A non-neurotoxic vsv vector recombinant oncolytic virus carrying a marburg virus defective g protein

Abstract

The application discloses a non-neurotoxic VSV vector recombinant oncolytic virus carrying a Marburg virus defective G protein, and is characterized in that: a coding gene of a wild-type VSV G protein is replaced by a coding gene of a Marburg virus defective G protein through reverse genetic manipulation technology, so that a recombinant oncolytic virus VSV-MARG-ΔMLD capable of autonomous replication is obtained. The application has both non-neurotoxicity and high oncolytic activity.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a non-neurotoxic VSV vector recombinant oncolytic virus carrying a Marburg virus-deficient G protein. Background Technology

[0002] Malignant tumors pose a serious threat to human health and life, with their incidence rate continuing to rise globally, becoming a significant public health challenge. Surgery, radiotherapy, and chemotherapy, as traditional treatments, have limited efficacy in patients with advanced or metastatic cancer, and are often accompanied by significant toxic side effects. In recent years, immune checkpoint inhibitors (ICIs) have shown breakthrough efficacy in some cancer types, but challenges such as low overall response rates and primary drug resistance remain. Therefore, developing novel, highly effective, and safe anti-tumor strategies is particularly urgent.

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

[0004] Marburg virus ( Marburg virus Marburg virus, also known as the green monkey virus, is named after the German city of Marburg. The virion is polymorphic, branching or coiling into U-shapes, figure-6 shapes, or rings. It is a deadly virus and the first filovirus discovered in humans, causing Marburg hemorrhagic fever. This virus is related to Ebola virus, belonging to the Filoviridae family, and also originated in Uganda and Kenya in Africa, causing a common disease in humans and other primates. Marburg virus was the first filovirus discovered; its genome is a single-stranded negative-sense RNA, approximately 19 kb long, encoding seven viral proteins. The Marburg virus envelope contains specific glycoproteins that bind to specific receptors on the surface of human cells, allowing it to enter the cell and replicate. The toxic effects of these viral proteins lead to apoptosis (apoptosis).

[0005] Vesicular stomatitis virus (VSV) is a non-pathogenic, negative-sense RNA enveloped virus that possesses several characteristics ideal for an oncolytic virus vector: low pre-existing immunity in the human population, no genome integration into the host chromosome, short replication cycle, good immunogenicity, and ease of genetic manipulation. However, preclinical studies have shown that VSV can induce significant neurotoxicity in rodent and non-human primate models (especially when inoculated via the central nervous system), and this safety concern severely limits its translation to clinical applications.

[0006] The neurotoxicity of wild-type VSV is mainly related to the cytotoxicity of its matrix protein (M protein) and the neurotropic properties of its glycoprotein (G protein). Therefore, modifying these two genes has become a key strategy for reducing neurotoxicity. However, M protein mutants often suffer from excessively reduced toxicity, leading to rapid clearance in vivo or decreased replication and spread in tumor tissues, thus affecting their oncolytic efficacy. Currently, there is an urgent need to develop a VSV vector that possesses both good safety (no neurotoxicity) and retains highly efficient oncolytic activity to advance this field. Summary of the Invention

[0007] The purpose of this invention is to provide a non-neurotoxic VSV vector recombinant oncolytic virus carrying a Marburg virus-deficient G protein, which has both non-neurotoxicity and high oncolytic activity.

[0008] The technical solution adopted by this invention to solve its technical problem is:

[0009] A neurotoxic recombinant oncolytic virus (VSV) carrying a Marburg virus-deficient G protein was obtained by rescuing the autonomously replicating recombinant oncolytic virus VSV-MARG-ΔMLD through reverse genetics manipulation, where the G protein coding gene of wild-type VSV was replaced with the coding gene of the Marburg virus-deficient G protein. The amino acid sequence of the Marburg virus-deficient G protein is shown in SEQ ID No. 26.

[0010] Preferably, the wild-type VSV strain is the Indiana strain.

[0011] Preferably, the sequence of the gene encoding the Marburg virus defective G protein is shown in SEQ ID No. 1. This sequence is a codon-optimized sequence.

[0012] The specific preparation method is as follows:

[0013] (1) Constructing the VSV-MARG-ΔMLD plasmid: First, insert the VSV genome into the BAC vector, and 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.

[0014] (2) Rescue of recombinant virus: The first cell to be infected was infected with a poxvirus expressing T7 polymerase. The infected cells were co-transfected with pBAC-VSV-MARG-ΔMLD plasmid, pN, pP, pL and pG plasmid. The supernatant of the diseased cells was collected 48 h after transfection. The supernatant was used to infect the second cell to be infected. After the virus was amplified, the supernatant of the diseased cells was collected to obtain the recombinant oncolytic virus of the VSV vector.

[0015] The first cell type to be infected was BHK21 cells;

[0016] The second cell to be infected is Vero cell.

[0017] As a preferred method, the mass ratio of pBAC-VSV-MARG-ΔMLD, pN, pP, pL and pG plasmids during co-transfection is 10:3:5:1:3.

[0018] The application of the aforementioned non-neurotoxic VSV vector recombinant oncolytic virus in the preparation of antitumor drugs. Tumors include gastrointestinal tumors, head and neck tumors, breast cancer, lymphoma, uterine cancer, ovarian cancer, bladder cancer, liver cancer, lung cancer, osteosarcoma, and melanoma.

[0019] A recombinant oncolytic virus modified with antitumor enhancement is formed by inserting antitumor enhancing factors into the aforementioned neurotoxic VSV vector recombinant oncolytic virus as the vector backbone. The antitumor enhancing factors include tumor-targeting regulatory elements, immunomodulatory factors, and tumor antigens. Tumor-targeting regulatory elements include tumor-specific promoters such as hTERT, survivin, and AFP, as well as gene fragments targeting the surface of tumor cells, such as anti-HER2 antibody fragments; immunomodulatory factors include GMCSF, IL12, IL15, IL7, anti-PD-1 / PD-L1 antibody fragments, anti-CTLA-4 antibody fragments, chemokines CCL5, CXCL9 / 10 / 11, and co-stimulatory molecules 4-1BBL; tumor antigens include NY-ESO-1, gp100, and CEA.

[0020] The beneficial effects of this invention are:

[0021] Excellent safety profile: Animal studies have shown that when mice were inoculated via the intracranial route, the virus did not induce significant neurotoxic symptoms (such as significant weight loss, paralysis, convulsions, death, etc.).

[0022] Mass production capability: The virus can replicate efficiently on Vero cells, with titers reaching 10-1. 8.75 TCID 50 / mL, meeting the requirements of industrial production.

[0023] Significant oncolytic effect: In mouse models, it can effectively inhibit tumor growth and significantly prolong the survival of tumor-bearing mice, demonstrating excellent therapeutic potential. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of the construction of a recombinant oncolytic virus molecule using a VSV vector; in the diagram, GP represents an envelope glycoprotein inserted into another virus.

[0025] Figure 2 This is a graph showing the change in body weight of mice after intracranial challenge with VSV vector recombinant oncolytic virus;

[0026] Figure 3 This is a graph showing the survival rate of mice after intracranial challenge with VSV vector recombinant oncolytic virus;

[0027] Figure 4 This is a graph showing the change in body weight of mice after intracranial challenge with VSV-MARG-ΔMLD recombinant virus;

[0028] Figure 5 This is a graph showing the survival rate of mice after intracranial challenge with VSV-MARG-ΔMLD recombinant virus;

[0029] Figure 6 This refers to the tumor-suppressive effect of VSV-MARG-ΔMLD recombinant virus on a mouse tumor model;

[0030] Figure 7 This refers to the change in body weight in mice after intratumoral administration of the VSV-MARG-ΔMLD recombinant virus. Detailed Implementation

[0031] The technical solution of the present invention will be further described in detail below through specific embodiments.

[0032] In this invention, unless otherwise specified, all raw materials and equipment used are commercially available or commonly used in the field. The methods described in the following embodiments are conventional methods in the field, unless otherwise specified.

[0033] Example 1: Preparation of VSV-vectored recombinant oncolytic virus (VSV-MARG) (virus construction and rescue)

[0034] To screen for potentially neurotoxic recombinant VSV vector oncolytic viruses, we used a vesicular stomatitis virus (VSV) vector, deleted the gene for its surface envelope protein (G protein), and inserted the gene sequences of the envelope proteins from various strains, including Hepatitis C Virus 1a type (HCV, GeneBank accession number M62321), Andes orthohantavirus (ANDV, GeneBank accession number NC_003467), Rift Valley fever virus (RVFV, GeneBank accession number ABD38819), dengue viruses (DENV, GeneBank accession number AY243469), Oropouche virus (OROV, GeneBank accession number NC_005775), and feline infectious peritonitis virus (FIPV, GeneBank). The following viruses were identified: Junin Virus (JUNV, GeneBank accession number U70799), Seoul Virus (SEOV, GeneBank accession number S47716), Chikungunya Virus (CHIK-V, GeneBank accession number AF369024), Borna Virus (BDV, GeneBank accession number U04608.1), and Marburg Virus (MARV, GeneBank accession number CAA82539). Recombinant viruses of each VSV vector were constructed using reverse genetics technology. Figure 1 ).

[0035] The vesicular stomatitis virus strain used in this embodiment is the Indiana strain.

[0036] For the construction of full-length plasmids 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 (corresponding one-to-one with the viruses above), after the corresponding G protein genes were synthesized, the synthesized gene fragments were amplified by PCR according to the Primer Star enzyme instructions. The primer sequence information for amplification is shown in Table 1.

[0037] Table 1 Amplification Primer Sequences

[0038]

[0039]

[0040] The amplified gene fragments corresponding to the G protein of the above-mentioned strains were cloned into the G protein gene position in the BAC-VSV vector through homologous recombination (Uniclone One StepSeamless Cloning Kit) and the ORF region of the G protein gene was replaced to form a full-length plasmid of recombinant virus carrying the G protein of other strains.

[0041] The specific plasmid construction process is as follows: 1. PCR amplification using DNA polymerase (Primer Star) to obtain the corresponding fragments; 2. Recombination of each fragment using homologous recombinase (Uniclone One Step Seamless Cloning Kit) and transformation into competent cells; 3. Picking single colonies and performing bacterial PCR using universal vector primers and Taq enzyme, and sending the PCR product with the correct band size for detection; 4. Extracting plasmids from the correctly sequenced colony clones.

[0042] The virus rescue method is as follows: BHK-21 cells were infected with poxvirus expressing T7 polymerase, and then co-transfected with the full-length plasmids constructed above and the helper plasmids expressing VSV-N, VSV-P, VSV-L, and VSV-G (pN, pP, pL, and pG; the helper plasmids were constructed by inserting the corresponding VSV protein coding sequence into the pBluescript II SK(+) vector) (the mass ratio of the full-length plasmid, pN, pP, pL, and pG plasmids was 10:3:5:1:3). After 48 h, the cells and supernatant were collected, filtered through a 0.22 μm filter, and the supernatant was used for later use. The viral stock solution was inoculated into new Vero cells, and the cells were observed to show cytopathic effects. If cytopathic effects were observed, the cells and culture medium were collected again and subjected to three freeze-thaw cycles. The mixture was then filtered through a 0.45 μm filter, aliquoted, and stored at -80°C to obtain the viral stock solution. The collected recombinant viruses were named VSV-HCG, VSV-ANDG, VSV-RVFG, VSV-DENG, VSV-OROG, VSV-FIPG, VSV-JUNG, VSV-SEOG, VSV-CHIKG, VSV-BDG, and VSV-MARG, respectively.

[0043] Recombinant virus titers were measured using the Reed-Muench method. Viruses were serially diluted 10-fold and inoculated into 96-well plates coated with Vero cells. After 48 hours of incubation, cytopathic effects were observed, and the number of positive and negative wells was recorded. The viral TCID was calculated. 50 The viral titers of the recombinant virus in this embodiment of the invention after passage stabilization were measured as follows (Table 2).

[0044] Table 2. Recombinant virus titers of each VSV vector

[0045] .

[0046] Example 2: Evaluation of neurotoxicity of VSV recombinant virus

[0047] To investigate the neurotoxicity of the successfully rescued recombinant VSV vector virus strains from Example 1, we evaluated them via intracranial injection in mice. Since VSV virus also possesses potential neurotoxicity, we used wild-type VSV-WT as a positive control and established a negative control (water for injection group). Wild-type VSV-WT and each recombinant virus candidate strain were directly inoculated into the mouse brain at a dose of 10... 5 TCID 50 / mouse, this dose of wild-type VSV virus inoculation induced experimental central nervous system infection symptoms in mice. Phenotypic symptoms, body weight, and survival rate of mice were recorded after challenge to evaluate the safety of each recombinant VSV vector virus.

[0048] Changes in mouse body weight and survival status after challenge are as follows: Figure 2 and Figure 3 As shown. The results indicated that after intracranial inoculation with the three strains VSV-CHIKG, VSV-BDG, and VSV-MARG, mice exhibited similar symptoms to the positive control VSV-WT group, with significant weight loss, and each group of mice experienced varying degrees of mortality. Figure 3 The results showed that all three recombinant viruses successfully rescued in Example 1 were neurotoxic.

[0049] Example 3: Molecular construction and virus rescue of VSV-MARG-ΔMLD

[0050] The Marburg virus (MARV) G protein contains a mucin-like domain (MLD) at positions 290-422, which is highly glycosylated. This domain can induce immune evasion and enhance viral infectivity. To investigate the effect of this region on the neurotoxicity of VSV-MARG, we constructed a plasmid lacking the MLD domain from the VSV-MARG genome sequence, named pBAC-VSV-MARG-ΔMLD, and performed virus rescue.

[0051] Molecular construction: For the construction of pBAC-VSV-MARG-ΔMLD plasmid, the successfully constructed pBAC-VSV-MARG plasmid was used as a template. The upstream and downstream fragments containing homologous arms in the MLD region were amplified using the primers in Table 3, and then homologous recombination was performed with the BAC-VSV vector.

[0052] Table 3. PCR primers required for constructing the pBAC-VSV-MARG-ΔMLD plasmid

[0053] .

[0054] The homologous recombination and virus rescue methods for plasmid construction were the same as in Example 1. Through reverse genetics rescue, the VSV-MARG-ΔMLD virus strain was successfully rescued, and its titer was measured to be 10. 8.75 TCID 50 / ml.

[0055] Example 4: Validation of neurotoxicity of VSV-MARG-ΔMLD virus

[0056] The neurotoxicity of VSV-MARG-ΔMLD virus was verified using a mouse intracranial injection model. Water for injection was set as a negative control and VSV-MARG as a positive control. The experimental method was the same as in Example 2.

[0057] Experimental results showed that after intracranial inoculation of mice, the body weight of mice in the positive control VSV-MARG group decreased significantly. Figure 4 Furthermore, the survival rate decreased, and no mice survived on day 7. Figure 5 Mice in the VSV-MARG-ΔMLD group showed no significant decrease in body weight, similar to the negative control group. Figure 4 No mice died. Figure 5 The survival rate was 100%, indicating that the VSV-MARG-ΔMLD virus strain has no neurotoxicity.

[0058] Example 5: The recombinant VSV-MARG-ΔMLD strain exhibits tumor-suppressive activity in a mouse tumor model.

[0059] To evaluate the potential therapeutic effect of the VSV-MARG-ΔMLD recombinant strain on tumors, a mouse colon cancer CT26 cell xenograft model was used to evaluate its oncolytic activity. Sixteen female BALB / c mice aged 6 to 8 weeks were selected, and each mouse was subcutaneously inoculated with CT26 cells (2 × 10⁻⁶ cells) on the right side. 6 (Number of tumors per 100 μL). Tumor growth was observed daily after inoculation. Tumor volume was measured once visible to the naked eye, with digital calipers measuring the volume three times every two days. The volume was calculated using the following formula: Tumor volume = 1 / 2 × a × b² (where a represents the maximum diameter in millimeters; b represents the minimum diameter in millimeters). On day 7 post-inoculation, when the tumor volume reached 50–80 mm³, mice were randomly divided into two groups of eight mice each. Intratumoral drug administration was initiated, with PBS used as a negative control. The administration regimen was: each mouse was administered 10 μL of PBS. 6.5 TCID 50 / 100 μL, administered once every 2 days for a total of 3 doses. Tumor volume was measured every 2 days after administration.

[0060] The results of the mouse xenograft model showed that ( Figures 6-7 Compared with the PBS control group, VSV-MARG-ΔMLD showed significant tumor inhibition. After intratumoral injection, the tumor growth volume of mice was significantly inhibited, and the body weight did not change significantly, indicating that VSV-MARG-ΔMLD has good safety and efficacy as an oncolytic virus.

[0061] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the present invention in any way. Other variations and modifications are possible without departing from the technical solutions described in the claims.

[0062] SEQ ID No.1

[0063]

[0064] SEQ ID No.26

[0065] MKTTCLFISLILIQGIKTLPILEIASNNQPQNVDSVCSGTLQKTEDVHLMGFTLSGQKVADSPLEASKRWAFRTGVPPKNVEYTEGEEAKTCYNISVTDPSGKSLLLDPPTNIRDYPKCKTIHHIQGQNPHAQGIALHLWGAFFLYDRIASTTMYRGRVFTEGNIAAMIVNKTVHKMIFSRQGQGYRHMNLTSTNKYWTSNNGTQTNDTGCFGALQEYNSTKNQTCAPSKIPSPLPTARPEIKPTSTPTDATTLNTTDPNNDDEDLITSGSGSGEQEPYTTSDAVTKQGPTTQHLVYFRKKRSILWREGDMFPFLDGLINAPIDFDPVPNTKTIFDESSSSGASAEEDQHASPNISLTLSYFPNINENTAYSGENENDCDAELRIWSVQEDDLAAGLSWIPFFGPGIEGLYTAGLIKNQNNLVCRLRRLANQTAKSLELLLRVTTEERTFSLINRHAIDFLLTRWGGTCKVLGPDCCIGIEDLSRNISEQIDQIKKDEQKEGTGWGLGGKWWTSDWGVLTNLGILLLLSIAVLIALSCICRIFTKYIG。

Claims

1. A neurotoxic VSV vector recombinant oncolytic virus carrying a Marburg virus-deficient G protein, characterized in that, The recombinant oncolytic virus VSV-MARG-ΔMLD, which can replicate autonomously, was obtained by replacing the G protein coding gene of wild-type VSV with the coding gene of Marburg virus defective G protein through reverse genetics. The sequence of the coding gene of Marburg virus defective G protein is shown in SEQ ID No.

1.

2. The neurotoxic VSV vector recombinant oncolytic virus according to claim 1, characterized in that, The wild-type VSV strain is the Indiana strain.

3. The neurotoxic VSV vector recombinant oncolytic virus according to claim 1, characterized in that, The specific preparation method is as follows: (1) Constructing the VSV-MARG-ΔMLD plasmid: First, insert the VSV genome into the BAC vector, and 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) Rescue of recombinant virus: The first cell to be infected was infected with a poxvirus expressing T7 polymerase. The infected cells were co-transfected with pBAC-VSV-MARG-ΔMLD plasmid, pN, pP, pL and pG plasmid. The supernatant of the diseased cells was collected 48 h after transfection. The supernatant was used to infect the second cell to be infected. After the virus was amplified, the supernatant of the diseased cells was collected to obtain the recombinant oncolytic virus of the VSV vector. The first cell type to be infected was BHK21 cells; The second cell to be infected is Vero cell.

4. The neurotoxic VSV vector recombinant oncolytic virus according to claim 3, characterized in that, The mass ratio of pBAC-VSV-MARG-ΔMLD, pN, pP, pL and pG plasmids during co-transfection is 10:3:5:1:

3.

5. The use of a non-neurotoxic VSV vector recombinant oncolytic virus as described in claim 1 in the preparation of an anti-colon cancer drug.

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