Title: Efficient delivery of structurally diverse protein cargo into mammalian cells by a bacterial toxin

Biography:

My laboratory has a keen interest in understanding the molecular mechanisms for disease and in applying this information towards the development of new therapeutics. In particular, our current research focuses on bacterial toxins; on their role in disease, on their druggability, and on their potential as drug-delivery vectors. My postdoctoral training in bacterial toxin pathogenesis at Harvard Medical School with Dr. John Collier provided much of the foundation for my understanding of bacterial toxin structure and function. Following my postdoctoral studies, I accepted a position as a Sr. Scientist at Merck & Company, where I led teams of scientists in drug discovery programs destined for clinical development. With this background in bacterial pathogenesis and drug discovery, I re-entered academia in 2011 at the University of Toronto and The Hospital for Sick Children. Since coming to Sickkids, I have built a team of talented trainees and world-renowned collaborators locally and abroad. 

Abstract

Given the vast array of applications for protein-based tools and therapeutics inside cells, there is great interest in developing safe and efficient protein delivery platforms that direct biologics into cells. To date, numerous approaches been investigated to facilitate protein entry into the cytoplasm of cells; however, though each capable of delivering protein cargo into cells to varying degrees, general mechanism-based limitations exist for these platforms. In particular, selectivity and/or efficiency remain elusive features for most platforms owing to their shared nonspecific mode of interaction with membranes. Protein toxins, which use host cell-surface receptors to initiate entry into cells, are attractive vectors to consider given their natural tendency to delivery proteins into specific cells with high efficiency. The paucity of development efforts for toxins as protein delivery vectors stem from early studies, which suggested that delivery was restricted to a select few cargo that were largely unfolded prior to translocation; and that the cargo itself greatly diminished the efficiency of translocation of the system. Through careful engineering of the platform, we show that neither of these assertions is true. We show that the diphtheria toxin platform is capable of delivering proteins that are over 100-kDa in size, and of varying structures and stability with exquisite efficiency. In fact, to our surprise, we found that diphtheria toxin could deliver the hyper-stable passenger protein mCherry, which we calculated to have a melting temperature greater than 90 degrees under the translocation conditions, suggesting that even folded proteins could be delivered into cells. Through a rigorous set of experiments we trace the misleading early results to affects of cargo on the readout of translocation, rather than the efficiency of translocation. We also provide functional evidence that the delivered cargo is functional. Using a-amylase as cargo we show that cytosolic glycogen is degraded in a dose- and time-dependent manner.