Patent Description:
Human angiotensin-converting enzyme II (ACE2) is a type I transmembrane glycoprotein expressed by target cells. The ACE2 consists of <NUM> amino acids, where amino acids <NUM> to <NUM> are located extracellularly and called extracellular ACE2; amino acids <NUM> to <NUM> are located in a transmembrane region and called transmembrane ACE2; and amino acids <NUM> to <NUM> are located intracellularly and called intracellular ACE2.

The structure of a coronavirus includes a single-stranded ribonucleic acid (ssRNA), a spike protein (S protein), a membrane protein (M protein), an envelope protein (E protein), and a nucleocapsid protein (N protein), where an N-terminus of the S protein includes an N-terminus domain (S1-NTD) and a receptor-binding domain (S1-RBD).

The coronavirus binds to the ACE2 of a target cell through its S <NUM>-RBD, and undergoes membrane fusion and endocytosis, such that the coronavirus enters the target cell through an ACE2 channel. This shows that coronavirus RBD and target cell ACE2 have a ligand-receptor relationship. According to this, an ACE2 polypeptide is designed as a targeted delivery vector, and the ACE2 polypeptide and an RNAi sequence are synthesized into nCoVshRNA-2ACE2 for targeted delivery of shRNA by the ACE2. Due to cell penetrating peptide properties of the ACE2, an siRNA drug in the nCoVshRNA-2ACE2 is subjected to targeted delivery by the ACE2 and passes through the ACE2 channel of the target cell, playing a specific role in targeting virus-infected cells; since ACE2 can combine with the virus RBD, nCoVshRNA-2ACE2 combines with the virus to form a complex, such that the siRNA drug can enter target cells with virus infection, playing a characteristic role in virus-infected cells. In addition, since the ACE2 can bind to the RBD, nCoVshRNA-2ACE2 can block the virus RBD, thereby blocking virus infection of the target cells.

RNA interference (RNAi), as an efficient sequence-specific gene silencing technology, is bringing unimaginable application prospects to the treatment of diseases, and a variety of siRNA drugs have been approved and marketed by the Food and Drug Administration (FDA). However, currently siRNA in the siRNA drugs is generally designed using a single strain, the siRNA drugs are prepared using a single-stranded siRNA (antisense RNA), or non-specific delivery of the siRNA drugs is conducted using non-targeted delivery vectors. Moreover, the siRNA generally designed using a single strain may be off-target and ineffective due to the constantly mutating coronavirus. Therefore, it is necessary to compare mutated strains, so as to preferably select an siRNA that is consensus to each strain and does not change with the virus mutation, thereby preparing a broad-spectrum siRNA drug for the mutated and mutating strains. If a non-targeted delivery vector is used, the siRNA drugs may be delivered to cells that are less susceptible to coronavirus infection, since the cells do not express ACE2. Accordingly, it is necessary to design a targeted delivery vector that can specifically deliver the siRNA to target cells. More importantly, according to an RNAi mechanism reported by Hre A et al. , a double-stranded RNA prepared by mixing the sense RNA and antisense RNA has an efficiency of silencing homologous mRNAs over <NUM> times higher than that of the single-stranded RNA (antisense RNA). This shows that a correct and effective RNAi technology should prepare drugs using the double-stranded RNA (shRNA) containing the sense and antisense siRNAs, rather than the single-stranded siRNA.

Small interfering RNA, or siRNA, is delivered to the cytoplasm to regulate gene expression in a manner that participates in RNAi, thus specifically degrading a complementary target messenger RNA (mRNA). Since siRNA is difficult to pass through the cell membrane and is easily degraded by RNases, traditionally the siRNA is transported and protected non-specifically by methods such as lipid nanoparticle encapsulation. In the present disclosure, the siRNA is synthesized into a shRNA, and an ACE2 polypeptide is ligated to double-strand ends of the shRNA to achieve specific transport and protection of the siRNA.

In the prevention and treatment of COVID-<NUM>, if nCoVsiRNA can be stably and specifically delivered to target organs, target tissues, and target cells in sequence with a suitable targeted delivery vector. The nCoVsiRNA can localize to target cells, cross the target cell membrane, and be released into the target cytoplasm, which is broadly effective against a variety of variant strains. As a result, a targeted gene therapy for the COVID-<NUM> can be better developed.

To solve the above problems, in the present disclosure, a targeted drug nCoVshRNA·2ACE2 is designed and synthesized.

<NPL> the plausibility of using aptamers. siRNAs, and aptamer-siRNA chimeras against the SARS-CoV-<NUM> based on their previously established effectiveness.

<CIT> discloses a siRNA inhibiting novel corona virus gene expression and a pharmaceutical composition.

An objective of the present disclosure is to provide a drug nCoVshRNA-2ACE2 that delivers a shRNA by using a targeted delivery vector ACE2, and a synthesis method and use thereof. In the present disclosure, the bivalent ACE2 can bind to a RBD to neutralize a virus and deliver the shRNA in a targeted manner, and make the shRNA delivered by the ACE2 to indirectly bind to the RBD to form a complex of "shRNA-ACE2-RBD-virus", such that the shRNA is targeted to be delivered by the complex, and enters target cells with virus infection, thus playing a broad-spectrum and targeted role in resisting variant strains.

The objective of the present disclosure is achieved by a synthesis method according to claim <NUM>.

A target siRNA against the variant strain is selected, a shRNA is synthesized, and then an ACE2 polypeptide is separately extended and ligated to double-strand ends of the shRNA to synthesize a targeted drug nCoVshRNA-2ACE2 of a COVID-<NUM> virus.

Screening of a target against variant strains: the siRNA is selected from a consensus gene of various pathogenic coronaviruses and variant strains thereof, where the consensus gene includes a conserved gene, an ultra-conserved gene, and/or a conserved microsatellite, such that the siRNA is a common target of each variant strain that does not change with virus mutation, with a broad-spectrum effect in resisting the variant strains.

Synthesis of an anti-variant strain target siRNA: the siRNA is synthesized into two complementary oligonucleotide siRNAs of <NUM> nt to <NUM> nt, and a base sequence that acts as a spacer is synthesized.

Synthesis of a shRNA: the two complementary oligonucleotide siRNAs and the base sequence that acts as a spacer are further synthesized into a double-stranded small hairpin shRNA with a loop that is in a middle position and separated by the base sequence.

Preferred siRNA: an interference vector is constructed, and mRNA expression, protein expression, and interference effect of the shRNA are detected; after conducting siRNA design, synthesis, screening, iterative design, and verification, the siRNA with a high silencing efficiency is preferably selected.

Synthesis of preferred siRNAs and shRNAs: siRNAs and shRNAs are synthesized using sequences of the preferred siRNA according to the method above, including chemical modifications to increase stability and avoid off-target.

Synthesis of an ACE2 polypeptide or protein: the synthesized ACE2 includes but is not limited to a full-length ACE2, a transmembrane ACE2 of amino acids <NUM> to <NUM>, an intracellular ACE2 of amino acids <NUM> to <NUM>, an extracellular ACE2 of amino acids <NUM> to <NUM>, and an amino acid sequence codon-optimized ACE2 polypeptide or protein.

Synthesis of nCoVshRNA·2ACE2: the shRNA is ligated by a coupling method using a disulfide bond, a phosphodiester bond, a phosphorodithioate bond, a thioether bond, an oxime bond, an amide bond, and a maleimide-thiol bond with the ACE2 to synthesize a compound; alternatively, ACE2-shRNA-ACE2 is directly synthesized from the amino acid level according to a nucleotide sequence of the shRNA and an amino acid sequence of the ACE2.

Purification of the nCoVshRNA-2ACE2: the purification is conducted by high-performance liquid chromatography, reversed high-performance liquid chromatography, or ion exchange chromatography.

Liposome modification of the nCoVshRNA-2ACE2: a liposome-modified compound is prepared by adsorbing the negatively-charged shRNA to a positively-charged liposome; a PEG-internalized liposome-modified compound is prepared by thiolation of an amino group of the ACE2 to form a maleimide-thiol bond between a thiol group and maleamide of the liposome; a liposome-modified compound is prepared by forming a carbamate bond between the amino group of the ACE2 and the liposome; and a liposome-modified compound is prepared by ligating the ACE2 or an ACE2 fragment to the liposome-modified siRNA.

Verification of the nCoVshRNA·2ACE2: an antiviral effect of the nCoVshRNA-2ACE2 on two or more different variant strains is detected at the cellular level in vitro, and it is observed whether it has a broad-spectrum anti-variant strain effect targeting the conserved gene; it is tested that whether the nCoVshRNA·2ACE2 has an effect in targeted delivery of the shRNA in animals and whether the nCoVshRNA-2ACE2 can stimulate the host to produce ACE2-Ab.

The present disclosure has the following beneficial effects:
In the present disclosure, an siRNA drug is designed that delivers shRNA by targeting ACE2.

In the present disclosure, a difference between the drug and traditional siRNA drugs is that: from various coronaviruses and their variant strains, a common target siRNA of each strain that does not change with the virus mutation is selected, such that the siRNA has a broad-spectrum anti-variant strain effect.

In the present disclosure, a difference between the drug and the traditional siRNA drugs is that: the traditional siRNA drugs adopt a sense strand siRNA or an antisense strand siRNA, that is, a single-stranded siRNA; in the present disclosure, shRNA is synthesized from the siRNA of sense and antisense strands. According to an RNAi mechanism, double-stranded RNA has a more efficient RNAi effect. Therefore, the present disclosure has a more accurate design that is more in line with the RNAi mechanism.

In the present disclosure, a distinguishing feature of the drug from the traditional siRNA drugs is that: based on the characteristics of coronavirus infection through the binding of RBD to ACE2 expressed by host susceptible cells, the nCoVshRNA-2ACE2 that delivers shRNA by targeting ACE2 is designed, which consists of a short hairpin shRNA region and a double-stranded ACE2 region. ACE2 plays the role of delivering shRNA and binding coronavirus RBD. A "shRNA-ACE2-RBD-virus" complex formed by the ACE2 and virus RBD can make shRNA enter the target cells with virus infection. This method of co-delivering shRNA with the ACE2 and virus can avoid a side effect of non-specific delivery of the shRNA to uninfected cells, such that shRNA can produce an anti-variant strain effect against the virus-infected cells.

Since the siRNA/shRNA is negatively charged and lipid-soluble, with poor permeability and stability; after synthesizing the siRNA/shRNA with the ACE2 into the nCoVshRNA-2ACE2, the permeability, stability, and easy delivery of the siRNA/shRNA are optimized.

Furthermore, the nCoVshRNA-2ACE2 is synthesized with <NUM> molecules of the ACE2 and <NUM> molecule of the shRNA, where the bivalent ACE2 can bind to the virus RBD, thereby neutralizing the virus and blocking the virus from infecting target cells.

Furthermore, the nCoVshRNA-2ACE2 includes two ACE2 molecules and one shRNA molecule. Since the shRNA is not only antiviral, but also an oligonucleotide immunologic adjuvant, nCoVshRNA-2ACE2 is more antigenic than the single-molecule ACE2, which can stimulate the host to produce higher titer of ACE2-Ab. The ACE2-Ab competes with the virus for binding to an ACE2 receptor of the target cell, thereby enabling the nCoVshRNA-2ACE2 (ACE2-Ab) to block viral infection.

Furthermore, liposome modification can make the shRNA release slowly in vivo, prolong a drug efficacy, and play a role in endocytosis into plasma, and can be used as an immunologic adjuvant to enhance an ACE2 immune effect in the nCoVshRNA-2ACE2.

Furthermore, in vitro cell experiments show that the nCoVshRNA·2ACE2 is effective against two different variant strains simultaneously, indicating an anti-variant strain effect by targeting conserved genes; in vivo animal experiments show that the nCoVshRNA-2ACE2 has an effect in targeted delivery of the RNAi in animals, and the stimulation-derived ACE2-Ab can inhibit virus infection by blocking ACE2 receptors.

In <FIG>, after conserved gene screening, broad-spectrum anti-variant strain target siRNA screening by targeting conserved genes, shRNA synthesis, and ACE2 synthesis, the nCoVshRNA·2ACE2 or ACE2shRNA is finally synthesized.

In <FIG>, <FIG> is a loop, <NUM> is a shRNA formed by two complementary sense and antisense strands, and <NUM> is two ACE2 polypeptides (proteins); the two ACE2 polypeptides are ligated to the sense and antisense strands of the shRNA, respectively. shRNA is protected by ACE2 and then delivered by the ACE2 to the virus RBD or the ACE2 receptor channel, and then specifically enters the target cytoplasm with the virus RBD through the ACE2 channel to degrade the target gene of virus.

In <FIG>, <FIG> is the loop formed by the base sequence between the sense and antisense strands of the small hairpin shRNA; <NUM> is the shRNA formed by complementary combination of two sense and antisense strands of siRNA, where the siRNA takes the conserved gene of coronavirus as an interference target and has a targeted gene therapy effect of broad-spectrum anti-variant strain properties; <NUM> is the ACE2 ligated to the sense and antisense strands of the shRNA; <NUM> is the coronavirus; <NUM> is the RBD of a coronavirus S protein; <NUM> is the cells; <NUM> is the expressed ACE2 receptors; and <NUM> is the cells that do not express ACE2. As shown in <FIG>, the nCoVshRNA·2ACE2 is composed of the loop <NUM>, the shRNA <NUM>, and the ACE2 <NUM>; the coronavirus <NUM> infects cells <NUM> through specific binding of its RBD <NUM> to ACE2 receptor <NUM>, but the coronavirus <NUM> does not infect cells <NUM> that do not express ACE2; ACE2 <NUM> in the nCoVshRNA-2ACE2, like ACE2 <NUM> expressed by cells <NUM>, can also bind to the RBD <NUM> of coronavirus <NUM>, such that the ACE2 <NUM> can compete with the ACE2 <NUM> for binding to the RBD <NUM>, such that the ACE2 <NUM> has an ability to inhibit the virus <NUM> from infecting the cells <NUM>; the nCoVshRNA-2ACE2 plays the role of a vaccine in a later stage, since the ACE2 <NUM> can stimulate the host to produce anti-ACE2, and the anti-ACE2 can block the ACE2 <NUM>, thereby inhibiting the virus <NUM> from infecting cells <NUM>; more importantly, through bridging of the ACE2 <NUM>, a complex of "shRNA <NUM>-ACE2 <NUM>-RBD <NUM>-virus <NUM>" is formed, allowing shRNA <NUM> to enter cell <NUM> with virus <NUM>, targeting to interfere with the replication of virus <NUM> in cells <NUM>, avoiding toxic and side effects caused by the non-specific entry of the shRNA <NUM> into cells <NUM> that are not infected with virus <NUM>.

In <FIG>, <FIG> and <FIG> are the loop and shRNA wrapped by liposome <NUM>, respectively, where the shRNA is formed by the complementary combination of two sense and antisense strands of siRNA, and the siRNA takes the coronavirus conserved gene as an interference target; <NUM> is ACE2 exposed outside the liposome <NUM>, plays a role in targeted delivery; liposome <NUM> protects shRNA and induces endocytosis; <NUM> is polyethylene glycol (PEG), the PEG can release siRNA slowly with long-term circulation. As shown in <FIG>, a complex of ACE2-shRNA/Lip is formed.

In <FIG>, <FIG> is the siRNA encapsulated by liposomes, <NUM> is a liposome layer, <NUM> is a PEG layer, and <NUM> is the ACE2. The siRNA acts as RNAi; the liposome protects siRNA and induces endocytosis; the PEG enables slow release and long-term circulation of siRNA; and the ACE2 is in the outermost layer and plays a role of targeted delivery of the siRNA. As shown in <FIG>, a complex of ACE2-siRNA/Lip is formed.

Below in conjunction with accompanying drawings <NUM>, <NUM>, <NUM>, <NUM> and <NUM>, the specific implementation method of the present disclosure is described in detail, but these exemplary descriptions do not constitute any limitation to the protection scope defined by the claims of the present disclosure.

As shown in <FIG> of the technical circuit, a whole genome sequence (cDNA) of β-coronavirus (especially COVID-<NUM> virus and its variant strains) is downloaded from the Genbank database (http://www. gov/genome/), the longest common subsequence is searched in the whole genome sequence to obtain the ultra-conserved genes or conserved genes; with Clustal W software, sequence alignment is conducted on the whole genome downloaded from the Genbank database, the similarity between different sequences is detected, and conserved microsatellite sequences are screened; with MEGA6. <NUM> molecular evolution genetic analysis software, an amino acid germline molecular evolution tree is constructed by using neighbor-joining (N-J) on the downloaded coronavirus amino acid sequence, and molecular variation characteristics of the amino acid sequence were analyzed and optimized, so as to infer conserved genes sequence.

The following three longest and second longest ultra-conserved subsequences are obtained, with a length of <NUM> bp to <NUM> bp, which are comparable to a length of small RNAs, but these three subsequences are not included in higher organisms, especially human beings. A specific sequence is as follows:.

The following three longest and second longest conserved subsequences are obtained, with a length of <NUM> bp to <NUM> bp, which are comparable to a length of small RNAs, but these three conserved subsequences are not included in higher organisms, especially human beings:.

The following five conserved microsatellite loci with repeated nucleotides are obtained, where microsatellites are CTCTCT, AGAGAG, AAAAAAA, TATATA, and CACACA, respectively:.

A whole genome sequence (cDNA) of β-coronavirus (especially the COVID-<NUM> virus and its variant strains) is downloaded from the Genbank database (http://www. gov/genome/); with Ambion's shRNA online design software (http://www. com/techlib/misc/siRNAtools. html) or DSIR and the like, multiple siRNA candidates are obtained with a length of about <NUM> nt; based on a Tm value of RNA binding and specificity alignment results, the siRNA is preferably selected. Therefore, RNAi sequences (siRNAs) of each strain and common RNAi sequences (common target siRNAs) of each strain are obtained from the E gene, M gene, N gene, ORF1ab gene, and S gene of <NUM> COVID-<NUM> virus strains and their variant strains that are discovered so far. The common siRNAs of each strain are shown in Table <NUM>, with sequences marked as SEQ ID NO: <NUM> to SEQ ID NO: <NUM>. For example, the siRNAs (SEQ ID NO: <NUM> to SEQ ID NO: <NUM>) marked in Tables <NUM> to <NUM> are the common target siRNAs of NC_045512. <NUM>, Delta strain, and Omicron strain, and the siRNAs without marking are their respective RNAi sequences (siRNAs). It is seen that although the earliest NC_045512. <NUM> strain has mutated into the Delta strain and the recent Omicron strain, each strain, except for its own unique targeting interference sequence siRNA (parts without marking), still remains unchanged and theoretically has common conserved sequences SEQ ID NO: <NUM> to SEQ ID NO: <NUM> with a targeted interference effect.

With the Clustal W software or other software, gene sequence alignment is conducted on the ultra-conserved genes, conserved genes, and conserved microsatellites with conventionally-screened siRNAs, to detect a similarity between different sequences; multiple pairs of siRNAs are designed, which are the ultra-conserved genes, conserved genes, or conserved microsatellites, as well as RNAi target sites (siRNAs by targeting ultra-conserved genes, conserved genes, or conserved microsatellites are designed).

Through the above design, siRNAs that theoretically resist coronavirus variant strains are obtained with ultra-conserved genes, conserved genes, or conserved microsatellites as interference targets, named siRNA1/<NUM>/<NUM>/<NUM>.

According to the RNAi mechanism, when siRNA effectively interferes with the mRNA expression of the S gene, an S protein-deficient virus that loses infectivity is formed. When the siRNA effectively interferes with the mRNA expression of the N gene, the packaging and replication of the virus may be inhibited. When siRNA effectively interferes with the mRNA expression of ORF1a or ORF1b gene, the synthesis of viral RNA polymerase (RdRp) or protein processing enzyme (3CLpro) may be affected. However, the M and E genes are membrane genes of the virus, with an inhibitory effect of their gene defects on the virus being not obvious. Therefore, siRNA targeting N gene (SEQ ID NO: <NUM> to SEQ ID NO: <NUM>, SEQ ID NO: <NUM> to SEQ ID NO: <NUM>), siRNA targeting ORF1ab gene (SEQ ID NO: <NUM> to SEQ ID NO: <NUM>, SEQ ID NO: <NUM> to SEQ ID NO: <NUM>), and siRNA targeting S gene (SEQ ID NO: <NUM> to SEQ ID NO: <NUM>, SEQ ID NO: <NUM> to SEQ ID NO: <NUM>), and SEQ ID NO: <NUM> to SEQ ID NO: <NUM> and SEQ ID NO: <NUM> to SEQ ID NO: <NUM> are selected for synthesis. In addition, according to polyclonal restriction enzyme cleavage site of a pSilencer4. CMVneo interference vector, a shRNA template capable of expressing a hairpin structure is designed; each template is composed of two mostly complementary <NUM> bp single-stranded DNAs, and the single-stranded DNA can be annealed and complementary to form a double-stranded DNA with sticky ends of Bam HI and Hind III restriction sites for ligation with a linearized pSilencer4. According to the designed siRNA and its shRNA template, a company is commissioned to synthesize the siRNA.

The shRNA is ligated with the linearized interference vector pSilencer4. neo, and then identified to construct a shRNA expression plasmid, and transformed into DH5a to obtain the shRNA expression vector.

According to the synthesized siRNA/shRNA and its constructed expression plasmid, a corresponding target gene is selected for synthesis or PCR amplification, a fluorescent tag vector is constructed, and co-transfected into type II alveolar epithelial cells (AEC2s) or 293T cells separately with the shRNA expression plasmid, and the cells are identified. A conventional method of PCR amplification is as follows:.

Primer design: upstream and downstream primers are designed, a start codon is added at a <NUM>'-end of the upstream primer, and a homology arm is added to the <NUM>'-end of the primer for homologous recombination with a vector in order to clone an amplification product into pEGFP-N1.

Target gene amplification: gene amplification, product recovery, and purification are conducted according to a gene amplification reaction system and reaction conditions provided by a Shanghai Sangon kit to obtain the amplification product.

Linearization of pEGFP-N1: a DH5a strain containing a pEGFP-N1 plasmid is resuscitated, the plasmid is extracted by the kit, the concentration is determined, restriction digestion is conducted, and a linearized vector is identified and recovered by <NUM>% agarose gel electrophoresis.

An amplified target gene is ligated to a fluorescent tag vector (pEGFP-N1): the ligation is conducted with a GenScript's homologous recombination kit, and a ligated product can be stored at -<NUM> for later use or transformed immediately.

Identification of effects of a shRNA interference vector: the interference vector (pSilencer-shRNA) and the fluorescent tag vector (pEGFP-N/S/ORF1ab) are co-transfected into 293T cells, where the interference vector and the tag vector have a mass ratio of <NUM>:<NUM>, while a control is set up; the fusion expression of a GFP protein in the cells is observed <NUM> after the transfection, and an interference effect is evaluated according to a fluorescence intensity.

Flow cytometry: to quantitatively analyze the interference effects of different interference vectors, flow cytometry is conducted to analyze a proportion of fluorescent protein-expressing cells in the total number of cells.

Westernbolt analysis: (<NUM>) cell collection and lysis: cells are lysed with RIPA; (<NUM>) SDS-PAGE protein electrophoresis: an SDS-PAGE gel is prepared, a sample is added to an equal volume of a <NUM>×SDS buffer, boiled in boiling water for <NUM>, treated in an ice bath for <NUM>, and then centrifuged at <NUM>,<NUM>×g for <NUM>; (<NUM>) Western blot detection: after conducting transferring, blocking, primary antibody binding, washing, secondary antibody binding, and color development, results are observed.

RT-PCR detection of mRNA: relative fluorescence quantitative RT-PCR is conducted to detect a relative expression level of the target genes in transfected cells; according to a standard curve, a copy number of the target gene and a B-actin reference gene is converted from a CT value; a relative expression level of the viral gene mRNA (the number of copies of the target gene/the number of copies of the B-actin) is corrected with the B-actin reference gene, such that an interference effect is quantitatively evaluated.

After design, synthesis, screening, iterative design, resynthesis, and verification at the cellular level, siRNAs with a high silencing efficiency are obtained. The siRNAs have sequences of SEQ ID NO: <NUM> (named shRNA1, the same below), SEQ ID NO: <NUM> (shRNA2), SEQ ID NO: <NUM> (shRNA3), SEQ ID NO: <NUM> (shRNA4) and SEQ ID NO: <NUM> (shRNA5) targeting the N gene, SEQ ID NO: <NUM> (shRNA6) and SEQ ID NO: <NUM> (shRNA7) targeting the ORF1ab gene, and SEQ ID NO: <NUM> (shRNA8) targeting the S gene, with silencing efficiencies of <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, and <NUM>%, respectively.

According to the common targets (SEQ ID NO: <NUM> to SEQ ID NO: <NUM>), SEQ ID NO: <NUM> (shRNA3), SEQ ID NO: <NUM> (shRNA5), and SEQ ID NO: <NUM> (shRNA8) with a shorter target gene sequence are selected, synthesized by a biological company. Each shRNA is synthesized with <NUM> complementary oligonucleotide siRNAs of <NUM> nt to <NUM> nt, and synthesizes a <NUM> nt base sequence that acts as a spacer; the siRNA (drug) and the base sequence are ligated into a double-stranded small hairpin shRNA with a loop formed by separation of intermediate base sequences; each single strand of the double-stranded shRNA can be ligated to an ACE2 polypeptide or protein, respectively.

For example, SEQ ID NO: <NUM> (shRNA1), SEQ ID NO: <NUM> (shRNA2), and SEQ ID NO: <NUM> (shRNA3) are synthesized into <NUM>'-ttaatacgacctctctgttggattttgacattcaagagatgtcaaatccaacagagaggtcgtattaa-<NUM>' (shRNA1) (SEQ ID NO: <NUM>), <NUM>'-ggttcgcaacttcacacagagtttcaagagaactctgtgtgaagttgcgaacc-<NUM>' ( shRNA2) (SEQ ID NO: <NUM>) and <NUM>'-ggtt cggt tgtta tatac gata ttcaagaga tatc gtata taaca accg aacc-<NUM>' (shRNA3) (SEQ ID NO: <NUM>), where "TTCAAGAGA" is a loop, and left and right sides thereof are complementary sense and antisense strands, and then a <NUM>'-and/or <NUM>'-end of the shRNA is ligated with the ACE2 protein or polypeptide separately. Similarly, preferably other siRNAs with a high silencing efficiency are synthesized into shRNAs.

The human ACE2 gene sequence information was searched from the GeneBank database (http://www. gov/genbank); the ACE2 consists of <NUM> amino acids, where amino acids <NUM> to <NUM> (SEQ ID NO: <NUM>) are located extracellularly, amino acids <NUM> to <NUM> (SEQ ID NO: <NUM>) are located in a transmembrane region, and amino acids <NUM> to <NUM> (SEQ ID NO: <NUM>) are located intracellularly. Among the <NUM> amino acids that make up the ACE2 protein, leucine accounts for <NUM>%, cysteine and histidine account for <NUM>% and <NUM>%, respectively, and negatively-charged amino acid residues (aspartate + glutamate) and positively-charged amino acid residues (arginine + lysine) as a balance. SARS-CoV and SARS-CoV <NUM> interact with an extracellular catalytic domain of the ACE2 through virus RBD. This interaction can lead to endocytosis and membrane fusion, such that the SARS-CoV enters cells that highly express ACE2 or contain ACE2 receptors.

ACE2 is a lipid-soluble type I transmembrane glycoprotein with an amino-terminal catalytic domain and a carboxyl-terminal domain. The N-terminus is outside the cell membrane and the C-terminus is inside the cell membrane. This glycoprotein is divided into an N-terminal signal peptide region, a carboxypeptidase activation region, and a transmembrane region. When the Spike protein of coronavirus contacts a tip of a subdomain I of the catalytic domain of ACE2 (without affecting a subdomain II and a peptidase activity), an outer domain of the ACE2 is cleaved and the transmembrane domain is internalized, enabling further virus-host cell fusion. Therefore, it is believed that the transmembrane region is involved in the transport of virus-receptor complexes from the cell membrane to the cytoplasm.

From an amino acid sequence of ACE2 and a receptor function of ACE2, it can be seen that there are ACE2 receptor channels in the cell wall and cell membrane of ACE2-expressing cells. Full-length ACE2, transmembrane region ACE2, intracellular ACE2, and extracellular ACE2 each are a membrane-penetrating polypeptide with a function of targeted delivery of siRNA. The N-terminus of extracellular ACE2 has a function of neutralizing virus by binding to the coronavirus RBD. Therefore, full-length ACE2, extracellular ACE2 of amino acids <NUM> to <NUM>, transmembrane ACE2 of amino acids <NUM> to <NUM>, or intracellular ACE2 of amino acids <NUM> to <NUM>, or ACE2 with optimized amino acid sequences can be designed and synthesized as a targeted delivery vector for siRNA.

Two amino acids are dehydrated and condensed to form peptide bonds, and multiple amino acid residues are ligated by the peptide bonds to form polypeptides. A company can be entrusted to automatically synthesize peptides by a peptide synthesizer. Basically, amino acids are added in order according to a sequence of the polypeptide to be synthesized, such that the peptide chain is gradually extended from the C-terminal to the N-terminal residues; each amino acid residue is required to be condensed in the form of protection at one end and activation at the other end, and temporary protection groups on the amino group are removed after each round of peptide chain elongation, until all amino acid sequences of the target polypeptide are condensed. At present, a commonly used reaction for solid-phase synthesis of the polypeptides includes: in a closed explosion-proof glass reactor, amino acids are continuously added from the C-terminus-carboxy terminus to the N-terminus-amino terminus according to a known sequence, and synthesis is conducted to finally obtain the polypeptides. A synthesis method includes: (<NUM>) deprotection: removing the protective group of the amino group with an alkaline solvent; (<NUM>) activation and cross-linking: activating a carboxyl group of a next amino acid, cross-linking an activated single carboxyl group with a free amino group to form a peptide bond; and repeating these two steps until the polypeptide is synthesized.

Take the synthesis of shRNA and extracellular, transmembrane or intracellular ACE2 as an example: one end of the sense and antisense strands of the synthesized siRNA/shRNA is ligated to the loop (<NUM>'-TTCAAGAGA-<NUM>'), and the other end is ligated to ACE2 (C-terminus), so as to obtain a structure of "ACE2-siRNA sense strand-loop-siRNA antisense strand-ACE2", which is expressed as "2ACE2-shRNA". The two complementary sense and antisense strands can form a double strand, but the two ACE2 polypeptides cannot form the double strand. As shown in <FIG>, there is a hairpin ligation product shRNA with two ACE2 polypeptides, two siRNA sense and antisense strands, and a loop. Since the virus RBD infects cells through the C-terminus binding to the N-terminus of ACE2, and the binding of ACE2 polypeptide to siRNA can increase the permeability, stability and interference effect of siRNA. Accordingly, as shown in <FIG>, ACE2 in this design can neutralize the virus and prevent virus infection, and can deliver siRNA/shRNA to the virus RBD in a targeted manner to form a complex of "shRNA-ACE2-RBD-virus". As a result, the shRNA is delivered by the virus, and the shRNA enters the target cells with virus infection, playing a role of targeted interference.

According to the design (<FIG>), the entrusted company adopts the sequences of extracellular ACE2, SEQ ID NO: <NUM> (shRNA3), transmembrane ACE2, SEQ ID NO: <NUM> (shRNA5), and SEQ ID NO: <NUM> (shRNA8), by a conventional synthesis method of oligonucleotide, the polypeptide (ACE2) and oligonucleotide (shRNA/siRNA) are coupled to form a conjugate with a carboxyhydrazone bond, a disulfide bond, a phosphodiester bond, a phosphorodithioate bond, a thioether bond, an oxime bond, an amide bond, and a maleimide-thiol bond. The sense strand (<NUM>'-end and <NUM>'-end) or antisense strand (<NUM>'-end) of polypeptides and oligonucleotides can be non-covalently or covalently cross-linked with a firmer covalent bond, a looser ionic bond, a hydrophobic bond, or the carboxyhydrazone bonds with a spacer arm to synthesize a polypeptide-oligonucleotide conjugate (POCs). At present, the POCs are generally synthesized by covalent crosslinking-liquid phase fragment synthesis, and various POCs are prepared. The method includes the following steps: synthesizing a polypeptide and an oligonucleotide separately on a solid-phase substrate, simultaneously peeling the polypeptide and the oligonucleotide from the solid-phase substrate, and coupling peeled polypeptide and oligonucleotide in a solution by a reactive group. Synthesis of POCs mainly includes: (<NUM>) Maleimide-thiol bond coupling: maleimide is modified on the polypeptide or oligonucleotide, thiol is modified on another monomer, and the two monomers are added into a same solution to obtain the POCs after a reaction. (<NUM>) Disulfide bond or thioether bond coupling: <NUM>'- or <NUM>'-positions of the oligonucleotide is modified with a thiol group, and then reacted with a polypeptide whose C-terminus is modified with a bromoacetyl group in a buffer solution of pH <NUM>; the disulfide bond coupling can be directly oxidized by two thiol groups, or the thiol group can be activated by an activator such as dipyridyl disulfide and then coupled with another oligomer containing a thiol group; the disulfide bonds are commonly used to synthesize a conjugate of siRNA and polypeptides. (<NUM>) Oxime bond coupling: the aldehyde group reacts with the amino group to produce oxime; the reaction conditions are mild, with a high reaction efficiency, and a coupling product of double-stranded DNA and a specific polypeptide can be directly generated; meanwhile, two polypeptides can be simultaneously ligated to the <NUM>'- and <NUM>'-end of the nucleic acid through an oxime bond, by a bifunctional oligonucleotide with a polypeptide or a carbohydrate. This method does not require various protection processes and can be completed in one step, which is used to synthesize a "peptide-oligonucleotide-peptide" product. Specifically, the aldehyde group is introduced into the <NUM>'- and <NUM>'-end of the oligonucleotide, and then reacted with a hydroxylamine-modified polypeptide to obtain a "peptide-oligonucleotide-peptide" with a high yield. This one-step reaction of bifunctionalized oligonucleotides with polypeptides does not require any protection strategies and cross-linking reagents, and has a high yield under the slightly acidic environment. (<NUM>) Amide bond coupling: an oligomer containing activated carboxylic acid or thioester is reacted with another polymer modified with an amino group to obtain a product. (<NUM>) Hydrazone bond coupling: a hydrazine group is introduced into the polypeptide, a citric acid buffer with a pH value of <NUM> to <NUM> is added, and the mixture is reacted with an oligonucleotide modified with an acetaldehyde group. Thus, POCs ligated by hydrazone bonds are obtained and expressed as "2ACE2-shRNA3", "2ACE2-shRNA5", and "2ACE2-shRNA8", respectively.

Chromatographic methods are most commonly used for purification and analysis of the conjugate of peptides and oligonucleotides. According to complexity of the conjugates, different chromatographic methods should be selected for separation. The main methods include high-performance liquid chromatography (HPLC), reverse high-performance liquid chromatography (RP-HPLC), ion exchange chromatography (IEC, generally anion exchange chromatography), or two or more of which are used in series. Specifically, according to operating instructions, 2ACE2-shRNA3, 2ACE2-shRNA5, and 2ACE2-shRNA8 are extracted.

Similarly, shRNA (SEQ ID NO: <NUM> to SEQ ID NO: <NUM>) can be ligated with intracellular ACE2 or codon-optimized ACE2 polypeptide to form compounds, including but not limited to "transmembrane ACE2-shRNA-transmembrane ACE2", and "intracellular ACE2 -shRNA-intracellular ACE2"; alternatively, the shRNA/siRNA is inserted into the middle of ACE2 polypeptide to synthesize "transmembrane ACE2-shRNA-extracellular ACE2", and "intracellular ACE2-shRNA-extracellular ACE2"; similarly, compounds with ACE2 or ACE2-optimized polypeptides as targeted delivery vectors are designed, including but not limited to "ACE2-siRNA, extracellular ACE2-siRNA, transmembrane ACE2-siRNA, and intracellular ACE2-siRNA".

Liposome (Lip) modification includes: adsorption of positively-charged liposomes with negatively-charged shRNA, formation of the thiol-maleimide bond with thiol and liposomes by thiolation of amino groups in the ACE2 polypeptides, or formation of the carbamate bond with amino groups of ACE2 and liposomes (<FIG>).

DOTAP (MW=<NUM>): <NUM>/ml, <NUM> of an N-<NUM>-(<NUM>,<NUM>-di-oleoyloxy)propyl)-N,N,N-trimethylammoniumethyl sulfate (DOTAP) powder was added into a <NUM> volumetric flask, and a chloroform solution was added to reach a scale line.

Chol (MW=<NUM>): <NUM>/ml, <NUM> of a cholesterol (Chol) powder was added into a <NUM> volumetric flask, and a chloroform solution was added to reach a scale line.

m-PEG2000-DSPE (MW=<NUM>): <NUM>/ml, <NUM> of a methoxy-polyethylene glycol-distearoyl phosphatidyl-ethanolamine (m-PEG2000-DSPE) powder was added to <NUM> of DEPC water, and dissolved completely by vortexing and sonication for <NUM>.

Mal-PEG2000-DSPE (MW=<NUM>): <NUM>/ml, <NUM> of a maleimide derivatized polyethylene glycol-distearoyl phosphatidyl-ethanolamine (Mal-PEG2000-DSPE) powder was added to <NUM> of DEPC water, and dissolved by vortexing and sonication for <NUM>.

The liposome DOTAP/Chol was prepared by a lipid-film method, at a lipid concentration of <NUM>. The method included the following steps: according to an amount of preparing <NUM> of the liposome DOTAP/Chol, <NUM> to <NUM> of chloroform solutions of DOTAP and Chol were separately added to a <NUM> conical flask at a ratio of DOTAP: Chol=<NUM>:<NUM> (M:M); vacuum rotary evaporation was conducted at <NUM> for <NUM> to <NUM> to obtain a uniform lipid film, and traces of chloroform were blown out with high-purity nitrogen; <NUM> of DEPC water was added to shake to wash the lipid film from a bottle wall, to obtain a lipid suspension; after being fully hydrated, the lipid film was sonicated for <NUM>, and extruded through <NUM>, <NUM>, <NUM>, and <NUM> of polycarbonate membranes in sequence for <NUM> to <NUM> times in each size, so as to obtain the liposome DOTAP/Chol.

<NUM>-IT (Traut'S reagent) was a common reagent for protein thiolation, and the thiolation was conducted at an N amino group of the ACE2 protein. The thiolation included the following steps: 2ACE2-shRNA and <NUM>-IT (Traut'S reagent, <NUM>-iminothiolane-HCl) were mixed evenly (the <NUM>-IT and the 2ACE2-shRNA have a molar ratio of <NUM>: <NUM>), and reacted at a room temperature for <NUM>; excess <NUM>-IT was removed by dialysis with a sufficient amount of the dialysate (<NUM>×PBS, <NUM> EDTA, pH=<NUM>) that was stored at <NUM> for each time, where the dialysis was conducted overnight by magnetic stirring and the dialysate was changed after <NUM> to <NUM>; a protein concentration and a degree of thiolation were determined by a BCA method and an Ellman method, respectively.

To increase a circulation time and targeting specificity of the liposomes, various liposome-modified complexes prepared in (A) were further PEGylated and ACE2-modified to obtain a PEGylated liposome-modified complex with RBD as a ligand.

<NUM>µl (while setting <NUM>µl and <NUM>µl as a control) of <NUM>/ml MAL-DSPE-PEG was inserted into the liposome-modified complex 2ACE2-shRNA/ch1, siRNA/ch1, or shRNA/ch1 prepared in (A) separately (mixing the two separately), incubated in a <NUM> water bath for <NUM>, and allowed to stand for <NUM> at a room temperature; about <NUM>µg of thiolated ACE2 or 2ACE2-shRNA/ch1 was added to cross-link the thiol group on the thiolated amino group in the ACE2 with the maleimide in MAL-DSPE-PEG, to obtain ACE2-modified and PEGylated (PEGylation had PEG concentrations of <NUM> mol% PEG, <NUM> mol% PEG, and <NUM> mol% PEG, respectively). That is, the complex was electrostatically adsorbed to the liposome DOTAP/Chol with siRNA, shRNA, and/or 2ACE2-shRNA, wrapped on an outer layer with the MAL-DSPE-PEG, and then ligated with the ACE2 or 2ACE2-shRNA by the MAL-DSPE-PEG. The prepared liposome (ch)-modified complexes included ACE2-siRNA/ch1, ACE2-shRNA/ch1, ACE2-(2ACE2-shRNA/ch1), (2ACE2-shRNA)-siRNA/ch1, (2ACE2-shRNA)-shRNA/ch1, and (2ACE2-shRNA)-(2ACE2-shRNA/ch1), abbreviated as ACE2-siRNA/ch1, ACE2-shRNA/ch1, 3ACE2-shRNA/ch1, 2ACE2-shRNA/siRNA/ch1, 2ACE2-2shRNA/ch1, and 4ACE2-2shRNA/ch1, respectively. As shown in <FIG>, the ACE2 played a role in targeted delivery of siRNA/shRNA; the liposome and the PEG functioned as protection of siRNA/shRNA, slow release of siRNA/shRNA, and intracellular transfection of siRNA/shRNA, or an immunologic adjuvant.

When pH is greater than <NUM>, the amino group of ACE2 reacted with pNP-PEG-DPPE (PEG-PE) to form a stable carbamate bond conjugate, which was then inserted into an outer membrane of the liposomes in a targeted and quantitative manner to obtain a liposome-modified compound. In this example, ACE2 fragments and siRNA were used to prepare a liposome-modified compound for ACE2-targeted delivery of siRNA (ACE2-siRNA).

ACE2 or its fragments were synthesized using the ACE2 synthesis method described above.

<NUM> of a <NUM>/mL DPPE chloroform solution was placed in a <NUM> round-bottomed flask, and <NUM> of triethylamine (TEA) was added dropwise; about <NUM> of a polyethylene glycol <NUM> di-p-nitrophenyl carbonate [(pNP)<NUM>-PEG<NUM>] chloroform solution with a concentration of <NUM>/mL was added to an obtained mixture; the mixture was blown with nitrogen, sealed, protected from light, stirred magnetically overnight at a room temperature, the solvent was removed by evaporation under reduced pressure, and residual chloroform was removed in vacuum; <NUM> of a <NUM> mol/L HCl aqueous solution was added, and a transparent micellar solution was formed by an ultrasonic treatment. With the <NUM> mol/L HCl aqueous solution as an eluent, separation was conducted by CL-4B Sepharose to remove unreacted (pNP)<NUM>-PEG<NUM> and released pNP; an eluate containing pNP-PEG-DPPE micelles was collected, lyophilized, and pNP-PEG-DPPE was characterized and quantified by TLC, HPLC, MS and NMR.

(<NUM>) Synthesis of ACE2-PEG-DPPE: <NUM> of the pNP-PEG-DPPE was dissolved in <NUM> of chloroform, placed in a <NUM> flask, and the chloroform was removed under reduced pressure on a rotary evaporator to form a lipid film, and the residual chloroform was removed by vacuum; <NUM> of ACE2 was dissolved in <NUM> of <NUM> mol/L HCl, a resulting mixture was added to a flask coated with the lipid film on the inner wall, incubated at a room temperature for <NUM>, and shaken gently to disperse the lipid film. <NUM> of a <NUM> mum/L (pH <NUM>) Tris buffer was added to a suspension, mixed well, and incubated at <NUM> overnight under nitrogen protection. The samples were placed in a dialysis bag with a molecular mass of <NUM> kD, dialyzed in the <NUM> mmol/L (pH <NUM>) Tris buffer for about <NUM>, and then dialyzed in deionized water at <NUM> for <NUM>; the solution in the bag was lyophilized and stored in a -<NUM> refrigerator.

(<NUM>) Synthesis of ACE2-siRNA/liposomal: chloroform solutions of egg yolk phospholipid (ePC), cholesterol (Ch), distearoyl ethanolamine-polyethylene glycol <NUM> (PEG<NUM>-DSPE), and dioleoyl trimethylamine propane (DOTAP) were mixed in a molar ratio of <NUM>:<NUM>:<NUM>:<NUM>; to label the lipid film, Rho-PE with a molar ratio of <NUM>% to a total lipid mass was added in the above mixture, and chloroform was removed under reduced pressure to form the lipid film. A certain amount of the siRNA was dissolved in DEPC-treated ultrapure water, where an amount of the siRNA should ensure that the positive charge of DOTAP was completely neutralized. The phospholipid film was hydrated with an aqueous solution containing siRNA in a <NUM> water bath for <NUM>, to form an siRNA-encapsulated liposome. The preliminarily formed liposome was passed through <NUM> and <NUM> of polycarbonate nuclear pore membranes (Whatman) <NUM> times with a manual extrusion device (Avanti Polar Lipids), to prepare the liposomes with a uniform particle size. An appropriate amount of the ACE2-PEG-DPPE was dissolved in methanol, placed in a flask, and blow-dried with nitrogen to form a film; the prepared liposome suspension was added and treated in a water bath at <NUM> for <NUM>, such that the ACE2-PEG-DPPE was inserted into an outer membrane of the liposome and the ACE2 accounted for <NUM>% to <NUM>% of the total lipid (the molar ratio could be adjusted appropriately); a liposome (ch/lip) complex ACE2-shRNA/siRNA/lip as shown in <FIG> was prepared after dynamic laser scattering, cryo-etching electron microscopy, and nucleic acid electrophoresis.

The nCoVshRNA-2ACE2 was synthesized from the ACE2 or its polypeptides with the shRNA/siRNA, and then further modified by the liposomes.

The virus strains were added to a DMEM medium (<NUM>% FBS) of Vero E6 cells grown to <NUM>% confluence, and incubated in a <NUM>, <NUM>% CO<NUM> incubator for <NUM> d to <NUM> d; when an cytopathic effect (CPE) occurred, the virus was isolated and then prepared by a medium into a <NUM><NUM> TCID<NUM>/ml to <NUM><NUM> TCID<NUM>/ml virus solution for later use. According to this, virus solutions of two variant strains B. <NUM> and B. <NUM> of the COVID-<NUM> virus were prepared separately to verify whether the nCoVshRNA-2ACE2 was effective against two or more variant viruses containing a same conserved gene, so as to prove whether the shRNA/siRNA of the present disclosure had a broad-spectrum antiviral effect by targeting the conserved gene.

An experimental group and a control group were set up to test an effect of the compounds against the B. <NUM> and B. Each group was inoculated with a <NUM>-well plate, and <NUM>×<NUM><NUM> Vero-E6 cells and <NUM> of a DMEM medium (<NUM>% FBS) were added to each well, and then incubated in a <NUM> and <NUM>% CO<NUM> incubator to <NUM>% confluence, followed by changing the medium; meanwhile, the tested compound and the virus solutions of B. <NUM>, and B. <NUM> strains were added.

The experimental groups included: a 2ACE2-shRNA3 group (<NUM> nmol 2ACE2-shRNA3 + <NUM> virus solution), a 2ACE2-shRNA3/ch1 group (<NUM> nmol 2ACE2-shRNA3/ch1 + <NUM> virus solution), a 4ACE2-2shRNA3/ch1 group (<NUM> nmol 4ACE2-2shRNA3/ch1 + <NUM> virus solution), an ACE2-siRNA3/lip group (<NUM> nmol ACE2-siRNA3/lip + <NUM> virus solution); the control group included: a naked shRNA3 group (<NUM> nmol naked shRNA3 + <NUM> virus solution), a naked siRNA3 group (<NUM> nmol naked siRNA3 + <NUM> virus solution), an ACE2 control group (<NUM> nmol ACE2 + <NUM> virus solution), a positive control group (<NUM> virus solution), and a negative control group (<NUM> DMEM medium) (Tables <NUM> to <NUM>). Similarly, experiments with 2ACE2-shRNA5 and 2ACE2-shRNA8 were conducted (Tables 1a to 6a).

After <NUM>, <NUM>, and <NUM> of incubation, a supernatant was collected from each group, and then diluted at <NUM>:<NUM>, <NUM>:<NUM>, <NUM>:<NUM>, <NUM>:<NUM>, <NUM>:<NUM>, <NUM>:<NUM>, <NUM>:<NUM>, and <NUM>:<NUM> to conduct RT-PCR detection.

Viral nucleic acid extraction and nucleic acid (ORF1ab/N) detection were conducted according to kit instructions.

As shown in Table <NUM>, after culturing the cells in each group for <NUM>, a viral RNA titer of the negative control group was negative, a viral RNA titer of the positive control group was <NUM>:<NUM>, and viral RNA titers of the other groups each were <NUM>:<NUM>.

As shown in Table <NUM>, after culturing the cells in each group for <NUM>, a viral RNA titer of the negative control group was still negative, and a viral RNA titer of each control group was <NUM>:<NUM> to <NUM>:<NUM>; while viral RNA titers of the experimental groups each were <NUM>:<NUM>, which were significantly lower than that of the positive control group and the ACE2 control group (p<<NUM>).

As shown in Table <NUM>, after culturing the cells in each group for <NUM>, a viral RNA titer in the negative control group was still negative, a viral RNA titer of each control group was greater than <NUM>:<NUM>, and a viral RNA titer of the control group was greater than <NUM>:<NUM> to <NUM>:<NUM>; while a viral RNA titer of the experimental group was <NUM>:<NUM> to <NUM>:<NUM>, which was significantly lower than that of the control group (p<<NUM>).

Tables <NUM> to <NUM> showed that the experimental group had an obvious anti-B. <NUM> effect, indicating that shRNA or siRNA ligated to ACE2 could be delivered to target cells for RNA interference. However, the shRNA or siRNA that was not ligated to ACE2 could not enter the target cells, and could not play a role of RNA interference extracellularly. The results in Tables 1a to 3a were basically consistent with those in Tables <NUM> to <NUM>.

As shown in Table <NUM>, after culturing the cells in each group for <NUM>, a viral RNA titer of the negative control group was negative, viral RNA titers of the positive control group and the ACE2 control group each were <NUM>:<NUM>, and viral RNA titers of the other groups each were <NUM>:<NUM>. As shown in Table <NUM>, after culturing the cells in each group for <NUM>, a viral RNA titer of the negative control group was still negative, and a viral RNA titer of each control group was <NUM>:<NUM> to <NUM>:<NUM>; while viral RNA titers of the <NUM> experimental groups were <NUM>:<NUM> to <NUM>:<NUM>, which were significantly lower than that in the control group (p<<NUM>). As shown in Table <NUM>, after culturing the cells in each group for <NUM>, a viral RNA titer of the negative control group was still negative, and a viral RNA titer of each control group was <NUM>:<NUM> to <NUM>:<NUM>; while viral RNA titers of the <NUM> experimental groups were <NUM>:<NUM> in <NUM> groups and <NUM>:<NUM> in the other <NUM> groups, which were still significantly different from that in the control group (p<<NUM>). Tables <NUM> to <NUM> showed that the experimental group had an obvious anti-B. <NUM> effect, indicating that shRNA or siRNA ligated to ACE2 could be delivered to target cells for RNA interference. However, the shRNA or siRNA that was not ligated to RBD could not enter the target cells, and could not play a role of RNA interference. The results in Tables 4a to 6a were basically consistent with those in Tables <NUM> to <NUM>. Tables <NUM> to <NUM> and 1a to 6a showed that the experimental group (nCoVshRNA·2ACE2) had anti-B. <NUM> and anti-B. <NUM> effects, indicating that the nCoVshRNA·2ACE2 targeting conserved genes in the experimental group had a broad-spectrum anti-variant strain effect.

Animal grouping: female BALB/c mice in an SPF grade of <NUM> to <NUM> weeks and about <NUM> were selected; taking the 2ACE2-shRNA3 as an example, the mice were randomly divided into a 2ACE2-shRNA3 (shRNA5/<NUM>) group (inoculated with 2ACE2-shRNA3 + B. <NUM>), a 2ACE2-shRNA3/chl group (inoculated with 2ACE2-shRNA3/chl + B. <NUM>), a 4ACE2-2shRNA3/chl group (inoculated with 4ACE2-2shRNA3/chl + B. <NUM>), an ACE2-siRNA3/lip group (inoculated with ACE2-siRNA3/lip + B. <NUM>), a shRNA3 group (inoculated with shRNA3 + B. <NUM>), a positive control group (inoculated with B. <NUM> + normal saline), and a negative control group (inoculated with normal saline only), with <NUM> mice in each group.

Animal inoculation: the mice each were inoculated with <NUM>µl of a B. <NUM> strain virus solution with a titer of <NUM><NUM>/ml TCID<NUM> by nasal spray, while the negative control group was inoculated with <NUM>µl of a normal saline by nasal spray. The mice were anesthetized by intraperitoneal injection of a <NUM>% chloral hydrate solution, and <NUM> nmol of the 2ACE2-shRNA, 2ACE2-shRNA/ch1, 4ACE2-2shRNA/ch1, ACE2-siRNA/lip, and shRNA were slowly injected into the trachea of the mice separately. On the 7th day after infection, <NUM> mice in each group were sacrificed for virus detection; the remaining <NUM> mice in each group were used to observe antibodies.

A <NUM>% homogenate was prepared from a lung tissue of the mice sacrificed on the 7th day after infection, and <NUM> pl of the homogenate was centrifuged to remove a supernatant, the homogenate was diluted <NUM>-fold successively, and inoculated in a <NUM>-well plate with VeroE6 growing in a single layer at <NUM>µl per well and <NUM> wells per dilution; the homogenate was gently shaken, adsorbed at <NUM> for <NUM>, washed with a Hank's solution, added with a medium, and then incubated in a <NUM> CO<NUM> incubator; a cytopathic effect (CPE) was observed, and a percentage of VeroE6 TCID<NUM> in each group was calculated, where a higher percentage meant a higher virus content (Tables <NUM> to <NUM>).

As was seen from Tables <NUM> to <NUM>, percentages of VeroE6 TCID<NUM> (<NUM><NUM> in each well) induced by lung homogenate in each group were as follows: 2ACE2-shRNA3 group was <NUM>%, 2ACE2-shRNA3/ch1 group was <NUM>%, 4ACE2-2shRNA3/ch1 group was <NUM>%, ACE2-siRNA3/lip group was <NUM>%, shRNA3 group was <NUM>%, positive control group was <NUM>%, and negative control group was <NUM>%. Since RNAi mainly occurred in the cytoplasm, shRNA3 in the shRNA3 group was not easy to pass through the cell membrane, so as to have a little effect on the RNAi, and results were consistent with the positive control group; meanwhile, the shRNA3 in the 2ACE2-shRNA3 group, 2ACE2-shRNA3/ch1 group, 4ACE2-2shRNA3/ch1 group, and ACE2-siRNA3/lip group had a better RNAi effect due to targeted delivery of the ACE2 to the target cytoplasm, and the percentage of VeroE6 TCID<NUM> was significantly different from that of the positive control group (p<<NUM>).

A TCID<NUM> assay was conducted separately on the 2ACE2-shRNA8 and 2ACE2-shRNA8/ch1 according to the TCID<NUM> assay method above. The results showed that the 2ACE2-shRNA8 group, 2ACE2-shRNA8/ch1 group, shRNA8 group, positive control group, and negative control group had TCID<NUM> of <NUM>%, <NUM>%, <NUM>%, <NUM>%, and <NUM>%, respectively, and the TCID<NUM> of experimental groups was significantly lower than that of the positive control group.

The venous blood of the remaining <NUM> mice in each group was collected at the 2nd, 4th, and 6th weeks, a serum was separated, and sera of a same week in each group were mixed, and then stored at -<NUM> for future use. The ACE2-Ab was determined by a double-antigen sandwich method according to instructions of the kit.

It was seen from Table <NUM> that the ACE2-Ab in the lung tissue homogenate of mice in each experimental group (with ACE2) was significantly higher than that in the control group (without ACE2) (p<<NUM>), indicating that the ACE2 of experimental groups could stimulate the mice to produce ACE2-Ab, and a content of the ACE2-Ab was related to ACE2 molecular composition and liposome modification.

The 2ACE2-shRNA group, 2ACE2-shRNA/ch1 group, 4ACE2-2shRNA/ch1 group, and ACE2-siRNA/lip group were used as experimental groups (all containing ACE2-Ab), while the shRNA group was used as a control group (containing virus but not ACE2-Ab). The sera of 2nd, 4th, and 6th week in each group after ACE2-Ab detection were mixed, a mixed serum in each group were double-diluted, and <NUM>µl of each diluted serum was inoculated in a <NUM>-well plate with VeroE6 growing in a single layer; each experimental group was simultaneously inoculated with <NUM>µl of an undiluted mixed serum of the shRNA group, while the negative control group was inoculated with <NUM>µl of a normal saline only. The samples in each group were shaken and mixed well, adsorbed at <NUM> for <NUM>, washed with the Hank's solution, added with a medium, and incubated in a <NUM> CO<NUM> incubator; a cytopathic effect (CPE) was observed within <NUM> week, where "+" indicated the normal cell growth, as shown in Table <NUM>.

As was seen from Table <NUM>, since the negative control group was not inoculated with virus, there was no CPE in each well of VeroE6; the positive control group was inoculated with the virus without ACE2-Ab neutralization (the mixed serum in shRNA3 group was not diluted), such that VeroE6 in each well produced CPE; since the shRNA3 group had no neutralization effect of ACE2-Ab, VeroE6 did not have CPE only after a virus-containing self-serum was diluted at <NUM>:<NUM>; though the experimental groups were also inoculated with the virus like the positive control group, but due to the neutralization effect of ACE2-Ab, the VeroE6 showed CPE only when the serum (ACE2-Ab content) was diluted at not less than <NUM>:<NUM>. It showed that the ACE2-Ab could neutralize the viral receptor ACE2 on the surface of VeroE6 cells, thereby producing an antiviral effect.

Claim 1:
An in vitro synthesis method of a targeted drug nCoVshRNA-2ACE2, comprising the following steps: synthesizing a shRNA with a sense strand and an antisense strand of an RNAi sequence, extending a terminus of the sense strand and/or the antisense strand constituting the shRNA, and ligating to a ACE2 polypeptide to synthesize a drug that delivers the shRNA by the ACE2 polypeptide, wherein the RNAi sequence is a consensus conserved gene of a COVID-<NUM> virus and a variant strain thereof, such that the shRNA synthesized with the sense strand and the antisense strand of the RNAi sequence is targeted to interfere with the conserved gene, resulting in a broadspectrum RNAi effect.