Use of engineered Jurona virus (JURV) as a tumor-lytic virus platform against human cancer

Abstract

The present disclosure provides a composition comprising a recombinant polynucleotide encoding a Jurona virus, an infectious particle, a pharmaceutical composition, and a cell comprising the same, as well as methods and systems for producing a recombinant Jurona virus.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 344,395, filed May 20, 2022, the content of which is hereby incorporated by reference in its entirety. Statement Regarding Federally Sponsored Research This invention was made with government support under grant CA234324 awarded by the National Cancer Institute. The government has certain rights in this invention. Sequence Listing This application is accompanied by a sequence listing submitted as an ASCII text file named "169852_00112_Sequence_Listing.xml", size 110,996 bytes, created on May 3, 2023. The sequence listing is submitted electronically via EFS - Web with this application and is hereby incorporated by reference in its entirety into this specification.

Background Art

[0002] Oncolytic viruses are viruses that preferentially infect and kill cancer cells and represent a promising advance in cancer treatment. Currently, several viral platforms, including vesicular stomatitis virus (VSV), are being studied as oncolytic viruses. VSV is a widely studied vector and has advanced to early - stage clinical trials. However, concerns regarding hepatotoxicity and neurotoxicity may impede its clinical deployment for the treatment of human cancers. In recent years, other vesiculovirus vectors have also been developed, but most have not been able to achieve the efficacy levels of VSV in pre - clinical models of human cancer. Accordingly, there remains a need in the art for novel oncolytic viruses.

Summary of the Invention

[0003] In one aspect of the present disclosure, there are provided constructs comprising a promoter operably linked to a polynucleotide encoding a Jurona virus genome or a sense strand copy of the Jurona virus genome, and compositions comprising such constructs. The Jurona virus has a negative-sense single-stranded RNA genome, and the construct comprises a full-length antisense genome and, when transfected into mammalian cells, will enable the production of a negative-sense viral genome and the production of infectious virus. The polynucleotide encoding the Jurona virus genome may comprise SEQ ID NOs: 1-5, and optionally the leader sequence of SEQ ID NO: 6 and / or the trailer sequence of SEQ ID NO: 7. The polynucleotide encoding the Jurona virus genome may further comprise at least one of SEQ ID NOs: 8-11, 21, and 22 as an intergenic region. In some embodiments, the polynucleotide encoding the Jurona virus genome is or comprises SEQ ID NO: 12 (JURV-XN-2). The polynucleotide encoding the Jurona virus genome may further comprise 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 may be encoded by the polynucleotide of SEQ ID NO: 13 (JURV-eGFP). In another aspect of the disclosure, a further composition or construct is provided. A portion of the construct comprises a codon-optimized polynucleotide encoding at least one rhabdovirus protein selected from the group consisting of glycoprotein (G), nucleoprotein (N), phosphoprotein (P), RNA-directed RNA polymerase L protein (L), and matrix protein (M), operably linked to a promoter for expression in mammalian cells. The polynucleotide encoding the G protein comprises SEQ ID NO: 4, the polynucleotide encoding the N protein comprises SEQ ID NO: 1, the polynucleotide encoding the P protein comprises SEQ ID NO: 2, the polynucleotide encoding the M protein comprises SEQ ID NO: 3, the polynucleotide encoding the L protein comprises SEQ ID NO: 5, the composition may comprise a plasmid, and more than one polynucleotide encoding viral polypeptides may be encoded in a single construct. Such a construct may be contained in the genome of a cell, or may be transiently transfected into a cell to produce viral proteins and enable the packaging and production of infectious virus. Accordingly, cells containing the constructs described herein are also provided.

[0004] In another aspect of the disclosure, infectious particles are provided. The infectious particles may comprise a rhabdovirus genome comprising the negative-sense RNA of SEQ ID NO: 12. The infectious particles can be produced by the methods provided in the Examples, and recombinant infectious virus particles can be generated using the constructs and cells provided herein. In another aspect of the disclosure, additional infectious particles are provided. The infectious particles are produced by transfecting cells with a composition comprising a codon-optimized polynucleotide encoding at least one rhabdovirus protein selected from the group consisting of G, N, P, L, and M, optionally 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, the polynucleotide encoding the L protein may comprise SEQ ID NO: 5, and the composition may comprise a plasmid. Also, the cells are transfected with a construct encoding a negative-sense genomic copy of a rhabdovirus operably linked to a promoter capable of generating a genomic copy of the virus.

[0005] In another aspect of the disclosure, a pharmaceutical composition is provided. The pharmaceutical composition comprises an infectious particle comprising a negative-sense RNA of SEQ ID NO: 12 or a rhabdovirus genome having at least 95% sequence identity to SEQ ID NO: 12, and a pharmaceutically acceptable carrier or excipient. In some embodiments, the pharmaceutical composition comprises an infectious particle produced by transfecting cells with a composition comprising a codon-optimized polynucleotide encoding at least one rhabdovirus protein selected from the group consisting of G, N, P, L, and M, operably linked to a promoter for expression in mammalian cells, and a pharmaceutically acceptable carrier or excipient.

[0006] In other aspects of the present disclosure, methods are provided for treating a cell proliferative disease or disorder in a subject in need thereof. Such methods include administering to a subject a pharmaceutical composition comprising an infectious particle comprising a negative-sense RNA of SEQ ID NO: 12 or a Juno virus genome comprising a sequence having at least 95% identity to SEQ ID NO: 12, and a pharmaceutically acceptable carrier or excipient. The infectious particle is produced by transfecting cells with a composition comprising a codon-optimized polynucleotide encoding at least one Juno virus protein selected from the group consisting of G, N, P, L, and M, operably linked to a promoter for expression in mammalian cells, and a pharmaceutically acceptable carrier or excipient. The cell proliferative disease or disorder may be cancer, and may be selected from hepatocellular carcinoma, cholangiocarcinoma, breast cancer, colorectal cancer, prostate cancer, and reticulosarcoma. The breast cancer may be HER2-negative. The cancer may be local or metastatic. The subject may have a suppressed immune system. Such methods may further include 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 an inhibitor of PD-1, an inhibitor of PD-L1, an inhibitor of CTLA-4, and an inhibitor of LAG-3.

[0007] In another aspect of the disclosure, cells are provided. The cells are operably linked to a polynucleotide encoding a Zaire ebolavirus genome and include a promoter that enables the production of a negative-sense viral genome when transfected into mammalian cells. In some embodiments, the promoter is the T7 promoter. The cells may include a codon-optimized polynucleotide encoding at least one Zaire ebolavirus protein selected from the group consisting of G, N, P, L, and M, operably linked to a promoter for expression in mammalian cells. The cells may further or alternatively include infectious particles comprising a Zaire ebolavirus genome comprising the negative-sense RNA of SEQ ID NO: 12, and the infectious particles can be produced by transfecting the cells with a composition comprising a codon-optimized polynucleotide encoding at least one Zaire ebolavirus protein selected from the group consisting of G, N, P, L, and M, operably linked to a promoter for expression in mammalian cells, and a composition capable of encoding a full-length genome and producing negative-sense genomic RNA.

[0008] In another aspect of the present disclosure, a method for generating a recombinant rhabdovirus is provided. Such methods include a step of introducing into a cell at least one composition that comprises a promoter operably linked to a polynucleotide encoding a rhabdovirus genome and that enables the production of a negative-sense viral genome when transfected into a mammalian cell; a step of enabling the cell to express one or more rhabdovirus proteins selected from the group consisting of G, M, N, L, and P; a step of incubating the cell for a time sufficient to generate a recombinant rhabdovirus; and a step of recovering the virus produced by the cell. The cell may comprise a polynucleotide encoding a rhabdovirus genome operably linked to a promoter that enables the production of a negative-sense viral genome when transfected into a mammalian cell. The one or more rhabdovirus proteins may include the rhabdovirus N, P, and L proteins. The one or more rhabdovirus 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 comprise 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 further comprise a heterologous polynucleotide encoding a protein not naturally associated with the rhabdovirus.

[0009] In another aspect of the disclosure, a system for generating recombinant Junin virus is provided. Such a system comprises: a) one or more vectors comprising polynucleotides encoding at least three Junin virus proteins selected from the group consisting of G, N, P, L, and M, each operably linked to a promoter so as to be capable of expressing at least three proteins in mammalian cells; b) a vector comprising a polynucleotide encoding a negative-sense Junin virus genome, operably linked to a promoter so as to be capable of producing a negative-sense Junin virus genome in mammalian cells; and optionally (c) mammalian cells capable of expressing the Junin virus proteins of (a) and the negative-sense Junin virus genome of (b) to produce recombinant Junin virus. The cells may comprise T7 RNA polymerase, and / or at least one of the promoters may be a T7 promoter. In some embodiments, the cells are BHK-1 cells, Vero cells, or HEK-293 cells. The one or more vectors may comprise polynucleotides encoding at least the Junin virus N, P, and L proteins, operably linked to a promoter. The polynucleotides encoding at least three Junin 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 the negative-sense Junin virus genome described herein. The one or more vectors encoding at least three Junin virus proteins may comprise the compositions provided herein.

[0010] In other aspects of the disclosure, kits are provided. In some embodiments, the kit comprises a composition having a promoter operably linked to a polynucleotide encoding a Jurona virus genome, which enables the production of a negative-sense viral genome when transfected into mammalian cells. The promoter may be a T7 promoter. The polynucleotide encoding the Jurona virus genome may comprise at least one of SEQ ID NOs: 1-5, the leader sequence of SEQ ID NO: 6, the trailer sequence of SEQ ID NO: 7, and / or the intergenic regions of SEQ ID NOs: 8-11, 21, and 22. In some embodiments, the polynucleotide encoding the Jurona virus genome comprises SEQ ID NO: 12 (JURV-XN-2). The polynucleotide encoding the Jurona virus genome may further comprise a heterologous polynucleotide capable of encoding a polypeptide not naturally associated with the Jurona virus, and the polypeptide may be a reporter polypeptide that may be encoded by the polynucleotide of SEQ ID NO: 13 (JURV-eGFP). The kit may alternatively or additionally comprise any of the compositions or infectious particles disclosed herein. The kit may further comprise an immunotherapy that may be a checkpoint inhibitor therapy. The checkpoint inhibitor therapy may be selected from the group consisting of an inhibitor of PD-1, an inhibitor of PD-L1, an inhibitor of CTLA-4, and an inhibitor of LAG-3. The kit may further comprise a receptor tyrosine kinase inhibitor that may be an inhibitor of IFN-α and / or pazopanib.

Brief Description of the Drawings

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[0012] Hepatocellular carcinoma (HCC) is a major cause of cancer incidence and mortality worldwide. 1 Most HCC patients are diagnosed with advanced disease and have limited treatment options remaining. Current approaches for HCC patients who cannot undergo surgery or liver transplantation include cytotoxic therapies, targeted therapies, and immune checkpoint inhibitors. 23 However, long-term disease control is not achieved with these treatments, and HCC has become one of the cancers with the most unmet clinical needs worldwide. Oncolytic viruses (OVs) are powerful anticancer agents. They do not replicate in normal cells but preferentially amplify their genomes in tumor cells that cannot activate cell-based antiviral defense mechanisms. 6,7 Because OVs have multifaceted anticancer activities, including direct tumor cell killing ability and immunomodulatory properties, they are attracting increasing attention in immuno-oncology. 1,2 Among the reported OVs, members of the family Rhabdoviridae 2,3 have been intensively investigated for their potential applications as therapeutic agents for many years. The present invention relates to Jurovirus (JURV), a new member of the family Rhabdoviridae, in the treatment of HCC. 4,5 This example shows that JURV induces a potent cytolytic effect against HCC cell lysis in vitro and in animal models. Furthermore, JURV induces systemic antitumor immunity and results in tumor growth inhibition against both injected and non-injected tumors in an autologous HCC model. Additionally, the combination of JURV and immune checkpoint blockade antibodies significantly modulates the tumor microenvironment by promoting the activation of tumor-specific cytotoxic T cells. Such compelling data indicate that the JURV provided herein can be used as a novel oncolytic virus therapy platform against HCC and possibly other cancers as well.

[0013] The present invention provides compositions, constructs, infectious particles, pharmaceutical compositions, treatment methods, and systems related to the novel Jurona virus of the present disclosure and its use for treating cancer.

[0014] Compositions: Jurona virus (JURV) is non-pathogenic and is closely related to the Indiana strain of vesicular stomatitis virus (Figure 1A), but is genetically distinct. 1,2 The JURV genome is a 10,993 bp linear negative-sense RNA and has five specific vesiculovirus genes (in the 3' to 5' direction for the negative-sense RNA genome and thus 5' to 3' for the positive-sense complementary RNA or complementary DNA): matrix (M), nucleoprotein (N), phosphoprotein (P), glycoprotein (G), and polymerase (L). 1Refer to Figure 3A, which shows the schematic structure of the JURV genome from 3' to 5'. Similar to other negative-sense RNA viruses, such as vesicular stomatitis virus (VSV), influenza virus, Ebola virus, and rabies virus, the viral genome encodes an RNA-dependent RNA polymerase called the L protein in JURV. Therefore, the L protein can directly synthesize "mRNA" from the viral negative-sense RNA genome. Thus, it should be understood that each embodiment of the compositions of the present disclosure also contemplates complementary sense polynucleotides, such as complementary sense RNA to the polynucleotides of the present disclosure, single-stranded or double-stranded cDNA containing the polynucleotides of the present disclosure.

[0015] As used herein, "negative-sense RNA genome" refers to a single-stranded RNA of a virus having genetic content that is the antisense strand of viral mRNA, as understood in the art. In a more general sense, "negative-sense" may refer to being reverse complementary to both the positive-sense strand and RNA transcripts.

[0016] In a first aspect, the construct comprises a promoter operably linked to a polynucleotide encoding a full-length Junin virus genome. The construct is DNA, but the promoter is linked such that production of a negative-sense viral genome is possible when transfected into mammalian cells. The construct includes, but is not limited to, any composition containing DNA, such as plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, transposons, viral or viral vectors, recombinant chromosomal genomic DNA, or any other construct that can be used by one of ordinary skill in the art.

[0017] To generate functional viral particles, the construct comprises a polynucleotide encoding the 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 that is responsive to RNA polymerase derived from bacteriophage T7. As used herein, "leader sequence" refers to the region of a messenger RNA (mRNA) molecule that precedes the coding sequence of a gene, "trailer sequence" refers to the segment of the 3' end of the mRNA that follows the signal to terminate translation, which may be untranslated and may exclude the polyA tail, and "intergenic sequence" refers to the sequence of DNA located between genes. The construct may comprise a polynucleotide encoding a JURV N protein having at least 90% sequence identity with SEQ ID NO: 1. The construct may comprise a polynucleotide encoding a JURV P protein having at least 90% sequence identity with SEQ ID NO: 2. The construct may comprise a polynucleotide encoding a JURV M protein having at least 90% sequence identity with SEQ ID NO: 3. The construct may comprise a polynucleotide encoding a JURV G protein having at least 90% sequence identity with SEQ ID NO: 4. The construct may comprise a polynucleotide encoding a JURV L protein having at least 90% sequence identity with 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 therewith. As discussed above, the construct encoding a functional recombinant JURV must comprise a leader sequence and a trailer sequence that may have the sequences of SEQ ID NOs: 6 and 7, respectively. Exemplary intergenic regions include SEQ ID NOs: 8-11, which must be present in a specific order in the polynucleotide composition. Figure 3A shows a schematic diagram of the negative-sense JURV genome having polynucleotides encoding the JURV N, P, M, G, and L genes from 3' to 5'.Therefore, in the order from 3' to 5', the intergenic regions having SEQ ID NOs: 8-11 need to be present in the composition in the following arrangement: SEQ ID NO: 8 is between N and P, SEQ ID NO: 9 is between P and M, SEQ ID NO: 10 is between M and G, and SEQ ID NO: 11 is between G and L. However, it should be understood that the intergenic regions can be modified without significantly affecting the ability of the composition to be used for the generation of functional viruses, and such modifications are contemplated as part of the present disclosure. Additionally, it should be understood that the order of the JURV genes can be changed, which may result in a decrease in virus production.

[0018] The construct may also contain SEQ ID NO: 12, also called JURV-XN-2, which is a polynucleotide encoding the nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and RNA-directed RNA polymerase L protein (L), and was codon-optimized by the inventors for expression in mammalian cells from a laboratory-adapted JURV virus clone. In addition to the aforementioned modifications from wild-type JURV, the inventors incorporated an intergenic region derived from vesicular stomatitis virus into SEQ ID NO: 12.

[0019] The constructs of the present disclosure further comprise a heterologous polynucleotide. As used herein, "heterologous polynucleotide" refers to a polynucleotide that encodes a protein ("heterologous protein") that is not found in wild-type Juno virus (i.e., is non-native or "not naturally associated"). Suitable heterologous proteins include, but are not limited to, reporter proteins and antigenic proteins. A "reporter protein" can refer to a protein that is expressed when certain conditions are met (e.g., when a gene is expressed). An "antigenic protein" can refer to a protein that is recognized by the immune system. The reporter protein may be a fluorescent protein. As used herein, "fluorescent protein" refers to 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. An "antigenic protein" is a protein that can serve as an antigen (i.e., a substance that induces an immune response). Suitable antigen polypeptides include, but are not limited to, viral antigens, bacterial antigens, fungal antigens, parasitic antigens, and tumor-specific antigens. In an example, GFP was encoded in the recombinant viral genome as a heterologous protein. The composition comprises SEQ ID NO: 13 and a polynucleotide encoding JURV tagged with GFP.

[0020] The heterologous protein may be a viral antigen. Suitable viral antigens include proteins produced by viruses such as coronavirus, alphavirus, flavivirus, adenovirus, herpesvirus, poxvirus, parvovirus, reovirus, picornavirus, togavirus, orthomyxovirus, rhabdovirus, retrovirus, hepadnavirus, herpesvirus, rhinovirus, cytomegalovirus, Kaposi's sarcoma virus, human papillomavirus (HPV), human immunodeficiency virus (HIV), herpes simplex virus, herpesvirus 1, herpesvirus 2, herpesvirus 6, herpesvirus 7, herpesvirus 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, and Zika virus.

[0021] In another aspect of the present disclosure, there is provided a construct comprising a codon-optimized polynucleotide encoding at least one Ebola 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 may encode the N protein and may include SEQ ID NO: 1. The polynucleotide may encode the P protein and may include SEQ ID NO: 2. The polynucleotide may encode the M protein and may include SEQ ID NO: 3. The polynucleotide may encode the G protein and may include SEQ ID NO: 4. The polynucleotide may encode the L protein and may include SEQ ID NO: 5.

[0022] As used herein, the term "codon optimization" refers to a protein encoded by a nucleic acid triplet (i.e., a codon), wherein the composition of the codon has been improved based on various criteria without changing the amino acid sequence of the protein. Examples of such criteria include optimization of expression in the organism in which the protein is to be expressed (e.g., expression in mammalian cells). Nucleic acids generally refer to polymers that include nucleotides or nucleotide analogs joined together through backbone linkages such as, but not limited to, phosphodiester linkages. Examples of nucleic acids include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) such as viral genomic RNA, messenger RNA (mRNA), and transfer RNA (tRNA). Typically, polymeric nucleic acids, e.g., nucleic acid molecules containing three or more nucleotides, are linear molecules in which adjacent nucleotides are linked to each other via phosphodiester linkages. The term "nucleic acid" refers to individual nucleic acid residues (e.g., nucleotides and / or nucleosides). In some embodiments, "nucleic acid" refers to an oligonucleotide chain containing three or more individual nucleotide residues.

[0023] As used herein, the terms "oligonucleotide" and "polynucleotide" can be used interchangeably to refer to a polymer of nucleotides (e.g., a contiguous sequence of at least three nucleotides). In some embodiments, "nucleic acid" includes RNA as well as single-stranded and / or double-stranded DNA. Nucleic acids can be natural, for example, in the context of a genome, transcript, mRNA, tRNA, rRNA, siRNA, snRNA, plasmid, cosmid, chromosome, chromatid, or other native nucleic acid molecule. On the other hand, a nucleic acid molecule can be a non-natural molecule, such as recombinant DNA or RNA, artificial chromosome, engineered genome, or fragments thereof, or synthetic DNA, RNA, DNA / RNA hybrid, or can contain non-natural nucleotides or nucleosides. Further, the terms "nucleic acid", "DNA", "RNA", and / or similar terms include nucleic acid analogs, i.e., analogs having something other than a phosphodiester backbone. Nucleic acids can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. Where appropriate, for example, in the case of chemically synthesized molecules, nucleic acids can contain nucleoside analogs such as chemically modified bases or sugars, and analogs having backbone modifications. Nucleic acid sequences are presented in the 5' to 3' direction unless otherwise indicated.

[0024] As used herein, the terms "complementary" or "complementarity" are used with respect to "polynucleotides" and "oligonucleotides" (interchangeable terms that refer to sequences of nucleotides) that are related by base pairing rules. For example, the sequence "5'-C-A-G-T" is complementary to the sequence "5'-A-C-T-G". The nucleic acids, proteins, and / or other compositions described herein can be purified. As used herein, "purified" means separated from most of the other compounds or entities, and includes being partially purified or substantially purified. Purity can be expressed on a mass-to-mass scale and can be determined using various analytical techniques, including but not limited to mass spectrometry, HPLC, etc. As used herein, "operably linked" refers to the functional relationship between two or more nucleic acid (e.g., DNA) segments. Typically, this refers to the functional relationship between a transcriptional regulatory element (promoter) and the sequence being transcribed. For example, a promoter is operably linked to a coding sequence if it stimulates or modulates transcription of the coding sequence in an appropriate cell. Generally, a promoter transcriptional regulatory element that is operably linked to a sequence is physically adjacent to the sequence being transcribed, i.e., is cis-acting. However, some transcriptional regulatory elements, such as enhancers, need not be physically adjacent or even in close proximity to the coding sequence whose transcription they enhance. The terms "protein", "polypeptide", and "peptide" are used interchangeably herein to refer to a polymer of amino acids. A "protein" typically includes a polymer of natural 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).

[0025] Infectious particle: In another aspect of the disclosure, infectious particles are provided. The infectious particles are generated by transfecting cells with a composition, such as a construct provided herein, that is operably linked to a polynucleotide encoding a full-length antisense journavirus genome and includes a promoter that enables the production of a negative-sense virus genome. The cells may be mammalian cells, and if the cells also produce some of the necessary journavirus proteins, infectious particles will be generated. The cells may be engineered to produce the JURV proteins necessary for manufacturing journavirus infectious particles by transiently transfecting a construct that encodes and is capable of producing the necessary proteins along with a composition capable of generating the viral genome, or the cells may be stably engineered to produce the viral proteins. The proteins can be produced only after induction of an inducible promoter that drives the production of the viral proteins. As used herein, "infectious particle" refers to any particle capable of causing infection of an organism or cell. Exemplary infectious particles include, but are not limited to, virus particles or virions. As used herein, the terms "virus," "virus particle," and "virion" are used interchangeably.

[0026] An infectious particle will contain some of the viral proteins necessary to produce viral particles and viral genomes. The infectious particle may contain JURV proteins including the N, P, and / or L proteins. Also, the infectious particle may contain the N, P, M, and G proteins. Alternatively, the infectious particle may contain the N, P, M, L, and G proteins. Further, the infectious particle contains a negative-sense RNA genome encoding the mRNA of 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. The infectious particle may contain a negative-sense RNA genome capable of encoding all of SEQ ID NOs: 1-5. Also, the negative-sense RNA genome of the infectious particle may contain a leader sequence and a trailer sequence. The leader sequence and the trailer sequence may be encoded by cDNA sequences containing SEQ ID NOs: 6 and 7, respectively. The negative-sense RNA genome of the infectious particle may contain an intergenic region. Exemplary intergenic regions may be encoded by SEQ ID NOs: 8-11, and they need to be present in a specific order in the polynucleotide composition. Figure 3A shows a schematic diagram of a negative-sense JURV genome having a polynucleotide encoding the JURV N, P, M, G, and L genes from 3' to 5'. In the order from 3' to 5', when the intergenic regions having SEQ ID NOs: 8-11 are present in the infectious particle, they need to be present in the following arrangement: SEQ ID NO: 8 is between N and P, SEQ ID NO: 9 is between P and M, SEQ ID NO: 10 is between M and G, and SEQ ID NO: 11 is between G and L. The infectious particle may contain a negative-sense RNA of the polynucleotide of SEQ ID NO: 12.

[0027] The infectious particles of the present disclosure may further contain 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 contains the polynucleotide of SEQ ID NO: 13. The inclusion of the reporter protein in the virus enables the detection of infected cells. An antigen may be included, and infection by the virus or infectious particle enables the expression of the antigen in cells and the induction of an immune response against the antigen delivered together with the infectious particle. The infectious particles described herein contain a codon-optimized polynucleotide encoding at least one rhabdovirus 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 contain SEQ ID NO: 4, the polynucleotide encoding the N protein may contain SEQ ID NO: 1, the polynucleotide encoding the P protein may contain SEQ ID NO: 2, the polynucleotide encoding the M protein may contain SEQ ID NO: 3, and the polynucleotide encoding the L protein may contain SEQ ID NO: 5.

[0028] Pharmaceutical composition: In another aspect of the present disclosure, a pharmaceutical composition is provided. The pharmaceutical composition contains infectious particles containing a rhabdovirus genome. The rhabdovirus particles may be infectious and, when transfected into mammalian cells, can enable the further production of a negative-sense virus genome. The composition may further contain a pharmaceutically acceptable carrier. The infectious particles are the infectious particles described above and may contain at least the N, P, and L proteins of rhabdovirus, or alternatively at least the N, P, M, and G proteins, or alternatively the N, P, M, G, and L proteins of rhabdovirus. 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, acetate, phosphate), preservatives (e.g., thimerosal, benzyl alcohol, parabens), solubilizers (e.g., glycerol, polyethylene glycol), emulsifiers, liposomes, and nanoparticles. The pharmaceutically acceptable carrier may be an aqueous or non-aqueous solution, suspension, or emulsion. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and organic esters for injection such as ethyl oleate. Aqueous carriers include isotonic solutions, alcohol / aqueous solutions, emulsions, or suspensions, including saline and buffered media.

[0029] The pharmaceutical composition of the present invention may further contain additives such as albumin or gelatin, detergents (e.g., Tween20, Tween80, Pluronic F68, bile salts), antioxidants (e.g., ascorbic acid, sodium bisulfite), bulking agents, or tonicity modifiers (e.g., lactose, mannitol), etc. to prevent absorption on the surface. The components of the composition may be covalently attached to a polymer (e.g., polyethylene glycol), form a complex with metal ions, or be incorporated within or onto the particle preparations of polymer compounds (e.g., polylactic acid, polyglycolic acid, hydrogels, etc.), or liposomes, microemulsions, micelles, multilamellar, or multiple membrane vesicles, erythrocyte ghosts, or spheroplasts. The composition can also be formulated in a lipophilic depot (e.g., fatty acids, waxes, oils) for controlled or sustained release.

[0030] In addition, the pharmaceutical composition may contain an adjuvant for increasing 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, and zirconium. Exemplary gel-based adjuvants include aluminum gel-based adjuvants and acemannan. Exemplary carbohydrate adjuvants include inulin-derived adjuvants (e.g., gamma inulin, algammulin), 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. Also, suitable adjuvants include, but are not limited to, any FDA-approved adjuvants including aluminum salts (alum) and squalene oil-in-water emulsion-based MF59 (Wadman 2005 (Novartis)) and AS03 (GlaxoSmithKline).

[0031] Method for treating a cell proliferative disease or disorder: As shown in the examples, administration of Jurona virus (JURV) to non-tumor-bearing mice did not cause a significant change in body weight, caused a slight reduction in lymphocyte count, and caused little change in monocyte and neutrophil counts in the case of intranasal administration (Figure 4A) or intravenous administration (Figure 4B) (Figures 4F-4H). Therefore, JURV is non-pathogenic. In the examples provided herein, JURV was able to reduce tumor volume and extend the survival time of tumor-bearing mice. Administration of JURV reduced the tumor volume of mice bearing Hepa1-6 (mouse hepatoma) tumors and RM-1 (prostate cancer) tumors, as shown in FIG. 5, and extended the survival time of mice bearing EMT-6 (breast cancer) tumors, CT26 (mouse colorectal cancer) tumors, and RM-1 tumors. Further, FIG. 6 shows that mice transplanted with HepC3 (hepatocellular carcinoma) cells showed a significant reduction in tumor volume compared to control-treated animals after JURV treatment.

[0032] Accordingly, a method for treating a cell proliferative disease or disorder is provided. As used herein, "cell proliferative disease or disorder" is any disease or disorder characterized by uncontrolled or abnormal cell proliferation or division. Exemplary cell proliferative diseases and disorders include, but are not limited to, cancer, carcinoma in situ, lymphoproliferative disorders such as chronic lymphocytic leukemia, and myeloproliferative disorders such as polycythemia vera. Such methods include administering to a subject a pharmaceutical composition comprising an infectious particle comprising a jurona virus genome and a pharmaceutically acceptable carrier for treating a cell proliferative disease or disorder. The cell proliferative disease or disorder may be cancer, and may be selected from hepatocellular carcinoma, cholangiocarcinoma, breast cancer, colorectal cancer, prostate cancer, and sarcoma. The breast cancer may be HER2 negative. The cancer may be local or metastatic. "Local" cancer can refer to a cancer that has not spread from its original (primary) site within the subject's body. "Metastatic" cancer can refer to a cancer that has spread from its original (primary) site within the subject's body to another (secondary) site within the subject's body. The subject may have a suppressed immune system. A suppressed immune system can be identified or determined by means known in the art and may include identifying a reduction in white blood cells, monocytes, lymphocytes, neutrophils, and / or other immune cells.

[0033] The pharmaceutical composition may be administered to a subject in combination with another agent having similar or different biological activities. For example, such a method may further comprise administering an immunotherapy to the subject before, simultaneously with, or after administration of infectious particles comprising a Jurona virus genome or other composition. The immunotherapy may be checkpoint inhibitor therapy. The checkpoint inhibitor therapy may be selected from the group consisting of inhibitors of PD-1, inhibitors of PD-L1, inhibitors of CTLA-4, and inhibitors of LAG-3 (CD223). The 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. Monoclonal antibodies specific for CTLA4, such as oncolytic adenovirus vectors encoding monoclonal antibodies specific for CTLA4 (or antibodies encoded thereby), can be used. Such a method may further comprise administering an inhibitor of IFN-α to the subject before, simultaneously with, or after administration of infectious particles comprising a Jurona virus genome or other composition. Such a method may further comprise administering a receptor tyrosine kinase inhibitor (such as pazopanib) to the subject before, simultaneously with, or after administration of infectious particles comprising a Jurona virus genome or other composition.

[0034] Such methods include administering to a subject a therapeutically effective amount of a pharmaceutical composition comprising Juno virus-infected particles. As used herein, the term "therapeutically effective amount" refers to the amount of viral particles or pharmaceutical formulation sufficient to reduce one or more signs or symptoms of a cell proliferative disease or disorder in a subject. Exemplary signs or symptoms of cell proliferative diseases that can be "treated" or "reduced" by the methods of the present disclosure include, but are not limited to, reduction of tumor volume, remission of disease, cure of disease, reduction of the number of tumors, weight gain, increased appetite, and the like. In addition, for each type of cell proliferative disease treated by the methods of the present disclosure, there is a disease-specific outcome that represents an effective treatment. For example, in the case of hepatocellular carcinoma, a patient being treated by the present method may experience increased appetite, disappearance of pain or fullness under the right rib of the body, reduction of nausea or vomiting, and remission of jaundice, among others. Other disease-specific signs and symptoms that can be treated or reduced by the present method are well known in the art.

[0035] As used herein, the terms "administering" and "administration" refer to any method for providing a pharmaceutical preparation 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 can be administered intranasally, intramuscularly, or intratumorally. The pharmaceutical composition can be administered as a single dose or multiple doses. For example, the pharmaceutical composition can be administered more than once at intervals of 4 hours, 6 hours, 8 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 1 week, 2 weeks, or more than 3 weeks. For example, in the examples, the viral particles were administered intratumorally once a week for 3 weeks. The dose for each mouse in each administration was 1×10 7 TCID 50 . One of ordinary skill in the art will be able to calculate the dosage for administration depending on the tumor to be treated and the subject. Thus, in some embodiments, the viral particles are administered to the subject at least twice. The "subject" to which the present method is applied may be any vertebrate. Suitable vertebrates include, but are not limited to, humans, cows, horses, sheep, pigs, goats, rabbits, dogs, cats, bats, mice, and rats. In certain embodiments, such methods may be performed on laboratory animals (e.g., mice and rats) for research purposes. In other embodiments, such methods are used to treat commercially important livestock (e.g., cows, horses, pigs, rabbits, goats, sheep, and chickens) or companion animals (e.g., cats and dogs). In a preferred embodiment, the subject is a human.

[0036] Cell: In another aspect of the present disclosure, cells are provided. The cells include a promoter operably linked to a polynucleotide encoding a Juno virus genome, enabling the production of a negative-sense virus genome. The cells may further include a construct for producing the viral proteins necessary to enable the assembly of infectious particles, or may be genetically engineered as such. The cells may be engineered to produce the viral proteins necessary for the assembly of infectious particles after induction. Those skilled in the art will understand that such cells may be engineered by the stable integration of a construct comprising an inducible promoter operably linked to a polynucleotide encoding the necessary Juno virus protein selected from the group consisting of the N, P, M, G, and L proteins of the Juno virus, provided herein as SEQ ID NOs: 1-5, respectively. Alternatively, the cells may be transfected with one or more plasmids to enable the production of the necessary proteins.

[0037] In some embodiments, the cell comprises SEQ ID NO: 12, also referred to as JURV-XN-2, a polynucleotide encoding the negative-sense RNA genome of the codon-optimized mammalian cell-adapted Junin virus 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-directed RNA polymerase L protein (L), which have been codon-optimized for expression in mammalian cells from a laboratory-adapted JURV virus clone. In addition to the above-described modifications from wild-type JURV, the inventors incorporated an intergenic region derived from vesicular stomatitis virus into SEQ ID NO: 12. The composition may further comprise a polynucleotide encoding a heterologous protein, the heterologous protein may be GFP, and the composition may comprise the sequence of SEQ ID NO: 13. Also included are sequences having at least 95%, 96%, 97%, 98%, and 99% identity to SEQ ID NO: 12 or SEQ ID NO: 13.

[0038] Method for producing virus particles: The present invention also provides a method for producing the virus particles described herein. The inventors have found that cells expressing an exogenous polymerase can be transfected with a polynucleotide encoding the full-length JURV genome, i.e., the M, P, L, G, and N proteins, the leader and trailer sequences, and the intergenic regions, and an additional promoter operably linked to one or more polynucleotides encoding JURV proteins, such that production of JURV in mammalian cell culture can be effected. See, for example, FIG. 2. Thus, when the polynucleotide is transfected into cells having an exogenous polymerase, e.g., T7 polymerase transcribes important JURV transcripts that are translated into the functional proteins necessary for the efficient assembly of virus particles. The polynucleotide encoding the complete JURV genome and the additional promoter operably linked to one or more polynucleotides encoding JURV proteins necessary for JURV assembly and functional JURV virus particle formation may be encoded by a single polynucleotide. In other embodiments, the complete JURV genome may be encoded by one polynucleotide, and the polynucleotides encoding one or more JURV proteins necessary for virus assembly may be encoded by one or more separate polynucleotide molecules. Importantly, for virus assembly to occur in the cells, the complete JURV genome must be present together with one or more JURV proteins selected from the M, P, L, G, and N proteins encoded by one or more polynucleotides operably linked to a promoter.

[0039] Accordingly, in some embodiments, such methods include introducing a composition comprising a promoter operably linked to a polynucleotide encoding a Junin virus genome, which, when transfected into mammalian cells, enables the production of a negative-sense viral genome; enabling the cell to express one or more Junin virus proteins selected from the group consisting of G, M, N, L, and P; incubating the cell for a time sufficient to generate recombinant Junin virus; and recovering the virus produced by the cell. One or more JURV proteins required for infectious particle production may include the N, P, and L proteins. The one or more Junin 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 comprise T7 polymerase.

[0040] The polynucleotide encoding the Junin virus genome may additionally comprise a heterologous polynucleotide encoding a protein not naturally associated with Junin virus. The heterologous polynucleotide may encode an antigen or a reporter protein, as described above. As used herein, the terms "transfecting" and "transfection" refer to the process of artificially introducing a nucleic acid (DNA or RNA) into a cell. Transfection can be carried out under natural or artificial conditions. Suitable transfection methods include, but are not limited to, lipofection, bacteriophage or virus infection, electroporation, heat shock, microinjection, and particle bombardment. As used herein, the terms "infecting" and "infection" refer to the process of introducing a virus into a cell. A cell may be infected with a virus by simply contacting the cell with viral particles.

[0041] The cell line used in this method is a eukaryotic cell line. Suitable eukaryotic cells include, but are not limited to, mammalian cells or chicken cells. The cells may be cells that are being cultured. Suitable mammalian cells include, but are not limited to, BHK-21 cells, MDCK cells, A549 cells, CHO cells, HEK293 cells, HEK293T cells, HeLa cells, NS0 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 cells are BHK-21 cells and can express T7 polymerase. The method for producing viral particles may further include an additional step of recovering the jourona virus from the cells. In embodiments using cultured cells, such methods may further include, for example, the step of recovering the supernatant of the culture by centrifugation or pipetting. The jourona virus recovered from the cells can be further isolated or purified from the cells and the medium by methods known to those skilled in the art, such as density gradient centrifugation.

[0042] System for generating recombinant jourona virus: Systems are also provided for generating recombinant Juno virus. Such systems include: a) one or more vectors comprising polynucleotides encoding at least three Juno virus proteins selected from the group consisting of G, N, P, L, and M, each operably linked to a promoter such that at least three proteins are capable of being expressed in mammalian cells; b) a vector comprising a polynucleotide comprising a negative-sense Juno virus genome operably linked to a promoter such that a negative-sense Juno virus genome is capable of being produced in mammalian cells. Accordingly, the systems of the present disclosure enable efficient production of recombinant JURV and may further comprise mammalian cells that enable expression of the Juno virus proteins of (a) and the negative-sense Juno virus genome of step (b) to produce recombinant Juno virus. The cells of the system may comprise T7 RNA polymerase. The cells may be BHK-21 cells. The one or more vectors comprise polynucleotides encoding Juno virus N, P, and L proteins operably linked to a promoter. The polynucleotide may comprise any one of SEQ ID NOs: 1-5. The one or more vectors may comprise a codon-optimized polynucleotide encoding at least one Juno 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 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. The one or more vectors comprise a promoter operably linked to a polynucleotide encoding a Juno virus genome and a polynucleotide that enables production of a negative-sense virus genome when transfected into mammalian cells.

[0043] Kit for treating a cell proliferative disease or disorder: A kit is provided. The kit includes an infectious particle containing a Jurovirus genome and a pharmaceutical composition containing an optional pharmaceutically acceptable carrier. The kit may further include immunotherapy. The immunotherapy may be checkpoint inhibitor therapy. The checkpoint inhibitor therapy can be selected from the group consisting of an inhibitor of PD-1, an inhibitor of PD-L1, an inhibitor of CTLA-4, and an inhibitor of LAG-3 (CD223). The 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, a tumor-lytic adenovirus vector encoding a monoclonal antibody specific for CTLA4, such as a human monoclonal antibody specific for CTLA4 (or the antibody encoded thereby), can be used. The kit may further include an inhibitor of IFN-α. The kit may further include a receptor tyrosine kinase inhibitor (such as pazopanib).

[0044] The present disclosure is not limited to the specific details of the structures, arrangements of components, or method steps shown herein. The compositions and methods disclosed herein can be made, implemented, used, practiced, and / or formed in various ways that will be apparent to those skilled in the art in light of the following disclosure. The expressions and terms used herein are for illustrative purposes only and should not be regarded as limiting the claims. In this specification and the claims, the ordinal indicators such as first, second, third, etc. used to refer to various structures or method steps are not intended to be construed as indicating any particular structure or step or any particular order or configuration of such structures or steps. All of the methods described herein can be performed in any suitable order, unless otherwise indicated herein or otherwise clearly inconsistent with the context. The use of any and all examples or exemplary language (e.g., "etc.") provided herein is intended merely to facilitate the disclosure and does not suggest any limitation to the scope of the disclosure unless otherwise claimed. Neither the language in this specification nor the structures shown in the drawings should be construed as indicating that any non-claimed element is essential to the practice of the subject matter of the disclosure. The use of the terms "including", "comprising", or "having" and variations thereof in this specification is intended to include the recited elements and their equivalents, as well as additional elements. Embodiments described as "including", "comprising", or "having" a particular element are also contemplated as "consisting essentially of" and "consisting of" such a particular element.

[0045] The recitation of a range of values herein is merely intended to serve as a convenient method for referring individually to each separate value falling within the range, unless otherwise specified herein. Each separate value is incorporated herein as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, values such as 2% to 40%, 10% to 30%, or 1% to 3% are intended to be explicitly recited herein. These are merely examples of what is specifically intended, and all possible combinations of numerical values between them, including the recited minimum and maximum values, should be considered to be explicitly stated in this disclosure. The use of the word "about" to describe a particular recited amount or range of amounts is intended to refer to values that are very close to the recited amount, taking into account manufacturing tolerances, equipment and human error in making measurements, and the like, or values that would be naturally considered to be included in the recited amount. All percentages referring to amounts are by mass unless otherwise indicated.

[0046] Any reference, including any non-patent or patent document cited herein, is not to be taken as an admission that it constitutes prior art. In particular, it will be understood that any reference to a document herein does not constitute an admission that such a document forms part of the common general knowledge in the art in the United States or any other country, unless otherwise stated. Any discussion of a reference is to state what the authors have asserted, and the applicants reserve the right to question the accuracy and validity of any of the documents cited herein. All references cited herein are incorporated by reference in their entirety, unless otherwise expressly indicated. In the event of any inconsistency between any definition and / or description in the cited document, the present disclosure shall prevail. The following examples are intended to be illustrative only and are not intended to be limiting of the invention or the scope of the appended claims.

Examples

[0047] The following examples demonstrate the generation of recombinant Jurona virus and its use in the treatment of cell proliferative diseases. Example 1: Characterization of Jurona virus (JURV) and its cytopathic effect in tumor cells Oncolytic viruses provide multimodal antitumor activities ranging from selective tumor cell death to promotion of systemic antitumor immunity, and can make them formidable adversaries 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 in humans. In this example, the inventors characterized JURV and demonstrated its cytopathic effect in tumor cells. Results Characterization and generation of oncolytic recombinant Jurona virus (JURV) Jurona virus (JURV) is non-pathogenic and is closely related to the Indiana strain of vesicular stomatitis virus, but is genetically distinct (Figure 1). 1、2 . JURV, which belongs to the order Mononegavirales, family Rhabdoviridae, and genus Vesiculovirus (composed of approximately 5,000 members), has an envelope, is bullet-shaped, and is approximately 180 nm in length and 75 nm in width (Figure 1A). Vesicular stomatitis virus (the prototype Vesiculovirus) has a negative-strand RNA linear genome of approximately 11 kb in size and encodes five proteins (Figure 1B). JURV was trained to selectively infect tumor cells using the method described in Figure 2. Figure 2 shows a schematic diagram of the method used to derive a laboratory-adapted Jurona virus clone (Figure 2A), and the process of developing a reverse genetics-based method for generating recombinant Jurona virus (Figure 2B). The JURV genome is a 10,993-bp linear RNA and has five specific Vesiculovirus genes (in the 3' to 5' direction): nucleoprotein (JURV-N), phosphoprotein (JURV-P), matrix (JURV-M), glycoprotein (JURV-G), and polymerase (JURV-L) (Figure 3A). 1There is a highly conserved intergenic region containing SEQ ID NO: 11 between the JURV-M region and the JURV-G region.

[0048] JURV induced a robust cytopathic effect (CPE) in tumor cells To evaluate the cytotoxicity of JURV in vitro, the inventors performed a CPE assay on three human HCC cells (Hep3B, PLC, Huh7) and two mouse HCC cells (Hepa1-6, RILWT) (FIGS. 3B-3E). The inventors infected monolayers of human and mouse liver cancer cells with JURV, VSV, and MORV at MOIs of 10, 1, and 0.1, respectively, and measured cell viability 72 hours after infection. JURV, MORV, and VSV were able to lyse cancer cells and showed relatively similar lytic activities in the tested cancer cell lines (FIG. 3B). These results indicate that JURV can selectively infect and lyse tumor cells in vitro without affecting normal cells, but that the infectivity of JURV is similar to that of VSV and MORV, and it can grow at a relatively high titer and infect Vero cells (FIG. 3E).

[0049] High-dose intranasal administration of JURV is not associated with neurotoxicity or hepatotoxicity To determine whether JURV has a causal relationship with brain damage and neurotoxicity in an animal model, immunocompetent mice were inoculated with JURV (low dose: 1×10 7 TCID 50 ; high dose: 1×10 8 TCID 50Two intranasal (IN) and intravenous (IV) administrations of 8 TCID 50 ) were given (Figure 4). Animals in the control group were treated with phosphate-buffered saline (PBS). To evaluate short-term toxicity, three mice per group were sacrificed three days after infection, and blood, brain, liver, and spleen tissues were collected for further analysis. The remaining animals were monitored for signs of toxicity by a board-certified veterinarian for 45 days. We found that the low and high IN and IV doses of JURV had good tolerability and manageability in all groups (Figures 4A–4D). However, it is important to note that in this study, we used a laboratory-adapted attenuated strain (serial passage) of JURV, which is known to be significantly attenuated compared to the wild-type virus. In the JURV groups, weight loss three days after treatment was not statistically significant, but weight consistently increased from seven days after IN and IV administrations until the end of the study. Consistent with minimal effects on body weight, thorough pathological examination of brain and liver sections from mice treated with the highest IN and IV doses of virus (1 × 10 8 TCID 50 ) showed that JURV did not cause obvious signs of toxicity (Figures 4B, 4C). Analysis of complete blood counts three days after treatment with JURV revealed a decrease in the numbers of white blood cells, lymphocytes, granulocytes, and platelets (Figures 4E–4H). In addition to virus-mediated hematological changes, these changes in blood cell counts may have been caused by hemolysis due to terminal cardiac puncture, which is known to interfere with blood cell counts and other blood parameters.

[0050] JURV induced tumor regression and improved survival in murine HCC (Hepa1-6), breast cancer (EMT6), colon cancer (CT26), and prostate cancer (RM-1). To evaluate the antitumor efficacy of JURV across multiple murine cancer types, Hepa1-6 (HCC), EMT6 (breast), CT26 (colon), and RM-1 (prostate) cell lines were subcutaneously implanted into female C57BL / 6J mice (n = 7 / group) (Jackson Laboratories) (Figure 5). After tumors reached a treatable size (80–120 mm 3 ), 1 × 107 TCID 50 Three (once a week) intratumoral (IT) injections of JURV in units were administered. Tumor volume and survival rate were recorded. In the Hepa1-6 model (p = 0.006), JURV induced significant tumor growth inhibition. However, in the CT26 (p = 0.0355), EMT6 (p = 0.0396), and RM-1 (p = 0.0094) models, JURV mediated a significant gain in survival rate compared to PBS (Figure 5). The inventors found no adverse events or virus-related toxicities. The mechanism behind the heterogeneity of the antitumor efficacy of JURV in animal models and human liver cancer is worthy of further investigation.

[0051] Antitumor activity of JURV against local and untreated distant mouse HCC Oncolytic vesicular virus exerts its antitumor effect by inducing direct cytotoxicity of tumor cells and stimulating the host antitumor immune response (Figure 6). Therefore, to understand whether the injection of JURV is associated with an immune response against tumor cells, the inventors subcutaneously implanted Hepa1-6 cells into C57BL / 6J mice (n = 7 / group). When the tumors reached 80 - 120 mm 3 upon reaching, the mice were given JURV (1×10 7 TCID 50) received mouse anti-PD-1 (10 mg / kg once daily for 14 days), or three IT injections of JURV + anti-PD-1. The inventors evaluated the changes in tumor volume and immune cell profile of Hepa1-6 tumors at the end of the study (EOS). Compared with PBS, tumor volume significantly decreased in the JURV (p = 0.008) treatment group, anti-PD-1 (p = 0.0047) group, and JURV + anti-PD-1 (p = 0.0023) group. Interestingly, the tumor growth arrest induced by JURV was similar to that by anti-PD-1, and the combination of JURV + anti-PD-1 was not better than JURV alone (Figure 4.A). In addition, the inventors observed tumor regression in both JURV-treated tumors and untreated tumors in mice bearing Hepa1-6 in the right and left flanks (Figure 6B). By flow cytometry analysis, CD4+PD-1+ T cells significantly increased in the double flank (JURV) group compared with the JURV + anti-PD1 treatment group (Figure 6K). CD8+CD44+ T cells significantly increased in the anti-PD1 group compared with the PBS group, JURV + anti-PD1, and double flank (JURV) (Figure 6I). In the anti-PD-1 group, the proliferation of CD8+(CD8+Ki67+) T cells significantly increased compared with the PBS group and double flank (JURV) (Figure 6G). CD8+PD1+ T cells significantly increased in the anti-PD1 group compared with JURV and JURV + anti-PD1. In addition, CD8+PD1+ T cells significantly increased in the double flank (JURV) group compared with the JURVV + anti-PD1 group (Figure 6H). NK cells of JURV and anti-PD1 dramatically increased compared with the JURV + anti-PD1 and double flank (JURV) groups (Figure 6D). M1 macrophages in the anti-PD1 group and double flank (JURV) group significantly decreased compared with the PBS control group. M2 macrophages in the anti-PD1 group significantly decreased compared with the PBS group and JURVV (Figure 6C, 6F, 6N, 6O). These results suggest that IT administration of JURV is associated with an effective and robust anti-tumor immune response mainly by CD8 + T cell-mediated cytotoxicity.

[0052] Low-dose JURV effectively reduced the tumor burden in HCC xenografts To evaluate the cancer-killing properties of JURV particles in vivo, the inventors subcutaneously transplanted bioluminescent human HCC cells (Hep3B) into the right flank of immunodeficient mice (Figure 7). When the tumors reached 80 - 120 mm 3 , three IT doses of PBS or JURV (1×10 7 TCID 50 ) were administered to the mice (Figure 7A). Bioluminescence imaging on days 7, 14, and 21 correlated with tumor growth inhibition in Hep3B mice (Figures 7B - 7D). Thus, measurement of tumor size in the JURV group versus the PBS group showed significant tumor regression in the JURV cohort compared to PBS.

[0053] Substances and Methods Cell Lines In this study, a panel of three human hepatocellular carcinoma (HCC) cell lines (Hep3B, PLC, Huh7) and two mouse HCC cell lines (Hepa1 - 6, RILWT) was used. All cell lines were cultured at 37°C, 5% CO2 in medium supplemented with L - glutamine and antibiotics (100 μg / ml -1 penicillin and 100 μg / ml -1 streptomycin). All HCC cells were maintained in Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS). BHK - 21 cells and Vero cells were obtained from the American Type Culture Collection (ATCC; Manassas, VA). Hep3B, PLC, and Huh7, and Hepa1 - 6 were purchased from ATCC. RILWT (gift from Dr. Dan Duda) is a clone derived from the RIL - 175 cell line and was propagated in DMEM with 10% FBS.

[0054] Oncolytic Viruses The inventors obtained JURV from the World Reference Center for Emerging Viruses and Arboviruses (WRCEVA) at the University of Texas Medical Branch (UTMB). A laboratory-adapted virus clone of JURV was generated by serial plaque purification in Vero cells (ATCC). RNA sequencing was applied to confirm the full-length JURV genome (Iowa State University Veterinary Laboratory). The full-length JURV genome (10,993 nucleotides), which consists of genes encoding the nucleoprotein (JURV-N), phosphoprotein (JURV-P), matrix protein (JURV-M), glycoprotein (JURV-G), and RNA-directed RNA polymerase L protein (JURV-L), was codon-optimized for expression in mammalian cells, synthesized from the laboratory-adapted JURV virus clone (Genscript), and subcloned into a plasmid (JURV-XN2). However, the inventors inserted five genes of the JURV intergenic region derived from VSV in between to increase the translation of viral proteins. JURV-XN2 and JURV-eGFP (having the GFP gene between the G gene and the L gene) were used together with helper plasmids (JURV-P, JURV-N, and JURV-L) to generate recombinant JURV and JURV-GFP as previously described 50 . Such plasmids were used to express the anti-genomic sense RNA of JURV under the bacteriophage T7 promoter to generate recombinant JURV. However, in in vivo studies, the inventors used the laboratory strain of JURV instead of recombinant JURV. JURV and JURV-GFP were rescued in BHK-21 cells derived from the plasmids (JURV-XN2 and JURV-eGFP). All viruses were rescued in the vaccinia rescue system and propagated and titrated in BHK-21 cells as previously described 3、4。The inventors used several concentrations of JURV-XN2 and JURV-eGFP. 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). Prior to in vitro and in vivo studies, purified JURV particles were obtained using sucrose density gradient centrifugation.

[0055] A representative JURV sequence includes SEQ ID NO: 12 of the present disclosure, and the plasmid map JURV-XN-2 (SEQ ID NO: 14) containing the said sequence can be found in FIG. 8. A representative JURV sequence with a green fluorescent protein fusion includes SEQ ID NO: 13 of the present disclosure, and the plasmid map JURV-GFP (SEQ ID NO: 15) containing the said sequence can be found in 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 FIGS. 10A - 10E respectively.

[0056] Cell viability assay In all cytotoxicity assays (96-well format), cells (1.5×10 4 cells) were infected with laboratory-based strains of JURV or MORV or VSV at MOIs of 10, 1, and 0.1 in serum-free Gibco Minimum Essential Medium (Opti-MEM). Cell viability was determined using the Cell Titer 96 AQueous One Solution cell proliferation assay. Data were generated as the mean ± SEM of six replicates from six independent experiments. Visualization of virus-induced cytopathic effects in cholangiocarcinoma cells Using JURV, adherent cells in 6-well plates (5×10 5Cells were infected with 0.1 multiplicity of infection (MOI). Cells were incubated at 37 °C until analysis. At 72 hours post-infection, cells were fixed with 5% glutaraldehyde and stained with 0.1% crystal violet to visualize the morphological features of cell viability and remaining adhesion. Photographs of representative areas were taken.

[0057] Animal experiments The inventors conducted the in vivo evaluations described below according to a protocol approved by the Mayo Clinic Institutional Animal Care and Use Committee. Toxicity and biodistribution of virus administered via the nasal route To determine whether treatment with JURV can be associated with neurotoxicity or hepatotoxicity, female C57BL / 6J mice (N = 36, n = 6 mice per group) including controls were administered PBS (25 μl per nostril) or doses of JURV (1×10 7 、1×10 8 TCID 50 ) intranasally (IN) or intravenously (IV). To detect signs of toxicity, body weight, body temperature, behavior, and clinical signs were monitored by a board-certified veterinarian at least three times a week. Three days after infection, three 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 three times a week during the study period.

[0058] Blood tests Blood was collected from the submandibular vein (cheek bleed) on day 3 and by cardiac puncture on day 45. Blood was collected in BD Microtainer tubes with ethylenediaminetetraacetic acid or lithium heparin (Becton, Dickinson and Company) for complete blood count measurements and in BD Microtainer SST tubes (Becton, Dickinson, and Company) for serum analysis. Complete blood count analysis was performed on a Piccolo Xpress chemistry analyzer (Abaxis), and blood chemistry analysis was performed on a VetScan HM5 blood analyzer (Abaxis).

[0059] In vivo efficacy study in human CCA and HCC xenograft models To evaluate the in vivo therapeutic efficacy of oncolytic JURV in subcutaneous xenograft models of mouse tumors and human hepatocellular carcinoma, tumor cells (2×10 6 cells) were subcutaneously inoculated into the right flanks of female athymic nude (NU / J) mice (n = 7 mice per group) (Jackson Laboratories). When the tumors reached an average size of 80 - 120 mm 3 , the mice were randomized into treatment groups and dosed within 24 hours of randomization. Each mouse received three intratumoral injections (50 μl containing PBS or 1×10 7 TCID 50 units of JURV or PBS) at one-week intervals. Tumor volume and body weight were monitored. If side effects were observed, or if the tumor size became larger than 2,000 mm 3 , the mice were euthanized. Tumor volume was calculated using the following formula: (longest diameter * shortest diameter 2 ) / 2. Tumor images were taken before resection and tumor mass was recorded after resection.

[0060] Analysis of tumor-infiltrating immune cells After resection, tumors from 5 mice per group were dissociated using a gentleMACS Octo Dissociator (Miltenyi) according to the manufacturer's protocol. CD45 (TIL) mouse microbeads (Miltenyi) were used to isolate CD45 +Cells were isolated. The cells were incubated with Fixable Viability Stain 510 (BD Horizon) for 15 minutes, followed by incubation with anti-Fc blocking reagent (Miltenyi) for 10 minutes prior to surface staining. The cells were stained and then data were acquired using a MACSQuant Analyzer 10 optical bench flow cytometer (Miltenyi). All antibodies were used according to the manufacturer's recommendations. Fluorescence Minus One controls were used in each independent experiment to establish gating. For intracellular staining of granzyme B, the cells were stained with an intracellular staining kit (Miltenyi). Analysis was performed using FlowJo (TreeStar). Forward scatter and side scatter were used to exclude cell debris and doublets.

[0061] Flow cytometry analysis antibodies The following antibodies were used for flow cytometry analysis: CD45-FITC (Catalog number 553079, BD Biosciences), CD3-BUV395 (Catalog number 563565, BD Biosciences), CD4-BUV737 (Catalog number 612761, BD Biosciences), CD8-Percp-Cy5.5 (Catalog number 45-0081-82, eBioscience), CD44-BV711 (Catalog number 103057, Biolegend), CD335-PE / Dazzle594 (Catalog number 137630, Biolegend), PD-1-PE (Catalog number 551892, BD Biosciences), Ki67*-BV605 (Catalog number 652413, Biolegend), Granzyme B*-APC (Catalog number 366408, Biolegend), IFN-γ*-BV421 (Catalog number 563376, BD Biosciences), CD11b-PE-Cy7 (Catalog number 101216, Biolegend), F4 / 80-BV510 (Catalog number 123135, Biolegend), CD206-AF700 (Catalog number 141734, Biolegend), I-A / I-E-BV786 (Catalog number 743875, BD Biosciences), and L / D-efluor780 (Catalog number 65-0865-18, eBioscience).

[0062] Histopathological analysis The evaluation of any abnormal changes in the brain and liver was determined by histopathological evaluation of H&E-stained images by a board-certified pathologist. The percentage of tumor necrosis area was measured using HALO v3.1.1076.379.

[0063] Statistical analysis All values were expressed as mean ± standard deviation, and the results were analyzed using the statistical software of GraphPad Prism version 8 (GraphPad Software) by one-way analysis of variance, followed by Tukey's test for multiple comparisons and the Kaplan-Meier method for survival rates. A p-value of less than.05 was considered significant.

[0064] References 1. Walker, P.J., et al. Evolution of genome size and complexity in the rhabdoviridae. PLoS Pathog 11, e1004664 (2015). 2. Amarasinghe, G.K., et al. Taxonomy of the order Mononegavirales: update 2017. Arch Virol 162, 2493-2504 (2017). 3. Lawson, N.D., Stillman, E.A., Whitt, M.A. & Rose, J.K. Recombinant vesicular stomatitis viruses from DNA. Proc Natl Acad Sci U S A 92, 4477-4481 (1995). 4. Whelan, S.P., Ball, L.A., Barr, J.N. & Wertz, G.T. Efficient recovery of infectious vesicular stomatitis virus entirely from cDNA clones. Proc Natl Acad Sci U S A 92, 8388-8392 (1995).

[0065] Example 2: Comprehensive proteogenomic analysis of the antitumor immune activity of a novel oncolytic vesiculovirus in hepatocellular carcinoma In this example, the inventors show that intratumoral (IT) administration of Jurona virus (JURV) induces dynamic tumor regression in human HCC xenograft and syngeneic models. Furthermore, IT injection of JURV induces the mobilization and activation of cytotoxic T lymphocytes (CTLs), reduces tumor-associated macrophage (TAM) infiltration, and is associated with delayed tumor growth in both local and distant mouse HCC tumors in the syngeneic model. Moreover, co-administration of JURV and anti-PD-1 antibody shows a synergistic effect, modulating the tumor microenvironment (TME) by increasing tumor-infiltrating CD4+ T cells and depleting CD8+ PD-1+ and NK cells. Mechanistically, the inventors' analysis reveals that JURV and anti-PD-1 antibody activate different effectors of the immune system but have complementary anti-tumor activities. Additionally, the inventors' results suggest that the abscopal effect induced by JURV is likely mediated by the activation of several tumor suppressor genes and the mechanism of regulating the T helper cell response. The inventors' research supports the further development of JURV as a novel immunoviral therapy platform for hepatocellular carcinoma.

[0066] Results In vitro cytotoxic activity of JURV in HCC cells Genomic analysis of Jurona virus (JURV) showed the same genomic constitution as that of vesicular stomatitis virus (VSV) and Morleton virus (MORV) (Figure 11). These are two other members of the 6 Rhabdoviridae family that have been previously investigated for their oncolytic ability in human cancers 7To investigate whether JURV has similar cancer cell killing ability in vitro, the inventors infected monolayers of human and mouse HCC cells including HEP3B, PLC, HuH7, HEPA1-6, and RILWT with JURV, VSV, or MORV at multiplicity of infection (MOI) of 0.1, 1, or 10, respectively (FIGS. 12A-12E). The inventors performed a cytotoxic MTS cell viability assay 72 hours post-infection to determine the susceptibility to virus infection and the extent of virus-induced cancer cell death. The cell viability assay results are summarized in Table 1. Regardless of MOI, a significant decrease in cell viability (about 30%) was observed in HEP3B cells in response to infection with JURV, VSV, and MORV (FIG. 12A). Differences in virus-induced cell death were also confirmed in PLC, HuH7, HEPA1-6, and RILWT cells (FIGS. 12B-12E). In PLC cells, JURV and VSV induced similar cytotoxicity rates (about 25%) at MOIs of 10, 1, and 0.1, while the cell viability of MORV-infected cells was twice as high (about 50%). HuH7, HEPA1-6, and RILWT cells showed lower susceptibility to JURV infection (about 24%) than MORV and VSV, except for RILWT cells treated with JURV at MOIs of 1 and 10. Subsequently, the inventors infected HCC cells with JURV at an MOI of 0.1. Three days after incubation, the cells were stained with crystal violet to evaluate the comparative cytotoxic effect of JURV in infected cancer cells. The inventors observed that viable adherent cells remained (<50%) in HuH7 after infection, while adherent cells were completely lost in HEP3B, PLC, HEPA1-6, and RILWT (>90%), suggesting that MTS may have slightly underestimated the oncolytic effect of JURV (see FIG. 3E). Furthermore, analysis of JURV kinetics showed a 1000-fold increase in virus titer approximately 10 hours after infection (see FIG. 3E). This indicates the high permissiveness and robust replication ability of JURV in HCC cells. The inventors' results indicate that JURV, VSV, and MORV can efficiently infect and lyse mouse and human HCC cells.However, the basis for the differences observed in virus-mediated HCC cell killing is worthy of investigation in future studies.

[0067]

Table 1-1

Table 1-2

[0068] In vitro cell viability of JURV-infected HCC cells pretreated with type I IFN-α The tumor microenvironment (TME) is a site of complex interactions between cancer cells, normal tissues, and diverse components of the immune system. 8 Detection of viral pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs) present on most immune cells induces the production of numerous cytokines, including IFNα / β, which activate hundreds of interferon-stimulated genes (ISGs). 9、10 Activation of ISGs triggers innate antiviral mechanisms and generates an adaptive cellular response to viral infection. Interestingly, studies have shown that type I IFN signaling pathway deficiencies often occur concurrently with carcinogenesis and create the conditions necessary for tumor-selective virus replication and lysis by many oncolytic viruses (OVs). 11、12 However, immune and non-cancer cells in the TME can produce type I IFN-α / β upon sensing the presence of the virus. This can prematurely impair the oncolytic activity of OVs if tumor cells are responsive to the effects of exogenous IFN. 11、13Therefore, to determine the impact of type I IFN on the outcome of JURV infection, the inventors compared the susceptibility of monolayers of human (HEP3B) and mouse HCC (HEPA1-6) cells pretreated with species-specific IFN-α to JURV infection. Specifically, as shown in the method and Figure 13A, the inventors treated HEP3B cells and HEPA1-6 cells with serial concentrations of IFN-α and subsequently infected them with JURV at MOIs of 10, 0.1, or 0.01. Cell viability (MTS) was evaluated 72 hours after infection. The inventors' data show that treatment with IFN-α did not shield human HCC (HEP3B) cells and mouse HCC (HEPA1-6) cells from JURV-induced cytopathic effects (30 - 75% cell death). However, the inventors confirmed that there were differences in the response to IFN-α among the treated HCC cells. At an MOI of 1, a concentration of 500 U / mL of IFN-α killed approximately 20% of HEPA1-6 (Figure 13B). In contrast, the same treatment resulted in approximately 75% cell death in HEP3B cells (Figure 13A). Overall, there were no statistical differences in cell viability among the different treatment groups. This indicates that treatment with IFN-α did not significantly alter JURV oncolytic activity, as shown in the inventors' previous studies 7、14 , suggesting that there may be a defect in the IFN pathway in such HCC cells.

[0069] In vivo safety evaluation of JURV According to reports, intranasal (IN) administration of wild-type VSV to mice has been shown to result in significant weight loss and lethality approximately 3 days after infection 15~17 . Since JURV shares the same genomic structure as VSV, the inventors attempted 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, the inventors used 1 × 10 6 TCID 50Two doses of wild-type JURV that were 10 to 100 times higher than the dose of 15~17 were selected. To mimic the natural route of systemic VSV infection in non-tumor-bearing healthy experimental mice, 1×10 7 or 1×10 8 TCID 50 of JURV was administered intranasally or intravenously (IV). As a control group, the inventors 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 collected and subjected to H&E staining to evaluate short-term toxicity. In the virus treatment group, compared to the control group, the body weight of the mice decreased by 10 to 15% on day 3 (Figures 14A and 14B). However, upon review by experts of histological slides, no obvious abnormalities were found in brain, liver, or spleen sections of animals treated with either the low-dose or high-dose JURV cohort (Figures 14C and 14D). In mice treated with JURV, there was transient leukopenia, including a decrease in white blood cells and lymphocytes (Figures 14E - 14H). Viral infection is characterized by leukopenia in humans and animals and is well-documented for systemic VSV infection 21 . According to the inventors' data, in mice infected with JURV, there were no significant differences in body weight or clinical signs (i.e., paralysis, death, ruffled fur) compared to PBS treatment.

[0070] Toxicoproteomic analysis of JURV As discussed above, the lack of changes in brain and liver tissues recovered 3 days after JURV infection indicated severe toxicity, including neurotoxicity or hepatotoxicity, at any of the virus doses tested. Next, the inventors performed quantitative proteomic analysis of these tissues to identify potential biological changes associated with neuroprotection and anti-hepatotoxicity in mice infected with high-dose JURV. The inventors used Ingenuity Pathway Analysis (IPA) to identify biologically enriched processes and signaling pathways of differentially expressed proteins (DEPs) with significantly altered expression in the brains and livers of mice infected with JURV. 1.0×10 8 TCID 50Of the 4,253 analyzed DEPs in the brains of mice treated with JURV, 127 DEPs showed significantly altered expression compared to the control group (fold change > 2, p-value < 0.055). Such DEPs included 60 upregulated DEPs and 67 downregulated DEPs (Figures 15A and 15B). Of the 2,400 analyzed DEPs in the livers of the same mice, 87 DEPs were upregulated and 123 DEPs were downregulated (Figures 15C and 15D). To understand the dynamics of biological changes in such tissues, the inventors further examined the top 10 upregulated and downregulated DEPs (Figure 15E), as well as the five most important signaling pathways in the brain (Figure 15G). The inventors used the National Library of Medicine (NLM) database to match these proteins, except for Prps1l3, with human orthologs (Table 2). This enabled the inventors to gain insights into changes in protein expression in response to JURV infection.

[0071]

Table 2-1

Table 2-2

[0072] Gamma complex-associated protein 4 (Tubgcp4), which functions in microtubule (MT) nucleation at the centrosome, was the most significantly upregulated protein in the brains of JURV-treated mice (Figure 15E). Interestingly, studies have shown that MT alterations are essential for the formation of viral intracellular replication compartments, 22 suggesting that JURV induces MT changes in brain cells to promote viral replication. However, other upregulated proteins also included orthologs of the human leukocyte antigen (HLA) complex molecules H2-Q7, H2-Q8, H2-Q6, and H2-L, HLA-A, -B, -C, -E, -G, and -F (Table 2). Several studies have shown that IFN-γ produced during viral infection upregulates the expression of mammalian HLA-A, -B, -C.23、24 HLA-A, -B, and -C are specialized for antigen presentation to cytotoxic T cells and are thus very important components of the cell-mediated adaptive immune response against pathogens, including viruses and bacteria. In contrast, HLA-E, -G, and -F have been reported to downregulate inflammation and immune responses. 25、26 。

[0073] These findings suggest that the host immune response was well regulated in the brain after high-dose JURV administration and that viral infection was resolved rapidly and effectively. Surprisingly, the inventors found among the top upregulated proteins, IFIT3 (interferon-induced protein with tetratricopeptide repeats 3) 27,28 and Stat1 (signal transducer and activator of transcription 1). Both proteins have been shown to enhance the immune response. However, Lgals9 (lectin, galactose-binding, soluble 9) and HPx (haptoglobin) act as suppressors of excessive inflammatory and immune responses. 29、30 。In addition, Nenf (neurotrophic factor), which functions in 31~33 neuron protection, was also upregulated in JURV-treated brain tissue. The five most enriched predicted signaling pathways were PRPP biosynthesis, LXR / RXR activation, RH / H in influenza pathogenesis, FXR / RXR activation, and acute-phase response signaling (Figure 15G).

[0074] Based on the altered expression of DEPs used in the IPA analysis, the inventors propose that activation of RH / H in influenza pathogenesis and acute-phase response signaling occur first and are linked to the activation of PRPP biosynthesis, which is essential for establishing immunity against the virus. In contrast, activation of LXR / RXR and FXR / RXR is thought to represent an early response for the infected mouse to return to homeostasis. Similarly, the top 10 DEPs upregulated in the liver, such as Isg15 34 , Cmpk2 35 , Uba7 36exerts major cellular antiviral functions (Figure 15F). IPA analysis of DEPs in the liver infected with JURV (Figure 15H) also predicted activation of interferon signaling, mitochondrial dysfunction, and acute-phase response signaling pathways. All of these are related to the sensing of viral infection and the induction of innate and adaptive immune responses against it. The inventors found that the changes in DEPs after administration of 1×10 7 TCID 50 of JURV were similar to those when 10-fold higher doses (1×10 8 TCID 50 ) of the virus were administered (Figures 16A - 16D). Furthermore, analysis of the top 10 upregulated DEPs and predicted activation pathways (IPA) in the brain (Figure 16E) and liver (Figure 16G) were all immune responses against virus infection-related proteins and pathways (Figures 16G and 16H). Collectively, these data strongly indicate that multiple mechanisms coexist to control and eliminate JURV infection and simultaneously prevent neuronal damage. These results indicate that host cellular machinery senses JURV, controls virus infection, eliminates the virus from the brain, and simultaneously prevents neuronal damage through multiple mechanisms. Additionally, this indicates that intranasal administration of high doses of JURV did not induce severe neurotoxicity or physical impairment in these mice.

[0075] In vivo antitumor efficacy of JURV in an isogenic HCC model First, the inventors evaluated the efficacy of JURV when combined with an anti-PD-1 antibody using a subcutaneous syngeneic HCC model, compared to anti-PD-1 alone or JURV alone. Therapeutic efficacy was evaluated in Hepa1-6 tumors grafted onto the right flank of immunocompetent mice. An extended dosing regimen (Figure 17B) was used, including an intratumoral (IT) dose of JURV (3 doses) in the group treated with JURV and an intraperitoneal dose of anti-PD-1 (6 doses) in the immune checkpoint blockade group. 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) (Figure 17A). The antitumor effect of anti-PD-1 therapy was equivalent to that of JURV (p<0.0001) (Figure 17A). However, the inventors found that the delay in tumor growth in mice treated with JURV combined with an anti-PD-1 antibody was not more significant than that in the case of JURV alone or anti-PD-1 alone (p<0.001). Further analysis of the individual tumor volumes of the mice showed that tumors were eliminated (>70%) in almost all mice in the JURV-treated group and the anti-PD-1-treated group compared to the JURV group combined with an anti-PD-1 antibody (>50%) (Figure 18A).

[0076] Next, the inventors attempted to investigate whether local administration of JURV could induce a systemic antitumor effect that could affect both local and distant tumor development. The inventors subcutaneously transplanted HEPA1-6 tumors into the right and left flanks of immunocompetent mice. However, the inventors performed local IT injection of JURV only into the right flank, leaving the left flank untreated. The inventors' data showed that IT injection of JURV triggered antitumor activity and reduced the growth of HEPA1-6 tumors in both the right and left flanks (Figure 17C, Figure 18B). Furthermore, starting from day 18, the inventors observed a dramatic decrease in the tumor volume of the left flank of mice that had not been inoculated with the virus. These results are consistent with the induction of systemic antitumor activity commonly observed with oncolytic viruses in clinical trials and animal models 3,37In addition, the inventors confirmed a slight decrease in mouse body weight 1 day after administration of the final dose of JURV, but the mice recovered immediately and no adverse events were confirmed until the end of the study (day 28) (Figure 18C). In addition, one mouse death was found in the control group (PBS), but there was no significant difference in survival rate between the different groups (Figures 18D and 18E). Evaluation of the levels of serum biomarkers associated with virus treatment-related liver and kidney toxicity showed that the mice did not experience severe virus-induced toxicity. Such primary investigations have highlighted the ability of JURV to induce antitumor activity against local and distant non-injected tumors without causing harmful effects on normal tissues, which is important for targeting oligometastatic or metastatic diseases.

[0077] In vivo antitumor efficacy of JURV in various mouse solid tumor models To evaluate the efficacy of JURV-based therapy in other solid tumor models, the inventors transplanted several mouse tumor cells into immunocompetent mice. Such models include breast cancer (EMT6, Figures 19A - 19B), rectal cancer (CT26, Figures 19E - 19F), sarcoma (A20, Figures 19G - 19H), breast cancer (B16-F10, Figures 19I - 19J), and prostate cancer (RM-1, Figures 19C - 19D). As described in the Methods section, when the tumors reached a treatable size, the mice were given 50 μL of PBS or 1×10 7 TCID 50A single IT injection of JURV was administered (Figure 19). The inventors found heterogeneity in the response to JURV in such models. The inventors' data showed that JURV induced significant tumor growth delay in B16-F10 (p = 0.02) and CT26 (p = 0.03), but not in the A20, EMT6, and RM-1 models (Figures 19E and 19I). Interestingly, JURV provided a survival benefit only in the RM-1 model (Figure 19D). However, due to the high malignancy in these syngeneic tumor models, a significant number of animals reached tumor burden and had to be sacrificed before the end of the study in this research. Overall, the tolerance to treatment in mice was good and drug-related toxicity was not present in this study. These data indicate that JURV exhibits anti-tumor activity across multiple tumor types. However, the variability in its activity suggests that combination therapy of JURV with other anti-cancer drugs may be able to provide a more long-term therapeutic effect.

[0078] JURV in combination with a PD-1 blocker significantly modulates the immune components of the tumor microenvironment in murine HCC In many studies, vesicular virus has been shown to selectively infect tumor cells, replicate in tumor cells, lyse tumor cells, and modulate local and systemic anti-tumor immune responses 38Considering that administration of JURV, anti-PD-1 antibody, or a combination of JURV and anti-PD-1 antibody induced significant tumor growth delay in a subcutaneous syngeneic HCC model, the inventors initiated an investigation and comparison of the changes in immune responses associated with these different treatment regimens. As expected, the inventors' data showed that the frequency of tumor-infiltrating lymphocytes (TILs) changed dramatically after administration of these therapies to mice. Intratumoral administration of JURV significantly decreased a subset of F4 / 80-TILs (p<0.001) (Figure 20B) and increased the distribution of M2-like macrophages (p<0.01) (Figure 17K), natural killer (NK) cells (p<0.01) (Figure 17L), and natural killer T (NKT) (p<0.01) cells (Figure 20A) compared to control (PBS), anti-PD-1, and double flank (left). This indicates viral replication and phagocytosis of infected tumor cells 39 Treatment with anti-PD-1 antibody was correlated with intratumoral accumulation of cytotoxic CD8+ T cells, mainly CD8+Ki67+ (p<0.01) (Figure 17D), CD8+PD-1+ (p<0.01) (Figure 17E), and CD8+CD44+ (p<0.001) (Figure 17F) compared to PBS, JURV, and double flank (left). The inventors also confirmed a significant decrease in TIL macrophages (p<0.001) (anti-PD-1 vs. PBS) (Figure 17I), M1 (p<0.01) (anti-PD-1 vs. PBS) (Figure 17J), and M2-like (p<0.01) (anti-PD-1 vs. JURV) (Figure 17K). M2-like macrophages, together with myeloid cells, are known to abrogate antitumor 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 the responsive CTLs in the anti-PD-1 group are tumor-specific and may contain effector memory cells in the tumor 42Furthermore, the inventors found that when JURV and anti-PD-1 antibody were combined, the tumor microenvironment (TME) was significantly modulated as shown by an increase in CD4+ (p<0.001) (Figure 17G), F4 / 80- (p<0.01) (Figure 20B), CD4+PD-1+ (p<0.01) (Figure 17H), CD11b+ (p<0.01) (Figure 20D), CD11b- (p<0.001) (Figure 20E), and a decrease in CD8+PD-1+ (p<0.001) (Figure 17E), NK (p<0.001) (Figure 17L), compared to JURV, anti-PD-1, and dual flank (left). No significant change in serum IFN-β was observed in mice treated with JURV, anti-PD-1, and JURV in combination with anti-PD-1 antibody, and dual flank (Figure 22). Collectively, the inventors' data suggest that the combination of JURV and anti-PD-1 antibody induces anti-tumor immunity through the recruitment and tumor infiltration of cytotoxic effector T (CTL) cells 43,44 , inhibiting immunosuppression, which is a characteristic of a durable response to immunotherapy.

[0079] Multi-omics analysis identified the major molecular mechanisms of the anti-tumor activity of JURV in vivo To determine the effect of IT administration of JURV on the gene expression profile of tumors, the inventors investigated the transcriptome of mouse HCC tumors treated with 3 doses of JURV. The limma-voom method 45 was used to analyze differentially expressed genes (DEGs). The inventors' data (Figures 22A and 22B) showed that out of 22,786 genes, 203 DEGs were upregulated and 463 DEGs were downregulated (fold change >2, p-value <0.055). Some of the 10 upregulated DEGs, Myo3a 46 , Cd209c 47 , Trim67 48 , St8sia2 49 , and Wnt5b 50is associated with the immune response pathway (Figure 22C). Many of the enriched cellular signaling pathways identified by IPA analysis, such as B cell receptor signaling, IL-15 signaling, and phagosome formation, are related to the activation of the host innate and adaptive immune responses (Figure 22D). Furthermore, to better understand the mechanism of JURV-induced antitumor activity, the inventors analyzed DEPs and DEGs from transcriptome and proteome data. Among the associated DEG / DEPs, the inventors identified the top 30 enriched features significantly upregulated or downregulated in the JURV group compared to the control group (PBS). Among the upregulated features, S1pr3 51 , Tnpo1 52 , Psmb10 53 , Ddt 54 , Ncor2 55 , Slc04c1 56 have been identified in inflammation, host immune responses against microorganisms (viruses, bacteria), and tumorigenesis.

[0080] Similarly, the inventors' data show that IP administration of anti-PD-1 antibody is associated with significant transcriptional and proteomic changes in the TME and promotes antitumor immune responses. Indeed, the inventors found that in HEPA1-6 tumors, out of 22,786 genes, 860 DEGs were upregulated and 241 DEGs were downregulated (Figures 23A and 23B). Prps1l1 57 , Cstdc4 58 , and Ppbp 59 among the top 10 DEGs upregulated in the anti-PD-1 therapy group compared to the control group (PBS) (Figure 23C) are all involved in inflammation, adaptive immune responses, and cell survival and proliferation. The most enriched pathways predicted by IPA analysis (Figure 23D) (i.e., phagosome, communication between innate and adaptive immune cells) are immune-related pathways. Integrated analysis of the associated DEP / DEG features revealed Tmod3 60 , Nfkb2 61 , Dapk3 62 , Nipsnap3b, which are the main effectors of the pathways regulating immunosuppression, angiogenesis, inflammation, and antitumor immunity.63 、Sart3 64 、and Adgrl4 65 showed upregulation. Such research reveals the potential molecular mechanisms involved in JURV and / or anti-PD1-induced antitumor activity.

[0081] Multi-omics analysis predicts that anti-tumor immunity is effectively activated by combining JURV with anti-PD-1 antibody. The inventors analyzed the transcriptional profiles of mouse HCC and identified dysregulated genes and pathways in tumors after treatment with JURV, anti-PD-1 antibody, and the combination of JURV and anti-PD-1 antibody, compared with the control group (PBS). The inventors analyzed 22,786 genes between the control group (PBS) and the combination of JURV and anti-PD-1 antibody. The inventors found that 323 DEGs were upregulated and 778 DEGs were downregulated (Figures 24A and 24B). Cox20 66 、Dpf3 67 、Trp63 68 、Flg2 69 、Ush2a 70 、and Cdh24 71 The top 10 upregulated DEGs, including, are effector genes associated with tumor suppression and immune cell infiltration mechanisms (Figure 24C). IPA analysis identified enriched pathways (i.e., hepatic fibrosis / hepatic stellate cell activation, oxytocin signaling, calcium signaling pathway). All of these are mainly associated with the activation and regulation of immune cells (Figure 24D). Integrated analysis of related DEP / DEGs revealed Cdk5r1 72 、Ptgdr2 73 、Crip2 74 、Tardnp 75Highlighted the upregulation of genes associated with immunotherapy, autophagy, tumor suppression, and sensitivity to immune responses, among others. Furthermore, investigation of the unique and common DEGs between PBS vs JURV, PBS vs anti-PD-1, and PBS vs JURV + anti-PD-1 (Figure 25) revealed relatively little overlap between differentially expressed genes (both upregulated and downregulated) among these three groups, suggesting that there are differences in the mechanisms of antitumor responses to these therapies. This may be driven by differences in the types of TILs activated by these two treatment modalities and mobilized and transported to the tumor site, or between the two delivery routes (IP and IT).

[0082] Furthermore, the inventors performed gene ontology (GO) term enrichment analysis on 35 upregulated or downregulated DEGs common to all three datasets (Table 3). Some mitogen-activated protein kinases (MAPKs) known to be upregulated during viral infection 76 pathways, macrophage migration inhibitory factor (MIF) involved in inflammation and immune responses 77 were found to be enriched, as well as necroptosis signaling and the vascular endothelial growth factor (VEGF) family. These data indicate that JURV therapy and anti-PD-1 therapy activate important and complementary pathways involved in natural and adaptive antitumor immunity, leading to the control and regression of tumor growth, despite presenting different mechanisms of antitumor activity.

[0083]

Table 3

[0084] JURV induces antitumor immunity targeting both local and distant tumors Previous reports have shown that oncolytic viruses can induce virus-mediated local and systemic antitumor immune activation through the mobilization of class I MHC-restricted virus-specific and tumor-specific CTLs 78、79。To better understand the mechanism underlying the observed abscopal effect of JURV on distant tumors, the inventors analyzed the transcriptome and proteome of uninjected (left flank) HEPA1-6 tumors. Among the 21,260 genes analyzed, the inventors found that 1165 DEGs were upregulated and 361 DEGs were downregulated in the treatment group compared to the PBS control group (Figures 26A and 26B). The top 10 upregulated genes are listed in Table 4. Tent5b 80 , Per1 81、82 , Tubb4b-ps1 83 , Dbp 84、85 , and Cry2 85、86 . Many of the top upregulated genes, including, play important tumor suppressor activities in mammalian cells (Figure 26C). The inventors also found that Fgfr2 was upregulated in their dataset. On the other hand, overexpression of Fgfr2 and its fusion partners has been associated with several advanced cancers, including HCC cholangiocarcinoma, so the Fgfr2 pathway is an attractive target for liver cancer 87~93 . Conversely, studies have shown that treatment with pazopanib, a multi-targeted receptor tyrosine kinase inhibitor, resulted in better outcomes in gastric tumors with Fgfr2 amplification 90 . The exact role of Fgfr2 in response to immunotherapy has not yet been determined and is particularly worthy of further investigation in viral therapy. Cry2 has been associated with a better prognosis in ERC / HER2-tumors 85、86 . The efficacy of JURV in the treatment of ERC / HER2-tumors is also worthy of future investigation. To the inventors' knowledge, the biological functions of the Gm6614, Rn7s2, and D930015M05Rik genes have not yet been defined (Table 4). IPA analysis made it possible to identify the most enriched canonical pathways in this dataset (Figure 26D). These enriched biological pathways include T helper cell differentiation, granulocyte adhesion, extravasation, B cell signaling, Th1 and Th2 activation signaling, and communication signaling between innate and adaptive immune cells, the T helper cell pathway 94Modulation has been shown to play a very important role in the abscopal effect induced by IT injection of JURV (Figure 26D). By comparison of DEP / DEG, it was further confirmed that most upregulated features including Anxa3 95 , Hspg2 96 , Cyp2e1 97 , and Map1lc3a 98 are either therapeutic targets for immunotherapy or function as tumor suppressor genes. In addition, to evaluate the relationship between local antitumor immunity and systemic antitumor immunity in mouse tumors, the inventors performed GO term enrichment analysis of DEGs between JURV-injected HEPA1-6 tumors and non-injected HEPA1-6 tumors. The top enriched canonical pathways identified by analysis of DEGs between JURV and double flank (i.e., inhibition of ARE-mediated mRNA decay, FAT10 signaling, EIF2 signaling) are associated with response to immunotherapy, activation of the MAPK pathway, and apoptosis (Figure 27). These results indicate that JURV can effectively prime antitumor immunity against local and distant tumors in the HCC models studied.

[0085]

Table 4

[0086] IT administration of JURV mediates robust antitumor efficacy in the HEP3B xenograft HCC model The inventors previously showed that the in vitro production of type I IFN or responsiveness to viral kinetics by infected cancer cell lines does not necessarily correlate with the in vivo efficacy of oncolysis 7To determine whether JURV can induce tumor lysis-dependent tumor cell death in vivo, the inventors injected three doses of JURV into HEP3B xenografts by IT injection. The inventors used luciferase-tagged HEP3B cells to monitor tumor growth during the first three weeks of treatment. Compared to the control group (PBS), bioluminescence imaging showed significant (p<0.0001) tumor inhibition in JURV-treated mice (Figs. 28A-28C), which was visible from the first week after injection until the end of the study. In addition to luciferase activity, comparison of tumor volumes between PBS and JURV showed that JURV triggered a significant (p<0.0001) delay in tumor growth (Figs. 28D, 29A). The inventors then performed a proteomic analysis of tumor tissues to determine the changes that occurred after IT delivery of JURV in HEP3B tumors. Analysis of a total of 2,088 proteins showed that in JURV tumors compared to PBS tumors, 860 DEPs were upregulated and 241 DEPs were downregulated (Figs. 28E and 28F). Among the top 10 upregulated DEPs, the inventors identified 99 , COL6A3 100 , HSPG2 101 , NAMPT 102 , STAT1 103 , and VIM 104 as proteins associated with activation of the mTORC2 / AKT pathway, cancer prognosis and treatment, regulation of tumor growth, and cancer cell stiffness. Body weight decreased in the JURV group from day 19 (by approximately 15%) (Fig. 29B). One mouse was found to be moribund on day 18, but since the other mice did not experience toxicity, the inventors attributed this to an isolated adverse event due to the severely immunodeficient nature 105 of the NOD-SCID mouse model (Fig. 29C). The inventors' findings indicate that JURV efficiently infects and lyses human HCC cells in human HEP3B xenografts in vivo, thereby further increasing interest in the use of new immunoviral therapies in human HCC.

[0087] Discussion Rhabdovirus has several advantageous properties compared to other oncolytic virus vector platforms, including ease of genetic manipulation and low seropositivity rates within the population due to not using humans as natural hosts. 5 In addition to episomes and rapid kinetic cycles in tumor cells, most vesiculoviruses can encode large transgenes while maintaining the ability to infect, replicate in, and induce apoptosis in a wide range of cancer cells. 4 .

[0088] In recent decades, vectors derived from vesicular stomatitis virus (VSV), the prototype rhabdovirus family, have advanced through various stages of clinical trials against various human cancers. Although the therapeutic efficacy of VSV-based vectors has been confirmed in multiple studies, barriers to FDA approval and clinical application still remain. Such obstacles include multiple reports regarding VSV-induced neurotoxicity, hepatotoxicity, and rapid elimination by the host immune system. In previous studies by the present inventors, the anti-tumor ability of the non-VSV rhabdovirus MORV, which has natural tumor selectivity and hypersensitivity to type I interferon responses that are defective in 3 / 4 of all cancers, has been described. To expand the spectrum of oncolytic virus backbones, in this study, the inventors investigated the potential anti-cancer effects of Jurona virus (JURV), a genetically distinct member of the same virus family, as an anti-cancer agent. 6,7 1 type interferon response 7 is explained.

[0089] The inventors evaluated the antitumor effects of JURV in human and mouse hepatocellular carcinoma (HCC) cell lines and a mouse model of HCC in vitro. JURV efficiently infected all HCC cells tested, although there was variability in cytolytic activity. Further investigation of the mechanisms of in vitro HCC killing differences can provide insights into responses to JURV-based therapies in HCC and potentially other solid tumors. Concerns about VSV-induced encephalitis and hepatotoxicity led to the development of attenuated recombinant VSV platforms using various viral engineering techniques. Although these vectors showed some improvement in safety, reduced oncolytic activity compared to parental VSV has also been reported due to impaired intratumoral replication ability.

[0090] In this study, the inventors demonstrated that the naturally attenuated JURV exhibits all the characteristics required for a potent immunoviral therapy, such as a strong cytolytic effect and rapid replication cycle in tumor cells, sensitivity to type I IFN, and, more importantly, the absence of long-term neurotoxicity and hepatotoxicity in mice. Analysis of the toxicoproteome of brain tissue showed activation of multiple mechanisms that limit viral spread and prevent brain damage, which are involved in pathogen clearance and neuron protection. Furthermore, the inventors' data showed that JURV stimulates multiple mechanisms of the innate and adaptive immune responses and is associated with delayed tumor growth in an autochthonous HCC model. The combination of JURV and anti-PD-1 therapy significantly modulates the TME by enhancing the infiltration of cytotoxic T cells through the activation and recruitment of different immune system effectors with complementary antitumor activities. Additionally, the inventors demonstrated that JURV efficiently induces tumor lysis-mediated tumor growth delay in human HCC xenografts. Analysis of the 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 revealed upregulation of immune-related genes and identified enriched pathways involved in inflammation, regulation of immunosuppression, angiogenesis, and antitumor immunity. Interestingly, the inventors found that IT administration of JURV is associated with an abscopal effect in bilateral murine HCC. To elucidate the mechanisms contributing to the abscopal effect, the inventors used a proteomic analysis approach on untreated tumors transplanted at distant sites in animals. The inventors' results indicate that JURV triggered the activation of several tumor suppressor genes, suggesting that the tumor suppressor pathway may play a very important role in the abscopal effect in their animal model. In addition, the inventors' data suggest that cellular antitumor immunity via activation of the T helper cell pathway may also play a major role in the JURV-induced abscopal effect.

[0091] In summary, the results of the present inventors indicate that JURV potently infects HCC cells in vitro and in animal models, inducing tumor lysis. Also, JURV-infected tumor cells prime anti-tumor immunity (targeting primary and distant tumors), which is shown to be enhanced by the addition of anti-PD-1 antibody.

[0092] Method Experimental design Such experiments were conducted to provide new and highly important mechanistic insights into the safety and efficacy of oncolytic JURV in HCC tumor models. This will enable rational study designs for using JURV as monotherapy or together with other cancer therapies in early or late HCC to obtain long-term responses that are likely to be additive or perhaps synergistic in the clinical setting. All animals were randomly assigned to different study groups in a non-blinded manner. The mean tumor volume (mm 3 ) + SEM at randomization was set to 80 - 120 mm 3 . Tumor volume (or its log transformation) was evaluated in relation to time using a mixed linear regression model with time, treatment effect, and their interaction as independent variables. The inventors used a random effect to account for the within-subject correlation due to repeated measurements. The slope was interpreted as the tumor growth rate (or logarithm) over time and compared between groups. Kaplan-Meier curve analysis was used to identify the proportion of tumor-bearing mice surviving for a certain period after treatment. The n values and statistical methods are shown in the statistical analysis section.

[0093] Cell line In this study, a panel of three human hepatocellular carcinoma (HCC) cell lines (Hep3B, PLC, Huh7) and two mouse HCC cell lines (HEPA1-6, RILWT) was used. In addition, the inventors used several mouse solid tumor cells, including breast cancer cells (EMT6), colon cancer cells (CT26), reticulum sarcoma cells (A20), skin melanoma cells (B16-F10), and prostate cancer cells (RM-1). All cell lines were cultured at 37 °C with 5% CO2 in medium supplemented with antibiotic agents (100 μg ml−1 penicillin and 100 μg ml−1 streptomycin). Hep3B, PLC, and HuH7 were maintained in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS). The inventors maintained HEPA1-6, RILWT, BHK-21 (baby hamster kidney fibroblasts), and Vero (African green monkey kidney) cells in DMEM with 10% fetal bovine serum (FBS). BHK-21 cells and Vero cells were obtained from the American Type Culture Collection (Manassas, VA). The inventors purchased Hep3B, PLC, HuH7, HEPA1-6, 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.

[0094] Oncolytic virus The inventors obtained Junin virus (JURV) from the World Reference Center for Emerging Viruses and Arboviruses (WRCEVA), University of Texas Medical Branch (UTMB). Laboratory-adapted JURV virus clones were generated by serial plaque purification in Vero cells (ATCC, Manassas, VA). RNA sequencing was applied to confirm the full-length JURV genome (10,993 bp) as previously described 4 , as described by Lawson et al. Infectious JURV was generated as described by Lawson et al. 14、The full-length cDNA clone (Genscript, USA) containing the genes encoding nucleoprotein (JURV-N), phosphoprotein (JURV-P), matrix protein (JURV-M), glycoprotein (JURV-G), and RNA-directed RNA polymerase L protein (JURV-L) was recovered. Vesicular stomatitis virus (VSV) was rescued from the pXN2 cDNA plasmid, and the virus stock was amplified in BHK-21 cells. Prior to in vitro and in vivo studies, purified virus particles (VSV, MORV, and recombinant JURV) were obtained using sucrose density gradient centrifugation.

[0095] Amplification of virus stock Virus amplification was performed by infecting confluent (approximately 80%) Vero cells in a T-175 flask with JURV, MORV, or VSV at a low multiplicity of infection (MOI) of 0.001. At 48 hours post-infection or when the cytopathic effect (CPE) became observable, the supernatant of virus-infected cells was collected from the flask. The virus stock was purified using 10 - 40% sucrose density gradient ultracentrifugation followed by dialysis. The titer (TCID 50 ) of each virus was determined by the Spearman - Karber algorithm using serial virus dilutions in BHK-21 cells.

[0096] Cell viability assay In all cytotoxicity assays (96-well format), 1.5×10 4 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). Cell viability was determined using the Cell Titer 96 AQueous One Solution cell proliferation assay (Promega Corp, Madison, Wisconsin, USA). Data were generated as the mean ± SEM of six replicates from two independent experiments.

[0097] Crystal violet assay In a 6-well plate, 500,000 cells were infected with tumor-lytic JURV at an MOI of 0.1 for 1 hour. The supernatant of the virus-infected cells was removed, the cells were washed with PBS, and incubated at 37°C until analysis. 72 hours after infection, the cells were fixed with 5% glutaraldehyde and stained with 0.1% crystal violet to visualize the cell morphological features and residual adhesion indicating cell viability. Photographs of representative areas were taken.

[0098] One-step virus growth kinetics 200,000 HCC cells were seeded into each well of a 6-well plate containing 2 mL of complete DMEM. After an overnight rest, the inventors infected the cells with JURV at an MOI of 0.1 for 1 hour. The supernatant of the virus-infected cells was removed, the cells were washed with PBS, and fresh medium was added. Supernatants were collected at 10, 24, 48, and 72 hours and stored at -80°C. The virus titer (TCID 50 ) was determined by serial dilution of the supernatant against Vero cells. Data were generated as the mean + / - SEM of two independent experiments.

[0099] Interferon sensitivity assay HCC cells were seeded into a 96-well plate at 2.0×10 4Cells were seeded at a density of cells / well and cultured overnight. Twenty-four hours after infection, the cells were pretreated with various concentrations of universal type I IFN-α directly added to the culture medium. After overnight incubation, fresh medium containing universal type I IFN-α (catalog number 11105-1; PBL Assay Science, USA) was added, and the cells were infected with JURV at an MOI of 0.01. Cell viability was evaluated using the Cell Titer 96 AQueous One Solution cell proliferation assay (Promega Corp, Madison, Wisconsin, USA). Absorbance measurements at 490 nm were normalized to the maximum reading per cell line representing 100% viability. Data from three independent experiments are shown. In all cell viability experiments, absorbance was read using a Cytation3 plate reader (BioTeK, Winooski, Vermont, USA). Data are presented as the mean + / − SEM of triplicates from three independent experiments.

[0100] Mouse Six- to eight-week-old female C57BL6 / J mice (strain number: 000664), BALB / cJ (strain number: 000651) mice, and NOD.Cg-Prkdc scid / J mice (strain number: 001303) were purchased from Jackson Laboratories. 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 sufficient staff of veterinarians and veterinary technicians who oversee and support the care of animals throughout the study. All animal experiments were conducted and approved in accordance with the regulations of the Institutional Animal Care and Use Committee at the University of Arkansas for Medical Sciences.

[0101] Analysis of virus-induced adverse events in mice Female C57BL / 6J mice (N = 6 mice / group) were administered phosphate-buffered saline (PBS), medium and high doses (1×10 7 TCID 50The virus of ( 8 TCID 50 ) or the virus at a high dose (1×10 ) was administered intranasally (25 μL per nostril) or intravenously (50 μL / mouse). To detect signs of toxicity, body weight, body temperature, behavior, and clinical signs were monitored by a board-certified veterinarian at least three times a week. However, three days after infection, three mice and tissues per group (blood, brain, liver, and spleen) were collected for short-term toxicity evaluation and in vivo virus distribution. The remaining mice were monitored for 30 days.

[0102] Short-term toxicological analysis of blood components On the third day after treatment, blood was collected from the submandibular vein (cheek bleed) and cardiac puncture. Blood was collected into BD Microtainer tubes (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) with ethylenediaminetetraacetic acid or lithium heparin for complete blood count (CBC), and into BD Microtainer SST tubes (Becton, Dickinson, and Company) for serum analysis. CBC analysis was performed on an Abaxis Piccolo Xpress chemistry analyzer (Abaxis, Union City, CA, USA), and blood chemistry analysis was performed on a VetScan HM5 blood analyzer (Abaxis).

[0103] Toxicoproteomics analysis Three days after JURV inoculation, mouse brain and liver tissues were collected, dehydrated using a gradually increasing ethanol concentration, embedded in paraffin, and formalin-fixed paraffin-embedded (FFPE) blocks were prepared as previously described. 126 The tissue blocks were sectioned into 3 - 5 10-μm sections and subjected to the deparaffinization procedure for FFPE tissues. 127 The FFPE samples were deparaffinized with xylene, the tissues were lysed with sodium dodecyl sulfate, and then the filter-aided sample preparation method 128It was used together with sequence-determination grade-modified porcine trypsin (Promega) to reduce, alkylate, and digest all proteins. Trypsin peptides were separated by reverse-phase XSelect CSH C18 2.5 μm resin (Waters) on an in-line 150×0.075 mm column using an UltiMate 3000 RSLCnano device (Thermo). Peptides were eluted using a 60-minute gradient from a 98:2 ratio to a 65:35 ratio (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 subsequently subjected to mass spectrometry (MS) using an Orbitrap Exploris 480 mass spectrometer (Thermo). MS data were obtained at a resolution of 120,000 over the range of 375 - 1500 m / z using a Fourier transform MS (FTMS) analyzer in profile mode. After HCD activation, the FTMS analyzer was used in centroid mode at a resolution of 15,000 and a normalized collision energy of 30% over the normal mass range to obtain MS / MS data. Proteins were identified by database search using the MaxQuant (Max Planck Institute) label-free quantification method with a parent ion tolerance of 2.5 ppm and a fragment ion tolerance of 20 ppm. Scaffold Q+S (Proteome Software) was used to validate MS / MS-based peptide and protein identification. Protein identification could be established with a false discovery rate of less than 1% and was accepted if it could contain at least two identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm 129 assigned by.

[0104] In vivo efficacy of oncolytic JURV in a syngeneic mouse model of HCC To evaluate the in vivo therapeutic efficacy of oncolytic JURV in a syngeneic mouse HCC model, 1×10 in 100 μL of chilled RPMI 6Individual HEPA1-6 cells were subcutaneously injected into the right flank of immunocompetent female C57BL6 / J mice (n = 7 / group; Jackson Laboratory) using a 1 mL syringe. Mice were monitored weekly for palpable tumors or any changes in appearance or behavior. Once the average tumor reached a treatable size (80 - 120 mm 3 ), the mice were randomized into their respective study groups and dosed within 24 hours of randomization. On days 0, 7, and 14, mice received a 50 μL IT injection of PBS (control group) or 1 × 10 7 TCID 50 units of JURV (test substance group). Groups receiving anti-PD-1 therapy or the combination of JURV + anti-PD-1 also received 50 μL of anti-PD-1 antibody intraperitoneally (IP) twice weekly for 3 weeks. To establish syngeneic bilateral (dual flank) HCC tumors, first HEPA1-6 cells (1 × 10 6 cells / mouse) were subcutaneously grafted into the right flank. These became tumors in approximately 14 days and were classified as "primary" tumors. At the same time, the inventors performed a remote HEPA1-6 tumor graft injection (1 × 10 6 cells / mouse) into the left flank of these mice. Mice in the dual flank group received only a 50 μL IT injection of 1 × 10 7 TCID 50 units of JURV once weekly for 3 weeks. After randomization and initiation of treatment, tumor volume and body weight were measured twice weekly using digital calipers and a balance. Tumor volume was calculated using digital calipers with the following formula: (longest diameter * shortest diameter2) / 2. During the first week of treatment and after each injection, mice were monitored daily for up to 72 hours for signs of recovery. If weight loss exceeded 20%, if tumor size exceeded 2,000 m 3 , or if there were side effects from the treatment, the mice were euthanized. Mice were sacrificed 28 days after the first JURV dose administration, at which time tumors and blood were collected for downstream analysis.

[0105] HEP3B xenograft model Female NOD.Cg-Prkdc scidHep3B cells tagged with the firefly luciferase reporter gene were subcutaneously inoculated into the right flank of female BALB / cJ mice (n = 6 / group). When the average tumor volume reached 80 - 120 mm 3 3, 1 × 10 7 TCID 50 of JURV in 50 μL or 50 μL of PBS was administered to the mice once a week for 3 weeks. Tumor volume was measured twice a week until the end of the study (day 21) or the humane endpoint described above. The inventors also recorded the mouse body weight and clinical observations twice a week.

[0106] Antitumor effect of JURV against multiple solid tumors EMT6 (breast cancer) cells, CT26 (colon cancer) cells, and A20 (reticulum cell sarcoma) cells were subcutaneously inoculated into the right flank of female BALB / cJ mice (n = 6 - 8 / group). B16F10 melanoma cells were transplanted into the right flank of female C57BL6 / J mice. RM-1 (prostate cancer) cells were grafted into the right flank of male C57BL6 / J mice. When the average tumor volume reached 80 - 120 mm 3 3, 1 × 10 7 TCID 50 of JURV in a single 50 μL or 50 μL of PBS was administered to the mice (n = 6 - 8 / group). Tumor volume was measured twice a week until the end of the study (day 21) or the humane endpoint described above. The inventors also recorded the mouse body weight and clinical observations twice a week.

[0107] Bioluminescence imaging Tumor-bearing (HEP3B) mice were anesthetized with isoflurane, and virus-induced changes in tumor growth were imaged once a week (day 0, day 7, day 14) using an IVIS Xenogen imaging device. Anesthesia was induced in an induction chamber (2 - 5% isoflurane), and then the mice were placed inside the imaging instrument, and a nose cone connected to a vaporizer was attached to maintain isoflurane (1.5 - 2%) during the procedure. This concentration range provides an anesthetic level that prevents movement of the animals during scanning. If the respiratory rate increases or decreases, the isoflurane concentration is increased or decreased. The inventors used a heated animal bed, heated pads, and, if necessary, a heating lamp to ensure that body temperature was maintained both before and during imaging. Each mouse received an intraperitoneal injection of D-luciferin (Sigma-Aldrich #L9504; 150 mg / kg body weight in a volume of 10 μl / g body weight, prepared in sterile water). The stomach of the anesthetized mouse was placed in the IVIS Xenogen imaging device. The time required to image the mice in each group was less than 10 minutes. This was a non-invasive imaging procedure, and the inventors did not require restraint.

[0108] Analysis of tumor-infiltrating immune cells Hepa1-6 tumors (n = 3 samples / group) were excised and dissociated using a mouse tumor dissociation kit (Miltenyi, catalog number 130-096-730) together with a gentleMACS™ Octo Dissociator (Miltenyi) according to the manufacturer's protocol. Mouse CD45 (TIL) microbeads (Miltenyi) were used to isolate CD45 +Cells were isolated. The cells were incubated with Fixable Viability Stain 510 for 15 minutes at 4°C and then incubated with an anti-Fc blocking reagent (Biolegend, catalog number 101320) for 10 minutes prior to surface staining. The cells were stained and then data were obtained using a BD LSRFortessa X-20 flow cytometer. All antibodies were used according to the manufacturer's recommendations. Fluorescence Minus One controls were used in each independent experiment to establish gating. For intracellular staining of granzyme B, the cells were stained using an intracellular staining kit (Miltenyi) and analyzed using FlowJo™ (TreeStar). Forward scatter and side scatter cytometry were used to exclude cell debris and doublets.

[0109] Flow Cytometry Antibody Analysis 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), I-A / I-E-BV786 (Catalog No. 743875, BD Biosciences), and L / D-efluor780 (Catalog No. 65-0865-18, eBioscience). The complete list of antibodies used can be found in Table 5.

[0110]

Table 5

[0111] RNA Sequencing of Mouse HCC Tumors Hepa1-6 (n = 3 samples / group) FFPE scrolls were processed using the Quick-DNA / RNA FFPE Miniprep Kit (Catalog No. R1009, Zymo Research) with on-column DNase digestion for DNA and RNA extraction. The mass concentration of RNA was evaluated using the Qubit RNA Broad Range Assay Kit (Catalog No. Q10211; Invitrogen) with a Qubit 4 fluorometer (Catalog No. Q33238; Invitrogen). RNA quality was evaluated using a standard sensitivity RNA analysis kit (Catalog No. DNF-471-0500; Agilent) with a Fragment Analyzer system (Catalog No. M5310AA; Agilent). Sequencing libraries were prepared using TruSeq Stranded Total RNA Library Prep Gold (Catalog No. 20020599; Illumina). Fragmentation time was determined using the RNA DV200 score. Libraries were evaluated using the Qubit 1X dsDNA HS Assay Kit (Catalog No. Q33231; Invitrogen) with a Qubit 4 fluorometer (Catalog No. Q33238; Invitrogen). Library fragment size was evaluated using a high-sensitivity NGS fragment analysis kit (Catalog No. DNF-474-0500; Agilent) with a Fragment Analyzer system (Catalog No. M5310AA; Agilent). Libraries were functionally verified using the KAPA Universal Library Quantification Kit (Catalog No. 07960140001; Roche). Sequencing was performed using a 200-cycle S1 flow cell of the NovaSeq 6000 sequencer (Illumina) to generate paired-end reads (2 × 100 bp).

[0112] Bioinformatics analysis The inventors investigated the mRNA and protein expression profiles of Hepa1-6 tumors treated with PBS, JURV, anti-PD-1, or JURV + anti-PD-1. Three replicates were used for each of the untreated group (PBS) and the treatment groups. Tumor samples were sequenced on an NGS platform. Subsequently, files containing sequencing reads (FASTQ) were examined using MultiQC for quality control (QC). 130 The Cutadapt tool trims off the Illumina adapter and low-quality bases at the end. After quality control, reads were aligned to the mouse reference genome (mm10 / GRCm38) using the HISAT2 aligner. 131 Subsequently, reads mapped to RefSeq genes with features were counted. The inventors created a count matrix from the sequence reads using HTSeq-count. 132 Genes with low counts across samples affect the false discovery rate and thus reduce the ability to detect differentially expressed genes. Therefore, prior to identifying differentially expressed genes, the inventors excluded genes with low expression using the module of the limma-voom tool. 45 Subsequently, the inventors normalized the counts by using TMM normalization, which is the weighted trimmed mean of log-expression ratios used to scale the counts of samples. 133 Finally, the inventors fit a linear model with limma, determined the differentially expressed genes, and represented the data as mean ± standard error of the mean. All p-values were corrected using Benjamini-Hochberg FDR adjustment for multiple comparisons. After identifying the differentially expressed genes, an Ingenuity Pathway Analysis tool was used to perform enriched pathways to gain biological insights. Statistical differences between groups were evaluated using the non-parametric Mann-Whitney U test R module.

[0113] Integration of transcriptomics and proteomics The limma-normalized transcript expression levels and normalized protein intensities were integrated using two independent methods. First, as previously described 134 , the mixOmics package (Omics Data Integration Project R package, version 6.1.1) was implemented to create a heatmap of the relevant DEP / DEGs. Next, the MOGSA package was used to create a heatmap of the top 30 up- or down-regulated DEP / DEGs among the various groups 135 .

[0114] Blood chemistry and cytokines Blood chemistry analysis was performed using an Abaxis Piccolo Xpress chemistry analyzer (Abaxis) to evaluate hepatotoxicity (i.e., aspartate transaminase, alkaline phosphatase, albumin), nephrotoxicity (i.e., creatinine, blood urea nitrogen), and serum electrolytes. A mouse IFN-beta SimpleStep ELISA kit (catalog number ab252363; Abcam) was used to perform a mouse type I interferon-beta assay.

[0115] TUNEL assay immunohistochemistry Tumor tissue sections were subjected to the terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling (TUNEL) assay using an In Situ Cell Death Detection Kit (Roche Diagnostics, Indianapolis, Indiana) according to the manufacturer's protocol. After staining, cells were counterstained with 4’,6-diamidino-2-phenylindole (DAPI) to visualize cell nuclei, mounted under a coverslip with Prolong® Antifade Kit (Invitrogen, Carlsbad, California), and data were acquired using an Olympus IX-81 inverted microscope (Olympus America, Center Valley, Pennsylvania) equipped with a Hamamatsu ORCA-ER monochrome camera (Hamamatsu Photonics K.K., Hamamatsu City, Japan). Image analysis was performed using SlideBook6.2 software. For quantification, 10 independent fields were collected for each well (each n), and the mean optical density (MOD) of the fluorescein (TUNEL) channel or the co-localized pixel area was recorded.

[0116] Statistical analysis All values were expressed as mean ± standard error of the mean, and the results were analyzed using the statistical software of GraphPad Prism version 8 (GraphPad Software) by one-way analysis of variance, followed by Tukey's test or Benjamini-Hochberg FDR adjustment for multiple comparisons, and t-test for comparing group means, and the Kaplan-Meier method for survival rates. A p-value of less than 0.05 was considered statistically significant.

[0117] References JPEG2025522278000009.jpg142163 JPEG2025522278000010.jpg207163 JPEG2025522278000011.jpg216160 JPEG2025522278000012.jpg207162 JPEG2025522278000013.jpg215163 JPEG2025522278000014.jpg215161 JPEG2025522278000015.jpg214163 JPEG2025522278000016.jpg214162 JPEG2025522278000017.jpg201162 JPEG2025522278000018.jpg213164 JPEG2025522278000019.jpg100164

Claims

**Claim 1** A construct comprising a promoter operably linked to a polynucleotide encoding a full-length antisense Jun virus genome, which enables the production of a negative-sense virus genome when transfected into mammalian cells. **Claim 2** The construct according to claim 1, wherein the promoter is a T7 promoter. **Claim 3** The construct according to claim 1 or 2, wherein the polynucleotide encoding the Jun virus genome comprises SEQ ID NOs: 1 to 5. **Claim 4** The construct according to any one of claims 1 to 3, wherein the polynucleotide encoding the Jun virus genome further comprises a leader sequence of SEQ ID NO: 6 and / or a trailer sequence of SEQ ID NO:

7. **Claim 5** The construct according to any one of claims 1 to 4, wherein the polynucleotide encoding the Jun virus genome further comprises at least one of SEQ ID NOs: 8 to 11, 21, and 22 as an intergenic region. **Claim 6** The construct according to claim 1 or 2, wherein the polynucleotide encoding the Jun virus genome comprises SEQ ID NO: 12 (JURV-XN-2) or a sequence having at least 95% identity with SEQ ID NO:

12. **Claim 7** The construct according to any one of claims 1 to 5, wherein the polynucleotide encoding the Jun virus genome further comprises a heterologous polynucleotide capable of encoding a polypeptide not naturally associated with the Jun virus. **Claim 8** The construct according to claim 7, wherein the polypeptide is a reporter polypeptide. **Claim 9** The construct according to claim 8, wherein the reporter polypeptide is a fluorescent protein. **Claim 10** The construct according to claim 9, wherein the polynucleotide comprises SEQ ID NO: 13 (JURV-eGFP) or a sequence having at least 95% identity with SEQ ID NO:

13. **Claim 11** A construct comprising a codon-optimized polynucleotide encoding at least one Jun virus protein selected from the group consisting of glycoprotein (G), nucleoprotein (N), phosphoprotein (P), RNA-directed RNA polymerase L protein (L), and matrix protein (M), operably linked to a promoter for expression in mammalian cells. **Claim 12** The construct according to claim 11, wherein the polynucleotide encodes the G protein and contains SEQ ID NO:

4.

13. The construct according to claim 11, wherein the polynucleotide encodes the N protein and contains SEQ ID NO:

1.

14. The construct according to claim 11, wherein the polynucleotide encodes the P protein and contains SEQ ID NO:

2.

15. The construct according to claim 11, wherein the polynucleotide encodes the M protein and contains SEQ ID NO:

3.

16. The construct according to claim 11, wherein the polynucleotide encodes the L protein and contains SEQ ID NO:

5.

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

18. A cell comprising at least one of the constructs 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 to 10 and at least two of the constructs according to claims 11 to 17.

20. An infectious particle comprising the negative-sense RNA of SEQ ID NO: 12 or a Juno virus genome having 95% identity with SEQ ID NO:

12.

21. An infectious particle produced by transfecting a cell with the 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 to 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 a therapeutically effective amount of a Juno virus or the composition according to claim 22 to the subject to treat the cell proliferative disease or disorder.

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

12.

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

26. The method according to claim 25, wherein the cancer is selected from hepatocellular carcinoma, cholangiocarcinoma, breast cancer, colorectal cancer, prostate cancer, and reticulosarcoma.

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

28. The method according to claim 27, wherein the breast cancer is HER2-negative.

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

30. The method according to any one of claims 23 to 29, wherein the Durovirus or composition is administered intratumorally to a local tumor.

31. The method according to any one of claims 23 to 30, wherein the cancer is metastatic.

32. The method according to any one of claims 23 to 31, wherein the immune system of the subject is suppressed.

33. The method according to any one of claims 23 to 32, wherein the Durovirus or composition is administered intranasally, intramuscularly, intratumorally, intravenously, or subcutaneously.

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

35. The method according to claim 34, wherein the immunotherapy is checkpoint inhibitor therapy.

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

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

38. The method according to any one of claims 23 to 37, further comprising the step of administering a receptor tyrosine kinase inhibitor.

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

40. A method for generating a recombinant Durovirus, comprising introducing at least one of the constructs according to claims 1 to 10 into a cell; enabling the cell to express one or more Durovirus proteins selected from the group consisting of G, M, N, L, and P; incubating the cell to generate a recombinant Durovirus; and recovering the virus produced by the cell.

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

42. The method according to claim 40 or 41, wherein the one or more Durovirus proteins comprise Durovirus N, P, and L proteins.

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

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

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

46. The method according to any one of claims 40 to 45, wherein the construct further comprises a heterologous polynucleotide encoding a protein not naturally associated with rhabdovirus.

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

48. The system according to claim 47, further comprising mammalian cells that enable expression of the rhabdovirus protein of (a) and the negative-sense rhabdovirus genome of (b) to produce the recombinant rhabdovirus.

49. The system according to claim 48, wherein the mammalian cells comprise T7 RNA polymerase.

50. The system according to claim 48 or 49, wherein the mammalian cells are BHK-1 cells.

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

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

53. The system according to any one of claims 47 to 52, wherein the polynucleotide encoding the at least three deltavirus proteins is codon-optimized for expression in mammalian cells.

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

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

56. The system according to any one of claims 47 to 55, wherein the one or more constructs encoding at least three deltavirus proteins comprise the construct according to any one of claims 11 to 17.

57. A kit comprising at least one of the constructs according to any one of claims 1 to 17.

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

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

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

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

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