Reversibly neutralized perfringolysin O for intracellular delivery of macromolecules/

Abstract

With an increasing understanding of the molecular bases of disease, macromolecules such as proteins and siRNA can potentially be used as therapeutics, modulating biological function with high specificity to reverse pathological progression. However, while certain contexts require manipulating the biology inside the cell, the large size and charge of macromolecules prevent them from spontaneously crossing the cell membrane. For proteins, this problem limits the scope of diseases that are potentially addressable. For siRNA, this prevents access to the cellular machinery responsible for executing gene silencing in the first place. Thus, a safe and effective method to deliver proteins and siRNA into the cytoplasm is desired to fully enable their therapeutic potential. In this thesis, we describe the development of an intracellular delivery system based on a bacterial pore-forming protein, and demonstrate its efficacy in vitro using protein and siRNA payloads. Perfringolysin O (PFO) is a member of the cholesterol-dependent cytolysin (CDC) family of bacterial toxins whose pores, reaching up to 30nm in diameter, allow the passage of large molecules without a specialized transport mechanism. However, the creation of such pores on the cell membrane is accompanied by cytotoxicity, which limits the practical use of these proteins as a delivery tool. Thus, we developed a strategy to selectively activate PFO in endosomal compartments to minimize cytotoxicity. Specifically, we engineered a neutralizing binder against PFO on the fibronection scaffold. The binder was designed to have a higher affinity for PFO at neutral pH, inhibiting pore-formation on the cell membrane, and a lower affinity at acidic pH, promoting pore-formation on endosomal membranes. Fusing this binder to an antibody against EGFR allowed specific targeting and internalization. Using a protein payload-the ribosome-inactivating protein gelonin-administered in trans, we demonstrated that this strategy enables efficient delivery with high specificity and low toxicity, increasing the therapeutic window of PFO by orders of magnitude in vitro. One advantage of this delivery system is its modularity, as the payload administered in trans is readily swappable with other molecules of interest. Thus, we next demonstrated that the neutralized PFObased system can also be used for intracellular delivery of siRNA. For this application, we engineered a targeted siRNA carrier based on the dsRNA-binding protein p19 of the Carnation Italian Ringspot Virus (CIRV). In particular, we matured the affinity of p19 to create clones with some of the highest affinities for siRNA reported to date. Higher affinity correlated with higher potency, with the tightest-binding p19 mutant enabling silencing of a reporter gene with pM concentrations of siRNA in vitro. This increase in potency was partially due to increased uptake of siRNA. However, we also observed that the high-affinity clones enable stronger silencing even When each clone internalizes similar numbers of siRNA. This observation suggests that prolonging the association of siRNA and its carrier inside the cell may be a strategy for further improving the efficiency of silencing. Overall, the work described in this thesis demonstrates how neutralizing binders can be used to control the activity of potent membrane-disrupting agents, to deliver exogenous macromolecules into the cytoplasm with low toxicity. Further optimization of the neutralized PFO-based system for in vivo use will enhance its utility as a viable therapeutic strategy.