at low concentrations. To this end, we have developed and characterized a tandem-dimerized variant of the p53tet domain (Figure 2). This construct, termed p53tetTD, retains a dimeric structure at concentrations at which wildtype p53tet is monomeric (Figure 3).⁹ This is an example of a highly stabilized oligomeric scaffold that would resist dissociation at dilute concentrations.

Figure 1. Short film highlighting the import and nuclear localization of one of our p53tet peptide delivery vectors (10R-p53tet) into CHO cells. The peptide 10R-p53tet is labeled with an N-terminal fluorescein group (green color). The film was assembled using merged phase contrast and confocal images of live, unfixed CHO cells exposed to the peptide over a one-hour period¹.

Figure 2. (A) The oligomeric structure of the tetramerization domain of the human p53 tumour suppressor (p53tet, residues 325 to 355). (B) Predicted structure of a tandem-dimerized p53tet domain (linker shown in blue), p53tetTD^2.

Figure 3. Comparative oligomeric stability of p53tetTD (open squares) relative to wildtype p53tet (solid squares) under physiological conditions. T50 is the transition temperature at which each oligomer is half-dissociated^2.

References (Click here)