An intranasal ASO therapeutic targeting SARS-CoV-2

Abstract

The COVID-19 pandemic is exacting an increasing toll worldwide, with new SARS-CoV-2 variants emerging that exhibit higher infectivity rates and that may partially evade vaccine and antibody immunity. Rapid deployment of non-invasive therapeutic avenues capable of preventing infection by all SARS-CoV-2 variants could complement current vaccination efforts and help turn the tide on the COVID-19 pandemic. Here, we describe a novel therapeutic strategy targeting the SARS-CoV-2 RNA using locked nucleic acid antisense oligonucleotides (LNA ASOs). We identify an LNA ASO binding to the 5' leader sequence of SARS-CoV-2 that disrupts a highly conserved stem-loop structure with nanomolar efficacy in preventing viral replication in human cells. Daily intranasal administration of this LNA ASO in the COVID-19 mouse model potently suppresses viral replication (>80-fold) in the lungs of infected mice. We find that the LNA ASO is efficacious in countering all SARS-CoV-2 "variants of concern" tested both in vitro and in vivo. Hence, inhaled LNA ASOs targeting SARS-CoV-2 represents a promising therapeutic approach to reduce or prevent transmission and decrease severity of COVID-19 in infected individuals. LNA ASOs are chemically stable and can be flexibly modified to target different viral RNA sequences and could be stockpiled for future coronavirus pandemics.

Fig. 1. In vitro screening of LNA ASOs targeting SARS-CoV-2.

a Schematic representation of the genome structure and the regions targeted by LNA ASOs. b, c The SARS-CoV-2 WA1 strain-infected Huh-7 cells treated with LNA ASO (100 nM) and cell culture medium were collected at 48 hpi. Viral RNA levels were analyzed by RT-qPCR for LNA ASO screening. Each LNA ASO was tested in duplicate and compared with in vitro Mixmer control LNA ASO or Gapmer control LNA ASO. d Dose-dependent effects of 5’-ASO#26 were evaluated in infected Huh-7 cells with increasing doses (N = 3) of the LNA ASO (as indicated) by RT-qPCR. The exact p-values stated in the following order: Nucleocapsid group and then Spike group ((Mixmer Ctrl vs. 5’_ASO#26), at 5 nM: P = 0.8279, **** P < 0.0001; at 10 nM: **P = 0.0012, *** P = 0.0002; at 20 nM: ***P = 0.0007, ****P < 0.0001; at 50 nM: *** P = 0.0002, **** P < 0.0001; at 100 nM: **** P < 0.0001, **** P < 0.0001. e The infectious virus was measured by TCID₅₀ assay. The infected Huh-7 cells with different doses (N = 3) of LNA ASO treatment were collected at 48 hpi. The exact p-values stated here: at 5 nM, *** P = 0.0002; at 10 nM, *** P = 0.0003; at 20 nM, **** P < 0.0001; at 50 nM, **** P < 0.0001; at 100 nM, **** P < 0.0001. For d and e, one-way ANOVA with Dunnett’s test was used to determine significance (** P < 0.01, *** P < 0.001, **** P < 0.0001, P > 0.05, ns, not significant). Source data are provided as a Source Data file.

Fig. 2. 5’-ASO#26 disrupts stem loop 1 (SL1) of SARS-CoV-2.

a Structural model of SL1-4 predicted from the DMS-MaPseq of in vitro-transcribed 5’leader RNA with the addition of control LNA ASO (left) and 5’-ASO#26 (right). Nucleotides are color-coded by normalized DMS signal. The 5’-ASO#26- sequence is shown in magenta and the binding site on the viral genome is highlighted with a pink frame; PCR primer binding site, where DMS information is unavailable, is highlighted with a grey frame. b Per-nucleotide-difference in DMS reactivity (Δ DMS reactivity) between in vitro-transcribed SARS-CoV-2 5’-leader RNA versus following the addition of control LNA ASO (top left), and versus titration with 5’ -ASO#26 at 0.1× (top right), 0.45× (middle left), 0.7× (middle right), 1× (bottom left) and 10× (bottom right) molar ratio of 5’-ASO#26. c Per-nucleotide-difference in DMS reactivity (Δ DMS reactivity) between Huh-7 cells transfected with control LNA ASO and those with 5’ -ASO#26, collected 6 (top) and 12 (bottom) hours after SARS-CoV-2 infection. For b, c only nucleotides from positions 26 to 79 in the SARS-CoV-2 genome are included. Each bar represents one nucleotide, and the 5’-ASO#26 target region is coloured in pink. Source data are provided as a Source Data file.

Fig. 3. Evaluation of lung delivery efficacy and potential immune-stimulatory effects of LNA ASOs.

a Representative 5’-ASO#26 FISH in mice lung. Scale bar = 100 μm. The FISH assay was repeated three times. b, c Representative flow cytometry result of alveolar neutrophils and macrophages in BALF collected from mice with three days-treatment of LNA ASOs or Saline; one day-treatment with LPS was used as a positive control. Neutrophils were identified as Ly-6G+ and CD11b+ and macrophages were identified as F4/80+. d) Multiplex cytokine assay in BALF collected from mice with three days-treatment of LNA ASOs or Saline (N = 5 for Saline, Mixmer Control and 5’-ASO#26 and N = 4 for LPS group, symbols represent mean ± SD). The exact p-values are: BCA-1/CXCL13(Saline vs. LPS), **** P < 0.0001; ENA-78/CXCL5(Saline vs. LPS), ***P < 0.001; CCL11(Saline vs. Mixmer Ctrl), * P = 0.0424; CCL11(Saline vs. LPS), **P = 0.0028; GM-CSF(Saline vs. LPS), **P = 0.0092; INF-gamma(Saline vs. LPS), ***P = 0.0005; IL-1beta(Saline vs. LPS), **P = 0.0036; IL-6(Saline vs. LPS), ****P < 0.0001; IL-10(Saline vs. Mixmer Ctrl), * P = 0.0286; IL-16(Saline vs. LPS), ****P < 0.0001; IP-10/CXCL10(Saline vs. LPS), ****P < 0.0001; I-TAC/CXCL11(Saline vs. LPS), * P = 0.0402; KC/CXCL1(Saline vs. LPS), *** P = 0.0004; MCP-1/CCL2(Saline vs. LPS), ***P = 0.0009; MCP-2/CCL7(Saline vs. LPS), ****P < 0.0001; MDC/CCL22(Saline vs. Mixmer Ctrl), **P = 0.0011; MIP-1alpha/CCL3(Saline vs. LPS), ****P < 0.0001, MIP-1beta/CCL4(Saline vs. LPS), **** P < 0.0001; MIP-1a/CCL20(Saline vs. LPS), ****P < 0.0001; RANTES/CCL5(Saline vs. LPS), ****P < 0.0001;SCYB16/CXCL16(Saline vs. Mixmer Ctrl), ** P = 0.0084; SCYB16/CXCL16(Saline vs. 5’-ASO#26), *P = 0.0341; SCYB16/CXCL16(Saline vs. LPS), **P = 0.0040; TARC/CCL17(Saline vs. Mixmer Ctrl), * P = 0.0448; TARC/CCL17(Saline vs. Mixmer Ctrl), *** P = 0.0009; TNF-alpha(Saline vs. LPS), ***P = 0.0001. For d), one-way ANOVA with Dunnett’s test was used to determine significance (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001). Source data are provided as a Source Data file.

Fig. 4. Once-daily treatment of 5′-ASO#26 represses the generation of infectious virus in mice.

a Treatment course of once-daily 5’-ASO#26 administration. The red arrow indicates the inoculation of 1 × 10⁴ TCID50 SARS-CoV-2 intranasally in mice. Mice were treated once-daily with saline (vehicle) or 400 µg LNA ASO (dissolved in saline). Treatment on the day of infection was carried out at 6 hpi. b The viral burden in lungs of mice treated with Saline (N = 5) or LNA ASO (N = 5) was measured by TCID₅₀ assay using lung homogenates. *P = 0.0427. Center line, median; box limits, upper and lower quartiles; plot limits, maximum and minimum in the boxplot. c The levels of viral RNAs encoding Nucleocapsid protein (N) (*P = 0.0246) and Spike (S) (*P = 0.0121) were measured in mouse lungs by RT-qPCR (N = 5). For b and c, center line, median; box limits, upper and lower quartiles; plot limits, maximum and minimum in the boxplot. Student t-test was used to determine significance (*P < 0.05). d IHC staining of SARS-CoV-2 N in all infected K18-hACE2 mice with or without LNA ASO treatment. Scale bar = 2 mm. The staining was repeated in lung collected from two independent batches of mice. e Expression changes of SARS-CoV-2 infection-upregulated genes in Saline- and LNA ASO-treated groups. Columns represent samples and rows represent genes. Colors indicate expression levels (log2 RPKM) relative to average expression across all samples. f Gene set enrichment analysis (GSEA) of Hallmark gene sets enriched in lungs of Saline- or LNA ASO-treated mice. Terms were ranked by the false discovery rate (q value). Source data are provided as a Source Data file.

Fig. 5. Prophylaxis and treatment of SARS-CoV-2 variant strains with 5′-ASO#26.

a Dose-dependent effects of 5′-ASO#26 on inhibition of viral replication of SARS-CoV-2 WA1 and B.1.351 strains were evaluated in infected Huh-7 cells by RT-qPCR of Nucleocapsid protein (N) and Spike (S) RNA (N = 3, symbols represent mean ± SD). One-way ANOVA with Dunnett’s test was used to determine significance. For WA1 (Nucleocapsid), Mixmer Ctrl vs. 5 nM, * P = 0.0496; Mixmer Ctrl vs. 10 nM, *** P = 0.0003. For B.1.351 (Nucleocapsid), Mixmer Ctrl vs. 5 nM, ***P = 0.0001. For WA1(Spike), Mixmer Ctrl vs. 5 nM, **P = 0.0033 and groups marked with **** are P < 0.0001. b, d and f The viral burden in lungs of mice treated with Saline (N = 5) and 5′-ASO#26 (N = 5) was measured by TCID₅₀ assay using lung homogenates. Student t-test was used to determine significance. In b *P = 0.0394 c and in f, ** P = 0.0060. c, e and g Weight change of mice in b, d and f was monitored (N = 5 for control or ASO-treated group in each experiment, symbols represent mean ± SD). Two-way ANOVA was used to determine significance, in e ** P = 0.0027, ****P < 0.0001. h Mice were administered with different treatment regimens of 5′-ASO#26 as indicated. Treatment on the day of infection was carried out at 6 hpi. The viral burden in lungs of mice in each group (N = 5) was measured by TCID₅₀ assay using lung homogenates. One-way ANOVA with Dunnett’s test was used to determine significance (*P = 0.0293). i Mice were administered with different treatment regimens of 5′-ASO#26 as indicated. Treatment on the day of infection was carried out at 2 hpi. The viral burden in lungs of mice in each group (N = 7) was measured by TCID₅₀ assay using lung homogenates. One-way ANOVA with Dunnett’s test was used to determine significance (****P < 0.0001). j Survival graph of mice administered with different treatment regimens of 5′-ASO#26 as indicated. Each group contain 5 male and 5 female K18-hACE2 mice. Log-rank (Mantel-Cox) test was used to determine significance and p value threshold was corrected by Bonferroni Correction. For b, d, f, h and i, center line, median; box limits, upper and lower quartiles; plot limits, maximum and minimum in the boxplot. Source data are provided as a Source Data file.