A SINV infectious vector and its application in the preparation of anti-tumor drugs

Abstract

The present invention discloses a SINV infectious vector and its application in the preparation of anti-tumor drugs, wherein the SINV infectious vector uses pSINV as a backbone vector, and granulocyte-macrophage colony stimulating factor GM-CSF is connected to the backbone vector. The SINV infectious vector of the present invention can more stably express the inserted exogenous gene, and the biological characteristics of the virus are not significantly different from those of the wild type; the SINV virus particles of the present invention can replicate in cervical cancer Hela tumor cells and liver cancer Hep3B tumor cells, selectively infect tumor cells, and achieve a killing effect on tumors in an immunodeficient mouse model, which has important practical significance and wide application value for applied research and basic research such as oncolytic treatment of cervical cancer and liver cancer.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a SINV infectious vector and application thereof in the preparation of anti-tumor drugs. Background Art

[0002] At present, the traditional technical means for cancer treatment are mainly surgery, chemotherapy, radiotherapy, etc. Although these methods can control tumor growth to a certain extent, they also have certain limitations. Surgical treatment often cannot completely eliminate tumor cells, and the response of postoperative wound healing may also lead to the growth of metastatic tumors; radiotherapy and chemotherapy are prone to cause tumor cell tolerance and recurrence, leading to poor prognosis. Therefore, new strategies for cancer treatment are urgently needed.

[0003] As a new type of tumor cell biological therapy, oncolytic virus therapy has brought new development hope for the treatment of malignant tumors. Oncolytic viruses can selectively kill tumor cells without damaging normal tissues and cells. In addition to their own tumor-killing effects, oncolytic viruses can also carry a variety of exogenous genes to achieve biological regulation. Oncolytic viruses can also induce local and systemic specific anti-tumor immunity, triggering acquired and adaptive immune responses in the body.

[0004] Sindbis virus (SINV) belongs to the Togaviridae family and the Alphavirus genus. Its genome is a single-stranded positive-strand RNA with a length of about 12 kb, encoding a total of 5 structural proteins (capsid, E3, E2, 6K and E1) and 4 non-structural proteins (NSP1, NSP2, NSP3 and NSP4). In nature, Sindbis virus is mainly transmitted in vertebrates such as birds and mammals through mosquito bites. It has a wide host range, including humans, mice, monkeys, etc. This infection characteristic can make it a vector for mediating exogenous genes into the host. Oncolytic virus therapy, which replicates in tumor cells, selectively infects tumor cells and then kills tumor cells, is currently one of the most promising tumor immunotherapy methods. Summary of the invention

[0005] In view of the defects in the prior art, the present invention proposes a SINV infectious vector that stably expresses GM-CSF, its virus particles and applications, and evaluates the effects of the virus particles in the subcutaneous tumor model of Hela mice with cervical cancer and the subcutaneous tumor model of Hep3B mice with liver cancer. The present invention introduces mutations in the NSP1 gene of Sindbis virus - the nucleotide sequence No. 853 of the NSP1 gene mutates from G to A, and the corresponding amino acid sequence No. 285 mutates from Gly to Ser. The introduction of mutations can increase the stability of foreign gene insertion. The present invention successfully prepared Sindbis virus particles that can more stably express the granulocyte-macrophage colony stimulating factor (GM-CSF) gene by transfecting cells with the mutated Sindbis virus infectious vector, and successfully applied them to the oncolytic treatment of the Hela mouse model of cervical cancer and the Hep3B mouse model of liver cancer.

[0006] The invention provides a SINV infectious vector for stably expressing GM-CSF. The SINV infectious vector uses pSINV as a backbone vector, and granulocyte-macrophage colony stimulating factor GM-CSF is connected to the backbone vector; the SINV infectious vector is sequentially connected with a UBC promoter, a 5'UTR, a nucleotide sequence of an NSP1 gene, a nucleotide sequence of an NSP2 gene, a nucleotide sequence of an NSP3 gene, a nucleotide sequence of an NSP4 gene, a nucleotide sequence of a C gene, a nucleotide sequence of an E3 gene, a nucleotide sequence of an E2 gene, a nucleotide sequence of a 6K gene, a nucleotide sequence of an E1 gene, a nucleotide sequence of a GM-CSF gene, and a 3'UTR.

[0007] Furthermore, the nucleotide sequence of the NSP1 gene is shown in SEQ ID NO.14.

[0008] Furthermore, the nucleotide sequence of the SINV infectious vector is shown in SEQ ID NO.13.

[0009] The present invention also provides a method for preparing Sindbis virus particles, using the SINV infectious vector to transfect cells.

[0010] Furthermore, the cells are BHK-21 cells.

[0011] The present invention also provides a Sindbis virus particle, which is prepared by transfecting cells with the SINV infectious vector.

[0012] The present invention also provides the use of the Sindbis virus particles in preparing a drug for treating cervical cancer.

[0013] The present invention also provides the use of the Sindbis virus particles in preparing drugs for treating liver cancer.

[0014] In summary, compared with the prior art, the present invention achieves the following technical effects:

[0015] 1. The present invention provides a Sindbis virus infectious vector that stably expresses the GM-CSF gene. It does not require the steps of in vitro transcription and RNA transfection of cells. The cells can be directly transfected with the Sindbis virus infectious vector, saving time and operation steps. The SINV infectious vector of the present invention can more stably express the inserted foreign gene, and the biological characteristics of the virus are not significantly different from those of the wild type, which is helpful for conducting research on the Sindbis virus infectious vector as a new type of oncolytic virus.

[0016] 2. The present invention is of great value for studying the application prospects of Sindbis virus as a new type of oncolytic virus. It also has important practical significance and broad application value for applied research and basic research (such as virus replication and killing mechanism in tumor cells) such as oncolytic treatment of cervical cancer and liver cancer.

[0017] 3. The Sindbis virus particles prepared by the present invention can exert a significant tumor-killing effect on solid tumors in Hela cell immunodeficient mouse models within 7 days.

[0018] 4. The Sindbis virus particles prepared by the present invention can exert a significant tumor-killing effect on solid tumors in Hep3B cell immunodeficient mouse models within 7 days.

[0019] 5. Oncolytic virus therapy uses genetic engineering to modify oncolytic viruses so that they retain the ability to replicate and are delivered to tumor cells in a targeted manner to kill tumor cells and achieve the purpose of treatment. Suitable oncolytic viruses are of great significance for tumor treatment. The SINV virus particles of the present invention can replicate in cervical cancer Hela tumor cells and liver cancer Hep3B tumor cells, selectively infect tumor cells, and achieve tumor killing effects in immunodeficient mouse models. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0021] Figure 1The present invention is a schematic diagram of the construction of an adaptive mutant Sindbis virus infectious vector expressing GM-CSF and a wild-type Sindbis virus vector expressing GM-CSF, as well as a schematic diagram of the structures of pSINV-GM-CSF and pSINV-G285S-GM-CSF, wherein NSP1-4, C, E3, E2, 6K and E1 are all Sindbis virus proteins.

[0022] Figure 2 The one-step growth curve of the adaptively mutated Sindbis virus infectious vector carrying the GM-CSF gene of the present invention (A), the virus growth curve of BHK-21 cells infected with 0.1 MOI (B), and the virus growth curve of BHK-21 cells infected with 1 MOI (C).

[0023] Figure 3 The adaptively mutated Sindbis virus particles carrying the GM-CSF gene of the present invention infect Hela cells in vitro, and the pathological changes of the cells at different time points after virus infection with different MOIs; wherein A is the pathological changes of the cells after crystal violet staining, and B is the pathological changes of the cells at different time points.

[0024] Figure 4 The present invention is a Sindbis virus particle carrying the GM-CSF gene after adaptive mutation and continuously passaged on Hela cells to determine the expression stability of the exogenously inserted GM-CSF protein.

[0025] Figure 5 The present invention shows the oncolytic treatment of solid tumors in an immunodeficient mouse model implanted subcutaneously with Hela cells by using the adaptively mutated Sindbis virus particles carrying the GM-CSF gene; wherein A is an immunodeficient mouse model implanted subcutaneously with Hela cells and a drug administration process, B is the tumor volume of mice in the experimental group and the control group 7 days after drug administration, C is a curve chart of the changes in the tumor volume of mice in the experimental group and the control group within 7 days after the start of drug administration, and D is the HE staining results of tumor tissues of the two groups of mice 7 days after drug administration.

[0026] Figure 6 The adaptively mutated Sindbis virus particles carrying the GM-CSF gene of the present invention infect Hep3B cells in vitro, and the pathological changes of the cells at different time points after virus infection with different MOIs; wherein A is the pathological changes of the cells at different time points, and B is the pathological changes of the cells after crystal violet staining.

[0027] Figure 7 The adaptively mutated Sindbis virus particles carrying the GM-CSF gene of the present invention are continuously passaged on Hep3B cells to examine the expression stability of the exogenously inserted GM-CSF protein.

[0028] Figure 8 The Sindbis virus particles carrying the GM-CSF gene after adaptive mutation of the present invention are applied to the oncolytic treatment of solid tumors in an immunodeficient mouse model implanted subcutaneously with Hep3B cells; wherein A is an immunodeficient mouse model implanted subcutaneously with Hep3B cells and a drug administration process, B is the tumor volume of mice in the experimental group and the control group 7 days after drug administration, C is a curve chart of the changes in the tumor volume of mice in the experimental group and the control group within 7 days after the start of drug administration, and D is the HE staining results of tumor tissues of the two groups of mice 7 days after drug administration. DETAILED DESCRIPTION

[0029] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only embodiments of a part of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work should fall within the scope of protection of the present invention.

[0030] Unless otherwise specified, the technical solutions described in the present invention are all conventional technologies in the art.

[0031] The mice used in the examples were purchased from Hunan Slake Jingda Experimental Animal Co., Ltd.

[0032] Example 1

[0033] The adaptively mutated Sindbis virus infectious vector stably expressing the granulocyte-macrophage colony stimulating factor gene of the present invention is prepared by the following steps:

[0034] Both pSINV-GM-CSF and pSINV-G285S-GM-CSF use the UBC promoter to initiate the transcription and translation of viral structural and nonstructural proteins to complete viral packaging. The GM-CSF gene was amplified using the primers shown in SEQ ID NO.1 and SEQ ID NO.2, and the nucleotide sequence of the amplified GM-CSF fragment is shown in SEQ ID NO.3;

[0035] The PCR reaction system was 50 μl: 5× Reaction Buffer: 10 μl, 10 mM d NTPs: 1 μl, 10 μM Forward Primer: 2.5 μl, 10 μM Reverse Primer: 2.5 μl, Template DNA: 0.5 μl, DNA Polymerase: 0.5 μl, Nuclease-Free Water: 33 μl; the amplification conditions were: 98°C 60 s, 98°C 10 s, 55°C 15 s, 72°C 60 s, 72°C 10 min, 16°C 10 min, 30 cycles;

[0036] pSINV-EGFP was digested with ApaI and NotI (plasmid pSINV-EGFP has been disclosed in the Chinese invention patent application "CN202111117573.9 Sindbis virus vector and its viral particles and applications in neural circuits"), and then the amplified GM-CSF fragment SEQ ID NO.3 was inserted into pSINV-EGFP using the Vazyme homologous recombination kit. The recombinant product was transformed into competent HB101, and the clones identified as positive by PCR were cultured and the plasmids were extracted for sequencing. The clones with correct sequencing were named pSINV-GM-CSF.

[0037] The two fragments shown in SEQ ID NO.8 and SEQ ID NO.9 were amplified respectively using primers shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, SEQ ID NO.7, and then fused into the fragment shown in SEQ ID NO.10, thereby introducing a point mutation in the nucleotide sequence at position 853 of NSP1, and the corresponding amino acid sequence at position 285 was mutated from Gly to Ser (the nucleotide sequence of the NSP1 gene after mutation is shown in SEQ ID NO.14). The PCR reaction system was 50 μl: 5× Reaction Buffer: 10 μl, 10 mM dNTPs: 1 μl, 10 μM Forward Primer: 2.5 μl, 10 μM Reverse Primer: 2.5 μl, Template DNA: 0.5 μl, DNA Polymerase: 0.5 μl, Nuclease-Free Water: 33 μl; the amplification conditions were: 98°C for 60 s, 98°C for 10 s, 55°C for 15 s, 72°C for 60 s, 72°C for 10 min, 16°C for 10 min, 30 cycles;

[0038] pSINV-GM-CSF was digested with PacI and BglII, and then the fusion fragment shown in SEQ ID NO.10 was inserted into pSINV-GM-CSF using a Vazyme homologous recombination kit. The recombinant product was transformed into competent HB101, and the clones identified as positive by PCR were cultured and the plasmids were extracted for sequencing. The clone with the correct mutation was named pSINV-G285S-GM-CSF, and its nucleotide sequence is shown in SEQ ID NO.13.

[0039] Example 2

[0040] Fluorescence expression and one-step growth curve of the adaptive mutant Sindbis virus infectious vector carrying the GM-CSF gene and the wild-type Sindbis virus infectious vector carrying the GM-CSF gene after transfection:

[0041] After extracting the pSINV-G285S-GM-CSF and pSINV-GM-CSF plasmids prepared in Example 1 using a plasmid extraction kit, lipofectamine 2000 (Thermo Fisher) was used to transfect BHK-21 cells respectively, and cultured in a 37°C, 5% (v / v) CO2 incubator. The generated virus infected BHK-21 cells, and the cell state was observed by an inverted fluorescence microscope at different time points. The cells showed obvious cytopathic conditions after 48 hours. After a portion of the virus supernatant collected at different time points was packaged, a portion was taken to detect the virus titer of the supernatant collected at different time points using the double-layer plaque method, and a one-step growth curve was drawn and compared with the virus of the wild-type GM-CSF. The one-step growth curve of the infectious clone is shown in FIG. Figure 2 The obtained viruses were used to infect BHK-21 cells at 0.1 MOI and 1 MOI, respectively. The growth curves of the two viruses on the cells are shown in Figure 2 As shown in B and C in the figure. The virus supernatant collected at the 48h infection time point was packaged and stored at -80°C for later use in subsequent experiments. After infection in this example, Sindbis virus particles carrying the GM-CSF gene after adaptive mutation prepared by the SINV-G285S-GM-CSF infectious vector and wild-type Sindbis virus particles carrying the GM-CSF gene can be obtained.

[0042] Example 3

[0043] The infection of Sindbis virus particles carrying GM-CSF gene after adaptive mutation of the present invention on Hela cells and the killing of Hela cells:

[0044] The virus supernatant stored in Example 2 was used to infect fresh Hela cells at 1 MOI and 0.1 MOI, respectively. The pathological changes of the cells were observed after 48 hours and 72 hours, and compared with the cells of the control group that were not infected with the Sindbis virus infectious vector. The results are as follows: Figure 3 As shown in B, cells at 1MOI and 0.1MOI showed obvious cytopathic effects. 0.05% crystal violet staining solution was used for staining. Figure 3 As shown in A, it was found that the cells in the 1MOI and 0.1MOI groups showed obvious cytopathic effects. The above results show that the Sindbis virus particles carrying the GM-CSF gene after adaptive mutation can infect Hela cells and have a significant killing effect on them.

[0045] Example 4

[0046] Expression of Sindbis virus particles carrying GM-CSF gene after adaptive mutation of the present invention in Hela cells:

[0047] The virus supernatant stored in Example 2 was recorded as P0 generation. The virus particles of P0 generation were then used to infect fresh Hela cells at 1 MOI. The supernatant obtained after 24 hours was recorded as P1 generation. The supernatant was cultured continuously until the virus supernatant of P5 generation was obtained. RNA was extracted from the virus supernatant of each generation, and one-step RT-PCR amplification was performed using primers shown in SEQ ID NO.11 and SEQ ID NO.12 to detect the loss of the inserted gene. The results are shown in FIG. Figure 4 As shown (M in the figure is a DNA molecular weight standard), the Sindbis virus particles carrying the GM-CSF gene after adaptive mutation can stably express foreign genes in Hela cells.

[0048] Example 5

[0049] The Sindbis virus particles carrying the GM-CSF gene after adaptive mutation of the present invention are applied to solid tumors in immunodeficient mouse models implanted subcutaneously with Hela cells:

[0050] Hela cells were resuspended in PBS and cultured at 2×10 6 The nude mice were grown for 7 days on the 8th day after subcutaneous injection in 3-4 week old Nu / Nu female nude mice. One group was used as the experimental group and injected intratumorally with the virus suspension resuspended in PBS after the virus supernatant obtained in Example 2 was concentrated. 1×10 7 PFU / 100uL / mouse, and the other group was injected with 100uL PBS as the control group.

[0051] The length and width of the mouse tumor were measured every day before and after administration, and the tumor volume of the model mouse was calculated, tumor volume = 1 / 2 × length (mm) × width (mm) × width (mm). After administration, the tumor volume of the experimental group mice decreased significantly, but the tumor volume of the mice in the PBS control group continued to increase. The results are as follows Figure 5 As shown in B and C. Tumor tissues were obtained 7 days after administration, and it was observed that the tumor volume of mice in the PBS control group was significantly larger than that of mice in the drug treatment experimental group, indicating that the Sindbis virus particles carrying the GM-CSF gene after adaptive mutation had a significant tumor-killing effect when applied to the solid tumor model of immunodeficient mice implanted subcutaneously with Hela cells.

[0052] HE staining of the tumor tissues of the two groups of mice revealed that Figure 5 As shown in D in the figure, the mice in the drug treatment experimental group showed obvious abnormalities in the overall structure of the tumor tissue. Large areas of tumor cells were obviously necrotic in the tissue, and only part of the cell nuclear outline remained in the necrotic area, as shown by the arrows in the figure. No obvious inflammatory cell infiltration was observed in the tissue, indicating that the SINV infectious vector stably expressing the GM-CSF gene of the present invention has a significant tumoricidal effect on cervical cancer cells.

[0053] Example 6

[0054] The infection of Sindbis virus particles carrying GM-CSF gene after adaptive mutation of the present invention on Hep3B cells and the killing of Hep3B cells:

[0055] The virus supernatant stored in Example 2 was used to infect fresh Hep3B cells at 1 MOI and 0.1 MOI, respectively. The pathological changes of the cells were observed after 48 and 72 hours, and compared with the cells of the control group that were not infected with the Sindbis virus infectious vector. The results are as follows: Figure 6 As shown in A, cells at 1MOI and 0.1MOI showed obvious cytopathic effects. 0.05% crystal violet staining solution was used for staining. Figure 6 As shown in B, it was found that the cells in the 1MOI and 0.1MOI groups showed obvious cytopathic effects. The above results show that the Sindbis virus particles carrying the GM-CSF gene after adaptive mutation can infect Hep3B cells and have a significant killing effect on them.

[0056] Example 7

[0057] Expression of Sindbis virus particles carrying GM-CSF gene in Hep3B cells after adaptive mutation of the present invention:

[0058] The viral supernatant obtained by transfection in Example 2 was recorded as the P0 generation. The P0 generation virus particles were then used to infect fresh Hep3B cells at an MOI of 1. The supernatant obtained 24 hours later was recorded as the P1 generation. The supernatant was cultured continuously until the P5 generation viral supernatant was obtained. RNA was extracted from the viral supernatant of each generation, and one-step RT-PCR amplification was performed using the primers shown in SEQ ID NO.11 and SEQ ID NO.12 to detect the loss of the inserted gene. The results are shown in FIG. Figure 7 As shown, the Sindbis virus particles carrying the GM-CSF gene after adaptive mutation can stably express the exogenous gene in Hep3B cells.

[0059] Example 8

[0060] The adaptively mutated Sindbis virus particles carrying the GM-CSF gene of the present invention are applied to solid tumors in immunodeficient mouse models implanted subcutaneously with Hep3B cells:

[0061] Hep3B cells were resuspended in PBS and cultured at 2×10 6 The nude mice were grown on the nude mice for 14 days and then randomly divided into two groups on the 15th day. One group was used as the experimental group and injected intratumorally with the virus suspension obtained in Example 2 after concentration and resuspended in PBS. 1×10 7 PFU / 100uL / mouse, and the other group was injected with 100uL PBS as the control group.

[0062] The length and width of the mouse tumor were measured every day before and after administration, and the tumor volume of the model mouse was calculated, tumor volume = 1 / 2 × length (mm) × width (mm) × width (mm). After administration, the tumor volume of the experimental group mice decreased significantly, but the tumor volume of the mice in the PBS control group continued to increase. The results are as follows Figure 8 As shown in B and C. Tumor tissues were taken 7 days after administration, and it was observed that the tumor volume of mice in the PBS control group was significantly larger than that of mice in the drug treatment group, indicating that the Sindbis virus particles carrying the GM-CSF gene after adaptive mutation had a significant tumor-killing effect when applied to the solid tumor model of immunodeficient mice implanted subcutaneously with Hep3B cells.

[0063] HE staining of the tumor tissues of the two groups of mice revealed that Figure 8 As shown in D, the mice in the drug treatment experimental group showed obvious abnormalities in the overall structure of the tumor tissue, and large areas of tumor cells were obviously necrotic in the tissue, and no obvious inflammatory cell infiltration was observed in the tissue, indicating that the SINV infectious vector stably expressing the GM-CSF gene of the present invention has a significant tumoricidal effect on liver cancer cells.

[0064] In summary of the above embodiments, the present invention provides a Sindbis virus infectious vector that stably expresses the granulocyte-macrophage colony stimulating factor (GM-CSF) gene, and this infectious vector has obvious tumor killing effect when applied to cervical cancer cells, cervical cancer mouse subcutaneous solid tumor model, liver cancer cells and liver cancer mouse subcutaneous solid tumor model; it has wide application value in the establishment of drug screening platform, establishment of animal model, analysis of the mechanism of drug killing cervical cancer cells, analysis of virus replication and anti-tumor mechanism in cervical cancer cells, analysis of the mechanism of drug killing liver cancer cells, analysis of virus replication and anti-tumor mechanism in liver cancer cells, etc.

[0065] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A SINV infectious vector, characterized in that: The SINV infectious vector uses pSINV as a backbone vector, and granulocyte-macrophage colony stimulating factor GM-CSF is connected to the backbone vector; The SINV infectious vector is sequentially connected with UBC promoter, 5'UTR, nucleotide sequence of NSP1 gene, nucleotide sequence of NSP2 gene, nucleotide sequence of NSP3 gene, nucleotide sequence of NSP4 gene, nucleotide sequence of C gene, nucleotide sequence of E3 gene, nucleotide sequence of E2 gene, nucleotide sequence of 6K gene, nucleotide sequence of E1 gene, nucleotide sequence of GM-CSF gene, and 3'UTR; The nucleotide sequence of the SINV infectious vector is shown in SEQ ID NO.

13.

2. The SINV infectious vector according to claim 1, characterized in that The nucleotide sequence of the NSP1 gene is shown in SEQ ID NO.

14.

3. A method for preparing Sindbis virus particles, characterized in that: Cells are transfected using the SINV infectious vector of claim 1.

4. The method for preparing Sindbis virus particles according to claim 3, characterized in that: The cells are BHK-21 cells.

5. A Sindbis virus particle, characterized in that The method is described in claim 3.

6. Use of the Sindbis virus particles according to claim 5 in the preparation of a drug for treating cervical cancer.

7. Use of the Sindbis virus particles according to claim 5 in the preparation of drugs for treating liver cancer.

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