WO2022199690A1 - 一种siRNA药物、药物组合物、siRNA-小分子药物偶联物及其应用 - Google Patents

一种siRNA药物、药物组合物、siRNA-小分子药物偶联物及其应用 Download PDF

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WO2022199690A1
WO2022199690A1 PCT/CN2022/083042 CN2022083042W WO2022199690A1 WO 2022199690 A1 WO2022199690 A1 WO 2022199690A1 CN 2022083042 W CN2022083042 W CN 2022083042W WO 2022199690 A1 WO2022199690 A1 WO 2022199690A1
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sirna
influenza virus
seq
influenza
drug
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French (fr)
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陆阳
王志远
唐盛高
徐军
路阳
林冠权
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圣诺生物医药技术(苏州)有限公司
圣诺生物医药技术(广州)有限公司
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Priority to US18/284,025 priority Critical patent/US20240156966A1/en
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Definitions

  • the invention belongs to the technical field of biomedicine, and in particular relates to a siRNA drug, a pharmaceutical composition, a siRNA-small molecule drug conjugate and an application thereof.
  • Influenza viruses belong to the Orthomyxoviridae family of single-stranded negative-stranded RNA viruses [1] , and their genomes are divided into multiple parts, with different host ranges and pathogenicity.
  • influenza A, B, and C viruses also known as influenza A, B, and C, respectively
  • Influenza A viruses can infect a variety of avian and mammalian hosts, while influenza B viruses are almost exclusively human.
  • Influenza A virus has caused widespread concern because of its pandemic.
  • the structure of influenza virus consists of three parts: core protein, envelope protein and matrix protein.
  • HA hemagglutinin
  • NA matrix protein 1
  • M2 proton channel protein
  • NP nucleoprotein
  • NS1 and PB2 non- Structural protein 1
  • NEP nuclear export protein
  • PB1-F2, PB1-N40 and PA-X proteins
  • Influenza A viruses are further classified by HA and NA subtypes, with 18 subtypes for HA and 11 subtypes for NA.
  • H1N1 and H3N2 are human influenza viruses
  • H5N1 and H7N9 are avian influenza viruses.
  • HA and NA frequently undergo point mutations (antigenic drift) in seasonal influenza, and gene rearrangements (antigenic transfer) between human and avian viruses may cause pandemics [3] .
  • Influenza is a serious problem that affects human health for a long time. Influenza virus infects millions of people every year and causes 250,000 to 500,000 deaths worldwide [4] . Despite the availability of vaccines and antiviral drugs, influenza still has serious health, economic and social impacts. Because the virus continues to evolve, current vaccines provide only limited protection against influenza. Currently, widespread resistance to adamantanes exists in circulating viruses, and neuraminidase (NA) inhibitors (NAIs) are the only effective antiviral drugs available in most countries. However, NAI is not a perfect solution for influenza virus. For example, the seasonal influenza A (H1N1) virus circulating around the world in 2008-2009 was resistant to oseltamivir and had significant side effects [5] . Regardless, influenza virus infection remains a threat to human health and society. Therefore, the development and clinical application of novel antiviral drugs with different mechanisms of action are crucial.
  • H1N1 seasonal influenza A
  • H1N1 seasonal influenza A virus circulating around the world in 2008-
  • the drugs that have been marketed against influenza A virus infection include: viral neuraminidase (NA) inhibitors Relenza (zanamivir), Tamiflu (oseltamivir phosphate), Inavir (lanimvir octanoic acid) ester) and Rapivab (peramivir); M2 ion channel blockers amantadine (Amantadine) and rimantadine (Rimantadine); viral polymerase inhibitor Favipiravir (Favipiravir); broad-spectrum antiviral drug Bavirin (Ribavirin) and Abidol (Arbidol) and so on.
  • NA viral neuraminidase
  • Relenza zanamivir
  • Tamiflu oseltamivir phosphate
  • Inavir lanimvir octanoic acid ester
  • Rapivab peramivir
  • viral neuraminidase inhibitors still face problems such as oral availability, drug resistance, and induction of cytokine storms.
  • the long-term, extensive, and extensive use of adamantane drugs has resulted in severe drug resistance of most influenza A viruses [6] .
  • the adverse reaction data of favipiravir is not perfect, and there is also the problem of drug resistance [7] .
  • oseltamivir Tamiflu [8] .
  • oseltamivir Tamiflu
  • Pharmaceutical companies such as Roche have conducted many clinical trials of oseltamivir, but until recently data from some early clinical trials were available as pdf scans of edited reports, while Not provided as actual raw data, and therefore cannot be analyzed in more detail by other investigators.
  • reports include median viral shedding curves for both placebo and drug-treated influenza virus infections, often indicating high efficacy of early treatment.
  • median dropout curves may not accurately represent an individual's effect on a drug.
  • TCAD oral oseltamivir 75 mg, amantadine 100 mg, and ribavirin 600 mg
  • oseltamivir Wei [10] .
  • TCAD oral oseltamivir 75 mg, amantadine 100 mg, and ribavirin 600 mg
  • oseltamivir Wei [10] .
  • TCAD oral oseltamivir 75 mg, amantadine 100 mg, and ribavirin 600 mg
  • oseltamivir Wei [10] oseltamivir Wei [10] .
  • TCAD had a significantly greater antiviral effect than oseltamivir alone (40.0% of TCAD treated patients had detectable viral RNA on day 3)
  • 50.0% of oseltamivir alone treated patients had detectable viral RNA
  • Serious adverse events and hospitalizations occurred in the TCAD group ratio is higher.
  • this triple-drug regimen failed to improve
  • Favipiravir (T-705) was first developed in Japan and was approved for influenza pandemic preparedness in Japan on March 24, 2014. Since favipiravir increases the risk of teratogenicity and embryotoxicity, only a conditional marketing authorization has been obtained, and strict regulations have been established for its production and clinical use [11] . Therefore, favipiravir is only indicated for patients with novel or recurrent pandemic influenza virus infection for which other influenza antiviral drugs are ineffective or insufficiently effective. In other countries, favipiravir is still in clinical research.
  • Favipiravir is phosphorylated in infected cells, converted to its active form, recognized by RNA-dependent RNA polymerase (RdRP) as a purine analog, and efficiently integrated into neoplastic as guanosine and adenosine analogs. in the generated RNA chain [12] .
  • RdRP RNA-dependent RNA polymerase
  • RNAi Nucleic acid interference
  • silencing effect is related to small nucleotide duplex interfering RNAs (siRNAs) of 21-23bp and dsRNA-specific endoribonuclease III (called Dicer), which is responsible for cleaving dsRNAs into siRNAs, Trigger RNAi silencing mechanisms.
  • siRNAs small nucleotide duplex interfering RNAs
  • Dicer dsRNA-specific endoribonuclease III
  • siRNA binds to specific proteins/enzymes to form RNA-induced silencing complex (RISC), the sense strand in siRNA is detached, and the antisense strand targets and binds to sequence-specific mRNA [14] . Subsequently, enzymes in RISC cleave the target mRNA by about 12 nucleotides from the 3' end of the siRNA strand, resulting in mRNA degradation and gene silencing.
  • RISC RNA-induced silencing complex
  • RNAi technology has become a powerful tool for antiviral infection. Compared with small molecule chemical drugs, siRNA has many advantages [15] .
  • siRNA "drugs" can be rapidly synthesized and scaled up for production.
  • different siRNAs targeting another viral sequence can be used, or even two or more siRNA molecules targeting different genes of the influenza virus can be used simultaneously.
  • siRNA is water-soluble, which is very beneficial for drug utilization.
  • siRNA against influenza A virus infection also showed good results.
  • siRNA had a significant inhibitory effect on H1N1 virus, and the specific siRNA could inhibit the expression of influenza virus mRNA by about 50%.
  • Plaque assay showed that siRNA could reduce the influenza virus titer. to 1/200 [16] .
  • siRNA can also inhibit the proliferation of influenza virus in animals and improve the protection rate of animals.
  • mice intravenously with siRNA decreased after H1N1 challenge. Then, 18 days after a single injection of one siRNA or two siRNAs into mice, the protection rate of a single siRNA was 80%-90%, while the survival rate of mice treated with both siRNAs reached 100% [18] .
  • Another study injected siRNA into the nasal cavity and intravenously into mice at the same time, and found that mRNA and protein were inhibited by more than 90%, and inflammatory factors were also significantly reduced [19] .
  • influenza viruses have the ability to mutate rapidly and can easily develop drug resistance through mutation.
  • H1N1 pandemic was caused by a recombinant virus between human influenza virus and avian influenza virus strains; similarly, the "swine flu" H1N1 influenza pandemic that has occurred continuously in recent years is also Due to a series of recombination events between human influenza A H3N2, swine influenza H1N1 and avian influenza H1N2 [20] .
  • the purpose of the present invention is to provide a siRNA molecule that efficiently and specifically inhibits influenza virus replication and a new siRNA drug, pharmaceutical composition, and siRNA-small molecule drug conjugate for preventing and treating influenza virus infection.
  • the technical scheme adopted in the present invention is:
  • a first aspect of the present invention provides an siRNA molecule that inhibits influenza virus replication
  • the siRNA molecule includes a sense strand and an antisense strand
  • the sequence of the sense strand is selected from SEQ ID No. 1-16, SEQ ID No. 20- 54.
  • Any one of SEQ ID No. 56-69, SEQ ID No. 71-91, SEQ ID No. 93, SEQ ID No. 94, and the antisense strand is selected from SEQ ID No. 98-113, SEQ ID No. 98-113, SEQ ID No. One of ID No. 117-151, SEQ ID No. 153-166, SEQ ID No. 168-188, SEQ ID No. 190, SEQ ID No. 191 complementary to the sense strand.
  • a second aspect of the present invention provides an siRNA drug for preventing or treating influenza virus infection, the siRNA drug comprising an active ingredient, and the active ingredient is one or more of the siRNA molecules of claim 1 .
  • the active ingredient further includes one or more other siRNA molecules that inhibit influenza virus replication.
  • sequences of the sense strands of the other siRNA molecules that inhibit influenza virus replication are selected from SEQ ID No.17-19, SEQ ID No.55, SEQ ID No.70, SEQ ID No.92, SEQ ID No. Any one of .95 to 97, the antisense strands of the other siRNA molecules that inhibit influenza virus replication are selected from SEQ ID No. 114 to 116, SEQ ID No. 152, SEQ ID No. 167, SEQ ID No. 189 , one of SEQ ID Nos. 192-194 complementary to the sense strands of the other siRNA molecules that inhibit influenza virus replication.
  • a third aspect of the present invention provides a pharmaceutical composition for preventing or treating influenza virus infection, wherein the active ingredients of the pharmaceutical composition include an siRNA molecule that inhibits replication of influenza virus and another molecule, and the other molecule includes One or more of siRNA molecules that inhibit the expression of PD-1, siRNA molecules that inhibit the expression of PD-L1, anti-influenza virus small molecule compounds, influenza mRNA vaccines, or anti-influenza virus monoclonal antibodies.
  • the siRNA molecules for inhibiting influenza virus replication are designed for conserved gene sequences among different strains of influenza A virus, including one or more of H1N1, H5N1, H7N9, or H3N2 Subtype; the siRNA molecules that inhibit influenza virus replication block the life cycle of virus replication by targeting and inhibiting the expression of key genes related to influenza virus invasion, replication, assembly or release, reducing virus titer, and inhibiting infection until complete clearance Virus.
  • the siRNA molecules that inhibit influenza virus replication are selected from one or more of the following siRNA molecules: any one of SEQ ID No. 1 to 97, and the antisense strand is selected from SEQ ID No. 98 to One of 194 complementary to the sense strand.
  • the siRNA molecule for inhibiting the expression of PD-1 is designed based on the homologous sequence between the human PD-1 gene and the mouse PD-1 gene, and the siRNA molecule for inhibiting the expression of PD-L1 is based on the human PD-1 gene - Homologous sequences between the L1 gene and the mouse PD-L1 gene were designed.
  • the homologous sequence refers to a DNA sequence whose sequence is confirmed to be 100% identical after the two genes of human and mouse are aligned.
  • siRNA molecules targeting PD-L1 are small interfering nucleotides that specifically inhibit the expression of human programmed death 1 (PD1) ligand 1 (PD-L1).
  • PD1 programmed death 1
  • PD-L1 has a significant effect on the immune system of the body, and can inhibit the function of T cells during virus infection, or cause T cell exhaustion.
  • the siRNA molecules that inhibit the expression of PD-1 are selected from one or more of the following siRNA molecules: the sequence of the sense strand is selected from any one of SEQ ID Nos. One complementary to the sense strand from SEQ ID Nos. 207-218.
  • the siRNA for inhibiting the expression of PD-L1 is selected from one or more of the following siRNA molecules: the sequence of the sense strand is selected from any one of SEQ ID No. 219-230, and the antisense strand is selected from One of SEQ ID Nos. 231 to 242 complementary to the sense strand.
  • influenza mRNA vaccine is a messenger RNA vaccine designed according to the influenza virus gene sequence.
  • influenza virus gene is a gene encoding a viral structural protein and/or a gene encoding a non-structural egg, further preferably, the gene encoding a viral structural protein is selected from PB2, PB1, PA, HA, NP, One or more of NA, M1, or M2, and the non-structural protein-encoding gene is NS1 and/or NS2.
  • the mRNA vaccine in addition to containing specific viral gene sequences, also contains elements necessary for translation in cells.
  • the elements include, but are not limited to, untranslated regions (UTRs) at both ends, cap structures at the 3' end and polyA tails at the 5' end.
  • UTRs untranslated regions
  • the anti-influenza virus small molecule compound is a specific influenza virus inhibitor and/or a broad-spectrum antiviral small molecule compound.
  • the specific influenza virus inhibitor is selected from M2 ion channel blockers, NA (neuraminidase) inhibitors, PA (polymeric alpha-PA subunits) inhibitors and PB2 (polymeric beta PB2 subunits) ) one or more of the inhibitors.
  • the broad-spectrum antiviral small molecule compound is selected from one or more of ribavirin, nitazoxanide, arbidol hydrochloride, and favipiravir.
  • the small molecule compound is a water-soluble compound.
  • the small molecule compound has good stability after being dissolved in an aqueous solution, and can still maintain a certain activity after being atomized.
  • a fourth aspect of the present invention provides an siRNA-small molecule drug conjugate, wherein the siRNA-small molecule drug conjugate is formed by covalently coupling an siRNA molecule that inhibits influenza virus replication and an anti-influenza small molecule drug.
  • the small molecule compound is a small molecule containing a nucleotide base structure.
  • the chemical bond is a covalent bond, an ionic bond or a metallic bond.
  • the siRNA molecules for inhibiting influenza virus replication are designed for conserved gene sequences among different strains of influenza A virus, including one or more of H1N1, H5N1, H7N9, or H3N2 Subtype; the siRNA molecules that inhibit influenza virus replication block the life cycle of virus replication by targeting and inhibiting the expression of key genes related to influenza virus invasion, replication, assembly or release, reducing virus titer, and inhibiting infection until complete clearance Virus.
  • the siRNA molecule that inhibits the replication of influenza virus is selected from any one of SEQ ID Nos. 1 to 97, and the antisense strand is selected from one of SEQ ID No. 98 to 194 that is complementary to the sense strand.
  • the anti-influenza virus small molecule compound is a specific influenza virus inhibitor and/or a broad-spectrum antiviral small molecule compound.
  • the specific influenza virus inhibitor is selected from one or more of M2 ion channel blockers, NA inhibitors, PA inhibitors and PB2 inhibitors.
  • the broad-spectrum antiviral small molecule compound is selected from one or more of ribavirin, nitazoxanide, arbidol hydrochloride, and favipiravir.
  • the siRNA molecule for inhibiting influenza virus replication and the anti-influenza virus small molecule compound are linked through respective active groups, or by introducing Linker into the siRNA molecule for inhibiting influenza virus replication, the active group of Linker is utilized. The group is coupled with the anti-influenza virus small molecule compound.
  • the reactive group includes one or more of amino group, carboxyl group, hydroxyl group, phosphoric acid group, epoxy group, aldehyde group and isocyanate group.
  • a fifth aspect of the present invention provides an application of the siRNA-small molecule drug conjugate in the preparation of a drug for preventing or treating influenza virus infection.
  • the siRNA drug for preventing or treating influenza virus infection the pharmaceutical composition for preventing or treating influenza virus infection, or the siRNA-small molecule drug conjugate and pharmaceutically acceptable
  • the accepted carrier forms a formulation
  • the pharmaceutically acceptable carrier is selected from one or more of saline, saccharides, polypeptides, high molecular polymers, lipids, creams, gels, micellar materials, or metal nanoparticles kind.
  • the high molecular polymer is the polypeptide based high molecular polymer.
  • the polypeptide-based high molecular polymer is a cationic polypeptide composed of histidine and lysine.
  • the polypeptide-based high molecular polymer is HKP(H3K4b) and/or HKP(+H) branched polypeptide.
  • the siRNA drug, or the pharmaceutical composition, or the siRNA-small molecule drug conjugate and the pharmaceutically acceptable carrier form a nano-formulation; the nano-formulation is an oral dose, Injections, or nebulized inhalers.
  • the dosage form of the nano-formulation is an aerosol inhalation preparation, and the preparation is delivered to the disease by intravenous injection, oral administration, subcutaneous injection, intramuscular injection, aerosol inhalation, intranasal, etc. inhibitory effect.
  • the drug is delivered to the lower respiratory tract and the lungs by inhalation to inhibit the replication of influenza virus.
  • the siRNA medicine for preventing or treating influenza virus infection of the present invention, or the pharmaceutical composition for preventing or treating influenza virus infection, or the application, are directed against influenza virus G4EA H1N1 strain, One or more of the H1N1 strain, the H5N1 strain, the H7N9 strain, or the H3N2 strain. ,
  • the present invention is based on siRNA molecules that inhibit influenza virus replication, and combines siRNA molecules with other types of anti-influenza virus drugs.
  • This combination strategy aims to provide an effective and complementary strategy for the treatment of influenza virus infection, and this treatment Strategies are more effective than each therapy alone.
  • each siRNA proven to have a significant inhibitory effect on influenza virus with a marketed or clinically validated anti-influenza virus small molecule drug, and evaluating its efficacy in cells, rodents, and non-human primates Broad-spectrum efficacy in infection models such as influenza virus.
  • the pharmaceutical composition described in the present invention may be a combination of a siRNA molecule that inhibits influenza virus replication and another molecule in a specific ratio to form a mixed solution and then administered in the same manner.
  • the specific ratio is determined according to the concentrations required for the two molecules to exert their drug effects, especially the blood drug concentrations.
  • the drug concentration is determined according to the data results of preclinical studies.
  • the drug concentration is determined according to the data of preclinical, clinical experiments and clinical applications.
  • the composition mixed in the specific ratio also considers the synergistic effect and interaction between the drugs.
  • the pharmaceutical composition described in the present invention can be the siRNA molecule that inhibits the replication of influenza virus as a single drug solution, another molecule as another single drug solution, and the two drug solutions are used as a combination.
  • the two separate drug solutions are dissolved in the same or similar solvent.
  • the two separate drug solutions are dissolved in different solvents.
  • the composition composed of the two separate drug solutions can be administered simultaneously or at different times. Further preferably, the two separate drug solutions are administered sequentially at approximately the same time, or are administered interspersed at different times.
  • the siRNA drug molecule, the pharmaceutical composition, and the siRNA-small molecule drug conjugate can also be combined with a pharmaceutically acceptable nano-introduction carrier conjugate to form a nano-drug.
  • the nano-drug carrier is combined with the various molecules through electrostatic interaction, hydrogen bond and van der Waals force to form a stable uncoupled nano-polymer.
  • the nano-introduction carrier can encapsulate the siRNA molecule that inhibits the replication of influenza virus and another molecule at the same time, or encapsulate the two molecules separately.
  • the nano-introduction carrier wraps two molecules at the same time to form nano-drug particles with uniform particle size.
  • Said separately encapsulating two kinds of molecules can use exactly the same nano-introduction carrier to encapsulate two kinds of molecules respectively to form nanoparticles with the same or different particle size, or can use different nano-introducer carriers to encapsulate two kinds of molecules respectively to form particle size same or different nanoparticles.
  • the nano-drug particles are polymers that are stably suspended in the form of particles in a specific solvent, and their diameters range from a few nanometers to hundreds or even thousands of nanometers.
  • the diameter of the nanoparticles is 30-300 nm, and further preferably, the size of the nanoparticles is 50-150 nm.
  • the nanomedicine can be administered by atomization inhalation, intravenous injection, subcutaneous injection, intramuscular injection, oral administration and the like.
  • it can be atomized into droplets by an ultrasonic atomization device, and administered by inhalation to reach the lower respiratory tract and lungs to inhibit the replication of influenza virus.
  • the two nanoparticles can be administered in the same way or in different ways.
  • the present invention has the following advantages compared with the prior art:
  • the present invention provides siRNA molecules that can efficiently and specifically inhibit influenza virus replication, has a significant inhibitory effect on influenza viruses, and provides more options for preparing medicines for preventing or treating influenza virus infection, based on the siRNA molecules that inhibit influenza virus replication , can prepare more new siRNA drugs for the prevention and treatment of influenza virus infection, or combine with other types of anti-influenza virus drugs to prepare pharmaceutical compositions based on siRNA molecules and siRNA-small molecule drug conjugates, which are used for influenza
  • the treatment of viral infections offers potent and complementary strategies with broad-spectrum efficacy against influenza viruses and other infection models in cells, rodents, and non-human primates.
  • FIG. 1 Anti-influenza virus siRNA molecules can effectively inhibit virus replication in in vitro cell experiments.
  • A shows the titer of H5N1 influenza virus hemagglutinin (HA) in the cell supernatant after different treatments
  • B is the TCID 50 (half tissue culture infectious dose) of the H5N1 virus in the cell supernatant after different treatments, where the The siRNA sequence (80nM) had the best inhibitory effect on H5N1 influenza virus.
  • C is the titer of H1N1 influenza virus hemagglutinin (HA) in the cell supernatant after different treatments
  • D is the TCID 50 (half tissue culture infectious dose) of H7N9 virus in the cell supernatant after different treatments.
  • siRNA molecules such as M1-1, PA-19, NP-15 can effectively inhibit H1N1 virus infection, and the siRNA sequence (80nM) of M1-1 has a significant inhibitory effect on H7N9.
  • FIG. 1 Anti-influenza virus siRNA molecules attenuate the death of mice caused by influenza virus infection.
  • A shows the animal grouping, dosage and mode of administration
  • Figure B shows the survival curve of mice in each group.
  • the medium dose of M1-1siRNA intravenous injection group has a good inhibitory effect on the virus, and the survival rate (70%) of the mice on the 15th day was significantly higher than that of the negative siRNA control group, which was also better than that of the negative siRNA control group.
  • FIG. 3 Expression rates of PD-1 or PD-L1 gene in different cells after siRNA transfection.
  • a and B show the target genes PD-1 and PD-L1 after transfection of MCF-7 (breast cancer), BxPC3 (pancreatic cancer) and HepG2 (liver cancer) cells with siRNAs designed against PD-1 and PD-L1 genes, respectively expression rate.
  • FIG. 4 Inhibition of PD-1 expression by siRNA can activate immune cells to secrete cytokines. After transfection of mouse RAW264.7 macrophages with siRNA against PD-1, the concentration of TNF- ⁇ in the cell culture supernatant was significantly increased (see Figure 4A), and the concentration of TNF- ⁇ in the cell lysate also increased (No statistically significant difference, see Figure 4B).
  • FIG. 5 Schematic diagram of the combined anti-influenza siRNA and mRNA vaccine for A virus infection.
  • siRNA vaccine Using mRNA vaccine, it can be expressed into influenza virus-specific protein/polypeptide fragments in the body, activate the immune mechanism against influenza virus infection, and reduce the probability of virus infection. At the same time, inhibiting the replication of the virus in cells by siRNA blocks the life cycle of the virus.
  • Figure 6 Conjugation of zanamivir to siRNA molecules.
  • the superphosphate group and the hydroxyl group are condensed into phosphate ester, or the zanamivir is connected to the end of the siRNA by the way of Linker.
  • FIG. 1 Conjugation of peramivir to siRNA molecules.
  • the carboxyl group of peramivir can be linked to siRNA through Linker.
  • Figure 8 Conjugation of oseltamivir to siRNA molecules.
  • the hydroxyl group at one end of Linker reacts with the phosphate group at the end of the siRNA, and the epoxy group at the other end undergoes a nucleophilic reaction with the amino group of oseltamivir.
  • A-192558 and A-315675 Conjugation of A-192558 and A-315675 to siRNA molecules.
  • A-192558 contains modifiable amino and carboxyl groups, and then the epoxy group at one end of the Linker reacts with the amino group, and the hydroxyl group at the other end reacts with the phosphate group of the siRNA; hydrogen halide can also be used to add carbon-carbon double bonds, and then Linked to Linker via substitution reaction.
  • Aerosol (short duration, short interval) administration can efficiently deliver siRNA molecules into the lungs.
  • A shows the distribution of siRNA molecules in the lung after aerosol inhalation
  • B shows the inhibitory effect of siRNA molecules on lung target genes.
  • Aerosol inhalation (long duration long interval) administration can effectively deliver siRNA molecules into the lungs.
  • A shows the distribution of siRNA molecules in the lungs when aerosol inhalation is completed, and B shows the distribution of siRNA molecules in the lungs when aerosol inhalation is completed 24 hours later.
  • FIG. 12 The aerosol inhalation administration of siRNA has no significant toxic and side effects.
  • A nebulization inhalation administration has no significant effect on the body weight of mice;
  • B pulmonary interleukin-6 (IL-6) has no significant change after nebulization administration;
  • C pulmonary TNF- ⁇ has no significant change after nebulization administration .
  • MDCK was cultured in medium containing 10% MEM, and expanded at a ratio of 1:1 to maintain cell viability. 12-18 hours before transfection, MDCK cells were added to a 24-well plate at 2.4 ⁇ 10 5 cells/well, and transfection was performed when the cell density reached about 80%. Transfection Various siRNAs were transfected according to the instructions of Lipofectamine 2000 lipofection reagent (Life Technologies). The oseltamivir control drug was added directly to the medium at a final concentration of 125 ⁇ M. Twenty-four hours after transfection (or adding oseltamivir), the cell culture plate was washed three times with PBS, and 400 ⁇ L/well of OPTI-MEM was added.
  • the virus was diluted with OPTI-MEM and inoculated with 100 ⁇ L/well. Placed in a cell culture incubator for 1 h, in which the cell plate was gently shaken every 15 min. After 1 hour, the virus solution was aspirated, washed with PBS, and then replaced with 1 mL of OPTI-MEM containing 0.5% antibiotics in each well, and cultured in a 37°C cell incubator for 3 days. 48 hours after inoculation, 200 ⁇ L/well of cell supernatant was collected for the determination of HA titer and TCID 50 (half the tissue culture infectious dose). The determination of HA titer is carried out by conventional methods.
  • the TCID 50 assay was performed as follows: each sample was diluted with 1% MEM in 8 dilutions, and each dilution was seeded in 4 wells. Discard the waste solution of the 96-well plate, wash it three times with PBS, and insert the virus solution into the 96-well plate at an amount of 100 ⁇ L/well. Statistically calculated TCID50 . The results are shown in Figure 1.
  • the different doses of siRNA molecules described in the patent of the present invention have different degrees of inhibitory effect on influenza A virus subtypes such as H1N1, H5N1, and H7N9. Among them, M1-1 has the best inhibitory effect. close to oseltamivir.
  • siRNA molecules For PD-1 and PD-L1 genes, after selecting the homologous sequences of human and mouse to design siRNA molecules, we commissioned the synthesis of these siRNA molecules (Suzhou Beixin), and then used human tumor cell lines MCF-1, BxPC3 and HepG2 to determine these siRNAs Inhibitory effect of molecules on PD-1 or PD-L1 expression. A specific amount of cells were inoculated into 6-well plates or 12-well plates and cultured for more than 6 hours to make the cells adherent. Then, Lipofectamine 2000 liposome transfection reagent was used to transfect various siRNAs according to the operating instructions, and then cultured for 48 hours.
  • RNA tissue/cell RNA rapid extraction kit, Beijing Jumeimei
  • concentration was determined with a micro UV spectrophotometer (MicroDrop, Bio-DEL), and 100-500 ng of total RNA was taken for reverse transcription (first-strand cDNA reverse transcription). Transcription kit, Beijing Poly America).
  • fluorescence quantitative PCR amplification kit Realtime PCR Super mix (SYBRgreen, with anti-Taq), Beijing Jumeimei
  • SYBRgreen Realtime PCR Super mix
  • Talaq anti-Taq
  • Beijing Jumeimei was used to analyze and detect on fluorescent PCR instrument (QuantStudio 3, ABI). The results are shown in Figure 3.
  • the siRNA designed for PD-1 or PD-L1 can inhibit the expression of corresponding target genes to varying degrees. Accordingly, the most efficient siRNA molecule for each target gene can be selected.
  • a siRNA molecule of PD-1 (PD-1-10) was selected and transfected into mouse RAW264.7 macrophage cell line, and the cell culture supernatant and cells were measured after 28 hours of culture.
  • TNF- ⁇ tumor necrosis factor- ⁇
  • Figure 4A the concentration of TNF- ⁇ was significantly increased
  • Figure 4B the content of TNF- ⁇ in the protein lysate was also increased. It can be seen that the use of siRNA to inhibit the expression of PD-1 can activate the function of immune cells. While using anti-influenza siRNA to inhibit virus infection in mammals, the use of PD-1 siRNA to activate the body's immune system can effectively strengthen anti-virus immunity and more effectively remove viruses.
  • siRNA is an efficient antiviral method, and mRNA vaccines have been clinically proven to effectively protect the human body from the infection of the new coronavirus (SARS-CoV-2). It is used in combination with influenza mRNA vaccine to achieve the purpose of efficient and specific elimination of pathogens.
  • SARS-CoV-2 new coronavirus
  • an mRNA containing a cap structure, a 5'-untranslated region, an open reading frame (ORF), a 3'-untranslated region and a polyadenylation (PolyA) tail Vaccine wherein ORF is a gene sequence encoding a specific protein of influenza virus.
  • ORF is a gene sequence encoding a specific protein of influenza virus.
  • APC antigen presenting cells
  • siRNA nanopharmaceutical preparation is administered to the mammal.
  • the antiviral siRNA molecule enters the body cells, it forms an RNA-induced silencing complex (RISC) with a specific enzyme/protein.
  • RISC RNA-induced silencing complex
  • the antisense strand carries the entire complex and binds to the viral RNA, degrading the viral RNA through the RNAi mechanism, thereby preventing the expression of virus-specific genes into proteins/enzymes, and the virus cannot complete the replication life cycle.
  • RISC RNA-induced silencing complex
  • the combined application of mRNA vaccine and siRNA molecule can effectively block the infection and replication of influenza virus.
  • Zanamvir contains hydroxyl, carboxyl and guanidine active groups, and Zanamvir is obtained by substituting the hydroxyl group at the C-4 position in DANA with a guanidine group.
  • the guanidino group can be combined with two amino acids Glu119, Glu227 or Asp115 in the S2 region of influenza virus neuraminidase (NA) to improve the enzyme inhibitory activity in vitro. Therefore, the guanidine group plays an important role in the inhibition of NA, and the integrity of the guanidine group should be maintained as much as possible.
  • the present invention designs two modification methods to combine with antiviral siRNA: one is to condense phosphate groups and hydroxyl groups into phosphate esters, and block Zanamvir in the siRNA molecule (Fig. 6, ⁇ [Zanamvir] Mivir] ⁇ ); Another method is to connect Zanamvir to the end of siRNA by linking compound (Linker) or directly.
  • Linker such as PEG
  • the phosphate group in the 5' end of siRNA can be directly coupled to the hydroxyl group of Zanamvir
  • a Linker such as PEG
  • one end is connected to the carboxyl group of Zanamvir, and the other end is connected to the carboxyl group of Zanamvir.
  • DANA and FANA have similar structural formulas, and can be combined with siRNA by the above two methods to screen out binding substances that meet the therapeutic effect.
  • Peramivir contains guanidine, hydroxyl and carboxyl active groups.
  • the hydroxyl group can be condensed with the phosphate group at the 5' end of the siRNA or the hydroxyl group at the 3' end of the siRNA, and the retained carboxyl group can be combined with Arg292, Arg371 and Arg118 in the S1 region of the influenza virus NA.
  • the carboxyl group can be connected to siRNA through Linker, as shown in Figure 7, peramivir RWJ-270201-siRNA, the amino group at one end of the Linker reacts with the carboxyl group to form an amide bond, and the hydroxyl group at the other end reacts with the phosphate group to form a phosphate ester; It contains responsive groups such as disulfide bonds, which can be cleaved under specific conditions, and the drug and siRNA will have a synergistic effect to improve the therapeutic effect. While the cyclopentane derivatives and cyclopentanamide derivatives of its drugs retain the guanidino group, siRNA can be coupled by the above method.
  • the amino group of Oseltamivir is the main functional group and active group. Amino groups can react with epoxy groups, aldehyde groups, isocyanate groups, and carboxyl groups. As shown in Figure 8, in oseltamivir-siRNA, the hydroxyl group at one end of Linker reacts with the phosphate group at the end of the siRNA, and the epoxy group at the other end undergoes a nucleophilic reaction with the amino group of Oseltamivir, among which the secondary amine is less basic than the primary amine. , but can still bind to the carboxyl group of Glu119, Glu227 or Asp115 in the S2 region of influenza virus NA protein.
  • the active groups that can be modified are amino and carboxyl groups.
  • the epoxy group at one end of Linker reacts with amino group, and the hydroxyl group at the other end reacts with phosphate group of siRNA (as shown in Figure 9, A-192558-siRNA).
  • the retained carboxyl group can react with Arg292 and Arg371 in S1 region of NA. Binds to the guanidino group of Arg118. If the carboxyl group is used for coupling, the retained amino group is bound to the carboxyl group of Glu119, Glu227 or Asp115 in the S2 region of NA.
  • the modifiable groups are carbon-carbon double bonds and carboxyl groups, and carbon-carbon double bonds can undergo addition reactions with water, halogen, etc., or with carbon-carbon double bonds Bonds, carbon-carbon triple bonds, and other olefins and alkynes undergo polymerization reactions.
  • A-315675-siRNA uses hydrogen halide to add carbon-carbon double bond, and then connects to Linker through substitution reaction.
  • Embodiment 5 Anti-influenza virus siRNA molecule and mRNA vaccine polypeptide nanomedicine preparation
  • the invention adopts polymers, especially histidine-lysine copolymer (HKP), to encapsulate nucleic acid drug molecules, including siRNA and mRNA, to prepare nano-drug particles.
  • the HKP and siRNA molecules form nanoparticles, wherein the nanoparticles are about 30 nm to about 300 nm in diameter.
  • HKP and siRNA molecules self-assemble into nanoparticles or can be formulated into nanoparticles.
  • the HKP and the mRNA vaccine molecules form nanoparticles, wherein the nanoparticles are from about 30 nm to about 400 nm in diameter.
  • the HKP is H3K(+H)4b, which can self-assemble with mRNA molecules to form nanoparticles or can be formulated into nanoparticles.
  • HKP can be used to encapsulate siRNA and mRNA molecules at the same time to form nanoparticles with a diameter of 30nm-400nm or even larger.
  • the present invention establishes a series of assay methods to characterize the physicochemical properties of nanomedicine preparations, including particle size, surface potential, morphological studies, loading efficiency of mRNA or siRNA, biological activity, etc.
  • a Nano Zetasizer Nano ZS (Malvern Instruments, UK) was used to measure the size and potential of the nanopharmaceutical formulation particles.
  • the real-time quantitative fluorescence PCR method is used to determine the inhibitory effect of siRNA on the expression of viral target genes.
  • the expressed proteins or polypeptides are identified and quantified by RPHPLC using analytical column C18 (2S0mm x 2.1mm; Phenomenex).
  • Embodiment 6 atomization inhalation administration is used for the prevention and treatment of respiratory virus infection disease
  • the siRNA (unlabeled or fluorescently labeled) targeting specific target genes was passed through the mouth and nose by aerosol inhalation. into the respiratory system.
  • the mice were placed in a closed chamber, and the nanomedicine preparation was placed in the atomization cup. After the spray port of the atomizer was airtightly connected to the chamber, the power supply was turned on for atomization for a certain period of time, and the situation of siRNA entering the lungs and its effect on the lungs were determined. Inhibition efficiency of target gene expression.
  • the fluorescently labeled siRNA (AF647-siRNA, Qiagen) or the siRNA against Cyclophilin-B (Suzhou Beixin) was nebulized using an ultrasonic nebulizer (ALC) or a hand-held nebulizer (ZYM), and the entry into the lungs of mice was determined. efficiency of the department.
  • the drug is administered by atomization with a short duration and a short interval. The drug is given once (2 mL), and then atomized: first, the atomization chamber is filled with about Then stop for 20 seconds, atomize for 10 seconds, stop for 20 seconds, atomize for 10 seconds, stop for 20 seconds.
  • the administration was nebulized with a long duration, long interval, and administered once (2 mL, fully nebulized): first, the nebulization chamber was filled for about 1 minute, then stopped for 1 minute , atomization for 1 minute, stop for 1 minute, atomization for 1 minute, stop for 1 minute. This process was repeated until all the liquid medicines were nebulized; when the nebulization administration was completed or 24 hours after administration, some mice were sacrificed, the lungs were isolated, and the siRNA fluorescence was measured.
  • siRNA can be detected in the lungs, indicating that after the siRNA enters the lungs, it can be detected in the lungs. Continue in the lung for a certain period of time to ensure full effect, wherein, Figure 11 shows the experimental results using 250 ⁇ g of siRNA against Cyclophilin-B.
  • the negative siRNA in the examples and accompanying drawings of the present application is an siRNA that does not target any gene.
  • siRNA small interfering nucleotide
  • the siRNA molecule for suppressing influenza A virus is a molecule designed for the conserved gene sequences of influenza A viruses of various subtypes, including but not limited to the G4EA H1N1 virus strain (A /swine/Hebei/0116/2017 (H1N1) and A/swine/Jiangsu/J004/2018 (H1N1, etc.), H1N1 strains (A/PuertoRico/8/1934 and A/California/07/2009, etc.), H5N1 Virus strain (A/Vietnam/1194/2004 etc.), H7N9 virus strain (A/Shanghai/CN02/2013 etc.) and H3N2 virus strain (A/Texas/50/2012 etc.) etc.
  • G4EA H1N1 virus strain A /swine/Hebei/0116/2017 (H1N1) and A/swine/Jiangsu/J004/2018 (
  • the siRNA molecules are designed against the homologous sequences of the above strains.
  • the length of the siRNA molecule is 19-30 base pairs, preferably, the length of the siRNA molecule is 21 base pairs or 25 base pairs.
  • the GC content of the siRNA molecule is 30-70%, preferably, the GC content of the siRNA molecule is 40-60%.
  • NP-6 19+dTdT CGGACGAAAAGGCAACGAAdTdT 61 UUCGUUGCCUUUUCGUCCGdTdT 158 NP-7 19+dTdT CGAAAAGGCAACGAACCCGdTdT 62 CGGGUUCGUUGCCUUUUCGdTdT 159 NP-8 19+dTdT GAAAAGGCAACGAACCCGAdTdT 63 UCGGGUUCGUUGCCUUUUCdTdT 160 NP-9 19+dTdT CCUUUGACAUGAGUAAUGAdTdT 64 UCAUUACUCAUGUCAAAGGdTdT 161 NP-10 19+dTdT CUUAUUUCUUCGGAGACAAdTdT 65 UUGUCUCCGAAGAAAUAAGdTdT 162 NP-11 25 GAGUCUUCGAGCUCUCGGACGAAAA 66 UUUUCGUCCGAGAGCUCGAAGACUC 163 NP-12 25 CGAAAA
  • the PD-1/PD-L1 signaling pathway is important for antiviral immune effects and can affect the severity of immune pathological damage caused by pathogen infection.
  • programmed death 1 PD1
  • CD8+ T cells CD8+ T cells
  • regulatory T cells also highly express inhibitory molecules such as PD1 during chronic viral infection, which may be related to increased viral load or increased inhibition of antiviral T cell responses.
  • virus-specific T cells rapidly upregulate the co-inhibitory receptor PD-1 upon antigen recognition, and directly upregulate PD-L1 on hematopoietic and non-hematopoietic cells via PRR signaling or by inducing IFN and other inflammatory
  • the release of cytokines indirectly upregulates PD-L1.
  • Viruses can also control the balance of the immune system, preventing an effective antiviral immune response to help the persistence of pathogens in an organism. After blocking regulatory T cells and depleting the PD1/PD-L1 signaling pathway on the surface of CD8+ T cells, the function of depleting CD8+ T cells can be reversed, which brings new targeted therapy strategies for the treatment of chronic viral infectious diseases. opportunity.
  • RSV respiratory syncytial virus
  • PD-L1 was significantly up-regulated in H9N2 virus-infected pulmonary microvascular endothelial cells (RPMECs), and viral infection-induced PD-L1 expression transmits a negative signal to migrating T cells, thereby This results in down-regulation of antiviral cytokines and reduced production of cytotoxic proteins [29] .
  • RPMECs pulmonary microvascular endothelial cells
  • the composition of the present invention also comprises a siRNA molecule that specifically inhibits the expression of PD-1 or PD-L1 gene in specific cells of the host, and the siRNA molecule is selected from Table 2 and Sequences in Table 3.
  • the siRNA molecule that specifically inhibits the expression of PD-1 or PD-L1 gene can inhibit the expression of PD-1 or PD-L1 gene in specific cells after reaching a specific part of the body, thereby enhancing the function of virus-specific T cells, And it has a synergistic effect with the siRNA molecule against influenza A virus infection, effectively inhibiting the virus infection and completely eliminating the virus in the body.
  • the siRNA molecule that inhibits the expression of PD-1 or PD-L1 gene has the function of inhibiting the expression of human PD-1 or PD-L1 gene, and also has the function of inhibiting the expression of mouse PD-1 or PD-L1 gene.
  • influenza virus There are two existing strategies against influenza viruses: vaccines and small-molecule anti-influenza drugs. Influenza vaccination is the most effective way to prevent influenza. Now there are also trivalent inactivated vaccines and live attenuated vaccines on the market, but vaccines need to be reconfigured every year to deal with antigenic variation, and their development cycle is long and the cost is high. These shortcomings make small molecules Drugs have become the main means of prevention and treatment of influenza.
  • Antiviral small molecule compounds currently on the market or in clinical stage mainly include specific influenza virus inhibitors (M2 ion channel blockers, NA inhibitors, PA inhibitors and PB2 inhibitors) and some broad-spectrum antiviral drugs (ribavirin) warin, nitazoxanide, arbidol hydrochloride, favipiravir, etc.).
  • M2 ion channel blockers NA inhibitors, PA inhibitors and PB2 inhibitors
  • ribavirin broad-spectrum antiviral drugs
  • combination therapy has become a major development direction for the treatment of influenza virus infection.
  • Clinical research on the combination therapy of drugs has been carried out continuously.
  • the anti-influenza A virus infection siRNA molecule and the anti-influenza A small molecule compound are used in combination to exert a synergistic antiviral effect through their different action mechanisms.
  • the composition comprises an anti-influenza A virus infection siRNA molecule and an anti-influenza A small molecule compound, wherein the siRNA molecule and the small molecule compound target and inhibit viral internal proteins and viral external proteins respectively.
  • the siRNA molecule against influenza A virus infection targets the expression of internal viral proteins such as PA, PB1, PB2 or NP gene, and the small molecule compound is oseltamivir that inhibits viral external proteins such as NA, HA, and M proteins , arbidol and amantadine and other molecules.
  • the anti-influenza A virus infection siRNA molecule targets the expression of the genes of external proteins such as NA, HA, and M proteins, and the small molecule compound is favipiravir or Favipiravir that inhibits internal proteins such as PA, PB1, PB2 or NP. Naproxen and other molecules.
  • PB2-11 siRNA (sense strand is 5'-GAAACGAAAACGGGACUCUAGCAUA-3') is used in combination with the NA inhibitor oseltamivir to simultaneously inhibit the viral polymerase gene and neuraminidase.
  • an siRNA molecule that inhibits the M1 protein is used in combination with the influenza virus polymerase inhibitor favipiravir to simultaneously inhibit two different genes/proteins expression or function.
  • NA-1 sense strand is 5'-GUCUUGGCCAGACGGGUGCUdTdT-3'
  • siRNA molecule that inhibits polymerase PA gene siRNA molecule that inhibits polymerase PA gene
  • ribavir Forest combined use.
  • mRNA vaccines carry genetic information encoding viral antigens, but they do not integrate with the host cell genome or interact with DNA and therefore pose no mutational risk to the host. Also, mRNA vaccines do not contain viral particles. So the mRNA vaccine itself does not induce the disease it prevents.
  • mRNA therapy including vaccine
  • the modification of specific nucleosides in the mRNA sequence and the development of various RNA packaging and introduction systems have greatly promoted the development of mRNA vaccines [26] .
  • Much evidence shows that, compared with DNA vaccines, which are also nucleic acid vaccines, mRNA not only mediates better transfection efficiency and longer protein expression time, but also has significant advantages because mRNA does not need to enter the nucleus to function.
  • mRNA vaccines can also be used as an effective means of preventing influenza virus infection.
  • Traditional flu vaccines generally consist of proteins found in influenza viruses that "train" a patient's immune system to develop mechanisms to fight influenza virus infection.
  • influenza viruses mutate very quickly, often altering these proteins and rendering vaccines ineffective. That's why the flu vaccine changes every year and doesn't always keep people from getting sick.
  • the use of mRNA vaccines to fight influenza has significant advantages over traditional vaccines. In a recent study, mRNA vaccines against H7N9 and H10N8 influenza A induced robust humoral immune responses and were well tolerated [27] .
  • the anti-influenza virus siRNA molecule is used in combination with the influenza virus mRNA vaccine, which can effectively prevent and treat various influenza A virus infections. Since siRNA and mRNA are both RNA molecules, only different in length and single and double strands, both can be packaged with the same type of nano-introduction carrier to prepare a mixed nano-drug preparation, which has broad application prospects in clinical treatment.
  • influenza virus mRNA vaccine designed based on the HA gene sequence is used in combination with an siRNA molecule that inhibits the M1 protein (sense strand is 5'-UACGCUGCAGUCCCUCGCUCACUGGG-3') to activate the body's antiviral immunity and enhance antiviral At the same time of immune cell viability, the expression of M1 gene is inhibited.
  • influenza virus mRNA vaccine designed based on the viral nucleoprotein NP gene sequence is used in combination with PB2-11 siRNA (sense strand is 5'-GAAACGAAAACGGGACUCUAGCAUA-3') that inhibits the PB2 protein.
  • nucleic acid-based small molecules can increase endogenous and improve silencing efficiency and inhibition rate.
  • the siRNA of PR8-M1 was linked to a ribozyme-catalyzed degrading nucleic acid sequence to form an siRNA-ribozyme chimera, which further enhanced the ability of the siRNA to degrade nucleic acid, and the silencing efficiency of this optimized siRNA improved by a factor of four [30] .
  • Another study found that after adding a sequence with immunostimulatory function (5'-UGUGU-3') to the 5' end of NP-siRNA, the inhibition rate of influenza virus reached 80%, which is four times the inhibition efficiency of siRNA alone.
  • NP-1496 siRNA into a vector containing endogenous microRNA (miRNA) significantly enhanced its endogenous nature.
  • miRNA endogenous microRNA
  • shRNAmir-NP endogenous microRNA
  • the NP protein was completely inhibited, and the virus titer was reduced to about 1/100 of the control [32] .
  • the coupling between these same types of molecules can effectively provide the ability to fight influenza virus infection and replication, but is also limited by the similarity between the molecules and cannot exert the maximum synergistic effect.
  • the present invention comprises a novel compound molecule formed by covalently coupling an anti-influenza virus siRNA molecule and an anti-influenza A small molecule compound.
  • the siRNA molecules include the siRNA molecules in Table 1.
  • the anti-influenza A small molecule compounds include but are not limited to specific influenza virus inhibitors (M2 ion channel blockers, NA inhibitors, PA inhibitors and PB2 inhibitors) and broad-spectrum antiviral drugs.
  • the siRNA molecule and the anti-influenza A small molecule compound can be directly connected through their respective active groups, or can be coupled by introducing a linker to couple the two molecules by using the active group of the linker.
  • the reactive groups include, but are not limited to, amino groups, carboxyl groups, hydroxyl groups, phosphoric acid groups, epoxy groups, aldehyde groups, isocyanate groups, and the like. Covalent bonds can be formed between the reactive groups through addition reactions, polymerization reactions, condensation reactions, and the like.
  • a phosphate group and a hydroxyl group into a phosphate ester, block zanamivir (Zanamvir) in the siRNA molecule, or connect Zanamvir to the siRNA molecule through a linker (such as polyethylene glycol, etc.). the ends of the siRNA.
  • a linker such as polyethylene glycol, etc.
  • the amino group at one end of the linker reacts with the carboxyl group of Peramivir to form an amide bond, and the hydroxyl group at the other end reacts with the phosphate group of the siRNA to form a phosphate ester.
  • the hydroxyl group at one end of the linker reacts with the phosphate group at the end of the siRNA, and the epoxy group at the other end undergoes a nucleophilic reaction with the amino group of Oseltamivir.
  • the epoxy group at one end of the linker reacts with the amino group of the NA inhibitor A-192558, and the hydroxyl group at the other end reacts with the phosphate group of the siRNA.
  • a pharmaceutically acceptable carrier is used as an introduction (delivery) system for siRNA drugs or siRNA drug-based compositions
  • pharmaceutically acceptable carriers generally include saline, sugars, polypeptides, polymers, lipids, creams, gels , micellar materials and metal nanoparticles.
  • the carrier is a histidine-lysine copolymer (high molecular weight polymer) described in US Pat. The entire contents are incorporated herein by reference.
  • the HKP vector is H3K4b, H3K(+H)4b, H2K4b or H3K(+N)4b, these HKPs have a lysine backbone whose four branches comprise multiple repeated histidines, lysines or asparagine.
  • HKP is H3K4b with the following structure:
  • HKP is H3K(+H)4b with the following structure:
  • HKP is H2K4b with the following structure:
  • HKP is H3K(+N)4b with the following structure:
  • a kind of nano-drug preparation that is formed by HKP and siRNA medicine or the composition based on siRNA medicine, described HKP carries positive charge, and siRNA, the composition of siRNA and siRNA, the composition of siRNA and mRNA vaccine etc. carry negative charge, when When the HKP aqueous solution is mixed with siRNA or siRNA drug-based composition in a specific mass ratio (eg, 4:1), the nanoparticles will self-assemble.
  • the average diameter of the nanoparticles is in the range of 30-400 nm, and further preferably, the size of the nanoparticles is 50-150 nm.
  • the present invention also includes methods for preventing or treating influenza A virus infection using anti-influenza virus siRNA molecules and pharmaceutical compositions based on these siRAN molecules.
  • "treating” or “treating” refers to reducing the severity of or curing influenza A disease.
  • a therapeutically effective amount of the composition of the present invention is administered to a mammal.
  • the mammal is a human, rodent (eg, rat, mouse, or guinea pig), ferret, or non-human primate (eg, monkey).
  • the mammal is an experimental animal, such as a rodent.
  • the mammal is a non-human primate, such as a monkey.
  • the mammal is a human.
  • a "therapeutically effective amount" is an amount that prevents, reduces the severity of, or cures, influenza A infection.
  • a therapeutically effective amount of the pharmaceutical composition administered to a human comprises from about 0.1 mg of siRNA molecules per kilogram of human body weight to about 10 mg of siRNA molecules per kilogram of human body weight.
  • a therapeutically effective amount of the pharmaceutical composition administered to a human comprises from about 0.1 mg of the siRNA molecular composition per kilogram of human body weight to about 100 mg of the siRNA molecular composition per kilogram of human body weight.
  • the route of administration can be determined by one of skill in the art. These routes include intranasal administration, airway instillation, inhalation administration, eg, by use of aerosolized spray devices. In some embodiments, routes of administration also include injection instillation and intraperitoneal, intravenous, intradermal, intravaginal, and subcutaneous administration.
  • the nanopharmaceutical formulation is delivered to the virus-infected lower respiratory tract or lungs by inhalation administration or intravenous injection. Further preferably, the pharmaceutical formulation is introduced into the virally infected lower respiratory tract or lungs by aerosol inhalation administration.
  • Trehampati N Yas AK. Immunomodulation of T regulatory cells in hepatitis B virus-associated inflammation and cancer. Scandinavian Journal of Immunology. 2017;85:175-181.

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Abstract

提供了一种siRNA药物、药物组合物、siRNA-小分子药物偶联物及其应用。该siRNA分子通过靶向抑制流感病毒关键基因的表达而阻断病毒复制生命周期，降低病毒感染并最终清除病毒。基于该siRNA的药物，可以经由雾化装置雾化成液滴，以吸入方式给药到达下呼吸道和肺部，抑制流感病毒的复制，药物组合物能够与通过各自不同的作用机制发挥协同抗病毒效果。

Description

一种siRNA药物、药物组合物、siRNA-小分子药物偶联物及其应用 技术领域
本发明属于生物医药技术领域，具体涉及一种siRNA药物、药物组合物、siRNA-小分子药物偶联物及其应用。
背景技术
流感病毒属于正粘病毒科的单股负链RNA病毒 [1]，其基因组分为多个部分，宿主范围和致病性各不相同。有A、B和C型流感病毒(分别又称为甲、乙、丙型流感病毒)可以感染人类，其中甲型流感病毒的毒性最高。甲型流感病毒可感染多种禽类和哺乳动物宿主，而乙型流感病毒几乎只能感染人类。由于甲型流感病毒引起大流行，因此引起了人们的广泛关注。流感病毒的结构包含三个部分：核心蛋白，包膜蛋白和基质蛋白。这些蛋白是血凝素(HA)，神经氨酸酶(NA)，基质蛋白1(M1)，质子通道蛋白(M2)，核蛋白(NP)，RNA聚合酶(PA，PB1和PB2)，非结构蛋白1(NS1)和核输出蛋白(NEP，NS2)。此外，在某些菌株中发现了一些蛋白质(例如PB1-F2、PB1-N40和PA-X) [2]。甲型流感病毒按HA和NA亚型进一步分类，HA有18个亚型，NA有11个亚型。例如，H1N1和H3N2是人类流感病毒，而H5N1和H7N9是禽流感病毒。HA和NA在季节性流感中经常发生点突变(抗原漂移)，人类和禽类病毒之间的基因重排(抗原转移)可能会引起大流行 [3]。
流感是一个长期影响人类健康的严重问题，每年流感病毒感染数以百万计的人类，且导致全球25-50万人死亡 [4]。尽管已有疫苗和抗病毒药，但流感仍对健康、经济和社会造成严重影响。因为病毒不断进化，目前的疫苗只能提供有限的流感防护。当前，在流行的病毒中存在对金刚烷类药物的广泛耐药性，在大多数国家，神经氨酸酶(NA)抑制剂(NAI)成为唯一可用的有效抗病毒药。但是，NAI也并非流感病毒的完美解决方案，例如在2008-2009年全球流通的季节性流感A(H1N1)病毒就对奥司他韦有耐药性，而且毒副作用显著 [5]。无论如何，流感病毒感染仍然是一个对人类健康和社会的威胁。因此，具有不同作用机理的新型抗病毒药物的开发和临床应用至关重要。
目前，已上市的抗甲型流感病毒感染的药物包括：病毒神经氨酸酶(NA)抑制剂Relenza(扎那米韦)、Tamiflu(磷酸奥司他韦)、Inavir(拉尼米韦辛酸酯)和Rapivab(帕拉米韦)；M2离子通道阻滞剂金刚烷胺(Amantadine)和金刚乙胺(Rimantadine)；病毒聚合酶抑制剂法匹拉韦(Favipiravir)；广谱抗病毒药物利巴韦林(Ribavirin)和阿比多尔(Arbidol)等。但是，上述药物分别存在各种局限性。例如，病毒神经氨酸酶抑制剂依然面临一些问题，例如口服有效性、耐药性以及诱导细胞因子风暴。金刚烷类药物的长期、广泛、大量使用，导致多数甲型流感病毒产生了严重的耐药性 [6]。而法匹拉韦的不良反应数据不完善，且同样存在耐药性的问题 [7]。
在各种可用于治疗流感感染的抗病毒药物中，最常用的药物之一是奥司他韦(Tamiflu) [8]。尽管奥司他韦作为神经氨酸酶抑制剂的作用机理已广为人知，但奥司他韦对人类流感病毒动力学的影响一直存在争议。罗氏(Roche)等制药公司已经进行了奥司他韦的许多临床试验，但是直到最近，虽然可以得到一些早期临床试验的数据，但这些数据是作为编辑过的报告的pdf扫描给出的，而不是作为实际的原始数据提供的，因此无法由其他研究者进行更详细的分析。通常，此类报告包括安慰剂和药物治疗的流感病毒感染的中值病毒脱落曲线，通常表明早期治疗具有很高的疗效。但是，中值脱落曲线可能无法准确地代表个体对药物的影响。
在过去的流感研究中，较老的PB1转录酶抑制剂利巴韦林已通过口服、气雾剂或静脉内给药，但尚未显示出令人信服的临床疗效 [9]。在一项双盲随机对照试验(RCT)中，对口服金刚烷胺、利巴韦林和奥司他韦的组合(称为三联抗病毒药物或TCAD)进行了测试，在临床前模型(包括使用病毒的模型)中，其三联抗病毒药物的疗效优于单药或双药，包括对耐金刚烷胺的毒株也是如此。在症状发作后5天内出现流感并发症风险较高的门诊患者，每天两次(BID)随机分配至TCAD(口服奥司他韦75mg，金刚烷胺100mg 和利巴韦林600mg)或奥司他韦 [10]。在394名经证实流感病毒感染的病人中，TCAD与奥司他韦单药相比具有显着更大的抗病毒作用(在第3天，TCAD治疗者中40.0％的人能检测到病毒RNA，而奥司他韦单独治疗者中50.0％的人可检测到病毒RNA)，但也有某些疾病的效果并不是很好，可能与TCAD方案的副作用有关，TCAD组发生严重的不良事件和住院的比例更高。因此，与单独使用奥司他韦相比，在流感并发症风险增加的门诊患者中，这种三联药物治疗方案未能改善临床疗效。
法匹拉韦(T-705)最早由日本研发，并于2014年3月24日在日本获准用于流感大流行防范。由于法匹拉韦增加了致畸性和胚胎毒性的风险，因此只是获得了有条件的销售许可，并对其生产和临床使用制定了严格的规定 [11]。因此，法匹拉韦仅适用于其他流感抗病毒药无效或不够有效的新型或复发性大流行性流感病毒感染的患者。在其他国家/地区，法匹拉韦仍处于临床研究阶段。法匹拉韦在受感染的细胞中发生磷酸化，转变成其活性形式，被RNA依赖性RNA聚合酶(RdRP)识别为嘌呤类似物，并作为鸟苷和腺苷类似物有效地整合到新生成的RNA链中 [12]。
核酸干扰(RNAi)技术首先在线虫中被发现，当时发现与特定基因互补的双链RNA(dsRNA)在沉默相应基因表达方面比单独一条链更有效 [13]。随后的研究表明，沉默效应与21-23bp的小核苷酸双链体干扰RNA(siRNA)及dsRNA特异性内切核糖核酸酶III(称为Dicer)有关，后者负责将dsRNA切割成siRNA，触发RNAi沉默机制。具体而言，siRNA与特定蛋白/酶结合形成RNA诱导沉默复合物(RISC)，siRNA中的正义链脱离，反义链靶向序列特异性的mRNA并与之结合 [14]。随后，RISC中的酶将靶mRNA从siRNA链的3'末端切割约12个核苷酸，从而导致mRNA降解，达到基因沉默的目的。
RNAi技术已经成为抗病毒感染的利器，相对于小分子化学药物，siRNA具有许多优势 [15]。首先，可以快速合成siRNA“药物”并扩大规模以进行生产。其次，在对一种siRNA产生病毒抗性的情况下，可以使用靶向另一种病毒序列的不同siRNA，甚至可以同时使用两种以上针对流感病毒不同基因的siRNA分子。第三，无论siRNA序列如何，所有siRNA都使用相同的合成化学方法，因此使用相同的制造工艺，非常容易将两种siRNA组合在一起使用。此外，与许多具有药理活性的有机化合物不同，siRNA是水溶性的，对药物的利用非常有利。
采用siRNA来对抗甲型流感病毒感染也显示了良好的效果。在一项采用MDCK细胞的研究中，观察到siRNA对H1N1病毒具有明显的抑制作用，特异性siRNA可以抑制大约50％流感病毒mRNA的表达，噬菌斑试验表明，siRNA可以将流感病毒滴度降低至1/200 [16]。研究人员将设计的19bp的siRNA分子长度延长到27bp，表明增加siRNA的长度可将H1N1和H3N2多重菌株抑制60％以上，并且在48h时抑制作用最为明显 [17]。siRNA也可以抑制流感病毒在动物体内的增殖，提高对动物的保护率。一项研究向小鼠静脉内注射siRNA，H1N1攻击后病毒滴度降低。然后，在向小鼠单次注射一种siRNA或两种siRNA后18天，单个siRNA的保护率为80％-90％，而两种siRNA治疗的小鼠存活率达100％ [18]。另一项研究将siRNA注入鼻腔并同时静脉内注入小鼠，结果发现mRNA和蛋白质被抑制了90％以上，并且炎症因子也显着减少 [19]。
然而众所周知，流感病毒具有快速变异的能力，很容易通过突变产生耐药性。流感病毒抗原多变性的机制有两种：(1)病毒HA和NA基因容易发生突变抗原漂移，导致形成新的抗原(从而避免了先前存在的宿主免疫力)，造成漂移的主要原因是病毒聚合酶容易出错；(2)在同一宿主内的两种不同流感病毒之间基因片段重排引起抗原转移，产生了新的病毒株。有研究认为，1918年的甲型H1N1流感大流行是由于人流感病毒和禽流感病毒株之间的重组病毒引起的；同样，近些年不断发生的“猪流感”甲型H1N1流感大流行也是由于人甲型流感H3N2、猪流感H1N1和禽流感H1N2之间的一系列重组事件所致 [20]。
由于流感病毒的快速突变能力对药物产生耐药性，因此采用两种以上的药物联合治疗成为一种有效的应对流感病毒的方式。在体外细胞 [21]和小鼠实验 [22-23]已证明法匹拉韦和NA抑制剂的协同作用。一项法维拉韦的IIa期临床试验表明，法维拉韦和奥司他韦的联合治疗可能会加速18岁及以上患有严重流感的住院患者的临床康复 [24]。但是，各种小分子化合物之间的联合使用，由于药物之间的结构、作用机制、生物利用度、半衰期等有较多的相似之处，导致这些药物组合使用并不能达到非常显著的协同效果。
发明内容
本发明的目的是提供一种高效特异性抑制流感病毒复制的siRNA分子及新的用于预防和治疗流感病毒感染的siRNA药物、药物组合物、siRNA-小分子药物偶联物。
为达到上述目的，本发明采用的技术方案是：
本发明第一方面提供一种抑制流感病毒复制的siRNA分子，所述siRNA分子包括正义链和反义链，所述正义链的序列选自SEQ ID No.1～16、SEQ ID No.20～54、SEQ ID No.56～69、SEQ ID No.71～91、SEQ ID No.93、SEQ ID No.94中的任意一条，所述反义链选自SEQ ID No.98～113、SEQ ID No.117～151、SEQ ID No.153～166、SEQ ID No.168～188、SEQ ID No.190、SEQ ID No.191中与所述正义链互补的一条。
本发明第二方面提供一种用于预防或治疗流感病毒感染的siRNA药物，所述siRNA药物包括活性成分，所述活性成分为权利要求1所述的siRNA分子中的一种或多种。
优选地，所述活性成分还包括一种或多种其他抑制流感病毒复制的siRNA分子。
进一步优选地，所述其他抑制流感病毒复制的siRNA分子的正义链的序列选自SEQ ID No.17～19、SEQ ID No.55、SEQ ID No.70、SEQ ID No.92、SEQ ID No.95～97中的任意一条，所述其他抑制流感病毒复制的siRNA分子的反义链选自SEQ ID No.114～116、SEQ ID No.152、SEQ ID No.167、SEQ ID No.189、SEQ ID No.192～194中与所述其他抑制流感病毒复制的siRNA分子的正义链互补的一条。
本发明第三方面提供一种用于预防或治疗流感病毒感染的药物组合物，所述药物组合物的活性成分包括抑制流感病毒复制的siRNA分子和另一种分子，所述另一种分子包括抑制PD-1表达的siRNA分子、抑制PD-L 1表达的siRNA分子、抗流感病毒小分子化合物、流感mRNA疫苗、或抗流感病毒单克隆抗体中的一种或多种。
优选地，所述抑制流感病毒复制的siRNA分子针对甲型流感病毒不同毒株之间的保守基因序列设计，所述甲型流感病毒包括H1N1、H5N1、H7N9、或H3N2中的一种或多种亚型；所述抑制流感病毒复制的siRNA分子通过靶向抑制流感病毒与侵入、复制、组装或释放相关的关键基因的表达而阻断病毒复制生命周期，降低病毒效价，抑制感染直至彻底清除病毒。
优选地，所述抑制流感病毒复制的siRNA分子选自以下siRNA分子中的一种或多种：选自SEQ ID No.1～97中的任意一条，反义链选自SEQ ID No.98～194中与正义链互补的一条。
优选地，所述抑制PD-1表达的siRNA分子是根据人PD-1基因和小鼠PD-1基因之间的同源序列设计的，所述抑制PD-L1表达的siRNA分子是根据人PD-L1基因和小鼠PD-L1基因之间的同源序列设计的。
进一步优选地，所述同源序列是指人和小鼠的两个基因通过比对之后确认序列100％相同的DNA序列。
针对PD-L1的siRNA分子是特异性抑制人程序性死亡因子1(PD1)配体1(PD-L1)表达的小干扰核苷酸。所述PD-L1对机体免疫系统具有显著影响，可以抑制病毒感染过程中的T细胞功能，或者导致T细胞耗竭。
根据一些实施方式，所述抑制PD-1表达的siRNA分子选自以下siRNA分子中的一种或多种：正义链的序列选自SEQ ID No.195～206中的任意一条，反义链选自SEQ ID No.207～218中与正义链互补的一条。
根据一些实施方式，所述抑制PD-L1表达的siRNA选自以下siRNA分子中的一种或多种：正义链的序列选自SEQ ID No.219～230中的任意一条，反义链选自SEQ ID No.231～242中与正义链互补的一条。
优选地，所述的流感mRNA疫苗是根据流感病毒基因序列设计的信使核糖核酸疫苗。
进一步优选地，所述流感病毒基因为编码病毒结构蛋白的基因和/或编码非结构蛋的基因，进一步优选地，所述编码病毒结构蛋白的基因选自PB2、PB1、PA、HA、NP、NA、M1、或M2中的一种或多种，所述编码非结构蛋白基因为NS1和/或NS2。
优选地，所述mRNA疫苗，除了包含特定的病毒基因序列，还含有在细胞内翻译所必需的元件。
进一步优选地，所述元件包括但不限于两端的非翻译区(UTR)、3’端的帽子结构和5’端的polyA尾巴。
优选地，所述抗流感病毒小分子化合物为特异性的流感病毒抑制剂和/或广谱抗病毒小分子化合物。
进一步优选地，所述特异性的流感病毒抑制剂选自M2离子通道阻滞剂、NA(神经氨酸酶)抑制剂、 PA(聚合美PA亚单位)抑制剂和PB2(聚合美PB2亚单位)抑制剂中的一种或多种。
进一步优选地，所述广谱抗病毒小分子化合物选自利巴韦林、硝唑尼特、盐酸阿比多尔、法匹拉韦中的一种或多种。
优选地，所述小分子化合物为水溶性化合物。
进一步优选地，所述小分子化合物溶解在水溶液后具有较好的稳定性，经过雾化后依然可以保持一定的活性。
本发明第四方面提供一种siRNA-小分子药物偶联物，所述siRNA-小分子药物偶联物由抑制流感病毒复制的siRNA分子与抗流感小分子药物通过共价键偶联形成。
优选地，所述小分子化合物为含有核苷酸基础结构的小分子。
优选地，所述化学键为共价键、离子键或金属键。
优选地，所述抑制流感病毒复制的siRNA分子针对甲型流感病毒不同毒株之间的保守基因序列设计，所述甲型流感病毒包括H1N1、H5N1、H7N9、或H3N2中的一种或多种亚型；所述抑制流感病毒复制的siRNA分子通过靶向抑制流感病毒与侵入、复制、组装或释放相关的关键基因的表达而阻断病毒复制生命周期，降低病毒效价，抑制感染直至彻底清除病毒。
优选地，所述抑制流感病毒复制的siRNA分子选自SEQ ID No.1～97中的任意一条，反义链选自SEQ ID No.98～194中与正义链互补的一条。
优选地，所述抗流感病毒小分子化合物为特异性的流感病毒抑制剂和/或广谱抗病毒小分子化合物。
进一步优选地，所述特异性的流感病毒抑制剂选自M2离子通道阻滞剂、NA抑制剂、PA抑制剂和PB2抑制剂中的一种或多种。
进一步优选地，所述广谱抗病毒小分子化合物选自利巴韦林、硝唑尼特、盐酸阿比多尔、法匹拉韦中的一种或多种。
优选地，所述抑制流感病毒复制的siRNA分子与所述抗流感病毒小分子化合物通过各自的活性基团连接，或通过在所述抑制流感病毒复制的siRNA分子中引入Linker，利用Linker的活性基团与所述抗流感病毒小分子化合物偶联。
优选地，所述活性基团包括氨基、羧基、羟基、磷酸基、环氧基、醛基、异氰酸酯基中的一种或多种。
本发明第五方面提供一种所述的siRNA-小分子药物偶联物在制备用于预防或治疗流感病毒感染的药物中的应用。
优选地，所述的用于预防或治疗流感病毒感染的siRNA药物、所述的用于预防或治疗流感病毒感染的药物组合物、或所述的siRNA-小分子药物偶联物与药学上可接受的载体形成制剂，所述药学上可接受的载体选自盐水、糖、多肽、高分子聚合物、脂质、乳膏、凝胶、胶束材料、或金属纳米颗粒中的一种或多种。
进一步优选地，所述高分子聚合物为所述多肽类高分子聚合物。
再进一步优选地，所述多肽类高分子聚合物是由组氨酸和赖氨酸组成的阳离子多肽。
根据一些具体实施方式，所述多肽类高分子聚合物为HKP(H3K4b)和/或HKP(+H)分支状多肽。
再进一步优选地，所述siRNA药物、或所述药物组合物、或所述的siRNA-小分子药物偶联物与所述药学上可接受的载体形成纳米制剂；所述纳米制剂为口服剂、注射剂、或雾化吸入剂。
根据一些具体实施方式，所述纳米制剂的剂型为雾化吸入制剂，所述制剂通过静脉注射、口服、皮下注射、肌肉注射、雾化吸入、鼻内等方式输送到疾病不为，发挥对病毒的抑制效果。
优选地，所述纳米制剂通过超声雾化给药装置雾化后，通过吸入方式将药物输送到下呼吸道和肺部，抑制流感病毒复制。
本发明所述的用于预防或治疗流感病毒感染的siRNA药物、或所述的用于预防或治疗流感病毒感染的药物组合物、或所述的应用，针对的流感病毒为G4EA H1N1病毒株、H1N1病毒株、H5N1病毒株、H7N9病毒株、或H3N2病毒株中的一种或多种。、
本发明以抑制流感病毒复制的siRNA分子为基础，将siRNA分子与其他类型的抗流感病毒药物联合， 这种组合策略旨在为流感病毒感染的治疗提供有效的和互补的策略，而这种治疗策略比单独的每种疗法会更有效。通过将对每种经过证实对流感病毒具有显著抑制效果的siRNA与已经上市或经过临床验证的抗流感病毒小分子药物联合使用，并评估其在细胞、啮齿类动物、非人类灵长类动物中对流感病毒等感染模型中的广谱功效。
本发明中所述的药物组合物，可以是将抑制流感病毒复制的siRNA分子和另一种分子以特定比例组合，形成混合溶液后以相同的方式给药。所述特定比例，是根据两种分子发挥药效所需要的浓度，特别是血药浓度而确定的。所述抑制流感病毒复制的siRNA分子，根据临床前研究的数据结果确定药物浓度。所述另一种分子，根据临床前、临床实验、临床应用的数据确定药物浓度。所述特定比例混合的组合物，还考虑药物之间的协同作用和相互作用。
本发明中所述的药物组合物，可以是抑制流感病毒复制的siRNA分子作为一种单独的药物溶液，另一种分子作为另一种单独的药物溶液，两种药物溶液作为组合使用。所述两种单独的药物溶液，采用相同或相似的溶剂溶解。所述两种单独的药物溶液，采用不同的溶剂溶解。所述两种单独的药物溶液组成的组合物，可以同时给药，也可以不同时间给药。进一步优选地，两种单独的药物溶液在几乎相同的时间先后给药，或在不同的时间穿插给药。
本发明中，所述的siRNA药物分子、所述的药物组合物、所述的siRNA-小分子药物偶联还可以与一种药学上可接受的纳米导入载体偶联物形成纳米药物。所述纳米药物载体与所述各种分子通过静电相互作用、氢键、范德华力结合，形成稳定的非偶联的纳米聚合物。所述纳米导入载体可以同时包裹抑制流感病毒复制的siRNA分子和另一种分子，也单独分别包裹两种分子。优选地，所述纳米导入载体同时包裹两种分子形成粒度均匀的纳米药物颗粒。所述单独分别包裹两种分子，可以使用完全相同的纳米导入载体分别包裹两种分子，形成粒径相同或不同的纳米颗粒，也可以使用不同的纳米导入载体分别包裹两种分子，形成粒径相同或不同的纳米颗粒。
所述纳米药物颗粒，是一种在特定溶剂中以颗粒状形式稳定悬浮的聚合物，其直径大小从几纳米到几百甚至上千纳米。优选地，所述纳米颗粒直径为30-300纳米，进一步优选地，所述纳米颗粒大小为50-150纳米。所述纳米药物可以通过雾化吸入、静脉注射、皮下注射、肌肉注射、口服等方式给药。优选地，可以通过超声雾化装置雾化成液滴，以吸入方式给药到达下呼吸道和肺部，抑制流感病毒的复制。所述两种分子单独制备纳米颗粒时，两种纳米颗粒可以通过相同的方式给药，也可以通过不同的方式给药。
由于上述技术方案运用，本发明与现有技术相比具有下列优点：
本发明提供了高效特异性抑制流感病毒复制的siRNA分子，对流感病毒具有显著抑制效果，为制备用于预防或治疗流感病毒感染的药物提供更多选择，以抑制流感病毒复制的siRNA分子为基础，能够制备更多新的用于预防和治疗流感病毒感染的siRNA药物，或与其他类型的抗流感病毒药物联合，制备基于siRNA分子的药物组合物以及siRNA-小分子药物偶联物，为流感病毒感染的治疗提供有效的和互补的策略，在细胞、啮齿类动物、非人类灵长类动物中对流感病毒等感染模型中的广谱功效。
附图说明
附图1.抗流感病毒siRNA分子可以在体外细胞实验中有效抑制病毒的复制。A显示了不同处理后细胞上清内H5N1流感病毒血凝素(HA)的效价，B为不同处理后细胞上清内H5N1病毒的TCID 50(半数组织培养感染剂量)，其中M1-1的siRNA序列(80nM)对H5N1流感病毒抑制效果最佳。C为不同处理后细胞上清内H1N1流感病毒血凝素(HA)的效价，D为不同处理后细胞上清内H7N9病毒的TCID 50(半数组织培养感染剂量)。可以看出，M1-1、PA-19、NP-15等siRNA分子可以有效抑制H1N1病毒感染，M1-1的siRNA序列(80nM)对H7N9有显著的抑制效果。
附图2.抗流感病毒siRNA分子减轻流感病毒感染引起的小鼠死亡。A显示了动物分组、给药剂量及给药方式，B图显示为各组小鼠的存活曲线。从图中可以看出，中剂量的M1-1siRNA静脉注射给药组对病毒有很好的抑制效果，第15天小鼠的存活率(70％)显著高于阴性siRNA对照组，也优于达菲(图中的奥司他韦)治疗组。
附图3.siRNA转染后不同细胞中PD-1或PD-L1基因的表达率。A和B分别显示了针对PD-1和PD-L1基因设计的siRNA转染MCF-7(乳腺癌)、BxPC3(胰腺癌)和HepG2(肝癌)细胞后，靶标基因PD-1和PD-L1的表达率。
附图4.采用siRNA抑制PD-1表达可激活免疫细胞分泌细胞因子。针对PD-1的siRNA，转染小鼠RAW264.7巨噬细胞后，细胞培养上清中TNF-α的浓度显著升高(见图4A)，细胞裂解液中TNF-α的浓度也升高(无统计显著性差异，见图4B)。
附图5.抗流感病毒siRNA和mRNA疫苗联合对甲型病毒感染的示意图。采用mRNA疫苗，可以在机体内表达成流感病毒特定蛋白/多肽片段，激活抗流感病毒感染的免疫机制，降低病毒感染几率。同时，通过siRNA来抑制病毒在细胞内的复制阻断病毒的生命周期。
附图6.扎那米韦与siRNA分子的偶联。过磷酸基和羟基缩合成磷酸酯的方式、或者通过Linker的方式把扎那米韦连接到siRNA的末端。
附图7.帕拉米韦与siRNA分子的偶联。帕拉米韦的羧基可通过Linker与siRNA连接。
附图8.奥司他韦与siRNA分子的偶联。通过Linker一端的羟基与siRNA末端的磷酸基反应，另一端的环氧基与奥司他韦的氨基发生亲核反应。
附图9.A-192558和A-315675与siRNA分子的偶联。A-192558含有可改性的氨基和羧基，再通过Linker的一端的环氧基与氨基反应，另一端的羟基与siRNA的磷酸基反应；也可以利用卤化氢与碳碳双键加成，再通过取代反应与Linker连接。
附图10.雾化吸入(短持续短间隔)给药可以将siRNA分子有效导入肺部。A显示了雾化吸入siRNA分子完成后在肺部的分布情况，B显示siRNA分子对肺部靶标基因的抑制效果。
附图11.雾化吸入(长持续长间隔)给药可以将siRNA分子有效导入肺部。A显示了雾化吸入siRNA分子完成时在肺部的分布情况，B显示雾化吸入24小时后siRNA分子完成时在肺部的分布情况。
附图12.siRNA的雾化吸入给药无显著毒副作用。A，雾化吸入给药对小鼠体重无显著影响；B，雾化给药后肺部白介素6(IL-6)无显著变化；C，雾化给药后肺部TNF-α无显著变化。
具体实施例
实施例1、高效抑制病毒感染的siRNA分子
MDCK用含10％MEM的培养基培养，按1:1的比例扩增培养，保持细胞活力。转染前12-18小时将MDCK细胞按照2.4×10 5个细胞/孔加入24孔板内，待细胞密度达80％左右时进行转染。转染按照Lipofectamine 2000脂质体转染试剂(Life Technologies)说明书转染各种siRNA。奥司他韦对照药物直接加入培养基内，终浓度为125μM。转染(或加入奥司他韦)后24小时，先对细胞培养板用PBS洗3次，添加400μL/孔的OPTI-MEM。优化病毒感染剂量后(选定MOI＝0.01)，用OPTI-MEM对病毒进行稀释，100μL/孔接毒。置于细胞培养箱内吸附1h，其中每隔15min轻轻晃动细胞板。1小时后吸弃病毒液，用PBS洗净后换成每孔内1mL含0.5％抗生素的OPTI-MEM，置于37℃细胞培养箱中培养3天。接毒后的48小时收取细胞上清200μL/孔，用于HA效价和TCID 50(半数组织培养感染剂量)的测定。HA效价测定按常规方法实行。TCID 50测定方法如下：将每个样品用1％MEM稀释8个稀释度，每个稀释度接种4孔。弃96孔板废液，用PBS洗3次，按100μL/孔的量将病毒液接入96孔板内，晃匀后将板子放入37℃的细胞培养箱培养3天，观察细胞病变，统计计算TCID 50。结果如图1所示，本发明专利所述的不同剂量的siRNA分子对H1N1、H5N1、H7N9等甲型流感病毒亚型都有不同程度的抑制效果，其中以M1-1的抑制效果最好，与奥司他韦接近。
在小鼠体内动物实验中，采用静脉注射和呼吸道雾化给药两种方式对抗流感的候选药物进行有效性评估(图2A)。采用高致病性禽流感H5N1毒株进行攻毒(30个LD 50)，确保“病毒感染组”小鼠能够大部分甚至全部死亡。结果表明(图2B)，静脉注射中剂量或低剂量的M1-1，存活率(分别为70％和60％)显著高于静脉注射阴性对照组(NC)和病毒感染组的存活率(分比为37.5％和20％)。由此可见，本发明所述的抗流感病毒siRNA分子可以显著抑制多种甲型流感病毒在体内外的感染和复制过程。
实施例2、靶向抑制PD-1和PD-L1基因表达的siRNA分子的筛选与验证
针对PD-1和PD-L1基因，选择人鼠同源的序列设计siRNA分子后，委托合成这些siRNA分子(苏州贝信)，然后采用人的肿瘤细胞系MCF-1、BxPC3和HepG2确定这些siRNA分子对PD-1或PD-L1表达的抑制效果。特定量的细胞接种6孔板或12孔板培养6小时以上使细胞贴壁，然后采用Lipofectamine 2000脂质体转染试剂，根据操作说明书转染各种siRNA，转然后培养48小时，提取细胞的总RNA(组织/细胞RNA快速提取试剂盒，北京聚合美)后，用微量紫外分光光度计(MicroDrop，Bio-DEL)测定浓度，取100-500ng的总RNA进行逆转录(第一链cDNA反转录试剂盒，北京聚合美)。最后，采用荧光定量PCR扩增试剂盒(Realtime PCR Super mix(SYBRgreen,with anti-Taq)，北京聚合美)，在荧光PCR仪(QuantStudio 3，ABI)上分析检测。结果如图3所示，针对PD-1或PD-L1设计的siRNA，可以不同程度的抑制相应靶标基因的表达。据此，可以选定针对每个靶标基因的最有效率的siRNA分子。
为了分析PD-1的免疫激活作用，选取PD-1的一种siRNA分子(PD-1-10)转染到小鼠RAW264.7巨噬细胞系中，培养28小时后测定细胞培养上清和细胞裂解液(蛋白裂解液)中肿瘤坏死因子-α(TNF-α)的浓度。结果如图4所示，PD-1处理过的小鼠巨噬细胞培养上清中，TNF-α浓度显著升高(图4A)，而蛋白裂解液中的TNF-α含量也有所提高(无统计显著性差异，图4B)。由此可见，采用siRNA抑制PD-1表达后，可以激活免疫细胞功能。在哺乳动物体内用抗流感病毒siRNA抑制病毒感染的同时，采用PD-1的siRNA来激活机体免疫系统，可以有效加强抗病毒的免疫能力，更有效地清除病毒。
实施例3、采用siRNA分子和mRNA疫苗联合抑制流感病毒
不少研究已经证实siRNA是一种高效的抗病毒手段，而mRNA疫苗已经经过临床应用证实可以有效保护人体免受新型冠状病毒(SARS-CoV-2)的感染，本发明将抗流感病毒siRNA分子与流感mRNA疫苗联合使用，以此达到高效特异性清除病原体的目的。在本实施例中，如图5所示，一种含有帽子结构、5’-非翻译区、开放阅读框(ORF)、3’-非翻译区和多聚腺苷酸(PolyA)尾巴的mRNA疫苗，其中ORF为编码流感病毒特定蛋白的基因序列。将mRNA与HKP多肽纳米导入系统以特定比例混合，制备成纳米颗粒，通过肌肉注射到哺乳动物体内后，mRNA翻译成蛋白质，分泌到胞外后被抗原递呈细胞(APC)识别，刺激机体免疫系统，形成抗病毒免疫保护机制。
在给予mRNA治疗的同时或者之后特定的时间，给予哺乳动物有效剂量的siRNA纳米药物制剂，抗病毒siRNA分子进入机体细胞内后，与特定的酶/蛋白质形成RNA诱导沉默复合物(RISC)，正义链解离后，反义链携带整个复合物与病毒RNA结合，通过RNAi作用机制将病毒RNA降解，从而阻止病毒特定基因表达成蛋白质/酶，病毒无法完成复制生命周期。通过mRNA疫苗与siRNA分子的联合应用，可以高效阻断流感病毒的感染和复制。
实施例4、抗流感病毒siRNA分子与抗流感药物的偶联
扎那米韦(Zanamvir)药物含有羟基、羧基和胍基活性基团，Zanamvir是通过胍基取代DANA中的C-4位的羟基得到的。其中，胍基可与流感病毒神经氨酸酶(NA)中S2区域的两个氨基酸Glu119、Glu227或Asp115结合，提高体外抑酶活性。所以胍基对于NA的抑制具有重要的作用，要尽可能保持胍基完整性。为此，本发明设计两种改性方式与抗病毒siRNA结合：一种是通过磷酸基和羟基缩合成磷酸酯的方式，把Zanamvir嵌段在siRNA分子中(图6，～～～[扎那米韦]～～～)；另一种方法是通过连接化合物(Linker)或直接把Zanamvir连接到siRNA的末端。本实施例的一方面，如siRNA的5’末端中的磷酸基可直接与Zanamvir的羟基偶联；本实施例的另一方面，引入Linker(如PEG)，一端与Zanamvir的羧基连接，另一端与siRNA的5’末端的磷酸基连接(图6，扎那米韦-siRNA)或3’末端的羟基连接。DANA和FANA结构式相似，可通过上述两种方法与siRNA结合，筛选出符合治疗效果的结合性物质。
帕拉米韦(Peramivir，RWJ-270201)结构式中，含有胍基、羟基和羧基活性基团。其中羟基可与siRNA的5’末端的磷酸基或3’末端的羟基缩合，保留的羧基可以与流感病毒NA中S1区的Arg292、Arg371和Arg118结合。羧基可通过Linker与siRNA连接，如图7中帕拉米韦RWJ-270201-siRNA，Linker一端的氨基与羧基反应生成酰胺键，另一端的羟基与磷酸基反应生成磷酸酯；此外，Linker中可含有二硫键等响应性基团， 在特定情况下裂解，药物与siRNA两者产生协同作用，提高治疗效果。而其药物的环戊烷衍生物和环戊烷酰胺衍生物，保留胍基的情况下，可通过上述方法偶联siRNA。
奥司他韦(Oseltamivir)的氨基是主要的功能基团及活性基团。氨基可与环氧基、醛基、异氰酸酯基、羧基反应。如图8所示，奥司他韦-siRNA，Linker一端的羟基与siRNA末端的磷酸基反应，另一端的环氧基与Oseltamivir的氨基发生亲核反应，其中，仲胺的碱性虽不及伯胺，但仍可与流感病毒NA蛋白的S2区域的Glu119、Glu227或Asp115的羧基结合。
A-192558结构中，可改性的活性基团为氨基和羧基。利用氨基改性，Linker一端的环氧基与氨基反应，另一端的羟基与siRNA的磷酸基反应(如图9，A-192558-siRNA)，保留的羧基可与NA中S1区的Arg292、Arg371和Arg118的胍基结合。如利用羧基偶联，则保留的氨基与NA中S2区域的Glu119、Glu227或Asp115的羧基结合。另一种抗流感小分子药物A-315675结构中，可改性的基团有碳碳双键和羧基，而碳碳双键可与水、卤素等发生加成反应，也可与碳碳双键、碳碳三键等烯烃及炔烃物质发生聚合反应。如图9中的A-315675-siRNA，利用卤化氢与碳碳双键加成，再通过取代反应与Linker连接。
实施例5、抗流感病毒siRNA分子与mRNA疫苗的多肽纳米药物制剂
本发明采用聚合物、特别是组氨酸-赖氨酸共聚物(HKP)来包裹核酸药物分子，包括siRNA和mRNA，制备纳米药物颗粒。在本实施例的一个方面，HKP与siRNA分子形成纳米颗粒，其中纳米颗粒的直径为约30nm至约300nm。其中所述HKP为H3K(+H)4b，包含结构(R)K(R))-K(R)-(R)K(X)，其中R＝KHHHKHHHKHHHHKHHHK，K＝赖氨酸，和H＝组氨酸。HKP和siRNA分子自组装成纳米颗粒或可以配制成纳米颗粒。在本实施例的另一个方面，HKP与mRNA疫苗分子形成纳米颗粒，其中纳米颗粒的直径为约30nm至约400nm。所述HKP为H3K(+H)4b，可以和mRNA分子自组装成纳米颗粒或可以配制成纳米颗粒。在本实施例的另一个方面，可以采用HKP同时包裹siRNA和mRNA分子，形成直径为30nm-400nm甚至更大的纳米颗粒。
采用HKP多肽包裹siRNA或/和mRNA形成纳米颗粒后，本发明建立了一系列的测定方法来表征纳米药物制剂的理化性质，包括粒径、表面电位、形态学研究、mRNA或siRNA的加载效率、生物学活性等。在本实施例的一个方面，采用纳米Zetasizer Nano ZS(英国马尔文仪器公司)来测定纳米药物制剂颗粒的大小和电位。在本实施例的另一个方面，采用实时定量荧光PCR方法测定siRNA对病毒靶标基因表达的抑制作用。在本实施例的另一个方面，将mRNA纳米药物处理细胞后，其所表达的蛋白或多肽通过RPHPLC使用分析柱C18(2S0mm x 2.1mm；Phenomenex)进行鉴定和定量。
实施例6、雾化吸入给药用于呼吸道病毒感染疾病防治
为了检测HKP多肽与siRNA构成的纳米药物制剂在雾化吸入给药治疗呼吸道/肺部疾病的发展潜力，采用雾化吸入的方式将针对特定靶标基因的siRNA(无标记或荧光标记)通过口鼻输送到呼吸系统内。将小鼠置于密闭的腔室内，将纳米药物制剂置于雾化杯内，雾化器喷雾口与腔体密闭连接后，开通电源雾化一定时间后，测定siRNA进入肺部的情况以及对靶标基因表达的抑制效率。
采用超声雾化器(ALC)或手持式雾化器(ZYM)对荧光标记的siRNA(AF647-siRNA，Qiagen)或者针对Cyclophilin-B的siRNA(苏州贝信)进行雾化，测定进入小鼠肺部的效率。在本实施例的一方面，采用持续时间短、间隔时间短的方式雾化给药，药物一次给完(2mL)，雾化：首先，将雾化室充满约
Figure PCTCN2022083042-appb-000001
Figure PCTCN2022083042-appb-000002
然后停止20秒，雾化10秒，停止20秒，雾化10秒，停止20秒。雾化和停止过程循环，直到全部雾化完成；雾化给药完成后，处死部分小鼠，分离肺，测定siRNA荧光；雾化给药后24小时，处死部分小鼠，分离肺，提取组织RNA，PCR测定Cyclophilin-B基因表达情况。如图10所示，药物可以有效达到肺部(富集在肺泡部位)，并且对靶标基因有显著的抑制效果，其中，图10A为采用250μg针对Cyclophilin-B的siRNA的实验结果。
在本实施例的另一方面，采用持续时间长、间隔时间长的方式雾化给药，给药一次(2mL，完全雾化)：首先，将雾化室填充约1分钟，然后停止1分钟，雾化1分钟，停止1分钟，雾化1分钟，停止1分钟。循环这一过程直到所有药液完成雾化；雾化给药完成时或者给药后24小时，处死部分小鼠，分离肺，测定 siRNA荧光。如图11所示，无论是雾化给药完成后马上检测(图11A)，还是给药后24小时检测(图11B)，都可以在肺部检测到siRNA，表明siRNA进入肺部后，可以持续在肺部一定时间，确保充分发挥作用，其中，图11为采用250μg针对Cyclophilin-B的siRNA的实验结果。
在本实施例的另一方面，测定了siRNA雾化吸入给药(手持式雾化吸入器ZYM)后对机体的不良影响。结果表明，雾化给药对小鼠小鼠体重无显著影响(图12A)；雾化给药后不同时间，测定肺部炎症因子情况，表明IL-6和TNF-a均未有显著变化(图12B-C)，其中，图12为采用针对Cyclophilin-B的siRNA的实验结果，高剂量组的siRNA的用量为500μg，低剂量组的siRNA的用量为250μg。由此可见，siRNA雾化吸入后无显著毒副作用，可以用于治疗各种呼吸道和肺部疾病，特别是诸如甲型流感病毒感染这类呼吸道病毒感染疾病。
本申请各实施例和附图中的阴性siRNA为一种不针对任何基因的siRNA。
上述实施例只为说明本发明的技术构思及特点，其目的在于让熟悉此项技术的人士能够了解本发明的内容并据以实施，并不能以此限制本发明的保护范围。凡根据本发明精神实质所作的等效变化或修饰，都应涵盖在本发明的保护范围之内。
发明内容详细说明
抗甲型流感病毒感染的siRNA分子
一种可以有效抑制甲型流感病毒的小干扰核苷酸(siRNA)分子，所述分子选自表1中的新的siRNA分子。所述抑制甲型流感病毒的siRNA分子，是针对多种亚型的甲型流感病毒的保守基因序列设计的分子，包括但不限于高度适应感染人类的“所有特征”的G4EA H1N1病毒株(A/swine/Hebei/0116/2017(H1N1)和A/swine/Jiangsu/J004/2018(H1N1)等)、H1N1病毒株(A/PuertoRico/8/1934和A/California/07/2009等)、H5N1病毒株(A/Vietnam/1194/2004等)、H7N9病毒株(A/Shanghai/CN02/2013等)和H3N2病毒株(A/Texas/50/2012等)等。所述siRNA分子针对上述病毒株的同源序列设计。所述siRNA分子长度为19-30个碱基对，优选的，所述siRNA分子长度为21个碱基对或者25个碱基对长度。所述siRNA分子的GC含量为30-70％，优选的，所述siRNA分子的GC含量为40-60％。
表1、广谱抑制甲型流感病毒的siRNA分子
siRNA名称 长度 正义链(5’-3’)SEQ ID No. 反义链(5’-3’)SEQ ID No.
PB2-1 19+dTdT GUGGACCAUAUGGCCAUAAdTdT 1 UUAUGGCCAUAUGGUCCACdTdT 98
PB2-2 19+dTdT CGGGACUCUAGCAUACUUAdTdT 2 UAAGUAUGCUAGAGUCCCGdTdT 99
PB2-3 19+dTdT GACUCUAGCAUACUUACUGdTdT 3 CAGUAAGUAUGCUAGAGUCdTdT 100
PB2-4 19+dTdT CUCUAGCAUACUUACUGACdTdT 4 GUCAGUAAGUAUGCUAGAGdTdT 101
PB2-5 19+dTdT CUAGCAUACUUACUGACAGdTdT 5 CUGUCAGUAAGUAUGCUAGdTdT 102
PB2-6 19+dTdT GCAUACUUACUGACAGCCAdTdT 6 UGGCUGUCAGUAAGUAUGCdTdT 103
PB2-7 19+dTdT CUUACUGACAGCCAGACAGdTdT 7 CUGUCUGGCUGUCAGUAAGdTdT 104
PB2-8 19+dTdT GCCAGACAGCGACCAAAAGdTdT 8 CUUUUGGUCGCUGUCUGGCdTdT 105
PB2-9 19+dTdT GCGACCAAAAGAAUUCGGAdTdT 9 UCCGAAUUCUUUUGGUCGCdTdT 106
PB2-10 19+dTdT GAAUUCGGAUGGCCAUCAAdTdT 10 UUGAUGGCCAUCCGAAUUCdTdT 107
PB2-11 25 GAAACGAAAACGGGACUCUAGCAUA 11 UAUGCUAGAGUCCCGUUUUCGUUUC 108
PB2-12 25 CGAAAACGGGACUCUAGCAUACUUA 12 UAAGUAUGCUAGAGUCCCGUUUUCG 109
PB2-13 25 GCAUACUUACUGACAGCCAGACAGC 13 GCUGUCUGGCUGUCAGUAAGUAUGC 110
PB2-14 25 CUUACUGACAGCCAGACAGCGACCA 14 UGGUCGCUGUCUGGCUGUCAGUAAG 111
PB2-15 25 GACAGCCAGACAGCGACCAAAAGAA 15 UUCUUUUGGUCGCUGUCUGGCUGUC 112
PB2-16 25 CCAAAAGAAUUCGGAUGGCCAUCAA 16 UUGAUGGCCAUCCGAAUUCUUUUGG 113
PB2-17 25 AUGGCCAUCCGAAUUCUUUUGGUCG 17 CGACCAAAAGAAUUCGGAUGGCCAU 114
PB2-18 25 UUGAUGGCCAUCCGAAUUCUUUUGG 18 CCAAAAGAAUUCGGAUGGCCAUCAA 115
PB2-19 19+dTdT GGGAACAGAUGUACACUCCdTdT 19 GGAGUGUACAUCUGUUCCCdTdT 116
PB1-1 19+dTdT CACAUUCCCUUAUACUGGAdTdT 20 UCCAGUAUAAGGGAAUGUGdTdT 117
PB1-2 19+dTdT CCUCCAUACAGCCAUGGAAdTdT 21 UUCCAUGGCUGUAUGGAGGdTdT 118
PB1-3 19+dTdT CAGCCAUGGAACAGGAACAdTdT 22 UGUUCCUGUUCCAUGGCUGdTdT 119
PB1-4 19+dTdT CCAUGGAACAGGAACAGGAdTdT 23 UCCUGUUCCUGUUCCAUGGdTdT 120
PB1-5 19+dTdT GAACAGGAACAGGAUACACdTdT 24 GUGUAUCCUGUUCCUGUUCdTdT 121
PB1-6 19+dTdT GAUGAUGGGCAUGUUCAACdTdT 25 GUUGAACAUGCCCAUCAUCdTdT 122
PB1-7 19+dTdT GUGGAGGCCAUGGUGUCUAdTdT 26 UAGACACCAUGGCCUCCACdTdT 123
PB1-8 19+dTdT GAGAUCAUGAAGAUCUGUUdTdT 27 AACAGAUCUUCAUGAUCUCdTdT 124
PB1-9 19+dTdT GAUCAUGAAGAUCUGUUCCdTdT 28 GGAACAGAUCUUCAUGAUCdTdT 125
PB1-10 19+dTdT CAUGAAGAUCUGUUCCACCdTdT 29 GGUGGAACAGAUCUUCAUGdTdT 126
PB1-11 19+dTdT GAAGAUCUGUUCCACCAUUdTdT 30 AAUGGUGGAACAGAUCUUCdTdT 127
PB1-12 25 CUUAUACUGGAGAUCCUCCAUACAG 31 CUGUAUGGAGGAUCUCCAGUAUAAG 128
PB1-13 25 CUGGAGAUCCUCCAUACAGCCAUGG 32 CCAUGGCUGUAUGGAGGAUCUCCAG 129
PB1-14 25 GCCAUGGAACAGGAACAGGAUACAC 33 GUGUAUCCUGUUCCUGUUCCAUGGC 130
PB1-15 25 CAUGGUGGAGGCCAUGGUGUCUAGG 34 CCUAGACACCAUGGCCUCCACCAUG 131
PB1-16 25 GAUCAUGAAGAUCUGUUCCACCAUU 35 AAUGGUGGAACAGAUCUUCAUGAUC 132
PB1-17 25 CAUGAAGAUCUGUUCCACCAUUGAA 36 UUCAAUGGUGGAACAGAUCUUCAUG 133
PA-1 19+dTdT GGGAUUCCUUUCGUCAGUCdTdT 37 GACUGACGAAAGGAAUCCCdTdT 134
PA-2 19+dTdT GAUUCCUUUCGUCAGUCCGdTdT 38 CGGACUGACGAAAGGAAUCdTdT 135
PA-3 19+dTdT GCCUGAUUAAUGAUCCCUGdTdT 39 CAGGGAUCAUUAAUCAGGCdTdT 136
PA-4 19+dTdT CUGAUUAAUGAUCCCUGGGdTdT 40 CCCAGGGAUCAUUAAUCAGdTdT 137
PA-5 19+dTdT GAUUAAUGAUCCCUGGGUUdTdT 41 AACCCAGGGAUCAUUAAUCdTdT 138
PA-6 19+dTdT GAUCCCUGGGUUUUGCUUAdTdT 42 UAAGCAAAACCCAGGGAUCdTdT 139
PA-7 19+dTdT CUGGGUUUUGCUUAAUGCAdTdT 43 UGCAUUAAGCAAAACCCAGdTdT 140
PA-8 19+dTdT CUUAAUGCAUCUUGGUUCAdTdT 44 UGAACCAAGAUGCAUUAAGdTdT 141
PA-9 19+dTdT GCAUCUUGGUUCAACUCCUdTdT 45 AGGAGUUGAACCAAGAUGCdTdT 142
PA-10 19+dTdT CUUGGUUCAACUCCUUCCUdTdT 46 AGGAAGGAGUUGAACCAAGdTdT 143
PA-11 19+dTdT GUUCAACUCCUUCCUCACAdTdT 47 UGUGAGGAAGGAGUUGAACdTdT 144
PA-12 19+dTdT CUCCUUCCUCACACAUGCAdTdT 48 UGCAUGUGUGAGGAAGGAGdTdT 145
PA-13 25 GAAAGAAUAUGGGGAAGAUCCGAAA 49 UUUCGGAUCUUCCCCAUAUUCUUUC 146
PA-14 25 GCCUGAUUAAUGAUCCCUGGGUUUU 50 AAAACCCAGGGAUCAUUAAUCAGGC 147
PA-15 25 CUUAAUGCAUCUUGGUUCAACUCCU 51 AGGAGUUGAACCAAGAUGCAUUAAG 148
PA-16 25 GCAUCUUGGUUCAACUCCUUCCUCA 52 UGAGGAAGGAGUUGAACCAAGAUGC 149
PA-17 25 CUUGGUUCAACUCCUUCCUCACACA 53 UGUGUGAGGAAGGAGUUGAACCAAG 150
PA-18 25 GUUCAACUCCUUCCUCACACAUGCA 54 UGCAUGUGUGAGGAAGGAGUUGAAC 151
PA-19 19+dTdT GCAAUUGAGGAGUGCCUGAdTdT 55 UCAGGCACUCCUCAAUUGCdTdT 152
NP-1 19+dTdT GAGUCUUCGAGCUCUCGGAdTdT 56 UCCGAGAGCUCGAAGACUCdTdT 153
NP-2 19+dTdT CUUCGAGCUCUCGGACGAAdTdT 57 UUCGUCCGAGAGCUCGAAGdTdT 154
NP-3 19+dTdT CGAGCUCUCGGACGAAAAGdTdT 58 CUUUUCGUCCGAGAGCUCGdTdT 155
NP-4 19+dTdT GCUCUCGGACGAAAAGGCAdTdT 59 UGCCUUUUCGUCCGAGAGCdTdT 156
NP-5 19+dTdT CUCGGACGAAAAGGCAACGdTdT 60 CGUUGCCUUUUCGUCCGAGdTdT 157
NP-6 19+dTdT CGGACGAAAAGGCAACGAAdTdT 61 UUCGUUGCCUUUUCGUCCGdTdT 158
NP-7 19+dTdT CGAAAAGGCAACGAACCCGdTdT 62 CGGGUUCGUUGCCUUUUCGdTdT 159
NP-8 19+dTdT GAAAAGGCAACGAACCCGAdTdT 63 UCGGGUUCGUUGCCUUUUCdTdT 160
NP-9 19+dTdT CCUUUGACAUGAGUAAUGAdTdT 64 UCAUUACUCAUGUCAAAGGdTdT 161
NP-10 19+dTdT CUUAUUUCUUCGGAGACAAdTdT 65 UUGUCUCCGAAGAAAUAAGdTdT 162
NP-11 25 GAGUCUUCGAGCUCUCGGACGAAAA 66 UUUUCGUCCGAGAGCUCGAAGACUC 163
NP-12 25 CGAAAAGGCAACGAACCCGAUCGUG 67 CACGAUCGGGUUCGUUGCCUUUUCG 164
NP-13 25 GAAAAGGCAACGAACCCGAUCGUGC 68 GCACGAUCGGGUUCGUUGCCUUUUC 165
NP-14 25 CUUAUUUCUUCGGAGACAAUGCAGA 69 UCUGCAUUGUCUCCGAAGAAAUAAG 166
NP-15 19+dTdT CUCCGAAGAAAUAAGAUCCdTdT 70 GGAUCUUAUUUCUUCGGAGdTdT 167
NA-1 19+dTdT GUCUUGGCCAGACGGUGCUdTdT 71 AGCACCGUCUGGCCAAGACdTdT 168
NA-2 19+dTdT GGCCAGACGGUGCUGAGUUdTdT 72 AACUCAGCACCGUCUGGCCdTdT 169
NA-3 19+dTdT GACGGUGCUGAGUUGCCAUdTdT 73 AUGGCAACUCAGCACCGUCdTdT 170
NA-4 25 GGCCAGACGGUGCUGAGUUGCCAUU 74 AAUGGCAACUCAGCACCGUCUGGCC 171
M-1 19+dTdT GUCUUCUAACCGAGGUCGAdTdT 75 UCGACCUCGGUUAGAAGACdTdT 172
M-2 19+dTdT CUUCUAACCGAGGUCGAAAdTdT 76 UUUCGACCUCGGUUAGAAGdTdT 173
M-3 19+dTdT CUAACCGAGGUCGAAACGUdTdT 77 ACGUUUCGACCUCGGUUAGdTdT 174
M-4 19+dTdT CCGAGGUCGAAACGUACGUdTdT 78 ACGUACGUUUCGACCUCGGdTdT 175
M-5 19+dTdT CGAGGUCGAAACGUACGUUdTdT 79 AACGUACGUUUCGACCUCGdTdT 176
M-6 19+dTdT CCCUCAAAGCCGAGAUCGCdTdT 80 GCGAUCUCGGCUUUGAGGGdTdT 177
M-7 19+dTdT GGCUAAAGACAAGACCAAUdTdT 81 AUUGGUCUUGUCUUUAGCCdTdT 178
M-8 19+dTdT CACGCUCACCGUGCCCAGUdTdT 82 ACUGGGCACGGUGAGCGUGdTdT 179
M-9 19+dTdT CGCUCACCGUGCCCAGUGAdTdT 83 UCACUGGGCACGGUGAGCGdTdT 180
M-10 19+dTdT CGUGCCCAGUGAGCGAGGAdTdT 84 UCCUCGCUCACUGGGCACGdTdT 181
M-11 19+dTdT CCAGUGAGCGAGGACUGCAdTdT 85 UGCAGUCCUCGCUCACUGGdTdT 182
M-12 19+dTdT GAGCGAGGACUGCAGCGUAdTdT 86 UACGCUGCAGUCCUCGCUCdTdT 183
M-13 19+dTdT GGACUGCAGCGUAGACGCUdTdT 87 AGCGUCUACGCUGCAGUCCdTdT 184
M-14 25 GUCUUCUAACCGAGGUCGAAACGUA 88 UACGUUUCGACCUCGGUUAGAAGAC 185
M-15 25 CACGCUCACCGUGCCCAGUGAGCGA 89 UCGCUCACUGGGCACGGUGAGCGUG 186
M-16 25 GCUCACCGUGCCCAGUGAGCGAGGA 90 UCCUCGCUCACUGGGCACGGUGAGC 187
M-17 25 CCGUGCCCAGUGAGCGAGGACUGCA 91 UGCAGUCCUCGCUCACUGGGCACGG 188
M-18 25 CCCAGUGAGCGAGGACUGCAGCGUA 92 UACGCUGCAGUCCUCGCUCACUGGG 189
M-19 25 CAGUGAGCGAGGACUGCAGCGUAGA 93 UCUACGCUGCAGUCCUCGCUCACUG 190
M-20 25 GACUGCAGCGUAGACGCUUUGUCCA 94 UGGACAAAGCGUCUACGCUGCAGUC 191
M-21 19+dTdT UUCGACCUCGGUUAGAAGAdTdT 95 UCUUCUAACCGAGGUCGAAdTdT 192
M1-1 25 UACGCUGCAGUCCUCGCUCACUGGG 96 CCCAGUGAGCGAGGACUGCAGCGUA 193
M2-1 19+dTdT AUCCACAGCAUUCUGCUGUdTdT 97 ACAGCAGAAUGCUGUGGAUdTdT 194
与PD-L1的siRNA分子的组合
PD-1/PD-L1信号通路对于抗病毒免疫效应具有重要性，并且能够影响病原体感染机体所致免疫病理损伤的严重程度。在慢性病毒感染中，程序性死亡因子1(PD1)高表达于CD8+T细胞表面，这是CD8+T细胞耗竭的标志之一。近年来研究发现在慢性病毒感染中调节性T细胞也高表达PD1等抑制性分子，并可能与病毒载量增加或抗病毒T细胞反应的抑制作用增加有关。在病毒感染的急性期，病毒特异性T细胞在识 别抗原后迅速上调共抑制受体PD-1，并通过PRR信号直接上调造血和非造血细胞上的PD-L1或通过诱导IFN和其他炎性细胞因子的释放间接上调PD-L1。病毒还可以控制免疫系统的平衡，从而阻止有效的抗病毒免疫反应，来帮助病原体在生物体内的持续存在。阻断调节性T细胞与耗竭CD8+T细胞表面PD1/PD-L1信号通路后，可以逆转耗竭CD8+T细胞的功能，这给慢性病毒感染性疾病治疗的靶向治疗策略带来了新的契机。
一项研究表明，呼吸道合胞病毒(RSV)诱导支气管上皮细胞上的PD-L1表达，进而抑制了局部CD8 +T细胞的抗病毒作用 [25]，表明病毒感染期间会影响上皮细胞会与T细胞的相互作用，这对病毒感染和复制有利。先前也有研究表明，在感染的急性期，乙型肝炎病毒感染会显着增加效应T细胞上PD-1的表达，然后才变为持久或潜伏的慢性感染 [28]。而在最近的一项研究中，感染H9N2病毒的肺微血管内皮细胞(RPMEC)中PD-L1的水平显着上调，病毒感染诱导的PD-L1表达将负信号传递给正在迁移的T细胞，从而导致抗病毒细胞因子的下调和细胞毒性蛋白产生的减少 [29]。
本发明所述组合物除了抗甲型流感病毒感染的siRNA分子，还包含一种特异性抑制宿主特定细胞内PD-1或PD-L1基因表达的siRNA分子，所述siRNA分子选自表2和表3中的序列。所述特异性抑制PD-1或PD-L1基因表达的siRNA分子在到机体特定部位后，可以抑制特定细胞内PD-1或PD-L1基因的表达，从而增强病毒特异性T细胞的功能，并与抗甲型流感病毒感染的siRNA分子产生协同效应，有效抑制病毒感染，彻底清除机体内的病毒。优选的，所述抑制PD-1或PD-L1基因表达的siRNA分子具备抑制人PD-1或PD-L1基因表达的功能，也具备抑制小鼠PD-1或PD-L1基因表达的功能。
表2、抑制PD-1表达的siRNA分子
编号 正义链(5'-3')SEQ ID No. 反义链(5'-3')SEQ ID No.
PD-1-1 ACCCUGCUCAGCCUGCACAAGGUGG 195 CCACCUUGUGCAGGCUGAGCAGGGU 207
PD-1-2 CCCUGCUCAGCCUGCACAAGGUGGA 196 UCCACCUUGUGCAGGCUGAGCAGGG 208
PD-1-3 CUGUCCUGCAGCUCCUCCCUCUGCC 197 GGCAGAGGGAGGAGCUGCAGGACAG 209
PD-1-4 UGUCCUGCAGCUCCUCCCUCUGCCA 198 UGGCAGAGGGAGGAGCUGCAGGACA 210
PD-1-5 UCCUGCAGCUCCUCCCUCUGCCAGC 199 GCUGGCAGAGGGAGGAGCUGCAGGA 211
PD-1-6 CCUGCAGCUCCUCCCUCUGCCAGCG 200 CGCUGGCAGAGGGAGGAGCUGCAGG 212
PD-1-7 CUGCAGCUCCUCCCUCUGCCAGCGC 201 GCGCUGGCAGAGGGAGGAGCUGCAG 213
PD-1-8 UGCAGCUCCUCCCUCUGCCAGCGCU 202 AGCGCUGGCAGAGGGAGGAGCUGCA 214
PD-1-9 GCAGCUCCUCCCUCUGCCAGCGCUG 203 CAGCGCUGGCAGAGGGAGGAGCUGC 215
PD-1-10 CAGCUCCUCCCUCUGCCAGCGCUGU 204 ACAGCGCUGGCAGAGGGAGGAGCUG 216
PD-1-11 AGCUCCUCCCUCUGCCAGCGCUGUG 205 CACAGCGCUGGCAGAGGGAGGAGCU 217
PD-1-12 UCCUCCCUCUGCCAGCGCUGUGACA 206 UGUCACAGCGCUGGCAGAGGGAGGA 218
表3、抑制PD-L1表达的siRNA分子
Figure PCTCN2022083042-appb-000003
Figure PCTCN2022083042-appb-000004
与抗流感病毒小分子化合物的组合
对抗流感病毒的现有策略有两种：疫苗和小分子抗流感药物。接种流感疫苗是预防流感最有效方法，现在也有三价灭活疫苗和减毒活疫苗上市，但疫苗每年都需要重新配置以应对抗原变异，且其研发周期长、成本高，这些缺点使得小分子药物成为防治流感的主要手段。目前上市或处于临床阶段的抗病毒小分子化合物主要包括特异性流感病毒抑制剂(M2离子通道阻滞剂、NA抑制剂、PA抑制剂和PB2抑制剂)和一些广谱抗病毒药物(利巴韦林、硝唑尼特、盐酸阿比多尔、法匹拉韦等)。然而，病毒对这些药物快速出现耐药性、生物利用度限制、不良反应等问题限制了这些药物的广泛使用，同时也使联合用药成为治疗流感病毒感染的一大发展方向，各种小分子化药的联合治疗的临床研究不断展开。但是，各种小分子化合物之间的联合使用，由于药物之间的结构、作用机制、生物利用度、半衰期等有较多的相似之处，导致这些药物组合使用并不能达到非常显著的协同效果，需要采用结构和机制显著不同、理化性质差异较为显著的药物进行联合，只有这种新的组合物，才能真正多角度、多层次显著抑制病毒的生命周期，形成巨大的协同效应。
本发明将所述的抗甲型流感病毒感染的siRNA分子与抗甲型流感小分子化合物联合使用，通过各自不同的作用机制发挥协同抗病毒效果。优选的，所述包括抗甲型流感病毒感染的siRNA分子与抗甲型流感小分子化合物的组合物，siRNA分子与小分子化合物分别靶向抑制病毒内部蛋白和病毒外部蛋白。所述抗甲型流感病毒感染的siRNA分子靶向病毒内部蛋白如PA、PB1、PB2或NP基因的表达，所述小分子化合物为抑制病毒外部蛋白如NA、HA、M蛋白的奥司他韦、阿比多尔和金刚烷胺等分子。所述抗甲型流感病毒感染的siRNA分子靶向外部蛋白如NA、HA、M蛋白的基因的表达，所述小分子化合物为抑制内部蛋白如PA、PB1、PB2或NP的法匹拉韦或萘普生等分子。
在一种实施方式中，将PB2-11siRNA(正义链为5’-GAAACGAAAACGGGACUCUAGCAUA-3’)与NA抑制剂奥司他韦联合使用，同时抑制病毒的聚合美基因和神经氨酸酶。
在另一种实施方式中，将抑制M1蛋白的siRNA分子(正义链为5’-UACGCUGCAGUCCUCGCUCACUGGG-3’)与流感病毒聚合酶抑制剂法匹拉韦联合使用，同时抑制两种不同的基因/蛋白的表达或功能。
在另一种实施方式中，将NA-1(正义链为5’-GUCUUGGCCAGACGGUGCUdTdT-3’)siRNA、抑制聚合酶PA基因的siRNA分子(正义链为5’-GCAAUUGAGGAGUGCCUGAdTdT-3’)及利巴韦林联合使用。
与流感mRNA疫苗的组合
mRNA疫苗携带编码病毒抗原的遗传信息，但它们不与宿主细胞基因组整合或与DNA相互作用，因此不会对宿主造成突变风险。而且，mRNA疫苗不含病毒颗粒。因此mRNA疫苗本身不会诱发它所预防的疾病。近年来，mRNA治疗(包括疫苗)技术取得重大进展，对mRNA序列中特定的核苷进行修饰，开发各种RNA包装和导入系统，大大促进了mRNA疫苗的发展 [26]。许多证据表明，与同为核酸疫苗的DNA疫苗相比，mRNA不仅能介导更优的转染效率和更长的蛋白表达时间，还因为mRNA无需进入细胞核即可发挥功能而具有显著优势。
mRNA疫苗也可以作为一种预防流感病毒感染的有效手段。传统的流感疫苗一般是由流感病毒中发现的蛋白质组成，这些蛋白可以“训练”患者的免疫系统，形成对抗流感病毒感染的机制。然而，流感病毒突变非常快，经常会改变这些蛋白质并使疫苗失效。这就是为什么流感疫苗每年都会发生变化，并且不能总是阻止人们生病的原因。采用mRNA疫苗来对抗流感，相比传统疫苗有显著优势。在一项最近的研究中，针对H7N9和H10N8甲型流感的mRNA疫苗诱发强大的体液免疫反应，且耐受性良好 [27]。
本发明将抗流感病毒siRNA分子与流感病毒mRNA疫苗组合使用，可以高效预防和治疗各种甲型流感病毒感染。由于siRNA和mRNA同为RNA分子，只是在长度和单双链上有所不同，两者均可以采用同样类型的纳米导入载体包裹，制备成混合的纳米药物制剂，在临床治疗有广泛应用前景。
在一种实施方式中，将基于HA基因序列设计的流感病毒mRNA疫苗与抑制M1蛋白的siRNA分子(正义链为5’-UACGCUGCAGUCCUCGCUCACUGGG-3’)联合使用，在激活机体抗病毒免疫、增强抗病毒免疫细胞活 力的同时，抑制M1基因的表达。
在另一种实施方式中，基于病毒核蛋白NP基因序列设计的流感病毒mRNA疫苗与抑制PB2蛋白的PB2-11siRNA(正义链为5’-GAAACGAAAACGGGACUCUAGCAUA-3’)联合使用。
抗流感siRNA分子与小分子化合物的偶联
siRNA与功能不同的核酸分子、基于核酸的小分子的偶联可以增加内源性，提高沉默效率和抑制率。在一项研究中，将PR8-M1的siRNA与核酶催化的降解性核酸序列连接起来，形成siRNA-核酶嵌合体，从而进一步提高了siRNA降解核酸的能力，这一优化的siRNA的沉默效率提高了四倍 [30]。另有研究发现，将具有免疫刺激功能的序列(5’-UGUGU-3’)添加到NP-siRNA的5’末端后，对流感病毒的抑制率达到80％，是单独的siRNA抑制效率的四倍 [31]。将NP-1496siRNA构建到含有内源性microRNA(miRNA)(形成shRNAmir-NP)的载体中，显著增强了其内源性。用PR8活病毒株感染后，NP蛋白被完全抑制，病毒效价降低到对照租的1/100左右 [32]。这些同类型分子之间的偶联，可以有效提供对抗流感病毒感染和复制的能力，但同样受限于分子之间的相似性而无法发挥最大的协同效应。
本发明包含一种将抗流感病毒siRNA分子与抗甲型流感小分子化合物共价偶联形成的新的化合物分子。所述siRNA分子包括表1中的siRNA分子。所述抗甲型流感小分子化合物包括但不限于特异性流感病毒抑制剂(M2离子通道阻滞剂、NA抑制剂、PA抑制剂和PB2抑制剂)和广谱抗病毒药物。所述siRNA分子与抗甲型流感小分子化合物可以直接通过各自的活性基团连接，也可以通过引入连接器(Linker)，利用连接器的活性基团将两种分子偶联。所述活性基团包括但不限于氨基、羧基、羟基、磷酸基、环氧基、醛基、异氰酸酯基等。所述活性基团之间可以通过加成反应、聚合反应、缩合反应等形成共价键。
在一种实施方式中，通过磷酸基和羟基缩合成磷酸酯的方式，把扎那米韦(Zanamvir)嵌段在siRNA分子中，或者通过连接器(如聚乙二醇等)把Zanamvir连接到siRNA的末端。
在另一种实施方式中，采用linker一端的氨基与帕拉米韦(Peramivir)的羧基反应生成酰胺键，另一端的羟基与siRNA的磷酸基反应生成磷酸酯。
在另一种实施方式中，linker一端的羟基与siRNA末端的磷酸基反应，另一端的环氧基与奥司他韦(Oseltamivir)的氨基发生亲核反应。
在另一种实施方式中，linker一端的环氧基与NA抑制剂A-192558的氨基反应，另一端的羟基与siRNA的磷酸基反应。
纳米导入(递送)载体与纳米药物制剂
一种药学上可接受的载体作为siRNA药物或基于siRNA药物的组合物的导入(递送)系统，药学上可接受的载体一般包括盐水、糖、多肽、聚合物、脂质、乳膏、凝胶、胶束材料和金属纳米颗粒。在一项实施方案中，所述载体是一种组氨酸-赖氨酸共聚物(高分子聚合物)，这种共聚物描述于美国专利号7070807B2、7163695B2和7772201B2的多项专利中，其全部内容通过引用并入本文。优选的，所述HKP载体是H3K4b、H3K(+H)4b、H2K4b或H3K(+N)4b，这些HKP具有赖氨酸主干，其四个分支包含多个重复的组氨酸、赖氨酸或天冬酰胺。
在一项实施方案中，HKP为H3K4b，结构如下：
(R)K(R)-K(R)-(R)K(X)，其中R＝KHHHKHHHKHHHKHHHK，或R＝KHHHKHHHNHHHNHHHN，X＝C(0)NH2，K＝赖氨酸，H＝组氨酸，N＝天冬酰胺。
在另一项实施方案中，HKP为H3K(+H)4b，结构如下：
(R)K(R)-K(R)-(R)K(X)，其中R＝KHHHKHHHKHHHHKHHHK，X＝C(0)NH2，K＝赖氨酸，H＝组氨酸。
在另一项实施方案中，HKP为H2K4b，结构如下：
(R)K(R)-K(R)-(R)K(X)，其中R＝KHKHHKHHKHHKHHKHHKHK，X＝C(0)NH2，K＝赖氨酸，H＝组氨酸。
在另一项实施方案中，HKP为H3K(+N)4b，结构如下：
(R)K(R)-K(R)-(R)K(X)，其中R＝KHHHHKHHHKHHHNHHHN，X＝C(0)NH2，K＝赖氨酸，H＝组氨酸，N＝天冬酰胺。
一种由HKP与siRNA药物或基于siRNA药物的组合物形成的纳米药物制剂，所述HKP携带正电荷，而siRNA、 siRNA与siRNA的组合物、siRNA与mRNA疫苗的组合物等携带负电荷，当HKP水溶液与siRNA或基于siRNA药物的组合物按特定质量比(如4:1)混合时，纳米颗粒会自组装形成。所述纳米颗粒的平均直径在30-400纳米的范围内，进一步优选的，所述纳米颗粒大小为50-150纳米。
雾化吸入给药
本发明还包括使用将抗流感病毒siRNA分子和基于这些siRAN分子的药物组合物，用于预防或治疗甲型流感病毒感染的方法。如本文所用，“治疗”或“治疗”是指降低甲型流感病毒感染疾病的严重程度或治愈甲型流感病毒感染疾病。将治疗有效量的本发明组合物给予哺乳动物。在一项实施方案中，哺乳动物是人、啮齿动物(例如大鼠，小鼠或豚鼠)、雪貂或非人灵长类动物(例如猴)。在该实施方案的一方面，哺乳动物是实验动物，例如啮齿动物。在该实施方案的另一方面，哺乳动物是非人灵长类动物，例如猴子。在该实施方案的另一方面，哺乳动物是人。如本文所用，“治疗有效量”是预防，降低甲型流感病毒感染疾病的严重性或治愈甲型流感病毒感染疾病的量。
在一项实施方案中，给予人的治疗有效量的药物组合物包含每千克人体重约0.1mg的siRNA分子至每千克人体重约10mg的siRNA分子。
在该实施方案的另一方面，给予人的治疗有效量的药物组合物包含每千克人体重约0.1mg的siRNA分子组合物至每千克人体重约100mg的siRNA分子组合物。
鉴于本文包含的给药，给药途径可由本领域技术人员确定。这些途径包括鼻内给药、气道滴注、吸入给药，例如通过使用雾化喷雾装置。在一些实施方案中，给药途径还包括注射滴注和腹膜内、静脉内、皮内、阴道内和皮下给药。优选的，通过吸入给药或静脉注射的方式将纳米药物制剂输送到病毒感染的下呼吸道或肺部。进一步优选的，通过雾化吸入给药将药物制剂导入到病毒感染的下呼吸道或肺部。
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Claims (15)

  1. 一种抑制流感病毒复制的siRNA分子，其特征在于：所述siRNA分子包括正义链和反义链，所述正义链的序列选自SEQ ID No.1～16、SEQ ID No.20～54、SEQ ID No.56～69、SEQ ID No.71～91、SEQ ID No.93、SEQ ID No.94中的任意一条，所述反义链选自SEQ ID No.98～113、SEQ ID No.117～151、SEQ ID No.153～166、SEQ ID No.168～188、SEQ ID No.190、SEQ ID No.191中与所述正义链互补的一条。
  2. 一种用于预防或治疗流感病毒感染的siRNA药物，其特征在于：所述siRNA药物包括活性成分，所述活性成分包括权利要求1所述的siRNA分子中的一种或多种。
  3. 根据权利要求2所述的用于预防或治疗流感病毒感染的siRNA药物，其特征在于：所述活性成分还包括一种或多种其他抑制流感病毒复制的siRNA分子；优选地，所述其他抑制流感病毒复制的siRNA分子的正义链的序列选自SEQ ID No.17～19、SEQ ID No.55、SEQ ID No.70、SEQ ID No.92、SEQ ID No.95～97中的任意一条，所述其他抑制流感病毒复制的siRNA分子的反义链选自SEQ ID No.114～116、SEQ ID No.152、SEQ ID No.167、SEQ ID No.189、SEQ ID No.192～194中与所述其他抑制流感病毒复制的siRNA分子的正义链互补的一条。
  4. 一种用于预防或治疗流感病毒感染的药物组合物，其特征在于：所述药物组合物的活性成分包括抑制流感病毒复制的siRNA分子和另一种分子，所述另一种分子包括抑制PD-1表达的siRNA分子、抑制PD-L1表达的siRNA分子、抗流感病毒小分子化合物、流感mRNA疫苗、或抗流感病毒单克隆抗体中的一种或多种。
  5. 根据权利要求4所述的用于预防或治疗流感病毒感染的药物组合物，其特征在于：所述抑制流感病毒复制的siRNA分子针对甲型流感病毒不同毒株之间的保守基因序列设计，所述甲型流感病毒包括H1N1、H5N1、H7N9、或H3N2中的一种或多种亚型；所述抑制流感病毒复制的siRNA分子通过靶向抑制流感病毒与侵入、复制、组装或释放相关的关键基因的表达而阻断病毒复制生命周期，降低病毒效价，抑制感染直至彻底清除病毒。
  6. 根据权利要求5所述的用于预防或治疗流感病毒感染的药物组合物，其特征在于：所述抑制流感病毒复制的siRNA分子选自以下siRNA分子中的一种或多种：正义链的序列选自SEQ ID No.1～97中的任意一条，反义链选自SEQ ID No.98～194中与正义链互补的一条。
  7. 根据权利要求4所述的用于预防或治疗流感病毒感染的药物组合物，其特征在于：所述抗流感病毒小分子化合物为特异性的流感病毒抑制剂和/或广谱抗病毒小分子化合物，优选地，所述特异性的流感病毒抑制剂选自M2离子通道阻滞剂、NA抑制剂、PA抑制剂和PB2抑制剂中的一种或多种，所述广谱抗病毒小分子化合物选自利巴韦林、硝唑尼特、盐酸阿比多尔、法匹拉韦、扎那米韦、帕拉米韦中的一种或多种。
  8. 根据权利要求4所述的用于预防或治疗流感病毒感染的药物组合物，其特征在于：所述抑制流感病毒复制的siRNA分子与所述抗流感病毒小分子化合物通过各自的活性基团连接，或通过在所述抑制流感病毒复制的siRNA分子中引入Linker，利用Linker的活性基团与所述抗流感病毒小分子化合物偶联；所述活性基团包括氨基、羧基、羟基、磷酸基、环氧基、醛基、异氰酸酯基中的一种或多种。
  9. 根据权利要求4所述的用于预防或治疗流感病毒感染的药物组合物，其特征在于：所述抑制PD-1表达的siRNA分子是根据人PD-1基因和小鼠PD-1基因之间的同源序列设计的，所述抑制PD-L1表达的siRNA分子是根据人PD-L1基因和小鼠PD-L1基因之间的同源序列设计的，优选的，所述同源序列是指人和小鼠的两个基因通过比对之后确认序列100％相同的DNA序列；
    所述的流感mRNA疫苗是根据流感病毒基因序列设计的信使核糖核酸疫苗，优选地，所述流感病毒基因为编码病毒结构蛋白的基因和/或编码非结构蛋的基因，进一步优选地，所述编码病毒结构蛋白的基因选自PB2、PB1、PA、HA、NP、NA、M1、或M2中的一种或多种，所述编码非结构蛋白基因为NS1和/或NS2；
    所述抗流感病毒小分子化合物为特异性的流感病毒抑制剂和/或广谱抗病毒小分子化合物，优选地，所述特异性的流感病毒抑制剂选自M2离子通道阻滞剂、NA抑制剂、PA抑制剂和PB2抑制剂中的一种或多种，所述广谱抗病毒小分子化合物选自利巴韦林、硝唑尼特、盐酸阿比多尔、法匹拉韦中的一种或多种。
  10. 根据权利要求4所述的用于预防或治疗流感病毒感染的药物组合物，其特征在于：所述抑制PD-1 表达的siRNA分子选自以下siRNA分子中的一种或多种：正义链的序列选自SEQ ID No.195～206中的任意一条，反义链选自SEQ ID No.207～218中与正义链互补的一条；
    所述抑制PD-L1表达的siRNA选自以下siRNA分子中的一种或多种：正义链的序列选自SEQ ID No.219～230中的任意一条，反义链选自SEQ ID No.231～242中与正义链互补的一条。
  11. 一种siRNA-小分子药物偶联物，其特征在于：所述siRNA-小分子药物偶联物由抑制流感病毒复制的siRNA分子与抗流感小分子药物通过共价键偶联形成。
  12. 一种如权利要求11所述的siRNA-小分子药物偶联物在制备用于预防或治疗流感病毒感染的药物中的应用。
  13. 根据权利要求2所述的用于预防或治疗流感病毒感染的siRNA药物、或权利要求4所述的用于预防或治疗流感病毒感染的药物组合物、或权利要求11所述的siRNA-小分子药物偶联物，其特征在于：所述用于预防或治疗流感病毒感染的siRNA药物、所述用于预防或治疗流感病毒感染的药物组合物、或所述siRNA-小分子药物偶联物与药学上可接受的载体形成制剂，所述药学上可接受的载体选自盐水、糖、多肽、高分子聚合物、脂质、乳膏、凝胶、胶束材料、或金属纳米颗粒中的一种或多种，优选地，所述高分子聚合物为所述多肽类高分子聚合物。
  14. 根据权利要求13所述的siRNA药物、或所述的药物组合物、或所述的siRNA-小分子药物偶联物，其特征在于：所述制剂为纳米药物制剂；所述纳米药物制剂的剂型为口服剂、注射剂、或雾化吸入剂。
  15. 根据权利要求2所述的用于预防或治疗流感病毒感染的siRNA药物、或权利要求4所述的用于预防或治疗流感病毒感染的药物组合物、或权利要求12所述的应用，其特征在于：所述流感病毒为G4 EA H1N1病毒株、H1N1病毒株、H5N1病毒株、H7N9病毒株、或H3N2病毒株中的一种或多种。
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