WO2021212892A1 - 广谱抗病毒药物及其应用 - Google Patents

广谱抗病毒药物及其应用 Download PDF

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WO2021212892A1
WO2021212892A1 PCT/CN2020/139898 CN2020139898W WO2021212892A1 WO 2021212892 A1 WO2021212892 A1 WO 2021212892A1 CN 2020139898 W CN2020139898 W CN 2020139898W WO 2021212892 A1 WO2021212892 A1 WO 2021212892A1
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methylation
virus
rna
sensitive
cells
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PCT/CN2020/139898
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French (fr)
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陈奇涵
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南京迈西可生物科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses

Definitions

  • the present invention relates to antiviral drugs, especially broad-spectrum antiviral drugs including 2'-O-methylation-sensitive RNA-related enzymes (such as MgR protein).
  • broad-spectrum antiviral drugs including 2'-O-methylation-sensitive RNA-related enzymes (such as MgR protein).
  • Viral diseases have the characteristics of high incidence and rapid spread, posing a major threat to poultry, livestock and human health.
  • Typical viral diseases that affect human health include AIDS (HIV), viral hepatitis, severe acute respiratory syndrome (SARS), influenza A (H1N1), hand, foot and mouth disease, etc.
  • SARS-CoV-2 The new coronavirus (SARS-CoV-2) currently circulating globally has infected more than one million people, caused tens of thousands of deaths, and caused serious social and economic problems.
  • the present disclosure provides an antiviral drug, which includes a nucleic acid sequence encoding a 2'-O-methylation-sensitive RNA-related enzyme or a 2'-O-methylation-sensitive RNA-related enzyme.
  • the 2'-O-methylation sensitive RNA-related enzyme is ribonuclease and/or RNA double-stranded helicase.
  • the 2'-O-methylation sensitive RNA-related enzyme is a 3'-5' direction ribonuclease and/or RNA double-stranded helicase.
  • the 2'-O-methylation-sensitive RNA-related enzymes include the amino acid sequence shown in SEQ ID NO: 3 or 4; or, the 2'-O-methylation-sensitive RNA-related enzymes include An amino acid sequence that has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 3 or 4 and has ribonuclease and/or helicase activity that is sensitive to 2'-O-methylation.
  • the amino acid sequence of the 2'-O-methylation sensitive RNA-related enzyme is compared with the amino acid sequence shown in SEQ ID NO: 3, and includes the D284A mutation.
  • the antiviral drug includes an expression vector carrying a nucleic acid sequence encoding the 2'-O-methylation-sensitive RNA-related enzyme.
  • the virus is a DNA virus and/or RNA virus.
  • the virus is an animal virus and/or a plant virus
  • the virus is MCMV virus, EV71 virus, or HSV-1 virus.
  • the antiviral drug is a broad-spectrum antiviral drug.
  • the present disclosure provides 2'-O-methylation-sensitive RNA-related enzymes or 2'-O-methylation-sensitive RNA-related enzymes encoding nucleic acid sequences prepared for use in the prevention or treatment of viruses in a subject. Use in medicines that cause diseases.
  • the 2'-O-methylation sensitive RNA-related enzyme is ribonuclease and/or RNA double-stranded helicase.
  • the 2'-O-methylation sensitive RNA-related enzyme is a 3'-5' direction ribonuclease and/or RNA double-stranded helicase.
  • the 2'-O-methylation-sensitive RNA-related enzymes include the amino acid sequence shown in SEQ ID NO: 3 or 4; or, the 2'-O-methylation-sensitive RNA-related enzymes include An amino acid sequence that has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 3 or 4 and has ribonuclease and/or helicase activity that is sensitive to 2'-O-methylation.
  • the amino acid sequence of the 2'-O-methylation sensitive RNA-related enzyme is compared with the amino acid sequence shown in SEQ ID NO: 3, and includes the D284A mutation.
  • the nucleic acid sequence encoding the 2'-O-methylation-sensitive RNA-related enzyme is located on an expression vector.
  • the virus is a DNA virus and/or RNA virus.
  • the virus is an animal virus and/or a plant virus.
  • the virus is MCMV virus, EV71 virus, or HSV-1 virus.
  • the drug is a broad-spectrum antiviral drug.
  • the subject is an animal or plant.
  • the subject is a mammal.
  • the subject is a human.
  • the present disclosure provides a method for preventing or treating a disease caused by a virus in a subject, including a therapeutically effective amount of 2'-O-methylation-sensitive RNA-related enzymes, or 2'-O-methyl
  • a method for preventing or treating a disease caused by a virus in a subject including a therapeutically effective amount of 2'-O-methylation-sensitive RNA-related enzymes, or 2'-O-methyl
  • the nucleic acid sequence encoding the chemosensitive RNA-related enzyme is administered to the subject.
  • the 2'-O-methylation sensitive RNA-related enzyme is ribonuclease and/or RNA double-stranded helicase.
  • the 2'-O-methylation sensitive RNA-related enzyme is a 3'-5' direction ribonuclease and/or RNA double-stranded helicase.
  • the 2'-O-methylation-sensitive RNA-related enzymes include the amino acid sequence shown in SEQ ID NO: 3 or 4; or, the 2'-O-methylation-sensitive RNA-related enzymes include An amino acid sequence that has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 3 or 4 and has ribonuclease and/or helicase activity that is sensitive to 2'-O-methylation.
  • the amino acid sequence of the 2'-O-methylation sensitive RNA-related enzyme is compared with the amino acid sequence shown in SEQ ID NO: 3, and includes the D284A mutation.
  • the nucleic acid sequence encoding the 2'-O-methylation-sensitive RNA-related enzyme is located on an expression vector.
  • the virus is a DNA virus and/or RNA virus.
  • the virus is an animal virus and/or a plant virus.
  • the virus is MCMV virus, EV71 virus, or HSV-1 virus.
  • the subject is at risk of being infected with two or more viruses, or the subject is infected by two or more viruses.
  • the subject is an animal or plant.
  • the subject is a mammal.
  • the subject is a human.
  • the present disclosure provides methods for preventing or treating diseases caused by viruses in a subject, including:
  • the 2'-O-methylation-sensitive RNA-related enzyme is the 2'-O-methylation-sensitive RNA-related enzyme mentioned in any one of claims 1-36.
  • the cell is the cell mentioned in any one of claims 1-36.
  • the virus is the virus mentioned in any one of claims 1-36.
  • the subject is the subject mentioned in any one of claims 1-36.
  • antiviral cells which include 2'-O-methylation-sensitive RNA-related enzymes or are modified to express 2'-O-methylation-sensitive RNA-related enzymes.
  • the 2'-O-methylation-sensitive RNA-related enzyme is the aforementioned 2'-O-methylation-sensitive RNA-related enzyme.
  • the antiviral cell is the cell mentioned above.
  • the antiviral cell is a CHO-K1 cell, Hela cell, or Vero cell.
  • the present disclosure provides the use of the above-mentioned antiviral cells in the preparation of drugs for preventing or treating diseases caused by viruses.
  • the present disclosure provides antiviral non-human animals or plants, which include the above-mentioned antiviral cells.
  • the present disclosure provides a method for preserving cells, which includes contacting the cells with 2'-O-methylation-sensitive RNA-related enzymes, or modifying the cells so that they can express 2'-O -Methylation-sensitive RNA-related enzymes.
  • the present disclosure provides a method for inhibiting virus replication in a cell, which includes contacting the cell with a 2'-O-methylation-sensitive RNA-related enzyme, or modifying the cell so that it can express 2'-O-methylation-sensitive RNA-related enzymes.
  • Figure 1 shows the results of MgR protein expression and purification and in vitro activity test.
  • Figure 2 shows the growth of cells transfected with plasmids expressing MgR or its mutants. From left to right are HEK293, HeLa, and CHO-K1 cells.
  • Figure 3 shows the amount of mRNA encoding the housekeeping gene GADPH in each experimental group of HeLa cells.
  • Figure 4 is a photo of microscope observation 24 hours after CHO-K1 cells were transfected with plasmid K.
  • Figure 5 shows the inhibitory effect of MgR and its mutants on MCMV virus in CHO-K1 cells.
  • Figure 6 shows the inhibitory effect of MgR and its mutants on the EV-71 virus in HeLa (left) and Vero (right) cells.
  • Figure 7 shows the inhibitory effect of MgR and its mutants on HSV-1 virus in HeLa (left) and Vero (right) cells.
  • Figure 8 shows the inhibitory effects of MgR and its mutants on the MCMV virus in the liver (left) and lung (right) of mice.
  • “And/or” means to include any one of the two elements before and after it, or to include both at the same time.
  • a and/or B means to include A, or include B, or include both A and B.
  • Virus is a special living body that can parasitize in cells, and it does not have a cellular structure.
  • the size of the virus is very small, and the structure is extremely simple.
  • a virus can be composed only of nucleic acid (viral genome) and a protein coat, and a slightly more complicated virus can also have a viral envelope formed by lipids and proteins.
  • the virus exists in the form of virus particles, with a certain size, shape, chemical composition, and physical and chemical properties. Like biological macromolecules, they generally do not exhibit any vital characteristics.
  • virus particles are infectious, that is, they have the ability to enter host cells under certain conditions.
  • the host cell's macromolecular synthesis device is used to produce progeny viral genomes and coat proteins, which are assembled into progeny viral particles and released outside the cell. This mode of reproduction is also commonly referred to as "replication" of viruses.
  • the virus can be divided into single-stranded DNA virus (for example, parvovirus, M13 phage), double-stranded DNA virus (for example, herpes virus, adenovirus, pox virus, hepatotropic DNA virus), and single-stranded RNA
  • single-stranded DNA virus for example, parvovirus, M13 phage
  • double-stranded DNA virus for example, herpes virus, adenovirus, pox virus, hepatotropic DNA virus
  • single-stranded RNA There are four main types of viruses (for example, coronavirus, HIV, Ebola virus, most plant viruses), and double-stranded RNA viruses (for example, reovirus).
  • Single-stranded RNA viruses and single-stranded DNA viruses can be subdivided into positive-strand RNA viruses and negative-strand RNA viruses, and positive-strand DNA viruses according to whether the virus performs protein translation based on the nucleotide sequence of the RNA or DNA strand. And negative-strand DNA viruses.
  • Antiviral refers to the ability of a drug to interfere with, inhibit or eliminate the virus from producing its progeny virus particles. This can be achieved in a variety of ways, such as preventing the virus from infecting cells, inhibiting the transcription of the viral genome in the cell, and interfering with the assembly and release of viral particles.
  • the detection of the "antiviral” ability of the compound or drug can be carried out by any means known to those skilled in the art, for example, by PCR to detect the change in the amount of viral nucleic acid in a viral infection sample.
  • Broad-spectrum antiviral refers to the ability of a drug to interfere, inhibit or eliminate two or more viruses to produce their progeny viruses.
  • the broad-spectrum antiviral drug is effective against at least one DNA virus and at least one RNA virus, or the broad-spectrum antiviral drug is effective against at least one animal virus and at least one plant virus.
  • RNA-related enzymes means that the enzymatic activity of the enzyme depends on its binding to the RNA chain and the enzymatic activity is affected by 2'-O-methylation in the RNA chain (for example , The enzymatic activity is inhibited).
  • the enzyme is a 2'-O-methylation-sensitive ribonuclease (Ribonuclease, RNase), that is, it can catalyze the hydrolysis of RNA strands into single nucleotides or small nucleotide fragments, which can be mainly divided into There are two types of endonuclease and exonuclease; and when there is 2'-O-methylation on the RNA molecule, its endonuclease or exonuclease catalytic activity on the RNA molecule is inhibited.
  • RNase 2'-O-methylation-sensitive ribonuclease
  • the 2'-O-methylation sensitive RNase is a 3'-5' direction exonuclease
  • 2'-O-methylated nucleotides are present in the RNA molecule
  • its hydrolysis of the RNA molecule Stay at a certain position downstream (3' direction) of the 2'-O-methylated nucleotide, for example, 2 or 1 nucleotide position downstream, or stay at the 2'-O-methylated nucleotide Location.
  • the enzyme is a 2'-O-methylation-sensitive helicase, which can play the role of RNA double-strand unwinding in the presence of ATP; and when there is 2'-O-methyl in the RNA molecule The unwinding effect of nucleotides is inhibited.
  • the enzyme has both ribonuclease activity and RNA double-strand helicase activity, and is affected by 2'-O-methylation in the RNA strand.
  • “Ribonuclease RNase R” has a well-known meaning in the art, and refers to a type of exo-ribonuclease existing in bacteria, including multiple family members.
  • the members of the RNase R family are widely distributed in species and have the highest similarity with the RNase II family. They are usually exonucleases that degrade RNA molecules from the 3'end to the 5'end. Previous studies have suggested that members of the RNase R family may be involved in the degradation of foreign RNA fragments, the post-transcriptional modification of self-RNA (such as tRNA), and the protection of cells under stress conditions.
  • the RNase R isolated from Mycoplasma genitalium is called "MgR" or "MgR protein” in this article. Mycoplasma genitalium has a very small genome, and the MgR is currently the only exonuclease identified in it.
  • the amino acid sequence of MgR is shown in SEQ ID NO: 3.
  • the enzyme not only has 3'-5' direction single-stranded RNA hydrolase activity, but also 3'-5' direction double-stranded RNA helicase activity, and these activities are affected by 2'-O-methylation.
  • removing up to 81 amino acids from the amino terminal of MgR did not affect the above-mentioned activity of the enzyme. Therefore, when referring to MgR in this article, these truncated forms of the enzyme (also called truncated enzymes) can also be included.
  • Encoding nucleic acid sequence refers to a DNA sequence corresponding to a protein or polypeptide.
  • the DNA sequence determines the amino acid sequence of the protein or polypeptide in a triplet codon after being transcribed and translated in a cell.
  • the coding nucleic acid sequence can be located on any strand of a double-stranded DNA molecule. It is well known in the art that when the amino acid sequence of a protein is known, it is easy to obtain its encoding nucleic acid sequence based on the triplet codon. In order to allow the encoding nucleic acid sequence to be efficiently expressed in a specific host cell, the optimal encoding nucleic acid sequence can also be determined according to the codon degeneracy and the codon preference of the host cell.
  • sequence identity refers to the amount of identity between two amino acid sequences (such as a query sequence and a reference sequence), generally expressed as a percentage .
  • sequence alignment is performed and gaps (if any) are introduced. If at a certain alignment position, the amino acids in the two sequences are the same, it is considered that the two sequences are identical or matched at that position; if the amino acids in the two sequences are different, it is considered that they are inconsistent or mismatched at that position. In some algorithms, the number of matching positions is divided by the total number of positions in the alignment window to obtain sequence identity.
  • the number of gaps and/or the length of the gaps are also taken into account.
  • the published alignment software BLAST (which can be found on the webpage ncbi.nlm.nih.gov) can be used to obtain the best sequence alignment by using the default settings and calculate the difference between the two amino acid sequences. Sequence identity.
  • MgR herein may also include its natural mutants or homologs in other species.
  • those skilled in the art know that some conservative changes or modifications can be made to the amino acid sequence of the enzyme, such as the substitution, deletion, or addition of one or several amino acid residues, while basically retaining the enzymatic activity. Or modifications are also included in the scope of the present invention. Therefore, in some embodiments, the MgR used includes the amino acid sequence shown in SEQ ID NO:3. In other embodiments, the MgR used may include at least 80% sequence identity with SEQ ID NO: 3 (e.g., at least 85%, at least 87%, at least 89%, at least 91%, at least 93%, at least 95%).
  • the MgR used includes the amino acid sequence shown in SEQ ID NO:4.
  • the MgR used may include at least 80% sequence identity with SEQ ID NO: 4 (e.g., at least 85%, at least 87%, at least 89%, at least 91%, at least 93%, at least 95%). %, at least 97%, at least 99% identity) and also has an amino acid sequence with exonuclease activity and/or RNA double-stranded helicase activity.
  • “Expression vector” refers to a nucleic acid molecule containing various expression elements for expressing a target protein in a host cell.
  • these expression elements usually include promoters, enhancers, polyadenylation signal sequences, and the like.
  • the expression vector In order to facilitate amplification in E. coli, the expression vector usually also includes an E. coli replicon sequence.
  • the expression vector may also include antibiotic resistance genes or selectable marker genes for screening (for example, ampicillin resistance gene (AmpR), thymidine kinase gene (TK), kanamycin resistance gene (KanR), new Resistance gene (NeoR), etc.) and a multiple cloning site (MCS) for the insertion of the coding sequence of the target protein.
  • antibiotic resistance genes or selectable marker genes for screening (for example, ampicillin resistance gene (AmpR), thymidine kinase gene (TK), kanamycin resistance gene (KanR), new Resistance gene (NeoR), etc.) and a multiple cloning site (MCS) for the insertion of the coding sequence of the target protein.
  • a variety of expression vectors can be used in the prior art.
  • common expression vectors include plasmid expression vectors and virus expression vectors.
  • Prevention refers to avoiding, reducing, or delaying the appearance of a specific disease or disease-related symptoms in a subject, and the disease or disease-related symptoms have not appeared before the relevant drug is administered. "Prevention” does not need to completely prevent the appearance of diseases or disease-related symptoms. For example, after the administration of related drugs, it can reduce the subject's risk of specific diseases or disease-related symptoms, or reduce the severity of related symptoms that appear later. All can be considered as “preventing” the emergence or development of the disease. “Treatment” refers to alleviating, alleviating, ameliorating, or inhibiting (e.g., preventing development) a disease that the subject has already exhibited or has experienced.
  • treatment can include “cure” the disease, but in most cases it is not necessary to completely eliminate all its symptoms, for example, administration of related drugs leads to at least one symptom of the subject attenuated or eliminated, It can be considered that the subject has been treated.
  • Subject refers to any individual in need of diagnosis, prognosis, or treatment.
  • the individual may be a patient with a disease or a healthy individual. This term can often be used interchangeably with "patient”, “test subject”, “treatment subject” and so on.
  • subjects include animals and plants.
  • Animal subjects include vertebrates, particularly mammals, for example, rats, mice, rabbits, cats, cows, and the like. Especially preferably, the animal subject is a human.
  • Plant subjects include seed plants, such as grasses, legumes, and the like.
  • “Therapeutically effective amount” refers to an amount sufficient to cause a desired biological or medical response in a subject, and can be generally determined by those skilled in the art according to factors such as the route of administration, the subject's weight, age, and the condition of the disease. When used for preventive purposes, the "therapeutically effective amount” can also be considered as the “prophylactically effective amount.” For example, a typical daily dose may range from 0.01 ⁇ g to 100 mg of the active pharmaceutical ingredient per kg body weight of the subject.
  • MgR isolated from Mycoplasma genitalium has the ability to inhibit the replication of multiple viruses (including DNA viruses and RNA viruses) in a variety of cells, and MgR itself does not It affects cell growth and has no obvious toxicity to cells.
  • this article provides applications related to the antiviral ability of 2'-O-methylation-sensitive RNA-related enzymes.
  • antiviral drugs including 2'-O-methylation-sensitive RNA-related enzymes can be used for virus control in animals and plants.
  • the nucleic acid sequence encoding the 2'-O-methylation-sensitive RNA-related enzyme can be integrated into the host cell genome by genetic engineering means, thereby giving the host cell long-term antiviral ability.
  • Those skilled in the art can understand that when the host cell is a non-human animal germ cell, a virus-resistant non-human transgenic animal can be obtained; and when the host cell is a plant cell, a virus-resistant transgenic plant can be obtained.
  • non-germ cells for example, by culturing human non-germ cells in vitro and contacting or expressing 2'-O-methylation-sensitive RNA-related enzymes, they can be transfused into patients in need after gaining antiviral ability.
  • bone marrow-derived cells such as lymphocytes, such as NK cells, CTL cells, etc.
  • the non-germ cells can be from the patient themselves and are transfused back after obtaining the antiviral ability; or the non-germ cells can be from other healthy donors, and the non-germ cells can be transfused into other patients after obtaining the antiviral ability (HLA may be required before transfusion) Matching inspection).
  • a drug including a 2'-O-methylation sensitive RNA-related enzyme or its encoding nucleic acid sequence is directly administered to the patient.
  • the modes of administration include injection administration (including intravenous injection, intramuscular injection, subcutaneous injection, etc.), mucosal administration, transdermal administration, and the like.
  • the 2'-O-methylation sensitive RNA-related enzyme encoding nucleic acid sequence is used for administration, it is usually inserted into an appropriate expression vector and then administered with the expression vector.
  • Methods for expressing foreign genes in humans are known in the art, including, but not limited to, liposome-mediated and viral vector (such as retrovirus and adenovirus)-mediated gene transfer and expression.
  • the recombinant plasmid was identified by Nco I and Hind III enzyme digestion and agarose gel electrophoresis. Invitrogen was commissioned to sequence the recombinant plasmid, and the sequencing result was analyzed using BioEdit software. The result was the same as the designed sequence, indicating that the recombinant bacteria was successfully constructed.
  • the bacterial cells were lysed by ultrasonic method (6W output for 8 minutes, 20 seconds on and 20 seconds off), and the supernatant was separated by 25000g centrifugation.
  • the supernatant and nickel resin Nickel resin, ThermoFisher
  • the recombinant protein was eluted with a lysis buffer solution containing 200mM imidazole, diluted to 0.1M NaCl and concentrated to 2mg/ml with a centrifuge tube.
  • the mass and concentration of the full-length MgR recombinant protein with the 6his tag were determined by SDS-PAGE (see the electrophoresis diagram on the left in Figure 1).
  • the sequence of the recombinant protein is shown in SEQ ID NO: 3 and 4, respectively (the 6his tag is not shown). Unless otherwise specified, the following examples are all performed using full-length MgR with 6his tags.
  • RNA substrate 5'-Fam-UAACCUAUGAAGNmNNUNmNNCUC-3' (SEQ ID NO: 5)
  • N is a mixture of four conventional bases
  • the nucleotides at positions 13 and 17 are modified by 2'-O-methylation.
  • the reaction system is 10 ⁇ L, containing 20 mM Tris-HCl (pH 8.0), 100 mM KCl, 0.01 mM ZnCl 2 , 0.2 ⁇ g substrate and corresponding amount of MgR protein.
  • the reaction mixture was incubated at 37°C for 30 minutes and inactivated at 85°C for 10 minutes. After using 15% Urea-PAGE gel to separate the digested products, they were photographed by the gel imaging system (Tanon 3500).
  • the mutant D284A encoding the above-mentioned MgR and its hydrolase activity removal mutant was inserted into the plasmid backbone PX330, and then the green fluorescent protein coding gene was inserted, and the two plasmids were named K and KM for subsequent cell experiments. middle.
  • K and KM the two plasmids were named K and KM for subsequent cell experiments. middle.
  • NC a plasmid expressing only GFP with PX330 as the backbone and named it NC.
  • HEK293, HeLa and CHO-K1 cells were cultured in high-sugar DMEM medium containing 10% FBS and 100 U/ml penicillin streptomycin, and the culture conditions were 37° C. and 5% CO2 saturated humidity. After the cell growth is stable, use lipo2000 to transfer the plasmids NC, K, and KM into different groups, and use the untreated group as a mock, and count and count the cells at 12, 24, 36 and 48 hours after transfection. , Used to measure the growth status of cells. The results are shown in Figure 2.
  • the CHO-K1 cells (ATCC) were cultured in high-sugar DMEM culture medium containing 10% FBS and 100 U/ml penicillin streptomycin, and the culture conditions were 37° C. and 5% CO 2 saturated humidity.
  • the cell density is about 60%
  • the plasmid K and KM and the mock group without any treatment are respectively transfected, with four parallel groups in each group, so that the final concentration of the plasmid is 1 ⁇ g/mL.
  • the cells were collected at 0h, 12h, 24h, and 48h after infection, washed twice with PBS before collecting the cells, and used the blood/cell/tissue genomic DNA extraction kit (DP304, Tiangen Biology) Extract total cell DNA, and use qPCR to detect and count the number of virus copies.
  • the results are shown in Figure 5 as a histogram and a graph respectively.
  • the MgR encoded by plasmid K and KM and its mutant D284A have a significant inhibitory effect on MCMV virus in CHO-K1 cells, and the inhibitory effect of wild-type MgR is stronger than Mutant D284A.
  • HeLa and Vero cells were cultured in high-sugar DMEM culture medium containing 10% FBS and 100 U/ml penicillin streptomycin, and the culture conditions were 37° C. and 5% CO 2 saturated humidity.
  • the cell density is about 60%
  • the plasmids NC, K and KM and the mock group without any treatment are respectively transfected, and each group has four parallels, so that the final concentration of the plasmid is 1 ⁇ g/mL.
  • HeLa and Vero cells were cultured in high-sugar DMEM culture medium containing 10% FBS and 100 U/ml penicillin streptomycin, and the culture conditions were 37° C. and 5% CO 2 saturated humidity.
  • the cell density is about 60%
  • the plasmids NC, K and KM and the mock group without any treatment are respectively transfected, and each group has four parallels, so that the final concentration of the plasmid is 1 ⁇ g/mL.
  • Plasmid K, KM and an equal volume of PBS solution were injected into the tail vein at a dosage of 5 mg/kg based on the body weight of each experimental mouse. After 48 hours, each mouse was intraperitoneally injected with MCMV virus at a dosage of 5,000,000 pfu/mouse. Then, the liver and lungs of the mice were collected on D1 (24 hours after virus injection), D3 (72 hours after virus injection), D5 (120 hours after virus injection), and washed with PBS solution. After that, the blood/cell/tissue genomic DNA extraction kit (DP304, Tiangen Bio) was used to extract the total DNA of the cells.
  • DP304 blood/cell/tissue genomic DNA extraction kit
  • mice injected with plasmid K and KM showed significant inhibitory effect on MCMV virus in the liver and lung, and the inhibitory effect of wild-type MgR was stronger than that of the wild-type MgR. Mutant D284A.
  • MgR and its mutant D284A are non-toxic in cells and can inhibit the replication of a variety of viruses (including DNA viruses and RNA) in cells.
  • viruses including DNA viruses and RNA
  • the possible mechanism of action is that these enzymes are sensitive to 2'-O-methylation modifications in RNA molecules, and animal cells (including human cells) and plant cells have abundant 2'-O-methylation modifications in their own RNA. It is basically non-toxic to these host cells; on the contrary, viral RNA genomes or RNA produced by viruses often lack this modification and are susceptible to the influence of these enzymes, resulting in viral inhibition.
  • MgR in the cell
  • the mutant D284A lacks hydrolase activity, but since it basically does not affect the helicase activity, its effect in inhibiting virus replication is similar to that of the wild type. Therefore, we speculate that other mutants of MgR that retain helicase activity should also have antiviral effects.

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Abstract

本公开提供了抗病毒药物，其包括2'-O-甲基化敏感RNA相关酶或2'-O-甲基化敏感RNA相关酶的编码核酸序列。本公开还提供了包括该2'-O-甲基化敏感RNA相关酶或2'-O-甲基化敏感RNA相关酶的编码核酸序列的制药用途。优选地，该2'-O-甲基化敏感RNA相关酶为来自生殖支原体的核糖核酸酶MgR。本公开提供的抗病毒药物具有广谱抗病毒效果，并且基本上无细胞毒性，可用于防止病毒对动植物和人的感染。

Description

广谱抗病毒药物及其应用 技术领域
本发明涉及抗病毒药物，尤其是包括2’-O-甲基化敏感RNA相关酶(例如MgR蛋白)的广谱抗病毒药物。
背景技术
病毒性疾病具有发病率高和传播快的特点，对家禽、家畜以及人类健康都构成了重大威胁。影响人类健康的典型病毒性疾病包括艾滋病(HIV)、病毒性肝炎、重症急性呼吸综合征(SARS)、甲型流感(H1N1)、手足口病等。目前正在全球流行的新型冠状病毒(SARS-CoV-2)已感染一百多万人，导致数万人死亡，引起了严重的社会和经济问题。
目前，病毒感染的防治主要依赖于疫苗接种和抗病毒药物，但疫苗和抗病毒药物的研制和应用都面临着相当多的困难。就疫苗来说，这种难度部分地体现在针对不同病毒研制特异性疫苗的周期较长，对于新发病毒性疾病的预防和治疗不能及时有效。另外，由于病毒种类繁多，同一种病毒还可能存在多种亚型，而研制的疫苗通常只能特异性的针对某种病毒或某种病毒亚型，限制了其应用范围。大多数抗病毒药物是通过抑制病毒基因组的转录来干扰病毒复制。通常，这些抗病毒药物抑制参与病毒基因组转录的特定蛋白，例如聚合酶或转录酶，然而，由于病毒主要依赖于宿主细胞来进行病毒基因组复制，因此这些药物往往会产生不希望的毒性。此外，由于抗病毒药物的高度特异性，病毒基因组中的小突变经常足以产生对该药物具有抗性的病毒株。
由于目前可获得的疫苗和抗病毒药物都受限于一些缺陷，临床上对于许多病毒引起的感染都没有行之有效的预防疫苗与治疗方法，因此亟需寻找新的广谱抗病毒药物。
发明内容
在一方面，本公开提供了一种抗病毒药物，其包括2’-O-甲基化敏感RNA相关酶或2’-O-甲基化敏感RNA相关酶的编码核酸序列。
在一些实施方案中，所述2’-O-甲基化敏感RNA相关酶为核糖核酸酶和/或RNA双链解旋酶。
在一些实施方案中，所述2’-O-甲基化敏感RNA相关酶为3’-5’方向核糖核酸酶和/或RNA双链解旋酶。
在一些实施方案中，所述2’-O-甲基化敏感RNA相关酶包括SEQ ID NO：3或4所示氨基酸序列；或者，所述2’-O-甲基化敏感RNA相关酶包括与SEQ ID NO：3或4所示氨基酸序列有至少90％序列一致性的氨基酸序列并且具有对2’-O-甲基化敏感的核糖核酸酶和/或解旋酶活性。
在一些实施方案中，所述2’-O-甲基化敏感RNA相关酶的氨基酸序列与SEQ ID NO：3所示氨基酸序列相比，包括D284A突变。
在一些实施方案中，所述抗病毒药物包括携带有所述2’-O-甲基化敏感RNA相关酶的编码核酸序列的表达载体。
在一些实施方案中，所述病毒为DNA病毒和/或RNA病毒。
在一些实施方案中，所述病毒为动物病毒和/或植物病毒
在一些实施方案中，所述病毒为MCMV病毒、EV71病毒或HSV-1病毒。
在一些实施方案中，所述抗病毒药物为广谱抗病毒药物。
另一方面，本公开提供了2’-O-甲基化敏感RNA相关酶或2’-O-甲基化敏感RNA相关酶的编码核酸序列在制备用于在受试者中预防或治疗病毒引起的疾病的药物中的用途。
在一些实施方案中，所述2’-O-甲基化敏感RNA相关酶为核糖核酸酶和/或RNA双链解旋酶。
在一些实施方案中，所述2’-O-甲基化敏感RNA相关酶为3’-5’方向核糖核酸酶和/或RNA双链解旋酶。
在一些实施方案中，所述2’-O-甲基化敏感RNA相关酶包括SEQ ID NO：3或4所示氨基酸序列；或者，所述2’-O-甲基化敏感RNA相关酶包括与SEQ ID NO：3或4所示氨基酸序列有至少90％序列一致性的氨基酸序列并且具有对2’-O-甲基化敏感的核糖核酸酶和/或解旋酶活性。
在一些实施方案中，所述2’-O-甲基化敏感RNA相关酶的氨基酸序列与SEQ ID NO：3所示氨基酸序列相比，包括D284A突变。
在一些实施方案中，所述2’-O-甲基化敏感RNA相关酶的编码核酸序列位于表达载体上。
在一些实施方案中，所述病毒为DNA病毒和/或RNA病毒。
在一些实施方案中，所述病毒为动物病毒和/或植物病毒。
在一些实施方案中，所述病毒为MCMV病毒、EV71病毒或HSV-1病毒。
在一些实施方案中，所述药物为广谱抗病毒药物。
在一些实施方案中，所述受试者为动物或植物。
在一些实施方案中，所述受试者为哺乳动物。
在一些实施方案中，所述受试者为人。
另一方面，本公开提供了在受试者中预防或治疗病毒引起的疾病的方法，包括以治疗有效量的2’-O-甲基化敏感RNA相关酶、或2’-O-甲基化敏感RNA相关酶的编码核酸序列向所述受试者给药。
在一些实施方案中，所述2’-O-甲基化敏感RNA相关酶为核糖核酸酶和/或RNA双链解旋酶。
在一些实施方案中，所述2’-O-甲基化敏感RNA相关酶为3’-5’方向核糖核酸酶和/或RNA双链解旋酶。
在一些实施方案中，所述2’-O-甲基化敏感RNA相关酶包括SEQ ID NO：3或4所示氨基酸序列；或者，所述2’-O-甲基化敏感RNA相关酶包括与SEQ ID NO：3或4所示氨基酸序列有至少90％序列一致性的氨基酸序列并且具有对2’-O-甲基化敏感的核糖核酸酶和/或解旋酶活性。
在一些实施方案中，所述2’-O-甲基化敏感RNA相关酶的氨基酸序列与SEQ ID NO：3所示氨基酸序列相比，包括D284A突变。
在一些实施方案中，所述2’-O-甲基化敏感RNA相关酶的编码核酸序列位于表达载体上。
在一些实施方案中，所述病毒为DNA病毒和/或RNA病毒。
在一些实施方案中，所述病毒为动物病毒和/或植物病毒。
在一些实施方案中，所述病毒为MCMV病毒、EV71病毒或HSV-1病毒。
在一些实施方案中，所述受试者有感染两种或两种以上病毒的风险，或者所述受试者被两种或两种以上病毒感染。
在一些实施方案中，所述受试者为动物或植物。
在一些实施方案中，所述受试者为哺乳动物。
在一些实施方案中，所述受试者为人。
另一方面，本公开提供了在受试者中预防或治疗病毒引起的疾病的方法，包括：
1)从所述受试者取出部分细胞；
2)让所述细胞与2’-O-甲基化敏感RNA相关酶接触，或者将所述细胞进行修饰以使其能够表达2’-O-甲基化敏感RNA相关酶；以及
3)将所述细胞回输到所述受试者体内。
在一些实施方案中，所述2’-O-甲基化敏感RNA相关酶为权利要求1-36任一项提及的2’-O-甲基化敏感RNA相关酶。
在一些实施方案中，所述细胞为权利要求1-36任一项提及的细胞。
在一些实施方案中，所述病毒为权利要求1-36任一项提及的病毒。
在一些实施方案中，所述受试者为权利要求1-36任一项提及的受试者。
另一方面，本公开提供了抗病毒细胞，其包括2’-O-甲基化敏感RNA相关酶或经修饰而能够表达2’-O-甲基化敏感RNA相关酶。
在一些实施方案中，所述2’-O-甲基化敏感RNA相关酶为上文提及的2’-O-甲基化敏感RNA相关酶。
在一些实施方案中，所述抗病毒细胞为上文提及的细胞。
在一些实施方案中，所述抗病毒细胞为CHO-K1细胞、Hela细胞、或Vero细胞。
另一方面，本公开提供了上述抗病毒细胞在制备用于预防或治疗病毒引起的疾病的药物中的用途。
另一方面，本公开提供了抗病毒非人动物或植物，其包括上述抗病毒细胞。
另一方面，本公开提供了保存细胞的方法，其包括让所述细胞与2’-O-甲基化敏感RNA相关酶接触，或者将所述细胞进行修饰以使其能够表达2’-O-甲基化敏感RNA相关酶。
另一方面，本公开提供了抑制病毒在细胞中复制的方法，其包括让所述细胞与2’-O-甲基化敏感RNA相关酶接触，或者将所述细胞进行修饰以使其能够表达2’-O-甲基化敏感RNA相关酶。
附图说明
图1显示了MgR蛋白表达纯化及体外活性测试结果。
图2显示了细胞转染表达MgR或其突变体的质粒后的生长情况。从左到右分别为HEK293、HeLa、和CHO-K1细胞。
图3显示了编码管家基因GADPH的mRNA的量在HeLa细胞中各实验组的情况。
图4为CHO-K1细胞转染质粒K后24小时显微镜观察照片。
图5显示了MgR及其突变体在CHO-K1细胞内对MCMV病毒的抑制作用。
图6显示了MgR及其突变体对于HeLa(左)和Vero(右)细胞内EV-71病毒的抑制作用。
图7显示了MgR及其突变体对于HeLa(左)和Vero(右)细胞内HSV-1病毒的抑制作用。
图8显示了MgR及其突变体在小鼠的肝脏(左)和肺(右)中对MCMV病毒的抑制作用。
具体实施方式
除非另有说明，本文使用的所有技术和科学术语具有本领域普通技术人员所通常理解的含义。除非另有说明，本文中所用的细胞培养、生物化学、细胞生物学等操作 步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供一些术语的定义和解释。
“和/或”指包括其前后两元素的任何一个或者同时包括二者。例如，“A和/或B”指包括A，或者包括B，或者同时包括A和B。
“病毒”为一种可在细胞内寄生的特殊生命体，其不具有细胞结构。病毒体积非常微小，结构也及其简单。例如，病毒可以仅由核酸(病毒基因组)和蛋白质外壳构成，稍复杂的病毒还可以具有脂类和蛋白形成的病毒包膜。在细胞外，病毒以病毒颗粒的形式存在，有一定的大小、形态、化学组成和理化性质，如同生物大分子一般不表现任何生命特征。但是，病毒颗粒具有感染性，即具有在一定条件下进入宿主细胞的能力。一旦病毒进入细胞，病毒颗粒解体，释放出的病毒基因组就具有繁殖性：利用宿主细胞的大分子合成装置产生子代病毒基因组和外壳蛋白质，它们装配成子代病毒颗粒，释放到细胞外。这种繁殖方式通常也称为病毒的“复制”。依据病毒核酸的不同，可以将病毒分为单链DNA病毒(例如，细小病毒、M13噬菌体)、双链DNA病毒(例如，疱疹病毒、腺病毒、痘病毒、嗜肝DNA病毒)、单链RNA病毒(例如，冠状病毒、HIV、埃博拉病毒、大多数的植物病毒)、双链RNA病毒(例如，呼肠孤病毒)4种主要类型。单链RNA病毒和单链DNA病毒可以再按病毒是否依据该RNA或DNA链的核苷酸序列进行蛋白质的翻译，将它们细分为正链RNA病毒和负链RNA病毒，以及正链DNA病毒和负链DNA病毒。
“抗病毒”指药物具有干扰、抑制或消除病毒生成其子代病毒颗粒的能力。这可以通过多种途径实现，例如，阻止病毒感染细胞、抑制病毒基因组在细胞内的转录、干扰病毒颗粒的组装和释放等。化合物或药物的“抗病毒”能力的检测可通过本领域技术人员已知的任何方式进行，例如通过PCR检测病毒感染样品中病毒核酸数量的变化。
“广谱抗病毒”指药物具有干扰、抑制或消除两种或两种以上病毒生成其子代病毒的能力。优选地，广谱抗病毒药物对至少一种DNA病毒和至少一种RNA病毒有效，或者，广谱抗病毒药物对至少一种动物病毒和至少一种植物病毒有效。
“2’-O-甲基化敏感RNA相关酶”，指该酶的酶学活性有赖于其与RNA链的结合并且酶学活性受RNA链中2’-O-甲基化的影响(例如，酶学活性被抑制)。在一些情形中，该酶为2’-O-甲基化敏感的核糖核酸酶(Ribonuclease，RNase)，即其能够催化RNA链水解为单个核苷酸或小核苷酸片段，可主要分为内切酶与外切酶两大类；而当在RNA分子上存在2’-O-甲基化时，其对该RNA分子的内切酶或外切酶催化活性受到抑制。例如，当该2’-O-甲基化敏感RNase为3’-5’方向外切酶，并且RNA分子中存在2’-O-甲基化核苷酸时，其对该RNA分子的水解停留在2’-O-甲基化核苷酸下游(3’方向)某个位置，例如下游2个或1个核苷酸位置，或者停留在该2’-O-甲基化核苷酸位置。在另一些情形中，该酶为2’-O-甲基化敏感的解旋酶，其能够在ATP存在下发挥RNA双链解 旋作用；而当RNA分子中存在2’-O-甲基化核苷酸时，其解旋作用被抑制。在另一些情形中，该酶同时具有核糖核酸酶活性和RNA双链解旋酶活性，并受RNA链中2’-O-甲基化的影响。“核糖核酸酶RNase R”具有本领域公知的含义，指细菌中存在的一类核糖核酸外切酶，包括多个家族成员。RNase R家族成员在物种中分布广泛种类繁多，与RNase II家族相似度最高，通常为从3’端向5’端降解RNA分子的外切酶。之前的研究认为，RNase R家族成员可能与降解外源RNA片段、参与自身RNA(如tRNA)的转录后修饰、以及在应激定条件下保护细胞有关。本文将从生殖支原体(Mycoplasma genitalium)分离出的RNase R称为“MgR”或“MgR蛋白”。生殖支原体具有非常小的基因组，该MgR也是目前在其中唯一鉴定出的外切核糖核酸酶。MgR的氨基酸序列如SEQ ID NO：3所示。该酶不仅具有3’-5’方向单链RNA水解酶活性，还具有3’-5’方向双链RNA解旋酶活性，并且这些活性受2’-O-甲基化的影响。我们发现，去除MgR的氨基端多至81个氨基酸，并不影响该酶的上述活性。因此，在本文中，提到MgR时也可包括该酶的这些截短形式(也称为截短体酶)。
“编码核酸序列”指与蛋白质或多肽对应的DNA序列，该DNA序列在细胞内经转录和翻译后以三联体密码子方式决定了该蛋白质或多肽的氨基酸序列。编码核酸序列可以位于双链DNA分子的任意一条链上。本领域公知的是，在已知蛋白质的氨基酸序列情况下，容易根据三联体密码子得到其编码核酸序列。为了让该编码核酸序列在特定宿主细胞中高效表达，还可以根据密码子简并性，并结合该宿主细胞的密码子偏好，确定最佳的编码核酸序列。
提及氨基酸序列时，术语“序列一致性(sequence identity)”(也称为”序列同一性”)指两氨基酸序列(例如查询序列和参照序列)之间一致性程度的量，一般以百分比表示。通常，在计算两氨基酸序列之间的一致性百分比之前，先进行序列比对(alignment)并引入缺口(gap)(如果有的话)。如果在某个比对位置，两序列中的氨基酸相同，则认为两序列在该位置一致或匹配；两序列中的氨基酸不同，则认为在该位置不一致或错配。在一些算法中，用匹配位置数除以比对窗口中的位置总数以获得序列一致性。在另一些算法中，还将缺口数量和/或缺口长度考虑在内。出于本发明的目的，可以采用公开的比对软件BLAST(可在网页ncbi.nlm.nih.gov找到)，通过使用缺省设置来获得最佳序列比对并计算出两氨基酸序列之间的序列一致性。
本文中MgR还可以包括其天然突变体或者其它物种中的同源物。另外，本领域技术人员已知，可以在基本保留酶学活性的情况下，对酶的氨基酸序列进行一些保守性改动或修饰，例如一个或几个氨基酸残基的取代、缺失、添加，这些改动或修饰也包括在本发明的范围内。因此，在一些实施方案中，所采用的MgR包括SEQ ID NO：3所示的氨基酸序列。在另一些实施方案中，所采用的MgR可包括与SEQ ID NO：3至少有80％序列一致性(例如至少85％、至少87％、至少89％、至少91％、至少93％、 至少95％、至少97％、至少99％一致性)并且也具有核糖核酸外切酶活性和/或RNA双链解旋酶活性的氨基酸序列。或者，在一些实施方案中，所采用的MgR包括SEQ ID NO：4所示的氨基酸序列。在另一些实施方案中，所采用的MgR可包括与SEQ ID NO：4至少有80％序列一致性(例如至少85％、至少87％、至少89％、至少91％、至少93％、至少95％、至少97％、至少99％一致性)并且也具有核糖核酸外切酶活性和/或RNA双链解旋酶活性的氨基酸序列。
“表达载体(expression vector)”指包含各种表达元件以用于在宿主细胞中表达目的蛋白的核酸分子。对于用于在真核细胞中表达目的蛋白的表达载体，这些表达元件通常包括启动子、增强子、多腺苷酸化信号序列等。为了方便在大肠杆菌中扩增，该表达载体通常还包括大肠杆菌复制子序列。此外，表达载体还可包括用于筛选的抗生素抗性基因或选择标记基因(例如氨苄青霉素抗性基因(AmpR)，胸苷激酶基因(TK)，卡那霉素抗性基因(KanR)，新霉素抗性基因(NeoR)等)和用于目的蛋白编码序列插入的多克隆位点(MCS)。现有技术中已有多种表达载体可以使用，例如常见的表达载体有质粒表达载体和病毒表达载体。
“预防”指避免、减少、或延迟受试者中特定疾病或疾病相关症状的出现，并且在相关药物给药前这种疾病或疾病相关症状的还没有出现。“预防”并非需要完全阻止疾病或疾病相关症状的出现，例如，在相关药物给药后可以减小受试者出现特定疾病或疾病相关症状的风险，或者减弱后来出现的相关症状的严重程度，均可认为是“预防”了该疾病的出现或发展。“治疗”指缓解、减轻、改善、或抑制(例如，阻止发展)受试者已经表现出的或曾经出现过的疾病。就具体疾病来说，“治疗”可以包括“治愈”该疾病，但大多数情况下并不需要完全消除其所有症状，例如，以相关药物给药导致受试者至少一种症状减弱或消除，则可认为是对受试者进行了治疗。
“受试者”指需要诊断、预后或治疗的任何个体，该个体可以为疾病患者，也可能为健康个体。该术语通常可以和与“患者”、“检测对象”、“治疗对象”等互换使用。在本文中，受试者包括动物和植物。动物受试者包括脊椎动物，特别是哺乳动物，例如，大鼠、小鼠、兔、猫、牛等。尤其优选地，动物受试者为人。植物受试者包括种子植物，例如禾本科植物、豆科植物等。
“治疗有效量”指足以在受试者体内引起期望的生物学或医学反应的的量，可通常由本领域技术人员根据给药途径、受试者的体重、年龄、病情等因素而确定。当用于预防目的时，“治疗有效量”也可以认为是“预防有效量”。例如，典型的日剂量范围可以为受试者每kg体重0.01μg至100mg药物活性成分。
本文的一些技术方案至少部分地基于我们意外地发现从生殖支原体(Mycoplasma genitalium)分离出的MgR在多种细胞中具有抑制多种病毒(包括DNA病毒和RNA病毒)复制的能力，并且MgR本身不影响细胞生长，对细胞无明显毒性。
相应地，本文提供了与2’-O-甲基化敏感RNA相关酶的抗病毒能力有关的应用。例如，包括2’-O-甲基化敏感RNA相关酶的抗病毒药物可用于动物和植物的病毒防治。在一些实施方案中，可通过基因工程手段将2’-O-甲基化敏感RNA相关酶的编码核酸序列整合进宿主细胞基因组中，从而赋予宿主细胞长期抗病毒能力。本领域技术人员可以理解，当该宿主细胞为非人动物生殖细胞时，可以获得抗病毒非人转基因动物；而当该宿主细胞为植物细胞时，可以获得抗病毒转基因植物。
对于与人类抗病毒预防和治疗相关的应用，可通过多种方式进行。
在一些实施方案中，可以例如通过在体外培养人非生殖细胞，并使其接触或表达2’-O-甲基化敏感RNA相关酶，在获得抗病毒能力后输入有需要的患者体内，这尤其适用于一些骨髓来源细胞，例如淋巴细胞，如NK细胞、CTL细胞等。所述非生殖细胞可以来自患者自身，在获得抗病毒能力后回输；或者所述非生殖细胞可以来自其他健康供者，在在获得抗病毒能力后输入其他患者(可能输入前还需要做HLA配型检测)。
在另一些实施方案中，将包括2’-O-甲基化敏感RNA相关酶或其编码核酸序列的药物直接向患者给药。给药方式包括注射给药(包括静脉注射、肌内注射、皮下注射等)、粘膜给药、透皮给药等。当采用2’-O-甲基化敏感RNA相关酶的编码核酸序列给药时，通常是将其插入适当的表达载体中，再以该表达载体给药。将外源基因在人体内表达的方法是本领域已知的，例如包括但不限于脂质体介导和病毒载体(例如逆转录病毒和腺病毒)介导的基因转移和表达。
以下通过具体实施例对本发明进一步详细说明。
实施例1蛋白的克隆表达和纯化
委托Invitrogen公司合成MgR基因序列及去除前81个氨基酸的截短体基因序列(SEQ ID NO：1和2)，并在基因片段5’端引入Nco Ⅰ内切酶位点，3’端引入Hind Ⅲ内切酶位点。将合成的基因片段和pET28a(+)载体分别经Nco Ⅰ与Hind Ⅲ双酶切，使用T4 DNA连接酶连接基因片段和载体片段，常规转化DH5α感受态细胞(天根生化科技(北京)有限公司)。根据卡那霉素抗性筛选阳性克隆，提取质粒。重组质粒经Nco Ⅰ与Hind Ⅲ双酶切和琼脂糖凝胶电泳鉴定，委托Invitrogen公司对重组质粒进行序列测定，使用BioEdit软件对测序结果进行分析，结果与设计序列相同，说明重组菌构建成功。
将获得的阳性克隆质粒转化到E.ColiBL21(DE3)感受态细胞(天根生化科技(北京)有限公司)内，在含有100μg/mL卡纳霉素的LB培养基中37℃培养过夜，之后转移到1L的同样的LB培养基中37℃培养到OD＝0.6左右。之后培养基被降温到4℃，加入0.5mM的IPTG诱导表达约16小时。菌体通过4000g离心收集，用裂解缓冲液(50mM  Tris pH＝7.0，1M NaCl,20％甘油，10mM TCEP)重新悬浮。菌体由超声方法裂解(6W输出8分钟，20秒开20秒关)，上清液通过25000g离心分离。将上清液和镍树脂(Nickel resin，ThermoFisher)在4℃下共置1小时，然后通过重力柱并用40mL裂解缓冲溶液洗涤。重组蛋白用含有200mM咪唑的裂解缓冲溶液洗脱，稀释至0.1M的NaCl并用离心管浓缩至2mg/ml。带6his标签的全长MgR重组蛋白的质量和浓度通过SDS-PAGE确定(参见图1中左侧电泳图)。
重组蛋白的序列分别如SEQ ID NO：3和4所示(未显示6his标签)。除非另有说明，以下实施例均采用带6his标签的全长MgR进行。
Figure PCTCN2020139898-appb-000001
Figure PCTCN2020139898-appb-000002
Figure PCTCN2020139898-appb-000003
Figure PCTCN2020139898-appb-000004
Figure PCTCN2020139898-appb-000005
Figure PCTCN2020139898-appb-000006
Figure PCTCN2020139898-appb-000007
实施例2 MgR体外活性测试
委托金斯瑞生物科技有限公司合成长度为22个核苷酸的单链RNA底物(5’-Fam-UAACCUAUGAAGNmNNUNmNNCUC-3’(SEQ ID NO：5))，其中5’端由荧光基团FAM标记便于检测，N为四种常规碱基的混合，在第13和17号位置的核苷酸被2’-O-甲基化修饰。反应体系为10μL，包含20mMTris-HCl(pH 8.0)，100mM KCl，0.01mM ZnCl 2、0.2μg底物和相应量的MgR蛋白。将反应混合物在37℃下孵育30分钟，85℃灭活10分钟。使用15％Urea-PAGE凝胶分离消化产物后，通过凝胶图像系统(Tanon 3500)拍照。
电泳结果如图1右侧所示。由结果可见，实施例1得到的MgR水解RNA的活性会明显被2’-O-甲基化修饰的核苷酸抑制，而让这样的水解活性停留在修饰位点下游一位的核苷酸处(本实验中具体表现为长度18和14个核苷酸的产物)。由此推测，该RNA水解酶对修饰丰富的宿主RNA(人、其他动物、植物)影响有限，而可能对缺乏修饰的病毒RNA或病毒产生的RNA有强烈抑制作用。
实施例3 MgR及其突变体的细胞内安全性试验测试
将编码上述MgR及其水解酶活性去除突变体D284A分别插入到质粒骨架PX330中，并在其后插入绿色荧光蛋白的编码基因，将这两个质粒分别命名为K和KM用作后续的细胞实验中。作为对照，我们也构建了以PX330为骨架只表达GFP的质粒，并命名为NC。
将HEK293、HeLa和CHO-K1细胞(ATCC公司)培养在含10％FBS、青链霉素各100U/ml的高糖DMEM培养液中，培养条件是37℃、5％CO2饱和湿度。待细胞生长稳定之后，利用lipo2000将质粒NC、K和KM分别转入不同组别，并以未做任何处理的组作为mock，在转染12、24、36和48小时对细胞进行计数并统计，用来衡量细胞的生长状态。结果见图2。由图中可见，在3种细胞内，K和KM与NC相比，细胞的生长并没受到明显的影响，说明K和KM所编码的MgR及其突变体D284A在细胞内对细胞生长无影响，因此这些蛋白在细胞内的毒性极低。
另外，我们选取了HeLa细胞系的各实验组做进一步测试。具体的，我们利用流式细胞仪从每个Mock、NC、K和KM中分离了60000个细胞，提取总RNA，并利用qPCR对管家基因GADPH的mRNA进行定量。从图3结果可以看到，各组的GADPH的mRNA在含量上没有明显的差异，可看到MgR及其突变体D284A在细胞内对内源性的RNA没有明显影响。
实施例4 MgR细胞内分布测试
针对实施例3中的实验组CHO-K1细胞系转染质粒K的组，我们取转染后24小时的细胞在显微镜下进行观察。结果见图4。图中的红色为用染料染色的线粒体，作为标志表征细胞质的位置；而绿色则是GFP发出的荧光，表征与GFP融合在一起的MgR的位置。由图可见，MgR分布于细胞质和细胞核内，说明其对可能存在于细胞质和/或细胞核内的病毒RNA均具有水解活性或双链解旋活性。
实施例5 MgR及其突变体对CHO-K1细胞内MCMV病毒(DNA病毒)抑制作用测试
将CHO-K1细胞(ATCC公司)培养在含10％FBS、青链霉素各100U/ml的高糖DMEM培养液中，培养条件是37℃、5％CO 2饱和湿度。待细胞密度在60％左右分别转染质粒K和KM以及不做任何处理的mock组，每组四个平行，使质粒终浓度为1μg/mL。转染24H后，用MCMV病毒感染(感染复数MOI＝4)细胞，收集感染后0h,12h,24h,48h细胞，收集细胞之前用PBS洗两遍，用血液/细胞/组织基因组DNA提取试剂盒(DP304，天根生物)提取细胞总DNA，并利用qPCR检测病毒拷贝数并统计。结果分别以柱状图和曲线图显示在图5中。与未做处理直接感染病毒的组相比，质粒K和KM所编码的MgR及其突变体D284A在CHO-K1细胞内均对MCMV病毒有明显的抑制作用，并且野生型MgR的抑制作用要强于突变体D284A。
实施例6 MgR及其突变体在HeLa和Vero细胞中对于EV-71病毒(RNA(+)病毒)的抑制作用测试
将HeLa和Vero细胞(ATCC公司)培养在含10％FBS、青链霉素各100U/ml的高糖DMEM培养液中，培养条件是37℃、5％CO 2饱和湿度。待细胞密度在60％左右分别转染质粒NC、K和KM以及不做任何处理的mock组，每组四个平行，使质粒终浓度为1μg/mL。转染24H后，用EV-71病毒感染(感染复数MOI＝4)细胞，在恒温箱中孵育1小时后使用不含FBS的培养基清洗细胞，去除游离病毒，然后加入新鲜培养基，加入10％FBS。收集感染后48h细胞，收集细胞之前用PBS洗两遍，用血液/细胞/组织基因组DNA提取试剂盒(DP304，天根生物)提取细胞总DNA，并利用qPCR检测病毒拷贝数并统计(图6)。如结果所示，与感染病毒的NC组和未做处理直接感染病毒的mock组相比，质粒K和KM所编码的MgR及其突变体D284A在HeLa和Vero细胞内均对EV-71病毒有明显的抑制作用，并且野生型MgR的抑制作用要强于突变体D284A。
实施例7 MgR及其突变体在HeLa和Vero细胞中对于HSV-1病毒(DNA病毒)的抑制作用测试
将HeLa和Vero细胞(ATCC公司)培养在含10％FBS、青链霉素各100U/ml的高糖DMEM培养液中，培养条件是37℃、5％CO 2饱和湿度。待细胞密度在60％左右分别转染质粒NC、K和KM以及不做任何处理的mock组，每组四个平行，使质粒终浓度为1μg/mL。转染24H后，用HSV-1病毒感染(感染复数MOI＝4)细胞，在恒温箱中孵育1小时后使用不含FBS的培养基清洗细胞，去除游离病毒，然后加入新鲜培养基，加入10％FBS。收集感染后48h细胞，收集细胞之前用PBS洗两遍，用血液/细胞/组织基因组DNA提取试剂盒(DP304，天根生物)提取细胞总DNA，并利用qPCR检测病毒拷贝数并统计(图7)。如结果所示，与感染病毒的NC组和未做处理直接感染病毒的mock组相比，质粒K和KM所编码的MgR及其突变体D284A在HeLa和Vero细胞内均对HSV-1病毒有明显的抑制作用，并且野生型MgR的抑制作用要强于突变体D284A。
实施例8 MgR及其突变体在小鼠体内对MCMV病毒的抑制作用测试
将质粒K、KM及等体积的PBS溶液按照5mg/kg的用量基于各实验小鼠的体重进行尾静脉注射。48小时以后，对每只小鼠进行MCMV病毒的腹腔注射，用量为5000000pfu/只。之后在D1(注射病毒24小时后)、D3(注射病毒72小时后)、D5(注射病毒120小时后)收集小鼠的肝脏和肺，用PBS溶液洗净。之后，用血液/细胞/组织基 因组DNA提取试剂盒(DP304，天根生物)提取细胞总DNA，分别以0.5μg来自肝脏的总DNA和0.1μg来自肺的总DNA作为模版，利用qPCR对各组检测病毒拷贝数并统计。结果如图8所示，与注射PBS并感染病毒的组相比，注射质粒K和KM的小鼠在肝脏和肺都呈现出对MCMV病毒明显的抑制作用，并且野生型MgR的抑制作用要强于突变体D284A。
以上研究表明，MgR及其突变体D284A在细胞内无毒性，并且可抑制多种病毒(包括DNA病毒和RNA)在细胞内复制。可能的作用机理在于：这些酶对RNA分子中的2’-O-甲基化修饰敏感，而动物细胞(包括人细胞)和植物细胞自身RNA中2’-O-甲基化修饰丰富，因此对这些宿主细胞本身基本无毒性；与之相反，病毒RNA基因组或病毒产生的RNA往往缺乏这种修饰而易于受到这些酶的影响，从而产生病毒抑制作用。另外，从MgR在细胞内发分布研究(实施例4)可知，其分布于细胞质和细胞核内，从而能对存在于细胞质和/或细胞核内的病毒RNA均具水解和/或解旋活性，也可能是MgR及其突变体具有广谱抗病毒能力的部分原因。
突变体D284A相对于野生型缺乏水解酶活性，但由于基本上不影响解旋酶活性，在抑制病毒复制方面效果与野生型类似。因此，我们推测，MgR的保留了解旋酶活性的其他突变体也应具有抗病毒效果。

Claims (49)

  1. 抗病毒药物，包括2’-O-甲基化敏感RNA相关酶或2’-O-甲基化敏感RNA相关酶的编码核酸序列。
  2. 如权利要求1所述的抗病毒药物，其中所述2’-O-甲基化敏感RNA相关酶为核糖核酸酶和/或RNA双链解旋酶。
  3. 如权利要求1或2所述的抗病毒药物，其中所述2’-O-甲基化敏感RNA相关酶为3’-5’方向核糖核酸酶和/或RNA双链解旋酶。
  4. 如权利要求1-3任一项所述的抗病毒药物，其中所述2’-O-甲基化敏感RNA相关酶包括SEQ ID NO：3或4所示氨基酸序列；或者，所述2’-O-甲基化敏感RNA相关酶包括与SEQ ID NO：3或4所示氨基酸序列有至少90％序列一致性的氨基酸序列并且具有对2’-O-甲基化敏感的核糖核酸酶和/或解旋酶活性。
  5. 如权利要求1-4任一项所述的抗病毒药物，其中所述2’-O-甲基化敏感RNA相关酶的氨基酸序列与SEQ ID NO：3所示氨基酸序列相比，包括D284A突变。
  6. 如权利要求1-5任一项所述的抗病毒药物，其包括携带有所述2’-O-甲基化敏感RNA相关酶的编码核酸序列的表达载体。
  7. 如权利要求1-6任一项所述的抗病毒药物，其中所述病毒为DNA病毒和/或RNA病毒。
  8. 如权利要求1-7任一项所述的抗病毒药物，其中所述病毒为动物病毒和/或植物病毒
  9. 如权利要求1-8任一项所述的抗病毒药物，其中所述病毒为MCMV病毒、EV71病毒或HSV-1病毒。
  10. 如权利要求1-9任一项所述的抗病毒药物，其为广谱抗病毒药物。
  11. 2’-O-甲基化敏感RNA相关酶或2’-O-甲基化敏感RNA相关酶的编码核酸序列在制备用于在受试者中预防或治疗病毒引起的疾病的药物中的用途。
  12. 如权利要求11所述的用途，其中所述2’-O-甲基化敏感RNA相关酶为核糖核酸酶和/或RNA双链解旋酶。
  13. 如权利要求11或12所述的用途，其中所述2’-O-甲基化敏感RNA相关酶为3’-5’方向核糖核酸酶和/或RNA双链解旋酶。
  14. 如权利要求11-13任一项所述的用途，其中所述2’-O-甲基化敏感RNA相关酶包括SEQ ID NO：3或4所示氨基酸序列；或者，所述2’-O-甲基化敏感RNA相关酶包括与SEQ ID NO：3或4所示氨基酸序列有至少90％序列一致性的氨基酸序列并且具有对2’-O-甲基化敏感的核糖核酸酶和/或解旋酶活性。
  15. 如权利要求11-14任一项所述的用途，其中所述2’-O-甲基化敏感RNA相关酶的氨基酸序列与SEQ ID NO：3所示氨基酸序列相比，包括D284A突变。
  16. 如权利要求11-15任一项所述的用途，其中所述2’-O-甲基化敏感RNA相关酶的编码核酸序列位于表达载体上。
  17. 如权利要求11-16任一项所述的用途，其中所述病毒为DNA病毒和/或RNA病毒。
  18. 如权利要求11-17任一项所述的用途，其中所述病毒为动物病毒和/或植物病毒。
  19. 如权利要求11-18任一项所述的用途，其中所述病毒为MCMV病毒、EV71病毒或HSV-1病毒。
  20. 如权利要求11-19任一项所述的用途，其中所述药物为广谱抗病毒药物。
  21. 如权利要求11-20任一项所述的用途，其中所述受试者为动物或植物。
  22. 如权利要求11-21任一项所述的用途，其中所述受试者为哺乳动物。
  23. 如权利要求11-21任一项所述的用途，其中所述受试者为人。
  24. 在受试者中预防或治疗病毒引起的疾病的方法，包括以治疗有效量的2’-O-甲基化敏感RNA相关酶、或2’-O-甲基化敏感RNA相关酶的编码核酸序列向所述受试者给药。
  25. 如权利要求24所述的方法，其中所述2’-O-甲基化敏感RNA相关酶为核糖核酸酶和/或RNA双链解旋酶。
  26. 如权利要求24或25所述的方法，其中所述2’-O-甲基化敏感RNA相关酶为3’-5’方向核糖核酸酶和/或RNA双链解旋酶。
  27. 如权利要求24-26任一项所述的方法，其中所述2’-O-甲基化敏感RNA相关酶包括SEQ ID NO：3或4所示氨基酸序列；或者，所述2’-O-甲基化敏感RNA相关酶包括与SEQ ID NO：3或4所示氨基酸序列有至少90％序列一致性的氨基酸序列并且具有对2’-O-甲基化敏感的核糖核酸酶和/或解旋酶活性。
  28. 如权利要求24-27任一项所述的方法，其中所述2’-O-甲基化敏感RNA相关酶的氨基酸序列与SEQ ID NO：3所示氨基酸序列相比，包括D284A突变。
  29. 如权利要求24-28任一项所述的方法，其中所述2’-O-甲基化敏感RNA相关酶的编码核酸序列位于表达载体上。
  30. 如权利要求24-29任一项所述的方法，其中所述病毒为DNA病毒和/或RNA病毒。
  31. 如权利要求24-30任一项所述的方法，其中所述病毒为动物病毒和/或植物病毒。
  32. 如权利要求24-31任一项所述的方法，其中所述病毒为MCMV病毒、EV71病毒或HSV-1病毒。
  33. 如权利要求24-32任一项所述的方法，其中所述受试者有感染两种或两种以上病毒的风险，或者所述受试者被两种或两种以上病毒感染。
  34. 如权利要求24-33任一项所述的方法，其中所述受试者为动物或植物。
  35. 如权利要求24-34任一项所述的方法，其中所述受试者为哺乳动物。
  36. 如权利要求24-35任一项所述的方法，其中所述受试者为人。
  37. 在受试者中预防或治疗病毒引起的疾病的方法，包括：
    1)从所述受试者取出部分细胞；
    2)让所述细胞与2’-O-甲基化敏感RNA相关酶接触，或者将所述细胞进行修饰以使其能够表达2’-O-甲基化敏感RNA相关酶；以及
    3)将所述细胞回输到所述受试者体内。
  38. 如权利要求37所述的方法，其中所述2’-O-甲基化敏感RNA相关酶为权利要求1-36任一项提及的2’-O-甲基化敏感RNA相关酶。
  39. 如权利要求37或38所述的方法，其中所述细胞为权利要求1-36任一项提及的细胞。
  40. 如权利要求37-39任一项所述的方法，其中所述病毒为权利要求1-36任一项提及的病毒。
  41. 如权利要求37-40任一项所述的方法，其中所述受试者为权利要求1-36任一项提及的受试者。
  42. 抗病毒细胞，包括2’-O-甲基化敏感RNA相关酶或经修饰而能够表达2’-O-甲基化敏感RNA相关酶。
  43. 如权利要求42所述的抗病毒细胞，其中所述2’-O-甲基化敏感RNA相关酶为权利要求1-36任一项提及的2’-O-甲基化敏感RNA相关酶。
  44. 如权利要求42或43所述的抗病毒细胞，其为权利要求1-36任一项提及的细胞。
  45. 如权利要求42-44任一项所述的抗病毒细胞，其为CHO-K1细胞、Hela细胞、或Vero细胞。
  46. 权利要求42-45任一项所述的抗病毒细胞在制备用于预防或治疗病毒引起的疾病的药物中的用途。
  47. 抗病毒非人动物或植物，其包括权利要求42-45任一项所述的抗病毒细胞。
  48. 保存细胞的方法，包括让所述细胞与2’-O-甲基化敏感RNA相关酶接触，或者将所述细胞进行修饰以使其能够表达2’-O-甲基化敏感RNA相关酶。
  49. 抑制病毒在细胞中复制的方法，包括让所述细胞与2’-O-甲基化敏感RNA相关酶接触，或者将所述细胞进行修饰以使其能够表达2’-O-甲基化敏感RNA相关酶。
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