ONCOLYTIC VIRUS AND APPLICATION THEREOF, AND DRUG FOR TREATING CANCER

Abstract

Provided are an oncolytic virus and an application thereof, and a drug for treating a cancer. A first regulatory element and a second regulatory element are inserted into the genome of the oncolytic virus. The first regulatory element comprises a tumor-specific promoter and a first nucleic acid sequence, which is driven by the cancer cell specific promoter to express a specific protease in tumor cells; the second regulatory element comprises a second nucleic acid sequence for encoding an extracellular secretion signal peptide and a third nucleic acid sequence for encoding a specific cleavage site. The oncolytic virus can be replicated in tumor cells effectively to kill tumor cells while being safe to non-cancer cells.

Claims

1-21. (canceled)

22. An oncolytic virus, wherein a first regulatory element is inserted into the viral genome thereof, wherein the first regulatory element comprises a tumor-specific promoter and a first nucleic acid sequence. The first nucleic acid sequence is driven by the tumor-specific promoter to express the specific protease in target tumor cells; a second regulatory element is further inserted into the viral genome, wherein the second regulatory element comprises a second nucleic acid sequence for encoding a specific cleavage site; and the specific cleavage site is recognized and cleaved by the specific protease; and the second regulatory element is located between the first and second codons of the regulated essential gene of the oncolytic virus.

23. The oncolytic virus of claim 22, wherein the specific protease is selected from the group including human rhinovirus 3C protease, thrombin, factor Xa protease, tobacco etch virus protease or recombinant PreScission protease.

24. The oncolytic virus of claim 22, wherein the extracellular secretion signal peptide is either interferon α2, interleukin 2, human serum albumin, human immunoglobulin heavy chain extracellular secretion signal peptide, or luciferase extracellular secretion signal peptide derived from marine copepods.

25. The oncolytic virus of claim 22, wherein the second regulatory element is located between the first and second codons of the open reading frame of the regulated essential gene of the oncolytic virus.

26. The oncolytic virus of claim 22, wherein the first regulatory element further comprises an enhancer, and the enhancer is located between the tumor-specific promoter and the encoding sequence of the specific protease, the enhancer is used to enhance expression of the specific protease in target tumor cells; and preferably, the enhancer is either CMV enhancer or SV40 enhancer.

27. The oncolytic virus of claim 22, wherein the target tumor cells are lung cancer, liver cancer, breast cancer, gastric cancer, prostate cancer, brain tumor, human colon cancer, cervical cancer, kidney cancer, ovarian cancer, head and neck cancer, melanoma, pancreatic cancer, or esophageal cancer cells.

28. The oncolytic virus of claim 22, wherein the tumor-specific promoter is selected from the group consisting of telomerase reverse transcriptase, human epidermal growth factor receptor-2, E2F1, osteocalcin, carcinoembryonic antigen, survivin and ceruloplasmin promoters.

29. The oncolytic virus of claim 22, wherein the oncolytic virus is selected from the group consisting of herpes simplex virus, adenovirus, vaccinia virus, new castle disease virus, poliovirus, coxsackie virus, measles virus, mumps virus, vesicular stomatitis virus and influenza virus.

30. The oncolytic virus of claim 22, wherein the second regulatory element is inserted between the first and second codons of one or more essential genes of the oncolytic virus.

31. The oncolytic virus of claim 22, wherein the oncolytic virus is herpes simplex virus type 1, the essential gene is ICP27, the tumor-specific promoter is telomerase reverse transcriptase promoter, the specific protease is human rhinovirus 3C protease, the extracellular secretion signal peptide is interferon α2 signal peptide, the amino acid sequence of the specific cleavage site is LEVLFQGP; the second regulatory element is located between the first and second codons of the open reading frame of the regulated essential gene; and the first regulatory element is located downstream the regulated essential gene.

32. The oncolytic virus of claim 31, wherein the nucleotide sequence of the telomerase reverse transcriptase promoter is shown in SEQ ID NO: 4; the amino acid sequence of the human rhinovirus 3C protease is shown in SEQ ID NO: 5; the nucleotide sequence of the open reading frame of the human rhinovirus 3C protease is shown in SEQ ID NO: 6; The amino acid sequence of interferon α2 extracellular secretion signal peptide is shown in SEQ ID NO: 7; the nucleotide sequence of the second nucleic acid sequence is shown in SEQ ID NO: 8; and the nucleotide sequence of the third nucleic acid sequence is shown in SEQ ID NO: 9.

33. The oncolytic virus of claim 22, wherein the insertion of the first regulatory element is located between two genes of the oncolytic virus.

34. The oncolytic virus of claim 33, wherein the two genes can both be essential genes, or one is an essential gene and the other is a non-essential gene.

35. A nucleic acid fragment for preparing the oncolytic virus of claim 22, wherein the nucleic acid fragment consists of the 5′ UTR of the regulated essential gene, ATG, the second regulatory element, the remaining portion of the open reading frame of the regulated essential gene without ATG, an exogenous Poly(A) and the first regulatory element followed by the 3′ UTR of the regulated essential gene. The 5′ and 3′ UTRs provide the sequence basis for homologous recombination between plasmid DNA and the parental viral genome for generation of the oncolytic viruses provided in the disclosure.

36. A drug for treating tumors, wherein the drug comprises the oncolytic virus of claim 1 and pharmaceutically acceptable excipients.

37. The drug of claim 36, wherein the drug further comprises gene therapy drug or vaccine.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0070] Exemplary features of the present disclosure, its nature and various advantages will be apparent from the accompanying drawings and the following detailed description of various embodiments. Non-limiting and non-exhaustive embodiments are described with reference to the accompanying drawings. The sizes and relative positions of elements in the drawings are not necessarily drawn to scale. For example, the shapes of various elements are selected, enlarged, and positioned to improve drawing legibility. The particular shapes of the elements as drawn have been selected for ease of recognition in the drawings. One or more embodiments are described hereinafter with reference to the accompanying drawings in which

[0071] FIG. 1: Schematic representation of the genome structure of the recombinant oncolytic virus provided in the present disclosure. A: generalized genome structure of oncolytic viruses provided in this disclosure, wherein the first regulatory element was located downstream the regulated essential gene, and the second regulatory element is located between the first and second codons of the open reading frame of the regulated essential gene. B: a specific embodiment of A, wherein the virus was HSV-1, the tumor-specific promoter was hTERT promoter, the enhancer was CMV enhancer, and the specific protease was HRV-3C protease, the regulated essential gene was ICP27, the extracellular secretion signal peptide was interferon α2 signal peptide; the specific cleavage site was specifically recognized and cleaved by HRV-3C protease. The oncolytic virus constructed was named as oHSV-BJS.

[0072] FIG. 2: Schematic showing of the parental plasmid pcDNA3.1-EGFP unitized for constructing a plasmid expressing HSV-1 ICP27. In the plasmid, EGFP is constitutively expressed under the control of CMV promoter and the plasmid contains the neomycin-resistant gene expression sequence

[0073] FIG. 3: ICP27 expression from oncolytic virus oHSV-BJS in African green monkey kidney cells (Vero, normal cells). Vero cells were infected with 3 MOI (virus/cell) HSV-1 wild-type virus KOS or oncolytic virus oHSV-BJS. One day later, the cells were collected, RNAs and proteins were isolated. ICP27 mRNA was detected by reverse transcription combined with semi-quantitative PCR (A in the FIG.), and ICP27 protein detected by Western blotting (B in the FIG.).

[0074] FIG. 4: Expression of HRV-3C in four tumor cells. Four tumor cells were infected with 3 MOI oncolytic virus oHSV-BJS or KOS. 24 hours after infection, total RNA and protein were isolated. HRV-3C mRNA was analyzed by semi-quantitative PCR, and t ICP27 protein detected by Western blotting. A: HRV-3C mRNA; B: ICP27 protein.

[0075] FIG. 5: Replication kinetics of wild-type virus KOS and oncolytic virus oHSV-BJS in tumor cells. Four tumor cells were infected with 0.1 MOI KOS or oHSV-BJS, respectively. At different day after infection, the cells and culture medium were collected. The virus titer for each viral stock was determined. A: cervical tumor Hela cells; B: cervical squamous carcinoma siHa cells; C: breast cancer SK-BR3 cells; D: breast cancer ME-180 cells.

[0076] FIG. 6: Inhibition of tumor growth by oncolytic virus oHSV-BJS in animal tumor models. Tumor animal models were established. After the tumor grew to 50-80 mm.sup.3, the oncolytic virus was injected into the tumor every 3 days for a total of 3 times. PBS (without oncolytic virus) was injected as a negative control. After the oncolytic virus was injected, the tumor size was tested twice a week. When the negative control animals needed to be euthanized, the experiment ended. A tumor growth curve was plotted based on the tumor size (A: lung cancer; B: gastric cancer; C: liver cancer; D: rectal cancer), and the relative inhibition rate (E) was calculated by comparing the tumor size in the test group at the end of the test with that observed in the negative control.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0077] In order to demonstrate the features of the present disclosure, its nature and various advantages, exemplary embodiments were executed and are described in details below. All experiments were conducted using standard methods as described in literature. Reagents were purchased from commercial providers and used according to the instructions of the manufacturer.

[0078] As used herein, terms “base sequence” and “nucleotide sequence” can be used interchangeably, and generally refer to the composition and order of nucleotides arranged in DNA or RNA.

[0079] The term “primer” refers to a synthetic oligonucleotide, which is required for de novo nucleic acid synthesis. After binding to a polynucleotide template, the primer is extended in 5′ to 3′ direction along the template catalyzed by DNA polymerase, hereby producing an extended duplex. Nucleotide addition during the extension is determined by the sequence of the template. A primer is typically 18-23 nucleotides in length. However, a primer length is determined by several factors including the nucleotide composition and the melting point of the primer, and the downstream application of the PCR product after amplified.

[0080] The term “promoter” generally refers to a DNA sequence that is located upstream the coding region of a gene, can be specifically identified and bound to by an RNA polymerase, and is required by transcription.

[0081] The term “enhancer” refers to a DNA sequence that increases transcription frequency of the gene interlocked therewith. The enhancer enhances the transcription by increasing the activity of a promoter. An enhancer may be located either at the 5′ or the 3′end of a gene, and even may exist as an intron within a gene. An enhancer might significantly affect gene expression, which might increase the gene transcription by 10-200 folds, or even by thousand times.

[0082] Terms “subject”, “individual”, and “patient” can be used interchangeably herein, and refer to a vertebrate, preferably a mammal, most preferably human. The mammal comprises, but is not limited to, mouse, ape, human, domesticated animal, or farm-raised livestock.

[0083] The features and its nature of the present disclosure are described in detail below with reference to examples.

Example 1

[0084] The oncolytic virus provided in this example was generated by genetically engineering wild-type herpes simplex virus type 1 KOS. The genome of the oncolytic virus oHSV-BJS contains the following elements refer to FIG. 1B).

[0085] (1) A first regulatory element is located downstream the essential gene ICP27 of the oncolytic virus; and the first regulatory element includes: tumor specific promoter, namely hTERT promoter, an enhancer, namely CMV enhancer, a nucleic acid sequence for encoding the specific protease, namely human rhinovirus 3C protease (HRV-3C protease) and BGH Poly(A).

[0086] Herein, the base sequence of hTERT promoter is shown in SEQ ID NO: 4;

[0087] the base sequence of the CMV enhancer is shown in SEQ ID NO: 10;

[0088] the nucleic acid fragment encoding HRV-3C protease is shown in SEQ ID NO: 6; and

[0089] the amino acid sequence of HRV-3C protease is shown in SEQ ID NO: 5.

[0090] (2) A second regulatory element is located between the first and second codons of the open reading frame of the essential gene ICP27 of the oncolytic virus; and the second regulatory element includes: the second nucleic acid sequence for encoding an extracellular secretion signal peptide, namely the interferon α2 signal peptide, and the third nucleic acid sequence for encoding the specific cleavage site sequence.

[0091] Herein, the amino acid sequence of the interferon α2 signal peptide is shown in SEQ ID NO: 7;

[0092] the nucleotide sequence of the second nucleic acid sequence for encoding the interferon α2 signal peptide is shown in SEQ ID NO: 8;

[0093] the amino acid sequence of the specific cleavage site is LEVLFQGP; and

[0094] the nucleotide sequence of the third nucleic acid sequence for encoding the specific cleavage site, is TTAGAAGTTCTTTTTCAAGGTCCT.

[0095] When the oncolytic virus infects normal cells, the HRV-3C protease is not expressed. Therefore, the specific cleavage site will be not cleaved, and the interferon α2 extracellular secretion signal peptide will direct the secretion of the ICP27 fusion protein to the outside of the cells, resulting in no viral replication. Therefore, the virus is safe to normal cells. When the oncolytic virus infects tumor cells, the HRV-3C protease is specifically expressed under the control of hTERT promoter, and the specific cleavage site will be recognized and cleaved by the expressed HRV-3C protease, and the ICP27 protein can be partitioned and localized normally in the tumor cells. Therefore, the oncolytic virus replicates normally and kill target tumor cells.

[0096] The preparation of the above-mentioned oncolytic virus oHSV-BJS provided in this example was as follows.

[0097] (1) Preparation of Complementing Cells Expressing HSV-1 ICP27

[0098] (a) plasmid construction: Using the DNA of wild-type herpes simplex virus type 1 KOS as a template, the encoding region of ICP27 was amplified by PCR, and inserted into HindIII and XbaI sites of the plasm id pcDNA3.1-EGFP (FIG. 2) to replace EGFP. The recombinant plasmid was named as ICP27 expression plasmid. The expression of ICP27 from the plasm id is driven under the control of CMV promoter.

[0099] (b) G418 dose determination for selection: Vero cells were treated with G418 of different concentrations, the culture medium containing G418 was replaced every three days with media containing G418 of different concentrations, and cell death was monitored every day. The minimal concentration of G418 required for all the cells to die after 6 days of G418 treatment was determined. Such a concentration of G418 (500 μg/ml) was utilized for complementing cell establishment.

[0100] (c) Cell line establishment: 3.5×10.sup.5 Vero cells were seeded into each well of a 6-well cell culture plate and cultured overnight in an antibiotic-free culture medium, and 4 μg of the ICP27 expression plasmid DNA obtained from step (a) were transfected into cells in each well using Lipofectamine 2000. After 24 hours of culture, cells in each well were harvested, and diluted by 20, 40, or 60-fold. Cells were cultured in the culture medium containing 500 μg/ml G418, and the medium replaced with fresh medium containing G418 every 3 days. After 6-7 times of medium change, the clones were collected and propagated step by step from the 24-well plate to T150 tissue culture flasks. Subsequently, the protein was isolated, and the expression of ICP27 detected by western blotting. The Cells with the highest level of ICP27 expression were selected as the complementing cells to support the growth and replication of replication-defective viruses in which ICP27 are not expressed; the cells were named as C.sub.ICP27

[0101] The complementing C.sub.ICP27 cells have been preserved at China Center for Type Culture Collection (CCTCC), Wuhan University, Luojiashan, Wuchang, Wuhan City on Apr. 24, 2019 with a preservation number of CCTCC NO. C201974.

[0102] (2) Preparation of the Parental Virus HSV-EGFP

[0103] The wild-type type 1 herpes simplex virus KOS was used as the starting material. The recombinant parental virus HSV-EGFP was obtained by homologous recombination between plasmid and KOS genome. In HSV-EGFP, HSV-ICP27 was replaced by EGFP. HSV-EGFP served as the parental virus for generating the oncolytic viruses provided in this disclosure. The detailed manipulations were as the follows.

[0104] (a) the first nucleic acid fragment was synthesized and its nucleotide sequence is shown in SEQ ID NO. 1:

[0105] The fragment includes the following elements: ICP27 5′ sequence, CMV promoter, Kozak sequence, EGFP encoding frame, BGH Poly(A) and ICP27 3′ sequence.

[0106] Sequence seen in SEQ ID NO. 1:

[0107] site 1-6: irrelevant sequence, increasing the end length to facilitate enzyme digestion;

[0108] site 7-12: Xho1 site, C/TCGAG;

[0109] site 13-575: ICP27 5′ UTR without ICP27 endogenous promoter;

[0110] site 576-1163: CMV promoter;

[0111] site 1164-1174: interval sequence;

[0112] site 1175-1180: Kozak sequence, increasing protein expression;

[0113] site 1181-1900: EGFP encoding frame;

[0114] site 1901-2145: BGH Poly(A);

[0115] site 2146-2667: ICP27 3′ UTR;

[0116] site 2668-2673: HindIII site, A/AGCTT; and

[0117] site 2674-2679: irrelevant sequence, increasing the end length to facilitate enzyme digestion.

[0118] (b) the first fragment was cleaved and ligated to the HindIII and Xho1 sites of the pcDNA3.1-EGFP plasmid, and the resulting recombinant plasmid was named as EGFP expression plasmid.

[0119] (c) 3.5×10.sup.5 complementing C.sub.ICP27 cells were seeded into each well of 6-well cell culture plate and cultured overnight in an antibiotic-free culture medium.

[0120] (d) the cells were infected with 0.1, 0.5, 1, 3 MOI wild-type virus KOS (virus/cell), respectively. One hour later, the above-mentioned EGFP expression plasmid (4 μg DNA/well) was transfected into the cells using Lipofectamine 2000. After 4 hours of incubation, the transfection mixture was replaced with complete medium. When all the cells became spherical, the cells and culture medium were collected. The mixture was centrifuged after three cycles of freeze-thawing and the supernatant collected, The virus stocks were diluted, and infected complementing C.sub.ICP27 cells. Viruses were separated using plaque separation method. 4-5 days later, the virus plaque with the strongest green fluorescence was selected and picked under a fluorescence microscope. The obtained virus plaque was subjected to 2 or 3 rounds of screening to obtain pure virus plaques. The virus was propagated. The recombinant virus with ICP27 replaced by EGFP was named as HSV-EGFP. HSV-EGFP served as the parental virus for generation of oncolytic virus oHSV-BJS provided in this disclosure.

[0121] (3) Construction of Recombinant Plasm Id Containing the First Expression Regulatory Element and the Second Regulatory Element

[0122] (a) the TA cloning plasmid was modified such that the multiple cloning site only contains the XhoI site, and the resulting plasmid named as TA-XhoI plasmid.

[0123] (b) the second nucleic acid fragment was synthesized with sequence shown in SEQ ID NO:2. The fragment includes the following elements:

[0124] ICP27 5′ UTR including the endogenous promoter, ATG, the second nucleic acid sequence for encoding interferon α2 extracellular secretion signal peptide, the third nucleic acid sequence for encoding the specific cleavage site recognized and cleaved by HRV-3C protease, ICP27 open reading frame sequence without ATP, SV40 Poly(A), ICP27 3′ UTR, wherein there was a HindIII site between SV40 Poly(A) and ICP27 3′ UTR, which is used to insert the first regulatory element. Detailed information of each element in the fragment is as follows:

[0125] site 1-6: Xho1 site;

[0126] site 7-677: ICP27 5′ UTR containing endogenous ICP27 promoter;

[0127] site 678-746: the second nucleic acid sequence for encoding the interferon α2 signal peptide;

[0128] site 747-770: the third nucleic acid sequence for encoding the specific cleavage site of HRV-3C protease;

[0129] site 771-2306: ICP27 open reading frame (without start codon ATG);

[0130] site 2307-2753: SV40 Poly(A);

[0131] site 2754-2759: HindIII site;

[0132] site 2760-3279: ICP27 3′ UTR; and

[0133] site 3280-3285: Xho1 site.

[0134] The second nucleic acid fragment was cleaved by Xho 1, ligated into the Xho1 site of plasmid TA-XhoI, and the resulting plasmid was named as pTA-XhoI-S-ICP27.

[0135] (c) the third nucleic acid fragment was synthesized with sequence shown in SEQ ID NO:3. It contains the following elements: hTERT promoter, CMV enhancer, Kozak sequence, the nucleic acid sequence for encoding the HRV-3C protease and SV40 Poly(A). Detailed information of each element in the fragment is as follows:

[0136] site 1-6: HindIII site;

[0137] site 7-432: hTERT promoter;

[0138] site 433-527: CMV enhancer;

[0139] site 528-539: Kozak sequence;

[0140] site 540-1088: nucleic acid sequence for encoding HRV-3C protease;

[0141] site 1089-1544: SV40 Poly(A); and

[0142] site 1545-1550: HindIII site.

[0143] The third nucleic acid fragment was cleaved by HindIII and ligated into pTA-XhoI-S-ICP27, and the obtained plasmid was named as pTA-XhoI-S-ICP27-3C plasmid.

[0144] (4) Construction of Recombinant Oncolytic Herpes Viruses

[0145] (a) 3.5×10.sup.5 complementing C.sub.ICP27 cells were seeded into each well of a 6-well cell culture plate and cultured overnight in an antibiotic-free culture medium.

[0146] (b) the complementing C.sub.ICP27 cells were infected with 0.1, 0.5, 1, 3 MOI (virus/cell) of the parent virus HSV-EGFP, respectively. After 1 hour incubation, pTA-XhoI-S-ICP27-3C DNA (4 μg DNA/well) was transfected into the cells using Lipofectamine 2000. After 4 hours of incubation, the transfection mixture was replaced with complete medium. When all the cells became spherical, the cells and culture medium were collected. The mixture was centrifuged after three cycles of freeze-thawing, the supernatant collected. Virus stocks were diluted, and infected the complementing C.sub.ICP27 cells. The viruses were isolated using plaque separation method. 4-5 days after infection, virus plaques without green fluorescence under a fluorescence microscope were picked. The obtained virus plaques were subjected to 2 or 3 rounds of screening to obtain pure virus plaques, the virus was propagated and expanded, the infected cell DNA was isolated, and the recombinant oncolytic viruses were confirmed by PCR amplification and sequencing, and the oncolytic virus was named as oHSV-BJS.

[0147] The recombinant oncolytic virus oHSV-BJS was preserved in the Chinese Center of Type Culture Collection (CCTCC), a Wuhan University, Luojiashan, Wuchang, Wuhan, China on Apr. 24, 2019. The preservation number is CCTCC NO: V201920.

Experimental Example 1

[0148] Detection of the expression of ICP27 from the recombinant oncolytic virus oHSV-BJS in Vero cells

[0149] Method: Vero was infected with 3 MOI wild-type KOS and oncolytic virus oHSV-BJS, respectively. One day after infection, cells were collected, RNAs and proteins were isolated. ICP27 mRNA was detected by reverse transcription combined with semi-quantitative PCR, and tICP27 protein detected by Western blotting. For mRNA and protein detection, β-actin was used as the loading control.

[0150] Results: oHSV-BJS there was no significant difference in ICP27 mRNA level in oHSV-BJS-infected vero cells compared to KOS-infected vero cells (FIG. 3A). However, ICP27 protein in Vero cells infected with oHSV-BJS was significantly lower than that observed in Vero cells infected with KOS (FIG. 3B)). The results suggest ICP27 protein once produced is secreted to the outside of the cells.

Experimental Example 2

[0151] Detection of the Expression of HRV-3C Protease and ICP27 in Tumor Cells

[0152] Method: Four tumor cells were infected with 3 MOI oncolytic virus oHSV-BJS or KOS. 24 hours after infection, total RNA and protein were isolated. HRV-3C mRNA was analyzed by semi-quantitative PCR, and t ICP27 protein detected by Western blotting. For mRNA and protein detection, β-actin was used as the loading control.

[0153] Results: HRV-3C mRNA was expressed to a detectable level in all the four tumor cells infected with oHSV-BJS (FIG. 4A). ICP27 protein was accumulated to a detectable level in all the four tumor cells infected with the oncolytic virus oHSV-BJS. Moreover, there was no significant difference in ICP27 protein between oncolytic virus oHSV-BJS and KOS infected cells for a given cell type (FIG. 4B).

[0154] The results demonstrate that HRV-3C was specifically expressed in tumor cells and cleaved ICP27 fusion protein.

Experimental Example 3

[0155] Replication Kinetics of Recombinant Oncolytic Virus oHSV-BJS

[0156] Method: tumor cells were infected with 0.1 MOI KOS or oHSV-BJS, respectively. At different day after infection, the cells and culture medium were collected, and the viruses remaining in the cells were released into the culture medium through three cycles of −80/37° C. freeze-thawing. The complementing C.sub.ICP27 cells were then infected with the virus, and the virus titer (plaque forming unit/ml, PFU/ml) was determined by plaque assay.

[0157] Results: with the replication kinetics of oHSV-BJS are quite different from one cell to another cell type. But for a given cell type, there is no significant difference in viral replication in bother oHSV-BJS-infected and KOS-infected cells (FIG. 5A-D). the results indicate that genetic modification used for generating the oncolytic virus provided in this disclosure does not significantly alter the replication capacity in tumor cells.

Experimental Example 4

[0158] The Ability of Recombinant Oncolytic Virus oHSV-BJS to Kill Tumor Cells

[0159] Methods: tumor cells were infected by MOI 0.25 or 0.5 KOS or oHSV-BJS respectively. Cell viability was assayed at different day after infection

[0160] 0.25 or 0.5 MOI oHSV-BJS shows a varied ability to kill different tumor cells. But for a given cell type, the efficiency of oHSV-BJS in killing cells was basically the same as that observed for KOS (Table 1-4). the results indicate that oncolytic virus oHSV-BJS retains the ability of wild-type KOS virus to kill tumor cells.

TABLE-US-00001 TABLE 1 Viability of (%) of Hela cells infected with oHSV-BJS at different day after infection virus (MOI 0.25) oHSV-BJS KOS the first day  58 ± 3.0  46 ± 2.0 the second day  38 ± 2.0  29 ± 1.4 the third day  17 ± 0.8  11 ± 0.6 the fourth day   4 ± 0.3 0 virus (MOI 0.5) oHSV-BJS KOS the first day  52 ± 2.7  39 ± 1.8 the second day  28 ± 1.8  22 ± 1.1 the third day   9 ± 0.1   5 ± 0.3 the fourth day 0 0

TABLE-US-00002 TABLE 2 Viability (%) of siHA cells infected with oHSV-BJS at different day after infection virus (MOI 0.25) oHSV-BJS KOS the first day  22 ± 1.4  16 ± 1.0 the second day   6 ± 0.4 0 the third day 0 0 virus (MOI 0.5) oHSV-BJS KOS the first day   8 ± 0.4   4 ± 0.3 the second day   1 ± 0.1 0 the third day 0 0

TABLE-US-00003 TABLE 3 Viability (%) of SK-BR3 cells infected with oHSV-BJS at different day after infection virus (MOI 0.25) oHSV-BJS KOS the first day  21 ± 1.3 6 ± 0.4 the second day 0 0 the third day 0 0 virus (MOI 0.5) oHSV-BJS KOS the first day   3 ± 0.1 5 ± 0.3 the second day 0 0 the third day 0 0

TABLE-US-00004 TABLE 4 viability (%) of ME-180 cells infected with oHSV-BJS at different day after infection virus (MOI 0.25) oHSV-BJS KOS the first day  12 ± 0.8  10 ± 0.5 the second day   4 ± 0.3 0 the third day 0 0 virus (MOI 0.5) oHSV-BJS KOS the first day   5 ± 0.5   5 ± 0.4 the second day   1 ± 0.10 0 the third day 0 0

Experimental Example 5

[0161] Effect of Recombinant Oncolytic Virus oHSV-BJS on the Viability of Normal Cells

[0162] Methods: Vero cells or primary human corneal epidermal cells were infected with oncolytic virus oHSV-BJS (2 MOI) or wild virus KOS (0.5 MOI) respectively. Viability of the cells infected with oHSV-BJS were assayed 3 days after infection while the viability of the cells infected with wild virus KOS were measured 2 days after infection. All Vero cells or primary human corneal epidermal cells died 2 days after KOS infection. But the viability of the cells infected with the oncolytic virus oHSV-BJS was basically the same as that of observed for mock treatment (not treated) (Table 5). The results indicate that oncolytic virus oHSV-BJS is safe to normal cells.

TABLE-US-00005 TABLE 5 viability (%) of normal cells 3 days after oHSV-BJS infection or 2 days after KOS infection virus oHSV-BJS KOS untreated Vero cells 95 ± 2 0 98 ± 2 corneal epidermal cells 97 ± 2 0 97 ± 1

Experimental Example 6

[0163] Detection of the Inhibition of Tumor Growth by Oncolytic Virus oHSV-BJS In Vivo

[0164] Method: To establish animal tumor models of lung cancer (A549 cells), gastric cancer (NCI-N87 cells), liver cancer (SK-HEP-1 cells) and rectal cancer (HCT-8), tumor cells were subcutaneously inoculated into mice. When the tumors grew to 40-120 mm.sup.3, oncolytic virus oHSV-BJS was injected into the tumors once every 3 days for a total of 3 times of injection, 1.2×10.sup.7 infection units (suspended in 40μl PBS) were injected for each time at multiple sites. Mice injected with PBS (without oncolytic virus) were used as negative control, with 8 animals in each experiment group. After the injection of oncolytic virus, the tumor sizes were measured twice a week (the relative tumor size was defined as 1 at the first injection) for a total of 17-32 days (depending on the time when the animal in the negative control group needed to be euthanized). The tumor growth curve was plotted according to the tumor size. At the end of the experiment, the tumor size was measured and compared with that in the negative control, and the inhibition rate was calculated.

inhibition rate (%)=(tumor volume of negative control group−tumor volume of test group)/tumor volume in negative control group×100%.

[0165] oHSV-BJS slowed down the growth of lung, gastric, liver and rectal tumors (FIG. 6A-D). The inhibition of oHSV-BJS on lung, gastric, liver and rectal tumor were 45%, 37%, 26% and 49%, respectively (FIG. 6E). The results showed that oHSV-BJS can inhibit the proliferation of various tumors.

[0166] The foregoing descriptions are only typical examples of the present disclosure, and are not intended to limit the scope of the present disclosure. Any modification, equivalent replacement, improvement, etc. made within the concept and principle of the present disclosure shall be included in the protection scope of the present disclosure.

INDUSTRIAL APPLICABILITY

[0167] The oncolytic virus provided by the present disclosure can be produced on industrial scale. The oncolytic virus has not deleted any genes (either essential or non-essential genes) from its original genome. It can replicate with high capability and kill tumor cells, which can be used to treat cancer and it is safe to non-cancer cells.