AU2020103637A4 - Recombinant oncolytic herpes simplex virus type ii and its pharmaceutical composition - Google Patents

Recombinant oncolytic herpes simplex virus type ii and its pharmaceutical composition Download PDF

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AU2020103637A4
AU2020103637A4 AU2020103637A AU2020103637A AU2020103637A4 AU 2020103637 A4 AU2020103637 A4 AU 2020103637A4 AU 2020103637 A AU2020103637 A AU 2020103637A AU 2020103637 A AU2020103637 A AU 2020103637A AU 2020103637 A4 AU2020103637 A4 AU 2020103637A4
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Abstract

The present disclosure provides a recombinant oncolytic herpes simplex virus type 11 and its pharmaceutical composition. The recombinant oncolytic herpes simplex virus type 11, with two ICP34.5 genes and one ICP47 gene knocked out, are inserted human telomerase chemotactic gene SLC-Te-Fc expression cassette into the two ICP34.5 gene sites respectively, and the SLC-Te-Fc expression cassette is successively connected with CMV promoter and secondary lymphoid tissue chemokines SLC, telomerase Te, Fc fragment of antibody and Bovine growth hormone polyadenosine sequence BGHpA. Oncolytic HSV not only has a strong oncolytic effect, but also can be used as a gene carrier to carry and express tumor associated antigens efficiently as therapeutic immune gene vaccine. The combination of virus and chemotherapy also overcomes the weakness that oncolytic HSV alone cannot strongly induce specific anti-tumor immunity.

Description

Recombinant Oncolytic Herpes Simplex Virus Type II and Its Pharmaceutical Composition
Technical Field The present disclosure relates to the field of biomedicine, in particular to a recombinant oncolytic herpes simplex virus type II and its pharmaceutical composition.
Background Art Herpes simplex virus (HSV) is a 154 KB double stranded DNA virus that can replicate in the nucleus of infected host. HSV vector has the following advantages: 1) extensive host cells; 2) high virus titer; 3) large capacity of foreign gene. The disadvantage of HSV vector is its toxicity.
For example, ICP47 protein affects the function of transporters (TAP1 and TAP2) related to antigen processing, and hinders the antigen presentation process of MHC I molecules. Therefore, elimination of ICP47 gene is beneficial to enhance immune response. In addition, removing ICP47 gene increased the expression of US I1gene downstream. US I1could resist the inhibition of PKR on virus growth and reproduction. 1CP34.5 is a neurotoxic factor. The virus without 1CP34.5 can selectively kill tumor cells. The mechanism is that 1CP34.5 can resist PKR inactivation of ef-2a, and make the virus propagate smoothly. Therefore, when the virus without 1CP34.5 enters normal cells, it induces the activation of IFN/PKR pathway, which leads to the inactivation of ef-2a, the failure of viral protein synthesis and the inhibition of viral reproduction. However, the IFN/PKR signaling pathway in most tumor cells was impaired. Therefore, the virus without 1CP34.5 can grow and reproduce in tumor cells. The ICP6 gene encodes ribonucleotide reductase, which provides an essential precursor for viral DNA replication and is not essential in dividing cells (such as tumor cells).
As we all know, the disadvantage of DNA nucleic acid vaccine such as CADV is the low transfection efficiency in vivo, but if the virus vector with high transfection efficiency is used to carry CADV, this shortcoming can be overcome. Oncolytic HSV not only has a strong oncolytic effect, but also can be used as a gene carrier to carry and express tumor associated antigens efficiently as therapeutic immune gene vaccine. The combination of the two also overcomes the weakness that oncolytic HSV alone cannot strongly induce specific anti-tumor immunity.
In 2010, the China patent application No. 2010101162753 disclosed a recombinant herpes simplex virus type II vector, preparation method, recombinant virus, pharmaceutical composition and application thereof The inventor Liu Binlei deleted the 1CP34.5 gene and ICP47 gene from the genome of wild type II herpes simplex virus HG52 strain, and inserted Human Granulocyte Macrophage Colony Stimulating Factor Expression Cassette at the site where the 1CP34.5 gene was deleted. The recombinant virus obtained by the CN 2010101162753 has the characteristics of good safety, high oncolytic activity and strong anti-tumor immune response.
Although GM-CSF expressed by oHSV2-GM-CSF can promote the maturation of DC cells, tumor associated antigens (such as telomerase antigen) released by viral oncolysis may not be effectively captured and presented by DC cells. oHSV2-SLC-Te-Fc solves this problem. The SLC-Te-Fc fusion protein contains the sequence of the tumor broad-spectrum antigen telomerase
(Te, also known as TERT). Its SLC attracts immune cells (DC, T lymphocytes, etc.) to the tumor site, while FC can bind to the corresponding receptors on the DC, so that DC can capture Te antigen efficiently and present it to T cells, thus activating specific anti-tumor immune response more effectively.
Summary The technical problem to be solved by the disclosure is to provide a recombinant oncolytic herpes simplex virus type II and its pharmaceutical composition. The recombinant oncolytic herpes simplex virus type II also overcomes the weakness that oHSV cannot strongly induce specific anti-tumor immunity alone.
To solve the technical problem, the disclosure provides a recombinant oncolytic herpes simplex virus type II, with two ICP34.5 genes and one ICP47 gene knocked out, wherein the two ICP34.5 gene sites are inserted a human telomerase chemotactic gene SLC-Te-Fc expression cassette respectively, and the SLC-Te-Fc expression cassette is successively connected with CMV promoter and secondary lymphoid tissue chemokines SLC, telomerase Te, Fc fragment of antibody and Bovine growth hormone polyadenosine sequence BGHpA; The virus was classified as Herpes SimplexVirus Type2 and was deposited in the general microbiology center of China General Microbiological Culture Collection Center on January 3, 2014 in Beijing, and Its deposit number is CGMCC No. 8709.
The disclosure also provides a preparation method for recombinant oncolytic herpes simplex virus type II, comprising the following steps: (1) Deleting the ICP47 gene of wild type II herpes simplex virus HG52 strain to construct HG52dICP47 recombinant herpes simplex virus type II: a. Extracting the genomic DNA of herpes simplex virus type II HG52 strain; b. Constructing the plasmid pdICP47H2 containing the upstream flanking region sequence and the downstream flanking region sequence of ICP47 gene: b1. The upstream flanking region sequence and downstream flanking region sequence of ICP47 gene were amplified by PCR using the genomic DNA obtained from step a as template and the primers as shown below;
Forward 14 6554 AGAGTCACGACGCATTTGCCC1 46 574 Amplification of the primer upstream flanking region147755 flankigegine Reverse ATACGATCTCGTCGACCGGGG of1ICP47 gene pie primer 14 8 033 148 053 Forward CATGGTGTCCCGTCCACGAAG Amplification of The downstream flanking primer Reverse 1492 11 GGTTCGTGGTAATGAGATGCC 49 9 region of1ICP47 gene pie primer b2. Inserting the upstream flanking region of PCR product amplified by step b Iinto SmaI site of plasmid pSP73 to obtain the plasmid pSP73ICP47US; b3. Inserting the downstream flanking region of PCR product amplified by step b Iinto SmaI site of pSP73 plasmid to obtain plasmid pICP47DS; b4. The downstream flanking region sequence is cut out of the plasmid pICP47DS obtained from step b3 by restriction endonucleases Sac and BamHI, and inserted into the BglII site of the plasmid pSP73ICP47US obtained in step b2, then the plasmid pdICP47H2 containing the upstream flanking region sequence and downstream flanking region sequence of ICP47 gene is obtained; b5. Inserting the green fluorescent protein expression cassette controlled by cytomegalovirus IE promoter into the EcoRV site of the plasmid pdICP47H2 obtained in step b4, then the plasmid pdICP47H2-GFP is obtained; c. Constructing a recombinant herpes simplex virus type II HG52dICP47 with ICP47 gene deleted: cl. Co-transfecting the full-length viral DNA obtained from step A and the plasmid pdICP47H2-GFP obtained in step B into BHK cells, Homologous recombination between the ICP47 gene on the full-length viral DNA and the green fluorescent protein expression cassette on the plasmid pdICP47H2-GFP, resulting in the green fluorescence of the recombinant virus spots; c2. Selecting the green fluorescent spots to purify recombinant herpes simplex virus type II HG52dICP47-GFP; c3. Extracting the genomic DNA of recombinant herpes simplex virus type II HG52dICP47-GFP; c4. Co-transfecting the full-length viral DNA of the recombinant herpes simplex virus type II HG52dICP47-GFP obtained in step c3 and the plasmid pdICP47H2 obtained in step b4 into BHK cells, the green fluorescent protein expression cassette on the recombinant herpes simplex virus type II HG52dICP47-GFP is homologous recombined and eliminated; c5. Selecting non-fluorescent virus spots and purifying the recombinant type II herpes simplex virus HG52dICP47; (2) Removing the ICP34.5 gene and constructing the recombinant herpes simplex virus type II HG52dICP47d34.5-GFP; A. Extracting the genomic DNA of recombinant herpes simplex virus type II HG52dICP47; B. Constructing the plasmid pH2dI34.5 containing the upstream flanking region and downstream flanking region of ICP34.5 gene: B. The upstream flanking region sequence and downstream flanking region sequence of ICP34.5 gene were amplified by PCR using the genomic DNA obtained from step a as template and the primers as shown below; 4376 Amplification of Forward AAATCAGCTG 13CGGTGAAGGTCGTCGTCAGAG the upstream primer 41 flanking region Reverse AAATTCTAGA 1 GCCGGCTTCCCGGTATGGTAA1 56 of ICP34.5 gene primer 12 6 9 6 3
Amplification of Forward AAATGATATC16943 CAGCCCGGGCCGTGTTGCGGG the downstream primer flanking region Reverse AAATAGATCT CTCTGACCTGAGTGCAGGTTA12 76 2 0
of ICP34.5 gene primer B2. Inserting the upstream flanking region of PCR product amplified by step B1 into PvuII/XbaI site of plasmid pSP72 to obtain plasmid pSP72H2d34.5US; B3. Inserting the downstream flanking region sequence of PCR product amplified by step BI into EcoRV/BglII site of plasmid pSP72H2d34.5US obtained in step B2, and the plasmid pH2d34.5 containing upstream flanking region and downstream flanking region sequence of ICP34.5 gene is obtained;
B4 . Inserting the green fluorescent protein expression cassette controlled by cytomegalovirus IE promoter into EcoRV site of plasmid pH2d34.5 obtained in step B3, the plasmid pH2d34.5GFP is obtained; C. Constructing a recombinant herpes simplex virus typeII HG52dICP47d34.5 with ICP34.5 gene deleted: C1. Co-transfecting the genomic DNA of recombinant herpes simplex virus type II HG52dICP47 obtained from step A and plasmid pH2d34.5GFP obtained from step B into BHK cells, Homologous recombination between the ICP34.5 gene on the genomic DNA and the green fluorescent protein expression cassette on plasmid pH2d34.5GFP, resulting in the green fluorescence of the recombinant virus spots; C2. Selecting green fluorescent spots to purify recombinant herpes simplex virus type II HG52dICP47d34.5-GFP; C3. Extracting genomic DNA of recombinant herpes simplex virus type II HG52dICP47d34.5-GFP; C4. Co-transfecting the genomic DNA of recombinant herpes simplex virus type II HG52dICP47d34.5-GF obtained from step C3 and the plasmid pH2d34.5 obtained from step B3 into BHK cells, the green fluorescent protein expression cassette of recombinant herpes simplex virus type II HG52dICP47d34.5GFP is homologous recombined and eliminated; C5. Selecting non fluorescent spots and purifying the recombinant herpes simplex virus type II HG52dICP47d34.5; (3) Constructing the recombinant herpes simplex virus type II vector HG52dICP47dICP34.5-SLC-Te-Fc by inserting the gene of human telomerase chemoattractant gene SLC-Te-Fc expression cassette; i. Extracting the genomic DNA of recombinant herpes simplex virus type II HG52dICP47d34.5 obtained from step C5; ii. Inserting the expression cassette of telomerase chemoattractant gene SLC-Te-Fc controlled by cytomegalovirus IE promoter into EcoRV site of plasmid pH2d34.5 obtained in step B3, and the plasmid pH2d34.5-SLC-Te-Fc is obtained; iii. Co-transfecting the recombinant herpes simplex virus type II HG52dICP47d34.5 genomic DNA obtained in step i and the plasmid pH2d34.5-SLC-Te-Fc obtained in step ii into BHK cells, and the human telomerase chemoattractant gene SLC-Te-Fc is expressed by the homologous recombinant virus spots; iv. Obtaining the recombinant oncolytic herpes simplex virus type II HG52dICP47dICP34.5-SLC-Te-Fc, named oHSV2-SLC-Te-Fc/oHSV2-CADV, that is purified from the positive spots detected by ELISA. As a preferred scheme, the preparation method comprises the following steps: I. Extracting the full-length viral DNA of recombinant herpes simplex virus type II HG52dICP47d34.5-GFP obtained from step C2; II. Inserting the human granulocyte macrophage colony stimulating factor expression cassette controlled by cytomegalovirus IE promoter into EcoRV site of plasmid pH2d34.5 obtained in step B3 to obtain plasmid pH2d34.5- SLC-Te-Fc; III. Co-transfecting the recombinant herpes simplex virus type II HG52dICP47d34.5-GFP and the plasmid pH2d34.5- SLC-Te-Fc obtained in step II into BHK cells, the human granulocyte macrophage colony-stimulating factor is expressed by the homologous recombinant virus spots which is no-fluorescence; IV. Selecting non fluorescent spots and purifying the recombinant oncolytic herpes simplex virus type II HG52dICP47dICP34.5-SLC-Te-Fc. Further, the disclosure also provides applications of the recombinant oncolytic herpes simplex virus type II according to claim 1 in the preparation of gene drug for tumor treatment. For example, the gene drug is a combination drug of adriamycin and oHSV2-CADV.
Beneficial Effects
1. Using oncolytic HSV2 as a carrier, injects a chemotactic-antigen DNA vaccine (CADV) into the tumor and expresses it efficiently, which not only dissolves the tumor but also induces a tumor-specific immune response, so as to achieve the dual purpose of eliminating the primary tumor and metastasis. The structure of CADV is characterized by: (1) Secondary lymphoid tissue chemokine (SLC), which can chemotactic dendritic cells (DC) and T cells, is contained at the front of tumor-associated antigen genes. (2) At the back end of tumor-associated antigen gene contains Fc segment gene of IgG, SLC can actively recruit T cells, B cells, NK cells and DC cells positived for its receptor CCR7, while the Fc segment binds to the Fc receptor on the DC, so that the antigen is efficiently captured by the DC, and its efficiency is more than 1000 times that of the captured antigen by phagocytosis, thus significantly enhancing the immunogenicity of the vaccine. Oncolytic HSV not only has a strong oncolytic effect, but also can be used as a gene carrier to carry and highly express tumor-associated antigens as a therapeutic immune gene vaccine. The combination of virus and chemotherapy also overcomes the weakness that oncolytic HSV alone cannot strongly induce specific anti-tumor immunity.. 2. The present disclosure inserts the expression sequence of SLC-Te-Fc into the genome of oncolytic HSV2 (oHSV2 for short) to construct oHSV2-SLC-Te-Fc. Intratumoral injection of oHSV2-SLC-Te-Fc exerts potent anti-tumor effects through several main mechanisms: (1) The oHSV2-SLC-Te-Fc grows and propagates in tumor cells and has direct lysis effect on tumor cells. (2) Highly expressed SLC-Te-Fc is released to the extracellular space with the lysis and death of tumor cells. On the one hand, SLC recruits dendritic cell (DC) cells, on the other hand, Fc enables DC to capture TERT antigen efficiently and induce specific anti-tumor immune response. (3) The oHSV2-SLC-Te-Fc promotes the release of other tumor-associated antigens (TAA), which are captured and presented by dendritic cells (DC) to further enhance the specific anti-tumor immune response. (4) Although GM-CSF expressed by oHSV2-GM-CSF can promote DC maturation, tumor-associated antigens released by viral oncolysis, such as telomerase antigen, are not necessarily effectively captured and presented by DC cells. And oHSV2-SLC-Te-Fc solved this problem. The SLC-Te-Fc fusion protein has a sequence of the tumor broad-spectrum antigen Telomerase (Te, also known as TERT). Its SLC chemotaxis attracts immune cells(DC, T lymphocyte, etc.) to the tumor site, while Fc can bind to corresponding receptors on DC, enabling DC to efficiently capture Te antigen and present it to T cells, thereby more effectively activating specific anti-tumor immune responses.
Description of the Drawings
FIG. 1 is a diagram showing the effect of oHSV2-CADV in vivo. FIG. 2 is a diagram showing the tumor inhibition effect of different treatment groups.
Detailed Description of the Embodiments
The disclosure is further described in detail below in conjunction with specific embodiments, but the contents of the present invention are not limited to the following embodiments only. Embodiment 1 The preparation method for recombinant oncolytic herpes simplex virus type II, comprising the following steps: (1) Deleting the ICP47 gene of wild type II herpes simplex virus HG52 strain to construct HG52dICP47 recombinant herpes simplex virus type II: a. Extracting the genomic DNA of herpes simplex virus type II HG52 strain; b. Constructing the plasmid pdICP47H2 containing the upstream flanking region sequence and the downstream flanking region sequence of ICP47 gene: b1. The upstream flanking region sequence and downstream flanking region sequence of ICP47 gene were amplified by PCR using the genomic DNA obtained from step a as template and the primers as shown below;
Forward 14 65 54 AGAGTCACGACGCATTTGCCC1 46 574 Amplification of the primer upstream flanking region147755 Reverse ATACGATCTCGTCGACCGGGG ofICP47gene primer 14 80 33 148 053 Forward CATGGTGTCCCGTCCACGAAG Amplification of The downstream flanking primer Reverse 1492 11 GGTTCGTGGTAATGAGATGCC 49 9 region of1ICP47 gene primer b2. Inserting the upstream flanking region of PCR product amplified by step b Iinto SmaI site of plasmid pSP73 to obtain the plasmid pSP73ICP47US; b3. Inserting the downstream flanking region of PCR product amplified by step bl into SmaI site of pSP73 plasmid to obtain plasmid pICP47DS; b4. The downstream flanking region sequence is cut out of the plasmid pICP47DS obtained from step b3 by restriction endonucleases Sac and BamHI, and inserted into the BglII site of the plasmid pSP73ICP47US obtained in step b2, then the plasmid pdICP47H2 containing the upstream flanking region sequence and downstream flanking region sequence of ICP47 gene is obtained; b5. Inserting the green fluorescent protein expression cassette controlled by cytomegalovirus IE promoter into the EcoRV site of the plasmid pdICP47H2 obtained in step b4, then the plasmid pdICP47H2-GFP is obtained; c. Constructing a recombinant herpes simplex virus type II HG52dICP47 with ICP47 gene deleted: cl. Co-transfecting the full-length viral DNA obtained from step A and the plasmid pdICP47H2-GFP obtained in step B into BHK cells, Homologous recombination between the ICP47 gene on the full-length viral DNA and the green fluorescent protein expression cassette on the plasmid pdICP47H2-GFP, resulting in the green fluorescence of the recombinant virus spots; c2. Selecting the green fluorescent spots to purify recombinant herpes simplex virus type II HG52dICP47-GFP; c3. Extracting the genomic DNA of recombinant herpes simplex virus type II HG52dICP47-GFP; c4. Co-transfecting the full-length viral DNA of the recombinant herpes simplex virus type II HG52dICP47-GFP obtained in step c3 and the plasmid pdICP47H2 obtained in step b4 into BHK cells, the green fluorescent protein expression cassette on the recombinant herpes simplex virus type II HG52dICP47-GFP is homologous recombined and eliminated; c5. Selecting non-fluorescent virus spots and purifying the recombinant type II herpes simplex virus HG52dICP47; (2) Removing the ICP34.5 gene and constructing the recombinant herpes simplex virus type II HG52dICP47d34.5-GFP; A. Extracting the genomic DNA of recombinant herpes simplex virus type II HG52dICP47; B. Constructing the plasmid pH2dI34.5 containing the upstream flanking region and downstream flanking region of ICP34.5 gene: B. The upstream flanking region sequence and downstream flanking region sequence of ICP34.5 gene were amplified by PCR using the genomic DNA obtained from step a as template and the primers as shown below; 4376 Amplification of Forward AAATCAGCTG 13CGGTGAAGGTCGTCGTCAGAG the upstream primer 41 flanking region Reverse AAATTCTAGA 1 GCCGGCTTCCCGGTATGGTAA1 56 of ICP34.5 gene primer 12 6 9 6 3
Amplification of Forward AAATGATATC16943 CAGCCCGGGCCGTGTTGCGGG the downstream primer flanking region Reverse AAATAGATCT CTCTGACCTGAGTGCAGGTTA12 76 2 0
of ICP34.5 gene primer B2. Inserting the upstream flanking region of PCR product amplified by step B1 into PvuII/XbaI site of plasmid pSP72 to obtain plasmid pSP72H2d34.5US; B3. Inserting the downstream flanking region sequence of PCR product amplified by step BI into EcoRV/BglII site of plasmid pSP72H2d34.5US obtained in step B2, and the plasmid pH2d34.5 containing upstream flanking region and downstream flanking region sequence of ICP34.5 gene is obtained; B4 . Inserting the green fluorescent protein expression cassette controlled by cytomegalovirus IE promoter into EcoRV site of plasmid pH2d34.5 obtained in step B3, the plasmid pH2d34.5GFP is obtained; C. Constructing a recombinant herpes simplex virus typeII HG52dICP47d34.5 with ICP34.5 gene deleted: C1. Co-transfecting the genomic DNA of recombinant herpes simplex virus type II HG52dICP47 obtained from step A and plasmid pH2d34.5GFP obtained from step B into BHK cells, Homologous recombination between the ICP34.5 gene on the genomic DNA and the green fluorescent protein expression cassette on plasmid pH2d34.5GFP, resulting in the green fluorescence of the recombinant virus spots; C2. Selecting green fluorescent spots to purify recombinant herpes simplex virus type II HG52dICP47d34.5-GFP; C3. Extracting genomic DNA of recombinant herpes simplex virus type II HG52dICP47d34.5-GFP; C4. Co-transfecting the genomic DNA of recombinant herpes simplex virus type II HG52dICP47d34.5-GF obtained from step C3 and the plasmid pH2d34.5 obtained from step B3 into BHK cells, the green fluorescent protein expression cassette of recombinant herpes simplex virus type II HG52dICP47d34.5GFP is homologous recombined and eliminated; C5. Selecting non fluorescent spots and purifying the recombinant herpes simplex virus type II HG52dICP47d34.5; (3) Constructing the recombinant herpes simplex virus type II vector HG52dICP47dICP34.5-SLC-Te-Fc by inserting the gene of human telomerase chemoattractant gene SLC-Te-Fc expression cassette; i. Extracting the genomic DNA of recombinant herpes simplex virus type II HG52dICP47d34.5 obtained from step C5; ii. Inserting the expression cassette of telomerase chemoattractant gene SLC-Te-Fc controlled by cytomegalovirus IE promoter into EcoRV site of plasmid pH2d34.5 obtained in step B3, and the plasmid pH2d34.5-SLC-Te-Fc is obtained; iii. Co-transfecting the recombinant herpes simplex virus type II HG52dICP47d34.5 genomic DNA obtained in step i and the plasmid pH2d34.5-SLC-Te-Fc obtained in step ii into BHK cells, and the human telomerase chemoattractant gene SLC-Te-Fc is expressed by the homologous recombinant virus spots; iv. Obtaining the recombinant oncolytic herpes simplex virus type II HG52dICP47dICP34.5-SLC-Te-Fc, named oHSV2-SLC-Te-Fc/oHSV2-CADV, that is purified from the positive spots detected by ELISA.
The recombinant oncolytic herpes simplex virus type II oHSV2-CADV obtained from Example 1 was tested and verified. 1. Antitumor effect of oHSV2-CADV in vivo: Forty C57/BL mice were loaded with B16R (mouse B16 melanoma cell line stably expressing herpes simplex virus receptor) according to the number of1 X 105 cells/50tl/mouse. Five days later, the animals were grouped according to the average tumor volume as follows (10 mice/group): OHSV1-hGM-CSF(oHSV1), oHSV2-hGM-CSF(oHSV2), oHSV2-CADV(CADV) and control group (control). OHSV1-hGM-CSF and oHSV2-hGM-CSF were modified from herpes simplex virus type I 17+ strain and herpes simplex virus type II HG52 strain, respectively, including knockout of the neurovirulence gene ICP34.5, immunosuppressive gene ICP47, and insertion of the hGM-CSF expression cassette at the ICP34.5 site. Viral treatment doses were 2x106 PFU/200tl/mouse, while the control group was treated with serum-free medium, 200pl/mouse. Each treatment and Control groups were intratumoral injections. Viral and control treatments were given three times, the tumor size of mice was measured every other day on day 0 after the first treatment, and the survival time of mice was observed.
The experimental results are shown in Figure 1 and Table 1 below: Table 1: Median Survival oHSV1 oHSV2 CADV CONTROL Median survival 22 26 34 16 Tumor free 0/10 0/10 2/10 0/10 Compared with the control group (FIG. 1), the oHSV1, oHSV2 and CADV treatment groups could significantly prolong the survival of tumor-bearing mice. However, the CADV group had the longest median survival and had 2 tumor-free animals. No tumor-free animals were found in other groups. This experiment shows that the in vivo tumor inhibition effect of oHSV2-CADV is significantly better than that of the oHSV1 and oHSV2.
Embodiment 2 A virus injection containing oHSV2-CADV prepared by Example 1 , named oHSV2-CADV-injection. 1. Establishment of two animal models of tumors and in vivo oncolytic effect of the recombinant virus vector according to the present disclosure. 1) C57/BL and Balb/c female mice were purchased from the Laboratory Animal Center of the Chinese Academy of Medical Sciences at the age of 4-6 weeks, 16-20g/mouse (C57/BL was the melanoma tumor-bearing strain of B16R, BALB/c was the tumor-bearing strain of CT26); 2) Nodule melanoma cells (B16R) and colonic adenocareinoma cells (CT26) were selected and inoculated subcutaneously into the right axilla of mice with trocar. 105 tumor cells were injected subcutaneously into the flank of each mouse. When the tumor mass diameter was about 0.5-0.7 cm (5-7 days after injection), 10 mice were randomly divided into three groups (Experimental Group is oHSV2-CADV, Control Group is pSLC-Te-Fc DNA vaccine, Blank Group is control group). 3) Each group was injected intratumorally on the day 1, 4 and 7 with an injection volume of 100 microliters, and the Control Group are also electric-transduced to enhance the transduction efficiency of plasmid DNA on day 1, 14 and 28. Among them, the dosage of the oHSV2-CADV of the invention in the Experimental Group is 2.5 X108pfu/kg, the dosage of the plasmid DNA of the pSLC-Te-Fc DNA vaccine in the Control Group is 2.5 X mg/kg, and the Blank Group was injected a phosphate buffer of the same volume as the oHSV2-CADV of Experimental Group. After the last intratumoral administration, the observed tumor volume on the animal's body surface was measured weekly for 4 weeks, and then the animals were sacrificed by cervical dislocation. The results are shown in Table 2 below, and the tumor diameter is mm. Table 2: In vivo antitumor effect of the recombinant viral vector Before 2 Weeks After 4 Weeks After Treatment Treatment Treatment Melanoma Experimental Group 6.1±0.8 7.2±0.8 8.3±1.2 Control Group 6.0±0.7 11.6±1.2 16.7±3.8 Blank Group 6.1±0.6 13.5±1.8 18.1±2.9
Colonic Experimental Group 5.5±0.7 6.8±1.2 9.2±0.9 Adenocareinoma Control Group 5.4±0.6 9.5±1.3 17.9±4.1 Blank Group 5.6±0.5 10.9±1.5 16.7±3.2 The oHSV2-CADV vector has obvious anti-tumor effect on the two kinds of fast growing tumors. On the contrary, in the Blank Group, the tumor continued to grow, and the tumor diameter reached 1.5 cm at 4 weeks, and the control group had no obvious effect on tumor inhibition.
Synergistic effect of oHSV2-CADV combined with chemotherapy on breast cancer in mouse The mice bearing 4T1 (mouse breast cancer) cells were divided into 4 groups (8 mice in each group) after tumor emergence. The specific plan is shown in the table 3 below. In the doxorubicin (DOX group), caudal vein chemotherapy was given three times on day 0, 6 and 12, with a dose of 8 mg/kg/mouse/time. In the group using oHSV2-CADV alone (CADV for short), on the 2nd, 4th, 8th, 10th and 14th day, CADV (1 X 10 7pfu/100l mouse/time) was injected into the tumor with a 27G needle. The Control Group was given the same volume of solvent. In the combined chemotherapy group (DOX+CADV), DOX was given by tail vein chemotherapy on day 0, day 6 and day 12, and CADV intratumoral injection was given on day 2, 4, 8, 10 and 14. Table 3 Groups CADV (Intratumoral) DOX (vein) Dose (pfu) Day Dose (mg/kg) Day Control -
CADV 1 x 10 7 2,4,6,8,12 -
DOX - 8 0,6, 12 DOX + CADV 1 x 107 2, 4, 6,8,12 8 0,6,12 The changes of tumor diameter in each group were observed under the condition of CADV alone or DOX alone or in combination (see FIG. 2). The tumor growth of mice in the Control Group showed logarithmic growth, and the tumor growth was significantly inhibited in the CADV alone or DOX alone group, and the tumor growth inhibition effect was the most obvious in the combined treatment group(DOX+CADV). On the 24th day after treatment, there was no significant difference in tumor volume between CADV alone group (779.8±109.9 mm 3) and DOX alone group (708.5±91.6 mm 3) (P=0.2610), but they were significantly smaller than those in Control Group (1331.0±108.4 mm3; compared with the Control Group, the CADV group was P=0.0163, and the DOX group was P=0.0014). The average tumor volume in the combined chemotherapy group (DOX+CADV) was significantly lower than the other three groups (P<0.05). Therefore, in terms of tumor growth inhibition, viral therapy alone can achieve similar results as DOX alone. If these two methods are combined, the effect is obviously better than any single treatment, suggesting that oHSV2-CADV combined with chemotherapy may bring more benefits to patients.
The technologies not described are prior art. Although the above-mentioned embodiment has made a detailed description of the disclosure, it is only a part of the embodiment of the invention, not all the embodiments. People can obtain other embodiments according to the embodiment without creativity, and these embodiments belong to the protection scope of the disclosure.

Claims (6)

  1. WHAT IS CLAIMED IS: 1. A recombinant oncolytic herpes simplex virus type II, with two ICP34.5 genes and one ICP47 gene knocked out, wherein the two ICP34.5 gene sites are inserted a human telomerase chemotactic gene SLC-Te-Fc expression cassette respectively, and the SLC-Te-Fc expression cassette is successively connected with CMV promoter and secondary lymphoid tissue chemokines SLC, telomerase Te, Fc fragment of antibody and Bovine growth hormone polyadenosine sequence BGHpA; And Its deposit number is CGMCC No. 8709.
  2. 2. A preparation method for recombinant oncolytic herpes simplex virus type II of claim 1, comprising the following steps: (1) Deleting the ICP47 gene of wild type II herpes simplex virus HG52 strain to construct HG52dICP47 recombinant herpes simplex virus type II: a. Extracting the genomic DNA of herpes simplex virus type II HG52 strain; b. Constructing the plasmid pdICP47H2 containing the upstream flanking region sequence and the downstream flanking region sequence of ICP47 gene: b1. The upstream flanking region sequence and downstream flanking region sequence of ICP47 gene were amplified by PCR using the genomic DNA obtained from step a as template and the primers as shown below;
    46 574 Forward 14 6554 AGAGTCACGACGCATTTGCCC1 Amplification of the primer upstream flanking region147755 flankigegine Reverse ATACGATCTCGTCGACCGGGG of1ICP47 gene pie primer 14 8 033 148 053 Forward CATGGTGTCCCGTCCACGAAG Amplification of The downstream flanking primer Reverse 1492 11 GGTTCGTGGTAATGAGATGCC 49 9 region of1ICP47 gene pie primer b2. Inserting the upstream flanking region of PCR product amplified by step b Iinto SmaI site of plasmid pSP73 to obtain the plasmid pSP73ICP47US; b3. Inserting the downstream flanking region of PCR product amplified by step bl into SmaI site of pSP73 plasmid to obtain plasmid pICP47DS; b4. The downstream flanking region sequence is cut out of the plasmid pICP47DS obtained from step b3 by restriction endonucleases Sac and BamHI, and inserted into the BglII site of the plasmid pSP73ICP47US obtained in step b2, then the plasmid pdICP47H2 containing the upstream flanking region sequence and downstream flanking region sequence of ICP47 gene is obtained; b5. Inserting the green fluorescent protein expression cassette controlled by cytomegalovirus IE promoter into the EcoRV site of the plasmid pdICP47H2 obtained in step b4, then the plasmid pdICP47H2-GFP is obtained; c. Constructing a recombinant herpes simplex virus type II HG52dICP47 with ICP47 gene deleted: cl. Co-transfecting the full-length viral DNA obtained from step A and the plasmid pdICP47H2-GFP obtained in step B into BHK cells, Homologous recombination between the ICP47 gene on the full-length viral DNA and the green fluorescent protein expression cassette on the plasmid pdICP47H2-GFP, resulting in the green fluorescence of the recombinant virus spots; c2. Selecting the green fluorescent spots to purify recombinant herpes simplex virus type II HG52dICP47-GFP; c3. Extracting the genomic DNA of recombinant herpes simplex virus type II HG52dICP47-GFP; c4. Co-transfecting the full-length viral DNA of the recombinant herpes simplex virus type II HG52dICP47-GFP obtained in step c3 and the plasmid pdICP47H2 obtained in step b4 into BHK cells, the green fluorescent protein expression cassette on the recombinant herpes simplex virus type II HG52dICP47-GFP is homologous recombined and eliminated; c5. Selecting non-fluorescent virus spots and purifying the recombinant type II herpes simplex virus HG52dICP47; (2) Removing the ICP34.5 gene and constructing the recombinant herpes simplex virus type II HG52dICP47d34.5-GFP; A. Extracting the genomic DNA of recombinant herpes simplex virus type II HG52dICP47; B. Constructing the plasmid pH2dI34.5 containing the upstream flanking region and downstream flanking region of ICP34.5 gene: B. The upstream flanking region sequence and downstream flanking region sequence of ICP34.5 gene were amplified by PCR using the genomic DNA obtained from step a as template and the primers as shown below; 4376 Amplification of Forward AAATCAGCTG 13CGGTGAAGGTCGTCGTCAGAG the upstream primer 41 flanking region Reverse AAATTCTAGA 1 GCCGGCTTCCCGGTATGGTAA1 56 of ICP34.5 gene primer 12 6 9 6 3
    Amplification of Forward AAATGATATC16943 CAGCCCGGGCCGTGTTGCGGG the downstream primer flanking region Reverse AAATAGATCT CTCTGACCTGAGTGCAGGTTA12 76 2 0
    of ICP34.5 gene primer B2. Inserting the upstream flanking region of PCR product amplified by step B1 into PvuII/XbaI site of plasmid pSP72 to obtain plasmid pSP72H2d34.5US; B3. Inserting the downstream flanking region sequence of PCR product amplified by step BI into EcoRV/BglII site of plasmid pSP72H2d34.5US obtained in step B2, and the plasmid pH2d34.5 containing upstream flanking region and downstream flanking region sequence of ICP34.5 gene is obtained; B4 . Inserting the green fluorescent protein expression cassette controlled by cytomegalovirus IE promoter into EcoRV site of plasmid pH2d34.5 obtained in step B3, the plasmid pH2d34.5GFP is obtained; C. Constructing a recombinant herpes simplex virus typeII HG52dICP47d34.5 with ICP34.5 gene deleted: C1. Co-transfecting the genomic DNA of recombinant herpes simplex virus type II HG52dICP47 obtained from step A and plasmid pH2d34.5GFP obtained from step B into BHK cells, Homologous recombination between the ICP34.5 gene on the genomic DNA and the green fluorescent protein expression cassette on plasmid pH2d34.5GFP, resulting in the green fluorescence of the recombinant virus spots;
    C2. Selecting green fluorescent spots to purify recombinant herpes simplex virus type II HG52dICP47d34.5-GFP; C3. Extracting genomic DNA of recombinant herpes simplex virus type II HG52dICP47d34.5-GFP; C4. Co-transfecting the genomic DNA of recombinant herpes simplex virus type II HG52dICP47d34.5-GF obtained from step C3 and the plasmid pH2d34.5 obtained from step B3 into BHK cells, the green fluorescent protein expression cassette of recombinant herpes simplex virus type II HG52dICP47d34.5GFP is homologous recombined and eliminated; C5. Selecting non fluorescent spots and purifying the recombinant herpes simplex virus type II HG52dICP47d34.5; (3) Constructing the recombinant herpes simplex virus type II vector HG52dICP47dICP34.5-SLC-Te-Fc by inserting the gene of human telomerase chemoattractant gene SLC-Te-Fc expression cassette; i. Extracting the genomic DNA of recombinant herpes simplex virus type II HG52dICP47d34.5 obtained from step C5; ii. Inserting the expression cassette of telomerase chemoattractant gene SLC-Te-Fc controlled by cytomegalovirus IE promoter into EcoRV site of plasmid pH2d34.5 obtained in step B3, and the plasmid pH2d34.5-SLC-Te-Fc is obtained; iii. Co-transfecting the recombinant herpes simplex virus type II HG52dICP47d34.5 genomic DNA obtained in step i and the plasmid pH2d34.5-SLC-Te-Fc obtained in step ii into BHK cells, and the human telomerase chemoattractant gene SLC-Te-Fc is expressed by the homologous recombinant virus spots; iv. Obtaining the recombinant oncolytic herpes simplex virus type II HG52dICP47dICP34.5-SLC-Te-Fc, named oHSV2-SLC-Te-Fc/oHSV2-CADV, that is purified from the positive spots detected by ELISA.
  3. 3. The preparation method of claim 2, comprising: I. Extracting the full-length viral DNA of recombinant herpes simplex virus type II HG52dICP47d34.5-GFP obtained from step C2; II. Inserting the human granulocyte macrophage colony stimulating factor expression cassette controlled by cytomegalovirus IE promoter into EcoRV site of plasmid pH2d34.5 obtained in step B3 to obtain plasmid pH2d34.5- SLC-Te-Fc; III. Co-transfecting the recombinant herpes simplex virus type II HG52dICP47d34.5-GFP and the plasmid pH2d34.5- SLC-Te-Fc obtained in step II into BHK cells, the human granulocyte macrophage colony-stimulating factor is expressed by the homologous recombinant virus spots which is no-fluorescence; IV. Selecting non fluorescent spots and purifying the recombinant oncolytic herpes simplex virus type II HG52dICP47dICP34.5-SLC-Te-Fc.
  4. 4. The application of the recombinant oncolytic herpes simplex virus type II according to claim 1 in the preparation of gene drug for tumor treatment.
  5. 5. The application of claim 4, wherein the gene drug is a pharmaceutical composition containing oHSV2-CADV.
  6. 6. The application of claim 4, wherein the gene drug is a combination drug of adriamycin and oHSV2-CADV.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115707781A (en) * 2021-08-20 2023-02-21 广东东阳光药业有限公司 HSV (herpes simplex virus) vector and application thereof
CN115808528A (en) * 2022-09-21 2023-03-17 武汉滨会生物科技股份有限公司 Interaction method and application of oncolytic herpes simplex virus VP5 protein and human leukocyte antigen E

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115707781A (en) * 2021-08-20 2023-02-21 广东东阳光药业有限公司 HSV (herpes simplex virus) vector and application thereof
CN115808528A (en) * 2022-09-21 2023-03-17 武汉滨会生物科技股份有限公司 Interaction method and application of oncolytic herpes simplex virus VP5 protein and human leukocyte antigen E

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