key: cord-0808953-04rs27ck
authors: Gu, Se Hun; Yu, Chi Ho; Song, Youngjo; Kim, Na Young; Sim, Euni; Choi, Jun Young; Song, Dong Hyun; Hur, Gyeung Haeng; Shin, Young Kee; Jeong, Seong Tae
title: A Small interfering RNA lead targeting RNA-dependent RNA-polymerase effectively inhibit the SARS-CoV-2 infection in Golden Syrian hamster and Rhesus macaque
date: 2020-07-07
journal: bioRxiv
DOI: 10.1101/2020.07.07.190967
sha: 55a1740313bfbe198d6179cf507042e97092e2ba
doc_id: 808953
cord_uid: 04rs27ck

A small interfering RNA (siRNA) inhibitors have demonstrated the novel modality for suppressing infectious diseases. Sixty-one siRNA molecules, predicted by the bioinformatics programs, were screened for the possibility of treating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using an in vitro plaque assay. Among six siRNA leads with the efficacy of reducing plaque number, the siRNA targeting RNA-dependent RNA polymerase (RdRp) showed a reduction in SARS-CoV-2 infection-induced fever and virus titer in the Golden Syrian hamster and rhesus macaque. These results suggest the potential for RdRp targeting siRNA as a new treatment for the coronavirus disease 2019 (COVID-19).

mM L-glutamine, and antibiotics (penicillin/streptomycin) at 37°C for 3 days in a 5% CO 2 89 incubator. As determined using plaque assay, the infectivity titers of the SARS-CoV-2 stocks 90 were 2 × 10 6 plaque-forming units (PFU/mL). 91 92

In this study, we obtained the SARS-CoV-2 genome sequence of MT039890 from 94 (https://www.ncbi.nlm.nih.gov/nuccore/MT039890). We focused on seven regions of the 95 SARS-CoV-2 genome, namely the leader sequence, replicase polyprotein1a (pp1a), RdRp, 96 spike protein (S), nucleocapsid protein (N), membrane protein (M), and envelope protein (E) 97 to design specific siRNAs to inhibit viral replication. Potential siRNA sequences targeting 98 viral genes were predicted using the programs siDirect (http://sidirect2.rnai.jp/) and 99 VIRsiRNApred (http://crdd.osdd.net/servers/virsirnapred/). Among the siRNAs predicted, we 100 examined whether siRNA predicted sequences that targeted the sequence of SARS-CoV, to 101 identify the 21 siRNA sequences targeting the conserved region in coronaviruses. We further 102 selected 40 siRNA sequences from the rest of the predicted siRNAs that did not match 103 SARS-CoV according to two criteria: first, siRNAs that have a score of less than off-target 104 effects in two portals; second, siRNAs that have low mutation rate among variants from were 21-mer with dTdT at the 3´-overhang. The siRNAs were synthesized by Bioneer Co. 107 (Daejeon, Republic of Korea). 108

In vitro efficacy test using Vero E6 cells (siRNA transfection) 110

Vero E6 cells were inoculated into a 24-well plate, 1 mL each, through a 10% FBS 111 DMEM without penicillin/streptomycin to ensure that the 24-well plate is 80-90% confluent. 112

The next day, the Vero E6 cells were transfected with 100 nM siRNA using Lipofectamine 113

RNAiMAX transfection reagent (Invitrogen, USA), according to the manufacturer's 114 instructions. The cells were incubated at 37°C in 5% CO 2 for 3 h and washed with phosphate-115 buffered saline. Next, the SARS-CoV-2 stock of 1 × 10 5 PFU/mL was diluted at 1/100 into 116 1x10 3 PFU/mL. Next, 200 µL each was added to make the final 200 PFU. The Vero E6 cells 117

were then infected with SARS-CoV-2 for 1 h 30 min. After infection, the supernatant was 118 removed, changed with 2% FBS DMEM medium, and incubated at 37°C for 3 days in a 5% 119 CO 2 incubator. The cells were observed daily for CPE using a microscope (Zeiss, Germany). 120

To assess viral titers, a plaque assay was performed using Vero E6 cells in 6-well 123 culture plates. Briefly, subconfluent monolayers of Vero E6 cells were inoculated with 10-124 fold serial diluents and incubated at 37°C for 1 h 30 min in a 5% CO 2 incubator. After with 100 nM of the 61 siRNA sequences, the CPE and plaque assays were performed at 3 198 d.p.i. (Fig. 1a-1c) . At 3 d.p.i., all siRNA candidates reduced CPE in SARS-CoV-2 infected 199 Vero E6 cells, while CPE was observed in the control group (data not shown). Based on the 200 results of the CPE and plaque assays, we selected six siRNA leads, showing a reduction in 201 plaque number. Six siRNA leads were subjected to the NCBI blast to ensure that they did not 202 target any human genes. RdRp, pp1a, and N mRNAs were targeted by 3 siRNAs (Nos. 9, 14, 203 and 36), 2 siRNAs (No. 26 and 29), and 1 siRNA (No. 61), respectively. 204

The Nextstrain website was used to monitor the data on SARS-CoV-2 mutation and to 205 confirm whether the regions targeted by our siRNA leads were less mutated (as of June 26, 206 2020). Mutation rates in siRNA-targeted regions were 0-0.002%, indicating that siRNA leads 207 can be used to treat most SARS-CoV-2 strains. Hence, the six siRNA leads were effective in 208 reducing viral replication in vitro and less influenced by SARS-CoV-2 mutation (Table 1) . 209 210 Efficacy test of the No. 14 siRNA lead against SARS-CoV-2 211

With six siRNAs, we examined whether the replication of SARS-CoV-2 was inhibited 212 using a plaque reduction assay. Vero E6 cells were infected with 500 PFU of SARS-CoV-2 213 with or without siRNA candidates and the plaque assay was performed at 3 d.p.i. From the 214 results, lead siRNA No. 14 (out of the six candidates) showed the least plaque formation 215 (data not shown). To calculate the half-maximal effective concentration (EC 50 ), the Vero E6 216 cells infected with 500 PFU of SARS-CoV-2 were treated with the siRNA No. 14 in a dose-217 dependent manner (5-100 nM) (Fig. 2a-2i) . Then, the CPE assay was performed at different 218 time points. The CPE started to reduce with 20 nM of the siRNA No. 14, and cell 219 morphology was similar in cells treated with 100 nM of the siRNA No. 14. EC 50 was further 220 calculated using qRT-PCR to determine the copy number of SARS-CoV-2 (Fig. 2m) . From 221 the result, EC 50 of the siRNA No. 14 was approximately 9.7 nM at 2 d.p.i. in 500 PFU 222 SARS-CoV-2 infected Vero E6 cells. Recently, it was reported that EC 50 of the antiviral 223 drugs, remdesivir, chloroquine and nafamostat, known to be effective for . 224

To assess the therapeutic efficacy of siRNA in the hamsters, three groups were 228 formed composed of a control group and two treatment groups (Table 2) . At 2 d.p.i., the 229 control hamsters showed a hunched posture, ruffled hair, and mild cough. However, clinical 230 signs such as cough were not observed in the siRNA No. 14-treated hamsters. The three 231 rhesus macaques consisted of one control animal and two treatment animals (Table 3) . After 232 anesthetizing the animals, three male rhesus macaques were inoculated with 4 × 10 6 PFU 233 SARS-CoV-2, administered at a dose of 5 mL through intratracheal (4 mL) and intranasal (1 234 mL) instillation. After 4 and 24 h, we administered the siRNA No. 14 through the same route 235 at low (2 mg/kg; G2) and high (4 mg/kg; G3) doses, respectively. At 1 d.p.i., diarrhea was 236 observed in the viral control and body temperature was markedly elevated compared to the 237 base level (Fig. 3c) . SARS-CoV-2-infected rhesus macaques (G1) had elevated body 238 temperature, from 38.6-40.4°C, between 1 and 2 d.p.i. Interestingly, high-dose siRNA No. 239 14-treated animals (G3) showed a base level of body temperature over 3 days. All treated 240 animals displayed normal appetite and behavior. 241 242

To confirm infection of SARS-CoV-2 in the hamster, we inoculated each group with 244 4-40,000 PFU SARS-CoV-2 by the intranasal route. SARS-CoV-2 RNA was detected in all 245 hamsters, including the 4 PFU inoculation group (data not shown). We assessed the 246 therapeutic efficacy of the siRNA No. 14 in the hamsters and analyzed the lung of the 247 hamsters at 2 d.p.i. using qRT-PCR. The results showed that the number of viral RNA copies 248 from the lung tissue of the siRNA No. 14-treated hamsters markedly decreased by 249 approximately 10 4 viral copies compared to that of the control hamsters (Fig. 3a) . Next, we 

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investigated viral copies from the trachea of rhesus macaques at 3 d.p.i. using qRT-PCR. The 251number of viral RNA copies in the siRNA No. 14-treated rhesus macaques significantly 252 decreased by approximately 10 3 viral copies compared to that in the control animals (Fig. 3b) . 253These results indicate that siRNA No. 14 effectively inhibited SARS-CoV-2 infection and 254 replication in the lung and upper respiratory tract. In this study, to demonstrate the siRNAs that are effective for the inhibition of 280 SARS-CoV-2 infection, we tested 61 siRNA duplexes in Vero E6 cells. Among them, the best 281 lead, siRNA No. 14, showed strong inhibitory efficacy against SARS-CoV-2 infection and 282 replication in Vero E6 cells. To assess the inhibition efficacy of siRNA No. 14 for SARS-283CoV-2 in animals, we used Syrian hamsters and rhesus macaques. Our data showed that 284 SARS-CoV-2 viral RNA decreased in the siRNA No. 14-treated animals. Additional research 285 is warranted to determine the possibility of siRNA treatment for COVID-19. Further studies 286 would also be needed to address the safety and effective delivery methods of siRNA.