CN102191224A - Heavy targeted modification method for herpes simplex viruses and application thereof - Google Patents

Abstract

The invention belongs to the technical field of medicinal biotechnology and in particular relates to a heavy targeted modification method for herpes simplex viruses (HSV) and application thereof. In the method, surface glycoprotein of the HSV is chemically modified by using an allogenic targeted substance, particularly pegylation folic acid, so that the natural cell tropism is deprived and specifically transduced into tumor cells or tissues and does not infect other normal cells or tissues. By the method, the capacity of the HSV of specifically infecting the tumor cells can be effectively improved; immunoreaction and in vivo neutralization of antibodies are avoided; toxic or side effect is reduced; and safety is improved.

Description

A method for retargeting modification of herpes simplex virus and its application

technical field

The invention belongs to the technical field of medicine and biology, and in particular relates to a retargeting modification method of herpes simplex virus, which can effectively improve the ability of herpes simplex virus to specifically infect tumor cells, avoid immune reactions and neutralizing antibodies in vivo, and reduce Toxic and side effects, improve safety.

Background technique

Herpes simplex virus (HSV) is an enveloped double-stranded DNA virus, usually divided into type I (HSV-1) and type II (HSV-2) according to the difference in antigenicity. Type I herpes disease is mainly transmitted through close contact with the respiratory tract, skin and mucous membranes, and infects the skin mucous membranes and organs above the waist. Type II herpes virus mainly exists in women's cervix, vagina, vulva skin and men's penis, urethra, etc. It is the chief culprit that causes genital inflammation and herpes. Type I mainly causes infections of the skin, mucous membranes (oral mucosa) and organs (brain) other than the genitals, and type II mainly causes infections of the skin and mucous membranes of the genital area. The virus enters the body through the respiratory tract, oral cavity, genital mucous membrane and damaged skin, and resides in the normal mucous membrane, blood, saliva and sensory ganglion cells of the human body. When the body's resistance declines, such as fever, gastrointestinal dysfunction, menstruation, pregnancy, focal infection, and emotional changes, the latent HSV in the body is activated to cause the disease. Humans are the only natural host of herpes simplex virus, mainly through direct contact.

At present, HSV-1 has become an important tumor gene therapy virus carrier and oncolytic virus, the main reasons are: ① HSV-1 is an enveloped double-stranded DNA virus with a genome length of 152kb, which has a large capacity and can insert large foreign fragments , because half of the genes encoded by the HSV-1 genome are non-essential genes, which can be replaced by exogenous therapeutic genes; ② HSV-1 has a wide range of host cells, can infect quiescent and non-quiescent cells, and has high infection efficiency. Even when the multiplicity of infection (MOI) is very low, HSV can still infect about 70% of the cell population in vitro; ③ in infected host cells, the entire replication cycle process of HSV-1 can be completed within 20 hours, releasing Thousands of progeny virus particles are produced; ④ HSV-1 virus particles can not only carry out direct cell-cell transmission through cell membrane fusion, but also spread through extracellular space (extracellular space), which is especially useful for oncolytic viruses, because A low dose of virus can achieve efficient viral penetration (Viral Penetration) in solid tumors; ⑤ There are a variety of anti-HSV-1 drugs (such as acyclovir, famciclovir, etc.) clinically used to treat HSV-1 infection , and HSV-1 expresses the anti-tumor suicide gene - thymidine kinase (HSV thymidine kinasa, HSV-TK), which provides a safe mechanism for the anti-tumor effect of HSV-TK, because drugs can be used to shut down the replication of HSV-1 circulation; ⑥ HSV-1 can effectively infect a variety of experimental animals, which is conducive to the establishment of animal models and the translation of preclinical research results into clinical trials. Therefore, there are currently nearly a hundred programs related to HSV that have entered various phases of clinical research around the world.

However, like any other virus, HSV-1 has a wide range of host cells and does not exclusively infect tumor cells. How to specifically introduce oncolytic HSV into tumor cells in vitro, that is, cell transduction targeting (Cell Transduction Targeting), is an urgent problem to be solved in HSV-1-mediated tumor gene therapy and oncolytic HSV virus therapy. The current solution mainly relies on intratumoral injection, but this method has relatively large clinical limitations, especially for patients whose tumors have spread. Furthermore, oncolytic HSV also has strong immunogenicity, which is also a serious challenge for repeated administration. Therefore, it is necessary to carry out targeted modification of cell transduction on HSV-1, that is, re-targeting (Re-targeting).

Receptor-mediated targeted drug delivery is an important approach for targeted therapy. Since it was discovered in 1986 that folic acid (folate, FA) enters cells through the receptor-mediated endocytic pathway, the folate receptor (Folate Receptor, FR)-mediated targeted drug delivery system has made great progress. Compared with other receptor-mediated gene delivery methods, the folate receptor system has its unique advantages: ① FR is only highly expressed on the surface of tumor cell membranes, but almost not expressed in most normal tissues, so it has a good tumor Tissue-specific; ②The ligand of FR is folic acid, and FA is an essential vitamin for the human body, but the human body cannot synthesize FA, so exogenous FA needs to be ingested. Folic acid is easy to combine with other molecules through the carboxyl group without reducing its affinity; in addition, folic acid also has low immunogenicity, easy modification, small size, high chemical stability and biological stability, and physiological compatibility with organic solvents. Capacitance, low cost and other advantages; ③ FA has high affinity with FR, and through FR-mediated endocytosis, it can effectively take folic acid and folic acid conjugates bound to FR into cells. At present, folate receptor-mediated targeted drug delivery systems are widely used in gene drugs, protein drugs, chemotherapy drugs, toxin drugs, radiopharmaceuticals, monoclonal antibodies, etc., and have made good progress.

Usually, folic acid receptor-mediated gene drug delivery requires chemical cross-linking of ligands and cationic polymers, and then the gene drug is electrostatically combined with the conjugate to form a ligand-cation polymer-gene drug complex , through the action of receptor-ligand, gene medicine is introduced into target cells. In order to improve the efficiency of receptor-mediated gene transfer, polyethylene glycol (PEG) is usually used as a linker to more effectively present and mediate the combination of folic acid and folic acid receptors on the surface of tumor cells.

Generally, the polyethylene glycol modification of drugs, namely PEGylation, is to chemically couple activated polyethylene glycol to proteins, polypeptides, small molecule organic drugs, liposomes, etc. Since polyethylene glycol was first used to modify bovine serum albumin in 1977, pegylation technology has developed rapidly and has become an important means to improve the clinical effects of protein drugs and non-protein drugs. This is because PEG is safe, non-toxic, and has oil-water amphiphilicity. PEG is connected to the drug, which can increase the relative molecular weight of the drug, reduce the enzymatic hydrolysis of the drug, avoid rapid elimination in the metabolism of the kidney, prolong the half-life of the drug, increase the water solubility of the drug, enhance the stability of the drug, and the PEG chain Coating on the surface of the drug can shield the epitope of the drug and reduce the immunogenicity of the drug; at the same time, it changes the molecular structure of the drug to improve the properties of pharmacokinetics and pharmacodynamics, and increase the blood concentration of the drug at the site of action. At present, many PEGylated drugs (such as interferon, adenosine deaminase, doxorubicin, etc.) have been approved by the FDA to enter the market, and there are several PEGylated drugs in preclinical research and clinical trials. There are as many as ten kinds.

In recent years, some studies have begun to try the PEGylation of viruses (such as adenovirus) for gene therapy of tumors. In 1998, Welsh MJ tried PEG-encapsulating adenovirus to evade neutralizing antibodies. Jame M.Wilson et al. PEGylated adenovirus significantly enhanced its transduction efficiency and stability. Michael A.Barry et al. found that PEGylated adenovirus can effectively To reduce the natural immune response of adenovirus, Pastore L. et al. also found that PEGylated adenovirus significantly improved its safety. hinsaku Nakagawal et al. PEGylated adenoviruses with RGD short peptide targeting tumor neovascularization to significantly enhance their cell transduction efficiency and antibody evasion ability, while In Kyung Oh et al. found that folate immobilized (Folate Immobilized) PEGylated adenoviruses could re- Target tumor cells. In addition, Maria A. Croyle et al. also found that PEGylated lentiviral vectors can prevent their inactivation in serum. You-Kyoung Kim et al. also found that PEGylated baculoviral vectors can regulate transduction efficiency both in vivo and in vitro. Hong T. Le et al found that PEGylated adeno-associated virus can be used as a carrier for gene delivery and so on. Improving the ability of the virus to specifically infect tumor cells, while effectively avoiding the body's immune response to HSV-1, reducing toxic side effects, and increasing safety are the focus of researchers in this field.

Contents of the invention

The purpose of the present invention is to provide a new retargeting modification method of herpes simplex virus and its application. The present invention replaces the natural cell tropism of HSV with exogenous targeting substances, so that it can be specifically transduced into tumor cells or tissues without infecting normal cells or tissues, and thus makes HSV effective before reaching tumor cells Avoid clearance of neutralizing antibodies from the body.

Specifically, the retargeting modification method of a herpes simplex virus of the present invention is characterized in that it mainly uses the exogenous targeting substance folic acid-polyethylene glycol nanoparticles (folate-PEGylated Nanoparticles, FPN) to chemically modify Or modify HSV-1, through folic acid receptor exogenous targeting substances to achieve specific import into tumor cells, while preventing HSV-1 from being cleared by the body's immune system before reaching tumor cells.

The retargeted herpes simplex virus in the present invention is mainly selected from type I, followed by type II.

The retargeted herpes simplex virus is mainly recombinant herpes simplex virus, followed by non-recombinant herpes simplex virus (wild type). Wherein, the recombinant herpes simplex virus includes replication defective recombinant herpes simplex virus and conditional replication type oncolytic herpes simplex virus.

In the present invention, the natural cell tropism of the herpes simplex virus is replaced by an exogenous targeting substance, so that the herpes simplex virus is specifically transduced into tumor cells or tissues without infecting normal cells or tissues.

The exogenous targeting substance is mainly folic acid, followed by other tumor cell highly expressed receptor ligand molecules, including but not limited to: epidermal growth factor, vascular endothelial growth factor, estrogen, tumor necrosis factor-related apoptosis inducing ligand.

In the present invention, the combination of the exogenous targeting substance and the herpes simplex virus is mainly through mild chemical coupling, followed by biological and physical methods.

In the present invention, the target cells of the retargeted herpes simplex virus are mainly tumor cells, followed by neovascular endothelial cells of tumors.

The retargeting modification method of herpes simplex virus of the present invention comprises the following steps:

1. Culture and purification of HSV

Type I (HSV-1), or Type II (HSV-2) herpes simplex virus is used, either HSV-1 or HSV-2, including recombinant herpes simplex virus and non-recombinant herpes simplex virus (wild type). Among them, the herpes simplex virus is mainly a recombinant herpes simplex virus, including a replication-deficient recombinant herpes simplex virus and a conditional replication type oncolytic herpes simplex virus (such as MGH1, G207, G47, NV1020, AE618, 7134, etc.).

The culture of herpes simplex virus is preferentially carried out in African green monkey kidney cells (Vero cells) and its derivative cells (such as 2-2, 911, etc.), followed by amplification in 293 cells, CHO cells, Hela cells, etc.

Vero cells were routinely cultured, herpes simplex virus was inoculated according to the multiplicity of infection (MOI) of 0.01, and the culture was continued for 72 hours. The culture supernatant and cells were collected by conventional centrifugation, and the cells were lysed by conventional freeze lysis and the virus was collected by centrifugation. The above viruses were combined and stored in -80°C for purification.

The above preserved virus stock solution was purified by conventional sucrose density gradient centrifugation method, and the obtained purified virus particles were stored at -80°C for subsequent targeted modification.

Repeat the above production and purification process to the required amount of virus, usually the minimum virus amount is 1×10 ¹⁰ virus particles.

2. Synthesis and characterization of FA-PEG and polycation-PEG-FA

In the present invention, the exogenous targeting substance is mainly folic acid, followed by other tumor cell highly expressed receptor ligand molecules, including but not limited to: epidermal growth factor, vascular endothelial growth factor, estrogen, tumor necrosis factor-related apoptosis induction Ligand (TNF-related apoptosis-inducing ligand, TRAIL).

The combination of exogenous targeting substances and HSV-1 is mainly through mild chemical coupling to maintain virus viability. Usually, exogenous targeting substances need to be PEGylated before they can be mildly chemically coupled to HSV-1. In the present invention, folic acid-polyethylene glycol nanoparticles (FPN) are used to chemically modify or modify HSV-1. Therefore first synthesize FA-PEG (its synthesis process and chemical formula structure are shown in Figure 1), then use infrared spectroscopy, ¹ H NMR (Nuclear Magnetic Resonance, nuclear magnetic resonance) and other techniques to measure the structure and purity of the above-mentioned synthesized coupler, it is necessary The matrix-assisted time-of-flight mass spectrometer (MALDI-TOF) technique can be used for further characterization analysis. The FA-PEG conjugate with a purity of more than 95% can be used for subsequent targeted modification of HSV.

The combination of exogenous targeting substances and HSV-1 can also be biological and physical. First, polycation-PEG-FA is synthesized. In the present invention, natural aminopolysaccharide-chitosan (chitosan, CS) is selected as the polycation component. The chemical formula structure of the synthesized CS-PEG-FA is shown in FIG. 2 . The structure and degree of substitution of the synthesized CS-PEG-FA were confirmed by techniques such as infrared spectroscopy, ¹ H NMR and/or MALDI-TOF. The CS-PEG-FA conjugate with a purity of more than 95% can be used for subsequent targeted modification of HSV.

3. FA-PEGylation of herpes simplex virus

On the basis of preparation and purification of herpes simplex virus, the herpes simplex virus was chemically coupled with FA-PEG (Figure 3) and encapsulated with CS-PEG-FA (Figure 4), and determined by the fluorescamine method For the degree of modification of herpes simplex virus, a dynamic light scattering instrument (Dynamic light scattering instrument, DLS) was used to measure the particle size of the modified virus, and a Zeta potential meter was used to measure the surface charge of FA-PEG modified herpes simplex virus. At the same time, the titer of the modified HSV was determined by conventional plaque counting method.

4. In Vitro Cell Experiments

After HSV is modified by PEGylation, its natural cell transduction pathway is blocked. Whether the modified HSV can be taken up by cells and has the transduction specificity of tumor cells. In vitro cell experiments are used to test whether it can effectively reduce HSV immunogenicity.

In the present invention, folate receptor-overexpressed epithelial cancer cells KB and folate receptor-deficient lung cancer cells A549 were cultured and infected with FA-PEG-HSV and PEG-HSV respectively, while unmodified HSV was used as a control. The degree of virus infection is determined by the number of plaques caused by the virus, the cells expressing the virus protein are marked by immunofluorescence cytochemistry, the infected cells are counted by flow cytometry, and the FA-PEGylated HSV is compared with other HSV The similarities and differences between the above cells in infection. The results show that FA-PEGylated HSV (FA-PEG-HSV) can significantly improve the ability to target and transduce tumor cells by 3-10 times.

At the same time, macrophages RAW 264.1 were infected with FA-PEG-HSV, PEG-HSV and unmodified HSV respectively, and the secretion of IL-6 (Interleukin 6, IL-6) was detected by ELISA (macrophages secrete cytokine IL -6 secretion can directly reflect the degree of immune response), and compare the differences. In addition, the direct immune response difference of different viruses can also be indirectly reflected by measuring the DNA content of the HSV genome in infected cells. The total DNA of macrophage RAW 264.1 infected with different viruses was extracted, the HSV-TK gene fragment in the HSV-1 genome was amplified by PCR and quantitative PCR, and the similarities and differences between them were compared by electrophoresis. The results showed that FA-PEGylated HSV (FA-PEG-HSV) can effectively reduce the immunogenicity of herpes simplex virus by 50%-80%.

The experimental techniques and methods involved in the present invention can refer to known techniques in the art, and the obtained virus strains and experimental cell strains can be purchased through commercial channels.

The present invention uses exogenous targeting substances, especially chemically modifying or modifying the surface glycoprotein of herpes simplex virus HSV through pegylated folic acid, so that its natural cell tropism is lost, but is specifically transduced into tumor cells or tissues , No infection to other normal cells or tissues. The application of the method can effectively improve the ability of the herpes simplex virus to specifically infect tumor cells, avoid immune reactions and neutralizing antibodies in vivo, reduce toxic and side effects, and improve safety.

For ease of understanding, the present invention will be described in detail below through specific drawings and embodiments. It should be pointed out that the specific examples and accompanying drawings are only for illustration. Obviously, those skilled in the art can make various amendments and changes within the scope of the present invention according to the description herein. These amendments and Modifications are also included within the scope of the present invention.

Description of drawings

Figure 1 Schematic diagram of the synthesis and chemical formula structure of FA-PEG.

Fig. 2 Schematic diagram of the chemical formula structure of CS-PEG-FA.

Fig. 3 Schematic diagram of FA-PEG chemical coupling modification of HSV.

Fig. 4 Schematic diagram of CS-PEG-FA encapsulation modification of HSV.

Detailed ways

Example 1

Cultivation and purification of HSV-1

Vero cells were inoculated in a cell culture dish with a diameter of 12.5 cm. When the cells grew to 80% full, the cell culture medium was replaced with DMEM medium containing 5% newborn calf serum, and the virus HSV was inoculated with MOI=0.05. Continue culturing for 72 h at 37 °C in an incubator containing 5% _CO2 . The cell culture supernatant and cell debris were collected into sterile cryopreservation tubes and stored at -80°C.

The virus supernatant and cells collected above were repeatedly frozen and thawed three times, so as to fully lyse the cells and release the HSV virus particles in the cells. Put the virus liquid that had been frozen and thawed three times into a centrifuge tube, centrifuge at 5000 rpm at 4°C for 10 minutes, and collect the virus supernatant. Centrifuge at 12000 rpm at 4°C for 30 minutes to further remove cell debris in the virus supernatant. Add 25 ml of the preliminarily purified virus supernatant into a sterile ultracentrifuge tube (30 ml, Beckman Coulter). 5ml of HBSS solution containing 25% sucrose was carefully added to the bottom of the ultracentrifuge tube with a pipette, and ultracentrifuged at 25,000 rpm at 4°C for 4 hours (the centrifuge rotor model is Beckman SW28 rotor). After centrifugation, discard the supernatant and resuspend the HBSS pellet at the bottom of the ultracentrifuge tube with 250 l HBSS. Place the resuspended G207 virus solution on a shaker at 4°C and shake gently overnight. Centrifuge at 3000rpm at 4°C for 10 minutes to further remove cell debris. Part of the purified virus was taken out for titer determination, and the remaining virus was stored at -80°C.

Example 2

Synthesis and Characterization of FA-PEG

Folic acid and NHS form activated ester, that is, folic acid is dissolved in dimethyl sulfoxide (DMSO), reacted with dicyclohexylcarbodiimide (DCC), n-hydroxysuccinamide at a molar ratio of 1:2:2 , stirred for 18 hours, and the whole reaction was carried out under nitrogen protection. The resulting suspension was centrifuged at 15,000 rpm for 30 minutes to remove white insoluble matter and obtain a yellow supernatant.

Dissolve NH ₂ -PEG-COOH with a molar ratio of 1:8 to the above-mentioned folic acid activator in DMSO, react with the folic acid activator for 4 hours under the condition of nitrogen protection, and dissolve the obtained substance in DMSO and ultrapure water at 4 They were dialyzed for 3 days and 2 days respectively under the condition of ℃, and the final product was freeze-dried at -20 ℃.

Take a part of the FA-PEG freeze-dried product (about 5 mg) and dissolve it in deuterated DMSO, and detect it by nuclear magnetic resonance to confirm the reaction and structure.

Example 3

FA-PEGylation of herpes simplex virus

Dissolve methoxy-PEG-NHS (mPEG) in 2ml of HBSS, add HSV (the ratio of mPEG to HSV is 1PFU, add 1× ¹⁰⁷ mPEG molecules to HSV), and react under ice bath for 4 hours. The PEG-FA obtained after the reaction was dialyzed in ultrapure water at 4°C for 2 days, and the final product was stored at -80°C.

Dissolve FA-PEG-COOH in 2ml HBSS, add EDC and NHS for activation, the molar ratio is 1:10:10, react at room temperature for 1 hour, take the activated FA-PEG-COOH and dissolve it in 2ml HBSS, add HSV (The ratio of HSV to FA-PEG-COOH is 1 PFU of HSV added with 1×10 ⁷ FA-PEG-COOH molecules), reacted under ice bath for 4 hours. The PEG-FA obtained after the reaction was dialyzed in ultrapure water at 4°C for 2 days, and the final product FA-PEG-HSV was stored at -80°C.

The purity of FA-PEG-HSV was further detected, specifically: the fluorescein ammonia dye was dissolved in acetone to prepare a 7 mg/mL fluoresceinamide solution. Add 900 l of HSV or FA-PEG-HSV to 100 l of fluorescein dye, mix immediately, and incubate for 15 minutes in the dark. The fluorescence intensity was detected by a spectrophotometer (the excitation wavelength was 390 nm, and the emission wavelength was 475 nm), and a standard curve was drawn with PEG-diamine.

Example 4

Targeted modified HSV virus particle size and its zeta potential detection

HSV, PEG-HSV and FA-PEG-HSV were dissolved in ultrapure water, and the diameters of virus particles in each group were measured with a dynamic light scattering instrument (Dynamic lightscattering instrument, DLS, Malvern Company).

HSV, PEG-HSV and FA-PEG-HSV were dissolved in 2mL buffer HBSS (Hank's Balanced SaltSolution, the composition of which was 0.137M NaCl, 5.4mM KCl, 0.25mM Na ₂ HPO ₄ , 0.44mM KH ₂ PO ₄ , In 1.3mM CaCl ₂ , 1.0mM MgSO ₄ , 4.2mM NaHCO ₃ , pH 7.4), the surface zeta potential of each group of viruses was measured with a laser light scattering instrument (ZetasizerNano System, Malvern Company).

Example 5

Titer detection of HSV virus

Vero cells were seeded into 6-well plates at a density of 3 × ¹⁰⁵ cells/well. After 24 hours of inoculation, when the Vero cells grew to 100% full, the cell culture medium was removed, and the cells were washed once with PBS. Then add HSV virus or PEG-modified HSV virus that has been diluted 10-fold with DMEM medium in advance to each well. Incubate for 2 hours at 37 °C in an incubator containing 5% _CO2 to fully bind the virus to the cells. After 2 hours, use a pipette to suck off the virus that is not combined with the cells, add 2ml of DMEM medium containing 5% newborn calf serum to each well, and continue to cultivate at 37°C in an incubator containing 5% CO ₂ for 68 Hour. After 68 hours, suck off the cell supernatant with a pipette, add 1ml of 10% formaldehyde fixative solution to each well, incubate at room temperature for 30 minutes, suck off the 10% formaldehyde solution, and add 1ml of 10% crystal violet ( Shanghai Sangong) solution - stained Vero cells. After incubating at room temperature for 30 minutes, wash the Vero cells in the 6-well plate with water, count the number of plaques in each well, and calculate the PFU value of the virus according to the following formula.

PFU = number of plaques × virus dilution factor

Example 6

Targeted detection of modified HSV virus

KB and A549 cells were seeded in 24-well plates at 1×10 ⁵ cells/well for routine culture. One day later, the two cell lines were infected with HSV, PEG-HSV and FA-PEG-HSV at MOI=1, cultured at 37°C for 72 hours in an incubator containing 5% _CO2 , and the cells and cell culture medium were collected. Thaw three times to lyse the cells to release the progeny virus particles in the cells, and detect the number of PFU of the progeny after each group of virus infected cells.

Example 7

Determination of immunogenicity of HSV virus

Macrophages RAW 264.1 were seeded in a 6-well plate at a cell density of 1×10 ⁶ cells/well for routine culture. One day later, 2 ml of HSV, PEG-HSV and FA-PEG containing 2×10 ⁸ virus particles were used - Fresh culture medium of HSV was continued for 24 hours. The secreted amount of interleukin 6 (IL-6) in the cell culture was measured by conventional enzyme-linked immunosorbent assay (ELISA). Statistically compare the differences in the expression of IL-2 among the groups, so as to determine its immunogenicity.

Claims (10)

1. A retargeting modification method for herpes simplex virus, characterized in that, using an exogenous targeting substance to chemically modify or modify the herpes simplex virus HSV, and to specifically import tumor cells through the exogenous targeting substance to avoid herpes simplex virus Before the virus HSV reaches the tumor cells, it is cleared by the body's immune system through the following steps: 1) Cultivate and purify herpes simplex virus, and obtain a minimum virus amount of 1×1010 virus particles; 2) The exogenous targeting substance is combined with herpes simplex virus to obtain a conjugate with a purity of more than 95%; 3) Exogenous targeting substance of herpes simplex virus-PEGylation modification; 4) In vitro cell experiment verification. 2. The method according to claim 1, characterized in that, the retargeted herpes simplex virus is type I herpes simplex virus or type II herpes simplex virus. 3. The method according to claim 1, wherein the herpes simplex virus is a recombinant herpes simplex virus or a non-recombinant herpes simplex virus. 4. The method according to claim 3, wherein the recombinant herpes simplex virus comprises a replication-deficient recombinant herpes simplex virus and a conditional replication-type oncolytic herpes simplex virus. 5. The method according to claim 1, wherein the exogenous targeting substance is selected from folic acid or other ligand molecules of tumor cell highly expressed receptors. 6. The method according to claim 1, characterized in that the exogenous targeting substance is folic acid. 7. The method according to claim 5, characterized in that, the ligand molecules of other tumor cells highly expressing receptors include but not limited to: epidermal growth factor, vascular endothelial growth factor, estrogen, tumor necrosis factor-related apoptosis-inducing ligand. 8. The method according to claim 1, characterized in that the exogenous targeting substance is combined with the herpes simplex virus through chemical coupling, biological or physical means. 9. The method according to claim 1, characterized in that, the target cells of the retargeted herpes simplex virus are tumor cells or tumor neovascular endothelial cells. 10. The use of the re-targeted herpes simplex virus of claim 1 in the preparation of preparations that improve its ability to specifically infect tumor cells, avoid immune responses and virus neutralizing antibodies in vivo, reduce toxic and side effects, and improve safety.

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