Development of Locked Nucleic Acid Antisense Oligonucleotides Targeting Ebola Viral Proteins and Host Factor Niemann-Pick C1

Abstract

The Ebola virus is a zoonotic pathogen that can cause severe hemorrhagic fever in humans, with up to 90% lethality. The deadly 2014 Ebola outbreak quickly made an unprecedented impact on human lives. While several vaccines and therapeutics are under development, current approaches contain several limitations, such as virus mutational escape, need for formulation or refrigeration, poor scalability, long lead-time, and high cost. To address these challenges, we developed locked nucleic acid (LNA)-modified antisense oligonucleotides (ASOs) to target critical Ebola viral proteins and the human intracellular host protein Niemann-Pick C1 (NPC1), required for viral entry into infected cells. We generated noninfectious viral luciferase reporter assays to identify LNA ASOs that inhibit translation of Ebola viral proteins in vitro and in human cells. We demonstrated specific inhibition of key Ebola genes VP24 and nucleoprotein, which inhibit a proper immune response and promote Ebola virus replication, respectively. We also identified LNA ASOs targeting human host factor NPC1 and demonstrated reduced infection by chimeric vesicular stomatitis virus harboring the Ebola glycoprotein, which directly binds to NPC1 for viral infection. These results support further in vivo testing of LNA ASOs in infectious Ebola virus disease animal models as potential therapeutic modalities for treatment of Ebola.

Keywords: Ebola; Ebola virus (EBOV); Niemann-Pick C1 (NPC1); antisense oligonucleotides; locked nucleic acids.

FIG. 1.

pATG and ORFs of Ebola proteins used to design Ebola luciferase reporters for cellular studies. Ninety-eight base pairs of ORF sequence that was in frame with the pATG was fused with the luciferase gene in all reporters. In other words, 98 bp encompassing the start codons in the ORF of NP, VP24, VP35, and L were fused to luciferase. The pATG (the start site of the main ORF of the Ebola protein) is used as the main start site to express the in frame Luciferase-Ebola ORF fusion reporters. The uATGs and Luciferase ATG are either nonfunctional or mutated in the reporters. The pATG used in the reporters, their locations in the reporters, and LNA ASO target sites are highlighted in this figure. Regions encompassing the start codons in the ORF of (a) L, (b) VP24, (c) VP35, and (d) NP were fused to luciferase (98 bp of 5′UTR—luciferase). The start codon (ATG) of luciferase was mutated to CTG (alanine). Reporter expression was under the control of the CMV promoter. (e) LNA ASOs targeting Ebola proteins reduce expression of Ebola reporters in vitro and in HeLa cells. Schematic of LNA ASOs designed to target translation of Ebola fusion proteins: NP, VP24, VP35, and L. (f) Two micromolar of each LNA ASOs tested inhibited the translation of Ebola fusion proteins in vitro. (g) Two LNA ASOs targeting Ebola VP24 protein reduce expression of the Ebola VP24-luciferase fusion in HeLa cells, as determined by measurement of luminescence 24 h post-transfection. LNA30 targeting Ebola VP35 protein and LNA38 targeting Ebola L protein were used as negative controls. Neither demonstrated inhibition of Ebola VP24 protein, highlighting the specificity of LNA ASOs. IC50 values were calculated for LNAs tested in HeLa cells and are provided in Supplementary Table S2. (h) Two LNA ASOs targeting Ebola NP protein reduce expression of Ebola NP-luciferase fusion in HeLa cells, as determined by measurement of luminescence 24 h post-transfection. LNA30 targeting Ebola VP25 protein and LNA38 targeting Ebola L protein were used as negative controls. Neither demonstrated inhibition of Ebola NP protein, indicating specificity of LNA ASOs. *P < 0.05, **P < 0.01, ***P < 0.001. All experiments were performed in biological triplicates; each biological replicate was performed in technical triplicates. pATG, proximal ATG; ORF, open reading frame; NP, nucleoprotein; uATG, upstream ATG; LNA, locked nucleic acid; ASO, antisense oligonucleotide.

FIG. 2.

Titration of optimal concentration of LNA ASOs. A dose-dependent titration of LNA ASOs determined optimal concentration for specific and significant reduction of target Ebola genes. Individual coupled in vitro transcription/translation assays with rabbit reticulocyte lysate were performed at varying LNA ASO concentrations and fixed Ebola luciferase reporter concentrations (90 nM). Ebola luciferase reporter expression was measured and analyzed for each LNA ASO tested. (a) LNA28 targeting Ebola NP fusion protein dose dependently reduces expression of Ebola NP-luciferase fusion protein in vitro. (b) LNA29 targeting Ebola NP fusion protein dose dependently reduces expression of Ebola NP-luciferase fusion protein in vitro. (c) LNA30 targeting Ebola VP35 fusion protein dose dependently reduces expression of Ebola VP35-luciferase fusion protein in vitro. (d) LNA31 targeting Ebola VP35 fusion protein dose dependently reduces expression of Ebola VP35-luciferase fusion protein in vitro. Data were measured for LNA37, LNA36, and LNA38 (not reported); >50% knockdown of target reporters/fusion proteins was not observed until >2 μM of LNA ASO. IC50 values were calculated and are reported with graphs in Supplementary Fig. S4. *P < 0.05, **P < 0.01, ***P < 0.001.

FIG. 3.

LNA ASOs targeting NPC1 reduce expression of NPC1 mRNA in HeLa cells. (a) Six LNA ASOs targeting NPC1 were transfected into HeLa cells and RNA harvested 48 h later to measure mRNA levels. The negative control LNA ASO, siRNAs targeting NPC1, and nontargeting siRNAs were included as controls. LNA20, LNA21, and LNA22 demonstrated greatest reduction in NPC1 mRNA levels. (b) LNA ASOs targeting NPC1 reduce NPC1 protein levels in HeLa cells. One hundred nanomolar of LNA ASOs targeting NPC1 (LNA20, 21, and 22) were transfected into HeLa cells, and protein harvested 48 h later and NPC1 levels measured by immunoblotting. Tubulin was used as loading control. (c) LNA20 targeting NPC1 reduces expression of NPC1 mRNA in mouse macrophages. NPC1 siRNA was used as positive control. The negative control LNA24 and nontargeting siRNA were used as negative controls. (d) LNA20 targeting Npc1 reduces Npc1 protein levels in mouse macrophages as assessed by immunoblotting. β-Tubulin was used as loading control. Npc1 siRNA was used as positive control for Npc1 knockdown. Negative control LNA ASO and nontargeting siRNA controls did not reduce Npc1 levels. ***P < 0.001. Experiments were performed in biological triplicates; each biological replicate was performed in technical triplicates. NPC1, Niemann-Pick C1.

FIG. 4.

VSVluc-EboV GP infection is reduced in the presence of LNA ASOs that inhibit NPC1 expression. NPC1 LNA20 and the negative control LNA24 were transfected into HeLa cells, which were then infected with a VSV vector encoding Ebola-GP (VSVluc-EboV GP) after 48 h. VSVluc-EboV GP luciferase levels were measured after 48 h. VSVluc-EboV GP luciferase levels decreased significantly in a dose-dependent manner after treatment with 25, 50, and 100 nM of NPC1-targeting LNA20. LNA20 data are presented relative to LNA24 (control LNA). NPC1 siRNA used as positive control for NPC1 knockdown also showed significant decrease in VSVluc-EboV GP luciferase levels. Negative control LNA ASO and nontargeting siRNA controls did not reduce VSVluc-EboV GP luciferase levels. IC50 for LNA20 was calculated and provided in Supplementary Table S2. **P < 0.01, ***P < 0.001. Experiments were performed in three biological replicates (triplicate). GP, glycoprotein; VSV, vesicular stomatitis virus.