WO2023083315A1 - 一种以RBD靶向递送shRNA的nCOVsiRNA药物及合成方法和应用 - Google Patents

一种以RBD靶向递送shRNA的nCOVsiRNA药物及合成方法和应用 Download PDF

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WO2023083315A1
WO2023083315A1 PCT/CN2022/131458 CN2022131458W WO2023083315A1 WO 2023083315 A1 WO2023083315 A1 WO 2023083315A1 CN 2022131458 W CN2022131458 W CN 2022131458W WO 2023083315 A1 WO2023083315 A1 WO 2023083315A1
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rbd
shrna
sirna
seq
drug
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翁炳焕
黄荷凤
贺林
王伟平
朱智勇
姚航平
马端
林佳丽
陈敏
严恺
杨昊堃
应俊
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翁炳焕
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Priority claimed from CN202210116884.1A external-priority patent/CN114569712A/zh
Priority claimed from CN202210920201.8A external-priority patent/CN116254256A/zh
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/215—Coronaviridae, e.g. avian infectious bronchitis virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses

Definitions

  • the invention relates to a synthesis method of a chemical drug nCoVshRNA ⁇ 2RBD, which belongs to the field of biopharmaceuticals.
  • coronavirus includes single-stranded positive-strand nucleic acid (ssRNA), spike protein (S), membrane protein (M), envelope protein (E) and nucleocapsid protein (N), wherein the N-terminus of S protein is composed of domain (S1-NTD) and receptor binding domain (S1-RBD).
  • ACE2 Human angiotensin-converting enzyme II
  • coronavirus consisting of 805 amino acids, including a transmembrane region, an intracellular carboxy-terminus and an extracellular amino-terminus.
  • the coronavirus binds to the ACE2 of the target cell through its S1-RBD, and undergoes membrane fusion and endocytosis, allowing the virus to enter the target cell expressing ACE2 or containing an ACE2 channel.
  • RBD and ACE2 are the relationship between ligand and receptor. Based on this, the RBD polypeptide is designed as a targeted delivery carrier, and the drug is delivered to the target cells expressing ACE2 through RBD, so that the drug can exert its specificity only for virus-infected cells effect.
  • RNA interference RNA interference
  • siRNA drugs have been approved by the FDA for marketing.
  • the current siRNA drugs often use a single strain to design siRNA, use single-stranded siRNA (antisense RNA) to prepare siRNA drugs, or use non-target delivery vectors for non-specific delivery of siRNA drugs.
  • antisense RNA single-stranded siRNA
  • the above-mentioned siRNA designed based on a single strain will be off-target and invalid due to the mutation of the coronavirus.
  • siRNA shared by each strain is used to prepare broad-spectrum siRNA drugs against the mutated and mutated strains. If a non-targeted delivery vector is used, the siRNA drug will be delivered to cells that are not easily infected by the coronavirus because they do not express ACE2, resulting in side effects of non-targeted delivery. Therefore, it is necessary to design a drug that can specifically deliver siRNA to target cells Targeted delivery vehicle.
  • RNAi mechanism reported by HreA et al.
  • the efficiency of double-stranded RNA silencing homologous mRNA prepared by mixing sense RNA and antisense RNA is more than 100 times higher than that of single-stranded RNA (antisense RNA).
  • Effective RNAi technology should use double-stranded RNA (shRNA/dsRNA) containing both positive and antisense siRNA, while traditional siRNA drugs are not double-stranded RNA although they are based on the RNAi mechanism. Therefore, the synthetic shRNA of the present invention is used for RNAi treatment, so that according to the report of Hre A, it conforms to the RNAi mechanism in theory, and the RNAi effect is improved by more than 100 times.
  • siRNA Small interfering RNA
  • mRNA complementary target messenger RNA
  • RBD polypeptide is connected at the end of the shRNA double strand to achieve the purpose of specifically transporting and protecting siRNA.
  • siRNA/dsRNA/shRNA can induce inflammation induced by type I interferon (IFN- ⁇ / ⁇ ) or pro-inflammatory cytokines (IL6 and TNF ⁇ )-mediated immune response, in which IFN- ⁇ / ⁇ has broad-spectrum anti-virus, enhanced hematopoietic cell proliferation and immune regulation, and IL6 can induce B cell differentiation, produce immunoglobulin, promote T cells and hematopoietic stem cells
  • IFN- ⁇ / ⁇ type I interferon
  • IL6 and TNF ⁇ pro-inflammatory cytokines
  • IL6 can induce B cell differentiation, produce immunoglobulin, promote T cells and hematopoietic stem cells
  • siRNA/dsRNA/shRNA is an oligonucleotide immune adjuvant involved in immune activation.
  • This application synthesizes siRNA into shRNA and connects RBD polypeptide at the end of shRNA, so that the obtained nCoVshRNA ⁇ 2RBD contains dimeric RBD.
  • dimeric RBD has a more complex molecular structure and molecular weight than the original single-molecule RBD. Greater, especially the immune adjuvant effect of shRNA makes the vaccine more effective.
  • the present invention synthesizes siRNA into shRNA, and respectively extends and connects RBD polypeptides at the ends of the shRNA double strands, and synthesizes a chemical drug nCoVshRNA 2RBD that delivers shRNA with RBD targeting, in which shRNA is both an immune adjuvant and a broad-spectrum anti-mutation
  • the siRNA drug of the virus strain, and the RBD is not only a targeted delivery carrier, but also a dimer RBD vaccine, and can also compete with the virus for the ACE2 receptor, thereby competitively inhibiting the virus infection.
  • the purpose of the present invention is to provide a kind of nCOVsiRNA medicine that delivers shRNA with RBD targeting and its synthesis and application; Vaccines and the role of neutralizing ACE2 receptors, liposomes have the role of stabilizing shRNA, cell transfection and immune adjuvants.
  • Screening of targets against mutant strains Screen siRNA from the common genes of various pathogenic coronaviruses and their mutant strains, such common genes include conserved genes, ultra-conserved genes and/or conserved microsatellites, so that the screened siRNA is the common target of each mutant strain that does not change with virus mutation, so that it has a broad-spectrum anti-mutant strain effect.
  • Synthesis of target siRNA against mutant strains synthesize 2 complementary 21-25nt oligonucleotide siRNAs from the screened siRNA, and synthesize a base sequence that acts as a spacer.
  • Synthetic shRNA The synthesized 2 complementary oligonucleotide polypeptide siRNA and the base sequence that acts as a spacer are further synthesized into a small hairpin shRNA double strand spaced by an intermediate base sequence to form a loop.
  • siRNA is preferred: the synthetic shRNA is constructed as an interference vector, and its mRNA expression, protein expression and interference effect are detected. After siRNA design, synthesis, screening, iterative design and verification, siRNA with high silencing efficiency is preferred.
  • Synthesize preferred siRNA and shRNA Use preferred siRNA sequences to synthesize siRNA and shRNA as described above, including chemical modifications to increase stability and avoid off-target.
  • Synthesize or express RBD polypeptide or protein through RBD gene synthesize but not limited to the 319th to 510th amino acid sequence of the coronavirus S protein, the conserved amino acid sequence at but not limited to the N439, V483 and Q493 positions, and codon-optimized amino acid sequence.
  • Purification of compounds Purification of compounds by high performance liquid chromatography, reversed high performance liquid chromatography or ion exchange chromatography.
  • Liposome modification of compounds liposome modification compounds were prepared by negatively charged shRNA adsorbing positively charged liposomes; sulfhydryl groups and liposome maleimides were formed into maleimides by thiolation of RBD amino groups -Prepare PEG internalized liposome-modified compounds through sulfhydryl bonds; prepare liposome-modified compounds by forming carbamate bonds between the amino terminal of RBD and liposomes; prepare liposome-modified siRNA by linking RBD or RBD fragments to liposomes Compounds modified with body or lipid nanoparticles.
  • Compound verification test the antiviral effect of the compound on two or more different mutant strains at the cell level in vitro, and observe whether it has a broad-spectrum anti-mutant strain targeting conserved genes; detect whether the compound has a targeted effect in animals The RNAi effect of delivering shRNA, the immune effect of the vaccine, and whether it has the effect of immune enhancement.
  • shRNA plays the role of broad-spectrum anti-mutant strains and immune adjuvant
  • RBD plays the role of targeted delivery of shRNA, protection of shRNA, membrane-penetrating peptide and protein vaccine, and makes RBD and shRNA synergistically, further producing many new functions.
  • the distinguishing feature of the present invention from traditional siRNA drugs is that the present invention screens the common target siRNA of each strain that does not change with virus mutation from various coronaviruses and their mutant strains, so that this siRNA has a broad-spectrum anti-mutant strain effect .
  • siRNA medicines are prepared by using sense-strand siRNA or antisense-strand siRNA, that is, single-strand siRNA; while the present invention uses sense-antisense strand siRNA to synthesize shRNA.
  • the RNAi mechanism reported by HreA et al. the RNAi interference effect of double-stranded RNA is at least 100 times greater than that of single-stranded RNA. Therefore, the design of the present invention is more correct and more in line with the RNAi mechanism.
  • the distinguishing feature of the present invention from traditional siRNA drugs is that the present invention uses RBD as a carrier to deliver shRNA targetedly.
  • Coronavirus specifically infects target cells expressing ACE2, and currently there is no targeting vector that specifically delivers siRNA to virus-infected cells but not to uninfected cells.
  • the present invention connects RBD and shRNA, so that RBD can produce a new function of targeted delivery of shRNA, thereby avoiding the side effects of non-specific delivery.
  • siRNA/shRNA is negatively charged, fat-soluble, difficult to pass through the cell membrane, and easily degraded by nucleases, it is difficult to be delivered to the target cell plasma to produce RNAi.
  • RBD has the property of a membrane-penetrating peptide, it can protect shRNA from being degraded by nucleases and make it easier to be delivered to the cytoplasm through the target cell membrane.
  • the present invention connects 1 molecule of shRNA with 2 molecules of RBD to form a dimeric RBD vaccine, wherein the shRNA is an immune adjuvant.
  • the shRNA is an immune adjuvant.
  • the combined molecular weight of RBD and shRNA is more than double that of traditional single-molecule RBD vaccines, and the molecular structure is also more complex, so its immunogenicity is stronger.
  • the main components of the immune adjuvant are oligonucleotides and lipids
  • the siRNA or shRNA that is essentially an oligonucleotide has an immune adjuvant effect that enhances the immune effect of the RBD vaccine.
  • the liposome can also be used as an immune adjuvant to enhance the immune effect of the RBD protein.
  • the RBD in the chemical drug can compete with the RBD of the coronavirus to bind to the ACE2 receptor of the target cell, thereby inhibiting the virus infection.
  • the compound synthesized by 2 molecules of RBD and shRNA increases the molecular weight and structural complexity to enhance antigenicity.
  • the shRNA bound by RBD is not easily degraded by enzymes, easily passes through the cell membrane, and is easily delivered to the target cell plasma.
  • the design method of the present invention for preparing nCoV siRNA drugs by linking the ends of shRNA double strands to targeted delivery carrier RBD is expected to be applied to the preparation of siRNA gene therapy drugs for viruses, bacteria, tumors, and genetic diseases.
  • the nCoV siRNA drug of the present invention integrates a broad-spectrum anti-mutant strain targeted drug and a dimer S1-RBD vaccine.
  • the compound uses the coronavirus receptor binding domain S1-RBD as a carrier and delivers coronavirus to ACE2 expressing cells.
  • the broad-spectrum anti-mutant strain targeted drug refers to the design of siRNA with broad-spectrum anti-mutant strain effect based on the consensus RNAi sequence of coronavirus and its mutant strains, which does not change with virus mutation, and then synthesizes the siRNA shRNA; after the shRNA is linked to the S1-RBD polypeptide that is both a penetrating peptide and an ACE2 ligand, the membrane permeability and nuclease resistance stability of the shRNA are optimized, making it easier for the shRNA to be specifically delivered to Target cytoplasm expressing ACE2, specifically silencing target genes in ACE2 expressing cells susceptible to virus infection;
  • the dimeric S1-RBD vaccine refers to the connection of one molecule of shRNA with both broad-spectrum anti-variant strain and immune adjuvant effect and two molecules of S1-RBD polypeptide with protein antigen effect, and the synthetic molecular weight is higher.
  • a new technical line vaccine with large size, more complex structure and autoimmune adjuvant components.
  • Fig. 1 is the technical circuit diagram of preparation nCoVsiRNA medicine of the present invention
  • Fig. 2 is the synthetic and application schematic diagram of the present invention
  • Fig. 3 is the synthesizing compound schematic diagram of the present invention.
  • Fig. 4 is a schematic diagram of liposome-modified siRNA of the present invention.
  • Fig. 5 is a schematic diagram of liposome-modified shRNA of the present invention.
  • Fig. 6 is a schematic diagram of RBD targeted delivery of shRNA and competition between RBD and virus for ACE2 receptor of the present invention.
  • 1 is the siRNA that interferes with the conserved genes of coronaviruses; 2 is the sense and antisense strands of the small hairpin shRNA, and the shRNA is formed by annealing two complementary siRNAs; 3 is the sense and antisense strands in the shRNA.
  • 4 are two RBD polypeptides, which are respectively connected to the sense and antisense strands of shRNA through their N amino groups; 5 is the target cell expressing ACE2 receptor 6, and virus 10 is connected to the shRNA through the C of its RBD.
  • the RBD of the present invention is the same as the RBD of virus 10, and the C-terminal of RBD4 is also connected with the outer cell membrane of ACE2 receptor 6.
  • the N-terminus is combined to deliver shRNA2 to the target cell 5 expressing ACE2 receptor 6.
  • the target cell 5 is easy to infect the virus 10 because it contains ACE2 receptor 6; as shown in the target cell 9, the 7 and 8 represent the shRNA and RBD that enter the target cell plasma due to the targeted delivery of RBD4 and pass through the ACE2 receptor 6;
  • the shown shRNA15, long-chain RNA viruses 10 and 11 are also degraded into small fragments of RNA sequence 14 and inactivated, and RBD8 originally connected to shRNA7 is also dissociated into RBD12, thereby stimulating the host to produce anti-RBD12 antibodies 13.
  • 1 is a loop
  • 2 is an shRNA formed by two complementary sense and antisense strands
  • 3 is two RBD polypeptides (proteins), and the two RBDs are respectively connected to the shRNA sense and antisense strands.
  • shRNA is protected by RBD and delivered to the ACE2 receptor by RBD, and then specifically enters the target cell plasma through ACE2 receptor with RBD to degrade the viral target gene.
  • 1 is siRNA encapsulated by liposome
  • 2 is liposome layer
  • 3 is PEG layer
  • 4 is RBD.
  • siRNA plays the role of RNAi
  • liposome protects siRNA and causes endocytosis
  • PEG makes siRNA release slowly and circulate for a long time
  • RBD plays the dual role of targeted delivery of siRNA and vaccine.
  • 1 is a loop
  • 2 is an shRNA formed by two complementary sense and antisense strands
  • 3 is two RBD polypeptides (proteins), and the two RBDs are respectively connected to the shRNA sense and antisense strands.
  • 4 is the liposome layer
  • 5 is the PEG layer
  • shRNA2 is wrapped by liposome 4.
  • shRNA plays the role of RNAi
  • liposome protects shRNA and causes endocytosis
  • PEG enables slow release and long-term circulation of shRNA
  • RBD plays the dual role of targeted delivery and vaccine.
  • 1 is loop
  • 2 is shRNA
  • 3 is RBD polypeptide
  • 4 is ACE2 receptor
  • 5 is RBD of new coronavirus
  • 6 is new coronavirus
  • 7 is target cell expressing ACE2 receptor
  • 8 is not Cells expressing the ACE2 receptor.
  • the targeted delivery carrier RBD3 delivered the RNAi drug shRNA2 to target cells expressing ACE2 receptor 4, and the new coronavirus 6 also infected target cells 7 through its RBD5 binding to ACE2 receptor 4. Due to the targeted delivery of RBD3, shRNA2 specifically enters the target cells susceptible to virus infection 7 but not into cells not susceptible to virus infection 8, and at the same time, the RBD of the present invention competes with the viral RBD for the ACE2 receptor 4, thereby competitively inhibiting virus infection.
  • the technical circuit diagram 1 download the whole genome of the genus betacoronavirus (especially the novel coronavirus and its variant strains) from the Genbank database (http://www.NCBI.nlm.nih.gov/genome/) ( cDNA) sequence, search for the longest common subsequence in the whole genome sequence, and obtain ultra-conserved genes or conserved genes; use ClustalW software to compare the sequences of the whole genome downloaded from the Genbank database, detect the similarity between different sequences, and screen conserveed microsatellite sequence; using MEGA6.0 molecular evolutionary genetic analysis software, using the Neighbor-Jioning (N-J) method to construct an amino acid germline molecular evolution tree for the downloaded coronavirus amino acid sequence, and analyze and analyze the molecular variation characteristics of the amino acid sequence Optimize and deduce the conserved gene sequence as the basis for siRNA synthesis.
  • Genbank database http://www.NCBI.nlm.nih.gov/genome/
  • microsatellites were CTCTCT, AGAGAG, AAAAAAA, TATATA, and CACACA, respectively.
  • cDNA complete genome sequence of the ⁇ coronavirus (especially the new coronavirus and its variant strains) downloaded from the Genbank database (http://www.NCBI.nlm.nih.gov/genome/), using Ambion shRNA online design software ( http://www.ambion.com/techlib/misc/siRNAtools.html ) or DSIR and other software to obtain multiple siRNA candidate sequences with a length of about 19 nt, according to the Tm value and specificity of RNA binding Sex comparison results, preferably siRNA.
  • RNAi sequences siRNA
  • the common RNAi sequence of each strain that is, the consensus target siRNA
  • siRNAs marked with sequence numbers (SEQ ID NO.41-58) in Tables 2-5 are the common target siRNAs of NC_045512.2, Delta strain, and Omicron strain, and the siRNAs without sequence numbers are the respective RNAi sequences (siRNA), it can be seen that although the earliest NC_045512.2 strain mutated into the Delta strain and the recent Omicron strain, each strain remained unchanged except for their unique targeting interference sequence siRNA (unmarked part).
  • the commonly conserved sequence SEQ ID NO.41-58 that is variable and theoretically has the effect of targeting interference.
  • siRNA targeting ultra-conserved genes and conserved microsatellites S1/S2
  • siRNA S3/S4 targeting conserved genes and conserved microsatellites:
  • siRNAs theoretically anti-coronavirus mutant strains with ultra-conserved genes, conserved genes or conserved microsatellites as interference targets were obtained, named siRNA1/2/3/4.
  • RNAi mechanism when siRNA effectively interferes with the mRNA expression of the S gene, an S protein-deficient virus that loses infectivity will be formed.
  • siRNA effectively interferes with the mRNA expression of the N gene it will inhibit the packaging and replication of the virus.
  • siRNA effectively interferes with the mRNA expression of ORF1a or 1b gene it will affect the synthesis of viral RNA polymerase (RdRp) or protein processing enzyme (3CLpro).
  • RdRp viral RNA polymerase
  • 3CLpro protein processing enzyme
  • the present invention selects siRNA targeting N gene (SEQ ID NO.16 ⁇ 18, SEQ ID NO.49 ⁇ 51), siRNA targeting ORF1ab gene (SEQ ID NO.20 ⁇ 22, SEQ ID NO.52 ⁇ 54) ) and siRNA targeting S gene (SEQ ID NO.30-32, SEQ ID NO.56-58), and SEQ ID NO.1-2, SEQ ID NO.5-6 were synthesized.
  • an shRNA template capable of expressing a hairpin structure is designed. Each template is composed of two mostly complementary 55bp single-stranded DNAs, which can be formed after annealing and complementation. DNA duplex with BamH I and Hind III cohesive ends for ligation with linearized pSilencer4.1.CMV.neo. Then entrust the company to synthesize the designed siRNA and its shRNA template.
  • shRNA synthesized above was ligated and identified with the linearized interference vector pSilencer4.1.CMV.neo to construct shRNA expression plasmids and transformed into DH5a to obtain shRNA expression vectors respectively.
  • siRNA/shRNA According to the synthesized siRNA/shRNA and its constructed expression plasmid, select the corresponding target gene for synthesis or PCR amplification, then construct a fluorescent label vector, and co-transfect type II alveolar epithelial cells (AEC2s) or 293T with the shRNA expression plasmid cells for identification.
  • AEC2s co-transfect type II alveolar epithelial cells
  • 293T co-transfect type II alveolar epithelial cells
  • Primer design design upstream and downstream primers, add start codon at the 5' end of the upstream primer, and add a homology arm for homologous recombination with the vector to clone the amplified product into pEGFP-N1 at the 5' end of the primer .
  • Target gene amplification According to the gene amplification reaction system and reaction conditions provided by the Shanghai Sangon kit, the gene amplification, product recovery and purification are carried out to obtain the amplified product.
  • Linearization of pEGFP-N1 Resuscitate the DH5a strain containing pEGFP-N1 plasmid, extract the plasmid according to the kit, measure the concentration, perform enzyme digestion, identify and recover the linearized vector by 0.8% agarose gel electrophoresis.
  • pEGFP-N1 fluorescent tag carrier
  • the interference carrier pSilencer-shRNA
  • the fluorescent tag carrier pEGFP-N/S/ORF1ab
  • Flow cytometry detection In order to quantitatively analyze the interference effects of different interference vectors, flow cytometry was used to analyze the proportion of fluorescent protein expressing cells in the total number of cells.
  • RT-PCR detection of mRNA Relative fluorescence quantitative RT-PCR method was used to detect the relative expression of target genes in transfected cells. According to the standard curve, the CT value was used to convert the copy number of the target gene and B-actin internal reference gene, and the B-actin internal reference gene Genetic correction of the relative expression of viral gene mRNA (copy number of target gene/copy number of B-actin), and quantitative evaluation of the interference effect.
  • siRNAs with high silencing efficiency were obtained, and their sequences were SEQ ID NO.1 (named shRNA1, the same below), SEQ ID NO.2 ( shRNA2), SEQ ID NO.5 (shRNA3), and SEQ ID NO.16 (shRNA4), SEQ ID NO.49 (shRNA5) targeting the N gene, SEQ ID NO.21 (shRNA6) targeting the ORF1ab gene, SEQ ID NO.52 (shRNA7), SEQ ID NO.30 (shRNA8) targeting the S gene, the silencing efficiencies were 78%, 76%, 88%, 89%, 89%, 84%, 91%, 90%, respectively %.
  • each shRNA entrusts the biological company to synthesize 2 complementary 19-25nt oligonucleotide polypeptide siRNAs for each shRNA, and synthesize them as spacers
  • the base sequence of 9nt, and then the synthesized siRNA and the base sequence are connected into a small hairpin shRNA duplex separated by the middle base sequence to form a loop.
  • Each single strand of the synthesized shRNA duplex can be separately Link RBD polypeptide or protein.
  • synthesize 5'-ttaatacgacctctctgttggattttgacattcaagagatgtcaaatccaacagagagggtcgtattaa-3' (shRNA1, as shown in SEQ ID NO.78), 5'-ggttcgcaacttcacacagagtttcaagagaact, respectively, from SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.5.
  • TTCAAGAGA is a loop ring, and its left and right sides are respectively Complementary sense and antisense strands, and then link RBD protein or its polypeptide at its 3' and/or 5'.
  • siRNAs with high silencing efficiency are preferred, and shRNAs are synthesized respectively.
  • RBD (the amino acid sequence obtained by encoding the sequence shown in SEQ ID NO.59 or corresponding to the sequence shown in SEQ ID NO.59, the sequence shown in SEQ ID NO.59 is specifically: atgaatattacaaacttgtgcccttttggtgaagtttttaacgccaccagatttgcatctgtttatgcttggaacaggaagagaatcagcaactgtgttgctgattattctgtcctatataattccgcatc atttccactttttaagtgttatggagtgtctcctactaaattaaatgatctctgctttactaatgtctatgcagattcatttgtaattagaggtgatgaagtcagacaaatcgctccagggcaaactggaaagattgctgattataaattacagatgatgattttt
  • RBD protein vaccines are usually designed based on the characteristics of virus infection through the combination of RBD and ACE2, so the synthesized RBD is both a targeted delivery carrier and a protein vaccine.
  • FIG. 6 The schematic diagram of RBD targeted delivery of shRNA and competition between RBD and virus for ACE2 receptor of the present invention is shown in FIG. 6 .
  • 1 is loop
  • 2 is shRNA
  • 3 is RBD polypeptide
  • 4 is ACE2 receptor
  • 5 is RBD of new coronavirus
  • 6 is new coronavirus
  • 7 is target cell expressing ACE2 receptor
  • 8 is not Cells expressing the ACE2 receptor.
  • the targeted delivery carrier RBD 3 delivered the RNAi drug shRNA2 to target cells expressing ACE2 receptor 4, and the new coronavirus 6 also infected target cells 7 through its RBD5 binding to ACE2 receptor 4. Due to the targeted delivery of RBD 3, shRNA2 specifically enters the target cells susceptible to virus infection 7 but not into cells 8 that are not easily infected with viruses.
  • the RBD of the present invention competes with the viral RBD for the ACE2 receptor 4, thereby competitively inhibiting virus infection.
  • Amino acid sequence and synthesis design of RBD According to the Global Shared Avian Influenza Database (GISAID) and GenBank database, the S protein gene sequences of SARS-CoV, MERS, and SARS-CoV-2 were collected, and amino acid phylogenetic tree analysis was carried out, or sequenced Homology analysis determined the conserved amino acid sequence positions N439, V483 and Q493 in the RBD that can bind to the human ACE2 receptor and are not prone to variation.
  • GISAID Global Shared Avian Influenza Database
  • GenBank database the S protein gene sequences of SARS-CoV, MERS, and SARS-CoV-2 were collected, and amino acid phylogenetic tree analysis was carried out, or sequenced Homology analysis determined the conserved amino acid sequence positions N439, V483 and Q493 in the RBD that can bind to the human ACE2 receptor and are not prone to variation.
  • SARS CoV S protein consists of 1255 amino acids, it can be hydrolyzed into the S1 receptor binding region (RBD) and the S2 membrane fusion region, and the RBD is located at amino acids 319-510 of the S protein (AA319-510), Through its C-terminus binding to the N-terminus outside the cell membrane of ACE2, RBD can enter target cells through ACE2 alone, the removal of glycosylation of RBD S protein N-link will not affect the function of RBD S protein, and the RBD of SARS-CoV-2 ( aa.331-550), there are 3 N-glycosylation residues (N331, N343, N360), tryptophan, histidine, ornithine, lysine and arginine that make up the peptide chain Multiple N and other characteristics can design the synthesis of RBD and connect RBD with liposome or shRNA.
  • RBD Polypeptides are synthesized from amino acids, usually by dehydration condensation of two amino acids to form peptide bonds, and multiple amino acid residues are connected by peptide bonds to form polypeptides.
  • the company can be entrusted to use a peptide synthesizer to automatically synthesize the amino acid sequence at positions 319 to 510 of the S protein, the conserved amino acid sequence at positions N439, V483 and Q493 that can bind to ACE2 but is not prone to mutation, and codon-optimized amino acid sequence.
  • the basic method is to add amino acids one by one according to the amino acid sequence of the synthesized polypeptide, so that the peptide chain is gradually extended from the C-terminal to the N-terminal residues, requiring each amino acid residue to be condensed in the form of protection at one end and activation at the other end, and at each After one cycle of peptide chain elongation, the temporary protecting group on the amino group is removed until the entire amino acid sequence of the target polypeptide is condensed.
  • the reaction principle of the currently commonly used solid-phase synthesis of polypeptides is to continuously add the required amino acids in a closed explosion-proof glass reactor according to the known sequence from the C-terminus-carboxyl-terminus to the N-terminus-amino-terminus, and carry out the synthesis reaction. get the peptide.
  • the main steps include: 1 Deprotection: remove the protective group of the amino group with an alkaline solvent; 2 Activation and cross-linking: activate the carboxyl group of the next amino acid, and make the activated monomer carboxyl group cross-link with the free amino group to form a peptide bond.
  • the two-step reaction is repeated repeatedly until the peptide synthesis is completed.
  • shRNA is a gene therapy drug
  • RBD has the function of protein vaccine and targeted delivery of shRNA, so the synthetic compound (RBD-shRNA-RBD) is both a targeted drug and a vaccine.
  • RBD-shRNA-RBD design of RBD-shRNA-RBD: as shown in Figure 2 and Figure 3, according to the synthetic shRNA and RBD sequence, one end of the sense and antisense strand of the siRNA with the viral conserved gene as the interference target and the loop ring (5' -TTCAAGAGA-3'), and the other ends are respectively connected to the glycosylation site of the RBD S protein N connection to form "RBD-siRNA sense strand-loop ring-siRNA antisense strand-RBD".
  • the two complementary sense and antisense strands will form a double strand, but the two polypeptide RBDs will not form a double strand, so it is a hairpin connector shRNA with two polypeptide RBDs and a loop.
  • RBD can bind to the viral receptor ACE2 through the C-terminus and enter the target cell plasma through ACE2, and the combination of polypeptide and siRNA can increase the permeability, stability and interference effect of siRNA, so this design enables RBD to produce targeted delivery, penetration
  • the role of membrane peptides, protein antigens and competing receptors can not only stably and effectively deliver CoV siRNA to the target cytoplasm of virus infection for targeted interference, but also stimulate the body to produce immune antibodies as recombinant proteins, and can Competes with the virus to bind to the target cell receptor ACE2 to inhibit virus infection.
  • RBD-shRNA-RBD You can entrust the company to use the conventional synthesis method of polypeptides and oligonucleotides to synthesize polypeptides and oligonucleotides with oxime bonds, amide bonds, thioether bonds, disulfide bonds, and phosphoryl bond, hydrazone bond, ureide bond, phosphodiester bond, phosphorodithioate bond, maleimide-sulfhydryl bond, etc.
  • POCs polypeptide-oligonucleotide conjugates
  • the most commonly used method for synthesizing POCs is the covalent cross-linking-liquid phase fragment synthesis method, which has been widely used in the synthesis of various POCs.
  • the two composites are stripped from the solid matrix, and the stripped polypeptides and oligonucleotides are coupled in solution via reactive groups.
  • the synthesis of POCs mainly includes: 1Maleimide-sulfhydryl coupling: modify maleimide on the polypeptide or oligonucleotide, modify sulfhydryl on another monomer, and then add the two monomers into the same solution POCs can be obtained by reaction; 2 Disulfide bond or thioether bond coupling: The thioether bond coupling includes the reaction of thiol nucleophilic substitution of halogenated haloacetamide and the addition of thiol Michael to maleimide.
  • Disulfide bond coupling can be directly oxidized by two sulfhydryl groups, or activated by an activator such as bipyridyl disulfide and then coupled with another oligomer containing sulfhydryl groups. Disulfide bonds are often used to synthesize siRNA and polypeptides. Conjugates; 3 Oxime bond coupling: aldehyde groups and amino groups react with each other to produce oximes. The reaction conditions are mild and the reaction efficiency is high, and the coupling products of double-stranded DNA and specific polypeptides can be directly generated.
  • the optimized oligonucleotides and polypeptides or sugars are simultaneously connected to two polypeptides at the 5' and 3' ends of the nucleic acid through an oxime bond. This method does not require various protection processes and can be completed in one step.
  • the specific method is to introduce aldehyde groups at both the 5' and 3' of the oligonucleotide, and then react with the hydroxylamine-modified polypeptide to obtain "peptide-oligonucleotide-peptide", and The resulting yield is high, and the one-step reaction of this bifunctional oligonucleotide with the polypeptide does not require any protection strategy and cross-linking agent, and a higher yield can be obtained under slightly acidic conditions; 4 amide bond coupling Coupling: direct use of an oligomer containing activated carboxylic acid or thioester to react with another polymer modified with an amino group to obtain a product; ⁇ 5 citric acid buffer solution, and then react with oligonucleotides modified with acetaldehyde groups to obtain POCs linked by hydrazone bonds.
  • Chromatographic method has always been one of the most commonly used methods for purification and analysis of polypeptide and oligonucleotide conjugates. According to the complexity of the conjugate, different chromatographic methods need to be selected for separation. The main methods are high performance liquid chromatography (HPLC), reverse high performance liquid chromatography (RP-HPLC), ion exchange chromatography (IEC, usually anion exchange chromatography) ), or use two or more of them in series, according to the operating instructions.
  • HPLC high performance liquid chromatography
  • RP-HPLC reverse high performance liquid chromatography
  • IEC ion exchange chromatography
  • RBD including but not limited to siRNA drugs prepared with SEQ ID NO.1-58, RBD polypeptide or S protein polypeptide and sequence as SEQ ID NO.1-58
  • siRNA drugs prepared with SEQ ID NO.1-58, RBD polypeptide or S protein polypeptide and sequence as SEQ ID NO.1-58 can be obtained
  • the compounds synthesized in the present application are RBD-shRNA(1-8)-RBD, RBD-siRNA and S-siRNA.
  • the modification of liposomes includes the adsorption of positively charged liposomes with negatively charged shRNA, and the sulfhydrylation of RBD amino groups to form sulfhydryl groups with liposomes. Amine bonds or methods such as forming carbamate bonds with liposomes with the amino terminal of RBD. If encapsulated by liposome DOTAP/Chol, DC-chol/DOPE or Lip, RBD-shRNA(1-8)/Lip-RBD (abbreviated as RBD 2 -shRNA(1-8)/Lip), RBD- siRNA/Lip and S-siRNA/Lip.
  • Embodiment 1 prepare liposome modification compound with liposome DOTAP/Chol
  • dialysate
  • Embodiment 2 prepare liposome modification compound with liposomal
  • RBD-siRNA siRNA
  • RBD or its fragments were synthesized using the above-mentioned RBD synthesis method.
  • the sample was placed in a dialysis bag with a molecular weight of 5kD, dialyzed in 10mmol/L (pH 7.4) Tris buffer for about 4 hours, and then dialyzed with deionized water at 4°C for 24 hours, the solution in the bag was taken out, freeze-dried, and placed in- Store in a refrigerator at 20°C.
  • RBD-siRNA/liposomal the synthesis of ePC (egg yolk phospholipid), Ch (cholesterol), PEG 2000 -DSPE (distearoyl ethanolamine polyethylene glycol 2000) and DOTAP (dioleyl trimethylamine propane) Chloroform solution is mixed in molar ratio (60:34:3.0:3.0). If lipid film needs to be marked, 0.1% Rho-PE is added to the above mixture with a molar ratio of total lipid mass, and the chloroform is removed under reduced pressure to form a lipid film. . Dissolve a certain amount of siRNA in ultrapure water treated with DEPC, and the amount of siRNA should completely neutralize the positive charge of DOTAP.
  • ePC egg yolk phospholipid
  • Ch cholesterol
  • PEG 2000 -DSPE disistearoyl ethanolamine polyethylene glycol 2000
  • DOTAP dioleyl trimethylamine propane
  • the phospholipid membrane was hydrated with an aqueous solution containing siRNA for 30 min in a water bath at 50° C. to form liposomes encapsulating siRNA.
  • a manual extrusion device (Avanti Polar Lipids)
  • the initially formed liposomes were passed through 0.4 ⁇ m and 0.1 ⁇ m polycarbonate nuclear pore membranes (Whatman) 10 times to prepare liposomes with uniform particle size.
  • RBD-PEG-DPPE Dissolve an appropriate amount of RBD-PEG-DPPE in methanol, put it in a flask, dry it with nitrogen gas to form a film, add the prepared liposome suspension, and warm it in a water bath at 37°C for 2 hours, so that RBD-PEG-DPPE can be inserted into the liposome oriented on the outer membrane.
  • the molar ratio of RBD in the total lipid in the liposome is generally 0.5%-1.0% (can be adjusted appropriately).
  • the properties of RBD-modified siRNA-loaded polyethylene glycol-modified liposomes were examined by dynamic laser light scattering, cryo-etching electron microscopy, and nucleic acid electrophoresis.
  • virus fluids of the two mutant strains B.1.617.1 and B.1.617.2 of the new coronavirus were prepared respectively to verify whether the compound was effective against two or more mutant viruses containing the same conserved gene at the same time, so as to prove the Whether the invented shRNA has broad-spectrum antiviral effect targeting conserved genes.
  • An experimental group and a control group were respectively set up to test the effects of synthetic compounds RBD 2 -shRNA(1-8)/Lip, RBD-siRNA/Lip and S-siRNA/Lip against B.1.617.1 and B.1.617.2.
  • Each group was inoculated into 8-well plates, 2 ⁇ 105 Vero-E6 cells per well, 2mL DMEM culture solution (10% FBS), and cultured in a 36°C, 5% CO2 incubator to 30% confluence (after 24h ), replace the culture medium, and add the test compound, B.1.617.1 and B.1.617.2 strain virus liquid at the same time.
  • the experimental groups include: RBD2 -shRNA1(/lip) group (0.1nmol RBD2 -shRNA1(/lip)+0.6ml virus solution), RBD2 - shRNA2(/lip) group (0.1nmol RBD2 - shRNA2(/lip) lip)+0.6ml virus solution), and so on RBD 2 -shRNA8(/lip) group (0.1nmol RBD 2 -shRNA8(/lip)+0.6ml virus solution), RBD-siRNA(/lip) group (0.1nmol RBD-siRNA(/lip)+0.6ml virus solution); control group includes: naked shRNA1 group (0.1nmol naked shRNA1+0.6ml virus solution), naked shRNA2 group (0.1nmol naked shRNA2+0.6ml virus solution), naked shRNA3 group (0.1nmol naked shRNA3+0.6ml virus solution), naked siRNA group (0.1nmol naked siRNA+0.6ml virus solution), RBD control group (0.1nmol RBD+0.6ml virus solution), positive
  • the viral RNA detection result of the negative control group was still negative, and the RNA detection result titer of the positive control group was 1:2916, and the RNA detection result titer of 4 groups of control groups was 1: 972 to 1:2916, while the titer of the RNA test in the experimental group was 1:36 to 1:108, which was significantly lower than that in the control group (p ⁇ 0.01).
  • the viral RNA detection result of the negative control group was still negative
  • the RNA detection result titer of the positive control group was >1:8748
  • the RNA detection result titer of the control group was >1:2916 ⁇ 1:8748
  • the titer of RNA test results in the experimental group was 1:108-1:324, which was significantly lower than that in the control group (p ⁇ 0.01).
  • Tables 7-9 show that the experimental group has obvious anti-B.1.617.1 strain effect, indicating that shRNA or siRNA linked to RBD can be delivered to target cells for RNA interference, while shRNA or siRNA not linked to RBD cannot enter In the target cells, it cannot play the role of RNA interference.
  • RBD also has a certain antiviral effect.
  • RNA detection results of the 4 groups of control groups 3 The titer of the experimental group was 1:2916, while the titer of the RNA detection results of the 4 experimental groups was 1:108-1:324, which was significantly lower than that of the control group (p ⁇ 0.01). As shown in Table 12, after 72 hours of cell culture in each group, the virus RNA detection result of the negative control group was still negative, and the RNA detection result titer of the positive control group was > 1:8748.
  • RNA detection results of the 4 groups of control groups There are 3 groups whose titers are 1:8748 or above, and among the 4 experimental groups’ RNA detection results, 1 group has a titer of 1:972, and the third group has a titer of 1:324, which are still higher than those of the control group. There is a significant difference (p ⁇ 0.01).
  • Tables 10-12 show that the experimental group has obvious anti-B.1.617.2 strain effect, indicating that shRNA or siRNA linked to RBD can be delivered to target cells for RNA interference, while shRNA or siRNA not linked to RBD cannot enter In the target cell, it cannot play the role of RNA interference.
  • Tables 7 to 12 show that the experimental group has anti-B.1.617.1 and B.1.617.2 effects at the same time, indicating that the compound (shRNA) with the conserved gene as the interference target in the experimental group has a broad-spectrum anti-mutant strain effect.
  • Animal grouping SPF-grade female BALB/c mice aged 6-8 weeks and weighing about 40 grams were selected and randomly divided into RBD 2 -shRNA1 (RBD 2 -shRNA1-8)/Lip group (inoculated with RBD 2 -shRNA1/Lip+ B.1.617.2 strain), RBD-siRNA1/Lip group (inoculated with RBD-siRNA1/Lip+B.1.617.2 strain), RBD group (inoculated with RBD+B.1.617.2 strain), shRNA1/Lip group (inoculated with shRNA1/Lip+B.1.617.2 strain), shRNA1 group (vaccinated with shRNA1+B.1.617.2 strain) positive control group (vaccinated with B.1.617.2 strain+normal saline) and negative control group (only inoculated with normal saline) .
  • Animal inoculation 40 ⁇ l of B.1.617.2 strain virus liquid with a titer of 10 5 /ml TCID 50 was inoculated by nasal spray, and 40 ⁇ l of normal saline was inoculated by nasal spray in the negative control group.
  • Anesthetized by intraperitoneal injection of 5% chloral hydrate solution, respectively, 0.1nmol RBD 2 -shRNA1/Lip, RBD-siRNA1/Lip, RBD, shRNA1/Lip and shRNA1 were slowly injected into the mouse trachea to reset the tissue. Every day, 10 mice in each group were sacrificed for virus detection, and the other 10 mice were used for antibody observation.
  • the virus is detected by the percentage of the cell half infection dose (TCID 50 )
  • RNAi mainly occurs in the cytoplasm
  • the shRNA1 in the shRNA1 group is easily degraded by nucleases and difficult to pass through the cell membrane, so it hardly has the effect of RNAi; although the shRNA1 in the shRNA1/Lip group is protected by Lip, it is not easy to be degraded by nucleases and can pass through cell membrane, but the effect of RNAi is poor because it cannot specifically enter the target cells, and the percentage of VeroE6 half infection amount is 82.5%, which has no significant difference compared with the positive control group (p>0.05); while RBD 2 -shRNA1/ ShRNA1/siRNA1 in the Lip group and RBD-siRNA1/Lip group was delivered to the target cell plasma by RBD, so its RNAi effect was better, and half of the VeroE6 in the RBD 2 -shRNA1/Lip group and RBD-siRNA1/Lip group were infected Compared with the shRNA1/Lip group, the percentages of
  • RBD 2 -shRNA7/lip and RBD 2 -shRNA8/lip were selected for TCID 50 test, and it was found that RBD The TCID 50 of 2 -shRNA7/lip group, RBD 2 -shRNA8/lip group, positive control group and negative control group were 22.5%, 22.5%, 92.5% and 5.0%, respectively, and the TCID 50 results of the test group were significantly lower than those of the positive control group Group.
  • the detection cases of IgM, IgG and IgM+IgG in the RBD 2 -shRNA1/Lip group were 21 cases, 20 cases and 16 cases, respectively, which were more than 8 cases, 8 cases and 5 cases in the RBD group .
  • the compound in the RBD 2 -shRNA1/Lip group is synthesized by 2 molecules of RBD, 1 molecule of shRNA and Lip, it has a larger molecular weight and more complex molecular structure than the single molecule RBD of the RBD group, and shRNA and Lip also have the effect of immune adjuvants , so it is more antigenic and easier to produce antibodies.

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Abstract

提供了一种以RBD靶向递送shRNA的nCOVsiRNA药物及其合成方法，该药物是将源自新型冠状病毒受体结合域的RBD作为靶向递送载体，将源自新型冠状病毒及其变异毒株的各毒株共有RNAi序列的siRNA合成shRNA，并使shRNA正反义链分别连接RBD的N端，构成以RBD靶向递送shRNA的具有靶向基因药物和大分子疫苗双重作用的化合物。其中shRNA既是广谱抗病毒药物又是使RBD疫苗增效的免疫佐剂，RBD作为靶向递送载体可避免非靶向治疗的副作用，同时RBD又是蛋白疫苗，其免疫产生的抗-RBD能中和病毒、阻止病毒通过ACE2感染。

Description

一种以RBD靶向递送shRNA的nCOVsiRNA药物及合成方法和应用
本申请要求如下中国专利申请的优先权：于2021年11月11日提交中国专利局且申请号为“202111329883.7”的中国专利申请、于2022年02月08日提交中国专利局且申请号为“202210116884.1”的中国专利申请、于2022年05月01日提交中国专利局且申请号为“202210491811.0”的中国专利申请、于2022年06月09日提交中国专利局且申请号为“202210652488.0”的中国专利申请、以及于2022年08月01日提交中国专利局且申请号为“202210920201.8”的中国专利申请，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及一种化学药物nCoVshRNA·2RBD的合成方法，属于生物制药领域。
背景技术
冠状病毒的结构包括单股正链核酸(ssRNA)、刺突蛋白(S)、膜蛋白(M)、包膜蛋白(E)和核壳蛋白(N)，其中S蛋白的N端由结构域(S1-NTD)和受体结合域(S1-RBD)组成。
人血管紧张素转换酶II(ACE2)为易感染冠状病毒的靶细胞表达的I型跨膜糖蛋白，由805个氨基酸组成，包括跨膜区、胞内羧基端和胞外氨基端。冠状病毒通过其S1-RBD与靶细胞的ACE2结合，经胞膜融合和内吞，使病毒进入表达ACE2或含有ACE2通道的靶细胞。
说明RBD和ACE2为配体与受体的关系，据此将RBD多肽设计为靶向递送载体，通过RBD将药物靶向递送到表达ACE2的靶细胞，使药物发挥仅针对病毒感染细胞的特异性作用。
RNA干扰(RNA interference，RNAi)作为一种高效的序列特异性基因沉默技术，正在给疾病的治疗带来难以想象的应用前景，已经有多种siRNA药物被FDA审批上市。但目前的siRNA药物常以某单一毒株设计siRNA、应用单链siRNA(反义RNA)制备siRNA药物或以非靶向递送载体进行siRNA药物的非特异性递送。这就不断变异的冠状病毒而言，上述基于某单一毒株设计的siRNA会因冠状病毒变异而脱靶、失效，所以，须从已经变异的毒株中比对、优选出不随病毒变异而改变的各毒株共有的siRNA，用于制备针对已变异和将变异毒株的广谱siRNA药物。如果使用非靶向递送载体，则会将siRNA药物递送至因不表 达ACE2而不易被冠状病毒感染的细胞，产生非靶向递送的副作用，所以，需设计能将siRNA特异性递送给目标细胞的靶向递送载体。更重要的是，根据HreA等报道的RNAi机制，由正义RNA和反义RNA混合制备的双链RNA沉默同源mRNA的效率要比单链RNA(反义RNA)高100倍以上，说明正确、有效的RNAi技术应采用同时包含正、反义siRNA的双链RNA(shRNA/dsRNA)，而传统的siRNA药物虽基于RNAi机制，但不是双链RNA。所以本发明合成shRNA，用于RNAi治疗，使之根据Hre A的报道，在理论上符合RNAi机制、RNAi效果提高100倍以上。
小干扰RNA即siRNA，被递送到细胞浆的siRNA以参与RNAi的方式调节基因表达，特异性降解与之互补的靶信使RNA(mRNA)。由于siRNA自身很难穿过细胞膜、很容易被RNA酶降解，所以传统上常采用脂质纳米粒包裹等方法进行非特异性运输和保护siRNA。本申请将siRNA合成shRNA，并在shRNA双链末端连接RBD多肽，达到特异性运输和保护siRNA的目的。
现有技术主要根据新型冠状病毒毒刺蛋白S1-RBD设计疫苗。文献报道，灭活疫苗、亚单位疫苗的免疫原性较弱，应同时使用免疫佐剂，而siRNA/dsRNA/shRNA可引起由I型干扰素(IFN-α/β)或促炎症细胞因子(IL6和TNFα)介导的免疫应答，其中IFN-α/β具有广谱抗病毒、增强造血细胞增殖和免疫调节等作用，IL6具有诱导B细胞分化、产生免疫球蛋白、促进T细胞和造血干细胞增殖等作用，在本质上，siRNA/dsRNA/shRNA为参与免疫活化的寡核苷酸类免疫佐剂。本申请将siRNA合成shRNA并在shRNA末端连接RBD多肽，使所获得的nCoVshRNA·2RBD含有二聚体RBD，就RBD疫苗而言，二聚体RBD比原来的单分子RBD的分子结构更复杂、分子量更大，特别是shRNA的免疫佐剂作用，使疫苗的效果更好。
为了解决上述问题，本发明将siRNA合成shRNA，并在shRNA双链末端分别延伸连接RBD多肽，合成以RBD靶向递送shRNA的化学药物nCoVshRNA·2RBD，其中shRNA既是免疫佐剂又是广谱抗变异毒株的siRNA药物，而RBD既是靶向递送载体，又是二聚体RBD疫苗，还可与病毒竞争ACE2受体，从而竞争抑制病毒感染。
发明内容
本发明的目的是要提供一种以RBD靶向递送shRNA的nCOVsiRNA药物及 其合成和用途；在nCOVsiRNA药物中，shRNA具有靶向基因治疗和免疫佐剂的双重作用，RBD具有靶向递送、蛋白疫苗以及中和ACE2受体的作用，脂质体具有稳定shRNA、细胞转染和免疫佐剂的作用。
本发明的目的通过以下技术方案实施：
筛选抗变异毒株靶标siRNA，合成shRNA，进而在shRNA双链末端分别延接RBD多肽，合成集广谱抗新冠病毒的靶向药物和自身携带免疫佐剂的新型疫苗为一体的化学药物nCoVshRNA·2RBD。
抗变异毒株靶标的筛选：从各种致病性冠状病毒及其变异毒株的共有基因中筛选siRNA，这种共有基因包括保守基因、超保守基因和/或保守微卫星，使所筛选的siRNA为不随病毒变异而改变的各变异毒株的共同靶标，使之具有广谱抗变异毒株的作用。
合成抗变异毒株靶标siRNA：将筛选的siRNA合成2条互补的21-25nt的寡核苷酸siRNA，并合成起间隔作用的碱基序列。
合成shRNA：将已合成的2条互补寡核苷酸多肽siRNA和起间隔作用的碱基序列进一步合成由中间碱基序列间隔成loop环的小发夹shRNA双链。
优选siRNA：将合成的shRNA构建干扰载体，检测其mRNA表达、蛋白表达和干扰效果，经siRNA设计、合成、筛选、迭代设计和验证，优选具有高沉默效率的siRNA。
合成优选的siRNA和shRNA：采用优选的siRNA序列按上述所述合成siRNA、shRNA，包括为增加稳定性和避免脱靶进行的化学修饰。
合成或通过RBD基因表达RBD多肽或蛋白：合成位于但不限于冠状病毒S蛋白的第319～510位氨基酸序列、位于但不限于N439、V483和Q493位点的保守氨基酸序列及经密码子优化的氨基酸序列。
化学药物nCoVshRNA·2RBD的合成：以二硫键、磷酸二酯键、二硫代磷酸脂键、硫醚键、肟键、酰胺键或马来酰亚胺-巯基键等偶联方法将已合成的shRNA和RBD进行连接；或根据shRNA的核苷酸序列和RBD的氨基酸序列直接合成RBD-shRNA-RBD。
化合物的提纯：以高效液相色谱、反向高效液相色谱或离子交换色谱提纯化合物。
化合物的脂质体修饰：通过带负电荷的shRNA吸附带正电荷的脂质体制备 脂质体修饰化合物；通过RBD氨基的巯基化使巯基与脂质体的马来酰胺形成马来酰亚胺-巯基键制备PEG内化的脂质体修饰化合物；通过RBD氨基末端与脂质体形成氨甲酸酯键制备脂质体修饰化合物；通过RBD或RBD片段连接脂质体修饰的siRNA制备脂质体或脂质纳米粒修饰的化合物。
化合物的验证：在体外细胞水平检测化合物对2种或以上不同变异毒株的抗病毒效果，观察是否具有以保守基因为靶标的广谱抗变异毒株作用；检测化合物在动物体内是否具有靶向递送shRNA的RNAi作用、疫苗的免疫作用以及是否有免疫增强的效果。
本发明的有益效果在于：
首次发现源自冠状病毒受体结合域的新型靶向递送载体RBD和源自冠状病毒保守基因的广谱抗变异毒株靶标shRNA，并将RBD和shRNA合成为以RBD靶向递送shRNA的化学药物nCoVshRNA·2RBD。其中shRNA起广谱抗变异毒株和免疫佐剂的作用，RBD起靶向递送shRNA、保护shRNA、穿膜肽和蛋白疫苗的作用，并使RBD和shRNA相互增效，进一步产生诸多新功能。
本发明与传统siRNA药物的区别特征是：本发明从各种冠状病毒及其变异毒株中筛选不随病毒变异而改变的各毒株共同靶标siRNA，使这种siRNA具有广谱抗变异毒株作用。
本发明与传统siRNA药物的区别特征是：传统siRNA药物使用正义链siRNA或反义链siRNA制备，即使用单链siRNA制备；而本发明将正反义链siRNA合成shRNA进行使用。根据HreA等报道的RNAi机制，双链RNA的RNAi干扰效果要超过单链RNA的RNAi效果至少100倍。所以本发明的设计更正确、更符合RNAi机制。
本发明与传统siRNA药物的区别特征是：本发明以RBD为载体靶向递送shRNA。冠状病毒特异性感染表达ACE2的靶细胞，目前还没有将siRNA特异性递送给病毒感染细胞而不递送给未感染细胞的靶向载体。本发明根据RBD与ACE2的特殊关系，将RBD与shRNA进行连接，使RBD产生靶向递送shRNA的新功能，从而可避免非特异性递送的副作用。
因siRNA/shRNA带负电荷、脂溶性、不易通过细胞膜、极易被核酸酶降解，所以很难被递送到靶细胞浆产生RNAi。但本发明将shRNA与RBD多肽合成为化合物后，因RBD具有穿膜肽特性，所以能保护shRNA不被核酸酶降解，并使 之更易通过靶细胞膜被递送到胞浆。
本发明与传统RBD疫苗的区别特征是：本发明以1分子shRNA连接2分子RBD构成二聚体RBD疫苗，其中shRNA为免疫佐剂。以2分子RBD和1分子shRNA合成的化合物，其中RBD和shRNA相加的分子量要比传统单分子RBD疫苗的分子量增加1倍以上，分子结构也更复杂，所以其免疫原性更强。因为免疫佐剂的主要成分是寡核苷酸和脂质，所以本质为寡核苷酸的siRNA或shRNA具有增强RBD疫苗免疫效果的免疫佐剂作用。
脂质体除了使shRNA在体内缓慢释放、药效延长和内吞入浆等作用外，还能作为免疫佐剂增强RBD蛋白的免疫效果。
化学药物中的RBD能与冠状病毒的RBD竟争结合靶细胞的ACE2受体，从而起抑制病毒感染的作用。且2分子RBD与shRNA合成的化合物增加了分子量及结构复杂性从而增强抗原性，被RBD结合的shRNA不易被酶降解、易通过细胞膜、易被递送到靶细胞浆。
体外细胞实验表明合成后的化合物对2种不同变异毒株同时有效，说明具有以保守基因为靶标的抗变异毒株作用；体内动物实验表明合成后的化合物在动物体内具有靶向递送的RNAi作用、疫苗的免疫作用以及免疫增强的效果。
本发明以shRNA双链未端连接靶向递送载体RBD制备nCoVsiRNA药物的设计方法有望应用于病毒、细菌、肿瘤、遗传病等siRNA基因治疗药物的制备。
本发明的nCoVsiRNA药物集广谱抗变异毒株靶向药物和二聚体S1-RBD疫苗为一体，所述化合物以冠状病毒受体结合域S1-RBD为载体且向ACE2表达细胞靶向递送冠状病毒变异毒株的共有RNAi靶标shRNA；
所述广谱抗变异毒株靶向药物，是指基于冠状病毒及其变异毒株的共有RNAi序列设计不随病毒变异而改变的具有广谱抗变异毒株作用的siRNA，然后将所述siRNA合成shRNA；将所述shRNA和既是穿胞肽又是ACE2配体的S1-RBD多肽进行连接后，使shRNA的胞膜通透性和抗核酸酶稳定性得到优化，使shRNA更易被特异性递送至表达ACE2的靶细胞浆，特异性沉默易感染病毒的ACE2表达细胞内的靶基因；
所述二聚体S1-RBD疫苗，是指将既具有广谱抗变异毒株作用又具有免疫佐剂作用的1分子shRNA和具有蛋白质抗原作用的2分子S1-RBD多肽进行连接，合成分子量更大、结构更复杂并含有自身免疫佐剂成分的新型技术线路疫苗。
附图说明
图1是本发明制备nCoVsiRNA药物的技术线路图；
图2是本发明的合成和应用示意图；
图3是本发明合成的化合物示意图；
图4是本发明的脂质体修饰siRNA示意图；
图5是本发明的脂质体修饰shRNA示意图；
图6是本发明RBD靶向递送shRNA以及RBD与病毒竞争ACE2受体的示意图。
在图1中，经保守基因筛选、以保守基因为靶标的广谱抗变异毒株靶标siRNA筛选、shRNA合成、RBD合成，最终合成以RBD靶向递送的nCoVsiRNA药物。
在图2中，1是以冠状病毒保守基因为干扰靶标的siRNA；2是小发夹shRNA的正反义链，shRNA由2条互补的siRNA退火形成；3是shRNA中由间隔正反义链的碱基序列间隔形成的loop环；4是2条RBD多肽，通过其N氨基分别与shRNA的正反义链相连接；5是表达ACE2受体6的靶细胞，病毒10通过其RBD的C端与ACE2受体6的细胞膜外N端相结合而进入靶细胞5；本发明以RBD靶向递送shRNA的RBD与病毒10的RBD相同，也通过RBD4的C端与ACE2受体6的细胞膜外N端相结合，将shRNA2靶向递送到表达ACE2受体6的靶细胞5内，靶细胞5因含有ACE2受体6而易于感染病毒10；进一步如靶细胞9所示，其细胞内的7和8分别表示因RBD4的靶向递送并通过ACE2受体6而进入靶细胞浆的shRNA和RBD；然后如靶细胞16所示，随着靶细胞9所示的shRNA7被降解为靶细胞16所示的shRNA15，长链RNA的病毒10和11也被降解为小片段的RNA序列14而失活，而原先与shRNA7相连接的RBD8也被解离为RBD12，进而刺激宿主产生抗-RBD12的抗体13。
在图3中，1为loop环，2为由两条互补的正反义链形成的shRNA，3为两条RBD多肽(蛋白)，两条RBD分别与shRNA正反义链相连接。shRNA被RBD保护并被RBD靶向递送至ACE2受体，然后随RBD通过ACE2受体而特异性进入靶细胞浆，降解病毒靶基因。
在图4中，1为被脂质体包裹的siRNA，2为脂质体层，3为PEG层，4为RBD。其中siRNA起RNAi作用，脂质体起保护siRNA和引起细胞内吞作用，PEG使siRNA缓慢释放和长效循环，RBD起靶向递送siRNA和疫苗的双重作用。
在图5中，1为loop环，2为由两条互补的正反义链形成的shRNA，3为两 条RBD多肽(蛋白)，两条RBD分别与shRNA正反义链相连接。4为脂质体层，5为PEG层，shRNA2被脂质体4包裹。其中shRNA起RNAi作用，脂质体起保护shRNA和引起细胞内吞作用，PEG使shRNA缓慢释放和长效循环，RBD起靶向递送和疫苗的双重作用。
在图6中，1为loop环，2为shRNA，3为RBD多肽，4为ACE2受体，5为新冠病毒的RBD，6为新冠病毒，7为表达ACE2受体的靶细胞，8为不表达ACE2受体的细胞。靶向递送载体RBD3将RNAi药物shRNA2靶向递送给表达ACE2受体4的靶细胞7，而新冠病毒6也通过其RBD5与ACE2受体4结合感染靶细胞7。由于RBD3的靶向递送使shRNA2特异性进入易感染病毒的靶细胞7而不进入不易感染病毒的细胞8，同时本发明RBD与病毒RBD竞争ACE2受体4，从而竞争抑制病毒感染。
具体实施方式
下面结合图1，2、3、4、5和6，对本发明的具体实施方法作详细的举例描述，但这些范例性的描述并不对本发明权利要求所限定的保护范围构成任何限制。
一、设计以超保守基因、保守基因或保守微卫星为靶标的siRNA
1、超保守基因、保守基因及保守微卫星的设计
如技术线路图1所示，从Genbank数据库(http://www.NCBI.nlm.nih.gov/genome/)中下载β冠状病毒属(特别是新型冠状病毒及其变异毒株)全基因组(cDNA)序列，在全基因组序列中搜寻最长的公共子序列，获得超保守基因或保守基因；利用ClustalW软件对Genbank数据库下载的全基因组进行序列比对，检测不同序列之间的相似度，筛选保守微卫星序列；利用MEGA6.0分子进化遗传分析软件，用邻接法(Neighbour-Jioning，N-J)对下载的冠状病毒氨基酸序列构建氨基酸种系分子进化树，对氨基酸序列的分子变异特征进行分析和优化，推断保守基因序列，作为siRNA合成的依据。
获得以下3段最长及次长的超级保守子序列，其长度为22～30bp，与小RNA长度相当，但在高等生物特别是人类中不包含这3段子序列。具体序列如下：
SEQ ID NO.1(Subsequence 1)＝ttaatacgacctctctgttggattttgaca(30bp)；
SEQ ID NO.2(Subsequence 2)＝ggttcgcaacttcacaca gagt(22bp)；
SEQ ID NO.3(Subsequence 3)＝caggcgtttgttggttgattaa(22bp)。
获得以下3段最长及次长的保守子序列，它们的长度为22～30bp，与小RNA 长度相当，但在高等生物特别是人类中不包含这3段保守子序列：
SEQ ID NO.4(Subsequence 1)＝gttttacgacaacgatgttggtttaggaca(30bp)；
SEQ ID NO.5(Subsequence 2)＝ggttcggttgttatatacgata(22bp)；
SEQ ID NO.6(Subsequence 3)＝ggttcagagagtctcctattta(22bp)。
获得以下5个由核苷酸重复多次的保守微卫星位点，微卫星分别为CTCTCT、AGAGAG、AAAAAAA、TATATA、CACACA。
2、新冠病毒变异毒株共同靶标siRNA的设计
根据Genbank数据库(http://www.NCBI.nlm.nih.gov/genome/)下载的β冠状病毒属(特别是新型冠状病毒及其变异毒株)的完整基因组(cDNA)序列，利用Ambion公司的shRNA在线设计软件( http://www.ambion.com/techlib/misc/siRNAtools.html)或DSIR等软件，获得长度约为19nt的多个siRNA备选序列，根据RNA结合的Tm值及特异性比对结果，优选siRNA。由此可从目前已发现的18株新冠病毒及其变异毒株的E、M、N、ORF1ab和S基因中获得各毒株各自RNAi序列(siRNA)和各毒株共有RNAi序列即共有靶标siRNA，其中各毒株共有siRNA见表1所示，其序列标记为SEQ ID NO.7～40。例如，表2～5中已做序列号标记的siRNA(SEQ ID NO.41～58)为NC_045512.2、Delta株、Omicron株的共同靶标siRNA，未做序列号标记的siRNA为各自的RNAi序列(siRNA)，可见尽管最早出现的NC_045512.2株变异为Delta株和最近的Omicron株，但各毒株中除了各自独有的靶向干扰序列siRNA(未做序列标记部分)外，仍保持不变且理论上具有靶向干扰作用的共同保守序列SEQ ID NO.41～58。
表1 从18株新冠病毒变异毒株中筛选的共同靶标siRNA(SEQ ID NO.7～40)
Figure PCTCN2022131458-appb-000001
Figure PCTCN2022131458-appb-000002
表2 新冠病毒NC_045512.2、DELTA、OMICRON株E基因的siRNA候选序列
Figure PCTCN2022131458-appb-000003
表3 新冠病毒NC_045512.2、DELTA、OMICRON株M基因的siRNA候选序列
Figure PCTCN2022131458-appb-000004
表4 新冠病毒NC_045512.2、DELTA、OMICRON株N基因的siRNA候选序列
Figure PCTCN2022131458-appb-000005
Figure PCTCN2022131458-appb-000006
表5 新冠病毒NC_045512.2、DELTA、OMICRON株ORF1ab基因的siRNA候选序列
Figure PCTCN2022131458-appb-000007
表6 新冠病毒NC_045512.2、DELTA、OMICRON株S基因的siRNA候选序列
Figure PCTCN2022131458-appb-000008
Figure PCTCN2022131458-appb-000009
3、筛选以超保守基因、保守基因或保守微卫星为靶标的siRNA
利用Clustal W软件或其他软件，将上述设计的超保守基因、保守基因及保守微卫星与常规筛选的siRNA进行基因序列比对，检测不同序列之间的相似度，设计既为超保守基因、保守基因或保守微卫星，又为RNAi靶位点的多对siRNA(设计以超保守基因、保守基因或保守微卫星为靶标的siRNA)。
(1)以超保守基因和保守微卫星为靶标的siRNA(S1/S2)：
SEQ ID NO.1(Subsequence 1)＝ttaatacgacctctctgttggattttgaca(30bp)；
SEQ ID NO.2(Subsequence 2)＝ggttcgcaacttcacacagagt(22bp)；
(2)以保守基因和保守微卫星为靶标的siRNA(S3/S4)：
SEQ ID NO.5(Subsequence 3)＝ggttcggttgttatatacgata(22bp)；
SEQ ID NO.6(Subsequence 4)＝ggttcagagagtctcctattta(22bp)。
通过上述设计，获得以超保守基因、保守基因或保守微卫星为干扰靶标的在理论上抗冠状病毒变异毒株的siRNA，命名为siRNA1/2/3/4。
二、共同靶标siRNA的验证
1、合成siRNA/shRNA
根据RNAi机制，当siRNA有效干扰S基因的mRNA表达时，会形成失去感染性的S蛋白缺陷型病毒。当siRNA有效干扰N基因的mRNA表达时，会抑制病毒的包装、复制。当siRNA有效干扰ORF1a或1b基因的mRNA表达时,会影响病毒RNA聚合酶(RdRp)或蛋白质加工酶(3CLpro)的合成。而M和E基因是病毒的膜基因，其缺陷对病毒的抑制作用可能不明显。所以本发明选用靶向N 基因的siRNA(SEQ ID NO.16～18、SEQ ID NO.49～51)、靶向ORF1ab基因的siRNA(SEQ ID NO.20～22、SEQ ID NO.52～54)和靶向S基因的siRNA(SEQ ID NO.30～32、SEQ ID NO.56～58)，以及SEQ ID NO.1～2、SEQ ID NO.5～6进行合成。另外根据pSilencer4.1.CMV.neo干扰载体的多克隆酶切位点设计能表达发夹结构的shRNA模板，每个模板由两条大部分互补的55bp的单链DNA构成，退火互补后能形成带有BamH I和Hind III酶切位点粘性末端的DNA双链，用于与线性化的pSilencer4.1.CMV.neo的连接。然后按设计的siRNA及其shRNA模板，委托公司合成。
2、shRNA表达载体的构建
将上述合成的shRNA分别与线性化的干扰载体pSilencer4.1.CMV.neo进行连接和鉴定，构建shRNA表达质粒，转化DH5a，分别获得shRNA表达载体。
3、shRNA表达(干扰)载体的效果鉴定
根据合成的siRNA/shRNA及其构建的表达质粒，选择相应的靶基因进行合成或PCR扩增，然后构建荧光标签载体，并分别与shRNA表达质粒共转染II型肺泡上皮细胞(AEC2s)或293T细胞，进行鉴定。PCR扩增的常规方法如下：
引物设计：设计上、下游引物，在上游引物5'端添加起始密码，为将扩增产物克隆到pEGFP-N1中，引物的5'端添加用于与载体发生同源重组的同源臂。
靶基因扩增：按上海生工试剂盒所提供的基因扩增反应体系及反应条件进行基因扩增、产物回收和纯化，获得扩增产物。
pEGFP-N1的线性化：复苏含有pEGFP-N1质粒的DH5a菌种，按试剂盒提取质粒，测定浓度后进行酶切，0.8％琼脂糖凝胶电泳鉴定并回收线性化的载体。
将扩增的靶基因与荧光标签载体(pEGFP-N1)进行连接：使用金斯瑞公司的同源重组试剂盒进行连接，连接结束后可保存在-20℃备用或马上进行转化。
shRNA干扰载体的效果鉴定：分别将干扰载体(pSilencer-shRNA)和荧光标签载体(pEGFP-N/S/ORF1ab)共转染293T细胞，干扰载体与标签载体的质量比为1：2，同时设立对照，转染后48h观察细胞内GFP蛋白的融合表达，根据荧光强度评价干扰效果：
流式细胞检测：为定量分析不同干扰载体的干扰效果，用流式细胞术检测，分析荧光蛋白表达细胞在总细胞数中的比例。
Westernbolt分析：①细胞收集与裂解：以RIPA裂解细胞。②SDS-PAGE蛋 白电泳：制备SDS-PAGE胶，将样品加入等体积的2xSDS缓冲液，沸水煮5min，冰浴2min，12000xg，10min。③Westernblot检测：经转膜、封闭、一抗结合、洗涤、二抗结合和显色，观察结果。
RT-PCR检测mRNA：采用相对荧光定量RT-PCR法检测转染细胞中靶基因的相对表达量，根据标准曲线，由CT值换算目的基因以及B-actin内参基因拷贝数，以B-actin内参基因校正病毒基因mRNA相对表达量(目的基因拷贝数/B-actin拷贝数)，定量评价干扰效果。
4、获得高沉默效率的siRNA/shRNA
经上述设计、合成、筛选、迭代设计、再合成和细胞水平的验证，获得高沉默效率的siRNA，其序列分别为SEQ ID NO.1(命名为shRNA1，下同)、SEQ ID NO.2(shRNA2)、SEQ ID NO.5(shRNA3)，以及靶向N基因的SEQ ID NO.16(shRNA4)、SEQ ID NO.49(shRNA5)，靶向ORF1ab基因的SEQ ID NO.21(shRNA6)、SEQ ID NO.52(shRNA7)，靶向S基因的SEQ ID NO.30(shRNA8)，其沉默效率分别为78％、76％、88％、89％、89％、84％、91％、90％。
三、合成shRNA
根据上述筛选的共同靶标(SEQ ID NO.1～58，优选为shRNA1～8)，委托生物公司，每个shRNA合成2条互补的19～25nt的寡核苷酸多肽siRNA，以及合成起间隔作用的9nt的碱基序列，然后将所合成的siRNA和碱基序列连接成由中间碱基序列间隔成loop环的小发夹shRNA双链，所合成的shRNA双链的每条单链均可以分别连接RBD多肽或蛋白。例如，分别将SEQ ID NO.1、SEQ ID NO.2和SEQ ID NO.5合成5'-ttaatacgacctctctgttggattttgacattcaagagatgtcaaatccaacagagaggtcgtattaa-3'(shRNA1，具体如SEQ ID NO.78所示)、5'-ggttcgcaacttcacacagagtttcaagagaactctgtgtgaagttgcgaacc-3'(shRNA2，具体如SEQ ID NO.79所示)和5'-ggttcggttgttatatacgatattcaagagatatcgtatataacaaccgaacc-3'(shRNA3，具体如SEQ ID NO.80所示)，其中“TTCAAGAGA”为loop环，其左、右侧分别为互补的正、反义链，进而在其3'和/或5'连接RBD蛋白或其多肽。同理优选其他高沉默效率的siRNA，分别合成shRNA。
四、靶向递送载体RBD的设计和合成
RBD(由SEQ ID NO.59所示序列编码得到或与所示序列相对应的氨基酸序列，SEQ ID NO.59所示序列具体为： atgaatattacaaacttgtgcccttttggtgaagtttttaacgccaccagatttgcatctgtttatgcttggaacaggaagagaatcagcaactgtgttgctgattattctgtcctatataattccgcatcattttccacttttaagtgttatggagtgtctcctactaaattaaatgatctctgctttactaatgtctatgcagattcatttgtaattagaggtgatgaagtcagacaaatcgctccagggcaaactggaaagattgctgattataattataaattaccagatgattttacaggctgcgttatagcttggaattctaacaatcttgattctaaggttggtggtaattataattacctgtatagattgtttaggaagtctaatctcaaaccttttgagagagatatttcaactgaaatctatcaggccggtagcacaccttgtaatggtgttgaaggttttaattgttactttcctttacaatcatatggtttccaacccactaatggtgttggttaccaaccatacagagtagtagtactttcttttgaacttctacatgcaccagcaactgtttgtggacctaaaaagtctactaatttggttaaaaacaaatgtgtcaatttcaacttcaatggtttaacaggcacaggtgttcttactgagtctaacaaaaagtttctgcctttccaacaatttggcagagacattgctgacactactgatgctgtccgtgatccacagacacttgagtaa)多肽具有靶向递送载体和重组蛋白疫苗的双重作用。因为冠状病毒通过RBD特异性结合ACE2受体引起感染，RBD与ACE2是配体与受体的关系，所以可通过RBD将药物靶向递送给病毒感染细胞并进入胞浆。另外现有技术通常以病毒通过RBD与ACE2结合感染的特性设计RBD蛋白疫苗，所以合成的RBD既是靶向递送载体也是蛋白疫苗。本发明RBD靶向递送shRNA以及RBD与病毒竞争ACE2受体的示意图参见图6。在图6中，1为loop环，2为shRNA，3为RBD多肽，4为ACE2受体，5为新冠病毒的RBD，6为新冠病毒，7为表达ACE2受体的靶细胞，8为不表达ACE2受体的细胞。靶向递送载体RBD 3将RNAi药物shRNA2靶向递送给表达ACE2受体4的靶细胞7，而新冠病毒6也通过其RBD5与ACE2受体4结合感染靶细胞7。由于RBD 3的靶向递送使shRNA2特异性进入易感染病毒的靶细胞7而不进入不易感染病毒的细胞8，同时本发明RBD与病毒RBD竞争ACE2受体4，从而竞争抑制病毒感染。
1、RBD的氨基酸序列及合成设计：根据全球共享禽流感数据库(GISAID)和GenBank数据库收集SARS-CoV、MERS、SARS-CoV-2的S蛋白基因序列，进行氨基酸系统进化树分析，或经序列同源性分析，确定RBD中能与人ACE2受体结合并且不易发生变异的保守氨基酸序列位点N439、V483和Q493。另外根据SARS CoV S蛋白由1255个氨基酸组成、可被酶解为S1受体结合区(RBD)和S2膜融合区、RBD定位于S蛋白的第319到510位氨基酸(AA319-510)、RBD通过其C端与ACE2的细胞膜外N端结合、RBD能单独通过ACE2进入靶细胞、RBD S蛋白N连接的糖基化的去除不会影响RBD S蛋白的功能、SARS-CoV-2的RBD(aa.331-550)中有3个N-糖基化残基(N331，N343，N360)、组成肽链的色 氨酸、组氨酸、鸟氨酸、赖氨酸和精氨酸都有多个N等特点，可设计RBD的合成，以及将RBD与脂质体或shRNA进行连接。
2、RBD的合成：以氨基酸合成多肽通常是由两个氨基酸脱水缩合形成肽键，由多个氨基酸残基以肽键相连接形成多肽。可委托公司，采用多肽合成仪自动化合成位于S蛋白的第319～510位氨基酸序列、位于能与ACE2结合但不易发生变异的包含N439、V483和Q493位点的保守氨基酸序列以及经密码子优化的氨基酸序列。其基本方法是，按被合成多肽的氨基酸序列逐个加入氨基酸，使肽链从C端到N端残基逐步延长，要求每一个氨基酸残基以一端保护和另一端活化的形式缩合，并且在每一轮肽链延长循环后除去氨基上的临时性保护基团，直至目标多肽的全部氨基酸序列缩合完毕。目前常用的固相合成多肽的反应原理是在密闭的防爆玻璃反应器中使氨基酸按照已知的序列从C端-羧基端向N端-氨基端的顺序不断添加所需氨基酸，进行合成反应，最终得到多肽。其主要步骤包括：①去保护：用碱性溶剂去除氨基的保护基团；②激活和交联：活化下一个氨基酸的羧基，使活化的单体羧基与游离的氨基交联，形成肽键，反复循环这两步反应，直到多肽合成完成。
五、以shRNA和RBD合成化合物
以shRNA和RBD合成的化合物，因为shRNA为基因治疗药物，而RBD又具有蛋白疫苗和靶向递送shRNA的作用，所以合成的化合物(RBD-shRNA-RBD)既是靶向药物又是疫苗。
1、RBD-shRNA-RBD的设计：如图2和图3所示，根据合成的shRNA及RBD序列，将以病毒保守基因为干扰靶标的siRNA的正反义链的一端与loop环(5'-TTCAAGAGA-3’)相连，另一端分别与RBD S蛋白N连接的糖基化位点相连，连接成“RBD-siRNA正义链-loop环-siRNA反义链-RBD”。其中两条互补的正反义链会形成双链，但两条多肽RBD不会形成双链，所以是带有两条多肽RBD和loop环的发夹状连接物shRNA。因为RBD可通过C端结合病毒受体ACE2并通过ACE2进入靶细胞浆，以及多肽与siRNA的结合可增加siRNA的通透性、稳定性及干扰效果，所以本设计使RBD产生靶向递送、穿膜肽、蛋白抗原和竞争受体的作用，不但能稳定、有效地将CoVsiRNA靶向递送至病毒感染的靶细胞浆进行靶向干扰，而且自身能作为重组蛋白刺激机体产生免疫性抗体，以及能与病毒竞争结合靶细胞受体ACE2从而抑制病毒感染。
2、RBD-shRNA-RBD的合成：可委托公司，采用多肽与寡核苷酸的常规合成方法，将多肽和寡核苷酸以肟键、酰胺键、硫醚键、二硫键、磷酰键、腙键、酰脲键、磷酸二酯键、二硫代磷酸脂键、马来酰亚胺-巯基键等形式偶联成缀合物，包括多肽和寡核苷酸的正义链(5'末端、3'末端)或反义链(3'末端)以较牢固的共价键、较松散的离子键、疏水键或以带间隔臂的羧腙键进行非共价或共价交联，合成多肽-寡核苷酸偶联物(POCs)。目前最常用的合成POCs法是共价交联-液相片段合成法，已广泛应用于合成各种POCs，其主要步骤是：分别在固相基质上分别合成多肽和寡核苷酸，然后同时将两个合成物从固相基质上剥离，所剥离的多肽和寡核苷酸在溶液中通过反应活性基团进行偶联。合成POCs主要包括：①马来酰亚胺-巯基偶联：在多肽或寡核苷酸上修饰马来酰亚胺，在另一单体上修饰巯基，然后将两个单体加入同一溶液中即可反应得到POCs；②二硫键或硫醚键偶联：其中硫醚键偶联包括巯基亲核取代卤代乙酰胺上卤代物的反应和巯基Michael加成到马来酰亚胺两类；二硫键偶联可以通过两个巯基直接氧化，或先通过联吡啶二硫醚等活化剂将巯基活化再与另一含有巯基的寡聚物偶联，常用二硫键合成siRNA与多肽的偶联物；③肟键偶联：醛基与氨基相互反应产生肟，该反应条件温和、反应效率高，并且可以直接生成双链DNA与特定多肽的偶联产物，同时，还可以通过双功能化的寡核苷酸与多肽或糖类通过肟键在核酸的5’与3’端同时连接上两条多肽，该方法不需要各种保护过程并能一步完成，用于合成了“肽-寡核苷酸-肽”产物，具体方法为在寡核苷酸的5’和3’均引入醛基，再与羟基胺修饰的多肽反应，得到“肽-寡核苷酸-肽”，且所得产率较高，这种双官能团化的寡核苷酸与多肽的一步反应不需要任何保护策略和交联剂，在微酸的条件下就能得到较高的产率；④酰胺键偶联：直接利用含有活化羧酸或硫酯的寡聚物与另一修饰有氨基的聚合物反应得到产物；⑤腙键偶联：需要先将肼基引入到多肽上，然后加入pH值为3～5之间的柠檬酸缓冲溶液，再与修饰有乙酰醛基的寡核苷酸进行反应，才可得到以腙键连接的POCs。
3、RBD-shRNA-RBD的提纯：色谱方法一直是纯化和分析多肽与寡核苷酸偶联物的最常用的方法之一。根据偶联物的复杂程度需选择不同的色谱方法进行分离，主要方法有高效液相色谱(HPLC)、反向高效液相色谱(RP-HPLC)，离子交换色谱(IEC，通常为阴离子交换色谱)，或者将其中的两个或几个进行串联使用，具体按操作说明。
按照上述shRNA的筛选、合成及其与RBD的合成，可获得包括但不限于以SEQ ID NO.1～58制备的siRNA药物、以RBD多肽或S蛋白多肽与序列为SEQ ID NO.1～58的siRNA相连接构成的结合物RBD-shRNA-RBD、RBD-siRNA或S-siRNA，以及进而经脂质体修饰的复合物。
本申请合成的化合物为RBD-shRNA(1～8)-RBD、RBD-siRNA和S-siRNA。
六、化合物的脂质体修饰
如图4～5，脂质体(Liposomes，Lip)修饰包括以带负电荷的shRNA吸附带正电荷的脂质体、以RBD氨基的巯基化使巯基与脂质体形成巯基-马来酰亚胺键或以RBD氨基末端与脂质体形成氨甲酸酯键等方法。如经脂质体DOTAP/Chol、DC-chol/DOPE或Lip包裹，制成RBD-shRNA(1-8)/Lip-RBD(缩写为RBD 2-shRNA(1-8)/Lip)、RBD-siRNA/Lip和S-siRNA/Lip。
实施例1：以脂质体DOTAP/Chol制备脂质体修饰化合物
(1)RBD-shRNA-RBD的合成
采用本申请以shRNA和RBD合成的RBD-shRNA-RBD化合物。
(2)脂质溶液的配制
DOTAP(MW＝698.5)：10mg/ml，精确称取DOTAP[N-(2，3-二油酰氧基-1-丙基)三甲基甲磺酸铵：N-1-(2，3-di-oleoyloxy)propyl)-N，N，N-trimeth ylammoniumethyl sulfate]粉末100mg，加入10ml容量瓶中，加氯仿溶液至刻度线。
Chol(MW＝386)：5mg/ml，精确称取Chol[胆固醇：cholesterol]粉末50mg，加入10ml容量瓶中，加氯仿溶液至刻度线。
m-PEG 2000-DSPE(MW＝2787)：10mg/ml，称取m-PEG 2000-DSPE(甲氧基化聚乙二醇二硬脂酰磷脂酰乙醇胺：Methoxy-polyethylene glycol-distearoyl phosphatidyi-ethanolamine)粉末10mg，加入l ml DEPC水，涡旋后超声1min，使之彻底溶解。
Mal-PEG 2000-DSPE(MW＝2941.6)：10mg/ml，精确称取Mal-PEG2000-DSPE(Maleimide derivatized polyethylene glycol-distearoyl phosphatidyl-ethanolamine：马来酰亚胺化聚乙二醇二硬脂酰磷脂酰乙醇胺)粉末10mg，加1ml DEPC水，涡旋后超声1min，溶解。
(3)脂质体DOTAP/Chol的制备(薄膜水化法)
采用薄膜水化法(Lipid-film method)制备脂质体DOTAP/Chol，脂质浓度为10mM，主要步骤如下：按照制备1ml脂质体DOTAP/Chol的量，分别取DOTAP、Chol两种脂质的氯仿溶液，以DOTAP：Chol＝1：1(M：M)的比例加至500ml三角烧瓶，加3～4ml氯仿溶液；37℃真空旋转蒸发45～60min，使成为均匀的脂质薄膜，用高纯氮气吹尽痕量氯仿；加入1ml DEPC水振摇，将脂质薄膜从瓶壁洗下，得脂质混悬液；待充分水合后，超声lmin，依次通过400nm、200nm、80nm、50nm聚碳酸酯膜挤出，各10～20次，即得脂质体DOTAP/Chol。
(4)RBD-shRNA-RBD的巯基化
2-IT(Traut'S reagent)是蛋白巯基化的常用试剂，可在RBD S蛋白N连接的糖基化处进行巯基化，步骤如下：取RBD-shRNA-RBD和2-IT(Traut'S reagent，2-iminothiolane-HCl)，混合均匀(2-IT与RBD-shRNA-RBD的摩尔比为200：1)，室温条件下反应2h；通过透析法除去多余的2-IT，每次以足量透析液(1×PBS，5mM EDTA，pH＝7.4)，4℃保存，隔夜透析，小心低速磁力搅拌，6～8h换透析液，共2次；巯基化抗体的浓度和巯基化程度分别采用BCA法和Ellman法测定其蛋白浓度和巯基化程度。
(5)以脂质体DOTAP/Chol制备脂质体修饰化合物
(A)利用带负电荷的siRNA/shRNA吸附带正电荷的脂质体进行制备
①取120μl的DOTAP/Chol脂质体(10mM)，加入20μ1的RBD-shRNA-RBD(约2μg/μ1，40μg)，加DEPC水11μl，室温静置10min，获得脂质体包裹的RBD-shRNA-RBD复合物。
②取siRNA 90μ1(24μg，20mM)、shRNA 90μ1(24μg，10mM)和/或RBD-shRNA-RBD 100μ1(约10μg/μ1，200μg)，加DEPC水57.6μ1，室温静置10min，加入600μl的DOTAP/Chol脂质体(50mM)，获得脂质体包裹的siRNA、shRNA和/或RBD-shRNA-RBD复合物。
③将①制备的脂质体复合物与②制备的siRNA、shRNA和/或RBD-shRNA-RBD脂质体复合物等量混合，获RBD修饰的含游离siRNA/shRNA的脂质体包裹RBD-shRNA/siRNA-RBD复合物。
(B)以RBD中的巯基与PEG中的马来酰亚胺的交联反应进行制备
为了增长脂质体的循环时间和靶向特异性，对(A)制备的各种脂质体进行PEG化和进一步的RBD靶向修饰，获得PEG化和以RBD为配体的脂质体修饰 化合物。
分别取6.36μl、9.53μl和12.7μl的10mg/ml的MAL-DSPE-PEG，分别将其插入上述(A)制备的脂质体复合物RBD-shRNA/siRNA-RBD(分别将两者混合)，50℃水浴孵育10min，室温静置10min，然后加入约200μg经上述巯基化的RBD-shRNA-RBD，使RBD中巯基化氨基上的巯基与MAL-DSPE-PEG中的马来酰亚胺发生交联反应，分别获得RBD修饰的5mo1％PEG、7.5mo1％PEG和10mo1％PEG的PEG化脂质体复合物RBD-shRNA/siRNA-RBD，即该复合物为由脂质体DOTAP/Chol以静电吸附包裹siRNA、shRNA和/或RBD-shRNA-RBD，再以MAL-DSPE-PEG包裹在外层，最后以MAL-DSPE-PEG连接RBD-shRNA-RBD，其中RBD起靶向递送、蛋白抗原和稳定siRNA/shRNA的作用，脂质体和PEG起保护siRNA/shRNA、缓慢释放siRNA/shRNA/RBD、向胞内转染siRNA/shRNA或疫苗佐剂的作用。
实施例2：以脂质体liposomal制备脂质体修饰化合物
当pH＞8时，RBD的氨基末端与pNP-PEG-DPPE(PEG-PE)反应形成稳定的氨甲酸酯键偶联物，并定向定量地插入脂质体的外层膜，制成脂质体修饰化合物。本实施例以RBD片段和siRNA制备以RBD靶向递送siRNA(RBD-siRNA)的脂质体修饰化合物。
(1)RBD的合成
采用上述RBD合成方法合成RBD或其片段。
(2)pNP-PEG-DPPE的合成
取20mg/mL DPPE(二棕榈酰乙醇胺)氯仿溶液10ml，置50mL圆底烧瓶中，滴加三乙胺(TEA)0.65mL，取约4.0g浓度为200mg/mL的(pNP) 2-PEG 3400(聚乙二醇3400二对硝基苯碳酸酯)氯仿溶液加入上述混合液中，吹入氮气，密封，避光，于室温下磁力搅拌过夜，减压蒸干溶剂，真空除尽残留氯仿，加入0.01mol/L HCl水溶液100mL，超声处理形成透明的胶束溶液。0.01mol/L HCI水溶液为洗脱液，经CL-4B Sepharose进行分离，除去未反应的(pNP) 2-PEG 3400和释放的pNP，收集含pNP-PEG-DPPE胶束的洗脱液，冷冻干燥，用TLC、HPLC、MS和NMR对pNP-PEG-DPPE进行定性和定量。
(3)RBD-PEG-DPPE的合成：取pNP-PEG-DPPE 100mg溶于10mL氯仿中，置50mL烧瓶中，于旋转蒸发仪上减压除去氯仿，形成脂膜，真空除尽残留 氯仿，将25mg RBD溶于0.01mol/L HCl 4mL中，加入内壁涂布脂膜的烧瓶中，于室温孵育30min，轻摇使脂膜充分分散。于混悬液中加10mum/L(pH 9.0)Tris缓冲液20mL，混匀，氮气保护，于4℃孵育过夜。将样品置于分子质量为5kD的透析袋中，在10mmol/L(pH 7.4)Tris缓冲液中透析约4h，再用去离子水于4℃透析24h，取出袋内溶液，冷冻干燥，置-20℃冰箱内保存。
(4)RBD-siRNA/liposomal的合成：将ePC(蛋黄磷脂)、Ch(胆固醇)、PEG 2000-DSPE(二硬脂酰乙醇胺聚乙二醇2000)和DOTAP(二油酰三甲胺丙烷)的氯仿溶液按摩尔比(60:34:3.0:3.0)混合，如需标记脂膜，于上述混合液中加入占总脂质量摩尔比为0.1％Rho-PE，减压除尽氯仿，形成脂膜。将一定量的siRNA溶于经DEPC处理的超纯水中，siRNA用量应完全中和DOTAP所带的正电荷。于50℃水浴中用含siRNA的水溶液将上述磷脂膜水化30min，形成包裹siRNA的脂质体。用手动挤出装置(Avanti Polar Lipids)，将初步形成的脂质体分别过0.4μm和0.1μm的聚碳酸酯核孔膜(Whatman)10次，制备粒径均一的脂质体。取适量RBD-PEG-DPPE溶于甲醇中，置烧瓶，氮气吹干成膜，加入制备的脂质体悬液，于37℃水浴温浴2h，使RBD-PEG-DPPE定向地插入到脂质体的外层膜上。其中RBD在脂质体中占总脂质的摩尔比一般为0.5％～1.O％(可适当调整)。用动态激光散射、冰冻蚀刻电子显微镜、核酸电泳检验RBD修饰的包载siRNA的聚乙二醇修饰的脂质体的特性。
七、化合物(nCOVsiRNA)的验证
1、体外验证以保守基因为靶标的广谱抗病毒效果
(1)病毒液的准备
在Vero E6细胞生长至30％汇合度的DMEM培养液(10％FBS)中加入病毒株，36℃、5％CO 2培养箱培养5～7d，至出现细胞病变效应(CPE)时，分离病毒，用培养液配成10 3～10 5TCID 50/ml病毒液备用。据此分别准备新冠病毒的两种变异毒株B.1.617.1和B.1.617.2的病毒液，用于验证化合物是否对2种或以上含有相同保守基因的变异病毒同时有效，以证明本发明的shRNA是否具有以保守基因为靶标的广谱抗病毒效果。
(2)化合物(nCOVsiRNA)和病毒共培养
分别设立实验组和对照组，试验合成物RBD 2-shRNA(1-8)/Lip、RBD-siRNA/Lip和S-siRNA/Lip对抗B.1.617.1和B.1.617.2的效果。每组接种8 孔板，每孔2×10 5个Vero-E6细胞、2mL DMEM培养液(10％FBS)，置36℃、5％CO 2培养箱中培养至30％汇合度时(24h后)，更换培养液，同时加入试验化合物、B.1.617.1和B.1.617.2株病毒液。
其中实验组包括:RBD 2-shRNA1(/lip)组(0.1nmol RBD 2-shRNA1(/lip)+0.6ml病毒液)、RBD 2-shRNA2(/lip)组(0.1nmol RBD 2-shRNA2(/lip)+0.6ml病毒液)、依此类推RBD 2-shRNA8(/lip)组(0.1nmol RBD 2-shRNA8(/lip)+0.6ml病毒液)、RBD-siRNA(/lip)组(0.1nmol RBD-siRNA(/lip)+0.6ml病毒液)；对照组包括：naked shRNA1组(0.1nmol naked shRNA1+0.6ml病毒液)、naked shRNA2组(0.1nmol naked shRNA2+0.6ml病毒液)、naked shRNA3组(0.1nmol naked shRNA3+0.6ml病毒液)、naked siRNA组(0.1nmol naked siRNA+0.6ml病毒液)、RBD对照组(0.1nmol RBD+0.6ml病毒液)、阳性对照组(0.6ml病毒液)、阴性对照组(0.6ml DMEM培养液)(表1-6)。
继续培养，然后于培养1小时、24小时和72小时后每组各取上清液，以1:4、1:12、1:36、1:108、1:324、1:972、1:2916、1:8748倍稀释，进行RT-PCR检测。
(3)实时荧光RT-PCR检测各组病毒RNA
病毒核酸提取和核酸(ORF1ab/N)检测按试剂盒说明书操作。
(4)病毒RNA检测结果
①B.1.617.1株检测结果：如表7所示，各组细胞培养1h后，阴性对照组病毒RNA检测结果为阴性，阳性对照组RNA检测结果的滴度为1:36，其他各组RNA检测结果滴度均为1:12。如表8所示，各组细胞培养24h后，阴性对照组病毒RNA检测结果仍为阴性，阳性对照组RNA检测结果滴度为1:2916，4组对照组的RNA检测结果滴度为1:972～1:2916，而实验组的RNA检测结果滴度为1:36～1:108，明显低于对照组(p＜0.01)。如表9所示，各组细胞培养72h后，阴性对照组病毒RNA检测结果仍为阴性，阳性对照组RNA检测结果滴度＞1:8748，对照组的RNA检测结果滴度＞1:2916～1:8748，而实验组的RNA检测结果滴度为1:108～1:324，明显低于对照组(p＜0.01)。
表7a、表8a和表9a中RBD 2-shRNA(4-8)的实验结果与表7、表8和表9中RBD 2-shRNA(1-3)的结果一致，与阳性对照相比，均具有明显的抑制病毒作用。
表7～9说明，实验组具有明显的抗B.1.617.1株作用，说明与RBD连接的shRNA或siRNA能被递送至靶细胞内进行RNA干扰，而未与RBD连接的shRNA 或siRNA不能进入靶细胞内，不能发挥RNA干扰的作用，另外，RBD也有一定的抗病毒作用。
表7 化合物与B.1.617.1株共培养1小时培养液中病毒RNA RT-PCR检测结果(+/-)
Figure PCTCN2022131458-appb-000010
表7a RBD 2-shRNA(4-8)与B.1.617.1株共培养1小时培养液病毒RNA RT-PCR检测结果
Figure PCTCN2022131458-appb-000011
表8 化合物与B.1.617.1株共培养24小时培养液中病毒RNA RT-PCR检测结果(+/-)
Figure PCTCN2022131458-appb-000012
Figure PCTCN2022131458-appb-000013
表8a RBD 2-shRNA(4-8)与B.1.617.1株共培养24小时培养液病毒RNA RT-PCR检测结果
Figure PCTCN2022131458-appb-000014
表9 化合物与B.1.617.1株共培养72小时培养液中病毒RNA RT-PCR检测结果(+/-)
Figure PCTCN2022131458-appb-000015
表9a RBD 2-shRNA(4-8)与B.1.617.1株共培养72小时培养液病毒RNA RT-PCR检测结果
Figure PCTCN2022131458-appb-000016
Figure PCTCN2022131458-appb-000017
②B.1.617.2株检测结果：如表10所示，各组细胞培养1h后，阴性对照组病毒RNA检测结果为阴性，阳性对照组RNA检测结果的滴度为1:36，其他各组RNA检测结果滴度均为1:12～1：36。如表11所示，各组细胞培养24h后，阴性对照组病毒RNA检测结果仍为阴性，阳性对照组RNA检测结果滴度为1:2916，在4组对照组的的RNA检测结果中有3组的滴度为1:2916，而4组实验组的RNA检测结果滴度为1:108～1:324，明显低于对照组(p＜0.01)。如表12所示，各组细胞培养72h后，阴性对照组病毒RNA检测结果仍为阴性，阳性对照组RNA检测结果滴度＞1:8748，在4组对照组(naked)的RNA检测结果中有3组的滴度为1:8748或以上，在4组实验组的RNA检测结果中有1组的滴度为1:972、3组的滴度为1:324，与对照组相比仍有明显的差异(p＜0.01)。
表10a、表11a和表12a中RBD 2-shRNA(4-8)的实验结果与表10、表11和表12中RBD 2-shRNA(1-3)的结果一致，与阳性对照相比，均具有明显的抑制病毒作用。
表10～12说明，实验组具有明显的抗B.1.617.2株作用，说明与RBD连接的shRNA或siRNA能被递送至靶细胞内进行RNA干扰，而未与RBD连接的shRNA或siRNA不能进入靶细胞内，从而不能发挥RNA干扰的作用。
表7～12表明，实验组同时具有抗B.1.617.1和B.1.617.2的作用，说明实验组以保守基因为干扰靶标的化合物(shRNA)具有广谱抗变异毒株的效果。
表10 化合物与B.1.617.2株共培养1小时培养液中病毒RNA RT-PCR检测结果(+/-)
Figure PCTCN2022131458-appb-000018
Figure PCTCN2022131458-appb-000019
表10a RBD 2-shRNA(4-8)与B.1.617.2株共培养1小时培养液病毒RNA RT-PCR检测结果
Figure PCTCN2022131458-appb-000020
表11 化合物与B.1.617.2株共培养24小时培养液中病毒RNA RT-PCR检测结果(+/-)
Figure PCTCN2022131458-appb-000021
表11a RBD 2-shRNA(4-8)与B.1.617.2株共培养24小时培养液病毒RNA RT-PCR检测结果
Figure PCTCN2022131458-appb-000022
Figure PCTCN2022131458-appb-000023
表12 化合物与B.1.617.2株共培养72小时培养液中病毒RNA RT-PCR检测结果(+/-)
Figure PCTCN2022131458-appb-000024
表12a RBD 2-shRNA(4-8)与B.1.617.2株共培养24小时培养液病毒RNA RT-PCR检测结果
Figure PCTCN2022131458-appb-000025
2、动物体内验证RBD的靶向递送和免疫功能
(1)动物分组和接种
动物分组：选择6～8周龄、40克左右的SPF级雌性BALB/c小鼠，随机分为RBD 2-shRNA1(RBD 2-shRNA1～8)/Lip组(接种RBD 2-shRNA1/Lip+B.1.617.2 株)、RBD-siRNA1/Lip组(接种RBD-siRNA1/Lip+B.1.617.2株)、RBD组(接种RBD+B.1.617.2株)，shRNA1/Lip组(接种shRNA1/Lip+B.1.617.2株)，shRNA1组(接种shRNA1+B.1.617.2株)阳性对照组(接种B.1.617.2株+生理盐水)和阴性对照组(仅接种生理盐水)。
动物接种：经鼻腔喷雾接种40μl滴度为10 5/mlTCID 50的B.1.617.2株病毒液，阴性对照组经鼻腔喷雾接种40μl生理盐水。以腹腔注射5％水合氯醛溶液麻醉，分别将0.1nmol的RBD 2-shRNA1/Lip、RBD-siRNA1/Lip、RBD、shRNA1/Lip和shRNA1缓慢注入小鼠气管，复位组织，在感染后第7天每组处死10只小鼠，进行病毒检测，另10只用于观察抗体。
(2)以细胞半数感染量(TCID 50)的百分率检测病毒
将处死小鼠肺组织制备10％匀浆，取100pl离心后上清，以10倍递次稀释，接种于VeroE6单层生长的96孔板，每孔30μl，每个稀释度接种4个孔，轻摇匀浆，放37℃吸附lh，以Hank氏液清洗，加培养液，放37℃CO 2培养箱培养，观察细胞病变效应(CPE)，分别计算各组VeroE6细胞半数感染量(TCID 50)的百分率，百分率越大病毒含量越多，见表13～19。
表13 RBD 2-shRNA1/Lip组小鼠肺组织匀浆致VeroE6半数感染量的百分率
Figure PCTCN2022131458-appb-000026
表14 RBD-siRNA1/Lip组小鼠肺组织匀浆致VeroE6半数感染量的百分率
Figure PCTCN2022131458-appb-000027
表15 RBD组小鼠肺组织匀浆致VeroE6半数感染量的百分率
Figure PCTCN2022131458-appb-000028
Figure PCTCN2022131458-appb-000029
表16 shRNA1/Lip组小鼠肺组织匀浆致VeroE6半数感染量的百分率
Figure PCTCN2022131458-appb-000030
表17 shRNA1组小鼠肺组织匀浆致VeroE6半数感染量的百分率
Figure PCTCN2022131458-appb-000031
表18 阳性对照组小鼠肺组织匀浆致VeroE6半数感染量的百分率
Figure PCTCN2022131458-appb-000032
表19 阴性对照组小鼠肺组织匀浆致VeroE6半数感染量的百分率
Figure PCTCN2022131458-appb-000033
(3)RBD靶向递送的效果
从表13～19可知，各组小鼠肺匀浆致VeroE6半数感染量的百分率分别为RBD 2-shRNA1/Lip组20.0％、RBD-siRNA1/Lip组27.5％、RBD组87.5％、shRNA1/Lip组82.5％、shRNA1组95.0％、阳性对照组95.0％和阴性对照组2.5％。因为RNAi主要发生在细胞浆内，shRNA1组的shRNA1极易被核酸酶降解、不易通过细胞膜，所以几乎不起RNAi作用；shRNA1/Lip组的shRNA1虽被Lip保 护而不易被核酸酶降解并能通过细胞膜，但因不能特异性进入靶细胞而使RNAi效果较差，其VeroE6半数感染量的百分率达82.5％，与阳性对照组相比无显著性差异(p＞0.05)；而RBD 2-shRNA1/Lip组和RBD-siRNA1/Lip组中的shRNA1/siRNA1因以RBD靶向递送至靶细胞浆，所以其RNAi效果较好，RBD 2-shRNA1/Lip组和RBD-siRNA1/Lip组的VeroE6半数感染量的百分率与shRNA1/Lip组相比，均具有显著性差异(均为p＜0.05)。
参照上述TCID 50试验方法，根据shRNA7和shRNA8具有较高沉默效率(分别为90％和91％)的结果，选用RBD 2-shRNA7/lip和RBD 2-shRNA8/lip进行TCID 50试验，结果发现RBD 2-shRNA7/lip组、RBD 2-shRNA8/lip组、阳性对照组和阴性对照组的TCID 50分别为22.5％、22.5％、92.5％和5.0％，试验组的TCID 50结果明显低于阳性对照组。
(4)化合物(nCOVsiRNA)的免疫功能检测
采集RBD 2-shRNA1/Lip组和RBD组接种后第1、2、4、6周的10只小鼠静脉血，分离血清，保存于-20℃备用，按试剂盒操作，ELISA检测特异性抗体IgG和IgM(表20)。
表20 RBD 2-shRNA1/Lip组和RBD组各10只小鼠血清特异性抗体检测结果比较
Figure PCTCN2022131458-appb-000034
由表20可知，RBD 2-shRNA1/Lip组的IgM、IgG及IgM+IgG的检出例次分别为21例、20例和16例，分别多于RBD组的8例、8例和5例。因为RBD 2-shRNA1/Lip组的化合物由2分子RBD、1分子shRNA及Lip合成，比RBD组的单分子RBD的分子量更大、分子结构更复杂，而且shRNA及Lip又有免疫佐剂的作用，所以抗原性更强，更易于产生抗体。
尽管上述实施例对本发明做出了详尽的描述，但它仅仅是本发明一部分实施例而不是全部实施例，人们还可以根据本实施例在不经创造性前提下获得其他实施例，这些实施例都属于本发明保护范围。

Claims (23)

  1. 一种以RBD靶向递送shRNA的nCOVsiRNA药物，其特征在于，合成以RBD为载体靶向递送shRNA的化合物，包括分别合成shRNA和冠状病毒RBD，所述shRNA靶向新冠病毒共同靶标，所述RBD以ACE2为受体；然后将所述RBD分别连接到shRNA的正反义链上，构成以RBD为载体向ACE2靶向递送shRNA的RBD-shRNA-RBD化合物；进而以脂质体修饰。
  2. 根据权利要求1所述的一种以RBD靶向递送shRNA的nCOVsiRNA药物，其特征在于，所合成的化合物能使各组分相互增效，其中因RBD的靶向递送而使shRNA能发挥针对ACE2表达细胞的特异性作用；因RBD是蛋白抗原，所以被shRNA连接而构成的2个RBD分子因改变了分子结构和分子量而使其抗原性更强、使制备的以shRNA连接的二聚体RBD疫苗的免疫效果更好；因RBD多肽是穿膜肽，所以使被RBD连接的shRNA增加胞膜通透性和抗核酸酶稳定性；因RBD能结合ACE2受体而竞争抑制病毒RBD结合ACE2受体，从而竞争抑制病毒感染；shRNA和脂质体又是增强RBD疫苗免疫效果的免疫佐剂。
  3. 根据权利要求1所述的一种以RBD靶向递送shRNA的nCOVsiRNA药物，其特征在于，所述靶向新冠病毒共同靶标是指用于合成shRNA的siRNA选自数据库记载的各种致病性冠状病毒及其变异毒株的共有保守基因，使合成的shRNA靶向干扰所述共有基因，从而起广谱抗变异毒株的作用；所述共有基因包括但不限于超保守基因、保守基因和/或由保守微卫星拼接的基因。
  4. 根据权利要求1～3任意一项所述的一种以RBD靶向递送shRNA的nCOVsiRNA药物，其特征在于，所述合成shRNA包括：首先合成2条互补的约21～25nt的寡核苷酸多肽siRNA以及合成起间隔作用的碱基序列，然后将所述siRNA和碱基序列连接成由中间碱基序列间隔成loop环的小发夹shRNA双链；将所述RBD分别连接到shRNA的正反义链上，包括在所述shRNA双链的每条单链上各连接RBD。
  5. 根据权利要求4所述的一种以RBD靶向递送shRNA的nCOVsiRNA药物，其特征在于，所述siRNA序列包括SEQ ID NO.1～58中的一种或几种。
  6. 根据权利要求5所述的一种以RBD靶向递送shRNA的nCOVsiRNA药物，其特征在于，所述siRNA序列为SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.5、SEQ ID NO.7～10、SEQ ID NO.16～18、SEQ ID NO.20～22、SEQ ID NO.30～32、SEQ ID NO.41～58中的一种或几种。
  7. 根据权利要求6所述的一种以RBD靶向递送shRNA的nCOVsiRNA药 物，其特征在于，所述siRNA序列为SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.5、SEQ ID NO.8、SEQ ID NO.17、SEQ ID NO.30、SEQ ID NO.41和SEQ ID NO.50中的一种或几种。
  8. 根据权利要求1所述的一种以RBD靶向递送shRNA的nCOVsiRNA药物，其特征在于，所述RBD指能与ACE2结合的氨基酸序列，包括S蛋白的第319～510位氨基酸序列、能与ACE2结合但不易发生变异的N439、V483和Q493位点的保守氨基酸序列以及经密码子优化的核苷酸序列mRNA表达的氨基酸序列。
  9. 根据权利要求1所述的一种以RBD靶向递送shRNA的nCOVsiRNA药物，其特征在于，所述连接包括将RBD的糖基化N氨基与shRNA的反义链3'末端、正义链5'末端或3'末端进行连接，并包括以二硫键、磷酸二酯键、二硫代磷酸脂键、硫醚键、肟键、酰胺键或马来酰亚胺-巯基键的化学偶联或共价偶联进行连接。
  10. 根据权利要求1所述的一种以RBD靶向递送shRNA的nCOVsiRNA药物，其特征在于，所述nCOVsiRNA药物包括以RBD多肽或S蛋白多肽与siRNA相连接构成的结合物RBD-siRNA和S-siRNA，以及其脂质体修饰复合物RBD-siRNA/Lip和S-siRNA/Lip中的一种或几种。
  11. 一种siRNA药物的合成方法，其特征在于，将具有RNAi功能的正、反义链siRNA合成shRNA；然后在所述shRNA的正义链siRNA和/或反义链siRNA的未端延伸连接靶向递送载体，合成以shRNA双链连接载体进行shRNA靶向递送的siRNA药物。
  12. 一种化学药物nCoVshRNA·2RBD的合成方法，其特征在于，将具有RNAi功能的正、反义链siRNA合成shRNA，然后在合成的shRNA的正义链siRNA和/或反义链siRNA的未端连接靶向递送载体，合成以shRNA双链连接载体进行shRNA靶向递送的siRNA药物。
  13. 根据权利要求11或12所述的合成方法，其特征在于，所述siRNA选自病原体及其变异毒株的共有RNAi序列，使所述shRNA靶向干扰含有所述共有RNAi序列的病毒基因，从而产生抗变异毒株的广谱RNAi作用。
  14. 根据权利要求11或12所述的合成方法，其特征在于，所述合成shRNA包括：首先合成2条互补的长度为21～25nt的siRNA，以及合成起间隔作用的碱基序列；将所述siRNA和起间隔作用的碱基序列连接成由所述起间隔作用的碱基 序列间隔成中间为loop环的小发夹状shRNA；在所述小发夹状shRNA的每条单链上各连接靶向递送载体。
  15. 根据权利要求11或12所述的合成方法，其特征在于，所述shRNA的siRNA包括新冠病毒RNAi序列SEQ ID NO.1～58中的一种或几种。
  16. 根据权利要求15所述的合成方法，其特征在于，所述shRNA的siRNA包括新冠病毒共有RNAi序列SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.5、SEQ ID NO.7～10、SEQ ID NO.16～18、SEQ ID NO.20～22、SEQ ID NO.30～32、SEQ ID NO.41～58中的一种或几种。
  17. 根据权利要求16所述的合成方法，其特征在于，所述shRNA的siRNA为靶向冠状病毒N基因的SEQ ID NO.16～18和SEQ ID NO.49～51，靶向冠状病毒ORF1ab基因的SEQ ID NO.20～22和SEQ ID NO.52～54，以及靶向冠状病毒S基因的SEQ ID NO.30～32和SEQ ID NO.56～58中的一种或几种。
  18. 根据权利要求17所述的合成方法，其特征在于，所述合成shRNA的siRNA为靶向N基因的SEQ ID NO.16和SEQ ID NO.49，靶向ORF1ab基因的SEQ ID NO.21和SEQ ID NO.52，以及靶向S基因的SEQ ID NO.30中的一种或几种。
  19. 根据权利要求18所述的合成方法，其特征在于，所述RBD包括S蛋白的第319～510位氨基酸序列；所述RBD能与ACE2结合但不易发生变异的保守氨基酸序列；所述保守氨基酸序列包含N439、V483和Q493位点；或经密码子优化的核苷酸序列mRNA表达的RBD多肽。
  20. 根据权利要求11或12所述的合成方法，其特征在于，所述连接包括将RBD的糖基化N氨基与shRNA的反义链3'末端、正义链5'末端或3'末端进行连接；所述连接包括以二硫键、磷酸二酯键、二硫代磷酸脂键、硫醚键、肟键、酰胺键或马来酰亚胺-巯基键为连接健的化学偶联或共价偶联。
  21. 根据权利要求11或12所述的合成方法，其特征在于，在合成的shRNA的正义链siRNA和/或反义链siRNA的未端连接靶向递送载体，还包括将单链siRNA与多肽连接，所述多肽为RBD多肽或S蛋白多肽。
  22. 根据权利要求11或12所述的合成方法，其特征在于，在得到siRNA药物后，还包括对所述siRNA药物进行脂质体或脂质纳米粒修饰。
  23. 权利要求1～10任意一项所述的以RBD靶向递送shRNA的nCOVsiRNA药物或者权利要求11～22任意一项所述合成方法合成得到的siRNA药物在制备新冠疫苗或者制备预防和/或治疗新冠病毒病的药物中的应用。
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