Postexposure protection of guinea pigs against a lethal ebola virus challenge is conferred by RNA interference

Abstract

Background: Ebola virus (EBOV) infection causes a frequently fatal hemorrhagic fever (HF) that is refractory to treatment with currently available antiviral therapeutics. RNA interference represents a powerful, naturally occurring biological strategy for the inhibition of gene expression and has demonstrated utility in the inhibition of viral replication. Here, we describe the development of a potential therapy for EBOV infection that is based on small interfering RNAs (siRNAs).

Methods: Four siRNAs targeting the polymerase (L) gene of the Zaire species of EBOV (ZEBOV) were either complexed with polyethylenimine (PEI) or formulated in stable nucleic acid-lipid particles (SNALPs). Guinea pigs were treated with these siRNAs either before or after lethal ZEBOV challenge.

Results: Treatment of guinea pigs with a pool of the L gene-specific siRNAs delivered by PEI polyplexes reduced plasma viremia levels and partially protected the animals from death when administered shortly before the ZEBOV challenge. Evaluation of the same pool of siRNAs delivered using SNALPs proved that this system was more efficacious, as it completely protected guinea pigs against viremia and death when administered shortly after the ZEBOV challenge. Additional experiments showed that 1 of the 4 siRNAs alone could completely protect guinea pigs from a lethal ZEBOV challenge.

Conclusions: Further development of this technology has the potential to yield effective treatments for EBOV HF as well as for diseases caused by other agents that are considered to be biological threats.

Figures

Figure 1

Inhibition of the replication of the Zaire species of Ebola virus (ZEBOV) in Vero cells by a pool of 4 different small interfering RNAs (siRNAs) targeting individual regions of the ZEBOV polymerase (L) gene. Cells were transfected with either the siRNA pool or an equivalent dose of EbL-Scram1, an irrelevant scrambled sequence (SCR), as a control. At various time points after transfection (0, 24, and 48 h), the transfected cells were infected with ZEBOV, and cells and culture fluids were harvested 24 h later, for determination of the level of infectious virus by plaque assay

Figure 2

Inhibition of the replication of the Zaire species of Ebola virus (ZEBOV) in Vero cells, as demonstrated by immunofluorescence staining. Cells were transfected with either a pool of 4 different small interfering RNAs (siRNAs) targeting individual regions of the ZEBOV polymerase (L) gene or an equivalent dose of EbL-Scram1, an irrelevant scrambled sequence (SCR), as a control. Cells were counterstained with 4′,6′-diamidino-2-phenylindole and Evans blue, to aid visualization. ZEBOV-positive cells are identified by green fluorescence

Figure 3

Plasma viremia levels of inbred strain 13 guinea pigs 4 days after challenge with the Zaire species of Ebola virus (ZEBOV). Guinea pigs were treated 3 h before the ZEBOV challenge and 1, 2, and 4 days afterward with either a pool of 4 different small interfering RNAs (siRNAs) targeting individual regions of the ZEBOV polymerase (L) gene (n=5) or an equivalent dose of EbL-Scram1, an irrelevant scrambled sequence (SCR) (n=5). Data are means±SDs

Figure 4

In vivo clearance and biodistribution of stable nucleic acid–lipid particles (SNALPs). Shown are plasma clearance (A) and biodistribution (B and C) of [3H]cholesteryl oleyl ether–labeled SNALPs. Each guinea pig received a single intravenous injection of 0.75 mg/kg small interfering RNAs (siRNA) formulated as SNALPs. Biodistribution data were collected 24 h after injection. Data are means±SDs (for all tissues, n=5 guinea pigs, except for testes [n=3] and ovaries [n=2])

Figure 5

Antiviral efficacy of a pool of 4 different small interfering RNAs (siRNAs) targeting individual regions of the Zaire species of Ebola virus (ZEBOV) polymerase (L) gene and encapsulated in stable nucleic acid–lipid particles (SNALPs). Shown are plasma viremia levels (A) and survival rates (B) for inbred strain 13 guinea pigs after ZEBOV challenge. One hour after challenge and daily on days 1–6 thereafter, guinea pigs were treated, via the SNALP delivery system, with the siRNA pool (1.0 mg/kg) or an equivalent dose of EbL-Scram1, an irrelevant scrambled sequence (SCR). Plasma viremia levels were determined on day 7. Viremia data are means±SDs (n=5)

Figure 6

Antiviral efficacy of small interfering RNAs (siRNAs) targeting individual regions of the Zaire species of Ebola virus (ZEBOV) polymerase (L) gene and encapsulated in stable nucleic acid–lipid particles (SNALPs). Shown are plasma viremia levels (A) and survival rates (B) for inbred strain 13 guinea pigs after ZEBOV challenge. One hour after the challenge and daily on days 1–6 thereafter, guinea pigs were treated, via the SNALP delivery system, with a pool of 4 siRNA (0.75 mg/kg); an equivalent dose of EbL-Scram1, an irrelevant scrambled sequence (SCR); or 1 of the 4 siRNAs alone (EK1–EK4). Plasma viremia levels were determined on day 7. Viremia data are means±SDs (n=5). The P value is for the comparison between EK2 and SCR

Figure 7

Small interfering RNA (siRNA)–mediated cytokine induction in mice. Shown are serum interferon (IFN)–α (A) and IFN-β (B) levels 6 h after intravenous administration of 100 μg (∼5 mg/kg) of stable nucleic acid–lipid particles encapsulating either a pool of 4 different siRNAs that targeted individual regions of the Zaire species of Ebola virus polymerase (L) gene; an equivalent dose of EbL-Scram1, an irrelevant scrambled sequence (SCR); or 1 of the 4 siRNAs alone (EK1–EK4). Injection of PBS alone induced no detectable IFN-α or IFN-β. Note that injection of empty liposomes or naked siRNA alone also failed to induce detectable IFN-α or IFN-β (data not shown). Data are means±SDs (n=4)