A non-neurotoxic vsv vector recombinant oncolytic virus carrying sindbis virus g protein

Abstract

The application discloses a non-neurotoxic VSV vector recombinant oncolytic virus carrying a Sindbis virus G protein, and is a self-replicating recombinant oncolytic virus VSV-SING which is obtained by replacing the G protein coding gene of a wild type VSV with the G protein coding gene of a Sindbis virus through reverse genetic manipulation technology, and has 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 the Sindbis virus 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] Sindbis virus belongs to the genus Alphavirus of the family Togaviridae. It is an enveloped, single-stranded, positive-sense RNA virus. The virus particle is spherical, approximately 70-80 nanometers in diameter, with a genome RNA length of about 11.8-12.0 kb, encoding two main envelope glycoproteins (E1 and E2). The E2 protein is responsible for binding to host cell receptors, mediating viral invasion. Sindbis virus is a widely distributed arbovirus and, as an important scientific model, it has unique value for basic virological research.

[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 the Sindbis virus 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 VSV vector recombinant oncolytic virus carrying the Sindbis virus G protein was obtained by rescuing the autonomously replicating recombinant oncolytic virus VSV-SING through reverse genetics manipulation, where the G protein coding gene of wild-type VSV was replaced with the G protein coding gene of Sindbis virus. The amino acid sequence of the Sindbis virus G protein is shown in SEQ ID No. 30.

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

[0011] As a preferred embodiment, the gene sequence of the G protein encoding gene of Sindbis virus 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-SING 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 Sindbis virus G protein to obtain the pBAC-VSV-SING 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-SING 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-SING, 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, etc.; tumor antigens include NY-ESO-1, gp100, and CEA, etc.

[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. 8.25 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 the envelope protein inserted into other viruses.

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

[0027] Figure 4 This refers to the tumor-suppressive effect of VSV-SINVG recombinant oncolytic virus on a mouse tumor model.

[0028] Figure 5 This shows the weight changes in mice after intratumoral administration of VSV-SINVG recombinant oncolytic virus. Detailed Implementation

[0029] The technical solution of the present invention will be further described in detail below through specific embodiments.

[0030] In this invention, unless otherwise specified, all raw materials and equipment used are commercially available or commonly used in the field. Unless otherwise specified, the methods in the following embodiments are conventional methods in the field.

[0031] Example 1: Preparation of recombinant oncolytic virus using VSV vector (virus construction and rescue)

[0032] 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 protein from various strains, including Hepatitis C Virus 1a type (HCV, GeneBank accession number M62321), Andesorthohantavirus (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 Middle East respiratory syndrome-related coronavirus (MERS, GeneBank). Recombinant viruses of each VSV vector were constructed using reverse genetics technology. These included Sindbis virus (SINV, SEQ ID No. 1), Feline infectious peritonitis virus (FIPV, GeneBank accession number AFH55121), Junin virus (JUNV, GeneBank accession number U70799), Seoul virus (SEOV, GeneBank accession number S47716), Sabia virus (SABV, GeneBank accession number NC_006317), Chikungunya virus (CHIK-V, GeneBank accession number AF369024), Borna virus (BDV, GeneBank accession number U04608.1), and Marburg virus (MARV, GeneBank accession number CAA82539). Figure 1 ).

[0033] The vesicular stomatitis virus strain used in this embodiment is the Indiana strain.

[0034] For the construction of full-length plasmids pBAC-VSV-HCG, pBAC-VSV-ANDG, pBAC-VSV-RVFG, pBAC-VSV-DENG, pBAC-VSV-OROG, pBAC-VSV-MERS, pBAC-VSV-SING, pBAC-VSV-FIPG, pBAC-VSV-JUNG, pBAC-VSV-SEOG, pBAC-VSV-SABG, 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.

[0035] Table 1 Amplification Primer Sequences

[0036]

[0037]

[0038] 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.

[0039] 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.

[0040] 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-MERS, VSV-SING, VSV-FIPG, VSV-JUNG, VSV-SEOG, VSV-SABG, VSV-CHIKG, VSV-BDG, and VSV-MARG, respectively.

[0041] 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 Only five strains were rescued using reverse genetics viruses: VSV-SING, VSV-SABG, VSV-CHIKG, VSV-BDG, and VSV-MARG. The viral titers of the recombinant viruses in this embodiment of the invention after passage stabilization were measured as follows (Table 2).

[0042] Table 2. Recombinant virus titers of each VSV vector

[0043] .

[0044] Example 2: Evaluation of neurotoxicity of VSV-SING recombinant virus

[0045] To investigate the neurotoxicity of the recombinant VSV vector virus strain successfully rescued in Example 1, we evaluated it using intracranial injection of the virus into mice. Since VSV virus also has 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 brains of mice 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.

[0046] 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 After intracranial inoculation of mice with the two recombinant strains, VSV-SING and VSV-SABG, there was no significant change in mouse body weight, and no mice died. These results indicate that the VSV-SING and VSV-SABG recombinant viruses have no neurotoxicity and good safety profile.

[0047] Example 3: VSV-SING has tumor-suppressive effects in a mouse tumor model.

[0048] To evaluate the potential therapeutic effect of the recombinant strain on tumors, a mouse colon cancer CT26 cell xenograft model was used to evaluate the oncolytic activity of the neurotoxic-free recombinant strain constructed in Example 2. Eighteen 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 the tumor was visually perceptible, using calipers 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, intratumoral drug administration was initiated, using PBS as a negative control. The administration regimen was: 10 mg / 100 μL per mouse. 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.

[0049] The results of the mouse xenograft model showed that ( Figures 4-5 Compared with the negative control group, the VSV-SABG administration group showed some inhibition of tumor growth in mice, but the effect was not significant. In contrast, the VSV-SING administration group showed significant inhibition of tumor growth. Furthermore, there was no significant change in mouse body weight after intratumoral injection, indicating that VSV-SING has good safety as an oncolytic virus.

[0050] 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.

[0051] SEQ ID No.1

[0052]

[0053] SEQ ID No.30

[0054] MSAAPLVTAMCLLGNVSFPCDRPPTCYTREPSRALDILEENVNHEAYDTLLNAILRCGSSGRSKRSVIDDFTLTSPYLGTCSYCHHTVPCFSPVKIEQVWDEADDNTIRIQTSAQFGYDQSGAASANKYRYMSLKQDHTVKEGTMDDIKISTSGPCRRLSYKGYFLLAKCPPGDSVTVSIVSSKNGSFSATSCTLARKIKPKFVGREKYDLPPVHGKKIPCTVYDRLKETTAGYITMHRPRPHAYTSYLEESSGKVYAKPPSGKNITYECKCGDYKTGTVSTRTEITGCTAIKQCVAYKSDQTKWVFNSPDLIRHDDHTAQGKLHLPFKLIPSTCMVPVAHAPNVIHGFKHISLQLDTDHLTLLTTRRLGANPEPTTEWIVGKTVRNFTVDRDGLEYIWGNHEPVRVYAQESAPGDPHGWPHEIVQHYYHRHPVYTILAVASATVAMMIGVTVAVLCACKARRECLTPYALAPNAVIPTSLALLCCVRSANAETFTETMSYLWSNSQPFFWVQLCIPLAAFIVLMRCCSCCLPFLVVAGAYLAKVDAYEHATTVPNVPQIPYKALVERAGYAPLNLEITVMSSEVLPSTNQEYITCKFTTVVPSPKIKCCGSLECQPAAHADYTCKVFGGVYPFMWGGAQCFCDSENSQMSEAYVELSADCASDHAQAIKVHTAAMKVGLRIVYGNTTSFLDVYVNGVTPGTSKDLKVIAGPISASFTPFDHKVVIHRGLVYNYDFPEYGAMKPGAFGDIQATSLTSKDLIASTDIRLLKPSAKNVHVPYTQASSGFEMWKNNSGRPLQETAPFGCKIAVNPLRAVDCSYGNIPISIDIPNAAFIRTSDAPLVSTVKCEVSECTYSADFGGMATLQYVSDREGQCPVHSHSSTATLQESTVHVLEKGAVTVHFSTASPQANFIVSLCGKKTTCNAECKPPADHIVSTPHKNDQEFQAAISKTSWSWLFALFGGASSLLIIGLMIFACSMMLTSTRR。

Claims

1. A non-neurotoxic VSV vector recombinant oncolytic virus carrying the Sindbis virus G protein, characterized in that, The recombinant oncolytic virus VSV-SING, capable of autonomous replication, was obtained by replacing the G protein-coding gene of wild-type VSV with the G protein-coding gene of Sindbis virus through reverse genetics manipulation. The gene sequence of the G protein-coding gene of Sindbis virus 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-SING 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 Sindbis virus G protein to obtain the pBAC-VSV-SING 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-SING 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 a Vero cell.

4. The neurotoxic VSV vector recombinant oncolytic virus according to claim 3, characterized in that, The mass ratio of pBAC-VSV-SING, 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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