CN111876415A

CN111876415A - Use of RNAi agents in the prevention or treatment of coronavirus infection

Info

Publication number: CN111876415A
Application number: CN202010615818.XA
Authority: CN
Inventors: 李斌; 任盛
Original assignee: Wuhan Niufusi Biological Technology Co ltd
Current assignee: Wuhan Niufusi Biological Technology Co ltd; Wuhan Neurophth Biotechnology Ltd Co
Priority date: 2020-06-30
Filing date: 2020-06-30
Publication date: 2020-11-03

Abstract

The application relates to an oligonucleotide, which comprises a nucleotide sequence shown in any one of SEQ ID NO.1-50 and can specifically bind to the genome RNA or mRNA of the coronavirus.

Description

Use of RNAi agents in the prevention or treatment of coronavirus infection

Technical Field

The application relates to the field of biomedicine, in particular to an application of an RNAi (ribonucleic acid interfere) medicament in preventing or treating coronavirus infection.

Background

The use of RNAi (interfering RNA) against human viral diseases has advanced to some extent. In the antiviral aspect, the RNAi strategy against the respective specific target effectively suppresses the replication or transcription of the viral RNA. The development of the new coronavirus (SARS-CoV2), a coronavirus, in the beginning of the 21 st century, poses a serious threat to global public health, social stability and economic development. Thus, there is still an unmet medical need for prophylaxis or treatment against coronaviruses.

Disclosure of Invention

The present application provides an oligonucleotide comprising a nucleic acid sequence selected from the group consisting of:

(a) the nucleic acid sequence has a sequence identity of not less than 80% to a sequence selected from SEQ ID NOs: 1-50;

(b) the nucleic acid sequence has a sequence identity of not less than 85% to a sequence selected from the group consisting of SEQ ID NOs: 1-50;

(c) the nucleic acid sequence has no less than 90% identity to a sequence selected from SEQ ID NOs: 1-50; and

(d) the nucleic acid sequence has no less than 95% identity to a sequence selected from the group consisting of SEQ ID NOs: 1-50.

In certain embodiments, the oligonucleotide comprises a sequence selected from SEQ ID NOs: 1-50.

In certain embodiments, the oligonucleotide is a chemically modified oligonucleotide.

The present application also provides a viral vector comprising an oligonucleotide as described herein.

In certain embodiments, the viral vector is an adeno-associated virus, lentivirus, retrovirus, or adenovirus.

In certain embodiments, the viral vector comprises a U6 or H1 promoter, and expression of the oligonucleotide is regulated by the promoter.

In certain embodiments, the viral vector is an adeno-associated virus, and the serotype of the adeno-associated virus is one or more of a wild type and a mutant of AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh 10.

The application also provides an oligonucleotide as described herein or a viral vector as described herein for use in the prevention or treatment of a disease caused by a coronavirus infection.

In certain embodiments, the oligonucleotide or viral vector is capable of specifically binding to and degrading the genomic RNA or mRNA of the coronavirus.

In certain embodiments, the coronavirus is a atypical pneumonia virus (SARS-Cov), a middle east respiratory syndrome virus (MERS), or a novel coronavirus (SARS-Cov 2).

The present application also provides an oligonucleotide as described herein or a viral vector as described herein for use in the prevention or treatment of a disease caused by a coronavirus infection.

The application also provides the use of the oligonucleotide described herein or the viral vector described herein for the preparation of a medicament for the prevention or treatment of a disease caused by a coronavirus infection.

The present application also provides a pharmaceutical formulation comprising an oligonucleotide as described herein or a viral vector as described herein, and pharmaceutically acceptable carriers and excipients.

In certain embodiments, the pharmaceutical excipient is a nanocarrier and/or a liposome.

In certain embodiments, the pharmaceutical formulation is a liquid formulation.

The application also provides the use of a pharmaceutical formulation as described herein for the prevention or treatment of a disease caused by a coronavirus infection.

In certain embodiments, the pharmaceutical agent is capable of specifically binding to and degrading genomic RNA or mRNA of the coronavirus.

The present application also provides a pharmaceutical formulation as described herein for use in the prevention or treatment of a disease caused by a coronavirus infection.

The application also provides the use of the pharmaceutical formulation described herein in the manufacture of a medicament for the prevention or treatment of a disease caused by a coronavirus infection.

The nucleic acid molecules, vectors and/or pharmaceutical products of the present application have one or more of the following effects: can bind to the genomic RNA and/or mRNA of the new coronavirus, can degrade the viral RNA or inhibit translation or replication of the viral RNA, can prevent and/or inhibit infection by the new coronavirus, and/or can exert an anti-coronavirus effect.

Other aspects and advantages of the present application will be readily apparent to those skilled in the art from the following detailed description. Only exemplary embodiments of the present application have been shown and described in the following detailed description. As those skilled in the art will recognize, the disclosure of the present application enables those skilled in the art to make changes to the specific embodiments disclosed without departing from the spirit and scope of the invention as it is directed to the present application. Accordingly, the descriptions in the drawings and the specification of the present application are illustrative only and not limiting.

Drawings

The specific features of the invention to which this application relates are set forth in the appended claims. The features and advantages of the invention to which this application relates will be better understood by reference to the exemplary embodiments described in detail below and the accompanying drawings. The drawings are briefly described as follows:

FIG. 1 shows an AAV-shRNA-1 vector map comprising AAV 23 'ITR, U6 promoter, shRNA-1 targeting SARS-Cov2, and AAV 25' ITR as described herein.

FIG. 2 shows an AAV-shRNA-2 vector map comprising AAV 23 'ITR, U6 promoter, shRNA-2 targeting SARS-Cov2, and AAV 25' ITR as described herein.

FIG. 3 shows the mean luciferase activity and standard deviation of the results of a screening of 50 oligonucleotide sequences as described herein; co-transforming any one of oligonucleotides shRNA-1 to shRNA-50 targeting coronavirus sequences of different experimental groups or oligonucleotide shNC of random sequence of a control group 1 and dual-luciferase report plasmid containing a new coronavirus target sequence into 293 cells, and detecting luciferase activity after 24 hours.

FIG. 4 shows that RNAi agents described herein can inhibit infection of a new coronavirus in vitro; FIG. 4A shows the detection of titer of live viruses of human A549 cells infected with new coronavirus treated by control group 1 (AAV-shNC vector group with random sequence), experimental group A (AAV-shRNA-1 vector group described in this application), and experimental group B (AAV-shRNA-2 vector group described in this application); FIG. 4B shows the detection of RNA levels of viral polymerase RDRP from the treatment of human A549 cells infected with new coronavirus with control group 1 (AAV-shNC vector group with random sequences), experimental group A (AAV-shRNA-1 vector group described herein), experimental group B (AAV-shRNA-2 vector group described herein), and control group 2 (AAV-shRNA-80 vector group targeting new coronavirus sequences but having no effect on new coronavirus infection); FIG. 4C shows the detection of cell activity by control group 1 (AAV-shNC vector group with random sequences), experimental group A (AAV-shRNA-1 vector group described herein), and experimental group B (AAV-shRNA-2 vector group described herein); FIG. 4D shows the measurement of the titer of live viruses of human A549 cells infected with new coronaviruses after 12 hours (-12 hours), 6 hours (-6 hours) or simultaneously with (0 hours) infection of the cells with new coronaviruses, treated with control group 1 (AAV-shNC vector group of random sequences, only 0 hours), experimental group A (AAV-shRNA-1 vector group described in the present application), experimental group B (AAV-shRNA-2 vector group described in the present application), and control group 2 (AAV-shRNA-80 vector group targeting new coronaviruses but having no effect on infection with new coronaviruses), wherein the statistical significance is indicated by an asterisk: denotes P <0.05, n.s. no significant difference.

FIG. 5 shows that RNAi agents described herein can inhibit the infection of mice by new coronavirus; FIG. 5A shows the detection of the effect of treatment with control 1 (AAV-shNC vector group with random sequences), experimental A (AAV-shRNA-1 vector group described herein), experimental B (AAV-shRNA-2 vector group described herein), and control 2 (AAV-shRNA-80 vector group targeting a new coronavirus sequence but having no effect on infection with the new coronavirus) on the titer of the new coronavirus in the serum of mice; FIG. 5B shows the measurement of the effect of control 1 (AAV-shNC vector group with random sequences), experimental group A (AAV-shRNA-1 vector group described herein), experimental group B (AAV-shRNA-2 vector group described herein), and control 2 (AAV-shRNA-80 vector group targeting a new coronavirus sequence but having no effect on infection with the new coronavirus) on the body weight of mice; FIG. 5C shows the detection of the effect of treatment of control 1 (AAV-shNC vector group of random sequences), experimental group A (AAV-shRNA-1 vector group described herein), experimental group B (AAV-shRNA-2 vector group described herein), control 2 (AAV-shRNA-80 vector group targeting the neocoronavirus sequence but having no effect on the neocoronavirus infection) on viral RNA content in lung tissue of mice infected with the neocoronavirus, where statistical significance is indicated by asterisks: denotes P <0.05, n.s. no significant difference.

Detailed Description

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification.

Definition of terms

In the present application, the term "percent identity" in the context of two or more nucleic acid sequences generally refers to sequence identity between two molecules, e.g., between two nucleic acid molecules, e.g., between two DNA molecules or between two RNA molecules. When a subunit position is occupied by the same monomeric subunit in both molecules, for example when both DNA molecules are occupied by adenosine at a position, then they are homologous or identical at that position. Homology between two sequences is a direct function of the number of matched or homologous positions; for example, two sequences are 50% homologous when half the positions in the two sequences (e.g., 5 positions in a polymer 10 subunits long) are homologous; if 90% of the positions (e.g., 9 out of 10) are matched or homologous, then the two sequences are 90% homologous.

In this application, the term "oligonucleotide" generally refers to a polymer composed of a plurality of nucleotide residues (deoxyribonucleotides or ribonucleotides, or related structural variants or synthetic analogs thereof) linked by phosphodiester bonds (or related structural variants or synthetic analogs thereof). Oligonucleotides are generally short in length, typically having about 10-30 nucleotide residues.

In the present application, the term "pharmaceutically acceptable adjuvant" generally means and includes any and all solvents, dispersion media, coatings, excipients, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, which are physiologically compatible. The compositions may include pharmaceutically acceptable salts, such as acid addition salts or base addition salts (see, e.g., Berge et al (1977) Jpharm Sci66: 1-19). For example, a pharmaceutically acceptable adjuvant may be an excipient. For example, the excipient may be a nanocarrier or a liposome. For example, a pharmaceutical product comprising a pharmaceutically acceptable adjuvant may be a liquid formulation.

In the present application, the term "treatment" generally refers to interventions that attempt to alter the natural course of the treated subject and may be used prophylactically or during clinical pathology. Desirable effects include, but are not limited to, preventing occurrence or recurrence of a disease, alleviating symptoms, suppressing, reducing or inhibiting any direct or indirect pathological consequences of a disease, ameliorating or alleviating a disease state, and causing remission or improving prognosis.

In the present application, the term "coronavirus" generally refers to a positive-stranded RNA virus belonging to the family coronaviridae, the suborder coronaviridae, the order Neuroviridae, and divided into four genera, α, β, γ, and so on. The coronavirus is spherical or elliptical under electron microscope, and has regularly arranged saccular collagen fiber protrusions (such as S protein trimer) shaped like a crown. For example, the "coronavirus" may be Severe acute respiratory syndrome coronavirus (SARS-CoV), Zhongdong respiratory syndrome coronavirus (MERS-CoV), Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV2), human coronavirus 229E (HCoV-229E), human coronavirus OC43(HCoV-OC43), human coronavirus NL63(HCoV-NL63), and human coronavirus HKU1(HCoV-HKU 1). For example, the coronavirus may be SARS-CoV2, a positive-sense single-stranded RNA virus.

In the present application, the term "RNAi (RNA interference) agent" may generally include "siRNA" and/or "shRNA". "siRNA" is commonly referred to as short interfering RNA and cleaves long double-stranded RNA with ribonuclease to produce about 21nt duplexes with 3' -overhangs, which mediate sequence-specific mRNA degradation. "shRNA" (i.e., short hairpin RNA molecule) refers to an artificial single-stranded interfering RNA molecule that comprises both the sense and antisense strands of a "siRNA duplex" in a stem-loop or hairpin structure. The stem of this hairpin structure is typically in the range of 19 to 29 nucleotides in length, and the loop is typically in the range of 4 to 15 nucleotides in length. Typically, the shRNA encodes the shRNA molecule in a DNA expression vector under the control of an RNA polymerase III promoter (e.g., the U6 promoter). The siRNA is combined with a ribonuclease complex to form an RNA induced gene silencing complex (RISC), the complex depolymerizes siRNA double strands into single strands to activate the RISC depending on ATP energy release, the RISC is specifically combined with a homologous region of mRNA expressed by an exogenous gene, the RISC has the function of nuclease, the mRNA is cut at a combining part, the cutting sites are two ends which are complementarily combined with an antisense strand in the siRNA, and the cut broken mRNA is degraded immediately.

In the present application, the terms "specifically" and "binding" may generally refer to the binding of "siRNA" and/or "shRNA" to homologous regions of viral genomic RNA and/or mRNA to form sense and antisense complementary duplexes. The term "complementary" is used herein in accordance with its art-accepted meaning to refer to the ability of a particular base, nucleoside, nucleotide or nucleic acid to pair precisely. For example, adenine (a) and uracil (U) are complementary; (A) complementary to cytosine (T); guanine (G) and thymine (C) are complementary and are referred to in the art as watson-crick base pairing.

In the present application, the term "degradation" generally refers to the binding of siRNA to the ribonuclease complex to form an RNA-induced silencing complex (RISC), which specifically binds to homologous regions of viral genomic RNA and/or mRNA, and has the function of nuclease to cleave genomic RNA and/or mRNA at the binding site, i.e., at both ends of the binding site that are complementary to the antisense strand of siRNA, and then degrade the cleaved fragmented genomic RNA and/or mRNA.

In the present application, the terms "5 '" and "3'" are generally used to describe conventions for nucleic acid sequence features that are related to the position of the genetic element and/or the orientation of the event (5 'to 3'), e.g., transcription by RNA polymerase or translation by ribosomes, which proceed in the 5 'to 3' direction. Synonyms are upstream (5 ') and downstream (3'). Typically, DNA sequences, genetic maps, vector maps and RNA sequences are drawn from left to right 5 ' to 3 ' or in the 5 ' to 3 ' direction by arrows pointing in the 3 ' direction. Thus, when following this convention, 5 '(upstream) denotes the genetic element located on the left hand side, and 3' (downstream) denotes the genetic element located on the right hand side.

In the present application, the term "genomic RNA" generally refers to the genome of a virus composed of RNA. For example, the RNA that makes up the viral genome may be single-stranded or double-stranded, and may be a closed-loop molecule or a linear molecule. For example, the SARS-CoV2 genome can be about 29.9kb in full length and can possess a5 'end cap structure and a 3' end Poly A tail, as shown by data uploaded to the GISAID database (https:// www.gisaid.org /) by various institutions and hospitals worldwide. SARS-CoV2 may possess more than 10 Open Reading Frames (ORFs), wherein ORF1a and ORF1b may encode a total of 16 non-structural proteins (NSP), S, E, M, N may encode structural proteins, respectively, and the remaining ORFs may encode auxiliary non-structural proteins. NSP3 and NSP5 may be responsible for cleaving the polyprotein chain, NSP12 may be responsible for replication of the viral genome, and the S protein (Spike protein) may be responsible for recognizing host surface receptors and mediating fusion of the virus with the host cell membrane. For example, coronaviruses transcribe viral genomic RNA into mRNA for translation to produce viral proteins in the presence of a replicator/transcript.

In the present application, the term "promoter" generally refers to a region or sequence located upstream and/or downstream of the initiation of transcription that is involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. For example, the promoter may be the SV40 promoter, CMV promoter, EF 1-alpha promoter, RSV promoter, BROAD3 promoter, mouse rosa26 promoter, CAG, H1, and U6. For another example, one or more of the promoters may be CMV, CAG, and/or U6. For example, the promoter may be U6.

In the present application, the term "adeno-associated viral vector" or "AAV" generally refers to the adenovirus itself or a derivative thereof. Adeno-associated virus (AAV) generally refers to a class of single-stranded DNA viruses belonging to the genus dependovirus, the family parvoviridae. The AAV genome may comprise Inverted Terminal Repeats (ITRs) and two Open Reading Frames (ORFs) at both ends of a DNA strand. The open reading frame may include rep and cap. Rep consists of multiple overlapping genes encoding Rep proteins required for the AAV life cycle, cap contains overlapping nucleotide sequences encoding capsid proteins, which may include VP1, VP2, and VP 3. The capsid proteins interact to form the capsid. AAV has many common serotypes, 100 virus variants. In the present application, the AAV capsid, ITRs and other selected AAV components are selected from any AAV, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV8bp, AAV7M8 and AAVAnc80, variants of any known or mentioned AAV or AAV yet to be discovered or variants or mixtures thereof.

In the present application, the term "serotype" generally refers to the detection of epitopes on the capsid surface of adeno-associated viruses by serological methods and the typing of adeno-associated viruses. Adeno-associated viruses have a variety of common serotypes, 100 virus variants. In the present application, the AAV capsid, ITRs and other selected AAV components are selected from any AAV, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV8bp, AAV7M8 and AAVAnc80, variants of any known or mentioned AAV or AAV yet to be discovered or variants or mixtures thereof.

In the present application, the term "hybridizing" under "stringent conditions generally refers to membrane washing conditions after" hybridizing "of nucleotide sequences on a membrane. For example, in the art, a low stringency wash can be about 150ml of wash solution poured into a hybridization tube, the hybridization membrane placed in, and the shaking continued at room temperature for about 20 minutes, while a high stringency wash can be about 200ml of wash solution poured into a hybridization tube, the hybridization membrane placed in, and the shaking continued at 50 ℃ for about 20 minutes. The term "hybridizing" or "hybridization" is understood to mean that two nucleic acid strands (e.g., an oligonucleotide and a target nucleic acid) form hydrogen bonds between base pairs on opposite strands, thereby forming a duplex. The affinity of the binding between two nucleic acid strands is the strength of hybridization. Generally described by melting temperature (Tm), which is defined as the temperature at which half of the oligonucleotide is duplexed with the target nucleic acid.

In this application, the term "modified" oligonucleotide generally refers to a unit in a nucleic acid polymer that contains a modified base, sugar, or phosphate group or incorporates a non-natural moiety in its structure. Examples of non-natural nucleotides include nucleotides with modified nitrogen bases, e.g., alkylated or other substituents with groups not present in conventional nitrogen bases involved in Watson-Crick pairing. By way of illustration, and not limitation, modified nucleotides include those having a methyl, ethyl, benzyl, or butyl-benzyl substituted base.

In the present application, the term "liposome" generally refers to a vesicle composed of one or more centripetally arranged lipid bilayers, which encloses an aqueous phase. The aqueous phase typically contains the compound to be delivered to the cell, e.g., comprising a nucleic acid molecule and/or vector as described herein.

In the present application, the term "nanocarrier" generally refers to a delivery system that is being investigated as a drug, in particular as a possible sustained release system for targeting a drug to a specific site of action within a patient. The term "nanocarrier" is generally used to designate polymer-based particles having diameters in the nanometer range. Nanocarriers include particles of different structures, such as nanospheres and nanocapsules. Nanocarriers based on biocompatible and biodegradable polymers such as poly (alkyl cyanoacrylates) have been studied in the last three decades and are of particular interest for biomedical applications (cf. Couvreur et al, J Pharm Pharmacol,1979,31: 331-332; Vauthier et al, adv. drug Deliv. Rev.2003,55: 519-548). They can be prepared by miniemulsion polymerization (see, e.g., Reimold et al, Eur.J.Pharm.Biopharm.2008,70: 627-632; Vauthier et al, adv.drug Deliv.Rev.2003,55:519-548), and their surfaces can be modified in different ways that allow nanocarriers to accumulate in specific target organs or tissues (see Vauthier et al, adv.drug Deliv.Rev.2003,55: 519-548). In addition, it has been shown that nanocarriers coated with polysorbate 80 transport drugs that are not normally able to cross the blood brain barrier across this barrier (see WO 2007/088066; Kreuter et al, J.drug target.2002,10(4): 317-.

Detailed Description

Nucleic acid molecules

In one aspect, the present application provides a nucleic acid molecule, such as a siRNA and/or shRNA. For example, the siRNA molecule may be about 10 to 60 nucleotides or more (or nucleotide analogs), about 15 to 25 nucleotides (or nucleotide analogs), or about 19 to 23 nucleotides (or nucleotide analogs) in length. The nucleotide (or nucleotide analog) length of the siRNA molecule can be about 10-20, 20-30, 30-40, 40-50, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 nucleotides (or nucleotide analogs). For example, the siRNA molecule may be 19 nucleotides in length. For example, the siRNA molecule may be 20 nucleotides in length. It is understood that all ranges and values encompassed within the above-described ranges are within the scope of the invention. The siRNA may include a5 'terminal phosphate and a short overhang of about 1 or 2 nucleotides of a 3' terminal phosphate. For example, the nucleic acid molecules of the present application can be synthetic short hairpin rna (shRNA) or expressed shRNA.

In another aspect, siRNA and/or shRNA directed cleavage of target viral RNA can be highly sequence specific. For example, an siRNA and/or shRNA that contains a nucleotide sequence identical to a portion of a target viral RNA. For example, the nucleotide sequence of a nucleic acid molecule of the present application may comprise the nucleotide sequence set forth in any one of SEQ ID NO. 1-50. For example, the target virus may be a coronavirus. For example, the target virus may be selected from the group consisting of: atypical pneumonia virus (SARS-Cov), middle east respiratory syndrome virus (MERS-Cov) and novel coronavirus (SARS-Cov 2). For example, the target viral RNA can be genomic RNA and/or mRNA of the virus.

In another aspect, 100% sequence identity between the siRNA and/or shRNA and the target viral RNA is not necessary to practice the present application. The nucleic acid molecules of the present application may thus have sequence variations, which may be derived, for example, from genetic mutations, strain polymorphisms, or evolutionary divergences. For example, siRNAs and/or shRNAs that may carry insertions, deletions, and single point mutations relative to a target viral RNA are effective for specifically binding to the target viral RNA. For example, siRNA sequences with nucleotide analog substitutions or insertions are effective for specifically binding target viral RNA. For example, the nucleotide sequence of the nucleic acid molecules described herein may have no less than 80% identity to a sequence selected from the group consisting of SEQ ID NOs:1-50 and be effective for specifically binding to a target viral RNA. For example, the nucleotide sequence of the nucleic acid molecules described herein may have no less than 80%, no less than 81%, no less than 82%, no less than 83%, no less than 84%, no less than 85%, no less than 88%, no less than 90%, no less than 95%, no less than 96%, no less than 97%, no less than 98%, no less than 99% identity to a sequence selected from the group consisting of SEQ ID NOs:1-50, and be effective for specifically binding to the target viral RNA. For example, the siRNA and/or shRNA are effective to specifically bind to the target viral RNA, and may substantially cause degradation of the target viral RNA.

In another aspect, the nucleic acid molecules of the present application can be provided as double-stranded molecules. For example, the siRNA of the present application may be provided as a double stranded molecule. For example, the shrnas of the present application may be provided as double stranded molecules. For example, the shRNA of the present application may be provided as a stem-loop structure molecule having a double strand. The identity and complementarity of the siRNA and/or shRNA antisense strand can be determined relative to the target viral RNA.

In another aspect, the nucleic acid molecules of the present application can reduce viral gene expression in a host cell by at least partially specifically binding to a target viral transcript, resulting in disruption of the target viral RNA by host organelle or proteosome degradation. For example, the target viral RNA can include genomic RNA and/or mRNA. The nucleic acid molecules of the present application may comprise a sequence that may be complementary to a portion of a target viral RNA to mediate RNA interference (RNAi) as defined herein, i.e. the nucleic acid molecules of the present application may be specific enough to bind to viral genomic RNA and/or mRNA to initiate degradation of the target viral RNA sequence. The nucleic acid molecules of the present application can be designed such that all or part of the residues of the antisense strand are complementary to residues of the target viral RNA, or nucleotide substitutions can be made within the nucleic acid molecule to increase the stability of the nucleic acid molecule and/or enhance the processing activity of the nucleic acid molecule. For example, substitutions may be made within the RNA strand, or at residues at the ends of the RNA strand.

In another aspect, a nucleic acid molecule of the present application can have one or more modifications at one or more nucleotides. For example, a modified nucleotide may refer to a nucleic acid molecule that comprises a modified base, sugar, or phosphate group or incorporates a non-natural nucleotide in its structure. Examples of non-natural nucleotides can include nucleotides with modified nitrogen bases, e.g., alkylated or other substituents with groups not present in conventional nitrogen bases involved in Watson-Crick pairing. For example, modified nucleotides include those having a methyl, ethyl, benzyl, or butyl-benzyl substituted base. For example, the 3' -residues may be stabilized against degradation, e.g., they may be selected to consist of purine nucleotides, such as adenine or guanine nucleotides. For example, a pyrimidine nucleotide may be replaced by a modified analog, such as uracil replaced by 2' -deoxythymine, and the replacement does not affect specific binding to the target viral RNA. For example, no 2' hydroxyl group may be presentSignificantly enhancing ribozyme resistance of siRNA in tissue culture media. For example, the phosphodiester bond of a native RNA can be modified to include at least one nitrogen or sulfur heteroatom. For example, the phosphate group attached to the adjacent ribonucleotide may be replaced by a modification group such as a phosphorothioate group. For example, the 2' OH group may be substituted by a group selected from H, OR, R, halogen, SH, SR, NH₂、NHR、NR₂Or ON, wherein R is C₁-C₆Alkyl, alkenyl or alkynyl, halogen is F, CI, Br or I. For example, modified nucleobases may include, but are not limited to, uridine and/or cytidine modified at the 5-position, such as, 5- (2-amino) propyl uridine, 5-bromouridine; adenosine and/or guanosine modified at the 8-position, e.g., 8-bromoguanosine; a deazanucleotide, e.g., 7-deazaadenosine; o-and N-alkylated nucleotides, and the modification does not affect specific binding to the target viral RNA.

In one aspect, the nucleic acid molecules of the present application can be synthesized ex vivo or in vitro. The endogenous RNA polymerase of the cell may mediate transcription in vivo, or the cloned RNA polymerase may be used for transcription in vivo or in vitro. For transcription from a transgene or expression construct in vivo, regulatory regions (such as promoters, enhancers, silencers, or splice donors and acceptors) can be used to transcribe the siRNA or shRNA. Specific binding to target viral RNA can be achieved by transcription specific in an organ, tissue, or cell type, stimulation of environmental conditions (e.g., infection, stress, temperature, chemical inducers), and/or engineered transcription at developmental stage or age. By introducing the recombinant construct into a fertilized egg, an embryonic stem cell, or other pluripotent cell from a suitable organism, a transgenic organism can be produced that expresses siRNA from the recombinant construct. For example, siRNAs or shRNAs can also be enzymatically replicated and amplified by host cells (Alberts, et al, The Cell 452 (4 th edition. 2002)).

In another aspect, the nucleic acid molecules of the present application can be enzymatically or partially/completely organically synthesized, and any modified nucleotides can be introduced via in vitro enzymes or organic synthesis. For example, the siRNA or shRNA described herein can be chemically prepared. Methods for synthesizing RNA are known in the art, particularly for Verma and Eckstein, Ammul rev. biochem.67: 99-134 (1998). For example, the siRNA or shRNA described herein can be prepared by enzymatic digestion. For example, the sirnas or shrnas described herein can be prepared by enzymatic treatment of long dsrnas that have sufficient complementarity to the target viral RNA. Treatment of long dsrnas can be achieved in vitro, e.g., using a suitable cell lysis enzyme, siRNA or shRNA described herein can be subsequently purified via gel electrophoresis or gel filtration. For example, the sirnas or shrnas described herein can be purified from the mixture by solvent or resin extraction, precipitation, electrophoresis, chromatography, or combinations thereof. For example, the sirnas or shrnas described herein may be unpurified or minimally purified to avoid losses due to sample handling.

In another aspect, the siRNA or shRNA described herein can be prepared by enzymatic transcription from a synthetic DNA template or from a DNA plasmid isolated from a recombinant bacterium. For example, phage RNA polymerases such as T7, T3 or SP6 RNA polymerase (Milligan and Uhlenbeck, Methods enzymol.180: 51-62(1989)) may be used. The siRNA or shRNA described herein can be dried for storage or dissolved in an aqueous solution. The solution may contain buffers or salts to inhibit annealing, and/or to promote stabilization of the single strands.

According to the principle of RNAi technology, a person skilled in the art can reasonably conclude that RNAi drugs of the application with target spots corresponding to shRNA-1 to shRNA-50 may have similar effects on target RNAs of the novel coronavirus. According to the characteristics of different virus vectors, a person skilled in the art can reasonably conclude that shRNAs expressed by different types of virus vectors have similar anti-neocoronavirus effects with shRNAs expressed by AAV vectors. The effect of the RNAi drug of the present application against the coronavirus type new coronavirus (SARS-Cov2) is demonstrated by examples, while the RNAi drug of the present application targets the conserved sequence of coronavirus. For example, the conserved sequence may be the full length or a portion of a sequence that matches more than 20 (including 20) base pairs consecutively, obtained by aligning the disclosed sequences of different species of coronaviruses. For example, the conserved sequence may be a full-length or partial sequence that is a contiguous match of more than 20 (including 20) base pairs obtained by aligning any SARS-Cov2 sequence from GenBank accession number MT135041-MT135044 as a template with a plurality of published SARS-Cov and MERS sequences. For example, the conserved sequence may be the full length or part of a sequence that matches 20 or more (including 20) base pairs consecutively obtained by aligning a SARS-Cov2 sequence with a plurality of published SARS-Cov and MERS sequences using as a template a SARS-Cov2 sequence with GenBank accession number MT 135041. For example, the conserved sequence may be the full length or part of a sequence that is obtained by aligning a SARS-Cov2 sequence from GenBank accession number MT135043 as a template with a plurality of published SARS-Cov and MERS sequences and then continuously matching more than 20 (including 20) base pairs. For example, the conserved sequence may be the sequence targeted by the sequences shown in SEQ ID Nos. 1-50 and 53 of the present application. Through sequence comparison, different types of coronaviruses in the coronavirus family, such as SARS-Cov, SARS-Cov2, MERS and the like, are targets of the RNAi medicine, so that according to the characteristics of RNAi technology, a person skilled in the art can reasonably conclude that the RNAi medicine has similar effects on other coronaviruses with the same and/or complementary target sequences.

Reference nucleic acid molecule

In one aspect, the present application provides a reference nucleic acid molecule that can hybridize under stringent conditions to a nucleic acid molecule described herein. For example, hybridization under stringent conditions describes an interaction that maintains sufficient stability under highly stringent conditions as is recognized in the art. Guidance for performing hybridization reactions can be found, for example, in Current protocols in Molecular Biology (John Wiley & Sons, N.Y.,6.3.1-6.3.6,1989), the entire contents of which are incorporated herein by reference. See also Sambrook, Russell and Sambrook, molecular cloning A Laboratory Manual, 3 rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 2001. Aqueous and non-aqueous methods are described in the literature, either of which may be used. Generally, stringent conditions are defined to various degrees, such as low stringency conditions (e.g., 6 x sodium chloride/sodium citrate (SSC) at about 45 ℃ followed by two washes in 0.2 x SSC, 0.1% SDS at least 50 ℃ (for medium-low stringency conditions, the wash temperature can be raised to 55 ℃; medium stringency conditions (e.g., 6 x SSC at about 45 ℃ followed by one or more washes in 0.2 x SSC, 0.1% SDS at 60 ℃); high stringency hybridization conditions (e.g., 6 x SSC at about 45 ℃ followed by one or more washes in 0.2 x SSC, 0.1% SDS at 65 ℃); and very high stringency hybridization conditions (e.g., 0.5M sodium phosphate, 0.1% SDS at 65 ℃, followed by one or more washes in 0.2 x SSC, 1% SDS at 65 ℃), hybridization conditions that only hybridization under very high stringency conditions occur between sequences that are of very high complementarity, the parameters of conditions of varying degrees of stringency will generally vary depending on various factors, such as the length of the hybridizing sequence, whether it contains RNA or DNA, and the like. For example, shorter sequences (e.g., oligonucleotides) will hybridize at lower temperatures than longer sequences under high, medium, or low stringency conditions.

In another aspect, the reference nucleic acid molecule can comprise a nucleotide sequence set forth in any one of SEQ ID nos. 1-50.

Carrier

In one aspect, the present application provides a vector that can express one or more nucleic acid molecules comprising a nucleotide sequence as set forth herein that is sufficiently complementary to a portion of a target viral RNA to mediate RNAi, e.g., the vector can comprise a nucleotide sequence as set forth in any one of SEQ ID nos. 1-52. For example, the vector can be administered in vivo to express one or more copies of a nucleic acid molecule described herein to initiate RNAi treatment or prevention. For example, the synthetic siRNA or shRNA described herein may be expressed in a plasmid vector. For example, the plasmid may replicate in vivo. For example, the vector may be a viral vector. For example, the carrier may be selected from the group consisting of: adeno-associated virus, lentivirus, retrovirus, and adenovirus.

In another aspect, the vector may comprise one or more promoters. For example, one or more of the promoters may be selected from the group consisting of: CMV, CAG, H1, and U6. For example, the promoter may be U6. For example, the one or more U6 can be located anywhere in the vector and the U6 can initiate expression of a nucleic acid molecule described herein. For example, U6 may precede the sequence of the nucleic acid molecules described herein. For example, the 3 'end of the U6 is linked to the 5' end of a nucleic acid molecule described herein.

In another aspect, the vector may comprise one or more AAV inverted terminal repeat ITRs. For example, the one or more AAV inverted terminal repeat ITRs may be located anywhere in the vector, and the AAV inverted terminal repeats ITRs are capable of stabilizing the structure of the nucleic acid molecules described herein. For example, the AAV inverted terminal repeat ITRs can be located upstream and/or downstream from a near start site. For example, the 3 'end of the AAV inverted terminal repeat ITR can be linked to the 5' end of the promoter. For example, the 3 'end of one or more of the nucleic acid molecules set forth in SEQ ID Nos. 1-50 can be linked to the 5' end of the downstream AAV inverted terminal repeat ITR.

For example, the vectors of the present application may comprise 2 AAV inverted terminal repeat ITRs. For example, the 3 'end of the upstream AAV inverted terminal repeat ITR may be linked to the 5' end of the promoter. For example, the 3 'end of the nucleic acid molecule can be linked to the 5' end of the downstream AAV inverted terminal repeat ITRs. For example, the 3 'end of the upstream AAV inverted terminal repeat ITRs may be linked to the 5' end of the promoter and the 3 'end of the nucleic acid molecule may be linked to the 5' end of the downstream AAV inverted terminal repeat ITRs.

For example, the vectors of the present application may comprise the U6 promoter, one or more of the nucleic acid molecules shown in SEQ ID nos. 1-50, 2 AAV inverted terminal repeat ITRs. For example, the 3 'end of the upstream AAV inverted terminal repeat ITRs may be linked to the 5' end of the U6 promoter, the 3 'end of the U6 promoter may be linked to the 5' end of one or more of the nucleic acid molecules, and the 3 'end of the one or more nucleic acid molecules may be linked to the 5' end of the downstream AAV inverted terminal repeat ITRs.

For example, the AAV-shRNA-1 vector of the present application, as shown in SEQ ID No.51, comprises the U6 promoter, the shRNA-1 shown in SEQ ID No.1, 2 AAV inverted terminal repeats ITRs, the 3 'end of the upstream AAV inverted terminal repeats ITRs can be ligated to the 5' end of the U6 promoter, the 3 'end of the U6 promoter can be ligated to the 5' end of the shRNA-1, and the 3 'end of shRNA-1 can be ligated to the 5' end of the downstream AAV inverted terminal repeats ITRs.

For example, the AAV-shRNA-2 vector of the present application, as shown in SEQ ID No.52, comprises the U6 promoter, the shRNA-2 shown in SEQ ID No.2, 2 AAV inverted terminal repeats ITRs, the 3 'end of the upstream AAV inverted terminal repeat ITR may be ligated to the 5' end of the U6 promoter, the 3 'end of the U6 promoter may be ligated to the 5' end of the shRNA-2, and the 3 'end of the shRNA-2 may be ligated to the 5' end of the downstream AAV inverted terminal repeat ITR.

In another aspect, the vector may be selected from adeno-associated viruses. For example, the serotype of the vector may be selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV8bp, AAV7M8, AAVrh10, and/or AAVAnc80, or variants and combinations thereof. For example, the adeno-associated virus can be selected from AAV2, AAV5, AAV7, and/or AAV8, or a combination thereof. For example, the adeno-associated virus can be selected from AAV 2.

For example, the AAV2 vector can be AAV2/2, AAV2/5, AAV2/8, or AAV 2/9. For example, the AAV2 vector can comprise pAAV-RC5-Amp, RC8-cap, AAV2/8, AAV-helper-Amp, AAV-helper.

On the other hand, some target viral RNAs mutate rapidly and may result in even one nucleotide mismatch that may in some cases interfere with the RNAi of the present application. Thus, the present application provides vectors that can express a variety of siRNAs or shRNAs as described herein to increase the probability of homology sufficient to mediate RNAi. For example, the complementary sequences of these siRNAs or shRNAs described herein may be staggered along the target viral RNA or may be concentrated in a region of the target viral RNA. For example, various siRNAs or shRNAs described herein may be directed against a region of a target viral RNA that is about 200 nucleotides in length and includes 3' end of the target viral RNA. For example, the one or more RNAs expressed by the vector may be an siRNA or shRNA as described herein. The sirnas or shrnas described herein may be staggered along a portion of the target viral RNA or may target different portions of the target viral RNA. For example, the vector may encode about 3 sirnas or shrnas as described herein, and about 5 sirnas or shrnas as described herein. For example, the vector-encoded siRNA can be targeted to a conserved region of the target viral RNA nucleoprotein gene.

Host cell

In one aspect, the physical method of introducing the RNAi agent (e.g., siRNA, shRNA, or vector) of the application into a host cell can comprise injecting a solution containing the RNAi agent, bombarding particles covered with the RNAi agent, soaking the cell or organism in a solution of the RNAi agent, or electroporating the cell membrane in the presence of the RNAi agent. For example, a viral construct that includes a nucleic acid molecule described herein can be introduced into a cell to express the nucleic acid molecule described herein. For example, other methods known in the art for introducing nucleic acids into cells can be used, such as lipid-mediated transport of carriers, compound-mediated transport such as calcium phosphate, and the like.

In another aspect, the host cell can be infected with the target virus at the time of introduction of the RNAi agent described herein, or exposed to the target virus after introduction of the RNAi agent described herein. For example, the host cell may be derived from or contained in any organism, e.g., may be from the germline, a somatic cell, a totipotent or pluripotent, dividing or non-dividing, parenchymal or epithelial tissue, immortalized or mutated, and the like. For example, the host cell may be a stem cell or a differentiated cell.

In another aspect, quantification of gene expression in the host cell can show a similar amount of inhibition of accumulation or expression levels of the target viral RNA. For example, the efficacy of inhibition can be determined by assessing the amount of gene product in a cell, detecting RNA with hybridization probes having nucleotide sequences outside the region for inhibiting double-stranded RNA, or fluorescent quantitative PCR detection.

In another aspect, the host cell can incorporate an amount of one or more copies of the RNAi agent described in the application. For example, the host cell can incorporate higher doses (e.g., at least 5, 10, 100, 500, or 1000 copies per cell) of the RNAi agent described herein to produce more effective inhibition.

Pharmaceutical product

In one aspect, the pharmaceutical product can comprise one or more nucleic acid molecules of the present application. For example, the pharmaceutical product may comprise one or more carriers of the present application. For example, the nucleic acid molecule may be administered to a cell or subject as a pharmaceutical product alone; or the vector may be administered to the cell or subject separately as a pharmaceutical product. For example, the pharmaceutical product may be a lyophilized oligonucleotide or viral vector, which may be dissolved in a suitable diluent, such as sterile water, for administration.

In another aspect, the pharmaceutical product may comprise one or more nucleic acid molecules of the present application or one or more adjuvants of the present application, and a pharmaceutically acceptable adjuvant or excipient. A pharmaceutically acceptable adjuvant or excipient may refer to any ingredient that is not therapeutically active and has acceptable toxicity, such as buffers, solvents, tonicity agents, stabilizers, antioxidants, surfactant nanocarriers, and/or liposomes that may be used in formulating a pharmaceutical product. For example, the pharmaceutical product may be a liquid formulation.

For example, the pharmaceutical product may comprise one or more carriers of the present application. For example, a vector can comprise a nucleic acid molecule as described herein. For example, when the pharmaceutical product comprises a vector of the present application, the pharmaceutical product may be administered by administering the vector to a cell or subject. For example, when the pharmaceutical product comprises two or more vectors of the present application, the pharmaceutical product can be administered by administering the two or more vectors to the cell or subject simultaneously. For example, when the pharmaceutical product comprises two or more vectors of the present application, the pharmaceutical product may be administered by administering the two or more vectors to the cell or subject in any sequential order.

In the present application, the subject may include humans and non-human animals. For example, the subject may include, but is not limited to, a cat, dog, horse, pig, cow, sheep, rabbit, mouse, rat, or monkey; for example, the subject may comprise a DBA/2J mouse. In the present application, the cells may comprise bacterial cells (e.g., E.coli), yeast cells, or other eukaryotic cells, such as COS cells, Chinese Hamster Ovary (CHO) cells, HeLa cells, HEK293 cells, COS-1 cells, NS0 cells, or myeloma cells, 293T cells, Vero cells, or A549 cells.

For example, the pharmaceutical product may be introduced directly into the cell (i.e., intracellularly); or introduced extracellularly into a body cavity, into the intercellular matrix, into the circulation of the organism, introduced orally, inhaled, or introduced by bathing the cells or organism in a solution containing RNA. The pharmaceutical product may be introduced into the vascular or extravascular circulation, the blood or lymphatic system, and the cerebrospinal fluid.

Preparation of medicine for preventing or treating diseases

In one aspect, the present application provides the use of a vector, a nucleic acid molecule and/or a pharmaceutical product for the manufacture of a medicament for the treatment and/or prevention of a disease caused by a coronavirus. For example, the coronavirus may be selected from the group consisting of: atypical pneumonia virus (SARS-Cov), middle east respiratory syndrome virus (MERS-Cov) and novel coronavirus (SARS-Cov 2). For example, the disease is selected from the group consisting of: SARS, COVID-19 and respiratory syndrome of the middle east.

In one aspect, the present application provides a vector, a nucleic acid molecule and/or a pharmaceutical product for use in the treatment and/or prevention of a disease caused by a coronavirus. For example, the coronavirus may be selected from the group consisting of: atypical pneumonia virus (SARS-Cov), middle east respiratory syndrome virus (MERS-Cov) and novel coronavirus (SARS-Cov 2). For example, the disease is selected from the group consisting of: SARS, COVID-19 and respiratory syndrome of the middle east.

In one aspect, the present application provides the use of a vector, nucleic acid molecule and/or pharmaceutical product for the treatment and/or prevention of a disease caused by a coronavirus. For example, the coronavirus may be selected from the group consisting of: atypical pneumonia virus (SARS-Cov), middle east respiratory syndrome virus (MERS-Cov) and novel coronavirus (SARS-Cov 2). For example, the disease is selected from the group consisting of: SARS, COVID-19 and respiratory syndrome of the middle east.

In another aspect, the prevention may refer to administration that may occur prior to exhibiting symptoms typical of a viral infection, such as a viral infection. For example, administration may occur 6 hours before, 12 hours before, exhibiting viral infection. For example, the prophylactic effect can refer to a reduction in viral titer of a viral infection after administration of at least 1.5 fold, at least 2 fold, at least 2.5 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, at least 5 fold, at least 7 fold, at least 15 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 100 fold, relative to no administration or administration of a blank drug, of a pharmaceutical product described herein. For example, the prophylactic effect can refer to administration of a drug product described herein such that the RNA level of a viral gene of a viral infection is reduced by at least 1.5 fold, at least 2 fold, at least 2.5 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, at least 5 fold, at least 7 fold, at least 10 fold, at least 15 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 100 fold after administration relative to the absence or administration of a blank drug. For example, the viral gene may be the RdRp gene of a new coronavirus.

In another aspect, the treatment may refer to administration that may occur after exhibiting symptoms typical of a viral infection, such as a viral infection. For example, administration may occur at the same time as viral infection is manifested. For example, the therapeutic effect can refer to administration of a drug product described herein such that viral titer of the viral infection is reduced by at least 1.5 fold, at least 2 fold, at least 2.5 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, at least 5 fold, at least 7 fold, at least 15 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 100 fold after administration relative to no administration or administration of a blank drug. For example, the therapeutic effect can refer to administration of a drug product described herein such that the RNA level of a viral gene of a viral infection is reduced by at least 1.5 fold, at least 2 fold, at least 2.5 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, at least 5 fold, at least 7 fold, at least 10 fold, at least 15 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 100 fold after administration relative to the absence or administration of a blank drug. For example, the viral gene may be the RdRp gene of a new coronavirus.

Embodiments of the present application:

1. an isolated nucleic acid molecule comprising a nucleotide sequence set forth in any one of SEQ ID nos. 1-50.

2. The nucleic acid molecule according to embodiment 1, which specifically binds to genomic RNA and/or mRNA of a coronavirus.

3. The nucleic acid molecule according to any one of embodiments 1-2, which degrades genomic RNA and/or mRNA of a coronavirus.

4. The nucleic acid molecule according to any one of embodiments 2-3, wherein the coronavirus is selected from the group consisting of: atypical pneumonia virus (SARS-Cov), middle east respiratory syndrome virus (MERS-Cov) and novel coronavirus (SARS-Cov 2).

5. The nucleic acid molecule according to any one of embodiments 1 to 4, which is a double-stranded nucleic acid molecule.

6. The nucleic acid molecule according to any one of embodiments 1 to 5, which is an siRNA or shRNA.

7. The nucleic acid molecule according to any one of embodiments 1 to 6, which hybridizes under stringent conditions with a reference nucleic acid molecule comprising the nucleotide sequence set forth in any one of SEQ ID No.1 to 50.

8. The nucleic acid molecule according to any one of embodiments 1-7, wherein the oligonucleotide comprises a modification.

9. A vector comprising the nucleic acid molecule of any one of embodiments 1-8.

10. The vector of embodiment 9, comprising a viral vector.

11. The vector of embodiment 10, wherein the viral vector is selected from the group consisting of: adeno-associated virus, lentivirus, retrovirus, and adenovirus.

12. The vector according to any one of embodiments 9-11, wherein the vector comprises an adenoviral vector having a serotype selected from the group consisting of: AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAV9, and AAVrh 10.

13. The vector according to any one of embodiments 9-12, comprising a promoter.

14. The vector of embodiment 13, wherein the promoter is selected from the group consisting of: CMV, CAG, H1, and U6.

15. The vector according to any one of embodiments 9-14, comprising an AAV inverted terminal repeat ITR.

16. The vector of embodiment 15, wherein the 3 'end of the upstream AAV inverted terminal repeat ITRs is linked to the 5' end of the promoter.

17. The vector according to any one of embodiments 13-16, wherein the 3 'end of the promoter is linked to the 5' end of the nucleic acid molecule.

18. The vector according to any one of embodiments 15-17, wherein the 3 'end of the nucleic acid molecule is linked to the 5' end of the downstream AAV inverted terminal repeat ITRs.

19. The vector according to any one of embodiments 9-18, comprising a nucleotide sequence set forth in any one of SEQ ID nos. 1-52.

20. An isolated host cell comprising the vector of any one of embodiments 9-19.

21. A pharmaceutical product comprising a nucleic acid molecule according to any one of embodiments 1 to 8, and/or a vector according to any one of embodiments 9 to 19, and a pharmaceutically acceptable adjuvant.

22. The pharmaceutical product according to embodiment 21, wherein the pharmaceutically acceptable adjuvant comprises an excipient.

23. The pharmaceutical product of embodiment 22, wherein the excipient comprises a nanocarrier and/or a liposome.

24. A pharmaceutical product according to any one of embodiments 21-23, which is a liquid formulation.

25. A nucleic acid molecule according to any one of embodiments 1 to 8, a vector according to any one of embodiments 9 to 19, and/or a pharmaceutical product according to any one of embodiments 21 to 24 for use in the preparation of a medicament for the treatment and/or prevention of a disease caused by a coronavirus.

26. The use of embodiment 25, wherein the coronavirus is selected from the group consisting of: atypical pneumonia virus (SARS-Cov), middle east respiratory syndrome virus (MERS-Cov) and novel coronavirus (SARS-Cov 2).

27. The use according to any one of embodiments 25-26, wherein the disease is selected from the group consisting of: SARS, COVID-19 and respiratory syndrome of the middle east.

Examples

Example 1 RNAi drug inhibition of New coronavirus infection human A549 cells

AAV-shRNA-1 to AAV-shRNA-50 viral vectors. The vector genome comprises an AAV inverted terminal repeat ITR, a nucleic acid sequence selected from shRNA-1 to shRNA-50 (shown as SEQ ID Nos: 1-50), and an AAV inverted terminal repeat ITR, wherein the U6 promoter initiates transcription. As shown in FIG. 1, the AAV-shRNA-1 vector has a characteristic map (shown in SEQ ID No: 51), and as shown in FIG. 2, the AAV-shRNA-2 vector has a characteristic map (shown in SEQ ID No: 52). AAV-shRNA-3 to AAV-shRNA-50 vectors containing shRNA-3 to shRNA-50 sequences (shown in SEQ ID Nos: 3-50), AAV-shNC vector of control 1 containing a random sequence shNC, and AAV-shRNA-80 vector of control 2 containing shRNA-80 against a novel coronavirus sequence (shown in SEQ ID No: 53), but having No influence on infection with the novel coronavirus were constructed with reference to the above example.

Culture of mammalian cells (adherent). And (5) recovering the cells. Preparing warm water at 37-38 ℃, taking human A549 cells needing to be revived out of a liquid nitrogen tank, fixing the cryopreservation tube by using forceps for ophthalmic surgery, and quickly placing the cryopreservation tube in water to ensure that the cryopreservation tube is completely immersed in the water and uniformly heated until the cells in the cryopreservation tube are completely melted. The cryopreservation tube was sterilized with alcohol. 5ml of the cell culture medium T25-based cell culture flask was previously pipetted with a pipette, and the thawed cells were transferred to the cell flask with a new pipette and gently blown up once. Covering the cell bottle cap, placing the cell bottle in a cell culture box, and culturing at 37 deg.C with 5% CO₂And (5) standing and culturing. After 6-8 hours, the fresh medium was replaced to eliminate the effect of DMSO remaining in the cell culture on cell growth. And (5) carrying out cell passage. When the human a549 cells grew out of the T25 cell flask, the medium was aspirated with a pipette and discarded. 10ml of PBS was added, the cells were washed gently and then aspirated with a pipette and discarded. Sucking 1-1.5ml pancreatin with a pipette,covering the bottom of the cell bottle, placing the cell bottle at 37 deg.C and 5% CO₂The cell incubator is stationary for 3-5 minutes. Microscopic observation revealed that adherent cells rounded and all detached from the cell bottle wall. In the cell manipulation station, about 4ml of the medium was aspirated with a pipette, added to the cell vial, and gently whipped to break up the cells and neutralize the digestive effects of pancreatic enzymes. Sucking the uniformly blown cell suspension (about 1/3-2/3 in volume) with a pipette into another new cell bottle, adding 5ml of culture medium, placing in a cell culture box, maintaining at 37 deg.C and 5% CO₂The static culture was continued in the environment of (1).

And (4) detecting luciferase activity. At 48 hours post-transfection, the detection procedure was performed according to the instructions of Dual-GloTM luciferaseeassay system (Promega, USA) and the specific experimental procedures were as follows: the cell culture plate was removed from the incubator, the medium was aspirated off, washed once with PBS, lysed by adding the corresponding volume of PLB to the corresponding well plate, and incubated on a horizontal shaker at room temperature for 15 minutes. And (3) taking 20 mu l of cell lysate to a 96-well enzyme label plate, adding 100 mu l of luciferase detection reagent II (LARII), mixing uniformly, and detecting a luciferase (luciferase) chemiluminescence signal by using an enzyme label instrument. After the detection is finished, 100 mu l of Stop reagent (Stop Substrate) is added into each hole, the mixture is uniformly mixed, and a Renilla (Renilla) luciferase chemiluminescence signal is detected by a microplate reader. The data records the Ratio (Ratio) detected by the instrument, and the mean and standard deviation are calculated.

AAV-shRNA-1 to AAV-shRNA-50 plasmid vectors are respectively cotransformed with a luciferase report plasmid (synthesized by Kinry corporation) containing a new coronavirus target sequence into 293 cells, and AAV-shNC plasmid vectors with random sequences are used as a control group for 1 hour, and the inhibition effect of the plasmids on the new coronavirus target sequence is detected through luciferase activity after 48 hours. As shown in FIG. 3, the experimental groups of shRNA-1 to shRNA-50 all had different degrees of inhibitory effect on the target sequence of the novel coronavirus, compared to control group 1 of AAV-shNC vector containing random sequence.

New coronavirus (SARS-CoV2) infects A549 cells. Before the A549 cells are infected by the new coronavirus (SARS-CoV2, the construction method refers to Thao et al, "Rapid recovery of SARS-CoV-2 using a synthetic genetics plant," BioRxiv (2020) "), the complete culture medium containing fetal bovine serum is changed into a serum-free culture medium, the dose of infection of the new coronavirus is 0.05 or 0.02MOI, and after infection treatment is carried out for 24 hours, the cells and the supernatant containing the new coronavirus are collected for standby.

Reverse transcription fluorescent quantitative PCR detects the level of viral RNA. In the reverse transcription reaction system, as shown in Table 1, a dNTP (2 '-deoxynucleotide-5' -triphosphate) mixture contains four kinds of deoxynucleotides (dATP, dCTP, dGTP, dTTP), DEPC water is ultrapure water treated with DEPC (diethylpyrocarbonate) and sterilized at high temperature and high pressure; reverse transcription reaction conditions: the corresponding cDNA was obtained as template for the next RT-PCR reaction at 37 ℃ for 1 hour and 75 ℃ for 10 minutes.

TABLE 1 reverse transcription reaction System

RNA template	1.0μg
		Ribonuclease inhibitors	0.5μL
Oligomeric thymine primer	1μL
		5 × RT-PCR reaction buffer	5μL
10mM dNTP mixture	1μL
		Reverse transcriptase	1μL
Make up DEPC water to	25μL

RT-PCR reaction system, detection primer of target gene (new coronavirus RdRp (RNA-dependent RNA polymerase) gene) and internal reference (GAPDH) primer:

detection primer for RdRp 5'-CCAAGAAAAGGACGAAGATGACAAT-3' (forward primer),

5'-CGAGGTCTGCCATTGTGTATTTAGTAA-3' (reverse primer)

Internal control primer for GAPDH 5'-GGAAGGTGAAGGTCGGAGTCAACGG-3' (Forward primer)

5'-CTCGCTCCTGGAAGATGGTGATGGG-3' (reverse primer);

RT-PCR reaction system, as shown in Table 2; RT-PCR reaction procedure, as shown in Table 3.

TABLE 2 RT-PCR reaction System

SYBR green fluorescent quantitative PCR premix solution	10μL
		Detection primer (Forward + reverse)	1μL
cDNA template	1μL
		Make up water to	20μL

TABLE 3 RT-PCR reaction procedure

And (4) a virus titer determination method. The supernatant containing the new coronavirus (SARS-CoV2) is logarithmically diluted to different multiples, and virus solutions with different fold concentrations infect a single layer of Vero cells (2X 10 per well) in a 96-well plate⁴Individual cells). Cells were subsequently washed and cultured with fresh medium supplemented with 2% FBS. The cytopathic degree of the virus with different concentrations after infection for 3-5 days is recorded and scored, the cytopathic degree score of each concentration is independently repeated for at least 10 times, and the scoring person does not know the virus concentration used for infection in advance. Reed-Muench scoring, as is commonly used in the art, is used to determine the viral titer (TCID50) at which 50% of the cultured cells are infected.

The RNAi drug of the application inhibits the experiment that the new coronavirus infects human A549 cells. A549 cells were infected with 0.05MOI of the new coronavirus in a 6-well plate and treated with 6. mu.g of either RNAi drug of the present application or control RNAi (control 1 is AAV-shNC vector not directed to random sequences of the new coronavirus, control 2 is AAV-shRNA-80 plasmid directed to sequences of the new coronavirus but not affected by infection with the new coronavirus), and virus infection was examined 24 hours later. An experimental group A is an AAV-shRNA-1 vector which is transiently transfected aiming at a new coronavirus sequence and is described in the application, an experimental group B is an AAV-shRNA-2 vector which is transiently transfected aiming at the new coronavirus sequence and is described in the application, a control group 1 is an AAV-shNC vector of a random sequence, and a control group 2 is an AAV-shRNA-80 plasmid which aims at the new coronavirus sequence but has no influence on the infection of the new coronavirus.

The results are shown in FIG. 4. Fig. 4A shows that the titer of the new coronavirus is significantly reduced after the cells are treated by the experimental group a and the experimental group B, and fig. 4B shows that the RNA level of the RdRp gene of the new coronavirus in the cells is significantly reduced after the cells are treated by the experimental group a and the experimental group B, which indicates that the RNAi agent of the present application can inhibit the infection of the new coronavirus into the a549 cells. FIG. 4C shows that there was no significant change in cell viability following treatment of cells in groups A and B, indicating that RNAi drug treatment according to the present application was not significantly toxic to cells. Fig. 4D shows that when the new coronavirus infects a549 cells 12 hours ago, 6 hours ago, or the new coronavirus infects the cells, the titer of the new coronavirus is significantly reduced by treating the cells in advance in experimental group a and experimental group B using the RNAi agent of the present application and the control RNAi, indicating that there is a significant preventive effect on the new coronavirus. The results show that the RNAi medicine can effectively inhibit the new coronavirus from infecting cells, has an inhibition effect on the new coronavirus infection, and can play a role in preventing the new coronavirus infection.

Example 2 RNAi drug inhibition of New coronavirus infected mice

RNAi agents were tested for their ability to inhibit infection of mice with new coronavirus (ACE2 transgenic C57BL6 mice) in vivo. Preparation of 1X 10⁷TCID50/ml of new coronavirus. Configuration 1 × 10¹²The method comprises the following steps of taking a vg/ml AAV-shRNA-1 vector as an RNAi medicament of an experimental group A, taking an AAV-shRNA-2 vector as an RNAi medicament of an experimental group B, (wherein a control group 1 is an AAV-shNC vector with a random sequence, and a control group 2 is an AAV-shRNA-80 plasmid aiming at a new coronavirus sequence but having no influence on new coronavirus infection), injecting the control group medicament or the experimental group medicament and the new coronavirus into a mouse body through an abdominal cavity, observing the health condition of the mouse every day, weighing the mouse body, well recording the reconstruction, performing orbital blood collection, detecting the virus titer in mouse serum, killing the mouse on the 8 th day after virus infection, and analyzing the RNA content of the new coronavirus in lung tissues.

The results are shown in FIG. 5. Firstly, the influence of the RNAi drug treatment on the viral load in the mouse is detected, and the result of FIG. 5A shows that the viral titer in the serum of the mouse treated by the RNAi drug is remarkably reduced. Secondly, the influence of the RNAi drug on the health condition and the body weight of the mouse is verified, and the result of FIG. 5B shows that compared with the treatment of the control group 1 or the control group 2, the health condition and the body weight of the mouse are not significantly different after the RNAi drug treatment, which shows that the RNAi drug treatment has no obvious toxicity on the mouse. And then, detecting the content of the viral RNA in the lung tissue of the mouse after the mouse is killed on the 8 th day, wherein the result of FIG. 5C shows that the content of the viral RNA in the lung tissue of the mouse is obviously lower than that of a control group 1 or a control group 2 after the RNAi medicament treatment, and the RNAi medicament treatment can effectively inhibit the new coronavirus from infecting the mouse. By combining the results, the RNAi medicament provided by the application can block the new coronavirus from infecting mice and reduce the virus content in serum and lung tissues, so that the RNAi medicament provided by the application can effectively and efficiently inhibit the infection of the new coronavirus, discloses a new biological function of the undiscovered RNAi medicament, and can achieve the effect of resisting the infection of the new coronavirus in vivo and in vitro.

The foregoing detailed description is provided by way of illustration and example, and is not intended to limit the scope of the appended claims. Various modifications of the presently recited embodiments will be apparent to those of ordinary skill in the art and are intended to be within the scope of the appended claims and their equivalents.

Sequence listing

<110> Wuhan Newcastle Biotechnology Ltd

<120> effects of RNAi drugs in preventing or treating coronavirus infection

<130>0179-PA-003

<160>53

<170>PatentIn version 3.5

<210>1

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>shRNA-1

<400>1

gctagattcc ctaagagtga 20

<210>2

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>shRNA-2

<400>2

ggaaaggtta tggctgtagt 20

<210>3

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>shRNA-3

<400>3

gtaatgtcat ccctactata 20

<210>4

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>shRNA-4

<400>4

gtgtctctat ctgtagtact 20

<210>5

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>shRNA-5

<400>5

ggactgagac tgaccttact 20

<210>6

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>shRNA-6

<400>6

gatggtacac ttatgattga 20

<210>7

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>shRNA-7

<400>7

gcacactaga accagaatat 20

<210>8

<211>20

<212>DNA

<213> Artificial sequence (artificacial sequence)

<220>

<223>shRNA-8

<400>8

gaacaatgga acctagtaat 20

<210>9

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>shRNA-9

<400>9

ggcattcagt acggtcgta 19

<210>10

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>shRNA-10

<400>10

ggtaagatgg agagccttg 19

<210>11

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>shRNA-11

<400>11

caacctaaat agaggtatg 19

<210>12

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>shRNA-12

<400>12

ccaccttgta ggtttgtta 19

<210>13

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>shRNA-13

<400>13

ggtgactatg gtgatgctg 19

<210>14

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>shRNA-14

<400>14

gtatgccatt agtgcaaag 19

<210>15

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>shRNA-15

<400>15

catcctaatc aggagtatg 19

<210>16

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>shRNA-16

<400>16

tagccgccac tagaggagc 19

<210>17

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>shRNA-17

<400>17

ggccaaactg tcactaaga 19

<210>18

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>shRNA-18

<400>18

gaaattgacc gcctcaatg 19

<210>19

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>shRNA-19

<400>19

cctagtaata ggtttccta 19

<210>20

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>shRNA-20

<400>20

attggctact accgaagag 19

<210>21

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>shRNA-21

<400>21

gccttgaatt taggtgaaa 19

<210>22

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>shRNA-22

<400>22

gatgctccat atatagtgg 19

<210>23

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>shRNA-23

<400>23

gttctacttg caccattat 19

<210>24

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>shRNA-24

<400>24

gaagaagatt ggttagatg 19

<210>25

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>shRNA-25

<400>25

gtacttaatg agaagtgct 19

<210>26

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>shRNA-26

<400>26

gtgaggacta ttaaggtgt 19

<210>27

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>shRNA-27

<400>27

ggtcttaatg acaaccttc 19

<210>28

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>shRNA-28

<400>28

gttgcgtagt gatgtgcta 19

<210>29

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>shRNA-29

<400>29

gtcacaacat tgctttgat 19

<210>30

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>shRNA-30

<400>30

gagtcgaatg tacaactat 19

<210>31

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>shRNA-31

<400>31

gacgttcttg agtgtaatg 19

<210>32

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>shRNA-32

<400>32

gtgcacatca gtagtctta 19

<210>33

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>shRNA-33

<400>33

gctggtaatg caacagaag 19

<210>34

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>shRNA-34

<400>34

gatctcaatg gtaactggt 19

<210>35

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>shRNA-35

<400>35

gaccatgttg acatactag 19

<210>36

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>shRNA-36

<400>36

gctgtcacgg ccaatgtta 19

<210>37

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>shRNA-37

<400>37

gaagctataa gacatgtac 19

<210>38

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>shRNA-38

<400>38

gacatgtacg tgcatggat 19

<210>39

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>shRNA-39

<400>39

gtcttgaaat tccacgtag 19

<210>40

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>shRNA-40

<400>40

gctactgagg agacattta 19

<210>41

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>shRNA-41

<400>41

gtgctgtctg acagagaat 19

<210>42

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>shRNA-42

<400>42

gtgacacttg cagatgctg 19

<210>43

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>shRNA-43

<400>43

gctttaaaca cgcttgtta 19

<210>44

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>shRNA-44

<400>44

ggcataatga tgaatgtcg 19

<210>45

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>shRNA-45

<400>45

gctagtctta atggagtca 19

<210>46

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>shRNA-46

<400>46

gcacactttc ctcgtgaag 19

<210>47

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>shRNA-47

<400>47

gcacacgcct attaattta 19

<210>48

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>shRNA-48

<400>48

ggtgaaatca aggatgcta 19

<210>49

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>shRNA-49

<400>49

gaacatgtcc aaattcaca 19

<210>50

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>shRNA-50

<400>50

gctgatgagt acgaactta 19

<210>51

<211>5332

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>AAV-shRNA-1

<400>51

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120

aggggttcct gcggccgcac gcgtctagtt attaatagta atcaattacg gggtcattag 180

ttcatagccc atatatggag ttccggaggg cctatttccc atgattcctt catatttgca 240

tatacgatac aaggctgtta gagagataat tagaattaat ttgactgtaa acacaaagat 300

attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg cagttttaaa 360

attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt tcgatttctt 420

ggctttatat atcttgtgga aaggacgagg atcgctagat tccctaagag tgacacgagt 480

cactcttagg gaatctagct ttttgaattc taccggtacc gagggttaac aacaacactc 540

gagttaaggg cgaattcccg ataaggatct tcctagagca tggctacgta gataagtagc 600

atggcgggtt aatcattaac tacaaggaac ccctagtgat ggagttggcc actccctctc 660

tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg 720

cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt 780

attttctcct tacgcatctg tgcggtattt cacaccgcat acgtcaaagc aaccatagta 840

cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc 900

tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac 960

gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag 1020

tgctttacgg cacctcgacc ccaaaaaact tgatttgggt gatggttcac gtagtgggcc 1080

atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg 1140

actcttgttc caaactggaa caacactcaa ccctatctcg ggctattctt ttgatttata 1200

agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa 1260

cgcgaatttt aacaaaatat taacgtttac aattttatgg tgcactctca gtacaatctg 1320

ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg 1380

acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg 1440

catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat 1500

acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac 1560

ttttcgggga aatgtgttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc 1620

tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg 1680

gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggtgacag 1740

ttcctgttga gccacctcct tgtcagcatt tgatgttgtc agctttggat tttagccatt 1800

ctaataggtg agtagtggta catcattatt gtttcaattt gaaatggcta ctgacatatg 1860

atatggagca tctttcctta tgcttatttg ctatgtgtat attttctttg gtgaggtgtc 1920

tgctcagatc gtttgcccat tctttaattg ggttgttttc tattgagttt taacagttct 1980

ttgtgtattt tgaatgtaag tcccttatca ggtatgtccc ctgcaaatat gttctcctag 2040

tctgtagctt gtattgtcat tctcttgaca attcttttgc agagcagaag cgtctaattt 2100

taatgacaca gaacttatca attgcttctt tcaaggattg tgcttttggt gttgtatcta 2160

ataagccatc accaaactta aggtcatcta gattttctcc tacattatct tctagaagtt 2220

ttgtactcac tattcattta ggtctacaat aaacttggag ttaattttcg tgaaagccgt2280

aagggagctt ctaaggggga cacacactaa cttctttgag agtgggcagt gattgtgaaa 2340

ctctcagctg ggtcttacaa cagaccccag atccatcagg agagcccagc cttcagaacc 2400

catagtggaa tatgtttcat gcatacacgg aggagaatat gcagtcatat gctcacatgc 2460

atgtctgaac atgatcacac acatgaatat ggctatgtcc tgatccagag ttctctcatt 2520

ctctgtaagt gattctttcc ttcaacttct tcattaaggg atatattctc gttgtttagg 2580

tatttcagtg gggccggata ggaacttata tctaagtttt caggaaggtt aggtaccagg 2640

gccacggtct agtaatttag gtgagaagaa ctgagacata tatccaggtt tcaggtattt 2700

aaacgaggct ggattggagc acacatctag gtgttcagat attcaagtga agttgagttg 2760

gaagacacct gtccaggtgt ttagctcttc aggtggggct ggtatgaggt gagtgtcacc 2820

tgtctaggta tttaggcatt caagtaggct tggttggaga cagctgtcca gatacttact 2880

gggtctggat tgggggcttt tgttcaggtg ctcaggtatt aaagtggggc tggtgaagat 2940

gttgaagtgg aggggatacc tgttgaggtg tttacctatc caagtgggat gcactgggag 3000

cacctctgta aggtgtgtga ttggaaaggg ttggggaaca cttatttgtg tgtcagggaa 3060

caatctgtct agatatttat gtgtttaagg tgttggttag ggcagctgtc catgttttca 3120

ggtatttaag tgggtgaagt agagggcgct tggccagata ttccagtggg ctaatctggg 3180

acatttgtgt agatgtgaaa tgattatgat gggcctccag atttatcagc aataaaccag 3240

ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct 3300

attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt 3360

gttgccattg ctgcagccat gagattatca aaaaggatct tcacctagat ccttttcacg 3420

tagaaagcca gtccgcagaa acggtgctga ccccggatga atgtcagcta ctgggctatc 3480

tggacaaggg aaaacgcaag cgcaaagaga aagcaggtag cttgcagtgg gcttacatgg 3540

cgatagctag actgggcggt tttatggaca gcaagcgaac cggaattgcc agctggggcg 3600

ccctctggta aggttgggaa gccctgcaaa gtaaactgga tggctttctt gccgccaagg 3660

atctgatggc gcaggggatc aagctctgat caagagacag gatgaggatc gtttcgcatg 3720

attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc 3780

tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg 3840

caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcaa 3900

gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc 3960

gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat 4020

ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg 4080

cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc 4140

gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag 4200

catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgagcat gcccgacggc 4260

gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc 4320

cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata 4380

gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc 4440

gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac 4500

gagttcttct gactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt 4560

catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc 4620

ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct 4680

tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta 4740

ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc 4800

ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac 4860

ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct 4920

gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat 4980

aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg 5040

acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa 5100

gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg 5160

gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga 5220

cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc 5280

aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gt 5332

<210>52

<211>5332

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>AAV-shRNA-2

<400>52

cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt 60

ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120

aggggttcct gcggccgcac gcgtctagtt attaatagta atcaattacg gggtcattag 180

ttcatagccc atatatggag ttccggaggg cctatttccc atgattcctt catatttgca 240

tatacgatac aaggctgtta gagagataat tagaattaat ttgactgtaa acacaaagat 300

attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg cagttttaaa 360

attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt tcgatttctt 420

ggctttatat atcttgtgga aaggacgagg atcggaaagg ttatggctgt agtcacgaga 480

ctacagccat aacctttcct ttttgaattc taccggtacc gagggttaac aacaacactc 540

gagttaaggg cgaattcccg ataaggatct tcctagagca tggctacgta gataagtagc 600

atggcgggtt aatcattaac tacaaggaac ccctagtgat ggagttggcc actccctctc 660

tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg 720

cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt 780

attttctcct tacgcatctg tgcggtattt cacaccgcat acgtcaaagc aaccatagta 840

cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc 900

tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac 960

gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag 1020

tgctttacgg cacctcgacc ccaaaaaact tgatttgggt gatggttcac gtagtgggcc 1080

atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg 1140

actcttgttc caaactggaa caacactcaa ccctatctcg ggctattctt ttgatttata 1200

agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa 1260

cgcgaatttt aacaaaatat taacgtttac aattttatgg tgcactctca gtacaatctg 1320

ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg 1380

acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg 1440

catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat 1500

acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac 1560

ttttcgggga aatgtgttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc 1620

tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg 1680

gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggtgacag 1740

ttcctgttga gccacctcct tgtcagcatt tgatgttgtc agctttggat tttagccatt 1800

ctaataggtg agtagtggta catcattatt gtttcaattt gaaatggcta ctgacatatg 1860

atatggagca tctttcctta tgcttatttg ctatgtgtat attttctttg gtgaggtgtc 1920

tgctcagatc gtttgcccat tctttaattg ggttgttttc tattgagttt taacagttct 1980

ttgtgtattt tgaatgtaag tcccttatca ggtatgtccc ctgcaaatat gttctcctag 2040

tctgtagctt gtattgtcat tctcttgaca attcttttgc agagcagaag cgtctaattt 2100

taatgacaca gaacttatca attgcttctt tcaaggattg tgcttttggt gttgtatcta 2160

ataagccatc accaaactta aggtcatcta gattttctcc tacattatct tctagaagtt 2220

ttgtactcac tattcattta ggtctacaat aaacttggag ttaattttcg tgaaagccgt 2280

aagggagctt ctaaggggga cacacactaa cttctttgag agtgggcagt gattgtgaaa 2340

ctctcagctg ggtcttacaa cagaccccag atccatcagg agagcccagc cttcagaacc 2400

catagtggaa tatgtttcat gcatacacgg aggagaatat gcagtcatat gctcacatgc 2460

atgtctgaac atgatcacac acatgaatat ggctatgtcc tgatccagag ttctctcatt 2520

ctctgtaagt gattctttcc ttcaacttct tcattaaggg atatattctc gttgtttagg 2580

tatttcagtg gggccggata ggaacttata tctaagtttt caggaaggtt aggtaccagg 2640

gccacggtct agtaatttag gtgagaagaa ctgagacata tatccaggtt tcaggtattt 2700

aaacgaggct ggattggagc acacatctag gtgttcagat attcaagtga agttgagttg 2760

gaagacacct gtccaggtgt ttagctcttc aggtggggct ggtatgaggt gagtgtcacc 2820

tgtctaggta tttaggcatt caagtaggct tggttggaga cagctgtcca gatacttact 2880

gggtctggat tgggggcttt tgttcaggtg ctcaggtatt aaagtggggc tggtgaagat 2940

gttgaagtgg aggggatacc tgttgaggtg tttacctatc caagtgggat gcactgggag 3000

cacctctgta aggtgtgtga ttggaaaggg ttggggaaca cttatttgtg tgtcagggaa 3060

caatctgtct agatatttat gtgtttaagg tgttggttag ggcagctgtc catgttttca 3120

ggtatttaag tgggtgaagt agagggcgct tggccagata ttccagtggg ctaatctggg 3180

acatttgtgt agatgtgaaa tgattatgat gggcctccag atttatcagc aataaaccag 3240

ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct 3300

attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt 3360

gttgccattg ctgcagccat gagattatca aaaaggatct tcacctagat ccttttcacg 3420

tagaaagcca gtccgcagaa acggtgctga ccccggatga atgtcagcta ctgggctatc 3480

tggacaaggg aaaacgcaag cgcaaagaga aagcaggtag cttgcagtgg gcttacatgg 3540

cgatagctag actgggcggt tttatggaca gcaagcgaac cggaattgcc agctggggcg 3600

ccctctggta aggttgggaa gccctgcaaa gtaaactgga tggctttctt gccgccaagg 3660

atctgatggc gcaggggatc aagctctgat caagagacag gatgaggatc gtttcgcatg 3720

attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc 3780

tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg 3840

caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcaa 3900

gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc 3960

gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat 4020

ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg 4080

cggctgcata cgcttgatcc ggctacctgcccattcgacc accaagcgaa acatcgcatc 4140

gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag 4200

catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgagcat gcccgacggc 4260

gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc 4320

cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata 4380

gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc 4440

gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac 4500

gagttcttct gactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt 4560

catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc 4620

ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct 4680

tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta 4740

ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc 4800

ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac 4860

ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct 4920

gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat 4980

aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg 5040

acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa 5100

gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg 5160

gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga 5220

cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc 5280

aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gt 5332

<210>53

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223>shRNA-80

<400>53

tctatctgta gtactatgac 20

Claims

1. An oligonucleotide comprising a nucleic acid sequence selected from the group consisting of:

2. The oligonucleotide of claim 1, wherein the oligonucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 1-50.

3. The oligonucleotide of any one of claims 1-2, wherein the oligonucleotide is a chemically modified oligonucleotide.

4. A viral vector comprising the oligonucleotide of any one of claims 1-2.

5. The viral vector of claim 4, wherein the viral vector is an adeno-associated virus, a lentivirus, a retrovirus, or an adenovirus.

6. The viral vector according to any one of claims 4 to 5, wherein the viral vector comprises a U6 or H1 promoter and expression of the oligonucleotide is regulated by the promoter.

7. The viral vector of any one of claims 5 to 6, wherein the viral vector is an adeno-associated virus and the serotype of the adeno-associated virus is one or more of wild-type and mutant of AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh 10.

8. Use of an oligonucleotide according to any one of claims 1 to 3 or a viral vector according to any one of claims 4 to 7 for the prevention or treatment of a disease caused by a coronavirus infection.

9. The use of claim 8, wherein the oligonucleotide or viral vector is capable of specifically binding to and degrading the genomic RNA or mRNA of the coronavirus.

10. The use according to any one of claims 8 to 9, wherein the coronavirus is a severe acute respiratory syndrome virus (SARS-Cov), a middle east respiratory syndrome virus (MERS) or a novel coronavirus (SARS-Cov 2).

11. A pharmaceutical formulation comprising the oligonucleotide of any one of claims 1-3 or the viral vector of any one of claims 4-7, and pharmaceutically acceptable carriers and excipients.

12. The pharmaceutical formulation of claim 11, wherein the pharmaceutical excipient is a nanocarrier and/or a liposome.

13. The pharmaceutical formulation of claim 12, wherein the pharmaceutical formulation is a liquid formulation.

14. Use of a pharmaceutical preparation according to any one of claims 11 to 13 for the prophylaxis or treatment of a disease caused by a coronavirus infection.

15. The use of claim 14, wherein the pharmaceutical agent is capable of specifically binding to and degrading the genomic RNA or mRNA of the coronavirus.

16. The use according to any one of claims 14 to 15, wherein the coronavirus is a severe acute respiratory syndrome virus (SARS-Cov), a middle east respiratory syndrome virus (MERS) or a novel coronavirus (SARS-Cov 2).