WO2019149265A1 - 一种用于治疗肿瘤的伪狂犬病毒 - Google Patents

一种用于治疗肿瘤的伪狂犬病毒 Download PDF

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WO2019149265A1
WO2019149265A1 PCT/CN2019/074349 CN2019074349W WO2019149265A1 WO 2019149265 A1 WO2019149265 A1 WO 2019149265A1 CN 2019074349 W CN2019074349 W CN 2019074349W WO 2019149265 A1 WO2019149265 A1 WO 2019149265A1
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prv
cancer
tumor
sequence
virus
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PCT/CN2019/074349
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English (en)
French (fr)
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陈毅歆
王国松
袁权
曹佳莉
林丽娜
夏宁邵
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厦门大学
养生堂有限公司
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Priority to US16/966,345 priority Critical patent/US20220110988A1/en
Priority to CA3090825A priority patent/CA3090825A1/en
Priority to EP19747565.0A priority patent/EP3747997A4/en
Priority to JP2020542281A priority patent/JP7132339B2/ja
Priority to AU2019214179A priority patent/AU2019214179B2/en
Publication of WO2019149265A1 publication Critical patent/WO2019149265A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A61K35/763—Herpes virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from 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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16711—Varicellovirus, e.g. human herpesvirus 3, Varicella Zoster, pseudorabies
    • C12N2710/16721—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16711—Varicellovirus, e.g. human herpesvirus 3, Varicella Zoster, pseudorabies
    • C12N2710/16722—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16711—Varicellovirus, e.g. human herpesvirus 3, Varicella Zoster, pseudorabies
    • C12N2710/16732—Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent

Definitions

  • the invention relates to the field of viruses and the field of tumor therapy.
  • the invention relates to pseudorabies virus (PRV) or a modified form thereof, or a nucleic acid molecule comprising a genomic sequence of PRV or a modified form thereof, for use in treating a tumor (e.g., human) and/or reducing Or the use of inhibiting tumor recurrence, and the use in the manufacture of a medicament for treating a tumor in a subject (eg, a human) and/or reducing or inhibiting tumor recurrence.
  • PRV pseudorabies virus
  • the invention also relates to a method of treating a tumor and/or reducing or inhibiting tumor recurrence comprising administering to a subject in need thereof a PRV or a modified form thereof, or a nucleic acid molecule comprising a genomic sequence of PRV or a modified form thereof step.
  • the treatment of malignant tumors mainly includes surgery, chemotherapy and radiotherapy. These traditional therapies are not satisfactory for the treatment of metastatic tumors, and they can also cause great harm to the health of patients.
  • Oncolytic virus is a virus that can self-replicate in tumor cells, thereby killing, dissolving, or stagnating tumor cells.
  • oncolytic viruses When treated in vivo, oncolytic viruses exhibit selective specificity for tumor cells and can directly induce tumor cell death with little or no effect on normal cells; at the same time, oncolytic viruses can also stimulate the immune system. B lymphocytes react with T lymphocytes to indirectly kill tumor cells. Therefore, oncolytic virus is considered to be a promising treatment for cancer.
  • Pseudorabies virus also known as porcine herpesvirus type I, infectious medullary palsy virus, pruritus virus, and Aozhsky disease virus, belongs to the genus Herpesvirus subfamily varicella. It is a herpes virus that causes fever, itching (except pigs) and encephalomyelitis, which are the main symptoms of cattle, sheep, pigs, dogs and cats. Pigs are the natural host of pseudorabies virus. Symptoms of acute infectious diseases such as fever, diarrhea, dyspnea, encephalomyelitis, and reproductive disorders.
  • the pseudorabies virus has the following characteristics: First, as a double-stranded DNA virus, its genome does not integrate the genome into the host chromosome in the host; secondly, although the pseudorabies virus is prevalent in other lower mammals, it has not yet been officially The literature reports that it can infect humans and cause disease; again, pseudorabies virus does not contain oncogenes. At present, pseudorabies virus is widely used as a veterinary vaccine, and there is no report that any pseudorabies virus has oncolytic activity in the field.
  • PRV Perdorabies virus
  • Alpha herpesvirus subfamily Alpha herpesvirus subfamily
  • Varicellovirus varicella virus
  • the genome of the virus is linear double-stranded DNA with a size between 130 kd and 150 kd.
  • the genome of the virus consists of a unique long region sequence (UL) and a unique short region sequence (US) with terminal repeats (TRRs) and internal repeats (IRS) on both sides of the US.
  • UL unique long region sequence
  • US unique short region sequence
  • TRRs terminal repeats
  • IRS internal repeats
  • Genes encoding structural proteins include US2 (28K), US3 (PK), US4 (gG), US6 (gD), US7 (gI), US8 (gE), US9 (11K) and other genes located in the US region and located in the UL region.
  • Non-structural protein genes located in the UL region include UL54 capable of participating in transcriptional regulation.
  • PRV Perdorabies virus
  • PRV wild type PRV which can be isolated from a source in nature and not intentionally modified, and examples thereof include a prototype strain Bartha-K61 strain (BK61) And the strain HB-98, and various specimen isolates (for example, the isolates described in Example 1 of the present invention).
  • Wild type PRV genomic sequences are well known in the art and can be found in various public databases (e.g., GenBank database accession number: JF797217.1).
  • the term "oncolytic virus” refers to a virus that is capable of infecting tumor cells, replicating in tumor cells, causing tumor cell death, lysis, or preventing tumor cell growth.
  • the virus has minimal toxic effects on non-tumor cells.
  • tumor specific refers to selectively exhibiting biological function or activity within a tumor cell.
  • the term “tumor specific” when used to describe the kill selectivity of a virus, it means that the virus is capable of selectively killing tumor cells without killing or substantially killing non-tumor cells, or the virus Killing of tumor cells is more effective than killing of non-tumor cells.
  • the term "oncolysis activity" primarily includes tumor killing activity.
  • the oncolytic activity of the virus can usually be measured by its ability to infect tumor cells, the ability to replicate in tumor cells, and/or the ability to kill tumor cells.
  • the oncolytic activity of the virus can be determined by any method known in the art. For example, the ability of a virus to infect a tumor cell can be assessed by measuring the amount of virus required to infect a given percentage of tumor cells (eg, 50% of cells); the ability to replicate within a tumor cell can be measured by measuring the growth of the virus in the tumor cell. Evaluation; the ability to kill tumor cells can be evaluated by observing cytopathic effect (CPE) or measuring tumor cell activity.
  • CPE cytopathic effect
  • modified form of a virus refers to a modified virus obtained by modifying a wild type virus that retains the desired activity (eg, oncolytic activity) of the wild type virus.
  • a "modified form" of a PRV includes, but is not limited to, a modified PRV virus having one or more nucleotide substitutions, insertions or deletions compared to the genomic sequence of the wild-type PRV, and at least retained Oncolytic activity of PRV.
  • the modified form or modified PRV of the PRV of the invention is not limited by its mode of production.
  • a modified form of a PRV of the invention or a modified PRV can be produced by homologous recombination, or by culturing a host cell infected with the modified form or modified PRV.
  • EP0 protein refers to the early protein 0 of the PRV virus, which is encoded by the EP0 gene and is a transcriptional activator of early expression of the PRV virus.
  • the amino acid sequence of the EP0 protein is known and can be seen, for example, in public databases (e.g., EM64001.1).
  • the expression "does not express a functional EP0 protein” means that the viral or viral genome is unable to produce or express a biologically functional EPO protein when the virus or viral genome infects the cell.
  • the viral or viral genome may not produce or express an EPO protein at all due to a gene deletion, or may produce or express an EP0 protein that is not biologically functionally active due to a loss-of-function mutation.
  • Loss of function mutation refers to a mutation that results in the loss of its biologically functional activity by a protein encoded and expressed by the mutated gene.
  • Loss-of-function mutations include, but are not limited to, missense mutations, nonsense mutations, frameshift mutations, base deletions, base substitutions, base additions, and any combination thereof (eg, deletion or substitution or addition of a gene fragment), As long as the gene containing the loss-of-function mutation of the function cannot produce or express a protein having biologically functional activity.
  • cDNA sequence of PRV means a DNA sequence obtained by reverse transcription of mRNA transcribed from the viral genome as a template, which differs from the genomic sequence only in the cDNA sequence. Intron sequences in the genomic sequence are not included.
  • exogenous nucleotide sequence refers to an artificially introduced nucleotide sequence that is foreign to the original sequence.
  • Exogenous nucleotide sequences include, but are not limited to, any gene or nucleotide sequence not found in the viral genome.
  • the exogenous nucleotide sequence encodes a polypeptide having a therapeutic use, such as an immunomodulatory polypeptide, a cytokine, a chemokine, an anti-tumor protein or polypeptide, and the like.
  • immunomodulatory polypeptide refers to a polypeptide that modulates the function of immune cells, examples of which include, but are not limited to, CD40L, OX40L, inducible costimulatory molecules (ICOS), FTL3L, LIGHT, CD137L, CD70, 4-1BB, GITR, and CD28 (see, for example, Khalil D N, Smith E L, Brentjens R J, et al. The future of cancer treatment: immunomodulation, CARs and combination immunotherapy [J]. Nat Rev Clin Oncol, 2016, 13(5): 273-290).
  • cytokine has the meanings well known to those skilled in the art.
  • the oncolytic virus of the present invention when used to treat a tumor, it is particularly preferable that the cytokine is a cytokine which can be used for tumor treatment.
  • cytokines include, but are not limited to, interleukins (eg, IL-2, IL-12, and IL-15), interferons (eg, IFN ⁇ , IFN ⁇ , IFN ⁇ ), tumor necrosis factors (eg, TNF ⁇ ), colony stimulating factors ( For example, GM-CSF), and any combination thereof (see, for example, Ardolino M, Hsu J, Raulet D H. Cytokine treatment in cancer immunotherapy [J]. Oncotarget, 2015, 6(23): 19346-19347).
  • chemokine has the meanings well known to those skilled in the art. However, in the method of the present invention, when the oncolytic virus of the present invention is used to treat a tumor, it is particularly preferred that the cytokine is a chemokine capable of being used for tumor treatment.
  • chemokines include, but are not limited to, CCL2, RANTES, CCL7, CCL9, CCL10, CCL12, CCL15, CCL19, CCL21, CCL20, XCL-1, and any combination thereof (Homey B, Muller A, Zlotnik A. CHEMOKINES: AGENTS FOR THE IMMUNOTHERAPY OF CANCER [J]. Nat Rev Immunol, 2002, 2: 175-184).
  • anti-tumor protein or polypeptide refers to a protein or polypeptide having therapeutic tumor activity, including but not limited to: (1) toxic to cells, inhibition of cell proliferation, or induction of apoptosis.
  • proteins or polypeptides include, but are not limited to, thymidine kinase TK (TK/GCV), TRAIL, and FasL (see, for example, Candolfi M, King GD, Arabic AG, et al. Evaluation of proapototic transgenes to use in combination with Flt3L In an immune-stimulatory gene therapy approach for Glioblastoma multiforme (GBM) [J].
  • a protein or polypeptide having an immunotherapeutic effect such as an immunological checkpoint inhibitor, examples of which include But not limited to anti-PD-1 antibody, anti-PD-L1 antibody, anti-TIGIT antibody, anti-BTLA antibody, anti-CTLA-4 antibody, anti-Tim-3 antibody, anti-Lag-3 antibody, anti-CD137 antibody, anti-OX40 antibody, anti- GITR antibody, anti-CD73 antibody, anti-KIR antibody, anti-ICOS antibody, anti-CSF1R antibody (see, for example, Nolan E, Savas P, Policheni AN, et al. Combined immune checkpoint blockade as a therapeutic strategy for BRCA1-mutated br East cancer [J].
  • an immunological checkpoint inhibitor examples of which include But not limited to anti-PD-1 antibody, anti-PD-L1 antibody, anti-TIGIT antibody, anti-BTLA antibody, anti-CTLA-4 antibody, anti-Tim-3 antibody, anti-Lag-3 antibody, anti-CD137 antibody, anti-OX40 antibody, anti-
  • Tumor-specific targeting antibodies examples of which include, but are not limited to, anti-HER2 antibodies (such as Herceptin) , anti-CD20 antibody (such as rituximab), anti-VEGF antibody (such as bevacizumab), anti-EGFR antibody (such as cetuximab) or any combination thereof; (4) protein that inhibits tumor angiogenesis Or polypeptides, examples of which include, but are not limited to, anti-VEGF single-chain antibodies (scFv), VEGF-derived polypeptides (eg D (LPR), KSVRGKGKGQKRKRKKSRYK, etc.) and ATN-161 (see, eg, Rosca EV) , Koskimaki JE, Rivera CG, et al. Anti-angiogenic peptides for cancer therapeutics [J]. Curr Pharm Biotechnol, 2011, 12(8): 1101 - 1116; all
  • identity is used to mean the matching of sequences between two proteins/polypeptides or between two nucleic acids.
  • a position in the two sequences being compared is occupied by the same base or amino acid monomer subunit (for example, a position in each of the two DNA molecules is occupied by adenine, or two Each position in each of the proteins/polypeptides is occupied by lysine), then each molecule is identical at that position.
  • the "percent identity" between the two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions to be compared x 100. For example, if 6 of the 10 positions of the two sequences match, then the two sequences have 60% identity.
  • the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of a total of 6 positions match).
  • the comparison is made when the two sequences are aligned to produce maximum identity.
  • Such alignment can be achieved by, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48: 443-453, which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc.). It is also possible to use the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4: 11-17 (1988)) integrated into the ALIGN program (version 2.0), using the PAM 120 weight residue table.
  • the gap length penalty of 12 and the gap penalty of 4 were used to determine the percent identity between the two amino acid sequences.
  • the Needleman and Wunsch (J MoI Biol. 48: 444-453 (1970)) algorithms in the GAP program integrated into the GCG software package can be used, using the Blossum 62 matrix or The PAM250 matrix and the gap weight of 16, 14, 12, 10, 8, 6 or 4 and the length weight of 1, 2, 3, 4, 5 or 6 to determine the percent identity between two amino acid sequences .
  • vector refers to a nucleic acid vehicle into which a polynucleotide can be inserted.
  • a vector is referred to as an expression vector when the vector enables expression of the protein encoded by the inserted polynucleotide.
  • the vector can be introduced into the host cell by transformation, transduction or transfection, and the genetic material element carried thereby can be expressed in the host cell.
  • Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1 derived artificial chromosomes (PAC).
  • Phage such as lambda phage or M13 phage and animal virus.
  • Animal viruses useful as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, nipples Multi-tumor vacuolar virus (such as SV40).
  • a vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain an origin of replication.
  • promoter has the meaning well-known to those skilled in the art and refers to a non-coding nucleotide sequence located upstream of the gene that initiates expression of the downstream gene.
  • pharmaceutically acceptable carrier and/or excipient refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and the active ingredient.
  • a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and the active ingredient.
  • pH adjusting agents include, but are not limited to, phosphate buffers.
  • Surfactants include, but are not limited to, cationic, anionic or nonionic surfactants such as Tween-80.
  • Ionic strength enhancers include, but are not limited to, sodium chloride.
  • Agents that maintain osmotic pressure include, but are not limited to, sugars, NaCl, and the like.
  • Agents that delay absorption include, but are not limited to, monostearate and gelatin.
  • Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerin), and the like.
  • Adjuvants include, but are not limited to, aluminum adjuvants (e.g., aluminum hydroxide), Freund's adjuvant (e.g., complete Freund's adjuvant), and the like.
  • Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, and the like.
  • Stabilizers have the meaning commonly understood by those skilled in the art to stabilize the desired activity (eg, oncolytic activity) of the active ingredient in the drug, including but not limited to sodium glutamate, gelatin, SPGA, sugars (eg, sorbitol, Mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin or casein) or degradation products (such as lactalbumin hydrolysate) )Wait.
  • sugars eg, sorbitol, Mannitol, starch, sucrose, lactose, dextran, or glucose
  • amino acids such as glutamic acid, glycine
  • proteins such as dried whey, albumin or casein
  • degradation products such as lactalbumin hydrolysate
  • treating refers to treating or curing a disease (eg, a tumor), delaying the onset of symptoms of a disease (eg, a tumor), and/or delaying the progression of a disease (eg, a tumor).
  • a disease eg, a tumor
  • delaying the onset of symptoms of a disease eg, a tumor
  • delaying the progression of a disease eg, a tumor
  • a therapeutically effective amount refers to an amount that can effectively achieve the intended purpose.
  • a therapeutically effective amount can be an amount effective or sufficient to treat or cure a disease (eg, a tumor), delay the onset of symptoms of a disease (eg, a tumor), and/or delay the progression of a disease (eg, a tumor).
  • a disease eg, a tumor
  • delay the onset of symptoms of a disease eg, a tumor
  • a tumor e.g, a tumor
  • a tumor e.g., a tumor
  • Such an effective amount can be readily determined by one skilled in the art or by a physician, and can be related to the intended purpose (eg, treatment), the general health of the subject, age, sex, weight, severity of the disease to be treated, complications, The mode of administration is related. Determination of such an effective amount is well within the capabilities of those skilled in the art.
  • the term "subject” refers to a mammal, such as a primate mammal, such as a human.
  • the subject eg, a human
  • the inventors of the present application have surprisingly discovered that the pseudorabies virus (PRV) has a broad spectrum and remarkable tumor cell killing ability after extensive experiments and repeated explorations. Based on this finding, the inventors have developed a new oncolytic virus for treating tumors and a tumor treatment method based on the virus.
  • PRV pseudorabies virus
  • the invention provides a pseudorabies virus (PRV) or a modified form thereof or a nucleic acid molecule for use in treating a tumor in a subject and/or reducing or inhibiting tumor recurrence, or The use of a medicament for treating a tumor and/or reducing or inhibiting tumor recurrence in a subject; wherein the nucleic acid molecule comprises a sequence selected from the group consisting of:
  • the PRV is a wild-type PRV.
  • the PRV can be a strain isolated from an animal infected with pseudorabies virus (PRV).
  • the genomic sequence of the PRV or modified form thereof has at least 70%, at least 80%, at least 85%, at least 90%, and the nucleotide sequence set forth in SEQ ID NO: At least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.
  • the genomic sequence of the PRV or modified form thereof is the nucleotide sequence set forth in SEQ ID NO: 1.
  • the modified form is a modified PRV having one or more nucleotide substitutions, insertions or deletions in the genome as compared to wild-type PRV.
  • the modified PRV has one or more modifications selected from the group consisting of:
  • the modified PRV does not express a functional EPO protein.
  • functional expression of the protein can be prevented by engineering a gene encoding the EPO protein (EP0 gene).
  • EP0 gene a gene encoding the EPO protein
  • a loss-of-function mutation can be introduced into a gene encoding an EPO protein, or a gene encoding an EPO protein can be deleted or substituted into an exogenous nucleotide sequence (for example, a nucleotide sequence encoding a foreign protein), thereby preventing Functional expression of the protein of interest.
  • the genome of the modified PRV of the invention is modified such that the EP0 gene comprises a loss-of-function mutation (eg, addition, deletion, and/or substitution of one or more nucleotides) Alternatively, it is deleted or replaced with an exogenous nucleotide sequence (eg, a nucleotide sequence encoding a foreign protein).
  • the loss-of-function mutation is selected from the group consisting of a missense mutation, a nonsense mutation, a frameshift mutation, a base deletion, a base substitution, a base addition, and any combination thereof (eg, a gene fragment) Missing or replacing or adding).
  • the EP0 gene is deleted. In such embodiments, the modified PRV does not express an EPO protein. In certain preferred embodiments, the EP0 gene is replaced with an exogenous nucleotide sequence (eg, a nucleotide sequence encoding a foreign protein). In certain exemplary embodiments, the foreign protein is a fluorescent protein.
  • the genomic sequence of the modified PRV has at least 70%, at least 80%, at least 85%, at least 90%, compared to the nucleotide sequence set forth in SEQ ID NO: At least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.
  • the genomic sequence of the modified PRV is set forth in SEQ ID NO:4.
  • the modified PRV comprises a mutation, deletion or insertion of one or more nucleotides located in a non-coding region, such as a promoter.
  • the genome of the modified PRV comprises a modification in which the native promoter of one or more PRV genes is replaced with a tumor-specific promoter.
  • the native promoter of any PRV gene can be replaced with a tumor-specific promoter.
  • the tumor-specific promoter is selected from the group consisting of a human telomere reverse transcriptase (hTERT) promoter, an alpha-fetoprotein promoter, a carcinoembryonic antigen promoter, a prostate specific antigen promoter, and Survivin Promoter and cyclooxygenase COX-2 promoter.
  • the use of a tumor-specific promoter is advantageous in certain circumstances, for example, to enhance the tumor specificity of an oncolytic virus.
  • the activity of tumor-specific promoters is usually at a lower level, while in tumor cells its activity is at a higher level. Therefore, the use of tumor-specific promoters to regulate the transcriptional expression of some PRV key proteins allows the virus to acquire tumor-specific killing ability. Therefore, in some cases, replacing the native promoter of a key protein of PRV with a tumor-specific promoter can reduce the virulence of the virus against normal cells, without affecting its ability to kill tumor cells.
  • the modified PRV comprises an exogenous nucleotide sequence.
  • the exogenous nucleotide sequence encodes a foreign protein selected from the group consisting of a fluorescent protein, an immunomodulatory polypeptide, a cytokine, a chemokine, an anti-tumor protein, or a polypeptide.
  • the fluorescent protein is selected from the group consisting of green fluorescent protein, red fluorescent protein, blue fluorescent protein, yellow fluorescent protein, and any combination thereof.
  • the immunomodulatory polypeptide is selected from the group consisting of CD40L, OX40L, inducible costimulatory molecules (ICOS), FTL3L, LIGHT, CD137L, CD70, 4-1BB, GITR, CD28, and any combination thereof.
  • the cytokine is selected from the group consisting of an interleukin (eg, IL-2, IL-12, and IL-15), an interferon (eg, IFN ⁇ , IFN ⁇ , IFN ⁇ ), a tumor necrosis factor (eg, TNF ⁇ ) , colony stimulating factors (eg, GM-CSF), and any combination thereof.
  • an interleukin eg, IL-2, IL-12, and IL-15
  • an interferon eg, IFN ⁇ , IFN ⁇ , IFN ⁇
  • TNF ⁇ tumor necrosis factor
  • colony stimulating factors eg, GM-CSF
  • the chemokine is selected from the group consisting of CCL2, RANTES, CCL7, CCL9, CCL10, CCL12, CCL15, CCL19, CCL21, CCL20, XCL-1, and any combination thereof.
  • the anti-tumor protein or polypeptide is selected from the group consisting of: a cytotoxic peptide, such as thymidine kinase TK (TK/GCV), TRAIL, FasL, or any combination thereof; an immunological checkpoint inhibitor, such as an antibody PD-1 antibody, anti-PD-L1 antibody, anti-TIGIT antibody, anti-BTLA antibody, anti-CTLA-4 antibody, anti-Tim-3 antibody, anti-Lag-3 antibody, anti-CD137 antibody, anti-OX40 antibody, anti-GITR antibody, anti- CD73 antibody, anti-KIR antibody, anti-ICOS antibody, anti-CSF1R antibody or any combination thereof; tumor-specific targeting antibody, such as anti-HER2 antibody (such as Herceptin), anti-CD20 antibody (such as rituximab), anti- a VEGF antibody (such as bevacizumab), an anti-EGFR antibody (such as cetuximab), or any combination thereof; a protein or polypeptide, such as
  • the modified PRV comprises at least one insertion of an exogenous nucleotide sequence as described above and/or at least one of the untranslated regions (eg, a promoter) as described above. Mutation, deletion or insertion.
  • the modified PRV does not express a functional EPO protein and comprises at least one insertion of an exogenous nucleotide sequence as described above and/or at least one of which is located as described above Mutations, deletions or insertions of untranslated regions (eg promoters).
  • the modified PRV of the present invention can be obtained by techniques well known in the art.
  • a loss-of-function mutation can be introduced into a viral gene by deletion, substitution or insertion of a base to functionally inactivate the viral gene.
  • the viral gene is functionally inactivated by deletion (eg, deletion of the entire gene or portion thereof).
  • deletion eg, deletion of the entire gene or portion thereof.
  • at least 25%, at least 50%, at least 75%, or 100% of the sequence of the viral gene of interest can be deleted, or at least 10 bp, at least 100 bp, or at least 1000 bp of the viral gene sequence of interest can be deleted.
  • a frameshift mutation is caused by insertion or deletion of a base, thereby rendering the viral gene functionally inactive.
  • the viral gene is functionally inactivated by replacing the entire gene of interest or a portion thereof with an exogenous nucleotide sequence.
  • the modified PRV of the invention can be obtained by the CRISPR/Cas9 technology.
  • the CRISPR/Cas9 technology is known in the art, for example, see Ran FA, Feng Zhang et al., Nature, 2013, 2281-2308; which is incorporated herein by reference in its entirety.
  • the viral genome of the wild-type PRV is typically modified (eg, insertion of an exogenous nucleotide sequence, deletion or mutation of an endogenous gene or mutation in an untranslated region) to obtain a modified PRV. .
  • the PRV of the invention can be pretreated to reduce or eliminate the subject's immune response to the virus, wherein the pretreatment can comprise: packaging the PRV in a liposome or micelle, and / Alternatively, the protease capsid protein can be removed using a protease (eg, chymotrypsin or trypsin) to reduce host humoral and/or cellular immunity to the virus.
  • a protease eg, chymotrypsin or trypsin
  • a PRV or a modified form thereof as described herein can be continuously adapted for passage within a tumor cell.
  • the tumor cells can be tumor cell lines or tumor cell lines known in the art, or can be surgically resected or clinically performed from an individual (eg, a subject) having a tumor. Isolated tumor cells.
  • the PRV or a modified form thereof is continuously adaptively passaged within tumor cells obtained from an individual (eg, a subject) having a tumor.
  • the tumor cells are obtained by surgical resection or clinical isolation from an individual (eg, a subject) having a tumor.
  • the method of continuous adaptive passage comprises a plurality (eg, at least 5, at least 10, at least 15, at least 20) of cycles consisting of: 1) The virus infects the target tumor cells; 2) harvests the virus in the supernatant; and 3) reinfects the obtained virus with fresh target tumor cells.
  • the PRVs described above and modified forms thereof can be used in combination.
  • the medicament may include one or more of PRV and modified forms thereof.
  • the nucleic acid molecule consists of a genomic sequence or cDNA sequence of PRV or a modified form thereof as described herein, or a complement of the cDNA sequence.
  • the nucleic acid molecule has a genomic sequence of a PRV or a modified form thereof as described herein. In certain preferred embodiments, the nucleic acid molecule has a nucleotide sequence selected from the group consisting of:
  • nucleotide sequence identity (2) having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94 with the nucleotide sequence as shown in SEQ ID NO: 1 or 4. %, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% nucleotide sequence identity.
  • the nucleic acid molecule is a vector comprising a genomic sequence or cDNA sequence of a PRV or a modified form thereof as described herein, or a complement of the cDNA sequence (eg, a cloning vector or expression vector) ).
  • the nucleic acid molecule is a vector (eg, a cloning vector or an expression vector) comprising a cDNA sequence of a PRV or a modified form thereof as described herein, or a complement of the cDNA sequence.
  • the nucleic acid molecules of the invention can be delivered by any means known in the art, for example, by direct injection of a naked nucleic acid molecule (e.g., naked RNA), or by the use of a non-viral delivery system.
  • the non-viral delivery system can be obtained by preparation of various materials well known in the art including, but not limited to, "Yin H, et al. Nat Rev Genet. 2014 Aug; 15(8): 541-55. And various materials in "Riley MK, Vermerris W. Nanomaterials (Basel). 2017 Apr 28; 7 (5). pii: E94.”, all of which are incorporated herein by reference, for example, liposomes, inorganic nanoparticles (such as gold nanoparticles), polymers (such as PEG) and so on.
  • the medicament comprises a therapeutically effective amount of PRV and/or a modified form thereof, or a therapeutically effective amount of a nucleic acid molecule as described herein.
  • the drug can be in any form known in the medical arts.
  • the drug may be a tablet, a pill, a suspension, an emulsion, a solution, a gel, a capsule, a powder, a granule, an elixir, a lozenge, a suppository, an injection (including an injection, a lyophilized powder) Etc.
  • the drug is an injectable solution or a lyophilized powder.
  • the medicament further comprises a pharmaceutically acceptable carrier or excipient. In certain preferred embodiments, the medicament comprises a stabilizer.
  • the medicament optionally further comprises an additional pharmaceutically active agent.
  • the additional pharmaceutically active agent is a drug having anti-tumor activity, such as an additional oncolytic virus, chemotherapeutic agent or immunotherapeutic agent.
  • the additional oncolytic virus includes, but is not limited to, an adenovirus, parvovirus, reovirus, Newcastle disease virus, vesicular stomatitis virus, measles virus, or any combination thereof.
  • the chemotherapeutic agent includes, but is not limited to, 5-fluorouracil, mitomycin, methotrexate, hydroxyurea, cyclophosphamide, dacarbazine, mitoxantrone, anthracycline (such as epirubicin or more) [rectin], etoposide, a platinum compound (such as carboplatin or cisplatin), a taxane (such as paclitaxel or taxotere), or any combination thereof.
  • the immunotherapeutic agent includes, but is not limited to, an immunological checkpoint inhibitor (such as a PD-L1/PD-1 inhibitor or a CTLA-4 inhibitor), a tumor-specific targeting antibody (such as rituximab or Herceptin). ) or any combination thereof.
  • an immunological checkpoint inhibitor such as a PD-L1/PD-1 inhibitor or a CTLA-4 inhibitor
  • a tumor-specific targeting antibody such as rituximab or Herceptin.
  • the medicament comprises a unit dose of PRV and/or a modified form thereof, for example comprising at least 1 x 10 2 pfu, at least 1 x 10 3 pfu, at least 1 x 10 4 pfu, 1 x 10 5 pfu, 1 ⁇ 10 6 pfu, at least 1 ⁇ 10 7 pfu, at least 1 ⁇ 10 8 pfu, at least 1 ⁇ 10 9 pfu, at least 1 ⁇ 10 10 pfu, at least 1 ⁇ 10 11 pfu, at least 1 ⁇ 10 12 pfu At least 1 x 10 13 pfu, at least 1 x 10 14 pfu or at least 1 x 10 16 pfu of PRV and/or a modified form thereof.
  • the medicament comprises from 1 x 10 2 pfu to 1 x 10 17 pfu of PRV and/or a modified form thereof.
  • the medicament contains a unit dose of a nucleic acid molecule as described herein, for example, containing from 1 x 10 10 to 5 x 10 14 (eg, 3 x 10 10 to 3 x 10 14 ) viral genomes
  • a nucleic acid molecule as described herein, for example, containing from 1 x 10 10 to 5 x 10 14 (eg, 3 x 10 10 to 3 x 10 14 ) viral genomes
  • the nucleic acid molecule of the virus genome copies are examples of viruses.
  • the drug can be administered with additional therapies.
  • additional therapies can be any therapy known to be used in tumors, such as surgery, chemotherapy, radiation therapy, immunotherapy, hormone therapy or gene therapy. This additional therapy can be administered prior to, concurrently with, or after administration of the drug.
  • the tumor is selected from the group consisting of a glioma, a neuroblastoma, a gastric cancer, a liver cancer, a kidney cancer, a lung cancer, a breast cancer, a colon cancer, a lymphoma, an ovarian cancer, a cervical cancer, and an uterus.
  • the tumor recurrence is a recurrence of liver cancer.
  • the subject is a mammal, such as a human.
  • the invention provides a method of treating a tumor and/or reducing or inhibiting tumor recurrence comprising administering to a subject in need thereof an effective amount of PRV or a modified form thereof, or an effective amount of a nucleic acid molecule And wherein the nucleic acid molecule comprises a sequence selected from the group consisting of:
  • the subject is administered a PRV.
  • the PRV is a wild-type PRV.
  • the PRV can be a strain isolated from an animal infected with pseudorabies virus (PRV).
  • the genomic sequence of the PRV or modified form thereof has at least 70%, at least 80%, at least 85%, at least 90%, and the nucleotide sequence set forth in SEQ ID NO: At least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.
  • the genomic sequence of the PRV or modified form thereof is the nucleotide sequence set forth in SEQ ID NO: 1.
  • the subject is administered a modified form of PRV.
  • the modified form is a modified PRV having one or more nucleotide substitutions, insertions or deletions in the genome as compared to wild-type PRV.
  • the modified PRV has one or more modifications selected from the group consisting of:
  • the modified PRV does not express a functional EPO protein.
  • the genome of a modified PRV of the invention comprises a modification comprising an EP0 gene comprising a loss-of-function mutation (eg, addition, deletion, and/or substitution of one or more nucleotides) or It is deleted or replaced with an exogenous nucleotide sequence (for example, a nucleotide sequence encoding a foreign protein).
  • the loss-of-function mutation is selected from the group consisting of a missense mutation, a nonsense mutation, a frameshift mutation, a base deletion, a base substitution, a base addition, and any combination thereof (eg, a gene fragment) Missing or replacing or adding).
  • the EP0 gene is deleted. In such embodiments, the modified PRV does not express an EPO protein. In certain preferred embodiments, the EP0 gene is replaced with an exogenous nucleotide sequence (eg, a nucleotide sequence encoding a foreign protein).
  • the genomic sequence of the modified PRV has at least 70%, at least 80%, at least 85%, at least 90%, compared to the nucleotide sequence set forth in SEQ ID NO: At least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.
  • the genomic sequence of the modified PRV is set forth in SEQ ID NO:4.
  • the modified PRV comprises a mutation, deletion or insertion of one or more nucleotides located in a non-coding region, such as a promoter.
  • the genome of the modified PRV comprises a modification in which the native promoter of one or more PRV genes is replaced with a tumor-specific promoter.
  • the native promoter of any PRV gene can be replaced with a tumor-specific promoter.
  • the tumor-specific promoter is selected from the group consisting of a hTERT promoter, an alpha-fetoprotein promoter, a carcinoembryonic antigen promoter, a prostate-specific antigen promoter, a Survivin promoter, and a cyclooxygenase COX. -2 promoter.
  • the modified PRV comprises an exogenous nucleotide sequence.
  • the exogenous nucleotide sequence encodes a foreign protein selected from the group consisting of a fluorescent protein, an immunomodulatory polypeptide, a cytokine, a chemokine, an anti-tumor protein, or a polypeptide.
  • the fluorescent protein is selected from the group consisting of green fluorescent protein, red fluorescent protein, blue fluorescent protein, yellow fluorescent protein, and any combination thereof.
  • the immunomodulatory polypeptide is selected from the group consisting of CD40L, OX40L, inducible costimulatory molecules (ICOS), FTL3L, LIGHT, CD137L, CD70, 4-1BB, GITR, CD28, and any combination thereof.
  • the cytokine is selected from the group consisting of an interleukin (eg, IL-2, IL-12, and IL-15), an interferon (eg, IFN ⁇ , IFN ⁇ , IFN ⁇ ), a tumor necrosis factor (eg, TNF ⁇ ) , colony stimulating factors (eg, GM-CSF), and any combination thereof.
  • an interleukin eg, IL-2, IL-12, and IL-15
  • an interferon eg, IFN ⁇ , IFN ⁇ , IFN ⁇
  • TNF ⁇ tumor necrosis factor
  • colony stimulating factors eg, GM-CSF
  • the chemokine is selected from the group consisting of CCL2, RANTES, CCL7, CCL9, CCL10, CCL12, CCL15, CCL19, CCL21, CCL20, XCL-1, and any combination thereof.
  • the anti-tumor protein or polypeptide is selected from the group consisting of: a cytotoxic peptide, such as thymidine kinase TK (TK/GCV), TRAIL, FasL, or any combination thereof; an immunological checkpoint inhibitor, such as an antibody PD-1 antibody, anti-PD-L1 antibody, anti-TIGIT antibody, anti-BTLA antibody, anti-CTLA-4 antibody, anti-Tim-3 antibody, anti-Lag-3 antibody, anti-CD137 antibody, anti-OX40 antibody, anti-GITR antibody, anti- CD73 antibody, anti-KIR antibody, anti-ICOS antibody, anti-CSF1R antibody or any combination thereof; tumor-specific targeting antibody, such as anti-HER2 antibody (such as Herceptin), anti-CD20 antibody (such as rituximab), anti- a VEGF antibody (such as bevacizumab), an anti-EGFR antibody (such as cetuximab), or any combination thereof; a protein or polypeptide, such as
  • the modified PRV comprises at least one insertion of an exogenous nucleotide sequence as described above and/or at least one of the untranslated regions (eg, a promoter) as described above. Mutation, deletion or insertion.
  • the modified PRV does not express a functional EPO protein and comprises at least one insertion of an exogenous nucleotide sequence as described above and/or at least one of which is located as described above Mutations, deletions or insertions of untranslated regions (eg promoters).
  • the PRVs described above and modified forms thereof can be used in combination.
  • one or more of the PRV and its modified forms can be administered to the subject.
  • a nucleic acid molecule as described herein is administered to the subject.
  • the nucleic acid molecule consists of a genomic sequence or cDNA sequence of PRV or a modified form thereof as described herein, or a complement of the cDNA sequence.
  • the nucleic acid molecule has a genomic sequence of a PRV or a modified form thereof as described herein. In certain preferred embodiments, the nucleic acid molecule has a nucleotide sequence selected from the group consisting of:
  • nucleotide sequence identity (2) having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94 with the nucleotide sequence as shown in SEQ ID NO: 1 or 4. %, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% nucleotide sequence identity.
  • the nucleic acid molecule is a vector comprising a genomic sequence or cDNA sequence of a PRV or a modified form thereof as described herein, or a complement of the cDNA sequence (eg, a cloning vector or expression vector) ).
  • the nucleic acid molecule is a vector (eg, a cloning vector or an expression vector) comprising a cDNA sequence of a PRV or a modified form thereof as described herein, or a complement of the cDNA sequence.
  • a nucleic acid molecule as described herein can be delivered by any means known in the art, for example, by direct injection of a naked nucleic acid molecule (eg, naked RNA), or by a non-viral delivery system. ).
  • the non-viral delivery system can be obtained by preparation of various materials well known in the art including, but not limited to, "Yin H, et al. Nat Rev Genet. 2014 Aug; 15(8): 541-55. And various nanomaterials in "Riley MK, Vermerris W. Nanomaterials (Basel). 2017 Apr 28; 7(5). pii: E94.”, all of which are incorporated herein by reference, for example, liposome, inorganic nano Particles (such as gold nanoparticles), polymers (such as PEG), and the like.
  • the PRV and/or a modified form thereof, or a nucleic acid molecule as described herein can be formulated and administered as a pharmaceutical composition.
  • Such pharmaceutical compositions may comprise a therapeutically effective amount of PRV and/or a modified form thereof, or a therapeutically effective amount of a nucleic acid molecule as described herein.
  • the pharmaceutical composition can be in any form known in the medical arts.
  • the pharmaceutical composition may be a tablet, a pill, a suspension, an emulsion, a solution, a gel, a capsule, a powder, a granule, an elixir, a lozenge, a suppository, an injection (including an injection, lyophilization) Powder) and other forms.
  • the pharmaceutical composition is an injectable solution or a lyophilized powder.
  • the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient. In certain preferred embodiments, the pharmaceutical composition comprises a stabilizer.
  • the PRV and/or a modified form thereof, or a nucleic acid molecule as described herein can be administered to a subject in various suitable ways in the present invention.
  • the manner in which the PRV and/or its modified form, or the nucleic acid molecule as described herein, is administered depends on the location and type of tumor.
  • the virus or nucleic acid molecule is optionally administered by direct injection into the tumor (eg, an intratumoral injection); for tumors of the hematopoietic system, the virus can be administered by intravenous or other intravascular route.
  • the virus or nucleic acid molecule for a tumor that is not readily accessible in vivo (eg, a metastatic tumor), can be administered systemically throughout the body and thereby reach the tumor (eg, intravenous or intramuscular injection).
  • it can be administered subcutaneously, intraperitoneally, intrathecally (for example for brain tumors), topically (for example for melanoma), orally (for example for oral or esophageal cancer), nasally or by inhalation (for example for lung cancer)
  • the virus or nucleic acid molecule of the invention is administered by an equal route.
  • the PRVs of the invention and/or modified forms thereof, or nucleic acid molecules as described herein can be administered by intradermal, subcutaneous, intramuscular, intravenous, oral administration, and the like.
  • the method further comprises administering an additional pharmaceutically active agent having anti-tumor activity.
  • additional pharmaceutically active agents can be administered prior to, concurrently with, or subsequent to administration of the PRV and/or modified form thereof, or a nucleic acid molecule as described herein.
  • the additional pharmaceutically active agent comprises an additional oncolytic virus, chemotherapeutic agent, or immunotherapeutic agent.
  • the additional oncolytic virus includes, but is not limited to, an adenovirus, parvovirus, reovirus, Newcastle disease virus, vesicular stomatitis virus, measles virus, or any combination thereof.
  • the chemotherapeutic agent includes, but is not limited to, 5-fluorouracil, mitomycin, methotrexate, hydroxyurea, cyclophosphamide, dacarbazine, mitoxantrone, anthracycline (such as epirubicin or more) [rectin], etoposide, a platinum compound (such as carboplatin or cisplatin), a taxane (such as paclitaxel or taxotere), or any combination thereof.
  • the immunotherapeutic agent includes, but is not limited to, an immunological checkpoint inhibitor (such as a PD-L1/PD-1 inhibitor or a CTLA-4 inhibitor), a tumor-specific targeting antibody (such as rituximab or Herceptin). ) or any combination thereof.
  • an immunological checkpoint inhibitor such as a PD-L1/PD-1 inhibitor or a CTLA-4 inhibitor
  • a tumor-specific targeting antibody such as rituximab or Herceptin.
  • the PRV and/or a modified form thereof can be administered in any amount from 1 to 1 x 10 15 pfu/kg of the subject's body weight, for example at least 1 x 10 3 pfu/kg, at least 1 x 10 4 pfu/kg, 1 ⁇ 10 5 pfu/kg, 1 ⁇ 10 6 pfu/kg, at least 1 ⁇ 10 7 pfu/kg, at least 1 ⁇ 10 8 pfu/kg, at least 1 ⁇ 10 9 pfu/kg, at least PRV and/or a modified form thereof is administered in an amount of 1 x 10 10 pfu/kg, at least 1 x 10 11 pfu/kg or at least 1 x 10 12 pfu/kg of the subject's body weight.
  • any of the viral genome copies per kg of body weight may be between 1 x 10 10 and 5 x 10 14 (eg, 3 x 10 10 to 3 x 10 14 ).
  • a nucleic acid molecule as described herein is administered in an amount.
  • the PRV and/or its modified form can be administered three times daily, twice daily, once daily, once every two days, or once per week, or as herein
  • the nucleic acid molecule optionally as appropriate, repeats the dosing regimen as described above weekly or monthly.
  • the method further comprises administering an additional therapy.
  • additional therapies may be any therapy known to be used in tumors, such as surgery, chemotherapy, radiation therapy, immunotherapy, hormone therapy or gene therapy. This additional therapy can be administered prior to, concurrently with, or after administration of the methods described above.
  • the subject is a mammal, such as a human.
  • the tumor is selected from the group consisting of a glioma, a neuroblastoma, a gastric cancer, a liver cancer, a kidney cancer, a lung cancer, a breast cancer, a colon cancer, a lymphoma, an ovarian cancer, a cervical cancer, and an uterus.
  • the tumor recurrence is a recurrence of liver cancer.
  • the present invention is also a pharmaceutical composition
  • a pharmaceutical composition comprising a PRV and/or a modified form thereof as defined in the first aspect or the second aspect, or as in the first aspect or the second aspect A defined nucleic acid molecule.
  • the pharmaceutical composition can be in any form known in the medical arts.
  • the pharmaceutical composition may be a tablet, a pill, a suspension, an emulsion, a solution, a gel, a capsule, a powder, a granule, an elixir, a lozenge, a suppository, an injection (including an injection, lyophilization) Powder) and other forms.
  • the pharmaceutical composition is an injectable solution or a lyophilized powder.
  • the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient. In certain preferred embodiments, the pharmaceutical composition comprises a stabilizer.
  • the pharmaceutical composition optionally further comprises an additional pharmaceutically active agent.
  • the additional pharmaceutically active agent is a drug having anti-tumor activity, such as an additional oncolytic virus, chemotherapeutic agent or immunotherapeutic agent.
  • the pharmaceutical composition is for treating a tumor in a subject and/or reducing or inhibiting tumor recurrence.
  • the subject is a mammal, such as a human.
  • the tumor is selected from the group consisting of a glioma, a neuroblastoma, a gastric cancer, a liver cancer, a kidney cancer, a lung cancer, a breast cancer, a colon cancer, a lymphoma, an ovarian cancer, a cervical cancer, and an uterus.
  • the tumor recurrence is a recurrence of liver cancer.
  • the invention relates to a PRV and/or a modified form thereof as defined in the first or second aspect, or a nucleic acid molecule as defined in the first or second aspect, for use as drug.
  • the inventors of the present application have found for the first time that pseudorabies virus (PRV) has broad-spectrum tumor killing activity. Based on this finding, the present invention further provides a PRV-based oncolytic virus.
  • the oncolytic virus of the invention can be used alone for the treatment of tumors, can also be used as an auxiliary method for traditional tumor treatment, or as a treatment method in the absence of other treatment methods, and has great clinical value.
  • 1A-1C show the wild type PRV of human lung cancer cell line H1299, human liver cancer cell line BEL7402, human gastric cancer cell line BGC823, human colon cancer cell line HCT-116, mouse breast cancer cell line 4T1, human in Example 2.
  • Melanoma cell line MPWO human cervical cancer cell line SIHA, mouse kidney cancer cell line Renca, human ovarian cancer cell line A2780, human nasopharyngeal carcinoma cell line CNE1, glioma cell line GBM, human laryngeal carcinoma cell line Hep -2, a micrograph of the results of in vitro killing experiments of human pancreatic cancer cell line Panc-1, human lymphoma cell line A20, mouse prostate cancer cell line Tramp C2, human embryonic lung fibroblast MRC5, wherein MOCK indicates no infection Virus cells.
  • the results showed that PRV had significant oncolysis on human and mouse tumor cell lines after 72 hours of infection with a multiplicity of infection (MOI) of 1, but less killing of MRC5 in human non-t
  • Figure 2 shows the killing effect of wild-type PRV virus of Example 2 on the mouse kidney cancer cell line Renca. The results showed that the CPE of Renca cells infected with PRV for 24 hours was very obvious, and almost all of the lysis was killed by 48 hours.
  • Figure 3 shows the results of in vivo anti-tumor experiments of PRV-WT on human nasopharyngeal carcinoma model CNE1 (A), human Burkitt's lymphoma model Raji (B) and human glioma model GBM (C) in Example 3.
  • the results showed that in the challenge experiment group, the growth of tumors formed by SCID mice subcutaneously inoculated with CNE1, Raji and GBM cells was significantly slower, stagnant, and even lysed; in contrast, no oncolytic virus Tumors in the negative group (Mock) of the treatment maintained normal growth, and the tumor volume was significantly larger than that in the challenge experiment group.
  • Figure 4 shows PRV-WT versus mouse colon cancer model CT26 (Figure 4A), mouse liver cancer model Hep1-6 ( Figure 4B), mouse kidney cancer model Renca ( Figure 4C), mouse breast cancer in Example 3.
  • Figure 4D In vivo anti-tumor test results for model 4T1 (Fig. 4D). The results showed that PRV-WT exhibited significant therapeutic effects in the above mouse tumor models.
  • FIG 5 shows the results of safety evaluation of PRV-WT in the intravenous injection model of mice in Example 4.
  • the body weight and survival rate of mice were monitored by intravenous injection of PBS (A) or 1*10 7 PFU virus (B) in Bab/c mice.
  • the results showed that the weight of mice in PRV-WT group and PBS group showed the same trend.
  • there was no death in mice which confirmed that wild-type PRV-WT has very good safety in the mouse vein model.
  • Figure 6 shows the results of safety evaluation of wild-type PRV-WT in the intracranial injection model of mice in Example 4.
  • the B5/c mice were intracranially injected with 2*10 6 , 2*10 5 , 2*10 4 PFU PRV-WT to detect changes in survival rate of mice.
  • the results showed that the injected virus mice all died and showed certain Dose dependent. This result suggests that PRV-WT may have some neurotoxicity.
  • Fig. 7 shows the results of evaluation of the in vitro killing activity of PRV-del-EP0 against tumor cell lines and diploid cell lines (normal cell types) in Example 5.
  • Figure 7A shows the killing effect of PRV-del-EP0 (BK61-dEP0) on various tumor cell lines
  • Figure 7B shows PRV-del-EP0 (BK61-dEP0) against multiple diploid cell lines (normal cells)
  • PRV-del-EP0 has tumor killing activity comparable to that of PRV-WT, and the killing activity against normal cells is lowered.
  • Figure 8 shows the results of safety evaluation of PRV-del-EP0 in the intracranial injection model of mice in Example 5. Intracranial injection of 1*10 6 , 1*10 5 , 1*10 4 , 1*10 3 , 1*10 2 , 1*10 1 PFU of PRV-WT (A) or PRV-del-EP0 ( B), monitoring changes in mouse survival. The results show that PRV-del-EP0 has significantly improved in vivo safety compared to wild-type PRV.
  • Figures 9-10 show the therapeutic effect of PRV-del-EP0 on the mouse liver cancer model in Example 5.
  • Figure 9 shows the effect of PRV-del-EP0 on the tumor size of mouse liver cancer model mice
  • Figure 10 shows the effect of PRV-del-EP0 on the survival rate of mouse liver cancer model mice. The results showed that PRV-del-EP0 has significant antitumor activity comparable to PRV-WT.
  • Figure 11 shows the results of tumor recurrence evaluation of mice treated with PRV-WT and PRV-del-EP0 in Example 5. The results show that PRV-WT and PRV-del-EP0 can prevent tumor recurrence.
  • Throat swabs and anal swabs from sick pigs are from China, Xiamen City Center for Disease Control and Prevention, Porcine Embryonic Kidney Cells (PK-15; ATCC NumberCCL-33 TM ) from China, Xiamen University, National Infectious Disease Diagnosis
  • PK-15 Porcine Embryonic Kidney Cells
  • the Reagent and Vaccine Engineering Technology Research Center was preserved and cultured in DMEM medium supplemented with 10% fetal bovine serum, glutamine, penicillin and streptomycin.
  • PK-15 cells were plated in 24-well plates, the number of cells per well was 1 ⁇ 10 5 , and the growth solution (DMEM medium, 10% fetal bovine serum and glutamine, penicillin and streptomycin) was aspirated. The wells were replaced with 1 mL of maintenance solution (DMEM medium, 2% fetal bovine serum and glutamine, penicillin and streptomycin), followed by 50 ⁇ L of the sample supernatant per well except for the negative control wells, 37 ° C, 5% CO 2 incubator culture;
  • DMEM medium 10% fetal bovine serum and glutamine, penicillin and streptomycin
  • CPE cytopathic effect
  • the pseudorabies virus-characterized CPE is present in the inoculated cells within 7 days, the cells and supernatant are collected and frozen at -80 °C; if there is no CPE after 7 days, the cells are blindly transmitted;
  • the virus isolated from the clinical specimens was identified by PCR, and the pseudorabies virus-positive culture was selected for at least 3 viral plaque purification experiments; the cloned strain obtained from the virus plaques was also identified by PCR, and the pseudovirus was selected.
  • the rabies virus-positive cloned strain was subjected to the next round of cloning; a single viable strain of pseudorabies virus with strong growth activity was selected as the candidate strain of oncolytic virus.
  • This example shows that the wild type PRV strain can also be referred to as BK61-WT (SEQ ID NO: 1) as an example, showing how the PRV and its modified form for use in the present invention can be obtained by gene editing, and the specific method is as follows.
  • sgRNA design based on CRISPER/CAS9 technology using viral genome analysis to find the PAM site near the site where the foreign gene is to be inserted, designing the sgRNA according to the sequence near the PAM, so that the CAS9 protein can cleave the genome of the virus, and then In the use of homologous recombination, a new engineered virus is formed.
  • Modified form the key protein EP0 gene sequence of wild type PRV (the DNA sequence thereof is shown in SEQ ID NO: 2) is replaced with the green fluorescent protein (GFP) gene sequence (the DNA sequence thereof is shown in SEQ ID NO: 3), thereby obtaining a recombinant virus.
  • the genome designated PRV-del-EP0 may also be referred to as BK61-dEP0 (the DNA sequence of which is shown in SEQ ID NO: 4).
  • the sgRNA constructed above was transferred into 293T cells to form a stable cell strain, and then the target fragment was transferred into the cells, and then the PRV virus was infected, and a new recombinant virus was formed by intracellular homologous recombination technology.
  • progeny virus formed after infection of 293T is deactivated into PK-15 cells, and the recombinant progeny virus carries a fluorescent signal, which can be used for screening and isolation of progeny virus.
  • Virus This example used the PRV-WT (SEQ ID NO: 1) provided in Example 1.
  • HepaRG cells were cultured in WME medium (addition of 1.5% DMSO); AGS and TT were used in F-12K medium; SH-SY5Y was used in DMEM:F12 (1:1) medium; RD, C-33A, EBC-1, SK-MEL-1, J82 and DU145 use MEM medium, Raji, Daudi, 5637, 786-O, TE-1, Caski, NCI-H1299, NCI-H1703, NCI-H1975, NCI-H661, SGC7901, BGC823, RPMI-1640 medium was used for SW1116, HEp-2 and LNCap, and DMEM medium was used for other cells. These mediums were supplemented with 10% fetal bovine serum, glutamine and penicillin-streptomycin. All of the above cells were cultured under standard conditions of 37 ° C,
  • the RD cells were evenly spread on a 10 cm cell culture plate under the conditions of DMEM medium containing 10% fetal bovine serum and glutamine, penicillin and streptomycin, 37 ° C, 5% CO 2 , saturated humidity; When the confluency reached 90% or more, the cell culture medium was changed to 2% serum DMEM medium, and each plate was inoculated with 10 6 PFU of PRV-WT.
  • the RD cells were plated in 6-well plates which, a cell density of 10 5 / well; adherent cells to be covered after the 10-fold virus dilutions per well lOOul, infection of infected cells, vortexed every 15 minutes After 5 times, remove the supernatant.
  • the 2% agarose solution prepared with pbs was dissolved by heating, and then mixed with 10% serum DMEM medium in a volume ratio of 1:1, added to the cells, and after being cooled and solidified, the cells were inverted in an incubator. After three days of culture, 10% formaldehyde solution was added for fixation for 1 hour, and the gel was buckled out, and stained with crystal violet dye solution for 15 min, and the number of plaques was calculated to determine the titer of the virus.
  • Cell survival rate was measured using Cell Counting Kit-8 (CCK-8 kit; Shanghai Biyuntian Biotechnology Co., Ltd.) 72 hours after virus infection culture, as follows:
  • the adherent cells directly discard the original medium, and the suspended cells are carefully discarded after centrifugation, and then replaced with 100 ⁇ l of fresh serum-free medium per well; 10 ⁇ l of CCK-8 solution was added to the well, and an equal amount of CCK-8 solution was also added as a negative control in the blank medium; the cells were incubated at 37 ° C for 0.5-3 hours in a cell culture incubator at 0.5, 1, 2, and 3 hours, respectively.
  • a microplate reader was used to perform a detection at an absorbance of 450 nm, and a suitable time point of the absorbance range was selected as a reference for cell viability.
  • PRV-WT has a good killing effect on most of the detected tumor cells.
  • the virus has lung cancer, liver cancer, ovarian cancer, neuroblastoma, cervical cancer, lymphoma, and renal cancer.
  • the cell line killing is very significant.
  • PRV-WT has a certain killing effect on non-tumor cell lines including the human embryonic lung fibroblast cell line MRC-5.
  • Example 1 The PRV-WT provided in Example 1 was used in this example. Methods for virus culture and virus titer determination are described in Examples 2.2 and 2.3, respectively.
  • Cell line human nasopharyngeal carcinoma cell line CNE1, human Burkitt's lymphoma cell Raji ( Number: CCL-86 TM ), human glioma cell line GBM (patient tumor tissue isolated primary tumor cell line), mouse colon cancer cell CT26, mouse liver cancer cell Hep1-6, mouse kidney cancer cell Renca and Mouse breast cancer cells 4T1.
  • the above cells were cultured in DMEM medium except for Raji using RPMI-1640 medium, and the above medium was added with 10% fetal bovine serum, glutamine and penicillin-streptomycin double antibody. All of the above cells were cultured under standard conditions of 37 ° C, 5% CO 2 .
  • mice Female C.B17SCID mice or Bab/c mice of 6-8 weeks old were obtained from Shanghai Slack Laboratory Animals Co., Ltd.; according to the approved plan of Xiamen University Experimental Animal Center and Ethics Committee, The mice were housed under SPF conditions.
  • SCID mice were used, and the tumor cells for subcutaneous tumor formation were digested with 0.01% trypsin, and then resuspended into a single cell suspension using a cell culture medium containing 10% fetal bovine serum; The cell density of the suspension was 1000 g, and the cells were pelleted by centrifugation at 3 min, and then resuspended in an appropriate volume of PBS to reach about 10 6 -10 7 cells/100 ⁇ l of PBS; according to 10 6 -10 7 cells/100 ⁇ l PBS/point.
  • the tumor cells were inoculated subcutaneously with a syringe in the back of SCID mice.
  • the tumor-bearing SCID mice were randomly divided into experimental groups using PRV- WT (BK61) was treated with a negative control group (Mock).
  • PRV- WT PRV- WT
  • Mouse negative control group
  • Bab/c mice were used to inoculate tumor cells subcutaneously, and after 7-14 days, mice of about 100 mm 3 were selected for treatment.
  • Tumor size changes were measured and recorded every two days with vernier calipers. The calculation of tumor size was:
  • Tumor size (mm 3 ) tumor length value ⁇ (tumor width value) 2 /2.
  • the treatment results of PRV-WT on human tumor transplantation model and mouse tumor model are shown in Fig. 3 and Fig. 4, respectively.
  • the results showed that after treatment with PRV-WT, CNE1 (Fig. 3A), GBM (Fig. 3B), Raji (Fig. 3C), and CT26 (Fig. 4A), Hep1-6 (Fig. 4B), and Renca (Fig. 4C) were detected.
  • Virus This example uses the PRV-WT provided by Example 1. The methods of virus culture and virus titer determination were carried out in accordance with Examples 2.2 and 2.3, respectively.
  • This example uses PRV-del-EP0 (SEQ ID NO: 4) provided by Example 1.
  • the methods of virus culture and virus titer determination were carried out in accordance with Examples 2.2 and 2.3, respectively.
  • Fig. 7A shows the killing results of PRV-del-EP0 (BK61-dEP0) against various tumor cell lines, and the results showed that it substantially retained the killing effect comparable to the wild-type PRV of the parent strain.
  • Figure 7B shows the killing results of PRV-del-EP0 (BK61-dEP0) against various non-tumor cell lines.
  • PRV-del-EP0 pair diploid cell line was compared with the wild-type PRV of the parent strain. (normal cell line-like) showed significantly reduced killing activity.
  • PRV-del-EP0 not only retains the significant tumor killing activity of wild-type PRV, but also improves safety to a certain extent.
  • ICR mice were selected and intrapulmonarily injected with different doses of PRV-WT and PRV-del-EP0.
  • the doses of challenge titers were 1*10 6 , 1*10 5 , 1*10 4 , 1*10 3 , 1 . *10 2 , 1*10 1 PFU/only (4 per group), followed by daily survival of ICR mice in different doses of challenge groups.
  • the results of the PRV-WT (BK61-WT) group are shown in Fig. 8A
  • the results of the PRV-del-EP0 (BK61-dEP0) group are shown in Fig. 8B.
  • the tumor cells Hep1-6 used for immunization of mouse C57/B6 mice subcutaneously into tumor were digested with 0.01% trypsin, and then resuspended into a single cell suspension using a cell culture medium containing 10% fetal bovine serum; The cell density of the solution was 1000 g, and the cells were pelleted by centrifugation at 3 min, and then resuspended in an appropriate volume of PBS to reach about 10 6 -10 7 cells/100 ⁇ l of PBS; according to 10 6 -10 7 cells / 100 ⁇ l PBS / spot C57/B6 mice were inoculated with tumor cells by syringe in the back.
  • tumor-bearing SCID mice were randomly divided into 3 groups.
  • Intratumoral injection treatment was performed using PRV-WT (BK61-WT), PRV-del-EP0 (BK61-dEP0) and PBS, once every two days for a total of three treatments. Tumor size changes were measured and recorded every two days with vernier calipers. The calculation of tumor size was:
  • Tumor size (mm 3 ) tumor length value ⁇ (tumor width value) 2 /2.
  • mice in the PRV-WT and PRV-del-EP0 groups did not relapse, but all of the control mice that were not treated with PRV-WT and PRV-del-EP0 were found to have tumors. This result suggests that mice that have been cured by PRV-WT and PRV-del-EP0 have good anti-tumor immunity and can prevent tumor recurrence.

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Abstract

伪狂犬病毒(PRV)或其修饰形式，或包含PRV或其修饰形式的基因组序列或cDNA序列，或所述cDNA序列的互补序列的核酸分子，用于在受试者中治疗肿瘤和/或降低或抑制肿瘤复发的用途，以及在制备用于在受试者中治疗肿瘤和/或降低或抑制肿瘤复发的药物组合物中的用途。一种治疗肿瘤和/或降低或抑制肿瘤复发的方法，其包括向有此需要的受试者施用PRV或其修饰形式，或包含PRV或其修饰形式的基因组序列的核酸分子的步骤。

Description

一种用于治疗肿瘤的伪狂犬病毒 技术领域
本发明涉及病毒领域和肿瘤治疗领域。具体而言，本发明涉及伪狂犬病毒(PRV)或其修饰形式，或包含PRV或其修饰形式的基因组序列的核酸分子，用于在受试者(例如，人)中治疗肿瘤和/或降低或抑制肿瘤复发的用途，以及在制备用于在受试者(例如，人)中治疗肿瘤和/或降低或抑制肿瘤复发的药物中的用途。本发明还涉及一种治疗肿瘤和/或降低或抑制肿瘤复发的方法，其包括向有此需要的受试者施用PRV或其修饰形式，或包含PRV或其修饰形式的基因组序列的核酸分子的步骤。
背景技术
目前恶性肿瘤的治疗手段主要包括手术治疗、化疗和放疗。这些传统疗法对于已转移的肿瘤治疗效果不尽如人意，并且还会对患者身体健康产生伤害巨大。
溶瘤病毒是一种能够在肿瘤细胞当中自我复制，从而杀死、溶解肿瘤细胞，或者使肿瘤细胞生长停滞的病毒。当进行体内治疗时，溶瘤病毒表现出对肿瘤细胞的选择特异性，能够直接诱导肿瘤细胞死亡，而对正常细胞影响较小或无影响；同时，溶瘤病毒也能通过激发免疫系统当中的B淋巴细胞和T淋巴细胞反应，间接地杀伤肿瘤细胞。因此，溶瘤病毒被认为是一种比较有前景的肿瘤治疗手段。
伪狂犬病毒(Pseudorabies virus，PRV)又称猪疱疹病毒I型、传染性延髓麻痹病毒、奇痒症病毒、奥叶兹基氏病病毒，属于α疱疹病毒亚科水痘病毒属。是引起牛、羊、猪、犬和猫等多种家畜和野生动物发热、奇痒(猪除外)及脑脊髓炎为主要症状的疱疹病毒，猪是伪狂犬病毒的天然宿主，猪感染后出现发热、腹泻、呼吸困难、脑脊髓炎、繁殖障碍等急性传染病症状。伪狂犬病毒具备以下特点：首先作为双链DNA病毒，其基因组在宿主体内没有整合基因组到宿主染色体的过程；其次，伪狂犬病毒虽然在其他的一些低等哺乳动物中发生流行，但还没有正式的文献报道其能感染人及致病；再次，伪狂犬病毒不含致癌基因。目前，伪狂犬病毒被广泛使用于作为兽用疫苗等，目前本领域尚未有任何伪狂犬病毒具有溶瘤活性的报道。
发明内容
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术 人员所通常理解的含义。并且，本文中所用的细胞培养、病毒学、生物化学、细胞生物学、核酸化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，下面提供相关术语的定义和解释。
如本文中所使用的，术语“伪狂犬病毒(Pseudorabies virus，PRV)”是指，α疱疹病毒亚科(Alphaherpesvirinae)、水痘病毒属(Varicellovirus)的一种病毒。该病毒的基因组为线性双链DNA，其大小在130kd-150kd之间。该病毒的基因组包括一个独特的长区序列(UL)和一个独特的短区序列(US)，在US两侧还存在着末端重复序列(TRR)和内部的重复序列(IRS)。PRV的基因是根据他们所在的区域和发现顺序所命名的，但也可由他们编码的蛋白命名。编码结构蛋白的基因包括位于US区的US2(28K)、US3(PK)、US4(gG)、US6(gD)、US7(gI)、US8(gE)、US9(11K)等基因和位于UL区的UL9(OBP)、UL27(gB)、UL(gH)、UL(TK)、UL(gC)衣壳蛋白基因、DNA多聚酶基因等。位于UL区的非结构蛋白基因包括能够参与转录调节的UL54。在本发明中，表述“伪狂犬病毒(Pseudorabies virus，PRV)”意指野生型PRV，其可从自然中的来源分离并且没有被人为有意地修饰，其实例包括原型株Bartha-K61株(BK61)与HB-98株，以及各种标本分离株(例如，本发明实施例1所描述的分离株)。野生型PRV基因组序列是本领域熟知的，并且可参见各种公共数据库(例如，GenBank数据库登录号：JF797217.1)。
如本文中所使用的，术语“溶瘤病毒”是指，能够感染肿瘤细胞，在肿瘤细胞中复制，引起肿瘤细胞死亡、裂解或阻止肿瘤细胞生长的病毒。优选地，该病毒对非肿瘤细胞具有最小的毒性效应。
如本文中所使用的，术语“肿瘤特异性”是指，选择性地在肿瘤细胞内展现出生物功能或活性。例如，在本发明中，当术语“肿瘤特异性”用于描述病毒的杀伤选择性时，意指该病毒能够选择性杀伤肿瘤细胞而不杀伤或基本上不杀伤非肿瘤细胞，或者，该病毒对肿瘤细胞的杀伤比对非肿瘤细胞的杀伤更为有效。
如本文中所使用的，术语“溶瘤活性”主要包括肿瘤杀伤活性。当描述病毒的溶瘤活性时，通常可以通过其病毒感染肿瘤细胞的能力、在肿瘤细胞内复制的能力和/或杀伤肿瘤细胞的能力等指标衡量该病毒的溶瘤活性。病毒的溶瘤活性可以采用本领域已知的任何方法进行测定。例如，病毒感染肿瘤细胞的能力可以通过测量感染给定百分率的肿瘤细胞(例如50％细胞)所需要的病毒剂量进行评价；在肿瘤细胞内复制的能力可以通过测量病毒在肿瘤细胞内的生长情况进行评价；杀伤肿瘤细胞的能力可以通过观察致细胞 病变效应(CPE)或测量肿瘤细胞活性进行评价。
如本文中所使用的，术语病毒的“修饰形式”是指，对野生型病毒进行修饰而获得的经修饰的病毒，其保留了野生型病毒的期望的活性(例如，溶瘤活性)。在本发明中，PRV的“修饰形式”包括但不限于经修饰的PRV病毒，其与野生型PRV的基因组序列相比具有一个或多个核苷酸的替换、插入或删除，并且至少保留了PRV的溶瘤活性。应当理解的是，本发明的PRV的修饰形式或经修饰的PRV不受限于其生产方式。例如，本发明的PRV的修饰形式或经修饰的PRV可通过同源重组产生，也可通过培养感染了所述修饰形式或经修饰的PRV的宿主细胞而制备。
如本文中所使用的，术语“EP0蛋白”是指PRV病毒的early protein 0，其由EP0基因编码，并且为PRV病毒早期表达的转录激活因子。EP0蛋白的氨基酸序列是已知的，并可参见例如公共数据库(例如，EM64001.1)。
如本文中所使用的，表述“不表达功能性EP0蛋白”是指，当病毒或病毒基因组感染细胞后，所述病毒或病毒基因组不能产生或表达具有生物学功能活性的EP0蛋白。例如，所述病毒或病毒基因组可以因基因缺失而完全不产生或表达EP0蛋白，或者因功能丧失性突变而产生或表达不具有生物学功能活性的EP0蛋白。
如本文中所使用的，术语“功能丧失性突变”是指这样的突变，其导致突变基因所编码和表达的蛋白丧失了其生物学功能活性。功能丧失性突变包括但不限于，错义突变，无义突变，移码突变，碱基缺失，碱基置换，碱基添加，以及其任何组合(例如，基因片段的缺失或置换或添加)，只要包含该功能丧失性突变的基因不能产生或表达具有生物学功能活性的蛋白即可。
如本文中所使用的，表述“PRV的cDNA序列”意指，以该病毒基因组转录的mRNA为模板经逆转录而获得的DNA序列，其与所述基因组序列相比差异仅在于所述cDNA序列不包含所述基因组序列中的内含子序列。
如本文中所使用的，术语“外源核苷酸序列”是指，人工引入的核苷酸序列，其相对于原始序列而言是外来的。外源核苷酸序列包括但不限于，未在所述病毒基因组中发现的任何基因或核苷酸序列。然而，在某些情况下，优选地，外源核苷酸序列编码具有治疗用途的多肽，例如免疫调节多肽、细胞因子、趋化因子、抗肿瘤蛋白或多肽等。
如本文中所使用的，术语“免疫调节多肽”是指，能调节免疫细胞功能的多肽，其实例包括但不限于，CD40L、OX40L、可诱导共刺激分子(ICOS)、FTL3L、LIGHT、CD137L、CD70、4-1BB、GITR、和CD28(参见例如，Khalil D N,Smith E L, Brentjens R J,et al.The future of cancer treatment:immunomodulation,CARs and combination immunotherapy[J].Nat Rev Clin Oncol,2016,13(5):273-290)。
如本文中所使用的，术语“细胞因子”具有本领域技术人员公知的含义。然而，在本发明中，当使用本发明的溶瘤病毒来治疗肿瘤时，特别优选地，所述细胞因子为能够用于肿瘤治疗的细胞因子。“细胞因子”的实例包括但不限于，白介素(例如IL-2、IL-12和IL-15)、干扰素(例如IFNα、IFNβ、IFNγ)、肿瘤坏死因子(例如TNFα)、集落刺激因子(例如GM-CSF)，及其任何组合(参见例如，Ardolino M,Hsu J,Raulet D H.Cytokine treatment in cancer immunotherapy[J].Oncotarget,2015,6(23):19346-19347)。
如本文中所使用的，术语“趋化因子”具有本领域技术人员公知的含义。然而，在本发明的方法中，当使用本发明的溶瘤病毒来治疗肿瘤时，特别优选地，所述细胞因子为能够用于肿瘤治疗的趋化因子。“趋化因子”的实例包括但不限于，CCL2、RANTES、CCL7、CCL9、CCL10、CCL12、CCL15、CCL19、CCL21、CCL20、XCL-1，及其任何组合(Homey B,Muller A,Zlotnik A.CHEMOKINES:AGENTS FOR THE IMMUNOTHERAPY OF CANCER[J].Nat Rev Immunol,2002,2:175-184)。
如本文中所使用的，术语“抗肿瘤蛋白或多肽”是指，具有治疗肿瘤活性的蛋白或多肽，其包括但不限于：(1)对细胞具有毒性、可抑制细胞增殖或诱导细胞凋亡的蛋白或多肽，其实例包括但不限于胸苷激酶TK(TK/GCV)、TRAIL和FasL(参见例如，Candolfi M,King G D,Muhammad A G,et al.Evaluation of proapototic transgenes to use in combination with Flt3L in an immune-stimulatory gene therapy approach for Glioblastoma multiforme(GBM)[J].FASEB J,2008,22:1077.13)；(2)具有免疫治疗作用的蛋白或多肽，例如免疫检查点抑制剂，其实例包括但不限于抗PD-1抗体、抗PD-L1抗体、抗TIGIT抗体、抗BTLA抗体、抗CTLA-4抗体、抗Tim-3抗体、抗Lag-3抗体、抗CD137抗体、抗OX40抗体、抗GITR抗体、抗CD73抗体、抗KIR抗体、抗ICOS抗体、抗CSF1R抗体(参见例如，Nolan E,Savas P,Policheni A N,et al.Combined immune checkpoint blockade as a therapeutic strategy for BRCA1-mutated breast cancer[J].Science Trans Med,2017,9:eaal4922；其全部通过引用并入本文)；(3)肿瘤特异性靶向抗体，其实例包括但不限于抗HER2抗体(如赫赛汀)、抗CD20抗体(如利妥昔单抗)、抗VEGF抗体(如贝伐珠单抗)、抗EGFR抗体(如西妥昔单抗)或其任意组合；(4)抑制肿瘤血管生成的蛋白或多肽，其实例包括但不限于抗血管内皮生长因子(anti-VEGF)的单链抗体(scFv)、VEGF来源多肽(例如 D(LPR)、 KSVRGKGKGQKRKRKKSRYK等)和ATN-161(参见例如，Rosca E V，Koskimaki J E，Rivera C G，et al.Anti-angiogenic peptides for cancer therapeutics[J].Curr Pharm Biotechnol，2011,12(8):1101–1116；其全部通过引用并入本文)。
如本文中所使用的，术语“同一性”用于指两个蛋白/多肽之间或两个核酸之间序列的匹配情况。当两个进行比较的序列中的某个位置都被相同的碱基或氨基酸单体亚单元占据时(例如，两个DNA分子的每一个中的某个位置都被腺嘌呤占据，或两个蛋白/多肽的每一个中的某个位置都被赖氨酸占据)，那么各分子在该位置上是同一的。两个序列之间的“百分数同一性”是由这两个序列共有的匹配位置数目除以进行比较的位置数目×100的函数。例如，如果两个序列的10个位置中有6个匹配，那么这两个序列具有60％的同一性。例如，DNA序列CTGACT和CAGGTT共有50％的同一性(总共6个位置中有3个位置匹配)。通常，在将两个序列比对以产生最大同一性时进行比较。这样的比对可通过使用，例如，可通过计算机程序例如Align程序(DNAstar,Inc.)方便地进行的Needleman等人(1970)J.Mol.Biol.48：443-453的方法来实现。还可使用已整合入ALIGN程序(版本2.0)的E.Meyers和W.Miller(Comput.Appl Biosci.，4:11-17(1988))的算法，使用PAM120权重残基表(weight residue table)、12的缺口长度罚分和4的缺口罚分来测定两个氨基酸序列之间的百分数同一性。此外，可使用已整合入GCG软件包(可在www.gcg.com上获得)的GAP程序中的Needleman和Wunsch(J MoI Biol.48:444-453(1970))算法，使用Blossum 62矩阵或PAM250矩阵以及16、14、12、10、8、6或4的缺口权重(gap weight)和1、2、3、4、5或6的长度权重来测定两个氨基酸序列之间的百分数同一性。
如本文中所使用的，术语“载体(vector)”是指，可将多聚核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌粒；柯斯质粒；人工染色体，例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。可用作载体的动物病毒包括但不限于，逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可以含有多种控制表达的元件，包括但不限于，启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。
如本文中所使用的，术语“启动子”具有本领域技术人员公知的含义，其是指一段位于基因的上游能启动下游基因表达的非编码核苷酸序列。
如本文中所使用的，术语“药学上可接受的载体和/或赋形剂”是指，在药理学和/或生理学上与受试者和活性成分相容的载体和/或赋形剂，其是本领域公知的(参见例如Remington's Pharmaceutical Sciences.Edited by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995)，并且包括但不限于：pH调节剂，表面活性剂，离子强度增强剂，维持渗透压的试剂，延迟吸收的试剂，稀释剂，佐剂，防腐剂，稳定剂等。例如，pH调节剂包括但不限于磷酸盐缓冲液。表面活性剂包括但不限于阳离子，阴离子或者非离子型表面活性剂，例如Tween-80。离子强度增强剂包括但不限于氯化钠。维持渗透压的试剂包括但不限于糖、NaCl及其类似物。延迟吸收的试剂包括但不限于单硬脂酸盐和明胶。稀释剂包括但不限于水，水性缓冲液(如缓冲盐水)，醇和多元醇(如甘油)等。佐剂包括但不限于铝佐剂(例如氢氧化铝)，弗氏佐剂(例如完全弗氏佐剂)等。防腐剂包括但不限于各种抗细菌试剂和抗真菌试剂，例如硫柳汞，2-苯氧乙醇，对羟苯甲酸酯，三氯叔丁醇，苯酚，山梨酸等。稳定剂具有本领域技术人员通常理解的含义，其能够稳定药物中的活性成分的期望活性(例如溶瘤活性)，包括但不限于谷氨酸钠，明胶，SPGA，糖类(如山梨醇，甘露醇，淀粉，蔗糖，乳糖，葡聚糖，或葡萄糖)，氨基酸(如谷氨酸，甘氨酸)，蛋白质(如干燥乳清，白蛋白或酪蛋白)或其降解产物(如乳白蛋白水解物)等。
如本文中所使用的，术语“治疗”是指，治疗或治愈疾病(例如肿瘤)，延缓疾病(例如肿瘤)的症状的发作，和/或延缓疾病(例如肿瘤)的发展。
如本文中所使用的，术语“有效量”是指，可以有效实现预期目的的量。例如，治疗有效量可以是有效地或足以治疗或治愈疾病(例如肿瘤)，延缓疾病(例如肿瘤)症状的发作和/或延缓疾病(例如肿瘤)发展的量。这样的有效量可以由本领域技术人员或医生容易地确定，并且可以与预期目的(例如治疗)、受试者的一般健康状况、年龄、性别、体重、待治疗的疾病的严重程度、并发症、施用方式等相关。这样的有效量的确定完全在本领域技术人员的能力范围内。
如本文中使用的，术语“受试者”是指哺乳动物，例如灵长类哺乳动物，例如人。在某些实施方式中，所述受试者(例如人)患有肿瘤，或者，具有患有肿瘤的风险。
本申请的发明人经过大量实验和反复摸索，出人意料地发现，伪狂犬病毒(PRV)具有 广谱、显著的肿瘤细胞杀伤能力。基于这一发现，本发明人开发了新的用于治疗肿瘤的溶瘤病毒以及基于该病毒的肿瘤治疗方法。
因此，在第一方面，本发明提供了伪狂犬病毒(PRV)或其修饰形式或一种核酸分子，用于在受试者中治疗肿瘤和/或降低或抑制肿瘤复发的用途，或者用于制备在受试者中治疗肿瘤和/或降低或抑制肿瘤复发的药物的用途；其中，所述核酸分子包含选自下列的序列：
(1)PRV或其修饰形式的基因组序列或cDNA序列；
(2)所述cDNA序列的互补序列。
在某些优选的实施方案中，所述PRV为野生型PRV。在某些优选的实施方案中，所述PRV可以是从感染伪狂犬病毒(PRV)的动物体内分离得到的毒株。
在某些优选的实施方案中，所述PRV或其修饰形式的基因组序列与如SEQ ID NO：1所示的核苷酸序列具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性。在某些优选的实施方案中，所述PRV或其修饰形式的基因组序列为如SEQ ID NO：1所示的核苷酸序列。
在某些优选的实施方案中，所述修饰形式为经修饰的PRV，其与野生型PRV相比在基因组中具有一个或多个核苷酸的替换、插入或删除。
在某些优选的实施方案中，所述经修饰的PRV与野生型PRV相比，具有一个或多个选自下列的修饰：
(1)一个或多个内源基因的缺失或突变；
(2)位于非翻译区(例如启动子)中的一个或多个核苷酸的突变、缺失或插入；
(3)一个或多个外源核苷酸序列的插入；和
(4)上述三项的任意组合。
在某些优选的实施方案中，所述经修饰的PRV不表达功能性EP0蛋白。如本领域技术人员所熟知的，可通过对编码EP0蛋白的基因(EP0基因)进行改造来阻止所述蛋白的功能性表达。例如，可在编码EP0蛋白的基因中引入功能丧失性突变，或者将编码EP0蛋白的基因缺失或置换成外源核苷酸序列(例如，编码外源蛋白的核苷酸序列)，从而阻止所述目的蛋白的功能性表达。
因此，在某些优选的实施方案中，本发明的经修饰的PRV的基因组下述修饰：EP0基因包含功能丧失性突变(例如，一个或多个核苷酸的添加、缺失和/或置换)或者被缺 失或者被置换为外源核苷酸序列(例如，编码外源蛋白的核苷酸序列)。在某些优选的实施方案中，所述功能丧失性突变选自错义突变、无义突变、移码突变、碱基缺失、碱基置换、碱基添加、以及其任何组合(例如，基因片段的缺失或置换或添加)。
在某些优选的实施方案中，所述EP0基因被缺失。在此类实施方案中，所述经修饰的PRV不表达EP0蛋白。在某些优选的实施方案中，所述EP0基因被置换成外源核苷酸序列(例如，编码外源蛋白的核苷酸序列)。在某些实例性实施方案中，所述外源蛋白是荧光蛋白。
在某些优选的实施方案中，所述经修饰的PRV的基因组序列与SEQ ID NO：4所示的核苷酸序列相比具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性。在某些优选的实施方案中，所述经修饰的PRV的基因组序列如SEQ ID NO：4所示。
在某些优选的实施方案中，所述经修饰的PRV包括位于非编码区(如启动子)中的一个或多个核苷酸的突变、缺失或插入。
在某些优选的实施方案中，所述经修饰的PRV的基因组包含下述修饰：一个或多个PRV基因的原生启动子被替换为肿瘤特异性启动子。在本发明中，任意PRV基因的原生启动子均可被替换为肿瘤特异性启动子。在某些优选的实施方案中，所述肿瘤特异性启动子选自人端粒逆转录酶(hTERT)启动子、甲胎蛋白启动子、癌胚抗原启动子、前列腺特异性抗原启动子、Survivin启动子和环氧化酶COX-2启动子。
在本发明中，肿瘤特异性启动子的使用在某些情况下是有利的，例如有益于提高溶瘤病毒的肿瘤特异性。在正常细胞中，肿瘤特异性启动子的活性通常处于较低水平，而在肿瘤细胞中其活性处于较高水平。因此利用肿瘤特异性启动子来调控一些PRV关键蛋白的转录表达可以让病毒获得肿瘤特异性的杀伤能力。因此，在某些情况下，将PRV的关键蛋白的原生启动子换成肿瘤特异性启动子可以减少病毒对正常细胞的杀伤能力减小毒副作用，而不影响其对肿瘤细胞的杀伤能力。
在某些优选的实施方案中，所述经修饰的PRV包含外源核苷酸序列。
在某些优选的实施方案中，所述外源核苷酸序列编码选自下列的外源蛋白：荧光蛋白、免疫调节多肽、细胞因子、趋化因子、抗肿瘤蛋白或多肽。
在某些优选的实施方案中，所述荧光蛋白选自绿色荧光蛋白，红色荧光蛋白，蓝色荧光蛋白，黄色荧光蛋白，及其任何组合。
在某些优选的实施方案中，所述免疫调节多肽选自CD40L、OX40L、可诱导共刺激分子(ICOS)、FTL3L、LIGHT、CD137L、CD70、4-1BB、GITR、CD28，及其任何组合。
在某些优选的实施方案中，所述细胞因子选自白介素(例如IL-2、IL-12和IL-15)、干扰素(例如IFNα、IFNβ、IFNγ)、肿瘤坏死因子(例如TNFα)、集落刺激因子(例如GM-CSF)，及其任何组合。
在某些优选的实施方案中，所述趋化因子选自CCL2、RANTES、CCL7、CCL9、CCL10、CCL12、CCL15、CCL19、CCL21、CCL20、XCL-1，及其任何组合。
在某些优选的实施方案中，所述抗肿瘤蛋白或多肽选自：细胞毒性肽，例如胸苷激酶TK(TK/GCV)、TRAIL、FasL或其任意组合；免疫检查点抑制剂，例如抗PD-1抗体、抗PD-L1抗体、抗TIGIT抗体、抗BTLA抗体、抗CTLA-4抗体、抗Tim-3抗体、抗Lag-3抗体、抗CD137抗体、抗OX40抗体、抗GITR抗体、抗CD73抗体、抗KIR抗体、抗ICOS抗体、抗CSF1R抗体或其任意组合；肿瘤特异性靶向抗体，例如抗HER2抗体(如赫赛汀)、抗CD20抗体(如利妥昔单抗)、抗VEGF抗体(如贝伐珠单抗)、抗EGFR抗体(如西妥昔单抗)或其任意组合；抑制肿瘤血管生成的蛋白或多肽，例如抗血管内皮生长因子(anti-VEGF)抗体、VEGF来源多肽(如 D(LPR)、KSVRGKGKGQKRKRKKSRYK等)、ATN-161或其任意组合；以及上述各蛋白或多肽的任意组合。
在某些优选的实施方案中，所述经修饰的PRV包含至少一种如上所述的外源核苷酸序列的插入和/或至少一种如上所述的位于非翻译区(例如启动子)的突变、缺失或插入。
在某些优选的实施方案中，所述经修饰的PRV不表达功能性EP0蛋白，并且包含至少一种如上所述的外源核苷酸序列的插入和/或至少一种如上所述的位于非翻译区(例如启动子)的突变、缺失或插入。
在本申请中，可以通过本领域内众所周知的技术获得本发明的经修饰的PRV。例如，可以通过碱基的缺失、置换或插入，在病毒基因中引入功能丧失性突变，使病毒基因在功能上失活。在某些示例性实施方案中，通过缺失(例如缺失整个基因或其部分)，使病毒基因在功能上失活。在此类实施方案中，可缺失目的病毒基因序列的至少25％，至少50％，至少75％，或100％，或者可缺失至少10bp，至少100bp，或至少1000bp的目的病毒基因序列。在某些示例性实施方案中，通过碱基的插入或缺失造成移码突变，从而使病毒基因在功能上失活。在某些示例性实施方案中，通过将整个目的基因或其部 分置换为外源核苷酸序列，使病毒基因在功能上失活。
在某些优选的实施方案中，本发明的经修饰的PRV可以通过CRISPR/Cas9技术获得。所述CRISPR/Cas9技术是本领域已知的，例如可参见Ran FA,Feng Zhang et al.nature，2013,2281-2308；其全部通过引用并入本文。在此类实施方案中，通常对野生型PRV的病毒基因组进行修饰(例如，外源核苷酸序列的插入、内源基因的缺失或突变或位于非翻译区的突变)从而获得经修饰的PRV。
本发明的PRV或其修饰形式可以经预处理以减少或消除受试者对该病毒的免疫反应，其中所述预处理可以包括：将所述PRV包装在脂质体或胶束中，和/或使用蛋白酶(例如，糜蛋白酶或胰蛋白酶)去除病毒的衣壳蛋白以减小宿主对病毒的体液和/或细胞免疫。
在本发明中，可以将如本文所述的PRV或其修饰形式在肿瘤细胞内连续适应性传代。在某些优选的实施方案中，所述肿瘤细胞可以为本领域已知的肿瘤细胞系或肿瘤细胞株，也可以为从患有肿瘤的个体(例如，受试者)体内经手术切除或临床分离而得到的肿瘤细胞。在某些优选的实施方案中，将所述PRV或其修饰形式在获得自患有肿瘤的个体(例如，受试者)体内的肿瘤细胞内连续适应性传代。在某些优选的实施方案中，所述肿瘤细胞从患有肿瘤的个体(例如，受试者)体内经手术切除或临床分离而得到。在某些优选的实施方案中，所述连续适应性传代的方法包括多个(例如至少5个，至少10个，至少15个，至少20个)由下述过程所构成的周期：1)将病毒感染靶肿瘤细胞；2)收获上清中的病毒；和3)将获得的病毒重新感染新鲜靶肿瘤细胞。
在某些优选的实施方案中，可以组合使用如上所述的PRV和其修饰形式。因此，所述药物可以包括PRV以及其修饰形式中的一种或数种。
在某些优选的实施方案中，所述核酸分子由如本文所述的PRV或其修饰形式的基因组序列或cDNA序列，或所述cDNA序列的互补序列组成。
在某些优选的实施方案中，所述核酸分子具有如本文所述的PRV或其修饰形式的基因组序列。在某些优选的实施方案中，所述核酸分子具有选自下列的核苷酸序列：
(1)如SEQ ID NO：1或4所示的核苷酸序列；
(2)与如SEQ ID NO：1或4所示的核苷酸序列具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的核苷酸序列。
在某些优选的实施方案中，所述核酸分子为包含如本文所述的PRV或其修饰形式的基因组序列或cDNA序列，或所述cDNA序列的互补序列的载体(例如，克隆载体或表 达载体)。在某些优选的实施方案中，所述核酸分子为包含如本文所述的PRV或其修饰形式的cDNA序列，或所述cDNA序列的互补序列的载体(例如，克隆载体或表达载体)。
本发明的核酸分子可以通过本领域已知的任何方式进行递送，例如，直接注射裸露的核酸分子(例如，裸RNA)，或利用非病毒递送系统(non-viral delivery system)。所述非病毒递送系统可通过本领域熟知的各种材料制备获得，其中所述材料包括但不限于详细描述于“Yin H,et al.Nat Rev Genet.2014Aug；15(8):541-55.”以及“Riley MK,Vermerris W.Nanomaterials(Basel).2017Apr 28；7(5).pii:E94.”中的各种材料，其全部通过引用并入本文，例如脂质体、无机纳米粒子(如金纳米颗粒)、多聚物(如PEG)等等。
在某些优选的实施方案中，所述药物包含治疗有效量的PRV和/或其修饰形式，或治疗有效量的如本文所述的核酸分子。在某些优选的实施方案中，所述药物可以为医学领域已知的任何形式。例如，所述药物可以是片剂、丸剂、混悬剂、乳剂、溶液、凝胶剂、胶囊剂、粉剂、颗粒剂、酏剂、锭剂、栓剂、注射剂(包括注射液、冻干粉剂)等形式。在一些实施方案中，所述药物为注射液或冻干粉剂。
在某些优选的实施方案中，所述药物还包含药学上可接受的载体或赋形剂。在某些优选的实施方案中，所述药物包含稳定剂。
在某些优选的实施方案中，所述药物任选地还包含另外的药学活性剂。在一个优选的实施方案中，所述另外的药学活性剂是具有抗肿瘤活性的药物，例如另外的溶瘤病毒、化学治疗剂或免疫治疗剂。
在本发明中，所述另外的溶瘤病毒包括但不限于腺病毒、细小病毒、呼肠孤病毒、新城疫病毒、水疱性口炎病毒、麻疹病毒或其任意组合。所述化学治疗剂包括但不限于5-氟尿嘧啶、丝裂霉素、甲氨蝶呤、羟基脲、环磷酰胺、达卡巴嗪、米托蒽醌、蒽环类(如表柔比星或多柔比星)、依托泊苷、铂类化合物(如卡铂或顺铂)、紫杉烷类(如紫杉醇或紫杉特尔)或其任意组合。所述免疫治疗剂包括但不限于免疫检查点抑制剂(如PD-L1/PD-1抑制剂或CTLA-4抑制剂)、肿瘤特异性靶向抗体(如利妥昔单抗或赫赛汀)或其任意组合。
在某些优选的实施方案中，所述药物包含单位剂量的PRV和/或其修饰形式，例如包含至少1×10 2pfu、至少1×10 3pfu、至少1×10 4pfu、1×10 5pfu、1×10 6pfu、至少1×10 7pfu、至少1×10 8pfu、至少1×10 9pfu、至少1×10 10pfu、至少1×10 11pfu、至少1×10 12 pfu、至少1×10 13pfu、至少1×10 14pfu或至少1×10 16pfu的PRV和/或其修饰形式。在某些优选的实施方案中，所述药物包含1×10 2pfu～1×10 17pfu的PRV和/或其修饰形式。
在某些优选的实施方案中，所述药物含有单位剂量的如本文所述的核酸分子，例如含有1×10 10～5×10 14(例如，3×10 10～3×10 14)病毒基因组拷贝数(virus genome copies)的所述核酸分子。
在某些优选的实施方案中，可以将所述药物与额外的疗法一起施用。这种额外的疗法可以是已知用于肿瘤的任何疗法，例如手术、化学治疗、放射治疗、免疫治疗、激素治疗或基因治疗。这种额外的疗法可以在施用所述药物之前、同时或之后施用。
在某些优选的实施方案中，所述肿瘤选自神经胶质瘤、神经母细胞瘤、胃癌、肝癌、肾癌、肺癌、乳腺癌、结肠癌、淋巴瘤、卵巢癌、宫颈癌、子宫内膜癌、黑色素瘤、胰腺癌、成骨肉瘤、前列腺癌、鼻咽癌、鼻中隔鳞状细胞癌、喉癌、甲状腺癌、甲状腺导管癌、膀胱癌等。在某些示例性实施方案中，所述肿瘤复发是肝癌复发。
在某些优选的实施方案中，所述受试者为哺乳动物，例如人。
在第二方面，本发明提供了一种治疗肿瘤和/或降低或抑制肿瘤复发的方法，其包括向有此需要的受试者施用有效量的PRV或其修饰形式，或有效量的核酸分子的步骤；其中，所述核酸分子包含选自下列的序列：
(1)PRV或其修饰形式的基因组序列或cDNA序列；和
(2)所述cDNA序列的互补序列。
在某些优选的实施方案中，向所述受试者施用PRV。在某些优选的实施方案中，所述PRV为野生型PRV。在某些优选的实施方案中，所述PRV可以是从感染伪狂犬病毒(PRV)的动物体内分离得到的毒株。
在某些优选的实施方案中，所述PRV或其修饰形式的基因组序列与如SEQ ID NO：1所示的核苷酸序列具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性。在某些优选的实施方案中，所述PRV或其修饰形式的基因组序列为如SEQ ID NO：1所示的核苷酸序列。
在某些优选的实施方案中，向所述受试者施用PRV的修饰形式。在某些优选的实施方案中，所述修饰形式为经修饰的PRV，其与野生型PRV相比在基因组中具有一个或多个核苷酸的替换、插入或删除。
在某些优选的实施方案中，所述经修饰的PRV与野生型PRV相比，具有一个或多个选自下列的修饰：
(1)一个或多个内源基因的缺失或突变；
(2)位于非翻译区(例如启动子)中的一个或多个核苷酸的突变、缺失或插入；
(3)一个或多个外源核苷酸序列的插入；和
(4)上述三项的任意组合。
在某些优选的实施方案中，所述经修饰的PRV不表达功能性EP0蛋白。
在某些优选的实施方案中，本发明的经修饰的PRV的基因组包含下述修饰：EP0基因包含功能丧失性突变(例如，一个或多个核苷酸的添加、缺失和/或置换)或者被缺失或者被置换为外源核苷酸序列(例如，编码外源蛋白的核苷酸序列)。在某些优选的实施方案中，所述功能丧失性突变选自错义突变、无义突变、移码突变、碱基缺失、碱基置换、碱基添加、以及其任何组合(例如，基因片段的缺失或置换或添加)。
在某些优选的实施方案中，所述EP0基因被缺失。在此类实施方案中，所述经修饰的PRV不表达EP0蛋白。在某些优选的实施方案中，所述EP0基因被置换为外源核苷酸序列(例如，编码外源蛋白的核苷酸序列)。
在某些优选的实施方案中，所述经修饰的PRV的基因组序列与SEQ ID NO：4所示的核苷酸序列相比具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性。在某些优选的实施方案中，所述经修饰的PRV的基因组序列如SEQ ID NO：4所示。
在某些优选的实施方案中，所述经修饰的PRV包括位于非编码区(如启动子)中的一个或多个核苷酸的突变、缺失或插入。
在某些优选的实施方案中，所述经修饰的PRV的基因组包含下述修饰：一个或多个PRV基因的原生启动子被替换为肿瘤特异性启动子。在本发明中，任意PRV基因的原生启动子均可被替换为肿瘤特异性启动子。在某些优选的实施方案中，所述肿瘤特异性启动子选自hTERT启动子、甲胎蛋白启动子、癌胚抗原启动子、前列腺特异性抗原启动子、Survivin启动子和环氧化酶COX-2启动子。
在某些优选的实施方案中，所述经修饰的PRV包含外源核苷酸序列。
在某些优选的实施方案中，所述外源核苷酸序列编码选自下列的外源蛋白：荧光蛋白、免疫调节多肽、细胞因子、趋化因子、抗肿瘤蛋白或多肽。
在某些优选的实施方案中，所述荧光蛋白选自绿色荧光蛋白，红色荧光蛋白，蓝色荧光蛋白，黄色荧光蛋白，及其任何组合。
在某些优选的实施方案中，所述免疫调节多肽选自CD40L、OX40L、可诱导共刺激分子(ICOS)、FTL3L、LIGHT、CD137L、CD70、4-1BB、GITR、CD28，及其任何组合。
在某些优选的实施方案中，所述细胞因子选自白介素(例如IL-2、IL-12和IL-15)、干扰素(例如IFNα、IFNβ、IFNγ)、肿瘤坏死因子(例如TNFα)、集落刺激因子(例如GM-CSF)，及其任何组合。
在某些优选的实施方案中，所述趋化因子选自CCL2、RANTES、CCL7、CCL9、CCL10、CCL12、CCL15、CCL19、CCL21、CCL20、XCL-1，及其任何组合。
在某些优选的实施方案中，所述抗肿瘤蛋白或多肽选自：细胞毒性肽，例如胸苷激酶TK(TK/GCV)、TRAIL、FasL或其任意组合；免疫检查点抑制剂，例如抗PD-1抗体、抗PD-L1抗体、抗TIGIT抗体、抗BTLA抗体、抗CTLA-4抗体、抗Tim-3抗体、抗Lag-3抗体、抗CD137抗体、抗OX40抗体、抗GITR抗体、抗CD73抗体、抗KIR抗体、抗ICOS抗体、抗CSF1R抗体或其任意组合；肿瘤特异性靶向抗体，例如抗HER2抗体(如赫赛汀)、抗CD20抗体(如利妥昔单抗)、抗VEGF抗体(如贝伐珠单抗)、抗EGFR抗体(如西妥昔单抗)或其任意组合；抑制肿瘤血管生成的蛋白或多肽，例如抗血管内皮生长因子(anti-VEGF)抗体、VEGF来源多肽(如 D(LPR)、KSVRGKGKGQKRKRKKSRYK等)、ATN-161或其任意组合；以及上述各蛋白或多肽的任意组合。
在某些优选的实施方案中，所述经修饰的PRV包含至少一种如上所述的外源核苷酸序列的插入和/或至少一种如上所述的位于非翻译区(例如启动子)的突变、缺失或插入。
在某些优选的实施方案中，所述经修饰的PRV不表达功能性EP0蛋白，并且包含至少一种如上所述的外源核苷酸序列的插入和/或至少一种如上所述的位于非翻译区(例如启动子)的突变、缺失或插入。
在某些优选的实施方案中，可以组合使用如上所述的PRV和其修饰形式。因此，可以向受试者施用PRV以及其修饰形式中的一种或多种。
在某些优选的实施方案中，向所述受试者施用如本文所述的核酸分子。
在某些优选的实施方案中，所述核酸分子由如本文所述的PRV或其修饰形式的基因组序列或cDNA序列，或所述cDNA序列的互补序列组成。
在某些优选的实施方案中，所述核酸分子具有如本文所述的PRV或其修饰形式的基因组序列。在某些优选的实施方案中，所述核酸分子具有选自下列的核苷酸序列：
(1)如SEQ ID NO：1或4所示的核苷酸序列；
(2)与如SEQ ID NO：1或4所示的核苷酸序列具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的核苷酸序列。
在某些优选的实施方案中，所述核酸分子为包含如本文所述的PRV或其修饰形式的基因组序列或cDNA序列，或所述cDNA序列的互补序列的载体(例如，克隆载体或表达载体)。在某些优选的实施方案中，所述核酸分子为包含如本文所述的PRV或其修饰形式的cDNA序列，或所述cDNA序列的互补序列的载体(例如，克隆载体或表达载体)。
在本发明中，可以通过本领域已知的任何方式递送如本文所述的核酸分子，例如，直接注射裸露的核酸分子(例如，裸RNA)，或利用非病毒递送系统(non-viral delivery system)。所述非病毒递送系统可通过本领域熟知的各种材料制备获得，其中所述材料包括但不限于详细描述于“Yin H,et al.Nat Rev Genet.2014Aug；15(8):541-55.”以及“Riley MK,Vermerris W.Nanomaterials(Basel).2017Apr 28；7(5).pii:E94.”中的各种纳米材料，其全部通过引用并入本文，例如脂质体、无机纳米粒子(如金纳米颗粒)、多聚物(如PEG)等等。
在某些优选的实施方案中，可以将PRV和/或其修饰形式，或如本文所述的核酸分子，作为药物组合物进行配制和施用。这样的药物组合物可包含治疗有效量的PRV和/或其修饰形式，或治疗有效量的如本文所述的核酸分子。在某些优选的实施方案中，所述药物组合物可以是医学领域已知的任何形式。例如，所述药物组合物可以是片剂、丸剂、混悬剂、乳剂、溶液、凝胶剂、胶囊剂、粉剂、颗粒剂、酏剂、锭剂、栓剂、注射剂(包括注射液、冻干粉剂)等形式。在一些实施方案中，所述药物组合物为注射液或冻干粉剂。
在某些优选的实施方案中，所述药物组合物还包含药学上可接受的载体或赋形剂。在某些优选的实施方案中，所述药物组合物包含稳定剂。
在本发明中可以通过各种合适的方式，向受试者施用PRV和/或其修饰形式，或如本文所述的核酸分子。在某些情况下，PRV和/或其修饰形式，或如本文所述的核酸分子的施用方式取决于肿瘤的位置和类型。例如，对于容易接近的实体肿瘤，任选地通过直接 向肿瘤注射(例如，瘤内注射)来施用该病毒或核酸分子；对于造血系统的肿瘤，可以通过静脉内或其他血管内途径施用该病毒或核酸分子；对于体内不容易接近的肿瘤(例如转移瘤)，可以系统地施用该病毒或核酸分子以使其遍布全身并由此到达肿瘤(例如，静脉内或肌肉内注射)。任选地，可以经皮下、腹膜内、鞘内(例如对于脑部肿瘤)、局部(例如对于黑素瘤)、口服(例如对于口腔或食道癌)、经鼻或通过吸入喷雾(例如对于肺癌)等途径施用本发明的病毒或核酸分子。在某些优选的实施方案中，可以通过皮内注射、皮下注射、肌肉注射、静脉注射、口服给予等途径施用本发明的PRV和/或其修饰形式，或如本文所述的核酸分子。
在某些优选的实施方案中，所述方法还包括施用具有抗肿瘤活性的另外的药学活性剂。这种另外的药学活性剂可以在施用PRV和/或其修饰形式，或如本文所述的核酸分子之前、同时或之后施用。
在某些优选的实施方案中，所述另外的药学活性剂包括另外的溶瘤病毒、化学治疗剂或免疫治疗剂。
在本发明中，所述另外的溶瘤病毒包括但不限于腺病毒、细小病毒、呼肠孤病毒、新城疫病毒、水疱性口炎病毒、麻疹病毒或其任意组合。所述化学治疗剂包括但不限于5-氟尿嘧啶、丝裂霉素、甲氨蝶呤、羟基脲、环磷酰胺、达卡巴嗪、米托蒽醌、蒽环类(如表柔比星或多柔比星)、依托泊苷、铂类化合物(如卡铂或顺铂)、紫杉烷类(如紫杉醇或紫杉特尔)或其任意组合。所述免疫治疗剂包括但不限于免疫检查点抑制剂(如PD-L1/PD-1抑制剂或CTLA-4抑制剂)、肿瘤特异性靶向抗体(如利妥昔单抗或赫赛汀)或其任意组合。
在某些优选的实施方案中，可以以1～1×10 15pfu/kg受试者体重的任何量施用PRV和/或其修饰形式，例如以至少1×10 3pfu/kg、至少1×10 4pfu/kg、1×10 5pfu/kg、1×10 6pfu/kg、至少1×10 7pfu/kg、至少1×10 8pfu/kg、至少1×10 9pfu/kg、至少1×10 10pfu/kg、至少1×10 11pfu/kg或至少1×10 12pfu/kg受试者体重的量施用PRV和/或其修饰形式。在某些优选的实施方案中，可以以1×10 10～5×10 14(例如，3×10 10～3×10 14)病毒基因组拷贝数(virus genome copies)/kg受试者体重的任何量施用如本文所述的核酸分子。在某些优选的实施方案中，可以以每日3次、每日2次、每日1次、每两日1次或每周1次的方式施用PRV和/或其修饰形式，或如本文所述的核酸分子，任选地酌情每周或每月重复如上所述的给药方案。
在某些优选的实施方案中，所述方法还包括施用额外的疗法。这种额外的疗法可以 是已知用于肿瘤的任何疗法，例如手术、化学治疗、放射治疗、免疫治疗、激素治疗或基因治疗。这种额外的疗法可以在施用如上所述的方法之前、同时或之后施用。
在某些优选的实施方案中，所述受试者是哺乳动物，例如人。
在某些优选的实施方案中，所述肿瘤选自神经胶质瘤、神经母细胞瘤、胃癌、肝癌、肾癌、肺癌、乳腺癌、结肠癌、淋巴瘤、卵巢癌、宫颈癌、子宫内膜癌、黑色素瘤、胰腺癌、成骨肉瘤、前列腺癌、鼻咽癌、鼻中隔鳞状细胞癌、喉癌、甲状腺癌、甲状腺导管癌、膀胱癌等。在某些实例性实施方案中，所述肿瘤复发是肝癌复发。
在第三方面，本发明还涉及一种药物组合物，其包含如在第一方面或第二方面中所定义的PRV和/或其修饰形式，或如在第一方面或第二方面中所定义的核酸分子。
在某些优选的实施方案中，所述药物组合物可以为医学领域已知的任何形式。例如，所述药物组合物可以是片剂、丸剂、混悬剂、乳剂、溶液、凝胶剂、胶囊剂、粉剂、颗粒剂、酏剂、锭剂、栓剂、注射剂(包括注射液、冻干粉剂)等形式。在一些实施方案中，所述药物组合物为注射液或冻干粉剂。
在某些优选的实施方案中，所述药物组合物还包含药学上可接受的载体或赋形剂。在某些优选的实施方案中，所述药物组合物包含稳定剂。
在某些优选的实施方案中，所述药物组合物任选地还包含另外的药学活性剂。在一个优选的实施方案中，所述另外的药学活性剂是具有抗肿瘤活性的药物，例如另外的溶瘤病毒、化学治疗剂或免疫治疗剂。
在某些优选的实施方案中，所述药物组合物用于在受试者中治疗肿瘤和/或降低或抑制肿瘤复发。
在某些优选的实施方案中，所述受试者是哺乳动物，例如人。
在某些优选的实施方案中，所述肿瘤选自神经胶质瘤、神经母细胞瘤、胃癌、肝癌、肾癌、肺癌、乳腺癌、结肠癌、淋巴瘤、卵巢癌、宫颈癌、子宫内膜癌、黑色素瘤、胰腺癌、成骨肉瘤、前列腺癌、鼻咽癌、鼻中隔鳞状细胞癌、喉癌、甲状腺癌、甲状腺导管癌、膀胱癌等。在某些实例性实施方案中，所述肿瘤复发是肝癌复发。
在第四方面，本发明还涉及如在第一方面或第二方面中所定义的PRV和/或其修饰形式，或如在第一方面或第二方面中所定义的核酸分子，其用作药物。
发明的有益效果
与现有技术相比，本发明的技术方案至少具有以下有益效果：
本申请的发明人首次发现，伪狂犬病毒(PRV)具有广谱肿瘤杀伤活性。基于这一发现，本发明进一步提供了基于PRV的溶瘤病毒。本发明的溶瘤病毒可单独用于肿瘤的治疗，亦可用作传统肿瘤治疗的辅助方法，或作为缺少其他治疗方法时的治疗手段，具有重大的临床价值。
下面将结合附图和实施例对本发明的实施方案进行详细描述，但是本领域技术人员将理解，下列附图和实施例仅用于说明本发明，而不是对本发明的范围的限定。根据附图和优选实施方案的下列详细描述，本发明的各种目的和有利方面对于本领域技术人员来说将变得显然。
附图说明
图1A-1C显示了实施例2中野生型PRV对人肺癌细胞系H1299、人肝癌细胞系BEL7402、人胃癌细胞系BGC823、人结肠癌细胞系HCT-116、小鼠乳腺癌细胞系4T1、人黑色素瘤细胞系MPWO、人宫颈癌细胞系SIHA、小鼠肾癌细胞系Renca、人卵巢癌细胞系A2780、人鼻咽癌细胞系CNE1、神经胶质瘤细胞系GBM、人喉癌细胞系Hep-2、人胰腺癌细胞系Panc-1、人淋巴瘤细胞系A20、小鼠前列腺癌细胞系Tramp C2、人胚肺成纤维细胞MRC5的体外杀伤实验结果的显微镜照片，其中，MOCK表示未感染病毒的细胞。结果显示，在感染复数(MOI)为1感染72小时后，PRV对人和小鼠肿瘤细胞系均有显著的溶瘤作用，但对人非肿瘤细胞的MRC5杀伤较小。
图2显示了实施例2中野生型PRV病毒对小鼠肾癌细胞系Renca的杀伤效果。结果显示，PRV感染Renca细胞24小时出现CPE已十分明显，到48小时几乎全部裂解死亡。
图3显示了实施例3中PRV-WT对人鼻咽癌模型CNE1(A)、人Burkitt's淋巴瘤模型Raji(B)和人神经胶质瘤模型GBM(C)的体内抗肿瘤实验结果。结果显示，在攻毒实验组中，皮下分别接种了CNE1、Raji和GBM细胞的SCID小鼠所形成的肿瘤的生长明显变慢、停滞，甚至裂解消失；相比之下，未经溶瘤病毒治疗的阴性组(Mock)的肿瘤则保持正常生长，其肿瘤体积显著大于攻毒实验组。
图4显示了实施例3中PRV-WT对小鼠结肠癌模型CT26(图4A)、小鼠肝癌模型Hep1-6(图4B)、小鼠肾癌模型Renca(图4C)、小鼠乳腺癌模型4T1(图4D)的体 内抗肿瘤实验结果。结果显示，PRV-WT在上述小鼠肿瘤模型中均展现出显著的治疗效果。
图5显示了实施例4中PRV-WT在小鼠静脉注射模型中的安全性评价结果。通过对Bab/c小鼠静脉注射PBS(A)或1*10 7PFU病毒(B)，监测小鼠体重与生存率变化，结果显示PRV-WT组与PBS组小鼠体重呈现相同的变化趋势，同时无一小鼠死亡现象，证实野生型PRV-WT在小鼠静脉模型中具有十分好的安全性。
图6显示了实施例4中野生型PRV-WT在小鼠颅内注射模型中的安全性评价结果。通过对Bab/c小鼠颅内注射2*10 6，2*10 5，2*10 4PFU PRV-WT，检测小鼠生存率变化，结果显示注射病毒小鼠均相继死去，并呈现一定的剂量依赖性。这一结果提示PRV-WT可能具有一定的神经毒性。
图7显示了实施例5中PRV-del-EP0对肿瘤细胞系以及二倍体细胞系(类正常细胞系)的体外杀伤活性评价结果。图7A显示了PRV-del-EP0(BK61-dEP0)对多种肿瘤细胞系的杀伤结果，图7B显示了PRV-del-EP0(BK61-dEP0)对多种二倍体细胞系(类正常细胞系)的杀伤结果，结果显示，PRV-del-EP0具有与PRV-WT相当的肿瘤杀伤活性，并且对正常细胞的杀伤活性降低。
图8显示了实施例5中PRV-del-EP0在小鼠颅内注射模型中的安全性评价结果。对小鼠颅内注射1*10 6,1*10 5,1*10 4,1*10 3,1*10 2,1*10 1PFU的PRV-WT(A)或PRV-del-EP0(B)，监测小鼠生存率变化。结果显示，相比于野生型PRV，PRV-del-EP0具有显著提高的体内安全性。
图9-10显示了实施例5中PRV-del-EP0对小鼠肝癌模型的治疗效果。其中，图9显示了PRV-del-EP0对小鼠肝癌模型小鼠的肿瘤大小的影响；图10显示了PRV-del-EP0对小鼠肝癌模型小鼠的生存率的影响。结果显示，PRV-del-EP0具有与PRV-WT相当的显著的抗肿瘤活性。
图11显示了实施例5中经PRV-WT和PRV-del-EP0治愈小鼠的肿瘤复发率评估结果。结果显示，PRV-WT和PRV-del-EP0能够预防肿瘤的复发。
序列信息
本发明涉及的部分序列的信息提供于下面的表1中。
表1：序列的描述
Figure PCTCN2019074349-appb-000001
具体实施方式
现参照下列意在举例说明本发明(而非限定本发明)的实施例来描述本发明。
除非特别指明，本发明中所使用的分子生物学实验方法和免疫检测法，基本上参照J.Sambrook等人，分子克隆：实验室手册，第2版，冷泉港实验室出版社，1989，以及F.M.Ausubel等人，精编分子生物学实验指南，第3版，John Wiley&Sons，Inc.，1995中所述的方法进行；限制性内切酶的使用依照产品制造商推荐的条件。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。本领域技术人员知晓，实施例以举例方式描述本发明，且不意欲限制本发明所要求保护的范围。本文中提及的全部公开案和其他参考资料以其全文通过引用合并入本文。
实施例1：PRV及其修饰形式的获得及制备
1.1从标本中分离伪狂犬病毒病毒PRV
(1)病猪的咽拭子和肛拭子来源于中国，厦门市疾病预防与控制中心，猪胚肾细胞(PK-15；ATCC NumberCCL-33 TM)由中国，厦门大学，国家传染病诊断试剂与疫苗工程技术研究中心保存，使用添加有10％胎牛血清、谷氨酰胺、青霉素和链霉素的DMEM培养基进行培养。
(2)标本处理：将咽拭子和肛拭子在标本保存液中充分搅动，以洗下拭子上粘附的病毒及含有病毒的细胞等，随后将标本保存液在4℃条件下4000rpm高速离心30min；
(3)接种与观察：
A.将PK-15细胞铺24孔板，每孔细胞数量为1×10 5个，吸去生长液(DMEM培养基，10％胎牛血清以及谷氨酰胺、青霉素和链霉素)，每孔换上1mL的维持液(DMEM培养基，2％胎牛血清以及谷氨酰胺、青霉素和链霉素)，随后除了阴性对照 孔，每孔接种50μL的标本上清液，37℃，5％CO 2培养箱培养；
B.一周内每天在显微镜下观察细胞，记录接种孔内是否有特征性的细胞病变效应(CPE)的出现；
C.若7天内接种孔细胞出现伪狂犬病毒特征性CPE时，收集细胞及上清至-80℃冻存；若7天后仍无CPE出现，将细胞进行盲传；
D.盲传6代内出现细胞CPE的，收集细胞及上清至-80℃冻存；盲传6代后仍未出现CPE的，判定为阴性；
(4)病毒的分离与克隆化：
使用PCR鉴定从临床标本中分离获得的病毒，选取伪狂犬病毒阳性的培养物进行至少3次病毒空斑纯化实验；对每一轮由病毒空斑获得的克隆毒株同样使用PCR鉴定，选取伪狂犬病毒阳性的克隆毒株后进行下一轮克隆化；挑选生长活力强的伪狂犬病毒单一毒株作为溶瘤病毒候选毒株.
1.2基于CRISPER/CAS9技术对PRV基因编辑及其修饰形式的获得
本实施例以野生型PRV毒株也可以称为BK61-WT(SEQ ID NO:1)为示例，展示如何通过基因编辑方式获得用于本发明的PRV及其修饰形式，具体方法如下所述。
(1)基于CRISPER/CAS9技术的sgRNA设计：利用病毒基因组分析在要插入外源基因的位点附近找到PAM位点，根据PAM附近的序列设计sgRNA，以便CAS9蛋白可以切割开病毒的基因组，而后在利用同源重组，形成新的改造病毒。
(2)供体目的片段的构建：由于利用同源重组技术形成新的改造病毒，目的基因片段的获取，也是改造成功的关键，因此将目的片段送往上海生工进行合成。
修饰形式：将野生型PRV的关键蛋白EP0基因序列(其DNA序列见SEQ ID NO:2)替换为绿色荧光蛋白(GFP)基因序列(其DNA序列见SEQ ID NO:3)，从而获得重组病毒(命名为PRV-del-EP0也可称为BK61-dEP0)的基因组(其DNA序列见SEQ ID NO:4)。
(3)将上述构建的sgRNA转入293T细胞中形成稳定细胞株，再将目的片段转入细胞中，之后感染PRV病毒，利用胞内的同源重组技术，使其形成新的重组病毒。
(4)将感染293T后形成的子代病毒，去感染PK-15细胞，重组成功的子代病毒带有荧光信号，可用于子代病毒的筛选与分离。
实施例2：野生型PRV的体外抗肿瘤活性评价
2.1所使用的病毒与细胞系
(1)病毒：本实施例使用实施例1所提供的PRV-WT(SEQ ID NO:1)。
(2)细胞系：人横纹肌肉瘤细胞RD(
Figure PCTCN2019074349-appb-000002
Number:CCL-136 TM)；人结直肠癌细胞系SW1116(
Figure PCTCN2019074349-appb-000003
Number:CCL-233 TM)、SW480(
Figure PCTCN2019074349-appb-000004
Number:CCL-228 TM)和HT-29(
Figure PCTCN2019074349-appb-000005
Number:HTB-38 TM)；人胃癌细胞系AGS(
Figure PCTCN2019074349-appb-000006
Number:CRL-1739 TM)、SGC7901(CCTCC保藏编号:GDC150)、BGC823(CCTCC保藏编号:GDC151)和NCI-N87(
Figure PCTCN2019074349-appb-000007
Number:CRL-5822 TM)；人食管癌细胞系TE-1(购自中国科学院上海生命科学研究院细胞资源中心，编号:3131C0001000700089)；人小细胞肺癌细胞系DMS114(
Figure PCTCN2019074349-appb-000008
Number:CRL-2066 TM)；人非小细胞肺癌细胞系SPC-A-1(CCTCC保藏编号:GDC050)、NCI-H1975(
Figure PCTCN2019074349-appb-000009
Number:CRL-5908 TM)、NCI-H1299(
Figure PCTCN2019074349-appb-000010
Number:CRL-5803 TM)、A549(
Figure PCTCN2019074349-appb-000011
Number:CCL-185 TM)、NCI-H661(
Figure PCTCN2019074349-appb-000012
Number:HTB-183 TM)、EBC-1(Thermo Fisher Scientific,Catalog#:11875101)和NCI-H1703(
Figure PCTCN2019074349-appb-000013
Number:CRL-5889 TM)；人肝癌细胞系C3A(
Figure PCTCN2019074349-appb-000014
Number:CRL-10741 TM)、HepG2(
Figure PCTCN2019074349-appb-000015
Number:HB-8065 TM)、SMMC7721(购自中国医学科学院基础医学研究所基础医学细胞中心，编号:3111C0001CCC000087)、BEL7402(CCTCC保藏编号:GDC035)、BEL7404(购自中国科学院上海生命科学研究院细胞资源中心，编号:3131C0001000700064)、Huh7(CCTCC保藏编号:GDC134)和PLC/PRF/5(
Figure PCTCN2019074349-appb-000016
Number:CRL-8024 TM)；人卵巢癌细胞系SKOV3(
Figure PCTCN2019074349-appb-000017
Number:HTB-77 TM)和Caov3(
Figure PCTCN2019074349-appb-000018
Number:HTB-75 TM)；人子宫内膜癌细胞系Hec-1-A(
Figure PCTCN2019074349-appb-000019
Number:HTB-112 TM)、Hec-1-B(
Figure PCTCN2019074349-appb-000020
Number:HTB-113 TM)和Ishikawa(ECACC No.99040201)；人宫颈癌细胞系Hela(
Figure PCTCN2019074349-appb-000021
Number:CCL-2 TM)、Caski(
Figure PCTCN2019074349-appb-000022
Number:CRL-1550 TM)和C-33A(
Figure PCTCN2019074349-appb-000023
Number:HTB-31 TM)；人黑色素瘤细胞系SK-MEL-1(
Figure PCTCN2019074349-appb-000024
Number:HTB-67 TM)和MeWo(
Figure PCTCN2019074349-appb-000025
Number:HTB-65 TM)；人乳腺癌细胞系BcaP37(CCTCC保藏编号:GDC206)、BT-474(
Figure PCTCN2019074349-appb-000026
Number:HTB-20 TM)和MDA-MB-231(
Figure PCTCN2019074349-appb-000027
Number:HTB-26 TM)；人肾癌细胞系A-498(
Figure PCTCN2019074349-appb-000028
Number:HTB-44 TM)和786-O(
Figure PCTCN2019074349-appb-000029
Number:CRL-1932 TM)；人胰腺癌细胞系Capan-2(
Figure PCTCN2019074349-appb-000030
Number:HTB-80 TM)和HPAF-2(
Figure PCTCN2019074349-appb-000031
Number:CRL-1997 TM)；人成骨肉瘤细胞系U2OS(
Figure PCTCN2019074349-appb-000032
Number:HTB-96 TM)；人前列腺癌细胞系DU145(
Figure PCTCN2019074349-appb-000033
Number: HTB-81 TM)和LNCap(
Figure PCTCN2019074349-appb-000034
Number:CRL-1740 TM)；人神经胶质瘤细胞系GBM(病人肿瘤组织分离原代肿瘤细胞系)；人神经母瘤细胞系SH-SY5Y(
Figure PCTCN2019074349-appb-000035
Number:CRL-2266 TM)；人鼻咽癌细胞系CNE(购自中国医学科学院基础医学研究所基础医学细胞中心，编号:3131C0001000700013)；人鼻中隔鳞状癌细胞系RPMI 2650(
Figure PCTCN2019074349-appb-000036
Number:CCL-30 TM)；人喉癌细胞系HEp-2(
Figure PCTCN2019074349-appb-000037
Number:CCL-23 TM)；人甲状腺癌细胞系SW579(由国家传染病诊断试剂与疫苗工程技术研究中心保存)和人甲状腺导管癌细胞系TT(
Figure PCTCN2019074349-appb-000038
Number:CRL-1803 TM)；人膀胱癌细胞系J82(
Figure PCTCN2019074349-appb-000039
Number:HTB-1 TM)和5637(
Figure PCTCN2019074349-appb-000040
Number:HTB-9 TM)；人Burkitt's淋巴瘤细胞系Daudi(
Figure PCTCN2019074349-appb-000041
Number:CCL-213 TM)和Raji(
Figure PCTCN2019074349-appb-000042
Number:CCL-86 TM)；人正常细胞系包括：人皮肤角化细胞系HaCat(CCTCC保藏编号:GDC106)、人胚肺成纤维细胞系MRC-5(
Figure PCTCN2019074349-appb-000043
Number:CCL-171 TM)、人包皮成纤维细胞系HFF-1(
Figure PCTCN2019074349-appb-000044
Number:SCRC-1041 TM)、人前列腺基质细胞系WPMY-1(
Figure PCTCN2019074349-appb-000045
Number:CRL-2854 TM)、人脐静脉内皮细胞系HUVEC(Thermo Fisher Scientific,Catalog#:C01510C)以及分化后的人肝祖细胞系HepaRG(具原代肝细胞特征；Thermo Fisher Scientific,Catalog#:HPRGC10)。以上细胞均由中国，厦门大学，国家传染病诊断试剂与疫苗工程技术研究中心保存。HepaRG细胞在WME培养基(添加1.5％DMSO)中培养；AGS和TT使用F-12K培养基；SH-SY5Y使用DMEM:F12(1:1)培养基；RD、C-33A、EBC-1、SK-MEL-1、J82和DU145使用MEM培养基，Raji、Daudi、5637、786-O、TE-1、Caski、NCI-H1299、NCI-H1703、NCI-H1975、NCI-H661、SGC7901、BGC823、SW1116、HEp-2和LNCap使用RPMI-1640培养基，其他细胞都使用DMEM培养基，这些培养基均需添加10％的胎牛血清、谷氨酰胺与青霉素-链霉素双抗。以上所有细胞在37℃，5％CO 2的标准条件下培养。
2.2病毒的培养
将RD细胞均匀铺于10厘米细胞培养板上，培养条件为含有10％胎牛血清以及谷氨酰胺、青霉素和链霉素的DMEM培养基，37℃，5％CO 2，饱和湿度；待细胞汇合度达到90％以上时，将细胞培养基更换为2％血清的DMEM培养基，每板接种10 6PFU的PRV-WT。
继续培养24小时后，PRV-WT在RD细胞内增殖并引起细胞出现CPE；当90％以上细胞出现收缩变圆、颗粒感增加以及脱落裂解时，收获细胞及其培养液上清；经3次反复冻融后，收集培养上清进行离心除去细胞碎片，离心条件为4000rpm，10min，4℃； 最后，用0.22μm的一次性滤器(Millipore公司)过滤上清，除尽细胞碎片等杂质。
2.3病毒滴度的测定
将RD细胞铺于6孔板当中，细胞密度为10 5个/孔；待细胞贴壁长满之后，将病毒进行10倍梯度稀释，每孔100ul，感染感染细胞，每15分钟震荡混匀一次，5次之后，移去上清。将用pbs配制的2％琼脂糖溶液，加热溶解之后将其与10％血清的DMEM培养基按体积比1:1混匀，加入到细胞中，待其冷却凝固后倒扣在培养箱中，培养三天之后加入10％的甲醛溶液进行固定1小时，再将胶扣出，利用结晶紫染液进行染色15min，通过计算形成空斑的个数，来确定病毒的滴度。
2.4病毒的体外抗肿瘤实验
将人肿瘤细胞与正常细胞按照10 4个/孔接种至96孔板；待细胞贴壁，每孔更换为无血清的对应细胞培养基，并且分别接种MOI为10、1、0.1和0.01的病毒；随后，每天在显微镜下观察细胞是否产生CPE。
显微镜照片如图1A-1C所示。结果显示，在感染复数(MOI)为1感染72小时后，病毒感染组的肿瘤细胞数量明显减少，且明显皱缩、破裂等现象；而经病毒感染的非肿瘤细胞与Mock组的非肿瘤细胞相比，其细胞形态变化较小。上述结果表明，PRV对人和鼠多种肿瘤细胞系均有显著的溶瘤作用，但对非肿瘤细胞人胚肺成纤维细胞MRC5影响较小。此外，在MOI为1感染24小时后，Renca细胞的CPE已十分明显，到48小时几乎全部裂解死亡(图2)。
在病毒感染培养72小时后使用Cell Counting Kit-8(CCK-8试剂盒；上海碧云天生物技术有限公司)检测细胞存活率，具体方法如下：
在96孔细胞培养板中，贴壁细胞直接弃掉原培养基，悬浮细胞经离心后小心弃掉原培养基，随后更换为每孔100μl的新鲜无血清培养基；在接种细胞的孔中每孔加入10μl CCK-8溶液，同时在空白培养液中也加入等量的CCK-8溶液作为阴性对照；在细胞培养箱内37℃孵育0.5-3小时，在0.5、1、2、3小时分别利用酶标仪在吸光度为450nm进行一次检测，选取吸光度范围比较适宜的时间点作为细胞存活率的参考。PRV-WT对各细胞的CCK-8检测结果参见表2，其中，“-”表示病毒处理后的细胞存活率与MOCK组相比无显著性差异；“+”表示病毒处理后的细胞数量减少但存活率仍大于50％，且与MOCK组相比具有显著性差异；“++”表示病毒处理后的细胞存活率小于50％，且与MOCK组相比具有显著性差异。
细胞存活率的计算方法为：
Figure PCTCN2019074349-appb-000046
表2：PRV-WT体外抗肿瘤实验结果
Figure PCTCN2019074349-appb-000047
注：“-”表示病毒处理后的细胞存活率与MOCK组相比无显著性差异；“+”表示病毒 处理后的细胞数量减少但存活率仍大于50％，且与MOCK组相比具有显著性差异；“++”表示病毒处理后的细胞存活率小于50％，且与MOCK组相比具有显著性差异。
由表2可知，PRV-WT对大部分所检测的肿瘤细胞都有很好的杀伤作用，特别的，该病毒对肺癌、肝癌、卵巢癌、神经母细胞瘤、宫颈癌、淋巴瘤、肾癌等细胞系杀伤十分显著。另一方面，PRV-WT对非肿瘤细胞系包括人胚肺成纤维细胞系MRC-5存在一定杀伤。
实施例3：野生型PRV的体内抗肿瘤实验
3.1病毒、细胞系与实验动物
(1)病毒：本实施例中使用实施例1所提供的PRV-WT。病毒的培养与病毒滴度测定方法分别参见实施例2.2和2.3。
(2)细胞系：人鼻咽癌细胞系CNE1、人Burkitt's淋巴瘤细胞Raji(
Figure PCTCN2019074349-appb-000048
Number:CCL-86 TM)、人神经胶质瘤细胞系GBM(病人肿瘤组织分离原代肿瘤细胞系)、小鼠结肠癌细胞CT26、小鼠肝癌细胞Hep1-6、小鼠肾癌细胞Renca和小鼠乳腺癌细胞4T1。以上细胞除Raji使用RPMI-1640培养基，其他细胞均使用DMEM培养基培养，并且上述培养基均添加入10％的胎牛血清、谷氨酰胺与青霉素-链霉素双抗。以上所有细胞均在37℃，5％CO 2的标准条件下培养。
(3)实验动物：6-8周龄的雌性C.B17SCID小鼠或Bab/c小鼠来源于上海斯莱克实验动物有限责任公司；根据厦门大学实验动物中心与伦理委员会所批准的方案，将该小鼠在SPF条件下饲养。
3.2病毒的体内抗肿瘤实验
对于人肿瘤移植模型，使用SCID小鼠，将用于皮下成瘤的肿瘤细胞用0.01％胰蛋白酶消化后，再使用含10％的胎牛血清的细胞培养基重悬成单细胞悬液；计数悬液的细胞密度，1000g，3min离心沉淀细胞，再用适量体积的PBS重悬细胞，使达到约10 6-10 7个细胞/100μl PBS；按照10 6-10 7个细胞/100μl PBS/点在SCID小鼠背部皮下用注射器接种肿瘤细胞，待14-21天左右、肿瘤细胞在SCID小鼠皮下形成大约100mm 3的瘤块的时候，将荷瘤SCID小鼠随机分为实验组使用PRV-WT(BK61)与阴性对照组(Mock)进行治疗。对于小鼠肿瘤模型，使用Bab/c小鼠，皮下接种肿瘤细胞，7-14天后，选取100mm 3左右的小鼠进行治疗。
使用10 6TCID50/100μl无血清培养基/每瘤块的溶瘤病毒PRV-WT或等量无血清培养基瘤内注射处理，每两天注射1次，共处理5次。
每两天用游标卡尺测量并记录肿瘤大小变化，肿瘤大小的计算方法为：
肿瘤大小(mm 3)＝肿瘤长度数值×(肿瘤宽度数值) 2/2。
PRV-WT对人肿瘤移植模型和小鼠肿瘤模型的治疗结果分别如图3和图4所示。结果显示，使用PRV-WT进行治疗后，所检测的CNE1(图3A)、GBM(图3B)、Raji(图3C)以及CT26(图4A)、Hep1-6(图4B)、Renca(图4C)和4T1(图4D)肿瘤生长均出现逐渐变慢和停滞，甚至裂解消失；相比之下，阴性组(Mock)肿瘤则保持正常生长，其肿瘤大小显著大于实验组。
上述结果表明PRV-WT展现出显著有利的体内抗肿瘤活性。
实施例4：溶瘤病毒的安全性评价
4.1所用的病毒与实验动物
(1)病毒：本实施例使用由实施例1所提供的PRV-WT。病毒的培养与病毒滴度测定方法分别参照实施例2.2和2.3。
(2)实验动物：6-8周龄Bab/c小鼠来源于上海斯莱克实验动物有限责任公司；根据厦门大学实验动物中心与伦理委员会所批准的方案，将该小鼠在清洁级条件下饲养，随后用于伪狂犬病毒的体内毒力评估。
4.2病毒在小鼠体内的安全性评价
(1)选取Bab/c小鼠，并对其单次静脉注射PRV-WT或PBS，攻毒滴度剂量为10 7TCID50/只(每组6只)，随后每天监测攻毒组Bab/c小鼠的存活率和体重进行记录。注射PBS或PRV-WT后的小鼠体重统计结果分别如图5A-B所示，在攻毒后的15天内，攻毒组小鼠均未死亡，攻毒组动物体重增长趋势与PBS对照组一致，未见统计学差异(P>0.05)。这一结果表明，PRV-WT在小鼠静脉模型中具有十分好的安全性。
(2)选取Bab/c小鼠，并对其颅内注射不同剂量的PRV-WT，攻毒滴度剂量为2*10 6、2*10 5、2*10 4PFU/只(每组4只)，随后每天对不同剂量攻毒组Bab/c小鼠的存活率进行记录。结果如图6所示，小鼠陆陆续续出现死亡的现象，并且这种现象存在剂量依赖关系，说明PRV-WT具有一定的神经毒性。
实施例5：修饰型PRV的抗肿瘤活性及安全性评价
5.1所用的病毒：
本实施例使用由实施例1所提供的PRV-del-EP0(SEQ ID NO:4)。病毒的培养与病毒滴度测定方法分别参照实施例2.2和2.3。
5.2PRV-del-EP0的体外溶瘤活性评价
参照实施例2所描述的方法使用MOI＝1的PRV-del-EP0(BK61-dEP0)处理目的细胞，并使用CCK8法检测经PRV-del-EP0(BK61-dEP0)处理后的细胞存活率。图7A显示了PRV-del-EP0(BK61-dEP0)对多种肿瘤细胞系的杀伤结果，结果显示，其基本保留了与母本毒株野生型PRV相当的杀伤效果。图7B显示了PRV-del-EP0(BK61-dEP0)对多种非肿瘤细胞系的杀伤结果，结果显示，与母本毒株野生型PRV相比，PRV-del-EP0对二倍体细胞系(类正常细胞系)显示出显著降低的杀伤活性。以上结果表明，PRV-del-EP0不仅保留了野生型PRV显著的肿瘤杀伤活性，并且在一定程度上能提升了安全性。
5.3PRV-del-EP0的体内安全性评价
选取ICR小鼠并对其颅内注射不同剂量的PRV-WT与PRV-del-EP0，攻毒滴度剂量为1*10 6、1*10 5、1*10 4、1*10 3、1*10 2、1*10 1PFU/只(每组4只)，随后每天对不同剂量攻毒组ICR小鼠的存活率进行记录。PRV-WT(BK61-WT)组的结果如图8A所示，PRV-del-EP0(BK61-dEP0)组的结果如图8B所示。结果显示，PRV-WT组小鼠陆陆续续出现死亡的现象，1*10 6和1*10 5组小鼠全部死亡，1*10 4PFU组小鼠的存活率仅为25％；相比之下，PRV-del-EP0组只有1*10 6组小鼠出现死亡现象，其生存率为50％。以上结果表明，相比于野生型PRV，PRV-del-EP0具有显著提高的体内安全性。
5.4PRV-del-EP0的体内治疗效果评价
将用于免疫鼠C57/B6小鼠皮下成瘤的肿瘤细胞Hep1-6用0.01％胰蛋白酶消化后，再使用含10％的胎牛血清的细胞培养基重悬成单细胞悬液；计数悬液的细胞密度，1000g，3min离心沉淀细胞，再用适量体积的PBS重悬细胞，使达到约10 6-10 7个细胞/100μl PBS；按照10 6-10 7个细胞/100μl PBS/点在C57/B6小鼠背部皮下用注射器接种肿瘤细胞，待7-14天左右、肿瘤细胞在SCID小鼠皮下形成大约100mm 3的瘤块的时候，将荷瘤SCID小鼠随机分为3组，分别使用PRV-WT(BK61-WT)，PRV-del-EP0(BK61-dEP0)和PBS进行瘤内注射治疗，每两天注射1次，共处理3次。每两天用游标卡尺测量并记录肿瘤大小变化，肿瘤大小的计算方法为：
肿瘤大小(mm 3)＝肿瘤长度数值×(肿瘤宽度数值) 2/2。
结果如图9所示，PRV-WT、PRV-del-EP0组小鼠的肿瘤均能完全清除，表明PRV-del-EP0展现出了与野生株同样显著的体内治疗效果。
同时检测小鼠生存率，根据动物伦理，当小鼠肿瘤尺寸达到2000mm 3将其处死。结果如图10所示，经PRV-WT和PRV-del-EP0治疗均能够显著改善小鼠的生存率。
进一步对小鼠肿瘤复发率进行评估。具体而言，对上述PRV-WT和PRV-del-EP0组的治愈后小鼠再次进行肿瘤接种，接种细胞数为初始接种细胞量的10倍，同时以未经PRV-WT和PRV-del-EP0治疗过的小鼠为对照组(NC)，接种同等量肿瘤细胞，监测小鼠肿瘤生长情况，对复发肿瘤的小鼠只数进行统计，计算肿瘤复发率，肿瘤复发率＝(出现肿瘤的小鼠只数/进行肿瘤接种的小鼠总数)×100％。结果如图11所示，PRV-WT和PRV-del-EP0组小鼠均未复发肿瘤，而未经PRV-WT和PRV-del-EP0治疗过的对照组小鼠全部发现肿瘤。这一结果提示经PRV-WT和PRV-del-EP0治愈后的小鼠都具有很好的抗肿瘤免疫力，能预防肿瘤的复发。
尽管本发明的具体实施方式已经得到详细的描述，但本领域技术人员将理解：根据已经公布的所有教导，可以对细节进行各种修改和变动，并且这些改变均在本发明的保护范围之内。本发明的全部分为由所附权利要求及其任何等同物给出。

Claims (14)

  1. 伪狂犬病毒(PRV)或其修饰形式或一种核酸分子，用于在受试者中治疗肿瘤和/或降低或抑制肿瘤复发的用途，或者用于制备在受试者中治疗肿瘤和/或降低或抑制肿瘤复发的药物的用途；其中，所述核酸分子包含选自下列的序列：
    (1)PRV或其修饰形式的基因组序列或cDNA序列；
    (2)所述cDNA序列的互补序列。
  2. 权利要求1所述的用途，其中，所述PRV为野生型PRV；
    优选地，所述PRV是从感染伪狂犬病毒(PRV)的动物体内分离得到的毒株；
    优选地，所述PRV或其修饰形式的基因组序列与如SEQ ID NO：1所示的核苷酸序列具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性；更优选地，所述PRV或其修饰形式的基因组序列为如SEQ ID NO：1所示的核苷酸序列。
  3. 权利要求1或2所述的用途，其中，所述修饰形式为经修饰的PRV，其与野生型PRV相比在基因组中具有一个或多个核苷酸的替换、插入或删除；
    优选地，所述经修饰的PRV与野生型PRV相比，具有一个或多个选自下列的修饰：
    (1)一个或多个内源基因的缺失或突变；
    (2)位于非翻译区(例如启动子)中的一个或多个核苷酸的突变、缺失或插入；
    (3)一个或多个外源核苷酸序列的插入；和
    (4)上述三项的任意组合。
  4. 权利要求3所述的用途，其中，所述经修饰的PRV不表达功能性EP0蛋白；
    优选地，所述经修饰的PRV的基因组包含下述修饰：EP0基因包含功能丧失性突变(例如，一个或多个核苷酸的添加、缺失和/或置换)或者被缺失或者被置换为外源核苷酸序列(例如，编码外源蛋白的核苷酸序列)；
    优选地，所述功能丧失性突变选自错义突变、无义突变、移码突变、碱基缺失、碱基置换、碱基添加、以及其任何组合(例如，基因片段的缺失或置换或添加)；
    优选地，所述经修饰的PRV的基因组包含下述修饰：EP0基因被缺失或被置换为外源核苷酸序列(例如，编码外源蛋白的核苷酸序列)；
    优选地，所述经修饰的PRV的基因组序列与SEQ ID NO：4所示的核苷酸序列相比具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性；
    优选地，所述经修饰的PRV的基因组序列如SEQ ID NO：4所示。
  5. 权利要求3或4所述的用途，其中，所述经修饰的PRV包括位于非编码区(如启动子)中的一个或多个核苷酸的突变、缺失或插入；
    优选地，所述经修饰的PRV的基因组包含下述修饰：一个或多个PRV基因的原生启动子被替换为肿瘤特异性启动子。
  6. 权利要求3-5任一项所述的用途，其中，所述经修饰的PRV包含外源核苷酸序列；
    优选地，所述外源核苷酸序列编码选自下列的外源蛋白：荧光蛋白、免疫调节多肽、细胞因子、趋化因子、抗肿瘤蛋白或多肽。
  7. 权利要求1-6任一项所述的用途，其中，所述药物包括PRV以及其修饰形式中的一种或数种。
  8. 权利要求1-7任一项所述的用途，其中，所述核酸分子由所述PRV或其修饰形式的基因组序列或cDNA序列，或所述cDNA序列的互补序列组成；
    优选地，所述核酸分子具有所述PRV或其修饰形式的基因组序列；
    优选地，所述核酸分子具有选自下列的核苷酸序列：
    (1)如SEQ ID NO：1或4所示的核苷酸序列；
    (2)与如SEQ ID NO：1或4所示的核苷酸序列具有至少70％、至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的核苷酸序列；
    优选地，所述核酸分子具有如SEQ ID NO:1或4所示的核苷酸序列。
  9. 权利要求1-7任一项所述的用途，其中，所述核酸分子为包含所述PRV或其修饰形式的基因组序列或cDNA序列，或所述cDNA序列的互补序列的载体(例如，克隆载体或表达载体)；
    优选地，所述核酸分子为包含所述PRV或其修饰形式的cDNA序列，或所述cDNA序列的互补序列的载体(例如，克隆载体或表达载体)。
  10. 权利要求1-9任一项所述的用途，其中，所述药物包含治疗有效量的所述PRV和/或其修饰形式，或治疗有效量的所述核酸分子。
  11. 权利要求1-10任一项所述的用途，其中，所述药物还包含具有抗肿瘤活性的另外的药学活性剂，例如另外的溶瘤病毒、化学治疗剂或免疫治疗剂；
    优选地，所述另外的溶瘤病毒选自腺病毒、细小病毒、呼肠孤病毒、新城疫病毒、水疱性口炎病毒、麻疹病毒或其任意组合；
    优选地，所述化学治疗剂选自5-氟尿嘧啶、丝裂霉素、甲氨蝶呤、羟基脲、环磷酰胺、达卡巴嗪、米托蒽醌、蒽环类、依托泊苷、铂类化合物、紫杉烷类或其任意组合；
    优选地，所述免疫治疗剂选自免疫检查点抑制剂、肿瘤特异性靶向抗体或其任意组合。
  12. 权利要求1-11任一项所述的用途，其中，所述肿瘤选自神经胶质瘤、神经母细胞瘤、胃癌、肝癌、肾癌、肺癌、乳腺癌、结肠癌、淋巴瘤、卵巢癌、宫颈癌、子宫内膜癌、黑色素瘤、胰腺癌、成骨肉瘤、前列腺癌、鼻咽癌、鼻中隔鳞状细胞癌、喉癌、甲状腺癌、甲状腺导管癌和膀胱癌。
  13. 权利要求1-12任一项所述的用途，其中，所述受试者为哺乳动物，例如人。
  14. 一种治疗肿瘤和/或降低或抑制肿瘤复发的方法，其包括向有此需要的受试者施用有效量的如权利要求1-13任一项中所定义的PRV或其修饰形式，或有效量的如权利要求1-13任一项中所定义的核酸分子的步骤；
    优选地，所述肿瘤选自神经胶质瘤、神经母细胞瘤、胃癌、肝癌、肾癌、肺癌、乳腺癌、结肠癌、淋巴瘤、卵巢癌、宫颈癌、子宫内膜癌、黑色素瘤、胰腺癌、成骨肉瘤、前列腺癌、鼻咽癌、鼻中隔鳞状细胞癌、喉癌、甲状腺癌、甲状腺导管癌和膀胱癌；
    优选地，所述受试者为哺乳动物，例如人。
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