CN115707781A

CN115707781A - HSV (herpes simplex virus) vector and application thereof

Info

Publication number: CN115707781A
Application number: CN202211000529.4A
Authority: CN
Inventors: 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
Original assignee: Sunshine Lake Pharma Co Ltd
Current assignee: Sunshine Lake Pharma Co Ltd
Priority date: 2021-08-20
Filing date: 2022-08-19
Publication date: 2023-02-21
Also published as: WO2023020598A1; TW202313982A

Abstract

The invention provides an HSV (herpes Simplex Virus) vector and application thereof, wherein ICP47 and ICP34.5 genes of the HSV vector are silenced, and CCL19 genes are carried. The HSV viral vector disclosed by the embodiment of the invention can continuously and highly express a chemokine CCL19 and effectively inhibit tumors.

Description

HSV (herpes simplex virus) vector and application thereof

Technical Field

The invention relates to the field of biomedicine, in particular to an HSV (herpes Simplex Virus) vector and application thereof.

Background

Because of some limitations inherent in the molecular properties of CCL19, its clinical application is limited. Basically, these include stability problems such as acid degradation and the tendency to aggregate irreversibly under mild denaturing conditions, followed by 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 resulting in systemic toxicity and side effects such as fever, fatigue, nausea, vomiting, diarrhea, neurotoxicity and leukopenia.

The HSV1 carrying the chemotactic factor CCL19 not only enhances the targeting of the virus strain to tumor cells, but also further enhances the anti-tumor immune curative effect of the virus strain through the function of recruiting immune cells by the exogenous gene CCL19. By using the oncolytic virus for expressing the CCL19, the virus can be replicated in tumors in an intratumoral injection mode and can continuously express the CCL19 locally, so that the CCL19 which is rapidly cleared and causes systemic toxic and side effects through intravenous administration are avoided. The effects of CCL19 and CCR7 in anti-tumor treatment become research hotspots and make remarkable progress, the CCL and the CCR7 play a key role in chemotaxis of dendritic cells, CD4+ and CD8+ T cells to infiltrate tumors, mediate immune cells to release cytokines, inhibit tumor proliferation, migration and invasion and assist in treating tumors, and the combination of an anti-tumor drug and the CCL19 is helpful for finding out a new tumor treatment means and a gene vaccine, but no report related to the expression of the CCL19 by HSV1 virus exists at present.

Disclosure of Invention

The present application is based on the discovery and recognition by the inventors of the following problems:

at present, the clinical application of the chemotactic factor is limited by unstable product quality and frequent administration, and through a large number of experimental studies, the inventor surprisingly discovers that CCL19 can be better expressed in HSV virus compared with other chemotactic factors, particularly, when CCL19 gene is inserted into ICP34.5 site, the expression level of CCL19 is obviously improved, after the chemotactic factor CCL19 carrying double-copy mutation, the HSV virus vector can continuously express the chemotactic factor CCL19 at high level, the expression level of CCL19 is obviously higher than that of HSV virus carrying only single-copy mutation CCL19, oncolytic virus containing the HSV virus vector retains sensitivity and proliferation activity on tumor cells, further improves the expression level of CCL19, breaks through the limitation of low expression level, unstable expression level and frequent administration of the chemotactic factor, and has an effect of inhibiting tumors better than that of virus containing other chemotactic factor coding nucleic acid vectors.

In a first aspect of the invention, the invention features an HSV viral vector. According to an embodiment of the invention, the ICP47, ICP34.5 genes of the HSV viral vectors are silenced, and carry a CCL19 gene. The HSV1 virus is provided with double-copy ICP34.5 genes, and CCL19 genes are inserted into ICP34.5 gene sites, so that the neurotoxicity of the virus can be effectively reduced, and the anti-tumor selectivity of the virus is improved; after the HSV1 carries the double-copy chemokine CCL19, the targeting of the virus strain to tumor cells can be enhanced, and the anti-tumor curative effect of the virus strain is further enhanced by recruiting immune cells through the exogenous gene CCL19. By injecting the oncolytic virus expressing CCL19 in tumor, the virus can replicate in tumor cells and continuously express CCL19 locally, thus avoiding rapid clearance of CCL19 in intravenous administration and systemic toxic and side effects. Knocking out a replication nonessential gene ICP47 in an HSV genome, and improving the expression of MHC-1 on the surface of a tumor cell infected by a virus and the capability of presenting cell antigen; the inventor screens chemotactic factors highly expressed in HSV (herpes simplex virus), discovers that CCL19 can be better expressed in HSV virus compared with other chemotactic factors, and simultaneously explores an insertion site of CCL19, and unexpectedly discovers that the ICP34.5 gene in HSV genome is knocked out to ensure that HSV is selectively replicated in tumor cells and is not replicated and proliferated in normal cells, so that the medicine safety of the HSV virus can be improved, in addition, the nucleic acid for encoding the chemotactic factors is inserted into the site, so that the anti-tumor capacity of the virus containing the HSV virus vector can be effectively improved, the HSV virus vector can continuously and highly express the chemotactic factor CCL19, the CCL19 gene is inserted into the ICP34.5 gene site, the expression level of the CCL19 is remarkably improved, the killing capacity of the HSV virus on tumor cells is remarkably improved on the premise of not influencing the replication capacity in the tumor cells, and tumors can be effectively prevented or treated.

According to an embodiment of the present invention, the HSV viral vector may further comprise at least one of the following additional technical features:

according to an embodiment of the invention, two copies of the CCL19 gene are carried. The inventor screens the copy number of CCL19 gene carried by the expression vector, and finds that the CCL19 gene carried by the HSV virus vector according to the embodiment of the invention has two copies, so that the expression level of the CCL19 can be obviously improved, and the expression level of the CCL19 is obviously higher than that of HSV virus carrying only a single copy of the CCL19 gene.

According to an embodiment of the invention, the CCL19 gene has a 6.a > c mutation. Through base optimization, the expression level of the target gene in a host is improved on the premise of not changing the expressed amino acid.

The nucleic acid sequence of the wild-type CCL19 gene is shown below:

ATGGCCCTGCTACTGGCCCTCAGCCTGCTGGTTCTCTGGACTTCCCCAGCCCCAACTCTGAGTGGCACCAATGATGCTGAAGACTGCTGCCTGTCTGTGACCCAGAAACCCATCCCTGGGTACATCGTGAGGAACTTCCACTACCTTCTCATCAAGGATGGCTGCAGGGTGCCTGCTGTAGTGTTCACCACACTGAGGGGCCGCCAGCTCTGTGCACCCCCAGACCAGCCCTGGGTAGAACGCATCATCCAGAGACTGCAGAGGACCTCAGCCAAGATGAAGCGCCGCAGCAGTTAA(SEQ ID NO：1)。

the nucleic acid sequence of the CCL19 gene containing the 6.A > -C mutation is as follows:

ATGGCACTGCTGCTGGCCCTGTCCCTGCTGGTGCTGTGGACCTCTCCAGCACCCACCCTGAGCGGAACAAACGACGCAGAGGATTGCTGTCTGTCTGTGACACAGAAGCCTATCCCAGGCTACATCGTGAGGAATTTCCACTATCTGCTGATCAAGGACGGATGCAGGGTGCCAGCAGTGGTGTTTACCACACTGAGGGGCCGCCAGCTGTGCGCACCACCTGATCAGCCTTGGGTGGAGCGGATCATCCAGCGGCTGCAGAGAACCAGCGCCAAGATGAAGCGGAGAAGCTCCTGA(SEQ ID NO：2)。

according to an embodiment of the invention, the ICP34.5 gene silencing is achieved by knocking out nucleotides 135-723 of the ICP34.5 gene. The ICP34.5 gene sequence coding is coded by using the first nucleotide of the ICP34.5 gene starting codon as the 1 st nucleotide, the ICP34.5 gene sequence can refer to https:// www.ncbi.nlm.nih.gov/nuccore/NC _001806.2from =124834 and to =125861 and report = genbank strand = true, the specific sequence of ICP34.5 gene is as shown in SEQ ID NO:3, wherein the underlined part codes for the ICP34.5 gene sequence.

CCTCTGCACGCACATGCTTGCCTGTCAAACTCTACCACCCCGGCACGCTCTCTGTCTCCATGGCCCGCCGCCGCCATCGCGGCCCCCGCCGCCCCCGGCCGCCCGGGCCCACGGGCGCGGTCCCAACCGCACAGTCCCAGGTAACCTCCACGCCCAACTCGGAACCCGTGGTCAGGAGCGCGCCCGCGGCCGCatggcccgccgccgccatcgcggcccc cgccgcccccggccgcccgggcccacgggcgcggtcccaaccgcacagtcccaggtaacctccacgcccaactcgg aacccgtggtcaggagcgcgcccgcggccgccccgccgccgccccccgccagtgggcccccgccttcttgttcgct gctgctgcgccagtggctccacgttcccgagtccgcgtccgacgacgacgacgacgactggccggacagccccccg cccgagccggcgccagaggcccggcccaccgccgccgccccccgcccccggtccccaccgcccggcgcgggcccgg ggggcggggctaacccctcccaccccccctcacgccccttccgccttccgccgcgcctcgccctccgcctgcgcgt caccgcagagcacctggcgcgcctgcgcctgcgacgcgcgggcggggagggggcgccgaagccccccgcgaccccc gcgacccccgcgacccccacgcgggtgcgcttctcgccccacgtccgggtgcgccacctggtggtctgggcctcgg ccgcccgcctggcgcgccgcggctcgtgggcccgcgagcgggccgaccgggctcggttccggcgccgggtggcgga ggccgaggcggtcatcgggccgtgcctggggcccgaggcccgtgcccgggccctggcccgcggagccggcccggcg aactcggtctaaCGTTACACCCGAGGCGGCCTGGGTCTTCCGCGGAGCTCCCGGGAGCTCCGCACCAAGCCGCTCTCCGGAGAGACGATGGCAGGAGCCGCGCATATATACGCTTGGAGCCGGCCCGCCCCCGAGGCGGGCCCGCCCTCGGAGGGCGGGACTGGCCAATCGGCGGCCGCCAGCGCGGCGGGGCCCGGCCAACCAGCGTCCGCCGAGTCGTCGGGGCCCGGCCCACTGGGCGGTAACTCCCGCCCAGTGGGCCGGGCCGCCCACTTCCCGGTATGGTAATTAAAAACTTGCAGAGGCCTTGTTCCGCTTCCCGGTATGGTAATTAGAAACTCATTAATGGGCGGCCCCGGCCGCCCTTCCCGCTTCCGGCAATTCCCGCGGCCCTTAATGGGCAACCCCGGTATTCCCCGCCTCCCGCGCCGCGCGTAACCACTCCCCTGGGGTTCCGGGTTATGTTAATTGCTTTTTTGGCGGAACACACGGCCCCTCGCGCATTGGCCCGCGGGTCGCTCAATGAACCCGCATTGGTCCCCTGGGGTTCCGGGTATGGTAATGAGTTTCTTCGGGAAGGCGGGAAGCCCCGGGGCACCGACGCAGGCCAAGCCCCTGTTGCGTCGGCGGGAGGGGCATGCTAATGGGGTTCTTTGGGGGACACCGGGTTGGTCCCCCAAATCGGGGGCCGGGCCGTGCATGCTAATGATATTCTTTGGGGGCGCCGGGTTGGTCCCCGGGGACGGGGCCGCCCCGCGGTGGGCCTGCCTCCCCTGGGACGCGCGGCCATTGGGGGAATCGTCACTGCCGCCCCTTTGGGGAGGGGAAAGGCGTGGGGTATAAGTTAGCCCTGGCCCGACGGTCTGGTCGCATTTGCACCTCGGCACTCGGAGCGAGACGCAGCAGCCAGGCAGACTCGGGCCGCCCCCTCTCCGCATCACCACAGAAGCCCCGCCTACGTTGCGACCCCCAGGGACCCTCCGTCAGCGACCCTCCAGCCGCATACGACCCCCATGGAGCCCCGCCCCGGAGCGAGTACCCGCCGGCCTGAGGGCCGCCCCCAGCGCGAGGTGAGGGGCCGGGCGCCATGTCTGGGGCGCCATGTCTGGGGCGCCATGTCTGGGGCGCCATGTCTGGGGCGCCATGTTGGGGGGCGCCATGTTGGGGGGCGCCATGTTGGGGGACCCCCGACCCTTACACTGGAACCGGCCGCCATGTTGGGGGACCCCCACTCATACACGGGAGCCGGGCGCCATGTTGGGGCGCCATGTTAGGGGGCGTGGAACCCCGTGACACTATATATACAGGGACCGGGGGCGCCATGTTAGGGGGCGCGGAACCCCCTGACCCTATATATACAGGGACCGGGGTCGCCCTGTTAGGGGTCGCCATGTGACCCCCTGACTTTATATATACAGACCCCCAACACCTACACATGGCCCCTTTGACTCAGACGCAGGGCCCGGGGTCGCCGTGGGACCCCCCTGACTCATACACAGAGACACGCCCCCACAACAAACACACAGGGACCGGGGTCGCCGTGTTAGGGGGCGTGGTCCCCACTGACTCATACGCAGG(SEQ ID NO：3)。

According to an embodiment of the invention, the ICP47 gene silencing is achieved by knocking out nucleotides 3-266 of the ICP47 gene. The ICP47 gene sequence coding is coded by taking the first nucleotide of the start codon of the ICP47 gene as the 1 st position, the ICP47 gene sequence can be referred to https:// www.ncbi.nlm.nih.gov/nuccore/NC _001806.2report = genbank &from =46609 &47803 &strain = true, the specific sequence of the ICP47 gene is shown as SEQ ID NO:4, wherein the underlined part codes for the ICP47 gene sequence.

GACCGGCGGCGACCGTTGCGTGGACCGCTTCCTGCTCGTCGGGGCGACCGGCGGCGACCGTTGCGTGGACCGCTCCCTGCTCGTCGGGAAAAGCatgtcgtgggccctggaaatggcggacaccttcctggacaacatgcgggttgggcccaggacgtacgccgacgtacgcgatgagatcaataaaagggggcgtgaggaccgggaggcggccagaaccgccgtgcacgacccggagcgtcccctgctgcgctctcccgggctgctgcccgaaatcgcccccaacgcatccttgggtgtggcacatcgaagaaccggcgggaccgtgaccgacagtccccgtaatccggtaacccgttgaGTCCCGGGTACGACCATCACCCGAGTCTCTGGGCGGAGGGTGGTTCCCCCCCGTGTCTCTCG(SEQ ID NO：4)。

According to an embodiment of the present invention, the CCL19 gene is disposed between nucleotide 134 and nucleotide 724 of the ICP34.5 gene, such that the constructed HSV-1 carries CCL19.

According to an embodiment of the present invention, CMV and polyA are further included.

According to an embodiment of the invention, the CMV is operably linked to the CCL19 gene.

According to an embodiment of the present invention, the polyA is disposed between the 3' terminal nucleotide of the CCL19 gene and the 134 th nucleotide of the ICP34.5 gene.

According to an embodiment of the present invention, the viral vector has the nucleotide sequence shown in SEQ ID NO. 5.

AGCCCGGGCCCCCCGCGGGCTGAGACTAGCGAGTTAGACAGGCAAGCACTACTCGCCTCTGCACGCACATGCTTGCCTGTCAAACTCTACCACCCCGGCACGCTCTCTGTCTCCatggcccgccgccgccatcgcggcccccgccgcccccggccgcccgggcccacgggcgcggtcccaaccgcacagtcccaggtaacctccacgcccaactcggaacccgtggtcaggagcgcgcccgcggccgc[AAGCCATAGAGCCCACCGCATCCCCAGCATGCCTGCTATTGTCTTCCCAATCCTCCCCCTTGCTGTCCTGCCCCACCCCACCCCCCAGAATAGAATGACACCTACTCAGACAATGCGATGCAATTTCCTCATTTTATTAGGAAAGGACAGTGGGAGTGGCACCTTCCAGGGTCAAGGAAGGCACGGGGGAGGGGCAAACAACAGATGGCTGGCAACTAGAAGGCACAGTCGAGGCTGATCAGCGGGTTTAAACGGGCCCTCTAGACTCGAGCGGCCGCCACTGTGCTGGATATCTGTCAGGAGCTTCTCCGCTTCATCTTGGCGCTGGTTCTCTGCAGCCGCTGGATGATC CGCTCCACCCAAGGCTGATCAGGTGGTGCGCACAGCTGGCGGCCCCTCAGTGTGGTAAACACCACTGCTGGCACCC TGCATCCGTCCTTGATCAGCAGATAGTGGAAATTCCTCACGATGTAGCCTGGGATAGGCTTCTGTGTCACAGACAG ACAGCAATCCTCTGCGTCGTTTGTTCCGCTCAGGGTGGGTGCTGGAGAGGTCCACAGCACCAGCAGGGACAGGGCC AGCAGCAGTGCCATCCACACTGGACTAGTGGATCCGAGCTCGGTACCAAGCTTAAGTTTAAACGCTAGCCAGCTTGGGTCTCCCTATAGTGAGTCGTATTAATTTCGATAAGCCAGTAAGCAGTGGGTTCTCTAGTTAGCCAGAGAGCTCTGCTTATATAGACCTCCCACCGTACACGCCTACCGCCCATTTGCGTCAATGGGGCGGAGTTGTTACGACATTTTGGAAAGTCCCGTTGATTTTGGTGCCAAAACAAACTCCCATTGACGTCAATGGGGTGGAGACTTGGAAATCCCCGTGAGTCAAACCGCTATCCACGCCCATTGATGTACTGCCAAAACCGCATCACCATGGTAATAGCGATGACTAATACGTAGATGTACTGCCAAGTAGGAAAGTCCCATAAGGTCATGTACTGGGCATAATGCCAGGCGGGCCATTTACCGTCATTGACGTCAATAGGGGGCGTACTTGGCATATGATACACTTGATGTACTGCCAAGTGGGCAGTTTACCGTAAATACTCCACCCATTGACGTCAATGGAAAGTCCCTATTGGCGTTACTATGGGAACATACGTCATTATTGACGTCAATGGGCGGGGGTCGTTGGGCGGTCAGCCAGGCGGGCCATTTACCGTAAGTTATGTAACGCGGAACTCCATATATGGGCTATGAACTAATGACCCCGTAATTGATTACTATTAATAACTAGTCAATAATCAATGTC]GCGGCCGCCAGCGCGGCGGGGCCCGGCCAACCAGCGTCCGCCGAGTCGTCGGGGCCCGGCCCACTGGGCGGTAACTCCCGCCCAGTGGGCCGGGCCGCCCACTTCCCGGTATGGTAATTAAAAACTTGCAGAGGCCTTGTTCCGCTTCCCGGTATGGTAATTAGAAACTCATTAATGGGCGGCCCCGGCCGCCCTTCCCGCTTCCGGCAATTCCCGCGGCCCTTAATGGGCAACCCCGGTATTCCCCGCCTCCCGCGCCGCGCGTAACCACTCCCCTGGGGTTCCGGGTTATGTTAATTGCTTTTTTGGCGGAACACACGGCCCCTCGCGCATTGGCCCGCGGGTCGCTCAATGAACCCGCATTGGTCCCCTGGGGTTCCGGGTATGGTAATGAGTTTCTTCGGGAAGGCGGGAAGCCCCGGGGCACCGACGCAGGCCAAGCCCCTGTTGCGTCGGCGGGAGGGGCATGCTAATGGGGTTCTTTGGGGGACACCGGGTTGGTCCCCCAAATCGGGGGCCGGGCCGTGCATGCTAATGATATTCTTTGGGGGCGCCGGGTTGGTCCCCGGGGACGGGGCCGCCCCGCGGTGGGCCTGCCTCCCCTGGGACGCGCGGCCATTGGGGGAATCGTCACTGCCGCCCCTTTGGGGAGGGGAAAGGCGTGGGGTATAAGTTAGCCCTGGCCCGACGGTCTGGTCGCATTTGCACCTCGGCACTCGGAGCGAGACGCAGCAGCCAGGCAGACTCGGGCCGCCCCCTCTCCGCATCACCACAGAAGCCCCGCCTACGTTGCGACCCCCAGGGACCCTCCGTCAGCGACCCTCCAGCCGCATACGACCCCCATGGAGCCCCGCCCCGGAGCGAGTACCCGCCGGCCTGAGGGCCGCCCCCAGCGCGAGGTGAGGGGCCGGGCGCCATGTCTGGGGCGCCATGTCTGGGGCGCCATGTCTGGGGCGCCATGTCTGGGGCGCCATGTTGGGGGGCGCCATGTTGGGGGGCGCCATGTTGGGGGACCCCCGACCCTTACACTGGAACCGGCCGCCATGTTGGGGGACCCCCACTCATACACGGGAGCCGGGCGCCATGTTGGGGCGCCATGTTAGGGGGCGTGGAACCCCGTGACACTATATATACAGGGACCGGGGGCGCCATGTTAGGGGGCGCGGAACCCCCTGACCCTATATATACAGGGACCGGGGTCGCCCTGTTAGGGGTCGCCATGTGACCCCCTGACTTTATATATACAGACCCCCAACACCTACACATGGCCCCTTTGACTCAGACGCAGGGCCCGGGGTCGCCGTGGGACCCCCCTGACTCATACACAGAGACACGCCCCCACAACAAACACACAGGGACCGGGGTCGCCGTGTTAGGGGGCGTGGTCCCCACTGACTCATACGCAGG(SEQ ID NO:5)。

In a second aspect of the invention, an oncolytic virus is provided. According to an embodiment of the present invention, the HSV viral vector of the first aspect is carried. According to the embodiment of the invention, the oncolytic virus containing the HSV virus vector retains the sensitivity and the proliferation activity to tumor cells, and due to the characteristics of ICP34.5 gene, after CCL19 gene is inserted into ICP34.5 gene of HSV virus, the CCL19 expression level is obviously improved, and compared with the CCL19 expression level of HSV virus carrying CCL19 with other copy number, the HSV virus carrying double-copy CCL19 can be obviously improved, the oncolytic virus can continuously express chemokine CCL19 with high level, the lethality to tumor cells is obviously improved, and the tumor cells can be effectively inhibited.

According to an embodiment of the present invention, the above oncolytic virus may further comprise at least one of the following additional technical features:

according to an embodiment of the invention, the oncolytic virus is HSV-1. The inventor finds that HSV-2 has potential safety hazard and may cause virus infection of genitals, so that the use of HSV-1 can improve the safety of oncolytic virus in use.

According to an embodiment of the present invention, the HSV-1 includes at least one selected from the group consisting of an F strain, an HF strain, a KOS strain, a HrR strain, and a 17 strain.

In a third aspect of the invention, a pharmaceutical composition is provided. According to an embodiment of the invention, there is included an HSV viral vector according to the first aspect or an oncolytic virus according to the second aspect. The pharmaceutical composition provided by the embodiment of the invention is stable in quality, can take effect when a lower dose is used, and particularly has a very remarkable inhibition effect on recurrent and large-volume tumors.

According to an embodiment of the present invention, the above pharmaceutical composition may further comprise at least one of the following additional technical features:

according to an embodiment of the invention, the HSV viral vector or oncolytic virus comprises 10^5-10^12pfu per unit dose of the pharmaceutical composition.

In a fourth aspect, the present invention provides the use of an HSV viral vector according to the first aspect, an oncolytic virus according to the second aspect, or a pharmaceutical composition according to the third aspect, in the manufacture of a medicament. According to an embodiment of the invention, the medicament is for the treatment or prevention of a tumor. As mentioned above, the HSV viral vector of the first aspect and the oncolytic virus of the second aspect both can highly express the CCL19 under suitable conditions and are effective for treating or preventing tumors, and thus, a drug prepared by using the HSV viral vector, the oncolytic virus or the pharmaceutical composition can also be effective for treating or preventing tumors.

According to an embodiment of the present invention, the tumor includes at least one selected from the group consisting of lung cancer, liver cancer, pharyngeal squamous carcinoma, colon cancer, osteosarcoma, ovarian cancer, prostate cancer, glioma, melanoma, colorectal cancer, esophageal cancer, pancreatic cancer.

In a fourth aspect of the invention, a method of treating a tumor is presented. According to an embodiment of the invention, the method comprises administering to a subject in need thereof a therapeutically effective amount of the HSV viral vector of the first aspect or the oncolytic virus of the second aspect or the pharmaceutical composition of the third aspect. According to the method provided by the embodiment of the invention, the tumor can be effectively treated under the condition of low administration frequency.

According to an embodiment of the present invention, the method may further include at least one of the following additional technical features:

according to an embodiment of the invention, the individual in need thereof is a patient suffering from at least one of the following cancers: lung cancer, liver cancer, pharyngeal squamous carcinoma, colon cancer, osteosarcoma, ovarian cancer, prostatic cancer, glioma, melanoma, colorectal cancer, esophageal cancer and pancreatic cancer.

In a fifth aspect of the invention, the invention features a method of recruiting immune cells to a tumor. According to an embodiment of the invention, the method comprises contacting the tumor with the HSV viral vector of the first aspect or the recombinant oncolytic virus of the second aspect. As mentioned above, the HSV viral vector of the first aspect and the oncolytic virus of the second aspect can highly express the CCL19 under suitable conditions, the chemokine is a small molecule secretory protein capable of chemotactic immune cells for directional movement, CCL19 and its receptor (such as CCR 7) are expressed on dendritic cells, T cells and various tumor cells, and thus, the HSV viral vector or the oncolytic virus described in the present application can be effectively used to recruit immune cells to tumors, thereby effectively inhibiting tumors.

In a sixth aspect of the invention, a method of inhibiting tumor cell growth or promoting tumor cell death is provided. According to an embodiment of the invention, the method comprises contacting the tumor cell with the HSV viral vector of the first aspect or the recombinant oncolytic virus of the second aspect.

according to an embodiment of the invention, the tumor cell is selected from lung cancer, liver cancer, pharyngeal squamous carcinoma, colon cancer, osteosarcoma, ovarian cancer, prostate cancer, glioma, melanoma, colorectal cancer, esophageal cancer, pancreatic cancer.

According to an embodiment of the invention, the recombinant oncolytic virus is provided in a dose sufficient to cause death of the tumor cell.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Drawings

The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:

FIG. 1 is a graph showing the results of PCR after a first round of limiting dilution screening of recombinant oncolytic viruses obtained after a first round of recombination according to an embodiment of the present invention, wherein M is marker;1-32 are selected virus strain clones; +: a positive control (expressing hCCL19 gene) using hCCL19 recombinant virus as a template; d: treating with hCCL19-RL1-PMD18T plasmid as a template (expressing hCCL19hCCL19 gene); k: treatment with KOS-. DELTA.47 virus gene as a template (expressing ICP34.5 gene); water: negative control with water as template;

FIG. 2 is a graph showing the results of PCR after a second round of limiting dilution screening of recombinant oncolytic viruses obtained after a first round of recombination according to an embodiment of the present invention, wherein M is marker;1-28 for the second round screening selected 28 clones (1-13 for KOS-Delta 47-S1-gDNA-hCCL19-32,1-28 for KOS-Delta 47-S1-gDNA-hCCL 19-9); d: treating with hCCL19-RL1-PMD18T plasmid as template (expressing hCCL19 gene); k: treating with KOS-delta 47 virus gene as template (expressing ICP34.5 gene); water: negative control with water as template;

FIG. 3 is a diagram showing the results of PCR identification after propagation of a recombinant oncolytic virus obtained after a first round of recombination according to an embodiment of the present invention, wherein M is marker-DL5000;9-2: treating with KOS-delta 47-S1-gDNA-hCCL19-9-2 virus genome as template; d: treating with hCCL19-RL1-PMD18T plasmid as template (expressing hCCL19 gene); k: treating with KOS-delta 47 virus gene as template (expressing ICP34.5 gene); water: negative control with water as template;

FIG. 4 is a graph showing the results of PCR of the recombinant oncolytic viruses obtained after the second round of recombination after the first round of screening according to the embodiment of the present invention, wherein M is marker-DL2000;1-102 are selected 102 clones; 9-2: treating with KOS-delta 47-S1-gDNA-hCCL19-9-2 virus genome as template; d: treating with hCCL19-RL1-PMD18T plasmid as template (expressing hCCL19 gene); k: treatment with KOS-. DELTA.47 virus gene as a template (expressing ICP34.5 gene); water: negative control with water as template; KOS-delta 47-S5g-hCCL19 is a new virus strain obtained by 2 nd round of recombinant transfection of KOS-delta 47-S1-gDNA-hCCL 19-9-2;

FIG. 5 is a graph showing the results of a second round of screening PCR on recombinant oncolytic viruses obtained after a second round of recombination according to an embodiment of the present invention, wherein M is marker-DL2000; 4.5, 8, 9 … is: 60 positive viruses obtained by the first round of PCR screening; 9-2: treatment with the virus genome KOS-Delta 47-S1-gDNA-hCCL19-9-2 as a template (expressing hCCL19 gene and ICP34.5 gene); d: treating with hCCL19-RL1-PMD18T plasmid as template (expressing hCCL19 gene); k: treatment with KOS-DELTA 47 virus gene as a template (expressing ICP34.5 gene); water: water as negative control of the template; KOS-delta 47-S5g-hCCL19 is a new virus strain obtained by 2 nd round of recombinant transfection of KOS-delta 47-S1-gDNA-hCCL 19-9-2;

FIG. 6 is a diagram showing the results of PCR identification after propagation of a positive recombinant oncolytic virus obtained after the second round of recombination according to the embodiment of the invention, wherein M is marker-DL2000;9-2: treatment with the virus genome KOS-Delta 47-S1-gDNA-hCCL19-9-2 as a template (expressing hCCL19 gene and ICP34.5 gene); d: treating with hCCL19-RL1-PMD18T plasmid as template (expressing hCCL19 gene); 32-6: for the treatment with the KOS-delta 47-S1-hCCL19-32-6 virus gene as a template, a single copy insert virus clone (expressing the hCCL19 gene) is screened for 1 st round of recombination; water: water as negative control of the template; KOS-delta 47-S5g-hCCL19 is a new virus strain obtained by 2 nd round of recombinant transfection of KOS-delta 47-S1-gDNA-hCCL 19-9-2;

FIG. 7 is a graph showing the results of PCR obtained after amplification of the full length of the hCCL19 expression cassette, wherein M is marker-DL2000;9-2: treatment with the virus genome KOS-Delta 47-S1-gDNA-hCCL19-9-2 as a template (expressing hCCL19 gene and ICP34.5 gene); d: treating with hCCL19-RL1-PMD18T plasmid as template (expressing hCCL19 gene); 32-6: for the treatment with the KOS-delta 47-S1-hCCL19-32-6 virus gene as a template, a single copy insert virus clone (expressing hCCL 19) is screened for 1 st round of recombination; water: water as negative control of the template;

FIG. 8 is a graph showing the results of electrophoresis obtained after a first round of plaque purification of a positive recombinant oncolytic virus according to an embodiment of the invention, wherein M is marker-DL5000; d is the treatment with hCCL19-RL1-PMD18T plasmid as template to express hCCL19 gene; k is KOS-delta 47 virus genome as a template (expressing ICP34.5 gene); water: negative control with water template;

FIG. 9 is a graph showing the results of electrophoresis obtained after a second round of plaque purification of a positive recombinant oncolytic virus according to an embodiment of the invention, wherein M is marker-DL5000; d is the treatment with hCCL19-RL1-PMD18T plasmid as template to express hCCL19 gene; k is KOS-delta 47 virus genome as a template (expressing ICP34.5 gene); water: negative control with water template;

FIG. 10 is a graph showing the results of electrophoresis obtained after a third plaque purification run on a positive recombinant oncolytic virus according to an embodiment of the invention, wherein M is marker; d is the treatment with hCCL19-RL1-PMD18T plasmid as template to express hCCL19 gene; k is KOS-delta 47 virus genome as a template (expressing ICP34.5 gene); water: negative control with water template;

FIG. 11 is an electrophoretogram of a positive recombinant oncolytic virus according to an embodiment of the present invention, wherein the size of the amplified fragment of the recombinant virus KOS- Δ 47-hCCL19-05-02-02 is expected;

FIG. 12 is a diagram showing the results of screening positive clones for recombinant virus KOS-DELTA 47-hCCL19-05-02-02-02TA linkage;

FIG. 13 is a graph showing the results of the replication ability of KOS-ATCC and KOS-hCCL19 in 6 kinds of cancer cells according to the example of the present invention;

FIG. 14 is a graph showing the results of virus expression amounts detected after infection of cells with 2k virus and KOS-hCCL19 recombinant virus according to an embodiment of the present invention;

FIGS. 15 to 17 are graphs showing the results of the replication ability of KOS and hCCL19 in 20 kinds of cancer cells according to the example of the present invention; wherein KOS is a wild-type strain; hCCL19 (also KOS-hCCL 19) is a recombined virus strain;

FIG. 18 is a graph showing the results of tumor size change in a mouse model of NCI-H460 according to an embodiment of the present invention;

FIG. 19 is a graph of survival plots for groups of animals in a viral neurotoxicity assessment experiment according to an embodiment of the present invention;

FIG. 20 is a graph showing the results of changes in the size of the right-side (administration side) tumor in each group A20 mouse model according to the embodiment of the present invention;

FIG. 21 is a graph showing the results of changes in tumor size on the left side (non-administration side) in each group A20 mouse model according to the example of the present invention;

fig. 22 is a graph of group a20 mouse model mortality on day 28 after administration of the drug according to the example of the present invention.

Detailed Description

Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.

Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of the feature. In the description of the present invention, "a plurality" means at least two, e.g., two, three, etc., unless specifically limited otherwise.

In addition, the "chemokine" described in the present application is a kind of cytokine expressed on the cell membrane of immune cells and endothelial cells to chemotaxis various directional movement of immune cells, such as CCL19, CCL19 can chemotaxis dendritic cells, CD4 ⁺ And CD8+ cells infiltrate the tumor, mediate immune cells to release cytokines, inhibit tumor proliferation, migration and invasion and play a key role in assisting the treatment of the tumor.

As used herein, "operably linked" refers to the linkage of a foreign gene to a vector such that control elements, such as promoter sequences and the like, within the vector are capable of performing their intended function of regulating the transcription and translation of the foreign gene.

The "oncolytic virus" loses partial functional genes, so that the infection replication capacity of the oncolytic virus in normal cells is weakened, namely, the attenuated oncolytic virus, such as HSV-1 oncolytic virus of the application, ICP47 and ICP34.5 genes are knocked out, so that the oncolytic virus can only selectively replicate in tumor cells.

In the application, the inventor screens the chemokines needing to be inserted into the expression vector, and finds that the CCL19 chemokine is easier to express in the expression vector than other chemokines, and the HSV virus carrying the CCL19 has better oncolytic and immune effects and better tumor treatment or prevention effects than the viruses carrying other chemokines; the inventor further screens the insertion site of the CCL19 gene in HSV virus, and due to the characteristics of the ICP34.5 gene, the inventor finds that when the CCL19 gene is inserted into the ICP34.5 site, the gene expression level of the CCL19 of the obtained HSV virus is obviously improved compared with the virus in which the CCL19 is inserted into the site except the ICP34.5 gene, and the gene expression level of the CCL19 of the oncolytic virus containing the HSV expression vector is obviously improved and the safety is higher compared with the oncolytic virus containing the expression vector in which the CCL19 is inserted into the site except the ICP34.5 gene; meanwhile, the copy number of the CCL19 gene inserted into the expression vector is explored, the ICP34.5 gene with double copies in the expression vector is knocked out, after the CCL19 gene with double copies is inserted, the expression quantity of the CCL19 gene of the HSV expression vector is obviously higher than that of an HSV expression vector carrying a single-copy CCL19 gene, and the expression quantity of the CCL19 gene of the oncolytic protein containing the HSV expression vector is obviously higher than that of the oncolytic virus of the HSV expression vector containing the single-copy CCL19 gene.

In the following examples of the present application, "Δ 47 viral genome" refers to an HSV-1 oncolytic viral genome from which an ICP47 gene has been knocked out, and "KOS- Δ 47-S1-gDNA-hCCL19" refers to an insertion of hCCL19 based on KOS- Δ 47; the method comprises the steps of constructing a recombinant HSV-1 virus by using a CRISSPR/Cas9 system, and comprising 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.

The invention will now be described with reference to specific examples, which are intended to be illustrative only and not to be limiting in any way.

The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Example 1 first round of viral recombination and screening

In the present example, the inventors extracted the KOS- Δ 47 viral genome with phenol chloroform; transfecting 293T cells by using a Lipofectamine 3000 transfection reagent to obtain recombinant viruses; carrying out first round limiting dilution screening and second round limiting dilution screening on the recombinant viruses to obtain positive recombinant viruses, and carrying out amplification, genome extraction, PCR identification and sequencing on the positive recombinant viruses; the results show that: the recombinant virus obtained by the first round of virus recombination and screening is a single copy insertion, and the specific experimental operation is as follows:

1. phenol chloroform extraction of KOS-Delta 47 virus genome

Cultures of KOS- Δ 47 virus-infected vero cells were discarded supernatant, leaving adherent diseased-affected cells (-100% lesions) frozen at-80 ℃. Extracting a virus genome according to the following steps:

(1) add 1mL cell lysate to cells, shake evenly to lyse cells, scrape gently into 2mL centrifuge tube with cell scraper, ice-wash for 20min.

(2) To the product of step (1) was added 660. Mu.L of 5M NaCl, gently mixed and placed in a refrigerator at 4 ℃ overnight.

(3) Treating the product of step (2) under the following conditions: centrifuge at 12000rpm for 30min at 4 ℃.

(4) The centrifuged supernatant was pipetted with a tip into a fresh centrifuge tube, and an equal volume of phenol chloroform isoamyl alcohol (25.

(5) And (4) centrifuging the product obtained in the step (4) at 4 ℃ and 12000rpm for 10min, sucking a supernatant by using a tip removal gun head, and extracting the protein again.

(6) Adding pre-cooled 2 times volume of absolute ethyl alcohol into the extracted supernatant, gently mixing uniformly, and standing for 2 hours at-20 ℃.

(7) The product of step (6) was centrifuged at 12000rpm for 10min at 4 ℃, the supernatant was discarded, and the pellet was washed twice with 500 μ L of 75% ethanol (flicked rather than blown) at 12000rpm,10min at 4 ℃.

(8) The supernatant was discarded, 12000rpm,1min,4 ℃ and the liquid was blotted dry with a 10. Mu.L gun.

(9) To the product of step (7), 30. Mu.L of deionized water was added to dissolve the DNA.

2. Preparation of recombinant virus by transfecting 293T cell

After mixing the a and B (CRISPR/Cas 9 gene editing plasmid (34.5-S1-2 plasmid) + homologous repair donor plasmid (dornor DNA (hCCL 19-RL1-PMD 18T) + KOS- Δ 47 viral genome) in table 1 using Lipofectamine 3000 transfection reagent, cotransfecting into 6-well plate 293T cells to obtain a viral fluid containing recombinant viruses, wherein the 34.5-S1-2 plasmid is a plasmid containing gRNA, the plasmid is CRISPR/Cas9 gene editing plasmid for ICP34.5 locus, and the dornor DNA (hCCL 19-RL 1-18T) is a donor plasmid containing hCCL 19.

Table 1:

10% FBS/DMEM complete medium incubated at 37 ℃ was replaced before cell transfection, after which the cells were incubated in EP tubes with A and B mixed well (gently mixed, gently flicked with a hand) at room temperature for 15min, 300. Mu.L per well of the transfection complex was added dropwise to 293T cell cultures in 6-well plates for 6h, and the cells were cultured by changing fresh DMEM +10 FBS complete medium incubated at 6 ℃.

After 2 days of transfection, cytopathic effect was observed, the old culture medium was discarded, 1% FBS/DMEM complete culture medium incubated at 37 ℃ was added to 1mL, cells were collected, frozen and thawed repeatedly at minus 70 ℃ for 3 times, centrifuged at 4000rpm for 10min, and the supernatant was obtained as virus solution containing recombinant virus, named KOS-Delta 47-S1-gDNA-hCCL19-201224, and was used or frozen at-80 ℃.

3. Recombinant viruses were subjected to a first round of limiting dilution screening

And (3) subjecting the first round of recombinant viruses obtained in the step (2) to a first round of limiting dilution screening, wherein the specific operations are as follows:

the recombinant virus solution KOS-delta 47-S1-gDNA-hCCL19-201224 was diluted by 100-fold and 500-fold and added to a 96-well plate full of a monolayer Vero at 100. Mu.L/well, 2 pieces of 96-well plates per dilution, named: KOS-Delta 47-S1-gDNA-hCCL19-201224 (10) ^-2 ，①～②)、KOS-△47-S1-gDNA-hCCL19-201224(2×10 ^-3 (1) to (2)), the 96-well plate was incubated at 37 ℃ in a CO2 incubator 5%.

Cytopathic condition was observed after 72 hours, KOS-Delta47-S1-gDNA-hCCL19-201224(2×10 ^-3 (1) to (2)) all the wells showed significant lesions, and the lesions were detected in KOS-. DELTA.47-S1-gDNA-hCCL 19-201224 (2X 10) ^-3 (1)) crude extraction of viral gDNA from the supernatant of 96-well plates as follows:

mu.L of the virus solution + 8. Mu.L of gDNAextraction Buffer (proteinase K was added at 10. Mu.L/mL before use), mixed and reacted in a PCR apparatus at 55 ℃ 1h,95 ℃ 10min,16 ℃ infinity, as: KOS-Delta 47-S1-gDNA-hCCL 19-201227-2X 10 ^-3 - (No. 1) -1-96) for PCR amplification verification.

Wherein, the virus strain obtained after the first round of limiting dilution screening is subjected to crude extraction of virus gDNA, and the specific operations are as follows: mu.L of virus solution + 8. Mu.L of gDNA Extraction Buffer (proteinase K was added at 10. Mu.L/mL before use), mixed and reacted in a PCR instrument under PCR reaction conditions of 55 ℃ 1h,95 ℃ 10min and 16 ℃ infinity, and whether hCCL19 was inserted into the virus genome was verified by PCR amplification, wherein hCCL19 gene detection primers are shown in Table 2, and PCR amplification reaction systems are shown in Table 3.

Table 2:

table 3:

components	Volume of μ L
		Crude extraction of 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 ₂ O	3.75μL
		Total volume	10μL

The PCR results of the first round of limiting dilution screening of the recombinant viruses are shown in FIG. 1, and the PCR electrophoresis results show that KOS-Delta 47-S1-gDNA-hCCL19-9 and No. 32 suspected positive recombinant viruses are collected, and the virus solution is collected and named KOS-Delta 47-S1-gDNA-hCCL19-9 as KOS-Delta 47-S1gDNA-hCCL19-9-200227.

4. Recombinant viruses were subjected to a second round of limiting dilution screening

And (3) performing a second round of limiting dilution screening on the suspected positive recombinant viruses obtained in the step (3) after the first round of limiting dilution screening, wherein the specific operations are as follows:

the recombinant virus solution KOS-delta 47-S1gDNA-hCCL19-9-200227 is diluted by 1x10 ⁶ Diluted and added to a 96-well plate full of monolayer Vero cells at 100 μ L/well, named: KOS-Delta 47-S1gDNA-hCCL19-201231-1x10 ⁶ ，37℃，5％CO ₂ After 2 hours of culture, the virus solution was discarded, and 200. Mu.L/well of DPBS was added thereto to wash the cells for 1 time, and the cells were discarded, and 100. Mu.L/well of 1% FBS/DMEM was added thereto to continue the culture. After 96 hours of culture, the cytopathic condition was observed, KOS-Delta 47-S1gDNA-hCCL19-201231-1x10 ⁶ The lesion with 28 holes is obvious and is numbered as follows: s1 g-9-1-28, and virus gDNA crude extraction is carried out on the supernatant of the lesion-occurring hole by the same method.

Wherein, the virus strain (KOS-delta 47-S1-gDNA-hCCL 19-9) obtained after the second round of limiting dilution screening is subjected to virus gDNA crude extraction, and the operation is specifically as follows: mu.L of virus solution + 8. Mu.L of gDNAextraction Buffer (10. Mu.L/mL of proteinase K is added before use), and after mixing, the mixture reacts in a PCR instrument under the following reaction conditions: whether hCCL19 is inserted into the viral genome is verified by PCR amplification at 55 ℃ 1h,95 ℃ 10min and 16 ℃ infinity, wherein hCCL19 gene detection primers are shown in Table 2, and PCR amplification reaction systems are shown in Table 3.

The PCR result of the second round of limiting dilution screening of the recombinant viruses is shown in FIG. 2, and the PCR electrophoresis result shows that the suspected positive recombinant viruses No. KOS-delta 47-S1-gDNA-hCCL19-9-2 and KOS-delta 47-S1-gDNA-hCCL19-9-6 are selected; KOS-. DELTA.47-S1-gDNA-hCCL 19-9-2 was named KOS-. DELTA.47-hCCL 19-S1g-9-2 No. 210104.

5. Identification of recombinant oncolytic viruses from the second round of limiting dilution screening

And (4) identifying and sequencing the recombinant oncolytic virus obtained in the step (4), wherein the specific operations are as follows:

carrying out amplification propagation, genome extraction, PCR identification and sequencing on the positive recombinant virus (KOS-delta 47-S1-gDNA-hCCL 19-9-2), wherein the specific operation of genome extraction is as follows: propagating the recombinant virus KOS-Delta 47-hCCL19-S1 g-9-2-210104 with 6-well vero cells, adding 5. Mu.L of the virus, 37 ℃,5% of CO ₂ Culturing for 24 hours until the cells are completely diseased, collecting culture solution, namely KOS-delta 47-S1g-9-2-P01-210107, taking 200 mu L of virus solution, extracting virus genome by using a virus genome Extraction Kit Viral RNA/DNA Extraction Kit ver.5.0, and namely: KOS-delta 47-S1g-9-2-gDNA-210107, primers for detecting the sequence of the hCCL19 homologous arm in PCR amplification reaction are shown in Table 4, and PCR amplification reaction systems are shown in Table 5.

Table 4:

table 5:

the results of the propagation of the recombinant virus after the first round of virus recombination and the identification of PCR are shown in FIG. 3, wherein the genome of KOS-delta 47-S1g-9-2-gDNA-210107 (9-2 in FIG. 3) is KOS-delta 47-S1g-9-2-P01-210107, the bands before and after recombination are respectively cut and recovered, and are sent to Guangzhou Ai Jice sequencing (sanger sequencing), so that the band after recombination is confirmed, the hCCL19 gene expression cassette is completely and correctly sequenced, and the band before recombination is confirmed to be the band before KOS genome recombination; thus, the recombinant virus KOS-delta 47-S1g-9-2-P01-210107 obtained by the first round of virus recombination and screening is confirmed to be a single copy insertion.

Example 2 second round viral recombination and screening

This example was carried out by performing a second round of recombination and screening on KOS-. DELTA.47-S1-gDNA-hCCL 19-9-2 having a single copy of the insert obtained in example 1, and extracting the single copy of the recombinant viral genome with phenol chloroform; transfecting 293T cells by using a Lipofectamine 3000 transfection reagent to obtain recombinant viruses; carrying out limiting dilution, PCR screening in the 1 st round and PCR screening in the 2 nd round on the recombinant viruses to obtain positive recombinant viruses, carrying out amplification propagation, genome extraction, PCR identification and sequencing on the positive recombinant viruses, and specifically operating as follows:

1. phenol chloroform extraction of KOS-. DELTA.47-hCCL 19-9-2 (i.e., KOS-. DELTA.47-S1-gDNA-hCCL 19-9-2) viral genome

The vero cell culture infected with KOS-Delta 47-S1g-9-2-P01 (i.e., KOS-Delta 47-S1 g-9-2-P01-210107) virus was discarded from the supernatant, leaving adherent cells with a pathological effect (-100% lesion), frozen at-80 ℃ and extracted for viral genome in the same manner as above.

2. Preparation of recombinant virus by transfecting 293T cell

After mixing a and B (CRISPR/Cas 9 gene editing plasmid (Cas 9-sgRNA345-5 plasmid) + homologous repair donor plasmid (dorner DNA (hCCL 19-RL1-PMD 18T) + viral genome (KOS- Δ 47-hCCL 19-9-2)) in table 6 using Lipofectamine 3000 transfection reagent, cotransfected into 6-well plate 293T cells to obtain a viral fluid containing recombinant viruses, wherein Cas9-sgRNA345-5 is a plasmid containing grnas, is a CRISPR/Cas9 gene editing plasmid for ICP34.5 gene sites, and dorner DNA (hCCL 19-RL1-PMD 18T) is a donor plasmid containing hCCL 19.

Table 6:

the cell transfection procedure was as above, and the virus solution containing the recombinant virus was named KOS-DELTA 47-hCCL19-S5g-P00-210117 for use.

And (3) subjecting the second round of recombinant viruses obtained in the step (2) to a first round of limiting dilution screening, wherein the specific operations are as follows:

diluting the recombinant virus solution KOS-delta 47-hCCL19-S5g-P00-210117 by 400-fold, 1000-fold and 3000-fold dilution times, adding the diluted recombinant virus solution KOS-delta 47-hCCL19-S5g-P00-210117 into a 96-well plate full of single-layer Vero cells according to 100 mu L/well, wherein each dilution comprises 2 96-well plates, and the dilution is named as: KOS-Delta 47-S5g-hCCL 19-3000X/1000X/400X-P01-210117-01-02 at 37 ℃ 5% ₂ After 2 hours of incubation in the incubator, discard the virus solution, add 100. Mu.L/well DPBS wash plate, discard, add 100. Mu.L/well 1% FBS/DMEM, put 96 well plate at 37 ℃,5% ₂ Culturing in an incubator.

After the cells are cultured for 72 hours, cytopathic conditions are observed, 45-hole pathological changes exist in KOS-delta 47-S5g-hCCL19-3000X-P01-210117-01, 57-hole pathological changes exist in KOS-delta 47-S5g-hCCL19-3000X-P01-210117-02, and virus gDNA crude extraction is carried out on pathological change holes, wherein the method is recorded as follows: KOS-delta 47-S5g-hCCL 19-3000X-P01-210121-01-102 for PCR amplification verification.

Wherein, virus gDNA crude extraction is carried out on the virus strain obtained after the first round of limiting dilution screening, and PCR amplification is used for verifying whether the ICP34.5 gene sequence is successfully knocked out, wherein the ICP34.5 gene sequence detection primer is shown in Table 7, and the PCR amplification reaction system is shown in Table 8.

Table 7:

table 8:

composition (I)	Volume of
		Crude extraction of gDNA	0.5μL
MightyAmp DNA Polymerase	0.25μL
		2×MightyAmp Buffer Ver.2	5μL
LP-F2	0.25μL
		LP-R2	0.25μL
ddH ₂ O	3.75μL
		Total volume	10μL

The results of the 1 st round PCR screening after the second round of recombinant viruses are subjected to limiting dilution are shown in FIG. 4, and PCR electrophoresis results show that 60 positive recombinant viruses are obtained after limiting dilution.

4. Recombinant virus round 2 PCR screening

The 60 positive recombinant viruses obtained in the first round of limiting dilution in the step 3 of the example were subjected to round 2 PCR screening to verify whether hCCL19 was inserted into the viral genome, wherein hCCL19 gene detection primers are shown in Table 9 (for amplifying the full length of the hCCL19 gene expression cassette), and PCR amplification reaction systems are shown in Table 10.

Table 9:

table 10:

composition (I)	Volume of
		Crude extraction of 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 ₂ O	3.75μL
		Total volume	10μL

The results of the second round of recombinant viruses amplified after 2 nd round of PCR screening are shown in FIG. 5: PCR electrophoresis results show that the sample obviously only amplifying recombined bands is No. 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 and 101 in KOS-delta 47-S5g-hCCL19, and 47 positive recombined viruses are totally obtained.

5. Identification of Positive recombinant oncolytic viruses

carrying out amplification propagation, genome extraction, PCR identification and sequencing on positive recombinant viruses (4, 5, 8-10 and 12 in KOS-delta 47-S5g-hCCL 19), carrying out amplification propagation on the recombinant viruses, extracting viral genomes, and respectively naming the steps as follows: KOS-delta 47-S5g-hCCL19-04/05/08/09/10/12-gDNA-210125. The specific operation of genome extraction is as follows: expanding the cells with 6-well plate vero, adding 2. Mu.L of virus solution, 37 ℃,5% CO ₂ After culturing for 24 hours, the cells were completely diseased, and a culture medium was collected and named KOS-Delta 47-S5g-hCCL19-04/05/08/09/10/12210124, and stored at-70 ℃, 200. Mu.L of virus solution was taken, the genome was extracted using the Viral RNA/DNA Extraction Kit ver.5.0 Kit, and eluted with 50. Mu.L of sterile water and named: KOS-DELTA 47-S5g-hCCL19-04/05/08/09/10/12-gDNA-210125, the primers designed for the sequence of ICP34.5 gene in the PCR amplification reaction are shown in Table 7, and the PCR amplification reaction system is shown in Table 8. The PCR electrophoresis results are shown in FIG. 6, and show that: KOS-delta 47-S5g-hCCL19-04,05,08,09,10 and 12 fail to amplify knock-out fragments, and the recombinant virus successfully knocks out ICP34.5 gene.

Wherein, primers are designed according to the homologous arm sequences of the hCCL19 expression cassette, the full length of the hCCL19 expression cassette is amplified, the primer sequences are shown in tables 11 and 12, and the PCR amplification reaction system is shown in table 13.

Table 11:

table 12:

table 13:

composition (A)	Volume of
		gDNA	1.0μL
MightyAmp DNA Polymerase	1.0μL
		2×MightyAmp Buffer Ver.2	25.0μL
Primer F	1.0μL
		Primer R	1.0μL
ddH ₂ O	21.0μL
		Total volume	50μL

Wherein, the primer F is 345-HOM1-F2 in tables 11 and 12; the primer R is 345-HOM2-R2 or 345-flash-seq in Table 11 and Table 12.

As a result of PCR, the band of the sample No. KOS-. DELTA.47-S5 g-hCCL19-04,05,08,09,10,12 was single and consistent with the band of the hCCL19-RL1-pMD18T plasmid positive control sample, as shown in FIG. 7. And cutting the gel, recovering a band of KOS-delta 47-S5g-hCCL19-04,05,08,09,10,12, sending the band to Ai Ji biotechnology Limited Guangzhou for sequencing, and indicating that the sequencing results show that the gene expression cassette of the KOS-delta 47-S5g-hCCL19-04,05,08,09,10,12 recombinant virus hCCL19 is correctly sequenced, and the band is named as: KOS-delta 47-S5g-hCCL19-04/05/08/09/10/12-P01-210123, namely, the two-copy integrated KOS-delta 47-hCCL19 recombinant virus is successfully obtained through 2 rounds of recombination.

Example 3 first round plaque purification of Positive recombinant oncolytic viruses

1. Monoclonal preparation and genome extraction

Selecting KOS-delta 47-S5g-hCCL19-05/12-P01-210123 for 1-round plaque purification, diluting virus liquid to '5E + 03', '5E + 04', '5E + 05' by using 1% FBS/DMEM, abandoning 6-pore plate vero cell old culture solution, adding 300 mu L of virus diluent, carrying out 2-pore re-plating at each dilution degree, and continuing to culture after infection; after staining cells, observing the staining condition, selecting 5 virus spots of each virus strain, adding 200 mu L of virus liquid into full 6-well vero cells, culturing for 2 days until the cells are completely diseased, collecting the virus liquid, and naming as: KOS-delta 47-hCCL19-05-P01-01/02/03/04/05-210205, KOS-delta 47-hCCL19-12-P01-01/02/03/04/05-210205, and the Viral genes are extracted from 200 mu L of virus solution by using a kit Viral RNA/DNA Extraction kit 5.0, the specific operation of Extraction is carried out according to the kit instruction, and the extracted Viral genes are named as follows:

KOS-△47-hCCL19-05-01/02/03/04/05-gDNA-210205、KOS-△47-hCCL19-12-01/02/03/04/05-gDNA-210205。

2. PCR identification

The specific operation of PCR amplification is as follows: the sequences of the primers used are shown in tables 11 and 12, and the reaction system used is shown in Table 13.

The electrophoresis result of the KOS-. DELTA.47-hCCL 19-05/12 recombinant virus is shown in FIG. 8, and the electrophoresis result shows that the hCCL19 gene is expressed, but the ICP34.5 gene is not expressed, in both of KOS-. DELTA.47-hCCL 19-05-01/02/03/04/05 and KOS-. DELTA.47-hCCL 19-12-01/02/03/04/05.

3. Sequencing

And cutting the gel, recovering the bands of KOS-delta 47-hCCL19-05-01/02/03 and KOS-delta 47-hCCL19-12-01/02/03, and sending the bands to Guangzhou Ai Ji biotechnology limited for sequencing. The sequencing method comprises the following specific operations: according to the sequencing result of sanger sequencing, ai Ji-IGC 280537 and 6 clone sequencing prove that the sequencing of the recombinant virus and the hCCL19 gene expression cassette is completely correct, is consistent with a theoretical sequence and has good sequencing quality.

Example 4 second round plaque purification of recombinant oncolytic viruses

1. Monoclonal preparation and genome extraction

Selecting KOS-delta 47-hCCL19-05-02-P01-210205 and KOS-delta 47-hCCL19-12-03-P01-210205 to purify plaque for the 2 nd round, diluting the virus solution to be 2E +04, 2E +05 and 2E +06, performing virus infection operation in the same example 3, picking 5 virus plaques for each virus strain, performing propagation by using vero cells as hosts, culturing for 2 days until the cells are completely diseased, collecting the virus solution, and naming as follows: KOS-delta 47-hCCL19-05-02-P01-01/02/03/04/05-210303 and KOS-delta 47-hCCL19-12-03-P01-01/02/03/04/05-210303, 200 mu L of virus solution is taken, a Viral genome is extracted by using a Kit Viral RNA/DNA Extraction Kit ver.5.0, the specific operation is carried out according to the Kit instructions, and the extracted Viral genome is named as: KOS- 'DELTA' 47-hCCL19-05-02-01/02/03/04/05-gDNA-210303, KOS- 'DELTA' 47-hCCL19-12-03-01/02/03/04/05-gDNA-210303.

2. PCR identification

The electrophoresis result of the KOS-delta 47-hCCL19-05-02/12-03 recombinant virus is shown in FIG. 9, and the electrophoresis result shows that the sample bands of KOS-delta 47-hCCL19-05-02-01/02/03/04/05 and KOS-delta 47-hCCL19-12-03-01/02/03/04/05 are single and the bands are consistent with the band of the hCCL19-RL1-PMD18T plasmid positive control and are large bands after recombination after amplification by 2 pairs of primers.

3. Sequencing

And cutting the gel to recover a strip of KOS-delta 47-hCCL19-05-02-01/02/03 and KOS-delta 47-hCCL19-12-03-01/02/03, and sending the strip to Guangzhou Ai Ji biotechnology limited for sequencing. The sequencing method comprises the following specific operations: the sequencing result of sanger sequencing shows that Ai Ji-IGC 282403,6 clone sequencing confirms that the sequencing of the recombinant virus and the hCCL19 gene expression cassette is completely correct, is consistent with a theoretical sequence and has good sequencing quality.

Example 5 third round plaque purification of recombinant oncolytic viruses

1. Monoclonal preparation and genome extraction

Selecting KOS-delta 47-hCCL19-05-02-02 and KOS-delta 47-hCCL19-12-03-01 obtained in example 4 to carry out plaque purification of the 3 rd round, diluting the virus liquid to '5E + 05', '1E + 06', '2E + 06', '4E + 06', and carrying out virus infection operation as in example 3; each virus strain is picked up with 5 virus spots for propagation (vero cells are taken as hosts), after 2 days of culture, the cells are completely diseased, and virus fluid is collected, which is named as: KOS-delta 47-hCCL19-05-02-02-01/02/03/04/05-P01-210315, KOS-delta 47-hCCL19-12-03-01-01/02/03/04/05-P01-210315, 200. Mu.L of the virus solution was taken, and the Viral genome was extracted using a Kit Viral RNA/DNA Extraction Kit ver.5.0 and named: KOS- 'DELTA' 47-hCCL19-05-02-02-01/02/03/04/05-gDNA-210315, KOS- 'DELTA' 47-hCCL 19-12-03-01/02/03/04/05-gDNA-210315.

2. PCR identification

The genome extracted in step 1 was subjected to PCR amplification using the primer sequences shown in tables 7, 11, 12 and 14, and the reaction systems shown in tables 8, 15 and 16.

Table 14:

table 15:

composition (I)	Volume of
		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 ₂ O	21.0μL
		Total volume	50μL

Table 16:

composition (I)	Volume of
		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 ₂ O	21.0μL
		Total volume	50μL

Electrophoresis results of the recombinant viruses KOS-Delta 47-hCCL19-05-02-02 and KOS-Delta 47-hCCL19-12-03-01 are shown in FIG. 10, and show that after 2 pairs of primers are used for amplification, the electrophoresis results show that the sample bands of KOS-Delta 47-hCCL19-05-02-01/02/03/04/05 and KOS-Delta 47-hCCL 19-12-03-01/03/04/05 are single, and the bands are consistent with the bands of the hCCL19-RL1-PMD18T plasmid positive control and are large bands after recombination.

3. Sequencing

And cutting the gel, recovering the KOS-delta 47-hCCL19-05-02-02-01/02/04 and KOS-delta 47-hCCL19-12-03-01-01/02/03 bands, and sending the bands to Guangzhou Ai Ji biotechnology limited for sequencing. The specific operation of sequencing is as follows: and (5) carrying out sanger sequencing. Sequencing results show that Ai Ji-IGC 283714 and IGC283898 are correct in sequencing of the selected 6 clone expression frames, and the hCCL19 gene expression cassette is completely correct in sequencing, consistent with a theoretical sequence and good in sequencing quality.

Example 6: sequencing verification of recombinant virus KOS-delta 47-hCCL19-05-02-02

(ii) PCR amplification enzymes with high Fidelity enzymes respectively: (

Max DNA Polymerase, apexHF HS DNA Polymerase FS) to recombinant virus KOS-delta 47-hCCL19-05-02-02 gene expression frame sequence for TA cloning sequencing, the primer sequence is shown in Table 17, the reaction system is shown in Table 18.

Table 17:

table 18:

the PCR amplification procedure was: 3min at 98 ℃; (98 ℃ C. 10s,64 ℃ C. 15s,72 ℃ C. 3 min) for 30 cycles; 5min at 72 ℃;16 ℃ infinity, 1.5% agarose gel electrophoresis after completion, an electrophoretogram shown in figure 11, recovery by using a kit cycle-pure kit (omega), addition of A tail, T loading and transformation of large intestine competence DH5a, screening results of positive clones shown in figure 12, and sampling 6 positive clone bacterial liquids respectively to sequence by Guangzhou Ai Ji biotechnology Limited.

And (3) sequencing results: ai Ji IGC287528, IGC287406, IGC287407, high fidelity enzyme ApexHF HS DNA Polymerase FS selected clones: the 1,3,4,6,7 cloned gene expression frame is correct, the sequencing quality is good, the hCCL19 gene expression frame is correct, the hCCL19 gene expression frame is completely consistent with a theoretical sequence, and the sequencing quality is good; clones selected with the high fidelity enzyme Prime star max: the 3,5,6,7,8 cloned gene expression frame is correct, the sequencing quality is good, the hCCL19 gene expression frame is correct, the hCCL19 gene expression frame is completely consistent with a theoretical sequence, and the sequencing quality is good.

Example 7 viral cytotoxicity assay

The cell strains shown in Table 19 were cultured in vitro, seeded in 96-well culture plates at an appropriate cell density, and after overnight culture, two viruses were added at 12 gradient concentrations (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), respectively, and specific information of the two viruses was as shown in Table 20, followed by culturing for 24, 48, or 72h, respectively, and cell viability was examined according to the instructions of the CCK8 kit (purchased from Japan Co-Ron).

Table 19:

cell type	Cell name	Culture medium
			NCI-H460	Large cell lung cancer cell	RPMI-1640+10％FBS
Fadu	Human pharyngeal squamous carcinoma cell	MEM+10％FBS
			HepG2	Liver cancer cell	DMEM+10％FBS
Hep3B2.1-7	Liver cancer cell	DMEM+10％FBS
			HCT-116	Colon cancer cells	RPMI-1640+10％FBS
HT-29	Colon cancer cells	RPMI-1640+10％FBS
			SW620	Colon cancer cells	DMEM+10％FBS

Table 20:

sample name	Titer of the product	Source
			KOS-ATCC	8.33x10^7	SHS21054,P06
KOS-△47-hCCL19	7.76x10^7	SHS21054,P18

Note: KOS-ATCC is wild type virus; KOS-. DELTA.47-hCCL 19 is a 47 knockout virus having the hCCL19 gene inserted at position 34.5.

The IC50 values for the two viruses on different cells are shown in Table 21, according to the criteria for MOI IC50 < 0.6 after 72h incubation: from the results of virus killing, HT29, SW620, hepG2, hep3B2.1-7, NCI-H460 cells were oncolytic virus sensitive tumor cell lines, and compared with KOS-ATCC original virus strain, the KOS-hCCL19 recombinant virus strain still remained sensitive to HCT-116 cells.

Table 21:

note: N/A represents the non-linear fitting which cannot be made due to poor or unmeasured dose-effect relationship, and the number marked with a horizontal line represents that the fitting degree is poor (the fitting degree is less than 80 percent), and the corresponding MOI IC50 value is only used as a reference.

Example 8 test for viral replication Capacity

Taking the KOS-ATCC (namely the wild type virus) and the KOS-hCCL19 (HSV 1 carrying the hCCL 19) in the logarithmic growth phase, inoculating the cells 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 in a 6-well culture plate at proper cell density, counting the 6-well plate cells after culturing overnight, diluting virus mother liquor by using high-sugar DMEM or RPMI-1640 culture medium containing 1% inactivated FBS according to the number of the cells in each well, and preparing the virus solution with MOI = 0.1. Adding 300. Mu.L of virus solution to each well in sequence, subjecting to 37 ℃ and 5% CO ₂ Incubate under conditions, shake the plate every 15min to allow the virus to better adsorb the cells, and add 1mL of media after 1.25 h. After 2h, the medium was discarded, 2mL of medium was supplemented, and the cells were incubated in a CO2 incubator for 24h, 48h, or 72h, respectively.

And after the culture is finished, collecting virus liquid for virus titer determination. After freezing and thawing for three times (-80 ℃,37 ℃), the collected virus solution is diluted in a gradient manner, 300 mu L of Vero cells (6-hole plate) are taken to infect, the culture plate is shaken every 15min to enable the virus to better adsorb the cells, and 2mL of culture medium is added after 1.25 h. After 2h the medium was discarded and 300. Mu.L DMEM complete medium, 3mL2% methylcellulose immobilized virus was added. After 3-4 days of culture, the overlay medium was aspirated away, and 10% HCHO solution was added, 1 mL/well, and fixed for 20min. Then, the formaldehyde solution was aspirated off, and 1% crystal violet staining solution was added thereto at a concentration of 500. Mu.L/well for 30min. Finally, pouring off the staining solution, slowly washing the staining solution with tap water, reversely wiping the staining solution with absorbent paper, counting the plaques, and calculating the virus titer, wherein the virus titer = the virus dilution multiple (1000/300) × the number of the plaques.

This example evaluates the replication capacity of the original virus strain (KOS-ATCC) and the modified virus strain (KOS-hCCL 19) in 6 cancer cells (Hep3B2.1-7, hepG2, NCI-H460, SW620, HT29, HCT 116) and performs a T test on them, and the replication capacity of KOS-ATCC and KOS-hCCL19 in 6 cancer cells is shown in FIG. 13, in which the replication capacity of two viruses in different tumor cells is different, and the modified virus strain retains the proliferation activity on tumor cells as a whole.

Example 9 Virus expression amount test

In this example, a virus expression level experiment was performed on vero cells, and the specific experimental procedures were as follows:

9.1 test materials

The reagent materials are shown in tables 22 and 23:

table 22:

name (R)	Manufacturer of the product	Batch number	Rank of
				DMEM medium	Gibco	2186832、2186840	Biological grade
Trypan blue	Gibco	35050-061	Biological grade
				Fetal Bovine Serum (FBS)	Gibco	2175442p	Biological grade
DPBS	Gibco	223126	Biological grade
				Pancreatin	Gibco	2276876	Biological grade
P/S	Gibco	2289320	Biological grade

Table 23:

name (R)	Source	Culture medium
			vero Africa Green monkey kidney cell	ATCC	DMEM+10％FBS+1％P/S

9.2 test methods

9.2.1 cell culture

The vero cells were cultured and cultured in a 5% carbon dioxide incubator at 37 ℃. Cells were observed 1 time daily using an inverted microscope. Cell passage is carried out when the cell growth confluence rate in the culture dish reaches 80-90%: discarding the old culture solution, washing away the residual culture medium by using DPBS, adding 1mL of 0.25% pancreatin digestive juice, placing the mixture in an incubator for digestion for 2min, discarding pancreatin after the cells become round and float upwards, adding a new culture medium to stop digestion, and subculturing the mixture in a fresh sterile culture dish according to a certain proportion.

9.2.2 cell plating

Vero cells were removed from the cell incubator, the medium was aspirated, the residual medium was washed away with DPBS, 1mL of pancreatin was added to the dishes, digested for 2min in an incubator at 37 ℃, followed by aspiration of pancreatin, and the cells were resuspended in 10mL (10% FBS,1% P/S) of DMEM per dish and counted. The cells are plated in 6-well plates with plating density of 1x10 ^ 6/mL, and 2mL of cell sap per well are plated, and then the cells are placed into a cell culture box for further culture for 24 hours.

9.3 viral infection

9.3.1 cell count

Taking Vero cells cultured in a 6-well plate out of the cell incubator, selecting two wells for cell counting: the medium was aspirated off, the residual medium was washed off with DPBS, 500. Mu.L of pancreatin was added per well, digested in an incubator at 37 ℃ for 2min, followed by addition of 500. Mu.L of 10% FBS,1% P/S DMEM medium to terminate digestion, counted and the average number of cells per well was calculated.

9.3.2 viral dilution and viral infection

HSV1-hCCL19 and HSV1-2k viruses infect the cells according to MOI =0.01 and 0.1pfu/cell respectively, and 2-3 multiple holes are respectively made while blank control is arranged. Firstly, diluting HSV1-hCCL19 and HSV1-2k by 1000 times by using virus diluent, and then respectively preparing infectious virus culture media according to the infected MOI and the number of cells in each hole; 1mL of the prepared virus culture medium is added into each well for infection, and the blank control is replaced by virus diluent.

Viral infection 2h, shaking the plate 1 times every 20min, discarding the supernatant, changing to 10% FBS,1% P/S in DMEM medium, 2mL per well.

Culturing for 48 hours or 72 hours in the incubator, collecting the supernatant, centrifuging at 4500rpm for 5min at 4 ℃, taking the supernatant, subpackaging, and detecting the expression amount of hCCL19 by ELISA.

The expression of hCCL19 in the supernatant was determined according to the Human MIP-3b ELISA Kit (EhCCL 19).

Specific results are shown in FIG. 14, in which blank represents wells of blank medium, 2k represents HSV1 with ICP34.5 and ICP47 knocked out; the results showed that there was no significant difference in hCCL19 expression between 48h and 72h of viral infection, and that there was no significant difference in hCCL19 expression between cells infected with viruses using MOI =0.01 and 0.1pfu/cell under the same infection time conditions, but that the expression of hCCL19 was higher when cells were infected with viruses at MOI =0.01 pfu/cell.

Example 10 test for viral replication Capacity

The KOS-ATCC (i.e., the wild-type virus described above) and KOS-hCCL19 (hCCL 19-carrying HSV 1) were taken at logarithmic growth phase, and the replication ability was evaluated after infection of tumor cells with each of the two viruses. The tumor cells are CAL27 oral squamous 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 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 carcinoma cells, U87MG human brain glioma cells, skmel-28 human melanoma cells, LOVO intestinal carcinoma adenocarcinomas, T.Tn human esophageal carcinoma cells, calu-6 lung epithelial cell carcinomas and NCI-H226 (lung) squamous carcinoma cells.

The tumor cells were seeded in a 6-well culture plate at an appropriate cell density, and after overnight culture, the cells in the 6-well plate were counted, and the virus mother solution was diluted with high-sugar DMEM, RPMI-1640, or F12K medium containing 1% inactivated FBS according to the number of cells per well to prepare a virus solution with MOI = 0.02. Discarding the old culture medium from the cell plate, transferring the diluted two virus solutions to a cell plate containing different tumor cells in order of 1mL per well, at 7 deg.C, 5% ₂ Incubating in an incubator for 2h; discarding the supernatant after 2h of virus-infected cells, adding 2mL 1% of FBS per well in basal medium, standing at 37 ℃ and 5% of CO ₂ The incubator continues to culture for 24h, 48h or 72h.

After the culture is finished, virus liquid at two time points of 24h, 48h or 72h (re-collecting more than 50% of cytopathies) is collected according to the cytopathic condition, and the virus titer is determined. After three times of freeze thawing (-80 ℃ C., 37 ℃ C.), the collected virus solution was diluted in gradient, 300. Mu.L of Vero cells (6-well plate) were taken and shaken every 15min to allow the virus to better adsorb the cells, and after 75min of virus infection, 1mL of 1% inactivated FBS DMEM medium was added to each well and the cells were thawed at 37 ℃ C., 5% CO ₂ After 2h incubation in the incubator, the virus solution was aspirated away, 300. Mu.L of 1% inactivated FBS DMEM medium and 3mL of room temperature-equilibrated methylcellulose medium were added to each well, and the mixture was incubated at 37 ℃ and 5% CO ₂ And (5) culturing in an incubator. After 2 days of culture, cells were stained by uniformly adding 2mL of basal medium containing 0.01% neutral red. Standing at 37 deg.C, 5% CO ₂ Culturing in an incubator for more than 12 h. The aspirate pump discarded all fluid from the 6 well plate, plaque counts were performed, and viral titers were calculated, viral titer = viral dilution fold (1000/300) plaque number.

In this example, the replication capacities of the original virus strain (KOS-ATCC) and the modified virus strain (KOS-hCCL 19) in 20 cancer cells (CAL 27, A549, PC-3, CNE-2Z, panc-1, 143B, fadu, KYSE510, KB, ECA109, aspc, colo, SK-OV 829-3, hela, U87MG, skmel-28, LOVO, T.Tn, calu-6, and NCI-H226) were evaluated, the replication capacities of KOS-ATCC and KOS-hCCL19 in 20 cancer cells are shown in FIGS. 15 to 17, and the modified virus strain retains the proliferation activity on the tumor cells.

"KOS" and "KOS-ATCC" in FIGS. 1-17 each refer to the original virus strain; "hCCL19" and "KOS-hCCL19" both refer to engineered virus strains.

Example 11 anti-tumor Activity of viruses in animal models of xenograft tumors

NCI-H460 human lung cancer cells are inoculated under the skin of the right back of a BALB/c nude mouse, and a mouse tumor-bearing model is established. The size of the tumor to be detected is 100mm ³ On the left and right, the administration was carried out in groups of 8 animals each. In the NCI-H460 model, a KOS-hCCL19 high (KOS-hCCL 19 (high)) dose group and a KOS-hCCL19 low (KOS-hCCL 19 (low)) dose group are set, and the corresponding dose is 3.33E +06pfu/mouse/once and 3.33E +05pfu/mouse/once respectively. In the model, one virus Vehicle group (Vehicle group) was set at the same time. After the grouping, the drug was administered by intratumoral administration once every 3 days (

day

0, 3, and 6). NCI-H460 mouse model on 3 consecutive dosing. After the start of dosing, tumor size was measured 2 times per week, tumor changes were monitored, and the relative tumor growth inhibition rate TGI (TGI% = (1-T/C) × 100%; where T/C% is the relative tumor proliferation rate, at a certain time point, the percentage value of the relative tumor volume in the treated and control groups) was calculated.

This example evaluates KOS-hCCL19 for anti-tumor activity in an NCI-H460 human lung carcinoma xenograft tumor animal model. The in vivo anti-tumor effect exerted by the virus is shown in fig. 18. On the NCI-H460 model, at two dose levels of KOS-hCCL19 on day 20 after the start of dosing, the relative tumor suppression rate TGI was 69.44% (p < 0.001), 57.36% (p < 0.05), respectively. Overall, KOS-hCCL19 exhibited significant in vivo anti-tumor activity in the NCI-H460 human lung cancer model after administration.

Example 12 evaluation of neurotoxicity of viruses

Mixing KOS-WT (i.e. "KOS" orKOS-ATCC ") virus, KOS-hCCL19 virus respectively setting 5 different virus gradients, injecting the virus with certain concentration gradient into the intracranial (20 mu L/mouse) of female BALB/c mice respectively, observing the survival condition of animal animals, and calculating the half lethal dose LD of different viruses ₅₀ . Wherein, the virus dosage of each group corresponding to the KOS-WT virus is 5.00E +06pfu/mouse, 8.33E +05pfu/mouse, 1.39E +05pfu/mouse, 2.31E +04pfu/mouse, 3.86E +03pfu/mouse; the dosage of the virus corresponding to KOS-hCCL19 virus in each group is 4.64E +06pfu/mouse, 1.55E +06pfu/mouse, 5.16E +05pfu/mouse, 1.72E +05pfu/mouse and 5.73E +04pfu/mouse. A group of 1 viral Vehicle (Vehicle group) was also set up, with 6 animals per group. After the experiment is finished, the LD is carried out through SPSS-Probit ₅₀ And (4) calculating.

This example evaluates the neurotoxicity of wild-type KOS-WT strain and KOS-hCCL19 virus and counts the survival of each group of experimental animals within 14 days of dosing. As a result of the experiment, as shown in FIG. 19, no death of animals was observed in the Vehicle group, the numbers of deaths of animals in the KOS-WT virus group from the high to low groups were 6, 4, and 0, respectively, and the numbers of deaths of animals in the KOS-hCCL19 virus group from the high to low groups were 0, 2,1, 0, and 0, respectively. Calculating half lethal dose LD of different viruses according to animal death conditions under different administration gradients of the viruses ₅₀ . KOS-WT Virus, LD corresponding to KOS-hCCL19 Virus ₅₀ Are respectively 5.403E +, 04pfu,>4.64E+06pfu. Compared with a wild type KOS-WT strain, the KOS-hCCL19 virus obtained through modification has obvious reduction of neurotoxicity. Of the 3 viruses, the KOS-hCCL19 virus has the highest median lethality and the lowest toxicity, and the KOS-hCCL19 virus has an attenuation effect of more than 100 times that of the KOS-WT virus.

EXAMPLE 13 Effect of insertion site of Gene of interest on anti-tumor Activity of viruses

This example investigates the differences in anti-tumor activity of viruses with different hCCL19 insertion sites, wherein the viruses tested were

KOS-. DELTA.47-. DELTA.ICP 34.5-. DELTA.TK-hCCL 19, KOS-. DELTA.47-. DELTA.ICP 34.5-hCCL19, wherein KOS-. DELTA.47-. DELTA.ICP 34.5-. DELTA.TK-hCCL 19 was inserted at TK and KOS-. DELTA.47-. DELTA.ICP 34.5-hCCL19 was inserted at ICP34.5 (i.e., the recombinant virus of the present application, KOS-. DELTA.47-hCCL 19 or KOS-hCCL 19), and 1 virus Vehicle group (Vehicle group) was set as a control to obtain the target virus in accordance with examples 1 to 7. The specific experimental procedures were as follows:

a20 cells are subcutaneously inoculated on the left side and the right side of a mouse to establish a murine B cell lymphoma subcutaneous transplantation tumor model, and when the sizes of tumors on the left side and the right side are 100mm ³ At the left and right, the 3 viruses are administered in groups, and the dosage of each virus is 8.0E +07pfu/mouse/once; 8 animals per group; the right tumors were administered intratumorally on

days

0, 3,6, and 9, respectively, for a total of 4 doses. After the start of the administration, tumor size was measured every 3 days, tumor changes were monitored, and the relative tumor inhibition rate TGI (TGI% = (1-T/C) × 100%; where T/C% is the relative tumor proliferation rate, at a certain time point, the percentage value of the treated group and vehicle control group relative to the tumor volume) was calculated.

This example evaluated the antitumor activity of KOS-. DELTA.47-. DELTA.ICP 34.5-. DELTA.0 TK-hCCL19, KOS-. DELTA.147-. DELTA. 2ICP34.5-hCCL19 in an A20 xenograft animal model. The in vivo antitumor effect exerted by the virus is shown in fig. 20 (administration side) and fig. 21 (non-administration side). The results show that KOS- Δ 347- Δ 4ICP34.5- Δ TK-hCCL19, KOS- Δ 47- Δ ICP34.5-hCCL19 all significantly inhibited tumor growth in the right side of a20 tumor-bearing mice (TGI 64.24%,94.40%, respectively) by day 12 after dosing under the dosing regimen, and were statistically significant compared to the vehicle control group (p < 0.05). The left tumors, KOS-. DELTA.47-. DELTA.ICP 34.5-. DELTA.TK-hCCL 19 and KOS-. DELTA.47-. DELTA.ICP 34.5-hCCL19, which were not injected with intratumoral administration, also showed some degree of tumor growth inhibition (14.86%, 50.28% of TGI, respectively), but were not statistically different from each other in comparison with the photograph (p.p.<0.05). KOS-DELTA 47-DELTA ICP34.5-hCCL19 showed relatively good tumor suppression effect compared with KOS-DELTA 47-DELTA ICP 34.5-DELTA TK-hCCL19 on both the administration side and the non-administration side. By day 28 after dosing, 0 and 3 bilateral tumors of each mouse were cured in the groups KOS-Delta 47-Delta ICP 34.5-Delta TK-hCCL19 and KOS-Delta 47-Delta ICP34.5-hCCL19 (TV =0 mm), respectively ³ ) (ii) a Data display of the overall experimentKOS-delta 47-delta ICP 34.5-delta TK-hCCL19 and KOS-delta 47-delta ICP34.5-hCCL19 can obviously inhibit the tumor growth on the administration side of an A20 model under the administration dosage of 1.6E + 08pfu/mouse. At the same time, on the non-administration side, although there was no statistical difference compared with the control group, both test drugs also showed a certain tumor suppression effect. Compared with KOS-delta 47-delta ICP34.5-hCCL19 in terms of anti-tumor effect

KOS-Delta 47-Delta ICP 34.5-Delta TK-hCCL19 showed better tumor killing activity, indicating that different insertion sites influence the anti-tumor activity of the virus. The tumor inhibition of the non-administration side, KOS-delta 47-delta ICP 34.5-delta TK-hCCL19 and KOS-delta 47-delta ICP34.5-hCCL19, can generate certain remote anti-tumor activity after administration.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.

Claims

1. An HSV viral vector having silenced ICP47 and ICP34.5 genes and carrying a CCL19 gene.

2. The viral vector according to claim 1, characterized in that it carries two copies of the CCL19 gene.

3. The viral vector according to claim 1, wherein the CCL19 gene has a 6.a > -c mutation.

4. The viral vector according to claim 1, wherein the ICP34.5 gene silencing is achieved by knocking out nucleotides 135-723 of the ICP34.5 gene;

optionally, the ICP47 gene silencing is achieved by knockout of nucleotides 3 to 266 of the ICP47 gene.

5. The viral vector according to any one of claims 1 to 4, wherein the CCL19 gene is located between nucleotide 134 and nucleotide 724 of the ICP34.5 gene.

6. The viral vector of claim 5, further comprising CMV and polyA;

optionally, the CMV is operably linked to the CCL19 gene;

optionally, the polyA is disposed between the 3' terminal nucleotide of the CCL19 gene and nucleotide 134 of the ICP34.5 gene.

7. The HSV viral vector according to claim 1, having the nucleotide sequence shown in SEQ ID NO. 5.

8. An oncolytic virus carrying the HSV viral vector of any one of claims 1-7.

9. The oncolytic virus of claim 8, wherein the oncolytic virus is HSV-1;

optionally, the HSV-1 comprises at least one selected from the group consisting of a F strain, an HF strain, a KOS strain, a HrR strain, and a 17 strain.

10. A pharmaceutical composition comprising the HSV viral vector of any one of claims 1-7 or the oncolytic virus of claim 8 or 9.

11. The pharmaceutical composition of claim 10, wherein said HSV viral vector or oncolytic virus comprises 10^5 to 10^12pfu per unit dose of said pharmaceutical composition.

12. The pharmaceutical composition of claim 10 or 11, further comprising a pharmaceutically acceptable carrier.

13. Use of the HSV viral vector of any one of claims 1-7, the oncolytic virus of claim 8 or 9 or the pharmaceutical composition of any one of claims 10-12 for the manufacture of a medicament for the treatment or prevention of a tumor.

14. The use according to claim 13, wherein the tumor comprises at least one selected from the group consisting of lung cancer, liver cancer, pharyngeal squamous carcinoma, colon cancer, osteosarcoma, ovarian cancer, prostate cancer, glioma, melanoma, colorectal cancer, esophageal cancer, pancreatic cancer.