Cytosolic delivery of siRNA by ultra-high affinity dsRNA binding proteins.

Nicole J Yang,Lucy F Yang,Robert S Langer,Daniel G Anderson,Michael W Traxlmayr,Yao Yu,Fangdi Sun,Yingda Xu,K Dane Wittrup,Katie F Maass,Monique J Kauke

doi:10.1093/nar/gkx546

Abstract

Protein-based methods of siRNA delivery are capable of uniquely specific targeting, but are limited by technical challenges such as low potency or poor biophysical properties. Here, we engineered a series of ultra-high affinity siRNA binders based on the viral protein p19 and developed them into siRNA carriers targeted to the epidermal growth factor receptor (EGFR). Combined in trans with a previously described endosome-disrupting agent composed of the pore-forming protein Perfringolysin O (PFO), potent silencing was achieved in vitro with no detectable cytotoxicity. Despite concerns that excessively strong siRNA binding could prevent the discharge of siRNA from its carrier, higher affinity continually led to stronger silencing. We found that this improvement was due to both increased uptake of siRNA into the cell and improved pharmacodynamics inside the cell. Mathematical modeling predicted the existence of an affinity optimum that maximizes silencing, after which siRNA sequestration decreases potency. Our study characterizing the affinity dependence of silencing suggests that siRNA-carrier affinity can significantly affect the intracellular fate of siRNA and may serve as a handle for improving the efficiency of delivery. The two-agent delivery system presented here possesses notable biophysical properties and potency, and provide a platform for the cytosolic delivery of nucleic acids.

Highlights

Protein-based systems for delivering siRNA can potentially circumvent some of the challenges facing nanoparticlebased systems, such as accumulation in the liver [1,2]
While the non-cationic and non-particulate nature of this delivery system suggested that desirable pharmacokinetics and biodistribution properties could be achieved in vivo, we observed that siRNA dissociated rapidly from the double-stranded RNA binding domain (dsRBD) during circulation, negating the targeting functionalities of the carrier protein
Removing the targeting moiety from the p19 carriers decreased the silencing efficacy up to 1000-fold (Figure 3D), demonstrating that epidermal growth factor receptor (EGFR)-mediated internalization was essential for high potency. This result was expected, as we have previously shown that C225.2/PFOT490A,L491V predominantly permeablizes endosomal membranes following EGFR-mediated internalization [17]

Summary

Introduction

Protein-based systems for delivering siRNA can potentially circumvent some of the challenges facing nanoparticlebased systems, such as accumulation in the liver [1,2]. There exists multiple examples of protein-based delivery methods [3,4,5,6,7], they generally suffer from low potencies, complex preparation strategies, or poor pharmacokinetics and biodistribution properties stemming from high positive charge [8,9]. We reported a multi-agent delivery system that employed the dsRBD of PKR as a siRNA carrier. Administered together with an endosome-disrupting agent based on the pore-forming protein Perfringolysin O (PFO), and a receptor-clustering antibody that enhances siRNA uptake, efficient silencing was achieved in vitro [11]. While the non-cationic and non-particulate nature of this delivery system suggested that desirable pharmacokinetics and biodistribution properties could be achieved in vivo, we observed that siRNA dissociated rapidly from the dsRBD during circulation, negating the targeting functionalities of the carrier protein

Methods

Results

Conclusion