WO2022001080A1 - 一种重组单纯疱疹病毒及其构建方法 - Google Patents

一种重组单纯疱疹病毒及其构建方法 Download PDF

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WO2022001080A1
WO2022001080A1 PCT/CN2021/070373 CN2021070373W WO2022001080A1 WO 2022001080 A1 WO2022001080 A1 WO 2022001080A1 CN 2021070373 W CN2021070373 W CN 2021070373W WO 2022001080 A1 WO2022001080 A1 WO 2022001080A1
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herpes simplex
simplex virus
gene
cancer
ifn
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French (fr)
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张旭辉
罗显麟
冯翠娟
唐锐华
陈小锋
李文佳
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东莞市东阳光生物药研发有限公司
广东东阳光药业有限公司
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Definitions

  • the present invention relates to the fields of biotechnology and gene therapy, in particular, the present invention relates to a recombinant herpes simplex virus, a method for constructing a recombinant herpes simplex virus, and pharmaceutical use.
  • Oncolytic virus therapy is a novel tumor treatment method that utilizes viruses to replicate in tumor cells specifically, then kill tumor cells, and stimulate the body to produce specific anti-tumor immune responses. Compared with other tumor treatment methods, oncolytic virus therapy has the characteristics of high replication efficiency, good killing effect and less toxic and side effects, and has become a new hot spot in the field of tumor treatment research.
  • IFN- ⁇ interferon gamma
  • NK cells and CTL cells a well-known role
  • IFN- ⁇ can induce the expression of CXC chemokines such as MIG and IP-10, which have been shown to be critical for T cell infiltration in tumors.
  • IFN- ⁇ stimulates the expression of tumor antigen-presenting MHC molecules to increase the immunogenicity of tumor cells sex, making them more susceptible to immune recognition and destruction. IFN- ⁇ also shows direct anticancer activity by inhibiting cell proliferation and promoting apoptosis.
  • IFN- ⁇ stimulates the expression of tumor antigen-presenting MHC molecules to increase the immunogenicity of tumor cells, making them more susceptible to immune recognition and destruction. IFN- ⁇ also shows direct anti-cancer activity by inhibiting cell proliferation and promoting cell apoptosis.
  • HSV1 carrying IFN- ⁇ there is no report on HSV1 carrying IFN- ⁇ .
  • a new oncolytic virus HSV- 1IFN- ⁇ (+) has the advantages of high efficiency and strong specificity, and can also induce a specific anti-tumor immune response and improve the sensitivity of tumor cells.
  • the present application aims to solve one of the above technical problems in the background art at least to a certain extent.
  • the present invention proposes a recombinant herpes simplex virus.
  • the ICP47 and double-copy ICP34.5 genes of the recombinant herpes simplex virus are knocked out, and the coding frame of the IFN- ⁇ gene is set at the position of the UL23 gene, and the IFN- ⁇ is selected from hIFN- ⁇ or mIFN- ⁇ .
  • Knockout of the replication non-essential gene ICP47 in the HSV genome can improve the expression of MHC-I on the surface of virus-infected tumor cells and the ability to present cellular antigens; knockout of double-copy ICP34.5 can make herpes simplex virus (HSV) in The replication in normal cells is limited, and it selectively replicates in tumor cells, thereby improving the safety of HSV virus medication.
  • HSV herpes simplex virus
  • the recombinant herpes simplex virus can continue to express IFN- ⁇ at a high level, which not only reproduces and infects tumor cells with high selectivity, but also has immune regulation. It acts to ensure that tumor cells are completely removed by the body's own immune response.
  • the IFN- ⁇ may be hIFN- ⁇ or mIFN- ⁇ .
  • the above-mentioned recombinant herpes simplex virus may further include at least one of the following additional technical features:
  • the coding frame of the IFN- ⁇ gene is set between nucleotides 387-521 of the UL23 gene sequence. It should be noted that the sequence coding of the UL23 gene described in this application is based on the first nucleotide of the initiation codon of the UL23 gene as the first nucleotide sequence. For the sequence of the UL23 gene, please refer to:
  • the hIFN- ⁇ gene coding frame is set between the 387-521 nucleotides of the UL23 gene sequence by knocking out the 388-520 nucleotides of the UL23 gene sequence, and then the The coding frame of hIFN- ⁇ gene was inserted between nucleotides 387-521 of the UL23 gene sequence.
  • the promoter comprises at least one selected from the group consisting of CMV, CAG, EF1 ⁇ , Rous Sarcoma Virus Long Terminal Repeat (RSV LTR), and Metallothionein I (MTI).
  • the promoter is CMV.
  • the HSV is HSV1.
  • the inventors found that HSV2 may cause genital infection, and then selected HSV1 to further improve the safety.
  • the hIFN- ⁇ gene coding frame has the nucleotide sequence shown below, 1) the nucleotide sequence shown in SEQ ID NO: 1; 2) compared with 1), it has at least 70%, Nucleotide sequences that are at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identical.
  • the recombinant herpes simplex virus has the nucleotide sequence shown below, a) the nucleotide sequence shown in SEQ ID NO: 2; b) compared with a), it has at least 70%, Nucleotide sequences that are at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identical.
  • the mIFN- ⁇ gene coding frame has the nucleotide sequence shown below, 1) the nucleotide sequence shown in SEQ ID NO: 3; 2) compared with 1), it has at least 70%, Nucleotide sequences that are at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identical.
  • the recombinant herpes simplex virus has the nucleotide sequence shown below, a) the nucleotide sequence shown in SEQ ID NO: 4; b) compared with a), it has at least 70%, Nucleotide sequences that are at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identical.
  • the present invention provides a method for constructing a recombinant herpes simplex virus.
  • the construction method includes the following steps:
  • Step 1 Construction of PCDNA3.1(+)-CMV-IFN- ⁇ -poly(A) plasmid: PUC57simple-IFN- ⁇ plasmid was digested with NheI/NotI at the same time, and the fragment was recovered. .1(+) vector is connected to create a plasmid PCDNA3.1(+)-CMV-IFN- ⁇ -poly(A) in which CMV initiates IFN- ⁇ gene expression and terminates the poly(A) signal;
  • Step 2 Construction of pMD18-T-UL23-HOM1-HOM2: PCR amplification of the homology arm UL23-HOM1 fragment upstream of the virus UL23 gene, and connection with the linearized pMD18-T vector by means of homologous recombination to construct a successful plasmid Named pMD18-T-UL23-HOM1; PCR amplification of the homology arm UL23-HOM2 fragment downstream of the viral UL23 gene, the plasmid pMD18-T-UL23-HOM1 was digested with EcoRI enzyme, and UL23-HOM2 was converted by homologous recombination The fragment was ligated with the linearized pMD18-T-UL23-HOM1 vector, and the successfully constructed plasmid was named pMD18-T-UL23-HOM1-HOM2;
  • Step 3 Construction of pMD18-T-UL23-HOM1-HOM2-CMV-IFN- ⁇ -poly(A): Amplify the CMV-IFN- ⁇ -poly(A) sequence, digest the plasmid pMD18-T-UL23 with XbaI enzyme -HOM1-HOM2, the CMV-IFN- ⁇ -poly(A) fragment was ligated with the linearized pMD18-T-UL23-HOM1-HOM2 vector by homologous recombination, and the successfully constructed plasmid was named pMD18-T-UL23 -HOM1-HOM2-CMV-IFN- ⁇ -poly(A);
  • Step 4 Construct a targeting plasmid targeting the LacZ gene: anneal the primers targeting LacZ to each other, digest the plasmid CRISPR/Cas9-sgRNA plasmid with BbsI enzyme, and align the overlapping targeting primers with the linearized CRISPR/Cas9-sgRNA The vectors are connected by viscous complementation, and the targeting plasmid CRISPR/Cas9-sgRNA-LacZ-12 or CRISPR/Cas9-sgRNA-LacZ-11 is successfully constructed;
  • Step 5 Homologous recombination of herpes simplex virus type I KOS strain mediated by CRISPR/Cas9 gene editing system in 293FT cells: the homologous plasmid pMD18-T-UL23-HOM1-HOM2-CMV- hIFN- ⁇ -poly(A), the targeting plasmid targeting LacZ gene in step 4, ICP34.5 gene and ICP47 gene were knocked out, and the KOS genome in which the UL23 gene was inactivated was co-transfected into 293FT cells to obtain IFN- ⁇ -expressing KOS genome. Recombinant virus HSV-1IFN- ⁇ (+).
  • the IFN- ⁇ is selected from hIFN- ⁇ or mIFN- ⁇ .
  • a recombinant virus HSV-1 hIFN- ⁇ (+) expressing hIFN- ⁇ , or a recombinant virus HSV-1 mIFN- ⁇ (+) expressing mIFN- ⁇ can be constructed.
  • the recombinant herpes simplex virus constructed according to the method of the embodiment of the present invention can selectively kill tumor cells and improve the sensitivity to tumor cells.
  • the above method may further include at least one of the following additional technical features:
  • step 2 in the upstream and downstream homology arms in step 2,
  • the primer pair of the upstream flanking sequence includes: the upstream sequence hrR3UL23HOM1-F1 is CCTGCAGGTCGACGAGATGCCGTAGTCAGGTTTAGTTCGTCCGGC;
  • the downstream sequence hrR3UL23HOM1-R1 is GGGATCCTCTAGAGACGCGCGATACCTTATGGGCAGCATGAC;
  • the primer pair of the downstream flanking sequence includes: the upstream sequence hrR3UL23HOM2-F1 is CCGGGTACCGAGCTCCCCATTGTTATCTGGGCGCTTGTCATTACCAC;
  • the downstream sequence hrR3UL23HOM2-R1 is TATGACCATGATTACTCTGGAGCATCCGCACGACTGCGGTGATATTA.
  • the upstream sequence hrR3UL23HOM-F1 is AAGGTATCGCGCGTCTTCCATTGACGTCAATGGGTGGAGTATTTA;
  • the downstream sequence hrR3UL23HOM1-R1-2 is GGTACCCGGGGATCCACGGGGAAAGCCGGCGAACGTGGCGAGAAA.
  • the recombinant virus HSV-1IFN- ⁇ (+) in step 5 is purified by one or more rounds of picking virus plaques.
  • step 5 the recombinant virus HSV-1IFN- ⁇ (+) is purified by three rounds of picking virus plaques.
  • the present invention provides a recombinant cell.
  • the transgenic cells carry the recombinant herpes simplex virus described above or the recombinant herpes simplex virus constructed by the above-described construction method.
  • the recombinant cells according to the embodiments of the present invention are obtained by introducing the aforementioned recombinant herpes simplex virus into recipient cells. After the aforementioned recombinant herpes simplex virus is introduced into the recipient cells, the oncolytic virus can be present in the recipient cells. It is packaged and replicated in large numbers, and IFN- ⁇ is consistently expressed at high levels.
  • the present invention provides a pharmaceutical composition.
  • the pharmaceutical composition comprises the aforementioned recombinant herpes simplex virus, the aforementioned recombinant herpes simplex virus constructed by the aforementioned construction method, or the aforementioned recombinant cell.
  • the present invention proposes that the aforementioned recombinant herpes simplex virus, the aforementioned recombinant herpes simplex virus constructed by the aforementioned construction method, the aforementioned recombinant cell or the aforementioned pharmaceutical composition are prepared Use in a medicament for the treatment or prevention of tumors.
  • the drug is used to selectively kill tumor cells.
  • the present invention provides a method of treating cancer.
  • the method comprises administering a therapeutically effective amount of the aforementioned recombinant herpes simplex virus, the aforementioned recombinant herpes simplex virus constructed by the aforementioned construction method, the aforementioned recombinant cell or the aforementioned pharmaceutical composition to those in need. individual.
  • the individual in need is a patient suffering from at least one of the following cancers: ovarian cancer, breast cancer, lung cancer, nasopharyngeal cancer, liver cancer, stomach cancer, esophageal cancer, colorectal cancer, Pancreatic cancer, melanoma, skin cancer, prostate cancer, cervical cancer, leukemia or brain tumor.
  • Fig. 1 is a plasmid map of pMD18-T-UL23-HOM1-HOM2-CMV-hIFN- ⁇ -poly(A) according to an embodiment of the present invention
  • Fig. 2 is the PCR gel electrophoresis detection diagram of the recombinant virus constructed according to the embodiment of the present invention
  • Fig. 3 is the sequence structure diagram of the recombinant virus HSV-1-UL23-mIFN- ⁇ constructed according to the embodiment of the present invention
  • Figure 4 is a sequence diagram of the recombinant virus HSV-1-UL23-hlFN- ⁇ constructed according to the embodiment of the present invention.
  • Fig. 5 is the PCR gel electrophoresis detection diagram of the target site of the recombinant virus constructed according to the embodiment of the present invention.
  • Fig. 6 is the inhibition curve of mIFN- ⁇ , hIFN- ⁇ , 2K on Skmel-28 tumor cells according to an embodiment of the present invention
  • Fig. 7 is the inhibition curve of mIFN- ⁇ , hIFN- ⁇ and 2K on Skov3 tumor cells according to an embodiment of the present invention.
  • Fig. 8 is the inhibition curve of mIFN- ⁇ , hIFN- ⁇ and 2K on Fadu tumor cells according to an embodiment of the present invention.
  • Fig. 9 is the inhibition curve of mIFN- ⁇ , hIFN- ⁇ and 2K on Hep3B2.1-7 tumor cells according to an embodiment of the present invention.
  • Figure 10 is the inhibition curve of mIFN- ⁇ , hIFN- ⁇ and 2K on ASPC-1 tumor cells according to an embodiment of the present invention.
  • Figure 11 is the inhibition curve of mIFN- ⁇ , hIFN- ⁇ , 2K on BXPC-3 tumor cells according to an embodiment of the present invention.
  • restriction endonuclease NheI/NotI purchased from Takara; hIFN- ⁇ gene: synthesized by GenScript; PCDNA3.1(+): purchased from (Takara); pMD18-T: purchased from Takara Company; EcoRI: purchased from Takara Company; XbaI: purchased from Takara Company; CRISPR/Cas9-sgRNA: purchased from (Takara Company);
  • first and second are only used for descriptive purposes, and should not be construed as indicating or implying relative importance or implying the number of indicated technical features. Thus, a feature delimited with “first”, “second” may expressly or implicitly include at least one of that feature.
  • plurality means at least two, such as two, three, etc., unless otherwise expressly and specifically defined.
  • IFN- ⁇ is also called ⁇ -hIFN or immune interferon or ⁇ interferon, which is a lymphokine with immunoregulatory effect.
  • IFN- ⁇ can induce the expression of CXC chemokines such as MIG and IP-10, which have been shown to be critical for T cell infiltration in tumors.
  • IFN- ⁇ is the major product of Th1-mediated immune responses and coordinates Th1 effector mechanisms as further activation of innate immunity (macrophages and NK cells) in a positive feedback loop.
  • hIFN- ⁇ is human ⁇ interferon
  • mIFN- ⁇ is murine ⁇ interferon.
  • the "hIFN- ⁇ gene coding frame" described in this application refers to a nucleic acid sequence capable of expressing functional hIFN- ⁇ , that is, the hIFN- ⁇ segment encoded by the nucleic acid sequence is capable of realizing the hIFN- ⁇ function
  • the segment, or in other words, the hIFN- ⁇ segment encoded by the nucleic acid sequence is an essential functional region of hIFN- ⁇ .
  • ICP47 and ICP34.5 are knocked out means that the ICP47 and ICP34.5 genes are silenced. significantly reduced.
  • the "UL23 gene inactivation" mentioned in this application refers to insertion inactivation, that is, after inserting an exogenous gene at the position of the UL23 gene, the original function of the UL23 gene is lost.
  • operably linked in this application refers to linking an exogenous gene to a vector, so that control elements in the vector, such as promoter sequences, etc., can exert their intended functions to regulate the transcription and translation of the exogenous gene.
  • the following examples utilize the CRISPR/Cas9 system to construct recombinant HSV-1 virus, which specifically includes plasmid construction, recombinant screening, and virus identification.
  • the biological activity of the recombinant HSV-1 virus specifically includes the expression of hIFN- ⁇ or mIFN- ⁇ , and the ability of the virus to infect and kill tumor cells.
  • Step 1 Construction of pcDNA3.1(+)-CMV-hIFN- ⁇ -poly(A) plasmid: PUC57simple-hIFN- ⁇ plasmid was digested with NheI/NotI at the same time, the fragment was recovered, and the pCDNA3 digested with NheI/NotI was digested with NheI/NotI. .1(+) vector is connected to create a plasmid PCDNA3.1(+)-CMV-hIFN- ⁇ -poly(A) in which CMV initiates the expression of hIFN- ⁇ gene and terminates the poly(A) signal;
  • Step 2 Construction of pMD18-T-UL23-HOM1-HOM2: PCR amplification of the homology arm UL23-HOM1 fragment upstream of the virus UL23 gene, and connection with the linearized pMD18-T vector by means of homologous recombination to construct a successful plasmid Named pMD18-T-UL23-HOM1; PCR amplification of the homology arm UL23-HOM2 fragment downstream of the viral UL23 gene, the plasmid pMD18-T-UL23-HOM1 was digested with EcoRI enzyme, and UL23-HOM2 was converted by homologous recombination The fragment was ligated with the linearized pMD18-T-UL23-HOM1 vector, and the successfully constructed plasmid was named pMD18-T-UL23-HOM1-HOM2;
  • Step 3 Construction of pMD18-T-UL23-HOM1-HOM2-CMV-hIFN- ⁇ -poly(A): Amplify the CMV-hIFN- ⁇ -poly(A) sequence, digest the plasmid pMD18-T-UL23 with XbaI enzyme -HOM1-HOM2, the CMV-hIFN- ⁇ -poly(A) fragment was ligated with the linearized pMD18-T-UL23-HOM1-HOM2 vector by homologous recombination, and the successfully constructed plasmid was named pMD18-T-UL23 -HOM1-HOM2-CMV-hIFN- ⁇ -poly(A);
  • Step 4 Construction of CRISPR/Cas9-sgRNA-LacZ-12 targeting plasmid: anneal and overlap the primers targeting LacZ, digest the plasmid CRISPR/Cas9-sgRNA plasmid with BbsI enzyme, and align the overlapping targeting primers with the linearized CRISPR
  • the /Cas9-sgRNA vector was connected by cohesive complementation, and the successfully constructed plasmid was named CRISPR/Cas9-s gRNA-LacZ-12, abbreviated as gRNA-LacZ-12, code-named S12.
  • CRISPR/Cas9-sgRNA-LacZ-12 we also screened out another targeting plasmid CRISPR/Cas9-sgRNA-LacZ-11 (abbreviated as gRNA-LacZ-11, codenamed S11 ), CRISPR/Cas9-sgRNA-LacZ-12 and CRISPR/Cas9-sgRNA-LacZ-11 differ in the sgRNAs.
  • homologous recombination of herpes simplex virus type I KOS strain mediated by CRISPR/Cas9 gene editing system in 293FT cells the homologous plasmid pMD18-T-UL23-HOM1-HOM2-CMV- hIFN- ⁇ -poly(A), CRISPR/Cas9-sgRNA-LacZ-12 plasmid targeting LacZ gene in step 4, and KOS genome knocked out of ICP34.5 gene, UL23 gene and ICP47 gene were co-transfected into 293FT cells to obtain Recombinant virus 2k-HSV-1-UL23-hlFN- ⁇ (+) expressing hIFN- ⁇ .
  • Step 1. 293FT cells are spread in 12-well plate
  • the recombinant herpes simplex virus carrying mIFN- ⁇ was constructed according to the same method in this example.
  • gRNA-LacZ-11 and gRNA-LacZ-12 were used to construct the following four recombinant viruses:
  • Virus infection (6-well plate, dilution of virus stock solution is 5 ⁇ 10 7 )
  • the primer is UL23HOM1 left-F2/B48431UL24R;
  • PCR product size HSV-1-UL23-hlFN- ⁇ (4585bp), HSV-1-UL23-mIFN- ⁇ (4552bp).
  • PCR products were detected by gel electrophoresis. As shown in Figure 2, two p3-generation recombinant viruses HSV-1-UL23-mIFN- ⁇ (S11-5-1-1#, S11-7-2- 1#), two p3 generation recombinant viruses HSV-1-UL23-hIFN- ⁇ (S11-2-1-1#, S12-8-2-1#) were used as the final screened viruses (named in this application).
  • HSV-1-UL23-mIFN- ⁇ (S11-5-1-1#, S11-7-2-1#) were sequenced respectively, as shown in Figure 3, the sequencing verified that UL23HOM1 is 618bp from the left to UL23HOM2 is 500bp from the right The sequence is complete without mutation, indicating that mIFN- ⁇ has been successfully inserted into the HSV-1-virus genome.
  • HSV-1-UL23-hIFN- ⁇ (S11-5-1-1#, S11-7-2-1#) were sequenced respectively, as shown in Figure 4, the sequencing verified that UL23HOM1 is 618bp from the left to UL23HOM2 is 500bp from the right The sequence is complete without mutation, indicating that hIFN- ⁇ has been successfully inserted into the HSV-1-virus genome.
  • mIFN- ⁇ refers to P3 generation 2k-HSV-1-UL23-mIFN- ⁇
  • hIFN- ⁇ refers to P3 generation 2k-HSV-1-UL23-hIFN- ⁇
  • 2k refers to HSV-1 /2K
  • cells are Skmel-28 (human malignant melanoma cells), Skov3 (human ovarian cancer cells), Fadu (human pharyngeal squamous cell carcinoma cells), Hep3B2.1-7 (liver cancer cells), ASPC (pancreatic cancer cells), BXPC (human pancreatic cancer cells).
  • N/A represents the non-linear fitting that could not be made due to poor dose-response relationship or untested, in which the fitting degree ⁇ 80% means the fitting degree is poor, and the corresponding MOI IC 50 value is for reference only.
  • Figure 6 shows that the three viruses hIFN- ⁇ , mIFN- ⁇ and 2k all have good killing effects on Skmel-28 tumor cells.
  • the killing ability of hIFN- ⁇ virus is slightly stronger than that of mIFN- ⁇ virus at 24h and 48h, and at 72h
  • the cells are basically all infected.
  • Figure 7 shows that the three viruses hIFN- ⁇ , mIFN- ⁇ and 2k all have good killing effects on Skov3 tumor cells, and the IC50 values at 48h are all far less than 0.6, and the killing ability of hIFN- ⁇ and mIFN- ⁇ viruses is lower than that of Skov3. higher than 2k virus.
  • Figure 8 shows that Fadu, hIFN- ⁇ , mIFN- ⁇ and 2k viruses all have good killing effects on Skov3 tumor cells, and the three have comparable killing abilities to Skov3 tumor cells.
  • Figure 9 shows that the three viruses hIFN- ⁇ , mIFN- ⁇ and 2k all have good killing effect on Hep3B2.1-7 tumor cells.
  • the killing ability of hIFN- ⁇ virus is slightly stronger than that of the other two viruses at 48 h.
  • the fitted IC50 of mIFN- ⁇ was very low, the virus was very sensitive to Hep3B2.1-7 cells, and the cells were all infected at 48h.
  • Figure 10 shows that the three viruses hIFN- ⁇ , mIFN- ⁇ and 2k all have good killing effect on ASPC-1 tumor cells, and the killing ability of 2K at 24h is slightly stronger than that of mIFN- ⁇ or hIFN- ⁇ , and the cells are basically dead at 48h. All infected.
  • Figure 11 shows that the three viruses hIFN- ⁇ , mIFN- ⁇ and 2k all have good killing effects on BXPC-3 tumor cells. At 72h, hIFN- ⁇ and mIFN- ⁇ are more effective than 2k virus on BXPC-3 tumor cells. sensitive. (The curve fitting of 2k virus at 48h is poor, and the IC50 value is not representative).
  • Skmel-28 human malignant melanoma cells
  • Skov3 human ovarian cancer cells
  • Fadu human pharyngeal squamous cell carcinoma cells
  • Hep3B2.1-7 liver cancer cells
  • ASPC pancreatic cancer cells
  • BXPC Human pancreatic cancer cells

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Abstract

提供了一种重组单纯疱疹病毒及其构建方法和应用，通过将I型单纯疱疹病毒的ICP34.5基因、ICP47基因敲除，并在UL23基因上插入IFN-γ表达序列，构建得到的重组病毒，可有效降低其神经毒性及选择性在肿瘤细胞中感染复制增殖，并且提高对肿瘤细胞的敏感性。

Description

一种重组单纯疱疹病毒及其构建方法 技术领域
本发明涉及生物技术和基因治疗领域，具体地，本发明涉及一种重组单纯疱疹病毒、构建重组单纯疱疹病毒的方法以及制药用途。
背景技术
溶瘤病毒疗法是一种利用病毒特异性地在肿瘤细胞中复制继而杀伤肿瘤细胞，并刺激机体产生特异性抗肿瘤免疫反应的新型肿瘤治疗方法。相比其他肿瘤治疗方法，溶瘤病毒疗法具有复制高效、杀伤效果好和毒副作用小等特点，已经成为肿瘤治疗研究领域的新热点。
IFN-γ即干扰素γ，各种癌症类型影响的早期研究揭示了其广泛的抗肿瘤潜力。其中，最广为人知的作用是增强NK细胞和CTL细胞潜在抗肿瘤效应，IFN-γ可诱导CXC趋化因子如MIG和IP-10的表达，这已被证明对T细胞浸润肿瘤至关重要。由于肿瘤细胞表达的抗原与其未转化的细胞不同，例如，由基因突变引起的新抗原，过表达的细胞抗原或病毒抗原，IFN-γ刺激肿瘤抗原呈递MHC分子的表达以增加肿瘤细胞的免疫原性，使它们更容易受到免疫识别和破坏。IFN-γ还通过抑制细胞增殖和促进细胞凋亡显示出直接的抗癌活性。
IFN-γ刺激肿瘤抗原呈递MHC分子的表达以增加肿瘤细胞的免疫原性，使它们更容易受到免疫识别和破坏。IFN-γ还通过抑制细胞增殖和促进细胞凋亡显示出直接的抗癌活性，目前没有携带IFN-γ的HSV1的报道，将IFN-γ运用在抗肿瘤领域，构建的新型溶瘤病毒HSV-1IFN-γ(+)，具有高效、特异性强等优点，还可引起特异的抗肿瘤免疫反应，提升在肿瘤细胞的敏感性。
发明内容
本申请旨在至少在一定程度上解决上述背景技术中的技术问题之一。
为此，在本发明的第一方面，本发明提出了一种重组单纯疱疹病毒。所述重组单纯疱疹病毒的ICP47和双拷贝的ICP34.5基因被敲除，并将IFN-γ基因编码框设置于UL23基因位置，所述IFN-γ选自hIFN-γ或mIFN-γ。
敲除HSV基因组中的复制非必需基因ICP47，可提高被病毒感染的肿瘤细 胞表面MHC-I的表达和细胞抗原呈递的能力；敲除双拷贝ICP34.5，可使单纯疱疹病毒(HSV)在正常细胞内复制受限，选择性地在肿瘤细胞中复制，进而提高HSV病毒地用药安全性。
经试验验证，当IFN-γ基因编码框设置于HSV载体的UL23基因位置时，上述重组单纯疱疹病毒可持续高表达IFN-γ，不仅高选择地在肿瘤细胞内繁殖感染，而且还具有免疫调节作用，确保肿瘤细胞被机体自身的免疫反应彻底清除。
根据本发明的实施例，IFN-γ可以是hIFN-γ或mIFN-γ。
根据本发明的实施例，上述重组单纯疱疹病毒还可以进一步包括如下附加技术特征至少之一：
根据本发明的实施例，IFN-γ基因编码框设置于UL23基因序列的第387-521位核苷酸之间。需要说明的是，本申请描述的UL23基因序列编码是以UL23基因起始密码子的第一位核苷酸为第1位进行顺序编码的，UL23基因的序列可参考：
https://www.ncbi.nlm.nih.gov/nuccore/NC_001806.2？report＝genbank&from＝46 609&to＝47803&strand＝true。
根据本发明的具体实施例，hIFN-γ基因编码框设置于UL23基因序列的第387-521位核苷酸之间是通过将UL23基因序列的第388-520位核苷酸敲除，之后将hIFN-γ基因编码框插入UL23基因序列的第387-521位核苷酸之间获得的。
根据本发明的实施例，所述启动子包括选自CMV、CAG、EF1α、劳斯肉瘤氏病毒长末端重复序列(RSV LTR)、金属硫蛋白I(MTI)的至少之一。
根据本发明的实施例，所述启动子为CMV。
根据本发明的实施例，所述HSV为HSV1。发明人发现，HSV2可能引起生殖器感染，进而选择HSV1，安全性进一步提高。
根据本发明的实施例，hIFN-γ基因编码框具有如下所示的核苷酸序列，1)SEQ ID NO:1所示的核苷酸序列；2)与1)相比具有至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少99％同一性的核苷酸序列。
Figure PCTCN2021070373-appb-000001
Figure PCTCN2021070373-appb-000002
根据本发明的实施例，所述重组单纯疱疹病毒具有如下所示的核苷酸序列，a)SEQ ID NO:2所示的核苷酸序列；b)与a)相比具有至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少99％同一性的核苷酸序列。
Figure PCTCN2021070373-appb-000003
Figure PCTCN2021070373-appb-000004
根据本发明的实施例，mIFN-γ基因编码框具有如下所示的核苷酸序列，1)SEQ ID NO:3所示的核苷酸序列；2)与1)相比具有至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少99％同一性的核苷酸序列。
Figure PCTCN2021070373-appb-000005
根据本发明的实施例，所述重组单纯疱疹病毒具有如下所示的核苷酸序列，a)SEQ ID NO:4所示的核苷酸序列；b)与a)相比具有至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少99％同一性的核苷酸序列。
Figure PCTCN2021070373-appb-000006
Figure PCTCN2021070373-appb-000007
在本发明的第二方面，本发明提出了一种重组单纯疱疹病毒的构建方法。
根据本发明的实施例，所述构建方法包括如下步骤：
步骤一、构建PCDNA3.1(+)-CMV-IFN-γ-poly(A)质粒：用NheI/NotI同时酶切PUC57simple-IFN-γ质粒，将片段回收，与用NheI/NotI酶切的PCDNA3.1(+)载体连接，创建CMV启动IFN-γ基因表达、poly(A)信号终止的质粒PCDNA3.1(+)-CMV-IFN-γ-poly(A)；
步骤二、构建pMD18-T-UL23-HOM1-HOM2：PCR扩增病毒UL23基因上游的同源臂UL23-HOM1片段，利用同源重组的方式与线性化的pMD18-T载体连接，构建成功的质粒命名为pMD18-T-UL23-HOM1；PCR扩增病毒UL23基因下游的同源臂UL23-HOM2片段，用EcoRI酶酶切质粒pMD18-T-UL23-HOM1，使用同源重组的方式将UL23-HOM2片段与线性化的pMD18-T-UL23-HOM1载 体连接，构建成功的质粒命名为pMD18-T-UL23-HOM1-HOM2；
步骤三、构建pMD18-T-UL23-HOM1-HOM2-CMV-IFN-γ-poly(A)：扩增CMV-IFN-γ-poly(A)序列，用XbaI酶酶切质粒pMD18-T-UL23-HOM1-HOM2，使用同源重组的方式将CMV-IFN-γ-poly(A)片段与线性化的pMD18-T-UL23-HOM1-HOM2载体连接，构建成功的质粒命名为pMD18-T-UL23-HOM1-HOM2-CMV-IFN-γ-poly(A)；
步骤四、构建靶向LacZ基因的打靶质粒：将靶向LacZ的引物退火互搭，用BbsI酶酶切质粒CRISPR/Cas9-sgRNA质粒，将互搭的打靶引物与线性化的CRISPR/Cas9-sgRNA载体通过粘性互补的方式连接，成功构建出打靶质粒CRISPR/Cas9-sgRNA-LacZ-12或CRISPR/Cas9-sgRNA-LacZ-11；
步骤五、293FT细胞内CRISPR/Cas9基因编辑系统介导的同源重组I型单纯疱疹病毒KOS株：将步骤三中表达hIFN-γ的同源质粒pMD18-T-UL23-HOM1-HOM2-CMV-hIFN-γ-poly(A)、步骤四中靶向LacZ基因的打靶质粒与ICP34.5基因、ICP47基因被敲除，UL23基因失活的KOS基因组共同转染293FT细胞，获得表达IFN-γ的重组病毒HSV-1IFN-γ(+)。
本领域的技术人员可以理解，所述步骤一、二的顺序可互换。
根据本发明的实施例，上述方法中，所述IFN-γ选自hIFN-γ或mIFN-γ。
根据本发明实施例的方法可构建表达hIFN-γ的重组病毒HSV-1hIFN-γ(+)，或表达mIFN-γ的重组病毒HSV-1mIFN-γ(+)。
根据本发明实施例的方法构建的重组单纯疱疹病毒，可选择性杀伤肿瘤细胞，提升对肿瘤细胞的敏感性。
根据本发明的实施例，上述方法还可以进一步包括如下附加技术特征至少之一：
根据本发明的实施例，步骤二中的上下游同源臂中，
上游侧翼序列的引物对包括：上游序列hrR3UL23HOM1-F1为CCTGCAGGTCGACGAGATGCCGTAGTCAGGTTTAGTTCGTCCGGC；
下游序列hrR3UL23HOM1-R1为GGGATCCTCTAGAGACGCGCGATACCTTATGGGCAGCATGAC；
下游侧翼序列的引物对包括：上游序列hrR3UL23HOM2-F1为CCGGGTACCGAGCTCCCCATTGTTATCTGGGCGCTTGTCATTACCAC；
下游序列hrR3UL23HOM2-R1为TATGACCATGATTACTCTGGAGCATCCGCACGACTGCGGTGATATTA。
根据本发明的实施例，步骤三中的IFN-γ基因中，
上游序列hrR3UL23HOM-F1为AAGGTATCGCGCGTCTTCCATTGACGTCAATGGGTGGAGTATTTA；
下游序列hrR3UL23HOM1-R1-2为GGTACCCGGGGATCCACGGGGAAAGCCGGCGAACGTGGCGAGAAA。
根据本发明的实施例，步骤四中的LacZ基因打靶序列中，
Figure PCTCN2021070373-appb-000008
根据本发明的实施例，步骤五中重组病毒HSV-1IFN-γ(+)通过1轮或多轮挑取病毒斑的方法进行纯化。
根据本发明的实施例，步骤五中重组病毒HSV-1IFN-γ(+)通过3轮挑取病毒斑的方法进行纯化。
在本发明的第三方面，本发明提出了一种重组细胞。根据本发明的实施例，所述转基因细胞携带有前面所述的重组单纯疱疹病毒或前面所述的构建方法构建得到的重组单纯疱疹病毒。根据本发明实施例的重组细胞是通过将前面所述的重组单纯疱疹病毒导入受体细胞中获得的，前面所述的重组单纯疱疹病毒导入受体细胞后，溶瘤病毒可在受体细胞中包装并大量复制，并持续高表达IFN-γ。
在本发明的第四方面，本发明提出了一种药物组合物。根据本发明的实施例，所述药物组合物包括前面所述的重组单纯疱疹病毒、前面所述的构建方法构建的重组单纯疱疹病毒或前面所述的重组细胞。
在本发明的第五方面，本发明提出了前面所述的重组单纯疱疹病毒、前面所述的构建方法构建的重组单纯疱疹病毒、前面所述的重组细胞或前面所述的药物 组合物在制备药物中的用途，所述药物用于治疗或预防肿瘤。
根据本发明的实施例，所述药物用于选择性杀伤肿瘤细胞。
在本发明的第六方面，本发明提出了一种治疗癌症的方法。所述方法包括将治疗有效量的前面所述的重组单纯疱疹病毒、前面所述的构建方法构建的重组单纯疱疹病毒、前面所述的重组细胞或前面所述的药物组合物施用于有需要的个体。
根据本发明的实施例，所述有需要的个体为患有患有以下癌症中的至少之一的患者：卵巢癌、乳腺癌、肺癌、鼻咽癌、肝癌、胃癌、食道癌、结直肠癌、胰腺癌、黑色素瘤、皮肤癌、前列腺癌、宫颈癌、白血病或脑肿瘤。
本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本发明的实践了解到。
附图说明
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中：
图1是根据本发明实施例的pMD18-T-UL23-HOM1-HOM2-CMV-hIFN-γ-poly(A)质粒图谱；
图2是根据本发明实施例的构建的重组病毒的PCR凝胶电泳检测图；
图3是根据本发明实施例的构建的重组病毒HSV-1-UL23-mIFN-γ的测序结构图；
图4是根据本发明实施例的构建的重组病毒HSV-1-UL23-hIFN-γ的测序结构图；
图5是根据本发明实施例的构建的重组病毒的打靶位点的PCR凝胶电泳检测图；
图6是根据本发明实施例的mIFN-γ、hIFN-γ、2K对Skmel-28肿瘤细胞的抑制曲线；
图7是根据本发明实施例的mIFN-γ、hIFN-γ、2K对Skov3肿瘤细胞的抑制曲线；
图8是根据本发明实施例的mIFN-γ、hIFN-γ、2K对Fadu肿瘤细胞的抑制曲线；
图9是根据本发明实施例的mIFN-γ、hIFN-γ、2K对Hep3B2.1-7肿瘤细胞的抑制曲线；
图10是根据本发明实施例的mIFN-γ、hIFN-γ、2K对ASPC-1肿瘤细胞的抑制曲线；
图11是根据本发明实施例的mIFN-γ、hIFN-γ、2K对BXPC-3肿瘤细胞的抑制曲线。
具体实施方式
下面详细描述本发明的实施例，所述实施例的示例在附图中示出，其中自始至终相同或类似的标号表示相同或类似的元件或具有相同或类似功能的元件。下面通过参考附图描述的实施例是示例性的，旨在用于解释本发明，而不能理解为对本发明的限制。
本发明中，限制性核酸内切酶NheI/NotI：购自Takara公司；hIFN-γ基因：金斯瑞合成；PCDNA3.1(+)：购自(Takara公司)；pMD18-T：购自Takara公司；EcoRI：购自Takara公司；XbaI：购自Takara公司；CRISPR/Cas9-sgRNA：购自(Takara公司)；
此外，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括至少一个该特征。在本发明的描述中，“多个”的含义是至少两个，例如两个，三个等，除非另有明确具体的限定。
需要说明的是，本申请所述的“IFN-γ”又称γ-hIFN或免疫干扰素或γ干扰素，是一种具有免疫调节作用的淋巴因子。各种癌症类型影响的早期研究揭示了其广泛的抗肿瘤潜力。其中，最广为人知的作用是增强NK细胞和CTL细胞潜在抗肿瘤效应，IFN-γ可诱导CXC趋化因子如MIG和IP-10的表达，这已被证明对T细胞浸润肿瘤至关重要。此外，IFN-γ是Th1介导的免疫应答的主要产物，并协调Th1效应机制，作为正反馈环中先天免疫(巨噬细胞和NK细胞)的进一步激活。其中“hIFN-γ”是人γ干扰素，“mIFN-γ”是鼠γ干扰素。
本申请所述的“hIFN-γ基因编码框”是指能够表达功能性hIFN-γ的核酸序列，也就说，该核酸序列所编码的hIFN-γ区段是能够实现该hIFN-γ功能的区段，或者说，该核酸序列所编码的hIFN-γ区段是hIFN-γ必要功能区。
本申请所述的“ICP47和ICP34.5被敲除”是指ICP47和ICP34.5基因被沉默，这两种基因的表达量相比于未被敲除ICP47和ICP34.5的HSV-1，显著降低。
本申请所述的“UL23基因失活”是指插入失活，也就是说在UL23基因位置插入外源基因后，UL23基因的原有功能丧失。
本申请所述的“可操作地连接”是指将外源基因连接到载体上，使得载体内的控制元件，例如启动子序列等等，能够发挥其预期的调节外源基因的转录和翻译的功能。
以下实施例利用CRISPR/Cas9系统构建重组HSV-1病毒，具体包括质粒构建，重组筛选，及病毒鉴定等。重组HSV-1病毒的生物学活性，具体包括hIFN-γ或mIFN-γ的表达情况，病毒在肿瘤细胞上感染杀伤能力等。
实施例1 构建重组单纯疱疹病毒
1、单纯疱疹病毒重组靶向载体的构建
步骤一、构建PCDNA3.1(+)-CMV-hIFN-γ-poly(A)质粒：用NheI/NotI同时酶切PUC57simple-hIFN-γ质粒，将片段回收，与用NheI/NotI酶切的PCDNA3.1(+)载体连接，创建CMV启动hIFN-γ基因表达、poly(A)信号终止的质粒PCDNA3.1(+)-CMV-hIFN-γ-poly(A)；
步骤二、构建pMD18-T-UL23-HOM1-HOM2：PCR扩增病毒UL23基因上游的同源臂UL23-HOM1片段，利用同源重组的方式与线性化的pMD18-T载体连接，构建成功的质粒命名为pMD18-T-UL23-HOM1；PCR扩增病毒UL23基因下游的同源臂UL23-HOM2片段，用EcoRI酶酶切质粒pMD18-T-UL23-HOM1，使用同源重组的方式将UL23-HOM2片段与线性化的pMD18-T-UL23-HOM1载体连接，构建成功的质粒命名为pMD18-T-UL23-HOM1-HOM2；
步骤三、构建pMD18-T-UL23-HOM1-HOM2-CMV-hIFN-γ-poly(A)：扩增CMV-hIFN-γ-poly(A)序列，用XbaI酶酶切质粒pMD18-T-UL23-HOM1-HOM2，使用同源重组的方式将CMV-hIFN-γ-poly(A)片段与线性化的pMD18-T-UL23-HOM1-HOM2载体连接，构建成功的质粒命名为pMD18-T-UL23-HOM1-HOM2-CMV-hIFN-γ-poly(A)；
步骤四、构建CRISPR/Cas9-sgRNA-LacZ-12打靶质粒：将靶向LacZ的引物退火互搭，用BbsI酶酶切质粒CRISPR/Cas9-sgRNA质粒，将互搭的打靶引物与线性化的CRISPR/Cas9-sgRNA载体通过粘性互补的方式连接，构建成功的质粒命名为CRISPR/Cas9-s gRNA-LacZ-12，简写为gRNA-LacZ-12，代号为S12。
在本实施例中，除了CRISPR/Cas9-sgRNA-LacZ-12，我们还筛选到另一个效果较好的打靶质粒CRISPR/Cas9-sgRNA-LacZ-11(简写为gRNA-LacZ-11，代号为S11)，CRISPR/Cas9-sgRNA-LacZ-12和CRISPR/Cas9-sgRNA-LacZ-11的区别在于sgRNA不同。
2、单纯疱疹病毒的重组
293FT细胞内CRISPR/Cas9基因编辑系统介导的同源重组I型单纯疱疹病毒KOS株：将实施例1步骤三中表达hIFN-γ的同源质粒pMD18-T-UL23-HOM1-HOM2-CMV-hIFN-γ-poly(A)、步骤四中靶向LacZ基因的CRISPR/Cas9-sgRNA-LacZ-12质粒与ICP34.5基因、UL23基因、ICP47基因敲除的KOS基因组共同转染293FT细胞，获得表达hIFN-γ的重组病毒2k-HSV-1-UL23-hIFN-γ(+)。
具体来说，操作如下：
步骤一、293FT细胞铺12孔板
取293FT细胞悬浮液接种于十二孔板，每孔1ml，接种密度分别为10^4，放置于37℃、5％CO 2培养箱中培养，期间需要观察细胞生长密度，待细胞融合度达70％，进行转染。
步骤二、使用
Figure PCTCN2021070373-appb-000009
3000转染试剂进行转染
(1)使用
Figure PCTCN2021070373-appb-000010
培养基稀释
Figure PCTCN2021070373-appb-000011
试剂，然后充分混匀：
Figure PCTCN2021070373-appb-000012
(2)使用
Figure PCTCN2021070373-appb-000013
培养基稀释质粒，制备质粒预混液，然后添加P3000 TM试剂，然后充分混匀：
Figure PCTCN2021070373-appb-000014
Figure PCTCN2021070373-appb-000015
(3)在每管已稀释的
Figure PCTCN2021070373-appb-000016
3000试剂中加入稀释的DNA(体积比1:1)，充分混匀后室温孵育15min。
Figure PCTCN2021070373-appb-000017
(4)加入120μL DNA-脂质体复合物至293FT细胞中，37℃、5％CO2培养箱中孵育，4h后换一次液，放回培养箱继续培养。
(5)转染72h，将细胞和病毒一起收集。冻融三次。保存于-20℃。
依照本实施例同样的方法构建携带mIFN-γ的单纯疱疹重组病毒。
本次实验用两个打靶质粒(gRNA-LacZ-11和gRNA-LacZ-12)构建了以下4个重组病毒：
质粒 sgRNA-LacZ-11 sgRNA-LacZ-12
pMD18-T-UL23HOM-mIFN-γ + +
pMD18-T-UL23HOM-hIFN-γ + +
实施例2 HSV-1-UL23-h/mIFN-γ重组病毒的筛选纯化
(1)实验材料：
P2代重组病毒2k-HSV-1-UL23-mIFN-γ(S11-5-1#、S11-7-2#)、2k-HSV-1-UL23-hIFN-γ(S11-2-1#、S12-8-2#)(其中，2k表示敲除ICP47和ICP34.5，S11或S12表示打靶质粒的代号，5-1、7-2、2-1和8-2是病毒斑的代号，p2表示病毒代数)。
(2)重组病毒的第三轮筛选纯化
1、Vero细胞铺板(6孔板)
2、病毒感染(6孔板，病毒原液稀释度为5×10 7)
3、中性红/x-gal染色
4、挑白斑，每孔有1-3个斑
5、vero细胞扩增病毒(24孔板)
6、收取病毒
Figure PCTCN2021070373-appb-000018
实施例3 HSV-1-UL23-h/mIFN-γ重组病毒的PCR验证
对三轮纯化后单一病毒斑HSV-1-UL23-mIFN-γ、HSV-1-UL23-hIFN-γ(进行PCR验证。
实验材料：
P3代重组病毒2k-HSV-1-UL23-mIFN-γ(S11-5-1#、S11-7-2#)、2k-HSV-1-UL23-hIFN-γ(S11-2-1#、S12-8-2#)(其中，2k表示敲除ICP47和ICP34.5，S11或S12表示打靶质粒的代号，5-1、7-2、2-1和8-2是病毒斑的代号，p3表示病毒代数)。(备注：5-1/5-2为同一个孔，且该孔中仅有两个病毒斑)
1、PCR同源臂外臂
引物为UL23HOM1 left-F2/B48431UL24R；
PCR产物大小：HSV-1-UL23-hIFN-γ(4585bp)、HSV-1-UL23-mIFN-γ(4552bp)。
引物序列：
UL23HOM1 left-F2 ACCGGAGGGCTGTCGTGCATGGATATCA
B 48431 UL24 R ACGAGAACTGCGGTCGTTGTCCTAA
PCR体系：
组成 体积(μL)
5×PS GXL缓冲液 10.0
模板[HSV-1-UL23-hIFN-γ/mIFN-γ] 1.0
正向引物(UL23HOM1 left-F2) 1.0
反向因为u(B 48431 UL24 R) 1.0
primeSTAR GXL 1.0
dNTP混合物 4.0
ddH 2O 32.0
总体积 50.0
PCR程序：
98℃，3min；(98℃，10s；60℃，15s；68℃，3min)×35cycles；72℃，5min；12℃，forever。
测序结果与分析：PCR产物进行凝胶电泳检测，如图2所示，两个p3代重组病毒HSV-1-UL23-mIFN-γ(S11-5-1-1#、S11-7-2-1#)、两个p3代重组病毒HSV-1-UL23-hIFN-γ(S11-2-1-1#、S12-8-2-1#)作为最终筛选出的病毒(在本申请中命名为2k-HSV-1-UL23-mIFN-γ-S11-5-1-1-P3或2k-HSV-1-UL23-mIFN-γ-S11-7-2-1-P3或2k-HSV-1-UL23-hIFN-γ-S11-2-1-1-P3或2k-HSV-1-UL23-hIFN-γ-S11-8-2-1-P3)。分别对HSV-1-UL23-mIFN-γ(S11-5-1-1#、S11-7-2-1#)进行测序，如图3所示，测序验证UL23HOM1以左618bp到UL23HOM2以右500bp序列完整无突变，表明mIFN-γ已成功插入HSV-1-病毒基因组。分别对HSV-1-UL23-hIFN-γ(S11-5-1-1#、S11-7-2-1#)进行测序，如图4所示，测序验证UL23HOM1以左618bp到UL23HOM2以右500bp序列完整无突变，表明hIFN-γ已成功插入HSV-1-病毒基因组。
2、PCR打靶位点的LacZ基因
PCR体系：
Figure PCTCN2021070373-appb-000019
PCR程序：
98℃，3min；(98℃，10s；60℃，15s；68℃，1min)×35cycles；72℃，5min；12℃，forever。
测序结果与分析：PCR产物进行凝胶电泳检测，如图5所示，表明病毒较纯，未混有含有LacZ基因的病毒。
实施例4 IFN-γ病毒细胞毒性实验
(1)实验材料：mIFN-γ指代P3代2k-HSV-1-UL23-mIFN-γ；hIFN-γ指代P3代2k-HSV-1-UL23-hIFN-γ；2k指代HSV-1/2K；细胞为Skmel-28(人恶性黑色素瘤细胞)、Skov3(人卵巢癌细胞)、Fadu(人咽鳞癌细胞)、Hep3B2.1-7(肝癌细胞)、ASPC(胰腺癌细胞)、BXPC(人胰腺癌细胞)。
(2)细胞杀伤验证：以合适的细胞密度接种于96孔培养板，培养过夜后，分别加入7个梯度浓度(MOI＝10、5、1、0.5、0.1、0.05、0.01)的三种病毒，再分别培养24，48或72小时，依照CCK8试剂盒说明书进行细胞活力的检测。
(3)细胞杀伤结果：
hIFN-γ、mIFN-γ及2k三种病毒对不同肿瘤细胞的毒性或杀伤作用，结果如表1和图6-11所示。
表1：
Figure PCTCN2021070373-appb-000020
备注：N/A表示因量效关系差或未测，未能做出的非线性拟合，其中拟合度＜80％表示拟合度较差，其对应的MOI IC 50值仅作参考。
图6显示，hIFN-γ、mIFN-γ及2k三种病毒对Skmel-28肿瘤细胞均具有良好的杀伤作用，24h及48h时hIFN-γ病毒的杀伤能力稍强于mIFN-γ病毒，72h时细胞基本已全部感染。
图7显示，hIFN-γ、mIFN-γ及2k三种病毒对Skov3肿瘤细胞均具有良好的杀伤作用，48h时IC50值均远小于0.6，且hIFN-γ、mIFN-γ病毒的杀伤能力在Skov3上高于2k病毒。
图8显示，Fadu，hIFN-γ、mIFN-γ及2k三种病毒对Skov3肿瘤细胞均具有良好的杀伤作用，且三者对Skov3肿瘤细胞杀伤能力相当。
图9显示，hIFN-γ、mIFN-γ及2k三种病毒对Hep3B2.1-7肿瘤细胞均具有良好的杀伤作用，24h时hIFN-γ病毒的杀伤能力稍强于其他两种病毒，48h时拟合出的mIFN-γ的IC50非常低，病毒对Hep3B2.1-7细胞非常敏感，在48h时细胞已全部感染。
图10显示，hIFN-γ、mIFN-γ及2k三种病毒对ASPC-1肿瘤细胞均具有良好的杀伤作用，24h时杀伤能力2K稍强于mIFN-γ或hIFN-γ，48h时细胞基本已全部感染。
图11显示，hIFN-γ、mIFN-γ及2k三种病毒对BXPC-3肿瘤细胞均具有良好的杀伤作用，72h时hIFN-γ、mIFN-γ相较2k病毒对BXPC-3肿瘤细胞更为敏感。(48h时2k病毒曲线拟合较差，IC50值不具有代表性)。
综上，Skmel-28(人恶性黑色素瘤细胞)、Skov3(人卵巢癌细胞)、Fadu(人咽鳞癌细胞)、Hep3B2.1-7(肝癌细胞)、ASPC(胰腺癌细胞)、BXPC(人胰腺癌细胞)均为hIFN-γ或mIFN-γ敏感的细胞株，而且相较于2K病毒，mIFN-γ或hIFN-γ携带型病毒对Skmel-28、Skov3、Hep3B2.1-7、BXPC-3更为敏感。

Claims (16)

  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的ICP47和双拷贝的ICP34.5基因被敲除，并将IFN-γ编码框设置于UL23基因位置,所述IFN-γ选自hIFN-γ或mIFN-γ。
  2. 根据权利要求1所述的重组单纯疱疹病毒，其特征在于，IFN-γ基因编码框设置于UL23基因序列的第387-521位核苷酸之间位置。
  3. 根据权利要求1所述的重组单纯疱疹病毒，其特征在于，所述IFN-γ基因编码框与启动子可操作地连接，所述启动子包括选自CMV、CAG、EF1α、RSV LTR、MTI的至少之一。
  4. 根据权利要求1所述的重组单纯疱疹病毒，其特征在于，hIFN-γ基因编码框具有如下所示的核苷酸序列，
    1)SEQ ID NO:1所示的核苷酸序列；
    2)与1)相比具有至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少99％同源性的核苷酸序列；
    任选地，所述重组单纯疱疹病毒具有如下所示的核苷酸序列，
    a)SEQ ID NO:2所示的核苷酸序列；
    b)与a)相比具有至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少99％同源性的核苷酸序列。
  5. 根据权利要求1所述的重组单纯疱疹病毒，其特征在于，mIFN-γ基因编码框具有如下所示的核苷酸序列，
    1)SEQ ID NO:3所示的核苷酸序列；
    2)与1)相比具有至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少99％同源性的核苷酸序列；
    任选地，所述重组单纯疱疹病毒具有如下所示的核苷酸序列，
    a)SEQ ID NO:4所示的核苷酸序列；
    b)与a)相比具有至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少99％同源性的核苷酸。
  6. 一种重组单纯疱疹病毒的构建方法，其特征在于，包括如下步骤：
    步骤一、构建PCDNA3.1(+)-CMV-IFN-γ-poly(A)质粒：用NheI/NotI同时酶切PUC57simple-IFN-γ质粒，将片段回收，与用NheI/NotI酶切的PCDNA3.1(+)载体连接，创建CMV启动IFN-γ基因表达、poly(A)信号终止的质粒PCDNA3.1(+)-CMV-IFN-γ-poly(A)；
    步骤二、构建pMD18-T-UL23-HOM1-HOM2：PCR扩增病毒UL23基因上游的同源臂UL23-HOM1片段，利用同源重组的方式与线性化的pMD18-T载体连接，构建成功的质粒命名为pMD18-T-UL23-HOM1；PCR扩增病毒UL23基因下游的同源臂UL23-HOM2片段，用 EcoRI酶酶切质粒pMD18-T-UL23-HOM1，使用同源重组的方式将UL23-HOM2片段与线性化的pMD18-T-UL23-HOM1载体连接，构建成功的质粒命名为pMD18-T-UL23-HOM1-HOM2；
    步骤三、构建pMD18-T-UL23-HOM1-HOM2-CMV-IFN-γ-poly(A)：扩增CMV-IFN-γ-poly(A)序列，用XbaI酶酶切质粒pMD18-T-UL23-HOM1-HOM2，使用同源重组的方式将CMV-IFN-γ-poly(A)片段与线性化的pMD18-T-UL23-HOM1-HOM2载体连接，构建成功的质粒命名为pMD18-T-UL23-HOM1-HOM2-CMV-IFN-γ-poly(A)；
    步骤四、构建靶向LacZ基因的打靶质粒：将靶向LacZ的引物退火互搭，用BbsI酶酶切质粒CRISPR/Cas9-sgRNA质粒，将互搭的打靶引物与线性化的CRISPR/Cas9-sgRNA载体通过粘性互补的方式连接，成功构建出打靶质粒CRISPR/Cas9-sgRNA-LacZ-12或CRISPR/Cas9-sgRNA-LacZ-11；
    步骤五、293FT细胞内CRISPR/Cas9基因编辑系统介导的同源重组I型单纯疱疹病毒KOS株：将步骤三中表达IFN-γ的同源质粒pMD18-T-UL23-HOM1-HOM2-CMV-IFN-γ-poly(A)、步骤四中靶向LacZ基因的打靶质粒与ICP34.5基因、ICP47基因被敲除，UL23基因失活的KOS基因组共同转染293FT细胞，获得表达IFN-γ的重组病毒HSV-1 IFN-γ(+)；
    其中步骤一、二顺序可互换；所述IFN-γ选自hIFN-γ或mIFN-γ。
  7. 根据权利要求6所述的一种重组单纯疱疹病毒的构建方法，其特征在于，步骤二中的上下游同源臂中，
    上游侧翼序列的引物对包括：上游序列hrR3UL23HOM1-F1为CCTGCAGGTCGACGAGATGCCGTAGTCAGGTTTAGTTCGTCCGGC；
    下游序列hrR3UL23HOM1-R1为GGGATCCTCTAGAGACGCGCGATACCTTATGGGCAGCATGAC；
    下游侧翼序列的引物对包括：上游序列hrR3UL23HOM2-F1为CCGGGTACCGAGCTCCCCATTGTTATCTGGGCGCTTGTCATTACCAC；
    下游序列hrR3UL23HOM2-R1为TATGACCATGATTACTCTGGAGCATCCGCACGACTGCGGTGATATTA。
  8. 根据权利要求6所述的一种重组单纯疱疹病毒的构建方法，其特征在于，步骤三中的IFN-γ基因中，
    上游序列hrR3 UL23 HOM-F1为AAGGTATCGCGCGTCTTCCATTGACGTCAATGGGTGGAGTATTTA；
    下游序列hrR3UL23HOM1-R1-2为 GGTACCCGGGGATCCACGGGGAAAGCCGGCGAACGTGGCGAGAAA。
  9. 根据权利要求6所述的一种重组单纯疱疹病毒的构建方法，其特征在于，步骤四中的LacZ基因打靶序列中，
    上游序列sgRNA-Lac Z-12-F为CACCGGCCGGGGGCGGGGCATGTGC；
    下游序列sgRNA-Lac Z-12-R为AAACGCACATGCCCCGCCCCCGGCC；
    或上游序列sgRNA-Lac Z-11-F为CACCGGGTCTGCTTTCTGACAAACT；
    下游序列sgRNA-Lac Z-11-R为AAACAGTTTGTCAGAAAGCAGACCC。
  10. 根据权利要求6所述的一种重组单纯疱疹病毒的构建方法，其特征在于，步骤五中重组病毒HSV-1 IFN-γ(+)通过1轮或多轮挑取病毒斑的方法进行纯化。
  11. 一种重组细胞，其特征在于，所述重组细胞携带有权利要求1-5任一项所述的重组单纯疱疹病毒或权利要求6-10任一项所述的重组单纯疱疹病毒的构建方法构建的重组单纯疱疹病毒。
  12. 一种药物组合物，其特征在于，包括权利要求1-5任一项所述的重组单纯疱疹病毒、权利要求6-10任一项所述的重组单纯疱疹病毒的构建方法构建的重组单纯疱疹病毒或权利要求11所述的重组细胞。
  13. 权利要求1-5任一项所述的重组单纯疱疹病毒、权利要求6-10任一项所述的重组单纯疱疹病毒的构建方法构建的重组单纯疱疹病毒、权利要求11所述的重组细胞或权利要求12所述的药物组合物在制备用于治疗或预防肿瘤的药物中的用途，任选地，所述药物用于选择性杀伤肿瘤细胞。
  14. 根据权利要求13所述的用途，其特征在于，所述肿瘤为卵巢癌、乳腺癌、肺癌、鼻咽癌、肝癌、胃癌、食道癌、结直肠癌、胰腺癌、黑色素瘤、皮肤癌、前列腺癌、宫颈癌、白血病或脑肿瘤。
  15. 一种治疗癌症的方法，其特征在于，所述方法包括将治疗有效量的权利要求1-5任一项所述的重组单纯疱疹病毒、权利要求6-10任一项所述的重组单纯疱疹病毒的构建方法构建的重组单纯疱疹病毒、权利要求11所述的重组细胞或权利要求12所述的药物组合物施用于有需要的个体。
  16. 根据权利要求15所述的方法，所述有需要的个体为患有以下癌症中的至少之一的患者：卵巢癌、乳腺癌、肺癌、鼻咽癌、肝癌、胃癌、食道癌、结直肠癌、胰腺癌、黑色素瘤、皮肤癌、前列腺癌、宫颈癌、白血病或脑肿瘤。
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108841796A (zh) * 2017-06-15 2018-11-20 杭州睿可特生物科技有限公司 重组单纯疱疹病毒及其制备方法和应用
WO2019152868A1 (en) * 2018-02-01 2019-08-08 Editas Medicine, Inc. Crispr/cas-related methods and compositions for treating herpes simplex virus (hsv) related keratitis
WO2019236931A1 (en) * 2018-06-08 2019-12-12 The Board Of Trustees Of University Of Illinois Recombinant herpes simplex virus for cancer immunotherapy

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110982794B (zh) * 2020-03-05 2020-06-16 北京唯源立康生物科技有限公司 一种修饰的单纯疱疹病毒

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108841796A (zh) * 2017-06-15 2018-11-20 杭州睿可特生物科技有限公司 重组单纯疱疹病毒及其制备方法和应用
WO2019152868A1 (en) * 2018-02-01 2019-08-08 Editas Medicine, Inc. Crispr/cas-related methods and compositions for treating herpes simplex virus (hsv) related keratitis
WO2019236931A1 (en) * 2018-06-08 2019-12-12 The Board Of Trustees Of University Of Illinois Recombinant herpes simplex virus for cancer immunotherapy

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
HAO MENGRU; HUANG CHENGHAO; XIA NINGSHAO: "The Advances of Oncolytic Herpes Simplex Virus in Cancer Therapy", BINGDU XUEBAO - CHINESE JOURNAL OF VIROLOGY, ZHONGGUO WEISHENGWU XUCHUI, BEIJING,, CN, vol. 32, no. 4, 31 July 2016 (2016-07-31), CN , pages 516 - 522, XP009516731, ISSN: 1000-8721, DOI: 10.13242/j.cnki.bingduxuebao.002995 *
KANNO HIROSHI, HATTORI SATOSHI, SATO HIDEMITSU, MURATA HIDETOSHI, HUANG FU HUI, HAYASHI AKIMUNE, SUZUKI NORIYUKI, YAMAMOTO ISAO, K: "Experimental gene therapy against subcutaneously implanted glioma with a herpes simplex virus-defective vector expressing interferon-␥ Shin-ichi Miyatake, 147", CANCER GENE THERAPY, NATURE PUBLISHING GROUP US, NEW YORK, vol. 6, 1 March 1999 (1999-03-01), New York, pages 147 - 154, XP055886945, ISSN: 0929-1903, DOI: 10.1038/sj.cgt.7700008 *
MARTUZA R L; MALICK A; MARKERT J M; RUFFNER K L; COEN D M: "EXPERIMENTAL THERAPY OF HUMAN GLIOMA BY MEANS OF A GENETICALLY ENGINEERED VIRUS MUTANT", SCIENCE, AMERICAN ASSOCIATION FOR THE ADVANCEMENT OF SCIENCE, US, vol. 252., no. 5007, 10 May 1991 (1991-05-10), US , pages 854 - 856, XP002090037, ISSN: 0036-8075, DOI: 10.1126/science.1851332 *
YAHONG LONG, MI YANXIA; LI YUNCHUN: "Progess on Research of Herpes Simplex Virus Type 1 Mutants for Cancer Therapy", SHENGWU YIXUE GONGCHENGXUE ZAZHI = JOURNAL OF BIOMEDICAL ENGINEERING, SICHUAN DAXUE HUAXI YIYUAN, CN, vol. 25, no. 6, 1 December 2008 (2008-12-01), CN , pages 1446 - 1449, XP055887076, ISSN: 1001-5515 *

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