A recombinant herpes simplex virus and uses thereof

Abstract

The present application relates to the field of virology and tumor therapy. In particular, the present application provides a recombinant Herpes Simplex Virus (HSV) which is capable of specifically replicating at high levels in tumor cells and killing tumor cells, but replicating at low levels in normal cells, such that the recombinant Herpes Simplex Virus of the present application not only has a high killing power on tumor cells, but also has significantly reduced side effects (in particular neurotoxicity). Further, the present application relates to a viral vector constructed based on the recombinant Herpes Simplex Virus, a pharmaceutical composition comprising the recombinant Herpes Simplex Virus or the viral vector, and the use of the recombinant Herpes Simplex Virus or the viral vector. The recombinant Herpes Simplex Virus of the present application can be used to infect and kill tumor cells, and can be used to deliver a gene drug into tumor cells for gene therapy.

Description

[0001] This divisional application is based on the original Chinese patent application No. CN201810165563.4, filed on February 28, 2018, entitled "A Recombinant Herpes Simplex Virus and Uses Thereof". TECHNICAL FIELD

[0002] The present application relates to the field of virology and tumor therapy. In particular, the present application provides a recombinant Herpes Simplex Virus (HSV) which is able to specifically replicate at high levels in tumor cells and effectively kill tumor cells, but replicate at low levels in normal cells, such that the recombinant Herpes Simplex Virus of the present application not only has a high killing power on tumor cells, but also has significantly reduced side effects (in particular neurotoxicity). Further, the present application relates to a viral vector constructed based on the recombinant Herpes Simplex Virus, a pharmaceutical composition comprising the recombinant Herpes Simplex Virus or the viral vector, and uses of the recombinant Herpes Simplex Virus or the viral vector. The recombinant Herpes Simplex Virus of the present application can be used to infect and kill tumor cells, and can be used to deliver a gene drug into tumor cells for gene therapy. BACKGROUND

[0003] Radiotherapy, chemotherapy and targeted drugs are currently widely used in tumor treatment, but they all have problems such as incomplete treatment, large side effects, easy to produce drug resistance, unable to control tumor recurrence and metastasis, and so on, and the effect of tumor treatment is not ideal. Therefore, it is urgent to develop new and efficient tumor treatment methods. In recent years, people's understanding of the relationship between tumor and immunity has greatly improved, and tumor immunotherapy has developed rapidly. Among them, the use of oncolytic virus (OV) therapy as a new type of tumor immunotherapy method has attracted much attention (Lichty B D, Breitbach C J, Stojdl D F, et al. Going viral with cancer immunotherapy [J]. Nat Rev Cancer, 2014, 14(8): 559-567). Oncolytic virus is a kind of virus with tumor tropism, which can selectively replicate in tumor cells, while the proliferation in normal cells is limited. Oncolytic virus can replicate in tumor cells, leading to tumor cell lysis and death, and the amplified virus can continue to infect surrounding tumor cells, producing a cascade effect. In addition, oncolytic virus can release tumor antigens in the process of lysing tumor cells, stimulate the body to produce specific anti-tumor immunity against tumor antigens, and further expand the oncolytic effect of oncolytic virus. Oncolytic virus is the most popular new gene therapy drug in the field of malignant tumor treatment. Especially in the local control treatment of solid tumors, the infection and proliferation of oncolytic virus will not only lead to the ablation of the injection site tumor, but also the lysis of tumor cells will lead to the release of tumor antigens from tumor cells, thereby inducing the body to produce a systemic anti-tumor immune response to resist other parts of the tumor, which may be the key immune response of the body to systemically control tumor spread and metastasis (Russell S J, Peng K W, Bell J C. Oncolytic virotherapy [J]. Nat Biotechnol, 2012, 30(7): 658-670).

[0004] The current oncolytic virus can be divided into more than ten types according to the virus species. Among them, the oncolytic type I herpes simplex virus (HSV-1) has the advantages of large gene capacity, short replication cycle, high infection efficiency, and can insert multiple therapeutic genes, and has become the preferred gene engineering tumor treatment drug at home and abroad.

[0005] HSV-1 virus belongs to the Herpesviridae family, is a kind of DNA virus with envelope, and can cause herpes of the lips, eyes and facial skin in human. According to the epidemic research statistics, more than 60% of the population has been infected with HSV-1 virus. The genome of HSV-1 virus is composed of 152 kb double-stranded linear DNA, including two fragments connected with each other: long fragment and short fragment (Macdonald S J, Mostafa H H, Morrison L A, et al. Genome sequence of herpes simplex virus 1 strain KOS [J]. J Virol, 2012, 86(11): 6371-6372). The long fragment (L region) accounts for about 82% of the genome, while the short fragment (S region) accounts for about 18% of the genome, and the long fragment and the short fragment are connected together through the junction region. The L region contains a pair of inverted repeat fragments, and the segment between them is called unique segment U L ; the S region also has a pair of inverted repeat fragments, and the segment between them is called unique segment U s . At present, the whole genome sequencing of HSV-1 KOS strain has been completed. The genome of KOS virus strain contains a total of 152011 nucleotide bases, and contains a total of 72 genes encoding proteins, in which unique segment U L contains 56 genes, unique segment U s contains 12 genes, and, U L terminal inverted repeat segment (TR L ) and U L intermediate inverted repeat sequence (IR L ) each contains 3 same genes (ICP34.5, ICP0 and LAT, respectively), U S terminal inverted repeat segment (TR S ) and U S intermediate inverted repeat sequence (IR S ) each contains 1 same gene (ICP4).

[0006] HSV-1 synthesizes proteins in a cascade-regulated manner at the transcriptional level when HSV-1 replicates in large quantities. These proteins can be divided into three categories, α, β and γ, according to the order of their synthesis. HSV-1 virus first transcribes the α class genes, which encode five immediate-early proteins (IE proteins), including ICP0, ICP4, ICP22, ICP27 and ICP47, and then activates the β and γ class genes at the transcriptional level to promote the expression of viral early (E) and late (L) proteins. The immediate-early protein ICP0 can independently activate all classes of viral genes (IE, E and L) and various cellular genes (in some cases, the synergistic activation of ICP4 may be required). ICP0 not only interacts with various transcription factors or regulatory proteins in the cell to activate the transcription of certain genes in the host cell, but also regulates the expression of the viral genome and the transcription of viral genes through its ubiquitin ligase E3 domain (Kawaguchi Y, Bruni R, Roizman B. Interaction of herpes simplex virus 1 alpha regulatory protein ICP0 with elongation factor 1 delta: ICP0 affects translational machinery. J Virol, 1997, 71(2): 1019-1024). ICP4 and ICP27 are immediate-early proteins essential for viral replication (DeLuca N A, McCarthy A M, Schaffer PA. Isolation and characterization of deletion mutants of herpes simplex virus type 1 in the gene encoding immediate-early regulatory protein ICP4. J Virol, 1985, 56(2): 558-570; Sacks W R, Greene C C, Aschman D P, et al. Herpes simplex virus type 1 ICP27 is an essential regulatory protein. J Virol, 1985, 55(3): 796-805). ICP27 is a multifunctional regulatory protein that promotes the transcription of viral genes by interacting with RNA polymerase II. ICP27 can interact with ICP4 to coactivate the expression of early and late genes.ICP27 can also indirectly promote viral DNA replication by up-regulating viral replication-related genes. In addition, ICP27 also has the functions of inhibiting cell primary RNA splicing, promoting the transport and translation of viral mRNA, etc. ICP34.5 can reverse the effect of the antiviral protein PKR, so that the synthesis of host and viral proteins continues, thereby facilitating viral replication. In addition, ICP34.5 can also escape the host's antiviral response by regulating PP1 phosphatase activity (Randall G, Roizman B. Transcription of the derepressed open reading frame P of herpes simplex virus 1 precludes the expression of the antisense gamma(1)34.5 gene and may account for the attenuation of the mutant virus [J]. J Virol, 1997, 71(10): 7750-7757).

[0007] To date, a number of oncolytic HSV-1 viral vectors have been in preclinical or clinical studies worldwide, including HSV1716, which is obtained by knocking out the ICP34.5 gene in the R3616 mutant strain (derived from HSV-1 F strain) (MacKie RM, Stewart B, Brown S M. Intralesional injection of herpes simplex virus 1716 in metastatic melanoma [J]. Lancet, 2001, 357(9255): 525-526; and Papanastassiou V, Rampling R, Fraser M, et al. The potential for efficacy of the modified (ICP 34.5(-)) herpes simplex virus HSV1716 following intratumoural injection into human malignant glioma: a proof of principle study [J]. Gene Ther, 2002, 9(6): 398-406); G207, which is obtained by double knock-out of ICP34.5 / ICP6 genes in the R3616 mutant strain (Markert J M, Medlock M D, Rabkin S D, et al. Conditionally replicating herpes simplex virus mutant, G207 for the treatment of malignant glioma: results of a phase I trial [J]. Gene Ther, 2000, 7(10): 867-874; and Markert J M, Razdan S N, Kuo H C, et al. A phase 1 trial of oncolytic HSV-1, G207, given in combination with radiation for recurrent GBM demonstrates safety and radiographic responses [J]. Mol Ther, 2014, 22(5): 1048-1055); NV1020, which is obtained by deleting a single copy of ICP34.5 / ICP0 / ICP4 / UL56 genes (Gutermann A, Mayer E, von Dehn-Rothfelser K, et al. Efficacy of oncolytic herpesvirus NV1020 can be enhanced by combination with chemotherapeutics in colon carcinoma cells [J]. Hum Gene Ther, 2006, 17(12): 1241-1253; and Geevarghese S K, Geller D A, de Haan H A, et al. Phase I / II study of oncolytic herpes simplex virus NV1020 in patients with extensively pretreated refractory colorectal cancer metastatic to the liver [J]. Hum Gene Ther, 2010, 21(9): 1119-1128); and T-VEC, which is obtained by double knock-out of ICP34.5 / ICP47 genes in clinical HSV-1 isolate JS1 (Liu B L, Robinson M, Han Z Q, et al. ICP34.5 deleted herpes simplex virus with enhanced oncolytic, immune stimulating, and anti-tumour properties [J]. Gene Ther, 2003, 10(4): 292-303). The recombinant HSV-1 virus T-VEC of the American Amgen company made a breakthrough in the treatment of patients with advanced melanoma in the phase III clinical trial, becoming the first FDA-approved oncolytic virus treatment drug. However, the data show that this clinical study has only reached the primary endpoint of the durable response rate (DRR), but has not reached the secondary endpoint of improving overall survival (OS), although the T-VEC treatment group showed a strong favorable trend (Andtbacka R H, Kaufman H L, Collichio F, et al. Talimogene Laherparepvec Improves Durable Response Rate in Patients With Advanced Melanoma [J].J Clin Oncol, 2015, 33(25): 2780-2788). This is mainly because T-VEC has strong toxic side effects, and its first intratumoral treatment dose is only 10. 6 PFU virus, which greatly reduces the effect of tumor treatment and causes patients to miss the best opportunity for oncolytic therapy.

[0008] Although type I herpes simplex virus oncolytic therapy has made some progress in recent years, analysis of recombinant HSV-1 viruses that have entered clinical trials for tumor treatment shows that different oncolytic viruses have different genetic modifications, different oncolytic effects and safety, and thus their indications for treating tumors and their effects on treating tumors are not the same (Eager R M, Nemunaitis J. Clinical development directions in oncolytic viral therapy [J]. Cancer Gene Ther, 2011, 18(5): 305-317). In general, existing oncolytic viruses all have the defects of large toxic side effects, poor safety, and significantly limited therapeutic dose of oncolytic viruses, which poses a serious challenge to tumor treatment research (Liu T C, Galanis E, Kirn D. Clinical trial results with oncolytic virotherapy: a century of promise, a decade of progress [J]. Nat Clin Pract Oncol, 2007, 4(2): 101-117). The therapeutic effect of oncolytic viruses is positively correlated with the dose of virus administration. If the specificity and safety of oncolytic viruses are not high, the dose of oncolytic viruses must be reduced to avoid serious side effects to the body. This seriously affects the clinical treatment effect of oncolytic viruses and causes certain safety hazards. Take T-VEC of the American Amgen Company as an example. Drug toxicity / side effects are important factors limiting the clinical effect of T-VEC. Although scientists have been trying various methods, so far, no oncolytic virus that can both replicate at a high level in tumor cells and kill tumor cells without causing serious side effects to normal cells has been found. Therefore, it is still necessary to develop new oncolytic viruses to achieve low toxicity and high effectiveness of oncolytic virus therapy.

[0009] In order to overcome the above-mentioned defects of the recombinant herpes simplex virus in tumor gene therapy in the prior art, the inventors of the present application constructed a brand new recombinant herpes simplex virus which not only can replicate in tumor cells at a high level and has a high killing effect on tumor cells, but also has significantly reduced side effects (especially neurotoxicity), so that the recombinant herpes simplex virus of the present application not only maintains the high efficacy of oncolytic viruses, but also greatly improves the safety of oncolytic viruses. SUMMARY

[0010] In the present application, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by a person skilled in the art. And, the cell culture, molecular genetics, nucleic acid chemistry, immunology laboratory operation steps used herein are the conventional steps widely used in the corresponding fields. At the same time, in order to better understand the present application, the definitions and explanations of the related terms are provided as follows.

[0011] As used herein, the term "recombinant HSV virus" refers to an HSV virus which, compared with a wild-type HSV virus, comprises artificially introduced mutations. It should be understood that the recombinant HSV virus of the present application is not limited by its production method. For example, the recombinant HSV virus of the present application can be produced by homologous recombination, and can also be prepared by culturing a host cell infected with the recombinant HSV virus.

[0012] As used herein, the term "viral vector" refers to a nucleic acid carrier tool which is constructed based on a viral genome and can carry an exogenous nucleotide sequence. Generally, a viral vector can self-replicate and / or express the genes (endogenous and exogenous) it contains in a suitable host cell. The viral vector can comprise a complete wild-type viral genome, or a mutated or modified viral genome. However, for safety considerations, the viral vector generally preferably comprises a mutated or modified viral genome. Since the viral vector of the present application is derived from the viral genome of HSV, the viral vector of the present application can also be referred to as an HSV viral vector.

[0013] As used herein, the expression "not expressing a functional protein of interest" means that, after a virus or viral vector or viral genome infects a cell, the virus or viral vector or viral genome cannot produce or express a protein of interest having a biological functional activity. For example, the virus or viral vector or viral genome can not produce or express the protein of interest at all due to a gene deletion, or produce or express a protein of interest which does not have a biological functional activity due to a loss-of-function mutation.

[0014] As used herein, the term "loss-of-function mutation" refers to a mutation that results in a protein encoded and expressed by the mutated gene that loses its biologically functional activity. Loss-of-function mutations include, but are not limited to, missense mutations, nonsense mutations, frameshift mutations, base deletions, base substitutions, base additions, and any combination thereof (e.g., deletion or substitution or addition of a segment of a gene), as long as the gene comprising the loss-of-function mutation is unable to produce or express a protein having biologically functional activity.

[0015] As used herein, the term "essential gene" refers to a gene that is indispensable for the survival and replication of the HSV virus. Particular examples of such essential genes include, but are not limited to, the ICP27 gene (see, e.g., GenBank No. AFE62883.1), the ICP4 gene (see, e.g., GenBank No. AFE62888.1), the VP5 gene (see, e.g., GenBank No. AFE62846.1), the gL gene (see, e.g., GenBank No. AFE62828.1), the gH gene (see, e.g., GenBank No. AFE62849.1), the gD gene (see, e.g., GenBank No. AFE62894.1), the gK gene (see, e.g., GenBank No. AFE62882.1), the gB gene (see, e.g., GenBank No. AFE62855.1), the gN gene (see, e.g., GenBank No. AFE62878.1), the UL5 gene (see, e.g., GenBank No. AFE62832.1), the UL6 gene (see, e.g., GenBank No. AFE62833.1), the UL8 gene (see, e.g., GenBank No. AFE62835.1), the UL9 gene (see, e.g., GenBank No. AFE62836.1), the UL12 gene (see, e.g., GenBank No. AFE62839.1), the UL25 gene (see, e.g., GenBank No. AFE62852.1), the UL26 gene (see, e.g., GenBank No. AFE62853.1), the UL28 gene (see, e.g., GenBank No. AFE62856.1), the UL29 gene (see, e.g., GenBank No. AFE62857.1), the UL30 gene (see, e.g., GenBank No. AFE62858.1), the UL33 gene (see, e.g., GenBank No. AFE62861.1), the UL36 gene (see, e.g., GenBank No. AFE62864.1), the UL38 gene (see, e.g., GenBank No. AFE62866.1), the UL42 gene (see, e.g., GenBank No. AFE62870.1), the UL48 gene (see, e.g., GenBank No. AFE62876.1), the UL52 gene (see, e.g., GenBank No. AFE62881.1).Detailed descriptions of essential genes of HSV viruses can also be found, for example, in Roizman B, Knipe DM. Herpes simplex viruses and their replication. In: Knipe DM, Howley PM, editors. Fields Virology. 2. nd ed. Vol 2. Philadelphia, PA: Lippincot, Williams and Wilkins, 2001: 2399-2460; Subak- Sharpe JH, Dargan DJ. HSV molecular biology: general aspects of herpes simplex virus molecular biology. Virus Genes, 1998, 16(3): 239-251.

[0016] As used herein, the term "non-essential gene" refers to a gene that is not essential for the survival and replication of HSV virus. Generally, such genes in the HSV viral genome can be knocked out (deleted) or mutated without affecting the survival and replication ability of the HSV virus. Specific examples of such essential genes include, but are not limited to, the UL3 gene (see, e.g., GenBank No. AFE62830.1), the UL4 gene (see, e.g., GenBank No. AFE62831.1), the UL14 gene (see, e.g., GenBank No. AFE62841.1), the UL16 gene (see, e.g., GenBank No. AFE62843.1), the UL21 gene (see, e.g., GenBank No. AFE62848.1), the UL24 gene (see, e.g., GenBank No. AFE62851.1), the UL31 gene (see, e.g., GenBank No. AFE62859.1), the UL32 gene (see, e.g., GenBank No. AFE62860.1), the US3 gene (see, e.g., GenBank No. AFE62891.1), the UL51 gene (see, e.g., GenBank No. AFE62880.1), the UL55 gene (see, e.g., GenBank No. AFE62884.1), the UL56 gene (see, e.g., GenBank No. AFE62885.1), the US2 gene (see, e.g., GenBank No. AFE62890.1), the US12 gene (see, e.g., GenBank No. AFE62901.1; i.e., the ICP47 gene), and the LAT gene (see, e.g., GenBank No. JQ673480.1). Detailed descriptions of non-essential genes of HSV virus can also be found in, e.g., Roizman B, Knipe DM. Herpes simplex viruses and their replication. In: Knipe DM, Howley PM, editors. Fields Virology. 2 nd ed. Vol 2. Philadelphia, PA: Lippincot, Williams and Wilkins, 2001: 2399-2460; Subak-Sharpe JH, Dargan DJ. HSV molecular biology: general aspects of herpes simplex virus molecular biology. Virus Genes, 1998, 16(3): 239-251.

[0017] As used herein, the term "ICP0 protein" refers to the infected cell protein 0 of an HSV virus, which is encoded by the RL2 gene and is one of the immediate early gene products of an HSV virus. The amino acid sequence of the ICP0 protein is known and can be found, for example, in the public database NCBI (AFE62827.1).

[0018] As used herein, the term "ICP34.5 protein" refers to the infected cell protein 34.5 of an HSV virus, which is encoded by the RL1 gene and is one of the immediate early gene products of an HSV virus. The amino acid sequence of the ICP34.5 protein is known and can be found, for example, in the public database NCBI (AFE62826.1).

[0019] As used herein, the term "ICP27 protein" refers to the infected cell protein 27 of an HSV virus, which is encoded by the UL54 gene. The amino acid sequence of the ICP27 protein is known and can be found, for example, in the public database NCBI (AFE62883.1).

[0020] As used herein, the term "ICP4 protein" refers to the infected cell protein 4 of an HSV virus, which is encoded by the RS1 gene. The amino acid sequence of the ICP4 protein is known and can be found, for example, in the public database NCBI (AFE62888.1).

[0021] As used herein, the term "VP5 protein" refers to the major capsid protein of an HSV virus, which is encoded by the UL19 gene. The amino acid sequence of the VP5 protein is known and can be found, for example, in the public database NCBI (AFE62846.1).

[0022] As used herein, the term "ICP0 gene" refers to the nucleotide sequence in the HSV virus genome that encodes the ICP0 protein. As used herein, the term "ICP34.5 gene" refers to the nucleotide sequence in the HSV virus genome that encodes the ICP34.5 protein. As used herein, the term "ICP27 gene" refers to the nucleotide sequence in the HSV virus genome that encodes the ICP27 protein. As used herein, the term "ICP4 gene" refers to the nucleotide sequence in the HSV virus genome that encodes the ICP4 protein. As used herein, the term "VP5 gene" refers to the nucleotide sequence in the HSV virus genome that encodes the VP5 protein.

[0023] As used herein, the term "exogenous nucleotide sequence" refers to an artificially introduced nucleotide sequence that is foreign with respect to the original sequence. The exogenous nucleotide sequence includes, but is not limited to, any gene that is not found in the viral genome. However, in some cases, preferably, the exogenous nucleotide sequence encodes a polypeptide having therapeutic use, such as an immunomodulatory polypeptide, a cytokine, a chemokine, an antibody, and a cytotoxic peptide.

[0024] As used herein, the term "immunomodulatory polypeptide" refers to a polypeptide that can modulate the function of immune cells, examples of which include, but are not limited to, CD40L, OX40L, inducible costimulatory molecule (ICOS), FTL3L, LIGHT, CD137L, CD70, 4-1BB, GITR, and CD28 (see, e.g., Khalil D N, Smith E L, Brentjens R J, et al. The future of cancer treatment: immunomodulation, CARs and combination immunotherapy [J]. Nat Rev Clin Oncol, 2016, 13(5): 273-290).

[0025] As used herein, the term "cytokine" has the meaning well known to those skilled in the art. However, in the methods of the present application, when the recombinant virus of the present application is used to treat a tumor, particularly preferably, the cytokine is a cytokine that can be used for tumor treatment. Examples of "cytokine" include, but are not limited to, interleukins (e.g., IL-2, IL-12, and IL-15), interferons (e.g., IFNα, IFNβ, IFNγ), tumor necrosis factors (e.g., TNFα), colony stimulating factors (e.g., GM-CSF), and any combination thereof (see, e.g., Ardolino M, Hsu J, Raulet D H. Cytokine treatment in cancer immunotherapy [J]. Oncotarget, 2015, 6(23): 19346-19347).

[0026] As used herein, the term "chemokine" has the meaning well known to those skilled in the art. However, in the methods of the present application, when using the recombinant viruses of the present application to treat tumors, it is particularly preferred that the cytokine is a chemokine that can be used for tumor therapy. Examples of "chemokine" include, but are not limited to, CCL2, RANTES, CCL7, CCL9, CCL10, CCL12, CCL15, CCL19, CCL21, CCL20, XCL-1, and any combination thereof (Homey B, Muller A, Zlotnik A. CHEMOKINES: AGENTS FOR THE IMMUNOTHERAPY OF CANCER? [J]. Nat Rev Immunol, 2002, 2: 175-184).

[0027] As used herein, the term "cytotoxic peptide" refers to a polypeptide that is toxic to cells or can induce apoptosis of cells, examples of which include, but are not limited to, thymidine kinase TK (TK / GCV), TRAIL, and FasL (see, e.g., Candolfi M, King G D, Muhammad A G, et al. Evaluation of proapototic transgenes to use in combination with Flt3L in an immune-stimulatory gene therapy approach for Glioblastoma multiforme (GBM) [J]. FASEB J, 2008, 22: 1077.13).

[0028] As used herein, the term "antibody" has the meaning well known to those skilled in the art. However, in the methods of the present application, when the recombinant virus of the present application is used to treat a tumor, it is particularly preferred that the antibody is one that can be used for tumor treatment. Examples of "antibodies" include, but are not limited to, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-TIGIT antibodies, anti-BTLA antibodies, anti-CTLA-4 antibodies, anti-Tim-3 antibodies, anti-Lag-3 antibodies, anti-CD137 antibodies, anti-OX40 antibodies, anti-GITR antibodies, anti-CD73 antibodies, anti-KIR antibodies, anti-ICOS antibodies, anti-CSF1R antibodies, anti-EGFR antibodies, anti-VEGFR antibodies, anti-HER2 antibodies, and anti-PDGFR antibodies (see, e.g., Khalil D N, Smith E L, Brentjens R J, et al. The future of cancer treatment: immunomodulation, CARs and combination immunotherapy [J]. Nat Rev Clin Oncol, 2016, 13(5): 273-290; and Hughes P E, Caenepeel S, Wu L C. Targeted Therapy and Checkpoint Immunotherapy Combinations for the Treatment of Cancer [J]. Trends Immunol, 2016, 37(7): 462-476).

[0029] As used herein, the term "pharmaceutically acceptable carriers and / or excipients" refers to carriers and / or excipients that are compatible with the subject and the active ingredients in a pharmacological and / or physiological sense, which are well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and include, but are not limited to, pH adjusting agents, surfactants, adjuvants, ionic strength enhancers. For example, pH adjusting agents include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic or non-ionic surfactants, such as Tween-80; adjuvants include, but are not limited to, aluminum adjuvants (e.g., aluminum hydroxide), Freund's adjuvants (e.g., complete Freund's adjuvant); ionic strength enhancers include, but are not limited to, sodium chloride.

[0030] As used herein, the term "effective amount" means an amount that is sufficient to achieve or at least partially achieve a desired effect. For example, a prophylactically effective amount refers to an amount that is sufficient to prevent, deter, or delay the onset of a disease; a therapeutically effective amount refers to an amount that is sufficient to cure or at least partially arrest the disease and its complications in an already afflicted patient. Determining such an effective amount is well within the capability of those skilled in the art. For example, an amount effective for therapeutic purposes will depend on the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient, e.g., the age, weight, and sex of the patient, the mode of administration of the drug, and other therapies that the patient may be undergoing, etc.

[0031] To overcome the safety and side effect problems of the existing recombinant HSV viruses for tumor treatment, the inventors of the present application constructed a new recombinant HSV virus which does not express functional ICP0 and ICP34.5 proteins (e.g., double copies of ICP0 and ICP34.5 genes are deleted). The recombinant HSV virus of the present application has a high level of replication ability in tumor cells and can effectively kill a variety of tumor cells, but has significantly reduced replication ability and killing ability in normal cells. In addition, it has also been found that the recombinant HSV virus of the present application has significantly reduced neurotoxicity in animals and can be administered to animals at a significantly increased dose. Therefore, compared with the existing recombinant HSV viruses, the recombinant HSV virus of the present application not only maintains high oncolytic ability, but also has significantly improved safety, can be administered at a higher dose, and has a broad application prospect.

[0032] Recombinant HSV virus

[0033] Therefore, in one aspect, the present application provides a recombinant HSV virus which does not express functional ICP0 protein and ICP34.5 protein.

[0034] As well known to those skilled in the art, the functional expression of a protein of interest (e.g., ICP0 protein and / or ICP34.5 protein) can be prevented by modifying the gene encoding the protein of interest. For example, a loss-of-function mutation can be introduced into the gene encoding the protein of interest (e.g., ICP0 protein and / or ICP34.5 protein), or the gene encoding the protein of interest (e.g., ICP0 protein and / or ICP34.5 protein) is deleted or replaced with an exogenous nucleotide sequence (e.g., a nucleotide sequence encoding an exogenous protein), thereby preventing the functional expression of the protein of interest.

[0035] As known to those skilled in the art, the genome of HSV virus comprises 2 copies of the ICP0 gene and 2 copies of the ICP34.5 gene. Therefore, in order to block the functional expression of the ICP0 protein and the ICP34.5 protein in the recombinant HSV virus, it is necessary to modify both the 2 copies of the ICP0 gene and the 2 copies of the ICP34.5 gene. However, it is readily understood that the modifications of the 2 copies of the ICP0 gene and the 2 copies of the ICP34.5 gene (4 nucleotide segments) are independent of each other and can be the same or different.

[0036] Therefore, in certain preferred embodiments, the recombinant HSV virus has a genome wherein,

[0037] 2 copies of the ICP0 gene each independently comprise a loss-of-function mutation (e.g., addition, deletion, and / or substitution of one or more bases) or are deleted or replaced with an exogenous nucleotide sequence (e.g., a nucleotide sequence encoding an exogenous protein); and,

[0038] 2 copies of the ICP34.5 gene each independently comprise a loss-of-function mutation (e.g., addition, deletion, and / or substitution of one or more bases) or are deleted or replaced with an exogenous nucleotide sequence (e.g., a nucleotide sequence encoding an exogenous protein).

[0039] In certain preferred embodiments, the genome of the recombinant HSV virus comprises the following modifications:

[0040] 2 copies of the ICP0 gene each independently comprise a loss-of-function mutation (e.g., addition, deletion, and / or substitution of one or more bases) or are deleted or replaced with an exogenous nucleotide sequence (e.g., a nucleotide sequence encoding an exogenous protein); and,

[0041] 2 copies of the ICP34.5 gene each independently comprise a loss-offunction mutation (e.g., addition, deletion, and / or substitution of one or more bases) or are deleted or replaced with an exogenous nucleotide sequence (e.g., a nucleotide sequence encoding an exogenous protein).

[0042] In certain preferred embodiments, one copy of the ICP0 gene comprises a loss-of-function mutation (e.g., addition, deletion, and / or substitution of one or more bases), and the other copy of the ICP0 gene comprises a loss-of-function mutation (e.g., addition, deletion, and / or substitution of one or more bases) or is deleted or replaced with an exogenous nucleotide sequence (e.g., a nucleotide sequence encoding an exogenous protein). In certain preferred embodiments, one copy of the ICP0 gene is deleted, and the other copy of the ICP0 gene comprises a loss-of-function mutation (e.g., addition, deletion, and / or substitution of one or more bases) or is deleted or replaced with an exogenous nucleotide sequence (e.g., a nucleotide sequence encoding an exogenous protein). In certain preferred embodiments, one copy of the ICP0 gene is replaced with an exogenous nucleotide sequence (e.g., a nucleotide sequence encoding an exogenous protein), and the other copy of the ICP0 gene comprises a loss-of-function mutation (e.g., addition, deletion, and / or substitution of one or more bases) or is deleted or replaced with an exogenous nucleotide sequence (e.g., a nucleotide sequence encoding an exogenous protein).

[0043] In certain preferred embodiments, each of the 2 copies of the ICP0 gene independently comprises a loss-of-function mutation (e.g., addition, deletion, and / or substitution of one or more bases). In certain preferred embodiments, the 2 copies of the ICP0 gene comprise the same loss-of-function mutation. In certain preferred embodiments, the 2 copies of the ICP0 gene comprise different loss-of-function mutations. For example, in certain preferred embodiments, a first copy of the ICP0 gene comprises a first loss-of-function mutation, and a second copy of the ICP0 gene comprises a second loss-of-function mutation. The first loss-of-function mutation and the second loss-of-function mutation can be the same or different.

[0044] In certain preferred embodiments, the 2 copies of the ICP0 gene are deleted.

[0045] In certain preferred embodiments, each of the 2 copies of the ICP0 gene is independently replaced with an exogenous nucleotide sequence (e.g., a nucleotide sequence encoding an exogenous protein). In certain preferred embodiments, the 2 copies of the ICP0 gene are replaced with the same exogenous nucleotide sequence (e.g., a nucleotide sequence encoding an exogenous protein). In certain preferred embodiments, the 2 copies of the ICP0 gene are replaced with different exogenous nucleotide sequences (e.g., nucleotide sequences encoding exogenous proteins). For example, in certain preferred embodiments, a first copy of the ICP0 gene is replaced with a first exogenous nucleotide sequence, and a second copy of the ICP0 gene is replaced with a second exogenous nucleotide sequence. The first exogenous nucleotide sequence and the second exogenous nucleotide sequence can be the same or different.

[0046] In certain preferred embodiments, one copy of the ICP34.5 gene comprises a loss-of-function mutation (e.g., addition, deletion, and / or substitution of one or more bases) and the other copy of the ICP34.5 gene comprises a loss-of-function mutation (e.g., addition, deletion, and / or substitution of one or more bases) or is deleted or replaced with an exogenous nucleotide sequence (e.g., a nucleotide sequence encoding an exogenous protein). In certain preferred embodiments, one copy of the ICP34.5 gene is deleted and the other copy of the ICP34.5 gene comprises a loss-of-function mutation (e.g., addition, deletion, and / or substitution of one or more bases) or is deleted or replaced with an exogenous nucleotide sequence (e.g., a nucleotide sequence encoding an exogenous protein). In certain preferred embodiments, one copy of the ICP34.5 gene is replaced with an exogenous nucleotide sequence (e.g., a nucleotide sequence encoding an exogenous protein) and the other copy of the ICP34.5 gene comprises a loss-of-function mutation (e.g., addition, deletion, and / or substitution of one or more bases) or is deleted or replaced with an exogenous nucleotide sequence (e.g., a nucleotide sequence encoding an exogenous protein).

[0047] In certain preferred embodiments, each of the 2 copies of the ICP34.5 gene independently comprises a loss-of-function mutation (e.g., addition, deletion, and / or substitution of one or more bases). In certain preferred embodiments, the 2 copies of the ICP34.5 gene comprise the same loss-of-function mutation. In certain preferred embodiments, the 2 copies of the ICP34.5 gene comprise different loss-of-function mutations. For example, in certain preferred embodiments, a first copy of the ICP34.5 gene comprises a third loss-of-function mutation and a second copy of the ICP34.5 gene comprises a fourth loss-of-function mutation. The third loss-of-function mutation and the fourth loss-of-function mutation can be the same or different.

[0048] In certain preferred embodiments, the 2 copies of the ICP34.5 gene are deleted.

[0049] In certain preferred embodiments, each of the two copies of the ICP34.5 gene is independently replaced with an exogenous nucleotide sequence (e.g., a nucleotide sequence encoding an exogenous protein). In certain preferred embodiments, the two copies of the ICP34.5 gene are replaced with the same exogenous nucleotide sequence (e.g., a nucleotide sequence encoding an exogenous protein). In certain preferred embodiments, the two copies of the ICP34.5 gene are replaced with different exogenous nucleotide sequences (e.g., nucleotide sequences encoding exogenous proteins). For example, in certain preferred embodiments, a first copy of the ICP34.5 gene is replaced with a third exogenous nucleotide sequence, and a second copy of the ICP34.5 gene is replaced with a fourth exogenous nucleotide sequence. The third exogenous nucleotide sequence and the fourth exogenous nucleotide sequence can be the same or different.

[0050] In certain preferred embodiments, each of the two copies of the ICP0 gene independently comprises a loss-of-function mutation (e.g., an addition, a deletion, and / or a substitution of one or more bases), and each of the two copies of the ICP34.5 gene independently comprises a loss-of-function mutation (e.g., an addition, a deletion, and / or a substitution of one or more bases). For example, in certain preferred embodiments, a first copy of the ICP0 gene comprises a first loss-of-function mutation, a second copy of the ICP0 gene comprises a second loss-of-function mutation; and, a first copy of the ICP34.5 gene comprises a third loss-of-function mutation, a second copy of the ICP34.5 gene comprises a fourth loss-of-function mutation. The first loss-of-function mutation, the second loss-of-function mutation, the third loss-of-function mutation, and the fourth loss-of-function mutation can be the same or different.

[0051] In certain preferred embodiments, each of the two copies of the ICP0 gene independently comprises a loss-of-function mutation (e.g., an addition, a deletion, and / or a substitution of one or more bases), and the two copies of the ICP34.5 gene are deleted. For example, in certain preferred embodiments, a first copy of the ICP0 gene comprises a first loss-of-function mutation, a second copy of the ICP0 gene comprises a second loss-of-function mutation; and, the two copies of the ICP34.5 gene are deleted. The first loss-of-function mutation and the second loss-of-function mutation can be the same or different.

[0052] In certain preferred embodiments, the two copies of the ICP0 gene each independently comprise a loss-of-function mutation (e.g., addition, deletion, and / or substitution of one or more bases), and the two copies of the ICP34.5 gene each independently are replaced with an exogenous nucleotide sequence (e.g., a nucleotide sequence encoding an exogenous protein). For example, in certain preferred embodiments, a first copy of the ICP0 gene comprises a first loss-of-function mutation, a second copy of the ICP0 gene comprises a second loss-of-function mutation; and a first copy of the ICP34.5 gene is replaced with a third exogenous nucleotide sequence, a second copy of the ICP34.5 gene is replaced with a fourth exogenous nucleotide sequence. The first and second loss-of-function mutations can be the same or different. The third and fourth exogenous nucleotide sequences can be the same or different.

[0053] In certain preferred embodiments, the two copies of the ICP0 gene are deleted; and the two copies of the ICP34.5 gene each independently comprise a loss-of-function mutation (e.g., addition, deletion, and / or substitution of one or more bases). For example, in certain preferred embodiments, the two copies of the ICP0 gene are deleted; and a first copy of the ICP34.5 gene comprises a third loss-of-function mutation, a second copy of the ICP34.5 gene comprises a fourth loss-of-function mutation. The third and fourth loss-of-function mutations can be the same or different.

[0054] In certain preferred embodiments, the two copies of the ICP0 gene are deleted; and the two copies of the ICP34.5 gene are deleted. In such embodiments, the recombinant HSV virus does not express an ICP0 protein and an ICP34.5 protein. In certain preferred embodiments, the genome of the recombinant HSV virus lacks the base sequences between nt510 and nt5439 and the base sequences between nt120802 and nt125731 of the wild-type HSV-1 viral genome.

[0055] In certain preferred embodiments, the two copies of the ICP0 gene are deleted; and the two copies of the ICP34.5 gene each independently are replaced with an exogenous nucleotide sequence (e.g., a nucleotide sequence encoding an exogenous protein). For example, in certain preferred embodiments, the two copies of the ICP0 gene are deleted; and a first copy of the ICP34.5 gene is replaced with a third exogenous nucleotide sequence, a second copy of the ICP34.5 gene is replaced with a fourth exogenous nucleotide sequence. The third and fourth exogenous nucleotide sequences can be the same or different.

[0056] In certain preferred embodiments, the two copies of the ICP0 gene are each independently replaced with an exogenous nucleotide sequence (e.g., a nucleotide sequence encoding an exogenous protein), and the two copies of the ICP34.5 gene each independently comprise a loss-of-function mutation (e.g., addition, deletion, and / or substitution of one or more bases). For example, in certain preferred embodiments, a first copy of the ICP0 gene is replaced with a first exogenous nucleotide sequence, a second copy of the ICP0 gene is replaced with a second exogenous nucleotide sequence; and a first copy of the ICP34.5 gene comprises a third loss-of-function mutation, a second copy of the ICP34.5 gene comprises a fourth loss-of-function mutation. The first exogenous nucleotide sequence and the second exogenous nucleotide sequence can be the same or different. The third loss-of-function mutation and the fourth loss-of-function mutation can be the same or different.

[0057] In certain preferred embodiments, the two copies of the ICP0 gene are each independently replaced with an exogenous nucleotide sequence (e.g., a nucleotide sequence encoding an exogenous protein), and the two copies of the ICP34.5 gene are deleted. For example, in certain preferred embodiments, a first copy of the ICP0 gene is replaced with a first exogenous nucleotide sequence, a second copy of the ICP0 gene is replaced with a second exogenous nucleotide sequence; and the two copies of the ICP34.5 gene are deleted. The first exogenous nucleotide sequence and the second exogenous nucleotide sequence can be the same or different.

[0058] In certain preferred embodiments, the two copies of the ICP0 gene are each independently replaced with an exogenous nucleotide sequence (e.g., a nucleotide sequence encoding an exogenous protein), and the two copies of the ICP34.5 gene are each independently replaced with an exogenous nucleotide sequence (e.g., a nucleotide sequence encoding an exogenous protein). For example, in certain preferred embodiments, a first copy of the ICP0 gene is replaced with a first exogenous nucleotide sequence, a second copy of the ICP0 gene is replaced with a second exogenous nucleotide sequence; and a first copy of the ICP34.5 gene is replaced with a third exogenous nucleotide sequence, a second copy of the ICP34.5 gene is replaced with a fourth exogenous nucleotide sequence. The first exogenous nucleotide sequence, the second exogenous nucleotide sequence, the third exogenous nucleotide sequence, and the fourth exogenous nucleotide sequence can be the same or different.

[0059] In certain preferred embodiments, the first loss-of-function mutation, the second loss-of-function mutation, the third loss-of-function mutation, and the fourth loss-of-function mutation are each independently selected from the group consisting of a missense mutation, a nonsense mutation, a frameshift mutation, a deletion of a base, a substitution of a base, an addition of a base, and any combination thereof (e.g., a deletion or substitution or addition of a segment of a gene).

[0060] In certain preferred embodiments, the first, second, third, and fourth exogenous nucleotide sequences each independently encodes an exogenous protein selected from the group consisting of a fluorescent protein, an immunomodulatory polypeptide, a cytokine, a chemokine, an antibody, and a cytotoxic peptide.

[0061] In certain preferred embodiments, the fluorescent protein is selected from the group consisting of a green fluorescent protein (e.g., a green fluorescent protein having an amino acid sequence as set forth in SEQ ID NO: 7), a red fluorescent protein, a blue fluorescent protein, a yellow fluorescent protein, and any combination thereof.

[0062] In certain preferred embodiments, the immunomodulatory polypeptide is selected from the group consisting of CD40L, OX40L, inducible costimulatory molecule (ICOS), FTL3L, LIGHT, CD137L, CD70, 4-1BB, GITR, CD28, and any combination thereof.

[0063] In certain preferred embodiments, the cytokine is selected from the group consisting of an interleukin (e.g., IL-2, IL-12, and IL-15), an interferon (e.g., IFN a, IFN b, IFN g), a tumor necrosis factor (e.g., TNF a), a colony stimulating factor (e.g., GM-CSF), and any combination thereof.

[0064] In certain preferred embodiments, the chemokine is selected from the group consisting of CCL2, RANTES, CCL7, CCL9, CCL10, CCL12, CCL15, CCL19, CCL21, CCL20, XCL-1, and any combination thereof.

[0065] In certain preferred embodiments, the cytotoxic peptide is selected from the group consisting of thymidine kinase TK (TK / GCV), TRAIL, FasL, and any combination thereof.

[0066] In certain preferred embodiments, the antibody is selected from the group consisting of an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-TIGIT antibody, an anti-BTLA antibody, an anti-CTLA-4 antibody, an anti-Tim-3 antibody, an anti-Lag-3 antibody, an anti-CD137 antibody, an anti-OX40 antibody, an anti-GITR antibody, an anti-CD73 antibody, an anti-KIR antibody, an anti-ICOS antibody, an anti-CSF1R antibody, an anti-EGFR antibody, an anti-VEGFR antibody, an anti-HER2 antibody, an anti-PDGFR antibody, and any combination thereof.

[0067] In the past five years, antibody drugs targeting PD-1 have achieved great success in the clinic, and have been approved by FDA for the treatment of melanoma, lung cancer and other solid tumor patients. However, clinical studies have shown that anti-PD-1 antibodies are only effective for about 30% of solid tumor patients. This can be because T cells are difficult to penetrate into the interior of solid tumors and cannot act together with anti-PD-1 antibodies on tumor cells inside the solid tumors. Without being bound by theory, oncolytic HSV viruses can not only directly target and kill tumor cells, but also induce immune cells (e.g., T cells) to infiltrate tumors. Therefore, the use of the recombinant HSV virus of the present application in combination with anti-PD-1 antibodies can be particularly advantageous for improving the effectiveness of tumor treatment and has great application prospects in tumor immunotherapy. Thus, in certain preferred embodiments, the exogenous protein is an anti-PD-L1 antibody, an anti-PD-1 antibody, or any combination thereof. For example, the exogenous protein is an anti-PD-1 single-chain antibody.

[0068] In certain preferred embodiments, the recombinant HSV virus is a recombinant HSV-1 virus, a recombinant HSV-2 virus, or a HSV-1 / HSV-2 chimeric virus (i.e., a recombinant HSV virus whose genome contains both DNA derived from HSV-1 and DNA derived from HSV-2). In certain preferred embodiments, the recombinant HSV virus is derived from HSV-1 strain KOS.

[0069] In certain preferred embodiments, the recombinant HSV virus is capable of expressing a functional UL43 protein, a functional UL41 protein (i.e., a vhs protein), a functional UL48 protein (i.e., a VMW65 protein), or any combination thereof. In certain preferred embodiments, the recombinant HSV virus is capable of expressing a functional UL43 protein. In certain preferred embodiments, the recombinant HSV virus is capable of expressing a functional UL41 protein. In certain preferred embodiments, the recombinant HSV virus is capable of expressing a functional UL48 protein. In certain preferred embodiments, the recombinant HSV virus is capable of expressing a functional UL43 protein and a functional UL41 protein. In certain preferred embodiments, the recombinant HSV virus is capable of expressing a functional UL43 protein and a functional UL48 protein. In certain preferred embodiments, the recombinant HSV virus is capable of expressing a functional UL41 protein and a functional UL48 protein. In certain preferred embodiments, the recombinant HSV virus is capable of expressing a functional UL43 protein, a functional UL41 protein, and a functional UL48 protein.

[0070] In certain preferred embodiments, the genome of the recombinant HSV virus comprises a UL43 gene capable of expressing a functional UL43 protein, a UL41 gene capable of expressing a functional UL41 protein (i.e., a vhs gene), and / or, a UL48 gene capable of expressing a functional UL48 protein (i.e., a VMW65 gene). In certain preferred embodiments, the genome of the recombinant HSV virus comprises a UL43 gene capable of expressing a functional UL43 protein. In certain preferred embodiments, the genome of the recombinant HSV virus comprises a UL41 gene capable of expressing a functional UL41 protein. In certain preferred embodiments, the genome of the recombinant HSV virus comprises a UL48 gene capable of expressing a functional UL48 protein. In certain preferred embodiments, the genome of the recombinant HSV virus comprises a UL43 gene capable of expressing a functional UL43 protein, and a UL41 gene capable of expressing a functional UL41 protein. In certain preferred embodiments, the genome of the recombinant HSV virus comprises a UL43 gene capable of expressing a functional UL43 protein, and a UL48 gene capable of expressing a functional UL48 protein. In certain preferred embodiments, the genome of the recombinant HSV virus comprises a UL41 gene capable of expressing a functional UL41 protein, and a UL48 gene capable of expressing a functional UL48 protein. In certain preferred embodiments, the genome of the recombinant HSV virus comprises a UL43 gene capable of expressing a functional UL43 protein, a UL41 gene capable of expressing a functional UL41 protein, and a UL48 gene capable of expressing a functional UL48 protein.

[0071] In certain preferred embodiments, the genome of the recombinant HSV virus contains a UL43 gene, a UL41 gene (i.e., a vhs gene), and / or a UL48 gene (i.e., a VMW65 gene), and the UL43 gene, UL41 gene, and / or UL48 gene does not comprise a loss-of-function mutation. In certain preferred embodiments, the genome of the recombinant HSV virus contains a UL43 gene that does not comprise a loss-of-function mutation. In certain preferred embodiments, the genome of the recombinant HSV virus contains a UL41 gene that does not comprise a loss-of-function mutation. In certain preferred embodiments, the genome of the recombinant HSV virus contains a UL48 gene that does not comprise a loss-of-function mutation. In certain preferred embodiments, the genome of the recombinant HSV virus contains a UL43 gene and a UL41 gene that do not comprise a loss-of-function mutation. In certain preferred embodiments, the genome of the recombinant HSV virus contains a UL43 gene and a UL48 gene that do not comprise a loss-of-function mutation. In certain preferred embodiments, the genome of the recombinant HSV virus contains a UL41 gene and a UL48 gene that do not comprise a loss-of-function mutation. In certain preferred embodiments, the genome of the recombinant HSV virus contains a UL43 gene, a UL41 gene, and a UL48 gene that do not comprise a loss-of-function mutation.

[0072] In certain preferred embodiments, the genome of the recombinant HSV virus further comprises a modification in which one or more non-essential genes are deleted or mutated (e.g., comprise a loss-of-function mutation, or are replaced with an exogenous nucleotide sequence). In certain preferred embodiments, the non-essential gene is selected from the group consisting of the UL3 gene, the UL4 gene, the UL14 gene, the UL16 gene, the UL21 gene, the UL24 gene, the UL31 gene, the UL32 gene, the US3 gene, the UL51 gene, the UL55 gene, the UL56 gene, the US2 gene, the US12 gene (i.e., the ICP47 gene), the LAT gene, a nucleotide fragment corresponding to nt5853-nt7485 of JQ673480.1, and any combination thereof. In certain preferred embodiments, the UL3 gene is deleted or mutated (e.g., comprises a loss-of-function mutation, or is replaced with an exogenous nucleotide sequence) in the genome of the recombinant HSV virus. In certain preferred embodiments, the UL4 gene is deleted or mutated (e.g., comprises a loss-of-function mutation, or is replaced with an exogenous nucleotide sequence) in the genome of the recombinant HSV virus. In certain preferred embodiments, the UL14 gene is deleted or mutated (e.g., comprises a loss-of-function mutation, or is replaced with an exogenous nucleotide sequence) in the genome of the recombinant HSV virus. In certain preferred embodiments, the UL16 gene is deleted or mutated (e.g., comprises a loss-of-function mutation, or is replaced with an exogenous nucleotide sequence) in the genome of the recombinant HSV virus. In certain preferred embodiments, the UL21 gene is deleted or mutated (e.g., comprises a loss-of-function mutation, or is replaced with an exogenous nucleotide sequence) in the genome of the recombinant HSV virus. In certain preferred embodiments, the UL24 gene is deleted or mutated (e.g., comprises a loss-of-function mutation, or is replaced with an exogenous nucleotide sequence) in the genome of the recombinant HSV virus. In certain preferred embodiments, the UL31 gene is deleted or mutated (e.g., comprises a loss-of-function mutation, or is replaced with an exogenous nucleotide sequence) in the genome of the recombinant HSV virus. In certain preferred embodiments, the UL32 gene is deleted or mutated (e.g., comprises a loss-of-function mutation, or is replaced with an exogenous nucleotide sequence) in the genome of the recombinant HSV virus. In certain preferred embodiments, the US3 gene is deleted or mutated (e.g., comprises a loss-of-function mutation, or is replaced with an exogenous nucleotide sequence) in the genome of the recombinant HSV virus. In certain preferred embodiments, the UL51 gene is deleted or mutated (e.g., comprises a loss-of-function mutation, or is replaced with an exogenous nucleotide sequence) in the genome of the recombinant HSV virus. In certain preferred embodiments, the UL55 gene is deleted or mutated (e.g., comprises a loss-of-function mutation, or is replaced with an exogenous nucleotide sequence) in the genome of the recombinant HSV virus.In certain preferred embodiments, in the genome of the recombinant HSV virus, the UL56 gene is deleted or mutated (e.g., comprises a loss-of-function mutation, or is replaced with an exogenous nucleotide sequence). In certain preferred embodiments, in the genome of the recombinant HSV virus, the US2 gene is deleted or mutated (e.g., comprises a loss-of-function mutation, or is replaced with an exogenous nucleotide sequence). In certain preferred embodiments, in the genome of the recombinant HSV virus, the US12 gene is deleted or mutated (e.g., comprises a loss-of-function mutation, or is replaced with an exogenous nucleotide sequence). In certain preferred embodiments, in the genome of the recombinant HSV virus, the LAT gene is deleted or mutated (e.g., comprises a loss-of-function mutation, or is replaced with an exogenous nucleotide sequence). In certain preferred embodiments, in the genome of the recombinant HSV virus, a nucleotide fragment corresponding to nt5853-nt7485 of JQ673480.1 is deleted or mutated (e.g., comprises a loss-of-function mutation, or is replaced with an exogenous nucleotide sequence).

[0073] In certain preferred embodiments, the genome of the recombinant HSV virus further comprises the following modification: one or more of the UL55 gene, the US2 gene, the LAT gene, and a nucleotide fragment corresponding to nt5853-nt7485 of JQ673480.1 is deleted or mutated (e.g., comprises a loss-of-function mutation, or is replaced with an exogenous nucleotide sequence). In certain preferred embodiments, the genome of the recombinant HSV virus further comprises the following modification: the UL55 gene, the US2 gene, the LAT gene, or a nucleotide fragment corresponding to nt5853-nt7485 of JQ673480.1 is deleted or mutated (e.g., comprises a loss-of-function mutation, or is replaced with an exogenous nucleotide sequence).

[0074] In certain preferred embodiments, the essential genes of the recombinant HSV virus are not deleted and do not comprise loss-of-function mutations. In certain preferred embodiments, the coding sequences of the essential genes of the recombinant HSV virus are not deleted or mutated. In certain preferred embodiments, the recombinant HSV virus is capable of expressing all essential genes. In certain preferred embodiments, the genome of the recombinant HSV virus contains all essential genes, and none of the essential genes comprise loss-of-function mutations. In certain preferred embodiments, the genome of the recombinant HSV virus contains all essential genes, and the coding sequences of all essential genes do not comprise mutations. Essential genes are indispensable for the survival and replication of the HSV virus, and thus, generally, none of the essential genes comprise loss-of-function mutations in the recombinant HSV virus. However, it is readily understood that the promoters of such essential genes can be engineered (e.g., the native promoters of the essential genes are replaced with tumor-specific promoters, such as the promoter of hTERT), thereby further increasing the safety of the recombinant HSV virus without affecting the function / properties of the recombinant HSV virus of the present application. Thus, in certain preferred embodiments, the native promoters of one or more essential genes are replaced with tumor-specific promoters, such as the promoter of hTERT, in the genome of the recombinant HSV virus. In certain preferred embodiments, the essential genes are selected from the group consisting of the ICP27 gene, the ICP4 gene, the VP5 gene, the gL gene, the gH gene, the gD gene, the gK gene, the gB gene, the gN gene, the UL5 gene, the UL6 gene, the UL8 gene, the UL9 gene, the UL12 gene, the UL25 gene, the UL26 gene, the UL28 gene, the UL29 gene, the UL30 gene, the UL33 gene, the UL36 gene, the UL38 gene, the UL42 gene, the UL48 gene, the UL52 gene, and any combination thereof.

[0075] In certain preferred embodiments, the genome of the recombinant HSV virus contains all other genes of the wild-type HSV virus, in addition to the 2 copies of the ICP0 gene and the 2 copies of the ICP34.5 gene as described above, and none of the other genes comprise loss-of-function mutations. However, it is readily understood that the promoters of the other genes can be engineered (e.g., the native promoters are replaced with tumor-specific promoters, such as the promoter of hTERT), thereby further increasing the safety of the recombinant HSV virus without affecting the function / properties of the recombinant HSV virus of the present application. Thus, in certain preferred embodiments, the genome of the recombinant HSV virus further comprises the following modification: the native promoter of one or more HSV genes is replaced with a tumor-specific promoter, such as the promoter of hTERT. In certain preferred embodiments, the HSV genes are selected from the group consisting of the VP5 gene, the ICP27 gene, and the ICP4 gene.

[0076] In certain preferred embodiments, the genome of the recombinant HSV virus further comprises one or more modifications selected from the group consisting of:

[0077] (1) the native promoter of the VP5 gene is replaced with a tumor-specific promoter, such as the promoter of hTERT;

[0078] (2) the native promoter of the ICP27 gene is replaced with a tumor-specific promoter, such as the promoter of hTERT;

[0079] (3) the native promoter of the ICP4 gene is replaced with a tumor-specific promoter, such as the promoter of hTERT; and

[0080] (4) one or more of the UL55 gene, the US2 gene, the LAT gene, and a nucleotide fragment corresponding to nt 5853-nt 7485 of JQ673480.1 is deleted or mutated (e.g., comprises a loss-of-function mutation, or is replaced with an exogenous nucleotide sequence).

[0081] In certain preferred embodiments, the hTERT promoter has a sequence as set forth in SEQ ID NO: 5.

[0082] In addition, the recombinant HSV virus of the present application can be further modified to carry one or more exogenous nucleotide sequences. For example, in certain preferred embodiments, the genome of the recombinant HSV virus further comprises a fifth exogenous nucleotide sequence. In certain preferred embodiments, the fifth exogenous nucleotide sequence encodes an exogenous protein selected from the group consisting of a fluorescent protein, an immunomodulatory polypeptide, a cytokine, a chemokine, an antibody, and a cytotoxic peptide.

[0083] In the present application, loss-of-function mutations can be introduced into the various viral genes mentioned herein by techniques well known in the art. For example, a loss-of-function mutation can be introduced into a viral gene by deletion, substitution, or insertion of a base, rendering the viral gene functionally inactivated. In certain exemplary embodiments, a viral gene is rendered functionally inactivated by deletion (e.g., deletion of the entire gene or a portion thereof). In such embodiments, at least 25%, at least 50%, at least 75%, or 100% of the sequence of the viral gene of interest can be deleted, or at least 10 bp, at least 100 bp, or at least 1000 bp of the sequence of the viral gene of interest can be deleted. In certain exemplary embodiments, a viral gene is rendered functionally inactivated by insertion or deletion of a base that causes a frameshift mutation. In certain exemplary embodiments, a viral gene is rendered functionally inactivated by substitution of the entire gene of interest or a portion thereof with an exogenous nucleotide sequence.

[0084] Viral vector

[0085] In another aspect, the present application provides a viral vector comprising or consisting of the genome of a recombinant HSV virus according to the present application.

[0086] In another aspect, the present application provides a viral vector comprising or consisting of a mutated HSV genome that does not express a functional functional ICP0 protein and an ICP34.5 protein.

[0087] In certain preferred embodiments, in the mutated HSV genome,

[0088] 2 copies of the ICP0 gene each independently comprise a loss-of-function mutation (e.g., addition, deletion, and / or substitution of one or more bases) or are deleted or replaced with an exogenous nucleotide sequence (e.g., a nucleotide sequence encoding an exogenous protein); and,

[0089] 2 copies of the ICP34.5 gene each independently comprise a loss-of-function mutation (e.g., addition, deletion, and / or substitution of one or more bases) or are deleted or replaced with an exogenous nucleotide sequence (e.g., a nucleotide sequence encoding an exogenous protein).

[0090] In certain preferred embodiments, the mutated HSV genome comprises the following modifications:

[0091] 2 copies of the ICP0 gene each independently comprise a loss-of-function mutation (e.g., addition, deletion, and / or substitution of one or more bases) or are deleted or replaced with an exogenous nucleotide sequence (e.g., a nucleotide sequence encoding an exogenous protein); and,

[0092] 2 copies of the ICP34.5 gene each independently comprise a loss-offunction mutation (e.g., addition, deletion, and / or substitution of one or more bases) or are deleted or replaced with an exogenous nucleotide sequence (e.g., a nucleotide sequence encoding an exogenous protein).

[0093] In certain preferred embodiments, one copy of the ICP0 gene comprises a loss-of-function mutation (e.g., addition, deletion, and / or substitution of one or more bases), and the other copy of the ICP0 gene comprises a loss-of-function mutation (e.g., addition, deletion, and / or substitution of one or more bases) or is deleted or replaced with an exogenous nucleotide sequence (e.g., a nucleotide sequence encoding an exogenous protein). In certain preferred embodiments, one copy of the ICP0 gene is deleted, and the other copy of the ICP0 gene comprises a loss-of-function mutation (e.g., addition, deletion, and / or substitution of one or more bases) or is deleted or replaced with an exogenous nucleotide sequence (e.g., a nucleotide sequence encoding an exogenous protein). In certain preferred embodiments, one copy of the ICP0 gene is replaced with an exogenous nucleotide sequence (e.g., a nucleotide sequence encoding an exogenous protein), and the other copy of the ICP0 gene comprises a loss-of-function mutation (e.g., addition, deletion, and / or substitution of one or more bases) or is deleted or replaced with an exogenous nucleotide sequence (e.g., a nucleotide sequence encoding an exogenous protein).

[0094] In certain preferred embodiments, each of the 2 copies of the ICP0 gene independently comprises a loss-of-function mutation (e.g., addition, deletion, and / or substitution of one or more bases). In certain preferred embodiments, the 2 copies of the ICP0 gene comprise the same loss-of-function mutation. In certain preferred embodiments, the 2 copies of the ICP0 gene comprise different loss-of-function mutations. For example, in certain preferred embodiments, a first copy of the ICP0 gene comprises a first loss-of-function mutation, and a second copy of the ICP0 gene comprises a second loss-of-function mutation. The first loss-of-function mutation and the second loss-of-function mutation can be the same or different.

[0095] In certain preferred embodiments, the 2 copies of the ICP0 gene are deleted.

[0096] In certain preferred embodiments, each of the 2 copies of the ICP0 gene is replaced with an exogenous nucleotide sequence (e.g., a nucleotide sequence encoding an exogenous protein). In certain preferred embodiments, the 2 copies of the ICP0 gene are replaced with the same exogenous nucleotide sequence (e.g., a nucleotide sequence encoding an exogenous protein). In certain preferred embodiments, the 2 copies of the ICP0 gene are replaced with different exogenous nucleotide sequences (e.g., nucleotide sequences encoding exogenous proteins). For example, in certain preferred embodiments, a first copy of the ICP0 gene is replaced with a first exogenous nucleotide sequence, and a second copy of the ICP0 gene is replaced with a second exogenous nucleotide sequence. The first exogenous nucleotide sequence and the second exogenous nucleotide sequence can be the same or different.

[0097] In certain preferred embodiments, one copy of the ICP34.5 gene comprises a loss-of-function mutation (e.g., addition, deletion, and / or substitution of one or more bases) and the other copy of the ICP34.5 gene comprises a loss-of-function mutation (e.g., addition, deletion, and / or substitution of one or more bases) or is deleted or replaced with an exogenous nucleotide sequence (e.g., a nucleotide sequence encoding an exogenous protein). In certain preferred embodiments, one copy of the ICP34.5 gene is deleted and the other copy of the ICP34.5 gene comprises a loss-of-function mutation (e.g., addition, deletion, and / or substitution of one or more bases) or is deleted or replaced with an exogenous nucleotide sequence (e.g., a nucleotide sequence encoding an exogenous protein). In certain preferred embodiments, one copy of the ICP34.5 gene is replaced with an exogenous nucleotide sequence (e.g., a nucleotide sequence encoding an exogenous protein) and the other copy of the ICP34.5 gene comprises a loss-of-function mutation (e.g., addition, deletion, and / or substitution of one or more bases) or is deleted or replaced with an exogenous nucleotide sequence (e.g., a nucleotide sequence encoding an exogenous protein).

[0098] In certain preferred embodiments, each of the 2 copies of the ICP34.5 gene independently comprises a loss-of-function mutation (e.g., addition, deletion, and / or substitution of one or more bases). In certain preferred embodiments, the 2 copies of the ICP34.5 gene comprise the same loss-of-function mutation. In certain preferred embodiments, the 2 copies of the ICP34.5 gene comprise different loss-of-function mutations. For example, in certain preferred embodiments, a first copy of the ICP34.5 gene comprises a third loss-of-function mutation and a second copy of the ICP34.5 gene comprises a fourth loss-of-function mutation. The third loss-of-function mutation and the fourth loss-of-function mutation can be the same or different.

[0099] In certain preferred embodiments, the 2 copies of the ICP34.5 gene are deleted.

[0100] In certain preferred embodiments, each of the two copies of the ICP34.5 gene is independently replaced with an exogenous nucleotide sequence (e.g., a nucleotide sequence encoding an exogenous protein). In certain preferred embodiments, the two copies of the ICP34.5 gene are replaced with the same exogenous nucleotide sequence (e.g., a nucleotide sequence encoding an exogenous protein). In certain preferred embodiments, the two copies of the ICP34.5 gene are replaced with different exogenous nucleotide sequences (e.g., nucleotide sequences encoding exogenous proteins). For example, in certain preferred embodiments, a first copy of the ICP34.5 gene is replaced with a third exogenous nucleotide sequence, and a second copy of the ICP34.5 gene is replaced with a fourth exogenous nucleotide sequence. The third exogenous nucleotide sequence and the fourth exogenous nucleotide sequence can be the same or different.

[0101] In certain preferred embodiments, each of the two copies of the ICP0 gene independently comprises a loss-of-function mutation (e.g., an addition, a deletion, and / or a substitution of one or more bases), and each of the two copies of the ICP34.5 gene independently comprises a loss-of-function mutation (e.g., an addition, a deletion, and / or a substitution of one or more bases). For example, in certain preferred embodiments, a first copy of the ICP0 gene comprises a first loss-of-function mutation, a second copy of the ICP0 gene comprises a second loss-of-function mutation; and, a first copy of the ICP34.5 gene comprises a third loss-of-function mutation, a second copy of the ICP34.5 gene comprises a fourth loss-of-function mutation. The first loss-of-function mutation, the second loss-of-function mutation, the third loss-of-function mutation, and the fourth loss-of-function mutation can be the same or different.

[0102] In certain preferred embodiments, each of the two copies of the ICP0 gene independently comprises a loss-of-function mutation (e.g., an addition, a deletion, and / or a substitution of one or more bases), and the two copies of the ICP34.5 gene are deleted. For example, in certain preferred embodiments, a first copy of the ICP0 gene comprises a first loss-of-function mutation, a second copy of the ICP0 gene comprises a second loss-of-function mutation; and, the two copies of the ICP34.5 gene are deleted. The first loss-of-function mutation and the second loss-of-function mutation can be the same or different.

[0103] In certain preferred embodiments, the two copies of the ICP0 gene each independently comprise a loss-of-function mutation (e.g., addition, deletion, and / or substitution of one or more bases), and the two copies of the ICP34.5 gene each independently are replaced with an exogenous nucleotide sequence (e.g., a nucleotide sequence encoding an exogenous protein). For example, in certain preferred embodiments, a first copy of the ICP0 gene comprises a first loss-of-function mutation, a second copy of the ICP0 gene comprises a second loss-of-function mutation; and a first copy of the ICP34.5 gene is replaced with a third exogenous nucleotide sequence, a second copy of the ICP34.5 gene is replaced with a fourth exogenous nucleotide sequence. The first and second loss-of-function mutations can be the same or different. The third and fourth exogenous nucleotide sequences can be the same or different.

[0104] In certain preferred embodiments, the two copies of the ICP0 gene are deleted; and the two copies of the ICP34.5 gene each independently comprise a loss-of-function mutation (e.g., addition, deletion, and / or substitution of one or more bases). For example, in certain preferred embodiments, the two copies of the ICP0 gene are deleted; and a first copy of the ICP34.5 gene comprises a third loss-of-function mutation, a second copy of the ICP34.5 gene comprises a fourth loss-of-function mutation. The third and fourth loss-of-function mutations can be the same or different.

[0105] In certain preferred embodiments, the two copies of the ICP0 gene are deleted; and the two copies of the ICP34.5 gene are deleted. In such embodiments, the viral vector does not comprise a gene encoding an ICP0 protein and a gene encoding an ICP34.5 protein. In certain preferred embodiments, the mutated HSV genome lacks the base sequences between nt 510 and nt 5439 and the base sequences between nt 120802 and nt 125731 of a wild-type HSV-1 viral genome.

[0106] In certain preferred embodiments, the two copies of the ICP0 gene are deleted; and the two copies of the ICP34.5 gene each independently are replaced with an exogenous nucleotide sequence (e.g., a nucleotide sequence encoding an exogenous protein). For example, in certain preferred embodiments, the two copies of the ICP0 gene are deleted; and a first copy of the ICP34.5 gene is replaced with a third exogenous nucleotide sequence, a second copy of the ICP34.5 gene is replaced with a fourth exogenous nucleotide sequence. The third and fourth exogenous nucleotide sequences can be the same or different.

[0107] In certain preferred embodiments, the two copies of the ICP0 gene are each independently replaced with an exogenous nucleotide sequence (e.g., a nucleotide sequence encoding an exogenous protein), and the two copies of the ICP34.5 gene each independently comprise a loss-of-function mutation (e.g., addition, deletion, and / or substitution of one or more bases). For example, in certain preferred embodiments, a first copy of the ICP0 gene is replaced with a first exogenous nucleotide sequence, a second copy of the ICP0 gene is replaced with a second exogenous nucleotide sequence; and a first copy of the ICP34.5 gene comprises a third loss-of-function mutation, a second copy of the ICP34.5 gene comprises a fourth loss-of-function mutation. The first exogenous nucleotide sequence and the second exogenous nucleotide sequence can be the same or different. The third loss-of-function mutation and the fourth loss-of-function mutation can be the same or different.

[0108] In certain preferred embodiments, the two copies of the ICP0 gene are each independently replaced with an exogenous nucleotide sequence (e.g., a nucleotide sequence encoding an exogenous protein), and the two copies of the ICP34.5 gene are deleted. For example, in certain preferred embodiments, a first copy of the ICP0 gene is replaced with a first exogenous nucleotide sequence, a second copy of the ICP0 gene is replaced with a second exogenous nucleotide sequence; and the two copies of the ICP34.5 gene are deleted. The first exogenous nucleotide sequence and the second exogenous nucleotide sequence can be the same or different.

[0109] In certain preferred embodiments, the two copies of the ICP0 gene are each independently replaced with an exogenous nucleotide sequence (e.g., a nucleotide sequence encoding an exogenous protein), and the two copies of the ICP34.5 gene are each independently replaced with an exogenous nucleotide sequence (e.g., a nucleotide sequence encoding an exogenous protein). For example, in certain preferred embodiments, a first copy of the ICP0 gene is replaced with a first exogenous nucleotide sequence, a second copy of the ICP0 gene is replaced with a second exogenous nucleotide sequence; and a first copy of the ICP34.5 gene is replaced with a third exogenous nucleotide sequence, a second copy of the ICP34.5 gene is replaced with a fourth exogenous nucleotide sequence. The first exogenous nucleotide sequence, the second exogenous nucleotide sequence, the third exogenous nucleotide sequence, and the fourth exogenous nucleotide sequence can be the same or different.

[0110] In certain preferred embodiments, the first loss-of-function mutation, the second loss-of-function mutation, the third loss-of-function mutation, and the fourth loss-of-function mutation are each independently selected from the group consisting of a missense mutation, a nonsense mutation, a frameshift mutation, a base deletion, a base substitution, a base addition, and any combination thereof (e.g., deletion or replacement or addition of a gene fragment).

[0111] In certain preferred embodiments, the first, second, third, and fourth exogenous nucleotide sequences each independently encodes an exogenous protein selected from the group consisting of a fluorescent protein, an immunomodulatory polypeptide, a cytokine, a chemokine, an antibody, and a cytotoxic peptide.

[0112] In certain preferred embodiments, the fluorescent protein is selected from the group consisting of a green fluorescent protein (e.g., a green fluorescent protein having an amino acid sequence as set forth in SEQ ID NO: 7), a red fluorescent protein, a blue fluorescent protein, a yellow fluorescent protein, and any combination thereof.

[0113] In certain preferred embodiments, the immunomodulatory polypeptide is selected from the group consisting of CD40L, OX40L, inducible costimulatory molecule (ICOS), FTL3L, LIGHT, CD137L, CD70, 4-1BB, GITR, CD28, and any combination thereof.

[0114] In certain preferred embodiments, the cytokine is selected from the group consisting of an interleukin (e.g., IL-2, IL-12, and IL-15), an interferon (e.g., IFNa, IFP, IFN), a tumor necrosis factor (e.g., TNFa), a colony stimulating factor (e.g., GM-CSF), and any combination thereof.

[0115] In certain preferred embodiments, the chemokine is selected from the group consisting of CCL2, RANTES, CCL7, CCL9, CCL10, CCL12, CCL15, CCL19, CCL21, CCL20, XCL-1, and any combination thereof.

[0116] In certain preferred embodiments, the cytotoxic peptide is selected from the group consisting of thymidine kinase TK (TK / GCV), TRAIL, FasL, and any combination thereof.

[0117] In certain preferred embodiments, the antibody is selected from the group consisting of an anti-PD-1 antibody, an anti-PD-Ll antibody, an anti-TIGIT antibody, an anti-BTLA antibody, an anti-CTLA-4 antibody, an anti-Tim-3 antibody, an anti-Lag-3 antibody, an anti-CD137 antibody, an anti-OX40 antibody, an anti-GITR antibody, an anti-CD73 antibody, an anti-KIR antibody, an anti-ICOS antibody, an anti-CSF1R antibody, an anti-EGFR antibody, an anti-VEGFR antibody, an anti-HER2 antibody, an anti-PDGFR antibody, and any combination thereof.

[0118] In certain preferred embodiments, the exogenous protein is an anti-PD-Ll antibody, an anti-PD-1 antibody, or any combination thereof. For example, the exogenous protein is an anti-PD-1 single chain antibody.

[0119] In certain preferred embodiments, the mutated HSV genome is derived from the genome of an HSV-1 virus, an HSV-2 virus, or an HSV-1 / HSV-2 chimeric virus (i.e., a recombinant HSV virus whose genome contains both DNA derived from HSV-1 and DNA derived from HSV-2). In certain preferred embodiments, the mutated HSV genome is derived from the genome of HSV-1 strain KOS. In certain preferred embodiments, the mutated HSV genome is derived from the genome set forth in GenBank: JQ673480.1.

[0120] In certain preferred embodiments, the mutated HSV genome is capable of expressing a functional UL43 protein, a functional UL41 protein (i.e., a vhs protein), a functional UL48 protein (i.e., a VMW65 protein), or any combination thereof. In certain preferred embodiments, the mutated HSV genome is capable of expressing a functional UL43 protein. In certain preferred embodiments, the mutated HSV genome is capable of expressing a functional UL41 protein. In certain preferred embodiments, the mutated HSV genome is capable of expressing a functional UL48 protein. In certain preferred embodiments, the mutated HSV genome is capable of expressing a functional UL43 protein and a functional UL41 protein. In certain preferred embodiments, the mutated HSV genome is capable of expressing a functional UL43 protein and a functional UL48 protein. In certain preferred embodiments, the mutated HSV genome is capable of expressing a functional UL41 protein and a functional UL48 protein. In certain preferred embodiments, the mutated HSV genome is capable of expressing a functional UL43 protein, a functional UL41 protein, and a functional UL48 protein.

[0121] In certain preferred embodiments, the mutated HSV genome comprises a UL43 gene capable of expressing a functional UL43 protein, a UL41 gene capable of expressing a functional UL41 protein (i.e., a vhs gene), and / or, a UL48 gene capable of expressing a functional UL48 protein (i.e., a VMW65 gene). In certain preferred embodiments, the mutated HSV genome comprises a UL43 gene capable of expressing a functional UL43 protein. In certain preferred embodiments, the mutated HSV genome comprises a UL41 gene capable of expressing a functional UL41 protein. In certain preferred embodiments, the mutated HSV genome comprises a UL48 gene capable of expressing a functional UL48 protein. In certain preferred embodiments, the mutated HSV genome comprises a UL43 gene capable of expressing a functional UL43 protein, and a UL41 gene capable of expressing a functional UL41 protein. In certain preferred embodiments, the mutated HSV genome comprises a UL43 gene capable of expressing a functional UL43 protein, and a UL48 gene capable of expressing a functional UL48 protein. In certain preferred embodiments, the mutated HSV genome comprises a UL41 gene capable of expressing a functional UL41 protein, and a UL48 gene capable of expressing a functional UL48 protein. In certain preferred embodiments, the mutated HSV genome comprises a UL43 gene capable of expressing a functional UL43 protein, a UL41 gene capable of expressing a functional UL41 protein, and a UL48 gene capable of expressing a functional UL48 protein.

[0122] In certain preferred embodiments, the mutated HSV genome contains a UL43 gene, a UL41 gene (i.e., a vhs gene), and / or a UL48 gene (i.e., a VMW65 gene), and the UL43 gene, UL41 gene, and / or UL48 gene does not comprise a loss-of-function mutation. In certain preferred embodiments, the mutated HSV genome contains a UL43 gene that does not comprise a loss-of-function mutation. In certain preferred embodiments, the mutated HSV genome contains a UL41 gene that does not comprise a loss-of-function mutation. In certain preferred embodiments, the mutated HSV genome contains a UL48 gene that does not comprise a loss-of-function mutation. In certain preferred embodiments, the mutated HSV genome contains a UL43 gene and a UL41 gene that do not comprise a loss-of-function mutation. In certain preferred embodiments, the mutated HSV genome contains a UL43 gene and a UL48 gene that do not comprise a loss-of-function mutation. In certain preferred embodiments, the mutated HSV genome contains a UL41 gene and a UL48 gene that do not comprise a loss-of-function mutation. In certain preferred embodiments, the mutated HSV genome contains a UL43 gene, a UL41 gene, and a UL48 gene that do not comprise a loss-of-function mutation.

[0123] In certain preferred embodiments, the mutated HSV genome further comprises a modification in which one or more non-essential genes are deleted or mutated (e.g., comprise a loss-of-function mutation, or are replaced with an exogenous nucleotide sequence). In certain preferred embodiments, the non-essential gene is selected from the group consisting of a UL3 gene, a UL4 gene, a UL14 gene, a UL16 gene, a UL21 gene, a UL24 gene, a UL31 gene, a UL32 gene, a US3 gene, a UL51 gene, a UL55 gene, a UL56 gene, a US2 gene, a US12 gene (i.e., an ICP47 gene), a LAT gene, a nucleotide fragment corresponding to nt5853-nt7485 of JQ673480.1, and any combination thereof. In certain preferred embodiments, in the mutated HSV genome, the UL3 gene is deleted or mutated (e.g., comprises a loss-of-function mutation, or is replaced with an exogenous nucleotide sequence). In certain preferred embodiments, in the mutated HSV genome, the UL4 gene is deleted or mutated (e.g., comprises a loss-of-function mutation, or is replaced with an exogenous nucleotide sequence). In certain preferred embodiments, in the mutated HSV genome, the UL14 gene is deleted or mutated (e.g., comprises a loss-of-function mutation, or is replaced with an exogenous nucleotide sequence). In certain preferred embodiments, in the mutated HSV genome, the UL16 gene is deleted or mutated (e.g., comprises a loss-of-function mutation, or is replaced with an exogenous nucleotide sequence). In certain preferred embodiments, in the mutated HSV genome, the UL21 gene is deleted or mutated (e.g., comprises a loss-of-function mutation, or is replaced with an exogenous nucleotide sequence). In certain preferred embodiments, in the mutated HSV genome, the UL24 gene is deleted or mutated (e.g., comprises a loss-of-function mutation, or is replaced with an exogenous nucleotide sequence). In certain preferred embodiments, in the mutated HSV genome, the UL31 gene is deleted or mutated (e.g., comprises a loss-of-function mutation, or is replaced with an exogenous nucleotide sequence). In certain preferred embodiments, in the mutated HSV genome, the UL32 gene is deleted or mutated (e.g., comprises a loss-of-function mutation, or is replaced with an exogenous nucleotide sequence). In certain preferred embodiments, in the mutated HSV genome, the US3 gene is deleted or mutated (e.g., comprises a loss-of-function mutation, or is replaced with an exogenous nucleotide sequence). In certain preferred embodiments, in the mutated HSV genome, the UL51 gene is deleted or mutated (e.g., comprises a loss-of-function mutation, or is replaced with an exogenous nucleotide sequence). In certain preferred embodiments, in the mutated HSV genome, the UL55 gene is deleted or mutated (e.g., comprises a loss-of-function mutation, or is replaced with an exogenous nucleotide sequence).In certain preferred embodiments, in the mutated HSV genome, the UL56 gene is deleted or mutated (e.g., comprises a loss-of-function mutation, or is replaced with an exogenous nucleotide sequence). In certain preferred embodiments, in the mutated HSV genome, the US2 gene is deleted or mutated (e.g., comprises a loss-of-function mutation, or is replaced with an exogenous nucleotide sequence). In certain preferred embodiments, in the mutated HSV genome, the US12 gene is deleted or mutated (e.g., comprises a loss-of-function mutation, or is replaced with an exogenous nucleotide sequence). In certain preferred embodiments, in the mutated HSV genome, the LAT gene is deleted or mutated (e.g., comprises a loss-of-function mutation, or is replaced with an exogenous nucleotide sequence). In certain preferred embodiments, in the mutated HSV genome, a nucleotide fragment corresponding to nt5853-nt7485 of JQ673480.1 is deleted or mutated (e.g., comprises a loss-of-function mutation, or is replaced with an exogenous nucleotide sequence).

[0124] In certain preferred embodiments, the mutated HSV genome further comprises the following modification: one or more of the UL55 gene, the US2 gene, the LAT gene, and a nucleotide fragment corresponding to nt5853-nt7485 of JQ673480.1 is deleted or mutated (e.g., comprises a loss-of-function mutation, or is replaced with an exogenous nucleotide sequence). In certain preferred embodiments, the mutated HSV genome further comprises the following modification: the UL55 gene, the US2 gene, the LAT gene, or a nucleotide fragment corresponding to nt5853-nt7485 of JQ673480.1 is deleted or mutated (e.g., comprises a loss-of-function mutation, or is replaced with an exogenous nucleotide sequence).

[0125] In certain preferred embodiments, the essential genes in the mutated HSV genome are not deleted and do not comprise loss-of-function mutations. In certain preferred embodiments, the coding sequences of the essential genes in the mutated HSV genome are not deleted or mutated. In certain preferred embodiments, the mutated HSV genome is capable of expressing all essential genes. In certain preferred embodiments, the mutated HSV genome contains all essential genes and none of the essential genes comprise a loss-of-function mutation. In certain preferred embodiments, the mutated HSV genome contains all essential genes and the coding sequences of all essential genes do not comprise mutations. Essential genes are indispensable for the survival and replication of HSV viruses, and thus, generally, all essential genes do not comprise loss-of-function mutations in the genome of a recombinant HSV virus. However, it is readily appreciated that the promoters of such essential genes can be engineered (e.g., the native promoters of essential genes are replaced with tumor-specific promoters, such as the promoter of hTERT) to further improve the safety of the recombinant HSV virus without affecting the function / properties of the recombinant HSV virus of the present application. Thus, in certain preferred embodiments, the native promoters of one or more essential genes are replaced with tumor-specific promoters, such as the promoter of hTERT, in the mutated HSV genome. In certain preferred embodiments, the essential genes are selected from the group consisting of the ICP27 gene, the ICP4 gene, the VP5 gene, the gL gene, the gH gene, the gD gene, the gK gene, the gB gene, the gN gene, the UL5 gene, the UL6 gene, the UL8 gene, the UL9 gene, the UL12 gene, the UL25 gene, the UL26 gene, the UL28 gene, the UL29 gene, the UL30 gene, the UL33 gene, the UL36 gene, the UL38 gene, the UL42 gene, the UL48 gene, the UL52 gene, and any combination thereof.

[0126] In certain preferred embodiments, the mutated HSV genome contains all other genes of a wild-type HSV virus, and none of the other genes comprises a loss-of-function mutation, in addition to the 2 copies of the ICP0 gene and the 2 copies of the ICP34.5 gene as described above. However, it is readily appreciated that the promoters of the other genes can be engineered (e.g., the native promoters are replaced with tumor-specific promoters, such as the promoter of hTERT), so as to further improve the safety of the recombinant HSV virus without affecting the function / properties of the recombinant HSV virus of the present application. Thus, in certain preferred embodiments, the mutated HSV genome further comprises the following modification: the native promoter of one or more HSV genes is replaced with a tumor-specific promoter, such as the promoter of hTERT. In certain preferred embodiments, the HSV gene is selected from the group consisting of the VP5 gene, the ICP27 gene, and the ICP4 gene.

[0127] In certain preferred embodiments, the mutated HSV genome further comprises one or more modifications selected from the group consisting of:

[0128] (1) the native promoter of the VP5 gene is replaced with a tumor-specific promoter, such as the promoter of hTERT;

[0129] (2) the native promoter of the ICP27 gene is replaced with a tumor-specific promoter, such as the promoter of hTERT;

[0130] (3) the native promoter of the ICP4 gene is replaced with a tumor-specific promoter, such as the promoter of hTERT; and

[0131] (4) one or more of the UL55 gene, the US2 gene, the LAT gene, and the nucleotide fragment corresponding to nt5853-nt7485 of JQ673480.1 is deleted or mutated (e.g., comprises a loss-of-function mutation, or is replaced with an exogenous nucleotide sequence).

[0132] In certain preferred embodiments, the hTERT promoter has the sequence set forth in SEQ ID NO: 5.

[0133] In addition, the mutated HSV genome can be further modified to carry one or more exogenous nucleotide sequences. For example, in certain preferred embodiments, the mutated HSV genome further comprises a fifth exogenous nucleotide sequence. In certain preferred embodiments, the fifth exogenous nucleotide sequence encodes an exogenous protein selected from the group consisting of a fluorescent protein, an immunomodulatory polypeptide, a cytokine, a chemokine, an antibody, and a cytotoxic peptide.

[0134] Host cell

[0135] In another aspect, the present application provides a host cell infected with, or comprising a genome of, or transfected with, a recombinant HSV virus according to the present application. Such host cells include, but are not limited to, prokaryotic cells such as E. coli cells, and eukaryotic cells such as yeast cells, insect cells, plant cells and animal cells (e.g. mammalian cells, e.g. mouse cells, human cells, etc.). The recombinant HSV viruses of the present application have high replication capacity in tumor cells, but only replicate at low levels in normal cells. Thus, in certain particularly preferred embodiments, the cell is a tumor cell. Such tumor cells include, but are not limited to, lung cancer cells (e.g. H1299, H520, H1975, NCI-H358 and A549); liver cancer cells (e.g. Huh7, Hep3B, HepG2, GSG7701, SMMC7721, Hepa1-6, BEL7404, PLC / PRF and QGY7703); breast cancer cells (e.g. MADMB231, MCF7 and MADMB468); osteosarcoma cells (e.g. U2OS and SAOS2); ovarian cancer cells (e.g. SKOV3 and CAOV3); cervical cancer cells (e.g. SiHA and Hela); prostate cancer cells (e.g. PC-3); glioma cells (e.g. U87MG); melanoma cells (e.g. A375); colorectal cancer cells (e.g. HCT116) and pancreatic cancer cells (e.g. Panc-1).

[0136] Method of manufacture

[0137] In another aspect, the present application relates to a method of obtaining a recombinant HSV virus of the present application, comprising:

[0138] (1) culturing a host cell according to the present application;

[0139] (2) after the host cell becomes pathogenic, collecting and lysing the host cell to obtain a lysate of the host cell; and

[0140] (3) recovering a recombinant HSV virus of the present application from the lysate.

[0141] Pharmaceutical composition

[0142] In another aspect, the present application relates to a pharmaceutical composition comprising a recombinant HSV virus according to the present application, or a genome of a recombinant HSV virus according to the present application, or a viral vector according to the present application, and a pharmaceutically acceptable carrier or excipient. The pharmaceutical composition of the present application can be used for treating a tumor, for example, lung cancer, liver cancer, breast cancer, osteosarcoma, ovarian cancer, prostate cancer, glioma, melanoma, colorectal cancer, and pancreatic cancer.

[0143] The pharmaceutical composition of the present application can be administered by methods known in the art, for example, but not limited to, by injection. In certain preferred embodiments, the pharmaceutical composition of the present application is administered by injection, for example, intratumoral injection. In certain preferred embodiments, the pharmaceutical composition of the present application is a solution for injection or a lyophilized powder.

[0144] In certain preferred embodiments, the recombinant HSV virus or genome of a recombinant HSV virus or viral vector is present in a therapeutically effective amount, for example, a therapeutically effective amount for treating a tumor. In certain preferred embodiments, the pharmaceutical composition of the present application is present in unit dosage form. For example, but not intended to limit the present application, the amount of recombinant HSV virus contained in each unit dosage of the pharmaceutical composition can be 10 2 -10 9 pfu, for example, 10 2 -10 3 pfu, 10 3 -10 4 pfu, 10 4 -10 5 pfu, 10 5 -10 6 pfu, 10 6 -10 7 pfu, 10 7 -10 8 pfu, or 10 8 -10 9 pfu.

[0145] Use / method of use

[0146] The recombinant HSV virus of the present application can be used for treating various tumors. Thus, in another aspect, the present application relates to a method of treating a tumor, comprising administering to a subject in need thereof a therapeutically effective amount of the recombinant HSV virus of the present application or the viral vector of the present application or the pharmaceutical composition of the present application. In certain preferred embodiments, the tumor comprises, but is not limited to, lung cancer, liver cancer, breast cancer, osteosarcoma, ovarian cancer, prostate cancer, glioma, melanoma, colorectal cancer, and pancreatic cancer. In certain preferred embodiments, the subject is a mammal, such as a human. In certain preferred embodiments, the recombinant HSV virus of the present application or the viral vector of the present application or the pharmaceutical composition of the present application is administered to the subject by injection, such as intratumoral injection.

[0147] In another aspect, the present application relates to the use of the recombinant HSV virus of the present application or the viral vector of the present application for the manufacture of a pharmaceutical composition for treating a tumor in a subject. In certain preferred embodiments, the tumor comprises, but is not limited to, lung cancer, liver cancer, breast cancer, osteosarcoma, ovarian cancer, prostate cancer, glioma, melanoma, colorectal cancer, and pancreatic cancer. In certain preferred embodiments, the subject is a mammal, such as a human. In certain preferred embodiments, the pharmaceutical composition is administered by injection, such as intratumoral injection. In certain preferred embodiments, the pharmaceutical composition is a solution for injection or a lyophilized powder.

[0148] Advantages of the Invention

[0149] Compared with the recombinant herpes simplex virus in the prior art, the recombinant HSV virus of the present application has the following advantageous technical effects: the recombinant HSV virus of the present application has a high level of replication ability in tumor cells and can effectively kill a variety of tumor cells, but has significantly reduced replication ability and killing ability in normal cells. In addition, it has also been shown that the recombinant HSV virus of the present application has significantly reduced neurotoxicity in animals and can be administered to animals at a significantly increased dose. Therefore, compared with the existing recombinant HSV virus, the recombinant HSV virus of the present application not only maintains high oncolytic ability, but also has significantly improved safety, can be administered at a higher dose, and has a broad application prospect.

[0150] Description of Sequence Information

[0151] The information of the sequences involved in the present application is provided in Table 1.

[0152] Table 1: Sequence Information

[0153]

[0154] SEQ ID NO: 1

[0155]

[0156] SEQ ID NO: 2

[0157]

[0158] SEQ ID NO: 3

[0159] CCACCTGGTGTTTTGTCTCCACCATCGGCCTGACAGAGCTGTATTGTATTCTGCGGCGGGGCCCGGCCCCCAAGAACGCAGACAAGGCCGCCGCCCCGGGGCGATCCAAGGGGCTGTCGGGCGTCTGCGGGCGCTGTTGTTCCATCATCCTGTCGGGCATCGCAATGCGATTGTGTTATATCGCCGTGGTGGCCGGGGTGGTGCTCGTGGCGCTTCACTACGAGCAGGAGATCCAGAGGCGCCTGTTTGATGTATGACGTCACATCCAGGCCGGCGGAAACCGGAACGGCATATGCAAACTGGAAACTGTCCTGTCTTGGGGCCCACCCACCCGACGCGTCATATGTAAATGAAAATCGTTCCCCCGAGGCCATGTGTAGCCTGGATCCCAACGACCCCGCCCATGGGTCCCAATTGGCCGTCCCGTTACCAAGACCAACCCAGCCAGCGTATCCACCCCCGCCCGGGTCCCCGCGGAAGCGGAACGGTGTATGTGATATGCTAATTAAATACATGCCACGTACTTATGGTGTCTGATTGGTCCTTGTCTGTGCCGGAGGTG

[0160] SEQ ID NO: 4

[0161]

[0162] SEQ ID NO: 5

[0163] CTGCGCTGTCGGGGCCAGGCCGGGCTCCCAGTGGATTCGCGGGCACAGACGCCCAGGACCGCGCTTCCCACGTGGCGGAGGGACTGGGGACCCGGGCACCCGTCCTGCCCCTTCACCTTCCAGCTCCGCCTCCTCCGCGCGGACCCCGCCCCGTCCCGACCCCTCCCGGGTCCCCGGCCCAGCCCCCTCCGGGCCCTCCCAGCCCCTCCCCTTCCTTTCCGCGGCCCCGCCCTCTCCTCGCGGCGCGAGTTTCAGGCAGC

[0164] SEQ ID NO: 6

[0165]

[0166] SEQ ID NO: 7

[0167] MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK

[0168] SEQ ID NO: 8

[0169] DVLMTQTPLFLPVSLGDQASIFCRSSQNIVHINGNTYLEWYLQKPGQFPKLLMYKVSNRFFGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPWTFGGGTKLEIKGGGGSGGGGSGGGGSGGGGSSDVQVQESGPGLVKPSQSLSLTCTVTGSS ITSDFAWEWIRQFPGNKLECMGYIGYSGGTIYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCARWHGSSHWYFDVWGAGTTVTVSS

[0170] SEQ ID NO: 17

[0171]

[0172] SEQ ID NO: 18

[0173]

[0174] SEQ ID NO: 19

[0175]

[0176] SEQ ID NO: 20

[0177] ATGACAGCGACCCCCCTCACCAACCTGTTCTTACGGGCCCCGGACATAACCCACGTGGCCCCCCCTTACTGCCTCAACGCCACCTGGCAGGCCGAAACGGCCATGCACACCAGCAAAACGGACTCCGCTTGCGTGGCCGTGCGGAGTTACCTGGTCCGCGCCTCCTGTGAGACCAGCGGCACAATCCACTGCTTTTTCTTTGCGGTATACAAGGACACCCACCATACCCCTCCGCTGATTACCGAGCTCCGCAACTTTGCGGACCTGGTTAACCACCCGCCGGTCCTACGCGAACTGGAGGATAAGCGCGGGGTGCGGCTGCGGTGTGCGCGGCCGTTTAGCGTCGGGACGATTAAGGACGTCTCTGGGTCCGGCGCGTCCTCGGCGGGAGAGTACACGATAAACGGGATCGTGTACCACTGCCACTGTCGGTATCCGTTCTCAAAAACATGCTGGATGGGGGCCTCCGCGGCCCTACAGCACCTGCGCTCCATCAGCTCCAGCGGCATGGCCGCCCGCGCGGCAGAGCATCGACGCGTCAAGATTAAAATTAAGGCGTGA

[0178] SEQ ID NO: 21

[0179] CTACAGGGTGGTAACCGGATAGCAGATGTGAGGAAGTCTGGGCCGTTCGCCGCGAACGGCGATCAGAGGGTCCGTTTCTTGCGGACCACGGCCCGGTGATGTGGGTTGCTCGTCTAAAATCTCGGGCATACCCATACACGCACAACACGGACGCCGCACCGAATGGGACGTCGTAAGGGGGTGGGAGGTAGCTGGGTGGGGTTTGTGCAGAGCAATCAGGGACCGCAGCCAGCGCATACAATCGCGCTCCCGTCCGTTGGTCCCGGGCAGGACCACGCCGTACTGGTATTCGTACCGGCTGAGCAGGGTCTCCAGGGGGTGGTTGGGTGCCGCGGGGAACGGGGTCCACGCCACGGTCCACTCGGGCAAAAACCGAGTCGGCACGGCCCACGGTTCTCCCACCCACGCGTCTGGGGTCTTGATGGCGATAAATCTTACCCCGAGCCGGATTTTTTGGGCGTATTCGAGAAACGGCACACACAGATCCGCCGCGCCTACCACCCACAAGTGGTAGAGGCGAGGGGGGCTGGGTTGGTCTCGGTGCAACAGTCGGAAGCACGCCACGGCGTCCACGACCTCGGTGCTCTCCAAGGGGCTGTCCTCCGCAAACAGGCCCGTGGTGGTGTTTGGGGGGCAGCGACAGGACCTAGTGCGCACGATCGGGCGGGTGGGTTTGGGTAAGTCCATCAGCGGCTCGGCCAACCGTCGAAGGTTGGCCGGGCGAACGACGACCGGGGTACCCAGGGGTTCTGATGCCAAAATGCGGCACTGCCTAAGCAGGAAGCTCCACAGGGCCGGGCTTGCGTCGACGGAAGTCCGGGGCAGGGCGTTGTTCTGGTCAAGGAGGGTCATTACGTTGACGACAACAACGCCCAT

[0180] SEQ ID NO: 22

[0181]

[0182] SEQ ID NO:23

[0183]

[0184] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples, but it will be understood by those skilled in the art that the following drawings and examples are intended only to illustrate the present invention and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art based on the following detailed description of the accompanying drawings and preferred embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0185] Figure 1A The construction strategy of the recombinant virus OVN is shown.

[0186] Figure 1B The construction strategy of the recombinant virus OVH is shown.

[0187] Figure 2 Schematic representation of recombinant viruses HSV1716, NV1020, G207, OncoVex GM-CSF (T-VEC) and OVN contained genomic modifications; wherein, the symbol "×" indicates a deletion.

[0188] Figure 3 The schematic diagram shows the genome modifications contained in the recombinant viruses OVN, OVH and dICP0 compared with the virus strain KOS; wherein the symbol "×" represents a deletion.

[0189] Figure 4 The gel electrophoresis results of the products obtained by PCR using the genome of the virus strain KOS, OVN, OVH or dICP0 as a template and primers that specifically amplify the ICP0 gene, ICP34.5 gene, ICP27 gene or hTERT core promoter are shown.

[0190] Figure 5 The experimental results of real-time quantitative PCR analyzing IE gene expression (mRNA) of KOS, OVN, OVH or dICP0 are shown.

[0191] Figure 6 L-O2 cells were infected with KOS, OVN, OVH or dICP0 viruses at a multiplicity of infection of MOI=1 ( Figure 6 A) or U-2OS cells ( Figure 6 B) Virus titer after 48 h.

[0192] Figure 7 The viral titers at different time points (12 h, 24 h, 36 h, 48 h and 60 h after infection) after the monolayer of U-2OS cells was infected with the virus KOS, OVN, OVH or dICP0 at a multiplicity of infection of MOI = 0.01 are shown. Figure 7The results showed that the viruses KOS, OVN, OVH or dICP0 had essentially equivalent replication capabilities in tumor cells (e.g., U-2OS cells).

[0193] Figure 8 L-O2 cells were infected with KOS, OVN, OVH or dICP0 viruses at a multiplicity of infection of MOI=1 ( Figure 8 A) or U-2OS cells ( Figure 8 B) Cell viability after 72 h; MOCK represents cells not infected with the virus. Figure 8 The results showed that the viruses KOS, OVN, OVH and dICP0 had essentially equivalent cell-killing abilities against tumor cells (e.g., U-2OS cells), but the viruses OVN and OVH had significantly lower cell-killing abilities against normal cells (e.g., L-O2 cells) than those of viruses KOS and dICP0.

[0194] Figure 9 The cell survival rate of various tumor cells 48 hours after infection with the virus OVN or OVH is shown; among them, MOCK represents tumor cells that are not infected with the virus. Figure 9 The experimental results show that the recombinant viruses OVN and OVH can significantly kill a variety of tumor cells.

[0195] Figure 10 Shown are the survival rates of mice after intracranial injection of KOS, dICP0, OVN, or OVH viruses at the indicated doses; where Vehicle represents mice that were not injected with the virus.

[0196] Figure 11 Shown are the tumor volume-time curves of nude mice inoculated with Huh7 cells after treatment with OVN or OVH ( Figure 11 A) and survival rate-time curve ( Figure 11 B); DMEM represents mice that did not receive treatment.

[0197] Figure 12 Shows the left side tumor of the back of normal mice (C57BL / 6) inoculated with Hepa1-6 cells after receiving OVN or OVH treatment ( Figure 12 A) and a tumor on the right side of the back ( Figure 12 B) Tumor volume-time curve of mice receiving no treatment.

[0198] Figure 13The differences in the genomic structures of the recombinant viruses OVH, OVH1 and OVH2 and the recombinant virus OVN are schematically shown; among them, compared with the recombinant virus OVN, the native promoter sequence of the ICP27 gene of the recombinant virus OVH, the native promoter sequence of the VP5 gene of the recombinant virus OVH1, and the native promoter sequence of the ICP4 gene of the recombinant virus OVH2 are respectively replaced with the hTERT core promoter sequence.

[0199] Figure 14 The differences in the genomic structures of the recombinant viruses d34.5 / 0lacZ, OVN, OVN-GFP, OVN-PD-1-scfv, OVH-GFP, and OVH-PD-1-scfv are schematically shown; "NULL" indicates a deletion.

[0200] Figure 15 Shown are the results of fluorescence microscopy observation of U-2OS cells infected with the recombinant virus OVN-GFP or OVH-GFP.

[0201] Figure 16 The results show that after 24h or 48h infection of U-2OS cells with recombinant virus OVH or OVH-PD-1-scfv, the cell supernatant can inhibit the specific binding of PD-1 / PD-L1 ( Figure 16 A) and the interaction between cell supernatant and PD-1 protein ( Figure 16 B) Analysis results; wherein, MOCK represents tumor cells not infected with the virus.

[0202] Figure 17 Shows the left side tumor of the back of normal mice (C57BL / 6) inoculated with Hepa1-6 cells after treatment with OVH or OVH-PD-1-scfv ( Figure 17 A) and a tumor on the right side of the back ( Figure 17 B) Tumor volume-time curve of the mouse; Vehicle represents the mouse that did not receive treatment.

[0203] Figure 18 The viral titers are shown after U-2OS cells were infected with viruses OVN, OVN-dUL41, OVN-dUL43, OVN-dUL48, OVN-dUL55, OVN-dUS2, OVN-dLAT or OVN-dNF at a multiplicity of infection of MOI = 0.01 for 60 hours.

[0204] Figure 19The cell survival rates after 72h of infection with viruses OVN, OVN-dUL41, OVN-dUL43, OVN-dUL48, OVN-dUL55, OVN-dUS2, OVN-dLAT or OVN-dNF at MOI = 0.5, respectively, on normal cells (L-O2 cells; Figure 19 A) or tumor cells (U-2OS cells; Figure 19 B) are shown. DETAILED DESCRIPTION

[0205] The present application will now be described with reference to the following examples, which are intended to illustrate the application (but not to limit it).

[0206] Unless otherwise indicated, the experimental methods of molecular biology, virology, immunodetection, and zoology used in the present application are those conventional methods used by those skilled in the art. For example, the experimental methods of molecular biology can be performed according to the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, 1989, and F. M. Ausubel et al., Short Protocols in Molecular Biology, 3rd Ed., John Wiley & Sons, Inc., 1995. The reagents used in the examples (e.g., enzymes, plasmids, and primers) were purchased from commercial companies, and the various reagents (e.g., enzymes) were used according to the conditions recommended by the product manufacturer. Those skilled in the art will appreciate that the examples describe the present application by way of illustration, and are not intended to limit the scope of the present application as claimed.

[0207] Example 1. Construction of recombinant viruses OVN and OVH

[0208] (1.1) Culture and titer determination of herpes simplex virus type I (HSV-1)

[0209] The wild-type HSV-1 strain KOS was purchased from the American Type Culture Collection (ATCC) (Cat. No. VR-1493 TM ) and its whole genome information has been published in NCBI (GenBank: JQ673480.1). Cultured Vero cells (purchased from the American Type Culture Collection (ATCC) (Cat. No. CCL-81 TM ) were infected with the strain KOS at MOI = 0.1. After 48h, all the cells were collected with a cell scraper and centrifuged to remove the cell culture medium. The obtained cell pellet was resuspended in fresh complete medium and stored at -80°C. Subsequently, the cell suspension was subjected to repeated freeze-thawing (3 times) and then centrifuged to collect the supernatant, obtaining the virus solution. The virus solution was aliquoted and stored at -80°C.

[0210] The virus solution was diluted to 1 x 10 6U-2OS cells (purchased from ATCC, USA, catalog number HTB-96) were cultured at a density of 10 cells. TM ) were inoculated into a 6cm culture plate. After the cells grew into a monolayer, the virus solution obtained as above was serially diluted 10-fold gradients, and then the cells were infected with the virus solution (500μl) of each dilution gradient. After 75 minutes of infection, the cell culture medium was discarded, 5mL of fresh complete culture medium was added, and the cells were continued to be cultured. After 2 hours, 10mL of methylcellulose culture medium was added, and the culture plate was placed in an incubator and cultured for 2 days. Subsequently, a basic culture medium containing 0.01% neutral red was added to the culture plate, and the culture was continued for 12 hours. After the culture was completed, all cell culture medium was discarded, and the plaques on each culture plate were counted. According to the number of plaques on each culture plate and the dilution multiple of the virus solution, the virus titer was calculated according to the following formula: Virus titer (PFU / mL) = number of plaques per plate × 2 × virus dilution multiple.

[0211] (1.2) Construction of recombinant plasmid

[0212] Using restriction endonucleases SacI and PstI, the sequence (SEQ ID NO: 1) between base 33 (nt33) and base 5876 (nt5876) of the wild-type HSV-1 viral genome (GenBank: JQ673480.1) was cloned into the commercially available PUC57 vector (Shanghai Biotechnology) to obtain the plasmid PUC57-F0. Subsequently, using restriction endonucleases NcoI and SalI, the sequence between the NcoI and SalI restriction sites in the plasmid PUC57-F0 was replaced with the gene sequence of LacZ (SEQ ID NO: 2) to obtain the plasmid PUC57-d34.5 / 0lacZ. In addition, the sequence between the NcoI and SalI restriction sites in the plasmid PUC57-F0 was excised to obtain the PUC57-d34.5 / 0 plasmid.

[0213] (1.3) Construction and identification of recombinant viruses OVN and OVH

[0214] The construction strategies of recombinant viruses OVN and OVH are as follows: Figures 1A-1B shown.

[0215] (1.3.1) Construction of recombinant virus d34.5 / 0lacZ

[0216] 1×10 per well 5U-2OS cells were seeded in 24-well plates at a density of 1 x 105cells per well and incubated overnight at 37°C in a cell incubator. Recombinant plasmid PUC57-d34.5 / 0lacZ was transfected into U-2OS cells using transfection reagent lipofectamine 2000. After 24 h of transfection, cells were infected with viral strain KOS at a multiplicity of infection (MOI) of 3. After cytopathic effect was observed, cells were harvested. The harvested cells were lysed by repeated freeze-thawing, then centrifuged, and the supernatant was collected to obtain a virus solution. The virus titer of the obtained virus solution was determined.

[0217] The harvested virus was inoculated into culture plates in which U-2OS cells had been grown into a monolayer. After 2 days of incubation, the culture plates were uniformly added with basal medium containing 0.01% neutral red, 100 ug / mL X-gal, and the cells were further incubated for 12 hr. Subsequently, blue plaques that appeared on the culture plates were selected, and the virus obtained from the blue plaques was subjected to monoclonalization (3 times) to obtain recombinant virus d34.5 / 0lacZ. Sequencing verification confirmed that both copies of the ICP34.5 and ICP0 genes in the genome of the recombinant virus d34.5 / 0lacZ were replaced with the lacZ gene compared to viral strain KOS.

[0218] (1.3.2) Construction of recombinant virus OVN(d34.5 / 0)

[0219] U-2OS cells were seeded in 24-well plates at a density of 1 x 105 5 U-2OS cells were seeded in 24-well plates at a density of 1 x 105cells per well and incubated overnight at 37°C in a cell incubator. Recombinant plasmid PUC57-d34.5 / 0lacZ was transfected into U-2OS cells using transfection reagent lipofectamine 2000. After 24 h of transfection, cells were infected with viral strain KOS at a multiplicity of infection (MOI) of 3. After cytopathic effect was observed, cells were harvested. The harvested cells were lysed by repeated freeze-thawing, then centrifuged, and the supernatant was collected to obtain a virus solution. The virus titer of the obtained virus solution was determined.

[0220] The harvested virus was inoculated into U-2OS cells grown in monolayer in a culture plate. After 2 days of culture, the culture plate was uniformly added with basic culture medium containing 0.01% neutral red, 100 ug / mL X-gal, and the cells were continued to be cultured for 12 hr. Subsequently, the white plaques appeared on the culture plate were selected, and the virus obtained from the white plaques was monoclonalized (3 times), thereby obtaining the recombinant virus OVN(d34.5 / 0). Sequencing verification showed that, compared with the recombinant virus d34.5 / 0lacZ, both copies of the lacZ gene in the genome of the recombinant virus OVN(d34.5 / 0) were deleted; compared with the virus strain KOS, the recombinant virus OVN(d34.5 / 0) was deleted with the sequence (SEQ ID NO: 6) in the interval of nt510 to nt5439 and the sequence (SEQ ID NO: 6) in the interval of nt120802 to nt125731 of the wild-type HSV-1 genome (GenBank: JQ673480.1).

[0221] (1.3.3) Construction of the recombinant virus OVH

[0222] The sequence (SEQ ID NO: 3) between the 112861th base (nt112861) and the 113422th base (nt113422) of the wild-type HSV-1 virus genome (GenBank: JQ673480.1) was cloned into the commercially available PUC57 vector using the restriction endonucleases SacI and PmeI, thereby obtaining the plasmid PUC57-27p0. Subsequently, the sequence (SEQ ID NO: 4) between the 113590th base (nt113590) and the 115194th base (nt115194) of the wild-type HSV-1 virus genome (GenBank: JQ673480.1) was cloned into the plasmid PUC57-27p0 using the restriction endonucleases SpeI and PstI, thereby obtaining the plasmid PUC57-27p1. In the plasmid PUC57-27p1, the native promoter sequence of the ICP27 gene (nt113422 to nt113590 of the wild-type HSV-1 genome (GenBank: JQ673480.1)) has been deleted.

[0223] Subsequently, the sequence between the Pmel and Spel enzyme cutting sites in plasmid PUC57-27p1 was replaced with a LacZ expression sequence (SEQ ID NO: 2), thereby obtaining plasmid PUC57-27p / lacZ. In addition, the sequence between the Pmel and Spel enzyme cutting sites in plasmid PUC57-27p1 was also replaced with a core promoter sequence of human telomerase reverse transcriptase hTERT (SEQ ID NO: 5; see, Takakura M, Kyo S, Kanaya T, et al. Cloning of human telomerase catalytic subunit (hTERT) gene promoter and identification of proximal core promoter sequences essential for transcriptional activation in immortalized and cancer cells [J]. Cancer Res, 1999, 59(3):551-557), thereby obtaining plasmid PUC57-27p / htert. In plasmid PUC57-27p / htert, the ICP27 gene is regulated by a tumor-specific promoter (i.e., the hTERT core promoter).

[0224] Subsequently, referring to the construction methods described in (1.3.1) and (1.3.2), the hTERT core promoter sequence was introduced into the genome of recombinant virus OVN for regulating the ICP27 gene, using plasmids PUC57-27p / lacZ and PUC57-27p / htert, starting from recombinant virus OVN, thereby obtaining recombinant virus OVH. Sequencing verification showed that, compared with recombinant virus OVN, the native promoter sequence of the ICP27 gene (nt 113423 to nt 113589 of wild-type HSV-1 genome (GenBank: JQ673480.1)) in the genome of recombinant virus OVH has been replaced with the hTERT core promoter sequence (SEQ ID NO: 5).

[0225] (1.4) Comparison of recombinant viruses OVN and OVH with known recombinant HSV viruses

[0226] Currently, a variety of recombinant HSV viruses intended for tumor treatment have been developed, including, for example, HSV1716, NV1020, G207, OncoVex GM-CSF (T-VEC), etc. The genomic modifications contained in each of these recombinant HSV viruses and the recombinant viruses OVN and OVH of the present application are summarized in Table 2 below.

[0227] Table 2: Genomic modifications contained in various recombinant HSV viruses

[0228]

[0229] Figure 2 Figure 1 schematically illustrates the genomic modifications contained in the recombinant viruses HSV1716, NV1020, G207, OncoVex GM-CSF (T-VEC) and OVN, as compared to the viral strain KOS.

[0230] Example 2. Characterization of recombinant viruses OVN and OVH

[0231] Using the method described in Example 1, a recombinant virus dICP0 was constructed, which lacks double copies of the ICP0 gene as compared to the viral strain KOS. The viral strain KOS and the recombinant virus dICP0 were used as control viruses to characterize the recombinant viruses OVN and OVH. Figure 3 Figure 2 schematically illustrates the genomic modifications contained in the recombinant viruses OVN, OVH and dICP0 as compared to the viral strain KOS; wherein the recombinant virus dICP0 lacks double copies of the ICP0 gene as compared to the viral strain KOS; the recombinant virus OVN lacks double copies of the ICP34.5 gene and the ICP0 gene; the recombinant virus OVN lacks double copies of the ICP34.5 gene and the ICP0 gene, and the native promoter of the ICP27 gene is replaced by the hTERT core promoter.

[0232] The gene deletions in the recombinant viruses OVN, OVH and dICP0 were verified by PCR method. Briefly, the genome of the viral strain KOS, OVN, OVH or dICP0 was used as template for PCR with primers specific for amplifying the ICP0 gene, the ICP34.5 gene, the ICP27 gene or the hTERT core promoter, respectively. The primers used in the PCR are summarized in Table 3.

[0233] Table 3: Primer sequences

[0234]

[0235]

[0236] After the reaction, the PCR products were detected by gel electrophoresis. The results are shown in Figure 3. Figure 4 Figure 4 ​Gel electrophoresis results of products obtained by PCR using primers specific to amplify ICP0 gene, ICP34.5 gene, ICP27 gene or hTERT core promoter, respectively, with the genome of viral strain KOS, OVN, OVH or dICP0 as template; wherein lane 1 represents DNA molecular weight marker; lane 2 represents PCR product with viral strain KOS genome as template; lane 3 represents PCR product with genome of recombinant virus dICP0 as template; lane 4 represents PCR product with genome of recombinant virus OVN as template; lane 5 represents PCR product with genome of recombinant virus OVH as template; lane 6 represents PCR product with water as template.

[0237] Figure 4 The results show that the genome of viral strain KOS contains ICP0 gene, ICP34.5 gene and ICP27 gene, but does not contain hTERT core promoter; the genome of recombinant virus dICP0 contains ICP34.5 gene and ICP27 gene, but does not contain ICP0 gene and hTERT core promoter; the genome of recombinant virus OVN contains ICP27 gene, but does not contain ICP34.5 gene, ICP0 gene and hTERT core promoter; the genome of recombinant virus OVH contains ICP27 gene and hTERT core promoter, but does not contain ICP34.5 gene and ICP0 gene.

[0238] In addition, the gene expression (mRNA) after KOS, OVN, OVH or dICP0 infection of cells was also analyzed using real-time quantitative PCR. Briefly, host cells U-2 OS were infected with KOS, OVN, OVH and dICP0, respectively. After the cells were pathologically changed, the cells were harvested and total mRNA was extracted. The total mRNA was reverse transcribed into cDNA, and real-time quantitative PCR was performed using primers specific to amplify ICP0 gene, ICP34.5 gene or ICP27 gene, respectively. The results are shown in Figure 5

[0239] Figure 5 The experimental results of real-time quantitative PCR for analyzing IE gene expression (mRNA) of KOS, OVN, OVH or dICP0 are shown. Figure 5 ​The results showed that the virus strain KOS was able to express the ICP0 gene, ICP34.5 gene, and ICP27 gene; the recombinant virus dICP0 was able to express the ICP34.5 gene and ICP27 gene, but not the ICP0 gene; the recombinant viruses OVN and OVH were both able to express the ICP27 gene, but not the ICP34.5 gene and ICP0 gene. This indicates that the recombinant virus dICP0 has deleted both copies of the ICP0 gene and cannot express the ICP0 protein after infecting the host cell; and the recombinant viruses OVN and OVH have both deleted both copies of the ICP34.5 gene and ICP0 gene, and cannot express the ICP0 protein and ICP34.5 protein after infecting the host cell.

[0240] Example 3. Evaluation of the replication and killing abilities of recombinant viruses OVN / OVH

[0241] 5-7.5×10 6 Normal cells (L-O2 cells) and tumor cells (U-2OS cells) in good condition and in the logarithmic growth phase were inoculated into 6 cm culture plates at a density of cells / plate. Subsequently, the cultured cells were infected with the virus KOS, OVN, OVH or dICP0 at an infection multiplicity of MOI=1. After 48 hours of infection, the state of the cells was observed under a microscope and photographed for record. Subsequently, the virus-infected cells were digested, and the cell survival rate was calculated using the trypan blue staining method. Cell survival rate (%) = (number of living cells after virus infection) / (number of control cells not infected with the virus) × 100. Each group of experiments was repeated for 3 wells, and the experimental results were the average of 3 independent experiments. In addition, referring to the scheme described in Example 1, the virus titers at different time points after normal cells (L-O2 cells) and tumor cells (U-2OS cells) were infected with the virus KOS, OVN, OVH or dICP0 were determined. Each group of experiments was repeated for 3 wells, and the experimental results were the average of 3 independent experiments. The experimental results are shown in Figure 2. Figures 6-8 shown.

[0242] Figure 6 L-O2 cells were infected with KOS, OVN, OVH or dICP0 viruses at a multiplicity of infection of MOI=1 ( Figure 6 A) or U-2OS cells ( Figure 6 B) Virus titer after 48 h. Figure 6 The results of A showed that after infecting L-O2 cells, both viruses KOS and dICP0 replicated at high levels, and the virus titers of the two reached approximately 1.94×10 7 and 3.01×10 6 pfu / ml; however, the replication ability of viruses OVN and OVH decreased significantly, and the virus titers of the two were only about 2.14×105 and 1.85 x 10 3 pfu / ml. Figure 6 The results of B show that, after infecting U-2OS cells, viruses KOS, OVN, OVH and dICP0 all replicate at high levels, with their viral titers being 1.01-1.83 x 10 8 pfu / ml after 48h of infection. These results show that viruses KOS and dICP0 are able to replicate at high levels in both normal cells (e.g., L-O2 cells) and tumor cells (e.g., U-2OS cells); while viruses OVN and OVH are only able to replicate at high levels in tumor cells (e.g., U-2OS cells) (their replication ability is only slightly decreased compared to viruses KOS and dICP0), and their replication ability in normal cells (e.g., L-O2 cells) is significantly decreased. For example, in L-O2 cells, the replication ability of viruses OVN and OVH is decreased by about 90-fold and 10 4 fold, respectively, compared to virus KOS; and the replication ability of viruses OVN and OVH is decreased by about 14-fold and 1.6 x 10 3 fold, respectively, compared to virus dICP0.

[0243] Figure 7 The viral titers at different time points (12h, 24h, 36h, 48h and 60h post-infection) after infecting monolayer U-2OS cells with viruses KOS, OVN, OVH or dICP0 at a multiplicity of infection of MOI = 0.01 are shown. Figure 7 The results of B show that viruses KOS, OVN, OVH or dICP0 have substantially comparable replication abilities in tumor cells (e.g., U-2OS cells).

[0244] Figure 8 The cell survival rates after infecting L-O2 cells ( Figure 8 A) or U-2OS cells ( Figure 8 B) with viruses KOS, OVN, OVH or dICP0 at a multiplicity of infection of MOI = 1 for 72h are shown; where, MOCK represents cells not infected with viruses. Figure 8 The results of A show that, after 72h of infection, viruses KOS and dICP0 have a killing of about 91.5% and 89% on L-O2 cells, respectively (both have a significant killing on L-O2 cells); viruses OVN and OVH have a killing of about 42% and 5% on L-O2 cells, respectively (both have a significantly decreased killing ability on L-O2 cells). Figure 8The results of B show that, 72 h after infection, viruses KOS, OVN, OVH and dICP0 all can kill U-2OS cells at 100% (have very high killing ability to U-2OS cells). These results show that viruses KOS and dICP0 have very high killing activity to both normal cells (e.g. L-O2 cells) and tumor cells (e.g. U-2OS cells); while viruses OVN and OVH have high killing activity to tumor cells (e.g. U-2OS cells) (their killing ability is basically the same as viruses KOS and dICP0), but the killing ability to normal cells (e.g. L-O2 cells) is significantly reduced. For example, compared with virus KOS, the killing ability of viruses OVN and OVH is reduced by about 54.1% and 94.5%, respectively; compared with virus dICP0, the killing ability of viruses OVN and OVH is reduced by about 52.8% and 94.4%, respectively.

[0245] Figures 6-8 The experimental results of Table 1 show that, compared with wild-type virus KOS and recombinant virus dICP0, the replication ability and killing ability of recombinant viruses OVN and OVH in normal cells are significantly reduced, while the replication ability and killing ability in tumor cells are basically the same (or only slightly reduced). This shows that the recombinant viruses OVN and OVH of the present application not only can maintain high replication ability and high killing ability in tumor cells (have good anti-tumor activity), but also have significantly reduced virulence to normal cells. Therefore, the recombinant viruses OVN and OVH of the present application can be used for anti-tumor treatment, and have higher safety to normal cells, and can be used at a higher dose.

[0246] In addition, the killing ability of recombinant viruses OVN and OVH to various tumor cells was also determined by referring to the method as described above. Briefly, tumor cells in good condition in the logarithmic growth phase were inoculated in 6 cm culture plates at a density of 5-7.5 x 10 6 cells / plate. Subsequently, the cultured tumor cells were infected with viruses OVN or OVH at a multiplicity of infection (MOI) of 1. 48 h after infection, the tumor cells infected with viruses were digested, and the survival rate of tumor cells was calculated by trypan blue staining method. In this experiment, cells without virus infection were used as a control. Cell survival rate (%) = (number of living cells after virus infection) / (number of control cells without virus infection) x 100. Each group of experiments was set up in triplicate, and the experimental results were the average of 3 independent experiments. The experimental results are shown in Table 2. Figure 9

[0247] Figure 9 Table 2 shows the cell survival rate after various tumor cells were infected with viruses OVN or OVH for 48 h; wherein, MOCK represents tumor cells without virus infection. Figure 9 ​The experimental results showed that the recombinant viruses OVN and OVH can significantly kill a variety of tumor cells, including lung cancer cells H1299, H520, H1975, NCI-H358 and A549 (5 strains); liver cancer cells Huh7, Hep3B, HepG2, GSG7701, SMMC7721, Hepa1-6, BEL7404, PLC / PRF, QGY7703 (9 strains); breast cancer cells MADMB231, MCF7 , MADMB468 (3 strains); osteosarcoma cells U2OS and SAOS2 (2 strains); ovarian cancer cells SKOV3 and CAOV3 (2 strains); cervical cancer cells SiHA and Hela (2 strains); prostate cancer cells PC-3 (1 strain); glioma cells U87MG (1 strain); melanoma A375 (1 strain); colorectal cancer HCT116 (1 strain) and pancreatic cancer Panc1 (1 strain); and the tumor killing ability of OVN and OVH is comparable. These experimental results show that the recombinant viruses OVN and OVH of the present invention have good killing activity against various tumor cells and can be used for tumor treatment.

[0248] Example 4. Evaluation of neurotoxicity and in vivo safety of recombinant virus OVN / OVH

[0249] Herpes simplex virus has neurotoxicity and neurolatency, and its greatest harm is that it can infect the central nervous system of humans or animals, causing serious side effects such as encephalitis. Therefore, the most direct and sensitive way to evaluate the safety of herpes simplex virus is to inject the virus into the brain of young mice and assess the direct damage of the virus to the central nervous system of mice. In this embodiment, we utilized the mouse encephalitis model induced by intracranial injection of the virus to evaluate the neurotoxicity and safety of various recombinant HSV-1 viruses to mice.

[0250] Briefly, 4-6 week-old female BALB / c mice (n=10) were used as experimental subjects. 20 μl of virus was slowly injected intracranially into the left anterior lobe of the brain, near the junction of the coronal and sagittal sutures. After injection, the mice were observed daily for morbidity and survival. Figure 10 Shown are the survival rates of mice after intracranial injection of KOS, dICP0, OVN, or OVH viruses at the indicated doses; where Vehicle represents mice that were not injected with the virus.

[0251] The experimental results showed that when 1×10 4 When PFU wild-type virus KOS is injected, 100% of mice will experience moderate to severe side effects; after the onset of the disease, mice often have symptoms such as piloerection, anorexia, fear of cold, slow movement and even paralysis; and 100% of mice will die within 4-6 days after virus injection ( Figure 10 ). When 1×10 5When the virus dICP0 was injected at a high dose (1 x 10

[0252] When the virus OVN was injected intracranially at a high dose (1 x 10 7 PFU), no mice (0 / 10) died during the whole experimental period, and the survival rate of the mice was 100%. This indicates that the neurotoxicity of the virus OVN is significantly reduced, and the in vivo safety is significantly improved, compared with the wild-type virus KOS and the recombinant virus dICP0, and the use dose can be increased by at least 1000 times and 100 times, respectively.

[0253] When the virus OVN was injected intracranially at a higher dose (4 x 10 7 PFU), only one mouse (1 / 10) died during the whole experimental period, and the survival rate of the mice was 90%. This indicates that the virus OVN has a high median lethal dose of 4 x 10 7 PFU for mice, and has excellent in vivo safety.

[0254] When the virus OVH was injected intracranially at a dose of 4 x 10 7 PFU, no mice (0 / 10) died during the whole experimental period, and the survival rate of the mice was 100%. Moreover, and more importantly, the mice did not show any adverse reactions during the whole experimental period. This indicates that the neurotoxicity of the virus OVH is further significantly reduced, and the in vivo safety is further significantly improved, compared with the virus OVN.

[0255] The above experimental results show that the viruses OVN and OVH of the present application have low neurotoxicity, high in vivo safety, and have broad application prospects.

[0256] Example 5. Evaluation of the therapeutic potential of the recombinant viruses OVN / OVH

[0257] Tumor cells (Huh7 and Hepa1-6) were cultured in complete medium containing 10% calf serum in an incubator at 37°C with 5% CO2. When the cells grew to the logarithmic growth phase, the cells were digested with 0.05% trypsin and washed with PBS to obtain a cell suspension (cell density of 5 x 10 7 6 / mL) resuspended in PBS.

[0258] 0.1 mL of the Huh7 cell suspension was taken and inoculated subcutaneously on the right side of the back of 5-6-week-old nude mice (n = 8 / group). When the tumors on the back of the mice grew to 6 mm x 6 mm (tumor volume of about 100 mm 3 ), the mice were grouped (n = 8 / group), and treatment was started (day 0). The treatment regimen was as follows: intratumoral injection of 1 x 10 7PFU virus (OVN or OVH) or the same volume of DMEM (used as a control) was injected once every 3 days for a total of 3 injections.

[0259] Take 0.1mL of Hepa1-6 cell suspension and inoculate it subcutaneously on the left and right sides of the back of 5-week-old normal mice (C57BL / 6). When the tumor on the back of the mouse grows to 6mm×6mm (the tumor volume is about 100mm 3 ) were divided into groups and treatment was started. The treatment regimen was as follows: 1×10 7 PFU virus (OVN or OVH) or the same volume of DMEM was injected once every 3 days for a total of 3 injections.

[0260] The mice were monitored every 3 days and the tumor size was measured with an electronic vernier caliper. The tumor volume and tumor inhibition rate were calculated according to the following formula:

[0261] V(volume)=[L×(W) 2 ] / 2; L represents the major diameter, and W represents the minor diameter.

[0262] Tumor inhibition rate = (tumor volume of control group - tumor volume of experimental group) / tumor volume of control group × 100%.

[0263] The experimental results are summarized in Figures 11-12 middle.

[0264] Figure 11 Shown are the tumor volume-time curves of nude mice inoculated with Huh7 cells after treatment with OVN or OVH ( Figure 11 A) and survival rate-time curve ( Figure 11 B); DMEM represents mice that did not receive treatment. Figure 11 The results of A showed that after three virus injections, the recombinant viruses OVN and OVH significantly inhibited tumor growth; on the 21st day, the tumor inhibition rate of OVN reached 86.1%, and the tumor inhibition rate of OVH reached 78%. Figure 11 The results of B showed that after three virus injections, the recombinant viruses OVN and OVH significantly prolonged the survival time of tumor-bearing nude mice; on the 60th day, the nude mice in the control group died completely, while the nude mice that received the recombinant viruses OVN or OVH still had a survival rate of 75% after the end of the entire experiment.

[0265] Figure 12 Shows the left side tumor of the back of normal mice (C57BL / 6) inoculated with Hepa1-6 cells after receiving OVN or OVH treatment ( Figure 12 A) and a tumor on the right side of the back ( Figure 12 B) Tumor volume-time curve of mice receiving no treatment. Figure 12The results show that after three times of virus injection, the recombinant viruses OVN and OVH not only safely eliminated the tumor on the right side of the mouse back, but also eliminated the tumor on the left side of the back.

[0266] These experimental results demonstrate that the viruses OVN and OVH of the present application have significant potential for treating tumors in vivo, and have broad application prospects.

[0267] Example 6. Construction and characterization of other recombinant viruses (1)

[0268] In this example, based on the recombinant viruses OVN and OVH, a series of derivative recombinant viruses were constructed.

[0269] Referring to the methods described in Example 1 (especially 1.3.1-1.3.3), using the recombinant virus OVN as the starting virus, the hTERT core promoter sequence was introduced into the genome of the recombinant virus OVN using a recombinant plasmid to regulate the VP5 gene or the ICP4 gene, thereby obtaining the recombinant viruses OVH1 and OVH2.

[0270] Sequencing verification showed that compared with the recombinant virus OVN, in the genome of the recombinant virus OVH1, the native promoter sequence of the VP5 gene (nt40729-nt40475 of the wild-type HSV-1 genome (GenBank: JQ673480.1)) has been replaced by the hTERT core promoter sequence (SEQ ID NO: 5); in the genome of the recombinant virus OVH2, the native promoter sequence of the ICP4 gene (nt146151-nt146867 and nt131706-nt130990 of the wild-type HSV-1 genome (GenBank: JQ673480.1)) has been replaced by the hTERT core promoter sequence (SEQ ID NO: 5).

[0271] Figure 13 The genomic structure differences between the recombinant viruses OVH, OVH1 and OVH2 and the recombinant virus OVN are schematically shown; wherein compared with the recombinant virus OVN, the native promoter sequence of the ICP27 gene of the recombinant virus OVH, the native promoter sequence of the VP5 gene of the recombinant virus OVH1, and the native promoter sequence of the ICP4 gene of the recombinant virus OVH2 are replaced by the hTERT core promoter sequence, respectively.

[0272] In addition, referring to the method described in Example 1 (in particular, 1.3.1-1.3.3), using the recombinant virus d34.5 / 0lacZ as the starting virus, the nucleotide sequence encoding the GFP protein (SEQ ID NO: 7) and the nucleotide sequence encoding the anti-human PD-1 single-chain antibody (SEQ ID NO: 8) were introduced into the genome of the recombinant virus d34.5 / 0lacZ and replaced the lacZ gene, respectively, by using the recombinant plasmid, thereby obtaining the recombinant viruses OVN-GFP and OVN-PD-1-scfv. Sequencing verification showed that, compared with the recombinant virus d34.5 / 0lacZ, in the genome of the recombinant virus OVN-GFP, the two copies of the lacZ gene were replaced by the nucleotide sequence encoding the GFP protein (i.e., in the genome of the recombinant virus OVN-GFP, the sequence in the interval of nt510 to nt5439 and the sequence in the interval of nt120802 to nt125731 of the wild-type HSV-1 genome (GenBank: JQ673480.1) were replaced by the nucleotide sequence encoding the GFP protein); in the genome of the recombinant virus OVN-PD-1-scfv, the two copies of the lacZ gene were replaced by the nucleotide sequence encoding the PD-1 single-chain antibody (i.e., in the genome of the recombinant virus OVN-PD-1-scfv, the sequence in the interval of nt510 to nt5439 and the sequence in the interval of nt120802 to nt125731 of the wild-type HSV-1 genome (GenBank: JQ673480.1) were replaced by the nucleotide sequence encoding the PD-1 single-chain antibody).

[0273] Further, using the recombinant viruses OVN-GFP and OVN-PD-1-scfv as the starting viruses, the hTERT core promoter sequence was introduced into the genome of the starting viruses for regulating the ICP27 gene by using the plasmids PUC57-27p / lacZ and PUC57-27p / htert, thereby obtaining the recombinant viruses OVH-GFP and OVH-PD-1-scfv.

[0274] Sequencing verification showed that, compared with the recombinant virus OVN-GFP, the native promoter sequence of the ICP27 gene (nt 113423 to nt 113589 of the wild-type HSV-1 genome (GenBank: JQ673480.1)) in the genome of the recombinant virus OVH-GFP has been replaced by the hTERT core promoter sequence (SEQ ID NO: 5). Compared with the recombinant virus OVN-PD-1-scfv, the native promoter sequence of the ICP27 gene (nt 113423 to nt 113589 of the wild-type HSV-1 genome (GenBank: JQ673480.1)) in the genome of the recombinant virus OVH-PD-1-scfv has been replaced by the hTERT core promoter sequence (SEQ ID NO: 5).

[0275] Figure 14 The genomic structure differences of the recombinant viruses d34.5 / 0lacZ, OVN, OVN-GFP, OVN-PD-1-scfv, OVH-GFP and OVH-PD-1-scfv are schematically shown.

[0276] U-2OS cells (purchased from the American Type Culture Collection, item number HTB-96 6 ) were seeded in 6 cm culture plates at a density of 1 x 10 TM After the cells grew into a monolayer, the cells were infected with the recombinant viruses OVN-GFP and OVH-GFP, respectively. After the cells became cytopathic, the cells infected with the recombinant viruses OVN-GFP and OVH-GFP were observed under a fluorescence microscope. The results are shown in Figure 15 . Figure 15 The fluorescence microscope observation results of U-2OS cells infected with the recombinant viruses OVN-GFP or OVH-GFP are shown. Figure 15 The results show that the U-2OS cells infected with OVN-GFP and OVH-GFP can emit green fluorescence. This indicates that the recombinant viruses OVN-GFP and OVH-GFP can express GFP protein after infecting host cells.

[0277] U-2OS cells (purchased from the American Type Culture Collection, item number HTB-96 6 ) were seeded in 6 cm culture plates at a density of 1 x 10 TM) were inoculated in 6 cm culture plates. After the cells grew into a monolayer, the cells were infected with the recombinant viruses OVH and OVH-PD-1-scfv, respectively. The culture supernatants of the cells were harvested at 24 hours and 48 hours after the infection, respectively, for subsequent detection. The supernatants collected at 48 hours after the infection were serially diluted by 2-fold gradient, and the ability of each dilution of the supernatant to inhibit the interaction between PD-1 and PD-L1 was detected by an ELISA method based on the competition between the PD-1 single-chain antibody and the PD-L1 protein for binding to the PD-1 protein. In addition, the mutual binding ability between the supernatants collected at 24 hours and 48 hours after the infection and the PD-1 protein was determined by an ELISA method based on the reactivity between the PD-1 single-chain antibody and the PD-1 protein. The experimental results are shown in Figure 16 .

[0278] Figure 16 The results show the ability of the cell supernatant after the U-2OS cells were infected with the recombinant viruses OVH or OVH-PD-1-scfv for 24 hours or 48 hours to inhibit the specific binding of PD-1 / PD-L1 Figure 16 A) and the mutual interaction between the cell supernatant and the PD-1 protein Figure 16 B). MOCK represents tumor cells not infected with the viruses. Figure 16 The results show that the supernatant of the U-2OS cells infected with OVH-PD-1-scfv can inhibit the specific binding of PD-1 / PD-L1 and can specifically bind to PD-1. This indicates that the recombinant virus OVH-PD-1-scfv expresses the PD-1 single-chain antibody after infecting the host cells, which can bind to PD-1 and block the interaction between PD-1 / PD-L1.

[0279] Figures 15-16 The experimental results shown in indicate that the genomes of the recombinant viruses OVN and OVH of the present application can be used as viral vectors for carrying and expressing exogenous genes.

[0280] In addition, the ability of the recombinant viruses OVH and OVH-PD-1-scfv to treat tumors was also verified in normal mice (C57BL / 6) inoculated with Hepa1-6 cells according to the method described in Example 5. The results are shown in Figure 17 .

[0281] Figure 17 The tumor volume-time curves of the left dorsal tumor Figure 17 A) and the right dorsal tumor Figure 17 B) of normal mice (C57BL / 6) inoculated with Hepa1-6 cells after receiving OVH or OVH-PD-1-scfv treatment are shown. Vehicle represents mice not receiving treatment.Figure 17 The results show that after three virus injections, the recombinant viruses OVH and OVH-PD-1-scfv not only safely eliminated the tumor on the right side of the back of the mouse, but also eliminated the tumor on the left side of the back.

[0282] These experimental results demonstrate that the recombinant viruses OVH-PD-1-scfv and OVH of the present application have significant potential for treating tumors in vivo, and have broad application prospects.

[0283] Example 7. Construction and characterization of other recombinant viruses (2)

[0284] In this example, based on the recombinant virus OVN, a series of derived recombinant viruses were constructed. Briefly, referring to the methods described in Example 1 (in particular 1.3.1-1.3.3), using the recombinant virus OVN as the starting virus, the non-essential genes UL41, UL43, UL48, UL55, US2, LAT or NF in the genome of the recombinant virus OVN were deleted respectively by using the recombinant plasmid, thereby obtaining the recombinant viruses OVN-dUL41, OVN-dUL43, OVN-dUL48, OVN-dUL55, OVN-dUS2, OVN-dLAT and OVN-dNF.

[0285] Sequencing verification showed that, compared with the recombinant virus OVN, in the genome of the recombinant virus OVN-dUL41, the UL41 (vhs) gene (GenBank: AFE62869.1; corresponding to nt91088-nt92557 of the wild-type HSV-1 genome (GenBank: JQ673480.1)) has been deleted; in the genome of the recombinant virus OVN-dUL43, the UL43 gene (GenBank: AFE62871.1; corresponding to nt94721-nt95968 of the wild-type HSV-1 genome (GenBank: JQ673480.1)) has been deleted; in the genome of the recombinant virus OVN-dUL48, the UL48 (VMW65) gene (GenBank: AFE62876.1; corresponding to nt103527-nt104999 of the wild-type HSV-1 genome (GenBank: JQ673480.1)) has been deleted; in the genome of the recombinant virus OVN-dUL55, the UL55 gene (GenBank: AFE62884.1; corresponding to nt115418-nt115978 of the wild-type HSV-1 genome (GenBank: JQ673480.1)) has been deleted; in the genome of the recombinant virus OVN-dUS2, the US2 gene (GenBank: AFE62890.1; corresponding to nt133911-nt134786 of the wild-type HSV-1 genome (GenBank: JQ673480.1)) has been deleted; in the genome of the recombinant virus OVN-dLAT, the LAT gene (corresponding to nt4781-nt7062 of the wild-type HSV-1 genome (GenBank: JQ673480.1)) has been deleted; and, in the genome of the recombinant virus OVN-dNF, the nucleotide fragment (NF) (corresponding to nt5853-nt7485 of the wild-type HSV-1 genome (GenBank: JQ673480.1)) has been deleted.

[0286] In addition, the non-essential genes UL41, UL43, UL48, UL55, US2, LAT or NF in the genome of the recombinant virus OVN can also be deleted using CRISPR technology, for example, by designing specific sgRNA primers and using the commercially available LentiCRISPR v2 vector (Addgene).

[0287] Information about the non-essential genes UL41, UL43, UL48, UL55, US2, LAT and NF is also provided in Table 4.

[0288] Table 4. Information of non-essential genes

[0289] Gene name GenBank No. Location in genome SEQ ID NO: UL41 (vhs) AFE62869.1 nt91088-nt92557 17 UL43 AFE62871.1 nt94721-nt95968 18 UL48 (VMW65) AFE62876.1 nt103527-nt104999 19 UL55 AFE62884.1 nt115418-nt115978 20 US2 AFE62890.1 nt133911-nt134786 21 LAT From JQ673480.1 nt4781-nt7062 22 Nucleotide fragment (NF) From JQ673480.1 nt5853-nt7485 23

[0290] 5-7.5×10 6 Tumor cells (U-2OS cells) in good condition and in the logarithmic growth phase were inoculated into 6 cm culture plates at a density of cells / plate. Subsequently, the cultured cells were infected with recombinant viruses OVN, OVN-dUL41, OVN-dUL43, OVN-dUL48, OVN-dUL55, OVN-dUS2, OVN-dLAT or OVN-dNF at a multiplicity of infection of MOI=0.01. After 60 hours of infection, the viral titer of the above-mentioned recombinant virus was determined with reference to the protocol described in Example 1. Each group of experiments was repeated in 3 wells, and the experimental results are the average of 3 independent experiments. The experimental results are shown in Figure 2. Figure 18 shown.

[0291] Figure 18 The viral titers are shown after U-2OS cells were infected with viruses OVN, OVN-dUL41, OVN-dUL43, OVN-dUL48, OVN-dUL55, OVN-dUS2, OVN-dLAT or OVN-dNF at a multiplicity of infection of MOI = 0.01 for 60 hours. Figure 18 The results showed that after infecting U-2OS cells, the viruses OVN, OVN-dUL55, OVN-dUS2, OVN-dLAT and OVN-dNF all replicated at high levels: after 60 hours of infection, their viral titers were all between 1.01-1.18×10 8 pfu / ml, 10 8 pfu / ml level; while viruses OVN-dUL43, OVN-dUL41 and OVN-dUL48 all replicated at low levels: after 60 hours of infection, their viral titers were all less than 10 7 pfu / ml, 10 4 to 10 6 The virus titers of these recombinant viruses are also shown in Table 5.

[0292] Table 5: Virus titers of U-2OS cells after 60 h of infection with recombinant viruses

[0293]

[0294]

[0295] These results show that viruses OVN, OVN-dUL55, OVN-dUS2, OVN-dLAT and OVN-dNF are able to replicate at high levels in tumor cells (e.g., U-2OS cells); while viruses OVN-dUL43, OVN-dUL41 and OVN-dUL48 have significantly reduced replication ability in tumor cells (e.g., U-2OS cells). For example, in U-2OS cells, the replication ability of viruses OVN-dUL41, OVN-dUL43 and OVN-dUL48 is reduced by about 561-fold, 55-fold and 3 x 10 3 fold, respectively, as compared to virus OVN.

[0296] In addition, cultured normal cells (L-O2 cells) or tumor cells (U-2OS cells) were also infected with recombinant viruses OVN, OVN-dUL41, OVN-dUL43, OVN-dUL48, OVN-dUL55, OVN-dUS2, OVN-dLAT or OVN-dNF at an MOI of 0.5. The cell survival rate was determined 72 h after infection. Each group of experiments was set up in triplicate, and the experimental results were the average of three independent experiments. The experimental results are shown in Figure 19 .

[0297] Figure 19 The cell survival rate of normal cells (L-O2 cells; Figure 19 A) or tumor cells (U-2OS cells; Figure 19 B) 72 h after infection with viruses OVN, OVN-dUL41, OVN-dUL43, OVN-dUL48, OVN-dUL55, OVN-dUS2, OVN-dLAT or OVN-dNF at an MOI of 0.5 is shown.

[0298] Figure 19 The results of A show that the killing rate of viruses OVN-dUL41 and OVN-dUL48 on L-O2 cells is about 17.67% and 14.33%, respectively, 72 h after infection; both of them have stronger killing ability on L-O2 cells than virus OVN. The killing rate of viruses OVN-dUL43, OVN-dUL55, OVN-dUS2, OVN-dLAT or OVN-dNF on L-O2 cells is between 8.33% and 11.00%, which has no significant difference from virus OVN.

[0299] Figure 19The results of B show that, 72 h after infection, all of the viruses OVN, OVN-dUL55, OVN-dUS2, OVN-dLAT and OVN-dNF can kill U-2OS cells at 100% (i.e., have very strong killing ability to tumor cells). The killing ability of viruses OVN-dUL41, OVN-dUL43 and OVN-dUL48 to U-2OS cells is significantly weakened.

[0300] The killing rates of these recombinant viruses to L-O2 and U-2OS cells are also provided in Table 6.

[0301] Table 6: Killing rates of recombinant viruses to L-O2 cells and U-2OS cells

[0302]

[0303]

[0304] These results show that, similar to virus OVN, viruses OVN-dUL55, OVN-dUS2, OVN-dLAT and OVN-dNF only have very limited killing activity to normal cells (e.g., L-O2 cells) and have very high killing activity to tumor cells (e.g., U-2OS cells); this indicates that these four recombinant viruses have effects similar to virus OVN. Compared with virus OVN, viruses OVN-dUL41 and OVN-dUL48 not only have increased killing ability to normal cells (e.g., L-O2 cells), but also have significantly decreased killing ability to tumor cells (e.g., U-2OS cells); this indicates that these two recombinant viruses have increased toxicity to normal cells and decreased antitumor activity. Compared with virus OVN, virus OVN-dUL43 has no significantly increased killing activity to normal cells (e.g., L-O2 cells), but has significantly decreased killing activity to tumor cells (e.g., U-2OS cells).

[0305] Specifically, compared with virus OVN, viruses OVN-dUL41, OVN-dUL43 and OVN-dUL48 have increased killing ability to normal cells by about 7.67%, 1.00% and 4.33%, respectively. Compared with virus OVN, viruses OVN-dUL41, OVN-dUL43 and OVN-dUL48 have decreased killing ability to tumor cells by about 75.13%, 51.80% and 67.46%, respectively.

[0306] The above experimental results show that, in the recombinant HSV viruses of the present application, non-essential genes (e.g., UL55, US2, LAT and NF) other than UL41, UL43 and UL48 can be further modified, e.g., introducing loss-of-function mutations therein or deleting them.

[0307] While the specific embodiments of the application have been described in detail, those skilled in the art will appreciate that various modifications and alterations to the details can be made within the scope of the application as disclosed in the above teachings and that such modifications and alterations are intended to fall within the scope of the application. The full scope of the application is set forth in the claims that follow and any equivalents thereof.

Claims

1. A recombinant HSV virus which does not express a functional ICP0 protein and a ICP34.5 protein; but is capable of expressing a functional UL43 protein, a functional UL41 protein, and a functional UL48 protein; wherein, the recombinant HSV virus is a recombinant HSV-2 virus; wherein the genome of the recombinant HSV virus comprises the following modifications: each of the two copies of the ICP0 gene independently comprises a loss-of-function mutation or is deleted or replaced with an exogenous nucleotide sequence; and, each of the two copies of the ICP34.5 gene independently comprises a loss-of-function mutation or is deleted or replaced with an exogenous nucleotide sequence.

2. The recombinant HSV virus of claim 1, wherein, In the genome of the recombinant HSV virus, (a1) one copy of the ICP0 gene comprises a loss-of-function mutation and the other copy of the ICP0 gene comprises a loss-of-function mutation or is deleted or replaced with an exogenous nucleotide sequence; or (b1) one copy of the ICP0 gene is deleted and the other copy of the ICP0 gene comprises a loss-of-function mutation or is deleted or replaced with an exogenous nucleotide sequence; or (c1) one copy of the ICP0 gene is replaced with an exogenous nucleotide sequence and the other copy of the ICP0 gene comprises a loss-of-function mutation or is deleted or replaced with an exogenous nucleotide sequence; and (a2) one copy of the ICP34.5 gene comprises a loss-of-function mutation and the other copy of the ICP34.5 gene comprises a loss-of-function mutation or is deleted or replaced with an exogenous nucleotide sequence; or (b2) one copy of the ICP34.5 gene is deleted and the other copy of the ICP34.5 gene comprises a loss-of-function mutation or is deleted or replaced with an exogenous nucleotide sequence; or (c2) one copy of the ICP34.5 gene is replaced with an exogenous nucleotide sequence and the other copy of the ICP34.5 gene comprises a loss-of-function mutation or is deleted or replaced with an exogenous nucleotide sequence.

3. The recombinant HSV virus of claim 1, wherein, each of the loss-of-function mutations is independently selected from the group consisting of addition, deletion and / or substitution of one or more bases; and / or, each of the exogenous nucleotide sequences is independently a nucleotide sequence encoding an exogenous protein.

4. The recombinant HSV virus of claim 2, wherein, each of the loss-of-function mutations is independently selected from the group consisting of addition, deletion and / or substitution of one or more bases; and / or, each of the exogenous nucleotide sequences is independently a nucleotide sequence encoding an exogenous protein.

5. The recombinant HSV virus of claim 1, wherein, the two copies of the ICP0 gene comprise the same loss-of-function mutation or different loss-of-function mutations; or are both deleted; or are replaced with the same exogenous nucleotide sequence or different exogenous nucleotide sequences.

6. The recombinant HSV virus of claim 1, wherein, the two copies of the ICP34.5 gene comprise the same loss-of-function mutation or different loss-of-function mutations; or are both deleted; or are replaced with the same exogenous nucleotide sequence or different exogenous nucleotide sequences.

7. The recombinant HSV virus of claim 1, wherein, In the genome of the recombinant HSV virus, (1) each of the two copies of the ICP0 gene independently comprises a loss-of-function mutation and each of the two copies of the ICP34.5 gene independently comprises a loss-of-function mutation; or (2) each of the two copies of the ICP0 gene independently comprises a loss-of-function mutation and the two copies of the ICP34.5 gene are deleted; or (3) each of the two copies of the ICP0 gene is deleted and each of the two copies of the ICP34.5 gene comprises a loss-of-function mutation; or (4) each of the two copies of the ICP0 gene is replaced with an exogenous nucleotide sequence and each of the two copies of the ICP34.5 gene comprises a loss-of-function mutation. (3) the two copies of the ICP0 gene each independently comprise a loss-of-function mutation, and the two copies of the ICP34.5 gene each independently are replaced with an exogenous nucleotide sequence; or (4) the two copies of the ICP0 gene are deleted; and the two copies of the ICP34.5 gene each independently comprise a loss-of-function mutation; or (5) the two copies of the ICP0 gene are deleted; and the two copies of the ICP34.5 gene are deleted; or (6) the two copies of the ICP0 gene are deleted; and the two copies of the ICP34.5 gene each independently are replaced with an exogenous nucleotide sequence; or (7) the two copies of the ICP0 gene each independently are replaced with an exogenous nucleotide sequence, and the two copies of the ICP34.5 gene each independently comprise a loss-of-function mutation; or (8) the two copies of the ICP0 gene each independently are replaced with an exogenous nucleotide sequence, and the two copies of the ICP34.5 gene are deleted; or (9) the two copies of the ICP0 gene each independently are replaced with an exogenous nucleotide sequence, and the two copies of the ICP34.5 gene each independently are replaced with an exogenous nucleotide sequence.

8. The recombinant HSV virus of claim 7, wherein, each of the loss-of-function mutations is independently selected from the group consisting of addition, deletion, and / or substitution of one or more bases; and / or each of the exogenous nucleotide sequences is independently a nucleotide sequence encoding an exogenous protein.

9. The recombinant HSV virus of claim 1, wherein, in the genome of the recombinant HSV virus, (1) a first copy of the ICP0 gene comprises a first loss-of-function mutation, a second copy of the ICP0 gene comprises a second loss-of-function mutation; and a first copy of the ICP34.5 gene comprises a third loss-of-function mutation, a second copy of the ICP34.5 gene comprises a fourth loss-of-function mutation; or (2) a first copy of the ICP0 gene comprises a first loss-of-function mutation, a second copy of the ICP0 gene comprises a second loss-of-function mutation; and the two copies of the ICP34.5 gene are deleted; or (3) a first copy of the ICP0 gene comprises a first loss-of-function mutation, a second copy of the ICP0 gene comprises a second loss-of-function mutation; and a first copy of the ICP34.5 gene is replaced with a third exogenous nucleotide sequence, a second copy of the ICP34.5 gene is replaced with a fourth exogenous nucleotide sequence; or (4) the two copies of the ICP0 gene are deleted; and a first copy of the ICP34.5 gene comprises a third loss-of-function mutation, a second copy of the ICP34.5 gene comprises a fourth loss-of-function mutation; or (5) the two copies of the ICP0 gene are deleted; and the two copies of the ICP34.5 gene are deleted; or (6) the two copies of the ICP0 gene are deleted; and a first copy of the ICP34.5 gene is replaced with a third exogenous nucleotide sequence, a second copy of the ICP34.5 gene is replaced with a fourth exogenous nucleotide sequence; or (7) the two copies of the ICP0 gene are deleted; and the two copies of the ICP34.5 gene are deleted; or (8) the two copies of the ICP0 gene are deleted; and the two copies of the ICP34.5 gene are deleted; or (9) the two copies of the ICP0 gene are deleted; and the two copies of the ICP34.5 gene are deleted; or (7) the first copy of the ICP0 gene is replaced with a first exogenous nucleotide sequence, and the second copy of the ICP0 gene is replaced with a second exogenous nucleotide sequence; and, the first copy of the ICP34.5 gene comprises a third loss-of-function mutation, and the second copy of the ICP34.5 gene comprises a fourth loss-of-function mutation; or (8) the first copy of the ICP0 gene is replaced with a first exogenous nucleotide sequence, and the second copy of the ICP0 gene is replaced with a second exogenous nucleotide sequence; and, the 2 copies of the ICP34.5 gene are deleted; or (9) the first copy of the ICP0 gene is replaced with a first exogenous nucleotide sequence, and the second copy of the ICP0 gene is replaced with a second exogenous nucleotide sequence; and, the first copy of the ICP34.5 gene is replaced with a third exogenous nucleotide sequence, and the second copy of the ICP34.5 gene is replaced with a fourth exogenous nucleotide sequence.

10. The recombinant HSV virus of claim 9, wherein, The first loss-of-function mutation, the second loss-of-function mutation, the third loss-of-function mutation, and the fourth loss-of-function mutation are each independently selected from a missense mutation, a nonsense mutation, a frameshift mutation, a base deletion, a base substitution, a base addition, and any combination thereof.

11. The recombinant HSV virus of claim 9, wherein The first exogenous nucleotide sequence, the second exogenous nucleotide sequence, the third exogenous nucleotide sequence, and the fourth exogenous nucleotide sequence each independently encodes an exogenous protein selected from a fluorescent protein, an immunomodulatory polypeptide, a cytokine, a chemokine, an antibody, and a cytotoxic peptide.

12. The recombinant HSV virus of claim 10, wherein, The first loss-of-function mutation, the second loss-of-function mutation, the third loss-of-function mutation, and the fourth loss-of-function mutation are each independently selected from a deletion or a replacement or an addition of a gene fragment.

13. The recombinant HSV virus of claim 11, wherein, The recombinant HSV virus has one or more features selected from: (1) the fluorescent protein is selected from a green fluorescent protein, a red fluorescent protein, a blue fluorescent protein, and a yellow fluorescent protein; (2) the immunomodulatory polypeptide is selected from CD40L, OX40L, inducible costimulatory molecule, FTL3L, LIGHT, CD137L, CD70, 4-1BB, GITR, and CD28; (3) the cytokine is selected from an interleukin, an interferon, a tumor necrosis factor, and a colony-stimulating factor; (4) the chemokine is selected from CCL2, RANTES, CCL7, CCL9, CCL10, CCL12, CCL15, CCL19, CCL21, CCL20, and XCL-1; (5) the cytotoxic peptide is selected from thymidine kinase TK, TRAIL, and FasL; (6) the antibody is selected from an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-TIGIT antibody, an anti-BTLA antibody, an anti-CTLA-4 antibody, an anti-Tim-3 antibody, an anti-Lag-3 antibody, an anti-CD137 antibody, an anti-OX40 antibody, an anti-GITR antibody, an anti-CD73 antibody, an anti-KIR antibody, an anti-ICOS antibody, an anti-CSF1R antibody, an anti-EGFR antibody, an anti-VEGFR antibody, an anti-HER2 antibody, and an anti-PDGFR antibody.

14. The recombinant HSV virus of any one of claims 1-13, wherein, The genome of the recombinant HSV virus comprises a UL43 gene capable of expressing a functional UL43 protein, a UL41 gene capable of expressing a functional UL41 protein, and / or a UL48 gene capable of expressing a functional UL48 protein.

15. The recombinant HSV virus of claim 14, wherein, The recombinant HSV virus further has one or more features selected from the group consisting of: (1) one or more non-essential genes are deleted or mutated; (2) the essential genes of the recombinant HSV virus are not deleted and do not comprise loss-of-function mutations; (3) the genome of the recombinant HSV virus contains all other genes of a wild-type HSV virus except for 2 copies of the ICP0 gene and 2 copies of the ICP34.5 gene, and none of the other genes comprise loss-of-function mutations; and (4) the genome of the recombinant HSV virus further comprises the modification that the native promoter of one or more HSV genes is replaced by a tumor-specific promoter.

16. The recombinant HSV virus of claim 15, wherein, The recombinant HSV virus further has one or more features selected from the group consisting of: (1) the non-essential genes are selected from the group consisting of the UL3 gene, the UL4 gene, the UL14 gene, the UL16 gene, the UL21 gene, the UL24 gene, the UL31 gene, the UL32 gene, the US3 gene, the UL51 gene, the UL55 gene, the UL56 gene, the US2 gene, the US12 gene, the LAT gene, and a nucleotide fragment corresponding to nt 5853-nt 7485 of JQ673480.1; (2) the non-essential genes comprise loss-of-function mutations or are replaced by exogenous nucleotide sequences; (3) the essential genes are selected from the group consisting of the ICP27 gene, the ICP4 gene, the VP5 gene, the gL gene, the gH gene, the gD gene, the gK gene, the gB gene, the gN gene, the UL5 gene, the UL6 gene, the UL8 gene, the UL9 gene, the UL12 gene, the UL25 gene, the UL26 gene, the UL28 gene, the UL29 gene, the UL30 gene, the UL33 gene, the UL36 gene, the UL38 gene, the UL42 gene, the UL48 gene, and the UL52 gene; and (4) the tumor-specific promoter is the hTERT promoter.

17. The recombinant HSV virus of claim 14, wherein, The genome of the recombinant HSV virus further comprises one or more modifications selected from the group consisting of: (1) the native promoter of the VP5 gene is replaced by a tumor-specific promoter; (2) the native promoter of the ICP27 gene is replaced by a tumor-specific promoter; (3) the native promoter of the ICP4 gene is replaced by a tumor-specific promoter; and (4) one or more of the UL55 gene, the US2 gene, the LAT gene, and a nucleotide fragment corresponding to nt 5853-nt 7485 of JQ673480.1 are deleted or mutated.

18. The recombinant HSV virus of any one of claims 1-13, wherein, The recombinant HSV virus further comprises a fifth exogenous nucleotide sequence.

19. A viral vector comprising or consisting of the genome of a recombinant HSV virus according to any one of claims 1-18.

20. A viral vector comprising or consisting of a mutated HSV genome; said mutated HSV genome does not express functional ICP0 protein and functional ICP34.5 protein, but is capable of expressing functional UL43 protein, functional UL41 protein, functional UL48 protein, or any combination thereof; wherein said mutated HSV genome is derived from the genome of HSV-2 virus.

21. The viral vector of claim 20, wherein, said mutated HSV genome comprises the following modifications: 2 copies of ICP0 gene each independently comprises a loss-of-function mutation or is deleted or replaced with an exogenous nucleotide sequence; and, 2 copies of ICP34.5 gene each independently comprises a loss-of-function mutation or is deleted or replaced with an exogenous nucleotide sequence.

22. The viral vector of claim 20, in said mutated HSV genome, (a1) one copy of ICP0 gene comprises a loss-of-function mutation, and the other copy of ICP0 gene comprises a loss-of-function mutation or is deleted or replaced with an exogenous nucleotide sequence; or (b1) one copy of ICP0 gene is deleted, and the other copy of ICP0 gene comprises a loss-of-function mutation or is deleted or replaced with an exogenous nucleotide sequence; or (c1) one copy of ICP0 gene is replaced with an exogenous nucleotide sequence, and the other copy of ICP0 gene comprises a loss-of-function mutation or is deleted or replaced with an exogenous nucleotide sequence; and (a2) one copy of ICP34.5 gene comprises a loss-of-function mutation, and the other copy of ICP34.5 gene comprises a loss-of-function mutation or is deleted or replaced with an exogenous nucleotide sequence; or (b2) one copy of ICP34.5 gene is deleted, and the other copy of ICP34.5 gene comprises a loss-of-function mutation or is deleted or replaced with an exogenous nucleotide sequence; or (c2) one copy of ICP34.5 gene is replaced with an exogenous nucleotide sequence, and the other copy of ICP34.5 gene comprises a loss-of-function mutation or is deleted or replaced with an exogenous nucleotide sequence.

23. A host cell infected with, or comprising the genome of, or transfected with the recombinant HSV virus of any one of claims 1-18, or the viral vector of any one of claims 19-22.

24. The host cell of claim 23, wherein, said cell is a tumor cell.

25. The host cell of claim 24, wherein, said tumor cell is a lung cancer cell, a liver cancer cell, a breast cancer cell, an osteosarcoma cell, an ovarian cancer cell, a cervical cancer cell, a prostate cancer cell, a glioma cell, a melanoma cell, a colorectal cancer cell, and a pancreatic cancer cell.

26. A method of obtaining the recombinant HSV virus of any one of claims 1-18, comprising: (1) culturing the host cell according to claim 23; (2) after the host cell is pathologically changed, collecting and lysing the host cell to obtain a lysate of the host cell; and (3) recovering the recombinant HSV virus from the lysate.

27. A pharmaceutical composition comprising the recombinant HSV virus according to any one of claims 1-18, or the genome of the recombinant HSV virus according to any one of claims 1-18, or the viral vector according to any one of claims 19-22, and a pharmaceutically acceptable carrier or excipient.

28. The pharmaceutical composition of claim 27, wherein, The pharmaceutical composition has one or more features selected from the group consisting of: (1) the pharmaceutical composition is for treating a tumor; (2) the pharmaceutical composition is an injection solution or a lyophilized powder; (3) the pharmaceutical composition comprises a therapeutically effective amount of the recombinant HSV virus or the genome of the recombinant HSV virus or the viral vector; (4) the pharmaceutical composition is in unit dosage form; (5) 10 2 -10 9 pfu of recombinant HSV virus per unit dose of the pharmaceutical composition.

29. Use of the recombinant HSV virus according to any one of claims 1-18, or the viral vector according to any one of claims 19-22, for the preparation of a pharmaceutical composition for treating a tumor in a subject.

30. The use of claim 29, which has one or more features selected from the group consisting of: (1) the tumor is selected from the group consisting of lung cancer, liver cancer, breast cancer, osteosarcoma, ovarian cancer, prostate cancer, glioma, melanoma, colorectal cancer, and pancreatic cancer; (2) the subject is a mammal; (3) the subject is a human.

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