Use of engineered cinna virus (JURV) as oncolytic virus platform for human cancer

Abstract

The present disclosure provides compositions, infectious particles, pharmaceutical compositions and cells comprising the same comprising a recombinant polynucleotide encoding a cinrona virus, as well as methods and systems for preparing a recombinant cinrona virus.

Description

[0001] Priority declaration

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application No. 63 / 344,395, filed on May 20, 2022, the contents of which are incorporated by reference in their entirety.

[0004] Statement Regarding Federally Sponsored Research

[0005] This invention was made with government support under Grant CA234324 from the National Cancer Institute. The government has certain rights in this invention.

[0006] Sequence Listing

[0007] A sequence listing is attached to this application and is submitted as an ASCII text file of a sequence listing named "169852_00112_Sequence_Listing.xml", which is 110,996 bytes in size and was created on May 3, 2023. The sequence listing is submitted electronically with the application via EFS-Web and is incorporated herein by reference in its entirety. Background Art

[0008] Oncolytic viruses are viruses that preferentially infect and kill cancer cells, representing a promising advance in cancer treatment. Currently, several viral platforms are being studied as oncolytic viruses, including vesicular stomatitis virus (VSV). VSV is a widely studied vector and has entered early clinical studies. However, concerns about liver and neurotoxicity may hinder its clinical deployment for the treatment of human cancer. Other vesicular virus vectors have also been developed in recent years, but most have failed to achieve the potency level of VSV in preclinical models of human cancer. Therefore, there is still a need for new oncolytic viruses in the art. Summary of the invention

[0009] In one aspect of the present disclosure, a construct and a composition comprising the construct are provided, the construct comprising a promoter operably linked to a polynucleotide encoding a sense strand copy of a Jurona virus genome or a Jurona virus genome. Jurona virus has a negative sense single-stranded RNA genome, so that the construct will include a full-length antisense genome, and allows the production of a negative sense viral genome and infectious viruses when transfected into mammalian cells. The polynucleotide encoding the Jurona virus genome may include a leader sequence of SEQ ID NO: 1-5 and an optional SEQ ID NO: 6 and / or a trailing sequence of SEQ ID NO: 7. The polynucleotide encoding the Jurona virus genome may further include at least one of SEQ ID NO: 8-11, 21 and 22 as an intergenic region. In some embodiments, the polynucleotide encoding the Jurona virus genome is or includes SEQ ID NO: 12 (JURV-XN-2). The polynucleotide encoding the Jurona virus genome may further include a heterologous polynucleotide capable of encoding a polypeptide not naturally associated with the Jurona virus. In some embodiments, the polypeptide is a reporter polypeptide and can be encoded by a polynucleotide of SEQ ID NO: 13 (JURV-eGFP).

[0010] In another aspect of the present disclosure, additional compositions or constructs are provided. Some constructs include codon-optimized polynucleotides encoding at least one Churona virus protein selected from the group consisting of glycoprotein (G), nucleoprotein (N), phosphoprotein (P), RNA-guided RNA polymerase L protein (L) and matrix protein (M), and the polynucleotides are operably connected to a promoter for expression in mammalian cells. The polynucleotide encoding the G protein may include SEQ ID NO: 4, the polynucleotide encoding the N protein may include SEQ ID NO: 1, the polynucleotide encoding the P protein may include SEQ ID NO: 2, the polynucleotide encoding the M protein may include SEQ ID NO: 3, the polynucleotide encoding the L protein may include SEQ ID NO: 5, and the composition may include a plasmid, and more than one polynucleotide encoding viral polypeptides may be encoded on a single construct. Such constructs may be included in the genome of the cell, or may be transiently transfected into the cell to produce viral proteins and allow packaging and production of infectious viruses. Therefore, cells comprising constructs described herein are also provided.

[0011] In another aspect of the present disclosure, infectious particles are provided. The infectious particles may comprise a Churona virus genome containing a negative sense RNA of SEQ ID NO: 12. The infectious particles may be prepared by the methods provided in the Examples, and recombinant infectious virus particles may be generated using the constructs and cells provided herein.

[0012] In another aspect of the present disclosure, additional infectious particles are provided. Infectious particles are prepared by transfecting cells with a composition comprising codon-optimized polynucleotides, the polynucleotides encoding at least one Jurona virus protein selected from the group consisting of G, N, P, L and M, the polynucleotides being operably linked to a promoter for expression in mammalian cells. The polynucleotide encoding the G protein may include SEQ ID NO:4, the polynucleotide encoding the N protein may include SEQ ID NO:1, the polynucleotide encoding the P protein may include SEQ ID NO:2, the polynucleotide encoding the M protein may include SEQ ID NO:3, the polynucleotide encoding the L protein may include SEQ ID NO:5, and the composition may include a plasmid. Cells are also transfected with a construct encoding a negative sense genomic copy of the Jurona virus operably linked to a promoter capable of generating a genomic copy of the virus.

[0013] In another aspect of the present disclosure, a pharmaceutical composition is provided. The pharmaceutical composition comprises infectious particles and a pharmaceutically acceptable carrier or excipient, wherein the infectious particles comprise a Jurona virus genome, wherein the Jurona virus genome comprises a negative sense RNA from SEQ ID NO: 12 or having at least 95% sequence identity with SEQ ID NO: 12. In some embodiments, the pharmaceutical composition comprises infectious particles prepared by transfecting cells with a composition comprising a codon-optimized polynucleotide and a pharmaceutically acceptable carrier or excipient, wherein the codon-optimized polynucleotide encodes at least one Jurona virus protein selected from the group consisting of G, N, P, L and M, and the polynucleotide is operably linked to a promoter for expression in a mammalian cell.

[0014] In other aspects of the present disclosure, a method for treating a cell proliferative disease or condition in a subject in need thereof is provided. The method includes administering to a subject a pharmaceutical composition comprising an infectious particle and a pharmaceutically acceptable carrier or excipient to treat a cell proliferative disease or condition, the infectious particle comprising a Jurona virus genome, the Jurona virus genome comprising SEQ ID NO: 12 or a negative sense RNA having a sequence with at least 95% identity to SEQ ID NO: 12. Infectious particles can be prepared by transfecting cells with a composition comprising a codon-optimized polynucleotide and a pharmaceutically acceptable carrier or excipient, the polynucleotide encoding at least one Jurona virus protein selected from the group consisting of G, N, P, L and M, the polynucleotide being operably linked to a promoter for expression in a mammalian cell. The cell proliferative disease or condition can be cancer and can be selected from hepatocellular carcinoma, hepatobiliary carcinoma, breast cancer, colorectal cancer, prostate cancer and reticulum sarcoma. Breast cancer can be HER2 negative. Cancer can be local or metastatic. The subject may have a suppressed immune system. The method may further comprise administering to the subject an immunotherapy, which may be a checkpoint inhibitor therapy. The checkpoint inhibitor therapy may be selected from the group consisting of a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, and a LAG-3 inhibitor.

[0015] In another aspect of the present disclosure, a cell is provided. The cell comprises a promoter, the promoter being operably connected to a polynucleotide encoding a Jurona virus genome, and allowing the production of a negative-sense viral genome when transfected into a mammalian cell. In some embodiments, the promoter is a T7 promoter. The cell may comprise a codon-optimized polynucleotide encoding at least one Jurona virus protein selected from the group consisting of G, N, P, L and M, the polynucleotide being operably connected to a promoter for expression in a mammalian cell. The cell may further or alternatively comprise infectious particles, the infectious particles comprising the Jurona virus genome containing the negative-sense RNA of SEQ ID NO: 12, and the infectious particles may be prepared by transfecting cells with a composition comprising a codon-optimized polynucleotide operably connected to a promoter for expression in a mammalian cell and a composition encoding a full-length genome and capable of generating a negative-sense genomic RNA, the codon-optimized polynucleotide encoding at least one Jurona virus protein selected from the group consisting of G, N, P, L and M.

[0016] In another aspect of the present disclosure, a method for generating a recombinant Jurona virus is provided. The method includes introducing at least one composition into a cell, the composition comprising a polynucleotide operably linked to a coding Jurona virus genome, and a promoter allowing the generation of a negative sense viral genome when transfected into a mammalian cell; allowing the cell to express one or more Jurona virus proteins selected from the group consisting of G, M, N, L and P; incubating the cell for a sufficient time to generate a recombinant Jurona virus; and harvesting the virus generated by the cell. The cell may include a promoter operably linked to a polynucleotide encoding a Jurona virus genome, and allowing the generation of a negative sense viral genome when transfected into a mammalian cell. One or more Jurona virus proteins may include Jurona virus N, P and L proteins. One or more Jurona virus proteins may be encoded by one or more polynucleotides comprising SEQ ID NO: 1, 2 or 5. The promoter may be a T7 promoter, and the cell may include a T7 RNA polymerase. In some embodiments, the cell is a BHK-21 cell, a Vero cell or a HEK-293 cell. The introduced composition may additionally comprise a heterologous polynucleotide encoding a protein not naturally associated with Churona virus.

[0017] In another aspect of the present disclosure, a system for generating a recombinant Jurona virus is provided. The system comprises: a) one or more vectors comprising polynucleotides encoding at least three Jurona virus proteins selected from the group consisting of G, N, P, L and M, each of which is operably connected to a promoter to allow the expression of the at least three proteins in a mammalian cell; b) a vector comprising a polynucleotide encoding a negative sense Jurona virus genome, the polynucleotide being operably connected to a promoter to allow the negative sense Jurona virus genome to be produced in a mammalian cell, and may further comprise: (c) a mammalian cell capable of expressing the Jurona virus protein of (a) and the negative sense Jurona virus genome of (b) to produce a recombinant Jurona virus. The cell may comprise T7 RNA polymerase, and / or at least one promoter may be a T7 promoter. In some embodiments, the cell is a BHK-1 cell, a Vero cell or a HEK-293 cell. One or more vectors may comprise polynucleotides encoding at least Jurona virus N, P and L proteins operably connected to a promoter. The polynucleotide encoding the at least three Jurona virus proteins may be codon optimized for expression in mammalian cells and may comprise any one of SEQ ID NOs: 1-5. The vector may comprise a polynucleotide encoding a negative sense Jurona virus genome as described herein. One or more vectors encoding at least three Jurona virus proteins may comprise a composition as provided herein.

[0018] In other aspects of the present disclosure, a kit is provided. In some embodiments, the kit comprises a composition having a polynucleotide operably connected to the coding Jurona virus genome and a promoter allowing the production of a negative sense viral genome when transfected into a mammalian cell. The promoter may be a T7 promoter. The polynucleotide encoding the Jurona virus genome may include a leader sequence of SEQ ID NO: 1-5, SEQ ID NO: 6, a trailing sequence of SEQ ID NO: 7, and / or at least one of SEQ ID NO: 8-11, 21, and 22 as an intergenic region. In some embodiments, the polynucleotide encoding the Jurona virus genome includes SEQ ID NO: 12 (JURV-XN-2). The polynucleotide encoding the Jurona virus genome may further include a heterologous polynucleotide capable of encoding a polypeptide that is not naturally associated with the Jurona virus, and the polypeptide may be a reporter polypeptide that can be encoded by a polynucleotide of SEQ ID NO: 13 (JURV-eGFP). The kit may alternatively or additionally include any composition or infectious particles disclosed herein. The kit may further include immunotherapy, which may be a checkpoint inhibitor therapy. Checkpoint inhibitor therapy may be selected from the group consisting of PD-1 inhibitors, PD-L1 inhibitors, CTLA-4 inhibitors and LAG-3 inhibitors. The kit may further comprise an IFN-α inhibitor and / or a receptor tyrosine kinase inhibitor, which may be pazopanib. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Shows herpes simplex virus ( Figure 1 A) and the vesiculovirus genome ( Figure 1 B) Schematic diagram of the organization and structure.

[0020] Figure 2 Schematic diagram showing (A) the method used to obtain laboratory-adapted Jurona virus clones and (B) the process of developing a reverse genetics-based approach to generate recombinant Jurona virus.

[0021] Figure 3 (A) Schematic diagram of the genome of Jurona virus, (B) Infection of cultured tumor cells with JURV at MOI of 0.1, 1 and 10 and measurement of cell viability. (C) As in (B), cancer cells were infected with Moreton virus (MORV) at MOI of 0.1, 1 and 10 and cell viability was observed. (D) Cancer cells were infected with vesicular stomatitis virus (VSV) at MOI of 0.1, 1 and 10 and cell viability was observed. The data confirm that JURV has oncolytic function. (E) JURV can replicate in a variety of tumor cells and Vero cells.

[0022] Figure 4 Shown were intranasal (A) administration of PBS vehicle control or low dose 1×10 7 Tissue culture infectious dose 50 (TCID 50 ) units and high doses of 1×10 8 TCID 50 Body weight changes of JURV animals. Panel (B) shows 1×10 8 TCID 50 Infected animals exhibited relatively normal histological brain tissue on day 3 post infection (short-term toxicity). Panel (C) shows the body weight changes of animals subjected to intravenous administration of PBS vehicle control, low or high doses of JURV. Panel (D) shows the body weight changes of animals subjected to intravenous administration of PBS vehicle control, low or high doses of JURV on day 3 post infection (short-term toxicity). 8 TCID 50 The histology of the liver of infected animals showing relatively normal histology. Panel (E) shows the total white blood cell (WBC) counts in animals inoculated intranasally or intravenously with vehicle or low or high concentrations of JURV, indicating a moderate reduction in total WBC counts in infected animals. Panel (F) shows a reduction in total lymphocyte counts in infected animals. Panel (G) shows similar monocyte counts in infected animals compared to controls. Similarly, panel (H) shows no change in neutrophil counts in infected animals.

[0023] Figure 5 Shown are tumor volume and survival of animals implanted with Hepa 1-6, EMT6, CT26, and RM-1 tumor cells.

[0024] Figure 6 JURV was shown to have antitumor activity against local and untreated distant mouse hepatocellular carcinoma (HCC). Figure 6 A and Figure 6 B shows intratumoral injection of JURV (1×10 7 TCID 50 , once a week for 3 weeks) and immune checkpoint blockade (anti-PD-1, 10 mg / kg, twice a week for 3 weeks) triggered tumor regression in subcutaneous syngeneic HCC mice (Hepa 1-6). However, the JURV+anti-PD-1 combination was not superior to JURV as a monotherapy. In addition, ( Figure 6 B), In right flank and left flank tumor-bearing mice (where the right flank was injected), significant tumor growth delays were observed in both the right flank (injected flank) and left flank (uninjected), highlighting the ability of JURV to trigger an anti-tumor immune response capable of targeting and killing local tumors and distant metastases. ( Figure 6 C- Figure 6L), while weekly doses of JURV f were associated with a significant reduction in F4 / 80- (C) tumor-infiltrating lymphocyte (TIL) cells and increased NK (D), NKT (E) TIL, and M2-like (F) macrophages compared with control (PBS), anti-PD-1, and bilateral flanks (left uninjected flank). PD-1 blockade was significantly associated with intratumoral accumulation of cytotoxic CD8+ T cells, primarily CD8+ Ki67+ (G), CD8+ PD-1 (H), and CD8+ CD44+ (I), compared with control (PBS), JURV alone, and bilateral flanks (left). The combination of JURV+anti-PD-1 therapy profoundly modulates both adaptive and innate immunity, as shown by an increase in F4 / 80-CD4+(J) and CD4+PD-1+(K), CD11b+(L), CD11b-(M) compared to JURV, anti-PD-1, and bilateral flanks (left), and a decrease in CD8+PD-1+(N), NK. In addition, TIL macrophages (anti-PD-1 versus PBS), M1(O) (anti-PD-1 versus PBS), and M2-like (anti-PD-1 versus JURV) were also significantly reduced in the anti-PD-1 treatment group.

[0025] point Figure 7 (A) shows representative bioluminescent imaging of mice implanted with luminescent Hep3B tumor cells, with summary results presented graphically in panel (B), individual tumor curves presented in panel (C), and survival curves presented in (D).

[0026] Figure 8 The plasmid map of JURV-XN-2 comprising SEQ ID NO: 14 is shown.

[0027] Fig. 9 A plasmid map of JURV-GFP comprising SEQ ID NO: 15 is shown.

[0028] Fig.10 Plasmid maps of (A) JURV-N (SEQ ID NO: 16), (B) JURV-P (SEQ ID NO: 17), (C) JURV-M (SEQ ID NO: 18), (D) JURV-G (SEQ ID NO: 19), and (E) JURV-L (SEQ ID NO: 20) are shown.

[0029] Fig.11Schematic representations showing the antigenome structure of JURV (A), VSV (B) and MORV (C). The antigenome of JURV is typical of rhabdoviruses and consists of five major genes from the 3' to 5' antigenome direction: nucleoprotein (JURV-N), phosphoprotein (JURV-P), matrix (JURV-M), glycoprotein (JURV-G), polymerase (JURV-L). Schematic representations of the 3' to 5' antigenome organization of a) attenuated JURV, b) wild-type vesicular stomatitis virus (VSV) and c) Moreton virus (MORV). The intergenic regions (IGRs) at the gene junctions differ between the three viruses. (A) The antigenome organization of JURV consists of (3′ to 5′): a leader sequence (Le), followed by approximately 10,993 bases containing five structural genes (JURV-N, JURV-P, JURV-M, JURV-G, and JURV-L) separated by a highly conserved intergenic region (IGR), and a trailer sequence (Tr).

[0030] Fig.12 Oncolytic JURV was shown to effectively induce oncolysis in HCC cell lines. Monolayers of human HCC, HEP3B (A), PLC (B), HuH7 (C) and murine HCC, HEPA 1-6 (D) and RILWT (E) were incubated at 1.5 × 10 4 The density of 100 μg / well was inoculated in 96-well plates and infected with JURV, recombinant Moreton virus (MORV) and recombinant vesicular stomatitis virus (VSV) at an indicated multiplicity of infection (MOI) of 10, 1 and 0.1. The percentage of cell viability was determined using a colorimetric assay (MTS, Promega USA) 72 hours after infection. The discontinuous lines on the graph indicate the cutoff percentages of resistance (>50% cell viability above the line) and sensitivity (<50% cell viability below the line). Data were collected from multiple replicates in three independent experiments. Bars indicate mean ± SEM.

[0031] Fig.13 Toxicological pathological examinations of the oncolytic activity of JURV and the dose-dependent response to treatment with type I interferon oncolytic activity of JURV are shown. Effect of exogenous IFN-β on the outcome of JURV-induced tumor cell death. Infectious titers of (A) HEP3B and (B) HEPA 1-6 cell lines used as tumor models and MOI-dependent responses to species-specific exogenous type I IFN-β on (A) HEP3B and (B) HEPA 1-6 cell lines used as tumor models. Results are from three independent experiments and are plotted as mean ± SEM. 1×10 7 or 1×10 8TCID 50 JURV.

[0032] Fig.14 The effects of low and high doses of oncolytic JURV on the body weight and blood count of mice are shown. A single dose of 1×10 7 TCID 50 or 1×10 8 TCID 50 JURV. Body weights of IN and IV groups were recorded twice a week to assess drug-related toxicity. Three days after infection, three mice per group (IN and IV) were sacrificed and blood, brain, and liver were harvested to assess short-term toxicity. 7 TCID 50 ) and high and low (1×10 8 TCID 50 Changes in body weight and hematoxylin and eosin (H&E) staining (brain and liver) of mice treated with 100% (A, B) and 100% (C, D) doses of JURV. Complete blood cell counts, including leukocytes (E), lymphocytes (F), monocytes (G), and neutrophils (H), were performed to evaluate JURV-induced toxicity.

[0033] Fig.15 Toxicoproteomic analysis of brain and liver tissues of mice injected with high doses of oncolytic JURV is shown. Intranasal and intravenous administration of oncolytic JURV in mice caused changes in the expression of proteins related to sensing RNA viruses and antiviral signaling pathways. 8 TCID 50 Volcano plots of the differences in protein expression in the brain (A) and liver (C) of JURV-treated mice. 8 TCID 50 3D pie slices of the number of differentially expressed proteins (DEPs) in the brain (B) and liver (D) of JURV-infected mice. 8 TCID 50 Heatmaps of the top 20 DEPs upregulated or downregulated in the brain (E) and liver (F) of mice fed JURV. DEPs were identified using the limma-voom method. A fold change |logFC| ≥ 1 and a false discovery rate (FDR) of 0.055 were used as cutoff values. logFC was calculated using the difference between the mean of log2(JURV) and the mean of log2(PBS) (i.e., mean of log2(JURV) - mean of log2(PBS)). The results were shown by quantifying the expression of 1 × 108 TCID 50 Graphics of the top scoring canonical pathways significantly enriched in brain (G) and liver (H) by JURV treatment.

[0034] Fig.16 Toxicoproteomic analysis showing differentially expressed proteins in brain and liver tissues of healthy non-tumor-bearing mice injected with low doses of oncolytic JURV. 7 TCID 50 Volcano plots of the differences in protein expression in the brain (A) and liver (C) of JURV-treated mice. 7 TCID 50 3D pie charts of the number of differentially expressed proteins (DEPs) in the brain (B) and liver (D) of JURV-treated mice. 7 TCID 50 Heatmaps of the top 20 DEPs upregulated or downregulated in the brain (C) and liver (D) of mice fed JURV. DEPs were identified using the limma-voom method. A fold change |logFC| ≥ 1 and a false discovery rate (FDR) of 0.055 were used as cutoffs. logFC was calculated using the difference between the mean of log2(JURV) and the mean of log2(PBS) (i.e., mean of log2(JURV) - mean of log2(PBS)). 7 TCID 50 Graphic of the highest scoring canonical pathways significantly enriched by JURV processing.

[0035] Fig.17 Evaluation of the antitumor efficacy of oncolytic JURV as monotherapy or in combination with anti-PD-1 antibody in a murine HCC model is shown. HEPA 1-6 cells were implanted into the right flank of female C57BL6 / J (strain number: 000664) (n=7 / group; Jackson Laboratory). (A) After reaching 80-120 mm 3 Afterwards, mice were administered with PBS (vehicle), 1×10 7 TCID 5050 μL IT injection of JURV, anti-PD-1 therapy or a combination of JURV + anti-PD-1. Anti-PD-1 antibody (10 mg / kg) was given intraperitoneally (IP) twice a week for three weeks. (B) In the treatment regimen, tumor cells were subcutaneously implanted into the right flank of mice on day -14, and then PBS, JURV, anti-PD-1 antibody (αPD-1), JURV + anti-PD-1 were injected (inj.) into tumor-bearing mice on days 0, 7, and 14. Tumors were harvested at the end of the study for downstream analysis. (C) In the remote model (bilateral flank), HEPA 1-6 cells (1×10 6 The mice were implanted subcutaneously with HEPA 1-6 tumor grafts (1×10 cells / mouse) into the right flank and were classified as “primary” tumors. 6 Mice in the bilateral flank group received 1×10 7 TCID 50 Unit of JURV 50μLIT injection once a week for three weeks. Data are plotted as mean + / - SD, **p<0.001***p<0.0001. The area under the tumor growth curve was compared by one-way ANOVA with Holm-Sidak correction for type I error. We defined the day when the first JURV or PBS was injected into mice as day 0. The combination of JURV+anti-PD-1 therapy profoundly modulates the tumor microenvironment, as shown by changes in the frequency of (D) CD8+Ki67+, (E) CD8+PD-1+, (F) CD8+CD44+, (G) CD4+, (H) CD4+PD-1+, (I) macrophages, (J) M1-like macrophages, (K) M2-like macrophages and (L) NK cells. The submyeloid population representing macrophages is CD11b+F480+ cells, and the two markers used to separate M1 and M2 subpopulations are CD206 and IA / IE, which are called major histocompatibility class II (MHCII) in mice. The Bartlett test was used to test for homogeneity of variance and normality. If the p value of the Bartlett test is not less than P=0.05, ANOVA and two-sample t-tests are used to compare group means. If the p value of the Bartlett test is less than 0.05, Kruskal-Wallis and Wilcoxon rank sum tests are used to compare group means. These figures confirm the potential significant differences in the gated subpopulations in the CD45+ population determined by the p value.

[0036] Fig.18Shown are changes in body weight and survival of mice bearing Hepa 1-6 tumors treated with oncolytic JURV. (A) Individual tumor volumes of mice bearing HEPA 1-6 tumors between groups in the combination therapy study and (B) abscopal effect study. (C) Body weight of mice bearing HEPA 1-6 tumors between groups in the combination therapy study and abscopal effect study. (D) Kaplan-Meier survival analysis of mice bearing HEPA 1-6 tumors in the combination therapy study (E) and abscopal effect study. The statistical significance of the differences in survival curves between groups was evaluated using the log-rank (Mantel-Cox) test.

[0037] Fig.19 The antitumor efficacy of JURV was shown against a panel of murine solid tumor models. Murine EMT6 (breast cancer), CT26 (colon cancer), and A20 (reticulosarcoma) cells were implanted in the right flank (n=6-8 / group). Murine B16F10 melanoma cells were inoculated into the right flank of female C57BL6 / J mice. Murine RM-1 (prostate cancer) cells were transplanted into the right flank of male C57BL6 / J mice. Tumor volume was measured twice weekly using a digital caliper until the end of the study (day 21) or humane endpoint (>2,000mm 3 ). When the average tumor volume reaches 80-120mm 3 When 1×10 7 TCID 50 Single 50 μL IT injection of JURV or PBS. Data are plotted as mean + / - SD. Paired t-tests and Wilcoxen signed-rank tests (95% CI) were performed. Tumor volume and Kaplan-Meier survival analysis of EMT6 (a, b), RM-1 (c, d), CT26 (e, f), A20 (g, h) and B16-F10 (i, j) tumor-bearing mice. The statistical significance of the differences in survival curves between the groups was evaluated using the log-rank (Mantel-Cox) test.

[0038] Fig. 20Analysis of tumor-infiltrating immune cells after intratumoral injection of oncolytic JURV in murine HCC tumors is shown. Immunophenotyping of syngeneic HEPA 1-6 mice treated with PBS, JURV, anti-PD-1 therapy, JURV+anti-PD-1 antibody, and bilateral flank (abscopal effect) shows the percentage of tumor-infiltrating immune cells including natural killer T (A, NKT), F4 / 80- macrophages (B), CD45+ (C), CD11b+ (D), CD11b- (E), CD3+ (F), CD8+ (G), CD8+ granzyme+ (H), CD8+IFNg+ (I), CD4+CD44+ (J), CD4+IFNg+ (K), and CD4+KI67+ (L) cells. Bartlett's test was used to test for homogeneity of variance and normality. If the p-value of Bartlett's test was not less than P=0.05, ANOVA and two-sample t-test were used to compare group means. If the P value of the Bartlett test was less than 0.05, the Kruskal-Wallis and Wilcoxon rank sum tests were used to compare group means. These plots demonstrate potentially significant differences in gated subpopulations within the CD45+ population as determined by the P values.

[0039] Fig.21 Murine type I IFN expression in HEPA 1-6 loaded mice injected with PBS, JURV, anti-PD-1 antibody, and JURV+anti-PD-1 antibody is shown. (A) Levels of antiviral cytokines (IFN-β) were measured in the serum of mice treated with PBS, JURV, anti-PD-1 therapy, JURV+anti-PD-1 antibody, and bilateral flank injection.

[0040] Fig. 22 Proteogenomic changes in HCC in mice injected with oncolytic JURV are shown. (A) PBS versus JURV (1×10 7 TCID 50 ). (B) 3D pie plot of the number of differentially expressed genes (DEGs) between PBS and JURV. (C) Heat map of the top 20 DEGs up- or down-regulated by PBS relative to JURV. DEGs were identified using limma-voom. (D) Graph showing the highest scoring canonical pathways significantly enriched by treatment with PBS relative to JURV. MixOmics supervised analysis was performed between DEPs and DEGs based on Log2 fold change values. DEG Log2 fold change × DEP Log2 fold change > 0 and P values ​​< 0.05 for both DEG and DEP were considered as related DEG / DEP.

[0041] Fig.23Transcriptome and proteome analysis of anti-PD-1 therapy in murine HCC tumors is shown. (A) Volcano plot of mRNA expression differences between PBS and anti-PD-1 therapy (10 mg / kg, twice a week for 3 weeks). (B) 3D pie plot of the number of differentially expressed genes (DEGs) between PBS and anti-PD-1 antibody. (C) Heat map of the top 20 DEGs upregulated or downregulated by PBS relative to anti-PD-1 antibody. DEGs were determined using limma-voom. (D) Graph showing the highest scoring typical pathways significantly enriched by treatment with PBS relative to anti-PD-1 antibody. MixOmics supervised analysis was performed between DEPs and DEGs based on Log2 fold change values. DEG Log2 fold change × DEP Log2 fold change > 0 and P value of DEG and DEP < 0.05 were considered to be related DEG / DEP.

[0042] Fig.24 Proteogenomic responses of murine HCC to combined oncolytic JURV and anti-PD-1 therapy are shown. HEPA 1-6 tumors were harvested from mice treated with PBS or JURV + anti-PD-1 antibody. (A) PBS versus JURV (1×10 8 TCID 50 ×3)+anti-PD-1 antibody (10 mg / kg×6). (B) 3D pie plot of the number of differentially expressed genes (DEGs) between PBS and JURV+anti-PD-1 antibody. (C) Heat map of the top 20 DEGs upregulated or downregulated by PBS relative to JURV+anti-PD-1 antibody. DEGs were determined using the limma-voom method. Fold change |logFC| ≥ 1 and a false discovery rate (FDR) of 0.055 were used as cutoff values. logFC was calculated using the difference between the mean of log2(JURV+anti-PD-1) and the mean of log2(PBS) (i.e., the mean of log2(JURV+anti-PD-1)-the mean of log2(PBS)). (D) A graph showing the highest-scoring typical pathways that were significantly enriched by treatment with JURV+anti-PD-1 antibody. MixOmics supervised analysis was performed between differentially expressed proteins (DEPs) and DEGs based on the Log2 fold change values. DEG / DEP with Log2 fold change of DEG × Log2 fold change of DEP > 0 and P value of DEG and DEP < 0.05 were considered as relevant DEG / DEP. (E) Heat map of DEG / DEP expression of the 30 most up-regulated and down-regulated signature DEG / DEPs in JURV+anti-PD-1 antibody relative to PBS.

[0043] Fig.25Common DEGs between PBS vs. JURV vs. PBS vs. anti-PD-1 vs. PBS vs. JURV+anti-PD-1 in murine HCC are shown. Venn diagram of 35 DEGs in HEPA 1-6 tumors from three datasets (PBS vs. anti-PD-1, PBS vs. JURV+anti-PD-1, PBS vs. JURV) is shown.

[0044] Fig.26 Figure 3 shows a mechanistic interpretation of oncolytic JURV-mediated antitumor activity in local and distant murine HCC. We performed transcriptomic analysis of HEPA 1-6 tumors harvested from non-injected HEPA 1-6 tumors in the abscopal effector cohort. (A) Volcano plot of mRNA expression differences between PBS and flanks (non-injected tumors). (B) 3D pie plot of the number of differentially expressed genes (DEGs) between PBS and flanks (non-injected tumors). (C) Heat map of the top 20 DEGs upregulated or downregulated in PBS relative to flanks (non-injected tumors). Fig.24 DEGs were identified using the limma-voom method as described in (D). (D) A graph showing the top scoring canonical pathways significantly enriched by treatment with PBS relative to bilateral flanks (non-injected tumors). MixOmics supervised analysis was performed between DEPs and DEGs based on Log2 fold change values. DEG Log2 fold change × DEP Log2 fold change > 0 and P values ​​< 0.05 for both DEG and DEP were considered as related DEG / DEP.

[0045] Fig. 27 The top canonical pathways enriched in JURV relative to the bilateral flanks (abscopal effects) are shown. Comparison of the top immune-related canonical pathways enriched in JURV relative to the bilateral flanks.

[0046] Fig.28 We showed that oncolytic JURV induced robust virus-mediated oncolysis-dependent tumor growth delay in JURV-treated female NOD.Cg-Prkdc Hep3B xenografts. scid / J (strain number: 001303) mice (n=6 / group) were subcutaneously inoculated with HEP3B cells expressing human luciferase. (A) Photon emission from mice bearing subcutaneous HEP3B tumors. When the average tumor volume exceeded 80-120 mm 3 The mice were divided into two groups and received 1×10 7 TCID 50IT injection of PBS or JURV. Photon counts were obtained at weeks 1, 2, and 3. Photon emission at the right dorsal (B) and ventral (C) sides of JURV-treated mice was significantly (P<005) reduced compared with PBS. (D) Tumor volume was determined twice weekly using a digital caliper until humane endpoint, death, or end of study (day 21). Data are plotted as mean + / - SD. Paired t-tests and Wilcoxon signed-rank tests (95% CI) were performed. HEP3B tumors treated with PBS or JURV were harvested, and we analyzed changes in protein expression. (E) Compared with 1×10 7 TCID 50 Volcano plot of protein expression differences in JURV-treated HEP3B tumors. (F) Injection of PBS versus 1×10 7 TCID 50 3D pie chart of the number of differentially expressed proteins (DEPs) in JURV's HEP3B tumors. (G) The number of DEPs in JURV's HEP3B tumors was 1×10 7 TCID 50 Heat map of the top 20 DEPs up-regulated or down-regulated in HEP3B tumors from JURV. Fig. 22 DEPs were identified using the limma-voom method as described in . A fold change |logFC| ≥ 1 and a false discovery rate (FDR) of 0.055 were used as cutoff values. logFC was calculated using the difference between the mean of log2(JURV) and the mean of log2(PBS) (i.e., mean of log2(JURV) - mean of log2(PBS)). (H) Shows that the DEPs in HEP3B tumors were identified by using 1 × 10 7 TCID 50 Graphical representation of the highest scoring canonical pathways significantly enriched by JURV treatment.

[0047] Fig.29 Figure 2 shows the volume and survival of individual HEP3B tumors. The volume of individual tumors after treatment. Female NOD.Cg-Prkdcscid / J (strain number: 001303) mice (n=6 / group) were subcutaneously inoculated with HEP3B cells labeled with luciferase reporter protein. When the tumor volume was 80-120 mm 3 The mice were divided into two groups and received a dose of 1×10 7 TCID 50 =Injection of PBS or JURV IT (Day 0, Day 7, and Day 14). (A) Tumor volume and (B) body weight were recorded twice a week until humane endpoint or end of study (Day 21). (C) Kaplan-Meier survival analysis of HEP3B tumor-bearing mice. The log-rank (Mantel-Cox) test was used to evaluate the statistical significance of differences in survival curves between groups. DETAILED DESCRIPTION

[0048] Hepatocellular carcinoma (HCC) is a leading cause of cancer morbidity and mortality worldwide. 1 Most HCC patients are diagnosed with advanced disease and have limited treatment options. Current treatment options for HCC patients who are not suitable for surgery or liver transplantation include cytotoxic therapies, targeted therapies, and immune checkpoint inhibitors. 23 However, these treatments fail to achieve long-term disease control, making HCC one of the cancers with the highest unmet clinical needs worldwide.

[0049] Oncolytic viruses (OVs) are potent anticancer agents; they do not replicate in normal cells but instead preferentially amplify their genome in tumor cells, which are unable to activate their cell-based antiviral defense mechanisms. 6,7 OVs have become increasingly attractive in immuno-oncology due to their multifaceted anticancer activities, including the ability to directly kill tumor cells and their immunomodulatory properties. 1,2 Among the reported OVs, members of the family Rhabdoviridae have been the focus of research for many years. 2,3 The present invention relates to a new member of the Rhabdoviridae family, Jurona virus (JURV), for the treatment of HCC. 4,5 .

[0050] The examples demonstrate that JURV induces strong cytolytic effects in HCC cell lysis in vitro and in animal models. In addition, JURV induces systemic anti-tumor immunity, resulting in tumor growth inhibition of both injected and non-injected tumors in a syngeneic HCC model. In addition, the combination of JURV and immune checkpoint blocking antibodies profoundly modulates the tumor microenvironment by favoring the activation of tumor-specific cytotoxic T cells. These convincing data demonstrate that the JURV provided herein can be used as a novel oncolytic virus therapy platform for HCC and possibly other cancers.

[0051] The present invention provides compositions, constructs, infectious particles, pharmaceutical compositions, therapeutic methods and systems related to the novel Jurona virus of the present disclosure and their uses for treating cancer.

[0052] Composition:

[0053] Jurona virus (JURV) is nonpathogenic and closely related to the Indiana strain of vesicular stomatitis virus, but genetically distinct from it ( Figure 1 A). 1,2The JURV genome is a 10,993 bp linear negative-sense RNA with five specific vesiculovirus genes (from 3' to 5' in the negative-sense RNA genome and, therefore, from 5' to 3' in the positive-sense complementary RNA or complementary DNA): matrix (M), nucleoprotein (N), phosphoprotein (P), glycoprotein (G), and polymerase (L). 1 See also Figure 3 A, which shows the schematic organization of the JURV genome from 3' to 5'. Similar to other negative-sense RNA viruses (e.g., vesicular stomatitis virus (VSV), influenza virus, Ebola virus, rabies virus), the viral genome encodes an RNA-dependent RNA polymerase, referred to as the L protein in JURV. Therefore, the L protein can synthesize "mRNA" directly from the negative-sense RNA genome of the virus. Therefore, it should be understood that each embodiment of the disclosed composition also encompasses complementary sense polynucleotides, such as complementary sense RNAs to the disclosed polynucleotides, single-stranded or double-stranded cDNAs containing the disclosed polynucleotides.

[0054] As used herein, "negative sense RNA genome" refers to a single-stranded RNA of a virus whose genetic content is the antisense strand of the viral mRNA, as understood in the art. In a more general sense, "negative sense" can refer to the reverse complement of both the positive strand and RNA transcript.

[0055] In the first aspect, the construct comprises a promoter operably connected to a polynucleotide encoding a full-length Churona virus genome. The construct is a DNA, but a promoter is connected to allow negative sense viral genomes to be produced when transfected into mammalian cells. The construct includes any composition comprising DNA, including but not limited to plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, transposons, viruses or viral vectors, recombinant chromosome genomic DNA or any other construct available to those skilled in the art.

[0056] To generate functional viral particles, the construct comprises polynucleotides encoding M, N, P, G and / or L proteins (on the sense strand), a leader sequence, a trailer sequence and / or an appropriate intergenic region. The promoter may be a T7 promoter, which responds to RNA polymerase from T7 bacteriophage. As used herein, a "leader sequence" refers to a region in a messenger RNA (mRNA) molecule that precedes a gene coding sequence, a "trailer sequence" refers to a segment after a signal that terminates translation at the 3' end of the mRNA, and may be non-translated and may not include a poly A tail, and an "intergenic sequence" refers to a DNA sequence located between genes. The construct may comprise a polynucleotide encoding a JURV N protein having at least 90% sequence identity to SEQ ID NO: 1. The construct may comprise a polynucleotide encoding a JURV P protein having at least 90% sequence identity to SEQ ID NO: 2. The comp construct may comprise a polynucleotide encoding a JURV M protein having at least 90% sequence identity to SEQ ID NO: 3. The construct may comprise a polynucleotide encoding a JURV G protein having at least 90% sequence identity to SEQ ID NO:4. The construct may comprise a polynucleotide encoding a JURV L protein having at least 90% sequence identity to SEQ ID NO:5. The construct may comprise SEQ ID NOs:1-5 or sequences having at least 90%, 92%, 94%, 95%, 97%, 98%, 99% or 100% identity thereto. As discussed above, constructs encoding functional recombinant JURVs must comprise a leader and trailer sequence, which may have the sequences SEQ ID NOs:6 and 7, respectively. Exemplary intergenic regions may include SEQ ID NOs:8-11 and should be present in a specific order in the polynucleotide composition. Figure 3 A shows a schematic diagram of a negative sense JURV genome having polynucleotides encoding JURV N, P, M, G and L genes from 3' to 5'. Thus, ordered from 3' to 5', the intergenic regions having SEQ ID NOs: 8-11 should be present in the composition in the following arrangement: SEQ ID NO: 8 between N and P, SEQ ID NO: 9 between P and M, SEQ ID NO: 10 between M and G, and SEQ ID NO: 11 between G and L. However, it should be understood that modifications may be made to the intergenic regions without significantly affecting the ability of the composition to be used to generate functional virus, and such modifications are considered part of this disclosure. Additionally, it should be understood that the order of the JURV genes may be altered, although this may result in reduced virus production.

[0057] The construct may comprise SEQ ID NO: 12, also referred to as JURV-XN-2, which is a polynucleotide encoding a nucleoprotein (N), a phosphoprotein (P), a matrix protein (M), a glycoprotein (G), and an RNA-guided RNA polymerase L protein (L), which was codon-optimized by the inventors from a laboratory-adapted JURV virus clone for expression in mammalian cells. In addition to the aforementioned modifications from wild-type JURV, the inventors also incorporated an intergenic region from vesicular stomatitis virus into SEQ ID NO: 12.

[0058] The construct of the present disclosure further comprises heterologous polynucleotides. As used herein, "heterologous polynucleotides" refer to polynucleotides encoding proteins ("heterologous proteins") that are not found in Jurona virus in nature (i.e., non-natural or "unnaturally related"). Suitable heterologous proteins include, but are not limited to, reporter proteins and antigenic proteins. "Reporter protein" may refer to proteins expressed when certain conditions are met (e.g., when a gene is expressed). "Antigenic protein" may refer to proteins identified by the immune system. Reporter protein may be a fluorescent protein. As used herein, "fluorescent protein" is any protein that emits light when exposed to light. Exemplary fluorescent proteins include, but are not limited to, zsGreen, mRuby, mCherry, green fluorescent protein (GFP) and GFP variants (e.g., sfGFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), DsRed fluorescent protein, far-red fluorescent protein, orange fluorescent protein (OFP), blue fluorescent protein (BFP), cyan fluorescent protein (CFP), Kindling red protein and JRed. "Antigenic protein" is a protein that can serve as an antigen (i.e., a substance that induces an immune response). Suitable antigenic polypeptides may include, but are not limited to, viral antigens, bacterial antigens, fungal antigens, parasitic antigens, and tumor-specific antigens. In an embodiment, GFP is encoded as a heterologous protein in the recombinant viral genome, and the composition comprises a polynucleotide comprising SEQ ID NO: 13 and encoding a GFP-tagged JURV.

[0059] The heterologous protein can be a viral antigen. Suitable viral antigens include proteins produced by viruses such as coronaviruses, alphaviruses, flaviviruses, adenoviruses, herpes viruses, poxviruses, parvoviruses, reoviruses, picornaviruses, togaviruses, orthomyxoviruses, rhabdoviruses, retroviruses, hepadnaviruses, herpes viruses, rhinoviruses, cytomegaloviruses, Kaposi's sarcoma virus, human papillomavirus (HPV), human immunodeficiency virus (HIV), herpes simplex virus, herpes virus type 1, herpes virus type 2, herpes virus type 6, herpes virus type 7, herpes virus type 8, hepatitis A, hepatitis B, hepatitis C, measles, mumps, parvovirus, rabies virus, rubella virus, varicella zoster virus, Ebola virus, West Nile virus, yellow fever virus, dengue virus, rotavirus, Zika virus, etc.

[0060] In another aspect of the present disclosure, a construct is provided comprising a codon-optimized polynucleotide encoding at least one Churona virus protein selected from the group consisting of G, N, P, L and M, the polynucleotide being operably linked to a promoter for expression in a mammalian cell. The polynucleotide may encode the N protein and may comprise SEQ ID NO: 1. The polynucleotide may encode the P protein and may comprise SEQ ID NO: 2. The polynucleotide may encode the M protein and may comprise SEQ ID NO: 3. The polynucleotide may encode the G protein and may comprise SEQ ID NO: 4. The polynucleotide may encode the L protein and may comprise SEQ ID NO: 5.

[0061] As used herein, the term "codon-optimized" refers to a protein encoded by a nucleic acid triplet (i.e., codons) wherein the composition of the codons has been improved based on various criteria without changing the amino acid sequence of the protein. Criteria may include optimizing expression in the organism in which the protein will be expressed (e.g., expression in a mammalian cell).

[0062] Nucleic acid generally refers to a polymer comprising nucleotides or nucleotide analogs connected together by a backbone connection (such as but not limited to a phosphodiester bond). Nucleic acid includes deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), such as viral genomic RNA, messenger RNA (mRNA), transfer RNA (tRNA), etc. Generally, polymeric nucleic acids (such as nucleic acid molecules comprising three or more nucleotides) are linear molecules in which adjacent nucleotides are connected to each other via phosphodiester bonds. The term "nucleic acid" refers to a single nucleic acid residue (such as nucleotides and / or nucleosides). In some embodiments, "nucleic acid" refers to an oligonucleotide chain comprising three or more single nucleotide residues.

[0063] As used herein, the terms "oligonucleotide" and "polynucleotide" are interchangeably used to refer to nucleotide polymers (e.g., strings of at least three nucleotides). In some embodiments, "nucleic acid" encompasses RNA and single-stranded and / or double-stranded DNA. Nucleic acid can be naturally occurring, such as in the context of a genome, transcript, mRNA, tRNA, rRNA, siRNA, snRNA, plasmid, cosmid, chromosome, chromatid or other naturally occurring nucleic acid molecules. On the other hand, nucleic acid molecules can be non-naturally occurring molecules, such as recombinant DNA or RNA, artificial chromosomes, engineered genomes or fragments thereof, or synthetic DNA, RNA, DNA / RNA hybrids, or include non-naturally occurring nucleotides or nucleosides. In addition, the terms "nucleic acid", "DNA", "RNA" and / or similar terms include nucleic acid analogs, i.e., analogs with the exception of the phosphodiester backbone. Nucleic acids can be purified from natural sources, produced using a recombinant expression system, and optionally purified, chemically synthesized, etc. Where appropriate, for example, in the case of chemically synthesized molecules, nucleic acids can include nucleoside analogs, such as analogs with chemically modified bases or sugars, and backbone modifications. Unless otherwise indicated, nucleic acid sequences are presented in 5' to 3' direction.

[0064] As used herein, the terms "complementary" or "complementarity" are used to refer to "polynucleotides" and "oligonucleotides" (which are interchangeable terms referring to nucleotide sequences) that are related by the base pairing rules. For example, the sequence "5'-CAGT" is complementary to the sequence "5'-ACTG".

[0065] The nucleic acids, proteins and / or other compositions described herein can be purified. As used herein, "purified" means separated from most other compounds or entities and encompasses partially purified or substantially purified. Purity can be represented by a weight / weight measurement and can be determined using a variety of analytical techniques such as, but not limited to, mass spectrometry, HPLC, etc.

[0066] As used herein, "operably linked" refers to the functional relationship between two or more nucleic acid (e.g., DNA) segments. Typically, it refers to the functional relationship between a transcriptional regulatory element (promoter) and a transcribed sequence. For example, if a promoter stimulates or regulates the transcription of a coding sequence in an appropriate cell, the promoter is operably linked to the coding sequence. Typically, promoter transcriptional regulatory elements that are operably linked to a sequence are physically adjacent to the transcribed sequence, i.e., they are cis-acting. However, some transcriptional regulatory elements, such as enhancers, do not need to be physically adjacent to the coding sequence whose transcription they enhance or be positioned very closely to the coding sequence.

[0067] The terms "protein," "polypeptide," and "peptide" are used interchangeably herein to refer to polymers of amino acids. "Proteins" typically comprise polymers of naturally occurring amino acids (e.g., alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine).

[0068] Infectious particles:

[0069] In another aspect of the present disclosure, infectious particles are provided. Infectious particles can be generated by transfecting cells with compositions provided herein, such as constructs, which contain polynucleotides operably connected to the coding full-length antisense JURV genome and promoters that allow the generation of negative-sense viral genomes. These cells can be mammalian cells, and when the cells also produce several required JURV proteins, infectious particles will be generated. Cells can be engineered to generate required JURV proteins via transient transfection encoding and capable of generating the construct of the required protein and the composition capable of generating the viral genome, to make JURV infectious particles, or cells can be stably engineered to produce viral proteins. Proteins can be produced only after induction of the inducible promoter that drives the production of viral proteins. As used herein, "infectious particles" refer to any particles that can cause infection of an organism or cell. Exemplary infectious particles include, but are not limited to, viral particles or virions, etc. The terms "virus", "viral particles" and "virion" are used interchangeably herein.

[0070] Infectious particles will contain several viral proteins required to generate viral particles and viral genomes. Infectious particles may contain JURV proteins, including N, P and / or L proteins. Infectious particles may also contain N, P, M and G proteins. Alternatively, infectious particles may contain N, P, M, L and G proteins. Infectious particles also contain a negative sense RNA genome encoding mRNA for each of the following viral proteins: JURV N protein of SEQ ID NO: 1; JURV P protein of SEQ ID NO: 2; JURV M protein of SEQ ID NO: 3; JURV G protein of SEQ ID NO: 4; and JURV L protein of SEQ ID NO: 5. Infectious particles may contain a negative sense RNA genome capable of encoding all of SEQ ID NOs: 1-5. The negative sense RNA genome of the infectious particle may also contain a leader sequence and a trailer sequence. The leader sequence and the trailer sequence may be encoded by a cDNA sequence comprising SEQ ID NOs: 6 and 7, respectively. The negative sense RNA genome of the infectious particle may contain an intergenic region. Exemplary intergenic regions can be encoded by SEQ ID NOs: 8-11 and should be present in a specific order in the polynucleotide composition. Figure 3 A shows a schematic diagram of the negative sense JURV genome with polynucleotides encoding JURV N, P, M, G and L genes from 3' to 5'. The intergenic regions with SEQ ID NOs: 8-11, ordered from 3' to 5', if present in infectious particles, should be present in the following arrangement: SEQ ID NO: 8 between N and P, SEQ ID NO: 9 between P and M, SEQ ID NO: 10 between M and G, and SEQ ID NO: 11 between G and L. Infectious particles may contain negative sense RNA of the polynucleotide of SEQ ID NO: 12.

[0071] The infectious particles of the present disclosure may further comprise a heterologous polynucleotide. The heterologous polynucleotide may be a polynucleotide encoding an antigen or may encode a reporter protein. In some embodiments, the heterologous polynucleotide is GFP and the infectious particle comprises a polynucleotide of SEQ ID NO: 13. The inclusion of a reporter protein in the virus allows for detection of infected cells. Antigens may be included so that infection of the virus or infectious particles allows the antigen to be expressed in the cell and induces an immune response to the antigen delivered by the infectious particles.

[0072] The infectious particles described herein may comprise a codon-optimized polynucleotide encoding at least one Churona virus protein selected from the group consisting of G, N, P, L and M, operably linked to a promoter for expression in mammalian cells. The polynucleotide encoding the G protein may comprise SEQ ID NO: 4, the polynucleotide encoding the N protein may comprise SEQ ID NO: 1, the polynucleotide encoding the P protein may comprise SEQ ID NO: 2, the polynucleotide encoding the M protein may comprise SEQ ID NO: 3, and the polynucleotide encoding the L protein may comprise SEQ ID NO: 5.

[0073] Pharmaceutical composition:

[0074] In another aspect of the present disclosure, a pharmaceutical composition is provided. The pharmaceutical composition comprises infectious particles comprising a Jurona virus genome. Jurona virus particles may be infectious and allow further production of a negative sense viral genome when transfected into mammalian cells. The composition may further comprise a pharmaceutically acceptable carrier. Infectious particles are those infectious particles as described above, and may comprise at least N, P and L proteins of Jurona virus, or alternatively, at least N, P, M and G proteins, or alternatively, N, P, M, G and L proteins of Jurona virus.

[0075] The pharmaceutical composition comprises infectious particles and a pharmaceutically acceptable carrier or excipient. Pharmaceutically acceptable carriers are known in the art and include, but are not limited to, diluents (e.g., Tris-HCl, acetates, phosphates), preservatives (e.g., thimerosal, benzyl alcohol, parabens), solubilizers (e.g., glycerol, polyethylene glycerol), emulsifiers, liposomes, and nanoparticles. Pharmaceutically acceptable carriers can be aqueous or non-aqueous solutions, suspensions, or emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils (such as olive oil), and injectable organic esters (such as ethyl oleate). Aqueous carriers include isotonic solutions, alcohol / water solutions, emulsions, or suspensions, including saline and buffered media.

[0076] The pharmaceutical composition of the present invention may further comprise additives such as albumin or gelatin (to prevent absorption to the surface), detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile salts), antioxidants (e.g., ascorbic acid, sodium pyrosulfite), bulking substances, or tonicity modifiers (e.g., lactose, mannitol). The components of the composition may be covalently linked to polymers (e.g., polyethylene glycol), complexed with metal ions, or incorporated into or on particulate preparations of polymeric compounds (e.g., polylactic acid, polyglycolic acid, hydrogels, etc.), or incorporated into liposomes, microemulsions, micelles, milamellar or multilamellar vesicles, erythrocyte ghosts, or spheroplasts. The composition may also be formulated in lipophilic reservoirs (e.g., fatty acids, waxes, oils) for controlled or sustained release.

[0077] The pharmaceutical composition may also include an adjuvant to increase their immunogenicity. Suitable adjuvants include, but are not limited to, mineral salt adjuvants, gel-based adjuvants, carbohydrate adjuvants, cytokines or other immunostimulatory molecules. Exemplary mineral salt adjuvants include aluminum adjuvants, calcium salts (e.g., calcium phosphate), iron salts, and zirconium salts. Exemplary gel-based adjuvants include adjuvants based on aluminum gel and acemannan. Exemplary carbohydrate adjuvants include inulin-derived adjuvants (e.g., γ inulin, algamulin) and polysaccharides based on glucose and mannose (e.g., glucan, dextran, lentinan, glucomannan, galactomannan). Exemplary cytokines include IFN-γ, granulocyte-macrophage colony stimulating factor (GM-CSF), IL-2, and IL-12. Suitable adjuvants also include any FDA approved adjuvant, including but not limited to aluminum salts (alum) and squalene oil-in-water emulsion systems MF59 (Wadman 2005 (Novartis)) and AS03 (GlaxoSmithKline).

[0078] Methods for treating a cell proliferative disease or disorder:

[0079] As shown in the Examples, when intranasal ( Figure 4 A) or intravenous ( Figure 4 B) Administration of Jurona virus (JURV) to non-tumor-bearing mice did not cause significant changes in body weight and caused a slight decrease in lymphocyte counts and minor changes in monocyte and neutrophil counts ( Figure 4 F- Figure 4 H). Therefore, JURV is non-pathogenic.

[0080] In the examples provided herein, JURV was able to reduce tumor volume and prolong survival of tumor-bearing mice. JURV administration reduced tumor volume in mice bearing Hepa 1-6 (murine hepatoma) and RM-1 (prostate cancer) tumors, and prolonged survival of mice bearing EMT-6 (breast cancer), CT26 (murine colorectal cancer), and RM-1 tumors, as shown in Figure 2. Figure 5 as shown in . Figure 6 It was further demonstrated that mice implanted with HepC3 (hepatocellular carcinoma) cells had significantly reduced tumor volume after treatment with JURV compared to control treated animals.

[0081] Thus, methods for treating cell proliferative diseases or disorders are provided. As used herein, a "cell proliferative disease or disorder" is any disease or disorder characterized by uncontrolled or abnormal cell growth or division. Exemplary cell proliferative diseases and disorders include, but are not limited to, cancer, carcinoma in situ, lymphoproliferative disorders (e.g., chronic lymphocytic leukemia), myeloproliferative disorders (e.g., polycythemia vera), and the like.

[0082] The method includes administering to a subject a pharmaceutical composition comprising infectious particles and a pharmaceutically acceptable carrier to treat a cell proliferative disease or condition, wherein the infectious particles comprise a Jurona virus genome. The cell proliferative disease or condition may be a cancer and may be selected from hepatocellular carcinoma, hepatobiliary carcinoma, breast cancer, colorectal cancer, prostate cancer, and reticulosarcoma. Breast cancer may be HER2 negative. Cancer may be local or metastatic. "Local" cancer may refer to a cancer that has not yet spread from its original (primary) position in the subject. "Metastatic" cancer may refer to a cancer that has spread from its original (primary) position in the subject to another (secondary) position in the subject. The subject may have a suppressed immune system. The suppressed immune system may be identified or determined by methods known in the art, and may include identifying a decline in leukocytes, monocytes, lymphocytes, neutrophils, and / or other immune cells.

[0083] The pharmaceutical composition may be administered to a subject in combination with another agent having similar or different biological activity. For example, the method may further include administering immunotherapy to the subject before, simultaneously or after administering infectious particles or other compositions comprising the genome of the Jurona virus. Immunotherapy may be a checkpoint inhibitor therapy. Checkpoint inhibitor therapy may be selected from a group consisting of PD-1 inhibitors, PD-L1 inhibitors, CTLA-4 inhibitors and LAG-3 (CD223) inhibitors. Checkpoint inhibitor therapy is known in the art. Suitable PD-1 inhibitors for use in the methods described herein are known in the art and include, but are not limited to, anti-PD-1 antibodies and anti-PD-L1 antibodies. An oncolytic adenovirus vector (or an antibody encoded thereby) encoding a monoclonal antibody specific to CTLA4 (e.g., a human monoclonal antibody specific to CTLA4) may be used. The method may further include administering an IFN-α inhibitor to the subject before, simultaneously or after administering infectious particles or other compositions comprising the genome of the Jurona virus. The method may further include administering a receptor tyrosine kinase inhibitor (e.g., pazopanib) to the subject before, simultaneously or after administering infectious particles or other compositions comprising the genome of the Jurona virus.

[0084] The method includes administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising infectious particles of the Jurona virus. As used herein, the term "therapeutically effective amount" refers to the amount of viral particles or pharmaceutical preparations sufficient to alleviate one or more signs or symptoms of a cell proliferative disease or condition in a subject. Exemplary signs or symptoms of cell proliferative diseases that can be "treated" or "relieved" by the disclosed method include, but are not limited to, reduced tumor volume, disease remission, disease cure, reduced tumor number, weight gain, increased appetite, etc. In addition, for each type of cell proliferative disease treated by the disclosed method, there are disease-specific results representing effective treatment. For example, in the case of hepatocellular carcinoma, a subject treated by the method of the present invention may experience increased appetite, pain under the ribs on the right side of the body or disappearance of satiety, reduced nausea or vomiting, and relief of jaundice, etc. Other disease-specific signs and symptoms that can be treated or alleviated by this method are well known in the art.

[0085] As used herein, the terms "administering" and "administration" refer to any method of providing a drug formulation to a subject. Suitable routes of administration include, but are not limited to, intramuscular, intradermal, intranasal, oral, topical, parenteral, intravenous, subcutaneous, intrathecal, transdermal, nasopharyngeal, intratumoral, and transmucosal routes. The pharmaceutical composition may be administered intranasally, intramuscularly, or intratumorally. The pharmaceutical composition may be administered as a single dose or in multiple doses. For example, the pharmaceutical composition may be administered twice or more, at intervals of 4 hours, 6 hours, 8 hours, 12 hours, one day, two days, three days, four days, one week, two weeks, or three weeks or more. For example, in an embodiment, viral particles are administered intratumorally once a week for three weeks. The dose per mouse at each administration is 1×10 7 TCID 50 Those skilled in the art will be able to calculate the dosage to be administered depending on the tumor and subject being treated. Thus, in some embodiments, the viral particles are administered to the subject at least twice.

[0086] The "subject" to which the present method is applied can be any vertebrate. Suitable vertebrates include, but are not limited to, humans, cattle, horses, sheep, pigs, goats, rabbits, dogs, cats, bats, mice, and rats. In certain embodiments, the method may be performed on experimental animals (e.g., mice and rats) for research purposes. In other embodiments, the method is used to treat commercially important farm animals (e.g., cattle, horses, pigs, rabbits, goats, sheep, and chickens) or companion animals (e.g., cats and dogs). In a preferred embodiment, the subject is a human.

[0087] cell:

[0088] In another aspect of the present disclosure, cells are provided. The cell comprises a promoter operably linked to a polynucleotide encoding a Jurona virus genome, and allows the production of a negative sense viral genome. The cell may further comprise a construct or be genetically engineered to produce viral proteins required for the assembly of infectious particles. The cell may be engineered to produce viral proteins required for the assembly of infectious particles after induction. It will be appreciated by those skilled in the art that these cells can be engineered by the stable integration of a construct comprising an inducible promoter operably linked to a polynucleotide encoding a necessary Jurona virus protein, the Jurona virus protein being selected from the group consisting of N, P, M, G and L proteins of the Jurona virus provided herein as SEQ ID NO: 1-5, respectively. Alternatively, cells may be transfected with one or more plasmids that allow the production of essential proteins.

[0089] In some embodiments, the cell comprises SEQ ID NO: 12, also referred to as JURV-XN-2, which is a polynucleotide encoding a negative sense RNA genome of a codon-optimized, mammalian cell-adapted JURV virus as provided herein. SEQ ID NO: 12 encodes a negative sense RNA that can be transcribed by a viral RNA polymerase to produce a nucleoprotein (N), a phosphoprotein (P), a matrix protein (M), a glycoprotein (G), and an RNA-guided RNA polymerase L protein (L), which has been codon-optimized from a laboratory-adapted JURV viral clone for expression in mammalian cells. In addition to the aforementioned modifications from wild-type JURV, the inventors also incorporated an intergenic region from a vesicular stomatitis virus into SEQ ID NO: 12. The composition may further comprise a polynucleotide encoding a heterologous protein, and the heterologous protein may be GFP and the composition may comprise a SEQ ID NO: 13 sequence. Sequences having at least 95%, 96%, 97%, 98%, and 99% identity to SEQ ID NO: 12 or SEQ ID NO: 13 are also encompassed.

[0090] Methods for producing viral particles:

[0091] The present invention also provides methods for producing the viral particles described herein. The inventors have discovered that JURV can be produced in mammalian cell culture by transfecting cells expressing an exogenous polymerase with a polynucleotide encoding the full-length JURV genome (i.e., encoding the M, P, L, G, and N proteins), the leader and trailer sequences, and the intergenic regions, and the cells contain an additional promoter operably linked to one or more polynucleotides encoding the JURV proteins. See, e.g. Figure 2 . Thus, when the polynucleotide is transfected into a cell with an exogenous polymerase (e.g., T7 polymerase), the exogenous polymerase transcribes key JURV transcripts, which are translated into functional proteins required for efficient assembly of viral particles. A polynucleotide encoding a complete JURV genome and an additional promoter operably linked to one or more polynucleotides encoding JURV proteins required for JURV assembly and functional JURV viral particle formation can be encoded on a single polynucleotide. In other embodiments, the complete JURV genome can be encoded on one polynucleotide, and the polynucleotides encoding one or more JURV proteins required for viral assembly can be encoded on one or more separate polynucleotide molecules. Crucially, in order for viral assembly to occur in a cell, the complete JURV genome must be present together with one or more JURV proteins selected from M, P, L, G, and N proteins, which are encoded by one or more polynucleotides operably linked to a promoter.

[0092] Thus, in some embodiments, the method comprises introducing a composition comprising a promoter that is operably linked to a polynucleotide encoding a JURV genome and allows for the generation of a negative sense viral genome when transfected into a mammalian cell; allowing the cell to express one or more JURV proteins selected from the group consisting of G, M, N, L and P; incubating the cell for a sufficient time to generate a recombinant JURV; and harvesting the virus produced by the cell. The one or more JURV proteins required for the production of infectious particles may include N, P and L proteins. The one or more JURV proteins may be encoded by one or more polynucleotides comprising SEQ ID NO: 1, 2 or 5. The promoter may be a T7 promoter and the cell may include a T7 polymerase.

[0093] The polynucleotide encoding the Churona virus genome may further comprise a heterologous polynucleotide encoding a protein not naturally associated with Churona virus. The heterologous polynucleotide may encode an antigen or a reporter protein, as described above.

[0094] As used herein, the term "transfection" and "transfection" refers to the process of artificially introducing nucleic acid (DNA or RNA) into a cell. Transfection can be performed under natural or artificial conditions. Suitable transfection methods include, but are not limited to, lipofection, phage or virus infection, electroporation, heat shock, microinjection, and particle bombardment.

[0095] As used herein, the terms "infecting" and "infection" refer to the process of introducing a virus into a cell. A cell can be infected with a virus by simply contacting the cell with a viral particle.

[0096] The cell line used in the present method is a eukaryotic cell line. Suitable eukaryotic cells include but are not limited to mammalian cells or chicken cells. The cell can be a cell in culture. Suitable mammalian cells include but are not limited to BHK-21 cells, MDCK cells, A549 cells, CHO cells, HEK293 cells, HEK293T cells, HeLa cells, NSO cells, Sp2 / 0 cells, COS cells, BK cells, NIH3T3 cells, FRhL-2 cells, MRC-5 cells, WI-38 cells, CEF cells, CEK cells, DF-1 cells or Vero cells. In some embodiments, the cell is a BHK-21 cell and can express T7 polymerase.

[0097] The method for producing viral particles may further include an additional step involving harvesting the Jurona virus from the cells. In embodiments utilizing cultured cells, the method may further include harvesting the supernatant of the culture by, for example, centrifugation or pipetting. The Jurona virus harvested from the cells may be further separated or purified from the cells and culture medium via methods known to those skilled in the art, such as density gradient centrifugation.

[0098] System for generating recombinant Jurona virus:

[0099] A system for generating a recombinant JURV is also provided. The system comprises: a) one or more vectors comprising a polynucleotide encoding at least three JURV proteins selected from the group consisting of G, N, P, L and M, each of which is operably linked to a promoter to allow the expression of the at least three proteins in a mammalian cell; b) a vector comprising a polynucleotide including a negative sense JURV genome, the polynucleotide being operably linked to a promoter to allow the negative sense JURV genome to be produced in a mammalian cell. Therefore, the disclosed system allows for the effective production of recombinant JURV, and may further comprise: (c) a mammalian cell capable of expressing the JURV protein of (a) and the negative sense JURV genome of step (b) to produce a recombinant JURV. The cells of the system may comprise T7 RNA polymerase. The cell may be a BHK-21 cell. One or more vectors comprise polynucleotides encoding JURV N, P and L proteins operably linked to a promoter. The polynucleotide may comprise any one of SEQ ID NO: 1-5. One or more vectors may comprise a codon-optimized polynucleotide encoding at least one Churona virus protein selected from the group consisting of G, N, P, L and M, operably linked to a promoter for expression in a mammalian cell. The polynucleotide may encode the N protein of SEQ ID NO: 1. The polynucleotide may encode the P protein of SEQ ID NO: 2. The polynucleotide may encode the M protein of SEQ ID NO: 3. The polynucleotide may encode the G protein of SEQ ID NO: 4. The polynucleotide may encode the L protein of SEQ ID NO: 5.

[0100] One or more vectors comprise a polynucleotide comprising a promoter operably linked to a polynucleotide encoding a Churona virus genome, thereby allowing for production of a negative sense viral genome when transfected into mammalian cells.

[0101] Kits for treating a cell proliferative disease or disorder:

[0102] A kit is provided. The kit comprises a pharmaceutical composition comprising infectious particles containing a Jurona virus genome and an optional pharmaceutically acceptable carrier. The kit may further comprise immunotherapy. Immunotherapy may be a checkpoint inhibitor therapy. Checkpoint inhibitor therapy may be selected from a group consisting of PD-1 inhibitors, PD-L1 inhibitors, CTLA-4 inhibitors, and LAG-3 (CD223) inhibitors. Checkpoint inhibitor therapy is known in the art. Suitable PD-1 inhibitors for use in the methods described herein are known in the art and include, but are not limited to, anti-PD-1 antibodies and anti-PD-L1 antibodies. In some embodiments, an oncolytic adenovirus vector (or an antibody encoded thereby) encoding a monoclonal antibody specific for CTLA4 (e.g., a human monoclonal antibody specific for CTLA4) may be used. The kit may further comprise an IFN-α inhibitor. The kit may further comprise a receptor tyrosine kinase inhibitor (e.g., pazopanib).

[0103] The present disclosure is not limited to the specific details of the construction, arrangement of parts or method steps described herein. The compositions and methods disclosed herein can be manufactured, practiced, used, implemented and / or formed in various ways that are obvious to those skilled in the art based on the following disclosure. The wording and terminology used herein are for descriptive purposes only and should not be considered to limit the scope of the claims. Ordinal indicators such as first, second and third, as used in the specification and claims, refer to various structures or method steps and are not intended to be interpreted as indicating any specific structure or step, or any specific order or configuration of such structures or steps. Unless otherwise specified herein or the context is clearly contradictory, all methods described herein can be performed in any suitable order. Unless otherwise required, the use of any and all examples or exemplary terms provided herein (e.g., "such as") is intended only to promote the present disclosure and does not imply any limitation on the scope of the present disclosure. Any language in this specification and any structure shown in the drawings should not be interpreted as indicating that any unclaimed element is essential for the practice of the disclosed subject matter. The terms "including", "comprising" or "having" and their variations used herein are intended to cover the elements listed thereafter and their equivalents and additional elements. Embodiments recited as "comprising," "including," or "having" certain elements are also considered to "consist essentially of" and "consist of" those certain elements.

[0104] Unless otherwise indicated herein, the range of values ​​narrated herein is intended only as a simplified method of individually representing each individual value within this range, and each individual value is incorporated into this specification as if it were individually narrated herein. For example, if the concentration range is specified as 1% to 50%, then numerical values ​​such as 2% to 40%, 10% to 30% or 1% to 3% are intended to be explicitly listed in this specification. These are merely examples of explicit intent, and all possible combinations of numerical values ​​between the lowest and highest values ​​listed (and including the end values) are considered to be explicitly stated in this disclosure. The use of the word "about" to describe a specific narrated amount or range of amounts is intended to indicate that values ​​very close to the narrated amount are included in the amount, such as values ​​that may or naturally be considered due to manufacturing tolerances, instruments and human errors when forming measurements, etc. Unless otherwise indicated, all percentages involving amounts are by weight.

[0105] It is not acknowledged that any reference, including any non-patent or patent document cited in this specification, constitutes prior art. In particular, it should be understood that, unless otherwise stated, reference to any document herein does not constitute an admission that any of these documents form part of the common general knowledge in the art in the United States or any other country. Any discussion of references states the claims of their authors, and the applicant reserves the right to question the accuracy and relevance of any document cited herein. Unless otherwise expressly indicated, all references cited herein are fully incorporated by reference. If there is any difference between any definition and / or description found in the cited references, the present disclosure shall prevail.

[0106] The following examples are intended to be illustrative only and are not intended to limit the scope of the invention or the appended claims.

[0107] Example

[0108] The following examples demonstrate the generation of recombinant Churona virus and its use in treating cell proliferative diseases.

[0109] Example 1: Characterization of Jurona virus (JURV) and its cytopathic effect in tumor cells

[0110] Oncolytic viruses can provide multimodal anti-tumor activity from selective killing of tumor cells to promoting systemic anti-tumor immunity, making them a powerful enemy against cancer. Among them, several members of the Rhabdoviridae family are particularly attractive as oncolytic agents due to their natural tumor selectivity and non-pathogenicity to humans. In this example, we characterized JURV and confirmed its cytopathic effect in tumor cells.

[0111] result

[0112] Characterization and Generation of Oncolytic Recombinant Jurona Virus (JURV)

[0113] Jurona virus (JURV) is nonpathogenic and closely related to the Indiana strain of vesicular stomatitis virus, but genetically distinct from it ( Figure 1 ). 1,2 JURV belongs to the order Mononegavirales, family Rhabdoviridae, and genus Vesiculovirus (containing about 5000 members). It has an envelope and is bullet-shaped, about 180 nm long and 75 nm wide ( Figure 1 A). Vesicular stomatitis virus (prototype vesicular virus) has a negative-strand RNA linear genome, about 11 kb in size, and encodes 5 proteins ( Figure 1 B) Use Figure 2 The method shown in , trains JURV to selectively infect tumor cells, Figure 2 Methods for obtaining laboratory-adapted clones of Churona virus are shown ( Figure 2 A) and the process of developing a reverse genetics-based approach to generate recombinant Churona virus ( Figure 2 The JURV genome is a 10,993 bp linear RNA with five specific vesiculovirus genes (from 3' to 5' direction): nucleoprotein (JURV-N), phosphoprotein (JURV-P), matrix (JURV-M), glycoprotein (JURV-G), and polymerase (JURV-L) ( Figure 3 A). 1 Between the JURV-M region and the JURV-G region is a highly conserved intergenic region comprising SEQ ID NO:11.

[0114] JURV induces robust cytopathic effect (CPE) in tumor cells

[0115] To evaluate the in vitro cytotoxicity of JURV, we performed CPE assays on three human HCC cell lines (Hep3B, PLC, and Huh7) and two mouse HCC cell lines (Hepa 1-6 and RILWT). Figure 3 B- Figure 3 E). We infected human and mouse hepatoma monolayers with JURV, VSV, and MORV at MOIs of 10, 1, and 0.1; cell viability was measured 72 hours after infection. JURV, MORV, and VSV were able to lyse cancer cells and displayed relatively similar lytic activity in the cancer cell lines tested ( Figure 3 B). These results indicate that JURV can selectively infect and lyse tumor cells in vitro while sparing normal cells, although JURV has similar infectivity to VSV and MORV and can grow and infect Vero cells at fairly high titers ( Figure 3 E).

[0116] High-dose intranasal administration of JURV was not associated with neurotoxicity or hepatotoxicity

[0117] To determine whether JURV is causally involved in brain injury and neurotoxicity in animal models, immunocompetent mice were given two intranasal (IN) and intravenous (IV) administrations of JURV (low dose: 1 × 10 7 TCID 50 ; High dose: 1×10 8 TCID 50 )( Figure 4 ). Control animals were treated with phosphate-buffered saline (PBS). Three mice per group were sacrificed 3 days after infection to assess short-term toxicity; blood, brain, liver, and spleen tissues were collected for further analysis. The remaining animals were monitored for signs of toxicity for 45 days by a certified veterinarian. We found that low and high IN and IV doses of JURV were well tolerated and managed in all groups ( Figure 4 A- Figure 4 D). However, it is important to note that in this study we used an attenuated laboratory-adapted JURV strain (serial passaged), which is known to be significantly attenuated compared to the wild-type virus. In the JURV group, the decrease in body weight was not statistically significant 3 days after treatment; body weight continued to increase from 7 days after IN and IV administration until the end of the study. Consistent with the minimal effect on body weight, the highest IN and IV doses of virus (1 × 10 8 TCID 50 A thorough pathological review of brain and liver sections from mice treated with ) showed that JURV did not induce overt signs of toxicity ( Figure 4 B. Figure 4 C). Complete blood count analysis 3 days after treatment with JURV revealed a decrease in the number of leukocytes, lymphocytes, granulocytes, and platelets ( Figure 4 E- Figure 4 H) In addition to virally mediated hematologic changes, these changes in blood counts may also be caused by hemolysis from terminal cardiac puncture, which is known to interfere with blood counts and other hematologic parameters

[0118] JURV induces regression and improves survival in mouse HCC (Hepa 1-6), breast cancer (EMT6), colon cancer (CT26), and prostate cancer (RM-1)

[0119] To evaluate the antitumor efficacy of JURV in various murine cancer types, Hepa 1-6 (HCC), EMT6 (breast), CT26 (colon), and RM-1 (prostate) cell lines were implanted subcutaneously into female C57BL / 6J mice (n=7 / group) (Jackson Laboratories). Figure 5 When the tumor reaches a treatable size (80-120 mm 3 ) and then 1×10 7 TCID 50 The unit received 3 intratumoral (IT) injections of JURV (once a week). Tumor volume and survival were recorded. In the Hepa 1-6 (p = 0.006) model, JURV induced significant tumor growth inhibition. However, in the CT26 (p = 0.0355), EMT6 (p = 0.0396), RM-1 (p = 0.0094) models, JURV mediated a significant increase in survival compared to PBS ( Figure 5 We found no adverse events or virus-related toxicities. The mechanisms behind the heterogeneity of JURV's antitumor efficacy in animal models and human HCC warrant further investigation.

[0120] Antitumor activity of JURV against local and untreated distant murine HCC

[0121] Oncolytic vesicular viruses exert their antitumor effects by inducing direct cytotoxicity to tumor cells and stimulating host antitumor immune responses ( Figure 6 Therefore, to understand whether the injection of JURV leads to an immune response against tumor cells, we implanted Hepa 1-6 cells subcutaneously into C57BL / 6J mice (n=7 / group). Once the tumors reached 80-120 mm 3 Mice were treated with JURV (1×10 7 TCID 50 ) or murine anti-PD-1 (10 mg / kg per day for 14 days) or 3 IT injections of JURV+anti-PD-1. We evaluated changes in tumor volume and immune cell profiles in Hepa 1-6 tumors at the end of the study (EOS). Tumor volume was significantly reduced in the JURV (p=0.008) treated group, anti-PD-1 (p=0.0047) and JURV+anti-PD-1 (p=0.0023) groups compared with PBS. Interestingly, JURV-induced tumor growth arrest was similar to that of anti-PD-1, and the JURV+anti-PD-1 combination was not superior to JURV alone ( Figure 4 A). In addition, we observed tumor regression in JURV-treated and untreated tumors in mice bearing Hepa 1-6 in the right and left flanks ( Figure 6B). For flow cytometry analysis, CD4+PD-1+ T cells were significantly increased in the bilateral flanks (JURV) group compared with the JURV+anti-PD1 treatment group ( Figure 6 K). Compared with the PBS group, JURV+anti-PD1 group, and bilateral flank (JURV) group, CD8+CD44+T cells in the anti-PD1 group increased significantly ( Figure 6 I). Compared with the PBS group and the bilateral flank (JURV) group, the proliferation of CD8+ (CD8+Ki67+) T cells in the anti-PD-1 group was significantly increased ( Figure 6 G). CD8+PD1+T cells were significantly increased in the anti-PD1 group compared with those in the JURV and JURV+anti-PD1 groups; in addition, CD8+PD1+T cells were significantly increased in the bilateral flank (JURV) group compared with the JURVV+anti-PD1 group ( Figure 6 H). Compared with the JURV+anti-PD1 group and the bilateral flank (JURV) group, NK cells in the JURV and anti-PD1 groups were significantly increased ( Figure 6 D). Compared with the PBS control group, the M1 macrophages in the anti-PD1 group and the bilateral flank (JURV) group were significantly reduced; compared with the PBS group and JURVV group, the M2 macrophages in the anti-PD1 group were significantly reduced ( Figure 6 C. Figure 6 F. Figure 6 N. Figure 6 O). These results suggest that IT administration of JURV is associated with an effective and robust antitumor immune response, primarily through CD8 + T cell-mediated cytotoxicity.

[0122] Low-dose JURV effectively reduces tumor burden in HCC xenografts

[0123] To evaluate the in vivo cancer-killing properties of JURV particles, we implanted bioluminescent human HCC cells (Hep3B) subcutaneously into the right flank of immunodeficient mice ( Figure 7 Once the tumor reaches 80-120mm 3 Mice were given three IT doses of PBS or JURV (1×10 7 TCID 50 )( Figure 7 A). Bioluminescence imaging at days 7, 14, and 21 correlates with tumor growth inhibition in Hep3B mice ( Figure 7 B- Figure 7 D). Thus, measurement of tumor size in the JURV and PBS groups showed that tumors in the JURV group regressed significantly compared to PBS.

[0124] Materials and methods

[0125] Cell lines

[0126] A panel of three human hepatocellular carcinoma (HCC) cell lines (Hep3B, PLC, Huh7) and two murine HCC cell lines (Hepa 1-6, RILWT) were used in this study. All cell lines were cultured at 37°C, 5% CO2 in a humidified medium supplemented with L-glutamine and antibiotics (100 μg ml -1 Penicillin and 100 μg ml -1 All HCC cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS). BHK-21 and Vero cells were obtained from the American Type Culture Collection (ATCC; Manassas, VA). Hep3B, PLC and Huh7 and Hepa 1-6 were purchased from ATCC. RILWT (gift from Dr. Dan Duda) is a clone derived from the RIL-175 cell line grown in DMEM containing 10% FBS.

[0127] Oncolytic viruses

[0128] We obtained JURV from the World Reference Center for Emerging Viruses and Arboviruses (WRCEVA) at the University of Texas Medical Branch (UTMB). A laboratory-adapted JURV virus clone was generated by serial plaque purification of Vero cells (ATCC). RNA sequencing was used to confirm the full-length JURV genome (Iowa State University Veterinary Laboratory). The full-length JURV genome (10,993 nucleotides) containing genes encoding nucleoprotein (JURV-N), phosphoprotein (JURV-P), matrix protein (JURV-M), glycoprotein (JURV-G), and RNA-guided RNA polymerase L protein (JURV-L) was codon-optimized for expression in mammalian cells and synthesized (Genscript) from a laboratory-adapted JURV virus clone and subcloned into a plasmid (JURV-XN2). However, we inserted between the five genes of the JURV intergenic region derived from VSV to increase the translation of viral proteins. Recombinant JURV and JURV-GFP were generated using JURV-XN2 and JURV-eGFP (carrying the GFP gene between the G and L genes) and helper plasmids (JURV-P, JURV-N, and JURV-L) as described previously. 50 These plasmids were used to express the antigenomic positive-sense RNA of JURV under the bacteriophage T7 promoter to generate recombinant JURV; however, for in vivo studies, we used a laboratory strain of JURV rather than recombinant JURV. JURV and JURV-GFP were rescued from plasmids (JURV-XN2 and JURV-eGFP) on BHK-21 cells. All viruses were rescued using the vaccinia rescue system and propagated and titered on BHK-21 cells as previously described. 3,4 We used several concentrations of JURV-XN2 and JURV-eGFP, and only the following concentrations produced infectious clones: JURV-XN2 or JURV-eGFP (5 μg), JURV-N (5 μg), JURV-P (3 μg), JURV-M (2 μg), JURV-G (0.1 μg), and JURV-L (1 μg). Purified JURV particles were obtained using sucrose density gradient centrifugation prior to in vitro and in vivo studies.

[0129] A representative JURV sequence comprises SEQ ID NO: 12 of the present disclosure, and a plasmid map of JURV-XN-2 (SEQ ID NO: 14) comprising said sequence can be found at Figure 8 A representative JURV sequence with a green fluorescent protein fusion comprises SEQ ID NO: 13 of the present disclosure, and a plasmid map of JURV-GFP (SEQ ID NO: 15) comprising said sequence can be found at Fig. 9 The plasmid maps of JURV-N (SEQ ID NO: 16), JURV-P (SEQ ID NO: 17), JURV-M (SEQ ID NO: 18), JURV-G (SEQ ID NO: 19) and JURV-L (SEQ ID NO: 20) can be found in Fig.10 A- Fig.10 In E.

[0130] Cell viability assay

[0131] For all cytotoxicity assays (96-well format), cells were infected with laboratory-based JURV or MORV or VSV strains at MOIs of 10, 1, and 0.1 in serum-free Gibco Minimum Essential Medium (Opti-MEM) (1.5 × 10 4 ). Cell viability was determined using the Cell Titer 96 AQueous One Solution cell proliferation assay. Data were generated as mean ± SEM of 6 replicates from 6 independent experiments.

[0132] Emergence of virus-induced cytopathic effects in cholangiocarcinoma cells

[0133] Adherent cells in 6-well plates (5 × 10 cells per well) were infected with JURV at an MOI of 0.1. 5 Cells were incubated at 37°C until analysis. 72 hours post infection, cells were fixed with 5% glutaraldehyde and stained with 0.1% crystal violet to visualize cell morphology and residual adhesion indicative of cell viability. Photographs of representative areas were taken.

[0134] Animal studies

[0135] We performed the following in vivo evaluations according to protocols approved by the Mayo Clinic Institutional Animal Care and Use Committee.

[0136] Toxicology and biodistribution of viruses administered via the nasal route

[0137] To determine whether treatment with JURV might be associated with neurotoxicity or hepatotoxicity, female C57BL / 6J mice (N = 36; n = 6 mice per group) (including controls) were administered intranasally (IN) or intravenously (IV) (25 μl per nostril) with PBS or a dose (1 × 10 7 , 1×10 8 TCID 50 ) JURV. Body weight, temperature, behavior and clinical signs were monitored at least 3 times per week by a board-certified veterinarian to detect signs of toxicity. Three days after infection, 3 mice per group were sacrificed and tissues (brain and liver) were collected to evaluate short-term toxicity and viral biodistribution. The remaining mice were monitored for 45 days, and body weight and clinical observations were recorded at least 3 times per week during the study.

[0138] Blood tests

[0139] Blood was collected from the submandibular vein (cheek bleed) on day 3 and from cardiac puncture on day 45. Blood was collected in BD Microtainer tubes (Becton, Dickinson and Company) with ethylenediaminetetraacetic acid or lithium heparin for complete blood counts and BD Microtainer SST tubes (Becton, Dickinson, and Company) for serum analysis. Complete blood count analysis was performed in a Piccolo Xpress chemistry analyzer (Abaxis), and blood chemistry analysis was completed in a VetScan HM5 hematology analyzer (Abaxis).

[0140] In vivo efficacy studies in human CCA and HCC xenograft models

[0141] To evaluate the in vivo therapeutic efficacy of oncolytic JURV in murine tumor and human hepatocellular carcinoma subcutaneous xenograft models, tumor cells (2 × 10 6 The tumors were subcutaneously inoculated into the right flank of female athymic nude (NU / J) mice (n=7 mice / group) (Jackson Laboratories). 3 The mice were randomly divided into treatment groups when the average size of the mice was 1.5×10 50 μl and received drug administration within 24 hours after randomization. Each mouse received three intratumoral injections (50 μl containing PBS or 1×10 7 TCID 50 Units of JURV or PBS) were administered at 1-week intervals. Tumor volume and body weight were monitored. 3The mice were euthanized at 4 pm. The tumor volume was calculated using the following formula: (longest diameter*shortest diameter2) / 2. Tumor images were taken before resection and tumor weights were recorded after resection.

[0142] Analysis of tumor-infiltrating immune cells

[0143] After resection, tumors from 5 mice per group were dissociated using the gentleMACS Octo dissociator (Miltenyi) according to the manufacturer's protocol. CD45 (TIL) mouse microbeads (Miltenyi) were used to isolate CD45 + Cells. Cells were incubated with FixableViability Stain 510 (BD Horizon) for 15 minutes, followed by incubation with anti-Fc blocking reagent (Miltenyi) for 10 minutes before surface staining. Cells were stained and data were acquired on a MACSQuant Analyzer 10 optical mount flow cytometer (Miltenyi). All antibodies were used as recommended by the manufacturer. A Fluorescence Minus One control was used to establish gating for each independent experiment. For intracellular staining of granzyme B, cells were stained with an intracellular staining kit (Miltenyi). Analysis was performed using FlowJo (TreeStar). Forward and side scatter were used to exclude cell debris and doublets.

[0144] Antibodies for flow cytometry analysis

[0145] The following antibodies were used for flow cytometry analysis: CD45-FITC (Catalog No. 553079, BD Biosciences), CD3-BUV395 (Catalog No. 563565, BD Biosciences), CD4-BUV737 (Catalog No. 612761, BD Biosciences), CD8-Percp-Cy5.5 (Catalog No. 45-0081-82, eBioscience), CD44-BV711 (Catalog No. 103057, Biolegend), CD335-PE / Dazzle594 (Catalog No. 137630, Biolegend), PD-1-PE (Catalog No. 551892, BD Biosciences), Ki67*-BV605 (Catalog No. 652413, Biolegend), Granzyme B*-APC (Catalog No. 366408, Biolegend), IFN-γ*-BV421 (Catalog No. 563376, BD Biosciences), CD11b-PE-Cy7 (Catalog No. 101216, Biolegend), F4 / 80-BV510 (Catalog No. 123135, Biolegend), CD206-AF700 (Catalog No. 141734, Biolegend), IA / IE-BV786 (Catalog No. 743875, BD Biosciences) and L / D-efluor780 (Catalog No. 65-0865-18, eBioscience).

[0146] Histopathological analysis

[0147] Assessment of any abnormal changes in the brain and liver was determined by histopathological evaluation of H&E stained images reviewed by a board-certified pathologist. HALO v3.1.1076.379 was used to measure the percentage of tumor necrosis area.

[0148] Statistical analysis

[0149] All values ​​are expressed as mean ± standard deviation, and the results were analyzed by one-way ANOVA followed by Tukey's test for multiple comparisons and Kaplan-Meier method for survival using statistical software (GraphPad Software) in GraphPad Prism version 8. p values ​​less than 0.05 were considered significant.

[0150] References

[0151] 1. Walker, PJ, et al., Evolution of genome size and complexity in the rhabdoviridae. PLoS Pathog 11, e1004664 (2015).

[0152] 2. Amarasinghe, GK et al. Taxonomy of the order Mononegavirales: update 2017. Arch Virol 162, 2493-2504 (2017).

[0153] 3. Lawson, ND, Stillman, EA, Whitt, MA and Rose, JK Recombinant vesicular stomatitis viruses from DNA. Proc Natl Acad Sci USA 92, 4477-4481 (1995).

[0154] 4. Whelan, SP, Ball, LA, Barr, JN and Wertz, GT. Efficient recovery of infectious vesicular stomatitis virus entirely from cDNA clones. Proc Natl Acad Sci USA 92, 8388-8392 (1995).

[0155] Example 2: Comprehensive proteogenomic analysis of the anti-tumor immune activity of a novel oncolytic vesicular virus in hepatocellular carcinoma

[0156] In this example, we demonstrate that intratumoral (IT) administration of Jurona virus (JURV) induces dynamic tumor regression in human HCC xenograft models and syngeneic models. In addition, IT injection of JURV induces the recruitment and activation of cytotoxic T lymphocytes (CTLs) and reduces tumor-associated macrophage (TAM) infiltration, resulting in delayed tumor growth of local and distant murine HCC tumors in syngeneic models. In addition, when administered simultaneously, JURV and anti-PD-1 antibodies act synergistically to regulate the tumor microenvironment (TME) via increasing tumor-infiltrating CD4+T cells and exhausting CD8+PD-1+ and NK cells. Mechanistically, our analysis reveals that JURV and anti-PD-1 antibodies activate different effectors of the immune system, but have complementary antitumor activity. In addition, our results indicate that the remote effects induced by JURV may be mediated by the activation of several tumor suppressor genes and mechanisms regulating T helper cell responses. Our work supports the further development of JURV as a novel immunoviral therapy platform for hepatocellular carcinoma.

[0157] result

[0158] In vitro cytotoxic activity of JURV in HCC cells

[0159] Analysis of the Jurona virus (JURV) genome revealed a genome organization identical to that of vesicular stomatitis virus (VSV) and Moreton virus (MORV) ( Fig.11 ), two other members of the Rhabdoviridae family 6 , whose oncolytic potential in human cancers has been previously investigated. 7 To investigate whether JURV has similar in vitro cancer cell killing ability, we infected monolayers of human and mouse HCC cells, including HEP3B, PLC, HuH7, HEPA 1-6, and RILWT ( Fig.12 A- Fig.12 E). We performed a cytotoxic MTS cell viability assay assessment at 72 hours post-infection to determine susceptibility to viral infection and the extent of virus-induced cancer cell death. The results of the cell viability assay are summarized in Table 1. Regardless of the MOI, a significant decrease in cell viability (approximately 30%) was observed in HEP3B cells in response to JURV, VSV, and MORV infection ( Fig.12 A). Differences in virus-induced cell death were also noted in PLC, HuH7, HEPA 1-6, and RILWT cells ( Fig.12 B- Fig.12E). In PLC cells, while JURV and VSV induced similar rates of cytotoxicity at MOIs of 10, 1, and 0.1 (approximately 25%), cell viability was two-fold higher in MORV-infected cells (approximately 50%). HuH7, HEPA 1-6, and RILWT cells exhibited lower sensitivity to JURV infection than MORV and VSV, with the exception of RILWT cells treated with JURV at MOIs of 1 and 10 (approximately 24%). We then infected HCC cells with JURV at an MOI of 0.1. After three days of incubation, cells were stained with crystal violet to assess the comparative cytotoxic effects of JURV in infected cancer cells. We observed that while HuH7 still had residual (<50%) viable adherent cells after infection, adherent cells were completely lost in HEP3B, PLC, HEPA 1-6, and RILWT (>90%), indicating that MTS may slightly underestimate the oncolytic effect of JURV (see Figure 3 E). In addition, JURV kinetic analysis showed that the virus titer increased 1000-fold around 10 hours after infection (see Figure 3 E), indicating that JURV has high permissiveness and robust replication in HCC cells. Our results indicate that JURV, VSV, and MORV can effectively infect and lyse mouse and human HCC cells. However, the basis for the observed differences in HCC cell killing mediated by these viruses deserves to be explored in future studies.

[0160] Table 1. In vitro cell viability assay results.

[0161]

[0162]

[0163] In vitro cell viability of JURV-infected HCC cells pretreated with type I IFN-α

[0164] The tumor microenvironment (TME) is the site of complex interactions between cancer cells, healthy tissues, and different components of the immune system. 8 Detection of viral pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs) present in most immune cells induces the production of multiple cytokines, including IFNα / β, which activate hundreds of interferon-stimulated genes (ISGs). 9,10 Activation of ISGs triggers innate antiviral mechanisms and generates adaptive cellular responses to viral infection. Interestingly, studies have demonstrated that defects in the type I IFN signaling pathway are frequently consistent with carcinogenesis and create a necessary condition for tumor-selective viral replication and oncolysis by many oncolytic viruses (OVs). 11,12However, upon sensing the presence of the virus, immune cells and non-cancerous cells in the TME can produce type I IFN-α / β. If tumor cells respond to the effects of exogenous IFN, this could prematurely impair the oncolytic activity of OVs. 11,13 Therefore, to determine the effect of type I IFN on the outcome of JURV infection, we compared the susceptibility of monolayers of human (HEP3B) and murine HCC (HEPA 1-6) cells pretreated with species-specific IFN-α to JURV infection. Specifically, we treated HEP3B and HEPA 1-6 cells with a series of concentrations of IFN-α and then infected them with JURV at an MOI of 10, 0.1, or 0.01 as described in Methods and Fig.13 As shown in A. Cell viability was assessed seventy-two hours after infection (MTS). Our data show that treatment with IFN-α does not protect (30-75% cell death) human HCC (HEP3B) and mouse HCC (HEPA 1-6) cells from JURV-induced cytopathic effects. However, we noted differences in the response to IFN-α between the treated HCC cells. At an MOI of 1, IFN-α at a concentration of 500 U / mL killed approximately 20% of HEPA 1-6 ( Fig.13 B). In contrast, the same treatment produced approximately 75% cell killing in HEP3B cells ( Fig.13 A). Overall, there was no statistical difference in cell viability between the different treatment groups, suggesting that treatment with IFN-α did not significantly alter JURV oncolytic activity and that the IFN pathway may be defective in these HCC cells, as shown in our previous work. 7,14

[0165] In vivo safety assessment of JURV

[0166] Reports have shown that intranasal (IN) administration of wild-type VSV in mice results in significant weight loss and lethality approximately three days after infection. 15-17 Because JURV shares the same genomic structure as VSV, we sought to determine whether infection of mice with JURV could result in acute adverse events as described for VSV and VSV-derived vectors. 18-20 To increase the likelihood of observing adverse events, we selected two doses of wild-type JURV that were higher than the 1 × 10 dose associated with toxicity in wild-type VSV-infected mice. 6 TCID 50 Doses are 10 to 100 times higher. 15-17 1×10 7 or 1×10 8 TCID 50JURV was administered intranasally or intravenously (IV) to non-tumor-bearing healthy laboratory mice at a dose of 100 mg / kg to mimic the natural route of systemic VSV infection. As a control group, we administered PBS to the animals in parallel. Three days after infection, half of the mice were sacrificed, and blood and animal tissues (brain, liver, and spleen) were harvested and H&E stained to assess short-term toxicity. In the virus-treated groups, the body weight of mice decreased by 10-15% (95% CI, 2.3-3.7) on day 3 compared with the control group. Fig.14 A and Fig.14 B). However, expert review of histology slides revealed no obvious abnormalities in brain, liver, or spleen sections from animals treated with either the low- or high-dose JURV groups ( Fig.14 C and Fig.14 D). Mice treated with JURV developed transient leukopenia, including a decrease in leukocytes and lymphocytes ( Fig.14 E- Fig.14 H). Viral infection is characterized by leukopenia in humans and animals, and systemic VSV infection is well documented. 21 Our data did not show significant differences in body weight or clinical signs (ie, paralysis, mortality, matted hair) between JURV-infected and PBS-treated mice.

[0167] Toxicoproteomic analysis of JURV

[0168] As discussed above, the lack of changes in brain and liver tissues harvested three days after infection with JURV indicated the presence of severe toxicity, including neurotoxicity or hepatotoxicity at any dose of the tested virus. We then performed quantitative proteomic analysis of these tissues to identify potential biological changes associated with neuroprotection and anti-hepatotoxicity in mice infected with a high dose of JURV. We used Ingenuity Pathway Analysis (IPA) to identify enriched biological processes and signaling pathways of differentially expressed proteins (DEPs) with significantly altered expression in the brain and liver of mice infected with JURV.

[0169] Compared with the control, the 8 TCID 50 Of the 4,253 analyzed DEPs in the brains of JURV-treated mice, 127 DEPs had significantly altered expression (2-fold change > 2, p-value < 0.055). These DEPs included 60 upregulated DEPs and 67 downregulated DEPs ( Fig.15 A and Fig.15 B). Of the 2,400 DEPs analyzed in livers from the same mice, 87 DEPs were upregulated, while 123 DEPs were downregulated ( Fig.15 C and Fig.15D). To understand the dynamics of biological changes in these tissues, we further scrutinized the top ten up- and down-regulated DEPs ( Fig.15 E) and the five most important signal transduction pathways in the brain ( Fig.15 G). Using the National Library of Medicine (NLM) database, we matched these proteins to their human orthologs, with the exception of Prps1l3 (Table 2), which allowed us to gain insight into changes in protein expression in response to JURV infection.

[0170] Table 2. List of up-regulated and down-regulated DEPs in the brain.

[0171]

[0172]

[0173] Tubgcp4, a gamma complex-associated protein that plays a role in microtubule (MT) nucleation at the centrosome, was the most significantly upregulated protein in the brains of JURV-treated mice ( Fig.15 E). Interestingly, it has been shown that changes in MTs are essential for the formation of viral intracellular replication compartments 22, suggesting that JURV induces MT changes in brain cells to favor viral replication. However, other upregulated proteins include H2-Q7, H2-Q8, H2-Q6, and H2-L, which are orthologs of human leukocyte antigen (HLA) complex molecules (HLA-A, HLA-B, HLA-C, HLA-E, HLA-G, and HLA-F) (Table 2). Several studies have demonstrated that IFN-γ produced during viral infection upregulates HLA-A, HLA-B, and HLA-C expression in mammals. 23,24 HLA-A, HLA-B, and HLA-C are specifically responsible for presenting antigens to cytotoxic T cells; therefore, they are important components of cell-mediated adaptive immune responses against pathogens, including viruses and bacteria. In contrast, HLA-E, HLA-G, and HLA-F are reported to downregulate inflammatory and immune responses. 25,26

[0174] These findings suggest that after administration of high doses of JURV, the host immune response in the brain is well regulated and viral infection is resolved rapidly and efficiently. Surprisingly, we found that among the most upregulated proteins, IFIT3 (interferon-induced protein with tetratricopeptide repeats 3) 27,28 Both proteins, Stat1 (signal transducer and activator of transcription 1), have been shown to enhance immune responses; however, Lgals9 (soluble galactose-binding lectin 9) and HPx (hemopexin) act as suppressors of excessive inflammation and immune responses.29,30 In addition, Nenf (neurotrophic factor) plays a role in neuronal protection 31-33 The five most enriched signaling pathways predicted were PRPP biosynthesis, LXR / RXR activation, RH / H in influenza pathogenesis, FXR / RXR activation, and acute phase response signaling ( Fig.15 G).

[0175] Based on the changes in DEP expression used in the IPA analysis, we propose that activation of RH / H occurs initially in influenza pathogenesis and acute phase response signaling, leading to active PRPP biosynthesis that is critical for establishing immunity against the virus. In contrast, activation of LXR / RXR and FXR / RXR appears to indicate an initial response to restore homeostasis in infected mice. Similarly, the top 10 DEPs upregulated in the liver, such as Isg15 34 、Cmpk2 35 、Uba7 36 ) exhibits key cellular antiviral functions ( Fig.15 F). IPA analysis of DEP in JURV-infected liver ( Fig.15 H) also predicted activation of interferon signaling, mitochondrial dysfunction, and acute phase response signaling pathways (all of which are involved in the sensing and induction of innate and adaptive immune responses to viral infection). We found that administration of 1×10 7 TCID 50 The changes of DEP after JURV were similar to those of the 10-fold higher dose (1×10 8 TCID 50 ) Fig.16 A- Fig.16 D). In addition, analysis of the brain ( Fig.16 E) and liver ( Fig.16 The top 10 upregulated DEPs and predicted activation pathways (IPA) in G) are all immune responses to proteins and pathways related to viral infection ( Fig.16 G and Fig.16 H). In summary, these data strongly suggest that multiple mechanisms coexist to control and clear JURV infection and simultaneously prevent neuronal damage. These results show that the host cell apparatus senses JURV and multiple mechanisms coexist to control viral infection and clear the virus from the brain while preventing neuronal damage. In addition, it confirms that intranasal administration of high doses of JURV does not induce severe neurotoxicity or cause physical damage in these mice.

[0176] In vivo antitumor efficacy of JURV in a syngeneic HCC model

[0177] We first employed a subcutaneous syngeneic HCC model to evaluate the efficacy of JURV in combination with anti-PD-1 antibody compared with anti-PD-1 alone or JURV alone. Therapeutic efficacy was evaluated in Hepa 1-6 tumors transplanted into the right flank of immunocompetent mice. Extended dosing regimens, including intratumoral (IT) dosing of JURV (3 doses) for the group treated with JURV, and intraperitoneal dosing of anti-PD-1 (6 doses) for the immune checkpoint blockade group ( Fig.17 B). By day 28 after treatment, a significant (p<0.0001) delay in tumor growth was observed in JURV-treated mice compared to the control group (PBS) ( Fig.17 A). The anti-tumor effect of anti-PD-1 therapy was comparable to that of JURV (p<0.0001) ( Fig.17 A). However, we found that the tumor growth delay in mice treated with JURV in combination with anti-PD-1 antibody was not as significant as that in mice treated with JURV alone or anti-PD-1 alone (p<0.001). Further analysis of individual tumor volumes in mice showed that nearly all (>70%) mice in the JURV-treated group and the anti-PD-1-treated group eliminated their tumors compared to the JURV and anti-PD-1 antibody combination (>50%) group ( Fig.18 A).

[0178] We next sought to investigate whether local administration of JURV could induce systemic antitumor effects that could affect local and distant tumor development. We implanted HEPA 1-6 tumors subcutaneously in the left and right flanks of immunocompetent mice; however, we performed local IT injections of JURV only in the right flank, leaving the left side unaffected. Our data demonstrated that IT injection of JURV triggered antitumor activity that reduced the growth of HEPA 1-6 tumors in both left and right flanks ( Fig.17 C. Fig.18 B). In addition, we observed a dramatic decrease in tumor volume in the left flank of non-virus-vaccinated mice starting from day 18. These results are consistent with the induction of systemic antitumor activity commonly observed with oncolytic viruses in clinical trials and animal models. 3,37 We also noted that mice experienced a slight decrease in body weight one day after the last dose of JURV; however, the mice recovered rapidly and no adverse events were noted until the end of the study (Day 28). Fig.18 C). In addition, one mouse was found dead in the control (PBS) group, but there was no significant difference in survival between the different groups ( Fig.18 D and Fig.18E). Evaluation of serum levels of biomarkers associated with liver and kidney toxicity associated with viral treatment indicated that mice did not experience severe virus-induced toxicity. These preliminary studies highlight the potential of JURV to induce antitumor activity against local and distant non-injected tumors without causing deleterious effects in normal tissues, which is critical for targeting oligometastatic or metastatic disease.

[0179] In vivo antitumor efficacy of JURV in various mouse solid tumor models

[0180] To evaluate the efficacy of JURV-based therapies in other solid tumor models, we implanted several murine tumor cells in immunocompetent mice. These models included breast cancer (EMT6; Fig.19 A- Fig.19 B), colon cancer (CT26; Fig.19 E- Fig.19 F), reticulosarcoma (A20; Fig.19 G- Fig.19 H), breast cancer (B16-F10; Fig.19 I- Fig.19 J) and prostate cancer (RM-1; Fig.19 C- Fig.19 D) Once tumors reached a treatable size as described in the Methods section, mice were treated IT with either 50 μL PBS or 1×10 7 TCID 50 A single injection of JURV ( Fig.19 ). We found heterogeneity in the response to JURV in these models. Our data showed that JURV induced significant tumor growth delay in B16-F10 (p=0.02) and CT26 (p=0.03), but not in A20, EMT6, and RM-1 models ( Fig.19 E and Fig.19 I). Interestingly, JURV only provided a survival benefit for the RM-1 model ( Fig.19 D). However, due to the aggressive nature of these syngeneic tumor models, a large number of animals in the study reached tumor burden and had to be sacrificed before the end of the study. Overall, the mice tolerated the treatment well, and there was no drug-related toxicity in the study. These data confirm that JURV exerts antitumor activity in multiple tumor types; however, its variable activity suggests that combination therapy of JURV with other anticancer drugs may produce a durable therapeutic effect.

[0181] JURV combined with PD-1 blockade profoundly modulates immune components of the tumor microenvironment in mouse HCC

[0182] Many studies have demonstrated that vesicular viruses selectively infect, replicate, and lyse tumor cells and modulate local and systemic antitumor immune responses. 38 Given that JURV, anti-PD-1 antibody, or the combination of JURV and anti-PD-1 antibody induced significant tumor growth delay in a subcutaneous syngeneic HCC model, we set out to investigate and compare changes in immune responses associated with these different treatment regimens. As expected, our data showed dramatic changes in the frequency of tumor-infiltrating lymphocytes (TILs) following administration of these therapies to mice. Intratumoral administration of JURV significantly reduced (p<0.001) the F4 / 80-TIL subset ( TILs) compared to control (PBS), anti-PD-1, and bilateral flanks (left). Fig. 20 B) and increased (p<0.01) M2-like macrophages ( Fig.17 K), (p<0.01) natural killer (NK) cells ( Fig.17 L) and (p<0.01) natural killer T (NKT) cells ( Fig. 20 A) Distribution, indicating viral replication and phagocytosis of infected tumor cells. 39 Compared with PBS, JURV and bilateral flanks (left), treatment with anti-PD-1 antibody was associated with increased expression of cytotoxic CD8+ T cells (predominantly (p<0.01) CD8+Ki67+( Fig.17 D), (p<0.01)CD8+PD-1+( Fig.17 E) and (p<0.001) CD8+CD44+( Fig.17 F)) were associated with intratumoral accumulation of TIL macrophages (p<0.001) (anti-PD-1 vs. PBS) ( Fig.17 I), (p<0.01) M1 (anti-PD-1 vs. PBS) Fig.17 J) and (p<0.01) M2-like (anti-PD-1 vs. JURV) ( Fig.17 M2-like macrophages, together with myeloid cells, are known to disable anti-tumor immunity through the expression and interaction of PD-L1 and PD-1 on the surface of cytotoxic T cells. 40,41 These results suggest that responding CTLs in the anti-PD-1 group may be tumor-specific and contain effector memory cells in the tumor. 42 Furthermore, we found that combining JURV and anti-PD-1 antibody profoundly modulated the TME, as evidenced by the significant increase in CD4+ ( Fig.17 G), (p<0.01) F4 / 80-( Fig. 20 B), (p<0.01)CD4+PD-1+( Fig.17 H), (p<0.01)CD11b+( Fig. 20 D), (p<0.001) CD11b-( Fig. 20 E) and CD8+PD-1+(p<0.001)( Fig.17 E), (p<0.001) NK( Fig.17 No significant changes in serum IFN-β were observed in mice treated with JURV, anti-PD-1, and JURV combined with anti-PD-1 antibody and bilateral flanks ( Fig. 22 In conclusion, our data suggest that the combination of JURV and anti-PD-1 antibody induces antitumor immunity via recruitment and tumor infiltration of cytotoxic effector T (CTL) cells. 43,44 , thereby suppressing immunosuppression, a hallmark of a durable response to immunotherapy.

[0183] Multi-omics analysis identifies key molecular mechanisms of JURV's anti-tumor activity in vivo

[0184] To determine the effect of IT administration of JURV on tumor gene expression profiles, we examined the transcriptome of murine HCC tumors injected with three doses of JURV. Differentially expressed genes (DEGs) were analyzed using the limma-voom method. 45 Our data( Fig. 22 A and Fig. 22 B) shows that among 22,786 genes, 203 DEGs were up-regulated and 463 DEGs were down-regulated (2-fold change > 2, p-value < 0.055). Several DEGs among the 10 up-regulated DEGs (Myo3a 46 、Cd209c 47 、Trim67 48 、St8sia2 49 and Wnt5b 50 ) is related to immune response pathways ( Fig. 22 C). Many of the enriched cell signaling pathways identified by IPA analysis (such as B cell receptor signaling, IL-15 signaling, and phagosome formation) are associated with the activation of host innate and adaptive immune responses ( Fig. 22 D). In addition, to better understand the mechanism of JURV-induced antitumor activity, we analyzed DEPs and DEGs from transcriptomic and proteomic data. Among the relevant DEGs / DEPs, we identified the top 30 enriched features that were significantly up-regulated or down-regulated in the JURV group compared with the control group (PBS). Among the up-regulated features, S1pr3 has been identified in inflammation, host immune response against microorganisms (viruses, bacteria), and tumorigenesis. 51 、Tnpo1 52 、Psmb10 53 、Ddt 54、Ncor2 55 、Slc04c1 56 .

[0185] Likewise, our data showed that IP administration of anti-PD-1 antibodies was accompanied by significant transcriptional and proteomic changes in the TME, promoting antitumor immune responses. Indeed, we found that 860 DEGs were upregulated and 241 DEGs were downregulated out of 22,786 genes in HEPA 1-6 tumors ( Fig.23 A and Fig.23 B). All top 10 DEGs upregulated in the anti-PD-1 therapy group compared to the control (PBS) ( Fig.23 C) (including Prps1l1 57 、Cstdc4 58 and Ppbp 59 ) are involved in inflammation, adaptive immune response, and cell survival and proliferation. The most enriched pathways predicted by IPA analysis ( Fig.23 D) (i.e., phagosomal communication between innate and adaptive immune cells) is an immunity-related pathway. Comprehensive analysis of the relevant DEP / DEG signatures revealed Tmod3 60 、Nfkb2 61 、Dapk3 62 、Nipsnap3b 63 、Sart3 64 and Adgrl4 65 These studies reveal potential molecular mechanisms involved in the antitumor activity induced by JURV and / or anti-PD1.

[0186] Multi-omics analysis predicts that the combination of JURV and anti-PD-1 antibody effectively activates anti-tumor immunity

[0187] We analyzed the transcriptional profiles of mouse HCC to identify genes and pathways that were dysregulated in tumors after treatment with JURV, anti-PD-1 antibody, and the combination of JURV and anti-PD-1 antibody compared to control (PBS). We analyzed 22,786 genes between the control group (PBS) and the combination of JURV and anti-PD-1 antibody, and we found that 323 DEGs were upregulated, while 778 DEGs were downregulated ( Fig.24 A and Fig.24 B). The top 10 upregulated DEGs (including Cox20 66 、Dpf3 67 , Trp63 68 、Flg2 69 、Ush2a 70 and Cdh24 71) are effector genes associated with tumor suppression and immune cell infiltration mechanisms ( Fig.24 C). IPA analysis identified enriched pathways (i.e., liver fibrosis / hepatic stellate cell activation, oxytocin signaling, and calcium signaling pathways), which are mainly related to the activation and regulation of immune cells ( Fig.24 D). Integrative analysis of relevant DEPs / DEGs highlighted genes associated with susceptibility to immunotherapy, autophagy, tumor suppression, and immune response (such as Cdk5r1 72 、Ptgdr2 73 、Crip2 74 、Tardnp 75 In addition, the unique and common DEGs between PBS vs. JURV, PBS vs. anti-PD-1, and PBS vs. JURV+anti-PD-1 were examined ( Fig.25 ) revealed relatively little overlap among the commonly differentially expressed genes (both up-regulated and down-regulated) in the three groups, suggesting differences in the mechanisms of antitumor response to these therapies, which could be driven by differences in the types of TILs activated, recruited, and trafficked to tumor sites by these two treatment modalities or differences between the two delivery routes (IP and IT).

[0188] In addition, we performed gene ontology (GO) term enrichment analysis for 35 up- or down-regulated DEGs common to all three datasets (Table 3). Some mitogen-activated protein kinases (MAPKs) known to be up-regulated during viral infection 76 Pathway, macrophage migration inhibitory factor (MIF) involved in inflammation and immune response 77 , necroptosis signaling, and the vascular endothelial growth factor (VEGF) family were found to be enriched. These data suggest that although JURV and anti-PD-1 therapy display different mechanisms of antitumor activity, they activate important and complementary pathways involved in innate and adaptive antitumor immunity, leading to tumor growth control and regression.

[0189] Table 3. DEGs between PBS vs. JURV vs. PBS vs. anti-PD-1 vs. PBS vs. JURV+anti-PD-1 in murine HCC.

[0190]

[0191]

[0192] JURV induces antitumor immunity targeting local and distant tumors

[0193] Previous reports have demonstrated that oncolytic viruses can induce virus-mediated activation of local and systemic antitumor immunity by recruiting MHC class I-restricted virus-specific and tumor-specific CTLs.78,79 To better understand the mechanisms behind the abscopal effects observed with JURV on distant tumors, we analyzed the transcriptome and proteome of tumors (left flank) that were not injected with HEPA 1-6. Among the 21,260 analyzed genes, we found 1165 DEGs upregulated and 361 DEGs downregulated in the treated group compared to the PBS control group ( Fig.26 A and Fig.26 B) The top 10 up-regulated genes are listed in Table 4. Many of the most highly up-regulated genes, including Tent5b 80 、Per1 81,82 、Tubb4b-ps1 83 、Dbp 84,85 and Cry2 85,86 , which exerts important tumor suppressor activity in mammalian cells ( Fig.26 C). We also found that Fgfr2 was upregulated in our dataset. On one hand, overexpression of Fgfr2 and its fusion partners has been associated with several advanced cancers including HCC hepatobiliary carcinoma, making the Fgfr2 pathway an attractive target in liver cancer. 87-93 In contrast, the study showed that treatment with pazopanib, a multi-targeted receptor tyrosine kinase inhibitor, produced better outcomes in gastric tumors with Fgfr2 amplification. 90 The exact role of Fgfr2 in response to immunotherapy remains to be determined and warrants future study, especially in virotherapy.Cry2 is associated with better prognosis in ERC / HER2- tumors. 85,86 The efficacy of JURV in treating ERC / HER2- tumors also warrants future investigation. To our knowledge, the biological functions of the Gm6614, Rn7s2, and D930015M05Rik genes have not yet been defined (Table 4). IPA analysis allowed the identification of the most enriched canonical pathways in this dataset ( Fig.26 D) These enriched biological pathways include T helper cell differentiation, granulocyte adhesion, diapedesis, B cell signaling, Th1 and Th2 activation signaling, and communication signaling between innate and adaptive immune cells, indicating regulation of T helper cell pathways 94 Plays a crucial role in the abscopal effect induced by IT injection of JURV ( Fig.26 D) Comparison of DEP / DEG further confirmed that most of the upregulated features, including Anxa3 95 、Hspg2 96 、Cyp2e1 97 and Map1lc3a 98are therapeutic targets in immunotherapy or function as tumor suppressor genes. In addition, to evaluate the relationship between local and systemic antitumor immunity in murine tumors, we performed GO term enrichment analysis of DEGs between JURV-injected and non-injected HEPA 1-6 tumors. The most highly enriched canonical pathways identified by analyzing DEGs between JURV-injected and non-injected flanks (i.e., inhibition of ARE-mediated mRNA degradation, FAT10 signaling, EILF2 signaling) were associated with response to immunotherapy, activation of the MAPK pathway, and apoptosis ( Fig. 27 ). These results suggest that JURV can effectively elicit antitumor immunity against local and distant tumors in the HCC models studied.

[0194] Table 4. List of up-regulated and down-regulated DEGs in the bilateral flank.

[0195]

[0196]

[0197] IT administration of JURV mediates robust antitumor efficacy in the HEP3B xenograft model of HCC

[0198] We have previously demonstrated that the responsiveness of infected cancer cell lines to type I IFN production or viral kinetics in vitro does not consistently correlate with the in vivo efficacy of oncolytic agents. 7 To determine whether JURV can induce oncolysis-dependent tumor cell killing in vivo, we injected three doses of JURV IT into HEP3B xenografts. We used luciferase-labeled HEP3B cells to monitor tumor growth during the first three weeks of treatment. Bioluminescence imaging showed significant (p<0.0001) tumor inhibition in JURV-treated mice compared with the control group (PBS). Fig.28 A- Fig.28 C), which was visible from the first week after injection until the end of the study. In addition to luciferase activity, comparison of tumor volume between PBS and JURV also showed that JURV triggered a significant (p<0.0001) tumor growth delay ( Fig.28 D. Fig.29 A). We then performed proteomic analysis of tumor tissue to identify changes that occurred after IT delivery of JURV in HEP3B tumors. Analysis of a total of 2,088 proteins revealed that 860 DEPs were upregulated and 241 DEPs were downregulated in JURV-treated tumors relative to PBS ( Fig.28 E and Fig.28 F) Among the 10 most upregulated DEPs, we selected LCP1 99 、COL6A3 100 HSPG2101 、NAMPT 102 , STAT1 103 and VIM 104 Identified as a protein associated with mTORC2 / AKT pathway activation, cancer prognosis and treatment, tumor growth regulation, and cancer cell stiffness. Starting from day 19, the JURV group lost weight (about 15%) ( Fig.29 B) However, one mouse was found moribund on day 18, which we attribute to the severely immunodeficient nature of the NOD-SCID mouse model. 105 isolated adverse event, as no other mice experienced toxicity ( Fig.29 C) Our findings demonstrate that JURV efficiently infects and lyses human HCC cells in vivo in human HEP3B xenografts, further increasing enthusiasm for the use of new immunoviral therapies in human HCC.

[0199] discuss

[0200] Rhabdoviruses have several advantageous properties over other oncolytic viral vector platforms, including their amenability to genetic manipulation and the fact that they do not use humans as a natural host, which results in low seroprevalence in populations. 5 In addition to their episomal forms and rapid kinetics in tumor cells, most vesicular viruses encode large transgenes. 4 , while maintaining the ability to infect, replicate, and induce apoptosis in a large number of cancer cells.

[0201] In recent decades, vesicular stomatitis virus (VSV)-derived vectors (prototypical Rhabdoviridae) have been advanced to various stages of clinical testing against a variety of human cancers. Although multiple studies have confirmed the therapeutic efficacy of VSV-based vectors, barriers to FDA approval and clinical application remain. These barriers include several reports of VSV-induced neurotoxicity, hepatotoxicity, and rapid clearance by the host immune system. Our previous studies have described the antitumor potential of MORV. 7 , MORV is a non-VSV Rhabdoviridae virus with natural tumor selectivity and hypersensitivity to the type 1 interferon response, which is defective in 3 / 4 of all cancers. 6,7 To expand the oncolytic virus backbone spectrum, in this study we examined the potential anticancer effects of Jurona virus (JURV), a genetically distinct member of the same virus family, as a therapeutic agent for cancer.

[0202] We evaluated the antitumor effects of JURV in vitro in human and mouse hepatocellular carcinoma (HCC) cell lines and in HCC mouse models. JURV efficiently infected all HCC cells tested, with variability in cytolysis. Further investigation of the mechanisms of differential HCC killing in vitro may provide insights into the response to JURV-based therapies in HCC and potentially other solid tumors. Concerns about VSV-induced encephalitis and hepatotoxicity have prompted the development of attenuated recombinant VSV platforms using different viral engineering techniques. Although these vectors have shown improved safety to some extent, reduced oncolytic activity compared with the parental VSV has also been reported due to impaired intratumoral replication capacity.

[0203] In this study, we demonstrated that naturally attenuated JURV exhibits all the features required for potent immunoviral therapy, such as strong cytolytic effects and rapid replication cycles in tumor cells, sensitivity to type I IFN, and more importantly, no long-term neurotoxicity and hepatotoxicity in mice. Analysis of the toxicoproteome of brain tissue showed activation of multiple mechanisms involved in pathogen clearance and neuronal protection, which limited viral spread and prevented brain damage. In addition, our data confirmed that JURV stimulates multiple mechanisms of innate and adaptive immune responses, leading to tumor growth delay in isogenic HCC models. The combination of JURV and anti-PD-1 therapy significantly regulates TME by enhancing the infiltration of cytotoxic T cells via activation and recruitment of unique immune system effectors with complementary anti-tumor activity. In addition, we demonstrated that JURV effectively induces oncolytic-mediated tumor growth delay in human HCC xenografts. Analysis of mRNA and protein expression profiles in HCC tumors after administration of JURV, anti-PD-1 antibody, and the combination of JURV and anti-PD-1 therapy revealed upregulation of immune-related genes and identified enriched pathways involved in inflammation, immunosuppression regulation, angiogenesis, and antitumor immunity. Interestingly, we found that IT administration of JURV was associated with abscopal effects in bilateral murine HCC. To elucidate the mechanisms contributing to the abscopal effect, we employed a proteomic profiling approach on untreated tumors implanted at distant sites in animals. Our results showed that JURV triggered the activation of several tumor suppressor genes, suggesting that tumor suppressor pathways may play a key role in the abscopal effect in our animal model. In addition, our data suggest that cellular antitumor immunity via the activated T helper cell pathway may also be a key factor in the JURV-induced abscopal effect.

[0204] In conclusion, our results demonstrate that JURV potently infects HCC cells and induces oncolysis in vitro and in animal models. It also suggests that JURV-infected tumor cells elicit antitumor immunity (against both primary and distant tumors), which can be enhanced by the addition of anti-PD-1 antibodies.

[0205] method

[0206] Experimental design

[0207] These experiments were performed to provide new and critical mechanistic insights into the safety and efficacy of oncolytic JURV in HCC tumor models, which will enable the rational design of studies using JURV as monotherapy or in combination with other cancer therapies in early or advanced HCC to obtain long-term responses that may be additive or synergistic in the clinical setting. All animals were randomly assigned to different study groups in an open-label manner. The mean tumor volume (mm) per group at randomization was 3 )+SEM set at 80-120mm 3 Between. Using time, treatment effect and its interaction as independent variables, the relationship between tumor volume (or its logarithmic transformation) and time was evaluated by mixed linear regression model. We used random effects to explain the intra-subject correlation caused by repeated measurements. The slope was interpreted as the growth rate (or logarithm) of the tumor over time and compared between groups. The proportion of tumor-bearing mice that survived a specific time after treatment was identified using Kaplan-Meyer curve analysis. N values ​​and statistical methods are shown in the statistical analysis section.

[0208] Cell lines

[0209] A panel of three human hepatocellular carcinoma (HCC) cell lines (HEP3B, PLC, HuH7) and two murine HCC cell lines (HEPA 1-6, R1LWT) were used in this study. We also used several murine solid tumor cells, including breast cancer cells (EMT6), colon cancer cells (CT26), reticulosarcoma cells (A20), skin melanoma cells (B16-F10), and prostate cancer cells (RM-1). All cell lines were grown at 37°C, 5% CO2 in an RT-PCR solution supplemented with antibiotic reagents (100 μg ml -1 Penicillin and 100 μg ml -1Streptomycin). HEP3B, PLC, and HuH7 were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS). We maintained HEPA 1-6, RILWT, BHK-21 (baby hamster kidney fibroblasts), and Vero (African green monkey kidney) cells in DMEM containing 10% fetal bovine serum (FBS). BHK-21 and Vero cells were obtained from the American Type Culture Collection (Manassas, VA). We purchased HEP3B, PLC, HuH7, HEPA 1-6, EMT6, A20, CT26, BF16-F10, and RM-1 cells from the American Type Culture Collection (ATCC, Manassas, VA). The RILWT cell line was a gift from Dr. Dan G.Duda of MGH, Boston, MA.

[0210] Oncolytic viruses

[0211] We obtained Jurona virus (JURV) from the World Reference Center for Emerging Viruses and Arboviruses (WRCEVA) at the University of Texas Medical Branch (UTMB). A laboratory-adapted JURV viral clone was generated by serial plaque purification on Vero cells (ATCC, Manassas, VA). RNA sequencing was applied to confirm the full-length JURV genome (10,993 bp) as previously described. 4 As Lawson et al. 14 Infectious JURV was recovered from a full-length cDNA clone (Genscript, USA) containing genes encoding nucleoprotein (JURV-N), phosphoprotein (JURV-P), matrix protein (JURV-M), glycoprotein (JURV-G) and RNA-directed RNA polymerase L protein (JURV-L) as described above. Vesicular stomatitis virus (VSV) was rescued from the pXN2 cDNA plasmid and the virus stock was amplified on BHK-21 cells. Purified virus particles (VSV, MORV and recombinant JURV) were obtained using sucrose density gradient centrifugation prior to in vitro and in vivo studies.

[0212] Amplification of viral stock

[0213] Virus amplification was accomplished by infecting confluent (approximately 80%) Vero cells in T-175 flasks with JURV, MORV, or VSV at a low multiplicity of infection (MOI) of 0.001. Supernatants of virus-infected cells were collected from flasks forty-eight hours post-infection or when cytopathic effect (CPE) was observed. Virus stocks were purified using 10-40% sucrose density gradient ultracentrifugation followed by dialysis. Serial virus dilutions in BHK-21 cells were used for dialysis by Spearman-Kouycki. The algorithm determines the titer of each virus (TCID 50 ).

[0214] Cell viability assay

[0215] For all cytotoxicity assays (96-well format), 1.5 × 10 cells were infected with JURV, MORV, or VSV at the indicated MOIs of 10, 1, and 0.1 in serum-free Gibco Minimum Essential Medium (Opti-MEM). 4 Cell viability was determined using the Cell Titer 96 AQueous One Solution cell proliferation assay (Promega Corporation, Madison, Wisconsin, USA). Data were generated as the mean + / - SEM of six replicates from two independent experiments.

[0216] Crystal violet assay

[0217] Five hundred thousand cells were infected with oncolytic JURV at an MOI of 0.1 for 1 h in 6-well plates. The supernatant of virus-infected cells was removed, and the cells were washed with PBS and incubated at 37°C until analysis. 72 hours after infection, cells were fixed with 5% glutaraldehyde and stained with 0.1% crystal violet to visualize cell morphology and residual adhesion indicating cell viability. Photographs of representative areas were taken.

[0218] One-step virus growth kinetics

[0219] Two hundred thousand HCC cells were plated in 2 mL complete DMEM in each well of a 6-well plate. After overnight rest, we infected the cells with JURV at an MOI of 0.1 for 1 hour. The supernatant of the virus-infected cells was removed, and the cells were washed with PBS and fresh medium was added. At time points 10, 24, 48, and 72 hours, the supernatant was collected and stored at -80 °C. The virus titer (TCID 50 ). Data are generated as mean + / - SEM of two independent experiments.

[0220] Interferon sensitivity assay

[0221] HCC cells were cultured at 2.0×10 4The density of individual cells / well was seeded in 96-well plates and cultured overnight. 24 hours after infection, cells were pretreated with universal type I IFN-α of different concentrations and added directly to the culture medium. After overnight incubation, fresh culture medium containing universal type I IFN-α (Catalog No. 11105-1; PBL Assay Science, USA) was added, and cells were infected with JURV at an MOI of 0.01. Cell viability was assessed using Cell Titer 96AQueous One Solution cell proliferation assay (Promega, Madison, Wisconsin, USA). The absorbance measurements at 490nm were normalized to the maximum reading of each cell line, indicating 100% viability. Data from three independent experiments are shown. For all cell viability experiments, absorbance was read using Cytation 3 plate readers (BioTeK, Winooski, VT, USA). Data are expressed as mean + / - SEM of triplicates from three independent experiments.

[0222] Mouse

[0223] Female C57BL6 / J mice (strain number: 000664), BALB / cJ mice (strain number: 000651) and NOD.Cg-Prkdc scid / J mice (strain number: 001303) were purchased from Jackson Laboratories at 6-8 weeks of age. Male C57BL6 / J mice (strain number: 000664) were also obtained from Jackson Laboratories. All mice were housed in the Division of Laboratory Animal Medicine (DLAM) at the University of Arkansas for Medical Sciences (UAMS). DLAM has a full team of veterinarians and veterinary technicians who supervise and assist in animal care throughout the study. All animal studies were conducted in accordance with and approved by the Institutional Animal Care and Use Committee of the University of Arkansas for Medical Sciences.

[0224] Analysis of virus-induced adverse events in mice

[0225] Phosphate buffered saline (PBS), medium-high dose (1×10 7 TCID50 ) or high dose (1×10 8 TCID 50 ) virus. Body weight, temperature, behavior and clinical signs were monitored at least 3 times a week by a board-certified veterinarian to detect any signs of toxicity. However, three days after infection, three mice per group were sacrificed and tissues (blood, brain, liver and spleen) were collected to evaluate short-term toxicity and viral biodistribution. The remaining mice were monitored for thirty days.

[0226] Short-term toxicology analysis of blood components

[0227] Blood was collected from the submandibular vein (cheek bleed) and cardiac puncture on the 3rd day after treatment. Blood was collected in BD Microtainer tubes (Becton, Dickinson and Company, Franklin Lakes, New Jersey, USA) with ethylenediaminetetraacetic acid or lithium heparin for complete blood cell count (CBC), or BD Microtainer SST tubes (Becton, Dickinson, and Company) for serum analysis. CBC analysis was performed in Abaxis Piccolo Xpress chemistry analyzer (Abaxis, Union City, California, USA), and blood chemistry analysis was completed in VetScan HM5 hematology analyzer (Abaxis).

[0228] Toxicoproteomics analysis

[0229] Three days after JURV inoculation, mouse brain and liver tissues were harvested, dehydrated using increasing ethanol concentrations, and embedded in paraffin to become formalin-fixed paraffin-embedded (FFPE) blocks as previously described. 126 Tissue blocks were cut into 3-5 10 μm sections and subjected to the dewaxing procedure used for FFPE tissues. 127 Filter-aided sample preparation was used after deparaffinization of FFPE samples with xylene and tissue lysis in sodium dodecyl sulfate. 128Total protein was reduced, alkylated and digested with sequencing grade modified porcine trypsin (Promega). Tryptic peptides were separated by reverse phase XSelect CSHC18 2.5 μm resin (Waters) on an online 150×0.075 mm column using an UltiMate 3000RSLCnano system (Thermo). Peptides were eluted with a 60-minute gradient elution ratio of 98:2 to 65:35 (buffer A, 0.1% formic acid, 0.5% acetonitrile: buffer B, 0.1% formic acid, 99.9% acetonitrile). The eluted peptides were ionized by electrospray (2.4 kV) and then mass spectrometry (MS) analysis was performed on an Orbitrap Exploris 480 mass spectrometer (Thermo). MS data were collected in the range of 375 to 1500 m / z using a Fourier transform MS (FTMS) analyzer in profile mode at a resolution of 120,000. After HCD activation, MS / MS data were collected using an FTMS analyzer in centroid mode at a resolution of 15,000 and a normal mass range (normalized collision energy of 30%). Proteins were identified by database search using MaxQuant (Max Planck Institute) label-free quantification, with a parent ion tolerance of 2.5ppm and a fragment ion tolerance of 20ppm. Scaffold Q+S (ProteomeSoftware) was used to verify peptide and protein identification based on MS / MS. Protein identification was accepted if the error rate of protein identification was less than 1% and contained at least two identified peptides. Protein probability was assigned by the Protein Prophet algorithm. 129

[0230] In vivo efficacy of oncolytic JURV in a syngeneic mouse model of HCC

[0231] To evaluate the in vivo therapeutic efficacy of oncolytic JURV in a syngeneic mouse HCC model, 1 × 10 6 HEPA 1-6 cells were injected subcutaneously into the right flank of immunocompetent female C57BL6 / J mice (n=7 / group; Jackson Laboratory). Mice were monitored weekly for palpable tumors or any changes in appearance or behavior. When the average tumor reached a treatable size (80-120 mm 3 ), the mice were randomly divided into respective study groups and administered drugs within 24 hours of randomization. On days 0, 7, and 14, the mice were administered with PBS (control group) or 1×10 7 TCID 50The groups that received anti-PD-1 therapy or JURV + anti-PD-1 combination also received 50 μL of anti-PD-1 antibody intraperitoneally (IP) twice a week for three weeks. To establish isogenic bilateral (bilateral) HCC tumors, HEPA 1-6 cells (1×10 6 The mice were then implanted subcutaneously with HEPA1-6 tumor grafts (1×10 cells / mouse) into the right flank. These cells generated tumors within approximately 14 days and were classified as “primary” tumors. 6 Mice in the bilateral flank group received 1×10 7 TCID 50 50 μL IT injections of JURV were given once a week for three weeks. After randomization and initiation of treatment, tumor volume and body weight were measured twice a week using a digital caliper and a scale. Tumor volume was calculated using a digital caliper using the following formula: (longest diameter * shortest diameter2) / 2. Mice were monitored daily for signs of recovery during the first week of treatment and after each injection for up to 72 hours. When body weight loss exceeded 20%, when tumor size was greater than 2,000 m 3 Mice were euthanized when the first JURV dose was administered or when adverse effects of treatment occurred. Mice were sacrificed 28 days after the first JURV dose, at which time tumors and blood were collected for downstream analysis.

[0232] HEP3B xenograft model

[0233] For female NOD.Cg-Prkdc scid / J mice were subcutaneously inoculated with HEP3B cells labeled with firefly luciferase reporter gene on the right flank (n=6 / group). 3 The mice were given 1×10 7 TCID 50 JURV or 50 mL PBS in 50 μL IT injection for three weeks. Tumor volume was measured twice weekly until the end of the study (day 21), or humane endpoint as described above. We also recorded mouse body weights and clinical observations twice weekly.

[0234] Antitumor effects of JURV in various solid tumors

[0235] Female BALB / cJ mice were inoculated subcutaneously in the right flank with EMT6 (breast cancer), CT26 (colon cancer), and A20 (reticulosarcoma) cells (n=6-8 / group). B16F10 melanoma cells were implanted in the right flank of female C57BL6 / J mice. RM-1 (prostate cancer) cells were transplanted into the right flank of male C57BL6 / J mice. When the average tumor volume reached 80-120 mm 3 When 1×10 7 TCID 50 A single 50 μL IT injection of JURV or 50 μL PBS. Tumor volumes were measured twice weekly until the end of the study (day 21), or humane endpoint as described above. We also recorded mouse body weights and clinical observations twice weekly.

[0236] Bioluminescence imaging

[0237] Tumor-bearing (HEP3B) mice were anesthetized with isoflurane, and the changes in virus-induced tumor growth were imaged once a week (day 0, day 7, and day 14) using the IVIS Xenogen imaging system. Anesthesia was induced in an induction chamber (2-5% isoflurane), after which the mice were placed in an imaging instrument and equipped with a nose cone connected to a vaporizer to maintain isoflurane (1.5-2%) during the procedure. This concentration range produces an anesthesia level that prevents animals from moving during scanning. If the respiratory rate is accelerated or slowed, the isoflurane concentration is increased or decreased. We use heated animal beds, heating pads, and heating lamps when necessary to ensure that body temperature is maintained before imaging and during surgery. Each mouse received an intraperitoneal injection of D-luciferin (Sigma-Aldrich #L9504; 150 mg / kg body weight, a volume of 10 μl / g body weight, prepared in sterile water). The anesthetized mice were placed in a prone position in the IVIS Xenogen imaging system. The imaging time for each group of mice was less than 10 minutes. This is a non-invasive imaging procedure, and we do not need any restrictions.

[0238] Analysis of tumor-infiltrating immune cells

[0239] The PCR products were analyzed using gentleMACS according to the manufacturer's protocol. TM Hepa 1-6 tumors (n=3 samples / group) were excised and dissociated using the Mouse Tumor Dissociation Kit (Miltenyi, catalog number 130-096-730) in the Octo dissociator (Miltenyi). CD45 (TIL) microbeads (Miltenyi) were used to isolate CD45 +Cells. Cells were incubated with Fixable Viability Stain 510 for 15 minutes at 4°C, followed by incubation with anti-Fc blocking reagent (Biolegend, catalog number 101320) for 10 minutes before surface staining. Cells were stained and data were acquired using a BD LSRFortessa X-20 flow cytometer. All antibodies were used according to the manufacturer's recommendations. Fluorescence minus one controls were used to establish gating for each independent experiment. For intracellular staining of granzyme B, cells were stained using an intracellular staining kit (Miltenyi) and analyzed using FlowJo TM (TreeStar). Forward scatter and side scatter cytometry were used to exclude cell debris and doublets.

[0240] Flow cytometry antibody analysis

[0241] The following antibodies were used for flow cytometry analysis: CD45-FITC (Catalog No. 553079; BD Biosciences), CD3-BUV395 (Catalog No. 563565; BD Biosciences), CD4-BUV737 (Catalog No. 612761; BD Biosciences), CD8-Percp-Cy5.5 (Catalog No. 45-0081-82; eBioscience), CD44-BV711 (Catalog No. 103057; Biolegend), CD335-PE / Dazzle594 (Catalog No. 137630; Biolegend), PD-1-PE (Catalog No. 551892; BD Biosciences). Biosciences), Ki67*-BV605 (Catalog No. 652413; Biolegend), granzyme B*-APC (Catalog No. 366408; Biolegend), IFN-γ*-BV421 (Catalog No. 563376; BD Biosciences), CD11b-PE-Cy7 (Catalog No. 101216; Biolegend), F4 / 80-BV510 (Catalog No. 123135; Biolegend), CD206-AF700 (Catalog No. 141734; Biolegend), IA / IE-BV786 (Catalog No. 743875; BD Biosciences), and L / D-efluor780 (Catalog No. 65-0865-18; eBioscience). A complete list of the antibodies used can be found in Table 5

[0242] Table 5. Antibody list.

[0243]

[0244] RNA sequencing of murine HCC tumors

[0245] Hepa 1-6 (n=3 samples / group) FFPE scrolls were processed for DNA and RNA extraction using the Quick-DNA / RNA FFPE Miniprep Kit (Cat. No. R1009; Zymo Research) with on-column DNase digestion for RNA preparation. RNA mass concentration was assessed using the Qubit RNA Broad Range Assay Kit (Cat. No. Q10211; Invitrogen) with a Qubit 4 Fluorometer (Cat. No. Q33238; Invitrogen). RNA quality was assessed using the Standard Sensitivity RNA Analysis Kit (Cat. No. DNF-471-0500; Agilent) on a Fragment Analyzer System (Cat. No. M5310AA; Agilent). Sequencing libraries were prepared using TruSeq Stranded Total RNA Library Prep Gold (Cat. No. 20020599; Illumina). The RNADV200 score was used to determine the fragmentation time. The quality concentration of the library was assessed using the Qubit 1X dsDNA HS Assay Kit (Catalog No. Q33231; Invitrogen) with a Qubit 4 Fluorometer (Catalog No. Q33238; Invitrogen). The library fragment size was assessed using a High Sensitivity NGS Fragment Analysis Kit (Catalog No. DNF-474-0500; Agilent) on a fragment analyzer system (Catalog No. M5310AA; Agilent). The library was functionally validated using the KAPA Universal Library Quantification Kit (Catalog No. 07960140001; Roche). Sequencing was performed on a NovaSeq 6000 sequencing system (Illumina) with an S1 flow cell for 200 cycles to generate paired-end reads (2×100 bp).

[0246] Bioinformatics analysis

[0247] We examined the mRNA and protein expression profiles of Hepa 1-6 tumors treated with PBS, JURV, anti-PD-1, or JURV + anti-PD-1. Three replicates were used to analyze each of the untreated group (PBS) and the treated group. Tumor samples were sequenced on an NGS platform. Files containing sequencing reads (FASTQ) were then tested for quality control (QC) using MultiQC. 130 The Cutadapt tool trims low-quality bases from Illumina adapters and ends. After quality control, the HISAT2 aligner is used 131 Reads were aligned to the mouse reference genome (mm10 / GRCm38), and feature counts were used to count reads mapped to RefSeq genes. We used HTSeq-count to generate count matrices from sequence reads. 132 Genes with low counts in the samples affect the false discovery rate, thereby reducing the power to detect differentially expressed genes; therefore, before identifying differentially expressed genes, we used the limma-voom tool 45 The modules in filter out genes with low expression. Then, we normalize by using TMM 133 Counts were normalized, and TMM normalization is a weighted trimmed mean of the log expression ratio used to scale sample counts. Finally, we fit a linear model in limma to determine differentially expressed genes and present the data as mean ± standard error of the mean. All p-values ​​were corrected for multiple comparisons using the Benjamini-Hochberg FDR adjustment. After identifying differentially expressed genes, enriched pathways were performed using the Ingenuity Pathway Analyses tool to obtain biological insights. Statistical differences between groups were assessed using the nonparametric Mann-Whitney U test R module.

[0248] Integration of transcriptomics and proteomics

[0249] Two independent methods were used to integrate limma-normalized transcript expression levels and normalized protein intensities.First, the mixOmics package (Omics Data Integration Project R package, version 6.1.1) was implemented to generate heatmaps of correlated DEPs / DEGs as described previously. 134 Second, the MOGSA package was used to generate heat maps of the top 30 up- or down-regulated DEPs / DEGs among the groups. 135

[0250] Cytokines in blood chemistry

[0251] Blood chemistry analysis was performed using the Abaxis Piccolo Xpress chemistry analyzer (Abaxis) to assess liver toxicity (i.e., aspartate aminotransferase, alkaline phosphatase, albumin), renal toxicity (i.e., creatinine, blood urea nitrogen), and serum electrolytes. The murine type I interferon β assay was performed using the ELISA kit (Cat. No. ab252363; Abcam).

[0252] TUNEL assay immunohistochemistry

[0253] Tumor tissue sections were subjected to terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling (TUNEL) assay using the In Situ Cell Death Detection Kit (Roche Diagnostics, Indianapolis, IN) according to the manufacturer's protocol. After staining, cells were counterstained with 4',6-diamidino-2-phenylindole (DAPI) to visualize cell nuclei and stained with Antifade kit (Invitrogen, Carlsbad, CA) is sealed under the coverslip, and the Olympus IX-81 inverted microscope (Olympus America, Center Valley, PA) equipped with Hamamatsu ORCA-ER monochrome camera (Hamamatsu Photonics KK, Hamamatsu City, Japan) is used to collect images. Image analysis is performed using SlideBook 6.2 software. For quantification, 10 independent fields of view are collected in each well (each n), and the mean optical density (MOD) or co-localization area in pixels of the fluorescein (TUNEL) channel is recorded.

[0254] Statistical analysis

[0255] All values ​​are expressed as mean ± standard error of the mean, and the results were analyzed by one-way ANOVA followed by Tukey's test or Benjamini-Hochberg FDR adjustment for multiple comparisons and t-test for comparison of group means, and the Kaplan-Meier method for survival using statistical software in GraphPad Prism version 8 (GraphPad Software). A p value of less than 0.05 was considered statistically significant.

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Claims

1. A construct comprising a promoter operably linked to a polynucleotide encoding a full-length negative-sense Churona virus genome and allowing production of a negative-sense viral genome when transfected into mammalian cells.

2. The construct according to claim 1, wherein the promoter is a T7 promoter.

3. The construct according to claim 1 or 2, wherein the polynucleotide encoding the Churona virus genome comprises SEQ ID NO: 1-5.

4. A construct according to any one of the preceding claims, wherein the polynucleotide encoding the Churona virus genome further comprises a leading sequence of SEQ ID NO: 6 and / or a trailing sequence of SEQ ID NO:

7.

5. The construct according to any one of the preceding claims, wherein the polynucleotide encoding the Churona virus genome further comprises at least one of SEQ ID NOs: 8-11, 21 and 22 as an intergenic region.

6. The construct of claim 1 or 2, wherein the polynucleotide encoding the JURV genome comprises SEQ ID NO: 12 (JURV-XN-2) or a sequence having at least 95% identity to SEQ ID NO:

12.

7. The construct according to any one of claims 1-5, wherein the polynucleotide encoding the Churona virus genome further comprises a heterologous polynucleotide capable of encoding a polypeptide not naturally associated with Churona virus.

8. The construct of claim 7, wherein the polypeptide is a reporter polypeptide.

9. The construct of claim 8, wherein the reporter polypeptide is a fluorescent protein. 10 . The construct of claim 9 , wherein the polynucleotide comprises SEQ ID NO: 13 (JURV-eGFP) or a sequence having at least 95% identity to SEQ ID NO:

13.

11. A construct comprising a codon-optimized polynucleotide encoding at least one Churona virus protein selected from the group consisting of glycoprotein (G), nucleoprotein (N), phosphoprotein (P), RNA-guided RNA polymerase L protein (L) and matrix protein (M), wherein the polynucleotide is operably linked to a promoter for expression in mammalian cells.

12. The construct of claim 11, wherein the polynucleotide encodes the G protein and comprises SEQ ID NO:

4.

13. The construct of claim 11, wherein the polynucleotide encodes the N protein and comprises SEQ ID NO:

1.

14. The construct of claim 11, wherein the polynucleotide encodes the P protein and comprises SEQ ID NO:

2.

15. The construct of claim 11, wherein the polynucleotide encodes the M protein and comprises SEQ ID NO:

3.

16. The construct of claim 11, wherein the polynucleotide encodes the L protein and comprises SEQ ID NO:

5.

17. The construct according to any one of claims 11-16, wherein the construct is a plasmid.

18. A cell comprising at least one construct according to any one of claims 1 to 17.

19. The cell according to claim 18, wherein the cell comprises one of the constructs according to claims 1-10 and at least two of the constructs according to claims 11-17.

20. An infectious particle comprising a Churonavirus genome comprising SEQ ID NO: 12 or a negative sense RNA having 95% identity to SEQ ID NO:

12.

21. An infectious particle produced by transfecting a cell with a construct according to any one of claims 1 to 10.

22. A pharmaceutical composition comprising the infectious particle according to any one of claims 20-21 and a pharmaceutically acceptable carrier or excipient.

23. A method for treating a cell proliferative disease or disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a Churona virus or a composition according to claim 22 to treat the cell proliferative disease or disorder.

24. The method of claim 23, wherein the Churona virus comprises the nucleotide sequence of SEQ ID NO: 12 or a sequence having at least 95% identity to SEQ ID NO:

12.

25. The method of claim 23 or 24, wherein the cell proliferative disease or disorder is cancer.

26. The method of claim 25, wherein the cancer is selected from the group consisting of hepatocellular carcinoma, hepatocholangiocarcinoma, breast cancer, colorectal cancer, prostate cancer, and reticulosarcoma.

27. The method of claim 26, wherein the cancer is breast cancer.

28. The method of claim 27, wherein the breast cancer is HER2 negative.

29. The method of any one of claims 23-26, wherein the cancer is liver cancer.

30. The method of any one of claims 23-29, wherein the Churona virus or composition is administered intratumorally in a local tumor.

31. The method of any one of claims 23-30, wherein the cancer has metastasized.

32. The method of any one of claims 23-31, wherein the subject's immune system is suppressed.

33. The method of any one of claims 23-32, wherein the Churona virus or composition is administered intranasally, intramuscularly, intratumorally, intravenously, or subcutaneously.

34. The method of any one of claims 23-33, further comprising administering immunotherapy to the subject.

35. The method of claim 34, wherein the immunotherapy is a checkpoint inhibitor therapy.

36. The method of claim 35, wherein the checkpoint inhibitor therapy is selected from the group consisting of a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, and a LAG-3 inhibitor.

37. The method of any one of claims 23-36, further comprising administering an IFN-α inhibitor.

38. The method of any one of claims 23-37, further comprising administering a receptor tyrosine kinase inhibitor.

39. The method of claim 34, wherein the receptor tyrosine kinase inhibitor is pazopanib.

40. A method for producing a recombinant Jurona virus, comprising introducing at least one construct according to claims 1-10 into a cell; allowing the cell to express one or more Jurona virus proteins selected from the group consisting of G, M, N, L and P; incubating the cell to produce a recombinant Jurona virus; and harvesting the virus produced by the cell.

41. The method of claim 40, wherein the cell is a cell according to claim 18 or 19.

42. The method of claim 40 or 41, wherein the one or more Churona virus proteins include Churona virus N, P and L proteins.

43. The method of any one of claims 40-42, wherein the one or more Churona virus proteins are encoded by one or more polynucleotides comprising SEQ ID NO: 1, 2 and / or 5.

44. The method of any one of claims 40-43, wherein the promoter is a T7 promoter and the cell comprises a T7 RNA polymerase.

45. The method of any one of claims 40-44, wherein the cell is a BHK-21 cell, a Vero cell, or a HEK-293 cell.

46. ​​The method of any one of claims 40-45, wherein the construct further comprises a heterologous polynucleotide encoding a protein not naturally associated with Churona virus.

47. A system for generating a recombinant Churona virus, comprising: a) one or more constructs comprising polynucleotides encoding at least three Churona virus proteins selected from the group consisting of G, N, P, L and M, each of the polynucleotides being operably linked to a promoter to allow expression of the at least three proteins in mammalian cells; b) A construct comprising a polynucleotide encoding a negative-sense Churona virus genome operably linked to a promoter to allow production of the negative-sense Churona virus genome in mammalian cells.

48. The system of claim 47, further comprising: (c) a mammalian cell capable of expressing the Churona virus protein of (a) and the negative sense Churona virus genome of (b) to produce the recombinant Churona virus.

49. The system of claim 48, wherein the mammalian cell comprises T7 RNA polymerase.

50. The system of claim 48 or 49, wherein the mammalian cell is a BHK-1 cell.

51. The system of any one of claims 47-50, wherein the one or more vectors comprise polynucleotides encoding Churona virus N, P, and L proteins operably linked to a promoter.

52. The system of any one of claims 47-51, wherein at least one of the promoters is a T7 promoter.

53. The system of any one of claims 47-52, wherein the polynucleotides encoding the at least three Churona virus proteins are codon-optimized for expression in mammalian cells.

54. The system of claim 53, wherein the polynucleotide comprises at least one of SEQ ID NOs: 1-5.

55. The system of any one of claims 47-54, wherein the construct comprising the polynucleotide encoding the negative sense Churona virus genome is a construct according to any one of claims 1-10.

56. The system of any one of claims 47-55, wherein the one or more constructs encoding at least three Churona virus proteins comprise a construct of any one of claims 11-17.

57. A kit comprising at least one construct according to any one of claims 1-17.

58. The kit of claim 57, further comprising an immune checkpoint inhibitor selected from the group consisting of a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, and a LAG-3 inhibitor.

59. The kit according to any one of claims 57 or 58, further comprising an IFN-α inhibitor.

60. The kit of any one of claims 57-59, further comprising a receptor tyrosine kinase inhibitor.

61. The kit of claim 60, wherein the receptor tyrosine kinase inhibitor is pazopanib.

Images