Postexposure protection of non-human primates against a lethal Ebola virus challenge with RNA interference: a proof-of-concept study

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Summary

Background

We previously showed that small interfering RNAs (siRNAs) targeting the Zaire Ebola virus (ZEBOV) RNA polymerase L protein formulated in stable nucleic acid-lipid particles (SNALPs) completely protected guineapigs when administered shortly after a lethal ZEBOV challenge. Although rodent models of ZEBOV infection are useful for screening prospective countermeasures, they are frequently not useful for prediction of efficacy in the more stringent non-human primate models. We therefore assessed the efficacy of modified non-immunostimulatory siRNAs in a uniformly lethal non-human primate model of ZEBOV haemorrhagic fever.

Methods

A combination of modified siRNAs targeting the ZEBOV L polymerase (EK-1 mod), viral protein (VP) 24 (VP24-1160 mod), and VP35 (VP35-855 mod) were formulated in SNALPs. A group of macaques (n=3) was given these pooled anti-ZEBOV siRNAs (2 mg/kg per dose, bolus intravenous infusion) after 30 min, and on days 1, 3, and 5 after challenge with ZEBOV. A second group of macaques (n=4) was given the pooled anti-ZEBOV siRNAs after 30 min, and on days 1, 2, 3, 4, 5, and 6 after challenge with ZEBOV.

Findings

Two (66%) of three rhesus monkeys given four postexposure treatments of the pooled anti-ZEBOV siRNAs were protected from lethal ZEBOV infection, whereas all macaques given seven postexposure treatments were protected. The treatment regimen in the second study was well tolerated with minor changes in liver enzymes that might have been related to viral infection.

Interpretation

This complete postexposure protection against ZEBOV in non-human primates provides a model for the treatment of ZEBOV-induced haemorrhagic fever. These data show the potential of RNA interference as an effective postexposure treatment strategy for people infected with Ebola virus, and suggest that this strategy might also be useful for treatment of other emerging viral infections.

Funding

Defense Threat Reduction Agency.

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Most cited references 22

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Sequence-dependent stimulation of the mammalian innate immune response by synthetic siRNA.

Adam Judge, Vandana Sood, Janet Shaw … (2005)

Short interfering RNAs (siRNAs) that mediate specific gene silencing through RNA interference (RNAi) are widely used to study gene function and are also being developed for therapeutic applications. Many nucleic acids, including double- (dsRNA) and single-stranded RNA (ssRNA), can stimulate innate cytokine responses in mammals. Despite this, few studies have questioned whether siRNA may have a similar effect on the immune system. This could significantly influence the in vivo application of siRNA owing to off-target effects and toxicities associated with immune stimulation. Here we report that synthetic siRNAs formulated in nonviral delivery vehicles can be potent inducers of interferons and inflammatory cytokines both in vivo in mice and in vitro in human blood. The immunostimulatory activity of formulated siRNAs and the associated toxicities are dependent on the nucleotide sequence. We have identified putative immunostimulatory motifs that have allowed the design of siRNAs that can mediate RNAi but induce minimal immune activation.

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Design of noninflammatory synthetic siRNA mediating potent gene silencing in vivo.

Ian MacLachlan, Gurneet Bola, D Judge … (2006)

Targeted silencing of disease-associated genes by synthetic short interfering RNA (siRNA) holds considerable promise as a novel therapeutic strategy. However, unmodified siRNA can be potent triggers of the innate immune response, particularly when associated with delivery vehicles that facilitate intracellular uptake. This represents a significant barrier to the therapeutic development of siRNA due to toxicity and off-target gene effects associated with this inflammatory response. Here we show that immune stimulation by synthetic siRNA can be completely abrogated by selective incorporation of 2'-O-methyl (2'OMe) uridine or guanosine nucleosides into one strand of the siRNA duplex. These noninflammatory siRNA, containing less than 20% modified nucleotides, can be readily generated without disrupting their gene-silencing activity. We show that, coupled with an effective systemic delivery vehicle, 2'OMe-modified siRNA targeting apolipoprotein B (apoB) can mediate potent silencing of its target mRNA, causing significant decreases in serum apoB and cholesterol. This is achieved at therapeutically viable siRNA doses without cytokine induction, toxicity, or off-target effects associated with the use of unmodified siRNA. This approach to siRNA design and delivery should prove widely applicable and represents an important step in advancing synthetic siRNA into a broad range of therapeutic areas.

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Cationic lipid saturation influences intracellular delivery of encapsulated nucleic acids.

Melanie Palmer, Ian MacLachlan, K Bremner … (2005)

An analogous series of cationic lipids (1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane (DODMA), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA) and 1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane (DLenDMA)) possessing 0, 1, 2 or 3 double bonds per alkyl chain respectively, was synthesized to determine the correlation between lipid saturation, fusogenicity and efficiency of intracellular nucleic acid delivery. 31P-NMR analysis suggests that as saturation increases, from 2 to 0 double bonds, lamellar (L(alpha)) to reversed hexagonal (H(II)) phase transition temperature increases, indicating decreasing fusogenicity. This trend is largely reflected by the efficiency of gene silencing observed in vitro when the lipids are formulated as Stable Nucleic Acid Lipid Particles (SNALPs) encapsulating small inhibitory RNA (siRNA). Uptake experiments suggest that despite their lower gene silencing efficiency, the less fusogenic particles are more readily internalized by cells. Microscopic visualization of fluorescently labelled siRNA uptake was supported by quantitative data acquired using radiolabelled preparations. Since electrostatic binding is a precursor to uptake, the pKa of each cationic lipid was determined. The results support a transfection model in which endosomal release, mediated by fusion with the endosomal membrane, results in cytoplasmic translocation of the nucleic acid payload.

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Author and article information

Contributors

Thomas W Geisbert

Journal

Journal ID (nlm-ta): Lancet

Journal ID (iso-abbrev): Lancet

Title: Lancet (London, England)

Publisher: Elsevier Ltd.

ISSN (Print): 0140-6736

ISSN (Electronic): 1474-547X

Publication date PMC-release: 28 May 2010

Publication date (Print): 29 May-4 June 2010

Publication date (Electronic): 28 May 2010

Volume: 375

Issue: 9729

Pages: 1896-1905

Affiliations

[a ]National Emerging Infectious Diseases Laboratories Institute, Boston University School of Medicine, Boston, MA, USA

[b ]Department of Microbiology, Boston University School of Medicine, Boston, MA, USA

[c ]Department of Medicine, Boston University School of Medicine, Boston, MA, USA

[d ]Virology Division, US Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD, USA

[e ]Tekmira Pharmaceuticals, Burnaby, BC, Canada

Author notes

[* ]Correspondence to: Prof Thomas W Geisbert, Boston University School of Medicine, National Emerging Infectious Diseases Laboratories Institute, 620 Albany Street, Room 401B, Boston, MA 02118, USA geisbert@ 123456bu.edu

[‡]

Contributed equally to the report

Article

Publisher ID: S0140-6736(10)60357-1

DOI: 10.1016/S0140-6736(10)60357-1

PMC ID: 7138079

PubMed ID: 20511019

SO-VID: d836ba93-e790-420c-ab19-a6b7432b83fb

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Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.

Postexposure protection of non-human primates against a lethal Ebola virus challenge with RNA interference: a proof-of-concept study

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Interpretation

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Most cited references 22

Sequence-dependent stimulation of the mammalian innate immune response by synthetic siRNA.

Design of noninflammatory synthetic siRNA mediating potent gene silencing in vivo.

Cationic lipid saturation influences intracellular delivery of encapsulated nucleic acids.

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