key: cord-336522-y9nzsv95
authors: Rosenke, Kyle; Leventhal, Shanna; Moulton, Hong M.; Hatlevig, Susan; Hawman, David; Feldmann, Heinz; Stein, David A.
title: Inhibition of SARS-CoV-2 in Vero cell cultures by peptide-conjugated morpholino-oligomers
date: 2020-09-30
journal: bioRxiv
DOI: 10.1101/2020.09.29.319731
sha: 
doc_id: 336522
cord_uid: y9nzsv95

Background SARS-CoV-2 is the causative agent of COVID-19 and a pathogen of immense global public health importance. Development of innovative direct-acting antiviral agents is sorely needed to address this virus. Peptide-conjugated morpholino oligomers (PPMO) are antisense agents composed of a phosphordiamidate 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. Objectives and 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 and viral growth was measured with quantitative RT-PCR and TCID50 infectivity assays. 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 titers by up to 4-6 log10 in cell cultures at 48-72 hours post-infection, in a non-toxic and dose-responsive manner. Conclusion The data indicate that PPMO have the ability to potently and specifically suppress SARS-CoV-2 growth and are promising candidates for further pre-clinical development.

Background: SARS-CoV-2 is the causative agent of COVID-19 and a pathogen of immense 24 global public health importance. Development of innovative direct-acting antiviral agents is 25 sorely needed to address this virus. Peptide-conjugated morpholino oligomers (PPMO) are 26 antisense agents composed of a phosphordiamidate morpholino oligomer covalently conjugated 27 to a cell-penetrating peptide. PPMO require no delivery assistance to enter cells and are able to 28 reduce expression of targeted RNA through sequence-specific steric blocking. 29

Objectives and Methods: Five PPMO designed against sequences of genomic RNA in the 30 SARS-CoV-2 5'-untranslated region and a negative control PPMO of random sequence were 31 synthesized. Each PPMO was evaluated for its effect on the viability of uninfected cells and its 32 inhibitory effect on the replication of SARS-CoV-2 in Vero-E6 cell cultures. Cell viability was 33 evaluated with an ATP-based method and viral growth was measured with quantitative RT-PCR 34 and TCID50 infectivity assays. 35

Results: PPMO designed to base-pair with sequence in the 5'-terminal region or the leader 36 transcription regulatory sequence-region of SARS-CoV-2 genomic RNA were highly 37 efficacious, reducing viral titers by up to 4-6 log10 in cell cultures at 48-72 hours post-infection, 38 in a non-toxic and dose-responsive manner. 39 Standard Operating Protocols. 66 PPMO synthesis. PPMO were synthesized by covalently conjugating the CPP (RXR)4 (where R 67 is arginine and X is 6-aminohexanoic acid) to PMO (Gene Tools LLC, Philomath, Oregon) at 68 the 3' end through a noncleavable linker, by methods described previously [3] . 69

Cells and viruses. Vero-E6 cells (ATCC) were maintained in DMEM supplemented with 10% 70 fetal calf serum, 1 mM L-glutamine, 50 U/mL penicillin and 50 μg/mL streptomycin (growth 71 medium). All cell culture incubations were carried out at 37º C and 5% CO2. SARS-CoV-2 72 isolate nCoV-WA1-2020 was kindly provided by the Centers for Disease Control and Prevention 73 (Atlanta, Georgia, USA). Preparation and quantification of the virus followed methods 74 previously described [13] . Briefly, the original virus stock was propagated once at RML in Vero-75 E6 cells in DMEM supplemented with 2% fetal bovine serum containing L-glutamine and 76 antibiotics as above (infection medium). The virus stock used in the experiments was passage 4 77 and was confirmed by sequencing to be identical to the initial deposited GenBank sequence 78

Cell viability assay. Cell viability was assessed using CellTiterGlo (Promega). Vero Evaluation of virus quantity by qRT-PCR. Supernatants were harvested as described above 93 and viral RNA purified and quantified by using one-step quantitative reverse transcription PCR 94 (qRT-PCR) following previous described methods [13] . Briefly, total RNA was isolated with the 95 RNeasy Mini kit (Qiagen) and RT-PCR carried out using the One-Step RT-PCR kit (Qiagen) 96 according to the manufacturer's protocols. Copy numbers were calculated using standards 97 produced as previously described [13] . 98 TCID50 evaluations. Viral supernatants were serially diluted in DMEM and each dilution 99 sample was titrated in triplicate. Subsequently, 100 ul of each virus dilution was transferred to 100

Vero-E6 cells grown in 96-well plates containing 100ul DMEM. Following a seven day 101 incubation period, wells were scored for cytopathic effect (CPE 14, 15] . In this study, five PPMO were designed to target the 5' UTR 107 and first translation start site-region of SARS-CoV-2 positive sense genomic RNA ( Table 1) . Two of the PPMO target the 5'-terminal-region of the genome. Both 5'END-1 and 5'END-2 were 109 designed with the intention of interfering with pre-initiation of translation of genomic and 110 subgenomic mRNAs. 111

Two PPMO were designed to target the genomic 5'UTR region containing the putative SARS-112

CoV-2 leader-TRS core sequence (nt 70-75, 5'-ACGAAC-3') and thereby potentially interfere 113 with body-TRS to leader-TRS base-pairing, and/or with translocation of the 48S translation 114 preinitiation complex along the 5'UTR of the genomic and various subgenomic mRNAs. 115

Coronaviruses produce a set of nested mRNAs through the process of discontinuous subgenomic 116 mRNA synthesis. The TRS is a six-ten nt sequence that is critical in the production of negative 117 strand mRNA templates during this process [16, 17] . For SARS-CoV, the leader-TRS core 118 sequence consists of nt 67-72 of the genomic RNA sequence [18] , and most viral mRNAs possess 119 the same 72 nt 5' leader sequence. 120

The AUG PPMO was designed to target the translation initiation region for ORF1a/b, which codes 121 for the replicase polyprotein, with the intention to block translation initiation. 122 A negative control PPMO (NC) of random sequence was included (see Table 1 ), to control for 123 nonspecific effects of the PPMO chemistry. NC was screened using BLAST and contains little 124 significant homology to any primate, rodent or viral sequences. 125

Evaluation of PPMO cytotoxicity. To evaluate the effect of PPMO treatments on cell viability, 126 cells were treated under similar conditions to the antiviral assays described below, but in the 127 absence of virus. Cells were treated in triplicate with increasing doses of PPMO for 48 hours before 128 being assayed using a quantitative cell viability assay. At the concentrations used in the antiviral 129 assays described below, none of the PPMO produced more than 5% cytotoxic effect ( Figure 1A) .

PPMO on SARS-CoV-2 replication, Vero-E6 cells were treated with each of the six PPMO 132 described in Table 1 at 4, 8, and 16 µM for 5 hours before infection, then incubated without 133 PPMO after infection. Cell supernatants were collected at four time-points post-infection: 12, 24, 134 48, and 72 hours. Virus growth was evaluated by two methods, qRT-PCR and TCID50 infectivity 135 assay. Using an MOI of 0.01, virus growth rose steadily and reached peak growth at 72 hrs post-136 infection ( Fig. 1B and H) AUG PPMO was not nearly as effective as the other 4 antiviral PPMO used in this study ( Figure  146 1C and I). 147

We found that PPMO targeting the 5'terminal-region or leader-TRS-region were highly effective 149 at inhibiting the growth of SARS-CoV-2, whereas a PPMO targeting the polyprotein 1a/b AUG 150 translation start site region was not effective. It is unknown if the ineffective AUG-PPMO was 151 unable to bind to its target, or if duplexing occurred yet was relatively inconsequential.

To date, little sequence variation in the PPMO target sites in the 5'UTR of SARS-CoV-2 has 153 been identified. Of the whole-genome nucleotide sequences reported in GenBank, two genotypes 154 contain a single mismatch with the 5'END PPMO and one genome has a single mismatch with 155 the TRS PPMO [19] . Previous studies have shown that PPMO having a single base mismatch 156 with their target site retain approximately 90% of their activity compared to those having perfect 157 agreement [15, 20] . 158

This study demonstrates that PPMO targeted against SARS-CoV-2 can enter cells readily and 159 inhibit viral replication in a sequence-specific, dose-responsive and non-toxic manner. 

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