The CDC and NIAID have classified the filoviruses, Ebola virus (EBOV) and Marburg virus (MARV), as Category A priority pathogens. These viruses cause severe and often fatal hemorrhagic fever (HF) in humans and nonhuman primates with case fatality rates as high as 90% for some species or strains such as Zaire ebolavirus (ZEBOV). There are presently no approved active or passive therapeutic modalities for EBOV or MARV infections resulting from a natural outbreak, laboratory accident, or deliberate misuse. Recently, we demonstrated the first complete postexposure protection of nonhuman primates against a lethal ZEBOV challenge using a combination of small interfering RNA (siRNA) targeting the ZEBOV L polymerase, viral protein (VP) 24, and VP35 formulated in stable nucleic acid-lipid particles (SNALP). The goal of this proposal is to develop siRNA-based therapies for treating EBOV and MARV infections. Specifically, we will: (1) Identify candidate siRNAs targeting Ebola and Marburg virus genes and evaluate antiviral activity in vitro. siRNA will be designed using sequences for EBOV and MARV. When possible, conserved siRNAs between virus species or strains will be chosen based on multiple sequence alignments. Lead candidate siRNAs will be identified by testing their ability to inhibit replication of infectious EBOV and MARV in Vero cells and also by using a nonviral plasmid-based expression system. (2) Evaluate the immune stimulatory activity of modified siRNAs targeting Ebola and Marburg virus genes in mice. Candidate siRNAs that have sufficient antiviral activity against filoviruses will be modified by substituting 2'-O-methyl (2'OMe) guanosine and/or uridines in the sense and antisense strands to eliminate the immune stimulatory capacity of the siRNA. These modified siRNAs will be tested for their ability to inhibit replication of infectious EBOV and MARV in Vero cells and administered to mice to demonstrate that they do not activate a nonspecific immunostimulatory response. (3) Determine the efficacy of non-immunostimulatory siRNAs against Ebola and Marburg viruses in guinea pigs. Promising modified siRNA candidates that demonstrate significant inhibition of filoviral replication in vitro and do not activate an immunostimulatory response in mice will be manufactured at a scale sufficient to support animal studies. siRNAs delivered by SNALP will be administered to guinea pigs i.v. by retroorbital injection shortly after filoviral challenge to determine protective efficacy. (4) Determine the efficacy of non-immunostimulatory siRNAs against Ebola and Marburg viruses in nonhuman primates. Modified siRNA candidates that show complete protection of guinea pigs against either SEBOV, ZEBOV, or against each of four strains of MARV (MARV-Angola, MARV-Ci67, MARV-Musoke, and MARV-Ravn) will be evaluated in rhesus monkeys to determine the protective efficacy against each of these viruses when administered at various times after filovirus challenge.
The filoviruses, Ebola virus and Marburg virus, are enigmatic emerging pathogens that cause severe hemorrhagic fever in humans and nonhuman primates with case fatality rates ranging from 23-90 percent. The filoviruses are of importance to public health as there are no licensed vaccines or treatments available for use in humans and because of the concern that filoviruses could be used as agents of biological terrorism. The siRNA-based treatment strategy proposed here may well represent an effective countermeasure for treating filovirus infections.
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|Shabman, Reed S; Jabado, Omar J; Mire, Chad E et al. (2014) Deep sequencing identifies noncanonical editing of Ebola and Marburg virus RNAs in infected cells. MBio 5:e02011|
|Thi, Emily P; Mire, Chad E; Ursic-Bedoya, Raul et al. (2014) Marburg virus infection in nonhuman primates: Therapeutic treatment by lipid-encapsulated siRNA. Sci Transl Med 6:250ra116|