WO2021175293A1 - 一种单纯疱疹病毒及其用途 - Google Patents

一种单纯疱疹病毒及其用途 Download PDF

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WO2021175293A1
WO2021175293A1 PCT/CN2021/079114 CN2021079114W WO2021175293A1 WO 2021175293 A1 WO2021175293 A1 WO 2021175293A1 CN 2021079114 W CN2021079114 W CN 2021079114W WO 2021175293 A1 WO2021175293 A1 WO 2021175293A1
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gene
virus
herpes simplex
simplex virus
genes
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French (fr)
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赵婧姝
李小鹏
田超
周华
王田田
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北京唯源立康生物科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • 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
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    • 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
    • C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
    • C12N2710/16621—Viruses as such, e.g. new isolates, mutants or their genomic sequences
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    • 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
    • C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
    • C12N2710/16622—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
    • C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
    • C12N2710/16632—Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent
    • 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
    • C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
    • C12N2710/16641—Use of virus, viral particle or viral elements as a vector
    • C12N2710/16643—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
    • C12N2710/16661—Methods of inactivation or attenuation
    • C12N2710/16662—Methods of inactivation or attenuation by genetic engineering

Definitions

  • the present invention relates to a new type of herpes simplex virus, modification of the virus, and genetically modified herpes simplex virus and uses thereof.
  • Herpes Simplex Virus belongs to the Herpesvirus family, and is an enveloped double-stranded DNA virus, including two subtypes, type I (HSV-1) and type II (HSV-2).
  • Herpes simplex virus consists of four parts: core, capsid, interlayer protein and envelope.
  • the core of the virus is composed of thick double-stranded DNA, wound into a filament reel.
  • the capsid surrounded by DNA has an icosahedral structure with a diameter of 100-110 nm and is composed of 162 capsids.
  • the outer capsid is covered by interlayer proteins that are arranged irregularly and irregularly.
  • the outermost layer of the virus is a lipid bilayer membrane with short protrusions, and the diameter of the virus enveloping the membrane is 150-200nm.
  • the viral envelope is composed of 11 glycoproteins and at least two non-glycosylated proteins, including viral glycoprotein B (gB), viral glycoprotein C (gC), viral glycoprotein D (gD), viral glycoprotein G (gG) ) And viral glycoprotein M (gM).
  • the three-dimensional structure of the virus envelope glycoprotein is related to the virus's cell infection ability, which determines whether the virus can enter the host cell and the amount of virus that enters the host cell.
  • the HSV-1 virus has been used for gene therapy of major diseases such as tumors, degenerative diseases of the nervous system, genetic diseases and immune system diseases. Compared with other gene therapy virus vectors, the HSV-1 virus has many advantages.
  • HSV-1 the genome of HSV-1 is large and can carry large or multiple foreign genes [Roizman B. The function of herpes simplex virus genes: a primer for genetic engineering of novel vectors. [J]. Proceedings of the National Academy of Sciences, 1996, 93(21): 11307-11312].
  • the HSV-1 genome is 152kb long, while the adenovirus genome is only 35kb.
  • the more than 80 known genes in the HSV-1 genome about half are non-essential genes in in vitro culture and can be replaced by multiple exogenous therapeutic genes, and its maximum insertion amount of exogenous genes can reach 30-40 kb. This is especially important for the treatment of many diseases, especially diseases related to multiple genes.
  • HSV-1 is not integrated with cell DNA, replication is controllable, and safe. HSV-1 hardly produces life-threatening diseases in immunocompetent adults[ShenY,Nemunaitis J.Herpes simplex virus 1(HSV-1)for cancer treatment[J].Cancer Gene Therapy,2006,13(11):975 -992]. Many non-essential genes of HSV-1 are related to its neurotoxicity. Remove some non-essential genes in HSV-1, such as the ICP34.5 gene, so that HSV-1 can only selectively replicate in tumor cells, and normal tissue cells are not affected by it [Chou J, Kern E, Whitley R, et al. Mapping of herpes simple virus-1 neurovirulence to gamma 34.5, a gene nonessential for growth in culture[J].Science,1990,250(4985):1262-1266].
  • HSV-1 has become one of the most widely used viral vectors for gene therapy.
  • HSV-1 is usually transformed into oncolytic herpes simplex virus type 1 (oHSV-1) through genetic modification.
  • the main methods include knocking out certain genes and inserting Tumor therapy-related genes, etc.
  • oHSV-1 has been studied in preclinical and clinical trials, and a large number of results have shown that it has good safety and effectiveness.
  • the general strategy for the construction of gene-deleted oHSV-1 is to mutate or delete single or multiple genes of the virus to achieve its specific killing effect on tumors, such as genes such as ICP34.5, ICP6, ICPO, TK and UNG.
  • Talimogene laherparepvec, HSV1716, NV1020, G207, G47 ⁇ , etc. that have been listed and entered clinical trials have knocked out the ICP34.5 gene in HSV-1.
  • the oncolytic virus HSV1716 only knocks out the double copy of the ICP34.5 gene. In clinical trials for the treatment of refractory or relapsed high-grade glioma, it has shown tumor regression and no obvious toxicity or side effects have been found.
  • G207 knocked out the double-copy ICP34.5 gene while mutating the ICP6 gene.
  • Talimogene laherparepvec is the first oncolytic virus approved for marketing in the United States.
  • Type I herpes simplex virus (HSV-1) is considered to be a very promising virus in gene therapy due to its wide host cell range, high safety and neurotropism.
  • type I herpes simplex virus can be genetically modified into a non-replicating virus, and foreign genes can be introduced into the subject to treat related diseases.
  • HSV-1 carriers carrying ENK, GABA or endorphin genes can be injected to reduce pain caused by chronic inflammation and bone cancer and neuropathic pain, while carrying anti-inflammatory IL-4 and IL HSV-1 carriers such as -10 and IL-13 can also relieve acute and chronic pain [Goss J R, Krisky D M, Wechuck J B, et al. Herpes simplex virus-based nerve targeting gene therapy in pain management[J] .Journal of Pain Research,2014:71-79].
  • herpes simplex viruses currently used for gene delivery or treatment are laboratory virus strains that have been passaged in tissue culture cells for many years. Although these viruses can enter cells to deliver genes, they need to be improved in terms of characteristics required for disease treatment, such as the ability to transfect and express foreign genes.
  • the present application in order to overcome the problems existing in the prior art, provides a new HSV isolate, compared to the previously used continuous passage standard HSV-1 virus 17 strains (referred to as 17 virus strains or 17 strains),
  • the HSV-1 virus strain of the present invention has a greatly improved ability to infect human cells in vivo, and a significantly enhanced ability of intracellular replication/lysis, and is suitable as an oncolytic virus or as a carrier to destroy tumor cells through oncolytic action.
  • the new HSV-1 isolates provided by the present invention (referred to as strains HL-1, HL-1 strains or HL-1) have enhanced human tumor cell lines. copy.
  • strains HL-1, HL-1 strains or HL-1 have enhanced human tumor cell lines. copy.
  • ICP34.5 and/or ICP47 compared with the 17 strains of HSV-1 virus which also lacks ICP34.5 and/or ICP47, the ability of the virus strain HL-1 to replicate in human tumor cell lines and the The killing ability of tumor cells is significantly enhanced.
  • the modified HL-1 virus of the present invention was subsequently used to deliver genes with anti-tumor activity, which proved to further enhance the anti-tumor immune response activity and oncolytic effect.
  • this application also provides a non-replicating recombinant herpes virus
  • the non-replicating recombinant herpes virus is derived from the herpes simplex virus with CCTCC deposit number V201810, in which one or more of the immediate early genes are knocked out , Or through gene mutation to prevent or reduce its expression.
  • the transfection ability and the ability of expressing foreign genes of the non-replicating recombinant herpes virus of the present application are obviously enhanced. Therefore, the non-replicating recombinant herpes virus of the present application also has potential development value in introducing foreign genes into patients to treat diseases such as nervous system diseases, blood system diseases, and immune system diseases.
  • this application involves the following items on the one hand:
  • HSV-1 herpes simplex virus
  • herpes simplex virus After the herpes simplex virus has been modified, it has an enhanced ability to replicate in tumor cells compared to a reference laboratory strain with equivalent modifications;
  • the reference laboratory strain is the standard model strain HSV1 virus 17 strains.
  • the type 1 herpes simplex virus of item 1 which is a virus strain with CCTCC deposit number: V201810 (CCTCC: V201810).
  • the herpes simplex virus of item 1 which is an attenuated strain of the virus strain with CCTCC deposit number V201810 (CCTCC: V201810) or its progeny.
  • this application relates to a genetically engineered construct of a virus strain with CCTCC deposit number V201810 (CCTCC: V201810).
  • a herpes simplex virus derived from a virus strain with CCTCC deposit number V201810 (CCTCC: V201810), wherein the herpes simplex virus has ICP34.5 gene, ICP6 gene, ICP0 gene, ICP47 gene, US3 gene, UL56 gene , One or more of the functional VP16 gene, VHS gene, UNG gene, glycoprotein H gene, and thymidine kinase gene are destroyed or knocked out.
  • the herpes simplex virus of item 4 in which the ICP34.5 gene or/and the ICP47 gene are destroyed or knocked out.
  • composition containing the herpes simplex virus of items 1-5 is a composition containing the herpes simplex virus of items 1-5.
  • composition comprising the host cell of item 8.
  • composition of item 9 which is a cell culture.
  • HSV-1 herpes simplex virus type 1
  • herpes simplex virus After the herpes simplex virus has been modified, it has an enhanced ability to replicate in tumor cells compared to a reference laboratory strain with equivalent modifications;
  • the reference laboratory strain is the standard model strain HSV1 virus 17 strains.
  • Herpes simplex virus type 1 obtained by the preparation method of item 11 or 12.
  • cytokine is selected from one or more of the following: GM-CSF, G-CSF, M-CSF, IL-1, IL-2, IL-3, IL- 4. IL-5, IL-6, IL7, IL-8, IL-10, IL-12, IL-13, IL-15, IL-18, IL-21, IL-23, IFN- ⁇ , IFN- ⁇ , TGF- ⁇ and TNF- ⁇ .
  • the monoclonal antibody having the effect of preventing and/or treating tumors is selected from one or more of the following: PD1 monoclonal antibody, PD-L1 monoclonal antibody, PD-L2 monoclonal antibody, CTLA-4 Mab, CD80 Mab, CD28 Mab, CD137 Mab, CD137L Mab, OX40 Mab, OX40L Mab, CD27 Mab, CD70 Mab, CD40 Mab, CD40L Mab, LAG-3 Mab and TIM -3 monoclonal antibodies.
  • tumor antigen is selected from one or more of the following: PSA, MUC1, MAGE-1, MAGE-2, MAGE-3, MAGE-12, BAGE, GAGE and LAGE.
  • herpes simplex virus according to item 5 wherein the prodrug converting enzyme is cytosine deaminase or herpes simplex virus thymidine kinase.
  • cytokine polypeptide that stimulates an immune response is selected from one or more of the following: granulocyte macrophage colony stimulating factor (GMCSF), IL-2, IL12, IFN- ⁇ and TNFa.
  • GMCSF granulocyte macrophage colony stimulating factor
  • IL-2 granulocyte macrophage colony stimulating factor
  • IL12 IL12
  • IFN- ⁇ TNFa
  • the genetically modified herpes simplex virus according to item 16 wherein the one or more immediate early genes are selected from one, two, three or four of the coding genes of ICPO, ICP4, ICP22 and ICP27.
  • composition comprising the genetically modified herpes simplex virus of any one of items 1-17.
  • the herpes simplex virus vector described in item 1 which of the ICP34.5 gene, ICP6 gene, ICPO gene, ICP47 gene, US3 gene, UL56 gene, VHS gene, UNG gene, glycoprotein H gene, and thymidine kinase gene One or more are destroyed or knocked out.
  • herpes simplex virus vector according to item 1 or 2 in preparing a recombinant oncolytic virus, wherein one or more foreign genes are introduced into the recombinant oncolytic virus.
  • the one or more foreign gene insertions are selected from the following one or more gene deletion sites: ICP34.5 gene, ICP6 gene, ICPO gene, ICP47 gene, US3 Gene, UL56 gene, VHS gene, UNG gene, glycoprotein H gene, thymidine kinase gene.
  • exogenous gene is selected from one or more of the following: a gene encoding a cytokine that promotes an immune response, a gene encoding a tumor antigen, a gene encoding a tumor-preventing and/or treating tumor Antigenes, genes encoding prodrug convertase, tumor suppressor genes, antisense RNA or small RNA.
  • cytokine is selected from one or more of the following: GM-CSF, G-CSF, M-CSF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL7, IL-8, IL-10, IL-12, IL-13, IL-15, IL-18, IL-21, IL-23, IFN- ⁇ , IFN- ⁇ , TGF- ⁇ and TNF- ⁇ .
  • the monoclonal antibody having the effect of preventing and/or treating tumors is selected from one or more of the following: PD1 monoclonal antibody, PD-L1 monoclonal antibody, PD-L2 monoclonal antibody, CTLA-4 monoclonal antibody , CD80 mAb, CD28 mAb, CD137 mAb, CD137L mAb, OX40 mAb, OX40L mAb, CD27 mAb, CD70 mAb, CD40 mAb, CD40L mAb, LAG-3 mAb and TIM-3 Monoclonal antibody.
  • tumor antigen is a tumor-derived specific antigen, selected from one or more of the following: PSA, MUC1, MAGE-1, MAGE-2, MAGE-3, MAGE-12, BAGE, GAGE and LAGE.
  • prodrug converting enzyme is cytosine deaminase or herpes simplex virus thymidine kinase.
  • RNA or small RNA is an RNA fragment that blocks or down-regulates tumor overexpression of proto-oncogenes and metabolic genes.
  • cytokine that promotes an immune response is granulocyte macrophage colony stimulating factor (GMCSF), IL-2, IL12, IFN- ⁇ and/or TNFa.
  • GMCSF granulocyte macrophage colony stimulating factor
  • a method for preparing a recombinant oncolytic virus comprising introducing one or more exogenous genes into herpes simplex virus, wherein the herpes simplex virus is derived from the herpes simplex virus with CCTCC deposit number V201810.
  • any one of items 15-17, wherein the one or more foreign gene insertions are selected from one or more gene deletion sites selected from the following: ICP34.5 gene, ICP6 gene, ICPO gene, ICP47 Gene, US3 gene, UL56 gene, VHS gene, UNG gene, glycoprotein H gene, thymidine kinase gene.
  • the exogenous gene is selected from one or more of the following: a gene encoding a cytokine that promotes an immune response, a gene encoding a tumor antigen, a gene encoding a tumor-preventing and/or treating tumor Antigenes, genes encoding prodrug convertase, tumor suppressor genes, antisense RNA or small RNA.
  • cytokine is selected from one or more of the following: GM-CSF, G-CSF, M-CSF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL7, IL-8, IL-10, IL-12, IL-13, IL-15, IL-18, IL-21, IL-23, IFN- ⁇ , IFN- ⁇ , TGF- ⁇ and TNF- ⁇ .
  • the monoclonal antibody having the effect of preventing and/or treating tumors is selected from one or more of the following: PD1 monoclonal antibody, PD-L1 monoclonal antibody, PD-L2 monoclonal antibody, CTLA-4 monoclonal antibody , CD80 mAb, CD28 mAb, CD137 mAb, CD137L mAb, OX40 mAb, OX40L mAb, CD27 mAb, CD70 mAb, CD40 mAb, CD40L mAb, LAG-3 mAb and TIM-3 Monoclonal antibody.
  • tumor antigen is a tumor-derived specific antigen, selected from one or more of the following: PSA, MUC1, MAGE-1, MAGE-2, MAGE-3, MAGE-12, BAGE, GAGE and LAGE.
  • RNA or small RNA is an RNA fragment that blocks or down-regulates tumor overexpression of proto-oncogenes and metabolic genes.
  • cytokine polypeptide that stimulates an immune response is granulocyte macrophage colony stimulating factor (GMCSF), IL-2, IL12, IFN- ⁇ and/or TNFa.
  • GMCSF granulocyte macrophage colony stimulating factor
  • a recombinant oncolytic virus prepared by the method of any one of items 15-29.
  • composition comprising the recombinant oncolytic virus of item 30.
  • composition of item 31 wherein one, two, three or four foreign genes are inserted into the same site or different sites of the herpes simplex virus.
  • composition of item 31 or 32, wherein the recombinant oncolytic virus is a mixture of two, three, four or four recombinant oncolytic viruses prepared by the method of any one of items 15-29, wherein each The recombinant oncolytic virus introduced different foreign genes.
  • composition according to any one of items 31 to 33 which is a virus culture.
  • the invention relates to:
  • a genetically modified herpes simplex virus in which one or more of the immediate early genes are knocked out or destroyed, or its expression is prevented or reduced by gene mutations, thereby losing the ability to replicate.
  • the herpes simplex virus is derived from the CCTCC deposit number It is a virus strain of V201810, its attenuated strain or its descendants.
  • the herpes simplex virus according to item 1 wherein the one or more immediate early genes are one, two, three or four selected from the group consisting of ICPO, ICP4, ICP22 and ICP27 encoding genes.
  • the herpes simplex virus of item 1 or 2 which is derived from the virus strain with CCTCC deposit number V201810.
  • the herpes simplex virus according to any one of items 1-3, its ICP34.5 gene, ICP6 gene, ICPO gene, ICP47 gene, US3 gene, UL56 gene, functional VP16 gene, VHS gene, UNG gene, glycoprotein One or more of the H gene and thymidine kinase gene are destroyed or knocked out.
  • composition of item 10 which is a virus culture.
  • composition comprising the host cell of item 12.
  • composition of item 13 which is a cell culture.
  • herpes simplex virus with CCTCC deposit number V201810 or its attenuated variant or its progeny as a vector for introducing nerve growth factor, neurotransmitter and/or neurotrophic factor exogenous genes into cells.
  • herpes simplex virus according to any one of items 1-9 in the preparation of a medicine for treating or preventing central nervous system diseases or peripheral nervous system diseases.
  • peripheral nervous system diseases include motor neuron disease, chronic pain, and peripheral nerve damage.
  • herpes simplex virus vector comprising any one of items 1-9 in the preparation of a medicine for treating or preventing pain or nerve damage.
  • This application also relates to a non-replicating herpes simplex virus vector, in which one or more immediate early genes of the herpes simplex virus are knocked out or destroyed to lose the ability to replicate, or its expression is blocked or reduced by gene mutations and thus lost Copy ability.
  • the one or more immediate early genes are one, two, three, or four selected from the group consisting of ICP0, ICP4, ICP22, ICP47, and ICP27 encoding genes.
  • the herpes simplex virus is derived from the herpes simplex virus with CCTCC deposit number V201810 or its progeny.
  • the ICP34.5 gene, ICP6 gene, ICPO gene, ICP47 gene, US3 gene, UL56 gene, VP16 gene, VHS gene, UNG gene, glycoprotein H gene, thymidine kinase gene of the herpes simplex virus One or more of them are destroyed or knocked out.
  • the ICP34.5 gene of the herpes simplex virus is disrupted or knocked out.
  • the ICP47 gene of the herpes simplex virus is disrupted or knocked out.
  • the herpes simplex virus vector further comprises one or more foreign genes introduced.
  • the exogenous gene is a nerve growth factor, neurotransmitter and/or neurotrophic factor gene.
  • the exogenous gene is a coagulation factor gene.
  • This application also relates to a non-replicating herpes simplex virus, which is derived from the herpes simplex virus with CCTCC deposit number V201810, wherein one or more of the herpes simplex virus is knocked out or destroyed immediately and early genes, thereby losing the ability to replicate , Or its expression is prevented or reduced by genetic mutations, thereby losing the ability to replicate.
  • the one or more immediate early genes are one, two, three, or four selected from the group consisting of ICP0, ICP4, ICP22, ICP47, and ICP27 encoding genes.
  • the ICP34.5 gene, ICP6 gene, ICPO gene, ICP47 gene, US3 gene, UL56 gene, VP16 gene, VHS gene, UNG gene, glycoprotein H gene, thymidine kinase gene of the herpes simplex virus One or more of them are destroyed or knocked out.
  • the ICP34.5 gene of the herpes simplex virus is disrupted or knocked out.
  • the ICP47 gene of the herpes simplex virus is disrupted or knocked out.
  • exogenous genes such as nerve growth factor, neurotransmitter, neurotrophic factor, or coagulation factor are introduced into the virus.
  • Figure 1 25000 times electron micrograph of HL-1 strain.
  • FIG. 1 HL-1 and HSV (17) transfected HepG2 cells with different MOI (multiplicity of infection).
  • FIG. 3 HL-1 and HSV (17) transfected A375 cells with different MOIs.
  • FIG. 4 HL-1 and HSV (17) transfected A549 cells with different MOIs.
  • FIG. 5 HL-1 and HSV (17) transfected MCF-7 cells with different MOI.
  • FIG. 6 HL-1 and HSV (17) transfected Hela cells with different MOI.
  • FIG. 7 Transfection of U251 cells with HL-1 and HSV (17) at different MOIs.
  • FIG. 8 Vero cells were transfected with different MOIs of HL-1 and HSV (17).
  • Figure 9 Comparison of IC50 of HL-1 and HSV(17) transfected into different cells.
  • Figure 10 CCTCC knocked out ICP34.5 and ICP47 genes: IC50 determination results of V201810 virus strain and control virus 17 virus strain transfected into different cells.
  • FIG. 11 CCTCC expressing IL-12 exogenous gene: IC50 determination results of tumor cells transfected by V201810 virus strain and control virus 17 virus strain.
  • Figure 12 IC50 determination results of HL-1 virus strain recombinant virus and 17 virus strain recombinant virus transfected into tumor cells after insertion of IL-12 gene and PD1 monoclonal antibody gene.
  • Figure 13 shows the comparison of the transfection ability of the HL-1 strain and the 17 strains of recombinant viruses with the ICP34.5 and ICP4 genes knocked out to nerve cells.
  • Figure 14 Shows the comparison of the transfection ability of the HL-1 strain and the 17 strains of recombinant viruses with the ICP34.5, ICP4, and ICP27 genes knocked out on nerve cells.
  • Figure 15 Shows the transfection of human embryonic liver diploid cells by HL-1 strain and 17 strains of recombinant viruses with knockout of ICP34.5 and ICP4 genes and the expression of foreign genes in hepatocytes.
  • Figure 16 shows the transfection of human embryonic liver diploid cells by HL-1 strain and 17 strains of recombinant viruses with knockout of ICP34.5, ICP4 and ICP27 genes and the expression of foreign genes in liver cells.
  • Figure 17 Shows the effect of HSV-FVIII administration at 48 hours, 72 hours and 120 hours on APTT.
  • Figure 18 Shows the effect of HL1-ENK on the behavioral activity score of arthritis chronic pain rats, proving that HL1-ENK has a significant analgesic effect on arthritis chronic pain rats.
  • This application relates to a non-replicating herpes simplex virus with improved performance compared with laboratory model virus strains and its use as a vector.
  • the "oncolytic virus” mentioned in this application refers to a type of virus that infects and kills cancer cells. After the virus infects cancer cells, it destroys the infected cancer cells through oncolytic action, and at the same time releases new infectious virus particles or viruses. Body, destroy the remaining cancer cells. This type of virus gets its name from its oncolytic effect.
  • Herpes Simplex Virus is one of the first viruses (oncolytic viruses) chosen to selectively attack cancer cells because of its deep research foundation and relatively harmless in its natural state.
  • the virus of the present invention is herpes simplex virus
  • the virus may be derived from, for example, HSV-1 strain or HSV-2 strain or their derivative strains, preferably HSV-1.
  • Some embodiments of this application relate to a virus strain with CCTCC deposit number: V201810 (CCTCC: V201810).
  • the virus strain is referred to as HSV-1 virus HL-1 strain, or HL-1 strain for short, HL-1 or HL1.
  • Some embodiments of the present application relate to derived strains of HSV-1 strains, for example, the genome sequence homology of the virus strain with CCTCC deposit number: V201810 (CCTCC: V201810) is at least 70%, more preferably at least 80%, 85% , 90%, 95%, 98% sequence homology virus strains.
  • Herpes simplex virus vector refers to a herpes simplex virus carrying foreign genes.
  • Plaque forming unit refers to the number of viruses corresponding to the formation of plaques (plaques) on animal cells cultured in a single layer.
  • Immune checkpoint blocking (inhibitory) antibodies or “immune checkpoint blocking (inhibitory) monoclonal antibodies” refer to monoclonal antibodies that inhibit or block suppressive immune checkpoint molecules.
  • Immune checkpoint stimulating (agonistic) antibodies or “immune checkpoint stimulating (agonistic) monoclonal antibodies” refer to monoclonal antibodies that stimulate or agonize stimulating immune checkpoint molecules.
  • Immune checkpoints are the regulators of the immune system. Their role is to enhance the immune system's ability to eliminate foreigners or prevent the immune system from attacking cells indiscriminately, which is essential for immune regulation. Immune checkpoints are divided into inhibitory checkpoint molecules and stimulatory checkpoint molecules.
  • Inhibitory checkpoint molecules include but are not limited to: A2AR, B7-H3 (CD276), B7-H4, BTLA (CD272), CTLA-4 (CD152), IDO, KIR, LAG3, NOX2, PD1, TIM3, VISTA, CD47 ; Stimulant checkpoint molecules include but are not limited to: CD27, CD40, OX40, GITR, CD137, CD28, ICOS. Inhibitory checkpoint molecules and stimulatory checkpoint molecules are targets for cancer immunotherapy because they may be used in many types of cancer. 1.
  • the present invention relates to a herpes simplex virus, which is a herpes simplex virus type I (HSV-1) with a deposit number of CCTCC NO: V201810.
  • the type I herpes simplex virus (HSV-1) has one or more characteristics selected from the following: (a) compared with the reference laboratory strain, it has an enhanced ability to infect tumor cells, (b) compared with the reference Compared with laboratory strains, it has an enhanced ability to replicate in tumor cells, (c) compared with the reference laboratory strain, it has an enhanced ability to kill tumor cells; (d) the herpes simplex virus has been modified to have Compared with the reference laboratory strain with equivalent modification, it has an enhanced ability to infect tumor cells.
  • this application relates to an attenuated variant of herpes simplex virus type 1 with a deposit number of CCTCC NO: V201810.
  • the virus strain of the present invention is a "non-laboratory" strain and can be referred to as a "clinical” strain.
  • laboratory strains can easily distinguish laboratory strains from non-laboratory strains or clinical strains.
  • the key difference between laboratory strains and non-laboratory strains is that currently commonly used laboratory strains have been maintained in culture for a long time.
  • the appropriate cells are infected with the virus, the virus replicates in the cell, and then the virus is harvested; then fresh cells are reinfected.
  • This process constitutes a cycle of continuous passaging. In the case of HSV, each such cycle may take, for example, several days.
  • This continuous passage may lead to changes in the characteristics of the virus strain, such as selection of characteristics that are conducive to growth in culture (such as rapid replication), rather than selection. With features that are conducive to practical applications.
  • the virus strains of the present invention are non-laboratory strains because they are derived from strains newly isolated from infected individuals.
  • the strains of the present invention have been modified to eliminate toxicity but basically retain the ideal characteristics of the original clinical isolates from which they originated.
  • the virus of the present invention can effectively infect human target cells.
  • Such viruses have just been isolated from infected individuals and then screened for their ability to enhance replication in tumor cells and/or other cells in vitro and/or in vivo compared to standard laboratory strains. Compared with laboratory virus strains, this type of virus with improved characteristics is the virus of the present invention.
  • the identified viruses with the desired improved properties can then be engineered through mutations in the appropriate genes so that they can selectively kill tumor cells, or they can be mutated so that they can be used in non-oncolytic applications. The gene is delivered to the target tissue and the toxic effect is reduced. These modified viruses are also viruses of the present invention.
  • the virus of the present invention infects any tumor cell or replicates in it, kills tumor cells or in tissues than a reference laboratory strain with equivalent modification.
  • the ability to spread between cells is strong.
  • this stronger ability is a statistically significant stronger ability.
  • the ability of the virus strain of the present invention can be at most 1 time, 1.5 times, 2 times, 3 times, 4 times, 5 times, or the ability of the reference strain. 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, 50 times or 100 times.
  • the virus of the present invention basically has (ie retains) the ability of its unmodified clinical precursor strain.
  • the virus strain of the present invention preferably basically has its unmodified clinical precursor strain to infect any tumor cell or replicate in it, kill tumor cells or in tissues. The ability to spread between cells.
  • This application covers modified viruses in which the changed viral regions can either be eliminated (completely or partially) or become non-functional, or replaced by other sequences, especially by foreign gene sequences.
  • One or more genes can be made non-functional genes, and one or more foreign genes can be inserted.
  • the virus of the application is a modified non-laboratory oncolytic virus.
  • These viruses can be used in oncolysis to treat cancer. Such viruses infect tumor cells and replicate within the tumor cells, and then kill the tumor cells. Therefore, this type of virus is a virus capable of replication. It is best that they selectively have the ability to replicate in tumor cells. This means that they replicate in tumor cells but not in non-tumor cells, or they replicate more efficiently in tumor cells than in non-tumor cells. The determination of selective replication ability can be carried out by the assay described herein for measuring replication ability and tumor cell killing ability.
  • the virus of the present application can also be modified to lose its ability to replicate in any cell including tumor cells, but still has the ability to infect cells including tumor cells. The modified virus exerts a tumor-killing effect by expressing the tumor therapy gene carried in the cell. In cancer treatment, it is preferable to use an oncolytic virus that has the ability to replicate in tumor cells for treatment.
  • the oncolytic virus of the present invention is more capable of infecting tumor cells or replicating in tumor cells, killing tumor cells or spreading between cells in tissues than a reference laboratory strain with the same modification (preferably statistically significant) .
  • the ability of the virus to infect tumor cells can be quantified by measuring the virus dose required to infect a certain percentage of cells (for example, 50% or 80% of cells).
  • the ability to replicate in tumor cells can be determined by measuring the growth of the virus in the cell, for example, by measuring the presence of the virus in the cell within a time period of 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, or 72 hours or more.
  • the ability of the virus to kill tumor cells can be quantified by counting the number of viable cells retained for a given cell type at a given time point and MOI (multiplicity of infection) within a given time.
  • MOI multiplicity of infection
  • the number of cells that exclude trypan blue i.e., viable cells
  • Quantitative determination can also be performed by flow cytometry (FACS), MTT, CCK-8 or CTG method analysis.
  • the killing ability of tumor cells can also be determined in vivo, for example, by measuring the decrease in tumor volume caused by a specific virus.
  • a standard laboratory reference strain for comparison. Any suitable standard laboratory reference strain can be used. As far as HSV is concerned, it is best to use one or more of HSV-1 strain 17, HSV-1 strain F, or HSV-1 strain KOS.
  • the reference strain usually has the same modification as the test strain of the present invention, such as gene deletion and/or foreign gene insertion. For example, with regard to the HSV strain, if the ICP34.5, ICP6, and/or thymidine kinase (TK) coding genes have been rendered non-functional in the virus of the present invention, they are also made in the reference strain Becomes non-functional.
  • the reference strain is the laboratory standard model strain HSV-1 virus 17 strain, or HSV-1 virus 17 strain, HSV(17), 17 virus strain, 17 strain.
  • This application relates to the human herpes virus type I HL-1 strain used for the preservation of patent procedures at the China Type Culture Collection (CCTCC) (Address: China, Wuhan, Wuhan University) on July 30, 2019, and the deposit number It is CCTCC NO: V201810.
  • CTCC China Type Culture Collection
  • herpes virus type I type 1
  • type I type 1
  • herpes simplex virus type I type 1
  • type I type 1 herpes simplex virus
  • HSV-1 herpes simplex virus
  • the human herpes virus type I HL-1 strain can be abbreviated as HL-1 strain, HL1 strain, HL-1 or HL1 in this application.
  • Herpes simplex virus can be used as a gene delivery vector in the nervous system and other systems, and can also be used for oncolysis to treat cancer. However, in these two applications, the virus must be made defective so that it is no longer pathogenic, but it can still enter the cell and perform the required functions.
  • the HSV-1 genome has many genes that can be knocked out or mutated to confer safety and/or tumor targeting specificity. For example, one of the common features that distinguish cancer cells from most normal cells (after mitosis) is the continued proliferation of cancer cells and therefore maintenance of sufficient nucleotides for DNA replication.
  • HSV-1 contains genes involved in nucleotide metabolism (thymidine kinase, ribonucleotide reductase and uracil DNA glycosylase, etc.) allowing the virus to replicate in non-dividing cells lacking sufficient nucleotides. Knockout or mutation of these genes can endow the virus with specificity to dividing cells (tumor cells), and often attenuate the pathogenicity of the virus.
  • cells have multiple mechanisms to detect and inactivate invading viruses.
  • Part of the virus's genes ICP34.5, ICPO, UL56, etc.
  • the knockout or mutation of this part of the gene is also the same
  • the virus can be given specificity to dividing cells (tumor cells).
  • the HSV strain of the present invention is modified to lack ICP34.5 gene, ICP6 gene, ICPO gene, ICP47 gene, US3 gene, UL56 gene, functional VP16 gene, VHS gene, UNG gene, glycoprotein One or more of H gene and thymidine kinase gene.
  • the virus lacks the ICP34.5 gene.
  • the virus can be further modified to lack the ICP47 gene.
  • one or more of the ICP34.5, ICP47, Us3, ICPO, and UL56 genes are mutated to prepare oncolytic herpes simplex virus type 1, oHSV-1.
  • HSV Mutations For oncolytic therapy of cancer (the therapy may also include the delivery of genes that enhance the therapeutic effect), many mutations of HSV have been identified (Peters C. Designing Herpes Viruses as Oncolytics Molecular Therapy Oncolytics, 2015.), these of HSV Mutations still allow the virus to replicate in culture or actively dividing cells in the body (e.g., tumors), but prevent its effective replication in normal tissues. Such mutations include the destruction of genes encoding ICP34.5, ICP6, and thymidine kinase. So far, among these mutant viruses, viruses with ICP34.5 mutation or ICP34.5 mutation and, for example, ICP6 mutation, have shown the most advantageous safety. It has been shown that viruses lacking only ICP34.5 replicate in many tumor cell types in vitro and selectively replicate in mouse brain tumors without harming surrounding tissues.
  • Any other gene deletion/mutation virus that provides oncolytic properties ie, selective replication within the tumor compared to surrounding tissues is also a virus covered by this application.
  • the techniques known in the art and/or the techniques described herein can be used to insert foreign genes into the viruses of the present invention, for example, the virus with CCTCC deposit number V201810 (CCTCC: V201810).
  • the foreign gene is usually a gene that enhances the virus's ability to fight tumors. Therefore, any gene that confers anti-tumor properties to the virus can be inserted.
  • the exogenous gene may be a gene that can improve the immune response against tumor cells in a beneficial way, especially an immunostimulatory polypeptide.
  • This application relates to genetically modified herpes simplex virus and its application as a carrier in terms of non-oncolysis.
  • the vector of the genetically modified herpes simplex virus in the non-oncolytic application of the present application can be mutated, so that the expression of the immediate early gene of the virus can be minimized. Therefore, ICP4, ICP27, ICP22 and/or ICPO encoding genes can be inactivated or deleted alone or in combination, or mutations in the virion transactivation protein vmw65 include preventing/reducing its transactivation ability.
  • the ICP27, ICP0 and ICP4 coding genes are deleted (with or without additional ICP22 and/or ICP47 deletion/inactivation), or the ICP27 and ICP4 deletions are added Inactivation in vmw65, or ICP4 deletion plus inactivation in vmw65.
  • Some embodiments of the application relate to herpes simplex virus vectors that deliver foreign genes to the nervous system.
  • the virus according to this embodiment of the invention is generally stronger than a reference laboratory strain with equivalent modifications in its ability to infect neurons, its ability to spread between cells in neural tissue, or its ability to transport within axons.
  • the virus of the present invention is modified to lack one, two, three, or all of the functional ICP27 encoding gene, the functional ICP4 encoding gene, the functional ICPO encoding gene, or the functional ICP22 encoding gene.
  • the virus of the present invention lacks both a functional ICP4 encoding gene and a functional ICP27 encoding gene, and the virus of the present invention has an inactivating mutation in the vmw65 encoding gene that eliminates its transcriptional activation activity.
  • viruses can be used to treat peripheral nervous system diseases or central nervous system diseases. In the case of a central nervous system disease, it is particularly preferable to make at least two immediate early genes selected from ICP0, ICP4, ICP22, and ICP27 non-functional.
  • the virus of the present invention can also control the ICP34.5 gene, ICP6 gene, ICPO gene, ICP47 gene, US3 gene, UL56 gene, functional VP16 gene, VHS gene, UNG gene, glycoprotein H gene, One or more of the thymidine kinase genes are modified.
  • the insertion site of the foreign gene can be any one or more of the above-mentioned modified sites, and can also be the latent infection area (LAT) of the herpes simplex virus. When the herpes simplex virus enters the latent state, all genes are in a dormant state except for the genes in the LAT region. Inserting foreign genes into the LAT region will help the long-term expression of the genes. Therefore, when the virus of the present invention is used as a non-replicating gene therapy vector, it is preferable to insert the foreign gene into the LAT region.
  • LAT latent infection area
  • the replication-incompetent virus of the present invention can be used to deliver genes to individuals in need of gene therapy.
  • the herpes simplex virus of the present invention can be used to treat central nervous system diseases or peripheral nervous system diseases.
  • Central nervous system diseases used for treatment or prevention include neurodegenerative diseases.
  • the central nervous system disease used for treatment or prevention is stroke, Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, and Huntington's disease.
  • Preferred peripheral nervous system diseases for treatment or prevention include motor neuron disease, chronic pain, and peripheral nerve injury.
  • the virus of the present invention is administered to a subject suffering from pain, degenerative disease or nerve damage to improve the subject’s condition, such as reducing the severity of pain, slowing down the degeneration of nerve tissue, or Promote nerve regrowth.
  • the herpes simplex virus of the present invention can be used to treat blood system diseases.
  • blood system diseases include hemophilia.
  • the oncolytic virus of the present invention can be used to prepare medicines for the treatment of cancer.
  • the oncolytic virus preparation of the present invention can be used in the treatment of cancer, for example, by direct intratumoral injection.
  • the oncolytic virus preparation of the present invention can be used to treat any solid tumors in mammals, preferably humans.
  • the virus of the present invention can be administered to subjects suffering from the following diseases: liver cancer, melanoma, glioma, sarcoma, lung cancer, colorectal cancer, head and neck tumors, breast cancer, renal cell carcinoma, ovarian cancer , Cervical cancer, prostate cancer, stomach cancer, lymphoma, pancreatic cancer and bladder cancer.
  • this application relates to a recombinant oncolytic virus or modified oncolytic virus, which is characterized in that the recombinant oncolytic virus or modified oncolytic virus is derived from the CCTCC deposit number: V201810(CCTCC NO : V201810) type I herpes simplex virus, wherein the CCTCC: V201810 virus is genetically modified to make its ICP34.5 gene, ICP6 gene, ICP0 gene, ICP47 gene, US3 gene, UL56 gene, functional VP16 gene, and VHS gene One or more of the UNG gene, glycoprotein H gene, and thymidine kinase gene are knocked out, thereby inserting a foreign gene, such as a cytokine polypeptide that stimulates an immune response, at the position where one or more of the above-mentioned genes are knocked out.
  • a foreign gene such as a cytokine polypeptide that stimulates an immune response
  • an antibody polypeptide that can promote an immune response for example, a cytokine polypeptide that stimulates an immune response is granulocyte macrophage colony stimulating factor (GMCSF), IL-2, IL12, IFN- ⁇ or TNFa, and an antibody polypeptide that can promote an immune response is Immune checkpoint antibodies.
  • GMCSF granulocyte macrophage colony stimulating factor
  • IL-2 granulocyte macrophage colony stimulating factor
  • IL12 IL12
  • IFN- ⁇ or TNFa an antibody polypeptide that can promote an immune response
  • Immune checkpoint antibodies for example, a cytokine polypeptide that stimulates an immune response is granulocyte macrophage colony stimulating factor (GMCSF), IL-2, IL12, IFN- ⁇ or TNFa
  • Immune checkpoint antibodies for example, a cytokine polypeptide that stimulates an immune response is granulocyte macrophage colony stimulating factor (GMCSF), IL-2, IL12, IFN-
  • the recombinant oncolytic virus or modified oncolytic virus of the present invention can be used to prepare medicines for the treatment of cancer.
  • the oncolytic virus preparation of the present invention can be used in the treatment of cancer, for example, by direct intratumoral injection.
  • the oncolytic virus preparation of the present invention can be used to treat any solid tumors in mammals, preferably humans.
  • the virus of the present invention can be administered to subjects suffering from the following diseases: liver cancer, melanoma, glioma, sarcoma, lung cancer, colorectal cancer, head and neck tumors, breast cancer, renal cell carcinoma, ovarian cancer , Cervical cancer, prostate cancer, stomach cancer, lymphoma, pancreatic cancer and bladder cancer.
  • the present invention relates to the use of replication-incompetent viruses in gene delivery to individuals in need of gene therapy.
  • the genetically modified herpes simplex virus vector of the present invention can be used to treat central nervous system diseases or peripheral nervous system diseases.
  • Central nervous system diseases used for treatment or prevention include neurodegenerative diseases.
  • the central nervous system disease used for treatment or prevention is stroke, Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, and Huntington's disease.
  • Peripheral nervous system diseases used for treatment or prevention include motor neuron disease, chronic pain, and peripheral nerve damage.
  • the virus of the present invention is administered to a subject suffering from pain, degenerative disease or nerve damage to improve the subject’s condition, such as reducing the severity of pain, slowing down the degeneration of nerve tissue, or Promote nerve regrowth.
  • the herpes simplex virus of the present invention can be used to treat blood system diseases.
  • blood system diseases include hemophilia.
  • Some embodiments of the application relate to a pharmaceutical composition
  • a pharmaceutical composition comprising the virus of the application and a pharmaceutically acceptable carrier or diluent.
  • Suitable carriers and diluents are known in the art and include but are not limited to phosphate buffered saline solution.
  • this application relates to an article or kit, comprising a vial containing the aforementioned pharmaceutical composition and a package insert, the package insert including instructions on how to use the article or kit to treat diseases.
  • the pharmaceutical composition of the present application can be directly injected into target tissues, such as cancer tissues, for oncolytic treatment and/or gene delivery into cells to achieve therapeutic purposes.
  • the dose range of the virus administered by the pharmaceutical composition containing the virus or recombinant virus in the present application is 10 4 -10 12 pfu/ml, preferably 10 5 -10 8 pfu/ml, more preferably 10 6 -10 8 pfu/ml.
  • the injection amount of a pharmaceutical composition consisting essentially of the virus and a pharmaceutically acceptable suitable carrier or diluent is usually at most 10 ml, usually 1 -5ml, preferably 1-3ml. However, for certain oncolytic therapy applications, depending on the tumor and the site of inoculation, larger volumes above 10 ml may also be used.
  • the dosage can be determined according to various parameters, especially according to the age, weight and condition of the patient to be treated, the severity of the disease or condition, and the route of administration.
  • the preferred route of administration for cancer patients is direct injection into the tumor.
  • the virus can also be administered systemically, such as intravenous injection, or by injection, into the blood vessel of the tumor.
  • the optimal route of administration will depend on the location and size of the tumor.
  • the dosage can be determined according to various parameters, especially according to the location of the tumor, the size of the tumor, the age, weight and condition of the patient to be treated, and the route of administration.
  • the purpose of this section is to test and screen primary clinical isolates of HSV-1 and select the virus strain with the strongest oncolytic ability.
  • the virus strain with the best oncolytic effect was selected as the HL-1 strain.
  • HL-1 strain virus After HL-1 strain virus infects cells, cell debris is removed and observed under an electron microscope at 25,000 times. The figure shows that the virus has a typical HSV-1 appearance, and four components of genomic DNA, capsid, interlayer protein and envelope can be observed (see Figure 1).
  • HSV-1 herpes simplex virus
  • NC_001806.2 standard model strain HSV-1 virus 17 strain
  • the gD, gG, gI, gJ, gL, gM, and gN amino acid sequences in the envelope proteins of the HL-1 strain and the 17 strains differed by more than 1%, especially the amino acid sequence differences between gG and gI were more than 3%, of which gG reached 5.46% .
  • the membrane protein of the herpes simplex virus envelope is related to virus invasion, release, and direct transmission between cells, suggesting that the HL-1 strain is different from the 17 strains in infectivity.
  • ICP4 and ICP22 are quite different in amino acid sequence. Both exceed 1%.
  • ICP4 and ICP22 can stimulate the expression of early genes of the virus and DNA synthesis, and induce the expression of late genes. They are the key factors for HSV-1 gene expression to cause infection.
  • ICP34.5 is also a gene with a large difference between the HL-1 strain and the 17 genes, with an amino acid difference of 6.25%. ICP34.5 is an important neurotoxic factor of the HSV-1 virus, which plays a key role in the replication and pathogenicity of the virus.
  • the HL-1 virus strain was used for the preservation of patent procedures on July 30, 2019, and was deposited at the China Type Culture Collection (CCTCC) (Address: China, Wuhan, Wuhan University), and the deposit number is CCTCC NO: V201810 , The classification is named: "Human herpes virus type I HL-1 strain”.
  • the experimental cells include: Hep G2 human liver cancer cells, A-375 human skin cancer cells, A549 human lung cancer cells, MCF7 human breast cancer cells, HeLa human cervical cancer cells, U251 human glioma cells, and Vero monkey kidney cells.
  • the experimental method is as follows:
  • HL-1 and HSV (17) were transfected with different cells and observed under microscope
  • HL-1 and HSV (17) were transfected with different cells and observed under microscope
  • HL-1 and HSV(17) transfected HepG2 cells 48 hours later under the microscope results show that under the same MOI conditions, the transfection efficiency of HL-1 is higher than the transfection efficiency of HSV(17) (see figure 2).
  • HL-1 and HSV(17) transfected A549 cells 48 hours later under the microscope results showed that under the same MOI conditions, the transfection efficiency of HL-1 was significantly higher than HSV(17). It can be observed under the microscope that the number of HL-1 transfected cells is significantly greater than the number of HSV (17) transfected cells. At MOI 0.1, almost no normal cells are observed, but HSV (17) transfected cells are still visible Normal cells, and the number increases with the decrease of MOI (see Figure 4).
  • Vero cells are non-tumor cells and are sensitive cell lines of HSV virus. They are one of the commonly used production cells. Therefore, comparing the infection of HL-1 and HSV (17) on Vero cells is of great significance for production capacity.
  • FIG. 9 for the comparison of the IC50 of HL-1 and HSV(17) transfected into different cells.
  • the results in the figure show that the IC50 (MOI) of HL-1 is smaller than that of HSV (17), indicating that HL-1 has a stronger ability to kill cells than HSV (17).
  • This example aims to compare the difference in the ability of HL-1 and HSV (17) virus strains to transfect tumor cells.
  • HL-1 and HSV(17) were transfected with different tumor cells (HepG2, A375, A549, MCF-7, Hela and U251) and normal passage cells Vero cells.
  • the results of microscopic observation after 48 hours of transfection showed that, Although various types of cells have different susceptibility to viruses, the transfection efficiency of HL-1 is significantly higher than that of HSV (17) under the same MOI.
  • the IC50 values of HL-1 and HSV(17) transfected different cells were further measured by CCK-8 method. The results showed that the IC50 values of HL-1 transfected cells were all lower than HSV(17).
  • the HL-1 virus strain compared with the HSV (17) virus strain, the HL-1 virus strain has better infection efficiency and replication ability in cells, and has a stronger oncolytic effect.
  • the ICP34.5 gene and ICP47 gene of the virus strain with CCTCC preservation number V201810 (CCTCC: 201810) and the control virus 17 strain of the present invention were knocked out, and the knock-out results were compared.
  • Recombinant viruses HL1- ⁇ 34.5- ⁇ 47 and 17- ⁇ 34.5- ⁇ 47 have different ability to transfect tumor cells.
  • the experimental cells include Hep G2 human liver cancer cells, A375 human skin cancer cells, A549 human lung cancer cells, MCF7 human breast cancer cells, HeLa human cervical cancer cells, U251 human glioma cells, and Vero monkey kidney cells.
  • Example 4 The oncolytic effect of HL-1 virus strain as a carrier carrying foreign genes on tumor cells
  • Experimental cells include A375 human skin cancer cells, A549 human lung cancer cells, AGS human gastric cancer cells, AsPC-1 human pancreatic cancer cells, HeLa human cervical cancer cells, Hep G2 human liver cancer cells, HT-29 human colorectal cancer cells, MCF7 Human breast cancer cells, PC-3 human prostate cancer cells, T24 human bladder cancer cells, U-2OS human bone cancer cells and U-87 human glioma cells.
  • the ICP34.5 gene and ICP47 gene of the virus strain with CCTCC preservation number V201810 (CCTCC: V201810) and the control virus 17 strain of the present invention were knocked out, and inserted at the position where the ICP34.5 gene was knocked out Artificial chemical synthesis of foreign genes.
  • the exogenous gene includes the EF1 ⁇ promoter, the gene encoding IL-12 (Gene ID: 16159, 16160) and TK PolyA in sequence from the 5'end to the 3'end. Sequencing at Beijing Kinco Xinye Company confirmed that the above-mentioned coding gene was correctly inserted into the herpes simplex virus vector.
  • the successfully constructed recombinant virus vector and the above-mentioned cell strain were propagated under the conditions of 37°C and 5% CO 2.
  • the experimental results show the difference in the ability of HL-1 recombinant virus (HL1-IL12) and 17 strains of recombinant virus (17-IL12) to transfect tumor cells after inserting the IL-12 gene.
  • the HL-1 recombinant virus inserting the IL-12 gene corresponds to
  • the IC50 values of the 17 strains of recombinant virus inserted into the IL-12 gene to transfect tumor cells are all lower, indicating that the recombinant virus of the HL-1 strain inserted into the IL-12 gene has better infection efficiency and replication ability in tumor cells ,
  • the oncolytic effect is strong, and it has better application prospects as an oncolytic virus.
  • HL1-IL12 and HL1-mock were constructed to evaluate the oncolytic effect of oncolytic viruses on melanoma mice.
  • C57BL/6J mice were subcutaneously inoculated with B16F10 tumor cells to establish an allograft melanoma model.
  • the control group was phosphate solution, and the treatment groups were oxaliplatin (10mg/kg), the tested oncolytic virus HL1-mock(10 ⁇ 7pfu), HL1-IL12(10 ⁇ 7pfu), HL1-IL12(10 ⁇ 6pfu) and HL1-IL12 (10 ⁇ 5pfu), 10 mice in each group; the control group and the test oncolytic virus administration group were injected intratumorally, and the oxaliplatin administration group was injected intraperitoneally.
  • TGI% (1-T/C) ⁇ 100%.
  • T/C% is the relative tumor proliferation rate, that is, the percentage value of the relative tumor volume between the treatment group and the control group at a specific time point.
  • T and C are the relative tumor volume (RTV) of the treatment group and the control group at a specific time point, respectively.
  • RTV animal tumor volume after treatment/control tumor volume. According to the tumor inhibition rate (TGI) for efficacy evaluation.
  • the tumor inhibition rates are 6% and 20%, respectively.
  • the control group all died on the 14th day, and 3 mice in the HL1-IL12 (10 7 PFU) administration group had tumors disappeared and survived. Except for the above 3 mice, all other mice in the administration group died on the 21st day.
  • mice in the HL1-IL12 (10 7 PFU) group had tumors disappeared and survived until 91 days.
  • the mice were again inoculated with B16F10 tumor cells on the contralateral side of the mice for re-challenge.
  • the control group had 5 C57BL/6J mice inoculated subcutaneously B16F10 tumor cells, observe the tumor growth.
  • mice in the control group grew tumors and continued to grow on the 10th day, and the re-challenge group did not grow tumors, and continued observation did not grow tumors, which proved that the mice had tumor immunity after HL1-IL12 treatment.
  • the ICP34.5 gene and ICP47 gene of the virus strain with CCTCC preservation number V201810 were knocked out, and inserted at the position where the ICP34.5 gene was knocked out Artificial chemical synthesis of foreign genes.
  • the exogenous gene includes the CMV promoter, the gene encoding GM-CSF (Gene ID: 12981), the BGH PolyA, the EF1 ⁇ promoter, and the gene encoding IL-2 (Gene ID: 16183) from 5'end to 3'end. ) And TK PolyA. Sequencing at Beijing Sanbo Yuanzhi Company confirmed that the above-mentioned coding gene was correctly inserted into the herpes simplex virus vector.
  • C57BL/6 mice were subcutaneously inoculated with murine melanoma B16F10 cell line to construct animal models.
  • HL-1 recombinant virus treatment group HL-1 recombinant virus introduced with foreign genes GM-CSF and IL-2
  • Recombinant virus treatment group Recombinant virus of 17 virus strains introduced with foreign genes GM-CSF and IL-2
  • HSV-mock group oncolytic virus without inserting foreign genes and knocking out ICP34.5 gene and ICP47 gene
  • Negative control PBS was given.
  • Each group was given a total pfu virus were 10 6 pfu.
  • the relative tumor inhibition rate 14 days after administration was used as the criterion.
  • TGI% (1-T/C) ⁇ 100%.
  • T/C% is the relative tumor proliferation rate, that is, the percentage value of the relative tumor volume between the treatment group and the control group at a specific time point.
  • T and C are the relative tumor volume (RTV) of the treatment group and the control group at a specific time point, respectively.
  • RTV animal tumor volume after treatment/control tumor volume.
  • the ICP34.5 gene and ICP47 gene of the virus strain with CCTCC preservation number V201810 (CCTCC: V201810) and the control virus 17 strain of the present invention were knocked out, and inserted at the position where the ICP34.5 gene was knocked out
  • the exogenous gene of artificial chemical synthesis, the exogenous gene of PD1 monoclonal antibody from 5'end to 3'end includes: CMV promoter, gene encoding anti-PD1 antibody (J43, BioXCell), and BGHPolyA sequence.
  • C57BL/6J mice were subcutaneously inoculated with B16F10 tumor cells to establish an allograft melanoma model.
  • the control group was phosphate solution
  • the treatment group was PD1 monoclonal antibody (5mg/kg)
  • the tested oncolytic virus HL1-mock (10 7 pfu), HL1-PD1 (10 7 pfu), HL1-PD1 (10 6 pfu)
  • HL1-PD1 (10 5 pfu)
  • mice in each group were administered intratumorally
  • the PD1 monoclonal antibody administration group was administered intraperitoneally, as calculated as above Relative tumor inhibition rate.
  • HL1-PD1 oncolytic virus alone administration group has statistically significant differences in anti-tumor effects compared with the control group. (P ⁇ 0.05); HL1-PD1 high, medium and low dose groups have obvious dose effects, tumor suppression rates are 86%, 65%, and 51%, respectively.
  • the tumor suppression rates of HL1-mock group and PD1 monoclonal antibody group are respectively Are 34% and 42%.
  • the ICP34.5 gene and ICP47 gene of the virus strain with CCTCC preservation number V201810 (CCTCC: V201810) and the control virus 17 strain of the present invention were knocked out, and IL was inserted at the ICP34.5 and ICP47 genes, respectively -12 gene and PD1 monoclonal antibody gene, determine HL1-IL12+PD1 (HL-1 inserted IL-12 gene and PD1 monoclonal antibody gene), 17-IL12+PD1 (17 strains inserted IL-12 gene and PD1 monoclonal antibody gene) The IC50 of different cells were transfected respectively.
  • Experimental cells include A-375 human skin cancer cells, A549 human lung cancer cells, AGS human gastric cancer cells, AsPC-1 human pancreatic cancer cells, HeLa human cervical cancer cells, Hep G2 human liver cancer cells, and HT-29 human colorectal cancer cells. , MCF7 human breast cancer cells, PC-3 human prostate cancer cells, T24 human bladder cancer cells, U-2OS human bone cancer cells and U-87 human glioma cells.
  • This example aims to compare the difference in the ability to transfect tumor cells between the recombinant virus of the HL-1 virus strain and the recombinant virus of the 17 virus strain after inserting the IL-12 gene and the PD1 monoclonal antibody gene.
  • the IC50 values of tumor cells transfected by viruses were determined by the CCK-8 method.
  • the IC50 values of tumor cells transfected by different viruses were determined as shown in Figure 12.
  • This example aims to compare the ability of the non-replicating HL-1 virus knocked out of different immediate early genes and 17 strains to infect nerve cells to compare the non-replicating HL-1 herpes virus of the present invention as a gene vector with the standard in the prior art The effect of 17 strains of laboratory viruses.
  • the results of HL-1 recombinant virus and 17 strains of recombinant virus transfected into primary neural cells are shown in Figure 13 and Figure 14.
  • the experimental results showed that under different MOI transfection conditions, the HL-1 recombinant virus showed more blue spots in the primary neuronal cell culture, indicating that the HL-1 recombinant virus has the ability to transfect neuronal cells compared to the 17 strains of recombinant virus. Stronger, the ability to express foreign genes is also stronger.
  • the recombinant virus constructed according to the above methods 1 and 2 has lost the ability to replicate in nerve cells, but can still transfect nerve cells at a high level and express the foreign genes carried. Therefore, it is good for the treatment of neurological diseases as a gene therapy vector. Application prospects.
  • This example aims to compare the ability of the non-replicating HL-1 recombinant virus knocked out of different immediate early genes and 17 strains of recombinant virus to infect hepatocytes, so as to evaluate the potential of the non-replicating recombinant virus of the present invention as a gene therapy vector.
  • the non-replicating recombinant virus was constructed according to the methods 1 and 2 of Example 5 respectively, except that the reporter gene was replaced with the GFP gene.
  • CCC-HEL-1 human embryonic liver diploid cells
  • the results of HL-1 recombinant virus and 17 strains of recombinant virus transfected into CCC-HEL-1 cells are shown in Figure 15 and Figure 16.
  • the experimental results showed that under different MOI transfection conditions, the HL-1 recombinant virus showed more and brighter green fluorescence in CCC-HEL-1 cell culture, indicating that the HL-1 recombinant virus was transfected compared to the 17 recombinant viruses.
  • the ability to infect CCC-HEL-1 cells is stronger, and the ability to express foreign genes is stronger.
  • CCC-HEL-1 is a normal human embryonic liver diploid cell.
  • the recombinant virus constructed according to the above methods 1 and 2 has lost its ability to replicate in hepatocytes, but can still infect hepatocytes and express the foreign genes it carries.
  • the ability of HL-1 recombinant virus to exhibit high transfection and high expression of foreign genes carried in hepatocytes indicates that it can effectively deliver therapeutic genes into target cells as a vector to achieve the purpose of treating diseases.
  • Example 7 The therapeutic effect of HL-1 virus strain as a gene therapy vector carrying foreign genes
  • the ICP34.5 and ICP4 genes of the HL-1 virus were knocked out according to the method of application publication number CN105219739A, and the FVIII gene was inserted into the latent expression region (LAT region) of the HL-1 virus.
  • This example aims to observe the effect of HL1-FVIII on the coagulation function of the FVIII gene knockout mouse model at different time points.
  • the normal control group is C57BL/6 normal mice
  • the model control group and the HL1-FVIII group are FVIII gene knockout Mouse model (Shanghai Southern Model Biotechnology Co., Ltd.).
  • Intramuscular injection the HL1-FVIII group was given 10 7 pfu/only the HL1-FVIII non-replicating HL-1 recombinant virus prepared above, and the other two groups were given PBS. It is administered once every 3 days, and it is administered 3 times in a row. After 48 hours in the control group, blood was collected for APTT 48 hours, 72 hours and 120 hours later in the test group.
  • the activated partial thromboplastin time (APTT) reflects the sensitivity of the coagulation activity of the endogenous coagulation system.
  • the APTT values detected at different time periods are shown in Figure 17.
  • the high APTT value of the model control group indicates that the lack of factor FVIII in the body leads to an increase in clotting time.
  • the experimental results showed that the APTT value of the group after treatment with the non-replicating HL-1 recombinant virus HL1-FVIII was significantly shorter than that of the untreated group, which was close to the value of normal mice, indicating that the non-replicating HL-1 recombinant virus HL1-FVIII is in the small
  • the FVIII factor can be continuously expressed in mice, which has a good clinical application prospect in the treatment of hemophilia A.
  • the ICP34.5 and ICP4 genes of the HL-1 virus were knocked out according to the method of application publication number CN105219738A, and the enkephalin (ENK) gene was inserted into the latent expression region (LAT region) of the HL-1 virus.
  • This example aims to observe the sustained analgesic effect of HL1-ENK in arthritis chronic pain rats.
  • the model control group and test product group used complete Freund’s adjuvant induced arthritis chronic pain model in rats, with 6 rats in each group.
  • the right rear footpad was administered, the test group was given 10 7 pfu/only non-replicating recombinant virus HL1-ENK, and the other two groups were given PBS control.
  • the score interval is 1-5 points, 5 is divided into normal mouse behavioral ability, and 1 is divided into loss of normal movement ability and severely hindered behavior. The higher the score, the more normal the activity, and the chronic pain rats with arthritis will affect their behavior due to pain.
  • the behavioral activity scores of each experimental group are shown in Figure 18.
  • arthritis rats showed significant differences from the model control group 3 days after administration of the non-replicating recombinant virus HL1-ENK.
  • the analgesic effect was obvious, lasting more than 9 weeks, and the peak value Appears in 1-4 weeks, the behavioral score is not significantly different from that of normal rats.

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Abstract

提供了一种新的I型单纯疱疹病毒、基因改造的单纯疱疹病毒、包含该病毒的组合物、宿主细胞和细胞培养物，以及该病毒在治疗疾病中的用途。

Description

一种单纯疱疹病毒及其用途 技术领域
本发明涉及一种新型的单纯疱疹病毒、对该病毒的改造、以及基因改造的单纯疱疹病毒及其用途。
背景技术
单纯疱疹病毒(Herpes Simplex Virus，HSV)属于疱疹病毒科，是有包膜的双链DNA病毒，包括I型(HSV-1)和II型(HSV-2)两个亚型。
单纯疱疹病毒由核心、衣壳、间层蛋白和囊膜四部分组成。病毒核心由浓厚的双链DNA构成，缠绕成纤丝卷轴。DNA外包围的衣壳呈二十面体结构，直径为100-110nm，由162个壳粒组成。衣壳外由排列无规则、不定形的间层蛋白覆盖。病毒最外层是带短小突起的脂质双层囊膜，包裹囊膜的病毒直径为150-200nm。病毒囊膜由11种糖蛋白和至少两种非糖基化蛋白质组成，包括病毒糖蛋白B(gB)、病毒糖蛋白C(gC)、病毒糖蛋白D(gD)、病毒糖蛋白G(gG)和病毒糖蛋白M(gM)等。病毒囊膜糖蛋白的立体结构对病毒的细胞侵染能力有关，其决定了病毒能否进入宿主细胞及进入宿主细胞的病毒量。
目前，HSV-1病毒己经用于肿瘤、神经系统退行性疾病、遗传性疾病和免疫系统疾病等重大疾病的基因治疗，相比其它基因治疗病毒载体，HSV-1病毒具有众多优势。
首先，HSV-1的基因组较大，可携带较大或多个外源基因[Roizman B.The function of herpes simplex virus genes:a primer for genetic engineering of novel vectors.[J].Proceedings of the National Academy of Sciences,1996,93(21):11307-11312]。HSV-1基因组长达152kb，而腺病毒基因组仅有35kb。HSV-1基因组的80多个已知基因中，约半数在体外培养中为非必需基因，可以被多个外源性治疗基因所替换，它的最大外源基因插入量可以达到30-40kb。这对于治疗许多疾病，尤其是多个基因相关的疾病尤其重要。
HSV-1与细胞DNA不整合，复制可控制，安全性高。HSV-1在有免疫能力的成人几乎不产生威胁生命的疾病[ShenY,Nemunaitis J.Herpes simplex virus 1(HSV-1)for cancer treatment[J].Cancer Gene Therapy,2006,13(11):975-992]。HSV-1的许多非必需基因与其神经毒性有关。去掉HSV-1中某些非必需基因，如ICP34.5基因，使HSV-1只选择性在肿瘤细胞内复制，正常组织细胞不受其影响[Chou J,Kern E,Whitley R,et al.Mapping of herpes simplex virus-1 neurovirulence to gamma 34.5,a gene nonessential for growth in culture[J].Science,1990,250(4985):1262-1266]。
基于以上众多优点，HSV-1已成为现今基因治疗应用最广泛的病毒载体之一。在基因治疗研究中，HSV-1通常是通过基因改造的手段被改造成为溶瘤型I型单纯疱疹病毒(Oncolytic herpes simplex virus type 1，oHSV-1)，主要方法包括敲除某些基因和插入肿瘤治疗相关基因等。目前已有多种oHSV-1进行了临床前及临床试验研究，大量结果显示其具有良好的安全性和有效性。
基因缺失的oHSV-1构建的一般策略为突变或缺失病毒的单个或多个基因以实现其对肿瘤的特异性杀伤作用，如ICP34.5、ICP6、ICP0、TK和UNG等基因。目前已经上市和进入临床试验的Talimogene laherparepvec、HSV1716、NV1020、G207、G47Δ等都敲除了HSV-1中的ICP34.5基因。溶瘤病毒HSV1716仅敲除了双拷贝的ICP34.5基因，在针对难治或复发的高级别脑胶质瘤治疗的临床试验中显示肿瘤消退并且没有发现明显的毒性或副作用。G207在敲除双拷贝ICP34.5基因的同时使ICP6基因突变，在针对复发和进展的恶性胶质瘤治疗的临床试验中显示，在瘤内注射10 9pfu的G207后未出现严重不良反应，具有良好的安全性[Markert JM,Liechty PG,Wang W,et al.Phase Ib trial of mutant herpes simplex virus G207 inoculated pre-andpost-tumor resection for recurrent GBM[J].Molecular Therapy,2009,17(1):199-207]。Talimogene laherparepvec是美国第一个批准上市的溶瘤病毒，其敲除了HSV-1的ICP34.5和ICP47基因，Ⅲ期临床试验数据显示在恶性黑色素瘤治疗上客观缓解率为26.4％，具有较为显著的治疗效果和良好的安全性[Andtbacka R H I,Kaufman H L,Collichio F,et al.Talimogene Laherparepvec Improves Durable Response Rate in Patients With Advanced Melanoma[J].Journal of Clinical Oncology,2015,33(25):2780-2788]。这些临床 数据表明HSV-1在肿瘤的基因治疗具有良好安全性和巨大潜力。
Ⅰ型单纯疱疹病毒(HSV-1)由于其宿主细胞范围广、安全性高且具有嗜神经性，被认为是基因治疗中非常具有前景的一种病毒。例如，可将Ⅰ型单纯疱疹病毒通过基因改造成为非复制性病毒，将外源基因导入受试者体内治疗相关疾病。例如在疼痛治疗中，通过注射携带ENK、GABA或者内啡肽基因的HSV-1载体，可以减轻慢性炎性和骨癌引起的疼痛以及神经性疼痛，而携带抗炎性的IL-4、IL-10和IL-13等因子的HSV-1载体也可以缓解急慢性疼痛[Goss J R,Krisky D M,Wechuck J B,et al.Herpes simplex virus-based nerve targeting gene therapy in pain management[J].Journal of Pain Research,2014:71-79]。
目前使用的大多数用于基因传递或治疗的单纯疱疹病毒是已经在组织培养细胞内传代多年的实验室病毒株。这些病毒虽然可以进入细胞传递基因，但是在疾病治疗所需的特性例如转染和表达外源基因的能力等诸方面有待改进。
发明内容
本申请，一方面为了克服现有技术存在的问题，提供一种新的HSV分离株，相对先前一直使用的连续传代的标准模式株HSV-1病毒17株(简称17病毒株或17株)，本发明的HSV-1病毒株体内感染人细胞的能力大大改进、细胞内复制/裂解的能力显著增强，适于作为溶瘤病毒或作为载体通过溶瘤作用破坏肿瘤细胞。
与标准模式株HSV-1病毒17株相比，本发明提供的新的HSV-1分离株(简称毒株HL-1、HL-1株或HL-1)增强了在人肿瘤细胞系内的复制。在缺失ICP34.5和/或ICP47的情况下，毒株HL-1与其中也缺失ICP34.5和/或ICP47的HSV-1病毒17株相比，在人肿瘤细胞系内的复制能力、对肿瘤细胞的杀伤能力显著增强。
将本发明的经修饰的HL-1病毒随后用来传递具有抗肿瘤活性的基因，证明可进一步增强抗肿瘤免疫应答活性和溶瘤效果。
另一方面，本申请也提供了一种非复制性重组疱疹病毒，该非复制性重组疱疹病毒来源于CCTCC保藏号为V201810的单纯疱疹病毒，其中的一种或多种立即早期基因被敲除，或通过基因突变使其表达被阻止或降低。与现 有技术中的疱疹病毒相比，本申请的非复制性重组疱疹病毒转染能力和表达外源基因的能力明显增强。因此，本申请的非复制性重组疱疹病毒在将外源基因导入患者治疗例如神经系统疾病、血液系统疾病、免疫系统等疾病方面也具有潜在的开发价值。
具体地，本申请一方面涉及如下各项：
1.一种1型单纯疱疹病毒(HSV-1)，其具有选自如下的一种或多种特性：
(a)与参比实验室毒株相比，具有增强的感染肿瘤细胞能力；
(b)与参比实验室毒株相比，具有增强的肿瘤细胞内复制能力；
(c)与参比实验室毒株相比，具有增强的杀伤肿瘤细胞能力；
(d)该单纯疱疹病毒经修饰后，与具有等同修饰的参比实验室毒株相比，具有增强的感染肿瘤细胞能力；
(e)该单纯疱疹病毒经修饰后，与具有等同修饰的参比实验室毒株相比，具有增强的肿瘤细胞内复制能力；和
(f)该单纯疱疹病毒经修饰后，与具有等同修饰的参比实验室毒株相比，具有增强的杀伤肿瘤细胞能力，
其中，所述参比实验室毒株为标准模式株HSV1病毒17株。
2.项1的1型单纯疱疹病毒，其为CCTCC保藏号为：V201810(CCTCC：V201810)的病毒株。
3.项1的单纯疱疹病毒，其为CCTCC保藏号为V201810(CCTCC：V201810)的病毒株的减毒株或其后代。
在一些实施方案中，本申请涉及CCTCC保藏号为V201810(CCTCC：V201810)的病毒株的基因工程构建体。
4.一种单纯疱疹病毒，其来源于CCTCC保藏号为V201810(CCTCC：V201810)的病毒株，其中该单纯疱疹病毒中ICP34.5基因、ICP6基因、ICP0基因、ICP47基因、US3基因、UL56基因、功能性VP16基因、VHS基因、UNG基因、糖蛋白H基因、胸苷激酶基因中的一个或多个被破坏或敲除。
5.项4的单纯疱疹病毒，其中ICP34.5基因或/和ICP47基因被破坏或敲除。
6.包含项1-5的单纯疱疹病毒的组合物。
7.项6的组合物，其为病毒培养物。
8.由项1-5任一项的单纯疱疹病毒侵染的宿主细胞。
9.包含项8的宿主细胞的组合物。
10.项9的组合物，其为细胞培养物。
11.一种制备1型单纯疱疹病毒(HSV-1)的方法，包括
1)临床采集单纯疱疹病毒株；
2)筛选具有选自如下的一种或多种特性的病毒株：
(a)与参比实验室毒株相比，具有增强的感染肿瘤细胞能力；
(b)与参比实验室毒株相比，具有增强的肿瘤细胞内复制能力；
(c)与参比实验室毒株相比，具有增强的杀伤肿瘤细胞能力；
(d)该单纯疱疹病毒经修饰后，与具有等同修饰的参比实验室毒株相比，具有增强的感染肿瘤细胞能力；
(e)该单纯疱疹病毒经修饰后，与具有等同修饰的参比实验室毒株相比，具有增强的肿瘤细胞内复制能力；和
(f)该单纯疱疹病毒经修饰后，与具有等同修饰的参比实验室毒株相比，具有增强的杀伤肿瘤细胞能力，
其中，所述参比实验室毒株为标准模式株HSV1病毒17株。
12.项11的制备1型单纯疱疹病毒(HSV-1)的方法，包括
1)临床采集单纯疱疹病毒株；
2)筛选具有如下特性的病毒株：
(a)与参比实验室毒株相比，具有增强的感染肿瘤细胞能力；
(b)与参比实验室毒株相比，具有增强的肿瘤细胞内复制能力；和
(c)与参比实验室毒株相比，具有增强的杀伤肿瘤细胞能力。
13.通过项11或12的制备方法获得的1型单纯疱疹病毒(HSV-1)。
一方面，本申请还涉及如下各项：
1.一种基因改造的单纯疱疹病毒，来源于CCTCC保藏号为V201810的单纯疱疹病毒，其中选自如下的一个或多个基因被敲除或破坏：ICP34.5基因、ICP6基因、ICP0基因、ICP47基因、US3基因、UL56基因、功能性VP16基因、VHS基因、UNG基因、糖蛋白H基因和胸苷激酶基因。
2.项1所述的基因改造的单纯疱疹病毒，其中ICP34.5基因和/或ICP47基因被破坏或敲除。
3.项1或2所述的基因改造的单纯疱疹病毒，其中向所述病毒中导入一 种或多种外源基因。
4.项3所述的基因改造的单纯疱疹病毒，其中所述一种或多种外源基因插入选自如下的一处或多处基因缺失位点：ICP34.5基因、ICP6基因、ICP0基因、ICP47基因、US3基因、UL56基因、功能性VP16基因、VHS基因、UNG基因、糖蛋白H基因和胸苷激酶基因。
5.项4的基因改造的单纯疱疹病毒，其中所述外源基因选自如下的一种或多种：编码促进免疫应答的细胞因子的基因、编码肿瘤抗原的基因、编码具有预防和/或治疗肿瘤作用的单抗基因、编码前药转化酶的基因、肿瘤抑制基因、反义RNA或小RNA。
6.项5的单纯疱疹病毒，其中所述细胞因子选自如下的一种或多种：GM-CSF、G-CSF、M-CSF、IL-1、IL-2、IL-3、IL-4、IL-5、IL-6、IL7、IL-8、IL-10、IL-12、IL-13、IL-15、IL-18、IL-21、IL-23、IFN-α、IFN-γ、TGF-β和TNF-α。
7.项5的单纯疱疹病毒，其中具有预防和/或治疗肿瘤作用的单抗选自如下的一种或多种：PD1单抗、PD-L1单抗、PD-L2单抗、CTLA-4单抗、CD80单抗、CD28单抗、CD137单抗、CD137L单抗、OX40单抗、OX40L单抗、CD27单抗、CD70单抗、CD40单抗、CD40L单抗、LAG-3单抗和TIM-3单抗。
8.项5的单纯疱疹病毒，其中肿瘤抗原选自如下的一种或多种：PSA、MUC1、MAGE-1、MAGE-2、MAGE-3、MAGE-12、BAGE、GAGE和LAGE。
9.项5的单纯疱疹病毒，其中前药转化酶为胞嘧啶脱氨酶或单纯疱疹病毒胸苷激酶。
10.项5的单纯疱疹病毒，其中肿瘤抑制基因为P53或PTEN。
11.项5的单纯疱疹病毒，其中反义RNA或小RNA为阻断或下调肿瘤过表达的原癌基因、代谢基因的RNA片段。
12.项5的基因改造的单纯疱疹病毒，其中所述外源基因选自编码刺激免疫应答的细胞因子多肽和促进免疫应答的抗体多肽中的至少一种、二种、或三种。
13.项12的基因改造的单纯疱疹病毒，其中所述刺激免疫应答的细胞因子多肽选自如下的一种或多种：粒细胞巨噬细胞集落刺激因子(GMCSF)、IL-2，IL12，IFN-γ和TNFa。
14.项12的基因改造的单纯疱疹病毒，其中所述能够促进免疫应答的抗体多肽为免疫检查点抗体多肽。
15.项14所述的基因改造的单纯疱疹病毒，其中所述免疫检查点抗体为PD1抗体或PD-L1抗体。
16.项1-4任一项所述的基因改造的单纯疱疹病毒，还包括破坏或敲除一种或多种立即早期基因。
17.项16所述的基因改造的单纯疱疹病毒，所述一种或多种立即早期基因选自ICP0、ICP4、ICP22和ICP27编码基因中的一个、两个、三个或四个。
18.包含项1-17任一项的基因改造的单纯疱疹病毒的组合物。
19.项18的组合物，其中所述基因改造的单纯疱疹病毒在同一位点或不同位点插入一种、二种、三种或四种外源基因。
一方面，本申请还进一步涉及如下各项：
1.一种单纯疱疹病毒载体，其来源于CCTCC保藏号为V201810的单纯疱疹病毒。
2.项1所述的单纯疱疹病毒载体，其ICP34.5基因、ICP6基因、ICP0基因、ICP47基因、US3基因、UL56基因、VHS基因、UNG基因、糖蛋白H基因、胸苷激酶基因中的一个或多个被破坏或敲除。
3.项1或2所述的单纯疱疹病毒载体在制备重组溶瘤病毒中的用途，其中所述重组溶瘤病毒中导入了一种或多种外源基因。
4.项3所述的用途，其中所述一种或多种外源基因插入选自如下的一处或多处基因缺失位点：ICP34.5基因、ICP6基因、ICP0基因、ICP47基因、US3基因、UL56基因、VHS基因、UNG基因、糖蛋白H基因、胸苷激酶基因。
5.项4的用途，其中所述外源基因选自如下的一种或多种：编码促进免疫应答的细胞因子的基因、编码肿瘤抗原的基因、编码具有预防和/或治疗肿瘤作用的单抗基因、编码前药转化酶的基因、肿瘤抑制基因、反义RNA或小RNA。
6.项5的用途，其中所述细胞因子选自如下的一种或多种：GM-CSF、G-CSF、M-CSF、IL-1、IL-2、IL-3、IL-4、IL-5、IL-6、IL7、IL-8、IL-10、IL-12、IL-13、IL-15、IL-18、IL-21、IL-23、IFN-α、IFN-γ、TGF-β和TNF-α。
7.项5的用途，其中具有预防和/或治疗肿瘤作用的单抗选自如下的一种 或多种：PD1单抗、PD-L1单抗、PD-L2单抗、CTLA-4单抗、CD80单抗、CD28单抗、CD137单抗、CD137L单抗、OX40单抗、OX40L单抗、CD27单抗、CD70单抗、CD40单抗、CD40L单抗、LAG-3单抗和TIM-3单抗。
8.项5的用途，其中肿瘤抗原为肿瘤来源的特异性抗原，选自如下的一种或多种：PSA、MUC1、MAGE-1、MAGE-2、MAGE-3、MAGE-12、BAGE、GAGE和LAGE。
9.项5的用途，其中前药转化酶为胞嘧啶脱氨酶或单纯疱疹病毒胸苷激酶。
10.项5的用途，其中肿瘤抑制基因为P53或PTEN。
11.项5的用途，其中反义RNA或小RNA为阻断或下调肿瘤过表达的原癌基因、代谢基因的RNA片段。
12.项5的用途，其中所述促进免疫应答的细胞因子是粒细胞巨噬细胞集落刺激因子(GMCSF)、IL-2，IL12，IFN-γ和/或TNFa。
13.项5的用途，其中所述具有预防和/或治疗肿瘤作用的单抗为为免疫检查点抗体。
14.项13所述的用途，其中所述免疫检查点抗体为PD1抗体或PD-L1抗体。
15.一种制备重组溶瘤病毒的方法，包括将一种或多种外源基因导入单纯疱疹病毒，其中所述单纯疱疹病毒来源于CCTCC保藏号为V201810的单纯疱疹病毒。
16.项15所述的方法，其中所述单纯疱疹病毒中ICP34.5基因、ICP6基因、ICP0基因、ICP47基因、US3基因、UL56基因、VHS基因、UNG基因、糖蛋白H基因、胸苷激酶基因中的一个或多个被破坏或敲除。
17.项16所述的方法，其中所述单纯疱疹病毒缺失ICP34.5基因和/或ICP47基因。
18.项15-17任一项的方法，其中所述一种或多种外源基因插入选自如下的一处或多处基因缺失位点：ICP34.5基因、ICP6基因、ICP0基因、ICP47基因、US3基因、UL56基因、VHS基因、UNG基因、糖蛋白H基因、胸苷激酶基因。
19.项18的方法，其中所述外源基因选自如下的一种或多种：编码促进免疫应答的细胞因子的基因、编码肿瘤抗原的基因、编码具有预防和/或治疗 肿瘤作用的单抗基因、编码前药转化酶的基因、肿瘤抑制基因、反义RNA或小RNA。
20.项19的方法，其中所述细胞因子选自如下的一种或多种：GM-CSF、G-CSF、M-CSF、IL-1、IL-2、IL-3、IL-4、IL-5、IL-6、IL7、IL-8、IL-10、IL-12、IL-13、IL-15、IL-18、IL-21、IL-23、IFN-α、IFN-γ、TGF-β和TNF-α。
21.项19的方法，其中具有预防和/或治疗肿瘤作用的单抗选自如下的一种或多种：PD1单抗、PD-L1单抗、PD-L2单抗、CTLA-4单抗、CD80单抗、CD28单抗、CD137单抗、CD137L单抗、OX40单抗、OX40L单抗、CD27单抗、CD70单抗、CD40单抗、CD40L单抗、LAG-3单抗和TIM-3单抗。
22.项19的方法，其中肿瘤抗原为肿瘤来源的特异性抗原，选自如下的一种或多种：PSA、MUC1、MAGE-1、MAGE-2、MAGE-3、MAGE-12、BAGE、GAGE和LAGE。
23.项19的方法，其中前药转化酶为胞嘧啶脱氨酶或单纯疱疹病毒胸苷激酶。
24.项19的方法，其中肿瘤抑制基因为P53或PTEN。
25.项19的方法，其中反义RNA或小RNA为阻断或下调肿瘤过表达的原癌基因、代谢基因的RNA片段。
26.项18的方法，其中所述外源基因是编码刺激免疫应答的细胞因子多肽和/或促进免疫应答的抗体多肽。
27.项26的方法，其中所述刺激免疫应答的细胞因子多肽是粒细胞巨噬细胞集落刺激因子(GMCSF)、IL-2，IL12，IFN-γ和/或TNFa。
28.项26的方法，其中所述能够促进免疫应答的抗体多肽为免疫检查点抗体多肽。
29.项28所述的方法，其中所述免疫检查点抗体为PD1抗体或PD-L1抗体。
30.由项15-29任一项的方法制备的重组溶瘤病毒。
31.包含项30的重组溶瘤病毒的组合物。
32.项31的组合物，其中所述单纯疱疹病毒的同一位点或不同位点插入了一种、二种、三种或四种外源基因。
33.项31或32的组合物，其中所述重组溶瘤病毒为二种、三种、四种或 四种项15-29任一项的方法制备的重组溶瘤病毒的混合物，其中每种重组溶瘤病毒导入了不同的外源基因。
34.项31-33任一项的组合物，其为病毒培养物。
在一些实施方案中，本发明涉及：
1.一种基因改造的单纯疱疹病毒，其中一种或多种立即早期基因被敲除或破坏，或其表达通过基因突变被阻止或降低从而丧失复制能力，该单纯疱疹病毒来源于CCTCC保藏号为V201810的病毒株、其减毒株或其后代。
2.项1所述的单纯疱疹病毒，所述一种或多种立即早期基因是选自ICP0、ICP4、ICP22和ICP27编码基因中的一个、两个、三个或四个。
3.项1或2的单纯疱疹病毒，其来源于CCTCC保藏号为V201810的病毒株。
4.项1-3任一项所述的单纯疱疹病毒，其ICP34.5基因、ICP6基因、ICP0基因、ICP47基因、US3基因、UL56基因、功能性VP16基因、VHS基因、UNG基因、糖蛋白H基因、胸苷激酶基因中的一个或多个被破坏或敲除。
5.项4所述的单纯疱疹病毒，其ICP34.5基因被破坏或敲除。
6.项5所述的单纯疱疹病毒，其ICP47基因被破坏或敲除。
7.项1-6任一项所述的单纯疱疹病毒，其中引入了的一种或多种外源基因。
8.项7的单纯疱疹病毒，其中所述外源基因为神经生长因子、神经递质和/或神经营养因子基因。
9.项7的单纯疱疹病毒，其中所述外源基因为凝血因子基因。
10.包含项1-9任一项的单纯疱疹病毒的组合物。
11.项10的组合物，其为病毒培养物。
12.包含项1-9任一项的单纯疱疹病毒的宿主细胞。
13.包含项12的宿主细胞的组合物。
14.项13的组合物，其为细胞培养物。
15.CCTCC保藏号为V201810的单纯疱疹病毒或其减毒变体或其后代用作将神经生长因子、神经递质和/或神经营养因子外源基因导入细胞内的载体的用途。
16.CCTCC保藏号为V201810的单纯疱疹病毒或其减毒变体或其后代在制备治疗神经系统疾病、血液系统疾病、免疫系统疾病的药物中的用途。
17.项16的用途，其中所述血液系统疾病为凝血障碍疾病，例如血友病。
18.项1-9任一项的单纯疱疹病毒在制备治疗或预防中枢神经系统疾病或周围神经系统疾病的药物中的用途。
19.项18的用途，其中所述中枢神经系统疾病为中风、瘫痪或神经退行性疾病。
20.项19的用途，其中所述神经退行性疾病为帕金森氏病、阿兹海默症、肌萎缩侧索硬化症和亨廷顿病。
21.项18的用途，其中周围神经系统疾病包括运动神经元病、慢性疼痛和外周神经损伤。
22.包含项1-9任一项的单纯疱疹病毒载体在制备治疗或预防疼痛或神经损伤的药物中的用途。
23.项22的用途，其中所述治疗或预防疼痛或神经损伤包括减轻疼痛的严重程度或促进神经再生长。
本申请还涉及一种非复制性单纯疱疹病毒载体，其中该单纯疱疹病毒的一种或多种立即早期基因被敲除或破坏从而丧失复制能力，或其表达通过基因突变被阻止或降低从而丧失复制能力。在一些实施方案中，所述一种或多种立即早期基因是选自ICP0、ICP4、ICP22、ICP47和ICP27编码基因中的一种、两种、三种或四种。在一些实施方案中，所述单纯疱疹病毒来源于CCTCC保藏号为V201810的单纯疱疹病毒或其后代。在一些实施方案中，所述单纯疱疹病毒的ICP34.5基因、ICP6基因、ICP0基因、ICP47基因、US3基因、UL56基因、VP16基因、VHS基因、UNG基因、糖蛋白H基因、胸苷激酶基因中的一个或多个被破坏或敲除。在一些实施方案中，所述单纯疱疹病毒的ICP34.5基因被破坏或敲除。在一些实施方案中，所述单纯疱疹病毒的ICP47基因被破坏或敲除。在一些实施方案中，所述单纯疱疹病毒载体还包含引入的一种或多种外源基因。在一些实施方案中，所述外源基因为神经生长因子、神经递质和/或神经营养因子基因。在一些实施方案中，所述外源基因为凝血因子基因。
本申请还涉及一种非复制性单纯疱疹病毒，其来源于CCTCC保藏号为V201810的单纯疱疹病毒，其中，该单纯疱疹病毒的一种或多种立即早期基因被敲除或破坏从而丧失复制能力，或其表达通过基因突变被阻止或降低从 而丧失复制能力。在一些实施方案中，所述一种或多种立即早期基因是选自ICP0、ICP4、ICP22、ICP47和ICP27编码基因中的一种、两种、三种或四种。在一些实施方案中，所述单纯疱疹病毒的ICP34.5基因、ICP6基因、ICP0基因、ICP47基因、US3基因、UL56基因、VP16基因、VHS基因、UNG基因、糖蛋白H基因、胸苷激酶基因中的一个或多个被破坏或敲除。在一些实施方案中，所述单纯疱疹病毒的ICP34.5基因被破坏或敲除。在一些实施方案中，所述单纯疱疹病毒的ICP47基因被破坏或敲除。在一些实施方案中，所述病毒中导入了外源基因，例如神经生长因子、神经递质、神经营养因子或凝血因子。
附图说明
图1：HL-1株病毒25000倍电镜图。
图2：HL-1和HSV(17)不同MOI(multiplicity of infection)转染HepG2细胞。
图3：HL-1和HSV(17)不同MOI转染A375细胞。
图4：HL-1和HSV(17)不同MOI转染A549细胞。
图5：HL-1和HSV(17)不同MOI转染MCF-7细胞。
图6：HL-1和HSV(17)不同MOI转染Hela细胞。
图7：HL-1和HSV(17)不同MOI转染U251细胞。
图8：HL-1和HSV(17)不同MOI转染Vero细胞。
图9：HL-1和HSV(17)分别转染不同细胞的IC50比较。
图10：敲除ICP34.5和ICP47基因的CCTCC：V201810病毒株和对照病毒17病毒株转染不同细胞的IC50测定结果。
图11：表达IL-12外源基因的CCTCC：V201810病毒株和对照病毒17病毒株转染肿瘤细胞的IC50测定结果。
图12：插入IL-12基因和PD1单抗基因后HL-1病毒株重组病毒和17病毒株重组病毒转染肿瘤细胞的IC50测定结果。
图13：显示敲除了ICP34.5和ICP4基因的HL-1株和17株重组病毒对神经细胞的转染能力的比较。
图14：显示敲除了ICP34.5、ICP4以及ICP27基因的HL-1株和17株重组病毒对神经细胞的转染能力的比较。
图15：显示敲除了ICP34.5和ICP4基因的HL-1株和17株重组病毒对人胚肝二倍体细胞的转染和外源基因在肝细胞中的表达。
图16：显示敲除了ICP34.5、ICP4以及ICP27基因的HL-1株和17株重组病毒对人胚肝二倍体细胞的转染和外源基因在肝细胞中的表达。
图17：显示给药HSV-FⅧ48小时、72小时和120小时对APTT的影响。
图18：显示HL1-ENK对关节炎慢性疼痛大鼠的行为活动评分的影响，证明HL1-ENK对关节炎慢性疼痛大鼠有显著的镇痛效果。
具体实施方式
本申请涉及一种与实验室模式病毒株相比性能改善的非复制性单纯疱疹病毒以及其作为载体的用途。本申请所述“溶瘤病毒”是指一类感染和杀死癌细胞的病毒，该病毒感染癌细胞后通过溶瘤作用破坏受感染的癌细胞，同时释放出新的感染性病毒颗粒或病毒体，破坏剩余的癌细胞。此类病毒因其溶瘤作用而得名。
“单纯疱疹病毒(HSV)”因其研究基础深厚并且在其天然状态下相对无害，因此是最先选择用于选择性攻击癌细胞的病毒(溶瘤病毒)之一。当本发明的病毒为单纯疱疹病毒时，所述病毒可以来源于例如HSV-1毒株或HSV-2毒株或者它们的衍生毒株，优选是HSV-1。
本申请一些实施方案涉及CCTCC保藏号为：V201810(CCTCC：V201810)的病毒株。在本申请中，该病毒株称为HSV-1病毒HL-1毒株、或简称HL-1株、HL-1或HL1。
本申请一些实施方案涉及HSV-1毒株的衍生毒株，例如与CCTCC保藏号为：V201810(CCTCC：V201810)的病毒株基因组序列同源性为至少70％，更优选至少80％、85％、90％、95％、98％序列同源性的病毒株。
“单纯疱疹病毒载体”指携带外源基因的单纯疱疹病毒。
空斑形成单位(plaque forming unit)，缩写pfu，指在单层培养的动物细胞上形成空斑(噬斑)对应的病毒数。
“免疫检查点阻断(抑制)性抗体”或“免疫检查点阻断(抑制)性单克隆抗体”指抑制或阻断抑制性免疫检查点分子的单克隆抗体。“免疫检查点刺激(激动)性抗体”或“免疫检查点刺激(激动)性单克隆抗体”指刺激或激动刺激性免疫检查点分子的单克隆抗体。免疫检查点是免疫系统的监管者， 它们的作用体现在增强免疫系统的清除异己能力或者阻止免疫系统不加选择地攻击细胞，从而对免疫调节至关重要。免疫检查点分抑制性检查点分子和刺激性检查点分子。抑制性检查点分子包括但不限于：A2AR、B7-H3(CD276)、B7-H4、BTLA(CD272)、CTLA-4(CD152)、IDO、KIR、LAG3、NOX2、PD1、TIM3、VISTA、CD47；刺激性检查点分子包括但不限于：CD27、CD40、OX40、GITR、CD137、CD28、ICOS。抑制性检查点分子和刺激性检查点分子是癌症免疫疗法的靶标，因为它们可能用于多种类型的癌症。1.
1.本申请筛选的单纯疱疹病毒野生株HL-1
本发明在第一方面涉及一种单纯疱疹病毒，该单纯疱疹病毒是保藏号为CCTCC NO：V201810的I型单纯疱疹病毒(HSV-1)。该I型单纯疱疹病毒(HSV-1)具有选自如下的一种或多种特性：(a)与参比实验室毒株相比，具有增强的感染肿瘤细胞能力，(b)与参比实验室毒株相比，具有增强的肿瘤细胞内复制能力，(c)与参比实验室毒株相比，具有增强的杀伤肿瘤细胞能力；(d)该单纯疱疹病毒经修饰后，与具有等同修饰的参比实验室毒株相比，具有增强的感染肿瘤细胞能力，(e)该单纯疱疹病毒经修饰后，与具有等同修饰的参比实验室毒株相比，具有增强的肿瘤细胞内复制能力，和(f)该单纯疱疹病毒经修饰后，与具有等同修饰的参比实验室毒株相比，具有增强的杀伤肿瘤细胞能力。在一些实施方案中，本申请涉及保藏号为CCTCC NO：V201810的1型单纯疱疹病毒的减毒变体。
本发明病毒株是“非实验室”毒株，可以被称为“临床”毒株。本领域技术人员能够容易地将实验室毒株和非实验室毒株或临床毒株区分开来。实验室毒株和非实验室毒株之间的关键区别在于目前常用的实验室毒株已经在培养物中保持了相当长时间。为了使病毒生长和保持，用病毒感染合适的细胞，病毒在细胞内复制，然后收获病毒；然后重新感染新鲜细胞。该过程构成连续传代的一个周期。就HSV而论，每个这样的周期可能需要例如几天，这种连续传代可能导致病毒株特性的改变，例如发生有利于在培养物中生长的特性(例如快速复制)的选择，而不是选择与有利于实际应用的特性。
本发明病毒株是非实验室毒株，因为它们来源于从受感染个体中最新分离的毒株。本发明毒株经过修饰，消除了毒性但基本上保留了它们所来源的原始临床分离株的理想特性。
本发明的病毒能够有效感染人类靶细胞。这样的病毒刚从受感染个体中分离，然后根据与标准实验室毒株相比增强在体外和/或体内在肿瘤细胞和/或其它细胞内复制的所需能力进行筛选。与实验室病毒株相比，具有改进特性的这类病毒是本发明的病毒。然后可以对具有这种所需改进特性、经鉴定的病毒通过合适基因的突变进行工程改造，使它们可以选择性地杀伤肿瘤细胞，或者将其突变，使它们可以在非溶瘤性应用中将基因传递至靶组织而毒性效应降低。这些经修饰的病毒也是本发明的病毒。
本发明病毒与具有等同修饰的参比实验室毒株相比，本发明的病毒株比具有等同修饰的参比实验室毒株感染任何肿瘤细胞或在其中复制、杀伤肿瘤细胞或在组织中的细胞间传播的能力强。优选这种更强的能力是具统计学显著性的更强的能力。例如，按照本发明，就待测特性而论，本发明的病毒株的能力可以是所述参比毒株的能力的至多1倍、1.5倍、2倍、3倍、4倍、5倍、6倍、7倍、8倍、9倍、10倍、15倍、20倍、50倍或100倍。
本发明的病毒基本上具有(即保留)其未经修饰的临床前体毒株的能力。例如，就计划用来治疗肿瘤的溶瘤病毒而论，本发明的病毒株优选基本上具有其未经修饰的临床前体毒株感染任何肿瘤细胞或在其中复制、杀伤肿瘤细胞或在组织中的细胞间传播的能力。
2.本申请经修饰的溶瘤病毒
本申请涵盖经修饰的病毒，其中改变的病毒区域或者可以被消除(完全或部分)或者成为无功能的、或者被其它序列取代、尤其是被外源基因序列取代。可以使一种或多种基因成为无功能基因，并且可以插入一种或多种外源基因。
在一些实施方案中，本申请的病毒是经修饰的非实验室溶瘤病毒。这些病毒可用于溶瘤法治疗癌症。这类病毒感染肿瘤细胞并在所述肿瘤细胞内复制，随后杀伤所述肿瘤细胞。因此，这类病毒是具有复制能力的病毒。最好是它们选择性在肿瘤细胞内具有复制能力。这意味着它们在肿瘤细胞内复制而在非肿瘤细胞内不复制，或者它们在肿瘤细胞内比在非肿瘤细胞内更为有效地复制。选择性复制能力的测定可以通过本文所述用测定复制能力和肿瘤细胞杀伤能力的试验来进行。同时，本申请的病毒也可以经修饰失去其在任何细胞包括肿瘤细胞中的复制能力，但仍然具备感染细胞包括肿瘤细胞的能 力。经修饰的病毒通过在细胞内表达携带的肿瘤治疗基因从而发挥肿瘤杀伤作用。在癌症治疗当中，优选地采用在肿瘤细胞中具有复制能力的溶瘤病毒进行治疗。
优选本发明的溶瘤病毒比具有相同修饰的参比实验室毒株感染肿瘤细胞或在肿瘤细胞内复制、杀伤肿瘤细胞或在组织中的细胞间传播的能力强(优选具有统计学显著性)。
病毒感染肿瘤细胞的能力可以通过测量感染一定百分比细胞(例如50％或80％细胞)所需的病毒剂量进行定量。在肿瘤细胞内复制的能力可以通过测定病毒在细胞中的生长来确定，例如通过测量在6小时、12小时、24小时、36小时、48小时或72小时或更长时间的时间周期内在细胞内的病毒生长。病毒杀伤肿瘤细胞的能力可以通过对在规定时间内对于给定细胞类型在给定时间点和MOI(multiplicity of infection，感染复数)下保留的活细胞数进行计数来定量。可以使用本申请实施例中的细胞或细胞组合，也可以使用其它肿瘤细胞类型。为了对在给定时间点保留的活细胞数进行计数，可以对排除锥虫蓝的细胞(即活细胞)的数目进行计数。也可用流式细胞计数(FACS)、MTT、CCK-8或CTG方法分析进行定量测定。也可以体内测定肿瘤细胞杀伤能力，例如通过测量由于特定病毒引起的肿瘤体积的减小。
为了测定本发明病毒的特性，一般而言，最好使用标准实验室参比毒株进行比较。可以使用任何合适的标准实验室参比毒株。就HSV而论，最好使用HSV-1毒株17、HSV-1毒株F或HSV-1毒株KOS中的一种或多种。所述参比毒株通常具有与本发明待测毒株等同的修饰，例如基因缺失和/或外源基因插入。例如，就HSV毒株而论，如果在本发明的病毒中已经使ICP34.5、ICP6和/或胸苷激酶(TK)编码基因变成无功能的，则在参比毒株中也使它们变成无功能的。
在本申请实施例中，参比毒株为实验室标准模式株HSV-1病毒17株，或称HSV-1病毒17株、HSV(17)、17病毒株、17株。
本申请涉及在2019年7月30日，在中国典型培养物保藏中心(CCTCC)(地址：中国、武汉、武汉大学)、用于专利程序保藏的人疱疹病毒I型HL-1株、保藏号为CCTCC NO：V201810。
在本申请中，疱疹病毒I型(1型)、I型(1型)疱疹病毒、单纯疱疹病毒I型(1型)、I型(1型)单纯疱疹病毒、HSV-1含义相同，可互换使 用。人疱疹病毒I型HL-1株在本申请中可以简写为HL-1株、HL1株、HL-1或HL1。
单纯疱疹病毒(HSV)既可用作神经系统和其它系统中的基因传递载体，又可用于溶瘤法治疗癌症。然而，在这两种应用中，必须使病毒成为缺陷型的，使它不再具有致病性，但是使它仍能进入细胞并执行所需功能。
HSV-1基因组具有许多可以敲除或突变的基因赋予安全性和/或肿瘤靶向特异性。例如，将癌细胞与大部分正常细胞(有丝分裂后)相区别的普遍特征之一是癌细胞的持续增殖并因此维持足够的核苷酸用于DNA复制。HSV-1包含涉及核苷酸代谢的相关基因(胸苷激酶，核糖核苷酸还原酶和尿嘧啶DNA糖基化酶等)允许病毒在缺乏足够的核苷酸的非分裂细胞中复制。将这些基因进行敲除或突变可以赋予病毒对分裂细胞(肿瘤细胞)的特异性，并且也经常减弱病毒的致病性。此外，细胞中具有多种机制检测和失活入侵的病毒，病毒的部分基因(ICP34.5、ICP0和UL56等)可以逃避或者阻断细胞的抗病毒机制，该部分基因的敲除或突变同样可以赋予病毒对分裂细胞(肿瘤细胞)的特异性。
在一些实施方案中，对本发明HSV毒株进行修饰，使其缺乏ICP34.5基因、ICP6基因、ICP0基因、ICP47基因、US3基因、UL56基因、功能性VP16基因、VHS基因、UNG基因、糖蛋白H基因、胸苷激酶基因中的一个或多个。在一些实施方案中，所述病毒缺乏ICP34.5基因。在一些实施方案中，可以对所述病毒进行进一步修饰，使其缺乏ICP47基因。在一些实施方案中，突变ICP34.5、ICP47、Us3、ICP0、UL56基因中的一个或多个，制备溶瘤型I型单纯疱疹病毒(Oncolytic herpes simplex virus type 1，oHSV-1)。
对于溶瘤法治疗癌症(该疗法也可以包括传递增强所述治疗效应的基因)，已经鉴定出HSV的许多突变(Peters C.Designing Herpes Viruses as Oncolytics.Molecular Therapy Oncolytics,2015.)，HSV的这些突变仍允许病毒在培养物或在体内活跃分裂的细胞内(例如肿瘤内)复制，但是阻止其在正常组织内的有效复制。这样的突变包括破坏ICP34.5、ICP6和胸苷激酶的编码基因。迄今为止，在这些突变病毒中，具有ICP34.5突变或者ICP34.5突变以及例如ICP6突变的病毒已经显示最为有利的安全性。已经显示仅缺失ICP34.5的病毒在体外许多肿瘤细胞类型中复制并且在小鼠脑肿瘤内选择性 复制，而不伤害周围组织。
提供溶瘤特性(即与周围组织相比在肿瘤内选择性复制)的任何其它基因缺失/突变的病毒也是本申请涵盖的病毒。
可用本领域已知的技术和/或本文描述的技术，将外源基因插入到本发明的这类病毒中，例如CCTCC保藏号为V201810(CCTCC：V201810)的病毒中。在溶瘤病毒中，所述外源基因通常是增强所述病毒对抗肿瘤的能力的基因。因此可以插入赋予病毒抗肿瘤特性的任何基因。具体地说，所述外源基因可以是能够以有益方式改进针对肿瘤细胞的免疫应答的基因，尤其是免疫刺激多肽。
3.本申请经修饰的非复制性病毒
本申请涉及基因改造的单纯疱疹病毒及其作为载体在非溶瘤性方面的应用。本申请基因改造的单纯疱疹病毒在非溶瘤性应用中的载体，可以进行突变，使得病毒调节性立即早期基因的表达减至最小。因而，可以单独或联合使ICP4、ICP27、ICP22和/或ICP0编码基因失活或缺失，或者在病毒体反式激活蛋白vmw65中的突变包括阻止/降低其反式激活能力。在用于非溶瘤性应用的特别优选的实施方案中，使ICP27、ICP0和ICP4编码基因缺失(具有或不具有额外的ICP22和/或ICP47缺失/失活)，或者将ICP27和ICP4缺失加上vmw65中的失活，或者ICP4缺失加上vmw65中的失活。
本申请的一些实施方案涉及将外源基因传递至神经系统的单纯疱疹病毒载体。按照本发明这一实施方案的病毒通常比具有等同修饰的参比实验室毒株感染神经元的能力、在神经组织中的细胞间传播的能力或在轴突内运输的能力强。在这些实施方案中，对本发明病毒进行修饰，使其缺乏功能性ICP27编码基因、功能性ICP4编码基因、功能性ICP0编码基因或功能性ICP22编码基因中的一个、两个、三个或全部。在一些实施方案中，本发明的病毒既缺乏功能性ICP4编码基因，又缺乏功能性ICP27编码基因，并且本发明的病毒在vmw65编码基因中具有一个消除其转录激活活性的失活突变。这类病毒可以用来治疗周围神经系统疾病或中枢神经系统疾病。在中枢神经系统疾病的情况下，特别优选使选自ICP0、ICP4、ICP22和ICP27的至少两个立即早期基因变成无功能的。为了提高修饰病毒的安全性，本发明病毒还可以对ICP34.5基因、ICP6基因、ICP0基因、ICP47基因、US3基因、 UL56基因、功能性VP16基因、VHS基因、UNG基因、糖蛋白H基因、胸苷激酶基因中的一个或多个进行修饰。外源基因插入位点可以是上述修饰位点的任意一处或多处，也可以是单纯疱疹病毒的潜伏感染区域(LAT)。当单纯疱疹病毒进入潜伏状态时，除了LAT区基因处于活跃状态外，所有基因处于休眠状态，将外源基因插入LAT区有助于基因的长期表达。因此，当本发明的病毒用于非复制的基因治疗载体用途时，优选将外源基因插入LAT区。
本发明的无复制能力病毒可以用来将基因传递至需要基因治疗的个体。具体地说，本发明的单纯疱疹病毒可以用来治疗中枢神经系统疾病或周围神经系统疾病。用于治疗或预防的中枢神经系统疾病包括神经变性性疾病。在一些实施方案中，用于治疗或预防的中枢神经系统疾病是中风、帕金森氏病、阿兹海默症、肌萎缩侧索硬化症和亨廷顿病。用于治疗或预防的优选周围神经系统疾病包括运动神经元病、慢性疼痛和外周神经损伤。在一些实施方案中，将本发明的病毒给予患有疼痛、变性性疾病或神经损伤的受试者，改善所述受试者的病症，例如减轻疼痛的严重程度、减慢神经组织的变性或促进神经再生长。在一些实施方案中，本发明的单纯疱疹病毒可以用来治疗血液系统疾病。具体地，血液系统疾病包括血友病。
4.药物组合物、制品及其用途
本发明的溶瘤病毒可以用于制备用于治疗癌症的药品。具体地说，本发明的溶瘤病毒制剂可以用于癌症的治疗中，例如通过直接肿瘤内注射。本发明的溶瘤病毒制剂可以用来治疗哺乳动物、优选人体内的任何实体瘤。例如，可以将本发明的病毒给予患有以下疾病的受试者：肝癌、黑色素瘤、脑胶质瘤、肉瘤、肺癌、结直肠癌、头颈部肿瘤、乳腺癌、肾细胞癌、卵巢癌、宫颈癌、前列腺癌、胃癌、淋巴瘤、胰腺癌和膀胱癌。
在一些实施方案中，本申请涉及重组溶瘤病毒或称为经修饰的溶瘤病毒，其特征在于，该重组溶瘤病毒或经修饰的溶瘤病毒来源于CCTCC保藏号为：V201810(CCTCC NO：V201810)的I型单纯疱疹病毒，其中对该CCTCC：V201810病毒进行基因改造，使其ICP34.5基因、ICP6基因、ICP0基因、ICP47基因、US3基因、UL56基因、功能性VP16基因、VHS基因、UNG基因、糖蛋白H基因、胸苷激酶基因中的一个或多个被敲除，从而在 上述一处或多处基因敲除的位置插入外源基因，例如编码刺激免疫应答的细胞因子多肽或能够促进免疫应答的抗体多肽，例如刺激免疫应答的细胞因子多肽是粒细胞巨噬细胞集落刺激因子(GMCSF)、IL-2，IL12，IFN-γ或TNFa，能够促进免疫应答的抗体多肽为免疫检查点抗体。
本发明的重组溶瘤病毒或称为经修饰的溶瘤病毒可以用于制备用于治疗癌症的药品。具体地说，本发明的溶瘤病毒制剂可以用于癌症的治疗中，例如通过直接肿瘤内注射。本发明的溶瘤病毒制剂可以用来治疗哺乳动物、优选人体内的任何实体瘤。例如，可以将本发明的病毒给予患有以下疾病的受试者：肝癌、黑色素瘤、脑胶质瘤、肉瘤、肺癌、结直肠癌、头颈部肿瘤、乳腺癌、肾细胞癌、卵巢癌、宫颈癌、前列腺癌、胃癌、淋巴瘤、胰腺癌和膀胱癌。
本发明涉及无复制能力病毒在基因传递至需要基因治疗的个体方面的用途。在一些实施方案中，本发明的基因改造的单纯疱疹病毒载体可以用来治疗中枢神经系统疾病或周围神经系统疾病。用于治疗或预防的中枢神经系统疾病包括神经变性性疾病。在一些实施方案中，用于治疗或预防的中枢神经系统疾病是中风、帕金森氏病、阿兹海默症、肌萎缩侧索硬化症和亨廷顿病。用于治疗或预防的周围神经系统疾病包括运动神经元病、慢性疼痛和外周神经损伤。在一些实施方案中，将本发明的病毒给予患有疼痛、变性性疾病或神经损伤的受试者，改善所述受试者的病症，例如减轻疼痛的严重程度、减慢神经组织的变性或促进神经再生长。在一些实施方案中，本发明的单纯疱疹病毒可以用来治疗血液系统疾病。具体地，血液系统疾病包括血友病。
本申请的一些实施方案涉及包含本申请的病毒和药学上可接受的载体或稀释剂药物组合物。合适的载体和稀释剂是本领域已知的，包括但不限于磷酸缓冲盐溶液。
在一些实施方案中，本申请涉及制品或试剂盒，包含内装有前述药物组合物的小瓶和包装插页，所述包装插页包括如何使用该制品或试剂盒治疗疾病的使用说明书。
可以将所述本申请的药物组合物直接注射到靶组织中，例如癌症组织中，进行溶瘤法治疗和/或将基因传递至细胞内以实现治疗目的。本申请包含病毒或重组病毒的药物组合物给予的病毒剂量范围为10 4-10 12pfu/ml、优选10 5-10 8pfu/ml、更优选10 6-10 8pfu/ml。对于溶瘤法治疗或非溶瘤法治疗，当 注射时，基本上由所述病毒和一种药学上可接受的合适载体或稀释剂组成的药用组合物的注射用量通常至多10ml、通常1-5ml、优选1-3ml。然而，对于某些溶瘤法治疗应用，根据所述肿瘤和接种部位，也可以使用高于10ml的较大体积。
本领域技术人员根据受试者和疾病情况能够容易地确定最佳给药途径和剂量。所述剂量可以根据各种参数、尤其根据待治疗患者的年龄、体重和病症、疾病或病症的严重程度以及给药途径来确定。对于癌症患者优选的给药途径是直接注射到肿瘤内。也可以系统给予所述病毒，例如静脉注射，或通过注射将所述病毒给予给所述肿瘤供血的血管内。最适给药途径将取决于所述肿瘤的位置和大小。所述剂量可以根据各种参数、尤其是根据肿瘤的位置、肿瘤的大小、待治疗患者的年龄、体重和病症以及给药途径来确定。
实施例
实施例1 HL-1株病毒的获得
a)病毒的采集
招募63位患有复发性唇疱疹的健康志愿者，对疱疹液进行无菌采集。
b)病毒的筛选
本部分的目的是测试和筛选HSV-1初级临床分离株，选择溶瘤能力最强的病毒株。
每次使用5至8个病毒株同时进行实验。以HSV-1病毒17株为对照，将病毒株以相同的MOI感染六孔板中的细胞，平行试验，并在感染48小时之后观察病毒引起的CPE(cytopathic effect)现象并评价结晶紫染色后的空斑数量。CPE应为典型HSV-1感染引起，细胞变圆并由感染中心往外逐步脱落形成空斑。对CPE现象明显且染色后空斑数量高的病毒株进行评估。
经过如此方法评价并筛选出具有最佳溶瘤效果的病毒株为HL-1株。
c)HL-1病毒株的生物学特性分析
1)电镜观察
HL-1株病毒感染细胞后去除细胞碎片，在25000倍电镜下观察。图中显示病毒呈典型HSV-1外形，可观察到基因组DNA、衣壳、间层蛋白和囊膜四个组分(参见图1)。
2)HL-1病毒株基因组DNA测序结果分析
将I型单纯疱疹病毒(HSV-1)HL-1株基因组DNA交由上海欧易生物进行三代测序分析，比对HL-1株与标准模式株HSV-1病毒17株(NC_001806.2)的病毒基因组序列，结果显示两者的基因组序列存在1700多个碱基的差异，多个重要编码基因包括膜蛋白和立早基因等的氨基酸序列都存在较大差异，比对结果参见下表1和表2。
表1 HL-1株与17株膜蛋白氨基酸序列比对结果
Figure PCTCN2021079114-appb-000001
表2 HL-1株与17株病毒重要基因的氨基酸序列比对结果
Figure PCTCN2021079114-appb-000002
HL-1株与17株的包膜蛋白中gD、gG、gI、gJ、gL、gM和gN氨基酸序列差异超过1％，尤其是gG和gI的氨基酸序列差异超过3％，其中gG达到5.46％。单纯疱疹病毒包膜的膜蛋白与病毒侵入、释放、细胞间的直接传播有关，提示HL-1株与17株的侵染性差异。
对比立早基因ICP0、ICP4、ICP22、ICP27、ICP47以及重要基因ICP6、ICP34.5的氨基酸序列发现，HL-1株与17株的立早基因ICP4、ICP22和ICP47序列差异较大，氨基酸序列差异均超过1％。ICP4和ICP22作为HSV-1的立早基因，可以刺激病毒早期基因的表达和DNA合成，诱导晚期基因的表达，是HSV-1基因表达从而引发感染的关键因子。ICP34.5也是HL-1株和17株基因中差异较大的一个基因，其氨基酸差异达到6.25％。ICP34.5是HSV-1病毒重要的神经毒性因子，对病毒的复制和致病性起到了关键作用。
d)HL-1病毒株的保藏
将HL-1病毒株在2019年7月30日，用于专利程序的保藏，保藏于中国典型培养物保藏中心(CCTCC)(地址：中国、武汉、武汉大学)，保藏号为CCTCC NO：V201810，分类命名为：“人疱疹病毒I型HL-1株”。
实施例2 野生HL-1株和17株溶瘤效果的对比
本实施例提供HL-1和17株在不同细胞上的复制对比实验和结果以及它们转染肿瘤细胞的能力。实验细胞包括：Hep G2人肝癌细胞、A-375人皮肤癌细胞、A549人肺癌细胞、MCF7人乳腺癌细胞、HeLa人宫颈癌细胞、U251人胶质瘤细胞、Vero猴肾细胞。
实验方法如下：
1.HL-1和HSV(17)分别转染不同细胞后镜检观察
取处于对数生长期的上述细胞，消化后传代至六孔板中培养。24小时后弃去旧培养基，每孔加入培养基2ml。按照不同MOI数量的病毒进行转染，加入病毒后放入37℃二氧化碳培养箱培养48小时后取出镜下观察，拍照记录。
2.HL-1和HSV(17)分别转染不同细胞的IC50测定
取处于对数生长期的细胞，消化后传代至96孔板中。24小时后弃去旧培养基，分别加入9个稀释度的HL-1和HSV(17)病毒，100μl/孔，使得MOI为20、5、1.25、0.3125、0.0781、0.0195、0.0049、0.0012和0.0003， 设空白对照。37℃培养3天后弃去孔内所有液体，加入10％CCK-8试剂(Cell Counting Kit-8、Dojindo)，100μl/孔，37℃孵育1h，在450nm处测定样品OD值，根据拟合曲线计算IC50。
实验结果：
1.HL-1和HSV(17)分别转染不同细胞后镜检观察
1.1 HL-1和HSV(17)转染HepG2细胞镜检结果
HL-1和HSV(17)转染HepG2细胞48小时后显微镜下观察的结果显示，在各相同的MOI条件下，HL-1的转染效率都高于HSV(17)的转染效率(参见图2)。
1.2 HL-1和HSV(17)转染A375细胞镜检结果
HL-1和HSV(17)转染A375细胞48小时后显微镜下观察的结果显示，在各相同的MOI条件下，HL-1的转染效率都明显高于HSV(17)。甚至可在镜下观察到在转染病毒MOI 0.1的条件下，HL-1转染的细胞形态已经全部变圆，而在HSV(17)转染的细胞中仍可见正常细胞(参见图3)。
1.3 HL-1和HSV(17)转染A549细胞镜检结果
HL-1和HSV(17)转染A549细胞48小时后显微镜下观察的结果显示，在各相同的MOI条件下，HL-1的转染效率都明显高于HSV(17)。可在镜下观察到HL-1转染的细胞数量明显多于HSV(17)转染的细胞数量，在MOI 0.1时几乎没有观察到正常细胞，而HSV(17)转染的细胞中仍可见正常细胞，且数量随着MOI的减少而递增(参见图4)。
1.4 HL-1和HSV(17)转染MCF-7细胞镜检结果
根据以上实验中病毒转染细胞效果对比的经验，对于之后的实验只选取了MOI 0.1和MOI 0.01两个病毒浓度进行比较。
HL-1和HSV(17)转染MCF-7细胞48小时后显微镜下观察的结果显示HL-1的转染效率明显高于HSV(17)。HL-1转染的MCF-7细胞在MOI0.1的情况下细胞形态全部变圆，而HSV(17)转染的MCF-7细胞仍有部分正常细胞(参见图5)。
1.5 HL-1和HSV(17)转染Hela细胞镜检结果
HL-1和HSV(17)转染Hela细胞48小时后显微镜下观察的结果显示，在MOI 0.1条件下，HL-1的转染效率都明显高于HSV(17)。HL-1转染的 Hela细胞在MOI 0.1的情况下细胞形态大部分变圆，而HSV(17)转染的Hela细胞仍有一定数量的正常细胞(参见图6)。
1.6 HL-1和HSV(17)转染U251细胞镜检结果
HL-1和HSV(17)转染U251细胞48小时后显微镜下观察的结果显示，HL-1的转染效率明显高于HSV(17)。HL-1转染的U251细胞在MOI 0.01的情况下细胞形态大部分变圆，而HSV(17)转染的U251细胞只有很少细胞被病毒转染形态变圆(参见图7)。
1.7 HL-1和HSV(17)转染Vero细胞镜检结果
Vero细胞非肿瘤细胞，为HSV病毒的敏感细胞系，是常用的生产细胞之一，故比较HL-1和HSV(17)对Vero细胞的侵染情况对于生产产能来说具有重要意义。
HL-1和HSV(17)转染Vero细胞48小时后显微镜下观察的结果显示，在MOI 0.1和0.01的条件下，HL-1的转染效率都明显高于HSV(17)。HL-1转染的Vero细胞在病毒MOI 0.1的情况下细胞形态全部变圆，而HSV(17)转染的Vero细胞在MOI 0.1的情况下仍有部分正常细胞(参见图8)。
2.HL-1和HSV(17)分别转染不同细胞的IC50测定
HL-1和HSV(17)分别转染不同细胞的IC50比较参见图9。图中结果显示HL-1相比HSV(17)的IC50(MOI)均要小，表明HL-1针对细胞的杀伤能力强于HSV(17)。本实施例旨在比较HL-1和HSV(17)两种病毒株转染肿瘤细胞能力的差异。HL-1和HSV(17)转染不同的肿瘤细胞(HepG2、A375、A549、MCF-7、Hela和U251)以及正常传代细胞Vero细胞，在转染细胞48小时后显微镜下观察的结果显示，尽管各类细胞对病毒的敏感性不同，但在相同的MOI下HL-1的转染效率明显高于HSV(17)的转染效率。进一步通过CCK-8法测定HL-1和HSV(17)转染不同细胞的IC50值，结果显示HL-1转染细胞的IC50值均比HSV(17)小。综上所述，相比HSV(17)病毒株，HL-1病毒株在细胞中具有更好的侵染效率和复制能力，溶瘤效果更强。
实施例3 HSV(HL-1)重组病毒和HSV(17)重组病毒溶瘤效果对比
按照申请号CN1654667A的专利申请中记载的方法将本发明CCTCC保藏号为V201810(CCTCC：201810)的病毒株和对照病毒17病毒株的ICP34.5 基因和ICP47基因敲除，比较基因敲除后的重组病毒HL1-△34.5-△47和17-△34.5-△47转染肿瘤细胞能力差异。实验细胞包括Hep G2人肝癌细胞、A375人皮肤癌细胞、A549人肺癌细胞、MCF7人乳腺癌细胞、HeLa人宫颈癌细胞、U251人胶质瘤细胞、Vero猴肾细胞。
取处于对数生长期的细胞，消化后传代至96孔板中。24小时后弃去旧培养基，分别加入9个稀释度的不同病毒，100μl/孔，使得MOI为20、5、1.25、0.3125、0.0781、0.0195、0.0049、0.0012和0.0003，设空白对照。37℃培养3天后弃去孔内所有液体，加入10％CCK-8试剂，100μl/孔，37℃孵育1-2h，在450nm处测定样品OD值，根据拟合曲线计算IC50值。
两种重组病毒分别转染不同细胞的IC50比较结果参见图10。实验结果显示HL1-△34.5-△47转染细胞的IC50值均比17-△34.5-△47小，表明HL-1病毒株经过基因重组改构后，相比HSV(17)病毒株重组病毒在细胞中具有更好的侵染效率和复制能力，溶瘤效果强，作为溶瘤病毒具有更好应用前景。
实施例4 HL-1病毒株作为载体携带外源基因对肿瘤细胞的溶瘤作用
本实施例旨在比较HL-1重组病毒和17病毒株重组病毒携带外源基因的溶瘤作用。实验细胞包括A375人皮肤癌细胞、A549人肺癌细胞、AGS人胃癌细胞、AsPC-1人胰腺癌细胞、HeLa人宫颈癌细胞、Hep G2人肝癌细胞、HT-29人大肠结直肠癌细胞、MCF7人乳腺癌细胞、PC-3人前列腺癌细胞、T24人膀胱癌细胞、U-2OS人骨癌细胞和U-87人脑胶质瘤细胞。
1.表达IL-12外源基因的溶瘤病毒的溶瘤作用比较
按照实施例3的方法将本发明CCTCC保藏号为V201810(CCTCC：V201810)的病毒株和对照病毒17病毒株的ICP34.5基因和ICP47基因敲除，并在敲除ICP34.5基因的位置插入人工化学合成的外源基因。该外源基因从5’端至3’端依次包括：EF1α启动子、编码IL-12的基因(Gene ID：16159,16160)和TK PolyA。在北京擎科新业公司测序鉴定上述编码基因正确插入到单纯疱疹病毒载体中。构建成功的重组病毒载体与上述细胞株，在37℃、5％CO 2条件下增殖。
取处于对数生长期的细胞，消化后传代至96孔板中。24小时后弃去旧培养基，分别加入9个稀释度的不同病毒，100μl/孔，使得MOI为20、5、1.25、0.3125、0.0781、0.0195、0.0049、0.0012和0.0003，设空白对照。37℃ 培养3天后弃去孔内所有液体，加入10％CCK-8试剂，100μl/孔，37℃孵育1-2h，在450nm处测定样品OD值，根据拟合曲线计算IC50值。
不同病毒转染肿瘤细胞的IC50测定结果参见图11。实验结果显示了插入IL-12基因后HL-1重组病毒(HL1-IL12)和17株重组病毒(17-IL12)转染肿瘤细胞能力的差异，插入IL-12基因的HL-1重组病毒相比插入IL-12基因的17株重组病毒转染肿瘤细胞的IC50值均要小，表明插入IL-12基因的HL-1病毒株重组病毒在肿瘤细胞中具有更好的侵染效率和复制能力，溶瘤效果强，作为溶瘤病毒具有更好的应用前景。
2.导入外源基因IL-12的溶瘤病毒对黑色素瘤小鼠的溶瘤作用
按照实施例3和4的方法构建HL1-IL12和HL1-mock(HL1-△34.5-△47)，评估溶瘤病毒对黑色素瘤小鼠的溶瘤作用。C57BL/6J小鼠皮下接种B16F10肿瘤细胞，建立同种移植黑色素瘤模型。对照组为磷酸盐溶液，治疗组分别为奥沙利铂(10mg/kg)、受试溶瘤病毒HL1-mock(10^7pfu)，HL1-IL12(10^7pfu)，HL1-IL12(10^6pfu)和HL1-IL12(10^5pfu)，每组10只小鼠；对照组和受试溶瘤病毒给药组瘤内注射给药，奥沙利铂给药组腹腔注射给药。
相对肿瘤抑制率TGI(％)：TGI％＝(1-T/C)×100％。T/C％为相对肿瘤增殖率，即特定时间点治疗组和对照组相对肿瘤体积的百分比值。T和C分别为治疗组和对照组在特定时间点的相对肿瘤体积(RTV)。RTV＝治疗后动物瘤体积/对照组瘤体积。根据肿瘤抑制率(TGI)进行疗效评价。
研究结果：通过对小鼠在给药后第10天肿瘤平均体积分析，HL1-IL12(10 7PFU)肿瘤抑制率为76％、HL1-IL12(10 6PFU)肿瘤抑制率为47％、HL1-mock(10 7PFU)肿瘤抑制率为52％，三者相较于对照组在抗肿瘤作用上均具有统计学上的显著性差异(P<0.05)；而HL1-IL12(10 5PFU)与奥沙利铂(10mg/kg)单独给药组相较于对照组在抗肿瘤作用上没有统计学上的显著性差异(P>0.05)，肿瘤抑制率分别为6％和20％。对照组在第14天全部死亡，HL1-IL12(10 7PFU)给药组有3只小鼠肿瘤消失持续生存。除上述3只小鼠外，给药组其他小鼠均在第21天死亡。
剩余HL1-IL12(10 7PFU)组的3只小鼠肿瘤消失，持续存活至91天，再次在小鼠对侧接种B16F10肿瘤细胞进行再挑战，对照组设5只C57BL/6J小鼠皮下接种B16F10肿瘤细胞，观察肿瘤生长情况。
小鼠接种后对照组于第10天长出肿瘤且持续增大，再挑战组未长出肿瘤，持续观察仍未长出肿瘤，证明小鼠经HL1-IL12治疗后具有肿瘤免疫力。
3.表达GM-CSF和IL-2外源基因的溶瘤病毒的溶瘤作用
按照实施例3的方法将本发明CCTCC保藏号为V201810(CCTCC：V201810)的病毒株和对照病毒17病毒株的ICP34.5基因和ICP47基因敲除，并在敲除ICP34.5基因的位置插入人工化学合成的外源基因。该外源基因从5’端至3’端依次包括：CMV启动子、编码GM-CSF的基因(Gene ID：12981)、BGH PolyA、EF1α启动子、编码IL-2的基因(Gene ID：16183)和TK PolyA。在北京三博远志公司测序鉴定上述编码基因正确插入到单纯疱疹病毒载体中。
在C57BL/6小鼠皮下接种鼠源黑色素瘤B16F10细胞株构建动物模型。选取建模成功的小鼠，设置9组，每组5只小鼠，其中HL-1重组病毒治疗组：给予导入外源基因GM-CSF和IL-2的HL-1重组病毒；17病毒株重组病毒治疗组：给予导入外源基因GM-CSF和IL-2的17病毒株重组病毒；HSV-mock组：为不插入外源基因且敲除ICP34.5基因、ICP47基因的溶瘤病毒；阴性对照：给予PBS。各组给予病毒的总pfu均为10 6pfu。采用给药14天后的相对肿瘤抑制率作为判断标准。
相对肿瘤抑制率TGI(％)：TGI％＝(1-T/C)×100％。T/C％为相对肿瘤增殖率，即特定时间点治疗组和对照组相对肿瘤体积的百分比值。T和C分别为治疗组和对照组在特定时间点的相对肿瘤体积(RTV)。RTV＝治疗后动物瘤体积/对照组瘤体积。
实验结果：HL-1重组病毒治疗组相对肿瘤抑制率为79％；17病毒株重组病毒治疗组相对肿瘤抑制率为58％；HSV-mock组相对肿瘤抑制率为39％。
4.表达抗PD1单克隆抗体外源基因的溶瘤病毒的溶瘤作用
按实施例3的方法将本发明CCTCC保藏号为V201810(CCTCC：V201810)的病毒株和对照病毒17病毒株的ICP34.5基因和ICP47基因敲除，并在敲除ICP34.5基因的位置插入人工化学合成的外源基因，PD1单抗外源基因从5’端至3’端依次包括：CMV启动子、编码抗PD1抗体(J43,BioXCell)的基因、BGHPolyA序列。在C57BL/6J小鼠皮下接种B16F10肿瘤细胞，建立同种移植黑色素瘤模型。对照组为磷酸盐溶液，治疗组分别为PD1单抗(5mg/kg)、受试溶瘤病毒HL1-mock(10 7pfu)，HL1-PD1(10 7pfu)， HL1-PD1(10 6pfu)和HL1-PD1(10 5pfu)，每组10只小鼠；对照组和受试溶瘤病毒给药组瘤内注射给药，PD1单抗给药组腹腔注射给药，如上所述计算相对肿瘤抑制率。
研究结果：通过对小鼠在给药后第13天肿瘤平均体积分析，HL1-PD1各剂量组溶瘤病毒单独给药组相较于对照组在抗肿瘤作用上具有统计学上的显著性差异(P<0.05)；HL1-PD1高、中和低剂量组具有明显的剂量效应，肿瘤抑制率分别为86％、65％和51％，HL1-mock组和PD1单抗组的肿瘤抑制率分别为34％和42％。
5.表达IL-12和PD1单抗外源基因的溶瘤病毒的溶瘤作用
按实施例3的方法将本发明CCTCC保藏号为V201810(CCTCC：V201810)的病毒株和对照病毒17病毒株的ICP34.5基因和ICP47基因敲除，分别在ICP34.5和ICP47基因处插入IL-12基因和PD1单抗基因，测定HL1-IL12+PD1(HL-1插入IL-12基因和PD1单抗基因)、17-IL12+PD1(17株插入IL-12基因和PD1单抗基因)分别转染不同细胞的IC50。实验细胞包括A-375人皮肤癌细胞、A549人肺癌细胞、AGS人胃癌细胞、AsPC-1人胰腺癌细胞、HeLa人宫颈癌细胞、Hep G2人肝癌细胞、HT-29人大肠结直肠癌细胞、MCF7人乳腺癌细胞、PC-3人前列腺癌细胞、T24人膀胱癌细胞、U-2OS人骨癌细胞和U-87人脑胶质瘤细胞。
取处于对数生长期的细胞，消化后传代至96孔板中。24小时后弃去旧培养基，分别加入9个稀释度的不同病毒，100μl/孔，使得MOI为20、5、1.25、0.3125、0.0781、0.0195、0.0049、0.0012和0.0003，设空白对照。37℃培养3天后弃去孔内所有液体，加入10％CCK-8试剂，100μl/孔，37℃孵育1-2h，在450nm处测定样品OD值，根据拟合曲线计算IC50值。
本实施例旨在比较插入IL-12基因和PD1单抗基因后HL-1病毒株重组病毒和17病毒株重组病毒转染肿瘤细胞能力的差异。通过CCK-8法测定病毒转染肿瘤细胞的IC50值，不同病毒转染肿瘤细胞的IC50测定结果参见图12。结果显示插入IL-12基因和PD1单抗基因的HL-1重组病毒相比插入IL-12基因和PD1单抗基因的17株重组病毒转染肿瘤细胞的IC50值均要小，表明插入IL-12基因和PD1单抗基因的HL-1病毒株重组病毒在肿瘤细胞中具有更好的侵染效率和复制能力，溶瘤效果强。
实施例5 非复制性HSV(HL-1)病毒和HSV(17)病毒感染神经细胞能力的对比
本实施例旨在比较敲除不同立早基因的非复制性HL-1病毒和17株感染神经细胞的能力，以对比本发明非复制性HL-1疱疹病毒作为基因载体与现有技术中标准实验室病毒17株的效果。
分别按如下方法1和2构建非复制性病毒：
1.按照申请公布号CN105219739A的方法敲除了HL-1株和17株HSV病毒的ICP34.5和ICP4基因，并在病毒LAT区插入外源报告基因。该基因从5’端至3’端依次包括：CMV启动子、编码LacZ的基因和BGH PolyA。
2.按照申请公布号CN105219739A的方法敲除了HL-1株和17株HSV病毒的ICP34.5和ICP4基因，并在病毒LAT区插入外源报告基因，同时敲除ICP27基因(Howard M K,Kershaw T,Gibb B J,et al.High efficiency gene transfer to the central nervous system of rodents and primates using herpes virus vectors lacking functional ICP27 and ICP34.5.[J].Gene Therapy,1998,5(8):1137-1147.)。该基因从5’端至3’端依次包括：CMV启动子、编码LacZ的基因和BGH PolyA。
比较上述重组病毒在原代神经细胞上的转染和表达携带外源基因的能力。取E14大鼠皮层，切碎后胰酶消化，15分钟后加入完全培养基并用移液管吹打，将细胞接种于培养皿中并更换神经细胞培养基进行培养。按照不同MOI数量的病毒进行转染，加入病毒后放入37℃二氧化碳培养箱培养48小时后进行固定染色，取出镜下观察，拍照记录。
HL-1重组病毒和17株重组病毒转染原代神经细胞的结果见图13和图14。实验结果显示，在不同的MOI转染条件下，HL-1重组病毒在原代神经细胞培养物中显示更多的蓝斑，表明HL-1重组病毒相比17株重组病毒转染神经细胞的能力更强，携带外源基因的表达能力也更强。按照上述方法1和2构建的重组病毒失去了在神经细胞中的复制能力，但是仍然可以高水平转染神经细胞并高表达携带的外源基因，因此，作为基因治疗载体治疗神经系统疾病具有良好的应用前景。
实施例6 非复制性HSV(HL-1)重组病毒和HSV(17)重组病毒感染肝细胞能力的对比
本实施例旨在比较敲除不同立早基因的非复制性HL-1重组病毒和17株重组病毒感染肝细胞的能力，以评估本发明非复制性重组病毒作为基因治疗载体的潜力。
分别按实施例5的方法1和2构建非复制性重组病毒，不同的是报告基因更换为GFP基因。
比较上述重组病毒在CCC-HEL-1(人胚肝二倍体细胞)上的转染和表达携带外源基因的能力。将CCC-HEL-1细胞接种于六孔板中，按照不同MOI数量的病毒进行转染，加入病毒后放入37℃二氧化碳培养箱培养48小时，取出在荧光显微镜镜下观察，拍照记录。
HL-1重组病毒和17株重组病毒转染CCC-HEL-1细胞的结果见图15和图16。实验结果显示，在不同的MOI转染条件下，HL-1重组病毒在CCC-HEL-1细胞培养物中显示更多更亮的绿色荧光，表明HL-1重组病毒相比17株重组病毒转染CCC-HEL-1细胞的能力更强，携带外源基因的表达能力也更强。CCC-HEL-1是人胚肝二倍体正常细胞，按照上述方法1和2构建的重组病毒失去了在肝细胞中的复制能力，但是仍然可以感染肝细胞并表达携带的外源基因。HL-1重组病毒在肝细胞中展现的高转染和高表达其所携带的外源基因的能力表明其作为载体可以有效地将治疗基因传递入目的细胞中，实现治疗疾病的目的。
实施例7 HL-1病毒株作为基因治疗载体携带外源基因的治疗效果
1.HL1-FⅧ对FVIII基因敲除小鼠模型的促凝血效果
按照申请公布号CN105219739A的方法敲除了HL-1病毒的ICP34.5和ICP4基因，并在HL-1病毒的潜伏表达区(LAT区)插入了FⅧ基因。本实施例旨在观察HL1-FⅧ在不同时间点对FVIII基因敲除小鼠模型凝血功能的影响。
设置正常对照组、模型对照组和供试品组共3个实验组，每组实验动物5只，正常对照组为C57BL/6正常小鼠，模型对照组和HL1-FⅧ组为FVIII基因敲除小鼠模型(上海南方模式生物科技股份有限公司)。肌肉注射给药，HL1-FⅧ组给予10 7pfu/只上述制备的HL1-FⅧ非复制性HL-1重组病毒，另两组给予PBS。每3天给药1次，连续给药3次。对照组48小时后，供试品组48小时、72小时和120小时后采血检测APTT。
整个实验过程中，各组动物精神状况良好。活化部分凝血活酶时间(APTT)反映内源性凝血系统凝血活性的敏感度，不同时间段检测的APTT值参见图17。模型对照组的高APTT值表明体内FⅧ因子的缺乏导致的凝血时间增加。实验结果表明，经非复制性HL-1重组病毒HL1-FⅧ治疗后的小组相比未治疗组APTT值大幅缩短，接近正常小鼠值，表明非复制性HL-1重组病毒HL1-FⅧ在小鼠体内可以持续表达FⅧ因子，在A型血友病治疗上具有良好的临床应用前景。
2.HL1-ENK对关节炎慢性疼痛大鼠的镇痛效果
按照申请公布号CN105219738A的方法敲除了HL-1病毒的ICP34.5和ICP4基因，并在HL-1病毒的潜伏表达区(LAT区)插入了脑啡肽(ENK)基因。本实施例旨在观察HL1-ENK在关节炎慢性疼痛大鼠中的持续镇痛效果。
设置正常对照组、模型对照组和供试品组共3个实验组，模型对照组和供试品组采用完全弗氏佐剂诱导的大鼠关节炎慢性疼痛模型，每组6只。右后足垫给药，供试品组给予10 7pfu/只非复制性重组病毒HL1-ENK，另两组给予PBS对照。按照大鼠行为能力状态进行评分，评分区间为1-5分，5分为正常鼠行为能力，1分为丧失正常行动能力、行为严重受阻。分数越高活动越正常，患关节炎的慢性疼痛大鼠会由于疼痛而影响行为能力。各实验组行为活动评分见图18。
从大鼠行为能力打分的情况来看，关节炎大鼠在给药非复制性重组病毒HL1-ENK后3天与模型对照组出现显著性差异，镇痛效果明显，持续时间超过9周，峰值出现在1-4周，行为评分与正常大鼠无显著性差异。
以上详细描述了本发明的优选实施方式，但是，本发明并不限于此。在本发明的技术构思范围内，可以对本发明的技术方案进行多种简单变型，包括各个技术特征以任何其它的合适方式进行组合，这些简单变型和组合同样应当视为本发明所公开的内容，均属于本发明的保护范围。

Claims (25)

  1. 一种1型单纯疱疹病毒，其为CCTCC保藏号为：V201810(CCTCC：V201810)的病毒株、或其减毒株或其后代培养物。
  2. 一种基因改造的单纯疱疹病毒，其中该单纯疱疹病毒缺失选自如下的基因中的一种或多种：ICP34.5基因、ICP6基因、ICP0基因、ICP47基因、US3基因、UL56基因、功能性VP16基因、VHS基因、UNG基因、糖蛋白H基因和胸苷激酶基因，其中该单纯疱疹病毒来源于CCTCC保藏号为V201810的单纯疱疹病毒或其减毒株或其后代培养物。
  3. 权利要求2所述的基因改造的单纯疱疹病毒，其中所述修饰为所述单纯疱疹病毒缺失ICP34.5基因和/或ICP47基因。
  4. 权利要求2或3所述的基因改造的单纯疱疹病毒，其中所述修饰还包括向所述病毒中导入一种或多种外源基因。
  5. 权利要求4所述的基因改造的单纯疱疹病毒，其中所述一种或多种外源基因插入选自如下的一处或多处基因缺失位点：ICP34.5基因、ICP6基因、ICP0基因、ICP47基因、US3基因、UL56基因、VP16基因、VHS基因、UNG基因、糖蛋白H基因、胸苷激酶基因。
  6. 权利要求2-5任一项的基因改造的单纯疱疹病毒，其中所述外源基因选自如下的一种或多种：编码促进免疫应答的细胞因子的基因、编码肿瘤抗原的基因、编码具有预防和/或治疗肿瘤作用的单抗基因、编码前药转化酶的基因、肿瘤抑制基因、反义RNA或小RNA。
  7. 权利要求6的基因改造的单纯疱疹病毒，其中所述细胞因子选自如下的一种或多种：GM-CSF、G-CSF、M-CSF、IL-1、IL-2、IL-3、IL-4、IL-5、IL-6、IL7、IL-8、IL-10、IL-12、IL-13、IL-15、IL-18、IL-21、IL-23、IFN-α、IFN-γ、TGF-β和TNF-α。
  8. 权利要求6的基因改造的单纯疱疹病毒，其中具有预防和/或治疗肿瘤作用的单抗选自如下的一种或多种：PD1单抗、PD-L1单抗、PD-L2单抗、CTLA-4单抗、CD80单抗、CD28单抗、CD137单抗、CD137L单抗、OX40单抗、OX40L单抗、CD27单抗、CD70单抗、CD40单抗、CD40L单抗、LAG-3单抗和TIM-3单抗。
  9. 权利要求6的基因改造的单纯疱疹病毒，其中肿瘤抗原为肿瘤特异性抗原。
  10. 权利要求9的基因改造的单纯疱疹病毒，其中肿瘤特异性抗原选自如下的一种或多种：PSA、MUC1、MAGE-1、MAGE-2、MAGE-3、MAGE-12、BAGE、GAGE和LAGE。
  11. 权利要求4-10任一项的基因改造的单纯疱疹病毒，其中所述外源基因选自编码刺激免疫应答的细胞因子多肽和促进免疫应答的抗体多肽。
  12. 权利要求11的基因改造的单纯疱疹病毒，其中所述外源基因选自如下的一种或多种：粒细胞巨噬细胞集落刺激因子(GMCSF)、IL-2，IL12，IFN-γ、TNFa和免疫检查点抗体多肽。
  13. 权利要求2-5任一项的基因改造的单纯疱疹病毒，其中一种或多种立即早期基因被敲除或破坏从而丧失复制能力。
  14. 权利要求13所述的基因改造的单纯疱疹病毒，所述一种或多种立即早期基因是选自ICP0、ICP4、ICP22和ICP27编码基因中的一个、两个、三个或四个。
  15. 权利要求13或14的基因改造的单纯疱疹病毒，所述外源基因选自如下的一种或多种：神经生长因子、神经递质和神经营养因子基因。
  16. 权利要求13或14的基因改造的单纯疱疹病毒，所述外源基因为凝血因子基因。
  17. 一种用作载体的非复制性单纯疱疹病毒，其中选自如下的一种、二种或三种基因被敲除或破坏：ICP4、ICP27和ICP34.5，其中该单纯疱疹病毒来源于CCTCC保藏号为V201810的单纯疱疹病毒或其减毒株或其后代。
  18. 权利要求17的单纯疱疹病毒，其中还导入了一种或多种外源基因。
  19. 权利要求18的单纯疱疹病毒，其中所述外源基因选自如下的一种或多种：神经生长因子、神经递质、神经营养因子和凝血因子基因。
  20. 包含项1-19任一项的单纯疱疹病毒的组合物、病毒培养物、宿主细胞或宿主细胞培养物。
  21. 一种治疗癌症的方法，包括将权利要求1-12任一项的单纯疱疹病毒导入患有癌症的受试者体内。
  22. 一种治疗神经系统疾病、血液系统疾病或免疫系统疾病的方法，包括将权利要求15-19任一项的单纯疱疹病毒导入受试者体内。
  23. 权利要求22的方法，其中所述血液系统疾病为凝血障碍疾病。
  24. 权利要求22的方法，其中所述神经系统疾病为慢性疼痛、神经损伤、中风、瘫痪或神经退行性疾病。
  25. 权利要求24的方法，其中所述神经退行性疾病为帕金森氏病、阿兹海默症、肌萎缩侧索硬化症或亨廷顿病。
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