CN116144607A - Oncolytic virus and application thereof - Google Patents
Oncolytic virus and application thereof Download PDFInfo
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- CN116144607A CN116144607A CN202111382004.7A CN202111382004A CN116144607A CN 116144607 A CN116144607 A CN 116144607A CN 202111382004 A CN202111382004 A CN 202111382004A CN 116144607 A CN116144607 A CN 116144607A
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Abstract
The present invention relates to an oncolytic virus, which is a mutant herpes virus obtained by inactivating the US3 gene and/or TK gene in the wild type herpesvirus genome, which is an animal herpesvirus but not HSV, which is non-pathogenic to humans, capable of replication in tumor cells or tumor cell lines of human origin but not in normal cells or cell lines of human origin, and to the use thereof. Compared with wild type herpesvirus, the modified mutant herpesvirus has the advantages of enhanced replication efficiency in human tumor cells, enhanced capability of inducing human tumor cells to generate cytopathy, enhanced killing power to human tumor cells and enhanced capability of inducing natural immune response; can be used as an effective active ingredient for cracking tumor cells and can also be used as an expression vector and/or a delivery vector of tumor therapeutic active molecules.
Description
Technical Field
The invention belongs to the field of biological pharmacy, relates to an oncolytic virus, a preparation method and application thereof, and in particular relates to a mutant herpes virus modified by genetic modification and application thereof in tumor treatment.
Background
In recent years, the incidence and mortality rate of malignant tumors have rapidly risen, and have become one of the major diseases threatening human health. The global latest cancer burden data in 2020 shows that 1929 thousands of new cancer cases and 996 thousands of death cases are worldwide in 2020. Wherein 457 ten thousand people of new cancer in China occupy 23.7% of the world, and 300 ten thousand people of cancer death occupy 30% of the total cancer death. Cancer new onset and death people in China far exceed those in other countries of the world. Tumors have become the first killer in non-infectious diseases that lead to human death.
Tumorigenesis is a multi-step process involving multiple point mutations of the genome. Tumor treatment requires multiple schemes of combined treatment, and surgery, radiotherapy and chemotherapy are the most main treatment methods for tumors at present. However, the radiotherapy and chemotherapy have obvious toxic and side effects and limited effects, and are easy to cause tumor tolerance. Oncolytic virus treatment is a new treatment means, namely, tumor cells are specifically transfected by genetically modified live viruses, and the oncolytic virus treatment is nontoxic to normal tissues, so that the purpose of eliminating tumors is achieved. The anti-tumor mechanism of oncolytic viruses mainly comes from two aspects, namely, directly cracking tumor cells through infection, replicating progeny viruses and infecting surrounding malignant tumor cells, and secondly, stimulating the innate immunity and specific anti-tumor immunity of organisms. The mechanism by which oncolytic viruses kill tumor cells is different from chemoradiotherapy, so that oncolytic viruses may produce synergistic killing effects in combination with conventional treatment regimens (Liu S, dai M, you L, zhao Y.Advance in herpes simplex viruses for cancer therapy. Sci China Life Sci.2013;56 (4): 298-305.). The oncolytic viruses which are studied more at present are adenovirus, herpes simplex virus, measles virus, vaccinia virus, newcastle disease virus, vesicular stomatitis virus, parvovirus, reovirus and the like. There are various oncolytic viruses on the world, such as adenovirus H101 against nasopharyngeal carcinoma (An Kerui) which has been marketed in china, herpes simplex virus Imlygic (talimogenelaherparepevec) against melanoma, which is approved by the FDA in the united states, and the like.
Herpes simplex virus (HSV-1) is the first virus to be used for the treatment of tumors. The viral genome is easy to manipulate and can carry exogenous genes. Furthermore, many studies have shown that HSV in combination with radiotherapy or chemotherapy can produce synergistic killing. In 2015, T-VECs carrying human macrophage colony-stimulating factor (GM-CSF) with HSV-1 were approved by the U.S. food and drug administration and European drug administration for the treatment of melanoma. Oncolytic viruses can convert non-immunogenic 'cold' tumors into 'hot' tumors with immunogenicity, and can trigger immune cells and tumor-specific lymphocyte aggregation, thereby showing better curative effects. However, oncolytic viruses present a number of challenges during monotherapy or in combination with immunotherapy, limiting their successful use, especially in systemic administration, such as liver isolation, neutralization of blood neutralizing antibodies, physical barriers to infection and rapid clearance of the immune system (Moreno R.Mesenchyal stem cells and oncolytic viruses: joining forces against cancer.J Immunother cancer.2021;9 (2)).
The porcine herpes virus (SuidHerpesvirus 1, suHV-1) is a member of the genus varicella of the subfamily alpha herpes virus of the family herpesviridae, is a linear double stranded DNA virus and has a genome size of about 143kb. SuHV-1 has a wide range of hosts and can infect a wide variety of mammals, but is non-pathogenic to humans. SuHV-1 has the following advantages as an oncolytic virus: 1) Host cells are extensive; 2) The virus titer is high; 3) The capacity of the exogenous gene is large. SuHV-1 also has its unique advantages over other commonly used oncolytic viral vectors. Firstly, suHV-1 does not infect people, so that neutralizing antibodies do not exist in the treatment of human tumors, the rapid elimination of an immune system is weakened, and the curative effect is favorably exerted; secondly, suHV-1 is an animal virus, does not infect people, and has higher safety in treatment. In vivo experiments, suHV-1 infects and kills both mouse and human tumor cells and does not cause damage to the mice, indicating its potential for treating human tumors (Boldogkoi Z, bratincsak A, fodor I.evaluation of pseudorabies virus as a gene transfer vector and an oncolytic agent for human tumor cells. Anti-cancer research.2002;22 (4): 2153-9.). In addition, research shows that the expression of the virus gene is driven by the promoter of the gene HER-2/neu promoter which is highly expressed in bladder cancer, so that the recombinant SuHV-1 virus which can be conditionally replicated in bladder cancer cells is constructed, and has better curative effect on bladder cancer (Shau A-L, lin Y-P, shieh G-S, su C-H, wu W-L, tsai Y-S, et al, development of a conditionally replicating pseudorabies virus for HER-2/neu-overexpressing bladder cancer therapy.molecular therapy: the journal of the American Society of Gene therapy.2007;15 (1): 131-8.).
Although SuHV-1 shows great potential for oncolytic applications, it is not clear how oncolytic herpesviruses interact with the tumor, the immune system of the body, and how tumor cells resist oncolytic virus attacks, as the current results of oncolytic herpesvirus studies still have certain limitations, which are found to work only in a small fraction of patients in clinical trials and specific applications. Therefore, there is an urgent need in the art for herpesviruses that have greater targeting and safety, are capable of directly infecting and lysing tumor cells, and are also useful as expression vectors and delivery vectors for tumor killing active molecules, thereby enhancing anti-tumor effects.
Disclosure of Invention
In view of the above-mentioned drawbacks of the prior art, it is an object of the present invention to provide an oncolytic virus, which is a mutant herpes virus obtained by inactivating the US3 gene and/or TK gene in the wild-type herpesvirus genome, which is not HSV, is non-pathogenic to humans, is capable of replication in tumor cells or tumor cell lines of human origin, but not in normal cells or cell lines of human origin, and uses thereof. The modified mutant herpes virus has increased replication efficiency in human tumor cells, increased capacity to cause cytopathic effects in human tumor cells, increased killing of human tumor cells, and increased capacity to induce a natural immune response compared to wild-type herpes virus; can be used as an effective active ingredient for cracking tumor cells and can also be used as an expression vector and/or a delivery vector of tumor therapeutic active molecules.
The technical scheme adopted by the invention is as follows:
in one aspect, the invention provides an oncolytic virus which is a mutant herpes virus obtained by inactivating the US3 gene and/or TK gene in the genome of a wild-type herpes virus which is not HSV, is non-pathogenic to humans, is capable of replication in tumour cells or tumour cell lines of human origin, but not normal cells or cell lines of human origin.
Further, the oncolytic virus of the present invention is characterized in that the wild-type herpes virus is capable of causing cytopathic effects in, and/or killing, human tumor cells; preferably, the wild type herpes virus is porcine herpes virus1 (SuidHerpesvirus 1, suHV-1).
Further, the oncolytic virus of the present invention is characterized in that said mutant herpesvirus has improved or enhanced properties compared to a wild-type herpesvirus of at least one selected from the group consisting of:
A. replication efficiency in human tumor cells,
B. resulting in the ability of human tumor cells to produce cytopathic effects,
C. killing power of the human tumor cells,
D. the ability to induce a natural immune response,
E. security to the host.
Further, the oncolytic virus of the present invention is characterized in that the mutant herpesvirus further comprises inactivation of one or both of EPO, UL50 genes on the herpesvirus genome.
Further, the present invention is any one of the aforementioned oncolytic viruses, characterized in that the inactivation of the gene in said mutant herpesvirus comprises modification by deletion, insertion, substitution of nucleic acids such that the ability to encode the production of functional proteins is lost.
In a second aspect, the present invention provides a method of preparing an oncolytic virus comprising inactivating the US3 gene in a wild-type herpesvirus genome, said wild-type herpesvirus being non-HSV, non-pathogenic to humans, capable of replication in a tumour cell or tumour cell line of human origin, but not in a normal cell or cell line of human origin.
Further, the method for preparing an oncolytic virus according to the present invention is characterized in that the wild type herpes virus is capable of causing cytopathic effect of human tumor cells and/or killing human tumor cells; preferably, the wild type herpes virus is porcine herpes virus1 (SuidHerpesvirus 1, suHV-1).
Further, the method for preparing an oncolytic virus according to the present invention is characterized in that the mutant herpesvirus has an improved or enhanced performance compared to the wild type herpesvirus of at least one property selected from the group consisting of:
A. replication efficiency in human tumor cells,
B. resulting in the ability of human tumor cells to produce cytopathic effects,
C. killing power of the human tumor cells,
D. the ability to induce a natural immune response,
E. security to the host.
Further, the method for preparing an oncolytic virus according to the present invention is characterized in that the mutant herpesvirus also inactivates one or both of the EPO, UL50 genes on the genome of the wild type herpesvirus.
Further, the method for preparing an oncolytic virus of the present invention is characterized in that the inactivation of a gene in the mutant herpesvirus comprises the loss of the ability to encode a functional protein by deletion, insertion, substitution modification of a nucleic acid; the gene is knocked out, for example, by gene editing techniques such as Crispr/Cas.
In a third aspect, the invention provides a method of enhancing replication efficiency of a herpes virus in a human tumour cell, the ability to cause cytopathic effects in a human tumour cell, the ability to kill a human tumour cell, the ability to induce a innate immune response and/or the safety to a host, comprising inactivating the US3 gene and/or TK gene in the genome of the herpes virus.
Further, the method of the present invention is characterized by further comprising inactivating one or two genes selected from the group consisting of: EPO, UL50.
Further, the method according to any one of the preceding claims, characterized in that the inactivation of the gene comprises the loss of the ability to encode a functional protein by deletion, insertion, substitution modification of the nucleic acid; the gene is knocked out, for example, by gene editing techniques such as Crispr/Cas.
In a fourth aspect, the present invention provides the use of an oncolytic virus according to any one of the preceding claims for the manufacture of a medicament for the treatment of a tumor, wherein the oncolytic virus acts as an active ingredient for lysing tumor cells, inhibiting tumor growth.
Further, the application of the invention is characterized in that the tumor treatment medicine is prepared into injection, nose drops and spray.
Further, the use according to the invention is characterized in that said tumor therapeutic agent is an agent for the treatment of said tumor selected from the group consisting of: melanoma, non-small cell lung cancer, lung adenocarcinoma, esophageal cancer, liver cancer, stomach cancer, kidney cancer, bladder cancer, head and neck tumors, hodgkin's lymphoma, cervical cancer, breast cancer, colorectal cancer, colon cancer, nasopharyngeal cancer, ovarian cancer, prostate cancer, endometrial cancer, glioma, neuroendocrine tumor, malignant mesothelioma, non-hodgkin's lymphoma, merkel cell carcinoma, and solid tumors of all microsatellite highly unstable (MSI-H).
In a fifth aspect, the invention provides the use of an oncolytic virus according to any one of the preceding claims for the manufacture of a medicament for the treatment of a tumor, wherein the oncolytic virus acts as an expression vector and/or a targeted delivery vector for a therapeutically active molecule for a tumor.
Further, the application of the invention is characterized in that the tumor treatment medicine is prepared into injection, nose drops and spray.
Further, the use according to the invention is characterized in that said tumor therapeutic agent is an agent for the treatment of said tumor selected from the group consisting of: melanoma, non-small cell lung cancer, lung adenocarcinoma, esophageal cancer, liver cancer, stomach cancer, kidney cancer, bladder cancer, head and neck tumors, hodgkin's lymphoma, cervical cancer, breast cancer, colorectal cancer, colon cancer, nasopharyngeal cancer, ovarian cancer, prostate cancer, endometrial cancer, glioma, neuroendocrine tumor, malignant mesothelioma, non-hodgkin's lymphoma, merkel cell carcinoma, and solid tumors of all microsatellite highly unstable (MSI-H).
In a sixth aspect, the invention provides the use of an oncolytic virus of any one of the preceding claims in the manufacture of a medicament, wherein the oncolytic virus is used as an immunopotentiator for enhancing a natural immune response.
For a better understanding of the invention, some terms are first defined and other definitions are set forth throughout the detailed description.
As used herein, the term "oncolytic virus" refers to a virus that is capable of infecting, replicating in, causing death, lysis or preventing growth of tumor cells. Preferably, the virus has minimal toxic effects on non-tumor cells.
As used herein, the term "porcine herpes virus" SuidHerpesvirus 1, suHV-1, is a virus of the subfamily alpha herpes virus (Alphaheresvirina), varicella (Varicellovirus). The genome of the virus is linear double-stranded DNA, and the size of the virus is between 130kd and 150 kd. The genome of the virus comprises a unique long region sequence (UL) and a unique short region sequence (US), on both sides of which there are also terminal repeats (TRR) and Internal Repeats (IRS). The genes for SUHV-1 are named according to the region in which they are located and the order of discovery, but may also be named by the proteins they encode. Genes encoding structural proteins include genes such as US2 (28K), US3 (PK), US4 (gG), US6 (gD), US7 (gI), US8 (gE), US9 (11K) and the like in the US region, and genes such as UL9 (OBP), UL27 (gB), UL (gH), UL (TK), UL (gC) capsid protein genes, DNA polymerase genes and the like in the UL region.
As used herein, the term "US3" gene is an important gene for the innate immune response of SuHV-1 against host cells. US3 is able to down-regulate the expression of MHC class I (Major histocompatibility complex I) molecules. In addition, US3 is involved in the IFN-I related signaling pathway, and HSV-1 encoded US3 inhibits IFN alpha/beta production by phosphorylating IRF3 (Interferon regulatory factor 3) and P65 to inhibit their activation. Recent research results in the laboratory show that Bclaf1 is a new regulatory factor of an IFN-I signal channel, plays an important role in IFN-mediated antiviral immunity, and further reveals that alpha herpes virus conserved viral protein US3 escapes from a host natural immune response by degrading Bclaf1, and that US3 also participates in regulating apoptosis. SuHV-1US3 protein kinase has been shown to inhibit apoptosis through PI3K/Akt and NF-. Kappa.B signaling pathways (Chang C-D, lin P-Y, liao M-H, chang C-I, hsu J-L, yu F-L, et al support of apoptosis by pseudorabies virus Us3 protein kinase through the activation of PI3-K/Akt and NF-. Kappa.B pathway in research science.2013;95 (2): 764-74.). Apoptosis of infected cells is significantly promoted after deletion of US3, so that the killing effect of SuHV-1 on tumor cells can be enhanced by deletion of US 3. Furthermore, US3 is also associated with the neurovirulence of SuHV-1. US3 is capable of modulating the level of UL50 phosphorylation, and HSV-1UL50 phosphorylation can modulate viral virulence and genome integrity by compensating for lower dUTPase activity in cells of the central nervous system (Kato A, arii J, koyanagi Y, kawaguchi Y. Phosphor. Of herpes simplex virus 1dUTPase regulates viral virulence and genome integrity by compensating for low cellular dUTPase activity in the central nervous system.Journal of virology.2015;89 (1): 241-8.).
As used herein, the term "TK" gene encodes thymidine kinase, supports viral DNA replication, and plays an important role in virulence and neuroinvasiveness. Furthermore, TK is necessary for SuHV-1 to produce infectious virions in non-dividing cells. Therefore, after TK is deleted, suHV-1 can be enabled to specifically infect and kill malignant tumor cells which are rapidly replicated, normal cells are not damaged, and the targeting of tumor treatment is remarkably improved.
As used herein, the term "UL50" gene encodes dUTPase, and studies have shown that dUTPase active mutants of HSV-1 are capable of significantly reducing their neurotoxicity, neuro-invasiveness and their ability to activate from latency. (Pyles RB, sawtell NM, thompson RL. Herps simplex virus type 1dUTPase mutants are attenuated for neurovirulence,neuroinvasiveness,and reactivation from latency.J Virol.1992Nov;66 (11): 6706-13.) in addition, UL50 is involved in the innate immune response of the anti-host cells of the animal. Recent laboratory studies indicate that SuHV-1 and HSV-1 encoded UL50 degrade IFNAR1 in a manner independent of their dUTP enzymatic activity, thereby inhibiting STAT1 phosphorylation and ISGs expression, and IFN alpha also exhibits a stronger inhibition on UL50 deleted SUHV-1.
As used herein, the term "EP0" refers to the early protein 0 of a herpes virus, which is encoded by the EP0 gene and is a transcriptional activator of early expression of the herpes virus. The amino acid sequence of the EP0 protein is known and can be found, for example, in public databases.
As used herein, the term "oncolytic activity" includes primarily tumor killing activity. When describing the oncolytic activity of a virus, the oncolytic activity of the virus is typically measured by its ability to infect tumor cells, its ability to replicate within tumor cells, and/or its ability to kill tumor cells, among other indicators. The oncolytic activity of a virus can be measured using any method known in the art. For example, the ability of a virus to infect tumor cells can be assessed by measuring the amount of virus required to infect a given percentage of tumor cells (e.g., 50% cells); the ability to replicate in tumor cells can be assessed by measuring the growth of the virus in the tumor cells; the ability to kill tumor cells can be assessed by observing cytopathic effects (CPE) or measuring tumor cell activity.
As used herein, the term "treating" refers to treating or curing a disease (e.g., a tumor), delaying the onset of symptoms of a disease (e.g., a tumor), and/or delaying the progression of a disease (e.g., a tumor).
As used herein, the term "effective amount" refers to an amount that is effective to achieve the intended purpose. For example, a therapeutically effective amount may be an amount effective or sufficient to treat or cure a disease (e.g., a tumor), delay the onset of symptoms of a disease (e.g., a tumor), and/or delay the progression of a disease (e.g., a tumor). Such effective amounts can be readily determined by one of skill in the art or a physician, and can be related to the intended purpose (e.g., treatment), the general health of the subject, the age, sex, weight, severity of the disease to be treated, complications, mode of administration, and the like. Determination of such effective amounts is well within the ability of those skilled in the art.
As used herein, the term "subject" refers to a mammal, such as a primate mammal, e.g., a human. In certain embodiments, the subject (e.g., human) has, or is at risk of having, a tumor.
Compared with the prior art, the technical scheme of the invention has at least the following beneficial effects:
firstly, the invention constructs oncolytic virus by taking SuHV-1 as a framework, discovers that the oncolytic virus has the capacities of replicating in human tumor cells, causing the human tumor cells to generate cytopathy, specifically killing or cracking the human tumor cells and inducing natural immune response, and provides corresponding application on the basis.
Secondly, the invention carries out gene modification and structural transformation on SuHV-1, and particularly modifies US3 gene, TK gene, EP0 gene and UL50 gene on the genome, thereby not only improving the safety, but also not leading to the capability of cracking human tumor cells, thereby providing safer and more effective oncolytic viruses, particularly obviously improving the tumor inhibition capability of TK gene deletion mutant strain and obviously prolonging the survival time of tumor-bearing mice.
Thirdly, the invention researches the immunoregulation function of the SuHV-1 mutant strain, the capability of the genetically modified SuHV-1 mutant strain for stimulating nonspecific immune response is enhanced, and particularly, the deletion of the US3 gene is more obvious for CPE caused by tumor cells, and the capability of stimulating nonspecific immune response can be obviously improved.
Drawings
The above, as well as additional purposes, features, and advantages of exemplary embodiments of the present disclosure will become readily apparent from the following detailed description when read in conjunction with the accompanying drawings. Several embodiments of the present disclosure are illustrated by way of example, and not by way of limitation, in the figures of the accompanying drawings and in which like reference numerals refer to similar or corresponding parts and in which:
fig. 1: killing Activity of herpesvirus against human tumor cells and Normal cells
Infecting a human tumor cell line Hep2 and a human normal cell IRM90 with HSV-1 and porcine herpesvirus respectively; the results show that HSV-1 and the porcine herpesvirus have stronger killing ability to human tumor cells Hep2, but have no killing effect to normal cells. The results show that HSV-1 and the porcine herpesvirus have the activity of specifically killing human tumor cells.
Fig. 2: efficient replication of porcine herpesvirus in human tumor cell line Hep2 and porcine PK15 cell lines
Infecting human tumor cells Hep2 and pig kidney cells PK15 with HSV-1 and pig herpesvirus respectively; virus titers were measured at different time points and, similar to SUHV-1 infection of porcine kidney cells PK15, SUHV-1 replicated efficiently in Hep 2.
Fig. 3: westernBlot detection of SuHV-1 deleted strains
The results show successful knockdown of target genes on the SuHV-1 genome using the CRISPR/Cas9 system.
Fig. 4: cleavage of human tumor cells by wild strain and mutant deletion strain of porcine herpesvirus
FIG. 4A cleavage of Hep2 and PK15 by a porcine herpesvirus polygene deletion strain
FIG. 4B cleavage of Hep2 and PK15 by a deletion strain of the US3 gene of porcine herpesvirus
Through cell morphology observation after virus infection, both SUHV-1WT and the deletion toxin can crack and infect and kill Hep2 and PD15, and cell death detection shows that compared with WT, the killing capacity of double-deletion strain (delta TK delta US 3) and triple-deletion strain (delta TK delta US3 delta EP 0) on Hep2 is not obviously reduced. The delta US3 deleted strain has enhanced killing ability to both Hep2 and PK 15.
Fig. 5: safety of wild strain and mutant deletion strain of porcine herpesvirus on mice
FIG. 5A mortality of mice from nasal drops of DeltaUS 3 strain and DeltaTK strain
FIG. 5B mortality of mice by subcutaneous injection of DeltaUL 50 strain, deltaTK strain, deltaEP 0 strain
Fig. 6: wild strain and mutant deletion strain of herpesvirus of pig induce natural immune response
The SUHV-1 deletion toxin induced more interferon than WT, indicating a stronger innate immune response. Of these, the DeltaUS 3 strain induced the highest level of IFN- β at 8h of infection.
Fig. 7: growth inhibition of mouse tumor by pig herpesvirus single gene deletion strain
The three times of intratumoral injection treatment on the subcutaneous melanoma of the mice by using different pig herpesvirus monogenic deletion strains respectively has the effect of inhibiting the tumor growth, and has more obvious effects of inhibiting the tumor and prolonging the survival time of the mice compared with the delta TK strain treatment group.
Fig. 8: treatment effect of pig herpesvirus deletion strain on mouse tumor
FIG. 8A effect of DeltaTK DeltaUS 3 strain, deltaEP 0 DeltaUS 3 strain, deltaUS 3 DeltaUL 50 strain, deltaTK strain treatment on tumor volume of tumor-bearing mice
FIG. 8B effect of DeltaTK DeltaUS 3 strain, deltaEP 0 DeltaUS 3 strain, deltaUS 3 DeltaUL 50 strain, deltaTK strain treatment on survival of tumor-bearing mice
Compared with the double gene deletion strains of different porcine herpesviruses and TK single gene deletion strains, the three times of intratumoral injection treatment on the subcutaneous melanoma of the mice has the effect of inhibiting the tumor growth, and the effect of inhibiting the tumor of a treatment group is more obvious compared with the TK single gene deletion strain.
Fig. 9: treatment effect of pig herpesvirus deletion strains delta TK and delta EP0 on mouse tumor
FIG. 9A effect of treatment with DeltaTK strain and DeltaEP 0 strain on tumor volume in tumor-bearing mice
FIG. 9B effect of DeltaTK strain and DeltaEP 0 strain on the survival of tumor-bearing mice
The porcine herpesvirus EP0 deletion strain and the TK deletion strain are used for carrying out three times of intratumoral injection treatment on the subcutaneous melanoma of the mice, and both deletion strains have the effect of inhibiting the growth of tumors, and the effect of TK deletion strain treatment group tumor inhibition and the effect of prolonging the survival period of the mice are more obvious.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be further described in detail with reference to the accompanying drawings.
Example 1: suHV-1 targeted killing of human tumor cells
In order to detect the targeting of SuHV-1 to tumor cell killing, human hepatoma cells Hep2 and normal cells IRM90 were cultured in 12-well plates, and when the cell density reached 80%, hep2 and IRM92 cells were infected with 1MOI human herpes simplex virus HSV-1 and porcine herpes virus, respectively. The cells were inoculated at different time points, re-counted before inoculation, MOI was determined, and then cells were harvested at the same time points (samples were taken 0, 72, 96 hours after inoculation). 500g was centrifuged for 5min, the supernatant was discarded, the cells were resuspended in PBS, and 500g was centrifuged for 5min, the supernatant was discarded. The cell death of each group was analyzed by flow cytometry using PI staining.
As a result, as shown in FIG. 1, after HSV-1 and SuHV-1 infection of Hep2 cells, a large number of cells died; and only a small number of cells die after infection with IRM92 cells. The above results demonstrate that SuHV-1 can infect human tumor cells and induce cell death.
Example 2: suHV-1 replication in human tumor cells
Human hepatoma cells Hep2 and porcine kidney cells PK15 were cultured in 12-well plates, and when the cell density reached 80%, the cell counts, HSV-1 and SuHV-1, infected the Hep2 and PK15 cells at 1MOI, respectively. Cells and supernatants were harvested at 6, 12, 24, and 36 hours, respectively, and after 3 repeated freeze thawing, virus titers were determined by plaque assay, as shown in FIG. 2, and the results indicated that SuHV-1 was able to replicate efficiently on human tumor cells as HSV-1.
Example 3: construction of SuHV-1 Gene-deleted Virus Strain Using CRISPR/Cas9 System
The sgRNA is designed for the US3 gene, the TK gene, the EP0 gene and the UL50 gene respectively, and the Puc19-sgRNA vector is constructed after annealing. With a two plasmid system, one plasmid expresses Cas9 protein and the other plasmid is transcribed to produce sgRNA. The extracted viral genome and CRISPR/Cas9 system were co-transfected for viral rescue. The synthesis of sgRNA is designed by selecting two targets for each target gene, PK15 cells are co-transfected with a plasmid encoding Cas9, 2 plasmids for transcription of the sgRNA and a SuHV-1 genome, and after the cells are diseased, DNA is extracted from the cells. PCR amplification was performed using the identified primers and the amplified fragments were sequenced to verify the deletion of the target gene. For the target deletion strain successfully constructed by identification, hep2 cells were infected according to the method of example 2, and the virus was harvested 6 hours and 12 hours after infection, and WesternBlot detection verification (virus protein was detected using capsid protein VP5 antibody, EP0 antibody, US3 antibody, TK antibody; mock is a blank control, UV is an inactivated virus without replication function) was performed as shown in FIG. 3, and the results of FIG. 3 showed successful knockout of the target gene on the SuHV-1 genome using CRISPR/Cas9 system. The replication capacity of different deletion viruses can be detected by infecting Hep2 cells with different deletion viruses of single genes, and the expression of viral proteins can be detected within 6-12 hours, which shows that the deletion of US3 gene, TK gene, EP0 gene and UL50 gene does not influence the replication of SuHV-1 in tumor cells.
Example 4: cleavage and killing of SuHV-1 Gene-deleted Strain on human tumor cells
The SuHV-1WT and US3 deleted strains were infected with Hep2 and PK15 cells, respectively, and cytopathic effects of different treatment groups were observed after 48 hours, and both the SuHV-1WT and US3 deleted strains were able to lyse and kill Hep2 by cell morphology observation after virus infection (FIG. 4B).
The effect of double (Δtk Δul 50), triple (Δtk Δul50 Δep 0) on the killing capacity of Hep2 was examined according to the method of flow cytometry for cell death in example 1 (fig. 4A). The results show that compared with the WT, the killing ability of double-deficiency and triple-deficiency viruses on the Hep2 is not obviously reduced.
Example 5: safety of SuHV-1 Gene-deleted Strain on mice
To test the safety of different deletion toxins on mice, SUHV-1WT, deltaUS 3, deltaTK was tested at 5X10 5 TCID50 drops of nasal-challenge BALB/c mice, 6 per group. WT and Δus3 groups all died at 4dpi, 10dpi mice, respectively, while Δtk group mice died within 60dpi (fig. 5A). The result shows that the delta TK has higher safety on mice.
The safety of the different missing toxins of SUHV-1 was then further verified by subcutaneous injection. SUHV-1WT, deltaUL 50, deltaTK, deltaEP 0 at 5x10 5 TCID 50 Subcutaneous injection to combat toxicity, WT and Δul50 groups died all at 9dpi, 25dpi, respectively, while Δtk and Δep0 groups did not die within 40dpi (fig. 5B). The above results indicate that ΔTKand ΔEP0 have higher safety to mice.
Example 6: suHV-1 gene deletion strain can induce stronger natural immune response
Hep2 cells were cultured in 12-well plates and when the cell density reached 80%, hep2 cells were infected with different monogenic deleted SUHV-1 at1 MOI. Cells were harvested at 4 hours and 8 hours, total cellular RNA was extracted, and the expression level of IFN- β was detected by quantitative PCR (FIG. 6). The results indicate that SUHV-1 deleted toxin induces more interferon than WT, indicating that it induces a stronger innate immune response, particularly that US3 deleted toxin induces higher levels of IFN- β at 8h post-infection.
Example 7: tumor inhibition test of tumor-bearing mice
5X10 subcutaneous injections were administered ventrally to C57 mice 5 B16-F10 cells (100 ul in volume) reached a tumor volume of 100mm after approximately one week 3 Intratumoral injection of experimental group 1×10 7 TCID 50 The virus, control group was intratumorally injected with 100ul of PK15 cell culture broth, once every other day for three times. The size of tumor diameter was measured and recorded every 2/3 days and was expressed as 0.5 x length x (width) 2 To calculate and record tumor volumes.
Three SUHV-1 single gene deletion strains DeltaUS 3, deltaTK and DeltaUL 50 have inhibition effect (figure 7) on three times of intratumoral injection treatment on mice subcutaneous melanoma, and the effect of DeltaTK treatment group tumor inhibition (figure 7A) and mice survival prolongation (figure 7B) is more obvious.
Three SUHV-1 double-gene deletion strains and delta TK strains have inhibition effect on mice subcutaneous melanoma by three intratumoral injection treatments (figure 8), and compared with the delta TK treatment group, the tumor inhibition effect (figure 8A) is more obvious.
The SUHV-1 DeltaEP 0 strain and DeltaTK strain have inhibition effect on mice subcutaneous melanoma by three times of intratumoral injection treatment (figure 9), and compared with the DeltaTK strain, the treatment group has more obvious effects on tumor inhibition (figure 9A) and mice survival prolongation (figure 9B).
The foregoing description of the preferred embodiments of the present invention has been presented for purposes of clarity and understanding, and is not intended to limit the invention to the particular embodiments disclosed, but is intended to cover all modifications, alternatives, and improvements within the spirit and scope of the invention as outlined by the appended claims.
Claims (18)
1. An oncolytic virus which is a mutant herpes virus obtained by inactivating the US3 gene and/or TK gene in the genome of a wild type herpes virus which is an animal herpes virus, but not HSV, which is non-pathogenic to humans, capable of replication in tumour cells or tumour cell lines of human origin but not normal cells or cell lines of human origin.
2. Oncolytic virus according to claim 1, characterized in that the wild type herpes virus is capable of causing cytopathic effect of human tumour cells and/or killing human tumour cells; preferably, the wild type herpes virus is porcine herpes virus1 (Suid Herpesvirus1, suHV-1).
3. Oncolytic virus according to claim 1, characterized in that said mutant herpesvirus has an improved or enhanced performance compared to the wild-type herpesvirus of at least one property selected from the group consisting of:
A. replication efficiency in human tumor cells,
B. resulting in the ability of human tumor cells to produce cytopathic effects,
C. killing power of the human tumor cells,
D. the ability to induce a natural immune response,
E. security to the host.
4. The oncolytic virus of claim 1, wherein said mutant herpesvirus further comprises inactivation of one or both of EPO, UL50 genes on the herpesvirus genome.
5. Oncolytic virus according to any one of claims 1-4, wherein the inactivation of a gene in said mutant herpesvirus comprises a loss of the ability to encode a functional protein by deletion, insertion, substitution modification of a nucleic acid.
6. A method of preparing an oncolytic virus comprising inactivating the US3 gene in a wild-type herpesvirus genome, said wild-type herpesvirus being non-HSV, non-pathogenic to humans, capable of replication in a tumor cell or tumor cell line of human origin, but not in a normal cell or cell line of human origin.
7. The method of preparing an oncolytic virus of claim 6, wherein the wild-type herpes virus is capable of causing cytopathic effects in, and/or killing, human tumor cells; preferably, the wild type herpes virus is porcine herpes virus1 (Suid Herpesvirus1, suHV-1).
8. The method of preparing an oncolytic virus of claim 6, wherein said mutant herpesvirus has improved or enhanced properties compared to a wild-type herpesvirus of at least one member selected from the group consisting of:
A. replication efficiency in human tumor cells,
B. resulting in the ability of human tumor cells to produce cytopathic effects,
C. killing power of the human tumor cells,
D. the ability to induce a natural immune response,
E. security to the host.
9. The method of claim 6, wherein the mutant herpes virus further inactivates one or both of EPO, UL50 genes on the wild type herpes virus genome.
10. The method of preparing an oncolytic virus according to any one of claims 6-9, wherein the inactivation of a gene in said mutant herpesvirus comprises the loss of the ability to encode a functional protein by deletion, insertion, substitution modification of a nucleic acid; the gene is knocked out, for example, by gene editing techniques such as Crispr/Cas.
11. A method of enhancing the replication efficiency of a herpes virus in a human tumour cell, the ability to cause cytopathic effects in a human tumour cell, the ability to kill a human tumour cell, the ability to induce a innate immune response and/or the safety to a host, comprising inactivating the US3 gene and/or TK gene in the genome of the herpes virus.
12. The method of claim 11, further comprising inactivating one or both genes selected from the group consisting of: EPO, UL50.
13. The method of claim 11 or 12, wherein the inactivation of the gene comprises the loss of the ability to encode a functional protein by deletion, insertion, substitution modification of the nucleic acid; the gene is knocked out, for example, by gene editing techniques such as Crispr/Cas.
14. Use of the oncolytic virus of any one of claims 1-6 for the manufacture of a medicament for the treatment of tumors, wherein the oncolytic virus acts as an active ingredient for lysing tumor cells, inhibiting tumor growth.
15. Use of an oncolytic virus according to any one of claims 1-6 for the manufacture of a medicament for the treatment of a tumor, wherein the oncolytic virus is used as an expression vector and/or a targeted delivery vector for a therapeutically active molecule of a tumor.
16. Use of an oncolytic virus according to any one of claims 1-6 for the manufacture of a medicament for the treatment of a tumor, wherein the oncolytic virus is used as an immunopotentiator for enhancing the natural immune response of a tumor patient.
17. The use according to claim 14 or 15, characterized in that the tumor therapeutic agent is formulated as an injection, nasal drops, spray.
18. The use according to claim 14 or 15, characterized in that the tumor therapeutic agent is an agent for treating a tumor selected from the group consisting of: melanoma, non-small cell lung cancer, lung adenocarcinoma, esophageal cancer, liver cancer, stomach cancer, kidney cancer, bladder cancer, head and neck tumors, hodgkin's lymphoma, cervical cancer, breast cancer, colorectal cancer, colon cancer, nasopharyngeal cancer, ovarian cancer, prostate cancer, endometrial cancer, glioma, neuroendocrine tumor, malignant mesothelioma, non-hodgkin's lymphoma, merkel cell carcinoma, and solid tumors of all microsatellite highly unstable (MSI-H).
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