WO2023087464A1 - 基于中国hsv临床分离株的溶瘤病毒及其构建方法和应用 - Google Patents

基于中国hsv临床分离株的溶瘤病毒及其构建方法和应用 Download PDF

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WO2023087464A1
WO2023087464A1 PCT/CN2021/138484 CN2021138484W WO2023087464A1 WO 2023087464 A1 WO2023087464 A1 WO 2023087464A1 CN 2021138484 W CN2021138484 W CN 2021138484W WO 2023087464 A1 WO2023087464 A1 WO 2023087464A1
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gene
virus
vector
hsv1
tumor
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王华东
徐富强
应敏
苏鹏
刘奇
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Shenzhen Institute of Advanced Technology of CAS
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Definitions

  • the invention belongs to the field of biotechnology, relates to virology, molecular biology and oncology, and specifically relates to an oncolytic virus based on a Chinese HSV clinical isolate and a construction method and application thereof.
  • Oncolytic virus is a kind of natural or artificially modified virus that specifically replicates in tumor cells and kills tumors without harming normal cells.
  • Oncolytic viruses mainly achieve the purpose of treating tumors by directly killing tumor cells, inducing anti-tumor systemic immunity, and preventing tumor angiogenesis. They have the advantages of high killing efficiency, low toxicity and side effects, wide indications, and low cost.
  • OV vectors can be divided into two categories: one is DNA viral vectors, which include adenovirus, herpes simplex virus, poxvirus, and parvovirus; One is RNA viral vectors, which include reovirus, poliovirus, Newcastle disease virus, M1 virus, and vesicular stomatitis virus. Adenovirus and herpes virus are the two most widely used oncolytic viral vectors.
  • herpes simplex virus type 1 Herpes simplex virus simplex virus, HSV1 vectored oncolytic virus drug Imlygic (also known as T-VEC) was approved by the US FDA as a local treatment option for unresectable lesions in patients with melanoma. This is the first and only oncolytic virus therapy approved by the FDA, and it was also approved for marketing in Europe and Canada in 2016.
  • Daiichi Sankyo Co., Ltd.'s oncolytic HSV virus product G47 ⁇ was approved by the Japanese Ministry of Health, Labor and Welfare (MHLW) for the treatment of malignant glioma.
  • MHLW Japanese Ministry of Health, Labor and Welfare
  • the successive listing of two oHSV oncolytic viruses marks the maturity of oncolytic virus technology, and the world's major pharmaceutical giants have deployed oncolytic virus drug research and development through acquisitions or cooperation.
  • HSV-1 is widespread in the population, mainly causing orofacial herpes, and up to 90% of people at the age of 65 are positive for previous serum HSV infection.
  • clinical strains of HSV-1 i.e., strains sampled from individuals with cold sores
  • have substantial natural variation in evolutionary biological properties eg, infectivity, virulence, replication efficiency, etc.
  • oncolytic viruses based on herpes simplex virus in the world mostly uses typical HSV-1 "laboratory" standard strains such as 17+, KOS or F strains. These strains have been weakened by long-term continuous passage, or may not be suitable for cancer treatment.
  • Optimal oncolytic virus backbone T-VEC was initially screened after comparing two clinical isolates with the 17+ strain, both of which were more effective at killing human tumor cells than the 17+ strain, the most promising strain JS1 was screened and constructed to transform into T-VEC.
  • the present invention aims to provide an oncolytic virus based on Chinese HSV clinical isolates and its construction method and application.
  • the invention provides a herpes simplex virus HSV1-CH007 strain, and its preservation number is CCTCC NO: V202173.
  • the invention provides a virus vector, which is obtained by knocking out the double-copy gene of neurovirulence factor ⁇ 34.5 in the genome of herpes simplex virus HSV1-CH007 strain.
  • the viral vector inserts an exogenous gene expression cassette at the knockout ⁇ 34.5 gene locus; or,
  • the viral vector inserts an exogenous gene expression cassette at any site in the genome where the insertion does not affect the function;
  • the exogenous gene in the exogenous gene expression cassette is selected from genes expressing any target protein.
  • the invention provides the application of the viral vector in basic research of virology, viral vector with large carrying capacity, oncolytic virus, target gene delivery, nervous system targeting gene therapy drug, animal infection model establishment or antiviral drug screening.
  • the invention provides an oncolytic virus vector, which obtains a recombinant virus targeting tumor cell replication by knocking out the double-copy gene of the neurovirulence factor ⁇ 34.5 in the genome of the herpes simplex virus HSV1-CH007 strain.
  • the oncolytic effect is further enhanced by inserting a tumor therapy sequence gene expression cassette at any insertion site of the oncolytic virus vector genome that does not affect the function;
  • the tumor therapy sequence gene is selected from one or more of immune checkpoint protein antibody genes, tumor immunotherapy cytokine genes, and immune regulation enhancement genes;
  • the tumor therapeutic sequence gene expression cassette comprises a promoter and a tumor therapeutic sequence gene
  • the WPRE enhanced expression element is introduced downstream of the tumor therapy sequence gene
  • the tumor therapy sequence gene is an immune checkpoint protein PD1 antibody gene
  • a fluorescent protein expression cassette is also inserted at the knockout ⁇ 34.5 gene locus, which is convenient for recombinant virus screening and in vivo infection marker indication, and the transcription direction of the fluorescent protein expression cassette is consistent with the tumor treatment sequence gene expression cassette .
  • the invention provides an oncolytic virus, which comprises the oncolytic virus vector.
  • the invention provides an antitumor pharmaceutical composition, which comprises the oncolytic virus.
  • the invention provides a method for constructing an oncolytic virus vector, comprising knocking out the double-copy gene of neurovirulence factor ⁇ 34.5 in the genome of herpes simplex virus HSV1-CH007 strain;
  • it also includes inserting a tumor therapy sequence gene expression cassette at the knocked-out ⁇ 34.5 gene locus or any insertion site in the genome that does not affect the function.
  • the invention provides a method for constructing an oncolytic virus, comprising the following steps:
  • a tumor treatment sequence gene expression cassette into the recombinant vector 1, specifically: constructing a promoter (such as hUbC, CMV promoter and other promoters that can efficiently initiate transcription), a tumor treatment sequence gene (PD1 single-chain antibody gene) ) and the red fluorescent protein mCherry gene are bound and expressed by the self-cleaving peptide (2A) to replace the EGFP expression cassette of the recombinant targeting plasmid vector expressing green fluorescent protein to obtain the recombination of the expression tumor therapeutic sequence gene targeting plasmid vector;
  • a promoter such as hUbC, CMV promoter and other promoters that can efficiently initiate transcription
  • PD1 single-chain antibody gene PD1 single-chain antibody gene
  • red fluorescent protein mCherry gene are bound and expressed by the self-cleaving peptide (2A) to replace the EGFP expression cassette of the recombinant targeting plasmid vector expressing green fluorescent protein to obtain the recombination of the expression tumor
  • Co-transfect 293T cells with the recombinant targeting plasmid vector that knocks out the ⁇ 34.5 gene to express green fluorescent protein and the genomic DNA of HSV1-CH007 strain collect the supernatant after the cytopathic changes, infect Vero cells, and pick the green fluorescent virus spots , after more than 5 rounds of spot-picking purification, the recombinant virus expressing green fluorescent protein can be obtained;
  • the method includes: co-transfecting 293T cells with the recombinant targeting plasmid vector expressing the tumor treatment sequence gene and the recombinant HSV1-CH007 strain virus genomic DNA expressing green fluorescent protein, The supernatant was collected after the cell lesion, infected Vero cells, and red fluorescent virus spots were picked under a green fluorescent background, and obtained after more than 5 rounds of spot picking and purification.
  • the method for constructing the above-mentioned oncolytic virus of the present invention includes the following steps:
  • hUbC is the selected ubiquitin promoter with strong activation activity.
  • the hUbC-3EGFP fragment (length 3538bp) was digested with ClaI and KpnI and ligated into the pHSVCH007 ⁇ 34.5 vector to obtain the pHSV-CH007 ⁇ 34.5-hUbC-3EGFP vector ;
  • the WPRE-bGH PA fragment (846bp in length) was digested with KpnI and PacI and connected into the pHSV-CH007 ⁇ 34.5-hUbC-3EGFP vector to construct a recombinant targeting plasmid pHSV-CH007 ⁇ 34.5-hUbC-3EGFP expressing green fluorescent protein -WPRE-bGH PA vector (SEQ ID NO. 5).
  • a tumor therapy sequence gene expression cassette specifically:
  • Synthetic gene sequences for tumor treatment sequences such as the expression of PD1 antibody ( ⁇ PD1Ab) and granulocyte-macrophage colony-stimulating factor (GM-CSF), etc.
  • the FUGW plasmid (Addgene No. 14883) was selected as the construction backbone of the recombinant targeting vector.
  • This round of recombination uses the hUbC promoter fragment and the WPRE fragment as homology arms, which can replace the 3EGFP sequence in the above-mentioned targeting plasmid vector; the synthetic PD1 antibody Gene sequence ( ⁇ PD1 ) (SEQ ID NO.6), expression frame mCherry-P2A- ⁇ PD1Ab (SEQ ID NO.8) obtained by tandem self-cleaving peptide P2A and red fluorescent protein (mCherry) gene (SEQ ID NO.7) ; The expression frame fragment (length 1599bp) was cloned into the FUGW plasmid digested by BamHI and EcoRI by the Infusion method, and the original EGFP fluorescent gene was replaced to obtain the recombinant plasmid pFUW-hUbC-mCherry-P2A- ⁇ PD1Ab (SEQ ID NO. 9).
  • pHSV-CH007 ⁇ 34.5-hUbC-3EGFP-WPRE-bGH PA and HSV1-CH007 strain genomic DNA were co-transfected into 293T cells. After most of the cells showed cytopathic changes, the cells and supernatant were collected, and the centrifuged cell lysate or supernatant was used to infect Vero cells, and the cells were covered with 2 % fetal bovine serum, antibiotics and 1% agarose DMEM maintenance medium, observe the fluorescent expression of the infected cells after 1 day to determine whether the new virus recombination is successful; 2-3 days later, pick the spot-separated well-expressed green fluorescent virus plaque After serial 10-fold dilution, the new Vero cells were infected for the next round of spot-picking purification.
  • the construction of an oncolytic virus inserted into a tumor therapy sequence gene includes:
  • the recombinant plasmid pFUW-hUbC-mCherry-P2A- ⁇ PD1Ab and the oncolytic virus HSV CH007- ⁇ 34.5-hUbC-3EGFP-WPRE genomic DNA were co-transfected into 293T cells. After most of the cells appeared cytopathic, the cells were collected and The virus supernatant was used to infect Vero cells, the supernatant was removed, and the culture medium containing 2% fetal bovine serum, antibiotics and 1% agarose was placed on top. After 2-3 days, the spots were picked and separated to express red fluorescence.
  • HSV1 CH007- ⁇ 34.5-expressing green fluorescent protein was completely removed.
  • hUbC-3EGFP mother virus obtain monoclonal oncolytic virus HSV CH007- ⁇ 34.5-hUbC-mCherry-P2A-tumor therapeutic sequence gene sequence-WPRE carrying tumor therapeutic sequence gene sequence.
  • the Chinese clinical isolate of herpes simplex virus HSV1-CH007 provided by the present invention is isolated from the domestic popular HSV1 clinical strain, rather than the common laboratory standard strain imported from abroad for many times, and the Chinese clinical isolate HSV1-CH007 is used as the Oncolytic virus skeleton, the HSV oncolytic virus constructed through transformation and optimization is more suitable for the treatment of Chinese cancer patients.
  • the present invention uses HSV1-CH007 as the OV parent strain, and constructs an oncolytic virus based on HSV1-CH007 through a series of genetic transformations such as attenuation, tumor-targeted replication, and armed therapeutic genes, and the constructed oncolytic virus replicates With high efficiency, it can efficiently dissolve and kill solid tumors and metastatic tumors. Insert the gene expressing PD1 antibody ( ⁇ PD1Ab) into the genome of HSV1-CH007 oncolytic virus. When the oncolytic virus infects and lyses tumor cells, the virus will directly express immune checkpoint protein inhibitors to achieve synergistic tumor killing of oncolysis + immune activation effect. In vitro and in vivo experiments showed that the developed oHSV-CH007 oncolytic virus had good tumor cell infection efficiency and oncolytic effect.
  • the present invention can be used as a gene delivery virus with low toxicity and large capacity based on long-term and high expression of exogenous genes of HSV clinical strains
  • the vector can realize high-efficiency transduction and high-abundance expression of exogenous genes.
  • the virus vector modified based on the HSV1-CH007 clinical strain of the present invention is suitable for oncolytic viruses, gene delivery vectors for long-term and high expression of foreign genes, etc.
  • the low-virulence HSV1-CH007 vector has a wide range of application values in the nervous system targeted gene therapy, analysis of virus replication and pathogenic mechanism, establishment of animal infection models, and screening of antiviral drugs.
  • Figure 1 is a schematic diagram of the genome structure of HSV1-CH007- ⁇ 34.5-hUbC-3EGFP-WPRE;
  • Figure 2 is a cell map of HSV1-CH007- ⁇ 34.5-hUbC-3EGFP-WPRE virus recombination and spot-picking purification;
  • Fig. 3 is the molecular identification of the conditional replication type HSV1-CH007 recombinant virus genome of construction
  • Figure 4 is an analysis of the effect of the conditional replication type HSV1-CH007- ⁇ 34.5-hUbC-3EGFP-WPRE oncolytic virus backbone vector to lyse tumor cells;
  • Figure 5 is a schematic diagram of the genome structure of the constructed oncolytic virus oHSVCH007- ⁇ 34.5-hUbC-mCherry-P2A- ⁇ PD1Ab-WPRE (abbreviated as oHSVCH007- ⁇ 34.5- ⁇ PD1Ab-mCherry);
  • Figure 6 is a cell map of oHSVCH007- ⁇ 34.5- ⁇ PD1Ab-mCherry oncolytic virus recombinant spot picking and purification;
  • Figure 7 is the in vivo toxicity test of oHSVCH007- ⁇ 34.5- ⁇ PD1Ab-mCherry oncolytic virus
  • Figure 8 shows the construction, purification and exogenous fluorescent protein expression of U87MG-GFP malignant glioma cell line
  • Figure 9 shows the construction of U87MG-GFP malignant glioma intracranial tumor-bearing mouse model
  • Figure 10 shows that the oHSVCH007- ⁇ 34.5- ⁇ PD1Ab-mCherry oncolytic virus can effectively infect and lyse malignant glioma tissues.
  • HSV1-CH007 The screened Chinese clinical isolate of herpes simplex virus HSV1-CH007, and the low-passage strain wild-type virus after monoclonal purification were preserved in the China Center for Type Culture Collection (China Center for Type Culture Collection, referred to as CCTCC), the address is China.Wuhan.Wuhan University.
  • HSV1-CH007 is classified as herpes simplex virus Chinese clinical isolate HSV1-CH007, and the deposit number is CCTCC NO: V202173.
  • Example 1 Isolation and screening of herpes simplex virus clinical strain HSV1-CH007
  • the wild-type virus of isolated HSV1 clinical strain is relatively toxic, and all mice died within 3-5 days after intracranial injection after amplification and purification.
  • oncolytic virus drugs it is first necessary to attenuate the clinical strain of HSV1 and modify targeted replication to establish a safe viral vector.
  • a variety of synergistic strategies such as Chinese clinical virus strains, deletion of disease-causing genes, arming with immune enhancement factors, and carrying immune checkpoint protein antibodies, were designed and integrated to develop high-efficiency lytic viruses. Synergistic oHSV against solid tumors and metastases.
  • Knock out the double-copy ⁇ 34.5 gene Clone and sequence the ⁇ 34.5 gene and its upstream and downstream sequences of the clinical strain HSV1-CH007, and design primers to clone the upstream homology arm of the ⁇ 34.5 gene (UHA, 440bp in length, 78% GC content) (SEQ ID NO.1), the downstream homology arm (DHA, 500bp in length, 68% GC content) (SEQ ID NO.2); the cloned upstream and downstream homology arm fragments and fluorescent protein expression cassette fragments were connected into the pcDNA3-based 3.1+ ⁇ RL1 vector constructed with 1+ backbone; the restriction sites selected when cloning the upstream and downstream homology arm fragments were the pair of MluI and AgeI, the pair of SbfI and XbaI, respectively, and the obtained plasmid vector was named pHSV-CH007 ⁇ 34.5.
  • the vector introduces a multi-cloning site adapter in the middle of the upstream and downstream homology arms of the clone.
  • the six endonucleases introduced are -AgeI-ClaI-EcoRI-EcoRV-KpnI-PacI-SbfI-, which facilitates subsequent cloning of foreign genes Or expression control elements, etc. are inserted into the targeting vector.
  • UHA-F gtacgggccagatatacgcgtCGGGGCCGCGGGAGCGGGgggaggagc,
  • UHA-R TtaacccatcgatggaccggtGGAGACAGAGAGCGTGCCGgggtggtag;
  • DHA-F aatccttaattaaggcctgcaggCGTTACACCCGAGGCGGCctgggtcttc,
  • DHA-R ggtttaaacgggccctctagaCGGGCCAATGCGCGAGGGgccgtgtg.
  • the multi-fragment recombination used the multi-fragment infusion kit from Nanjing Novozyme Company.
  • the obtained vector was named pHSV-CH007 ⁇ 34.5.
  • hUbC-3EGFP eukaryotic expression cassette fragment
  • SEQ ID NO.3 eukaryotic expression cassette fragment with three green fluorescent genes in series was cloned, and the WPRE-bGH ploy ( A) Fragment (SEQ ID NO.4), hUbC is the selected ubiquitin promoter with strong activation activity.
  • the hUbC-3EGFP fragment (3538bp in length) was digested with ClaI and KpnI and ligated into the pHSV-CH007 ⁇ 34.5 vector to obtain the pHSV-CH007 ⁇ 34.5-hUbC-3EGFP vector;
  • the WPRE-bGH PA fragment (846bp in length) was digested with KpnI and PacI and ligated into the pHSV-CH007 ⁇ 34.5-hUbC-3EGFP vector to construct the recombinant targeting plasmid pHSV-CH007 ⁇ 34.5-hUbC-3EGFP-WPRE-bGH expressing green fluorescent protein PA vector (SEQ ID NO. 5).
  • Example 3 Tumor targeting, conditional replication HSV1-CH007 oncolytic virus recombination, spot-picking purification
  • HSV1-CH007 recombinant strain in which the double copy of the most important neurovirulence gene ⁇ 34.5 in the virus genome was completely knocked out, we extracted the targeting vector pHSV-CH007 ⁇ 34.5-hUbC-3EGFP-WPRE and the clinical strain virus HSV1-CH007 Genomic DNA was co-transfected into 293T cells cultured in a six-well plate, and the experimental method was carried out according to the instructions of Lipofectamine 2000 (Invitrogen): after most of the cells showed cytopathic changes, the medium was collected, and the cells were collected with PBS.
  • Lipofectamine 2000 Invitrogen
  • the supernatant and cell lysate were used to infect Vero cells in a six-well plate, respectively. After 1 h of infection, the supernatant was removed and the cells were overlaid with DMEM maintenance medium containing 2% fetal bovine serum, 1% antibiotics, and 1% agarose. After 1 day, observe whether the infected cells have fluorescent expression to determine whether the new virus has successfully recombined. After 2-3 days, pick spots to separate well-expressed green fluorescent virus spots, and infect new Vero cells after serial 10-fold dilution for the next round of spot picking. purification.
  • FIG. 1 shows the schematic diagram of the viral genome.
  • Figure 2 shows the images of virus recombination picking spots and completely purified cells. The upper row is the fluorescence image and bright field image of the recombinant virus spots (green fluorescence); the lower row is the fluorescence image of infected cells after the recombinant virus is completely purified. and brightfield images.
  • Embodiment 4 the amplification preparation of HSV1-CH007 recombinant virus
  • the titer of concentrated HSV CH007 recombinant virus was determined in Vero cells using a standard plaque assay, expressed in plaque-forming units per milliliter (PFU/mL).
  • the concentrated HSV1 CH007- ⁇ 34.5-hUbC-3EGFP-WPRE recombinant OV virus titer was determined to be about 5 ⁇ 10 9 PFU/mL.
  • Embodiment 5 Molecular Identification of Conditional Replication Type HSV1-CH007 Recombinant Virus Genome
  • HSV1 CH007- ⁇ 34.5-hUbC-3EGFP-WPRE recombinant virus (abbreviated as CH007 ⁇ 3-G in Figure 3), wild-type wtCH007 virus (positive control), and use the ⁇ 34.5 double that we constructed earlier based on the HSV1 H129 strain
  • the H306 virus whose copy gene has been completely knocked out (see the published paper J Chem Neuroanat. 2019;100:101662. doi: 10.1016/j.jchemneu.2019.101662) as a negative control, inactivated at 100°C for 10 minutes, and subsequently used for molecular identification of the ⁇ 34.5 gene.
  • Primers were designed with the ⁇ 34.5 ORF fragment (726bp), and the primer sequence was ⁇ 34.5-F: 5' ATGGCCCGCCGCCGCCGCCGCCATCGCGGCCCCCGCCGCCCCCGG 3' (SEQ ID NO. 10); ⁇ 34.5-R: 5' TTAGACCGAGTTCGCCGGGCCGGCTCCGCGGGCCAGGGCCCGGGC 3' (SEQ ID NO. 11).
  • the PCR reaction system is 50 ul, and the amount of inactivated virus sample is 1-5 ⁇ l, 2 ⁇ PrimeStar high- GC buffer 25 ⁇ l, 0.7 ⁇ l of 20 ⁇ M/ ⁇ l primers, 5 ⁇ l of dNTPs, 0.6 ⁇ l of Prime star HS high-fidelity enzyme, and 50 ⁇ l of sterilized water.
  • the PCR amplification conditions were: 98°C for 5min, (98°C for 30s, 60°C for 30s, 72°C for 1min) cycle 32 times, 72°C for 10min, 16°C for 30min.
  • the results of molecular identification are shown in Figure 3.
  • the positive control was the wild-type HSV1-CH007 virus, and a target band of the ⁇ 34.5 gene about 700 bp in length was amplified by PCR; the negative control (H306) had no band; while HSV1- CH007 recombinant virus HSV CH007- ⁇ 34.5-hUbC-3EGFP-WPRE (abbreviated as CH007 ⁇ 3-G in the figure) repeated experiments no matter whether 1 ⁇ l, 3 ⁇ l, or 5 ⁇ l concentrated virus was used for PCR reaction, the target band of ⁇ 34.5 was not amplified , the results showed that the double-copy ⁇ 34.5 gene in the HSV1 CH007- ⁇ 34.5-hUbC-3EGFP-WPRE recombinant oncolytic virus genome had been completely knocked out.
  • Example 6 Analysis of the oncolytic effect of the backbone of tumor-targeted and conditionally replicable HSV1 CH007- ⁇ 34.5-hUbC-3GFP oncolytic virus
  • the HSV1-CH007 clinical strain virus has been genetically modified to knock out the most important neurovirulence gene ⁇ 34.5, endowing the virus with the characteristics of only targeting tumor cells for replication and amplification in the body, and killing tumor cells, while infecting normal cells.
  • the body cells are replication deficient and cannot proliferate to produce progeny viruses.
  • the HSV1 CH007 recombinant virus was used to infect common tumor cell lines for testing.
  • Figure 4 shows the malignant glioblasts cultured in vitro
  • the reconstructed recombinant oncolytic virus HSV1 CH007- ⁇ 34.5-hUbC-3GFP can efficiently infect and kill malignant tumor cells.
  • Example 7 Construction of HSV1-CH007 oncolytic virus armed with PD1 antibody gene
  • the FUGW plasmid (Addgene No. 14883) was selected as the construction backbone of the recombinant targeting vector; the gene sequence of the synthetic PD1 antibody ( ⁇ PD1 ) (SEQ ID NO.6), through the self-cleaving peptide P2A and red fluorescent protein (mCherry) (SEQ ID NO.7) Gene tandem to obtain the expression cassette mCherry-P2A- ⁇ PD1Ab (SEQ ID NO.8); the expression cassette fragment (length 1599bp) was cloned into the FUGW plasmid digested with BamHI and EcoRI by the Infusion method, replacing the original EGFP fluorescent gene, obtained recombinant plasmid pFUW-hUbC-mCherry-P2A- ⁇ PD1Ab (SEQ ID NO.9).
  • the construction steps of the oncolytic virus carrying the PD1 antibody gene in the oHSV1-CH007 genome are as follows: combine the extracted targeting vector pFUW-hUbC-mCherry-P2A- ⁇ PD1Ab with the recombinant virus HSV1 CH007- ⁇ 34.5-hUbC-3GFP genome constructed above DNA co-transfected 293T cells, plasmid transfection expresses red fluorescent protein, and virus-infected cells express green fluorescent protein at the same time, if the recombination is successful, the mCherry-P2A- ⁇ PD1Ab gene sequence will be homologously recombined to replace the 3EGFP gene, and the new recombinant virus will only Express red fluorescent protein; collect the cell and virus supernatant after most of the cells appear cytopathic.
  • the Vero cells in a six-well plate were infected, the supernatant was removed after 1 hour, and the culture medium containing 2% fetal calf serum, antibiotics and 1% agarose was superimposed. After 2-3 days, pick and separate well-expressed red fluorescent spots, that is, pick red fluorescent spots in the green background field of view, and infect new Vero cells after serial 10-fold dilution for the next round of purification.
  • the HSV1 CH007- ⁇ 34.5-hUbC-3GFP parent virus expressing green fluorescent protein was completely removed, and the monoclonal oncolytic virus oHSV1 CH007- ⁇ 34.5 carrying the PD1 antibody gene was obtained -hUbC-mCherry-P2A- ⁇ PD1Ab-WPRE (abbreviated as oHSVCH007- ⁇ 34.5- ⁇ PD1Ab-mChe), the schematic diagram of the viral genome is shown in Figure 5, and the schematic diagram of virus recombinant picking and purification is shown in Figure 6.
  • Example 8 In vivo toxicity test of oHSVCH007- ⁇ 34.5- ⁇ PD1Ab-mChe oncolytic virus armed with PD1 antibody gene
  • the constructed oncolytic virus oHSVCH007- ⁇ 34.5- ⁇ PD1Ab-mChe was massively amplified, purified and concentrated, and the titer of the prepared oncolytic virus was determined to be 8 ⁇ 10 9 PFU/ml by plaque test. After intracranial injection of 2 ⁇ 10 6 PFU virus to infect the VTA brain region of the mouse, the mice were observed to be in good condition; 14 days later, the mice were perfused and the brain tissue slices were taken for observation. Areas LH, LHb, etc. were all well marked with red fluorescent light, and a certain inflammatory reaction was observed only at the injection site, and the neurons marked in other brain areas were in good condition (Figure 7).
  • HSV1-CH007 wild-type virus infected mice had obvious symptoms, and all mice died within one week.
  • the oHSVCH007- ⁇ 34.5- ⁇ PD1Ab-mChe oncolytic virus constructed by independent technology has low toxicity, and the foreign antibody protein carried by it is highly expressed, and the oncolytic virus that infects normal tissues of the body such as neurons is replication-deficient , will not proliferate and spread to kill animals.
  • Example 9 Construction of human malignant glioblastoma cell line U87MG-GFP expressing green fluorescent protein
  • the new U87MG stable transfection cell line that constitutively expresses the target protein was constructed by using the characteristic that the lentivirus will be integrated into the host cell genome; in order to facilitate the flow cytometric screening and purification of the new cell line, first construct and prepare the lentivirus LentiV expressing the green fluorescent protein -hUbC-EGFP-WPRE; human glioblastoma cell line U87MG can stably express green fluorescent protein after being infected with lentivirus, and a new type of malignant glioma cell line U87MG-GFP was obtained after flow cytometry purification; as shown in Figure 8 As shown, the prepared engineered cell line U87MG-GFP has a high purity, almost all cells stably express green fluorescent protein, and the cells are in good condition, which is consistent with wild-type U87MG,
  • the engineered U87MG-GFP cell line was cultivated, and the tumor cell suspension in the logarithmic growth phase was prepared; transplanted and inoculated in the CPU brain area of the right brain of BALB/c nude mice by brain stereotaxic method, the inoculation dose was 5 microliters, and the tumor cells The number was 1 ⁇ 10 6 ; the symptoms after transplantation of glioma in nude mice were continuously observed, and the tumor formation status of glioma was monitored using a small animal magnetic resonance imager. During magnetic resonance imaging (MRI) scanning, MSME (multi spin multi echo) T1-weight sequences were used to obtain functional images.
  • Figure 9 shows the results of MRI scans at the 4th week after inoculation of glioma cell lines.
  • mice were subjected to cardiac perfusion and brain tissue sampling after the MRI scan at the fourth week, and frozen sections were obtained to obtain brain tissue sections; after DAPI staining, the OLYMPUS VS120 whole slide scanning system was used for imaging.
  • the nude mice injected with U87MG-GFP formed large and obvious solid tumors in the CPU area, and because the modified glioma cells U87MG-GFP constitutively expressed green fluorescent protein, the tumor tissue showed bright green fluorescence (Fig. 9C).
  • the above results show that the engineered glioma cell line is in good condition, and the brain tumor model was successfully transplanted in vivo, laying a solid foundation for the evaluation of oHSV oncolytic virus effect.
  • Example 11 oHSVCH007- ⁇ 34.5- ⁇ PD1Ab-mChe oncolytic virus can efficiently lyse and kill malignant glioblastoma
  • oHSVCH007- ⁇ 34.5- ⁇ PD1Ab-mChe oncolytic virus on the established malignant glioma tumor-bearing animal model.
  • the above-mentioned engineered human astroglioblastoma (WHO grade IV) cell line U87MG-GFP was inoculated in the subcortical CPU brain area of BALB/c nude mice, and the brains of the mice were detected by nuclear magnetic resonance after 4 weeks. Tumors formed and showed invasive growth (Fig. 9C).
  • the prepared oHSVCH007- ⁇ 34.5- ⁇ PD1Ab-mChe oncolytic virus was injected into the malignant glioma tumor by intratumoral administration, and nude mice were perfused 14 days later, and brain tissue sections were taken for observation. It was found that oHSVCH007- ⁇ 34.5- ⁇ PD1Ab- The mChe oncolytic virus can effectively replicate and proliferate in glioblastoma, and obvious tissue cavitation and apoptotic inflammatory response appear in the tumor tissue area infected by the virus ( Figure 10).
  • the new oncolytic virus oHSVCH007- ⁇ 34.5- ⁇ PD1Ab-mChe oncolytic virus can efficiently lyse and kill malignant glioblastoma cells, and the volume of tumor tissue is significantly reduced compared with the control group of tumor-bearing mice injected with PBS buffer, which verifies the
  • the potent oncolytic virus constructed based on Chinese HSV clinical isolates has good safety and oncolytic activity.

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Abstract

一种单纯疱疹病毒临床分离株HSV1-CH007株，其保藏编号为CCTCC NO：V202173。该单纯疱疹病毒HSV1-CH007分离自中国流行的HSV1临床株，通过筛选以其作为溶瘤病毒骨架，经改造和优化所构建的HSV溶瘤病毒更适宜中国肿瘤患者。以HSV1-CH007作为溶瘤病毒亲本株，经减毒、肿瘤靶向复制、武装化治疗基因等系列遗传改造，构建获得基于HSV1-CH007临床株的新型溶瘤病毒，该溶瘤病毒毒性低，复制效率、安全性和有效性高，能够高效溶杀实体瘤和转移瘤。

Description

基于中国HSV临床分离株的溶瘤病毒及其构建方法和应用 技术领域
本发明属于生物技术领域，涉及病毒学、分子生物学及肿瘤学，具体涉及基于中国HSV临床分离株的溶瘤病毒及其构建方法和应用。
 背景技术
近年来，肿瘤免疫疗法已逐渐成为继手术、化疗、放疗之后的第四大肿瘤治疗手段。溶瘤病毒作为一种新型肿瘤免疫生物疗法，为恶性肿瘤的治疗带来了新希望。溶瘤病毒（oncolytic virus, OV）是一类天然或者经过人工修饰改造，特异在肿瘤细胞内复制杀伤肿瘤而不伤及正常细胞的病毒。溶瘤病毒主要通过直接杀伤肿瘤细胞、诱导机体抗肿瘤系统性免疫以及阻止肿瘤血管生成等多方面实现治疗肿瘤目的，具有杀伤效率高、毒副作用小、适应症广以及成本低等优势。另外，当其与免疫检查点抑制剂、放/化疗等联合应用时，具有协同效应，可进一步提高肿瘤治疗的效果，有着巨大的应用前景，成为未来肿瘤治疗的新方向。
利用病毒治疗肿瘤的概念已有100多年的历史。随着基因工程技术的不断发展，溶瘤病毒已经发展到第三代，如今有160多种不同的溶瘤病毒正在进行临床前及临床试验。全球基于十余类病毒载体在研发溶瘤病毒药物，根据基因组构成，OV载体可分为两大类：一类是DNA病毒载体，其包括腺病毒、单纯疱疹病毒、痘病毒和细小病毒等；一类是RNA病毒载体，其包括呼肠孤病毒、脊髓灰质炎病毒、新城疫病毒、M1病毒和水疱型口炎病毒等。腺病毒和疱疹病毒是目前应用最多的两类溶瘤病毒载体。
2015年基于单纯疱疹病毒1型（Herpes simplex virus, HSV1）载体的溶瘤病毒药物Imlygic（又名T-VEC）被美国FDA批准，作为黑色素瘤患者不可切除病灶的局部治疗方案。这是经过FDA批准的首个、也是唯一一个溶瘤病毒疗法，2016年在欧洲和加拿大也获批上市。2021年6月，日本第一三共株式会社的溶瘤HSV病毒产品G47∆被日本厚生劳动省（MHLW）批准，用于治疗恶性胶质细胞瘤。两种oHSV溶瘤病毒的相继上市标志着溶瘤病毒技术的成熟，全球各大制药巨头纷纷通过收购或合作方式布局溶瘤病毒药物研发。
HSV-1在人群中广泛流行，主要引起口面部疱疹，高达90%的人在65岁时检测出血清HSV既往感染阳性。然而，HSV-1临床株（即从患唇疱疹个体中取样分离的病毒株）在进化生物学特性（如感染力、毒力、复制效率等）方面存在实质性的自然变异。目前全球基于单纯疱疹病毒开发溶瘤病毒多是选用典型的HSV-1“实验室”标准株如17+、KOS或F株，这些毒株已通过长期的连续传代弱化，或可能不是癌症治疗的最佳溶瘤病毒骨架。T-VEC最初是在将两个临床分离株与17+毒株进行比较后筛选的，两株临床株病毒对人肿瘤细胞的杀伤效果均优于17+毒株，其中最有希望的毒株JS1被筛选并构建转化为T-VEC。
目前溶瘤病毒行业尚存在技术瓶颈难题，如溶瘤病毒安全性、临床治疗实体瘤/转移瘤效果不佳、肿瘤靶向性弱、抗体中和作用、载体大规模制备效率低等。随着相关技术的快速发展，病毒载体层面的问题日益凸显，亟待攻关研制高效治疗实体瘤的新型溶瘤病毒，OV病毒载体的深度改造和优化仍是研发核心内容。而且国外HSV-1流行感染株和中国人群流行的HSV-1感染株势必不同、存在实质性的自然变异，所以构建更适合国人和增效的新型HSV溶瘤病毒具有重要意义。
 技术问题
为了解决现有技术中的不足，本发明旨在提供一种基于中国HSV临床分离株的溶瘤病毒及其构建方法和应用。
 技术解决方案
本发明提供一种单纯疱疹病毒HSV1-CH007株，其保藏编号为CCTCC NO：V202173。
本发明提供一种病毒载体，其通过敲除单纯疱疹病毒HSV1-CH007株基因组中神经毒力因子γ34.5双拷贝基因得到。
进一步地，所述病毒载体在敲除的γ34.5基因座位插入外源基因表达盒；或，
所述病毒载体在其基因组任何插入不影响功能的位点插入外源基因表达盒；
所述外源基因表达盒中的外源基因选自表达任意目的蛋白的基因。
本发明提供所述的病毒载体在病毒学基础研究、大携载容量病毒载体、溶瘤病毒、目的基因递送、神经系统靶向基因治疗药物、动物感染模型建立或抗病毒药物筛选中的应用。
本发明提供一种溶瘤病毒载体，其通过敲除单纯疱疹病毒HSV1-CH007株基因组中神经毒力因子γ34.5双拷贝基因得到靶向肿瘤细胞内复制的重组病毒。
进一步地，在敲除的γ34.5基因座位插入肿瘤治疗序列基因表达盒，进一步增强溶瘤效果；或，
所述在溶瘤病毒载体基因组任何插入不影响功能的位点插入肿瘤治疗序列基因表达盒，进一步增强溶瘤效果；
优选地，所述肿瘤治疗序列基因选自免疫检查点蛋白抗体基因、肿瘤免疫治疗细胞因子基因和免疫调控增强基因中的一种或以上；
优选地，所述肿瘤治疗序列基因表达盒包含启动子和肿瘤治疗序列基因；
优选地，在肿瘤治疗序列基因下游引入WPRE增强表达元件；
优选地，所述肿瘤治疗序列基因为免疫检查点蛋白PD1抗体基因；
优选地，在敲除的γ34.5基因座位还插入荧光蛋白表达盒，便于重组病毒筛选及在体感染标记指示，所述荧光蛋白表达盒的转录方向与所述肿瘤治疗序列基因表达盒保持一致。
本发明提供一种溶瘤病毒，其包含所述的溶瘤病毒载体。
本发明提供一种抗肿瘤药物组合物，包含所述的溶瘤病毒。
本发明提供一种溶瘤病毒载体的构建方法，包括，敲除单纯疱疹病毒HSV1-CH007株基因组中神经毒力因子γ34.5双拷贝基因；
优选地，还包括在敲除的γ34.5基因座位或基因组任何插入不影响功能的位点插入肿瘤治疗序列基因表达盒。
本发明提供一种溶瘤病毒的构建方法，包括如下步骤：
（1）靶向载体构建
克隆γ34.5基因上下游同源臂，将克隆的上下游同源臂片段连入骨架载体，获得重组载体1；构建包括启动子（如hUbC、CMV启动子等）、绿色荧光蛋白基因和WPRE片段的EGFP表达盒（WPRE为土拨鼠肝炎病毒转录后调控元件，用于增强外源基因表达水平），连入重组载体1，获得表达绿色荧光蛋白的重组靶向质粒载体；
优选地，还包括在重组载体1插入肿瘤治疗序列基因表达盒，具体地：构建包括启动子（如hUbC、CMV启动子等高效启动转录的启动子）、肿瘤治疗序列基因（PD1单链抗体基因）和红色荧光蛋白mCherry基因通过自剪切肽（2A）绑定表达的肿瘤治疗序列基因表达盒，替换表达绿色荧光蛋白的重组靶向质粒载体的EGFP表达框，获得表达肿瘤治疗序列基因的重组靶向质粒载体；
（2）溶瘤病毒构建
将敲除γ34.5基因表达绿色荧光蛋白的重组靶向质粒载体与HSV1-CH007株基因组DNA共转染293T细胞，待细胞病变后收集上清，感染Vero细胞，挑斑绿色荧光的病毒嗜斑，经过5轮以上的挑斑纯化后即得表达绿色荧光蛋白的重组病毒；
优选地，插入肿瘤治疗序列基因的溶瘤病毒的构建，方法包括：将表达肿瘤治疗序列基因的重组靶向质粒载体与表达绿色荧光蛋白的重组HSV1-CH007株病毒基因组DNA共转染293T细胞，待细胞病变后收集上清，感染Vero细胞，在绿色荧光背景下挑斑红色荧光的病毒斑，经过5轮以上的挑斑纯化后即得。
进一步地，本发明上述溶瘤病毒的构建方法，包括如下步骤：
（1）靶向载体构建
克隆临床株HSV1-CH007 γ34.5基因上下游同源臂片段，设计引物克隆γ34.5基因上游同源臂（UHA，长440bp，GC含量78%）（SEQ ID NO.1），下游同源臂（DHA，长500bp，GC含量68%）（SEQ ID NO.2），将克隆的上下游同源臂片段以infusion技术连入基于pcDNA3.1+骨架构建的3.1+ΔRL1载体（实验室自主构建），克隆上下游同源臂片段选择的酶切位点分别为MluI和AgeI对，SbfI和XbaI对，在克隆的上下游同源臂中间引入一个多克隆位点接头，引入的6种内切酶分别是-AgeI-ClaI-EcoRI-EcoRV-KpnI-PacI-SbfI-，便于后续克隆外源基因或表达控制元件等插入靶向载体，获得的质粒载体命名为pHSV-CH007Δγ34.5。
以实验室自主构建的含hUbC-3EGFP表达框序列的质粒为模板，克隆三个绿色荧光基因串联的真核表达框片段hUbC-3EGFP（SEQ ID NO.3），以及WPRE-bGH PA片段（SEQ ID NO.4），hUbC是选用的启动活性强的泛素启动子，hUbC-3EGFP片段（长度3538bp）经ClaI、KpnI酶切连入pHSVCH007Δγ34.5载体获得pHSV-CH007Δγ34.5-hUbC-3EGFP载体；WPRE-bGH PA片段（长度846bp）经KpnI、PacI酶切连入pHSV-CH007Δγ34.5-hUbC-3EGFP载体，从而构建获得表达绿色荧光蛋白的重组靶向质粒pHSV-CH007Δγ34.5-hUbC-3EGFP-WPRE-bGH PA载体（SEQ ID NO.5）。
优选地，还包括插入肿瘤治疗序列基因表达盒，具体地：
合成肿瘤治疗序列基因序列，例如可以为表达PD1抗体（αPD1Ab）和粒细胞巨噬细胞集落刺激因子(GM-CSF)等。选择FUGW质粒（Addgene编号14883）作为重组靶向载体的构建骨架，此轮重组以hUbC启动子片段和WPRE片段作为同源臂，可替换掉上述靶向质粒载体中的3EGFP序列；合成PD1抗体的基因序列（ αPD1）（SEQ ID NO.6），通过自剪切肽P2A和红色荧光蛋白（mCherry）基因（SEQ ID NO.7）串联获得表达框mCherry-P2A-αPD1Ab（SEQ ID NO.8）；该表达框片段（长度1599bp）通过Infusion方法克隆入经BamHI、EcoRI双酶切的FUGW质粒，替换掉原来的EGFP荧光基因，获得重组质粒pFUW-hUbC-mCherry-P2A-αPD1Ab（SEQ ID NO.9）。
（2）溶瘤病毒构建
将pHSV-CH007Δγ34.5-hUbC-3EGFP-WPRE-bGH PA与HSV1-CH007株基因组DNA共转染293T细胞，待大部分细胞表现出细胞病变后，收集细胞和上清，用离心后的细胞裂解液或上清感染Vero细胞，在细胞上覆盖含有2%胎牛血清、抗生素和1%琼脂糖的DMEM维持培养基，1天后观察感染细胞有无荧光表达确定新型病毒是否重组成功；2-3天后，挑斑分离良好的表达绿色荧光的病毒嗜斑，连续10倍稀释后感染新的Vero细胞进行下一轮挑斑纯化，经过6轮嗜斑挑斑、单克隆纯化，完全去除无荧光表达的野生型临床株HSV1-CH007病毒，获得纯化的溶瘤病毒HSV CH007-Δγ34.5-hUbC-3EGFP-WPRE；
优选地，插入肿瘤治疗序列基因的溶瘤病毒的构建，方法包括：
将重组质粒pFUW-hUbC-mCherry-P2A-αPD1Ab与所述溶瘤病毒HSV CH007-Δγ34.5-hUbC-3EGFP-WPRE基因组DNA共转染293T细胞，待大部分细胞出现细胞病变后，收集细胞和病毒上清，感染Vero细胞，去除上清液，并上铺含有2%胎牛血清、抗生素和1%琼脂糖的培养基，2-3天后挑斑分离良好的表达红色荧光的嗜斑，即绿色背景视野中挑取红色荧光斑，连续10倍稀释后感染新的Vero细胞进行下一轮纯化，经过5轮挑斑、单克隆纯化，完全去除表达绿色荧光蛋白的HSV1 CH007-Δγ34.5-hUbC-3EGFP母本病毒，获得单克隆化的携带肿瘤治疗序列基因序列的溶瘤病毒HSV CH007-Δγ34.5-hUbC-mCherry-P2A-肿瘤治疗序列基因序列-WPRE。
 有益效果
（1）本发明提供的单纯疱疹病毒中国临床分离株HSV1-CH007分离自国内流行的HSV1临床株，而非国外引进的传代很多次的常见实验室标准株，以中国临床分离株HSV1-CH007作为溶瘤病毒骨架，经改造和优化所构建的HSV溶瘤病毒更适宜中国人肿瘤患者治疗。
（2）本发明以HSV1-CH007作为OV亲本株，经减毒、肿瘤靶向复制、武装化治疗基因等系列遗传改造，构建获得基于HSV1-CH007的溶瘤病毒，构建得到的溶瘤病毒复制效率高，能够高效溶杀实体瘤和转移瘤。在HSV1-CH007溶瘤病毒基因组中插入表达PD1抗体（αPD1Ab）的基因，溶瘤病毒感染裂解肿瘤细胞的同时，病毒会直接表达免疫检查点蛋白抑制剂，达到溶瘤+免疫激活的协同杀瘤效应。体外和体内实验表明研制的oHSV-CH007溶瘤病毒具有良好的肿瘤细胞感染效率和溶瘤效果。
（3）本发明敲除HSV1-CH007病毒株最主要的神经毒力基因γ34.5后，可作为一种低毒、大容量的基于HSV临床株的外源基因长效高表达的基因递送病毒载体，可实现外源基因的高效转导及高丰度表达。
（4）本发明基于HSV1-CH007临床株改造的病毒载体适于做溶瘤病毒、外源基因长效高表达的基因递送载体等。另外低毒HSV1-CH007载体在神经系统靶向基因治疗、病毒复制与致病机理分析、动物感染模型建立、抗病毒药物筛选等方面具有广泛的应用价值。
 附图说明
图1为HSV1-CH007-Δγ34.5-hUbC-3EGFP-WPRE基因组结构示意图；
图2为HSV1-CH007-Δγ34.5-hUbC-3EGFP-WPRE病毒重组、挑斑纯化的细胞图；
图3为构建的条件复制型HSV1-CH007重组病毒基因组分子鉴定；
图4为条件复制型HSV1-CH007-Δγ34.5-hUbC-3EGFP-WPRE溶瘤病毒骨架载体裂解肿瘤细胞效果分析；
图5为构建的溶瘤病毒oHSVCH007-Δγ34.5-hUbC-mCherry-P2A-αPD1Ab-WPRE（简写为oHSVCH007-Δγ34.5-αPD1Ab-mCherry）的基因组结构示意图；
图6为oHSVCH007-Δγ34.5-αPD1Ab-mCherry溶瘤病毒重组挑斑及纯化的细胞图；
图7为oHSVCH007-Δγ34.5-αPD1Ab-mCherry溶瘤病毒的在体毒性测试；
图8为U87MG-GFP恶性胶质瘤细胞株构建、纯化及外源荧光蛋白表达情况；
图9为构建U87MG-GFP恶性胶质瘤颅内荷瘤小鼠模型；
图10为oHSVCH007-Δγ34.5-αPD1Ab-mCherry溶瘤病毒能有效感染和溶杀恶性胶质瘤组织。
保藏说明
筛选出来的单纯疱疹病毒中国临床分离株HSV1-CH007，经单克隆纯化后的低传代株野生型病毒按要求和规定数量于2021年10月29日保藏于中国典型培养物保藏中心(China Center for Type Culture Collection，简称CCTCC)，地址为中国.武汉.武汉大学。HSV1-CH007分类命名为单纯疱疹病毒中国临床分离株HSV1-CH007，保藏编号为CCTCC NO：V202173。
 本发明的实施方式
为了更清楚地理解本发明，现参照下列实施例及附图进一步描述本发明。实施例仅用于解释而不以任何方式限制本发明。实施例中，各原始试剂材料均可商购获得，未注明具体条件的实验方法为所属领域熟知的常规方法和常规条件，或按照仪器制造商所建议的条件。
实施例1：单纯疱疹病毒临床株HSV1-CH007的分离、筛选
1、HSV临床株的分离与鉴定
我们通过与武汉大学人民医院、中国人民解放军中部战区总医院等临床医院皮肤科医生合作，从门诊口面部疱疹患者采样疱疹水疱液，经过培养、纯化、鉴定分离到18株HSV1型临床病毒株。通过克隆病毒gD、gB基因并测序，和从国外引进的F株、H129临床株比对基因序列均存在一定的碱基突变，而且分离的临床株间基因也存在差异，体现在进化生物学特性（如感染力、毒力、复制效率等）会存在差异，表明从中国健康人群分离的HSV临床株中比较和筛选溶瘤病毒亲本株很有必要。
2、HSV临床株的比较和筛选
我们从临床株筛选溶瘤病毒骨架载体的原则如下：①健康人群口面部疱疹来源；②基因型测序鉴定为HSV1型；③感染肿瘤细胞裂解活性高；④对阿昔洛韦敏感、无耐药性。通过系统比较临床毒株的复制效率、细胞毒性、裂解肿瘤细胞活性等，筛选出临床株CH007作为备选亲本株。单纯疱疹病毒临床株HSV1-CH007的保藏编号为CCTCC NO：V202173。
分离的HSV1临床株野生型病毒毒性还较大，经扩增纯化后颅内注射小鼠在3-5天内全部死亡。为研制溶瘤病毒药物，首先需要对HSV1临床株进行减毒和靶向复制改造等建立安全性的病毒载体。为开发更高效溶杀实体瘤的新型溶瘤病毒，设计集成采用中国临床病毒株、删除致病基因、免疫增强因子武装、携载免疫检查点蛋白抗体等多种增效策略，来研制高效溶杀实体瘤和转移瘤的增效oHSV。
实施例2：肿瘤靶向、条件复制型HSV1-CH007重组改造的靶向载体构建
（1）双拷贝神经毒力基因γ34.5敲除的靶向载体构建
敲除双拷贝γ34.5基因：克隆测序临床株HSV1-CH007 γ34.5基因及其上下游序列，设计引物克隆γ34.5基因上游同源臂（UHA，长440bp，GC含量78%）（SEQ ID NO.1），下游同源臂（DHA，长500bp，GC含量68%）（SEQ ID NO.2）；克隆的上下游同源臂片段以及荧光蛋白表达盒片段利用infusion技术连入基于pcDNA3.1+骨架构建的3.1+ΔRL1载体；克隆上下游同源臂片段时选择的酶切位点分别为MluI和AgeI对，SbfI和XbaI对，获得的质粒载体命名为pHSV-CH007Δγ34.5。该载体在克隆的上下游同源臂中间引入一个多克隆位点接头，引入的6种内切酶分别是-AgeI-ClaI-EcoRI-EcoRV-KpnI-PacI-SbfI-，便于后续克隆外源基因或表达控制元件等插入靶向载体。
具体步骤如下：
首先，分别使用以下引物扩增γ34.5 UHA和DHA片段：
UHA-F：gtacgggccagatatacgcgtCGGGGCCGCGGGAGCGGGgggaggagc，
UHA-R：TtaacccatcgatggaccggtGGAGACAGAGAGCGTGCCGgggtggtag；
DHA-F：aatccttaattaaggcctgcaggCGTTACACCCGAGGCGGCctgggtcttc，
DHA-R：ggtttaaacgggccctctagaCGGGCCAATGCGCGAGGGgccgtgtg。
多片段重组使用的是来自南京诺唯赞公司的多片段infusion试剂盒。将获得的载体命名为pHSV-CH007Δγ34.5。
（2）携载绿色荧光蛋白基因表达盒的重组靶向载体构建
以实验室自主构建的含hUbC-3EGFP表达框序列的质粒为模板，克隆三个绿色荧光基因串联的真核表达框片段（hUbC-3EGFP）（SEQ ID NO.3），以及WPRE-bGH ploy (A)片段（SEQ ID NO.4），hUbC是选用的启动活性强的泛素启动子。hUbC-3EGFP片段（长度3538bp）经ClaI、KpnI酶切连入pHSV-CH007Δγ34.5载体获得pHSV-CH007Δγ34.5-hUbC-3EGFP载体；WPRE-bGH PA片段（长度846bp）经KpnI、PacI酶切连入pHSV-CH007Δγ34.5-hUbC-3EGFP载体，从而构建获得表达绿色荧光蛋白的重组靶向质粒pHSV-CH007Δγ34.5-hUbC-3EGFP-WPRE-bGH PA载体（SEQ ID NO.5）。
实施例3：肿瘤靶向、条件复制型HSV1-CH007溶瘤病毒重组、挑斑纯化
双拷贝神经毒力基因γ34.5完全敲除、携带表达EGFP的HSV1- CH007重组病毒构建：
为了获得病毒基因组中最主要神经毒力基因γ34.5双拷贝完全敲除的HSV1-CH007重组株，我们抽提靶向载体pHSV-CH007Δγ34.5-hUbC-3EGFP-WPRE与临床株病毒HSV1-CH007基因组DNA共转染培养在六孔板中的293T细胞，实验方法按照Lipofectamine 2000 (Invitrogen)说明书进行：待大部分细胞表现出细胞病变后，收集培养基，用PBS收集细胞。经三轮冷冻-震荡-涡旋后，用上清和细胞裂解液分别感染六孔板中的Vero细胞。感染1小时后，去除上清液，在细胞上覆盖含有2%胎牛血清、1%抗生素和1%琼脂糖的DMEM维持培养基。1天后观察感染细胞有无荧光表达确定新型病毒是否重组成功，2-3天后，挑斑分离良好的表达绿色荧光的病毒嗜斑，连续10倍稀释后感染新的Vero细胞进行下一轮挑斑纯化。经过6轮嗜斑挑斑、单克隆纯化，完全去除无荧光表达的野生型临床株HSV1-CH007病毒，获得纯化的重组病毒HSV1 CH007-Δγ34.5-hUbC-3EGFP-WPRE，病毒基因组示意图如图1，病毒重组挑斑及完全纯化的细胞图如图2所示，上排为重组病毒斑（绿色荧光）的荧光图和明场图；下排为重组病毒完全挑纯后的感染细胞荧光图及明场图。
实施例4：HSV1-CH007重组病毒的扩增制备
纯化后的HSV1-CH007重组溶瘤病毒通过感染生长在10 cm平皿上的Vero细胞进行批量生产及纯化。将HSV1 CH007-Δγ34.5-hUbC-3EGFP-WPRE重组OV载体按照MOI=0.01感染Vero细胞，待细胞出现明显的变圆病变之后（大约3天），将含病毒的上清液收集到50mL离心管中，通过离心去除细胞碎片（6400 rpm，10分钟），上清液使用0.22 µm的滤膜过滤，最后使用贝克曼高速离心机进行浓缩（30000 rpm，3小时）；将浓缩后的病毒沉淀使用少量PBS (PH=7.4) 重悬，在4℃条件下过夜，并不断摇动；第2天混匀病毒液，接着将重悬的病毒溶液加到20%的蔗糖溶液上层进行超速离心（30000 rpm，3小时）、浓缩纯化；最后将溶解后的病毒进行分装，冻存在-80℃冰箱中。使用Vero细胞进行标准嗜斑实验来测定浓缩后HSV CH007重组病毒的滴度，滴度以每毫升的嗜斑形成单位（PFU/mL）表示。浓缩后的HSV1 CH007-Δγ34.5-hUbC-3EGFP-WPRE重组OV病毒滴度经测定约为5 × 10 9 PFU/mL。
实施例5：条件复制型HSV1-CH007重组病毒基因组分子鉴定
取浓缩纯化的HSV1 CH007-Δγ34.5-hUbC-3EGFP-WPRE重组病毒（图3中简写CH007Δ3-G）、野生型wtCH007病毒（阳性对照），以我们前期基于HSV1 H129株构建的γ34.5双拷贝基因已完全敲除的H306病毒（参看发表论文J Chem Neuroanat. 2019;100:101662. doi: 10.1016/j.jchemneu.2019.101662）作为阴性对照，在100℃灭活10分钟，后续用于γ34.5基因的分子鉴定。以γ34.5 ORF片段（726bp）设计引物，引物序列为γ34.5-F：5' ATGGCCCGCCGCCGCCGCCGCCATCGCGGCCCCCGCCGCCCCCGG 3' （SEQ ID NO.10）；γ34.5-R：5' TTAGACCGAGTTCGCCGGGCCGGCTCCGCGGGCCAGGGCCCGGGC 3' （SEQ ID NO.11）。由于γ34.5基因GC含量高达82%，需选用高GC buffer体系扩增γ34.5 ORF基因片段：PCR反应体系为50 ul，其中灭活病毒样用量为1-5 μl，2×PrimeStar high-GC buffer 25 μl，20 μM/μl引物各0.7 μl，dNTPs 5 μl，Prime star HS高保真酶0.6 μl，补灭菌水到50 μl。PCR扩增条件为：98℃ 5min，（98℃ 30s，60℃ 30s，72℃ 1min）循环32次，72℃延伸10min，16℃ 30min。分子鉴定结果如图3所示，阳性对照为野生型HSV1-CH007病毒，经PCR扩增出一条长700 bp左右的γ34.5基因目的条带；阴性对照（H306）无条带；而HSV1-CH007重组病毒HSV CH007-Δγ34.5-hUbC-3EGFP-WPRE（图中简写CH007Δ3-G）多次重复实验无论选用1μl、3μl、5μl浓缩病毒进行PCR反应均未扩增出γ34.5目的条带，结果说明HSV1 CH007-Δγ34.5-hUbC-3EGFP-WPRE重组溶瘤病毒基因组中的双拷贝γ34.5基因已被完全敲除。
实施例6：肿瘤靶向、条件复制型HSV1 CH007-Δγ34.5-hUbC-3GFP溶瘤病毒骨架溶瘤效果分析
HSV1-CH007临床株病毒通过遗传改造敲除最主要的神经毒力基因γ34.5，赋予了病毒在机体内只能靶向肿瘤细胞内复制扩增、裂杀肿瘤细胞的特性，而感染正常的机体细胞为复制缺陷型，不能增殖产生子代病毒。为检测构建的HSV1 CH007-Δγ34.5-hUbC-3GFP溶瘤病毒骨架的溶瘤效果，将HSV1 CH007重组病毒感染常见的肿瘤细胞株进行测试，图4所示为体外培养的恶性胶质母细胞瘤细胞株U87MG以及溶瘤病毒感染裂解细胞的结果：没有感染病毒的U87MG恶性胶质瘤细胞胞体呈现聚团以及长而明显的细胞突起和胞间网络化形态；将制备的HSV1 CH007-Δγ34.5-hUbC-3GFP溶瘤病毒按MOI=0.01感染U87MG细胞，感染后12小时就能观察到肿瘤细胞感染重组病毒后表达绿色荧光蛋白，24小时观测大部分肿瘤细胞均被感染、胞体呈绿色荧光；第2天后肿瘤细胞出现明显的细胞病变，细胞突起萎缩变圆；到第3天U87MG细胞全部圆缩，大部分细胞已凋亡（图4），结果表明基于临床分离的HSV1-CH007临床株改造构建的重组溶瘤病毒HSV1 CH007-Δγ34.5-hUbC-3GFP能高效的感染并裂杀恶性肿瘤细胞。
实施例7：武装化PD1抗体基因的HSV1-CH007溶瘤病毒构建
目前应用溶瘤病毒开展的临床治疗或临床试验中均发现OV联合免疫检查点抑制剂给药具有协同效应，溶瘤病毒能实现更好的溶瘤效果。我们设计通过遗传改造使HSV溶瘤病毒基因组本身直接携带抗PD-1蛋白的抗体基因，这样oHSV溶瘤病毒感染裂解肿瘤细胞的同时可在肿瘤细胞内组成性表达PD-1蛋白抗体，从而达到联合用药的增效目的，且能降低成本、简化临床联合给药途径，溶瘤病毒复制增殖和免疫检查点抑制剂表达均在感染的肿瘤细胞内完成，能更好的靶向协同实现高效溶杀肿瘤目的。选择FUGW质粒（Addgene编号14883）作为重组靶向载体的构建骨架；合成PD1抗体的基因序列（ αPD1）（SEQ ID NO.6），通过自剪切肽P2A和红色荧光蛋白（mCherry）（SEQ ID NO.7）基因串联获得表达框mCherry-P2A-αPD1Ab（SEQ ID NO.8）；该表达框片段（长度1599bp）通过Infusion方法克隆入经BamHI、EcoRI双酶切的FUGW质粒，替换掉原来的EGFP荧光基因，获得重组质粒pFUW-hUbC-mCherry-P2A-αPD1Ab（SEQ ID NO.9）。oHSV1-CH007基因组携载PD1抗体基因的溶瘤病毒构建步骤如下：将抽提的靶向载体pFUW-hUbC-mCherry-P2A-αPD1Ab与上述构建的重组病毒HSV1 CH007-Δγ34.5-hUbC-3GFP基因组DNA共转染293T细胞，质粒转染表达红色荧光蛋白，而病毒感染的细胞同时表达绿色荧光蛋白，如果重组成功mCherry-P2A-αPD1Ab基因序列会同源重组替换掉3EGFP基因，新重组病毒将只表达红色荧光蛋白；待大部分细胞出现细胞病变后，收集细胞和病毒上清。经三轮冷冻-震荡-涡旋后，感染六孔板的Vero细胞，1小时后去除上清液，并上铺含有2%胎牛血清、抗生素和1%琼脂糖的培养基。2-3天后挑斑分离良好的表达红色荧光的嗜斑，即绿色背景视野中挑取红色荧光斑，连续10倍稀释后感染新的Vero细胞进行下一轮纯化。经过5轮挑斑、单克隆纯化，完全去除表达绿色荧光蛋白的HSV1 CH007-Δγ34.5-hUbC-3GFP母本病毒，获得单克隆化的携带PD1抗体基因的溶瘤病毒oHSV1 CH007-Δγ34.5-hUbC-mCherry-P2A-αPD1Ab-WPRE（缩写为oHSVCH007-Δγ34.5-αPD1Ab-mChe），病毒基因组示意图如图5，病毒重组挑斑及纯化的细胞图如图6所示。
实施例8：武装化PD1抗体基因的oHSVCH007-Δγ34.5-αPD1Ab-mChe溶瘤病毒的在体毒性测试
将构建的溶瘤病毒oHSVCH007-Δγ34.5-αPD1Ab-mChe大量扩增、纯化浓缩，经嗜斑实验测定制备的溶瘤病毒滴度为8×10 9 PFU/ml。经颅内注射2×10 6 PFU病毒感染小鼠VTA脑区，观察小鼠一直状态良好；14天后灌流小鼠、取脑组织切片观察，可见在注射脑区VTA和病毒经末梢吸收感染的脑区LH、LHb等都有很好的红色荧光标记，只在注射部位观察到有一定炎症反应，其它脑区标记到的神经元都状态良好（图7）。相对的HSV1-CH007野生型病毒感染小鼠症状明显、一周内小鼠全部死亡。实验表明通过自主技术构建的oHSVCH007-Δγ34.5-αPD1Ab-mChe溶瘤病毒具有低毒性、携载的外源抗体蛋白等得到高效表达，感染机体正常组织如神经元细胞溶瘤病毒为复制缺陷型，不会增殖传播致死动物。
实施例9：构建表达绿色荧光蛋白的人源恶性胶质母细胞瘤细胞株U87MG-GFP
为构建新型肿瘤荷瘤动物模型测试oHSVCH007溶瘤病毒效果，我们建立了人源肿瘤细胞株工程化改造技术。利用慢病毒会整合到宿主细胞基因组的特性来构建组成性表达目的蛋白的新型U87MG稳转细胞株；为便于新细胞系的流式细胞筛选纯化，首先构建并制备表达绿色荧光蛋白的慢病毒LentiV-hUbC-EGFP-WPRE；人源胶质母细胞瘤细胞株U87MG感染慢病毒后能稳定表达绿色荧光蛋白，经流式分选纯化后获得新型恶性胶质瘤细胞株U87MG-GFP；如图8所示，制备的工程化细胞株U87MG-GFP纯度很高，几乎所有细胞都稳定表达绿色荧光蛋白，而且细胞状态良好，和野生型U87MG一致，保种到基于U87MG工程化改造的胶质瘤细胞库。
实施例10：利用遗传改造的U87MG-GFP建立恶性胶质母细胞瘤动物模型
培养工程化改造的U87MG-GFP细胞株，制备对数生长期的肿瘤细胞悬液；通过脑立体定位方式移植接种于BALB/c裸鼠右侧大脑CPU脑区，接种剂量5微升，肿瘤细胞数量为1×10 6个；连续观察裸鼠移植胶质瘤后症状，利用小动物磁共振成像仪监测胶质瘤的成瘤状态。磁共振成像（MRI）扫描过程中，使用MSME（multi spin multi echo ）T1-weight 序列获得功能像。胶质瘤细胞株接种后第4周核磁扫描结果如图9所示，未注射肿瘤细胞的健康裸鼠无肿瘤核磁信号（图9A），注射U87MG-GFP的裸鼠在CPU区域形成明显的肿瘤病灶（图9B），说明胶质母细胞瘤荷瘤动物建模成功。
为进一步评估胶质瘤建模情况，第四周核磁扫描后小鼠进行心脏灌流和脑组织取材，冰冻切片得到脑组织切片；经DAPI染色处理后，利用OLYMPUS VS120 全玻片扫描系统进行成像。如图9C所示，注射U87MG-GFP的裸鼠在CPU区域形成大而明显的实体肿瘤，因改造的胶质瘤细胞U87MG-GFP组成性表达绿色荧光蛋白，肿瘤组织呈现明亮的绿色荧光（图9C）。以上结果表明工程化改造的胶质瘤细胞株状态良好，在体移植造模脑肿瘤成功，为oHSV溶瘤病毒效果评价打下了坚实基础。
实施例11：oHSVCH007-Δγ34.5-αPD1Ab-mChe溶瘤病毒能高效溶杀恶性胶质母细胞瘤
我们将纯化制备的oHSVCH007-Δγ34.5-αPD1Ab-mChe溶瘤病毒在建立的恶性胶质瘤荷瘤动物模型上进行了测试。将上述工程化构建的人脑星形胶质母细胞瘤（WHO IV级）细胞株U87MG-GFP定位接种于BALB/c裸鼠皮层下CPU脑区，4周后经核磁共振检测小鼠大脑已成瘤、呈侵袭性生长（图9C）。制备的oHSVCH007-Δγ34.5-αPD1Ab-mChe溶瘤病毒通过瘤内给药方式注射到恶性胶质瘤瘤体内，14天后灌流裸鼠，取脑组织切片观察，发现oHSVCH007-Δγ34.5-αPD1Ab-mChe溶瘤病毒在胶质母细胞瘤体内能有效复制增殖，在病毒感染的肿瘤组织区域出现明显的组织空洞和凋亡炎症反应（图10），结果说明基于中国临床分离株HSV1-CH007构建的新型溶瘤病毒oHSVCH007-Δγ34.5-αPD1Ab-mChe溶瘤病毒能高效溶杀恶性胶质母细胞瘤细胞，和注射PBS缓冲液的对照组荷瘤小鼠相比肿瘤组织体积明显缩小，验证了基于中国HSV临床分离株构建的增效溶瘤病毒具有良好的安全性和溶瘤活性。
显然，上述实施例仅仅是为清楚地说明所作的举例，而并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引伸出的显而易见的变化或变动仍处于本发明创造的保护范围之中。

Claims (10)

  1. 一种单纯疱疹病毒HSV1-CH007株，其保藏编号为CCTCC NO：V202173。
  2. 一种病毒载体，其特征在于，通过敲除单纯疱疹病毒HSV1-CH007株基因组中神经毒力因子γ34.5双拷贝基因得到。
  3. 根据权利要求2所述的病毒载体，其特征在于，所述病毒载体在敲除的γ34.5基因座位插入外源基因表达盒；或，
    所述病毒载体在其基因组任何插入不影响功能的位点插入外源基因表达盒；
    所述外源基因表达盒中的外源基因选自表达任意目的蛋白的基因。
  4. 权利要求2所述的病毒载体在病毒学基础研究、大携载容量病毒载体、溶瘤病毒、目的基因递送、神经系统靶向基因治疗药物、动物感染模型建立或抗病毒药物筛选中的应用。
  5. 一种溶瘤病毒载体，其特征在于，通过敲除单纯疱疹病毒HSV1-CH007株基因组中神经毒力因子γ34.5双拷贝基因得到。
  6. 根据权利要求5所述的溶瘤病毒载体，其特征在于，在敲除的γ34.5基因座位插入肿瘤治疗序列基因表达盒；或，
    所述在溶瘤病毒载体基因组任何插入不影响功能的位点插入肿瘤治疗序列基因表达盒；
    优选地，所述肿瘤治疗序列基因选自免疫检查点蛋白抗体基因、肿瘤免疫治疗细胞因子基因和免疫调控增强基因中的一种或以上；
    优选地，所述肿瘤治疗序列基因表达盒包含启动子和肿瘤治疗序列基因；
    优选地，在肿瘤治疗序列基因下游引入WPRE增强表达元件；
    优选地，所述肿瘤治疗序列基因为免疫检查点蛋白PD1抗体基因；
    优选地，在敲除的γ34.5基因座位还插入荧光蛋白表达盒，所述荧光蛋白表达盒的转录方向与所述肿瘤治疗序列基因表达盒保持一致。
  7. 一种溶瘤病毒，其包含权利要求5或6所述的溶瘤病毒载体。
  8. 一种抗肿瘤药物组合物，其特征在于，包含权利要求7所述的溶瘤病毒。
  9. 一种溶瘤病毒载体的构建方法，其特征在于，敲除单纯疱疹病毒HSV1-CH007株基因组中神经毒力因子γ34.5双拷贝基因；
    优选地，还包括在敲除的γ34.5基因座位或基因组任何插入不影响功能的位点插入肿瘤治疗序列基因表达盒。
  10. 一种溶瘤病毒的构建方法，其特征在于，包括如下步骤：
    （1）靶向载体构建
    克隆γ34.5基因上下游同源臂，将克隆的上下游同源臂片段连入骨架载体，获得重组载体1；构建包括启动子、绿色荧光蛋白基因和WPRE片段的EGFP表达盒，连入重组载体1，获得表达绿色荧光蛋白的重组靶向质粒载体；
    优选地，还包括在重组载体1插入肿瘤治疗序列基因表达盒，具体地： 构建包括启动子、肿瘤治疗序列基因和红色荧光蛋白mCherry基因通过自剪切肽（2A）绑定表达的肿瘤治疗序列基因表达盒，替换表达绿色荧光蛋白的重组靶向质粒载体的EGFP表达框，获得表达肿瘤治疗序列基因的重组靶向质粒载体；
    （2）溶瘤病毒构建
    将敲除γ34.5基因表达绿色荧光蛋白的重组靶向质粒载体与HSV1-CH007株基因组DNA共转染293T细胞，待细胞病变后收集上清，感染Vero细胞，挑斑绿色荧光的病毒嗜斑，经过5轮以上的挑斑纯化后即得表达绿色荧光蛋白的重组病毒；
    优选地，插入肿瘤治疗序列基因的溶瘤病毒的构建，方法包括：将表达肿瘤治疗序列基因的重组靶向质粒载体与表达绿色荧光蛋白的重组HSV1-CH007株病毒基因组DNA共转染293T细胞，待细胞病变后收集上清，感染Vero细胞，在绿色荧光背景下挑斑红色荧光的病毒斑，经过5轮以上的挑斑纯化后即得。
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