1. ¹Research, Sirna Therapeutics Inc, Boulder, CO
2. ²Research, Protiva Biotherapeutics Inc, Burnaby, BC, Canada

^|[ast]|Shareholder of Protiva.

Abstract

RNA interference (RNAi) represents a powerful, naturally occurring biological strategy for inhibition of gene expression that has demonstrated utility in the inhibition of viral replication. However, the challenges associated with the effective in vivo delivery of siRNAs has been a major obstacle to their use. Here we describe the development of an effective siRNA based therapy for hepatitis virus infection.

siRNAs targeted to conserved sites in HBV RNA were designed incorporating specific chemical modifications designed to significantly prolong their stability in human serum. Cell culture studies revealed a high degree of gene silencing following treatment with modified siRNAs. To assess activity of the stabilized siRNAs in vivo, an HBV model was employed in which the siRNA and the HBV vector were co-delivered via hydrodynamic injection in mice. Serum HBV DNA, HBsAg and liver HBV RNA were reduced by more than 3 log|[circ]|10 compared to siRNA and saline controls. The observed decrease in serum HBV DNA was 1.5 log10 greater with a stabilized siRNA than unmodified siRNA. A more rigorous test of this approach utilized siRNA formulated in Stable Nucleic Acid Particles (SNALP). SNALP consist of siRNA fully encapsulated in a lipid bilayer containing a diffusible polyethylene glycol (PEG)-lipid conjugate. The PEG-lipid conjugates in the SNALP particle play an essential role during the formulation process, stabilizing the nascent particle and preventing aggregation in the vial. In the blood, the PEG-lipid shields the positive surface charge, preventing rapid clearance following intravenous injection. Following administration the PEG conjugate dissociates from the SNALP, revealing the positive charge and an increasingly fusogenic lipid bilayer, transforming the particle into a transfection-competent entity. HBV is known to replicate in the tissues of the reticuloendothelial system, specifically in hepatocytes of the liver. Pharmacokinetics and biodistribution studies utilizing radiolabelled SNALP designed for hepatocyte uptake in mice demonstrated accumulation of more than 45 percent of the injected dose in the liver 24 hours after intravenous administration. SNALP containing anti-HBV siRNA were evaluated by standard systemic intravenous administration commencing six days after administration of the HBV vector, once stable viral titres had been achieved. SNALP result in a 1-1.5 log reduction of HBV serum titers following three daily injections at 3 mg/kg. Further decreases in HBV serum titers are observed with subsequent weekly dosing, and treatment with the siRNAs in combination with lamuvidine yield additive antiviral effects. These experiments establish the strong potential impact that siRNAs could have on the extent of HBV infection, and underscore the importance of stabilization of siRNA against nuclease degradation. Further development and optimization of this technology has the potential to yield effective treatments against hepatitis virus infections.