CN116179602B

CN116179602B - A SINV infectious vector and its application in the preparation of anti-tumor drugs

Info

Publication number: CN116179602B
Application number: CN202211207752.6A
Authority: CN
Inventors: 贾凡; 施祥玮; 徐富强
Original assignee: Shenzhen Institute of Advanced Technology of CAS
Current assignee: Shenzhen Institute of Advanced Technology of CAS
Priority date: 2022-09-30
Filing date: 2022-09-30
Publication date: 2025-03-07
Anticipated expiration: 2042-09-30
Also published as: CN116179602A

Abstract

The invention discloses an SINV infectious carrier and application thereof in preparing antitumor drugs, wherein pSINV is used as a framework carrier, and granulocyte-macrophage colony stimulating factor GM-CSF is connected to the framework carrier. The SINV infectious vector can more stably express inserted exogenous genes, has no significant difference in virus biological characteristics compared with wild type, can replicate in cervical cancer Hela tumor cells and liver cancer Hep3B tumor cells, selectively infect the tumor cells, realizes the killing effect on tumors in an immunodeficiency mouse model, and has important practical significance and wide application value for application research and basic research of oncolytic treatment of cervical cancer and liver cancer and the like.

Description

SINV infectious vector and application thereof in preparation of antitumor drugs

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a SINV infectious vector and application thereof in preparation of antitumor drugs.

Background

At present, the traditional technical means for cancer treatment mainly comprise operation treatment, chemotherapy, radiotherapy and the like, and although the methods can control the tumor growth to a certain extent, certain limitations exist. The operation treatment often cannot completely remove the tumor cells, the reaction of wound healing after operation can also lead to the growth of metastatic tumors, and the radiotherapy and the chemotherapy easily cause the tolerance and the recurrence of the tumor cells, so that the prognosis is poor. Thus, new strategies for cancer treatment are urgently needed.

Oncolytic virus therapy is taken as a novel tumor cell biological therapy, and brings new development hope for the treatment of malignant tumors. Oncolytic viruses are capable of selectively killing tumor cells without damaging normal tissues and cells. Besides the tumor killing effect of the oncolytic virus, the oncolytic virus can also carry a plurality of exogenous genes to realize biological regulation and control effects, and the oncolytic virus can also induce local and systemic specific anti-tumor immunity to trigger an acquired immune response and an adaptive immune response in a body.

Sindbis virus (SINV) belongs to the family togaviridae, genus alphavirus, whose genome is a single-stranded positive strand RNA of about 12kb in length, and encodes a total of 5 structural proteins (capsid, E3, E2, 6K, and E1) and 4 non-structural proteins (NSP 1, NSP2, NSP3, and NSP 4). The sindbis virus is transmitted in nature mainly by mosquito bites in vertebrates such as birds and mammals, has a wide host range including human beings, mice, monkeys and the like, and can be used as a vector for mediating exogenous genes into the host body due to the infection characteristic. Oncolytic viral therapy, which replicates within a tumor cell, selectively infects the tumor cell and then kills the tumor cell, is one of the most promising tumor immunotherapeutic approaches at present.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a SINV infectious vector for stably expressing GM-CSF, a virus particle and application thereof, and the effect of the virus particle in a cervical cancer Hela mouse subcutaneous tumor model and a liver cancer Hep3B mouse subcutaneous tumor model is evaluated. The invention introduces mutation in NSP1 gene of sindbis virus, wherein the nucleotide sequence of 853 number of NSP1 gene is changed into A from G, the corresponding amino acid sequence of 285 number is changed into Ser from Gly, and the introduction of mutation can increase the insertion stability of exogenous gene. The invention successfully prepares sindbis virus particles capable of more stably expressing granulocyte-macrophage colony stimulating factor (GM-CSF) genes by using the mutated sindbis virus infectious vector transfected cells, and is successfully applied to oncolytic treatment of cervical cancer Hela mouse model and liver cancer Hep3B mouse model.

The invention provides an SINV infectious vector for stably expressing GM-CSF, which takes pSINV as a framework vector, and is connected with granulocyte-macrophage colony stimulating factor GM-CSF, and 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.

Further, the nucleotide sequence of the NSP1 gene is shown as SEQ ID NO. 14.

Further, the nucleotide sequence of the SINV infectious vector is shown as SEQ ID NO. 13.

The invention also provides a preparation method of SINV virus particles, and cells are transfected by using the SINV infectious vector.

Further, the cells are BHK-21 cells.

The invention also provides SINV virus particles, which are prepared by transfecting cells with the SINV infectious vector.

The invention also provides application of the Sindbis virus particles in preparing medicaments for treating cervical cancer.

The invention also provides application of the sindbis virus particles in preparing medicaments for treating liver cancer.

In conclusion, compared with the prior art, the invention achieves the following technical effects:

1. The invention provides a sindbis virus infectious vector for stably expressing GM-CSF gene, which does not need the steps of in vitro transcription and RNA transfection, and can directly transfect cells by the sindbis virus infectious vector, thereby saving time and operation steps. The SINV infectious vector can more stably express the inserted exogenous gene, has no significant difference in virus biological characteristics compared with a wild type, and is favorable for developing the research related to the sindbis virus infectious vector serving as a novel oncolytic virus.

2. The invention has important value for researching the Sindbis virus as a novel oncolytic virus application prospect, and has important practical significance and wide application value for application researches such as oncolytic treatment of cervical cancer and liver cancer and basic researches (such as replication of the virus in tumor cells, killing mechanism and the like).

3. The sindbis virus particle prepared by the invention can play an obvious role in killing tumors on solid tumors of a Hela cell immunodeficiency mouse model within 7 days.

4. The Sindbis virus particles prepared by the invention can play an obvious role in killing tumors on the solid tumors of the Hep3B cell immunodeficiency mouse model within 7 days.

5. Oncolytic virus therapy utilizes genetic engineering means to modify oncolytic viruses so as to ensure that the oncolytic viruses retain the replication capacity of the viruses, and the oncolytic viruses are delivered to tumor cells in a targeted manner to kill the tumor cells, thereby achieving the aim of treatment. The SINV virus particles can replicate in cervical cancer Hela tumor cells and liver cancer Hep3B tumor cells, selectively infect the tumor cells, and realize the killing effect on tumors in an immunodeficiency mouse model.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a schematic diagram of construction of an adaptive mutant Sindbis virus infectious vector of the invention expressing GM-CSF and a wild-type Sindbis virus vector expressing GM-CSF, and schematic diagrams of the structures of pSINV-GM-CSF and pSINV-G285S-GM-CSF, wherein NSP 1-4, C, E3, E2, 6K and E1 are Sindbis virus proteins.

FIG. 2 shows a one-step growth curve (A) 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, a virus growth curve (B) of BHK-21 cells infected at 0.1MOI, and a virus growth curve (C) of BHK-21 cells infected at 1MOI according to the present invention.

FIG. 3 shows the condition of the cells at different time points of different MOI-infected viruses after the adaptive mutation of the Sindbis virus particles carrying the GM-CSF gene of the invention are infected with Hela cells in vitro, wherein A is the condition of cytopathy after crystal violet staining, and B is the condition of the cells at different time points.

FIG. 4 shows the expression stability of exogenously inserted GM-CSF protein after adaptive mutation of the invention by serial passage of Sindbis virus particles carrying the GM-CSF gene on HeLa cells.

FIG. 5 shows the application of the sindbis virus particles carrying the GM-CSF gene after the adaptive mutation in the oncolytic treatment of solid tumors of an immunodeficiency mouse model subcutaneously planted by Hela cells, wherein A is the immunodeficiency mouse model subcutaneously planted by Hela cells and the administration flow, B is the tumor volumes of mice in an experimental group and a control group after 7 days of administration treatment, C is a graph showing the tumor volume change of mice in the experimental group and the control group within 7 days after the beginning of administration, and D is the HE staining result of tumor tissues of two groups of mice after 7 days of administration.

FIG. 6 shows the condition of the cells of different MOI-infected viruses at different time points after in vitro infection of Hep3B cells by the Sindbis virus particles carrying GM-CSF gene after the adaptive mutation, wherein A is the condition of the cells at different time points and B is the condition of cytopathy after crystal violet staining.

FIG. 7 expression stability of exogenously inserted GM-CSF protein after adaptive mutation of the invention by serial passaging of Sindbis virus particles carrying the GM-CSF gene on Hep3B cells.

FIG. 8 shows that the sindbis virus particle carrying the GM-CSF gene after the adaptive mutation is applied to the treatment condition of solid tumor dissolution of an immunodeficiency mouse model subcutaneously planted by Hep3B cells, wherein A is an immunodeficiency mouse model subcutaneously planted by Hep3B cells and a drug administration flow, B is the tumor volume of mice in an experimental group and a control group after 7 days of drug administration treatment, C is a graph of the tumor volume change condition of mice in the experimental group and the control group within 7 days after the beginning of drug administration, and D is the HE staining result of tumor tissues of the two groups of mice after 7 days of drug administration.

Detailed Description

In order that those skilled in the art will better understand the present invention, a technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in which it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, shall fall within the scope of the invention.

The technical scheme of the invention is conventional in the art unless specifically stated otherwise.

The mice used in the examples were purchased from Hunan Stokes Lemonda laboratory animals Co.

Example 1

The invention relates to an adaptive mutant sindbis virus infectious vector for stably expressing granulocyte-macrophage colony stimulating factor gene, which is prepared by the following steps:

pSINV-GM-CSF and pSINV-G285S-GM-CSF both start transcription and translation of viral structural proteins and non-structural proteins by UBC promoter, and complete packaging of the virus. Amplifying the GM-CSF gene by using primers shown in SEQ ID NO.1 and SEQ ID NO.2, wherein the nucleotide sequence of the amplified GM-CSF fragment is shown in SEQ ID NO. 3;

The PCR reaction system is 50μl：5×Reaction Buffer：10μl,10mM d NTPs：1μl,10μM Forward Primer：2.5μl,10μM Reverse Primer：2.5μl,Template DNA：0.5μl,DNA Polymerase：0.5μl,Nuclease-Free Water：33μl; amplification conditions of 98 ℃ for 60s,98 ℃ for 10s,55 ℃ for 15s,72 ℃ for 60s,72 ℃ for 10min,16 ℃ for 10min and 30 cycles;

The recombinant product was transformed into competent HB101 using ApaI and NotI cleavage pSINV-EGFP (plasmid pSINV-EGFP has been disclosed in the Chinese patent application "CN202111117573.9 Sindbis virus vector and its viral particles and use in neural circuits"), followed by insertion of the amplified GM-CSF fragment SEQ ID No.3 into pSINV-EGFP using Vazyme homologous recombination kit, culturing the clone identified as positive by PCR and extracting the plasmid for sequencing, the correctly sequenced clone being designated pSINV-GM-CSF.

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

The fusion fragment shown in SEQ ID NO.10 is inserted into pSINV-GM-CSF by using PacI and BglII enzyme digestion pSINV-GM-CSF, then a Vazyme homologous recombination kit is adopted, the recombinant product is transformed into competent HB101, clones which are identified as positive by PCR are cultivated, plasmids are extracted for sequencing, and the clone with the correct mutation after sequencing is named pSINV-G285S-GM-CSF, and the nucleotide sequence of the clone is shown in SEQ ID NO. 13.

Example 2

The invention relates to an adaptive mutation Sindbis virus infectious vector carrying GM-CSF gene, a fluorescent expression condition after transfection of a wild Sindbis virus infectious vector carrying GM-CSF gene and a one-step growth curve:

After extracting pSINV-G285S-GM-CSF and pSINV-GM-CSF plasmids prepared in example 1 with a plasmid extraction kit, BHK-21 cells were transfected with lipofectamine 2000 (Thermo Fisher) and cultured in a 37℃and 5% (v/v) CO ₂ incubator, respectively, and the resulting viruses infected BHK-21 cells, and at various time points, the cell states were observed by an inverted fluorescence microscope, and the cells appeared to be significantly cytopathic after 48 hours. After a part of the virus supernatant collected at different time points was split-charged, a part of the virus supernatant collected at different time points was measured by a double-layer plaque method, and a one-step growth curve was drawn, and compared with the virus of the wild type GM-CSF, the infectious clone one-step growth curve was shown as A in FIG. 2. The resulting viruses were infected with BHK-21 cells at 0.1MOI and 1MOI, respectively, and the growth curves of the two viruses on the cells are shown in FIG. 2 at B, C. The virus supernatant collected at the time point of 48 hours of infection is split-packed and stored at-80 ℃ for later use in subsequent experiments. Through the infection of this example, SINV-G285S-GM-CSF infectious vectors were obtained as adaptive mutated sindbis virus particles carrying GM-CSF gene and wild-type sindbis virus particles carrying GM-CSF gene.

Example 3

Infection of the GM-CSF gene-carrying Sindbis virus particles on Hela cells after the adaptive mutation and killing of the Hela cells:

The virus supernatant stored for standby in example 2 was infected with fresh Hela cells at 1MOI and 0.1MOI, respectively, and after 48 hours and 72 hours, the lesions of the cells were observed, and compared with the cells of the control group not infected with sindbis virus infectious vector, and as a result, as shown in B in fig. 3, the cells at 1MOI and 0.1MOI were both apparent cytopathic. Staining with 0.05% crystal violet staining solution, as shown in fig. 3, revealed that both 1MOI and 0.1MOI groups of cells exhibited significant cytopathic effects. The results show that the sindbis virus particles carrying the GM-CSF gene after the adaptive mutation can infect Hela cells and have obvious killing effect on the Hela cells.

Example 4

Expression of the inventive adapted mutated sindbis virus particles carrying the GM-CSF gene in Hela cells:

The viral supernatant stored for later use in example 2 was designated as P0 generation, the P0 generation viral particles were again infected with fresh Hela cells at 1MOI, the supernatant obtained after 24 hours was designated as P1 generation, and serial subculturing was performed sequentially until P5 generation viral supernatant was obtained. RNA is extracted from virus supernatant of each generation, primers shown as SEQ ID NO.11 and SEQ ID NO.12 are adopted for carrying out one-step RT-PCR amplification, the loss of inserted genes is detected, the result is shown in a graph (M is a DNA molecular weight standard in the graph) as shown in a graph 4, and the Sindbis virus particles carrying GM-CSF genes after adaptive mutation can stably express exogenous genes in Hela cells.

Example 5

The invention discloses application of the adaptive mutant sindbis virus particles carrying GM-CSF gene in solid tumors of immunodeficiency mouse model planted subcutaneously by Hela cells:

After the Hela cells were resuspended in PBS, nu/Nu female Nu mice of 3-4 weeks old were subcutaneously injected at 2×10 ⁶/mL, and after 7 days of growth on Nu mice, the model of Nu mice successfully modeled was randomly divided into two groups on day 8, one group was intratumorally injected with the virus suspension obtained in example 2 after concentration from the virus supernatant of PBS as an experimental group, and 1×10 ⁷ PFU/100 uL/d, and the other group was injected with 100uL of PBS as a control group.

Tumor volume of model mice was calculated by measuring the length and width of tumor of mice daily before and after administration, tumor volume=1/2×length (mm) ×width (mm). After administration, the tumor volume of the mice in the experimental group was significantly reduced, but the tumor volume of the mice in the PBS control group was still further increased, and the result is shown as B, C in fig. 5. Tumor tissues are taken 7 days after administration, the tumor volume of the PBS control group mice is observed to be obviously larger than that of the mice of the administration treatment experiment group, and the application of the adaptive mutated sindbis virus particles carrying the GM-CSF gene to the solid tumors of the immunodeficiency mouse model planted under the skin of Hela cells is proved to have obvious effect of killing the tumors.

After HE staining is carried out on tumor tissues of two groups of mice, as shown in D in fig. 5, the mice of the administration treatment experiment group have obvious tumor tissue overall structure abnormality, the tissues can obviously necrotize large tumor cells, only partial cell nucleus outline exists at the necrotic position, and as shown by the arrow of the figure, the tissues do not see obvious inflammatory cell infiltration, so that the SINV infectious vector for stably expressing GM-CSF gene has obvious tumor killing effect on cervical cancer cells.

Example 6

Infection of the Sindbis virus particle carrying GM-CSF gene on Hep3B cell after adaptive mutation and killing of Hep3B cell:

The virus supernatant stored in example 2 was used to infect fresh Hep3B cells at 1 and 0.1MOI, respectively, and after 48 and 72 hours the lesions of the cells were observed, as compared to the cells of the control group not infected with sindbis virus infectious vector, and as a result, as shown in fig. 6, the cells at 1 and 0.1MOI both showed significant cytopathic effects. Staining with 0.05% crystal violet staining solution, as shown in fig. 6B, found that both 1MOI and 0.1MOI groups of cells exhibited significant cytopathic effects. The results show that the Sindbis virus particle carrying GM-CSF gene after adaptive mutation can infect Hep3B cells and has obvious killing effect

Example 7

Expression of the Sindbis virus particles carrying the GM-CSF gene after adaptive mutation in Hep3B cells:

The viral supernatant obtained by transfection in example 2 was designated as P0 generation, the P0 generation viral particles were again infected with fresh Hep3B cells at 1MOI, the supernatant obtained after 24 hours was designated as P1 generation, and serial subculturing was performed sequentially until P5 generation viral supernatant was obtained. RNA is extracted from virus supernatant of each generation, primers shown in SEQ ID NO.11 and SEQ ID NO.12 are adopted for one-step RT-PCR amplification, the condition of inserted gene loss is detected, and the result is shown in figure 7, and the Sindbis virus particle carrying GM-CSF gene after adaptive mutation can stably express exogenous genes in Hep3B cells.

Example 8

The invention discloses application of the adaptive mutant sindbis virus particles carrying GM-CSF gene in the subcutaneous implantation of Hep3B cells in solid tumors of immunodeficiency mice model:

After Hep3B cells were resuspended in PBS, nu/Nu female nude mice of 3-4 weeks old were subcutaneously injected at 2×10 ⁶/mL, and after 14 days of growth on nude mice, the model of nude mice successfully molded was randomly divided into two groups on day 15, one group was intratumorally injected with the virus suspension obtained in example 2 after concentration of the virus supernatant resuspended in PBS as experimental group, 1×10 ⁷ PFU/100 uL/d, and the other group was injected with 100uL of PBS as control group.

Tumor volume of model mice was calculated by measuring the length and width of tumor of mice daily before and after administration, tumor volume=1/2×length (mm) ×width (mm). After administration, the tumor volume of the mice in the experimental group was significantly reduced, but the tumor volume of the mice in the PBS control group was still further increased, and the result is shown as B, C in fig. 8. Tumor tissues are taken 7 days after administration, the tumor volume of the PBS control group mice is observed to be obviously larger than that of the mice of the administration treatment group, and the application of the adaptive mutated Sindbis virus particles carrying the GM-CSF gene to the immunodeficiency mouse model solid tumors planted under the skin of the Hep3B cells is proved to have obvious effect of killing the tumors.

After HE staining is carried out on tumor tissues of two groups of mice, as shown in D in fig. 8, the mice of the administration treatment experiment group have obvious tumor tissue overall structure abnormality, the tissues can obviously necrotize large tumor cells, and the tissues do not obviously infiltrate inflammatory cells, which indicates that the SINV infectious vector for stably expressing GM-CSF gene has obvious tumor killing effect on liver cancer cells.

By combining the above embodiments, the invention provides a Sindbis virus infectious vector for stably expressing granulocyte-macrophage colony stimulating factor (GM-CSF) gene, and the infectious vector is applied to cervical cancer cells, cervical cancer mouse subcutaneous solid tumor models, liver cancer cells and liver cancer mouse subcutaneous solid tumor models, has obvious tumor killing effect, and has wide application value in the aspects of drug screening platform establishment, animal model establishment, drug killing cervical cancer cell action mechanism analysis, virus replication in cervical cancer cells and anti-tumor mechanism analysis, drug killing liver cancer cell action mechanism analysis, virus replication in liver cancer cells and anti-tumor mechanism analysis, and the like.

The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the 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 the UBC promoter, 5'UTR, the nucleotide sequence of the NSP1 gene, the nucleotide sequence of the NSP2 gene, the nucleotide sequence of the NSP3 gene, the nucleotide sequence of the NSP4 gene, the nucleotide sequence of the C gene, the nucleotide sequence of the E3 gene, the nucleotide sequence of the E2 gene, the nucleotide sequence of the 6K gene, the nucleotide sequence of the E1 gene, the nucleotide sequence of the GM-CSF gene, and the 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 with the SINV infectious vector according to claim 1.

4. The method for preparing Sindbis virus particles according to claim 3, wherein the cells are BHK-21 cells.

5. A Sindbis virus particle, characterized in that it is prepared by the preparation method according to 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.