STTR Phase I: A Platform Reagent for the Intracellular Delivery of Positively Charged Peptide

The broader impact/commercial potential of this Small Business Technology Transfer (STTR) project will be to develop an intracellular delivery technology for positively charged peptides for use in cell experiments in basic laboratory science and for delivery of peptide therapeutics for treatment of human disease. Peptides hold many advantages over the small molecule drugs that dominate the current healthcare market in terms of potency, specificity, and biocompatibility, and they are faster and easier to implement than genetic modifications for completion of hypothesis-driven studies. Peptides can inhibit enzymes and alter protein-protein interactions to have profound effects on cellular behavior. Despite these advantages, peptides suffer from poor intracellular uptake and entrapment within intracellular compartments, thereby preventing access to their intracellular target and limiting their potential as research tools and therapeutics. Positively charged cell penetrating peptides (CPPs) will be used to improve peptide uptake by cells. The proposed polyplex peptide delivery technology is designed to be a user-friendly reagent that further enhances CPP peptide uptake and dramatically improves potency and durability of action by enabling escape from intracellular compartments. The polyplex technology will fill this current gap in the delivery reagent market, bolstering peptide use and accelerating scientific advancement, and also enable peptide therapeutic approaches for intracellular targets. This STTR Phase I project proposes to leverage the properties of an anionic pH-responsive polymer to provide a more cost-effective, easy-to-use peptide delivery reagent that increases peptide uptake and longevity of action without cellular toxicity. The proposed work is focused on achieving the following aims: 1) Optimize a standardized formulation protocol that is effective for a broad range of peptides modified with positively charged cell-penetrating peptides by analyzing the physicochemical properties of a library of peptide-reagent formulations. 2) Verify polyplex-mediated peptide delivery efficiency as assessed by cellular peptide uptake across a broad spectrum of cell types, including both adherent and suspension cells as well as hard-to-transfect primary cell lines. 3) Establish advantages over competing delivery reagents in terms of ease of use, potency, duration of intracellular retention, and activity and biocompatibility. These aims will be achieved through the use of dynamic light scattering analysis, flow cytometric analysis of peptide uptake and retention, microscopy-based analysis of intracellular peptide localization, and in vitro cytotoxicity assays. Successful completion of these studies will establish the commercial viability of the peptide delivery reagent and enable the subsequent development of a prototype reagent kit for field testing.