Anti-Niemann Pick C1 Single-Stranded Oligonucleotides with Locked Nucleic Acids Potently Reduce Ebola Virus Infection In Vitro

Abstract

Ebola virus is the causative agent of Ebola virus disease, a severe, often fatal illness in humans. So far, there are no US Food and Drug Administration (FDA)-approved therapeutics directed against Ebola virus. Here, we selected the host factor Niemann-Pick C1 (NPC1), which has been shown to be essential for Ebola virus entry into host cytoplasm, as a therapeutic target for suppression by locked nucleic acid-modified antisense oligonucleotides. Screening of antisense oligonucleotides in human and murine cell lines led to identification of candidates with up to 94% knockdown efficiency and 50% inhibitory concentration (IC₅₀) values in the submicromolar range. Selected candidate oligonucleotides led to efficient NPC1 protein knockdown in vitro without alteration of cell viability. Furthermore, they did not have immune stimulatory activity in cell-based assays. Treatment of Ebola-virus-infected HeLa cells with the most promising candidates resulted in significant (>99%) virus titer reduction, indicating that antisense oligonucleotides against NPC1 are a promising therapeutic approach for treatment of Ebola virus infection.

Keywords: Ebola virus; LNA; NPC1; antisense oligonucleotide.

Figure 1

ASO Distribution on Human NPC1 mRNA All ASOs are depicted according to their location on the human NPC1 mRNA along the x axis. Distinct exons (red) and UTRs (green) are shown in the bottom part of the figure. The lengths of the ASOs are indicated on the y axis.

Figure 2

Screening of NPC1-Specific LNA-ASOs in Human and Murine Cell Lines (A and B) Human HeLa and THP1 cells (A) as well as murine cell line 4T1 (B) were treated with 10 μM of ASOs. After 3 days cell lysates were used to determine NPC1 and HPRT1 mRNA levels. (A) Shown is the correlation of residual NPC1 mRNA expression (means and SD of triplicate wells) after treatment in HeLa (x axis) versus THP1 cells (y axis). Values were normalized to HPRT1 and relative to the untreated control (set as 1; empty square). ASO 05HM is indicated as a filled diamond, ASO 28H as a filled triangle, Neg1 as an empty circle, and other NPC1-specific ASOs as filled circles. (B) Residual Npc1 mRNA expression in 4T1 cells after treatment with respective NPC1-specific ASOs or negative control Neg1. Values were normalized to Hprt1 and are shown relative to the untreated control (set as 1). Error bars show SD (triplicate wells). The number of mismatches to murine Npc1 sequence of human specific ASOs (H) are shown in different colors and patterns.

Figure 3

IC₅₀ Determination and Protein Knockdown Efficacy of ASOs 05HM and 28H (A–D) HeLa and 4T1 cells were used to generate dose-response curves by treating them with different concentrations of ASO 05HM (A and C) and 28H (B), respectively. On day 3, cell viability was determined using a CellTiter Blue Assay Kit (D). Then, cells were lysed and NPC1 and HPRT1 mRNA levels were determined. Values were normalized to the housekeeping gene HPRT1 and are shown relative to untreated cells (set as 100). IC₅₀ values were calculated using Prism 6 (GraphPad Software). Data are means and SD (triplicate wells). (E) HeLa cells were treated twice for 3 days with 10 μM ASO 05HM, 28H, negative control oligonucleotide Neg1 or were left untreated. On day 6, cells were lysed, and lysates were analyzed for NPC1 and Actin protein expression using SDS-PAGE and immunoblot analysis in duplicate wells.

Figure 4

NPC1-Specific ASOs 05HM and 28H Did Not Activate TLR9 Signaling (A) HEK-Blue hTLR9 cells were treated with ODN2006 or LNA-ASOs 05HM and 28H, respectively, with the indicated concentrations. After 20 h SEAP reporter activity was measured at 620 nm, using a microplate reader. (B) HEK cells expressing a mouse Tlr9 Nf-кb luciferase reporter plasmid were treated with the indicated concentrations of ODN1668 or LNA-ASO 05HM. After 20 h, the cells were treated with ONE-Glo EX reagent, and luminescence was measured at 560 nm. Values were normalized to untreated cells and are means with SD (triplicate wells).

Figure 5

NPC1-Specific ASO 05HM Did Not Stimulate Cytokine Release from Treated PBMCs (A–C) PBMCs from three different donors were treated with oligonucleotides and immune stimulatory agents (each condition in triplicate wells), with the indicated concentrations. The third day after treatment, supernatants were harvested and used for determination of cytokine release using ELISA: (A) IFNγ, (B) IL6, and (C) TNFα. Values were displayed as fold changes compared to untreated. Means and SDs were also indicated. ANOVA test was used to test for significant differences and p values were determined using Dunnett’s test in GraphPad Prism 7.04 Software.

Figure 6

NPC1-Specific ASOs 05HM and 28H Specifically Inhibit EBOV Replication (A and B) HeLa cells pretreated with the respective ASO were infected with EBOV at an MOI of 0.01. At 1 day after infection (p.i.), NPC1 (A) and EBOV (B) levels were quantified by RT-qPCR and normalized to the internal control α-tubulin. Shown is the fold change compared to untreated control (set at 100), which was calculated using the 2^−ΔΔCt method. Error bars show SD (n = 3, each in duplicate). Duplicates are labeled with identical symbol shapes. ANOVA was used to test for significant differences and p values were determined using Dunnett’s test, in GraphPad Prism 7.04 Software. See also Figure S2.