996. Development of an siRNA Based Therapy for Ebola Virus Infection

Abstract

Listen

Translate article

Ebola virus infection causes a severe and frequently fatal hemorrhagic fever that is refractory to treatment with currently available antiviral therapeutics. Ebola and the related Marburg virus are of concern as potential bioweapon (BW) threats since they have the potential for aerosol dissemination and weaponization. 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 siRNA based therapy for Ebola virus (EBOV) infection. Multiple siRNAs were designed targeting the viral polymerase (L) gene. When used either individually or in combination, siRNA inhibited EBOV replication in vitro in Vero and Vero E6 cells. A 60-99% reduction in production of infectious EBOV and 75-100% reduction in numbers of cells expressing EBOV protein was observed. Promising siRNA candidates demonstrating significant inhibition of viral replication in vitro were selected for formulation 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. EBOV is known to replicate in the tissues of the reticuloendothelial system, specifically in Kupffer cells of liver, and the monocytes, macrophages and dendritic cells of the peripheral blood and lymphoid tissues. Pharmacokinetics and biodistribution studies utilizing radiolabelled SNALP in mice demonstrated accumulation of more than 30 percent of the injected dose in the liver and 10 percent in the spleen 24 hours after intravenous administration. Fluorescent microscopy of tissue sections showed that SNALP containing Cy3 labelled siRNA were concentrated in the Kupffer cells and resident macrophages of the liver. Promising formulations were selected for evaluation in a guinea pig model of EBOV hemorrhagic fever. The ability of SNALP delivered siRNA to inhibit EVOV plasma viremia is compared to that of PEI polyplex. Further development and optimization of this technology has the potential to yield effective treatments against EBOV hemorrhagic fever (HF), Marburg virus (MARV) HF and other BW threat agents.