key: cord-0730945-3xlvpbia
authors: Su, Xiaoxuan; Ma, Wenxiao; Cheng, Boyang; Wang, Qian; Guo, Zefeng; Zhou, Demin; Tang, Xinjing
title: Efficient Inhibition of SARS-CoV-2 Using Chimeric Antisense Oligonucleotides through RNase L Activation
date: 2021-03-04
journal: bioRxiv
DOI: 10.1101/2021.03.04.433849
sha: 3666a73dd3659ef8d8949e64943d1d6f36392ce3
doc_id: 730945
cord_uid: 3xlvpbia

There is an urgent need for effective antiviral drugs to alleviate the current COVID-19 pandemic. Here, we rationally designed and developed chimeric antisense oligonucleotides to degrade envelope and spike RNAs of SARS-CoV-2. Each oligonucleotide comprises a 3’ antisense sequence for target recognition and a 5’-phosphorylated 2’-5’ poly(A)4 for guided ribonuclease L (RNase L) activation. Since RNase L can potently cleave single strand RNA during innate antiviral response, the improved degradation efficiency of chimeric oligonucleotides was twice as much as classic antisense oligonucleotides in Vero cells, for both SARS-CoV-2 RNA targets. In pseudovirus infection models, one of chimeric oligonucleotides targeting spike RNA achieved potent and broad-spectrum inhibition of both SARS-CoV-2 and its recently reported N501Y and/or ΔH69/ΔV70 mutants. These results showed that the constructed chimeric oligonucleotides could efficiently degrade pathogenic RNA of SARS-CoV-2 facilitated by immune activation, showing promising potentials as antiviral nucleic acid drugs for COVID-19.

Since the infection was first reported in 2019, severe acute respiratory syndrome coronavirus 2 29 (SARS-CoV-2) has continued to spread globally and caused the pandemic COVID-19 disease (1). 30 The current lack of highly effective antiviral drugs for SARS-CoV-2 has made the treatment of 31 infected patients more difficult, thus demanding more candidate options for drug discovery. Genomic positive-sense single-stranded RNA (ssRNA) and structural proteins participate in virus 33 packaging, which is an essential step in SARS-CoV-2 life cycle. Envelope (E), spike (S) and 34 membrane (M) proteins assemble the virus membrane in host cells infected by 35 3), and thus become ideal drug targets to intervene virus proliferation. 36 RNase L participates in innate antiviral response of vertebrate cells by cleaving UN^N sites located 37 in viral or cellular ssRNAs. Cytoplasmic RNase L monomer only displays weak catalytic cleavage 38 on the substrate. However, upon dimerization induced by its specific ligand 5 phosphorylated 2-39 5 polyA (such as 4A2-5), RNase L is highly activated and performs intense RNA cleavage (4). The 40 cleavage products can further bind to intracellular pattern recognition receptors (PRRs) to stimulate 41 the production of interferons (IFN) (5-7), which in turn induces the expression of interferon 42 stimulated genes (ISGs) including RNase L, to enhance the antiviral response (2, 7, 8) . Ubiquitous 43 activation of RNase L might cause widespread attenuation of basal mRNA and possible cell 44 apoptosis, especially at high doses of 4A2-5 (9-12) . Guided and controlled activation of RNase L 45 could otherwise achieve more specific target RNA degradation. RNA binding small molecules 46 conjugated with 4A2-5 have been reported to target highly-structured microRNA or RNA fragments 47 of virus genome (13, 14) , which contains particularly structured sequences. Nevertheless, the 48 selective binding between a small molecule and the specific region of pathogenic RNA is limited, 49 while the sequence-selective antisense oligonucleotides (ASO) will be more accessible and 50 effective to target viral RNA of interest. 51 ASO therapy has successfully targeted undruggable pathogenic genes of rare diseases and has been 52 developed against the infection of ssRNA viruses such as SARS-CoV (15) in a sequence-specific 53 manner. Chemical modifications on ASOs can further promote their nuclease resistance and/or 54 binding affinity to target RNA sequences, such as phosphorothioate (PS) linkages and 2'-O-methyl 55 (2'-OMe) substituents (16). Currently a few reports have raised the possibility of combining ASOs 56 with 4A2-5 for the treatment of tumors (17) and viral infections (12, 18) . Therefore, it is promising 57 to develop nucleic acid drugs in form of ASO-4A2-5 chimera targeting SARS-CoV-2 genomic 58 RNAs to inhibit virus infection. 59 Here, based on nucleic acid-hydrolysis targeting chimeras strategy, we developed chimeric Our study began with the selection of antisense oligonucleotides targeting specific genomic RNA 79 of SARS-CoV-2. After predicting RNA secondary structures of spike receptor binding domain (S-80 RBD) and envelope (E) protein of SARS-CoV-2, loops composed of more than 10 nucleotides 81 were selected as ideal target regions. In addition, considering the space required for RNase L 82 activation and substrate cleavage, the stem structure in 3 proximity of the selected loop was limited 83 to have less than 4 base pairs, and its 3 pairing end should have more than 1 RNase L cleavage 84 site (UN^N) in a bulge structure. As a result, antisense sequences complementary to the selected 85 loops were predicted with more than 70% probability of being efficient antisense strands as 86 evaluated by OligoWalk (19) and was synthesized through solid phase synthesis (Table S1 ).To 87 enhance nuclease resistance and binding affinity with their complementary viral RNA regions, 88 phosphorothioate (PS) linkages and 2-O-methyl (2-OMe) substituents were properly incorporated 89 into the chimeric structure, followed by the coupling of a poly 2-5 poly(A)4 ligand at 5 terminus 90 of the designed antisense sequence (15 nt) through a short PEG linker (Fig. 1B) .

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We first tested RNase L recruitment ability of Chimera-E-PO, an oligonucleotide modified with 5 92 native 4A2-5 ligand and complementary to a loop structure on Cy3-labeled partial E-RNA sequence 93 of SARS-CoV-2 (Table S1). As shown in Fig 1C, after incubating RNase L with Chimera-E-PO 94 or 4A2-5, in vitro cleavage of Cy3-labeled substrate RNA was analyzed in a denaturing PAGE gel. 95 Treatment of RNase L alone did not lead to the cleavage of substrate RNA, while additional 96 Chimera-E-PO treatment activated RNase L and produced cleavage bands in a manner different 97 from that of 4A2-5 treated group. The cleavage preferences of Chimera-E-PO for these specific 98 cleavage sites indicated its specific binding for RNA substrate. We first selected envelope gene featured with a relative short viral RNA sequence, and evaluated 102 E-RNA degradation efficiency using Chimera-E in Vero cells after co-transfection of pCAG-103 nCoV-E-FLAG plasmids. As we expected, treatment of 20 nM ASO-E alone could only partially 104 downregulate E-RNA level to 83% in comparison to the negative control, while treatment of 20 105 nM Chimera-E downregulated E-RNA level to 35%, 2-fold more efficiently than that of ASO-E as 106 measured by RT-qPCR ( Fig. 2A) . In addition, RNase L transcription level was also significantly 107 increased with higher concentration of Chimera-E (Fig. 2B ) which may further enhance the RNase 108 L induced sequence-specific degradation of E-RNA. This result showed that Chimera-E could 109 potently decreased intracellular E-RNA levels facilitated by RNase L activation, which inspired us 110 to develop 4A2-5-ASO chimeras for spike protein, a more promising target to inhibit SARS-CoV-111 2 infection.

The on-target effects of three previously designed chimeric oligonucleotides (Table S1) against 113 the spike RNA (S-RNA) of SARS-CoV-2 were evaluated in Vero cells using RT-qPCR (Fig. 3A) . 114 After co-transfection of pCAG-nCoV-S-FLAG plasmids and 80 nM oligonucleotides for 24 hours, 115 all chimeras (Chimera-S) and antisense oligonucleotides (ASO-S) decreased S-RNA down to less 116 than 50% level of negative control. Comparing ASO-S and Chimera-S containing the same 117 antisense oligonucleotide sequence, more than 2-fold enhancement of S-RNA degradation was 118 observed for Chimera-S that was able to activate endogenous RNase L by 4A2-5 moiety. Among Chimera-S4 was more potent to reduce titer of pseudovirus. At 40 nM and 80 nM, Chimera-S4 137 treatment reduced luminescence to 24% and 6% respectively, comparing to the corresponding 138 control group, while firefly luminescence was down to 45% and 14% for Chimera-S5, 50% and 139 28% for Chimera-S6 at the same concentrations (Fig. 3D) . Meanwhile, scrambled oligonucleotide 140 showed no inhibition of pseudovirus, confirming the on-target effect of Chimera-S. GFP level of 141 HEK293T-hACE2 was also monitored and Chimera-S4 also showed the most promising inhibition 142 efficiency ( Fig. 3E) , which was consistent with luciferase assay. These results clearly showed that 143 Chimera-S4 was the most effective and promising antiviral candidate among above Chimera-S 144 oligonucleotides and could be used for further assessment. 146 We further investigated the concentration dependence of Chimera S4 for S-RNA degradation. qPCR results showed that 20 nM Chimera-S4 induced a reduction of S-RNA up to 80%. Increasing 148 its concentration to 80 nM only led to slight enhancement of S-RNA reduction, but would cause 149 an approximately 2-fold up-regulation of RNase L expression (Fig. 4A, 4B) . Surprisingly, the titers 150 of SARS-CoV-2 pseudovirus dropped sharply from 60% to 6% when the concentration of 151 Chimera-S4 increased from 20 to 80 nM (Fig. 4C) . In comparison to the individual ASO-S4 and 152 4A2-5, Chimera-S4 degraded S-RNA in Vero cells with up to 4.5-and 2.1-fold higher efficiency at 153 Page 6 of 20 40 nM concentration (Fig. 4A ). In addition, 2.9-and 1.4-fold higher upregulation of RNase L were 154 also observed upon 80 nM Chimera S4 treatment (Fig. 4B) . Compared with physically mixed 4A2-155 5 and ASO-S4 (4A2-5 + ASO-S4), Chimera-S4 led to similar reduction of S-RNA in Vero cells at 156 20 nM ~ 80 nM concentrations. However, the result of luciferase assays showed that Chimera-S4 157 displayed 3.8-and 19.2-fold higher inhibitory effects on viral titers at 40 nM and 80 nM than those 158 of 4A2-5 + ASO-S4 group in HEK293T cells (Fig. 4C) . To reconfirm the efficiency of Chimera-159 S4, GFP expression in infected HEK293T-hACE2 cells was also analyzed by flow cytometry. The (Fig. S4) . luciferase assay showed that titer of all three 187 mutants were reduced to less than 20% after 48 hours treatment of 40 nM Chimera-S4 (Fig. 5A) , 188 which indicated a more robust inhibition of viral infection than those of 4A2-5, ASO-S4, 4A2-5 + 189 ASO-S4 and scrambled sequences. GFP fluorescence analysis in infected HEK293T-hACE2 cells 190 also displayed the same inhibiting manner as the firefly luciferase assay (Fig. 5B) . These results Spike, pspAX.2 and oligonucleotides were incubated for 48 h and GFP expression level was 392 analyzed by CytoFLEX flow cytometer (Beckman). 393 In order to confirm the titration of pseudovirus, HEK293T-hACE2 cells were seeded into 6-well 394 plates. After 24 h incubation, the medium was replaced by 1 mL fresh medium mixed with 1 mL 395 pseudovirus pLenti-FLuc-GFP filtrate. Cells were incubated for 48 h and GFP expression level 396 was analyzed by CytoFLEX flow cytometer (Beckman). 

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