Characterization of a Class of Cationic Peptides Able to Facilitate Efficient Protein Transduction in Vitro and in Vivo

Abstract

Protein transduction domains (PTDs), such as the third helix of the Drosophila Antennapedia homeobox gene (Antp) and the HIV TAT PTD, possess a characteristic positive charge on the basis of their enrichment for arginine and lysine residues. To determine whether cationic peptides are able to function as protein transduction domains, 12-mer peptide sequences from an M13 phage library were selected for synthesis on the basis of their varying cationic charge content. In addition, polylysine and polyarginine peptides were synthesized in order to assess the effect of charge contribution in protein transduction. Coupling of the biotinylated peptides to avidin-beta-galactosidase facilitated transduction in a wide variety of cell lines and primary cells, including islet beta-cells, synovial cells, polarized airway epithelial cells, dendritic cells, myoblasts, and tumor cells. Two of the peptides, PTD-4 and PTD-5, mediated transduction nearly 600-fold more efficiently than a random control peptide, but with an efficiency similar to the TAT PTD and the 12 mers of polylysine and polyarginine. Furthermore, confocal analysis of biotinylated peptide-streptavidin-Cy3 conjugates demonstrated that the internalized PTDs are found in both the nuclei and the cytoplasm of treated cells. When tested in vivo, the PTDs were able to facilitate efficient and rapid protein delivery into rabbit synovium and mouse solid tumors following intraarticular and intratumoral administration, respectively. These novel PTDs can be used to transfer therapeutic proteins and DNA for the treatment of a wide variety of diseases, including arthritis and cancer.

Protection of Islets by in SituPeptide-mediated Transduction of the IκB Kinase Inhibitor Nemo-binding Domain Peptide

“…Peptides were N-terminal conjugated to fluorescent probe using 6-carboxyfluorescein (6CF, Molecular Probes Inc., Eugene OR) and were subsequently purified and characterized by reversed-phase high performance liquid chromatography and mass spectrometry. The construction of the peptide-eGFP fusion protein has been described previously (53).…”

“…Previously we have identified a series of cationic peptides that were able to transduce certain cell types as or more efficiently than the Tat PTD (52). Moreover, we demonstrated that a specific PTD, termed PTD-5, was able to transduce human islets efficiently in culture (53).…”

“…In particular, we have demonstrated that human islets can be transduced with a specific PTD, PTD-5 marker protein complex in culture (53). To examine further the transduction efficiency of PTD-5 into isolated islets, mouse and human islets were incubated with fluorescently labeled PTD-5.…”

Protecting Pancreatic ß‐cells

“…Protein transduction has the distinctive feature of acting transiently without genetically modifying the target cells. Proteins or peptides fused to PTD-5, a protein transduction domain selected from an M13 phage peptide display library and to TAT/PTD, an 11-aa peptide derived from basic domain of TAT/HIV transactivator protein, can efficiently transduce islet cells (48,49). Furthermore, pancreatic duct infusion with the NFK-B inhibitor NBD peptide fused to the cationic protein transduction domain-PTD5 prior to islet isolation yielded islets with enhanced viability (50).…”

Gene- and cell-based therapeutics for type I diabetes mellitus

“…In the meantime, antigen nonspecific inflammation progresses within islets because of macrophage and DC secretion of soluble mediators of b-cell dysfunction and apoptosis activation. 233,234 Cationic liposomes 204,205,214 Peptide fusion domains 118,235 Therapeutics for type I diabetes mellitus R Bottino et al intact islet transduction. Equally unknown is the degree to which these vectors can contribute to post-transplantation inflammation.…”