Inhibition of SARS-CoV-2 growth in the lungs of mice by a peptide-conjugated morpholino oligomer targeting viral RNA

Abstract

Further development of direct-acting antiviral agents against human SARS-CoV-2 infections remains a public health priority. Here, we report that an antisense peptide-conjugated morpholino oligomer (PPMO) named 5'END-2, targeting a highly conserved sequence in the 5' UTR of SARS-CoV-2 genomic RNA, potently suppressed SARS-CoV-2 growth in vitro and in vivo. In HeLa-ACE 2 cells, 5'END-2 produced IC₅₀ values of between 40 nM and 1.15 μM in challenges using six genetically disparate strains of SARS-CoV-2, including JN.1. In vivo, using K18-hACE2 mice and the WA-1/2020 virus isolate, two doses of 5'END-2 at 10 mg/kg, administered intranasally on the day before and the day after infection, produced approximately 1.4 log10 virus titer reduction in lung tissue at 3 days post-infection. Under a similar dosing schedule, intratracheal administration of 1.0-2.0 mg/kg 5'END-2 produced over 3.5 log10 virus growth suppression in mouse lungs. Electrophoretic mobility shift assays characterized specific binding of 5'END-2 to its complementary target RNA. Furthermore, using reporter constructs containing SARS-CoV-2 5' UTR leader sequence, in an in-cell system, we observed that 5'END-2 could interfere with translation in a sequence-specific manner. The results demonstrate that direct pulmonary delivery of 5'END-2 PPMO is a promising antiviral strategy against SARS-CoV-2 infections and warrants further development.

Keywords: K-18-hACE2 mouse model; MT: Oligonucleotides: Therapies and Applications; PPMO; RNA virus; SARS-CoV-2; antisense; antiviral; intranasal delivery; intratracheal delivery; peptide-conjugated morpholino oligomer.

Graphical abstract

Figure 1

Heatmap of reference allele frequency in the 5′END-2 PPMO target region of different SARS-CoV-2 lineages The reference allele (GenBank: NC_045512) frequency at nt positions 1–30 of the SARS-CoV-2 genome was calculated from a dataset of complete and high-quality human-origin genome sequences, and visually represented in a heatmap. The reference allele sequence for nt 1–30 is shown in the top horizontal row. This terminal region of the viral 5′ UTR includes the target of the 5′END-2 PPMO, located from nt 5–29. As of January 8, 2024, the WHO has defined 27 variants of concern, variants of interest, and variants under monitoring. The vertical axis labels are indicative of the lineage names, which appear in order of their chronological appearance during the pandemic, with the accompanying numbers in parentheses representing the tally of genome sequences specific that were analyzed for each specific lineage.

Figure 2

5′END-2 PMO and PPMO duplex specifically and efficiently with target RNA (A) Schematic diagram of nt 1–36 of SARS-CoV-2 genome. This RNA (SL1) was produced by in vitro transcription and served as the analyte for the experiments of (B)–(E). The secondary structure of the diagram was produced by “RNAStructure” and the residues representing the 5′END-2 PMO or PPMO target site are shown in red. (B–E) EMSA gels of reactions containing SL1 and PMO/PPMO. A fixed concentration of SL1 RNA (2 μM) was used with titrations of the indicated PMO (B and C) or PPMO (D and E) from 0 to 16 μM, as described in detail in materials and methods. The reactions were run on native PAGE comprising 8% acylamide. A table indicating the overall lane-by-lane composition of the reactions is present below the gels. NC, negative control.

Figure 3

5′END-2 PPMO administered IN limits virus growth K18-hACE2 mice were treated with PBS, 5′END-2 PPMO, and NC705 (negative control) PPMO as indicated, via IN instillation at 24 h before and 18 h after IN administration of 5,000 ffu of WA-1/2020. MK-4482 (molnupravir), used as a positive control compound, was administered orally twice per day from 1 day before infection until 2 days post-infection. Viral load in the lungs was measured at 3 days post-infection by focus-forming assay, as described in materials and methods. Mean ± SD is shown (n = 5) and was analyzed by one-way ANOVA with Dunnett’s multiple comparisons (∗p < 0.05; ∗∗∗∗p < 0.0001).

Figure 4

5′END-2 PPMO administered via IT injection markedly limits virus growth in the lungs of K18-hACE2 mice and protects from weight loss (A–C) K18-hACE2 mice were treated with PBS or the indicated PPMO via IT administration at 24 h before and 18 h after IN administration of 5,000 ffu of SARS-CoV-2 (strain WA-1/2020). MK-4482 (molnupravir), used as a positive control compound, was administered orally twice per day from 1 day before infection until 2 days post-infection. Virus load in the lungs was measured at 3 days post-infection by focus-forming assay, as described in materials and methods, and charted. The experiments represented by the graphs in (A)–(C) were carried out under the same conditions (see materials and methods), but at independent times. The limit of virus detection (LOD) in the focus-forming assay was 100 ffu/g tissue, as represented by a dotted line. n = 5, except the experiment of (C), which tested 5’END-2 at various concentrations, where each group contained 3 mice. (D) Mice were treated with PBS, MK-4482, or PPMO, as indicated, under the same experimental conditions as described above. Body weights were measured daily for 10 days post-infection and represented as a percentage of the animal weight on the day of infection. By 8 days post-infection, all the animals in the NC705 had lost greater than 35% of their original body weight and were considered moribund. n = 5/group. Data shown as mean ± SD and analyzed by one-way ANOVA with Dunnett’s multiple comparisons (∗∗∗p < 0.001; ∗∗∗∗p < 0.0001).

Figure 5

The 5′END-2 PPMO restricts protein expression from a SARS-CoV-2 sequence-containing reporter construct, in a potent and specific manner (A) Schematic of the two reporter constructs used in this experiment. One construct (pSARS2-75/mCherry) contains the first 75 nt of SARS-CoV-2 genomic sequence (viral leader) fused to mCherry coding sequence, while the second (pRNDM-75/GFP) contains 75 nt of non-viral sequence (non-viral leader) fused to GFP coding sequence. Both constructs contain a CMV promoter. (B and C) In-cell translation assays. HEK293 cells were treated with PBS or PBS containing PPMO at the indicated concentrations for 2.5 h, then co-transfected with 500 ng of each reporter construct. At 24 h post-transfection, the cells were assayed for their mCherry and GFP levels by flow cytometry. Translation of the construct containing SARS-CoV-2 leader RNA (B) was markedly limited by the 5′END-2 PPMO, whereas the NC705 PPMO had no effect. Neither PPMO produced appreciable inhibition of translation of the non-viral leader (NC) construct. These experiments were run twice independently, and both trials are shown.