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Lipid nanoparticle siRNA treatment of Ebola-virus-Makona-infected nonhuman primates

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Abstract

The current outbreak of Ebola virus in West Africa is unprecedented, causing more cases and fatalities than all previous outbreaks combined, and has yet to be controlled1. Several post-exposure interventions have been employed under compassionate use to treat patients repatriated to Europe and the United States2. However, the in vivo efficacy of these interventions against the new outbreak strain of Ebola virus is unknown. Here we show that lipid-nanoparticle-encapsulated short interfering RNAs (siRNAs) rapidly adapted to target the Makona outbreak strain of Ebola virus are able to protect 100% of rhesus monkeys against lethal challenge when treatment was initiated at 3 days after exposure while animals were viraemic and clinically ill. Although all infected animals showed evidence of advanced disease including abnormal haematology, blood chemistry and coagulopathy, siRNA-treated animals had milder clinical features and fully recovered, while the untreated control animals succumbed to the disease. These results represent the first, to our knowledge, successful demonstration of therapeutic anti-Ebola virus efficacy against the new outbreak strain in nonhuman primates and highlight the rapid development of lipid-nanoparticle-delivered siRNA as a countermeasure against this highly lethal human disease.

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Figure 1: siEbola-3 is active against EBOV Makona target sequences.
Figure 2: siEbola-3 LNP treatment confers survival and reduces viral load.
Figure 3: EBOV Makona tissue pathology and antigen in NHPs untreated or treated with siEbola-3 LNP.

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Acknowledgements

We thank V. Borisevich for assistance with clinical pathology assays performed in the GNL BSL-4 laboratory. We also thank S. Klassen for his assistance with siRNA-LNP preparation. This study was supported by the Department of Health and Human Services, National Institutes of Health grant U19AI109711 to T.W.G. and I.M.

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Contributions

E.P.T. and J.Z.Z. designed the siRNA and did preparative work for dual luciferase reporter studies. E.P.T., N.M.S., J.Z.Z. and A.C.H.L. designed the dual luciferase reporter studies. J.Z.Z., T.R.B. and N.M.S. conducted the dual luciferase reporter studies and analysed the data. E.P.T., C.E.M., A.C.H.L., I.M. and T.W.G. conceived and designed the NHP study. C.E.M., J.B.G., D.J.D. and T.W.G. performed the NHP challenge and treatment experiments and conducted clinical observations of the animals. J.B.G., K.N.A. and D.J.D. performed the clinical pathology assays. J.B.G. performed the EBOV Makona infectivity assays. C.E.M. and K.N.A. performed the PCR assays. E.P.T., C.E.M., J.B.G., K.N.A., D.J.D., K.A.F., A.C.H.L., I.M. and T.W.G. analysed the data. K.A.F. performed histological and immunohistochemical analysis of the data. E.P.T., C.E.M., A.C.H.L. and T.W.G. wrote the paper. All authors had access to all of the data and approved the final version of the manuscript.

Corresponding author

Correspondence to Thomas W. Geisbert.

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Competing interests

A.C.H.L., I.M. and T.W.G. claim intellectual property regarding RNA interference for the treatment of filovirus infections. I.M. and T.W.G. are co-inventors on US Patent 7,838,658 ‘siRNA silencing of filovirus gene expression’ and A.C.H.L., I.M. and T.W.G. are co-inventors on US Patent 8,716,464 ‘Compositions and methods for silencing Ebola virus gene expression’.

Additional information

Opinions, interpretations, conclusions and recommendations are those of the authors and are not necessarily endorsed by the University of Texas Medical Branch.

Extended data figures and tables

Extended Data Figure 1 Antiviral activity of siEbola-3 in cells infected with EBOV Makona.

For comparison, siEbola-3 activity was also assessed against the central African EBOV Kikwit strain and siEbola-2 activity was evaluated against both EBOV strains. Data are viral RNA copies per milliltre of each sample normalized to untreated infected cells. Results are mean ± s.e.m. from one biological replicate, conducted in technical triplicate.

Extended Data Figure 2 siEbola-3 LNP treatment provides partial protection against EBOV Makona clinical pathologies, and infection with EBOV Makona infection induces a lesser degree of liver dysfunction compared to EBOV Kikwit infection.

a, No differences in viraemia levels were observed in untreated animals infected with EBOV Makona or Kikwit. be, Liver dysfunction markers. Normal values for uninfected NHPs ranges are GGT (40–115 U l−1), AST (20–45 U l−1), ALT (20–165 U l−1), ALP (130–500 U l−1). f, g, Protection against EBOV-Makona-induced CRE and BUN elevation was observed. Normal values for uninfected NHPs range from BUN (10–25 mg dl−1) and CRE (0.8–1.2 mg dl−1).

Extended Data Figure 3 Comparison of coagulation and haematology characteristics between untreated control animals infected with EBOV Makona or Kikwit.

a, b, Coagulopathies are not as marked in EBOV Makona infection when compared to historical EBOV Kikwit data. c, Lymphopenia is observed in all infected animals. d, Thrombocytopenia levels are similar between EBOV-Makona and EBOV-Kikwit-infected control animals.

Extended Data Table 1 Comparison of clinical signs progression between untreated rhesus macaques infected with EBOV Makona or EBOV Kikwit

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Thi, E., Mire, C., Lee, A. et al. Lipid nanoparticle siRNA treatment of Ebola-virus-Makona-infected nonhuman primates. Nature 521, 362–365 (2015). https://doi.org/10.1038/nature14442

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