WO2023020598A1 - Hsv病毒载体及其应用 - Google Patents

Hsv病毒载体及其应用 Download PDF

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WO2023020598A1
WO2023020598A1 PCT/CN2022/113446 CN2022113446W WO2023020598A1 WO 2023020598 A1 WO2023020598 A1 WO 2023020598A1 CN 2022113446 W CN2022113446 W CN 2022113446W WO 2023020598 A1 WO2023020598 A1 WO 2023020598A1
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virus
hccl19
gene
kos
hsv
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French (fr)
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张旭辉
何玉兰
何亮亮
刘秋燕
黄�俊
冯翠娟
邱健健
陈小锋
李文佳
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广东东阳光药业有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A61K35/763—Herpes virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
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    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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    • C12N15/869—Herpesviral vectors
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    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof

Definitions

  • the present invention relates to the field of biomedicine, in particular, the present invention relates to HSV virus vector and application thereof.
  • CCL19 Due to some limitations inherent in the molecular properties of CCL19, its clinical application is limited. Basically, these include stability issues, such as acid degradation, and a tendency to aggregate irreversibly under mildly denaturing conditions, with subsequent loss of biological activity. Furthermore, when administered intravenously, CCL19 is rapidly cleared from the blood, requiring frequent re-administration of high concentrations to elicit an effective response at the target site and lead to systemic toxicity and side effects such as fever, fatigue, nausea, vomiting, diarrhea, Neurotoxicity and leukopenia.
  • HSV1 to carry the chemokine CCL19 not only enhances the targeting of the virus strain to tumor cells, but also further enhances the anti-tumor immune efficacy of the virus strain through the function of exogenous gene CCL19 to recruit immune cells.
  • the use of oncolytic virus expressing CCL19 can make the virus replicate in the tumor and continuously express CCL19 locally through intratumoral injection, avoiding the rapid clearance of CCL19 and the systemic side effects caused by intravenous administration.
  • the role of CCL19 and CCR7 in anti-tumor therapy has become a research hotspot and has made significant progress.
  • chemokines are limited by unstable product quality and frequent administration.
  • the inventors were consciously surprised to find that CCL19 can be better expressed in HSV virus than other chemokines , especially when the CCL19 gene is inserted at the ICP34.5 site, the expression level of CCL19 is significantly increased.
  • the HSV virus vector can continuously express the chemokine CCL19 at a high level.
  • the expression level is significantly higher than that of the HSV virus carrying only a single copy of mutated CCL19, and the oncolytic virus containing the HSV virus vector retains the sensitivity and proliferative activity to tumor cells, and further improves the expression level of CCL19, breaking through the trend. Due to the low expression level of chemokine, the unstable expression level and the limitation of frequent administration, its tumor inhibitory effect is better than that of viruses containing other chemokine-encoding nucleic acid vectors.
  • the present invention proposes an HSV viral vector.
  • the ICP47 and ICP34.5 genes of the HSV virus vector are silenced, and the CCL19 gene is carried.
  • the HSV1 virus has a double copy of the ICP34.5 gene, and the insertion of the CCL19 gene at the ICP34.5 gene site can effectively reduce the neurotoxicity of the virus and improve the anti-tumor selectivity of the virus; HSV1 carrying a double copy of the chemokine CCL19 can enhance The targeting of the virus strain to tumor cells and the recruitment of immune cells through the exogenous gene CCL19 further enhanced the anti-tumor immune efficacy of the virus strain.
  • the virus By intratumoral injection of oncolytic virus expressing CCL19, the virus replicates in tumor cells and continuously expresses CCL19 locally, avoiding rapid clearance of CCL19 in intravenous administration and causing systemic toxic side effects.
  • Knocking out the replication non-essential gene ICP47 in the HSV genome can improve the expression of MHC-1 on the surface of tumor cells infected by the virus and the ability of cellular antigen presentation; the inventors screened highly expressed chemokines in HSV and found that CCL19 Compared with other chemokines, it can be better expressed in the HSV virus.
  • the inventors explored the insertion site of CCL19, and unexpectedly found that knocking out the ICP34.5 gene in the HSV genome makes HSV selectively in the tumor Replication in cells, instead of replication and proliferation in normal cells, can improve the drug safety of HSV virus, in addition, inserting the nucleic acid of encoding chemokine into this site can effectively improve the virus that contains the HSV virus vector.
  • the HSV viral vector according to the embodiment of the present invention can continuously and highly express the chemokine CCL19, insert the CCL19 gene in the ICP34.5 gene locus, the expression of CCL19 is significantly improved, and the HSV virus replicates in the tumor cells Under the premise that the ability is not affected, the lethality to tumor cells is significantly improved, which can effectively prevent or treat tumors.
  • the above-mentioned HSV viral vector may further include at least one of the following additional technical features:
  • two copies of the CCL19 gene are carried.
  • the inventor screened the copy number of the CCL19 gene carried by the expression vector, and found that the HSV viral vector carrying two copies of the CCL19 gene according to the embodiment of the present invention can significantly increase the expression level of CCL19, and the expression level of CCL19 is significantly higher than that of only HSV viruses carrying a single copy of the CCL19 gene.
  • the CCL19 gene has a 6.A>C mutation.
  • base optimization the expression level of the target gene in the host can be improved without changing the expressed amino acid.
  • the gene nucleic acid sequence of wild-type CCL19 is as follows:
  • the nucleic acid sequence of the CCL19 gene containing 6.A>C mutation is as follows:
  • the ICP34.5 gene silencing is achieved by knocking out nucleotides 135-723 of the ICP34.5 gene.
  • the sequence coding of the ICP34.5 gene is based on the first nucleotide of the start codon of the ICP34.5 gene as the first bit.
  • the ICP47 gene silencing is achieved by knocking out nucleotides 3-266 of the ICP47 gene.
  • the sequence code of the ICP47 gene is encoded with the first nucleotide of the start codon of the ICP47 gene as the first position.
  • the CCL19 gene is set between the 134th nucleotide and the 724th nucleotide of the ICP34.5 gene, so that the constructed HSV-1 carries CCL19.
  • CMV and polyA are further included.
  • the CMV is operably linked to the CCL19 gene.
  • the polyA is set between the 3' terminal nucleotide of the CCL19 gene and the 134th nucleotide of the ICP34.5 gene.
  • the viral vector has the nucleotide sequence shown in SEQ ID NO:5.
  • the present invention provides an oncolytic virus.
  • the HSV virus vector described in the first aspect is carried.
  • the oncolytic virus comprising the HSV virus vector retains the sensitivity and proliferative activity to tumor cells. Due to the characteristics of the ICP34.5 gene, after the CCL19 gene is inserted into the ICP34.5 gene of the HSV virus, CCL19 The expression level is significantly increased, and the expression level of HSV virus CCL19 carrying double copies of CCL19 is significantly improved compared with the expression level of HSV virus CCL19 carrying other copy numbers of CCL19. The lethality is significantly improved and can effectively inhibit tumor cells.
  • the above-mentioned oncolytic virus may further include at least one of the following additional technical features:
  • the oncolytic virus is HSV-1.
  • HSV-2 has potential safety hazards and may cause viral infection of the genitals. Therefore, the use of HSV-1 can improve the safety of oncolytic viruses.
  • the HSV-1 includes at least one selected from F strain, HF strain, KOS strain, HrR3 strain and 17 strain.
  • the present invention proposes a pharmaceutical composition.
  • it includes the HSV viral vector described in the first aspect or the oncolytic virus described in the second aspect.
  • the quality of the pharmaceutical composition according to the embodiment of the present invention is stable, and it can be effective when using a lower dose, especially the inhibitory effect on recurrent and large-volume tumors is very significant.
  • the above pharmaceutical composition may further include at least one of the following additional technical features:
  • each unit dose of the pharmaceutical composition contains 10 ⁇ 5-10 ⁇ 12 pfu of the HSV viral vector or oncolytic virus.
  • the present invention proposes the use of the HSV virus vector described in the first aspect, the oncolytic virus described in the second aspect or the pharmaceutical composition described in the third aspect in the preparation of medicines.
  • the drug is used for treating or preventing tumors.
  • both the HSV virus vector described in the first aspect and the oncolytic virus described in the second aspect can highly express the CCL19 under suitable conditions, which can effectively treat or prevent tumors. Therefore, using the HSV Drugs prepared from viral vectors, oncolytic viruses or pharmaceutical compositions can also effectively treat or prevent tumors.
  • the tumor includes cancers selected from lung cancer, liver cancer, pharyngeal squamous cell carcinoma, colon cancer, osteosarcoma, ovarian cancer, prostate cancer, glioma, melanoma, colorectal cancer, esophageal cancer, pancreatic cancer at least one of the .
  • the present invention provides a method for treating tumors.
  • the method includes administering a therapeutically effective amount of the HSV viral vector described in the first aspect or the oncolytic virus described in the second aspect or the pharmaceutical composition described in the third aspect to the patient in need. individual.
  • tumors can be effectively treated with less frequency of administration.
  • the above method may further include at least one of the following additional technical features:
  • the individual in need is a patient suffering from at least one of the following cancers: lung cancer, liver cancer, pharyngeal squamous cell carcinoma, colon cancer, osteosarcoma, ovarian cancer, prostate cancer, glioma, Melanoma, colorectal cancer, esophageal cancer, pancreatic cancer.
  • the present invention proposes a method of recruiting immune cells to a tumor.
  • the method comprises contacting the tumor with the HSV viral vector of the first aspect or the recombinant oncolytic virus of the second aspect.
  • both the HSV virus vector described in the first aspect and the oncolytic virus described in the second aspect can highly express the CCL19 under suitable conditions.
  • the small molecule secreted protein of CCL19 and its receptors (such as CCR7) are expressed on dendritic cells, T cells and various tumor cells, therefore, the HSV viral vector or oncolytic virus described in this application can be effectively utilized recruits immune cells to tumors, thereby effectively suppressing tumors.
  • the present invention provides a method for inhibiting tumor cell growth or promoting tumor cell death.
  • the method includes contacting the tumor cells with the HSV viral vector of the first aspect or the recombinant oncolytic virus of the second aspect.
  • the above method may further include at least one of the following additional technical features:
  • the tumor cells are selected from lung cancer, liver cancer, pharyngeal squamous cell carcinoma, colon cancer, osteosarcoma, ovarian cancer, prostate cancer, glioma, melanoma, colorectal cancer, esophageal cancer, pancreatic cancer .
  • the recombinant oncolytic virus is provided at a dose sufficient to cause death of the tumor cells.
  • Fig. 1 is a PCR result diagram obtained after the first round of recombination according to an embodiment of the present invention, and the recombinant oncolytic virus is subjected to the first round of limited dilution screening, wherein, M is a marker; 1-32 is a selected virus strain clone; +: The hCCL19 recombinant virus is used as a positive control template (expressing hCCL19 gene); D: the hCCL19-RL1-PMD18T plasmid is used as a template (expressing hccl19hCCL19 gene); K: the KOS- ⁇ 47 virus gene is used as a template (expressing ICP34.5 Gene) processing; water: negative control with water as template;
  • Fig. 2 is a PCR result graph obtained after the first round of recombination according to an embodiment of the present invention and the second round of limited dilution screening of the recombinant oncolytic virus, wherein, M is a marker; 1-28 are 28 selected in the second round of screening Cloning (1-13 is KOS- ⁇ 47-S1-gDNA-hCCL19-32, 1-28 is KOS- ⁇ 47-S1-gDNA-hCCL19-9); D: hCCL19-RL1-PMD18T plasmid as template (expression hCCL19 gene) treatment; K: treatment with KOS- ⁇ 47 virus gene as template (expressing ICP34.5 gene); water: negative control with water as template;
  • Figure 3 is a diagram of the PCR identification results of the recombinant oncolytic virus obtained after the first round of recombination according to the embodiment of the present invention, where M is marker-DL5000; 9-2: KOS- ⁇ 47-S1- gDNA-hCCL19-9-2 viral genome as a template; D: hCCL19-RL1-PMD18T plasmid as a template (expressing hCCL19 gene); K: KOS- ⁇ 47 viral gene as a template (expressing ICP34.5 gene ) processing; Water: take water as the negative control of the template;
  • Fig. 4 is a PCR result diagram of the recombinant oncolytic virus obtained after the second round of recombination according to an embodiment of the present invention after the first round of screening, wherein, M is marker-DL2000; 1-102 are 102 selected clones; 9 -2: KOS- ⁇ 47-S1-gDNA-hCCL19-9-2 viral genome as template; D: hCCL19-RL1-PMD18T plasmid as template (expressing hCCL19 gene); K: KOS- ⁇ 47 viral genes as template (expression of ICP34.5 gene) treatment; water: negative control using water as template; New virus strains obtained from 2 rounds of recombinant transfection;
  • Fig. 5 is a PCR result diagram of the recombinant oncolytic virus obtained after the second round of recombination according to an embodiment of the present invention after the second round of screening, wherein, M is marker-DL2000; 4, 5, 8, 9...101 are: 60 positive viruses obtained through the first round of PCR screening; 9-2: the treatment of KOS- ⁇ 47-S1-gDNA-hCCL19-9-2 virus genome as a template (expressing hCCL19 gene and ICP34.5 gene); D : treatment of hCCL19-RL1-PMD18T plasmid as template (expressing hCCL19 gene); K: treatment of KOS- ⁇ 47 virus gene as template (expression of ICP34.5 gene); water: negative control of water as template; KOS- ⁇ 47-S5g-hCCL19 is a new virus strain obtained by the second round of recombinant transfection of KOS- ⁇ 47-S1-gDNA-hCCL19-9-2;
  • FIG. 6 is a diagram of the PCR identification results of the positive recombinant oncolytic virus obtained after the second round of recombination according to the embodiment of the present invention after amplification, wherein, M is marker-DL2000; 9-2: is KOS- ⁇ 47-S1 -gDNA-hCCL19-9-2 virus genome is the treatment of template (express hCCL19 gene and ICP34.5 gene); D: is the treatment of hCCL19-RL1-PMD18T plasmid as template (express hCCL19 gene); 32-6: is KOS -The treatment of ⁇ 47-S1-hCCL19-32-6 virus gene as a template, for the first round of recombination screening to obtain a single copy inserted virus clone (expressing hCCL19 gene); water: water as the negative control of the template; KOS- ⁇ 47- S5g-hCCL19 is a new virus strain obtained from the second round of recombinant transfection of KOS- ⁇
  • Figure 7 is a PCR result diagram obtained after amplifying the full length of the hCCL19 expression cassette according to an embodiment of the present invention, wherein, M is marker-DL2000; 9-2: KOS- ⁇ 47-S1-gDNA-hCCL19-9-2 Viral genome as template (expression of hCCL19 gene and ICP34.5 gene); D: treatment of hCCL19-RL1-PMD18T plasmid as template (expression of hCCL19 gene); 32-6: treatment of KOS- ⁇ 47-S1-hCCL19- 32-6 The treatment of viral gene as a template, for the first round of recombination screening to obtain a single-copy inserted virus clone (expressing hCCL19); water: water is the negative control of the template;
  • Figure 8 is the electrophoresis results obtained after the first round of plaque purification of the positive recombinant oncolytic virus according to an embodiment of the present invention, wherein, M is marker-DL5000; D is the hCCL19-RL1-PMD18T plasmid as a template (expressing hCCL19 gene ) treatment; K is the treatment of KOS- ⁇ 47 virus genome as template (expression of ICP34.5 gene); water: as the negative control of water template;
  • Figure 9 is an electrophoresis result obtained after the second round of plaque purification of the positive recombinant oncolytic virus according to an embodiment of the present invention, wherein, M is marker-DL5000; D is hCCL19-RL1-PMD18T plasmid as a template (expressing hCCL19 gene ) treatment; K is the treatment of KOS- ⁇ 47 virus genome as template (expression of ICP34.5 gene); water: as the negative control of water template;
  • Figure 10 is the electrophoresis result obtained after the third round of plaque purification of the positive recombinant oncolytic virus according to the embodiment of the present invention, wherein, M is a marker; D is the hCCL19-RL1-PMD18T plasmid as a template (expressing hCCL19 gene) Treatment; K is the treatment of KOS- ⁇ 47 virus genome as template (expressing ICP34.5 gene); water: negative control with water template;
  • Figure 11 is the electrophoresis diagram of the positive recombinant oncolytic virus of the embodiment of the present invention, in which the size of the amplified fragment of the recombinant virus KOS- ⁇ 47-hCCL19-05-02-02-02 is in line with expectations;
  • Figure 12 is a diagram showing the screening results of positive clones connected with the recombinant virus KOS- ⁇ 47-hCCL19-05-02-02-02TA;
  • Fig. 13 is a result graph of the replication ability of KOS-ATCC and KOS-hCCL19 in six cancer cells according to an embodiment of the present invention
  • Figure 14 is a graph showing the results of virus expression levels detected after infection of cells with 2k virus and KOS-hCCL19 recombinant virus according to an embodiment of the present invention.
  • Fig. 15-Fig. 17 are graphs of the replication ability results of KOS and hCCL19 in 20 cancer cells according to an embodiment of the present invention; wherein KOS is a wild-type strain; hCCL19 (that is, KOS-hCCL19) is a recombined virus strain;
  • Figure 18 is a graph showing the results of tumor size changes in the NCI-H460 mouse model according to an embodiment of the present invention.
  • Fig. 19 is the survival curve of each group of animals in the virus neurotoxicity evaluation experiment according to the embodiment of the present invention.
  • Fig. 20 is a graph showing the change results of the tumor size on the right side (administration side) on the A20 mouse model of each group according to an embodiment of the present invention.
  • Fig. 21 is a graph showing the change results of tumor size on the left side (non-administered side) of each group of A20 mouse models according to an embodiment of the present invention.
  • Fig. 22 is a graph showing the death status of A20 mouse models in each group after 28 days of administration according to an embodiment of the present invention.
  • first and second are used for descriptive purposes only, and cannot be interpreted as indicating or implying relative importance or implicitly specifying the quantity of indicated technical features.
  • the features defined as “first” and “second” may explicitly or implicitly include at least one of these features.
  • “plurality” means at least two, such as two, three, etc., unless otherwise specifically defined.
  • chemokines are a kind of cytokines that are expressed on the cell membranes of immune cells and endothelial cells, and chemokines the directional movement of various immune cells, such as CCL19, which can chemoattract dendritic cells.
  • Cells, CD4 + and CD8+ cells infiltrate tumors, mediate immune cells to release cytokines, play a key role in inhibiting tumor proliferation, migration and invasion, and assisting in the treatment of tumors.
  • operably linked refers to linking the exogenous gene to the vector, so that the control elements in the vector, such as the promoter sequence, etc., can exert their expected functions of regulating the transcription and translation of the exogenous gene .
  • the "oncolytic virus” described in this application has lost some functional genes, which weakens its ability to infect and replicate in normal cells, that is, it is an attenuated oncolytic virus, such as the HSV-1 oncolytic virus of this application, ICP47
  • ICP34.5 gene is knocked out so that it can only replicate selectively in tumor cells.
  • the inventor screened the chemokines that need to be inserted in the expression vector, and found that the CCL19 chemokine was easier to express in the expression vector than other chemokines, and the HSV virus carrying CCL19 was compared with other chemokines.
  • Chemokine viruses have better oncolytic and immune effects, and better effects in treating or preventing tumors; the inventor further screened the insertion site of the CCL19 gene in the HSV virus.
  • the inventors found that when the CCL19 gene was inserted into the ICP34.5 site, the obtained HSV virus had a significant increase in the gene expression of CCL19 compared with the virus inserted into the CCL19 at a site other than the ICP34.5 gene, including all Compared with the oncolytic virus containing the expression vector inserted into CCL19 at a site other than the ICP34.5 gene, the oncolytic virus of the above-mentioned HSV expression vector has significantly improved gene expression of CCL19, and has higher safety; at the same time, The copy number of the CCL19 gene inserted in the expression vector was explored, and it was found that the double copy of the ICP34.5 gene in the expression vector was knocked out, and after the double copy of the CCL19 gene was inserted, the expression level of the HSV expression vector CCL19 gene was significantly higher than that of For the HSV expression vector carrying a single copy of the CCL19 gene, the expression level of the CCL19 gene containing the
  • ⁇ 47 virus genome refers to the HSV-1 oncolytic virus genome with the ICP47 gene knocked out
  • KOS- ⁇ 47-S1-gDNA-hCCL19 refers to the KOS- ⁇ 47 inserted on the basis of hCCL19; Construction of recombinant HSV-1 virus using CRISSPR/Cas9 system, including plasmid construction, preparation and screening of single-copy recombinant oncolytic virus, identification of single-copy virus, preparation and screening of double-copy recombinant oncolytic virus, and identification of double-copy virus Identification.
  • Example 1 The first round of viral recombination and screening
  • the inventor used phenol chloroform to extract the KOS- ⁇ 47 virus genome; used Lipofectamine 3000 transfection reagent to transfect 293T cells to obtain the recombinant virus; the recombinant virus was screened by the first round of limited dilution, and the second round of limited dilution screening , to obtain a positive recombinant virus, the positive recombinant virus was multiplied, genome extraction, PCR identification, and sequencing; the results showed that after the first round of virus recombination and screening, the recombinant virus obtained was a single-copy insertion, and the specific experimental operations were as follows:
  • step 2 The product of step 2 was processed under the following conditions: 4°C, 12000rpm centrifugation for 30min.
  • step 4 5Centrifuge the product of step 4 at 4°C, 12000rpm for 10min, absorb the supernatant with a tipped pipette, and extract the protein again.
  • step 7 Centrifuge the product of step 6 at 4°C, 12000rpm for 10min, discard the supernatant, and wash the pellet twice with 500 ⁇ L of 75% ethanol (lightly flicking instead of pipetting), 4°C, 12000rpm, 10min.
  • a and B in Table 1 (CRISPR/Cas9 gene editing plasmid (34.5-S1-2 plasmid)+homologous repair donor plasmid (dornor DNA(hCCL19-RL1-PMD18T)+KOS- ⁇ 47 viral genomes) were mixed and co-transfected into 293T cells in a 6-well plate to obtain a virus liquid containing recombinant virus.
  • the 34.5-S1-2 plasmid was a plasmid containing gRNA, which was aimed at the ICP34.5 gene locus
  • the CRISPR/Cas9 gene editing plasmid, dornor DNA is the donor plasmid containing hCCL19.
  • the first round of recombinant virus obtained in step 2 is screened by the first round of limited dilution, and the specific operations are as follows:
  • KOS- ⁇ 47-S1-gDNA-hCCL19-201224 Dilute the recombinant virus solution KOS- ⁇ 47-S1-gDNA-hCCL19-201224 according to the dilution factor of 100 times and 500 times, add 100 ⁇ L/well to a 96-well plate covered with a single layer of Vero, 2 pieces for each dilution 96-well plate, named: KOS- ⁇ 47-S1-gDNA-hCCL19-201224 (10 -2 , 1 ⁇ 2), KOS- ⁇ 47-S1-gDNA-hCCL19-201224 (2 ⁇ 10 -3 , 1 ⁇ 2 2) Place the 96-well plate in a 37°C, 5% CO2 incubator for culture.
  • KOS- ⁇ 47-S1-gDNA-hCCL19-201224 (2 ⁇ 10 -3 , 1 ⁇ 2) all wells have obvious lesions, and KOS- ⁇ 47-S1-gDNA-hCCL19-201224 (2 ⁇ 10 -3 , 1)
  • the supernatant on the 96-well plate was used for rough extraction of virus gDNA, the method is as follows:
  • virus strains obtained after the first round of limited dilution screening were subjected to rough extraction of viral gDNA.
  • the specific operation was: 2 ⁇ L virus solution + 8 ⁇ L gDNA Extraction Buffer (add proteinase K at 10 ⁇ L/mL before use), mix well and react in the PCR instrument
  • the PCR reaction conditions were 55°C for 1h, 95°C for 10min, and 16°C ⁇ .
  • PCR amplification was used to verify whether hCCL19 was inserted into the viral genome.
  • the primers for hCCL19 gene detection were shown in Table 2, and the PCR amplification reaction system was shown in Table 3.
  • PCR results of the first round of limited dilution screening of recombinant viruses are shown in Figure 1.
  • PCR electrophoresis results showed that KOS- ⁇ 47-S1-gDNA-hCCL19-9 and No. 32 were suspected positive recombinant viruses.
  • the virus liquid was collected and KOS- ⁇ 47-S1-gDNA-hCCL19-9 was named KOS- ⁇ 47-S1gDNA-hCCL19-9-200227.
  • the suspected positive recombinant virus after the first round of limited dilution screening obtained in step 3 is subjected to the second round of limited dilution screening, and the specific operations are as follows:
  • KOS- ⁇ 47-S1gDNA-hCCL19-9-200227 Dilute the recombinant virus liquid KOS- ⁇ 47-S1gDNA-hCCL19-9-200227 according to the dilution factor of 1 ⁇ 106 , add 100 ⁇ L/well into a 96-well plate covered with monolayer Vero cells, and name it: KOS- ⁇ 47- S1gDNA-hCCL19-201231-1x10 6 , 37°C, 5% CO 2 culture for 2 hours, discard the virus solution, add 200 ⁇ L/well DPBS to wash once, discard, add 100 ⁇ L/well 1% FBS/DMEM, continue to culture . After culturing for 96 hours, observe the cell lesions. KOS- ⁇ 47-S1gDNA-hCCL19-201231-1x10 6 had obvious lesions in 28 wells, numbered: s1g-9-1-28. gDNA crude extraction, the method is the same as above.
  • the virus strain (KOS- ⁇ 47-S1-gDNA-hCCL19-9) obtained after the second round of limited dilution screening was subjected to rough extraction of viral gDNA.
  • the specific operation was: 2 ⁇ L virus liquid + 8 ⁇ L gDNA Extraction Buffer (press 10 ⁇ L before use) Add proteinase K in /mL, mix well and react in a PCR instrument.
  • the reaction conditions are: 55°C for 1h, 95°C for 10min, and 16°C ⁇ .
  • the hCCL19 gene detection primers are shown in the table 2, the PCR amplification reaction system is shown in Table 3.
  • KOS- ⁇ 47-S1-gDNA-hCCL19-9-2 KOS- ⁇ 47-S1-gDNA-hCCL19-9- No. 6 suspected positive recombinant virus
  • KOS- ⁇ 47-S1-gDNA-hCCL19-9-2 was named KOS- ⁇ 47-hCCL19-S1g-9-2#-210104.
  • step 4 The recombinant oncolytic virus obtained in step 4 is identified and sequenced, and the specific operations are as follows:
  • the positive recombinant virus (KOS- ⁇ 47-S1-gDNA-hCCL19-9-2) was amplified, genome extracted, PCR identified, and sequenced.
  • the specific operation of genome extraction was: the recombinant virus KOS- ⁇ 47-hCCL19 -S1g-9-2#-210104 was expanded with 6-well plate vero cells, added 5 ⁇ L of virus, cultured at 37°C, 5% CO 2 for 24 hours, the cells were completely damaged, and the culture medium was collected and named KOS- ⁇ 47-S1g- 9-2-P01-210107, take 200 ⁇ L of the virus liquid, use the virus genome extraction kit Viral RNA/DNA Extraction Kit ver.5.0 to extract the virus genome, and name it: KOS- ⁇ 47-S1g-9-2-gDNA-210107,
  • Table 4 The primers for detecting the hCCL19 homology arm sequence in the PCR amplification reaction.
  • Element volume gDNA 1.0 ⁇ L MightyAmp DNA Polymerase 1.0 ⁇ L 2 ⁇ MightyAmp Buffer Ver.2 25.0 ⁇ L 345-HOM1-F2 1.0 ⁇ L 345-HOM2-R2 1.0 ⁇ L ddH 2 O 21.0 ⁇ L total capacity 50 ⁇ L
  • KOS- ⁇ 47-S1g-9-2-gDNA-210107 (9-2 in Figure 3) is the virus KOS- ⁇
  • the genome of 47-S1g-9-2-P01-210107 was gelled before and after the recombination and sent to Guangzhou Aiji for sequencing (sanger sequencing) to confirm the recombination bands, hCCL19 gene expression cassette sequencing was completely correct, and the pre-recombination band , confirmed as the band before KOS genome recombination; thus, it was confirmed that the recombinant virus KOS- ⁇ 47-S1g-9-2-P01-210107 obtained through the first round of virus recombination and screening was a single-copy insertion.
  • Example 2 The second round of virus recombination and screening
  • the second round of recombination and screening was performed on the single-copy inserted KOS- ⁇ 47-S1-gDNA-hCCL19-9-2 obtained in Example 1, and the single-copy recombinant virus genome was extracted with phenol-chloroform; transfected with Lipofectamine 3000 Reagents were transfected into 293T cells to obtain recombinant viruses; the recombinant viruses were limitedly diluted, the first round of PCR screening, the second round of PCR screening, and positive recombinant viruses were obtained, and the positive recombinant viruses were multiplied, genome extraction, PCR identification, and sequencing. Specific operations as follows:
  • KOS- ⁇ 47-S1g-9-2-P01 i.e., KOS- ⁇ 47-S1g-9-2-P01-210107
  • Effected cells ⁇ 100% lesions
  • CRISPR/Cas9 gene editing plasmid (Cas9-sgRNA345-5 plasmid)+homologous repair donor plasmid (dornor DNA (hCCL19-RL1-PMD18T)+viral genome ( KOS- ⁇ 47-hCCL19-9-2) were mixed and co-transfected into 293T cells in a 6-well plate to obtain virus liquid containing recombinant virus, in which Cas9-sgRNA345-5 was a plasmid containing gRNA, targeting ICP34.
  • CRISPR/Cas9 gene editing plasmid with 5 gene loci, dornor DNA (hCCL19-RL1-PMD18T) is the donor plasmid containing hCCL19.
  • the cell transfection operation was the same as above, and the virus solution containing the recombinant virus was named KOS- ⁇ 47-hCCL19-S5g-P00-210117 and set aside.
  • the second round of recombinant virus obtained in step 2 is screened by the first round of limited dilution, and the specific operations are as follows:
  • Two 96-well plates each, named: KOS- ⁇ 47-S5g-hCCL19-3000X/1000X/400X-P01-210117-01 ⁇ 02, cultivated at 37°C for 2 hours in a 5% CO2 incubator, discarded
  • For the virus solution add 100 ⁇ L/well DPBS to wash the plate, discard it, add 100 ⁇ L/well 1% FBS/DMEM, and place the 96-well plate in a 37°C, 5% CO 2 incubator for cultivation.
  • KOS- ⁇ 47-S5g-hCCL19-3000X-P01-210117-01 had lesions in 45 holes
  • KOS- ⁇ 47-S5g-hCCL19-3000X-P01-210117-02 had lesions.
  • virus gDNA was crudely extracted from the lesion wells using the same method as above, which was recorded as: KOS- ⁇ 47-S5g-hCCL19-3000X-P01-210121-01-102 for PCR amplification verification.
  • virus strains obtained after the first round of limited dilution screening were subjected to rough extraction of virus gDNA, and PCR amplification was used to verify whether the ICP34.5 gene sequence was knocked out successfully.
  • the primers for ICP34.5 gene sequence detection were as shown in Table 7.
  • the amplification reaction system is shown in Table 8.
  • the 60 positive recombinant viruses obtained after the first round of limited dilution in step 3 of this embodiment were screened by the second round of PCR to verify whether hCCL19 was inserted into the viral genome, and the hCCL19 gene detection primers were shown in Table 9 (amplification of the hCCL19 gene expression frame full length), the PCR amplification reaction system is shown in Table 10.
  • PCR electrophoresis results showed that the samples that only amplified the recombinant bands were 4 and 5 in KOS- ⁇ 47-S5g-hCCL19 , 8-10, 12, 15, 18, 19, 21, 26, 28-31, 36, 37, 43, 45, 47, 48, 53, 54, 56, 57, 59, 60, 63, 67, 68 , 71, 72, 77, 79-81, 84, 87, 88, 90, 93-98, and 101, a total of 47 positive recombinant viruses.
  • step 4 The recombinant oncolytic virus obtained in step 4 is identified and sequenced, and the specific operations are as follows:
  • the positive recombinant virus (4, 5, 8-10, 12 in KOS- ⁇ 47-S5g-hCCL19) was amplified, genome extracted, PCR identified, sequenced, the recombinant virus was amplified, and the viral genome was extracted, respectively named as : KOS- ⁇ 47-S5g-hCCL19-04/01/08/1510/12-gDNA-210125.
  • the specific operation of genome extraction is: use 6-well plate vero cells to proliferate, add 2 ⁇ L of virus solution, incubate at 37°C, 5% CO 2 for 24 hours, the cells are completely damaged, collect the culture medium, and name it KOS- ⁇ 47-S5g-hCCL19 -04/01/08/1510/12210124, stored at -70°C, took 200 ⁇ L of virus liquid, used the kit Viral RNA/DNA Extraction Kit ver.5.0 to extract the genome, eluted with 50 ⁇ L sterile water, and named them respectively : KOS- ⁇ 47-S5g-hCCL19-04/01/08/1510/12-gDNA-210125, carry out the sequence design primer of ICP34.5 gene in the PCR amplification reaction as shown in Table 7, PCR amplification reaction The system is shown in Table 8.
  • primers were designed according to the homology arm sequence of the hCCL19 expression cassette to amplify the full length of the hCCL19 expression cassette.
  • the primer sequences are shown in Tables 11 and 12, and the PCR amplification reaction system is shown in Table 13.
  • Element volume gDNA 1.0 ⁇ L MightyAmp DNA Polymerase 1.0 ⁇ L 2 ⁇ MightyAmp Buffer Ver.2 25.0 ⁇ L Primer F 1.0 ⁇ L Primer R 1.0 ⁇ L ddH 2 O 21.0 ⁇ L
  • primer F is 345-HOM1-F2 in Table 11 and Table 12
  • primer R is 345-HOM2-R2 or 345-flank-seq in Table 11 and Table 12.
  • KOS- ⁇ 47-S5g-hCCL19-04, 05, 08, 09, 10, and 12 samples have a single band, and the band size is the same as that of the hCCL19-RL1-pMD18T plasmid positive control sample unanimous.
  • the bands of KOS- ⁇ 47-S5g-hCCL19-04, 05, 08, 09, 10, and 12 were recovered by cutting the gel, and the samples were sent to Guangzhou Aiji Biotechnology Co., Ltd. for sequencing.
  • Example 3 The first round of plaque purification of positive recombinant oncolytic virus
  • KOS- ⁇ 47-hCCL19-05-P01 -01/02/03/04/05-210205 KOS- ⁇ 47-hCCL19-12-P01-01/02/03/04/05-210205
  • the specific operation of the extraction is carried out according to the kit instructions.
  • the extracted virus gene is named: KOS- ⁇ 47-hCCL19-05-01/02/03/04/05-gDNA-210205, KOS- ⁇ 47-hCCL19-12-01/02/03/04/05-gDNA-210205.
  • the electrophoresis results of KOS- ⁇ 47-hCCL19-05/12 recombinant virus are shown in Figure 8.
  • the electrophoresis results show that KOS- ⁇ 47-hCCL19-05-01/02/03/04/05, KOS- ⁇ 47-hCCL19 -12-01/02/03/04/05 all expressed hCCL19 gene but not ICP34.5 gene.
  • Example 4 The second round of plaque purification of recombinant oncolytic virus
  • Virus liquid named: KOS- ⁇ 47-hCCL19-05-02-P01-01/02/03/04/05-210303, KOS- ⁇ 47-hCCL19-12-03-P01-01/02/03/ 04/05-210303, take 200 ⁇ L of the virus liquid, use the kit Viral RNA/DNA Extraction Kit ver.5.0 to extract the virus genome, the specific operation is carried out according to the kit instructions, and the extracted virus genome is named: KOS- ⁇ 47-hCCL19- 05-02-01/02/03/04/05-gDNA-210303, KOS- ⁇ 47-hCCL19-12-03-01/02/03/04/05-gDNA-210303.
  • the electrophoresis results of the KOS- ⁇ 47-hCCL19-05-02/12-03 recombinant virus are shown in Figure 9.
  • the electrophoresis results showed that the KOS- ⁇ 47-hCCL19-05-02-01/02/ No. 03/04/05, KOS- ⁇ 47-hCCL19-12-03-01/02/03/04/05 samples all had a single band, which was consistent with the size of the hCCL19-RL1-PMD18T plasmid positive control , is the large band after recombination.
  • Example 5 The third round of plaque purification of recombinant oncolytic virus
  • the primer sequences used are shown in Table 7, Table 11, Table 12, and Table 14, and the reaction systems used are shown in Table 8, Table 15, and Table 16.
  • Element volume gDNA 1.0 ⁇ L MightyAmp DNA Polymerase 1.0 ⁇ L 2 ⁇ MightyAmp Buffer Ver.2 25.0 ⁇ L 345-HOM1-F2 1.0 ⁇ L 345-HOM2-R2 1.0 ⁇ L ddH 2 O 21.0 ⁇ L total capacity 50 ⁇ L
  • Element volume gDNA 1.0 ⁇ L MightyAmp DNA Polymerase 1.0 ⁇ L 2 ⁇ MightyAmp Buffer Ver.2 25.0 ⁇ L 345-HOM1-F2 1.0 ⁇ L 345-flank-seq-R3 1.0 ⁇ L ddH 2 O 21.0 ⁇ L
  • the electrophoresis results of recombinant viruses KOS- ⁇ 47-hCCL19-05-02-02 and KOS- ⁇ 47-hCCL19-12-03-01 are shown in Figure 10.
  • the electrophoresis results showed that KOS- ⁇ 47 -hCCL19-05-02-02-01/02/03/04/05, KOS- ⁇ 47-hCCL19-12-03-01/02/03/04/05 had a single band, with The hCCL19-RL1-PMD18T plasmid positive control had the same band size and was a large band after recombination.
  • Element volume template 1 ⁇ L PCR amplification enzyme (2X) 25 ⁇ L 345-HOM1-F2(10uM) 1 ⁇ L 345-flank-seq-R3 (10uM) 1 ⁇ L DMSO 2 ⁇ L ddH 2 0 20 ⁇ L total capacity 50 ⁇ L
  • the PCR amplification program is: 98°C for 3min; (98°C for 10s, 64°C for 15s, 72°C for 3min) for 30 cycles; 72°C for 5min; 11.
  • cycle-pure kit (omega)
  • A-tailing, T-loading, and transformation into colon-competent DH5a were carried out.
  • the results of positive clone screening are shown in Figure 12, and 6 positive clones were picked for sample delivery. Sequenced by Guangzhou Aiji Biotechnology Co., Ltd.
  • Sequencing results Aiji IGC287528, IGC287406, IGC287407, high-fidelity enzyme ApexHF HS DNA Polymerase FS Selected clones: 1, 3, 4, 6, 7 clones have correct gene expression frames, good sequencing quality, hCCL19 gene expression frames are correct, and theoretical The sequence is completely consistent and the sequencing quality is good; the clones selected by the high-fidelity enzyme Prime star max: 3, 5, 6, 7, and 8 clones have correct gene expression frames and good sequencing quality. The hCCL19 gene expression frames are correct and completely consistent with the theoretical sequence. good quality.
  • sample name Titer source KOS-ATCC 8.33x10 ⁇ 7 SHS21054,P06 KOS- ⁇ 47-hCCL19 7.76x10 ⁇ 7 SHS21054,P18
  • KOS-ATCC is a wild-type virus
  • KOS- ⁇ 47-hCCL19 is a virus in which 47 is knocked out and the hCCL19 gene is inserted at position 34.5.
  • the IC50 values of the two viruses on different cells are shown in Table 21. According to the judgment standard of MOI IC50 ⁇ 0.6 after 72h incubation: from the results of virus killing, HT29, SW620, hepG 2, Hep3B2.1-7, NCI-H460 The cells are oncolytic virus-sensitive tumor cell lines. Compared with the KOS-ATCC original virus strain, the KOS-hCCL19 recombinant virus strain still retains the sensitivity to HCT-116 cells.
  • N/A means that the nonlinear fitting cannot be made due to the poor or untested dose-effect relationship, and the number marked with a horizontal line indicates that the fitting degree is poor (fitting degree ⁇ 80%), which corresponds to MOI IC50 Values are for reference only.
  • the KOS-ATCC i.e. the aforementioned wild-type virus
  • KOS-hCCL19 HSV1 carrying hCCL19
  • the cells were Hep3B2.1-7 liver cancer cells, NCI-H460 large cell lung cancer cells, HepG2 liver cancer cells, HT29 colon cancer cells, HCT-116 human colon cancer cells, and SW620 human colon cancer cells were inoculated in 6-well culture plates at an appropriate cell density. After culturing overnight, the cells in the 6-well plates were counted.
  • the virus liquid was collected for virus titer determination. After freezing and thawing three times (-80°C, 37°C), serially dilute the collected virus solution, take 300 ⁇ L to infect Vero cells (6-well plate), shake the culture plate every 15 minutes to make the virus better adsorb the cells, and add 2 mL of Vero cells after 1.25 hours Medium. After 2 hours, the medium was discarded, and 300 ⁇ L of DMEM complete medium and 3 mL of 2% methylcellulose were added to fix the virus. After culturing for 3-4 days, discard the covering medium, add 10% HCHO solution, 1 mL/well, and fix for 20 min.
  • virus titer virus dilution factor * (1000/300) * number of plaques.
  • This example evaluates the replication ability of the original virus strain (KOS-ATCC) and the modified virus strain (KOS-hCCL19) in 6 cancer cell lines (Hep3B2.1-7, HepG2, NCI-H460, SW620, HT29, HCT116) A T-test was performed on them.
  • the replication abilities of KOS-ATCC and KOS-hCCL19 in six types of cancer cells are shown in Figure 13. Among them, the replication abilities of the two strains of viruses in different tumor cells are different. Overall, the modified virus strain retains the proliferative activity on tumor cells.
  • virus expression experiment was carried out in vero cells, and the specific experimental operations were as follows:
  • Cultivate vero cells in a 37°C, 5% carbon dioxide incubator Cells were observed once a day using an inverted microscope.
  • the confluence rate of the cells in the culture dish reaches 80-90%, subculture the cells: discard the old culture medium, wash off the residual medium with DPBS, add 1 mL of 0.25% trypsin digestion solution, put it in the incubator for digestion for 2 minutes, and wait for the cells to After becoming round and floating, discard the trypsin, add new medium to stop digestion, and transfer to a fresh sterile culture dish according to a certain ratio.
  • hCCL19 The expression of hCCL19 in the supernatant was determined according to the Human MIP-3b ELISA Kit (EhCCL19).
  • KOS-ATCC that is, the aforementioned wild-type virus
  • KOS-hCCL19 HSV1 carrying hCCL19
  • Tumor cells are CAL27 oral squamous cell carcinoma cells, A549 human lung cancer cells, PC-3 prostate cancer cells, CNE-2Z nasopharyngeal carcinoma cells, Panc-1 pancreatic cancer cells, 143B osteosarcoma cells, fadu human nasopharyngeal carcinoma cells, KYSE510 human Esophageal squamous cell carcinoma cells, KB human oral epidermoid carcinoma cells, ECA109 human esophageal phosphocarcinoma cells, Aspc human pancreatic cancer cells, colo829 human melanoma cells, SK-OV-3 ovarian cancer cells, Hela cervical cancer cells, U87MG human brain glue Gynecoma cells, skmel-28 human melanoma cells, LOVO intestinal carcinoma adenocarcinoma, T.Tn human esophageal carcinoma cells, Calu-6 lung epithelial cell carcinoma, and NCI-H226 (lung) squamous carcinoma cells.
  • the virus liquid at two time points in 24h, 48h or 72h (the cytopathy is more than 50% is collected again) is collected according to the cytopathic condition, and the virus titer is determined. After freezing and thawing three times (-80°C, 37°C), serially dilute the collected virus solution, take 300 ⁇ L to infect Vero cells (6-well plate), and shake the culture plate every 15 minutes to make the virus better adsorb the cells.
  • each Add 1mL 1% inactivated FBS DMEM medium to the well, incubate at 37°C, 5% CO 2 incubator for 2 hours, aspirate the virus liquid, then add 300 ⁇ L 1% inactivated FBS DMEM medium to each well and 3mL equilibrate to room temperature cultured in a 37°C, 5% CO 2 incubator. After culturing for 2 days, 2 mL of basal medium containing 0.01% neutral red was evenly added to stain the cells. Place in a 37°C, 5% CO 2 incubator for more than 12 hours. The suction pump discarded all the liquid in the 6-well plate, counted the plaques, and calculated the virus titer, virus titer virus dilution factor * (1000/300) * number of plaques.
  • This example evaluates the effect of the original virus strain (KOS-ATCC) and the transformed virus strain (KOS-hCCL19) on 20 cancer cell lines (CAL27, A549, PC-3, CNE-2Z, Panc-1, 143B, fadu, KYSE510 , KB, ECA109, Aspc, colo829, SK-OV-3, hela, U87MG, skmel-28, LOVO, T.Tn, Calu-6, NCI-H226), KOS-ATCC, KOS-hCCL19 in 20
  • the replication ability in the cancer cells is shown in Figures 15-17, and the modified virus strain retains the proliferative activity on tumor cells.
  • KOS and “KOS-ATCC” in Figure 1-17 both refer to the original virus strain; "hCCL19” and “KOS-hCCL19” both refer to the modified virus strain.
  • NCI-H460 human lung cancer cells were inoculated subcutaneously on the right back of BALB/c nude mice to establish a mouse tumor-bearing model.
  • the drugs are divided into groups, with 8 animals in each group.
  • the KOS-hCCL19 high (KOS-hCCL19(high)) dose group and the KOS-hCCL19 low dose (KOS-hCCL19(low)) group were set up, and the corresponding dosages were 3.33E+06pfu/mouse /once, 3.33E+05pfu/mouse/once.
  • a virus vehicle group (Vehicle group) is set at the same time.
  • TGI% (1-T/C) ⁇ 100%; where, T/C% is the relative tumor growth rate Rate, at a certain time point, the percentage value of the relative tumor volume in the treatment group and the control group).
  • This example evaluates the antitumor activity of KOS-hCCL19 on the NCI-H460 human lung cancer xenograft tumor animal model.
  • the in vivo anti-tumor effect played by the virus is shown in Figure 18.
  • TGI tumor inhibition rates
  • KOS-WT (“KOS” or "KOS-ATCC”) virus and KOS-hCCL19 virus were respectively set up in 5 different virus gradients, and the above-mentioned viruses with a certain concentration gradient were injected into the intracranial of female BALB/c mice ( 20 ⁇ L/mouse), and observe the survival of the animals, and calculate the LD 50 of different viruses.
  • the virus doses of each group corresponding to the KOS-WT virus are 5.00E+06pfu/mouse, 8.33E+05pfu/mouse, 1.39E+05pfu/mouse, 2.31E+04pfu/mouse, 3.86E +03pfu/mouse; KOS-hCCL19 virus corresponding to each group of virus doses were 4.64E+06pfu/mouse, 1.55E+06pfu/mouse, 5.16E+05pfu/mouse, 1.72E+05pfu/mouse, 5.73 E+04pfu/mouse.
  • a virus vehicle group (Vehicle group) was set up, with 6 animals in each group. After the experiment, the LD 50 was calculated by SPSS-Probit.
  • the neurotoxicity of wild-type KOS-WT strain and KOS-hCCL19 virus was evaluated, and the survival conditions of the above-mentioned experimental animals in each group were counted within 14 days after administration.
  • the specific experimental results are shown in Figure 19. No animal death was seen in the Vehicle group, and the number of animal deaths in the KOS-WT virus group from high to low doses were 6, 4, 4, 4, 0, and KOS-hCCL19 The number of animal deaths in the virus group from high to low groups were 0, 2, 1, 0, 0, respectively.
  • the median lethal dose LD 50 of different viruses was calculated according to the death of animals under different virus dosage gradients.
  • the LD 50 of KOS-WT virus and KOS-hCCL19 virus were 5.403E+04pfu and >4.64E+06pfu respectively.
  • the modified KOS-hCCL19 virus has significantly reduced neurotoxicity.
  • the KOS-hCCL19 virus had the highest half-lethal rate and the lowest toxicity.
  • the KOS-hCCL19 virus had about 100 times more attenuation effect than the KOS-WT virus.
  • Example 13 The effect of the target gene insertion site on the anti-tumor activity of the virus
  • TGI tumor growth inhibition
  • KOS- ⁇ 47- ⁇ ICP34.5- ⁇ TK-hCCL19 showed a relatively better tumor inhibitory effect on both the drug-administered side and the non-administered side .
  • KOS- ⁇ 47- ⁇ ICP34.5-hCCL19 showed better tumor killing activity than KOS- ⁇ 47- ⁇ ICP34.5- ⁇ TK-hCCL19, indicating that different insertion sites would affect Antitumor activity of viruses.
  • Tumor inhibition on the non-administered side after administration of KOS- ⁇ 47- ⁇ ICP34.5- ⁇ TK-hCCL19 and KOS- ⁇ 47- ⁇ ICP34.5-hCCL19, can produce certain distant anti-tumor activity.

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Abstract

一种HSV病毒载体及其应用，该HSV病毒载体的ICP47、ICP34.5基因沉默，以及携带CCL19基因。所述HSV病毒载体可以持续高表达趋化因子CCL19，有效抑制肿瘤。

Description

HSV病毒载体及其应用 技术领域
本发明涉及生物医药领域，具体地，本发明涉及HSV病毒载体及其应用。
背景技术
由于CCL19分子特性固有的一些限制，其临床应用受到限制。基本上，这些包括稳定性问题，例如酸降解，以及在温和变性条件下不可逆地聚集的趋势，随后生物活性的丧失。此外，当静脉内给药时，CCL19迅速从血液中清除，需要频繁重新给予高浓度，以在靶位点引起有效反应，并导致全身毒性和副作用，如发烧、疲劳、恶心、呕吐、腹泻、神经毒性和白细胞减少。
利用HSV1携带趋化因子CCL19不仅增强了病毒株对肿瘤细胞的靶向性，也通过外源基因CCL19募集免疫细胞的功能进一步增强了病毒株的抗肿瘤免疫疗效。利用表达CCL19的溶瘤病毒可以通过瘤内注射的方式使病毒在肿瘤中复制并在局部持续表达CCL19，避免通过静脉给药出现的CCL19迅速被清除及引起的全身性毒副作用。CCL19及CCR7在抗肿瘤治疗中的作用成为研究热点并取得显著进展，它们在趋化树突状细胞、CD4+和CD8+T细胞浸润肿瘤，介导免疫细胞释放细胞因子，抑制肿瘤增殖、迁移和侵袭以及协助治疗肿瘤过程中发挥关键作用，将抗肿瘤药物与CCL19联用有助于找到新的肿瘤治疗手段和基因疫苗，但目前没有采用HSV1病毒表达CCL19的相关报道。
发明内容
本申请是基于发明人对以下问题的发现和认识作出的：
目前，趋化因子的临床应用受到产品质量不稳定、需频繁给药的限制，发明人经过大量实验研究，惊喜地发现，CCL19相较于其它趋化因子可以更好的在HSV病毒中进行表达，尤其当CCL19基因插入在ICP34.5位点时，CCL19的表达量得到显著提高，当携带双拷贝突变后的趋化因子CCL19后，HSV病毒载体可以持续的高表达趋化因子CCL19，CCL19的表达量显著高于仅携带单拷贝突变后CCL19的HSV病毒，包含所述HSV病毒载体的溶瘤病毒保留了对肿瘤细胞的敏感性及增殖活性，并进一步提高了CCL19的表达量，突破了趋化因子表达量低、表达量不稳定以及频繁给药的限制，其抑制肿瘤的效果优于含有其它趋化因子编码核酸载体的病毒。
在本发明的第一方面，本发明提出了一种HSV病毒载体。根据本发明的实施例，所述HSV病毒载体的ICP47、ICP34.5基因沉默，以及携带CCL19基因。HSV1病毒中自带双拷贝ICP34.5基因，在ICP34.5基因位点插入CCL19基因可以有效降低病毒的神经毒性，同时提高病毒的抗肿瘤选择性；HSV1携带双拷贝趋化因子CCL19后可以增强病毒株对肿瘤细胞的靶向性，且通过外源基因CCL19募集免疫细胞进一步增强了病毒株的抗肿瘤免疫疗效。通过瘤内注射表达CCL19的溶瘤病毒，使病毒在肿瘤细胞中复制并在局部持续表达CCL19，避免静脉给药出现的CCL19被迅速清除及引起全身性毒副作用。敲除HSV基因组中的复制非必需基因ICP47，可提高被病毒感染的肿瘤细胞表面MHC-1的表达和细胞抗原呈递的能力；发明人对在HSV中高表达的趋化因子进行筛选，发现CCL19相较于其它趋化因子可以更好的在HSV病毒中进行表达，同时，发明人对CCL19的插入位点进行探索，意外地发现敲除HSV基因组中ICP34.5基因使得HSV有选择性地在肿瘤细胞中复制，而不在正常细胞内进行复制、增殖，由此可以提高HSV病毒的用药安全性，此外，将编码趋化因子的核酸插入该位点能够有效提高包含所述HSV病毒载体的病毒的抗肿瘤能力，根据本发明实施例的HSV病毒载体可以持续的高表达趋化因子CCL19，在ICP34.5基因位点中插入CCL19基因，CCL19的表达量得到显著提高，HSV病毒在肿瘤细胞内复制能力不受影响的前提 下，对于肿瘤细胞的杀伤力显著提高，可以有效的预防或治疗肿瘤。
根据本发明的实施例，上述HSV病毒载体还可以进一步包括如下附加技术特征至少之一：
根据本发明的实施例，携带两个拷贝的CCL19基因。发明人对所述表达载体携带CCL19基因的拷贝数进行筛选，发现根据本发明实施例的HSV病毒载体携带两个拷贝的CCL19基因可以显著提高CCL19的表达量，且CCL19的表达量显著高于只携带单拷贝CCL19基因的HSV病毒。
根据本发明的实施例，所述CCL19基因具有6.A>C突变。通过碱基优化，在不改变表达的氨基酸的前提下，提高目的基因在宿主中的表达水平。
野生型CCL19的基因核酸序列如下所示：
Figure PCTCN2022113446-appb-000001
含有6.A>C突变的所述CCL19基因核酸序列如下：
Figure PCTCN2022113446-appb-000002
根据本发明的实施例，所述ICP34.5基因沉默是通过敲除ICP34.5基因的第135-723核苷酸实现的。所述ICP34.5基因序列编码是以ICP34.5基因起始密码子的第一位核苷酸为第1位进行顺序编码的，ICP34.5基因的序列可参考https://www.ncbi.nlm.nih.gov/nuccore/NC_001806.2？from＝124834&to＝125861&report＝genbank&strand＝true，ICP34.5基因的具体序列如SEQ ID NO：3所示，其中，划线部分为所述ICP34.5基因序列编码。
Figure PCTCN2022113446-appb-000003
Figure PCTCN2022113446-appb-000004
根据本发明的实施例，所述ICP47基因沉默是通过敲除ICP47基因的第3-266核苷酸实现的。所述ICP47基因序列编码是以ICP47基因起始密码子的第一位核苷酸为第1位进行顺序编码的，ICP47基因的序列可参考https://www.ncbi.nlm.nih.gov/nuccore/NC_001806.2？report＝genbank&from＝46609&to＝47803&strand＝true，ICP47基因的具体序列如SEQ ID NO：4所示，其中，划线部分为所述ICP47基因序列编码。
Figure PCTCN2022113446-appb-000005
根据本发明的实施例，所述CCL19基因设置于ICP34.5基因的第134位核苷酸和第724位核苷酸之间，使得构建的HSV-1携带CCL19。
根据本发明的实施例，进一步包括CMV和polyA。
根据本发明的实施例，所述CMV与所述CCL19基因可操作地连接。
根据本发明的实施例，所述polyA设置于所述CCL19基因的3’端核苷酸与ICP34.5基因的第134位核苷酸之间。
根据本发明的实施例，所述病毒载体具有SEQ ID NO:5所示的核苷酸序列。
Figure PCTCN2022113446-appb-000006
Figure PCTCN2022113446-appb-000007
在本发明的第二方面，本发明提出了一种溶瘤病毒。根据本发明的实施例，携带第一方面所述的HSV病毒载体。根据本发明实施例包含所述HSV病毒载体的溶瘤病毒保留了对肿瘤细胞的敏感性及增殖活性，由于ICP34.5基因的特性，在HSV病毒的ICP34.5基因中插入CCL19基因后，CCL19表达量显著提高，且携带双拷贝CCL19的HSV病毒较携带其它拷贝数的CCL19的HSV病毒CCL19的表达量得到显著提高，所述溶瘤病毒可以持续的高表达趋化因子CCL19，对于肿瘤细胞的杀伤力显著提高，能够有效抑制肿瘤细胞。
根据本发明的实施例，上述溶瘤病毒还可以进一步包括如下附加技术特征至少之一：
根据本发明的实施例，所述溶瘤病毒为HSV-1。发明人发现，HSV-2存在安全隐患，可能会引起生殖器发生病毒感染，因此，使用HSV-1可提高溶瘤病毒使用时的安全性。
根据本发明的实施例，所述HSV-1包括选自F毒株、HF毒株、KOS毒株、HrR3毒株和17毒株的至少之一。
在本发明的第三方面，本发明提出了一种药物组合物。根据本发明的实施例，包括第一方面所述的HSV病毒载体或第二方面所述的溶瘤病毒。根据本发明实施例的药物组合物质量稳定，且在使用较低剂量时即可起效，尤其对复发性和大体积肿瘤的抑制效果非常显著。
根据本发明的实施例，上述药物组合物还可以进一步包括如下附加技术特征至少之一：
根据本发明的实施例，每单位剂量的所述药物组合物中包含10^5-10^12pfu的所述HSV病毒载体或溶瘤病毒。
在本发明的第四方面，本发明提出了第一方面所述的HSV病毒载体、第二方面所述的溶瘤病毒或第三方面所述的药物组合物在制备药物中的用途。根据本发明的实施例，所述药物用于治疗或预防肿瘤。如前所述，第一方面所述的HSV病毒载体、第二方面所述的溶瘤病毒均能够在合适的条件下高表达所述CCL19，可有效治疗或预防肿瘤，因此，利用所述HSV病毒载体、溶瘤病毒或药物组合物制备的药物同样能够有效治疗或预防肿瘤。
根据本发明的实施例，所述肿瘤包括选自肺癌、肝癌、咽鳞癌、结肠癌、骨肉瘤、卵巢癌、前列腺癌、神经胶质瘤、黑色素瘤、结直肠癌、食管癌、胰腺癌的至少之一。
在本发明的第四方面，本发明提出了一种治疗肿瘤的方法。根据本发明的实施例，所述方法包括将治疗有效量的第一方面所述的HSV病毒载体或第二方面所述的溶瘤病毒或第三方面所述的药物组合物施用于有需要的个体。根据本发明实施例的方法，可在施药频率较低的情况下有效治疗肿瘤。
根据本发明的实施例，上述方法还可以进一步包括如下附加技术特征至少之一：
根据本发明的实施例，所述有需要的个体为患有以下癌症中的至少之一的患者：肺癌、肝癌、咽鳞癌、结肠癌、骨肉瘤、卵巢癌、前列腺癌、神经胶质瘤、黑色素瘤、结直肠癌、食管癌、胰腺癌。
在本发明的第五方面，本发明提出了一种将免疫细胞募集至肿瘤的方法。根据本发明的实施例，所述方法包括使所述肿瘤与第一方面所述的HSV病毒载体或第二方面所述的重组溶瘤病毒接触。如前所述，第一方面所述的HSV病毒载体、第二方面所述的溶瘤病毒均能够在合适的条件下高表达所述CCL19，趋化因子是一类能趋化免疫细胞定向移动的小分子分泌蛋白，CCL19和其受体(如CCR7)，在树突状细胞、T细胞和多种肿瘤细胞上表达，因此，可以有效的的利用本申请所述HSV病毒载体或溶瘤病毒将免疫细胞募集至肿瘤，从而有效抑制肿瘤。
在本发明的第六方面，本发明提出了一种抑制肿瘤细胞生长或促进肿瘤细胞死亡的方法。根据本发明的实施例，所述方法包括使所述肿瘤细胞与第一方面所述的HSV病毒载体或第二方面所述的重组溶瘤病毒接触。
根据本发明的实施例，上述方法还可以进一步包括如下附加技术特征至少之一：
根据本发明的实施例，所述肿瘤细胞选自肺癌、肝癌、咽鳞癌、结肠癌、骨肉瘤、卵巢癌、前列腺癌、神经胶质瘤、黑色素瘤、结直肠癌、食管癌、胰腺癌。
根据本发明的实施例，所述重组溶瘤病毒以足以引起所述肿瘤细胞死亡的剂量提供。
本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本发明的实践了解到。
附图说明
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中：
图1是根据本发明实施例的第一轮重组后获得重组溶瘤病毒进行第一轮有限稀释筛选后的PCR结果图，其中，M为marker；1-32为挑选的病毒株克隆；+：为hCCL19重组病毒为模板的阳性对照(表达hCCL19基因)；D：为hCCL19-RL1-PMD18T质粒为模板(表达hccl19hCCL19基因)的处理；K：为KOS-△47病毒基因为模板(表达ICP34.5基因)的处理；水：以水为模板的阴性对照；
图2是根据本发明实施例的第一轮重组后获得重组溶瘤病毒进行第二轮有限稀释筛选后的PCR结果图，其中，M为marker；1-28为第二轮筛选挑选的28个克隆(1-13为KOS-△47-S1-gDNA-hCCL19-32，1-28为KOS-△47-S1-gDNA-hCCL19-9)；D：为hCCL19-RL1-PMD18T质粒为模板(表达hCCL19基因)的处理；K：以KOS-△47病毒基因为模板(表达ICP34.5基因)的处理；水：以水为模板的阴性对照；
图3是根据本发明实施例的第一轮重组后获得的重组溶瘤病毒进行扩繁后的PCR鉴定结果图，其中，M为marker-DL5000；9-2：为KOS-△47-S1-gDNA-hCCL19-9-2病毒基因组为模板的处理；D：为hCCL19-RL1-PMD18T质粒为模板(表达hCCL19基因)的处理；K：以KOS-△47病毒基因为模板(表达ICP34.5基因)的处理；水：以水为模板的阴性对照；
图4是根据本发明实施例的第二轮重组后获得的重组溶瘤病毒经第一轮筛选后的PCR结果图，其中，M为marker-DL2000；1-102为挑选的102个克隆；9-2：为KOS-△47-S1-gDNA-hCCL19-9-2病毒基因组为模板的处理；D：为hCCL19-RL1-PMD18T质粒为模板(表达hCCL19基因)的处理；K：为KOS-△47病毒基因为模板(表达ICP34.5基因)的处理；水：以水为模板的阴性对照；KOS-△47-S5g-hCCL19为KOS-△47-S1-gDNA-hCCL19-9-2经第2轮重组转染获得的新病毒株；
图5是根据本发明实施例的第二轮重组后获得的重组溶瘤病毒经第二轮筛选后的PCR结果图，其中，M为marker-DL2000；4、5、8、9…101为：经第一轮PCR筛选获得的60个阳性病毒；9-2：为KOS-△47-S1-gDNA-hCCL19-9-2病毒基因组为模板(表达hCCL19基因和ICP34.5基因)的处理；D：为hCCL19-RL1-PMD18T质粒为模板(表达hCCL19基因)的处理；K：为KOS-△47病毒基因为模板(表达ICP34.5基因)的处理；水：水为模板的阴性对照；KOS-△47-S5g-hCCL19为KOS-△47-S1-gDNA-hCCL19-9-2经第2轮重组转染获得的新病毒株；
图6是根据本发明实施例的第二轮重组后获得的阳性重组溶瘤病毒进行扩繁后的PCR鉴定结果图，其中，M为marker-DL2000；9-2：为KOS-△47-S1-gDNA-hCCL19-9-2病毒基因组为模板(表达hCCL19基因和ICP34.5基因)的处理；D：为hCCL19-RL1-PMD18T质粒为模板(表达hCCL19基因)的处理；32-6：为KOS-△47-S1-hCCL19-32-6病毒基因为模板的处理，为第1轮重组筛选到单拷贝插入病毒克隆(表达hCCL19基因)；水：水为模板的阴性对照；KOS-△47-S5g-hCCL19为KOS-△47-S1-gDNA-hCCL19-9-2经第2轮重组转染获得的新病毒株；
图7是根据本发明实施例的扩增hCCL19表达盒全长后获得的PCR结果图，其中，M为marker-DL2000；9-2：为KOS-△47-S1-gDNA-hCCL19-9-2病毒基因组为模板(表达hCCL19基因和ICP34.5基因)的处理； D：为hCCL19-RL1-PMD18T质粒为模板(表达hCCL19基因)的处理；32-6：为KOS-△47-S1-hCCL19-32-6病毒基因为模板的处理，为第1轮重组筛选到单拷贝插入病毒克隆(表达hCCL19)；水：水为模板的阴性对照；
图8是根据本发明实施例的阳性重组溶瘤病毒进行第一轮噬斑纯化后获得的电泳结果图，其中，M为marker-DL5000；D为hCCL19-RL1-PMD18T质粒为模板(表达hCCL19基因)的处理；K为KOS-△47病毒基因组为模板(表达ICP34.5基因)的处理；水：以为水模板的阴性对照；
图9是根据本发明实施例的阳性重组溶瘤病毒进行第二轮噬斑纯化后获得的电泳结果图，其中，M为marker-DL5000；D为hCCL19-RL1-PMD18T质粒为模板(表达hCCL19基因)的处理；K为KOS-△47病毒基因组为模板(表达ICP34.5基因)的处理；水：以为水模板的阴性对照；
图10是根据本发明实施例的阳性重组溶瘤病毒进行第三轮噬斑纯化后获得的电泳结果图，其中，M为marker；D为hCCL19-RL1-PMD18T质粒为模板(表达hCCL19基因)的处理；K为KOS-△47病毒基因组为模板(表达ICP34.5基因)的处理；水：以水模板的阴性对照；
图11是本发明实施例的阳性重组溶瘤病毒电泳图，其中重组病毒KOS-△47-hCCL19-05-02-02-02扩增片段大小符合预期；
图12是重组病毒KOS-△47-hCCL19-05-02-02-02TA连接阳性克隆筛选结果图；
图13是根据本发明实施例的KOS–ATCC、KOS-hCCL19在6种癌细胞中的复制能力结果图；
图14是根据本发明实施例的2k病毒和KOS-hCCL19重组病毒对细胞进行感染后检测到的病毒表达量的结果图；
图15-图17是根据本发明实施例的KOS、hCCL19在20种癌细胞中的复制能力结果图；其中KOS为野生型毒株；hCCL19(也即KOS-hCCL19)为重组后的病毒株；
图18是根据本发明实施例的NCI-H460小鼠模型的肿瘤大小变化结果图；
图19是根据本发明实施例的病毒神经毒性评估实验中各组动物的生存曲线图；
图20是根据本发明实施例的各组A20小鼠模型上右侧(给药侧)肿瘤大小的变化结果图；
图21是根据本发明实施例的各组A20小鼠模型上左侧(未给药侧)肿瘤大小的变化结果图；
图22是根据本发明实施例的给药第28天后各组A20小鼠模型死亡状况图。
具体实施方式
下面详细描述本发明的实施例，所述实施例的示例在附图中示出。下面通过参考附图描述的实施例是示例性的，旨在用于解释本发明，而不能理解为对本发明的限制。
此外，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括至少一个该特征。在本发明的描述中，“多个”的含义是至少两个，例如两个，三个等，除非另有明确具体的限定。
需要说明的是，本申请所述“趋化因子”是一类表达在免疫细胞和内皮细胞的细胞膜上，趋化各种免疫细胞定向运动的细胞因子，如CCL19，CCL19可趋化树突状细胞、CD4 +和CD8+细胞浸润肿瘤，介导免疫细胞释放细胞因子，抑制肿瘤增殖、迁移和侵袭以及协助治疗肿瘤过程中发挥关键作用。
本申请所述的“可操作地连接”是指将外源基因连接到载体上，使得载体内地控制元件，例如启动子序列等等，能够发挥其预期地调节外源基因地转录和翻译的功能。
本申请所述的“溶瘤病毒”丧失了部分功能性基因，使其在正常细胞中感染复制能力减弱，即为减毒的溶瘤病毒，如本申请种的HSV-1溶瘤病毒，ICP47与ICP34.5基因被敲除，使其只能选择性地在肿瘤细胞中复制。
本申请中，发明人对表达载体中需要插入的趋化因子进行筛选，发现CCL19趋化因子较其它趋化因子更容易在该表达载体中进行表达，且携带CCL19的HSV病毒相较于携带其它趋化因子的病毒具有更好的溶瘤和免疫的效果，治疗或预防肿瘤的效果更佳；发明人进一步对CCL19基因在HSV病毒中的插入位点进行筛选，由于ICP34.5基因的特性，发明人发现将CCL19基因插入在ICP34.5位点时，所获得的HSV病毒相较于在除ICP34.5基因以外的位点插入CCL19的病毒，其CCL19的基因表达量得到显著提高，包含所述HSV表达载体的溶瘤病毒相较于包含在除ICP34.5基因以外的位点插入CCL19的表达载体的溶瘤病毒，其CCL19的基因表达量得到显著提高，且安全性更高；同时，对表达载体中插入CCL19基因的拷贝数进行探索，发现将表达载体中双拷贝的ICP34.5基因进行敲除，插入双拷贝的CCL19基因后，所述HSV表达载体CCL19基因的表达量显著高于携带单拷贝CCL19基因的HSV表达载体，包含所述HSV表达载体的溶瘤蛋白的CCL19基因的表达量显著高于包含单拷贝CCL19基因的HSV表达载体的溶瘤病毒。
本申请以下实施例中“Δ47病毒基因组”是指敲除了ICP47基因的HSV-1溶瘤病毒基因组、“KOS-△47-S1-gDNA-hCCL19”是指在KOS-△47的基础上插入了hCCL19；利用CRISSPR/Cas9系统构建重组HSV-1病毒，包括质粒构建、单拷贝重组溶瘤病毒的制备和筛选、单拷贝病毒的鉴定、双拷贝重组溶瘤病毒的制备和筛选、双拷贝病毒的鉴定。
下面参考具体实施例，对本发明进行描述，需要说明的是，这些实施例仅仅是描述性的，而不以任何方式限制本发明。
实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1第一轮病毒重组及筛选
本实施例中发明人利用酚氯仿抽提KOS-△47病毒基因组；使用Lipofectamine 3000转染试剂进行转染293T细胞，得到重组病毒；重组病毒经第一轮有限稀释筛选，第二轮有限稀释筛选，获得阳性重组病毒，阳性重组病毒进行扩繁、基因组提取、PCR鉴定、测序；结果显示：经第一轮病毒重组及筛选，获得重组病毒为单拷贝插入，具体实验操作如下：
1、酚氯仿抽提KOS-△47病毒基因组
经KOS-△47病毒感染的vero细胞培养物，弃上清，留下贴壁已发生病变效应的细胞(～100％病变)，冻于-80℃。按以下步骤提取病毒基因组：
①向细胞中加入1mL细胞裂解液，均匀晃动，使细胞裂解，用细胞刮子轻轻刮入2mL离心管中，冰浴20min。
②向步骤①的产物中加入660μL的5M NaCl，轻轻混匀后置于4℃冰箱过夜。
③将步骤②的产物于下列条件中进行处理：4℃，12000rpm离心30min。
④用去尖枪头吸取离心后的上清于新离心管中，加入等体积的酚氯仿异戊醇(25:24:1)，抽提蛋白，轻轻上下颠倒离心管5min。
⑤将步骤④的产物于4℃，12000rpm离心10min，用去尖枪头吸取上清，再次抽提蛋白一遍。
⑥上述抽提后的上清液加入预冷的2倍体积的无水乙醇，轻轻混匀，-20℃静置2h。
⑦将步骤⑥的产物于4℃，12000rpm离心10min，弃上清，沉淀用500μL的75％乙醇洗涤两遍(轻轻弹而不是吹打)，4℃，12000rpm，10min。
⑧弃上清，12000rpm，1min，4℃，用10μL枪将液体吸干。
⑨向步骤⑦的产物中加入30μL去离子水，溶解DNA。
2、转染293T细胞制备重组病毒
使用Lipofectamine 3000转染试剂将表1中的A和B(CRISPR/Cas9基因编辑质粒(34.5-S1-2质粒)+同源修复供体质粒(dornor DNA(hCCL19-RL1-PMD18T)+KOS-△47病毒基因组)混合后，共转染至6孔板293T细胞中，得到含有重组病毒的病毒液，其中，34.5-S1-2质粒为包含gRNA的质粒，该质粒为针对ICP34.5基因位点的CRISPR/Cas9基因编辑质粒，dornor DNA(hCCL19-RL1-PMD18T)为包含hCCL19的供体质粒。
表1：
Figure PCTCN2022113446-appb-000008
细胞转染前，更换37℃温育的10％FBS/DMEM完全培养液，随后在EP管中将A和B混合均匀(混匀时轻柔，用手轻弹)静置于室温15min后，按每孔300μL，将转染复合物逐滴加入到6孔板中293T细胞培养物中培养6h后，换新鲜的经过温育的DMEM+10％FBS完全培养基，细胞继续培养。
转染2天后，观察细胞病变，弃旧培养液，加入1mL 37℃温育的1％FBS/DMEM完全培养液，收集细胞，负70度反复冻融3次后，4000rpm离心10min，取上清即为含有重组病毒的病毒液，命名为KOS-△47-S1-gDNA-hCCL19-201224，备用或冻于-80℃。
3、重组病毒进行第一轮有限稀释筛选
将步骤2获得的第一轮重组病毒经第一轮有限稀释筛选，具体操作如下：
将重组病毒液KOS-△47-S1-gDNA-hCCL19-201224按稀释倍数100倍和500倍稀释，按照100μL/孔，加入长满单层Vero的96孔板中，每个稀释度各2块96孔板，命名为：KOS-△47-S1-gDNA-hCCL19-201224(10 -2，①～②)、KOS-△47-S1-gDNA-hCCL19-201224(2×10 -3，①～②)，将96孔板置于37℃，5％CO2培养箱中培养。
72小时后观察细胞病变情况，KOS-△47-S1-gDNA-hCCL19-201224(2×10 -3，①～②)全部孔有明显病变，对KOS-△47-S1-gDNA-hCCL19-201224(2×10 -3，①)96孔板上清液进行病毒gDNA粗提，方法如下：
2μL病毒液+8μL gDNA Extraction Buffer(使用前按10μL/mL加入蛋白酶K)，混匀后PCR仪中反应，55℃1h，95℃10min，16℃∞，记为：KOS-△47-S1-gDNA-hCCL19-201227-2×10 -3-①-1-96号)，用于PCR扩增验证。
其中，第一轮有限稀释筛选后获得的病毒株进行病毒gDNA粗提，具体操作为：2μL病毒液+8μL gDNA Extraction Buffer(使用前按10μL/mL加入蛋白酶K)，混匀后PCR仪中反应，PCR反应条件为55℃1h，95℃10min，16℃∞，用PCR扩增验证hCCL19是否插入病毒基因组，其中hCCL19基因检测引物如表2所示，PCR扩增反应体系如表3所示。
表2：
Figure PCTCN2022113446-appb-000009
Figure PCTCN2022113446-appb-000010
表3：
组分 体积μL
粗提gDNA 0.5μL
MightyAmp DNA Polymerase 0.25μL
2×MightyAmp Buffer Ver.2 5μL
hCCL19-screen-F2 0.25μL
hCCL19-screen-R2 0.25μL
ddH 2O 3.75μL
总体积 10μL
重组病毒第一轮有限稀释筛选的PCR结果如图1所示，PCR电泳结果显示，KOS-△47-S1-gDNA-hCCL19-9、32号疑似阳性重组病毒，收取病毒液，将KOS-△47-S1-gDNA-hCCL19-9命名为KOS-△47-S1gDNA-hCCL19-9-200227。
4、重组病毒进行第二轮有限稀释筛选
将步骤3获得的第一轮有限稀释筛选后的疑似阳性重组病毒进行第二轮有限稀释筛选，具体操作如下：
将重组病毒液KOS-△47-S1gDNA-hCCL19-9-200227按稀释倍数1×10 6稀释，按照100μL/孔加入长满单层Vero细胞的96孔板中，命名为：KOS-△47-S1gDNA-hCCL19-201231-1x10 6，37℃，5％CO 2培养2小时后，弃病毒液，加入200μL/孔DPBS洗1遍，弃去，加入100μL/孔的1％FBS/DMEM，继续培养。培养96小时后观察细胞病变情况，KOS-△47-S1gDNA-hCCL19-201231-1x10 6有28孔病变明显，编号为：s1g-9-1～28号，对发生病变孔的上清液进行病毒gDNA粗提，方法同上。
其中，第二轮有限稀释筛选后获得的病毒株(KOS-△47-S1-gDNA-hCCL19-9)进行病毒gDNA粗提，具体操作为：2μL病毒液+8μL gDNA Extraction Buffer(使用前按10μL/mL加入蛋白酶K)，混匀后于PCR仪中反应，反应条件为：55℃1h，95℃10min，16℃∞，用PCR扩增验证hCCL19是否插入病毒基因组，其中hCCL19基因检测引物如表2所示，PCR扩增反应体系如表3所示。
重组病毒第二轮有限稀释筛选的PCR结果如图2所示，PCR电泳结果显示，KOS-△47-S1-gDNA-hCCL19-9-2、KOS-△47-S1-gDNA-hCCL19-9-6号疑似阳性重组病毒；将KOS-△47-S1-gDNA-hCCL19-9-2命名为KOS-△47-hCCL19-S1g-9-2号-210104。
5、对第二轮有限稀释筛选获得的重组溶瘤病毒进行鉴定
将步骤4获得的重组溶瘤病毒进行鉴定、测序，具体操作如下:
对阳性重组病毒(KOS-△47-S1-gDNA-hCCL19-9-2)进行扩繁、基因组提取、PCR鉴定、测序，其中，基因组提取的具体操作为：将重组病毒KOS-△47-hCCL19-S1g-9-2号-210104用6孔板vero细胞扩繁，加入5μL病毒，37℃，5％CO 2培养24小时，细胞完全病变，收集培养液，命名为KOS-△47-S1g-9-2-P01-210107，取200μL病毒液，使用病毒基因组提取试剂盒Viral RNA/DNA Extraction Kit ver.5.0提取病毒基因组，命名为：KOS-△47-S1g-9-2-gDNA-210107，进行PCR扩增反应中hCCL19同源臂序列检测引物如表4所示，PCR扩增反应体系如表5所示。
表4：
Figure PCTCN2022113446-appb-000011
Figure PCTCN2022113446-appb-000012
表5：
成分 体积
gDNA 1.0μL
MightyAmp DNA Polymerase 1.0μL
2×MightyAmp Buffer Ver.2 25.0μL
345-HOM1-F2 1.0μL
345-HOM2-R2 1.0μL
ddH 2O 21.0μL
总体积 50μL
第一轮病毒重组后重组病毒扩繁、PCR鉴定结果见图3所示，其中，KOS-△47-S1g-9-2-gDNA-210107(图3中的9-2)是病毒KOS-△47-S1g-9-2-P01-210107的基因组将重组前后条带分别切胶收回，送广州艾基测序(sanger测序)，确认重组后条带，hCCL19基因表达盒测序完全正确，重组前条带，确认为KOS基因组重组前条带；由此确认，经第一轮病毒重组及筛选获得的重组病毒KOS-△47-S1g-9-2-P01-210107为单拷贝插入。
实施例2第二轮病毒重组及筛选
本实施例对实施例1获得的单拷贝插入的KOS-△47-S1-gDNA-hCCL19-9-2进行第二轮重组和筛选，酚氯仿抽提单拷贝重组病毒基因组；使用Lipofectamine 3000转染试剂转染293T细胞，得到重组病毒；重组病毒经有限稀释，第1轮PCR筛选，第2轮PCR筛选，获得阳性重组病毒，阳性重组病毒进行扩繁、基因组提取、PCR鉴定、测序，具体操作如下：
1、酚氯仿抽提KOS-△47-hCCL19-9-2(即，KOS-△47-S1-gDNA-hCCL19-9-2)病毒基因组
经KOS-△47-S1g-9-2-P01(即，KOS-△47-S1g-9-2-P01-210107)病毒感染的vero细胞培养物，弃上清，留下贴壁已发生病变效应的细胞(～100％病变)，冻于-80℃，提取病毒基因组步骤同上。
2、转染293T细胞制备重组病毒
使用Lipofectamine 3000转染试剂将表6中的A和B(CRISPR/Cas9基因编辑质粒(Cas9-sgRNA345-5质粒)+同源修复供体质粒(dornor DNA(hCCL19-RL1-PMD18T)+病毒基因组(KOS-△47-hCCL19-9-2)混合后，共转染至6孔板293T细胞中，得到含有重组病毒的病毒液，其中，Cas9-sgRNA345-5为包含gRNA的质粒，是针对ICP34.5基因位点的CRISPR/Cas9基因编辑质粒，dornor DNA(hCCL19-RL1-PMD18T)为包含hCCL19的供体质粒。
表6：
Figure PCTCN2022113446-appb-000013
细胞转染操作同上，将含有重组病毒的病毒液，命名为KOS-△47-hCCL19-S5g-P00-210117，备用。
3、重组病毒进行第一轮有限稀释筛选
将步骤2获得的第二轮重组病毒经第一轮有限稀释筛选，具体操作如下：
将重组病毒液KOS-△47-hCCL19-S5g-P00-210117按稀释倍数400倍、1000倍、3000倍稀释，按照100μL/孔，加入长满单层Vero细胞的96孔板中，每个稀释度各2块96孔板，命名为：KOS-△47-S5g-hCCL19-3000X/1000X/400X-P01-210117-01～02，于37℃，5％CO 2培养箱培养2小时后，弃病毒液，加入100μL/孔DPBS洗板，弃去，加入100μL/孔1％FBS/DMEM，将96孔板置于37℃，5％CO 2培养箱培养。
将上述细胞培养72小时后观察细胞病变情况，KOS-△47-S5g-hCCL19-3000X-P01-210117-01有45孔病变，KOS-△47-S5g-hCCL19-3000X-P01-210117-02有57孔病变，对病变孔进行病毒gDNA粗提，方法同上，记为：KOS-△47-S5g-hCCL19-3000X-P01-210121-01～102号，用于PCR扩增验证。
其中，第一轮有限稀释筛选后获得的病毒株进行病毒gDNA粗提，用PCR扩增验证ICP34.5基因序列是否敲除成功，其中ICP34.5基因序列检测引物为如表7所示，PCR扩增反应体系如表8所示。
表7：
Figure PCTCN2022113446-appb-000014
表8：
成分 体积
粗提gDNA 0.5μL
MightyAmp DNA Polymerase 0.25μL
2×MightyAmp Buffer Ver.2 5μL
LP-F2 0.25μL
LP-R2 0.25μL
ddH 2O 3.75μL
总体积 10μL
第二轮重组病毒进行有限稀释后经第1轮PCR筛选结果如图4所示，PCR电泳结果显示，经过有限稀释后共获得60个阳性重组病毒。
4、重组病毒第2轮PCR筛选
将本实施例步骤3第一轮有限稀释后获得的60个阳性重组病毒经第2轮PCR筛选，验证hCCL19是否插入病毒基因组，其中hCCL19基因检测引物如表9所示(扩增hCCL19基因表达框全长)，PCR扩增反应体系如表10所示。
表9：
Figure PCTCN2022113446-appb-000015
表10:
成分 体积
粗提gDNA 0.5μL
MightyAmp DNA Polymerase 0.25μL
2×MightyAmp Buffer Ver.2 5μL
345-HOM1-F2 0.25μL
345-HOM2-R2 0.25μL
ddH 2O 3.75μL
总体积 10μL
第二轮重组病毒经第2轮PCR筛选后扩增的结果如图5所示：PCR电泳结果显示明显仅扩增重组后条带的样本为KOS-△47-S5g-hCCL19中的4、5、8-10、12、15、18、19、21、26、28-31、36、37、43、45、47、48、53、54、56、57、59、60、63、67、68、71、72、77、79-81、84、87、88、90、93-98、101号，共47个阳性重组病毒。
5、对阳性重组溶瘤病毒进行鉴定
将步骤4获得的重组溶瘤病毒进行鉴定、测序，具体操作如下:
对阳性重组病毒(KOS-△47-S5g-hCCL19中的4、5、8-10、12)进行扩繁、基因组提取、PCR鉴定、测序，将重组病毒扩繁，提取病毒基因组，分别命名为：KOS-△47-S5g-hCCL19-04/05/08/09/10/12-gDNA-210125。基因组提取的具体操作为：用6孔板vero细胞扩繁，加2μL病毒液，37℃，5％CO 2培养24小时，细胞完全病变，收集培养液，命名为KOS-△47-S5g-hCCL19-04/05/08/09/10/12210124，于-70℃保存，取200μL病毒液，使用试剂盒Viral RNA/DNA Extraction Kit ver.5.0提取基因组，用50μL无菌水洗脱，分别命名为：KOS-△47-S5g-hCCL19-04/05/08/09/10/12-gDNA-210125，进行PCR扩增反应中ICP34.5基因的序列设计引物如表7所示，PCR扩增反应体系如表8所示。PCR电泳结果如图6所示，PCR电泳结果显示：KOS-△47-S5g-hCCL19-04、05、08、09、10、12未能扩增到敲除片段，重组病毒成功敲除ICP34.5基因。
其中，根据hCCL19表达盒的同源臂序列设计引物，扩增hCCL19表达盒全长，引物序列如表11、12所示，PCR扩增反应体系如表13所示。
表11：
Figure PCTCN2022113446-appb-000016
表12：
Figure PCTCN2022113446-appb-000017
表13：
成分 体积
gDNA 1.0μL
MightyAmp DNA Polymerase 1.0μL
2×MightyAmp Buffer Ver.2 25.0μL
引物F 1.0μL
引物R 1.0μL
ddH 2O 21.0μL
总体积 50μL
其中，引物F为表11、表12中的345-HOM1-F2；引物R为表11、表12中的345-HOM2-R2或345-flank-seq。
PCR结果如图7所示，KOS-△47-S5g-hCCL19-04,05,08,09,10,12号样品条带单一，且条带与hCCL19-RL1-pMD18T质粒阳性对照样品条带大小一致。切胶回收KOS-△47-S5g-hCCL19-04,05,08,09,10,12的条带，送样至广州艾基生物技术有限公司测序，测序结果显示KOS-△47-S5g-hCCL19-04,05,08,09,10,12重组病毒hCCL19基因表达框测序均正确，将其命名为：KOS-△47-S5g-hCCL19-04/05/08/09/10/12-P01-210123，即经2轮重组，成功获得双拷贝整合KOS-△47-hCCL19重组病毒。
实施例3阳性重组溶瘤病毒第一轮噬斑纯化
1、单克隆制备及基因组提取
选取KOS-△47-S5g-hCCL19-05/12-P01-210123进行第1轮噬斑纯化，用1％的FBS/DMEM将病毒液分别稀释至“5E+03”、“5E+04”、“5E+05”，弃6孔板vero细胞旧培养液，加入300μL病毒稀释液，每个稀释度2个复孔，感染后，继续培养；对细胞进行染色后，观察染色情况，每个病毒株各挑取5个病毒斑，取200μL病毒液加入长满的6孔板vero细胞中，培养2天后，细胞已完全病变，收病毒液，命名为：KOS-△47-hCCL19-05-P01-01/02/03/04/05-210205、KOS-△47-hCCL19-12-P01-01/02/03/04/05-210205，取200μL病毒液用试剂盒Viral RNA/DNA Extraction Kit ver.5.0提取病毒基因，提取的具体操作按照试剂盒说明书进行，将提取后的病毒基因命名为：KOS-△47-hCCL19-05-01/02/03/04/05-gDNA-210205、KOS-△47-hCCL19-12-01/02/03/04/05-gDNA-210205。
2、PCR鉴定
PCR扩增的具体操作如下所示：所用引物序列如表11、表12所示，所用反应体系如表13所示。
KOS-△47-hCCL19-05/12号重组病毒电泳结果如图8所示，电泳结果显示KOS-△47-hCCL19-05-01/02/03/04/05号，KOS-△47-hCCL19-12-01/02/03/04/05号均表达hCCL19基因，而不表达ICP34.5基因。
3、测序
切胶回收KOS-△47-hCCL19-05-01/02/03、KOS-△47-hCCL19-12-01/02/03的条带，送样至广州艾基生物技术有限公司测序。测序的具体操作为：sanger测序，经测序结果，艾基-IGC280537，6个克隆测序确认为重组病毒，hCCL19基因表达盒测序完全正确，与理论序列一致，测序质量良好。
实施例4重组溶瘤病毒第二轮噬斑纯化
1、单克隆制备及基因组提取
选取KOS-△47-hCCL19-05-02-P01-210205、KOS-△47-hCCL19-12-03-P01-210205进行第2轮噬斑纯化，将病毒液稀释至“2E+04”、“2E+05”、“2E+06”，病毒感染操作同实施例3，每个病毒株各挑取5个病毒斑，以vero细胞为宿主进行扩繁，培养2天后，细胞已完全病变，收病毒液，命名为：KOS-△47-hCCL19-05-02-P01-01/02/03/04/05-210303、KOS-△47-hCCL19-12-03-P01-01/02/03/04/05-210303，取200μL病毒液，使用试剂盒Viral RNA/DNA Extraction Kit ver.5.0提取病毒基因组，具体操作按照试剂盒说明书进行，将提取的病毒基因组命名为：KOS-△47-hCCL19-05-02-01/02/03/04/05-gDNA-210303、KOS-△47-hCCL19-12-03-01/02/03/04/05-gDNA-210303。
2、PCR鉴定
PCR扩增的具体操作如下所示：所用引物序列如表11、表12所示，所用反应体系如表13所示。
KOS-△47-hCCL19-05-02/12-03号重组病毒电泳结果如图9所示，电泳结果显示经2对引物扩增，KOS- △47-hCCL19-05-02-01/02/03/04/05号，KOS-△47-hCCL19-12-03-01/02/03/04/05号样品条带均单一，条带与hCCL19-RL1-PMD18T质粒阳性对照品条带大小一致，为重组后大条带。
3、测序
切胶回收KOS-△47-hCCL19-05-02-01/02/03、KOS-△47-hCCL19-12-03-01/02/03的条带，送样至广州艾基生物技术有限公司测序。测序的具体操作为：sanger测序测序结果显示艾基-IGC282403，6个克隆测序确认为重组病毒，hCCL19基因表达盒测序完全正确，与理论序列一致，测序质量良好。
实施例5重组溶瘤病毒第三轮噬斑纯化
1、单克隆制备及基因组提取
选取实施例4中获得的KOS-△47-hCCL19-05-02-02、KOS-△47-hCCL19-12-03-01进行第3轮噬斑纯化，将病毒液稀释至“5E+05”、“1E+06”、“2E+06”、“4E+06”，病毒感染操作同实施例3；每个病毒株各挑取5个病毒斑，进行扩繁(vero细胞为宿主)，培养2天后，细胞已完全病变，收病毒液，命名为：KOS-△47-hCCL19-05-02-02-01/02/03/04/05-P01-210315、KOS-△47-hCCL19-12-03-01-01/02/03/04/05-P01-210315，取200μL病毒液，使用试剂盒Viral RNA/DNA Extraction Kit ver.5.0提取病毒基因组，命名为：KOS-△47-hCCL19-05-02-02-01/02/03/04/05-gDNA-210315、KOS-△47-hCCL19-12-03-01-01/02/03/04/05-gDNA-210315。
2、PCR鉴定
对于步骤1提取的基因组进行PCR扩增，所用引物序列如表7、表11、表12、表14所示，所用反应体系如表8、表15、表16所示。
表14：
Figure PCTCN2022113446-appb-000018
表15：
成分 体积
gDNA 1.0μL
MightyAmp DNA Polymerase 1.0μL
2×MightyAmp Buffer Ver.2 25.0μL
345-HOM1-F2 1.0μL
345-HOM2-R2 1.0μL
ddH 2O 21.0μL
总体积 50μL
表16：
成分 体积
gDNA 1.0μL
MightyAmp DNA Polymerase 1.0μL
2×MightyAmp Buffer Ver.2 25.0μL
345-HOM1-F2 1.0μL
345-flank-seq-R3 1.0μL
ddH 2O 21.0μL
总体积 50μL
KOS-△47-hCCL19-05-02-02和KOS-△47-hCCL19-12-03-01号重组病毒电泳结果如图10所示，电泳结果显示经2对引物扩增，KOS-△47-hCCL19-05-02-02-01/02/03/04/05号，KOS-△47-hCCL19-12-03-01/02/03/04/05号样品条带均单一，条带与hCCL19-RL1-PMD18T质粒阳性对照品条带大小一致，为重组后大条带。
3、测序
切胶回收KOS-△47-hCCL19-05-02-02-01/02/04、KOS-△47-hCCL19-12-03-01-01/02/03的条带，送样至广州艾基生物技术有限公司测序。测序的具体操作为：sanger测序。测序结果显示艾基-IGC283714，IGC283898，挑取的6个克隆表达框测序均正确，hCCL19基因表达盒测序完全正确，与理论序列一致，测序质量良好。
实施例6：重组病毒KOS-△47-hCCL19-05-02-02-02测序验证
分别使用高保真酶PCR扩增酶(
Figure PCTCN2022113446-appb-000019
Max DNA Polymerase、ApexHF HS DNA Polymerase FS)对重组病毒KOS-△47-hCCL19-05-02-02-02基因表达框序列进行TA克隆测序，引物序列如表17所示，反应体系如表18所示。
表17：
Figure PCTCN2022113446-appb-000020
表18：
成分 体积
模板 1μL
PCR扩增酶(2X) 25μL
345-HOM1-F2(10uM) 1μL
345-flank-seq-R3(10uM) 1μL
DMSO 2μL
ddH 20 20μL
总体积 50μL
PCR扩增程序为：98℃3min；(98℃10s，64℃15s，72℃3min)进行30个循环；72℃5min；16℃∞，完成后1.5％琼脂糖凝胶电泳，电泳图见图11，使用试剂盒cycle-pure kit(omega)进行回收后，进行加A尾、连T载、转化大肠感受态DH5a，阳性克隆筛选结果见图12，各挑取6个阳性克隆菌液送样至广州艾基生物技术有限公司测序。
测序结果：艾基IGC287528，IGC287406，IGC287407，高保真酶ApexHF HS DNA Polymerase FS挑选的克隆：1,3,4,6,7克隆基因表达框正确，测序质量良好，hCCL19基因表达框正确，与理论序列完全一致，测序质量良好；高保真酶Prime star max挑选的克隆：3,5,6,7,8克隆基因表达框正确，测序质量良好，hCCL19基因表达框正确，与理论序列完全一致，测序质量良好。
施例7病毒细胞毒性试验
体外培养表19所示的细胞株，以合适的细胞密度接种于96孔培养板，培养过夜后，分别加入12个梯度浓度(MOI＝20、5、1.25、0.3125、0.078125、0.01953125、0.004882813、0.001220703、0.000305176、7.62939E-05、1.90735E-05、4.76837E-06)的两种病毒，两种病毒的具体信息如表20所示，再分别培养24，48或72h，依照CCK8试剂盒(购自日本同仁)说明书进行细胞活力的检测。
表19：
细胞类型 细胞名称 培养基
NCI-H460 大细胞肺癌细胞 RPMI-1640+10％FBS
Fadu 人咽鳞癌细胞 MEM+10％FBS
HepG2 肝癌细胞 DMEM+10％FBS
Hep3B2.1-7 肝癌细胞 DMEM+10％FBS
HCT-116 结肠癌细胞 RPMI-1640+10％FBS
HT-29 结肠癌细胞 RPMI-1640+10％FBS
SW620 结肠癌细胞 DMEM+10％FBS
表20：
样品名称 滴度 来源
KOS-ATCC 8.33x10^7 SHS21054,P06
KOS-△47-hCCL19 7.76x10^7 SHS21054,P18
注：KOS-ATCC为野生型病毒；KOS-△47-hCCL19为敲除47，在34.5位置插入hCCL19基因的病毒。
两种病毒对不同细胞的IC50值如表21所示，按照72h孵育后MOI IC50＜0.6的判断标准：从病毒杀伤结果来看，HT29、SW620、hepG 2、Hep3B2.1-7、NCI-H460细胞为溶瘤病毒敏感肿瘤细胞株，与KOS-ATCC原始病毒株相比，KOS-hCCL19重组病毒株仍保留对HCT-116细胞的敏感性。
表21：
Figure PCTCN2022113446-appb-000021
Figure PCTCN2022113446-appb-000022
注：N/A表示因量效关系差或未测，未能做出的非线性拟合，标注横线的数字的表示拟合度较差(拟合度＜80％)，其对应MOI IC50值仅作参考。
实施例8病毒复制能力试验
取对数生长期的所述KOS–ATCC(即前面所述的野生型病毒)、KOS-hCCL19(携带hCCL19的HSV1)，细胞为Hep3B2.1-7肝癌细胞、NCI-H460大细胞肺癌细胞、HepG2肝癌细胞、HT29结肠癌细胞、HCT-116人结肠癌细胞和SW620人结肠癌细胞以合适的细胞密度接种于6孔培养板，培养过夜后，对6孔板细胞进行计数，根据每孔细胞数将病毒母液用含1％灭活FBS的高糖DMEM或RPMI-1640培养基稀释，配制成MOI＝0.1的病毒溶液。对应每孔依次加入300μL的病毒溶液，置于37℃，5％CO 2条件下孵育，每15min摇动培养板使得病毒更好的吸附细胞，1.25h后添加1mL的培养基。2h后弃掉培养基，再补充2mL的培养基，放置CO2培养箱中分别孵育24h、48h或72h。
培养结束后，收取病毒液进行病毒滴度测定。冻融三次(-80℃、37℃)后，梯度稀释收取的病毒液，取300μL感染Vero细胞(6孔板)，每15min摇动培养板使得病毒更好的吸附细胞，1.25h后添加2mL的培养基。2h后弃掉培养基，添加300μL的DMEM完全培养基、3mL 2％甲基纤维素固定病毒。培养3-4天后，吸弃覆盖培养基，加入10％HCHO溶液，1mL/孔，固定20min。之后吸掉甲醛溶液，加入1％结晶紫染色液，500μL/孔，染色30min。最后倒掉染色液，自来水缓缓冲洗干净，吸水纸倒扣擦干，进行空斑计数，并计算病毒滴度，病毒滴度＝病毒稀释倍数*(1000/300)*空斑数。
本实施例评价了原始病毒株(KOS-ATCC)与改造后病毒株(KOS-hCCL19)在6株癌细胞(Hep3B2.1-7，HepG2，NCI-H460，SW620，HT29，HCT116)的复制能力并对其进行了T检验，KOS-ATCC、KOS-hCCL19在6种癌细胞中的复制能力如图13所示，其中，其中两株病毒在不同肿瘤细胞的复制能力有所差异，整体来讲，改造后的病毒株保留了对肿瘤细胞的增殖活性。
实施例9病毒表达量试验
本实施例在vero细胞进行了病毒表达量实验，具体实验操作如下：
9.1试验材料
试剂材料如表22、23所示：
表22：
Figure PCTCN2022113446-appb-000023
表23：
名称 来源 培养基
vero非洲绿猴肾细胞 ATCC DMEM+10％FBS+1％P/S
9.2试验方法
9.2.1细胞培养
培养vero细胞，置于37℃、5％二氧化碳培养箱中培养。每日使用倒置显微镜观察细胞1次。当培养皿中的细胞生长汇合率到80～90％时进行细胞传代：弃去旧培养液，用DPBS洗掉残余培养基，加入0.25％胰酶消化液1mL，放培养箱消化2min，待细胞变圆﹑上浮后，弃去胰酶，加入新的培养基终止消化，按照一定的比例传代至新鲜无菌培养皿中培养。
9.2.2细胞铺板
从细胞培养箱中取出Vero细胞，吸掉培养基，用DPBS洗掉残余培养基，加入1ml胰酶于培养皿中，于37℃培养箱中消化2min，随后吸掉胰酶，每一皿细胞，用10mL(10％FBS,1％P/S)DMEM重悬细胞，并计数。将细胞铺于6孔板中，铺板密度为1*10^6个/mL，按每孔2mL细胞液进行铺板，完成后放入细胞培养箱中继续培养24小时。
9.3病毒感染
9.3.1细胞计数
从细胞培养箱中取出培养于6孔板的Vero细胞，选择两孔细胞计数：吸掉培养基，用DPBS洗掉残余培养基，每孔加入胰酶500μL，于37℃培养箱中消化2min，随后加入500μL 10％FBS，1％P/S的DMEM培养基终止消化，计数，求算平均每孔细胞数。
9.3.2病毒稀释及病毒感染
HSV1-hCCL19、HSV1-2k病毒分别按MOI＝0.01和0.1pfu/cell感染上述细胞，分别做2-3个复孔，同时设空白对照。先使用病毒稀释液分别将HSV1-hCCL19、HSV1-2k病毒稀释1000倍，再根据感染MOI和每孔细胞数，分别配制感染病毒培养基；取上述配制好病毒培养基每孔加1mL进行感染，空白对照用病毒稀释液代替。
病毒感染2h，每20min晃动板1次，弃去上清液，换成10％FBS，1％P/S的DMEM培养基，每孔2mL。
培养箱中继续培养48小时或72小时，收集上清液，4℃，4500rpm离心5min,取上清，分装后用ELISA检测hCCL19的表达量。
根据Human MIP-3b ELISA Kit(EhCCL19)对上清液hCCL19的表达进行测定。
具体结果如图14所示，其中，blank代表空白培养基的孔，2k代表敲除了ICP34.5和ICP47的HSV1； 结果显示，病毒感染48h和72h对hCCL19的表达无明显差异，相同感染时间条件下，病毒使用MOI＝0.01和0.1pfu/cell感染细胞，对hCCL19的表达无明显差异，但MOI＝0.01pfu/cell感染细胞时，病毒感染至48h，hCCL19的表达较高。
实施例10病毒复制能力试验
取对数生长期的所述KOS–ATCC(即前面所述的野生型病毒)、KOS-hCCL19(携带hCCL19的HSV1)，评价两个病毒分别感染肿瘤细胞后复制能力。肿瘤细胞为CAL27口腔鳞癌细胞、A549人肺癌细胞、PC-3前列腺癌细胞、CNE-2Z鼻咽癌细胞、Panc-1胰腺癌细胞、143B骨肉瘤细胞、fadu人鼻咽癌细胞、KYSE510人食管鳞癌细胞、KB人口腔表皮样癌细胞、ECA109人食管磷癌细胞、Aspc人胰腺癌细胞、colo829人黑色素瘤细胞、SK-OV-3卵巢癌细胞、hela宫颈癌细胞、U87MG人脑胶质瘤细胞、skmel-28人黑色素瘤细胞、LOVO肠癌腺癌、T.Tn人食道癌细胞、Calu-6肺上皮细胞癌和NCI-H226(肺)鳞状癌细胞。
将上述肿瘤细胞以合适的细胞密度接种于6孔培养板，培养过夜后，对6孔板细胞进行计数，根据每孔细胞数将病毒母液用含1％灭活FBS的高糖DMEM、RPMI-1640或F12K培养基稀释，配制成MOI＝0.02的病毒溶液。细胞板弃去旧培养液，分别将已稀释好的两种病毒液以每孔1mL按顺序转移至含有不同肿瘤细胞的细胞板，于7℃、5％CO 2培养箱孵育2h；病毒感染细胞2h后弃上清，每孔加2mL 1％FBS的基础培养基，放置37℃、5％CO 2培养箱继续培养24h、48h或者72h。
培养结束后，根据细胞病变情况收集24h、48h或者72h中的两个时间点(细胞病变50％以上再收样)的病毒液，并进行病毒滴度测定。冻融三次(-80℃、37℃)后，梯度稀释收取的病毒液，取300μL感染Vero细胞(6孔板)，每15min摇动培养板使得病毒更好的吸附细胞，病毒感染75min后，每孔加入1mL 1％灭活FBS的DMEM培养基，于37℃、5％CO 2培养箱培养2h后，吸弃病毒液，然后每孔添加300μL1％灭活FBS的DMEM培养基以及3mL平衡到室温的甲基纤维素培养基，置于37℃、5％CO 2培养箱培养。培养2天后，均匀加入2mL含有0.01％中性红的基础培养基对细胞进行染色。放置37℃、5％CO 2培养箱培养12h以上。吸液泵弃去6孔板所有液体，进行空斑计数，并计算病毒滴度，病毒滴度＝病毒稀释倍数*(1000/300)*空斑数。
本实施例评价了原始病毒株(KOS-ATCC)与改造后病毒株(KOS-hCCL19)在20株癌细胞(CAL27、A549、PC-3、CNE-2Z、Panc-1、143B、fadu、KYSE510、KB、ECA109、Aspc、colo829、SK-OV-3、hela、U87MG、skmel-28、LOVO、T.Tn、Calu-6、NCI-H226)的复制能力，KOS-ATCC、KOS-hCCL19在20种癌细胞中的复制能力如图15-17所示，改造后的病毒株保留了对肿瘤细胞的增殖活性。
图1-17中的“KOS”和“KOS-ATCC”均指代原始病毒株；“hCCL19”和“KOS-hCCL19”均指代改造后的病毒株。
实施例11病毒在异种移植瘤动物模型上的抗肿瘤活性
NCI-H460人肺癌细胞接种于BALB/c裸小鼠右侧背部皮下，建立小鼠荷瘤模型。待肿瘤大小长至100mm 3左右时，进行分组给药，每组8只动物。NCI-H460模型中，设置KOS-hCCL19高(KOS-hCCL19(high))剂量组、KOS-hCCL19低剂量(KOS-hCCL19(low))组，对应的给药剂量分别为3.33E+06pfu/mouse/once、3.33E+05pfu/mouse/once。模型中，同时设置一个病毒溶媒组(Vehicle组)。分组后，通过瘤内给药的方式进行给药，每3天(第0、3、6天)给药一次。NCI-H460小鼠模型上连续给药3次。开始给药后，每周2次测量肿瘤大小，监测肿瘤变化，并计算相对肿瘤生长抑制率TGI(TGI％＝(1-T/C)×100％；其中，T/C％为相对肿瘤增殖率，在某一时间点，治疗组和对照组相对肿瘤体积的百分比值)。
本实施例评价了KOS-hCCL19在NCI-H460人肺癌异种移植瘤动物模型上的抗肿瘤活性。病毒发挥的 体内抗肿瘤作用，如图18所示。NCI-H460模型上，在开始给药后第20天，KOS-hCCL19两个剂量水平下，相对肿瘤抑制率TGI分别为69.44％(p＜0.001)、57.36％(p＜0.05)。整体而言，KOS-hCCL19在NCI-H460人肺癌模型，给药后，能呈现显著体内抗肿瘤活性。
实施例12病毒的神经毒性评价
将KOS-WT(即“KOS”或“KOS-ATCC”)病毒，KOS-hCCL19病毒分别设置5个不同的病毒梯度，将上述一定浓度梯度的病毒分别注入雌性BALB/c小鼠的颅内(20μL/mouse)，后观察动物动物的存活情况，并计算不同病毒的半数致死量LD 50。其中，所述KOS-WT病毒对应的各组别的病毒给药量分别为5.00E+06pfu/mouse、8.33E+05pfu/mouse、1.39E+05pfu/mouse、2.31E+04pfu/mouse、3.86E+03pfu/mouse；KOS-hCCL19病毒对应的各组别的病毒给药量分别为4.64E+06pfu/mouse、1.55E+06pfu/mouse、5.16E+05pfu/mouse、1.72E+05pfu/mouse、5.73E+04pfu/mouse。同时设置1病毒溶媒组(Vehicle组)，每组6只动物。实验结束后，通过SPSS-Probit进行LD 50的计算。
本实施例评价野生型KOS-WT毒株及KOS-hCCL19病毒的神经毒性，统计给药截止14天内上述各组实验动物的生存情况。具体的实验结果如图19所示，Vehicle组未见动物死亡，KOS-WT病毒组给药量由高到低组别的动物死亡数分别为6、4、4、4、0，KOS-hCCL19病毒组给药量由高到低组别的动物死亡数分别为0、2、1、0、0。通过病毒不同给药梯度下动物死亡情况，计算不同病毒的半数致死量LD 50。KOS-WT病毒，KOS-hCCL19病毒对应的LD 50分别为5.403E+04pfu，>4.64E+06pfu。经过改造后获得的KOS-hCCL19病毒，相比野生型KOS-WT毒株，具有明显的神经毒性的降低。3种病毒中，KOS-hCCL19病毒的半数致死最高，毒性最低，KOS-hCCL19病毒较KOS-WT病毒大约有100倍以上的减毒作用。
实施例13目的基因插入位点对病毒抗肿瘤活性的影响
本实施例研究具有不同hCCL19插入位点的病毒的抗肿瘤活性差异，其中，测试的病毒为KOS-△47-△ICP34.5-△TK-hCCL19、KOS-△47-△ICP34.5-hCCL19，其中，KOS-△47-△ICP34.5-△TK-hCCL19为在TK处插入hCCL19，KOS-△47-△ICP34.5-hCCL19为在ICP34.5处插入hCCL19(也即本申请重组病毒，KOS-△47-hCCL19或KOS-hCCL19)，此外，还设置1个病毒溶媒组(Vehicle组)作为对照，获得目标病毒的方法参考实施例1-7。具体的实验操作如下：
小鼠左右两侧皮下接种A20细胞建立鼠源B细胞淋巴瘤皮下移植肿瘤模型，待左右两侧肿瘤大小长至100mm 3左右时，将上述3种病毒进行分组给药，各病毒给药量均为8.0E+07pfu/mouse/once；每组8只动物；分别于第0、3、6、9天通过瘤内给药的方式对右侧肿瘤进行给药，共给药4次。开始给药后，每3天一次测量肿瘤大小，监测肿瘤变化，并计算相对肿瘤抑制率TGI(TGI％＝(1-T/C)×100％；其中，T/C％为相对肿瘤增殖率，在某一时间点，治疗组和溶媒对照组相对肿瘤体积的百分比值)。
本实施例评价了KOS-△47-△ICP34.5-△TK-hCCL19、KOS-△47-△ICP34.5-hCCL19在A20同种移植瘤动物模型上的抗肿瘤活性。病毒发挥的体内抗肿瘤作用，如图20(给药侧)和图21(未给药侧)所示。结果显示，在给药方案下，截止给药后的第12天，KOS-△47-△ICP34.5-△TK-hCCL19、KOS-△47-△ICP34.5-hCCL19均较明显抑制A20荷瘤小鼠右侧肿瘤生长(TGI分别为64.24％，94.40％)，且相比溶媒对照组均有统计学意义(p＜0.05)。未进行瘤内给药注射的左侧肿瘤，KOS-△47-△ICP34.5-△TK-hCCL19和KOS-△47-△ICP34.5-hCCL19也表现出一定程度的肿瘤生长抑制作用(TGI分别为14.86％，50.28％)，但相比对照相，均无统计学差异(p<0.05)。KOS-△47-△ICP34.5-hCCL19相比于KOS-△47-△ICP34.5-△TK-hCCL19，无论在给药侧及未给药侧，均表现出相对较好的肿瘤抑制作用。截止给药后第28天，KOS-△47-△ICP34.5-△TK-hCCL19及KOS-△47-△ICP34.5-hCCL19组分别各有0、3只小鼠的双侧肿瘤被治愈(TV＝0mm 3)； 整体实验数据显示，KOS-△47-△ICP34.5-△TK-hCCL19与KOS-△47-△ICP34.5-hCCL19在1.6E+08pfu/mouse的给药剂量下，均能较明显抑制A20模型上给药侧的肿瘤生长。同时在未给药侧，虽相比对照组无统计学差异，两测试药也表现出一定的肿瘤抑制作用。在抗肿瘤效果上，KOS-△47-△ICP34.5-hCCL19相较KOS-△47-△ICP34.5-△TK-hCCL19表现出更好的肿瘤杀伤活性，说明不同的插入位点会影响病毒的抗肿瘤活性。未给药侧的肿瘤抑制，KOS-△47-△ICP34.5-△TK-hCCL19与KOS-△47-△ICP34.5-hCCL19给药后，并能产生一定远端的抗肿瘤活性。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。

Claims (14)

  1. 一种HSV病毒载体，其特征在于，所述HSV病毒载体的ICP47、ICP34.5基因沉默，以及携带CCL19基因。
  2. 根据权利要求1所述的病毒载体，其特征在于，携带两个拷贝的CCL19基因。
  3. 根据权利要求1所述的病毒载体，其特征在于，所述CCL19基因具有6.A>C突变。
  4. 根据权利要求1所述的病毒载体，其特征在于，所述ICP34.5基因沉默是通过敲除ICP34.5基因的第135-723核苷酸实现的；
    任选地，所述ICP47基因沉默是通过敲除ICP47基因的第3-266位核苷酸实现的。
  5. 根据权利要求1～4任一项所述的病毒载体，其特征在于，所述CCL19基因设置于ICP34.5基因的第134位核苷酸和第724位核苷酸之间。
  6. 根据权利要求5所述的病毒载体，其特征在于，进一步包括CMV和polyA；
    任选地，所述CMV与所述CCL19基因可操作地连接；
    任选地，所述polyA设置于所述CCL19基因的3’端核苷酸与ICP34.5基因的第134位核苷酸之间。
  7. 根据权利要求1所述的HSV病毒载体，其特征在于，所述病毒载体具有SEQ ID NO:5所示的核苷酸序列。
  8. 一种溶瘤病毒，其特征在于，携带权利要求1～7任一项所述的HSV病毒载体。
  9. 根据权利要求8所述的溶瘤病毒，其特征在于，所述溶瘤病毒为HSV-1；
    任选地，所述HSV-1包括选自F毒株、HF毒株、KOS毒株、HrR3毒株和17毒株的至少之一。
  10. 一种药物组合物，其特征在于，包括权利要求1～7任一项所述的HSV病毒载体或权利要求8或9所述的溶瘤病毒。
  11. 根据权利要求10所述的药物组合物，其特征在于，每单位剂量的所述药物组合物中包含10^5-10^12pfu的所述HSV病毒载体或溶瘤病毒。
  12. 根据权利要求10或11所述的药物组合物，其特征在于，进一步包括药物上可接受的载体。
  13. 根据权利要求1～7任一项所述的HSV病毒载体、权利要求8或9所述的溶瘤病毒或权利要求10～12任一项所述的药物组合物在制备药物中的用途，所述药物用于治疗或预防肿瘤。
  14. 根据权利要求13所述的用途，其特征在于，所述肿瘤包括选自肺癌、肝癌、咽鳞癌、结肠癌、骨肉瘤、卵巢癌、前列腺癌、神经胶质瘤、黑色素瘤、结直肠癌、食管癌、胰腺癌的至少之一。
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