Neurotoxicity-free VSV vector recombinant oncolytic virus carrying Marburg virus deficient G protein

Abstract

The invention discloses a neurotoxicity-free VSV vector recombinant oncolytic virus carrying Marburg virus deficient G protein, which is characterized in that a G protein coding gene of wild VSV is replaced by a coding gene of Marburg virus deficient G protein through a reverse genetic manipulation technology, so as to save and obtain a recombinant oncolytic virus VSV-MARG-delta MLD capable of being autonomously replicated. The invention has the advantages of no neurotoxicity and efficient oncolytic activity.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to a non-neurotoxic VSV vector recombinant oncolytic virus carrying a Marburg virus defective G protein. BACKGROUND

[0002] Malignant tumors seriously threaten human health and life, and its incidence continues to rise worldwide, which has become an important public health challenge. Surgery, radiotherapy and chemotherapy as traditional treatment methods have limited efficacy in advanced or metastatic patients, and radiotherapy and chemotherapy are often accompanied by significant toxic side effects. In recent years, immune checkpoint inhibitors (ICIs) have shown breakthrough efficacy in some cancer types, but still face challenges such as low overall response rate and primary drug resistance. Therefore, it is particularly urgent to develop new, efficient and safe anti-tumor strategies.

[0003] Oncolytic viruses (OVs) are a class of viruses that can specifically infect and replicate in tumor cells, and then lyse tumor cells and induce anti-tumor immune response. Based on its unique mechanism of action, oncolytic virus therapy has become an important direction in the field of tumor immunotherapy.

[0004] Marburg virus ( Marburg virus ), also known as green monkey virus, is named after the city of Marburg in Germany. The virus body is polymorphic, showing branches or coiling into U-shaped, "6"-shaped or ring-shaped. It is a deadly virus and the first filamentous virus discovered by humans, which can cause Marburg hemorrhagic fever. The virus and Ebola virus are of the same origin and belong to the Filoviridae family, and are also common diseases of humans and other primates. Marburg virus is the first linear virus discovered, with a single-stranded negative-strand RNA genome of about 19 kb, encoding 7 viral proteins. The envelope of Marburg virus contains specific glycoproteins that can bind to specific receptors on the surface of human cells to enter the cell for replication, and cause cell apoptosis through the toxicity of viral proteins.

[0005] Vesicular Stomatitis Virus (VSV) is a non-pathogenic negative-strand RNA enveloped virus with multiple characteristics as an ideal oncolytic virus vector: low pre-existing immunity in the human population, no integration of the genome into the host chromosome, short replication cycle, good immunogenicity, and easy genetic manipulation. However, preclinical studies have shown that VSV can cause significant neurotoxicity in rodent and non-human primate animal models, especially when administered via the central nervous system, which has seriously limited its clinical application.

[0006] The neurotoxicity of wild-type VSV is mainly related to the cytotoxicity of its matrix protein (M protein) and the neurotropic property of its glycoprotein (G protein). Therefore, modification of these two genes is a key strategy to reduce neurotoxicity. However, M protein mutants often have excessive reduction in virulence, leading to rapid clearance in vivo or reduced ability to replicate and spread in tumor tissues, thereby affecting their oncolytic effect. At present, there is an urgent need to develop a VSV vector that has good safety (no neurotoxicity) and retains high oncolytic activity, in order to promote the development of this field. SUMMARY

[0007] The purpose of the present application is to provide a neurotoxicity-free VSV vector recombinant oncolytic virus carrying a Marburg virus defective G protein, which has both neurotoxicity-free and high oncolytic activity.

[0008] The technical solution adopted by the present application to solve its technical problems is: A neurotoxicity-free VSV vector recombinant oncolytic virus carrying a Marburg virus defective G protein is obtained by reverse genetic manipulation technology, in which the G protein coding gene of wild-type VSV is replaced by the coding gene of Marburg virus defective G protein, thereby rescuing a recombinant oncolytic virus VSV-MARG-ΔMLD that can self-replicate. The amino acid sequence of the Marburg virus defective G protein is shown in SEQ ID No. 26.

[0009] As a preferred embodiment, the wild-type VSV strain is the Indiana strain.

[0010] As a preferred embodiment, the sequence of the coding gene of the Marburg virus defective G protein is shown in SEQ ID No. 1. This sequence is a codon-optimized sequence.

[0011] The preparation method is specifically as follows: (1) Constructing the VSV-MARG-ΔMLD plasmid: first insert the VSV genome into the BAC vector, then replace the gene sequence of the G protein on the VSV genome with the gene sequence encoding the Marburg virus defective G protein, to obtain the pBAC-VSV-MARG-ΔMLD plasmid; (2) Rescuing the recombinant virus: the first infected cells are infected with a vaccinia virus expressing T7 polymerase, the infected cells are co-transfected with the pBAC-VSV-MARG-ΔMLD plasmid, pN, pP, pL and pG plasmids, the supernatant of the diseased cells is collected 48 h after transfection, the second infected cells are infected with the supernatant, and the supernatant of the diseased cells is collected after amplification of the virus, to obtain the VSV vector recombinant oncolytic virus; The first infected cells are BHK21 cells; The second infected cells are Vero cells.

[0012] As preferred, the mass ratio of pBAC-VSV-MARG-ΔMLD, pN, pP, pL and pG plasmids is 10:3:5:1:3 when co-transfected.

[0013] The application of the non-neurotoxic VSV vector recombinant oncolytic virus in the preparation of an antitumor drug. The tumor includes a digestive tract tumor, a head and neck tumor, breast cancer, lymphoma, uterine cancer, ovarian cancer, bladder cancer, liver cancer, lung cancer, osteosarcoma and melanoma.

[0014] An antitumor enhanced modified recombinant oncolytic virus is formed by inserting an antitumor enhancer into the non-neurotoxic VSV vector recombinant oncolytic virus as a carrier skeleton. The antitumor enhancer includes a tumor targeting regulatory element, an immunomodulatory factor and a tumor antigen. The tumor targeting regulatory element is, for example, a tumor-specific promoter such as hTERT, survivin and AFP and a gene fragment targeting the surface of a tumor cell such as an anti-HER2 antibody fragment. The immunomodulatory factor is, for example, GMCSF, IL12, IL15, IL7, an anti-PD-1 / PD-L1 antibody fragment, an anti-CTLA-4 antibody fragment, a chemokine CCL5, CXCL9 / 10 / 11, a costimulatory molecule 4-1BBL and the like. The tumor antigen is, for example, NY-ESO-1, gp100 and CEA.

[0015] The present application has the following advantages: Excellent safety: animal experiments show that the virus does not cause significant neurotoxicity symptoms (such as significant weight loss, paralysis, convulsions, death and the like) when inoculated into mice through an intracranial route.

[0016] Mass producibility: the virus can be efficiently replicated on Vero cells, and the titer can reach 10 8.75 TCID 50 / mL, meeting the requirements of industrial production.

[0017] Significant oncolytic effect: in a mouse model, the virus can effectively inhibit tumor growth, significantly prolong the survival of tumor-bearing mice and exhibit excellent therapeutic potential. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 is a schematic diagram of the construction of a VSV vector recombinant oncolytic virus; in the diagram, GP represents a cyst membrane glycoprotein inserted into other viruses; Figure 2 is a graph of the change in the weight of mice after intracranial challenge with a VSV vector recombinant oncolytic virus; Figure 3 is a graph of the survival rate of mice after intracranial challenge with a VSV vector recombinant oncolytic virus; Figure 4Figure 2 is a graph of the body weight changes of mice after intracranial challenge of VSV-MARG-ΔMLD recombinant virus; Figure 5 Figure 3 is a graph of the survival rate of mice after intracranial challenge of VSV-MARG-ΔMLD recombinant virus; Figure 6 Figure 4 is a graph of the tumor inhibition effect of VSV-MARG-ΔMLD recombinant virus on a mouse tumor model; Figure 7 Figure 5 is a graph of the body weight changes of mice after intratumoral administration of VSV-MARG-ΔMLD recombinant virus. DETAILED DESCRIPTION

[0019] The technical solutions of the present application are further specifically described below through specific examples.

[0020] In the present application, unless specified, the raw materials and equipment used are commercially available or commonly used in the art. The methods in the following examples are conventional methods in the art, unless specified.

[0021] Example 1: Preparation of VSV vector recombinant oncolytic virus (VSV-MARG) (virus construction and rescue) To screen potential non-neurotoxic recombinant VSV oncolytic viruses, we utilized vesicular stomatitis virus (VSV) vectors, in which the surface glycoprotein (G protein) gene was deleted and replaced with the glycoprotein gene sequence of various strains, including Hepatitis C Virus 1a type (HCV, GeneBank Accession No. M62321), Andes orthohantavirus (ANDV, GeneBank Accession No. NC_003467), Rift Valley fever virus (RVFV, GeneBank Accession No. ABD38819), Dengue viruses (DENV, GeneBank Accession No. AY243469), Oropouche Virus (OROV, GeneBank Accession No. NC_005775), Feline infectious peritonitis virus (FIPV, GeneBank Accession No. AFH55121), Junin Virus (JUNV, GeneBank Accession No. U70799), Seoul Virus (SEOV, GeneBank Accession No. S47716), Chikungunya Virus (CHIK-V, GeneBank Accession No. AF369024), Borna Virus (BDV, GeneBank Accession No. U04608.1), and Marburg Virus (MARV, GeneBank Accession No. CAA82539). Recombinant viruses of each VSV vector were constructed using reverse genetics technology Figure 1 ).

[0022] The vesicular stomatitis virus strain used in this example was the Indiana strain.

[0023] For pBAC-VSV-HCG, pBAC-VSV-ANDG, pBAC-VSV-RVFG, pBAC-VSV-DENG, pBAC-VSV-OROG, pBAC-VSV-FIPG, pBAC-VSV-JUNG, pBAC-VSV-SEOG, pBAC-VSV-CHIKG, pBAC-VSV-BDG and pBAC-VSV-MARG full-length plasmids (corresponding to the viruses above one by one), after the corresponding G protein gene is synthesized, the synthesized gene fragment is respectively amplified by PCR according to the Primer Star enzyme instruction book, and the primer sequence information of amplification is shown in Table 1.

[0024] Table 1 Amplification primer sequence

[0025] The G protein corresponding to the above strain is amplified by homologous recombination (Uniclone One Step Seamless Cloning Kit) to be respectively cloned into the BAC-VSV vector in the G protein gene position and replace the ORF region of the G protein gene, forming a recombinant virus full-length plasmid of VSV carrying other strain G protein.

[0026] The construction process of each specific plasmid: 1. The corresponding fragment is obtained by PCR amplification using DNA polymerase (Primer Star); 2. The homologous recombination enzyme (Uniclone One Step Seamless Cloning Kit kit) is used for recombination and transformed into competent cells; 3. Single colony is picked and used universal vector primer and Taq enzyme for bacterial liquid PCR, and the PCR product with correct band size is sent for testing; 4. The plasmid of the colony with correct sequencing is extracted.

[0027] The virus rescue method is as follows: the vaccinia virus expressing T7 polymerase is used to infect BHK-21 cells, and then the constructed full-length plasmid and the helper plasmid (pN, pP, pL and pG, the helper plasmid is constructed by inserting the corresponding VSV protein coding sequence into the pBluescript II SK(+) vector by homologous recombination) expressing VSV-N, VSV-P, VSV-L and VSV-G are co-transfected (the mass ratio of the full-length plasmid, pN, pP, pL and pG plasmid is 10:3:5:1:3) into the infected BHK-21 cells, and the cells and supernatant are collected after 48 hours, filtered with a 0.22 μm filter, and the supernatant is used for standby. The virus stock solution is inoculated into new Vero cells, and whether the cultured cells show lesions is observed. If lesions appear, the cells and culture medium are collected and repeatedly frozen and thawed three times, filtered with a 0.45 μm filter, and stored at -80℃ after being divided and stored. The collected recombinant virus is named as VSV-HCG, VSV-ANDG, VSV-RVFG, VSV-DENG, VSV-OROG, VSV-FIPG, VSV-JUNG, VSV-SEOG, VSV-CHIKG, VSV-BDG and VSV-MARG, respectively.

[0028] The Reed-Muench method is used to measure the titer of the recombinant virus. The virus is diluted by 10 times gradient, inoculated into a 96-well plate coated with Vero cells, and cultured for 48 hours. The number of positive and negative wells is recorded, and the TCID50 of the virus is calculated. 50 After measurement, the titer of the recombinant virus of the embodiment of the application after stable passage is as follows (Table 2).

[0029] Table 2 Titer of VSV recombinant virus .

[0030] Example 2: Evaluation of the neurotoxicity of VSV recombinant virus In order to study the neurotoxicity of the successfully rescued recombinant VSV vector virus strain in Example 1, we used intracranial injection of virus in mice to evaluate it. Since VSV virus also has potential neurotoxicity, we used wild-type VSV-WT as a positive control, and set up a negative control (water injection group). Wild-type VSV-WT and each recombinant virus candidate were directly inoculated into the brain of mice, and the inoculation dose was 10 5 TCID 50 / each, which can cause experimental central nervous system infection symptoms in mice. After challenge, the phenotype, body weight and survival rate of mice were recorded to evaluate the safety of each recombinant VSV vector virus.

[0031] The body weight change and survival of mice after challenge are shown in Figure 2 and Figure 3 . The results show that after intracranial inoculation of the three strains of VSV-CHIKG, VSV-BDG and VSV-MARG, the mice show similar symptoms to the positive control VSV-WT group, with significant weight loss, and different degrees of death in each group of mice. Figure 3 The results show that the three recombinant viruses successfully rescued in Example 1 all have neurotoxicity.

[0032] Example 3: Molecular construction and virus rescue of VSV-MARG-ΔMLD The amino acids 290-422 of the Marburg virus (MARV) G protein are a mucin-like domain (MLD), which is highly glycosylated, and this domain can induce immune escape and enhance the infection efficiency of the virus. In order to explore the effect of this region on the neurotoxicity of VSV-MARG, we constructed a plasmid in which the MLD domain in the genome sequence of VSV-MARG was deleted, named pBAC-VSV-MARG-ΔMLD, and carried out virus rescue.

[0033] Molecular construction: For the construction of the pBAC-VSV-MARG-ΔMLD plasmid, the successfully constructed pBAC-VSV-MARG plasmid was used as a template, and the upper and lower fragments containing the homologous arms of the MLD region were amplified with the primers in Table 3, and then homologous recombination was carried out with the BAC-VSV vector.

[0034] Table 3 PCR primers required for construction of pBAC-VSV-MARG-ΔMLD plasmid .

[0035] The homologous recombination of plasmid construction and virus rescue method are the same as in Example 1. After reverse genetic rescue, the VSV-MARG-ΔMLD virus strain was successfully rescued, and the titer of the virus strain was measured to be 10 8.75 TCID 50 / ml.

[0036] Example 4: Neurotoxicity verification of VSV-MARG-ΔMLD virus The neurotoxicity of VSV-MARG-ΔMLD virus was verified by intracranial injection of mice, with water for injection as the negative control and VSV-MARG as the positive control, and the experimental method was the same as in Example 2.

[0037] The experimental results show that after intracranial inoculation of mice, the positive control VSV-MARG group of mice showed a significant decrease in body weight Figure 4 , and the survival rate decreased, with no mice surviving on the 7th dayFigure 5 ). And the VSV-MARG-ΔMLD group mice had no significant weight loss compared with the negative control group mice ( Figure 4 ), no mice died ( Figure 5 ), and the survival rate was 100%. This shows that the VSV-MARG-ΔMLD strain has no neurotoxicity.

[0038] Example 5: VSV-MARG-ΔMLD recombinant strain has tumor inhibition effect on mouse tumor model To evaluate the potential therapeutic effect of VSV-MARG-ΔMLD recombinant strain on tumors, the oncolytic effect was evaluated using a mouse colon cancer CT26 cell transplanted tumor model. Sixteen 6- to 8-week-old female BALB / c mice were selected, and each mouse was inoculated subcutaneously with CT26 cells (2 x 10 6 On the day after tumor inoculation, the tumor growth was observed daily, and when the tumor was visible to the naked eye, the tumor volume was measured every 2 days using a digital caliper 3 times, and the volume was calculated according to the following formula: tumor volume = 1 / 2 x a x b2 (a represents the maximum diameter in millimeters; b represents the minimum diameter in millimeters). On the 7th day after inoculation, when the tumor volume of the mice grew to 50-80 mm3, the mice were randomly divided into 2 groups, 8 mice in each group, and intratumoral administration was started, with PBS as a negative control. The administration scheme was as follows: each mouse was administered 10 6.5 TCID 50 / 100 μL, once every 2 days, for a total of 3 times. The tumor volume was measured every 2 days after administration.

[0039] The results of the mouse transplanted tumor model showed that ( Figure 6-7 ), compared with the PBS control group, VSV-MARG-ΔMLD had a significant tumor inhibition effect, and after intratumoral injection, the tumor volume of the mice was significantly inhibited, and there was no significant change in body weight, indicating that the VSV-MARG-ΔMLD as an oncolytic virus has good safety and effectiveness.

[0040] The above-described embodiments are only a preferred scheme of the present application and do not limit the present application in any form. Other variants and modifications can be made without exceeding the technical scheme recited in the claims.

[0041] SEQ ID No.1 SEQ ID No. 26 MKTTCLFISLILIQGIKTLPILEIASNNQPQNVDSVCSGTLQKTEDVHLMGFTLSGQKVADSPLEASKRWAFRTGVPPKNVEYTEGEEAKTCYNISVTDPSGKSLLLDPPTNIRDYPKCKTIHHIQGQNPHAQGIALHLWGAFFLYDRIASTTMYRGRVFTEGNIAAMIVNKTVHKMIFSRQGQGYRHMNLTSTNKYWTSNNGTQTNDTGCFGALQEYNSTKNQTCAPSKIPSPLPTARPEIKPTSTPTDATTLNTTDPNNDDEDLITSGSGSGEQEPYTTSDAVTKQGPTTQHLVYFRKKRSILWREGDMFPFLDGLINAPIDFDPVPNTKTIFDESSSSGASAEEDQHASPNISLTLSYFPNINENTAYSGENENDCDAELRIWSVQEDDLAAGLSWIPFFGPGIEGLYTAGLIKNQNNLVCRLRRLANQTAKSLELLLRVTTEERTFSLINRHAIDFLLTRWGGTCKVLGPDCCIGIEDLSRNISEQIDQIKKDEQKEGTGWGLGGKWWTSDWGVLTNLGILLLLSIAVLIALSCICRIFTKYIG.

Claims

1. A non-neurotoxic VSV vector oncolytic virus carrying a Marburg virus defective G protein, characterized in that, The VSV-MARG-ΔMLD is rescued by reverse genetic manipulation technology, in which the G protein coding gene of wild type VSV is replaced by the coding gene of Marburg virus defective G protein, thus obtaining a recombinant oncolytic virus VSV-MARG-ΔMLD which can self-replicate.

2. The non-neurotoxic VSV vector oncolytic virus of claim 1, wherein, The wild type VSV strain is Indiana strain.

3. The non-neurotoxic VSV vector oncolytic virus of claim 1, wherein, The sequence of the coding gene of Marburg virus defective G protein is shown in SEQ ID No.

1.

4. The non-neurotoxic VSV vector oncolytic virus of claim 1, wherein, The preparation method is specifically as follows: (1) constructing the VSV-MARG-ΔMLD plasmid: first, inserting the VSV genome into the BAC vector, then replacing the G protein gene sequence on the VSV genome with the gene sequence encoding Marburg virus defective G protein, to obtain the pBAC-VSV-MARG-ΔMLD plasmid; (2) rescuing the recombinant virus: the first to be infected cells are infected with the vaccinia virus expressing T7 polymerase, the cells after infection are co-transfected with the pBAC-VSV-MARG-ΔMLD plasmid, pN, pP, pL and pG plasmids, the supernatant of the diseased cells is collected 48 h after transfection, the second to be infected cells are infected with the supernatant, and the supernatant of the diseased cells is collected after amplifying the virus, to obtain the VSV vector recombinant oncolytic virus; The first to be infected cells are BHK21 cells. The second to be infected cells are Vero cells.

5. The non-neurotoxic VSV vector oncolytic virus of claim 4, wherein, The mass ratio of the pBAC-VSV-MARG-ΔMLD, pN, pP, pL and pG plasmids when co-transfected is 10:3:5:1:

3.

6. The non-neurotoxic VSV vector recombinant oncolytic virus according to claim 1 in the preparation of an antitumor drug.

7. An antitumor, super-modified, recombinant oncolytic virus, characterized in that, The non-neurotoxic VSV vector recombinant oncolytic virus according to claim 1 is used as a vector skeleton, and an anti-tumor enhancer is inserted.

8. The antitumor, super-modified, recombinant oncolytic virus according to claim 7, characterized in that, The anti-tumor enhancer includes tumor targeting regulatory elements, immune modulators, and tumor antigens.

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