Herpes simplex virus strain and application thereof

Abstract

The invention relates to the field of biology, in particular to a herpes simplex virus strain and application thereof as well as a progeny culture and an attenuated modified variant of the herpes simplex virus strain. The present application also relates to a pharmaceutical composition comprising said herpes simplex virus and to the therapeutic use of said pharmaceutical composition.

Description

Technical Field

[0001] The present application relates to a virus strain of herpes simplex virus. Compared with the standard model strain, the virus strain of the present application has stronger oncolytic efficiency and / or gene delivery ability. Background Art

[0002] Herpes simplex virus (HSV) belongs to the family of Herpesviridae and is a double-stranded linear DNA virus with an envelope. The core of the herpes virus is double-stranded DNA, which is wrapped by a capsid on the outside. The tegument is located between the capsid and the envelope, and the outermost layer is a double-layer lipid envelope structure. The envelope is composed of about 13 proteins encoded by viral genes, including viral glycoprotein B (gB), viral glycoprotein C (gC), viral glycoprotein D (gD), viral glycoprotein G (gG) and viral glycoprotein M (gM). The three-dimensional structure of these glycoproteins is related to the cell infection ability of the virus, which determines whether the virus can enter the host cell and the amount of virus entering the host cell.

[0003] HSV includes two serotypes, type I (HSV-1) and type II (HSV-2), and is one of the most widely studied DNA viruses. HSV-1 usually causes facial herpes, such as herpes labialis and oral herpes, and HSV-2 usually causes genital herpes. Humans are the natural host of herpes simplex virus (HSV). HSV infection is extremely common in the population. 80% to 90% of people who are infected with HSV for the first time are latently infected, most of them have no obvious symptoms, and only a few are overtly infected. The common clinical manifestations are local herpes on the mucous membranes or skin, which can be effectively treated with antiviral drugs.

[0004] HSV-1 has strong infectivity, short replication time, high oncolytic efficiency, and the viral genome of HSV-1 will not be integrated into the genome of the host cell, making it a safe viral vector. In addition, the genome of HSV is relatively large (about 150Kb), and some of its genes are not essential for viral replication, which provides sufficient space for inserting exogenous functional genes without limiting the packaging efficiency of the virus, making it a highly potential viral vector in the field of oncolytic virus therapy. Currently, two oncolytic virus drugs with HSV-1 as a vector have been approved for marketing, namely Talimogene laherparepvec and Teserpaturev / G47Δ.

[0005] Due to its good replication ability and safety, herpes simplex virus has been widely valued in tumor treatment. However, at present, most of the HSV vectors used in oncolytic herpes simplex virus drugs under clinical development are laboratory virus strains that have been propagated in tissue culture cells for many years. Although these viruses can enter cells to deliver genes, the virus's own oncolytic efficiency and gene delivery ability still need to be improved. Summary of the invention

[0006] The purpose of the present application is to overcome the problems existing in the prior art and to provide a new HSV virus strain. Compared with the currently commonly used standard model strain that has been continuously propagated (such as the HSV-1F strain, referred to as the F strain), the HSV-1 virus strain of the present application has significantly enhanced ability to infect human cells in vivo and the ability to replicate / lyse in cells, and is suitable as an oncolytic virus or as a viral vector to destroy tumor cells.

[0007] The present application also provides a host cell, which is infected with the HSV-1 virus described in the present application or contains the viral genome of the HSV-1 virus described in the present application.

[0008] The present application also provides a pharmaceutical composition, which includes the HSV-1 virus described in the present application and a pharmaceutically acceptable excipient.

[0009] The present application also provides use of the HSV-1 virus or pharmaceutical composition of the present application in the preparation of a drug for treating a tumor in a subject.

[0010] The present application also provides a method for treating a tumor in a subject, the method comprising administering a therapeutically effective amount of the HSV-1 virus or pharmaceutical composition of the present application to the subject.

[0011] Virus

[0012] The present application provides a herpes simplex virus strain having improved performance compared to a standard model strain. In some embodiments, the herpes simplex virus strain may be derived from, for example, an HSV-1 virus strain or an HSV-2 virus strain or a derivative virus strain thereof.

[0013] In some embodiments, the herpes simplex virus strain of the present application is an HSV-1 virus strain.

[0014] In some embodiments, the HSV-1 virus strain has been deposited in the China Center for Type Culture Collection (CCTCC) on May 5, 2023, and was classified and named: Herpes simplex virus type 1 HSV-1 / TQ-C7-1 (Herpes simplex virustype 1, HSV-1 / TQ-C7-1), with a collection number of CCTCC NO: V202331, and the collection address is: Wuhan University, Wuhan, China, 430072.

[0015] In some embodiments, the HSV-1 virus strain is a progeny culture of the virus strain with a deposit number of CCTCC NO: V202331.

[0016] In some embodiments, the HSV-1 virus strain has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the genome sequence of the virus strain with the deposit number of CCTCC NO: V202331.

[0017] In some embodiments, the HSV-1 strain expresses a gD protein that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 1. In some embodiments, the HSV-1 strain expresses the gD protein set forth in SEQ ID NO: 1.

[0018] In some embodiments, the HSV-1 strain expresses a gH protein that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 3. In some embodiments, the HSV-1 strain expresses a gH protein set forth in SEQ ID NO: 3.

[0019] In some embodiments, the HSV-1 strain expresses a gL protein that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 5. In some embodiments, the HSV-1 strain expresses a gL protein set forth in SEQ ID NO: 5.

[0020] In some embodiments, the HSV-1 strain expresses a gB protein that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 7. In some embodiments, the HSV-1 strain expresses a gB protein set forth in SEQ ID NO: 7.

[0021] In some embodiments, the HSV-1 strain expresses a UL11 protein that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 9. In some embodiments, the HSV-1 strain expresses a UL11 protein set forth in SEQ ID NO: 9.

[0022] In some embodiments, the HSV-1 strain expresses a gI protein that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 11. In some embodiments, the HSV-1 strain expresses a gI protein set forth in SEQ ID NO: 11.

[0023] In some embodiments, the HSV-1 strain expresses an ICPO protein that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 13. In some embodiments, the HSV-1 strain expresses an ICPO protein set forth in SEQ ID NO: 13.

[0024] In some embodiments, the HSV-1 strain expresses a gC protein that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 17. In some embodiments, the HSV-1 strain expresses a gC protein set forth in SEQ ID NO: 17.

[0025] In some embodiments, the HSV-1 strain expresses an ICP4 protein that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 19. In some embodiments, the HSV-1 strain expresses the ICP4 protein set forth in SEQ ID NO: 19.

[0026] In some embodiments, the HSV-1 strain comprises a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 18 or 20.

[0027] In some embodiments, the HSV-1 virus strain is an attenuated modified variant of herpes simplex virus type 1 as shown in any one of the following a) to k):

[0028] a) It is a virus strain with a deposit number of CCTCC NO: V202331 or a progeny culture thereof;

[0029] b) having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the genome sequence of the virus strain with the deposit number of CCTCC NO:: V202331;

[0030] c) expresses a gD protein that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 1, or expresses the gD protein set forth in SEQ ID NO: 1;

[0031] d) expresses a gH protein that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:3, or expresses the gH protein set forth in SEQ ID NO:3;

[0032] e) expresses a gL protein that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:5, or expresses the gL protein set forth in SEQ ID NO:5;

[0033] f) expresses a gB protein that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:7, or expresses the gB protein set forth in SEQ ID NO:7;

[0034] g) expresses a UL11 protein that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:9, or expresses the UL11 protein set forth in SEQ ID NO:9;

[0035] h) expresses a gl protein that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 11, or expresses the gl protein set forth in SEQ ID NO: 11;

[0036] i) it expresses an ICPO protein that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 13, or expresses the ICPO protein set forth in SEQ ID NO: 13;

[0037] j) expresses a gC protein that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 17, or expresses the gC protein set forth in SEQ ID NO: 17; or

[0038] k) expresses an ICP4 protein that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:19, or expresses the ICP4 protein set forth in SEQ ID NO:19.

[0039] In some embodiments, the attenuated modified variant does not express functional ICPO protein, ICP34.5 protein, ICP47 protein and / or ICP6 protein.

[0040] In some embodiments, the attenuated modified variant does not express functional ICP34.5 protein.

[0041] In some embodiments, the attenuated modified variant does not express functional ICP34.5 protein and / or ICP6 protein.

[0042] In some embodiments, the attenuated modified variant does not express functional ICP34.5 protein and / or ICP47 protein.

[0043] In some embodiments, the attenuated modified variant does not express functional ICP34.5 protein, ICP47 protein and / or ICP6 protein.

[0044] In some embodiments, the attenuated modified variant does not express functional ICP34.5 protein and ICP6 protein.

[0045] In some embodiments, the attenuated modified variant does not express functional ICP34.5 protein and ICP47 protein.

[0046] In some embodiments, the attenuated modified variant does not express functional ICP34.5 protein, ICP47 protein, and ICP6 protein.

[0047] As used herein, the term "ICP0 protein" refers to infected cell protein 0 of HSV virus, which is encoded by the RL2 gene and is one of the immediate early gene products of HSV virus. The amino acid sequence of ICPO protein is known and can be found in, for example, the public database NCBI (AFE62827.1).

[0048] As used herein, the term "ICP34.5 protein" refers to infected cell protein 34.5 of HSV virus, which is encoded by RL1 gene and is one of the immediate early gene products of HSV virus. The amino acid sequence of ICP34.5 protein is known and can be found in, for example, the public database NCBI (AFE62826.1).

[0049] As used herein, the term "ICP47 protein" refers to the TAP transporter inhibitor ICP47 of the HSV virus, which is encoded by the US12 gene. The amino acid sequence of the ICP47 protein is known and can be found in, for example, the public database NCBI (WKR19818.1).

[0050] As used herein, the term "ICP6 protein" refers to the ribonucleotide reductase subunit 1 of the HSV virus, which is encoded by the UL39 gene. The amino acid sequence of the ICP6 protein is known and can be found in, for example, the public database NCBI (AOY34171.1).

[0051] "Not expressing a functional target protein" means that after a virus, viral vector or viral genome infects a cell, the virus, viral vector or viral genome cannot produce or express a target protein with biological functional activity. For example, the virus, viral vector or viral genome may not produce or express the target protein at all due to gene deletion, or may produce or express a target protein without biological functional activity due to gene mutation. The deletion includes one or more parts of a missing gene or a complete gene. The mutation includes, but is not limited to, missense mutation, nonsense mutation, frameshift mutation, base deletion, base substitution, base addition or any combination thereof.

[0052] In some embodiments, the attenuated modified variant comprises a RL2 gene, a RL1 gene, a US12 gene and / or a UL39 gene that is mutated or deleted or replaced with an exogenous nucleic acid sequence.

[0053] In some embodiments, the attenuated modified variant comprises a RL2 gene that is mutated or deleted or replaced with an exogenous nucleic acid sequence. In some embodiments, the double copy of the RL2 gene is independently mutated or deleted or replaced with an exogenous nucleic acid sequence (e.g., 2 copies of the RL2 gene are deleted; or, 1 copy of the RL2 gene is deleted and 1 copy of the RL2 gene is replaced with an exogenous nucleic acid sequence).

[0054] In some embodiments, the attenuated modified variant comprises a RL1 gene that is mutated or deleted or replaced with an exogenous nucleic acid sequence. In some embodiments, the double copy of the RL1 gene is independently mutated or deleted or replaced with an exogenous nucleic acid sequence (e.g., 2 copies of the RL1 gene are deleted; or, 1 copy of the RL1 gene is deleted and 1 copy of the RL1 gene is replaced with an exogenous nucleic acid sequence).

[0055] In some embodiments, the attenuated modified variant comprises a US12 gene that is mutated or deleted or replaced with an exogenous nucleic acid sequence.

[0056] In some embodiments, the attenuated modified variant comprises a UL39 gene that is mutated or deleted or replaced with an exogenous nucleic acid sequence.

[0057] In some embodiments, the attenuated modified variant comprises a mutation or deletion or a RL1 gene and / or a UL39 gene substituted with an exogenous nucleic acid sequence. In some embodiments, the attenuated modified variant comprises a mutation or deletion or a RL1 gene and a UL39 gene substituted with an exogenous nucleic acid sequence.

[0058] In some embodiments, the attenuated modified variant comprises a mutation or deletion or a RL1 gene and / or a US12 gene that is replaced with an exogenous nucleic acid sequence. In some embodiments, the attenuated modified variant comprises a mutation or deletion or a RL1 gene and a US12 gene that are replaced with an exogenous nucleic acid sequence (e.g., 2 copies of the RL1 gene are deleted and the US12 gene is deleted; or, 2 copies of the RL1 gene are replaced with an exogenous nucleic acid sequence and the US12 gene is deleted; or, 2 copies of the RL1 gene are deleted and the US12 gene is replaced with an exogenous nucleic acid sequence).

[0059] In some embodiments, the attenuated modified variant comprises a mutation or a deletion or a replacement of the RL1 gene, the US12 gene and / or the UL39 gene with an exogenous nucleic acid sequence. In some embodiments, the attenuated modified variant comprises a mutation or a deletion or a replacement of the RL1 gene, the US12 gene and the UL39 gene with an exogenous nucleic acid sequence.

[0060] In some embodiments, the attenuated modified variant further comprises one or more mutations or deletions or non-essential genes replaced with exogenous nucleic acid sequences. "Non-essential genes" refer to genes that are not necessary for maintaining the survival and replication of the 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. In some embodiments, the non-essential genes include UL3 gene (see, e.g., GenBank No. AFE62830.1), UL4 gene (see, e.g., GenBank No. AFE62831.1), UL14 gene (see, e.g., GenBank No. AFE62841.1), UL16 gene (see, e.g., GenBank No. AFE62843.1), UL21 gene (see, e.g., GenBank No. AFE62848.1), UL24 gene (see, e.g., GenBank No. AFE62851.1), UL32 gene (see, e.g., GenBank No. AFE62860.1), UL51 gene (see, e.g., GenBank No. AFE62880.1), UL55 gene (see, e.g., GenBank No. AFE62884.1), UL56 gene (see, e.g., GenBank No. AFE62885.1), US2 gene (see, e.g., GenBank No. AFE62890.1), US3 gene (see, e.g., GenBank No. AFE62891.1), and / or LAT gene (see, e.g., GenBank No. JQ673480.1).

[0061] In some embodiments, the attenuated modified variant further comprises a US3 gene, a UL56 gene and / or a LAT gene that is mutated or deleted or replaced with an exogenous nucleic acid sequence.

[0062] ICP0 is a protein necessary for HSV-1 to be expressed and replicated in normal cells at a low multiplicity of infection. Disrupting the function of ICP0 protein can make the virus infect only tumor cells but not normal cells at a low multiplicity of infection.

[0063] ICP34.5 is a protein necessary for HSV-1 to proliferate in nerve cells. Disrupting the function of ICP34.5 protein can inhibit the replication of the virus in nerve cells and eliminate the neurotoxicity of the virus.

[0064] ICP6 is a subunit of HSV-1 ribonucleotide reductase and a protein necessary for viral DNA replication. Disrupting the function of ICP6 protein can significantly reduce the virus's ability to replicate in normal cells without affecting the virus's ability to replicate in tumor cells.

[0065] ICP47 can block MHC-mediated antigen peptide presentation and affect the recognition of the immune system by binding to the transporter associated with antigen processing (TAP) of host cells. Disrupting the function of ICP47 protein can weaken virus-mediated immune escape and enhance the immune system's recognition and attack on virus-infected tumor cells.

[0066] HSV-1 contains genes related to nucleotide metabolism (such as genes encoding thymidine kinase, ribonucleotide reductase, and uracil DNA glycosylase), which allow the virus to replicate in non-dividing cells that lack sufficient nucleotides. Disrupting these genes can enhance the specificity of the virus for dividing cells (tumor cells) and reduce the pathogenicity of the virus.

[0067] In some embodiments, the attenuated modified variant further comprises one or more deletions or mutations or is replaced with nucleotide metabolism-related genes of exogenous nucleic acid sequences. In some embodiments, the nucleotide metabolism-related genes include thymidine kinase encoding genes, ribonucleotide reductase encoding genes and uracil DNA glycosylase encoding genes.

[0068] In some embodiments, the HSV-1 virus strain of the present application comprises one or more exogenous nucleic acid sequences. The insertion site of the exogenous nucleic acid sequence can be any one or more of the above-mentioned modification sites, or can be the latent infection area (LAT) of herpes simplex virus.

[0069] In some embodiments, the exogenous nucleic acid sequence encodes a fluorescent protein, an immunomodulatory protein and / or a cytotoxic peptide. In some embodiments, the immunomodulatory protein includes an antibody or an antigen-binding fragment thereof, a tumor-associated antigen, a cytokine or other protein capable of regulating cellular immune function. In some embodiments, the expression of the exogenous protein enhances the anti-tumor effect of the HSV-1 virus.

[0070] In some embodiments, the antibody or antigen binding fragment thereof includes an immune checkpoint inhibitory antibody or antigen binding fragment thereof. In some embodiments, the immune checkpoint inhibitory antibody or antigen binding fragment thereof includes an anti-PD-1 antibody or antigen binding fragment thereof, an anti-PD-L1 antibody or antigen binding fragment thereof, and an anti-CTLA-4 antibody or antigen binding fragment thereof.

[0071] In some embodiments, the tumor-associated antigen refers to a protein produced in tumor cells and differentially expressed in cancer compared to corresponding normal tissues, including cancer antigen-125 (CA-125) and melanoma associated antigen (MAGE).

[0072] In some embodiments, the cytokines include interleukins (e.g., IL-1, IL-2, IL-12, IL-15, IL-18, IL-21, or IL-24), interferons (e.g., IFN-α), tumor necrosis factors (e.g., TNF-α, IFN-β, or IFN-γ), colony stimulating factors (e.g., GM-CSF), growth factors (e.g., VEGF), chemokines (e.g., CCL2, CCL5, or CCL7), immune co-stimulatory molecules (e.g., CD40 ligand, ICOS ligand, 4-1BB ligand, or OX40 ligand), and any combination thereof.

[0073] In some embodiments, the cytotoxic peptide refers to a peptide capable of inducing cell damage or death, including Fas ligand (FasL).

[0074] In some embodiments, the exogenous nucleic acid sequence is operably linked to a control sequence that allows the exogenous nucleic acid sequence to be transcribed and / or expressed in a cell in vivo. Preferably, the exogenous nucleic acid sequence is operably linked to a control sequence that allows the exogenous nucleic acid sequence to be transcribed and / or expressed in a tumor cell.

[0075] "Operably linked" means that two or more nucleic acid or amino acid sequence elements are physically linked in a manner that places them in a functional relationship with each other. For example, a promoter is operably linked to a coding sequence if it is capable of initiating or otherwise controlling / regulating the transcription and / or expression of the coding sequence, in which case the coding sequence is understood to be "under the control of the promoter". Typically, when two nucleic acid sequences are operably linked, they will be in the same orientation and usually in the same reading frame. They are usually contiguous, but this is not required.

[0076] In some embodiments, the control sequence comprises a promoter that allows the expression of the exogenous nucleic acid sequence and a signal for terminating transcription. In some embodiments, the promoter is an inducible promoter. In some embodiments, the promoter is a tumor-specific promoter.

[0077] Conventional cloning techniques known to those skilled in the art can be used to construct an expression cassette or other suitable construct comprising the exogenous nucleic acid sequence and control sequences.

[0078] In the present application, herpesvirus type I (type 1), herpesvirus type I (type 1), herpes simplex virus type I (type 1), herpes simplex virus type I (type 1), and HSV-1 have the same meaning and can be used interchangeably. Human herpesvirus type I TQ-C7-1 strain can be abbreviated as TQ-C7-1 virus strain, TQ-C7-1 strain or TQ-C7-1 in the present application.

[0079] Host cells

[0080] The present application provides a host cell, which is infected with the HSV-1 virus described in the present application or contains the viral genome of the HSV-1 virus described in the present application.

[0081] Generally speaking, the virus is able to self-replicate and / or express the genes (including endogenous and exogenous) contained in a suitable host cell. In some embodiments, suitable host cells include, but are not limited to, prokaryotic cells (e.g., E. coli cells) and eukaryotic cells (e.g., yeast cells, insect cells, plant cells, and animal cells). In some embodiments, suitable host cells include mammalian cells (e.g., CHO and VERO). The HSV-1 virus of the present application has a high replication capacity in tumor cells, but only replicates at a low level in normal cells. Therefore, in a preferred embodiment, suitable host cells include tumor cells. Such tumor cells include, but are not limited to, lung cancer cells (e.g., A549), liver cancer cells (e.g., Huh7, Hep3B, HepG2, SMMC7721, PLC / PRF / 5, and QGY7703), breast cancer cells (e.g., MADMB231, MCF7, and MDAMB468), osteosarcoma cells (e.g., U2OS), ovarian cancer cells (e.g., SKOV3 and CAOV3), cervical cancer cells (e.g., Hela), prostate cancer cells (e.g., PC-3), glioma cells (e.g., U87MG), melanoma cells (e.g., B16F10 and SK-MEL-28), colorectal cancer cells (e.g., HCT116 and HT-29), and pancreatic cancer cells (e.g., Panc-1).

[0082] Pharmaceutical composition

[0083] The present application provides a pharmaceutical composition comprising the HSV-1 virus described in the present application and one or more pharmaceutically acceptable excipients.

[0084] Under normal conditions of manufacture and storage, these pharmaceutical compositions are stable and protected from contamination by microorganisms such as bacteria and fungi.

[0085] Under common conditions of use, these pharmaceutical compositions are convenient to use. The pharmaceutical composition of the present application can be used by methods well known in the art, including but not limited to, oral or parenteral administration (e.g., intravenous, intratumoral, abdominal, intramuscular, intracranial, subcutaneous administration). In some embodiments, the drug combination of the present application is suitable for injection administration, such as intravenous, intratumoral, abdominal, intramuscular, intracranial, subcutaneous injection administration.

[0086] The HSV-1 virus described in the present application can be formulated with one or more pharmaceutically acceptable excipients in an appropriate amount to form a pharmaceutical composition suitable for injection. Pharmaceutical compositions suitable for injection include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.

[0087] The HSV-1 virus described in the present application can be formulated into other suitable dosage forms in appropriate amounts with one or more pharmaceutically acceptable excipients, including but not limited to tablets, buccal tablets, pills, capsules (e.g., hard capsules, soft capsules, enteric-coated capsules, microcapsules), elixirs, granules, syrups, granules, emulsions, suspensions, solutions, dispersions and sustained-release preparations for oral or parenteral administration.

[0088] use

[0089] The HSV-1 virus of the present application can be used to treat a variety of tumors. The present application also provides a method for treating a tumor in a subject, the method comprising administering a therapeutically effective amount of the HSV-1 virus of the present application or the pharmaceutical composition of the present application to the subject. The present application also provides the use of the HSV-1 virus of the present application or the pharmaceutical composition of the present application in the preparation of a drug for treating a tumor in a subject.

[0090] In some embodiments, the tumor is a solid tumor and / or a non-solid tumor. In some embodiments, the tumor is a recurrent, refractory, metastatic and / or advanced tumor. In some embodiments, the tumor is selected from head and neck cancer, nasopharyngeal cancer, oral cancer, esophageal cancer, pancreatic cancer, ovarian cancer, vaginal cancer, colorectal cancer, liver cancer, lung cancer (such as non-small cell lung cancer or small cell lung cancer), gastric cancer, kidney cancer, bladder cancer, breast cancer, uterine cancer, cervical cancer, prostate cancer, melanoma, mesothelioma, lymphoma, hematological tumor, brain glioma, neuroblastoma, glioblastoma, sarcoma, myeloma and / or osteosarcoma.

[0091] The HSV-1 virus of the present application can be administered alone or in combination with one or more additional therapeutic agents to a subject in need. In some embodiments, the HSV-1 virus of the present application can be administered before, during, and / or after the subject is administered an additional therapeutic agent. In some embodiments, the additional therapeutic agent can be a tumor therapeutic agent known in the art, for example, a chemotherapeutic agent, a radiotherapeutic agent, a cytokine, a vaccine, an immune activator, and / or an immune checkpoint inhibitor (e.g., an immune checkpoint inhibitory antibody or an antigen-binding fragment thereof).

[0092] The HSV-1 virus of the present application can also be used to treat central nervous system diseases or peripheral nervous system diseases. In some embodiments, the central nervous system diseases include stroke, Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis and Huntington's disease. In some embodiments, the peripheral nervous system diseases include motor neuron disease, chronic pain and peripheral nerve injury.

[0093] The HSV-1 virus of the present application can also be used to treat blood diseases. In some embodiments, the blood disease includes hemophilia.

[0094] Technical Effects

[0095] In some embodiments, the HSV-1 strain of the present application has one or more characteristics selected from the following:

[0096] (a) Compared with standard model strains (e.g., F strain), it has an enhanced ability to infect tumor cells.

[0097] (b) Compared with standard model strains (e.g., F strain), it has enhanced ability to replicate in tumor cells.

[0098] (c) Compared with standard model strains (e.g., F strain), it has enhanced ability to lyse / kill tumor cells.

[0099] (d) The replication capacity in tumor cells is higher than that in non-tumor cells.

[0100] The characteristics of the HSV-1 virus strain of the present application can be determined by any method known in the art.

[0101] The HSV-1 virus of the present application is suitable for being used as an oncolytic virus or as a viral vector to destroy tumor cells and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0102] Figure 1 The cytopathic effect caused by the TQ-C7-1 virus strain is shown.

[0103] Figure 2 An electron micrograph of the TQ-C7-1 virus strain at 200,000 times magnification is shown.

[0104] Figure 3 The mutation rate of the amino acid sequence of the important coding genes of the TQ-C7-1 virus strain is shown relative to that of the HSV-1F strain.

[0105] Figure 4 The oncolytic effect of the TQ-C7-1 virus strain on SK-MEL-28 cells is shown.

[0106] Figure 5The oncolytic effect of the TQ-C7-1 virus strain on HT-29 cells is shown.

[0107] Figure 6 The oncolytic effect of the TQ-C7-1 virus strain on A549 cells is shown.

[0108] Figure 7 The oncolytic effect of the TQ-C7-1 virus strain on U-87MG cells is shown.

[0109] Figure 8 The oncolytic effect of the TQ-C7-1 virus strain on HepG2 cells is shown.

[0110] Fig. 9 The oncolytic effect of the TQ-C7-1-hGM-CSF virus strain on SK-MEL-28 cells is shown.

[0111] Fig.10 The oncolytic effect of the TQ-C7-1-hGM-CSF virus strain on HT-29 cells is shown.

[0112] Fig.11 The oncolytic effect of the TQ-C7-1-hGM-CSF virus strain on U-87MG cells is shown.

[0113] Fig.12 The oncolytic effect of the TQ-C7-1-hGM-CSF virus strain on HepG2 cells is shown. DETAILED DESCRIPTION

[0114] Definition and Explanation

[0115] Unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the cell culture, molecular genetics, nucleic acid chemistry, and immunology laboratory procedures used herein are conventional procedures widely used in the corresponding fields. At the same time, in order to better understand this application, the definitions and explanations of the relevant terms are provided below.

[0116] "Oncolytic virus" refers to a type of virus that can specifically infect and kill tumor cells. Usually, after the virus infects tumor cells, it destroys the infected tumor cells through oncolysis, while releasing new infectious virus particles or virions to destroy the remaining tumor cells.

[0117] The term "viral vector" refers to a nucleic acid delivery vehicle constructed based on a viral genome and capable of carrying an exogenous nucleic acid sequence. Generally speaking, a viral vector is capable of self-replication and / or expression of the genes (endogenous and exogenous) contained therein in a suitable host cell. A viral vector may contain a complete wild-type viral genome, or a mutated or modified viral genome. However, for safety reasons, it is preferred to contain a mutated or modified viral genome.

[0118] As used herein, the term "sequence identity" or "percentage identity" in two nucleic acids or polypeptide comparisons refers to when using a nucleotide or amino acid residue sequence comparison algorithm or by visual inspection to measure, when comparing and comparing with maximum correspondence, they are similar or have the same sequence specific percentage number. That is to say, the identity of nucleotide or amino acid sequence can be defined by the following ratio, which is a ratio of two or more nucleotide or amino acid sequences to the maximum mode according to consistent nucleotide or amino acid number comparisons, and adding consistent nucleotide number or amino acid number when comparing in room as required, the ratio in all nucleotide or amino acid numbers of the comparison part. The comparison of sequence identity can be carried out in a variety of ways within the scope of this area, such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. The judgment / calculation of "sequence identity" or "percentage identity" can be based on any suitable region of the sequence.

[0119] The term "exogenous nucleic acid sequence" refers to an artificially introduced nucleic acid sequence that is foreign to the original sequence. Exogenous nucleic acid sequences include, but are not limited to, any gene not found in the viral genome.

[0120] The term "antibody" refers to an antigen binding protein having at least one antigen binding domain. The antibodies of the present application include monoclonal antibodies or antigen binding fragments thereof and antibody variants or antigen binding fragments thereof. Examples of antibodies include monospecific antibodies, bispecific antibodies, multispecific antibodies, Fab fragments, Fab' fragments, F(ab)'2 fragments, Fv fragments, isolated CDR regions, single-chain Fv molecules (scFv) and other antibody fragments known in the art. The antibody can be a murine antibody, a chimeric antibody, a humanized antibody or a fully human antibody. Unless otherwise indicated, the "antibody" of the present application includes a whole antibody and any antigen binding fragment or single chain thereof. Conventional "whole antibodies" are glycoproteins comprising two heavy (H) chains and two light (L) chains, and the heavy chains and light chains are connected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains, namely CH1, CH2 and CH3. Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region is composed of a domain CL. The VH and VL regions can also be divided into hypervariable regions, i.e., complementary determining regions (CDRs), and framework regions (FRs) with relatively conservative sequences. Each VH and VL is composed of three CDRs and four FRs, respectively, from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody can mediate the binding of an immunoglobulin to a host tissue or factor, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. At the same time, as will be appreciated by those skilled in the art, a special "whole antibody", such as a nanobody, has only a heavy (H) chain and no light (L) chain.

[0121] The "antigen-binding fragment" of an antibody refers to one or more fragments of an antibody that retain the function of specifically binding to an antigen. It has been demonstrated that the antigen-binding function of an antibody can be implemented by a fragment of the entire antibody. Examples encompassed in the "antigen-binding fragment" of an antibody include: (i) Fab fragment: a monovalent fragment consisting of VL, VH, CL and CH1 domains; (ii) F(ab')2 fragment, a bivalent fragment comprising two Fab fragments connected by a disulfide bridge in the hinge region; (iii) Fd fragment consisting of VH and CH1 domains; (iv) Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) dAb fragment consisting of a VH domain (see Ward et al., Nature. 341: 544-546 (1989)); (vi) isolated complementary determining regions (CDRs); and (vii) nanobodies, a heavy chain variable region comprising a single variable domain and two constant domains. In addition, although the two domains VL and VH of the Fv fragment are encoded by different genes, VH and VL can be connected into a single protein chain by a synthetic linker using a recombinant method, wherein VL and VH are paired to form a monovalent molecule (called single-chain Fv (scFv); see, for example, Bird et al., Science. 242: 423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. 85: 5879-5883 (1988)). These single-chain antibodies are also encompassed in the term antigen-binding fragment.

[0122] The term "pharmaceutical composition" refers to a mixture of one or more active ingredients and one or more pharmaceutically acceptable excipients. The purpose of a pharmaceutical composition is to facilitate administration of the active ingredients to a subject. As used herein, the terms "pharmaceutical composition" and "preparation" have the same meaning and are used interchangeably.

[0123] The term "excipient" refers to any ingredient other than the active ingredient (e.g., the HSV-1 virus of the present application). The choice of excipient will depend to a large extent on factors such as the specific mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form. As used herein, "pharmaceutically acceptable excipients" include, but are not limited to, excipients, diluents, fillers, binders, disintegrants, solubilizers, stabilizers, colorants, flavoring agents, surfactants, emulsifiers, buffers, or encapsulating materials. The amount of each excipient may vary within the conventional range in the art.

[0124] The term "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms which, within the scope of sound medical judgment, are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0125] In this article, the terms "subject", "patient" or "subject" are used interchangeably. "Subject", "patient" or "subject" includes any human or non-human animal. The term "non-human animal" includes, but is not limited to, vertebrates, such as non-human primates, sheep, dogs, and rodents (e.g., mice, rats, and guinea pigs). In some embodiments, the terms "subject", "patient" or "subject" are mammals. In some embodiments, the subject, patient or subject is a mouse. In some embodiments, the subject, patient or subject is a human.

[0126] The term "treatment" refers to an attempt to alter the natural course of the disease in the subject being treated, and may be a clinical intervention performed for prevention or during the course of clinical pathology. The desired effects of treatment include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating the symptoms of the disease, reducing any direct or indirect pathological consequences of the disease, preventing the metastasis of the disease, slowing the rate of progression of the disease, improving or alleviating the state of the disease, prolonging the frequency and duration of symptom-free periods, and regressing or improving the prognosis of the disease.

[0127] The term "therapeutically effective amount" means an amount that (i) treats a specific disease, condition, or disorder, (ii) alleviates, ameliorates, or eliminates one or more symptoms of a specific disease, condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of a specific disease, condition, or disorder described herein. The amount of active substance (e.g., HSV-1 virus of the present application) that constitutes a "therapeutically effective amount" may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the therapeutic agent or combination of therapeutic agents to elicit a desired response in the subject. The therapeutically effective amount may also be routinely determined by a person skilled in the art based on their own knowledge and the content of the present application.

[0128]

[00136] The terms "administering" or "administering" refer to the physical introduction of a therapeutic agent into a subject or body using any of a variety of methods and delivery systems known to those skilled in the art.

[0129] Plaque Forming Unit (PFU) refers to the number of viruses that form plaques on monolayer cultured animal cells.

[0130] "A", "an" and "the" include plural referents unless otherwise specified. Therefore, the terms "a" (or "an"), "one or more" or "at least one" are used interchangeably herein; unless otherwise specified, "or" means "and / or".

[0131] The present application also provides the following specific implementation schemes, but the protection scope of the present application is not limited thereto:

[0132] Embodiment 1. A herpes simplex virus type 1, wherein the herpes simplex virus type 1:

[0133] a) is a virus strain with a preservation number of CCTCC NO: V202331 or a progeny culture thereof,

[0134] b) expressing a gD protein having at least 90% identity to SEQ ID NO: 1,

[0135] c) expressing a gH protein having at least 90% identity to SEQ ID NO: 3,

[0136] d) expressing a gL protein having at least 90% identity to SEQ ID NO:5,

[0137] e) expressing a gB protein having at least 90% identity to SEQ ID NO: 7,

[0138] f) expressing a UL11 protein having at least 90% identity to SEQ ID NO:9,

[0139] g) expressing a gl protein having at least 90% identity to SEQ ID NO: 11,

[0140] h) expressing an ICPO protein having at least 90% identity to SEQ ID NO: 13,

[0141] i) expresses a gC protein having at least 90% identity to SEQ ID NO: 17, or

[0142] j) expressing an ICP4 protein that is at least 90% identical to SEQ ID NO:19.

[0143] Embodiment 2. A herpes simplex virus type 1, wherein the herpes simplex virus type 1 is an attenuated modified variant of the herpes simplex virus type 1 described in embodiment 1.

[0144] Embodiment 3. The herpes simplex virus type 1 according to embodiment 2, wherein the herpes simplex virus type 1 does not express functional ICP0 protein, ICP34.5 protein, ICP47 protein and / or ICP6 protein.

[0145] Embodiment 4. The herpes simplex virus type 1 according to embodiment 2 or 3, wherein the herpes simplex virus type 1 does not express functional ICP34.5 protein and ICP47 protein.

[0146] Embodiment 5. The herpes simplex virus type 1 according to any one of embodiments 2-4, wherein the herpes simplex virus type 1 comprises a RL2 gene, a RL1 gene, a US12 gene and / or a UL39 gene that is mutated or deleted or replaced with an exogenous nucleic acid sequence.

[0147] Embodiment 6. The herpes simplex virus type 1 according to any one of embodiments 2-5, wherein the herpes simplex virus type 1 comprises the RL1 gene and the US12 gene which are mutated or deleted or replaced with exogenous nucleic acid sequences.

[0148] Embodiment 7. The herpes simplex virus type 1 according to any one of embodiments 2-6, wherein the herpes simplex virus type 1 comprises one or more non-essential genes that are mutated or deleted or replaced with exogenous nucleic acid sequences.

[0149] Embodiment 8. The herpes simplex virus type 1 according to any one of Embodiments 2 to 6, wherein the non-essential gene comprises a UL3 gene, a UL4 gene, a UL14 gene, a UL16 gene, a UL21 gene, a UL24 gene, a UL32 gene, a UL51 gene, a UL55 gene, a UL56 gene, a US2 gene, a US3 gene and / or a LAT gene.

[0150] Embodiment 9. The herpes simplex virus type 1 according to any one of embodiments 2-8, wherein the non-essential gene comprises a US3 gene, a UL56 gene and / or a LAT gene.

[0151] Embodiment 10. The herpes simplex virus type 1 according to any one of embodiments 2-9, wherein the herpes simplex virus type 1 comprises one or more nucleotide metabolism-related genes that are deleted or mutated or replaced with exogenous nucleic acid sequences, and the nucleotide metabolism-related genes include thymidine kinase encoding gene, ribonucleotide reductase encoding gene and uracil DNA glycosylase encoding gene.

[0152] Embodiment 11. The herpes simplex virus type 1 according to any one of embodiments 1-10, wherein the herpes simplex virus type 1 comprises one or more exogenous nucleic acid sequences.

[0153] Embodiment 12. The herpes simplex virus type 1 according to embodiment 11, wherein the exogenous nucleic acid sequence encodes a fluorescent protein, an immunomodulatory protein and / or a cytotoxic peptide.

[0154] Embodiment 13. The herpes simplex virus type 1 according to embodiment 12, wherein the immunomodulatory protein comprises an antibody or an antigen-binding fragment thereof, a tumor-associated antigen, an interleukin, an interferon, a tumor necrosis factor, a colony stimulating factor, a growth factor, a chemokine, an immune co-stimulatory molecule, or any combination thereof.

[0155] Embodiment 14. The herpes simplex virus type 1 according to embodiment 12, wherein the immunomodulatory protein comprises an anti-PD-1 antibody or an antigen-binding fragment thereof, an anti-PD-L1 antibody or an antigen-binding fragment thereof, an anti-CTLA-4 antibody or an antigen-binding fragment thereof, CA-125, MAGE, IL-1, IL-2, IL-12, IL-15, IL-18, IL-21, IL-24, IFN-α, IFN-β, IFN-γ, TNF-α, GM-CSF, VEGF, CCL2, CCL5, CCL7, CD40 ligand, ICOS ligand, 4-1BB ligand, OX40 ligand, or any combination thereof.

[0156] Embodiment 15. A host cell, wherein the host cell is infected with the herpes simplex virus type 1 described in any one of embodiments 1-14 or comprises the viral genome of the herpes simplex virus type 1 described in any one of embodiments 1-14.

[0157] Embodiment 16. A pharmaceutical composition, wherein the pharmaceutical composition comprises the herpes simplex virus type 1 according to any one of Embodiments 1-14 and one or more pharmaceutically acceptable excipients.

[0158] Embodiment 17. Use of the herpes simplex virus type 1 according to any one of Embodiments 1 to 14 in the preparation of a medicament for treating tumors.

[0159] Embodiment 18. The use according to Embodiment 17, wherein the tumor comprises head and neck cancer, nasopharyngeal cancer, oral cancer, esophageal cancer, pancreatic cancer, ovarian cancer, vaginal cancer, colorectal cancer, liver cancer, lung cancer, gastric cancer, kidney cancer, bladder cancer, breast cancer, uterine cancer, cervical cancer, prostate cancer, melanoma, mesothelioma, lymphoma, hematological tumor, brain glioma, neuroblastoma, glioblastoma, sarcoma, myeloma and / or osteosarcoma.

[0160] Embodiment 19. The use according to embodiment 17 or 18, wherein the herpes simplex virus type 1 is administered in combination with one or more additional therapeutic agents.

[0161] Embodiment 20. The use according to embodiment 19, wherein the additional therapeutic agent comprises a chemotherapeutic agent, a radiotherapeutic agent, a cytokine, a vaccine, an immune activator and / or an immune checkpoint inhibitor. Example

[0162] The present application is further described in conjunction with the examples, but the contents of the examples do not limit the scope of the present application. The experimental methods in the examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the raw material or commodity manufacturers. The reagents without specifying the specific sources are all conventional reagents in the art or commercially available reagents.

[0163] Example 1 Characterization and preservation of TQ-C7-1 virus strain

[0164] 1) Virus characterization

[0165] a) Subtype identification: The gG region of the virus was PCR amplified and sequenced, and then compared with the gG region of the standard model strain HSV-1F strain (GenBank: GU734771.1) and HSV-2HG52 strain (GenBank: Z86099.2) for sequence alignment and homology analysis. The results showed that the TQ-C7-1 virus strain was an HSV-1 virus.

[0166] b) Oncolytic activity assay: VERO cells in the logarithmic growth phase were plated in 6-well plates at 1,000,000 cells / well, cultured in a 37°C cell culture incubator for 12 hours, the culture medium was discarded, and the virus was inoculated at an MOI of 0.1. The control group was not inoculated with the virus, and the cytopathic effect (CPE) caused by the virus was observed after 24 hours. The results showed that the TQ-C7-1 virus strain caused a strong cytopathic effect, and the cells became round and gradually fell off from the infection center to form plaques ( Figure 1 ).

[0167] 2) Deposit of TQ-C7-1 virus strain

[0168] TQ-C7-1 was deposited in the China Center for Type Culture Collection (CCTCC) on May 5, 2023, and was classified and named: Herpes simplex virus type 1 HSV-1 / TQ-C7-1 (Herpes simplex virus type 1, HSV-1 / TQ-C7-1), with the preservation number: CCTCC NO: V202331. The preservation address is: Wuhan University, Wuhan, China, 430072.

[0169] Example 2 Analysis of biological characteristics of TQ-C7-1 virus strain

[0170] The biological characteristics of the TQ-C7-1 virus strain were analyzed by electron microscopy observation and whole genome sequencing.

[0171] 1) Electron microscopy observation

[0172] The virus was purified using multi-mode chromatography filler Capto Core 700 filler (GE, Cat. No. 17548103), and the purified TQ-C7-1 virus strain was observed under a 200,000x electron microscope. The results showed that the virus had a typical HSV-1 appearance, and four components, genomic DNA, capsid, stroma, and envelope, could be observed ( Figure 2 ).

[0173] 2) Whole genome DNA sequencing and analysis of TQ-C7-1 virus strain

[0174] The genomic DNA of the TQ-C7-1 virus strain was submitted to Beijing Novogene Technology Co., Ltd. for third-generation sequencing. The viral genome sequences of the TQ-C7-1 virus strain and the standard model strain HSV-1F strain (GenBank: GU734771.1) were compared. The results showed that the base sequences of the genomes of the two strains were very different. Compared with the standard model strain HSV-1F strain, the protein coding region (CDS region) of the TQ-C7-1 virus strain had a total of 505 amino acid mutations (Table 1), among which the amino acid sequences of several important coding genes, including the envelope protein and envelope glycoprotein coding genes, had large mutations ( Figure 3 ).

[0175] Herpes simplex virus (HSV) binds to receptors on the surface of host cells through glycoproteins on the envelope, and then enters the host cell through the fusion of the viral envelope and the cell membrane. This process requires the participation of four key envelope glycoproteins: gD (encoded by US6), gH (encoded by UL22), gL (encoded by UL1) and gB (encoded by UL27). Compared with the standard model strain HSV-1F strain, the envelope glycoproteins gD, gH, gL and gB of the TQ-C7-1 virus strain have 3, 8, 6 and 5 amino acid mutations, respectively (the amino acid sequences are shown in SEQ ID NO: 1, 3, 5 and 7 of the present application, respectively). In addition, the gC (encoded by UL44) protein binds to receptors on cells to help the virus adsorb to the surface of host cells, and plays an important role in the process of viral infection and replication. Compared with the standard model strain HSV-1F strain, the envelope glycoprotein gC of the TQ-C7-1 virus strain has 5 amino acid mutations (the amino acid sequence is shown in SEQ ID NO: 17 of the present application).

[0176] UL11 is the smallest tegument protein. UL11 and its homologs play a key role in efficient viral replication and tegument assembly (Baines and Roizman, 1992; Ding et al., 2021). The UL11 of the standard model strain HSV-1F has only 96 amino acid residues, while the UL11 of the TQ-C7-1 virus strain has 261 amino acid residues (the amino acid sequence is shown in SEQ ID NO: 9 of the present application).

[0177] US7 encodes envelope glycoprotein I (gI), and gI and its homologs are key mediators of viral cell-to-cell spread and syncytium formation (Gui et al., 2022). The gI of the standard model strain HSV-1F has 390 amino acid residues, while the gI of the TQ-C7-1 virus strain has 410 amino acid residues (the amino acid sequence is shown in SEQ ID NO: 11 of the present application).

[0178] RL2 encodes an E3 ubiquitin ligase, i.e., infected cell protein 0 (ICP0). ICP0 and its homologs have the function of blocking NF-κb-mediated immunomodulatory cytokine transcription and blocking IRF3 and IRF7-induced antiviral signaling pathways. At the same time, it is also involved in the transcription of viral genes and promotes viral replication (Ma et al., 2018). The ICP0 of the standard model strain HSV-1F has 768 amino acid residues, while the ICP0 of the TQ-C7-1 virus strain has 776 amino acid residues (the amino acid sequence is shown in SEQ ID NO: 13 of the present application).

[0179] RS1 encodes infected cell protein 4 (ICP4), which plays a major regulatory role in HSV replication and transcription. The ICP4 of the TQ-C7-1 virus strain has 1295 amino acid residues (the amino acid sequence is shown in SEQ ID NO: 19 of the present application)

[0180] Table 1. Mutation rate of the amino acid sequence of the CDS region of the TQ-C7-1 virus strain relative to the HSV-1F strain

[0181]

[0182]

[0183] Example 3 Comparison of oncolytic effects of TQ-C7-1 virus strain and HSV-1F strain

[0184] The replication ability and cell killing activity of TQ-C7-1 virus strain and HSV-1F strain in different cancer cells were compared.

[0185] Experimental cells: including SK-MEL-28 human malignant melanoma cells, HT-29 human colorectal cancer cells, A549 human non-small cell lung cancer cells, U-87MG human brain astrocytic glioblastoma cells and HepG2 human liver cancer cells.

[0186] Experimental method: Take the experimental cells in the logarithmic growth phase, plate them in a 96-well plate at 10,000 cells / well, culture them in a 37°C cell culture incubator for 24 hours, discard the culture medium, inoculate the virus at MOI=0.1, and the control group is not inoculated with the virus and continues to be cultured in a 37°C cell culture incubator. The cell growth status is detected by a real-time label-free dynamic cell analyzer (Real Time Cellular Analysis, RTCA), and the killing activity is analyzed.

[0187] Experimental results: Both the TQ-C7-1 and HSV-1F strains showed significant killing activity against different tumor cells. Compared with the HSV-1F strain, the TQ-C7-1 strain had higher replication ability and cell killing activity ( Figure 4-8 ).

[0188] Example 4 Comparison of oncolytic effects of TQ-C7-1-hGM-CSF virus strain and T-VEC strain

[0189] The TQ-C7-1 virus strain was modified as follows: the double copies of the ICP34.5 gene and the ICP47 gene were deleted, and the human granulocyte colony stimulating factor (hGM-CSF) gene was inserted into the ICP34.5 coding locus to obtain the TQ-C7-1-hGM-CSF virus strain. The replication ability and cell killing activity of the TQ-C7-1-hGM-CSF virus strain and the T-VEC (trade name: IMLYGIC) strain in different cancer cells were compared.

[0190] Experimental cells: including SK-MEL-28 human malignant melanoma cells, HT-29 human colorectal cancer cells, U-87MG human brain astrocytoblastoma cells and HepG2 human liver cancer cells.

[0191] Experimental method: Take the experimental cells in the logarithmic growth phase, plate them in a 96-well plate at 10,000 cells / well, culture them in a 37°C cell culture incubator for 24 hours, discard the culture medium, and inoculate the virus at MOI=0.01, 0.03, 0.1, 0.3, 1, 3, and 10 respectively. The control group is not inoculated with the virus and continues to be cultured in a 37°C cell culture incubator. After 72 hours, the cell growth status is detected according to the CCL (Cell Counting-Lite) method, and the killing activity is analyzed. Killing rate (%) = 100% - [(experimental well - blank well) / (control well - blank well)] × 100%

[0192] Experimental results: Both the TQ-C7-1-hGM-CSF virus strain and the T-VEC virus strain showed significant killing activity against different tumor cells. Compared with the T-VEC virus strain, the TQ-C7-1-hGM-CSF virus strain had higher replication ability and cell killing activity in the above tumors ( Figure 9-12 ).

[0193] The sequence information of this application is summarized in the table below.

[0194]

[0195]

[0196]

[0197]

[0198]

[0199]

[0200]

[0201]

Claims

1. A herpes simplex virus type 1, wherein The herpes simplex virus type 1: a) is a virus strain with a preservation number of CCTCC NO: V202331 or a progeny culture thereof, b) expressing a gD protein having at least 90% identity to SEQ ID NO: 1, c) expressing a gH protein having at least 90% identity to SEQ ID NO: 3, d) expressing a gL protein having at least 90% identity to SEQ ID NO:5, e) expressing a gB protein having at least 90% identity to SEQ ID NO: 7, f) expressing a UL11 protein having at least 90% identity to SEQ ID NO:9, g) expressing a gl protein having at least 90% identity to SEQ ID NO: 11, h) expressing an ICPO protein having at least 90% identity to SEQ ID NO: 13, i) expresses a gC protein having at least 90% identity to SEQ ID NO: 17, or j) expressing an ICP4 protein that is at least 90% identical to SEQ ID NO:

19.

2. A herpes simplex virus type 1, wherein The herpes simplex virus type 1 is an attenuated modified variant of the herpes simplex virus type 1 according to claim 1.

3. The herpes simplex virus type 1 according to claim 2, wherein The herpes simplex virus type 1 does not express functional ICP0 protein, ICP34.5 protein, ICP47 protein and / or ICP6 protein.

4. The herpes simplex virus type 1 according to claim 2 or 3, wherein The herpes simplex virus type 1 does not express functional ICP34.5 protein and ICP47 protein.

5. The herpes simplex virus type 1 according to any one of claims 2 to 4, wherein The herpes simplex virus type 1 comprises a RL2 gene, a RL1 gene, a US12 gene and / or a UL39 gene which are mutated or deleted or replaced with an exogenous nucleic acid sequence.

6. The herpes simplex virus type 1 according to any one of claims 2 to 5, wherein The herpes simplex virus type 1 comprises RL1 gene and US12 gene which are mutated or deleted or replaced with foreign nucleic acid sequences.

7. The herpes simplex virus type 1 according to any one of claims 2 to 6, wherein The herpes simplex virus type 1 contains one or more mutations or deletions or non-essential genes that are replaced with foreign nucleic acid sequences.

8. The herpes simplex virus type 1 according to any one of claims 2 to 6, wherein The non-essential genes include UL3 gene, UL4 gene, UL14 gene, UL16 gene, UL21 gene, UL24 gene, UL32 gene, UL51 gene, UL55 gene, UL56 gene, US2 gene, US3 gene and / or LAT gene.

9. The herpes simplex virus type 1 according to any one of claims 2 to 8, wherein The non-essential genes include US3 gene, UL56 gene and / or LAT gene.

10. The herpes simplex virus type 1 according to any one of claims 2 to 9, wherein The herpes simplex virus type 1 contains one or more nucleotide metabolism-related genes that are deleted or mutated or replaced with exogenous nucleic acid sequences, and the nucleotide metabolism-related genes include thymidine kinase encoding gene, ribonucleotide reductase encoding gene and uracil DNA glycosylase encoding gene.

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