WO2018228538A1 - 重组单纯疱疹病毒及其制备方法和应用 - Google Patents

重组单纯疱疹病毒及其制备方法和应用 Download PDF

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WO2018228538A1
WO2018228538A1 PCT/CN2018/091530 CN2018091530W WO2018228538A1 WO 2018228538 A1 WO2018228538 A1 WO 2018228538A1 CN 2018091530 W CN2018091530 W CN 2018091530W WO 2018228538 A1 WO2018228538 A1 WO 2018228538A1
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herpes simplex
simplex virus
gene
cancer
tumor
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PCT/CN2018/091530
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French (fr)
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WO2018228538A9 (zh
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李小鹏
田超
赵婧姝
刘家家
周华
孙春阳
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杭州睿可特生物科技有限公司
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Priority to US16/620,738 priority Critical patent/US20200149067A1/en
Priority to EP18818513.6A priority patent/EP3640327A4/en
Publication of WO2018228538A1 publication Critical patent/WO2018228538A1/zh
Publication of WO2018228538A9 publication Critical patent/WO2018228538A9/zh

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    • C12N2710/16011—Herpesviridae
    • C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
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Definitions

  • the invention relates to the field of genetic engineering, in particular to a recombinant herpes simplex virus, a preparation method and application of the recombinant herpes simplex virus.
  • Oncolytic virus refers to a type of virus that selectively infects tumor cells and replicates in target cells, ultimately leading to tumor cell lysis and death. These viruses rely on their own specificity to replicate in tumor cells to lyse tumor cells. The virus released after cell lysis can further infect surrounding tumor cells, and has no destructive effect on normal cells and tissues, or has less influence. . Oncolytic virus has multiple anti-tumor mechanisms, including: 1. Direct lysis of tumor cells; 2. Destruction of tumor blood vessels; 3. Viral protein expressed by viral replication has direct cytotoxicity; 4. Anti-tumor immune response; 5. Enhanced tumor The sensitivity of cells to chemoradiotherapy.
  • Oncolytic viruses currently used for anti-tumor research include Herpes simplex virus (HSV), adenovirus and vaccinia virus.
  • HSV Herpes simplex virus
  • adenovirus adenovirus
  • vaccinia virus a virus that is a virus that is a virus that is a virus that is a virus that is a virus that is a virus that is a virus that is a virus that is a virus.
  • HSV Herpes simplex virus
  • adenovirus adenovirus
  • vaccinia virus vaccinia virus
  • Cytokines are signals that connect immune cells to each other and regulate the response of the immune system to antigens. Cytokines play an important role in tumor immunotherapy, which can directly stimulate the expansion of immune effector cells at the tumor site and enhance the recognition of tumor cells, while activating the immune response of the systemic system. A large number of animal tumor models validate the anti-tumor activity of cytokines. However, although cytokines can be treated by direct administration for anti-tumor treatment, large-scale injection of cytokines may bring side effects such as autoimmune diseases to the body.
  • the object of the present invention is to overcome the above-mentioned drawbacks of the prior art and to provide a recombinant herpes simplex virus and a preparation method and application thereof.
  • the recombinant herpes simplex virus provided by the invention has good anti-tumor effect, and has small side reaction after use, and has good application prospect.
  • the present invention provides a recombinant herpes simplex virus, wherein the recombinant herpes simplex virus comprises a vector and a foreign gene encoding at least two cytokines, wherein the vector is a deletion encoding ICP34. 5 and a gene for ICP47, and optionally a herpes simplex virus lacking at least one of genes encoding ICP6, TK and UNG, the insertion site of the foreign gene is a deletion coding on the vector encoding ICP34.5, ICP47 At least one of the positions of the genes of ICP6, TK and UNG.
  • the present invention also provides a method for the preparation of the herpes simplex virus described above, which comprises: knocking out genes encoding ICP34.5 and ICP47 in herpes simplex virus, and optionally knocking out ICP6, TK and At least one of the genes of UNG, and inserting a foreign gene encoding at least two cytokines, wherein the insertion site of the foreign gene is knock-out encoding ICP34.5, ICP47, ICP6, TK on the vector And at least one of the locations of the UNG gene.
  • the present invention provides the use of the above recombinant herpes simplex virus and/or herpes simplex virus prepared by the above method for the preparation of a medicament for preventing and/or treating a tumor.
  • the present invention encodes a gene encoding an infected cell polypeptide 34.5 (infected cell Polypetide 34.5, ICP34.5) and a gene encoding ICP47, and optionally a gene encoding ICP6, a gene encoding thymidine kinase (TK).
  • herpes simplex virus encoding at least one of the genes of uracil-N-glycosylase (UNG), and inserting a foreign gene encoding at least two cytokines at a position where the above gene is deleted
  • UNG uracil-N-glycosylase
  • a gene particularly a gene inserted into at least two of GM-CSF, IL-2 and IL-12, obtains a stably expressed recombinant herpes simplex virus.
  • the recombinant herpes simplex virus exhibits a good anti-tumor effect, especially for melanoma and breast cancer, and the method of local intratumoral injection can not only produce a targeted anti-tumor effect, but also has a small side reaction after use. Has a good application prospects.
  • the present invention provides a recombinant herpes simplex virus, wherein the recombinant herpes simplex virus comprises a vector and a foreign gene encoding at least two cytokines, wherein the vector lacks a gene encoding ICP34.5 and ICP47 And, optionally, a herpes simplex virus lacking at least one of the genes encoding ICP6, TK and UNG, the insertion site of the foreign gene encoding the ICP34.5, ICP47, ICP6, TK and At least one of the locations of the UNG gene.
  • the insertion site of the foreign gene is at least one of the positions of the deleted gene.
  • the insertion site is anywhere in the deletion, not the gene encoding UNG.
  • At least two cytokines can be ligated into the same site or inserted into different sites.
  • the cytokine is ligated into the same site after ligation.
  • the gene encoding ICP34.5, the gene encoding ICP47, the gene encoding ICP6, the gene encoding TK, and the gene encoding UNG are well known to those skilled in the art, and can also be logged into a related database. It is found that, for example, the related nucleotide sequence can be queried by logging in to the GenBank database, which are conventional technical means available to those skilled in the art, and the present invention will not be repeated herein.
  • the cytokine may be interleukin (IL), colony stimulating factor (CSF), interferon (IFN), tumor necrosis factor (TNF), transformation.
  • IL interleukin
  • CSF colony stimulating factor
  • IFN interferon
  • TNF tumor necrosis factor
  • At least two of growth factors, growth factors and chemokines, preferably GM-CSF, IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, TNF- ⁇ , IFN At least two of ⁇ , IFN- ⁇ and IFN- ⁇ , more preferably at least two of GM-CSF, IL-2 and IL-12.
  • the gene encoding the cytokine can be queried by logging in to the relevant database.
  • the related nucleic acid sequence can be queried by logging into the GenBank database.
  • the genes encoding the cytokines may be of different origin. For example, when the obtained recombinant herpes simplex virus is applied to a human, the gene encoding the cytokine may be human; when the obtained recombinant herpes simplex virus is applied to a mouse, the gene encoding the cytokine may be a mouse. Source.
  • the Gene ID of the gene encoding GM-CSF is 1437
  • the Gene ID of the gene encoding IL-2 is 3558
  • the Gene ID of the gene encoding IL-12A is 3592
  • the Gene ID of the gene encoding IL-12B is 3593
  • the Gene ID of the gene encoding TNF- ⁇ is 7124.
  • the Gene ID of the gene encoding GM-CSF is 12981
  • the Gene ID of the gene encoding IL-2 is 16183
  • the Gene ID of the gene encoding IL-12A is 16159.
  • the gene encoding IL-12B has a Gene ID of 16160 and the gene encoding TNF- ⁇ has a Gene ID of 21926.
  • the foreign gene of the present invention preferably further comprises a promoter, a start codon and a stop codon, and an optional ligation sequence and/or Or a PolyA sequence, in such a preferred case, when the recombinant herpes simplex virus infects a host cell and expresses a self-gene, it is capable of transcribed an independent and complete target mRNA fragment, thereby enabling efficient and independent targeting of the gene of interest.
  • the exogenous gene encoding one of the cytokines when at one position of the vector (for example, the position of the gene encoding ICP34.5 is deleted on the vector, or the position of the gene encoding ICP47 is deleted on the vector), at least two insertion codes are inserted.
  • the exogenous gene encoding one of the cytokines in order to separately express the foreign gene encoding one of the cytokines, further includes a ligation sequence therebetween.
  • the linker sequence is an IRES sequence.
  • the vector when at one position of the vector (eg, the position of the gene encoding ICP34.5 is deleted on the vector, or the position of the gene encoding ICP47 is deleted on the vector), at least two are inserted.
  • the foreign genes encoding one of the cytokines in order to independently express the foreign gene encoding one of the cytokines, each have an independent expression frame, and each of the independent expression cassettes has Promoters, sequences encoding cytokines, and PolyA sequences.
  • the kind of the promoter is not particularly limited as long as the transcription of the foreign gene can be controlled, and preferably, the promoter is selected from the group consisting of a CMV promoter, an EF1 ⁇ promoter, and an SV40. At least one of a promoter, an RSV promoter and an MMTV promoter, preferably a CMV promoter and/or an EF1 ⁇ promoter.
  • the foreign gene may further include a marker gene (for example, a gene encoding ⁇ -galactosidase, luciferase, green fluorescent protein or other fluorescent protein).
  • a marker gene for example, a gene encoding ⁇ -galactosidase, luciferase, green fluorescent protein or other fluorescent protein.
  • the foreign gene may also include related transcriptional regulatory sequences normally associated with the transcribed sequence, for example, a polyadenylation site, a Kozak sequence, a WPRE, and a downstream enhancer element. These are well known to those skilled in the art, and the present invention will not be described herein.
  • the type of the herpes simplex virus is not particularly limited and may be a routine selection in the art, but the herpes simplex virus is preferably herpes simplex type I for the purpose of better achieving stable expression of cytokines. virus.
  • the source of the herpes simplex virus is also not particularly limited in the present invention, and can be obtained by conventional commercial purchase or by laboratory separation.
  • the tumor treatment effect of the above recombinant herpes simplex virus of the present invention is significantly better than the recombinant herpes simplex virus in which only one cytokine is inserted alone, and the therapeutic effect produced by the technical scheme of inserting two or more cytokines is synergistic.
  • the present invention also provides a method for preparing a recombinant herpes simplex virus, which comprises: knocking out genes encoding ICP34.5 and ICP47 in herpes simplex virus, and optionally knocking out codes encoding ICP6, TK and At least one of the genes of UNG, and inserting a foreign gene encoding at least two cytokines, wherein the insertion site of the foreign gene is knock-out encoding ICP34.5, ICP47, ICP6, TK on the vector And at least one of the locations of the UNG gene.
  • the knockout of the above genes encoding ICP, TK and UNG can be carried out by various methods conventional in the art, and the present invention is not particularly limited thereto, for example, by means of homologous recombination, by site-specific knockout. The way; or, you can also knock out by means of CRISPR. Under the premise of understanding the object of the present invention and the viral vector used in the present invention, those skilled in the art can achieve knockout of the gene encoding the ICP according to conventional techniques grasped by those skilled in the art.
  • the insertion of the foreign gene may also be carried out by various methods conventional in the art, and the insertion may be carried out by inserting the gene of interest directly at a selected insertion site, for example, by inserting in a CRISPR manner. It is also possible to insert a part of the base sequence by means of homologous recombination to insert the gene of interest, and the latter is preferred in the present invention.
  • the recombinant herpes simplex virus like the normal herpes simplex virus, also needs to complete its life history in the host cell, and thus the passage of the recombinant virus requires propagation in the host cell.
  • the host cell may be any host cell capable of culturing the viral vector and/or recombinant virus of the present invention, for example, African green monkey kidney cells (Vero cells), hamster kidney cells (BHK cells), primary rabbit kidney cells, chickens. Embryonic cells, amnion cells, human cervical cancer cells (Hela cells), and human embryonic lung diploid fibroblasts (WI-38 cells).
  • the present invention provides a virus-infected cell, wherein the virus is the above-described recombinant herpes simplex virus, the cell having a gene encoding ICP34.5 and a gene encoding ICP47, and a selectable expression coding.
  • the virus is the above-described recombinant herpes simplex virus, the cell having a gene encoding ICP34.5 and a gene encoding ICP47, and a selectable expression coding.
  • the specific selection of the cells can be referred to the selection of the host cells as enumerated above, and will not be described herein.
  • the present invention also provides the use of the above recombinant herpes simplex virus and/or the recombinant herpes simplex virus prepared by the above method for preparing a medicament for preventing and/or treating a tumor.
  • the tumor is melanoma, glioma, head and neck tumor, liver cancer, ovarian cancer, prostate cancer, breast cancer, lung cancer, colorectal cancer, renal cell carcinoma, stomach cancer, pancreatic cancer, lymphoma and At least one of bladder cancers.
  • the present invention relates to a genetically engineered herpes simplex virus comprising a deletion of the ICP34.5 and ICP47 genes and a tandem introduction of at least two cytokine genes at a deletion site of the ICP34.5 and/or ICP47 gene,
  • the introduced gene is selected from the group consisting of GM-CSF, IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, TNF- ⁇ , IFN- ⁇ , IFN- ⁇ and IFN- ⁇ ,
  • the genetically engineered herpes simplex virus exhibits synergistic oncolytic effect (tumor therapeutic effect).
  • the term "synergistic" as used in the present invention means that the genetically engineered herpes simplex virus which introduces two or more cytokines which promote (enhance) an immune response in series exhibits an oncolytic effect (tumor therapeutic effect) greater than that introduced separately. Genetically engineered herpes simplex virus that promotes immune response to cytokines.
  • the "synergistic" effect of the oncolytic effect or the therapeutic effect of the tumor of the present invention is that the effect achieved by introducing the cytokine which enhances immunity in series is greater than the effect of the effect of introducing the cytokine separately.
  • the synergistic effect exhibited by the recombinant herpes simplex virus of the present invention or the genetically engineered herpes simplex virus is determined based on the method used in the examples of the present application. It can also be judged using other judgment methods in the art, for example, using the Chou and Talalay combination method and dose-effect analysis (Chou and Talalay (1984) Adv. Enzyme Regul. 22: 27-55; Chou and Talalay, "New Avenues in Developmental Cancer Chemotherapy", Academic Press, 1987, Chapter 2) Judgment.
  • the "tandem" introduction in the technical solution of the present invention means that two or more cytokines, for example, a cytokine that promotes (enhanced) an immune response is introduced into the same gene deletion site of herpes simplex virus, and the deletion site is selected from ICP34. 5.
  • One of ICP47, ICP6, TK and UNG, or two or more cytokines, such as cytokines that promote (enhance) immune response are introduced into different gene deletion sites of herpes simplex virus, and the deletion site is selected from the group consisting of ICP34.5, ICP47, ICP6, TK and UNG.
  • the genetically engineered herpes simplex virus of the present invention simultaneously carries two or more, preferably two, three or four genes encoding cytokines that promote (enhance) immune responses.
  • the recombinant herpes simplex virus or the genetically engineered herpes simplex virus introduces two, three or four of the following cytokines: GM-CSF, IL-2, IL -12, TNF- ⁇ and IFN- ⁇ .
  • the herpes simplex virus is a herpes simplex virus type I.
  • the present invention also relates to a stock solution and host cell comprising the recombinant herpes simplex virus or the genetically engineered herpes simplex virus, and a pharmaceutical composition.
  • a stock solution and host cell comprising the recombinant herpes simplex virus or the genetically engineered herpes simplex virus, and a pharmaceutical composition.
  • it also relates to the above-mentioned recombinant herpes simplex virus or genetically engineered herpes simplex virus stock solution and host cells and pharmaceutical compositions for preparing tumors, especially solid tumors, preferably melanoma, glioma, head and neck Uses in drugs for cancer, liver cancer, lung cancer, colorectal cancer, renal cell carcinoma, gastric cancer, pancreatic cancer, lymphoma, bladder cancer, ovarian cancer, prostate cancer, and breast cancer.
  • the present invention relates to a method for treating a tumor, particularly a solid tumor, preferably melanoma, glioma, head and neck tumor, liver cancer, lung cancer, colorectal cancer, renal cell carcinoma, stomach cancer, pancreatic cancer, A method of lymphoma, bladder cancer, ovarian cancer, prostate cancer, and breast cancer comprising administering to a subject an effective amount of the above-described recombinant herpes simplex virus or a genetically engineered herpes simplex virus.
  • the herpes simplex virus of the present invention is treated by topical administration of the tumor, preferably by introducing the herpes simplex virus into the tumor tissue (intratumoral administration) by catheter or injection.
  • Vero cells were purchased from ATCC under the order number CCL-81;
  • B16-BL6 cells and 4T1 cells are from the Academy of Military Medical Sciences.
  • H22 cells are derived from the cell bank of the China Center for Type Culture Collection.
  • Balb/c mice are from the Academy of Military Medical Sciences and Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.
  • This example is intended to illustrate the construction of the recombinant herpes simplex virus provided by the present invention.
  • the ICP34.5 gene and the ICP47 gene of wild-type HSV-1 virus were knocked out by the method described in the patent application No. 2004100064921, the number of the patent application No. CN1283803C, and in HSV. -1 virus knockout
  • the position of the ICP34.5 gene was inserted into a foreign chemically synthesized foreign gene, except that Vero cells were used as host cells in this example.
  • the exogenous gene includes, in order from the 5' end to the 3' end: a CMV promoter, a gene encoding GM-CSF (Gene ID: 12981), a BGH PolyA, an EF1 ⁇ promoter, a gene encoding IL-2 (Gene ID: 16183) ) and TK PolyA.
  • the GM-CSF and IL-2 encoding genes were correctly sequenced into the herpes simplex virus vector in Beijing Sanbo Yuanzhi Co., Ltd. to obtain a recombinant viral vector.
  • the successfully constructed recombinant viral vector was propagated on Vero host cells at 37 ° C, 5% CO 2 , and the multiplicity of infection was 0.1.
  • the cell debris was removed with a 0.65 ⁇ m filter, and then purified by high-speed centrifugation at 13,000 rpm to obtain a titer of A virus suspension of 1 x 10 8 pfu/mL was used for animal experiments.
  • This example is intended to illustrate the construction of the recombinant herpes simplex virus provided by the present invention.
  • the recombinant herpes simplex virus was constructed according to the method of Example 1, except that the inserted foreign gene included in the 5' to 3' end: CMV promoter, gene encoding GM-CSF (Gene ID: 12981) BGH PolyA, EF1 ⁇ promoter, gene encoding IL-12B (Gene ID: 16160), IRES sequence, gene encoding IL-12A (Gene ID: 16159) and TK PolyA.
  • This example is intended to illustrate the construction of the recombinant herpes simplex virus provided by the present invention.
  • the ICP34.5 gene and the ICP47 gene of the wild-type HSV-1 virus were knocked out according to the method described in the patent application No. 2004100064921, the authorization of the publication No. CN1283803C, and the position of the ICP34.5 gene of the HSV-1 virus was knocked out.
  • the exogenous gene 1 synthesized by artificial chemical synthesis was inserted, and the artificially synthesized exogenous gene 2 was inserted at the position of the ICP47 gene of the HSV-1 virus, except that Vero cells were used as host cells in the present example.
  • the exogenous gene 1 includes, in order from the 5' end to the 3' end, an EF1 ⁇ promoter, a gene encoding IL-2 (Gene ID: 16183), and TK PolyA.
  • the exogenous gene 2 includes, in order from the 5' end to the 3' end, a CMV promoter, a gene encoding GM-CSF (Gene ID: 12981), and BGH PolyA.
  • the GM-CSF and IL-2 encoding genes were correctly sequenced into the herpes simplex virus vector in Beijing Sanbo Yuanzhi Co., Ltd. to obtain a recombinant viral vector.
  • the successfully constructed recombinant viral vector was propagated on Vero host cells at 37 ° C, 5% CO 2 , and the multiplicity of infection was 0.1. After harvesting, the cell debris was removed with a 0.65 ⁇ m filter, and then purified by high speed centrifugation at 13,000 rpm to obtain 1 ⁇ 10. A virus suspension of 8 pfu/mL was used for animal experiments.
  • This example is intended to illustrate the construction of the recombinant herpes simplex virus provided by the present invention.
  • the ICP34.5 gene and the ICP47 gene of the wild-type HSV-1 virus were knocked out according to the method described in the patent application No. 2004100064921, the authorization of the publication No. CN1283803C, and the position of the ICP34.5 gene of the HSV-1 virus was knocked out.
  • the exogenous gene 1 synthesized by artificial chemical synthesis was inserted, and the exogenous gene 2 synthesized by artificial chemical synthesis was inserted at the position of the ICP47 gene of the HSV-1 virus.
  • the exogenous gene 1 includes, in order from the 5' end to the 3' end, an EF1 ⁇ promoter, a gene encoding IL-12B (Gene ID: 16160), an IRES sequence, a gene encoding IL-12A (Gene ID: 16159), and TK PolyA.
  • the exogenous gene 2 includes, in order from the 5' end to the 3' end: a CMV promoter, a gene encoding GM-CSF (Gene ID: 12981), and BGH PolyA.
  • the coding genes of GM-CSF, IL-12A and IL-12B were sequenced and identified in Beijing Sanbo Yuanzhi Company and inserted into the herpes simplex virus vector to obtain the recombinant virus vector.
  • the successfully constructed recombinant viral vector was propagated on Vero host cells at 37 ° C, 5% CO 2 , and the multiplicity of infection was 0.1. After harvesting, the cell debris was removed with a 0.65 ⁇ m filter, and then purified by high speed centrifugation at 13,000 rpm to obtain 1 ⁇ 10. A virus suspension of 8 pfu/mL was used for animal experiments.
  • This example is intended to illustrate the construction of the recombinant herpes simplex virus provided by the present invention.
  • the construction of the recombinant herpes simplex virus was carried out in accordance with the method of Example 1, except that the ICP34.5 gene, the ICP47 gene and the ICP6 gene of the wild-type HSV-1 virus were knocked out to obtain a recombinant viral vector.
  • This example is intended to illustrate the construction of the recombinant herpes simplex virus provided by the present invention.
  • the construction of the recombinant herpes simplex virus was carried out in accordance with the method of Example 1, except that the insertion site of the foreign gene was the position at which the ICP47 gene was knocked out by the HSV-1 virus.
  • This example is intended to illustrate the construction of the recombinant herpes simplex virus provided by the present invention.
  • the recombinant herpes simplex virus was constructed according to the method of Example 1, except that the inserted foreign gene included in the 5' to 3' end: CMV promoter, gene encoding GM-CSF (Gene ID: 12981) ), BGH PolyA, EF1 ⁇ promoter, gene encoding TNF- ⁇ (Gene ID: 21926) and TK PolyA.
  • This example is intended to illustrate the construction of the recombinant herpes simplex virus provided by the present invention.
  • the ICP34.5 gene and the ICP47 gene of the wild-type HSV-1 virus were knocked out according to the method described in the patent application No. 2004100064921, the authorization of the publication No. CN1283803C, and the position of the ICP34.5 gene of the HSV-1 virus was knocked out.
  • the exogenous gene 1 synthesized by artificial chemical synthesis was inserted, and the exogenous gene 2 synthesized by artificial chemical synthesis was inserted at the position of the ICP47 gene of the HSV-1 virus.
  • the exogenous gene 1 includes, in order from the 5' end to the 3' end, an EF1 ⁇ promoter, a gene encoding IL-12B (Gene ID: 16160), an IRES sequence, a gene encoding IL-12A (Gene ID: 16159), and TK PolyA.
  • the exogenous gene 2 includes, in order from the 5' end to the 3' end, a CMV promoter, a gene encoding TNF- ⁇ (Gene ID: 21926), and BGH PolyA.
  • the genes encoding IL-12A, IL-12B and TNF- ⁇ were sequenced and identified in Beijing Sanbo Yuanzhi Company and inserted into the herpes simplex virus vector to obtain the recombinant virus vector.
  • the successfully constructed recombinant viral vector was propagated on Vero host cells at 37 ° C, 5% CO 2 , and the multiplicity of infection was 0.1. After harvesting, the cell debris was removed with a 0.65 ⁇ m filter, and then purified by high speed centrifugation at 13,000 rpm to obtain 1 ⁇ 10. A virus suspension of 8 pfu/mL was used for animal experiments.
  • the recombinant herpes simplex virus was constructed according to the method of Example 1, except that the inserted foreign gene included in the 5' to 3' end: CMV promoter, gene encoding GM-CSF (Gene ID: 12981) ) and BGH PolyA.
  • the recombinant herpes simplex virus was constructed according to the method of Example 1, except that the inserted foreign gene included in the 5' to 3' end: EF1 ⁇ promoter, gene encoding IL-2 (Gene ID: 16183) ) and TK PolyA.
  • the recombinant herpes simplex virus was constructed according to the method of Example 1, except that the inserted foreign gene included in the 5' to the 3' end: EF1 ⁇ promoter, gene encoding IL-12B (Gene ID: 16160) ), IRES sequence, gene encoding IL-12A (Gene ID: 16159) and TK PolyA.
  • the recombinant herpes simplex virus was constructed according to the method of Example 3, except that the ICP47 gene and the ICP6 gene of the wild-type HSV-1 virus were knocked out, and the artificial insertion of the ICP6 gene at the position of the HSV-1 virus was inserted.
  • the chemically synthesized foreign gene 1 is inserted into the artificially synthesized foreign gene 2 at the position of the ICP47 gene of the HSV-1 virus.
  • the construction of the recombinant herpes simplex virus was carried out in accordance with the method of Example 1, except that only the ICP34.5 gene and the ICP47 gene of the wild-type HSV-1 virus were knocked out, and the foreign gene was not inserted.
  • the recombinant herpes simplex virus was constructed according to the method of Example 1, except that the inserted foreign gene included in the 5' to 3' end: CMV promoter, gene encoding TNF- ⁇ (Gene ID: 21926) ) and BGH PolyA.
  • B16-BL6 cells were seeded in Balb/c mice to establish a melanoma mouse model.
  • Experimental mice with successful model establishment were randomly divided into experimental groups 1-14 and control groups.
  • Experimental groups 1-14 were administered virus suspensions obtained in Examples 1-8 and Comparative Examples 1-6, respectively, for 10 mice per group.
  • Each group of experimental mice was intratumorally injected with the virus suspension once every 3 days for a total of 5 injections of 100 ⁇ L each time, and the control group was injected with 100 ⁇ L of PBS. After 14 days of administration, the therapeutic effect was evaluated according to the relative tumor inhibition rate and tumor delay time.
  • the tumor volume calculation formula was: long diameter ⁇ short diameter 2 /2, relative tumor volume was the tumor volume after treatment / pre-treatment animal tumor volume, relative tumor
  • the inhibition rate TGI% (1-T/C) ⁇ 100%.
  • T/C% is the relative tumor growth rate, that is, the relative tumor volume percentage value of the treatment group and the control group.
  • T and C are the relative tumor volumes of the treatment group and the control group, respectively.
  • Experimental group 3 (Example 3) 76 Experimental group 4 (Example 4) 78 Experimental group 5 (Example 5) 71 Experimental group 6 (Example 6) 73 Experimental group 7 (Example 7) 69 Experimental group 8 (Example 8) 47 Experimental group 9 (Comparative Example 1) 63 Experimental group 10 (Comparative Example 2) 55 Experimental group 11 (Comparative Example 3) 60 Experimental group 12 (Comparative Example 4) 67 Experimental group 13 (Comparative Example 5) 49 Experimental group 14 (Comparative Example 6) 52
  • Tumor Delay Time refers to the number of days in which the treatment group was delayed compared to the control group when the tumor grew to 1200 mm 3 .
  • T is the number of days required for the average tumor volume of the treatment group to reach 1200 mm 3
  • C is the number of days required for the average tumor volume of the control group to reach 1200 mm 3 .
  • the larger the TC value the longer the delay time, indicating that the drug effect is better; and vice versa.
  • the experimental results show that the herpes simplex virus of the present invention exhibits a synergistic effect in tumor delay time (TC), and the specific data is shown in Table 2.
  • Experimental group 11 (Comparative Example 3) 4 Experimental group 12 (Comparative Example 4) 4.5 Experimental group 13 (Comparative Example 5) 3.5 Experimental group 14 (Comparative Example 6) 4
  • the experimental animals in the experimental groups 1-8 had a good mental state, and there was no significant difference with the mental state of the experimental animals in the comparative examples, indicating that the herpes simplex virus introduced into the cytokine in series according to the present invention has a significantly enhanced oncolytic effect, but Side effects were not increased by the increase in the introduction of cytokines.
  • the tumor treatment evaluation method was the same as Test Example 1.
  • the virus suspensions obtained in Examples 1-8 and Comparative Examples 1-6 were injected intratumorally, and the therapeutic effects were evaluated according to the relative tumor inhibition rate (calculation formula and Test Example 1) 14 days after administration.
  • the experimental results show that the herpes simplex virus of the present invention exhibits a synergistic effect in terms of relative tumor inhibition rate, and the specific data is shown in Table 3.
  • Tumor Delay Time refers to the number of days in which the treatment group was delayed compared to the control group when the tumor grew to 1200 mm 3 .
  • T is the number of days required for the average tumor volume of the treatment group to reach 1200 mm 3
  • C is the number of days required for the average tumor volume of the control group to reach 1200 mm 3 .
  • the larger the TC value the longer the delay time, indicating that the drug effect is better; and vice versa.
  • the experimental results show that the herpes simplex virus of the present invention exhibits a synergistic effect in tumor delay time (TC), and the specific data is shown in Table 4.
  • the experimental animals in the experimental groups 1-8 had a good mental state, and there was no significant difference with the mental state of the experimental animals in the comparative examples, indicating that the herpes simplex virus introduced into the cytokine in series according to the present invention has a significantly enhanced oncolytic effect, but Side effects were not increased by the increase in the introduction of cytokines.
  • H22 cells were intraperitoneally injected into Balb/c mice after resuscitation. One week later, ascites was inoculated to establish a liver cancer mouse model.
  • the tumor treatment evaluation method was the same as Test Example 1.
  • the virus suspensions obtained in Example 1, Example 4 and Comparative Examples 1-3 were injected intratumorally, and the therapeutic effects were evaluated according to the relative tumor inhibition rate (calculation formula and test example 1) 14 days after the administration.
  • the experimental results show that the herpes simplex virus of the present invention exhibits a synergistic effect on the relative tumor inhibition rate, and the specific data is shown in Table 5.
  • the experimental animals of the above experimental groups 1 and 4 had a good mental state, and there was no significant difference between the experimental animals and the mental state of the experimental animals in the comparative example, indicating that the herpes simplex virus of the present invention was introduced into the cytokine in series, and the oncolytic effect was remarkably enhanced, but Side effects were not increased by the increase in the introduction of cytokines.
  • the herpes simplex virus provided by the present invention exhibits a good antitumor effect, particularly for melanoma, breast cancer and liver cancer, by local tumors.
  • the method of intra-injection can not only produce a targeted anti-tumor effect, but also has no obvious side reaction after use, and has a good application prospect.

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Abstract

提供了一种重组单纯疱疹病毒及其制备方法和应用。该重组单纯疱疹病毒包括载体和编码至少两种细胞因子的外源基因，所述载体为缺失编码ICP34.5和ICP47的基因、以及可选择的缺失编码ICP6、TK和UNG的基因中的至少一种的单纯疱疹病毒，所述外源基因的插入位点为所述载体上缺失编码ICP34.5、ICP47、ICP6、TK和UNG的基因的位置中的至少一处。

Description

重组单纯疱疹病毒及其制备方法和应用 技术领域
本发明涉及基因工程领域，具体涉及一种重组单纯疱疹病毒、所述重组单纯疱疹病毒的制备方法和应用。
背景技术
传统肿瘤治疗包括手术、放疗和化疗，但是传统肿瘤治疗通常具有有效率低、副反应大且复发率高的缺陷，因此，肿瘤免疫治疗由于具有较好的疗效和较低的副反应的优势，近年来显示出了良好的应用前景，多种免疫治疗方法获得了上市批准并取得了良好的疗效。在《科学》杂志评选的2013年年度10大科学突破排行榜中，肿瘤免疫治疗高居榜首。其中，溶瘤病毒在肿瘤免疫治疗中发挥了巨大作用。
溶瘤病毒是指能选择性感染肿瘤细胞并在靶细胞内复制，最终导致肿瘤细胞裂解和死亡的一类病毒。这类病毒依靠其本身的特异性在肿瘤细胞中复制来裂解肿瘤细胞，细胞裂解后释放出来的病毒又可以进一步感染周围的肿瘤细胞，同时对正常细胞和组织则没有破坏作用，或影响较小。溶瘤病毒具有多重抗肿瘤作用机制，包括：1、直接裂解肿瘤细胞；2、破坏肿瘤血管；3、病毒复制表达的病毒蛋白具有直接细胞毒性作用；4、抗肿瘤免疫反应；5、增强肿瘤细胞对放化疗的敏感性。目前常用于抗肿瘤研究的溶瘤病毒包括单纯疱疹病毒(Herpes simplex virus，HSV)、腺病毒和牛痘病毒等。然而，由于溶瘤病毒的基因结构较为复杂，人们对其性质了解尚不够透彻，使得其应用存在不稳定因素。并且，由于受到机体内环境的影响，溶瘤病毒在机体内的复制能力往往也不够理想。这些均严重影响着溶瘤病毒的抗肿瘤效果。
细胞因子是免疫细胞互相联系的信号，用于调节免疫系统对抗原的反 应。细胞因子在肿瘤免疫治疗中具有重要的作用，其可以直接刺激肿瘤部位的免疫效应细胞的扩增并增强对肿瘤细胞的识别，同时激活全身系统的免疫反应。大量的动物肿瘤模型验证了细胞因子所具有的抗肿瘤活性。然而，虽然细胞因子可以通过直接给药进行抗肿瘤治疗，但是细胞因子的大量注射可能会给机体带来自身免疫疾病等副反应。
并且，由于肿瘤细胞具有多种免疫逃逸机制，加上体内环境的复杂性，目前任何单一治疗策略均无法很好地消除肿瘤细胞，因此，十分有必要寻找新的抗肿瘤效果好且副反应少的抗肿瘤方法。
发明内容
本发明的目的是为了克服现有技术存在的上述缺陷，提供一种重组单纯疱疹病毒及其制备方法和应用。本发明提供的重组单纯疱疹病毒具有良好的抗肿瘤效果，并且使用后副反应小，具有良好的应用前景。
为了实现上述目的，第一方面，本发明提供了一种重组单纯疱疹病毒，其中，所述重组单纯疱疹病毒包括载体和编码至少两种细胞因子的外源基因，所述载体为缺失编码ICP34.5和ICP47的基因、以及可选择的缺失编码ICP6、TK和UNG的基因中的至少一种的单纯疱疹病毒，所述外源基因的插入位点为所述载体上缺失编码ICP34.5、ICP47、ICP6、TK和UNG的基因的位置中的至少一处。
第二方面，本发明还提供了用于制备上述单纯疱疹病毒的方法，该方法包括：将单纯疱疹病毒中编码ICP34.5和ICP47的基因敲除、以及可选择地敲除编码ICP6、TK和UNG的基因中的至少一种，并插入编码至少两种细胞因子的外源基因，其中，所述外源基因的插入位点为所述载体上敲除编码ICP34.5、ICP47、ICP6、TK和UNG的基因的位置中的至少一处。
第三方面，本发明还提供了上述重组单纯疱疹病毒和/或由上述方法制 备得到的单纯疱疹病毒在制备用于预防和/或治疗肿瘤的药物中的应用。
本发明通过以缺失编码感染细胞多肽34.5(infected cell Polypetide 34.5，ICP34.5)的基因和编码ICP47的基因、以及可选择的缺失编码ICP6的基因、编码胸苷激酶(thymidine kinase，TK)的基因和编码尿嘧啶N-糖基化酶(uracil-N-glycosylase，UNG)的基因中的至少一种的单纯疱疹病毒为载体，并在缺失上述基因的位置插入编码至少两种细胞因子的外源基因，特别是插入GM-CSF、IL-2和IL-12中的至少两种的基因，获得了稳定表达的重组单纯疱疹病毒。并且，该重组单纯疱疹病毒表现出良好的抗肿瘤效果，特别是对于黑色素细胞瘤和乳腺癌，通过局部瘤内注射的方式，不仅可以产生靶向抗肿瘤的效果，而且使用后副反应小，具有良好的应用前景。
具体实施方式
在本文中所披露的范围的端点和任何值都不限于该精确的范围或值，这些范围或值应当理解为包含接近这些范围或值的值。对于数值范围来说，各个范围的端点值之间、各个范围的端点值和单独的点值之间，以及单独的点值之间可以彼此组合而得到一个或多个新的数值范围，这些数值范围应被视为在本文中具体公开。
第一方面，本发明提供了一种重组单纯疱疹病毒，其中，所述重组单纯疱疹病毒包括载体和编码至少两种细胞因子的外源基因，所述载体为缺失编码ICP34.5和ICP47的基因、以及可选择的缺失编码ICP6、TK和UNG的基因中的至少一种的单纯疱疹病毒，所述外源基因的插入位点为所述载体上缺失编码ICP34.5、ICP47、ICP6、TK和UNG的基因的位置中的至少一处。
希望说明的是，所述外源基因的插入位点为缺失基因的位置中的至少一处。例如，如果编码ICP34.5、ICP47、ICP6和TK的基因缺失，插入位点为缺失处的任意一处，而不是编码UNG的基因处。至少两种细胞因子可以连 接后插入同一位点处，也可以分别插入不同位点。优选地，所述细胞因子经连接后插入同一位点。
在本发明中，所述编码ICP34.5的基因、编码ICP47的基因、编码ICP6的基因、编码TK的基因和编码UNG的基因均为本领域技术人员所公知，并且也能够通过登录相关的数据库查到，例如，可以通过登录GenBank数据库查询到相关的核苷酸序列，这些均是本领域技术人员具有的常规技术手段，本发明在此不再赘述。
根据本发明，所述细胞因子可以为白细胞介素(interleukin，IL)、集落刺激因子(colony stimulating factor，CSF)、干扰素(Interferon，IFN)、肿瘤坏死因子(tumor necrosis factor，TNF)、转化生长因子、生长因子和趋化因子中的至少两种，优选为GM-CSF、IL-2、IL-7、IL-12、IL-15、IL-18、IL-21、TNF-α、IFN-γ、IFN-α和IFN-β中的至少两种，更优选为GM-CSF、IL-2和IL-12中的至少两种。
在本发明中，编码细胞因子的基因均可以通过登录相关数据库查询到，例如，可以通过登录GenBank数据库查询到相关的核酸序列。另外，根据获得的重组单纯疱疹病毒的应用对象的不同，编码细胞因子的基因可以有不同的来源。例如，当获得的重组单纯疱疹病毒的应用对象为人时，编码细胞因子的基因可以是人源的；当获得的重组单纯疱疹病毒的应用对象为小鼠时，编码细胞因子的基因可以是小鼠源的。
具体的，当获得的重组单纯疱疹病毒的应用对象为人时，编码GM-CSF的基因的Gene ID为1437，编码IL-2的基因的Gene ID为3558，编码IL-12A的基因的Gene ID为3592，编码IL-12B的基因的Gene ID为3593，编码TNF-α的基因的Gene ID为7124。
当获得的重组单纯疱疹病毒的应用对象为小鼠时，编码GM-CSF的基因的Gene ID为12981，编码IL-2的基因的Gene ID为16183，编码IL-12A的 基因的Gene ID为16159，编码IL-12B的基因的Gene ID为16160，编码TNF-α的基因的Gene ID为21926。
根据本发明，为了能够有效地对编码细胞因子的基因进行独立地翻译和表达，本发明的外源基因还优选包括启动子、起始密码子和终止密码子，以及可选的连接序列和/或PolyA序列，在这样优选的情况下，在所述重组单纯疱疹病毒感染宿主细胞并进行自身基因的表达时，能够转录出独立的且完整的目的mRNA片段，从而能够有效且独立地对目的基因进行翻译。
在本发明的一种优选的实施方式中，当在载体的一个位点(如，载体上缺失编码ICP34.5的基因的位置，或载体上缺失编码ICP47的基因的位置)插入编码至少两种细胞因子的外源基因时，为了使编码其中一种细胞因子的外源基因各自单独表达，编码其中一种细胞因子的外源基因各自之间还包括连接序列。优选地，所述连接序列为IRES序列。
在本发明的另一种优选的实施方式中，当在载体的一个位点(如，载体上缺失编码ICP34.5的基因的位置，或载体上缺失编码ICP47的基因的位置)插入编码至少两种细胞因子的外源基因时，为了使编码其中一种细胞因子的外源基因各自单独表达，编码其中一种细胞因子的外源基因各自具有独立的表达框，且各独立的表达框分别具有启动子、编码细胞因子的序列和PolyA序列。
根据本发明，对所述启动子的种类没有特别的限定，只要可以控制外源基因的转录即可，在优选的情况下，其中，所述启动子选自CMV启动子、EF1α启动子、SV40启动子、RSV启动子和MMTV启动子中的至少一种，优选为CMV启动子和/或EF1α启动子。
在本发明中，所述外源基因还可以包括标记基因(例如，编码β-半乳糖苷酶、荧光素酶、绿色荧光蛋白或其它荧光蛋白的基因)。并且，所述外源基因还可以包括通常与转录序列相关的相关转录调控序列，例如，聚腺苷酸 化位点、Kozak序列、WPRE和下游增强子元件。这些均为本领域技术人员所公知，本发明在此不再赘述。
在本发明中，对所述单纯疱疹病毒的种类没有特别的限定，可以为本领域的常规选择，但是为了更好地实现稳定表达细胞因子的目的，所述单纯疱疹病毒优选为I型单纯疱疹病毒。本发明对所述单纯疱疹病毒的来源也没有特别的限定，可以通过常规的商购获得，也可以通过实验室自行分离获得。
本发明上述重组单纯疱疹病毒的肿瘤治疗效果显著优于单独使用仅插入一种细胞因子的重组单纯疱疹病毒，插入二种以上细胞因子的技术方案产生的治疗效果是协同的。
第二方面，本发明还提供了一种重组单纯疱疹病毒的制备方法，该方法包括：将单纯疱疹病毒中编码ICP34.5和ICP47的基因敲除、以及可选择地敲除编码ICP6、TK和UNG的基因中的至少一种，并插入编码至少两种细胞因子的外源基因，其中，所述外源基因的插入位点为所述载体上敲除编码ICP34.5、ICP47、ICP6、TK和UNG的基因的位置中的至少一处。
在本发明中，以上编码ICP、TK和UNG的基因的敲除可以采用本领域常规的各种方法，本发明对此并没有特别的限制，例如，通过同源重组的方式，通过定点敲除的方式；或者，还可以通过CRISPR的方式定点敲除。在了解了本发明的发明目的以及本发明使用的病毒载体的前提下，本领域技术人员根据其掌握的常规技术手段能够实现对所述编码ICP的基因的敲除。
在本发明中，外源基因的插入也可以采用本领域常规的各种方法，所述插入的方式可以为直接在选定的插入位点插入所述目的基因，例如，可以通过CRISPR的方式插入，也可以通过同源重组的方式替换部分碱基序列从而插入所述目的基因，本发明优选后者。
在本发明中，重组单纯疱疹病毒与正常单纯疱疹病毒一样，也需要在宿主细胞内完成其生活史，因此所述重组病毒的传代增殖需要在宿主细胞中进 行。所述宿主细胞可以是各种能够培养本发明病毒载体和/或重组病毒的宿主细胞，例如，非洲绿猴肾细胞(Vero细胞)、仓鼠肾细胞(BHK细胞)、原代兔肾细胞、鸡胚细胞、羊膜细胞、人宫颈癌细胞(Hela细胞)以及人胚肺二倍体成纤维细胞(WI-38细胞)。
另外，本发明还提供了一种感染有病毒的细胞，其中，所述病毒为上述重组单纯疱疹病毒，所述细胞具有表达编码ICP34.5的基因和编码ICP47的基因、以及可选择的表达编码ICP6、TK和UNG的基因中的至少一种的基因。
在本发明中，所述细胞的具体选择可以参照如上所列举的关于宿主细胞的选择，在此不再赘述。
第三方面，本发明还提供了上述重组单纯疱疹病毒和/或由上述方法制备得到的重组单纯疱疹病毒在制备用于预防和/或治疗肿瘤的药物中的应用。优选地，所述肿瘤为黑色素细胞瘤、脑胶质瘤、头颈部肿瘤、肝癌、卵巢癌、前列腺癌、乳腺癌、肺癌、结直肠癌、肾细胞癌、胃癌、胰腺癌、淋巴瘤和膀胱癌中的至少一种。
第四方面，本发明涉及一种基因工程改造的单纯疱疹病毒，包含ICP34.5和ICP47基因的缺失和在ICP34.5和/或ICP47基因缺失部位的至少二种细胞因子基因的串联导入，该导入的基因选自：GM-CSF、IL-2、IL-7、IL-12、IL-15、IL-18、IL-21、TNF-α、IFN-γ、IFN-α和IFN-β，其中，该基因工程改造的单纯疱疹病毒呈现协同溶瘤效果(肿瘤治疗效果)。本发明所使用的术语“协同”是指串联导入二种以上促进(增强)免疫反应的细胞因子的基因工程改造的单纯疱疹病毒所呈现的溶瘤效果(肿瘤治疗效果)大于分开使用导入一种促进免疫反应的细胞因子的基因工程改造的单纯疱疹病毒。
本发明所述的溶瘤效果或肿瘤治疗效果的“协同性”的证明是串联导入增强免疫的细胞因子时所实现的效果大于分开导入所述细胞因子所致效果 的和。本发明重组单纯疱疹病毒或基因工程改造的单纯疱疹病毒所呈现的协同效果基于本申请实施例所使用的方法确定。也可以使用本领域其它判断方法判断，例如使用Chou和Talalay组合法和剂量-效果分析法(Chou和Talalay(1984)Adv.Enzyme Regul.22：27-55；Chou和Talalay，“New Avenues in Developmental Cancer Chemotherapy”，Academic Press，1987，第2章)判断。
本发明的技术方案中所述“串联”导入是指两种以上的细胞因子，例如促进(增强)免疫反应的细胞因子导入单纯疱疹病毒的同一基因缺失位点，该缺失位点选自ICP34.5、ICP47、ICP6、TK和UNG中的一处，或者，两种以上的细胞因子，例如促进(增强)免疫反应的细胞因子导入单纯疱疹病毒的不同基因缺失位点，该缺失位点选自ICP34.5、ICP47、ICP6、TK和UNG。通过串联导入，本发明的基因工程改造的单纯疱疹病毒同时携带二种以上的、优选二种、三种、四种编码促进(增强)免疫反应的细胞因子的基因。
在本发明一个优选的实施方案中，重组单纯疱疹病毒或基因工程改造的单纯疱疹病毒导入了选自如下的细胞因子中的二种、三种或四种：GM-CSF、IL-2、IL-12、TNF-α和IFN-γ。
在优选的实施方案中，上述单纯疱疹病毒为I型单纯疱疹病毒。
第五方面，本发明还涉及包含上述重组单纯疱疹病毒或基因工程改造的单纯疱疹病毒的储存液和宿主细胞以及药物组合物。同时，还涉及上述重组单纯疱疹病毒或基因工程改造的单纯疱疹病毒的储存液和宿主细胞以及药物组合物在制备治疗肿瘤，尤其是实体瘤，优选黑色素细胞瘤、脑胶质瘤、头颈部肿瘤、肝癌、肺癌、结直肠癌、肾细胞癌、胃癌、胰腺癌、淋巴瘤、膀胱癌、卵巢癌、前列腺癌和乳腺癌的药物中的用途。
第六方面，本发明还涉及一种治疗肿瘤，尤其是实体瘤，优选黑色素细胞瘤、脑胶质瘤、头颈部肿瘤、肝癌、肺癌、结直肠癌、肾细胞癌、胃癌、胰腺癌、淋巴瘤、膀胱癌、卵巢癌、前列腺癌和乳腺癌的方法，包括给药受 试者有效量的上述重组单纯疱疹病毒或基因工程改造的单纯疱疹病毒。在优选实施方案中，本发明的单纯疱疹病毒通过肿瘤局部给药治疗，优选通过导管或注射，将所述单纯疱疹病毒导入肿瘤组织内(瘤内给药)进行治疗。
以下将通过实施例对本发明进行详细描述。
在以下实施例和对比例中：
Vero细胞购自ATCC，货号为CCL-81；
外源基因的人工化学合成由苏州金唯智生物科技有限公司进行；
B16-BL6细胞和4T1细胞来自军事医学科学院，H22细胞来源于中国典型培养物保藏中心细胞库，Balb/c小鼠来自军事医学科学院和北京维通利华实验动物技术有限公司。
实施例1
本实施例用于说明本发明提供的重组单纯疱疹病毒的构建。
按照申请号2004100064921，授权公告号CN1283803C的专利申请中记载的方法将野生型HSV-1病毒(其基因序列的GenBank号：NC_001806，下同)的ICP34.5基因和ICP47基因敲除，并在HSV-1病毒的敲除ICP34.5基因的位置插入人工化学合成的外源基因，所不同的是本实施例采用Vero细胞为宿主细胞。该外源基因从5’端至3’端依次包括：CMV启动子、编码GM-CSF的基因(Gene ID：12981)、BGH PolyA、EF1α启动子、编码IL-2的基因(Gene ID：16183)和TK PolyA。在北京三博远志公司测序鉴定GM-CSF和IL-2编码基因正确插入到单纯疱疹病毒载体中，以获得重组病毒载体。构建成功的重组病毒载体在Vero宿主细胞上于37℃、5％CO 2下增殖，感染复数为0.1，收获后用0.65μm滤器去除细胞碎片，然后在13000rpm条件下高速离心纯化，得到滴度为1×10 8pfu/mL的病毒悬液用于动物实验。
实施例2
本实施例用于说明本发明提供的重组单纯疱疹病毒的构建。
按照实施例1中的方法进行重组单纯疱疹病毒的构建，不同的是，插入的外源基因从5’端至3’端依次包括：CMV启动子、编码GM-CSF的基因(Gene ID：12981)、BGH PolyA、EF1α启动子、编码IL-12B的基因(Gene ID：16160)、IRES序列、编码IL-12A的基因(Gene ID：16159)和TK PolyA。
实施例3
本实施例用于说明本发明提供的重组单纯疱疹病毒的构建。
按照申请号2004100064921，授权公告号CN1283803C的专利申请中记载的方法将野生型HSV-1病毒的ICP34.5基因和ICP47基因敲除，并且，在HSV-1病毒的敲除ICP34.5基因的位置插入人工化学合成的外源基因1，在HSV-1病毒的敲除ICP47基因的位置插入人工化学合成的外源基因2，所不同的是本实施例采用Vero细胞为宿主细胞。其中，外源基因1从5’端至3’端依次包括：EF1α启动子、编码IL-2的基因(Gene ID：16183)和TK PolyA。外源基因2从5’端至3’端依次包括：CMV启动子、编码GM-CSF的基因(Gene ID：12981)、BGH PolyA。在北京三博远志公司测序鉴定GM-CSF和IL-2编码基因正确插入到单纯疱疹病毒载体中，以获得重组病毒载体。构建成功的重组病毒载体在Vero宿主细胞上于37℃、5％CO 2下增殖，感染复数为0.1，收获后用0.65μm滤器去除细胞碎片，然后在13000rpm条件下高速离心纯化，得到1×10 8pfu/mL的病毒悬液用于动物实验。
实施例4
本实施例用于说明本发明提供的重组单纯疱疹病毒的构建。
按照申请号2004100064921，授权公告号CN1283803C的专利申请中记载的方法将野生型HSV-1病毒的ICP34.5基因和ICP47基因敲除，并且，在HSV-1病毒的敲除ICP34.5基因的位置插入人工化学合成的外源基因1，在HSV-1病毒的敲除ICP47基因的位置插入人工化学合成的外源基因2。其中，外源基因1从5’端至3’端依次包括：EF1α启动子、编码IL-12B的基因(Gene  ID：16160)、IRES序列、编码IL-12A的基因(Gene ID：16159)和TK PolyA。外源基因2从5’端至3’端依次包括：CMV启动子、编码GM-CSF的基因(Gene ID：12981)和BGH PolyA。在北京三博远志公司测序鉴定GM-CSF、IL-12A和IL-12B的编码基因正确插入到单纯疱疹病毒载体中，以获得重组病毒载体。构建成功的重组病毒载体在Vero宿主细胞上于37℃、5％CO 2下增殖，感染复数为0.1，收获后用0.65μm滤器去除细胞碎片，然后在13000rpm条件下高速离心纯化，得到1×10 8pfu/mL的病毒悬液用于动物实验。
实施例5
本实施例用于说明本发明提供的重组单纯疱疹病毒的构建。
按照实施例1中的方法进行重组单纯疱疹病毒的构建，不同的是，将野生型HSV-1病毒的ICP34.5基因、ICP47基因和ICP6基因敲除，以获得重组病毒载体。
实施例6
本实施例用于说明本发明提供的重组单纯疱疹病毒的构建。
按照实施例1中的方法进行重组单纯疱疹病毒的构建，不同的是，外源基因的插入位点是HSV-1病毒的敲除ICP47基因的位置。
实施例7
本实施例用于说明本发明提供的重组单纯疱疹病毒的构建。
按照实施例1中的方法进行重组单纯疱疹病毒的构建，不同的是，插入的外源基因从5’端至3’端依次包括：CMV启动子、编码GM-CSF的基因(Gene ID：12981)、BGH PolyA、EF1α启动子、编码TNF-α的基因(Gene ID：21926)和TK PolyA。
实施例8
本实施例用于说明本发明提供的重组单纯疱疹病毒的构建。
按照申请号2004100064921，授权公告号CN1283803C的专利申请中记 载的方法将野生型HSV-1病毒的ICP34.5基因和ICP47基因敲除，并且，在HSV-1病毒的敲除ICP34.5基因的位置插入人工化学合成的外源基因1，在HSV-1病毒的敲除ICP47基因的位置插入人工化学合成的外源基因2。其中，外源基因1从5’端至3’端依次包括：EF1α启动子、编码IL-12B的基因(Gene ID：16160)、IRES序列、编码IL-12A的基因(Gene ID：16159)和TK PolyA。外源基因2从5’端至3’端依次包括：CMV启动子、编码TNF-α的基因(Gene ID：21926)和BGH PolyA。在北京三博远志公司测序鉴定IL-12A、IL-12B和TNF-α的编码基因正确插入到单纯疱疹病毒载体中，以获得重组病毒载体。构建成功的重组病毒载体在Vero宿主细胞上于37℃、5％CO 2下增殖，感染复数为0.1，收获后用0.65μm滤器去除细胞碎片，然后在13000rpm条件下高速离心纯化，得到1×10 8pfu/mL的病毒悬液用于动物实验。
对比例1
按照实施例1中的方法进行重组单纯疱疹病毒的构建，不同的是，插入的外源基因从5’端至3’端依次包括：CMV启动子、编码GM-CSF的基因(Gene ID：12981)和BGH PolyA。
对比例2
按照实施例1中的方法进行重组单纯疱疹病毒的构建，不同的是，插入的外源基因从5’端至3’端依次包括：EF1α启动子、编码IL-2的基因(Gene ID：16183)和TK PolyA。
对比例3
按照实施例1中的方法进行重组单纯疱疹病毒的构建，不同的是，插入的外源基因从5’端至3’端依次包括：EF1α启动子、编码IL-12B的基因(Gene ID：16160)、IRES序列、编码IL-12A的基因(Gene ID：16159)和TK PolyA。
对比例4
按照实施例3中的方法进行重组单纯疱疹病毒的构建，不同的是，将野 生型HSV-1病毒的ICP47基因和ICP6基因敲除，并在HSV-1病毒的敲除ICP6基因的位置插入人工化学合成的外源基因1，在HSV-1病毒的敲除ICP47基因的位置插入人工化学合成的外源基因2。
对比例5
按照实施例1中的方法进行重组单纯疱疹病毒的构建，不同的是，仅将野生型HSV-1病毒的ICP34.5基因和ICP47基因敲除，未插入外源基因。
对比例6
按照实施例1中的方法进行重组单纯疱疹病毒的构建，不同的是，插入的外源基因从5’端至3’端依次包括：CMV启动子、编码TNF-α的基因(Gene ID：21926)和BGH PolyA。
测试例1
将B16-BL6细胞接种于Balb/c小鼠，建立黑色素瘤小鼠模型。将模型建立成功的实验小鼠随机分成实验组1-14和对照组。实验组1-14分别给予实施例1-8和对比例1-6得到的病毒悬液，每组10只小鼠。各组实验小鼠每3天瘤内注射病毒悬液一次，共注射5次，每次给药100μL，对照组注射100μL PBS。给药14天后根据相对肿瘤抑制率和肿瘤延迟时间进行疗效评价，肿瘤体积计算公式为：长径×短径 2/2，相对肿瘤体积为治疗后动物瘤体积/治疗前动物瘤体积，相对肿瘤抑制率TGI％＝(1-T/C)×100％。T/C％为相对肿瘤增值率，即治疗组和对照组相对肿瘤体积百分比值。T和C分别为治疗组和对照组的相对肿瘤体积。实验结果显示，本发明单纯疱疹病毒在相对肿瘤抑制率方面呈现出协同效果，具体数据参见表1。
表1：
组别 TGI％
对照组(PBS) ---
实验组1(实施例1) 81
实验组2(实施例2) 85
实验组3(实施例3) 76
实验组4(实施例4) 78
实验组5(实施例5) 71
实验组6(实施例6) 73
实验组7(实施例7) 69
实验组8(实施例8) 47
实验组9(对比例1) 63
实验组10(对比例2) 55
实验组11(对比例3) 60
实验组12(对比例4) 67
实验组13(对比例5) 49
实验组14(对比例6) 52
注：“---”表示无任何肿瘤抑制作用，因此无法得到相对肿瘤抑制率。
本实验测定了本发明单纯疱疹病毒对肿瘤延迟时间(T-C)的影响。肿瘤延迟时间(T-C)指肿瘤生长至1200mm 3时，治疗组比对照组延迟的天数。T为治疗组的平均肿瘤体积达到1200mm 3时所需天数，C为对照组平均瘤体积达到1200mm 3时所需天数。T-C值越大，延迟时间越长，说明药效越好；反之亦然。实验结果显示，本发明单纯疱疹病毒在肿瘤延迟时间(T-C)方面呈现出协同效果，具体数据参见表2。
表2：
组别 T-C(天数)
对照组(PBS) ---
实验组1(实施例1) 10.5
实验组2(实施例2) 13
实验组3(实施例3) 8
实验组4(实施例4) 9
实验组5(实施例5) 5
实验组6(实施例6) 5
实验组7(实施例7) 4.5
实验组8(实施例8) 3
实验组9(对比例1) 4.5
实验组10(对比例2) 4
实验组11(对比例3) 4
实验组12(对比例4) 4.5
实验组13(对比例5) 3.5
实验组14(对比例6) 4
副作用评估：
在实验过程中，实验组1-8中的实验动物精神状态良好，与对比例中的实验动物精神状态无明显差异，说明本发明串联导入细胞因子的单纯疱疹病毒，溶瘤效果显著增强，但副作用没有因导入细胞因子的增加而增加。
测试例2
将4T1细胞接种于Balb/c小鼠，建立乳腺癌小鼠模型。肿瘤治疗评估方法同测试例1。瘤内注射实施例1-8和对比例1-6得到的病毒悬液，给药14天后根据相对肿瘤抑制率(计算公式同测试例1)进行疗效评价。实验结果显示，本发明单纯疱疹病毒在相对肿瘤抑制率方面呈现出协同效果，具体数据参见表3。
表3：
组别 TGI％
对照组(PBS) ---
实验组1(实施例1) 83
实验组2(实施例2) 89
实验组3(实施例3) 80
实验组4(实施例4) 78
实验组5(实施例5) 73
实验组6(实施例6) 73
实验组7(实施例7) 68
实验组8(实施例8) 51
实验组9(对比例1) 69
实验组10(对比例2) 58
实验组11(对比例3) 64
实验组12(对比例4) 61
实验组13(对比例5) 42
实验组14(对比例6) 55
注：“---”表示无任何肿瘤抑制作用，因此无法得到相对肿瘤抑制率。
本实验测定了本发明单纯疱疹病毒对肿瘤延迟时间(T-C)的影响。肿瘤延迟时间(T-C)指肿瘤生长至1200mm 3时，治疗组比对照组延迟的天数。T为治疗组的平均肿瘤体积达到1200mm 3时所需天数，C为对照组平均瘤体积达到1200mm 3时所需天数。T-C值越大，延迟时间越长，说明药效越好；反之亦然。实验结果显示，本发明单纯疱疹病毒在肿瘤延迟时间(T-C)方面呈现出协同效果，具体数据参见表4。
表4：
组别 T-C(天数)
对照组(PBS) ---
实验组1(实施例1) 13
实验组2(实施例2) 15
实验组3(实施例3) 11.5
实验组4(实施例4) 10
实验组5(实施例5) 7
实验组6(实施例6) 6.5
实验组7(实施例7) 5.5
实验组8(实施例8) 3.5
实验组9(对比例1) 5
实验组10(对比例2) 4
实验组11(对比例3) 5.5
实验组12(对比例4) 4.5
实验组13(对比例5) 3.5
实验组14(对比例6) 4
副作用评估：
在实验过程中，实验组1-8中的实验动物精神状态良好，与对比例中的实验动物精神状态无明显差异，说明本发明串联导入细胞因子的单纯疱疹病毒，溶瘤效果显著增强，但副作用没有因导入细胞因子的增加而增加。
测试例3
H22细胞复苏后腹腔注射于Balb/c小鼠，1周后抽取腹水接种建立肝癌小鼠模型。肿瘤治疗评估方法同测试例1。瘤内注射实施例1、实施例4和对比例1-3得到的病毒悬液，给药14天后根据相对肿瘤抑制率(计算公式同测试例1)进行疗效评价。实验结果显示，本发明单纯疱疹病毒在相对肿瘤抑制率方面呈现出协同效果，具体数据参见表5。
表5：
组别 TGI％
对照组(PBS) ---
实验组1(实施例1) 79
实验组4(实施例4) 77
实验组9(对比例1) 58
实验组10(对比例2) 57
实验组11(对比例3) 54
从以上可见，实施例组的肿瘤治疗效果明显优于对比例，呈现协同效应。
副作用评估：
在实验过程中，上述实验组1和4的实验动物精神状态良好，与对比例中的实验动物精神状态无明显差异，说明本发明串联导入细胞因子的单纯疱疹病毒，溶瘤效果显著增强，但副作用没有因导入细胞因子的增加而增加。
通过将以上实施例1-8与对比例1-6的结果相比较可知，本发明提供的单纯疱疹病毒表现出良好的抗肿瘤效果，特别是对于黑色素细胞瘤、乳腺癌和肝癌，通过局部瘤内注射的方式，不仅可以产生靶向抗肿瘤的效果，而且使用后没有产生明显的副反应，具有良好的应用前景。
以上详细描述了本发明的优选实施方式，但是，本发明并不限于此。在本发明的技术构思范围内，可以对本发明的技术方案进行多种简单变型，包括各个技术特征以任何其它的合适方式进行组合，这些简单变型和组合同样应当视为本发明所公开的内容，均属于本发明的保护范围。

Claims (21)

  1. 一种重组单纯疱疹病毒，其特征在于，所述重组单纯疱疹病毒包括载体和编码至少两种细胞因子的外源基因，所述载体为缺失编码ICP34.5和ICP47的基因、以及可选择的缺失编码ICP6、TK和UNG的基因中的至少一种的单纯疱疹病毒，所述外源基因的插入位点为所述载体上缺失编码ICP34.5、ICP47、ICP6、TK和UNG的基因的位置中的至少一处。
  2. 根据权利要求1所述的重组单纯疱疹病毒，其中，所述载体为缺失编码ICP34.5和ICP47的基因、以及可选择的缺失编码ICP6的基因的单纯疱疹病毒，所述外源基因的插入位点为所述载体上缺失编码ICP34.5和/或ICP47的基因的位置。
  3. 根据权利要求1所述的重组单纯疱疹病毒，其中，所述细胞因子为白细胞介素、集落刺激因子、干扰素、肿瘤坏死因子、转化生长因子、生长因子和趋化因子中的至少两种，优选为GM-CSF、IL-2、IL-7、IL-12、IL-15、IL-18、IL-21、TNF-α、IFN-γ、IFN-α和IFN-β中的至少两种，更优选为GM-CSF、IL-2和IL-12中的至少两种。
  4. 根据权利要求1所述的重组单纯疱疹病毒，其中，所述外源基因还包括启动子、起始密码子和终止密码子，以及可选的连接序列。
  5. 根据权利要求3所述的重组单纯疱疹病毒，其中，所述启动子选自CMV启动子、EF1α启动子、SV40启动子、RSV启动子和MMTV启动子中的至少一种，优选为CMV启动子和/或EF1α启动子。
  6. 根据权利要求1所述的重组单纯疱疹病毒，其中，所述单纯疱疹病毒为I型单纯疱疹病毒。
  7. 一种制备权利要求1-6中任意一项所述的单纯疱疹病毒的方法，其特征在于，该方法包括：将单纯疱疹病毒中编码ICP34.5和ICP47的基因敲除、以及可选择地敲除编码ICP6、TK和UNG的基因中的至少一种，并插入编码至少两种细胞因子的外源基因，其中，所述外源基因的插入位点为所 述载体上敲除编码ICP34.5、ICP47、ICP6、TK和UNG的基因的位置中的至少一处。
  8. 权利要求1-6中任意一项所述的重组单纯疱疹病毒和/或由权利要求7所述的方法制备得到的重组单纯疱疹病毒在制备用于预防和/或治疗肿瘤的药物中的应用。
  9. 根据权利要求8所述的应用，其中，所述肿瘤为黑色素细胞瘤、脑胶质瘤、头颈部肿瘤、肝癌、肺癌、结直肠癌、肾细胞癌、胃癌、胰腺癌、淋巴瘤、膀胱癌、卵巢癌、前列腺癌和乳腺癌中的至少一种。
  10. 一种基因工程改造的单纯疱疹病毒，包含ICP34.5和ICP47基因的缺失和在ICP34.5和/或ICP47基因缺失部位的至少二种细胞因子基因的串联导入，该导入的基因选自：GM-CSF、IL-2、IL-7、IL-12、IL-15、IL-18、IL-21、TNF-α、IFN-γ、IFN-α和IFN-β，其中，该基因工程改造的单纯疱疹病毒呈现协同溶瘤效果。
  11. 权利要求10的基因工程改造的单纯疱疹病毒，还包括选自如下的一种或多种基因的缺失：编码ICP6的基因、编码TK的基因和编码UNG的基因，其中所述细胞因子基因的串联导入位点为所述编码ICP34.5、ICP47、ICP6、TK和UNG的基因缺失位置中的至少一处。
  12. 根据权利要求10或11所述的基因工程改造的单纯疱疹病毒，其中，所述细胞因子选自如下的至少二种：GM-CSF、IL-2、IL-12、TNF-α和IFN-γ。
  13. 根据权利要求10-12任一项所述的基因工程改造的单纯疱疹病毒，其中，所述单纯疱疹病毒为I型单纯疱疹病毒。
  14. 包含权利要求1-6任一项所述的重组单纯疱疹病毒或权利要求10-13任一项的基因工程改造的单纯疱疹病毒的宿主细胞。
  15. 包含权利要求1-6任一项所述的重组单纯疱疹病毒或权利要求10-13任一项的基因工程改造的单纯疱疹病毒的病毒储存液。
  16. 包含权利要求14的宿主细胞或权利要求15的病毒储存液的药物组 合物。
  17. 权利要求10-13任一项的基因工程改造的单纯疱疹病毒或权利要求14的宿主细胞在制备用于治疗实体肿瘤的药物中的应用。
  18. 根据权利要求17所述的应用，其中，所述肿瘤为黑色素细胞瘤、脑胶质瘤、头颈部肿瘤、肝癌、肺癌、结直肠癌、肾细胞癌、胃癌、胰腺癌、淋巴瘤、膀胱癌、卵巢癌、前列腺癌和乳腺癌中的至少一种。
  19. 一种治疗肿瘤的方法，包括给药受试者有效量的权利要求1-6任一项的重组单纯疱疹病毒或权利要求10-13任一项的基因工程改造的单纯疱疹病毒。
  20. 权利要求19所述的方法，其中，所述肿瘤为黑色素细胞瘤、脑胶质瘤、头颈部肿瘤、肝癌、肺癌、结直肠癌、肾细胞癌、胃癌、胰腺癌、淋巴瘤、膀胱癌、卵巢癌、前列腺癌和乳腺癌中的至少一种。
  21. 权利要求19或20所述的方法，其中，所述单纯疱疹病毒通过导管或注射瘤内给药。
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