Inhibition of SARS-CoV-2 in Vero cell cultures by peptide-conjugated morpholino oligomers

Abstract

Background: As the causative agent of COVID-19, SARS-CoV-2 is a pathogen of immense importance to global public health. Development of innovative direct-acting antiviral agents is sorely needed to address this virus. Peptide-conjugated morpholino oligomers (PPMO) are antisense compounds composed of a phosphorodiamidate morpholino oligomer covalently conjugated to a cell-penetrating peptide. PPMO require no delivery assistance to enter cells and are able to reduce expression of targeted RNA through sequence-specific steric blocking.

Methods: Five PPMO designed against sequences of genomic RNA in the SARS-CoV-2 5'-untranslated region and a negative control PPMO of random sequence were synthesized. Each PPMO was evaluated for its effect on the viability of uninfected cells and its inhibitory effect on the replication of SARS-CoV-2 in Vero-E6 cell cultures. Cell viability was evaluated with an ATP-based method using a 48 h PPMO treatment time. Viral growth was measured with quantitative RT-PCR and TCID50 infectivity assays from experiments where cells received a 5 h PPMO treatment time.

Results: PPMO designed to base-pair with sequence in the 5' terminal region or the leader transcription regulatory sequence region of SARS-CoV-2 genomic RNA were highly efficacious, reducing viral titres by up to 4-6 log10 in cell cultures at 48-72 h post-infection, in a non-toxic and dose-responsive manner.

Conclusions: The data indicate that PPMO have the ability to potently and specifically suppress SARS-CoV-2 growth and are promising candidates for further preclinical development.

Figure 1.

Effect of PPMO on cell viability and SARS-CoV-2 growth. (a) Evaluation of the effect of PPMO treatment on cellular ATP level, as an indicator of cell viability, was carried out using uninfected cells incubated for 48 h with increasing concentrations of the indicated PPMO. ATP levels were determined via luminescence readings and are shown compared with PBS-treated cells. For each PPMO concentration, triplicate samples were assayed and the mean±SD is shown. (b–m) Growth curves of SARS-CoV-2. Vero-E6 cells were treated with the indicated concentration of PPMO for 5 h before infection with SARS-CoV-2 (moi of 0.01), then incubated without PPMO after infection. Cell supernatants were collected at 12, 24, 48 and 72 h post-infection and analysed by qRT–PCR (b–g) or TCID₅₀/mL endpoint dilution (h–m) using three technical repeats. Cells treated with PBS had titres similar to those shown for NC PPMO. The limit of virus detection for the TCID₅₀ assay was 10¹/mL. This experiment was carried out twice, under similar conditions, yielding similar results, and the results from a single experiment are shown. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.