Oncolytic vaccinia virus and recombinant virus and methods of using the same

Abstract

Provided herein are cloned strains of vaccinia virus that exhibit enhanced anti-tumor properties and / or reduced immunogenicity, and recombinant vaccinia viruses derived therefrom. Also provided herein are recombinant oncolytic virus strains that contain inactivated mutations in one or more viral genes, and / or one or more heterologous nucleic acids that each encode one or more heterologous gene products. The viruses provided herein, such as vaccinia virus including recombinant vaccinia virus, can be used as oncolytic virus therapy, such as oncolytic vaccinia virus therapy, for treating cancer. Also provided herein are pharmaceutical compositions, methods, and uses of viruses, such as vaccinia virus, for treating cancer, and nucleic acids encoding the viruses. TIFF2025522951000024.tif128143

Description

Technical Field

[0001] Cross - reference to related applications This application claims priority to U.S. Provisional Application No. 63 / 368,029, filed on July 8, 2022, entitled "ONCOLYTIC VACCINIA VIRUSES AND RECOMBINANT VIRUSES AND METHODS OF USE THEREOF", the entire content of which is incorporated by reference for all purposes.

[0002] Incorporation by reference of the Sequence Listing This application is filed together with a sequence listing in electronic format. The sequence listing is provided as a file entitled 773192000140SeqList.xml created on July 6, 2023, and its size is 6,262,578 bytes. The electronic format information of the sequence listing is incorporated by reference in its entirety.

[0003] Field The present disclosure provides cloned strains of vaccinia virus that exhibit enhanced antitumor properties and / or reduced immunogenicity, as well as recombinant vaccinia viruses derived therefrom. The vaccinia viruses of the present disclosure, including the recombinant vaccinia viruses, can be used as an oncolytic vaccinia virus therapy for treating cancer. The present disclosure also provides pharmaceutical compositions, methods, and uses of vaccinia viruses for treating cancer.

Background Art

[0004] Background Vaccinia is an oncolytic virus that accumulates in tumors. In some cases, an oncolytic virus (OV) is a virus that replicates selectively or more efficiently within cancer cells than in non-cancer cells. Oncolytic vaccinia viruses include recombinant viruses engineered from natural viruses by gene disruption or gene addition to improve anti-tumor properties such as tumor selectivity or preferential replication within tumor cells, host tropism, surface attachment, lysis, and spread. Among such recombinant vaccinia viruses are attenuated viruses in which one or more viral genes are modified, resulting in loss or reduced expression of the viral gene or inactivation of the viral protein. However, the effectiveness of oncolytic viruses is hampered by the strong immune response induced by the virus. Immune factors such as antibodies neutralize the virus by binding directly to the virus and preventing successful cell infection or by labeling the virus for destruction by complement or other immune cells. Therefore, there is still a need for an improved oncolytic vaccinia virus with reduced ability to induce antiviral defense and enhanced anti-tumor activity.

Summary of the Invention

[0005] Summary Provided herein is a recombinant oncolytic vaccinia virus comprising an inactivating mutation of B2R, a heterologous nucleic acid encoding interferon regulatory factor 3 (IRF3), and at least one heterologous nucleic acid encoding one or more cytokines and / or chemokines.

[0006] In some embodiments, at least one heterologous nucleic acid encoding one or more cytokines and / or chemokines comprises a heterologous nucleic acid encoding chemokine ligand 9 (CXCL9) and / or IL-12. In some of any such embodiments, at least one heterologous nucleic acid encoding one or more cytokines and / or chemokines is a heterologous nucleic acid encoding chemokine ligand 9 (CXCL9). In some of any such embodiments, at least one heterologous nucleic acid encoding one or more cytokines and / or chemokines is a heterologous nucleic acid encoding IL-12. In some of any embodiments, at least one heterologous nucleic acid encoding one or more cytokines and / or chemokines is a heterologous nucleic acid encoding CXCL9 and IL-12. In some embodiments, at least one heterologous nucleic acid encoding one or more cytokines and / or chemokines is a heterologous nucleic acid encoding CXCL9 and a heterologous nucleic acid encoding IL-12.

[0007] In some of any embodiments, CXCL9 is human CXCL9. In some embodiments, CXCL9 comprises the amino acid sequence set forth in SEQ ID NO:99, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:99. In some embodiments, the amino acid sequence of CXCL9 is set forth in SEQ ID NO:99. In some of any embodiments, CXCL9 is mouse CXCL9. In some embodiments, CXCL9 comprises the amino acid sequence set forth in SEQ ID NO:106, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:106. In some embodiments, the amino acid sequence of CXCL9 is set forth in SEQ ID NO:106.

[0008] In some embodiments of any aspect, IL-12 is human single-chain IL-12. In some embodiments, the single-chain IL-12 is composed of a human IL-12A (p35) subunit and a human IL-12B (p40) subunit optionally separated by a linker. In some embodiments, the single-chain IL-12 comprises the amino acid sequence set forth in SEQ ID NO:103, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:103. In some embodiments, the amino acid sequence of the single-chain IL-12 is set forth in SEQ ID NO:103. In some embodiments of any aspect, IL-12 is mouse single-chain IL-12. In some embodiments, the single-chain IL-12 is composed of a mouse IL-12A (p35) subunit and a mouse IL-12B (p40) subunit optionally separated by a linker. In some embodiments, the single-chain IL-12 comprises the amino acid sequence set forth in SEQ ID NO:102, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:102. In some embodiments, the amino acid sequence of the single-chain IL-12 is set forth in SEQ ID NO:102.

[0009] In some embodiments of any aspect, at least one heterologous nucleic acid encoding one or more cytokines and / or chemokines comprises a heterologous nucleic acid encoding IL-2. In some embodiments, IL-2 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 98, 100, 101, 104, and 105, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 98, 100, 101, 104, and 105. In some embodiments, IL-2 is set forth in SEQ ID NO: 105. In some embodiments, IL-2 is a superkine of the sequence set forth in SEQ ID NO: 105, or a sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 105.

[0010] In some embodiments of any aspect, IL-2 is an IL-2 supercytokine. In some embodiments, the IL-2 supercytokine is H9, H9T, MDNA11 or MDNA11T. In some embodiments of any aspect, the H9 IL-2 supercytokine comprises the amino acid sequence of SEQ ID NO: 100 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 100. In some embodiments of any aspect, the H9T IL-2 supercytokine comprises the amino acid sequence of SEQ ID NO: 104 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 104. In some embodiments of any aspect, the MDNA11 IL-2 supercytokine comprises the amino acid sequence of SEQ ID NO: 101 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 101. In some embodiments of any aspect, the MDNA11T IL-2 supercytokine comprises the amino acid sequence of SEQ ID NO: 98 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 98. In some embodiments of any aspect, the IL-2 supercytokine is MDNA11T, and MDNA11T comprises the amino acid sequence set forth in SEQ ID NO: 98 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 98.

[0011] In some embodiments of any aspect, the recombinant oncolytic virus further comprises one or more heterologous gene products selected from the group consisting of a complement inhibitor, a T cell evader or NK cell evader, an immune stimulatory protein, an anti-angiogenic protein, an interferon regulatory factor, an apoptosis-inducing protein, or any combination of the foregoing.

[0012] In some of any aspect, the inactivating mutation of B2R is a deletion of all or part of the B2R locus. In some of any aspect, the deletion is sufficient to render the encoded B2R gene product non-functional. In some aspects, the inactivating mutation of B2R is one or more amino acid substitutions in the encoded gene product. In some of any aspect, the inactivating mutation of B2R is characterized by the insertion of a heterologous nucleic acid into the B2R locus, for example, instead of a deletion of all or part of the B2R locus. In some aspects, the heterologous nucleic acid encodes IRF3 or a cytokine and / or chemokine. In some of any aspect, the inactivating mutation of B2R is due to the insertion of a heterologous nucleic acid encoding IRF3 into the B2R locus and / or at least one insertion of at least one of the heterologous nucleic acids encoding one or more cytokines and / or chemokines. In some of any aspect, the inactivating mutation of B2R is characterized by the insertion of a heterologous nucleic acid encoding chemokine ligand 9 (CXCL9) and / or IL-12 into the B2R locus.

[0013] In some of any aspects, the heterologous nucleic acid encoding IRF3 is inserted into the hemagglutinin (HA), J2R (thymidine kinase), F14.5L, A56R (hemagglutinin), vaccinia growth factor (VGF), A35R, A49R, A55R, B14R, C4L, C6L, C16L, NIL / N2L, E2L / E3L, K1L / K2L, K7L, superoxide dismutase locus, 7.5K, C2L-F3L, C4L-F1L, C7-K1L, B13R + B14R, A26L or I4L locus within the viral genome. In some of any aspects, at least one of at least one heterologous nucleic acid encoding one or more cytokines and / or chemokines is inserted into the HA, J2R, F14.5L, A56R, vaccinia growth factor, A35R, A49R, A55R, B14R, C4L, C6L, C16L, NIL / N2L, E2L / E3L, K1L / K2L, K7L, superoxide dismutase locus, 7.5K, C2L-F3L, C4L-F1L, C7-K1L, B13R + B14R, A26L or I4L locus within the viral genome. In some of any such aspects, the insertion is in place of a deletion of all or part of each locus.

[0014] In some of any aspects, the nucleic acid genome of the recombinant oncolytic vaccinia virus is modified from a parental vaccinia virus having a nucleic acid genome with at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:1. In some aspects, the nucleic acid genome of the recombinant oncolytic vaccinia virus is modified from a parental vaccinia virus having the nucleic acid genome set forth in SEQ ID NO:1.

[0015] In some of any aspect, the nucleic acid genome of the parental vaccinia virus is (i) a variant 017 open reading frame (ORF) encoding an amino acid sequence having at least 95% sequence identity to SEQ ID NO:57 and having a polar uncharged amino acid at position 66, optionally threonine (T) at position 66; (ii) a variant 038 (K5L) ORF comprising a nucleotide insertion that results in a frameshift mutation and in which the 038 (K5L) gene product is modified; (iii) a variant 059 (E2L) ORF having at least 95% sequence identity to SEQ ID NO:60 and encoding an amino acid sequence having a hydrophobic amino acid other than leucine at position 419, optionally phenylalanine (F) at position 419; (iv) a variant 104 (H4L) ORF having at least 95% sequence identity to SEQ ID NO:61 and encoding an amino acid sequence having a charged amino acid at position 591, optionally aspartic acid (D) at position 591; and (v) characterized by one or more of a variant 182 (A56R) ORF comprising a deletion of two nucleotides that results in a frameshift mutation and in which the 182 (A56R) ORF gene product is modified.

[0016] In some of any embodiments, the nucleic acid genome of the parental virus is characterized by one or more of the following: (i) guanine (G) at the position corresponding to position 7770 of SEQ ID NO: 1; (ii) thymine (T) at the position corresponding to position 15261 of SEQ ID NO: 1; (iii) G at the position corresponding to position 32136 of SEQ ID NO: 1; (iv) G at the position corresponding to position 49455 of SEQ ID NO: 1; (v) cytosine (C) at the position corresponding to position 92969 of SEQ ID NO: 1; (vi) the nucleic acid sequence CACTTATATAT at the position corresponding to positions 106870-106880 of SEQ ID NO: 1; (vii) the nucleic acid sequence GTTTTCATTA at the position corresponding to positions 111267-111276 of SEQ ID NO: 1; (viii) adenine (A) at the position corresponding to position 162715 of SEQ ID NO: 1; (ix) the nucleic acid sequence TACAGACACC at the position corresponding to positions 165844-185853 of SEQ ID NO: 1; and (x) C at the position corresponding to position 187805 of SEQ ID NO: 1.

[0017] In some embodiments of any aspect, the nucleic acid genome of the recombinant oncolytic vaccinia virus has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:1. In some embodiments of any aspect, the nucleic acid genome of the recombinant oncolytic vaccinia virus has at least 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:1. In some embodiments of any aspect, the nucleic acid genome of the recombinant oncolytic vaccinia virus has at least 95% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:1. In some embodiments of any aspect, the nucleic acid genome of the recombinant oncolytic vaccinia virus has at least 96% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:1. In some embodiments of any aspect, the nucleic acid genome of the recombinant oncolytic vaccinia virus has at least 97% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:1. In some embodiments of any aspect, the nucleic acid genome of the recombinant oncolytic vaccinia virus has at least 98% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:1. In some embodiments of any aspect, the nucleic acid genome of the recombinant oncolytic vaccinia virus has at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:1.

[0018] In some embodiments of any aspect, the heterologous nucleic acid encoding IRF3 is inserted into the J2R (thymidine kinase) locus within the viral genome, and at least one heterologous nucleic acid encoding one or more cytokines and / or chemokines includes the heterologous nucleic acids encoding CXCL9 and IL-12, and the heterologous nucleic acids encoding CXCL9 and IL-12 are inserted into the A56R locus within the viral genome.

[0019] In some of any embodiments, the nucleic acid genome of the recombinant oncolytic vaccinia virus comprises a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence of SEQ ID NO:85, or the nucleic acid sequence described in SEQ ID NO:85. In some embodiments, the nucleic acid genome of the recombinant oncolytic vaccinia virus is described in SEQ ID NO:85.

[0020] In some of any embodiments, the heterologous nucleic acid encoding IRF3 is inserted into the B2R (viral cGAMP-specific nuclease) locus within the viral genome, and at least one heterologous nucleic acid encoding one or more cytokines and / or chemokines comprises the heterologous nucleic acids encoding CXCL9 and IL-12, and the heterologous nucleic acids encoding CXCL9 and IL-12 are inserted into the A56R locus within the viral genome.

[0021] In some of any embodiments, the recombinant oncolytic vaccinia virus further comprises a heterologous nucleic acid encoding an apoptosis-inducing protein. In some embodiments, the apoptosis-inducing protein is an inducible death effector domain (iDED). In some of any embodiments, the iDED comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:27, or the amino acid sequence described in SEQ ID NO:27. In some embodiments, the iDED is described in SEQ ID NO:27. In some of any embodiments, the heterologous nucleic acid encoding the iDED is inserted into or instead of the J2R locus within the viral genome.

[0022] In some embodiments of any aspect, the nucleic acid genome of the recombinant oncolytic vaccinia virus comprises a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence of SEQ ID NO:86, or the nucleic acid sequence described in SEQ ID NO:86. In some embodiments, the nucleic acid genome of the recombinant oncolytic vaccinia virus is described in SEQ ID NO:86.

[0023] In some embodiments of any aspect, the recombinant oncolytic vaccinia virus further comprises a heterologous nucleic acid encoding one or more T cell evasion factor proteins or NK cell evasion factor proteins. In some embodiments, the one or more T cell evasion factor proteins or NK cell evasion factor proteins comprise a set of proteins encoded by vaccinia virus ORFs 012, 203 and 018 (CPXV012-203-018). In some embodiments, the set of proteins encoded by CPXV012-203-018 comprises (i) an amino acid sequence described in SEQ ID NO:20 (CPXV012), or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence described in SEQ ID NO:20, (ii) an amino acid sequence described in SEQ ID NO:21 (CPXV0203), or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence described in SEQ ID NO:21, and (iii) an amino acid sequence described in SEQ ID NO:22 (CPXV018), or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence described in SEQ ID NO:22.

[0024] In some of any aspects, the recombinant oncolytic vaccinia virus further comprises a heterologous nucleic acid encoding a complement inhibitor. In some aspects, the complement inhibitor is Borrelia burgdorferi complement regulatory-acquiring surface protein-2 (CRASP-2). In some aspects, the heterologous nucleic acid encoding CRASP-2 produces a fusion gene encoding a fusion protein and is fused with a viral membrane gene, optionally F14.5L, to produce the fusion protein. In some of any aspects, the fusion protein comprises CRASP-2 fused to a viral membrane protein encoded by the viral membrane gene. In some of any aspects, the viral membrane protein is F14.5L. In some aspects, the fusion is a fusion at the C-terminus of F14.5L.

[0025] In some of any aspects, the nucleic acid genome of the recombinant oncolytic vaccinia virus comprises a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence of SEQ ID NO:90, or the nucleic acid sequence described in SEQ ID NO:90. In some aspects, the nucleic acid genome of the recombinant oncolytic vaccinia virus is described in SEQ ID NO:90.

[0026] In some of any aspects, the heterologous nucleic acid encoding IRF3 is inserted into or instead of the B2R (viral cGAMP-specific nuclease) locus within the viral genome, and at least one heterologous nucleic acid encoding one or more cytokines and / or chemokines comprises a heterologous nucleic acid encoding IL-2, and IL-2 is the IL-2 supercytokine MDNA11T.

[0027] In some of any aspects, the recombinant oncolytic vaccinia virus further comprises a heterologous nucleic acid encoding an immunostimulatory protein and / or a heterologous nucleic acid encoding one or more anti-angiogenic proteins. In some aspects, the immunostimulatory protein is recombinant LIGHT. In some aspects, the recombinant LIGHT comprises the amino acid sequence set forth in SEQ ID NO:30, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:30. In some aspects, the recombinant LIGHT has the sequence set forth in SEQ ID NO:30.

[0028] In some of any aspects, one or more anti-angiogenic proteins comprise a VEGF inhibitor, an angiopoietin inhibitor, versikine, or a fusion protein of any two or more of the foregoing. In some of any aspects, one or more anti-angiogenic proteins comprise an anti-VEGF antibody and / or an anti-Ang2 antibody. In some of any aspects, one or more anti-angiogenic proteins are bispecific anti-VEGF / anti-Ang2 antibodies. In some aspects, the bispecific anti-VEGF / anti-Ang2 antibody comprises the amino acid sequence set forth in SEQ ID NO:23, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO:23. In some aspects, the bispecific anti-VEGF / anti-Ang2 antibody has the sequence set forth in SEQ ID NO:23.

[0029] In some of any aspects, the nucleic acid genome of the recombinant oncolytic vaccinia virus comprises the nucleic acid sequence of SEQ ID NO:88, or a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:88. In some aspects, the nucleic acid genome of the recombinant oncolytic vaccinia virus is set forth in SEQ ID NO:88.

[0030] In some of any aspects, one or more of the heterologous nucleic acids encoding any of the above heterologous gene products (e.g., IRF3, cytokines, chemokines or other heterologous gene products) are operably linked to a promoter. In some aspects, each of the one or more heterologous nucleic acids encoding a heterologous gene product is operably linked to a promoter. In some aspects, the promoter is selected from the group consisting of 7.5E, 7.5E / L, SSE, 11KL, SSL, SSEL, mH5 and LEO. In some of any aspects, each heterologous nucleic acid encoding a heterologous gene product is independently operably linked to a promoter, and optionally, each heterologous nucleic acid encoding a heterologous gene product is independently operably linked to a promoter selected from the group consisting of 7.5E, 7.5E / L, SSE, 11KL, SSL, SSEL, mH5 and LEO. In some of any aspects, the promoter is a poxvirus promoter or a variant or derivative thereof. In some of any aspects, the promoter is a vaccinia virus promoter. In some of any aspects, the promoter is selected from the group consisting of 7.5E, 7.5E / L, SSE, 11KL, SSL, SSEL, mH5 and LEO. In some of any aspects, the promoter has an amino acid sequence set forth in any one of SEQ ID NO:29, 53, 55, 68, 69, 70, 71 or 72. In some of any aspects, the promoter is a synthetic strong early promoter (SSE). In some of any aspects, the promoter comprises the sequence set forth in SEQ ID NO:29. In some of any aspects, the promoter is a strong early / late promoter (SEL). In some of any aspects, the promoter comprises the sequence set forth in SEQ ID NO:55. In some of any aspects, the promoter is mH5. In some of any aspects, the mH5 promoter comprises the sequence set forth in SEQ ID NO:53.

[0031] A recombinant oncolytic virus comprising an inactivating mutation of at least one viral gene and at least one heterologous nucleic acid encoding one or more heterologous gene products, wherein the one or more heterologous gene products are immunomodulatory proteins, complement inhibitors, T cell evasion factors or NK cell evasion factors, anti-angiogenic proteins, interferon regulatory factors or apoptosis-inducing proteins, or any combination of the foregoing or include them, is also provided herein.

[0032] In some of any aspect, the oncolytic virus is vaccinia virus, herpes simplex virus, vesicular stomatitis virus (VSV), Maraba virus (MARAV), measles virus (MV), adenovirus, myxoma virus, orf virus, parvovirus, araigpox virus, coxsackievirus, reovirus, Newcastle disease virus, Seneca Valley virus, Semliki Forest virus, mumps virus, influenza virus, echovirus, and poliovirus (PV).

[0033] In some of any aspect, the oncolytic virus is vaccinia virus. In some of any aspect, the nucleic acid genome of the recombinant oncolytic vaccinia virus is modified from a parental vaccinia virus having a nucleic acid genome with at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:1. In some of any aspect, the nucleic acid genome of the recombinant oncolytic vaccinia virus is modified from a parental vaccinia virus having a nucleic acid genome having the nucleic acid genome set forth in SEQ ID NO:1.

[0034] Also provided herein are recombinant oncolytic viruses comprising at least one heterologous nucleic acid encoding one or more heterologous gene products, wherein the one or more heterologous gene products are complement inhibitors, T cell evasion factors or NK cell evasion factors, immunomodulatory proteins, anti-angiogenic proteins, interferon regulatory factors, apoptosis-inducing proteins, or combinations thereof or include them.

[0035] Also provided herein are recombinant oncolytic viruses comprising a nucleic acid genome modified from a parental vaccinia virus genome having at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:1 and comprising at least one heterologous nucleic acid encoding one or more heterologous gene products inserted into the genome. Also provided herein are recombinant oncolytic viruses comprising a nucleic acid genome modified from a parental vaccinia virus genome having the nucleic acid sequence set forth in SEQ ID NO:1 and comprising at least one heterologous nucleic acid encoding one or more heterologous gene products inserted into the genome.

[0036] In some of any aspect, the nucleic acid genome of the parental vaccinia virus is: (i) a variant 017 open reading frame (ORF) encoding an amino acid sequence having at least 95% sequence identity to SEQ ID NO:57, with a polar uncharged amino acid at position 66, optionally threonine (T) at position 66; (ii) a variant 038 (K5L) ORF comprising a nucleotide insertion that results in a frameshift mutation and modifies the 038 (K5L) gene product; (iii) a variant 059 (E2L) ORF having at least 95% sequence identity to SEQ ID NO:60, encoding an amino acid sequence with a hydrophobic amino acid other than leucine at position 419, optionally phenylalanine (F) at position 419; (iv) a variant 104 (H4L) ORF having at least 95% sequence identity to SEQ ID NO:61, encoding an amino acid sequence with a charged amino acid at position 591, optionally aspartic acid (D) at position 591; and (v) characterized by one or more of the variant 182 (A56R) ORFs comprising a deletion of two nucleotides that results in a frameshift mutation and modifies the 182 (A56R) ORF gene product.

[0037] In some of any embodiments, the parental vaccinia virus genome is characterized by one or more of: (i) guanine (G) at a position corresponding to position 7770 of SEQ ID NO:1; (ii) thymine (T) at a position corresponding to position 15261 of SEQ ID NO:1; (iii) G at a position corresponding to position 32136 of SEQ ID NO:1; (iv) G at a position corresponding to position 49455 of SEQ ID NO:1; (v) cytosine (C) at a position corresponding to position 92969 of SEQ ID NO:1; (vi) the nucleic acid sequence CACTTATATAT at a position corresponding to positions 106870 - 106880 of SEQ ID NO:1; (vii) the nucleic acid sequence GTTTTCATTA at a position corresponding to positions 111267 - 111276 of SEQ ID NO:1; (viii) adenine (A) at a position corresponding to position 162715 of SEQ ID NO:1; (ix) the nucleic acid sequence TACAGACACC at a position corresponding to positions 165844 - 185853 of SEQ ID NO:1; and (x) C at a position corresponding to position 187805 of SEQ ID NO:1.

[0038] In some of any aspects, the nucleic acid genome of the recombinant oncolytic vaccinia virus has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:1. In some of any aspects, the nucleic acid genome of the recombinant oncolytic vaccinia virus has at least 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:1. In some of any of the aspects, the nucleic acid genome of the recombinant oncolytic vaccinia virus has at least 95% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:1. In some of any aspects, the nucleic acid genome of the recombinant oncolytic vaccinia virus has at least 96% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:1. In some of any aspects, the nucleic acid genome of the recombinant oncolytic vaccinia virus has at least 97% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:1. In some of any aspects, the nucleic acid genome of the recombinant oncolytic vaccinia virus has at least 98% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:1. In some of any aspects, the nucleic acid genome of the recombinant oncolytic vaccinia virus has at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:1.

[0039] In some of any aspect, the recombinant oncolytic virus is a recombinant oncolytic vaccinia virus, and the nucleic acid genome of the recombinant oncolytic vaccinia virus comprises: (i) a variant 017 open reading frame (ORF) encoding an amino acid sequence having at least 95% sequence identity to SEQ ID NO:57 and having a polar uncharged amino acid at position 66, optionally threonine (T) at position 66; (ii) a variant 038 (K5L) ORF comprising a nucleotide insertion that results in a frameshift mutation and modifies the 038 (K5L) gene product; (iii) a variant 059 (E2L) ORF having at least 95% sequence identity to SEQ ID NO:60 and encoding an amino acid sequence having a hydrophobic amino acid other than leucine at position 419, optionally phenylalanine (F) at position 419; (iv) a variant 104 (H4L) ORF having at least 95% sequence identity to SEQ ID NO:61 and encoding an amino acid sequence having a charged amino acid at position 591, optionally aspartic acid (D) at position 591; and (v) one or more of a variant 182 (A56R) ORF comprising a deletion of two nucleotides that results in a frameshift mutation and modifies the 182 (A56R) ORF gene product.

[0040] In some of any aspects, the recombinant oncolytic virus is a recombinant oncolytic vaccinia virus, and the nucleic acid genome of the recombinant oncolytic vaccinia virus comprises: (i) guanine (G) at a position corresponding to position 7770 of SEQ ID NO:1; (ii) thymine (T) at a position corresponding to position 15261 of SEQ ID NO:1; (iii) G at a position corresponding to position 32136 of SEQ ID NO:1; (iv) G at a position corresponding to position 49455 of SEQ ID NO:1; (v) cytosine (C) at a position corresponding to position 92969 of SEQ ID NO:1; (vi) a nucleic acid sequence CACTTATATAT at a position corresponding to positions 106870 - 106880 of SEQ ID NO:1; (vii) a nucleic acid sequence GTTTTCATTA at a position corresponding to positions 111267 - 111276 of SEQ ID NO:1; (viii) adenine (A) at a position corresponding to position 162715 of SEQ ID NO:1; (ix) a nucleic acid sequence TACAGACACC at a position corresponding to positions 165844 - 185853 of SEQ ID NO:1; and (x) C at a position corresponding to position 187805 of SEQ ID NO:1, characterized by one or more thereof.

[0041] In some of any aspects, at least one of at least one heterologous nucleic acid encoding one or more heterologous gene products is inserted into a non-essential gene or non-essential region within the genome of the virus. In some of any such aspects, the insertion is in place of deletion of all or part of the gene or region.

[0042] In some of any aspects, at least one of at least one heterologous nucleic acid encoding one or more heterologous gene products is inserted into hemagglutinin (HA), J2R (thymidine kinase), F14.5L, A56R (hemagglutinin), B2R, vaccinia growth factor (VGF), A35R, A49R, A55R, B14R, C4L, C6L, C16L, NIL / N2L, E2L / E3L, K1L / K2L, K7L, superoxide dismutase locus, 7.5K, C2L-F3L, C4L-F1L, C7-K1L, B13R+B14R, A26L or I4L locus in the genome of the virus. In some of any aspects, each of at least one heterologous nucleic acid encoding one or more heterologous gene products inserted into a non-essential gene or non-essential region in the genome of the virus is independently inserted into hemagglutinin (HA), J2R (thymidine kinase), F14.5L, A56R (hemagglutinin), B2R, vaccinia growth factor (VGF), A35R, A49R, A55R, B14R, C4L, C6L, C16L, NIL / N2L, E2L / E3L, K1L / K2L, K7L, superoxide dismutase locus, 7.5K, C2L-F3L, C4L-F1L, C7-K1L, B13R+B14R, A26L or I4L locus in the genome of the virus. In some of any aspects, at least one viral gene comprises one or more viral genes selected from the group consisting of hemagglutinin (HA), J2R (thymidine kinase), F14.5L, A56R (hemagglutinin), B2R, vaccinia growth factor (VGF), A35R, A49R, A55R, B14R, C4L, C6L, C16L, NIL / N2L, E2L / E3L, K1L / K2L, K7L, superoxide dismutase locus, 7.5K, C2L-F3L, C4L-F1L, C7-K1L, B13R+B14R, A26L and I4L, and any combination thereof. In some of any such aspects, the insertion is in place of deletion of all or part of each locus.

[0043] In some of any aspects, at least one viral locus into which at least one of at least one heterologous nucleic acid is inserted is (i) B2R; (ii) A35R; (iii) A35R and J2R; (iv) J2R; (v) B2R and J2R; (vi) A35R, B2R and J2R; (vii) B2R, J2R and A56R; or (viii) A35R, B2R, J2R and A56R or includes them.

[0044] In some of any aspects, one or more inactivation mutations of one or more of at least one viral gene are independently due to at least one insertion of at least one heterologous nucleic acid encoding one or more heterologous gene products, deletion of all or part of at least one viral gene, and / or one or more nucleic acid substitutions within at least one viral gene. In some of any aspects, one or more inactivation mutations of one or more of at least one viral gene are due to at least one insertion of at least one heterologous nucleic acid encoding one or more heterologous gene products and deletion of all or part of at least one viral gene, and the insertion is in place of deletion of all or part of the viral gene.

[0045] In some embodiments of any aspect, the inactivating mutation is a deletion of all or part of at least one viral gene. In some embodiments of any aspect, the deletion of at least one viral gene is a deletion of the entire gene ORF of the viral gene. In some embodiments of any aspect, the deletion is sufficient to render the encoded viral gene product non-functional. In some embodiments of any aspect, one or more inactivating mutations in at least one of the viral genes are characterized by the insertion of at least one of at least one heterologous nucleic acid encoding one or more heterologous gene products into the viral locus. In some embodiments of any aspect, at least one viral gene comprises B2R. In some embodiments of any aspect, at least one viral gene comprises J2R. In some embodiments of any aspect, at least one viral gene comprises A35R. In some embodiments of any aspect, at least one viral gene comprises A56R. In some embodiments of any aspect, at least one viral gene comprises B2R, J2R, and A35R. In some embodiments of any aspect, at least one viral gene comprises B2R, J2R, A35R, and A56R. In some embodiments of any aspect, at least one viral gene comprises B2R, J2R, and A56R.

[0046] In some embodiments of any aspect, at least one heterologous nucleic acid encoding one or more heterologous gene products is inserted into F14.5L or instead thereof, and / or at least one heterologous nucleic acid encoding one or more heterologous gene products is inserted into A35R or instead thereof, and / or at least one heterologous nucleic acid encoding one or more heterologous gene products is inserted into J2R or instead thereof.

[0047] In some embodiments of any aspect, at least one heterologous nucleic acid encoding one or more heterologous gene products comprises one or more heterologous nucleic acids each encoding one or more immunomodulatory proteins. In some embodiments of any aspect, one or more inactivating mutations in at least one viral gene are due to the insertion of one or more heterologous nucleic acids each encoding one or more immunomodulatory proteins. In some embodiments of any aspect, one or more immunomodulatory proteins comprise one or more immunostimulatory proteins. In some embodiments of any aspect, one or more immunomodulatory proteins comprise one or more cytokines and / or chemokines. In some embodiments of any aspect, one or more immunomodulatory proteins comprise one or more interferon regulatory factors. In some embodiments, the interferon regulatory factor is IRF3. In some embodiments of any aspect, one or more interferon regulatory factors are or comprise interferon regulatory factor 3 (IRF3). In some embodiments of any aspect, one or more immunomodulatory proteins comprise interferon regulatory factor 3 (IRF3) and one or more cytokines and / or chemokines.

[0048] In some embodiments of any aspect, one or more immunomodulatory proteins comprise one or more immunomodulatory proteins selected from the group consisting of LIGHT, IRF3, IL-2, IL-12, and CXCL9. In some embodiments of any aspect, CXCL9 is human CXCL9. In some embodiments of any aspect, CXCL9 is human CXCL9 and comprises the amino acid sequence set forth in SEQ ID NO:99, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO:99. In some embodiments of any aspect, CXCL9 is mouse CXCL9. In some embodiments of any aspect, CXCL9 is mouse CXCL9 and comprises the amino acid sequence set forth in SEQ ID NO:106, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO:106.

[0049] In some embodiments, IL-12 is human single-chain IL-12. In some embodiments, IL-12 is human single-chain IL-12 and comprises the amino acid sequence set forth in SEQ ID NO:103, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:103. In some embodiments, IL-12 is mouse single-chain IL-12. In some embodiments, IL-12 is mouse single-chain IL-12 and comprises the amino acid sequence set forth in SEQ ID NO:102, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:102.

[0050] In some embodiments, one or more immunomodulatory proteins comprise IRF3. In some embodiments, IRF3 is human IRF3 (hIRF3). In some embodiments, hIRF3 comprises the amino acid sequence set forth in SEQ ID NO:51, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:51. In some embodiments, IRF3 is mouse IRF3 (mIRF3). In some embodiments, mIRF3 comprises the amino acid sequence set forth in SEQ ID NO:52, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:52.

[0051] In some embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence set forth in any one of SEQ ID NO:49, 50, 80, 82, and 84-93, or a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence set forth in any one of SEQ ID NO:49, 50, 80, 82, and 84-93.

[0052] In some embodiments of any aspect, one or more immunomodulatory proteins comprise IRF3 and one or more immunomodulatory proteins selected from the group consisting of LIGHT, IL-2, IL-12, and CXCL9. In some embodiments of any aspect, one or more immunomodulatory proteins comprise IL-2. In some embodiments of any aspect, one or more immunomodulatory proteins comprise IL-12. In some embodiments of any aspect, one or more immunomodulatory proteins comprise LIGHT. In some embodiments of any aspect, one or more immunomodulatory proteins comprise CXCL9. In some embodiments of any aspect, one or more immunomodulatory proteins are or comprise (i) IRF3; (ii) LIGHT; (iii) IRF3 and LIGHT; (iv) IRF3 and IL-2; (v) IRF3, CXCL9, and IL-12; (vi) IRF3, LIGHT, and IL-2; (vii) IRF3 and CXCL9; or (viii) IRF3, CXCL9, and IL-2.

[0053] In some of any aspects, IL-2 is human IL-2. In some of any aspects, IL-2 is an IL-2 supercytokine. In some of any aspects, the IL-2 supercytokine is H9, H9T, MDNA11 or MDNA11T. In some of any aspects, the H9 IL-2 supercytokine comprises the amino acid sequence of SEQ ID NO:100 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:100. In some of any aspects, the H9T IL-2 supercytokine comprises the amino acid sequence of SEQ ID NO:104 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:104. In some of any aspects, the MDNA11 IL-2 supercytokine comprises the amino acid sequence of SEQ ID NO:101 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:101. In some of any aspects, the MDNA11T IL-2 supercytokine comprises the amino acid sequence of SEQ ID NO:98 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:98. In some of any aspects, the IL-2 supercytokine is MDNA11 or MDNA11T. In some of any aspects, the IL-2 supercytokine is MDNA11T, and MDNA11T comprises the amino acid sequence set forth in SEQ ID NO:98 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:98.

[0054] In some embodiments of any aspect, LIGHT is recombinant LIGHT. In some embodiments, the recombinant LIGHT is the human LIGHT protein or a variant thereof. In some embodiments of any aspect, the recombinant LIGHT comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 30. In some embodiments of any aspect, the recombinant LIGHT is a human LIGHT variant (hmLIGHT) that is a human LIGHT variant that binds to human and mouse LTβR and HVEM. In some embodiments of any aspect, the recombinant LIGHT comprises one or more mutations selected from the group consisting of threonine at position 138, glycine at position 160, glycine at position 221, and lysine at position 222. In some embodiments of any aspect, the recombinant LIGHT comprises the amino acid sequence set forth in SEQ ID NO: 25, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 25. In some embodiments of any aspect, the recombinant LIGHT comprises the sequence set forth in SEQ ID NO: 25.

[0055] In some embodiments of any aspect, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence set forth in any one of SEQ ID NOs: 11, 82, 87 and 88, or a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence set forth in any one of SEQ ID NOs: 11, 82, 87 and 88.

[0056] In some embodiments of any aspect, IL-12 is human IL-12. In some embodiments, the human IL-12 is human single-chain IL-12 (hscIL-12). In some embodiments, the hscIL-12 comprises the amino acid sequence set forth in SEQ ID NO: 103, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 103.

[0057] In some aspects of any embodiment, CXCL9 is human CXCL9. In some embodiments, human CXCL9 comprises the amino acid sequence set forth in SEQ ID NO:99, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:99.

[0058] In some aspects of any embodiment, at least one heterologous nucleic acid encoding one or more heterologous gene products comprises one or more heterologous nucleic acids each encoding an apoptosis-inducing protein. In some aspects of any embodiment, one or more inactivating mutations of one or more of the at least one viral gene are by insertion of one or more heterologous nucleic acids each encoding an apoptosis-inducing protein. In some aspects of any embodiment, the apoptosis-inducing protein comprises an apoptosis-promoting molecule fused to an FKBP variant capable of binding to a chemical inducer of dimerization (CID). In some aspects of any embodiment, the FKBP variant is FKBP-F36V. In some embodiments, FKBP-F36V comprises the amino acid sequence set forth in SEQ ID NO:56, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:56.

[0059] In some embodiments of any aspect, the chemical dimerization inducer is AP1903 (Rimiducid). In some embodiments, the apoptosis-promoting molecule is, or comprises, Fas, the death effector domain (DED) of Fas-associated death domain-containing protein (FADD), or caspase, and optionally, the caspase is caspase 9. In some embodiments of any aspect, the apoptosis-inducing protein is inducible DED (iDED). In some embodiments of any aspect, iDED comprises the amino acid sequence set forth in SEQ ID NO:27, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:27. In some embodiments of any aspect, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence set forth in SEQ ID NO:8 or 86, or a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:8 or 86.

[0060] In some embodiments of any aspect, the apoptosis-inducing protein is inducible Fas (iFas). In some embodiments of any aspect, iFas comprises the amino acid sequence set forth in SEQ ID NO:28, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:28. In some embodiments of any aspect, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence set forth in SEQ ID NO:9, or a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:9.

[0061] In some of any aspects, the apoptosis-inducing protein is inducible caspase 9 (iCas9). In some of any aspects, iCas9 comprises the amino acid sequence set forth in SEQ ID NO:26, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:26. In some of any aspects, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence set forth in SEQ ID NO:7, or a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:7.

[0062] In some of any aspects, at least one heterologous nucleic acid encoding one or more heterologous gene products comprises one or more heterologous nucleic acids encoding one or more T cell evasion factor proteins or NK cell evasion factor proteins, respectively. In some of any aspects, one or more inactivating mutations of one or more of the at least one viral gene are due to the insertion of one or more heterologous nucleic acids encoding one or more T cell evasion factor proteins or NK cell evasion factor proteins, respectively.

[0063] In some of any aspects, one or more T cell evasion factor proteins or NK cell evasion factor proteins include a set of proteins encoded by vaccinia virus ORFs 012, 203 and 018 (CPXV012-203-018). In some of any aspects, one or more T cell evasion factor proteins or NK cell evasion factor proteins are or include a set of proteins that are the CPXV012 protein, the CPXV203 protein and the CPXV018 protein. In some of any aspects, the set of proteins encoded by CPXV012-203-018 includes (i) the amino acid sequence set forth in SEQ ID NO:20 (CPXV012), or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:20, (ii) the amino acid sequence set forth in SEQ ID NO:21 (CPXV0203), or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:21, and (iii) the amino acid sequence set forth in SEQ ID NO:22 (CPXV018), or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:22. In some of any aspects, the set of proteins encoded by CPXV012-203-018 includes the amino acid sequences set forth in SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:22. In some of any aspects, the nucleic acid genome of the recombinant oncolytic virus includes the nucleic acid sequence set forth in any one of SEQ ID NO:10, 89 and 90, or a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence set forth in any one of SEQ ID NO:10, 89 and 90.

[0064] In some of any embodiments, at least one heterologous nucleic acid encoding one or more heterologous gene products comprises one or more heterologous nucleic acids each encoding one or more complement inhibitors. In some of any embodiments, one or more inactivating mutations of one or more of the at least one viral gene are due to the insertion of one or more heterologous nucleic acids each encoding one or more complement inhibitors.

[0065] In some of any embodiments, one or more complement inhibitors are Borrelia burgdorferi complement regulator-acquiring surface protein 2 (CRASP-2) and / or minimized complement regulator factor H (miniFH). In some of any embodiments, one or more complement inhibitors is or comprises CRASP-2. In some of any embodiments, CRASP-2 comprises the amino acid sequence set forth in SEQ ID NO:18, or has an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:18. In some of any embodiments, one or more complement inhibitors is or comprises miniFH. In some of any embodiments, miniFH comprises the amino acid sequence set forth in SEQ ID NO:19, or has an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:19.

[0066] In some of any aspects, one or more heterologous nucleic acids encoding one or more complement inhibitors are introduced into a viral membrane gene, optionally F14.5L, to produce a fusion gene encoding a fusion protein. In some aspects, the fusion protein comprises a complement inhibitor fused to a viral membrane protein encoded by the viral membrane gene. In some of any aspects, the viral membrane gene is F14.5L, and optionally, the fusion is a fusion at the C-terminus of the F14.5L protein. In some of any aspects, the fusion protein is incorporated into the outer membrane of intracellular mature virus (IMV). In some of any aspects, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence set forth in SEQ ID NO:5, or a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:5. In some of any aspects, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence set forth in SEQ ID NO:6, or a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:6. In some of any aspects, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence set forth in SEQ ID NO:89, or a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:89. In some of any aspects, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence set forth in SEQ ID NO:90, or a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:90.

[0067] In some embodiments of any aspect, at least one heterologous nucleic acid encoding one or more heterologous gene products comprises one or more heterologous nucleic acids each encoding one or more anti-angiogenic proteins. In some embodiments of any aspect, one or more inactivating mutations of one or more of the at least one viral gene are due to the insertion of one or more heterologous nucleic acids each encoding one or more anti-angiogenic proteins. In some embodiments of any aspect, one or more anti-angiogenic proteins are VEGF inhibitors, angiopoietin inhibitors, versican, or fusion proteins of any two or more of the foregoing. In some embodiments of any aspect, one or more anti-angiogenic proteins comprise a VEGF inhibitor and / or an angiopoietin inhibitor, optionally including an inhibitor of Ang2. In some embodiments of any aspect, one or more anti-angiogenic proteins comprise an anti-VEGF antibody and / or an anti-Ang2 antibody. In some embodiments of any aspect, the VEGF inhibitor is an anti-VEGF antibody, optionally an anti-VEGF single-chain antibody (scAb). In some embodiments of any aspect, the angiopoietin inhibitor is an anti-angiopoietin-2 (Ang2) antibody, optionally an anti-Ang2 single-chain antibody (scAb). In some embodiments of any aspect, one or more anti-angiogenic proteins are bispecific anti-VEGF / anti-Ang2 antibodies. In some embodiments of any aspect, the bispecific anti-VEGF / anti-Ang2 antibody comprises the amino acid sequence set forth in SEQ ID NO:23, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO:23. In some embodiments of any aspect, one or more anti-angiogenic proteins comprise versican. In some embodiments of any aspect, versican comprises the amino acid sequence set forth in SEQ ID NO:24, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO:24.In some embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises a nucleic acid sequence as set forth in any one of SEQ ID NOs: 13, 47, 82, 87, and 88, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence as set forth in any one of SEQ ID NOs: 13, 47, 82, 87, and 88.

[0068] In some embodiments, at least one heterologous nucleic acid encoding one or more heterologous gene products comprises one or more heterologous nucleic acids encoding one or more therapeutic or diagnostic agents, respectively. In some embodiments, one or more inactivating mutations in at least one viral gene are due to the insertion of one or more heterologous nucleic acids encoding one or more therapeutic or diagnostic agents, respectively.

[0069] In some embodiments, one or more therapeutic or diagnostic agents are selected from the group consisting of anti-cancer agents, anti-metastasis agents, anti-angiogenesis agents, immunomodulatory molecules, antigens, extracellular matrix degrading genes, genes for tissue regeneration and for reprogramming human somatic cells to pluripotency, enzymes that modify a substrate to produce a detectable product or signal or are detectable by an antibody, proteins that can bind to a contrast agent, genes for optical imaging or detection, genes for PET imaging, and genes for MRI imaging. In some embodiments, one or more therapeutic or diagnostic agents comprise therapeutic agents selected from the group consisting of hormones, growth factors, cytokines, chemokines, costimulatory molecules, ribozymes, transporter proteins, single-chain antibodies, antisense RNAs, prodrug converting enzymes, siRNAs, microRNAs, toxins, anti-tumor oligopeptides, mitosis-inhibiting proteins, anti-mitotic oligopeptides, anti-cancer polypeptide antibiotics, angiogenesis inhibitors, tumor suppressors, cytotoxic proteins, cell growth inhibitory proteins, and tissue factors.

[0070] In some embodiments of any aspect, at least one viral gene is or comprises A35R, and optionally, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence of SEQ ID NO:3, or a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:3.

[0071] In some embodiments of any aspect, at least one viral gene is or comprises A35R and J2R, and optionally, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence of SEQ ID NO:12, or a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:12.

[0072] In some embodiments of any aspect, at least one viral gene is or comprises J2R, and the inactivating mutation of J2R is by insertion of one or more heterologous nucleic acids each encoding one or more T cell evasion factor proteins or NK cell evasion factor proteins, and optionally, one or more T cell evasion factor proteins or NK cell evasion factor proteins comprise the set of proteins encoded by vaccinia virus ORFs 012, 203 and 018 (CPXV012 - 203 - 018), and at least one heterologous nucleic acid encoding one or more heterologous gene products is introduced into the viral membrane gene to produce a fusion gene encoding a fusion protein, and comprises one or more heterologous nucleic acids each encoding one or more complement inhibitors. In some embodiments, the viral membrane gene is F14.5L. In some embodiments, the fusion is a fusion at the C - terminus of the F14.5L protein. In some embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence of SEQ ID NO:10, or a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:10.

[0073] In some of any aspects, at least one viral gene is or includes J2R. In some aspects, the nucleic acid genome of the recombinant oncolytic virus includes a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence of SEQ ID NO:4, or the nucleic acid sequence described in SEQ ID NO:4.

[0074] In some of any aspects, at least one viral gene is or includes J2R and A35R, and the inactivating mutation of A35R is by insertion of one or more heterologous nucleic acids each encoding one or more immunomodulatory proteins. In some aspects, one or more immunomodulatory proteins are selected from the group consisting of LIGHT, IRF3, IL-2, IL-12 and CXCL9. In some aspects, one or more immunomodulatory proteins are LIGHT. In some aspects, the nucleic acid genome of the recombinant oncolytic virus includes a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence of SEQ ID NO:11, or the nucleic acid sequence described in SEQ ID NO:11.

[0075] In some of any aspects, at least one viral gene is or includes J2R and A35R, and the inactivating mutation of J2R is by insertion of one or more heterologous nucleic acids encoding one or more anti-angiogenic proteins. In some aspects, one or more anti-angiogenic proteins include an inhibitor or inhibitors of VEGF, and / or Ang2. In some aspects, one or more anti-angiogenic proteins are bispecific anti-VEGF / anti-Ang2 antibodies. In some aspects, the nucleic acid genome of the recombinant oncolytic virus includes a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence of SEQ ID NO:13, or the nucleic acid sequence described in SEQ ID NO:13.

[0076] In some embodiments of any aspect, at least one viral gene is or comprises J2R and A35R, the inactivating mutation of A35R is by insertion of one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, and the inactivating mutation of J2R is by insertion of one or more heterologous nucleic acids encoding one or more anti-angiogenic proteins. In some embodiments, one or more immunomodulatory proteins are selected from the group consisting of LIGHT, IRF3, IL-2, IL-12 and CXCL9. In some embodiments, one or more immunomodulatory proteins are LIGHT. In some embodiments, one or more anti-angiogenic proteins comprise an inhibitor or inhibitors of VEGF, and / or Ang2. In some embodiments, one or more anti-angiogenic proteins are bispecific anti-VEGF / anti-Ang2 antibodies. In some embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence of SEQ ID NO:47, or a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:47.

[0077] In some of any aspects, at least one viral gene is J2R or includes it, and the inactivating mutation of J2R is due to the insertion of one or more heterologous nucleic acids each encoding an apoptosis-inducing protein. In some aspects, the apoptosis-inducing protein is inducible DED (iDED), inducible Fas (iFas) or inducible Cas9 (iCas9). In some aspects, the nucleic acid genome of the recombinant oncolytic virus comprises a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 7, 8 or 9, or the nucleic acid sequence described in SEQ ID NO: 7, 8 or 9. In some aspects, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence of SEQ ID NO: 7. In some aspects, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence of SEQ ID NO: 8. In some aspects, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence of SEQ ID NO: 9.

[0078] In some of any aspects, at least one viral gene is J2R or includes it, and the inactivating mutation of J2R is due to the insertion of one or more heterologous nucleic acids each encoding one or more immunomodulatory proteins. In some aspects, the one or more immunomodulatory proteins are selected from the group consisting of LIGHT, IRF3, IL-2, IL-12 and CXCL9. In some aspects, the one or more immunomodulatory proteins is IRF3. In some aspects, the nucleic acid genome of the recombinant oncolytic virus comprises a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 49, 50 or 93, or the nucleic acid sequence described in SEQ ID NO: 49, 50 or 93.

[0079] In some of any aspects, at least one viral gene is or comprises J2R and B2R. In some aspects, the nucleic acid genome of the recombinant oncolytic virus comprises a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence of SEQ ID NO:48, or the nucleic acid sequence described in SEQ ID NO:48.

[0080] In some of any aspects, at least one viral gene is or comprises J2R and B2R.

[0081] In some of any aspects, at least one viral gene is or comprises J2R and B2R, and the inactivation mutation of J2R is by insertion of one or more heterologous nucleic acids each encoding one or more immunomodulatory proteins. In some aspects, the one or more immunomodulatory proteins are selected from the group consisting of LIGHT, IRF3, IL-2, IL-12 and CXCL9. In some aspects, the one or more immunomodulatory proteins are IRF3. In some aspects, the nucleic acid genome of the recombinant oncolytic virus comprises a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence of SEQ ID NO:80, or the nucleic acid sequence described in SEQ ID NO:80.

[0082] In some embodiments of any aspect, at least one viral gene is or comprises J2R, B2R, and A35R, and the inactivating mutation of J2R is by insertion of one or more heterologous nucleic acids encoding one or more anti-angiogenic proteins, the inactivating mutation of B2R is by insertion of one or more heterologous nucleic acids encoding one or more immunomodulatory proteins respectively, and the inactivating mutation of A35R is by insertion of one or more heterologous nucleic acids encoding one or more immunomodulatory proteins. In some embodiments, one or more anti-angiogenic proteins include an inhibitor or inhibitors of VEGF, and / or Ang2. In some embodiments, one or more anti-angiogenic proteins are bispecific anti-VEGF / anti-Ang2 antibodies. In some embodiments, the inactivating mutation of B2R is by insertion of one or more heterologous nucleic acids encoding one or more immunomodulatory proteins selected from the group consisting of LIGHT, IRF3, IL-2, IL-12, and CXCL9 respectively. In some embodiments, one or more immunomodulatory proteins are IRF3. In some embodiments, the inactivating mutation of A35R is by insertion of one or more heterologous nucleic acids encoding one or more immunomodulatory proteins selected from the group consisting of LIGHT, IRF3, IL-2, IL-12, and CXCL9. In some embodiments, one or more immunomodulatory proteins are LIGHT. In some embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO:82, or the nucleic acid sequence described in SEQ ID NO:82.

[0083] In some of any aspects, at least one viral gene is or includes J2R, B2R, and A56R, the inactivating mutation of J2R is by insertion of one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally, one or more immunomodulatory proteins are IRF3, the inactivating mutation of A56R is by insertion of one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally, one or more immunomodulatory proteins are IL-2. In some aspects, IL-2 is an IL-2 supercytokine. In some aspects, the IL-2 supercytokine is MDNA11. In some aspects, the nucleic acid genome of the recombinant oncolytic virus comprises a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO:84, or the nucleic acid sequence described in SEQ ID NO:84.

[0084] In some of any aspects, at least one viral gene is or includes J2R, B2R, and A56R, the inactivating mutation of J2R is by insertion of one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally, one or more immunomodulatory proteins are IRF3, the inactivating mutation of A56R is by insertion of one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally, one or more immunomodulatory proteins include two or more immunomodulatory proteins selected from the group consisting of LIGHT, IRF3, IL-2, IL-12, and CXCL9. In some aspects, the two or more immunomodulatory proteins include IL-12 and CXCL9. In some aspects, the nucleic acid genome of the recombinant oncolytic virus comprises a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO:85, or the nucleic acid sequence described in SEQ ID NO:85.

[0085] In some aspects of any embodiment, at least one viral gene is or comprises J2R, B2R, and A56R, the inactivating mutation of B2R is by insertion of one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally, one or more immunomodulatory proteins are IRF3, the inactivating mutation of A56R is by insertion of one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally, one or more immunomodulatory proteins comprise two or more immunomodulatory proteins selected from the group consisting of LIGHT, IRF3, IL-2, IL-12, and CXCL9, and the inactivating mutation of J2R is by insertion of one or more heterologous nucleic acids each encoding an apoptosis-inducing protein. In some embodiments, two or more immunomodulatory proteins comprise IL-12 and CXCL9. In some embodiments, the apoptosis-inducing protein is inducible DED (iDED). In some embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence of SEQ ID NO:86, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:86.

[0086] In some embodiments of any aspect, at least one viral gene is or comprises J2R, B2R, A35R, and A56R, and the inactivating mutation of J2R is by insertion of one or more heterologous nucleic acids encoding one or more anti-angiogenic proteins, optionally, one or more anti-angiogenic proteins include an inhibitor or inhibitors of VEGF, and / or Ang2, optionally, one or more anti-angiogenic proteins are bispecific anti-VEGF / anti-Ang2 antibodies, the inactivating mutation of B2R is by insertion of one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally, one or more immunomodulatory proteins are IRF3, the inactivating mutation of A35R is by insertion of one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally, one or more immunomodulatory proteins are selected from the group consisting of LIGHT, IRF3, IL-2, IL-12, and CXCL9, optionally, one or more immunomodulatory proteins are LIGHT, the inactivating mutation of A56R is by insertion of one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, and one or more immunomodulatory proteins are the IL-2 supercytokine MDNA11. In some embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence of SEQ ID NO:87, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:87.

[0087] In some embodiments of any aspect, at least one viral gene is or includes J2R, B2R, A35R, and A56R, and the inactivating mutation of J2R is by insertion of one or more heterologous nucleic acids encoding one or more anti-angiogenic proteins, optionally, one or more anti-angiogenic proteins include an inhibitor or inhibitors of VEGF, and / or Ang2, optionally, one or more anti-angiogenic proteins are bispecific anti-VEGF / anti-Ang2 antibodies, the inactivating mutation of B2R is by insertion of one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally, one or more immunomodulatory proteins are IRF3, the inactivating mutation of A35R is by insertion of one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally, one or more immunomodulatory proteins are selected from the group consisting of LIGHT, IRF3, IL-2, IL-12, and CXCL9, optionally, one or more immunomodulatory proteins are LIGHT, the inactivating mutation of A56R is by insertion of one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, and one or more immunomodulatory proteins are the IL-2 supercytokine MDNA11T. In some embodiments, MDNA11T includes the amino acid sequence set forth in SEQ ID NO:98. In some embodiments, the nucleic acid genome of the recombinant oncolytic virus includes the nucleic acid sequence of SEQ ID NO:88, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:88.

[0088] In some aspects of any embodiment, at least one viral gene is or includes J2R, B2R, and A56R, and the inactivating mutation of J2R is by insertion of one or more heterologous nucleic acids each encoding one or more T cell evasion factor proteins or NK cell evasion factor proteins, optionally, one or more T cell evasion factor proteins or NK cell evasion factor proteins include a set of proteins encoded by vaccinia virus ORFs 012, 203, and 018 (CPXV012-203-018), the inactivating mutation of B2R is by insertion of one or more heterologous nucleic acids each encoding one or more immunomodulatory proteins, optionally, one or more immunomodulatory proteins is IRF3, the inactivating mutation of A56R is by insertion of one or more heterologous nucleic acids each encoding one or more immunomodulatory proteins, optionally, one or more immunomodulatory proteins are selected from the group consisting of LIGHT, IRF3, IL-2, IL-12, and CXCL9, optionally, one or more immunomodulatory proteins are IL-2 supercytokines, optionally, MDNA11 or MDNA11T, and at least one heterologous nucleic acid encoding one or more heterologous gene products includes one or more heterologous nucleic acids each encoding one or more complement inhibitors, optionally CRASP-2, which are introduced into a viral membrane gene, optionally F14.5L, to produce a fusion gene encoding a fusion protein. In some embodiments, the fusion is a fusion at the C-terminus of the F14.5L protein. In some embodiments, the nucleic acid genome of the recombinant oncolytic virus includes a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO:89, or the nucleic acid sequence set forth in SEQ ID NO:89.

[0089] In some embodiments of any aspect, at least one viral gene is or comprises J2R, B2R, and A56R, and the inactivating mutation of J2R is by insertion of one or more heterologous nucleic acids encoding one or more T cell evasion factor proteins or NK cell evasion factor proteins, optionally, one or more T cell evasion factor proteins or NK cell evasion factor proteins comprise a set of proteins encoded by vaccinia virus ORFs 012, 203, and 018 (CPXV012 - 203 - 018), the inactivating mutation of B2R is by insertion of one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally, one or more immunomodulatory proteins is IRF3, the inactivating mutation of A56R is by insertion of one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally, one or more immunomodulatory proteins comprise two or more immunomodulatory proteins selected from the group consisting of LIGHT, IRF3, IL - 2, IL - 12, and CXCL9, optionally, two or more immunomodulatory proteins comprise IL - 12 and CXCL9, and at least one heterologous nucleic acid encoding one or more heterologous gene products comprises one or more heterologous nucleic acids encoding a complement inhibitor, optionally CRASP - 2, which are introduced into a viral membrane gene, optionally F14.5L, to produce a fusion gene encoding a fusion protein. In some embodiments, the fusion is at the C - terminus of the F14.5L protein. In some embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence of SEQ ID NO:90, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:90.

[0090] In some aspects of any embodiment, at least one viral gene is or includes B2R and J2R, and the inactivating mutation of J2R is by insertion of one or more heterologous nucleic acids each encoding one or more immunomodulatory proteins, optionally, one or more immunomodulatory proteins are selected from the group consisting of LIGHT, IRF3, IL-2, IL-12, and CXCL9, and optionally, one or more immunomodulatory proteins is IRF3. In some embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence of SEQ ID NO:91, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:91.

[0091] In some aspects of any embodiment, at least one viral gene is or includes B2R, J2R, and A56R, and the inactivating mutation of J2R is by insertion of one or more heterologous nucleic acids each encoding one or more immunomodulatory proteins, optionally, one or more immunomodulatory proteins are selected from the group consisting of LIGHT, IRF3, IL-2, IL-12, and CXCL9, and optionally, one or more immunomodulatory proteins is IRF3, and the inactivating mutation of A56R is by insertion of one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally, one or more immunomodulatory proteins include two or more immunomodulatory proteins selected from the group consisting of LIGHT, IRF3, IL-2, IL-12, and CXCL9, and optionally, the two or more immunomodulatory proteins include IL-12 and CXCL9. In some embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence of SEQ ID NO:92, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:92.

[0092] In some aspects of any embodiment, at least one viral gene is or includes J2R, and the inactivating mutation of J2R is by insertion of one or more heterologous nucleic acids each encoding one or more immunomodulatory proteins, optionally, one or more immunomodulatory proteins are selected from the group consisting of LIGHT, IRF3, IL-2, IL-12, and CXCL9, and optionally, one or more immunomodulatory proteins are IRF3. In some embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence of SEQ ID NO:93, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:93.

[0093] In some aspects of any embodiment, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence set forth in any one of SEQ ID NO:48, 80, 82, and 84 - 93, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence set forth in any one of SEQ ID NO:48, 80, 82, and 84 - 93. In some aspects of any embodiment, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence set forth in any one of SEQ ID NO:85, 86, 88, and 90, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence set forth in any one of SEQ ID NO:85, 86, 88, and 90. In some aspects of any embodiment, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence set forth in SEQ ID NO:85, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:85.

[0094] In some aspects of any embodiment, one or more of the heterologous nucleic acids encoding a heterologous gene product are operably linked to a promoter.

[0095] In some of any aspects, each of one or more heterologous nucleic acids encoding a heterologous gene product, operably linked to a promoter, is selected from the group consisting of 7.5E, 7.5E / L, SSE, 11KL, SSL, SSEL, mH5 and LEO. In some of any aspects, each heterologous nucleic acid encoding a heterologous gene product is independently operably linked to a promoter, and optionally, each heterologous nucleic acid encoding a heterologous gene product is independently operably linked to a promoter selected from the group consisting of 7.5E, 7.5E / L, SSE, 11KL, SSL, SSEL, mH5 and LEO. In some of any aspects, the promoter is a poxvirus promoter or a variant or derivative thereof. In some of any aspects, the promoter is a vaccinia virus promoter. In some of any aspects, the promoter is selected from the group consisting of 7.5E, 7.5E / L, SSE, 11KL, SSL, SSEL, mH5 and LEO. In some of any aspects, the promoter has an amino acid sequence set forth in any one of SEQ ID NO:29, 53, 55, 68, 69, 70, 71 or 72. In some of any aspects, the promoter is a synthetic strong early promoter (SSE). In some of any aspects, the SSE promoter comprises the sequence set forth in SEQ ID NO:29. In some of any aspects, the promoter is a strong early / late promoter (SEL). In some of any aspects, the SEL promoter comprises the sequence set forth in SEQ ID NO:55. In some of any aspects, the promoter is mH5. In some of any aspects, the mH5 promoter comprises the sequence set forth in SEQ ID NO:53.

[0096] An isolated cloned vaccinia virus (VACV) strain comprising a nucleic acid genome having at least 95% sequence identity with the nucleic acid sequence set forth in SEQ ID NO:1, wherein the nucleic acid genome has: (i) at least 95% sequence identity to SEQ ID NO:57 and encodes an amino acid sequence having a polar uncharged amino acid at position 66, optionally threonine (T) at position 66, in variant 017 open reading frame (ORF); (ii) a variant 038 (K5L) ORF comprising a nucleotide insertion that results in a frameshift mutation and modifies the 038 (K5L) gene product; (iii) at least 95% sequence identity to SEQ ID NO:60 and encodes an amino acid sequence having a hydrophobic amino acid other than leucine at position 419, optionally phenylalanine (F) at position 419, in variant 059 (E2L) ORF; (iv) at least 95% sequence identity to SEQ ID NO:61 and encodes an amino acid sequence having a charged amino acid at position 591, optionally aspartic acid (D) at position 591, in variant 104 (H4L) ORF; and (v) one or more of variant 182 (A56R) ORF, which comprises a two nucleotide deletion that results in a frameshift mutation and modifies the 182 (A56R) ORF gene product, is also provided herein.

[0097] In some embodiments of any recombinant oncolytic virus or any isolated cloned VACV strain, the nucleic acid genome is characterized by (i), and the variant 017 ORF encodes an amino acid sequence having at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:57. In some embodiments of any recombinant oncolytic virus or any isolated cloned VACV strain, the nucleic acid genome is characterized by (i), and the variant 017 ORF encodes the amino acid sequence set forth in SEQ ID NO:57. In some embodiments of any recombinant oncolytic virus or any isolated cloned VACV strain, the nucleic acid genome is characterized by (ii), the nucleotide insertion is guanine (G) corresponding to the insertion after nucleotide position 32135 of SEQ ID NO:1, and optionally, the variant 038 (K5L) ORF is as set forth in SEQ ID NO:58. In some embodiments of any recombinant oncolytic virus or any isolated cloned VACV strain, the nucleic acid genome is characterized by (ii), and the 038 (K5L) gene product is as set forth in SEQ ID NO:59. In some embodiments of any recombinant oncolytic virus or any isolated cloned VACV strain, the nucleic acid genome is characterized by (iii), and the variant 059 (E2L) ORF encodes an amino acid sequence having at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:60. In some embodiments of any recombinant oncolytic virus or any isolated cloned VACV strain, the nucleic acid genome is characterized by (iii), and the variant 059 (E2L) ORF encodes the amino acid sequence set forth in SEQ ID NO:60. In some embodiments of any recombinant oncolytic virus or any isolated cloned VACV strain, the nucleic acid genome is characterized by (iv), and the 104 (H4L) ORF encodes an amino acid sequence having at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:61.In some of any aspect of any recombinant oncolytic virus or any isolated cloned VACV strain, the nucleic acid genome is characterized by (iv), and the variant 104 (H4L) ORF encodes the amino acid sequence set forth in SEQ ID NO: 61. In some of any aspect of any recombinant oncolytic virus or any isolated cloned VACV strain, the nucleic acid genome is characterized by (v), the two-nucleotide deletion is a deletion of two consecutive nucleotides corresponding to the nucleotides after nucleotide position 165972 of SEQ ID NO: 2, and optionally, variant 182 (A56R) is set forth in SEQ ID NO: 62. In some of any aspect of any recombinant oncolytic virus or any isolated cloned VACV strain, the nucleic acid genome is characterized by (v), and the VACV protein is set forth in SEQ ID NO: 63.

[0098] In some of any aspect of any recombinant oncolytic virus or any isolated cloned VACV strain, the nucleic acid genome is characterized by any two of (i) - (v). In some of any aspect of any recombinant oncolytic virus or any isolated cloned VACV strain, the nucleic acid genome is characterized by any three of (i) - (v). In some of any aspect of any recombinant oncolytic virus or any isolated cloned VACV strain, the nucleic acid genome is characterized by any four of (i) - (v). In some of any aspect of any recombinant oncolytic virus or any isolated cloned VACV strain, the nucleic acid genome is characterized by each of (i) - (v).

[0099] An isolated cloned vaccinia virus (VACV) strain comprising a nucleic acid genome having at least 95% sequence identity with the nucleic acid sequence set forth in SEQ ID NO:1, wherein the nucleic acid genome comprises: (i) guanine (G) at the position corresponding to position 7770 of SEQ ID NO:1; (ii) thymine (T) at the position corresponding to position 15261 of SEQ ID NO:1; (iii) G at the position corresponding to position 32136 of SEQ ID NO:1; (iv) G at the position corresponding to position 49455 of SEQ ID NO:1; (v) cytosine (C) at the position corresponding to position 92969 of SEQ ID NO:1; (vi) the nucleic acid sequence CACTTATATAT at the position corresponding to positions 106870-106880 of SEQ ID NO:1; (vii) the nucleic acid sequence GTTTTCATTA at the position corresponding to positions 111267-111276 of SEQ ID NO:1; (viii) adenine (A) at the position corresponding to position 162715 of SEQ ID NO:1; (ix) the nucleic acid sequence TACAGACACC at the position corresponding to positions 165844-185853 of SEQ ID NO:1; and (x) C at the position corresponding to position 187805 of SEQ ID NO:1, wherein one or more of (i)-(x) are characteristic. Also provided herein is an isolated cloned vaccinia virus (VACV) strain.

[0100] In some of any aspect of any recombinant oncolytic virus or any isolated cloned VACV strain, the nucleic acid genome is characterized by any two of (i) to (x). In some of any aspect of any recombinant oncolytic virus or any isolated cloned VACV strain, the nucleic acid genome is characterized by any three of (i) to (x). In some of any aspect of any recombinant oncolytic virus or any isolated cloned VACV strain, the nucleic acid genome is characterized by any four of (i) to (x). In some of any aspect of any recombinant oncolytic virus or any isolated cloned VACV strain, the nucleic acid genome is characterized by any five of (i) to (x). In some of any aspect of any recombinant oncolytic virus or any isolated cloned VACV strain, the nucleic acid genome is characterized by any six of (i) to (x). In some of any aspect of any recombinant oncolytic virus or any isolated cloned VACV strain, the nucleic acid genome is characterized by any seven of (i) to (x). In some of any aspect of any recombinant oncolytic virus or any isolated cloned VACV strain, the nucleic acid genome is characterized by any eight of (i) to (x). In some of any aspect of any recombinant oncolytic virus or any isolated cloned VACV strain, the nucleic acid genome is characterized by any nine of (i) to (x). In some of any aspect of any recombinant oncolytic virus or any isolated cloned VACV strain, the nucleic acid genome is characterized by each of (i) to (x).

[0101] In some embodiments of any recombinant oncolytic virus or any isolated cloned VACV strain, the nucleic acid genome has at least 96% sequence identity with the nucleotide sequence set forth in SEQ ID NO:1. In some embodiments of any recombinant oncolytic virus or any isolated cloned VACV strain, the nucleic acid genome has at least 97% sequence identity with the nucleotide sequence set forth in SEQ ID NO:1. In some embodiments of any recombinant oncolytic virus or any isolated cloned VACV strain, the nucleic acid genome has at least 98% sequence identity with the nucleotide sequence set forth in SEQ ID NO:1. In some embodiments of any recombinant oncolytic virus or any isolated cloned VACV strain, the nucleic acid genome has at least 99% sequence identity with the nucleotide sequence set forth in SEQ ID NO:1.

[0102] Also provided herein is an isolated cloned vaccinia virus (VACV) strain comprising a nucleic acid genome having at least 99% sequence identity with the nucleotide sequence set forth in SEQ ID NO:1.

[0103] In some aspects of any recombinant oncolytic virus or any isolated clonal VACV strain, the nucleic acid genome has at least 99.5% sequence identity with the nucleotide sequence set forth in SEQ ID NO:1. In some aspects of any recombinant oncolytic virus or any isolated clonal VACV strain, the nucleic acid genome has at least 99.9% sequence identity with the nucleotide sequence set forth in SEQ ID NO:1. In some aspects of any recombinant oncolytic virus or any isolated clonal VACV strain, the nucleic acid genome has at least 99.95% sequence identity with the nucleotide sequence set forth in SEQ ID NO:1. In some aspects of any recombinant oncolytic virus or any isolated clonal VACV strain, the nucleic acid genome does not contain the nucleotide sequence set forth in SEQ ID NO:2. In some aspects of any recombinant oncolytic virus or any isolated clonal VACV strain, the nucleic acid genome is not modified to contain a non-viral heterologous nucleic acid containing an open reading frame encoding a non-viral heterologous protein. In some aspects of any recombinant oncolytic virus or any isolated clonal VACV strain, the nucleic acid genome is as set forth in SEQ ID NO:1.

[0104] In some of any aspects of any recombinant oncolytic virus or any isolated cloned VACV strain, the cloned VACV strain shows enhanced production of extracellular enveloped virions (EEV) after cell infection, optionally as determined by the percentage of EEV, where the percentage of EEV is determined by the formula: viral titer in supernatant / (viral titer in supernatant + viral titer in cell lysate) * 100. In some of any aspects of any recombinant oncolytic virus or any isolated cloned VACV strain, more than 5% of the infectious particles after cell infection are EEV. In some of any aspects of any recombinant oncolytic virus or any isolated cloned VACV strain, more than 10% of the infectious particles after cell infection are EEV. In some of any aspects of any recombinant oncolytic virus or any isolated cloned VACV strain, more than 15% of the infectious particles after cell infection are EEV. In some of any aspects, the recombinant oncolytic virus or cloned VACV strain shows enhanced production of extracellular enveloped virions (EEV) after cell infection, as determined by at least 5%, 10% or 15% of the infectious particles being EEV.

[0105] In some of any aspects of any recombinant oncolytic virus or any isolated cloned VACV strain, the virus shows oncolytic activity for killing tumor cells.

[0106] Also provided herein are VACV preparations comprising any isolated cloned VACV strain of any of the isolated cloned VACV strains provided herein.

[0107] Also provided herein are VACV preparations comprising any recombinant oncolytic vaccinia virus provided herein.

[0108] Also provided herein is a recombinant oncolytic virus preparation comprising any of the recombinant oncolytic viruses provided herein, wherein at least 70%, 80%, 90%, 95% or 98% of the virus particles in the preparation have the genomic sequence of a cloned recombinant oncolytic virus.

[0109] In some of any aspect, the VACV preparation is substantially homogeneous and the plurality of virus particles in the preparation have the genomic sequence of a cloned VACV strain.

[0110] In some of any aspect, at least 70% of the virus particles in the preparation have the genomic sequence of a cloned VACV strain. In some of any aspect, at least 80% of the virus particles in the preparation have the genomic sequence of a cloned VACV strain. In some of any aspect, at least 90% of the virus particles in the preparation have the genomic sequence of a cloned VACV strain. In some of any aspect, at least 95% of the virus particles in the preparation have the genomic sequence of a cloned VACV strain. In some of any aspect, at least 98% of the virus particles in the preparation have the genomic sequence of a cloned VACV strain.

[0111] Also provided herein is a pharmaceutical composition comprising any of the isolated VACV clone strains provided herein.

[0112] Also provided herein is a pharmaceutical composition comprising any of the VACV provided herein.

[0113] Also provided herein is a pharmaceutical composition comprising any of the recombinant oncolytic vaccinia viruses provided herein.

[0114] Also provided herein is a recombinant vaccinia virus (VACV) strain comprising the nucleic acid genome of any of the VACV clone strains provided herein, which comprises an inactivating mutation in at least one viral gene.

[0115] In some embodiments, the viral gene is selected from the group consisting of hemagglutinin (HA), J2R (thymidine kinase), F14.5L, A56R (hemagglutinin), B2R, vaccinia growth factor (VGF), A35R, A49R, A55R, B14R, C4L, C6L, C16L, NIL / N2L, E2L / E3L, K1L / K2L, K7L, superoxide dismutase locus, 7.5K, C2L-F3L, C4L-F1L, C7-K1L, B13R+B14R, A26L, and I4L. In some embodiments, the inactivation mutation is a deletion of all or part of at least one viral gene. In some embodiments, the deletion of at least one viral gene is a deletion of the entire gene ORF of the viral gene. In some embodiments, the deletion of at least one viral gene is a deletion of a part of the ORF of the viral gene, and the deletion is sufficient to render the encoded gene product non-functional.

[0116] In some embodiments, at least one viral gene is A35R or includes it.

[0117] In some embodiments, the nucleic acid genome of the recombinant VACV strain comprises the nucleic acid sequence set forth in SEQ ID NO:3, or a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the sequence set forth in SEQ ID NO:3.

[0118] In some embodiments, at least one viral gene is J2R or includes it.

[0119] In some embodiments, the nucleic acid genome of the recombinant VACV strain comprises the nucleic acid sequence set forth in SEQ ID NO:4, or a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the sequence set forth in SEQ ID NO:4.

[0120] In some of any aspects, at least one viral gene is or comprises B2R.

[0121] In some of any aspects, at least one viral gene is or comprises A35R and J2R.

[0122] In some of any aspects, the nucleic acid genome of the recombinant VACV strain comprises the nucleic acid sequence set forth in SEQ ID NO:12, or a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the sequence set forth in SEQ ID NO:12.

[0123] In some of any aspects, at least one viral gene is or comprises B2R and J2R.

[0124] In some of any aspects, the nucleic acid genome of the recombinant VACV strain comprises the nucleic acid sequence set forth in SEQ ID NO:48, or a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the sequence set forth in SEQ ID NO:48.

[0125] In some of any aspects, the nucleic acid genome of the recombinant oncolytic virus comprises a nucleic acid sequence described in any one of SEQ ID NOs: 48, 80, 82, and 84-93, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence described in any one of SEQ ID NOs: 48, 80, 82, and 84-93. In some of any aspects, the nucleic acid genome of the recombinant oncolytic virus comprises a nucleic acid sequence described in any one of SEQ ID NOs: 85, 86, 88, and 90, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence described in any one of SEQ ID NOs: 85, 86, 88, and 90. In some of any aspects, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence described in SEQ ID NO: 85, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence described in SEQ ID NO: 85.

[0126] Also provided herein are nucleic acids comprising the genome of any of the recombinant oncolytic viruses provided herein or any of the isolated VACV clone strains provided herein.

[0127] Also provided herein are recombinant oncolytic viruses comprising any of the nucleic acids of the recombinant oncolytic viruses provided herein.

[0128] In some of any aspects, the recombinant oncolytic virus is a recombinant oncolytic vaccinia virus. In some of any aspects, the recombinant oncolytic virus is a recombinant oncolytic vaccinia virus.

[0129] Also provided herein are pharmaceutical compositions comprising any of the recombinant VACV strains provided herein.

[0130] Also provided herein are pharmaceutical compositions comprising any of the recombinant oncolytic viruses provided herein, optionally wherein the recombinant oncolytic virus is a recombinant oncolytic vaccinia virus.

[0131] In some embodiments of any aspect, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0132] In some embodiments of any aspect, the pharmaceutical composition is formulated for intravenous, intratumoral, intraperitoneal, or intrathoracic administration. In some embodiments of any aspect, the pharmaceutical composition is formulated for intravenous administration. In some embodiments of any aspect, the pharmaceutical composition is a liquid composition. In some embodiments of any aspect, the pharmaceutical composition is lyophilized.

[0133] Also provided herein is a method of treating a proliferative disorder in a subject, comprising administering to the subject any of the recombinant oncolytic viruses provided herein, any of the isolated oncolytic viruses provided herein, or any of the pharmaceutical compositions provided herein.

[0134] In some embodiments, the proliferative disorder is a tumor or metastasis. In some embodiments of any aspect, the proliferative disorder is cancer. In some embodiments of any aspect, the cancer is pancreatic cancer, ovarian cancer, lung cancer, colon cancer, prostate cancer, cervical cancer, breast cancer, rectal cancer, renal (kidney) cancer, gastric cancer, esophageal cancer, liver (hepatic) cancer, endometrial cancer, bladder cancer, brain cancer, head and neck cancer, oral cancer (e.g., oral cavity cancer), cervical cancer, uterine cancer, thyroid cancer, testicular cancer, prostate cancer, skin cancer, e.g., melanoma, e.g., malignant melanoma, cholangiocarcinoma (bile duct cancer), thymic epithelial cancer, e.g., thymoma, leukemia, lymphoma, or multiple myeloma. In some embodiments of any aspect, the cancer is microsatellite stable (MSS) colorectal cancer.

[0135] In some embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence set forth in SEQ ID NO:8, or a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the sequence set forth in SEQ ID NO:8.

[0136] In some embodiments, the recombinant oncolytic virus or the isolated oncolytic virus is 1×10 5 pfu~1×10 14 administered in an amount of pfu.

[0137] In some embodiments, the method further comprises administering a second therapeutic agent for treating the proliferative disorder.

[0138] In some embodiments, the method further comprises another treatment selected from surgery, radiotherapy, immunosuppressive therapy, and administration of an anticancer agent. In some embodiments, the another treatment is administration of an anticancer agent selected from cytokines, chemokines, growth factors, photosensitizers, toxins, anticancer antibiotics, chemotherapy compounds, radionuclides, angiogenesis inhibitors, signal transduction modulators, antimetabolites, anticancer vaccines, anticancer oligopeptides, mitosis-inhibiting proteins, anti-mitotic oligopeptides, anticancer antibodies, anticancer antibiotics, immunotherapy agents, and combinations of any of the foregoing.

[0139] In some embodiments, the recombinant oncolytic virus or the isolated oncolytic virus is administered intravenously.

[0140] In some embodiments, the method further comprises administering AP1903 (rimiducid) to the subject.

[0141] In some embodiments, the recombinant oncolytic virus administered to the subject comprises a heterologous nucleic acid encoding an apoptosis-inducing protein.

[0142] In some embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence set forth in SEQ ID NO:8, or a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the sequence set forth in SEQ ID NO:8.

[0143] In some embodiments, the subject exhibits severe immunodeficiency and is susceptible to viral infection.

[0144] In some embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence set forth in any one of SEQ ID NO:48, 80, 82, and 84-93, or a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence set forth in any one of SEQ ID NO:48, 80, 82, and 84-93.

[0145] Also provided herein is a method of inhibiting viral replication, comprising the step of contacting a cell infected with a recombinant oncolytic virus with AP1903 (rimiducid), wherein the recombinant oncolytic virus comprises a heterologous nucleic acid encoding an apoptosis-inducing protein.

[0146] Also provided herein is a method of inhibiting viral replication, comprising the step of contacting a cell with AP1903 (rimiducid) and infecting the cell with any of the recombinant oncolytic viruses provided herein, any of the isolated oncolytic viruses provided herein, or any of the recombinant oncolytic viruses provided herein, for example, a clonal VACV strain.

[0147] In some embodiments, the contacting step is performed in vivo in the subject. In some embodiments, AP1903 (Rimiducid) has been administered to a subject previously administered a recombinant oncolytic virus comprising a heterologous nucleic acid encoding an apoptosis-inducing protein. In some embodiments, AP1903 (Rimiducid) has been administered to a subject previously administered any of the recombinant oncolytic viruses provided herein or any of the isolated oncolytic viruses provided herein.

[0148] Also provided herein is a method of inhibiting viral replication in a subject, comprising the step of administering AP1903 (Rimiducid) to the subject, wherein the subject has been previously administered a recombinant oncolytic virus comprising a heterologous nucleic acid encoding an apoptosis-inducing protein.

[0149] Also provided herein is a method of inhibiting viral replication in a subject, comprising the step of administering AP1903 (Rimiducid) to the subject, wherein the subject has been previously administered any of the recombinant oncolytic viruses provided herein or any of the isolated oncolytic viruses provided herein.

[0150] In some embodiments, the method preferentially inhibits viral replication in non-cancer cells. In some embodiments, the apoptosis-inducing protein is inducible DED (iDED). In some embodiments, the iDED comprises the amino acid sequence set forth in SEQ ID NO:27, or an amino acid sequence having at least 85%, 90% or 95% sequence identity to SEQ ID NO:27.

[0151] In some embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence set forth in SEQ ID NO:8, or a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the sequence set forth in SEQ ID NO:8.

[0152] In some embodiments of any aspect, the nucleic acid genome of the recombinant oncolytic virus comprises a nucleic acid sequence as set forth in any one of SEQ ID NOs: 48, 80, 82, and 84-93, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence as set forth in any one of SEQ ID NOs: 48, 80, 82, and 84-93. BRIEF DESCRIPTION OF THE DRAWINGS

[0153]

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Mode for Carrying Out the Invention

[0154] Detailed description Provided herein are isolated clone strains that exhibit superior antitumorigenic activity compared to other vaccinia viruses and an enhanced ability to evade the host immune system. In particular, the provided clone strains are clone isolates derived from the parental IHD-J obtained from ATCC® Catalog No. VR-156™. Also provided are preparations obtained from the propagation of such isolated clone strains. Recombinant vaccinia viruses are provided that are attenuated by modifications that delete or reduce the expression of viral genes or inactivate viral proteins. Recombinant viruses are also provided that are further improved to avoid the host antiviral defense or to further enhance antitumor activity. For example, such recombinant viruses include heterologous nucleic acids encoding proteins to evade complement system inhibition, to evade natural killer (NK) cells or T cells, to incorporate immune checkpoint molecules to enhance immune stimulatory activity, or to provide anti-angiogenic activity. The recombinant viruses provided herein include, for example, those equipped with a virus-inducing system for inhibiting virus replication as a safety strategy by mediating apoptosis in certain unwanted infected cells, such as healthy cells. Specifically, provided herein is a recombinant oncolytic vaccinia virus comprising an inactivating mutation of B2R, a heterologous nucleic acid encoding interferon regulatory factor 3 (IRF3), and at least one heterologous nucleic acid encoding one or more cytokines and / or chemokines. In some embodiments, the at least one heterologous nucleic acid encoding one or more cytokines and / or chemokines comprises a heterologous nucleic acid encoding chemokine ligand 9 (CXCL9) and / or IL-12.Specifically, provided herein is also a recombinant oncolytic virus comprising an inactivating mutation of at least one viral gene and at least one heterologous nucleic acid encoding one or more heterologous gene products, wherein the one or more heterologous gene products are an immunomodulatory protein, a complement inhibitor, a T cell evasion factor or an NK cell evasion factor, an anti-angiogenic protein, an interferon regulatory factor or an apoptosis-inducing protein, or any combination thereof or include them.

[0155] Oncolytic viruses (OVs) are viruses that replicate selectively or more efficiently in cancer cells than in non-cancer cells. In some cases, the ability to selectively infect cancer cells, replicate within cancer cells, and destroy cancer cells often results from the ability to exploit biochemical differences between healthy cells and transformed cells during infection without harming healthy cells. Cancer cells are characterized by disruption of the apoptotic pathway, acquisition of new abilities to evade the immune system, and the ability to proliferate indefinitely, all of which are features favorable for viral replication. Since OVs cause little off-target toxicity, they are an attractive option as one of the main challenges in cancer treatment is to kill malignant cells while minimizing toxic effects.

[0156] Oncolytic viruses can be divided into three main groups: (1) viruses that have the natural property of preferentially replicating in cancer cells while being non-pathogenic in humans, such as parvovirus, myxoma virus, Newcastle disease virus, and reovirus; (2) viruses that have been genetically engineered to ensure selective replication in cancer cells, such as adenovirus, HSV, and vesicular stomatitis virus; and (3) viruses that have been attenuated by in vitro propagation for safe use in humans. The latter group includes oncolytic viruses derived from vaccinia, which are also preferred for their efficient replication, cell lysis, spread, host range, and natural tropism for tumor tissue (Shen et al. (2004) Mol. Ther., 11:180). For example, vaccinia virus is more potent in replication and spread than adenoviral vectors.

[0157] Vaccinia virus (VV), the prototype member of the Orthopoxvirus genus, replicates in the cytoplasm of host cells. VV is a large, complex enveloped virus with a linear double-stranded DNA genome approximately 190,000 base pairs in length, consisting of a single continuous polynucleotide chain encoding approximately 250 genes that can potentially express over 200 proteins. See, e.g., McCraith al., (1982) PNAS, 97(9):4879-4884. Generally, non-segmented non-infectious genomes are arranged such that centrally located genes are essential (and thus conserved) for virus replication, whereas genes near the two termini confer more peripheral functions, such as host range and pathogenicity. Vaccinia virus performs differential gene expression by utilizing open reading frames (ORFs) arranged in a generally non-overlapping set. See, e.g., Traktman, P., Chapter 27, Poxvirus DNA Replication, pp. 775-798, in DNA Replication in Eukaryotic Cells, Cold Spring Harbor Laboratory Press (1996). The ability of VV for rapid replication results in efficient lysis of infected cells and spread to other tumor cells during successive replications, resulting in significant local destruction of tumors. The VV genome encodes approximately 250 genes and can accept up to 20 kb of foreign DNA, making it ideal as a gene delivery vehicle. Recombinant VV vectors have been developed to deliver eukaryotic genes, such as tumor-associated antigens, to tumors and thus promote the induction of the host immune system aimed at killing cancer cells. However, a limiting factor in using VV as a cancer therapy delivery vector is the strong neutralizing antibody response induced by injection of VV into the bloodstream, which limits the virus's ability to persist and spread and prevents re-administration of the vector. In some cases, neutralizing antibodies recognize and bind to viral glycoproteins with high affinity, preventing viral interaction with host cell receptors and resulting in virus neutralization.

[0158] Vaccinia virus replicates in the cytoplasm of infected cells, where in the cytoplasm of the infected cells, the assembly of progeny begins in a special area called the viral factory. During replication, three morphologically and antigenically distinct forms of the virus are produced, namely, intracellular mature virions (IMVs), intracellular enveloped virions (IEVs), and extracellular virions. A subset of IMVs, which are the first infectious progeny produced, are transported to the trans-Golgi network (TGN), where they are enclosed by two additional membranes to produce IEVs. IEVs are transported through the cytoplasm to the cell periphery, where the outermost membrane fuses with the plasma membrane to release a double-membrane form called EV. EVs remaining on the cell surface are called cell-associated enveloped virions (CEVs), and EVs that have detached from the cell surface are called extracellular enveloped virions (EEVs). IMVs are the most abundant infectious form and are thought to be responsible for spread between hosts, CEVs are thought to play some role in cell-to-cell spread, and EEVs are considered important for long-distance dissemination within the host organism. In particular, EEVs are involved in long-distance viral spread and dissemination in vivo. See, for example, Blasco et al., (1993) Journal of Virology, 67(6):3319-3325. The outer proteins of EEVs can induce protective immunity against the virus (Blaso and Moss (1992) J. Virol., 66:4170-4179). However, there is a large variation in the amount of EEVs produced by vaccinia virus strains.

[0159] Attenuated vaccinia virus strains are being developed for therapeutic and diagnostic uses. For example, attenuated viruses include recombinant viruses in which one or more viral genes are modified, resulting in loss or reduced expression of viral genes or inactivation of viral proteins. Vaccinia is a well-studied attenuated virus with antitumorigenic properties, yet many strains of vaccinia, including recombinant strains, exhibit variability in pathogenicity and safety, rendering many unsuitable for clinical use. Thus, there is a need for improved vaccinia strains with enhanced antitumorigenic properties and low cytotoxicity, which are highly desirable as an effective oncolytic therapy. The oncolytic viruses and methods described herein address this need.

[0160] Since viral replication generally correlates with the cancer cell killing effect, various approaches have been tested to improve OV antitumor activity, mainly focusing on viral replication and spread. However, other aspects of viral infection, such as enhancing the host antitumor immune response, inducing apoptosis, and controlling tumor angiogenesis, are also important aspects of cancer virotherapy (Davola, M.E. and K.L. Mossman (2019) Oncoimmunology 8(6):e1581528).

[0161] Isolated cloned viruses are provided herein that are derived from a vaccinia virus strain known as IHD-J (ATCC® Catalog No. VR-156™). IHD-J is closely related to the Western Reserve (WR) strain but is a vaccinia virus strain that exhibits 10 - 40 times more EEV than WR and spreads to cells much farther away more efficiently than WR (Blaso and Moss, 1992). However, strains that exhibit an increased range of spread may not exhibit sufficient antitumorigenic activity for oncolytic virus therapy.

[0162] The provided embodiments are based on the identification of a specific clonal isolate of IHD-J, called VIP02, which not only exhibits a high percentage of EEV but also the highest antitumorigenic activity among other clonal isolates from the same strain. Further, the results demonstrated that single intravenous delivery of the clonal isolate at a low dose significantly inhibited tumor growth in a murine syngeneic tumor model and showed potent in vitro tumor cell killing against multiple tumor cells in 2D and 3D cultures. A vaccinia virus strain having the sequence characteristics of the VIP02 clonal isolate is also provided herein.

[0163] The provided embodiments also relate to recombinant viruses capable of introducing heterologous nucleic acids into isolated clonal viruses with enhanced antitumorigenic properties in order to further enhance their antitumorigenic properties while minimizing their cytotoxic effects on healthy cells.

[0164] In some embodiments, the selected clonal strains and their recombinant-derived strains are oncolytic virus candidates for the diagnosis and treatment of tumors. In some embodiments, the isolated clonal strains of vaccinia, and their recombinant-derived strains, can be used as therapeutic viruses for the treatment of proliferative disorders including cancer, hyperplasia, metastasis, and tumors, and for use in other therapeutic and / or diagnostic methods described herein. In some other embodiments, the clonal strains can be used in methods of vaccination. In other embodiments, the isolated clonal strains and their recombinant-derived strains can be used as the parental vaccinia virus for generating recombinant oncolytic viruses.

[0165] All publications referred to in this application, including patent documents, scientific papers, and databases, are hereby incorporated by reference in their entirety for all purposes as if each individual publication were separately incorporated by reference. If the definitions set forth herein conflict with or otherwise are inconsistent with the definitions set forth in patents, patent applications, published patent applications, and other publications incorporated herein by reference, the definitions set forth herein shall control over the definitions incorporated herein by reference.

[0166] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0167] I. Isolated Clone Virus Strains and Attenuated Strains Thereof Provided herein are isolated clone vaccinia virus (VACV) strains of the vaccinia virus strain IHD-J (ATCC® Catalog No. VR-156™) or clones thereof that exhibit characteristics of virus strains isolated therefrom. The parental IHD-J strain has a heterologous sequence. It is found herein that certain vaccinia virus clones with enhanced antitumorigenic properties can be isolated from IHD-J parental vaccinia virus preparations or mixtures.

[0168] In some embodiments, the clone strains provided herein are present in virus preparations propagated from IHD-J. For example, the clone strain or its preparation can be obtained by isolating an IHD-J-derived clone isolate from a cell culture in which the parental IHD-J or its variant has been propagated. The clone isolates provided herein are obtained by passaging the IHD-J virus in confluent CV-1s from African green monkey kidney fibroblast cultures grown in 6-well plates infected with serial dilutions of the vaccinia virus strain.

[0169] In some embodiments, the cloned strain does not contain a non-viral heterologous nucleic acid containing an open reading frame encoding a non-viral heterologous protein. In other embodiments, the cloned strain can be used as a parental sequence for generating a recombinant virus modified by a heterologous nucleic acid encoding a non-viral heterologous protein.

[0170] In some embodiments, the IHD-J cloned strain provided herein is designated VIP02 and has the nucleotide sequence set forth in SEQ ID NO:1.

[0171] In some embodiments, provided herein is a recombinant oncolytic vaccinia virus comprising an inactivating mutation of B2R, a heterologous nucleic acid encoding interferon regulatory factor 3 (IRF3), and at least one heterologous nucleic acid encoding one or more cytokines and / or chemokines. In some embodiments, the at least one heterologous nucleic acid encoding one or more cytokines and / or chemokines comprises a heterologous nucleic acid encoding chemokine ligand 9 (CXCL9) and / or IL-12.

[0172] In some embodiments, provided herein is also a recombinant oncolytic virus comprising an inactivating mutation of at least one viral gene and at least one heterologous nucleic acid encoding one or more heterologous gene products, wherein the one or more heterologous gene products are immunomodulatory proteins, complement inhibitors, T cell evasion factors or NK cell evasion factors, anti-angiogenic proteins, interferon regulatory factors or apoptosis-inducing proteins, or any combination or inclusion thereof.

[0173] In some embodiments, the provided vaccinia virus clone strain has a nucleic acid genome having at least 95% sequence identity to SEQ ID NO:1. In some embodiments, the provided vaccinia virus clone strain has a nucleic acid genome having at least 96% sequence identity to SEQ ID NO:1. In some embodiments, the provided vaccinia virus clone strain has a nucleic acid genome having at least 97% sequence identity to SEQ ID NO:1. In some embodiments, the provided vaccinia virus clone strain has a nucleic acid genome having at least 98% sequence identity to SEQ ID NO:1. In some embodiments, the provided vaccinia virus clone strain has a nucleic acid genome having at least 99% sequence identity to SEQ ID NO:1. In some embodiments, the provided vaccinia virus clone strain has a nucleic acid genome having at least 99.1% sequence identity to SEQ ID NO:1. In some embodiments, the provided vaccinia virus clone strain has a nucleic acid genome having at least 99.2% sequence identity to SEQ ID NO:1. In some embodiments, the provided vaccinia virus clone strain has a nucleic acid genome having at least 99.3% sequence identity to SEQ ID NO:1. In some embodiments, the provided vaccinia virus clone strain has a nucleic acid genome having at least 99.4% sequence identity to SEQ ID NO:1. In some embodiments, the provided vaccinia virus clone strain has a nucleic acid genome having at least 99.5% sequence identity to SEQ ID NO:1. In some embodiments, the provided vaccinia virus clone strain has a nucleic acid genome having at least 99.6% sequence identity to SEQ ID NO:1. In some embodiments, the provided vaccinia virus clone strain has a nucleic acid genome having at least 99.7% sequence identity to SEQ ID NO:1. In some embodiments, the provided vaccinia virus clone strain has a nucleic acid genome having at least 99.8% sequence identity to SEQ ID NO:1.In some embodiments, the provided vaccinia virus clone strain has a nucleic acid genome with at least 99.9% sequence identity to SEQ ID NO:1.

[0174] In some of any such embodiments, the provided vaccinia virus clone strain does not have a nucleic acid genome with the amino acid sequence set forth in SEQ ID NO:2 (IHD-W1). In some embodiments, the provided clone strain has a nucleotide sequence with less than 100% sequence identity to SEQ ID NO:2, and at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequence identity to SEQ ID NO:2. In some embodiments, the provided clone strain has a nucleotide sequence that differs from SEQ ID NO:2 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides. Such IHD-J clone viruses provided herein include viruses that differ in one or more open reading frames (ORFs) compared to the IHD-W1 strain having the nucleotide sequence set forth in SEQ ID NO:2. For example, the IHD-J clone viruses provided herein include viruses that differ in one or more ORFs compared to the IHD-W1 strain having the amino acid sequence set forth in SEQ ID NO:2. The IHD-J clone virus strain provided herein can contain nucleotide deletions or mutations in any one or more nucleotides of any ORF compared to SEQ ID NO:2, or can contain additions or insertions of viral DNA compared to SEQ ID NO:2.

[0175] In some embodiments, the provided vaccinia virus clone strain has at least 95% sequence identity to SEQ ID NO:1 and has a nucleic acid genome that exhibits the sequence characteristics of SEQ ID NO:1. For example, as described in Table E1 herein, the exemplary VIP02 clone isolate is characterized by a deletion or mutation in one or more nucleotides compared to SEQ ID NO:2, including one or more mutations in the ORF of SEQ ID NO:2. With respect to the ORF, the ORF is numbered in a consecutive order starting from 001. In other embodiments, the vaccinia virus open reading frame can also be designated by capital letters indicating the HindIII restriction endonuclease fragment, numbers indicating the position within the HindIII fragment, and a letter (L or R) indicating the direction of transcription, e.g., as designated by K5L. The corresponding protein is designated by capital letters and numbers, e.g., K5. In some embodiments, the nucleotide changes are within the non-ORF regions of the sequence.

[0176] In some embodiments, the provided vaccinia virus clone strain has at least 95% sequence identity to SEQ ID NO:57 and comprises or is characterized by a variant 017 open reading frame (ORF) encoding an amino acid sequence containing an amino acid other than alanine at position 66. In some embodiments, the amino acid at position 66 is a polar uncharged amino acid. In some embodiments, the amino acid at position 66 is serine (S), threonine (T), asparagine (N) or glutamine (E). In some embodiments, the amino acid at position 66 is T. In some embodiments, the provided clone strain comprises a variant 017 ORF having an A66T mutation compared to the 017 ORF set forth in SEQ ID NO:2. In some embodiments, the variant 017 ORF contains any of the above amino acid changes at position 66 and encodes an amino acid sequence having at least 96% sequence identity to SEQ ID NO:57. In some embodiments, the variant 017 ORF contains any of the above amino acid changes at position 66 and encodes an amino acid sequence having at least 97% sequence identity to SEQ ID NO:57. In some embodiments, the variant 017 ORF contains any of the above amino acid changes at position 66 and encodes an amino acid sequence having at least 98% sequence identity to SEQ ID NO:57. In some embodiments, the variant 017 ORF contains any of the above amino acid changes at position 66 and encodes an amino acid sequence having at least 99% sequence identity to SEQ ID NO:57. In some embodiments, the variant 017 ORF has the sequence set forth in SEQ ID NO:57. In some embodiments, such a vaccinia virus clone strain has a nucleic acid genome having at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.99% sequence identity to SEQ ID NO:1.

[0177] In some embodiments, the provided clone strain is a variant 038(K5L) ORF having a nucleotide insertion that results in a frameshift mutation, comprising or characterized by a variant 038(K5L) ORF in which the 038(K5L) gene product is modified. In some embodiments, the nucleotide insertion is an insertion of guanine (G) corresponding to the insertion after nucleotide position 32135 of SEQ ID NO:1. In some embodiments, the full-length sequence of the 038(K5L) gene product is set forth in SEQ ID NO:59. In some embodiments, the variant 038(K5L) ORF is set forth in SEQ ID NO:58. In some embodiments, such a vaccinia virus clone strain has a nucleic acid genome having at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.99% sequence identity to SEQ ID NO:1. In some embodiments, the variant 038(K5L) ORF is characterized by being modified as compared to the nucleic acid set forth in SEQ ID NO:73, or the amino acid sequence set forth in SEQ ID NO:74.

[0178] In some embodiments, the provided clone strain comprises, or is characterized by, variant 059(E2L) encoding an amino acid sequence having at least 95% sequence identity to SEQ ID NO:60 and containing an amino acid other than leucine at position 419. In some embodiments, the amino acid at position 419 is a hydrophobic amino acid other than leucine. In some embodiments, the amino acid at position 419 is alanine (A), valine (V), isoleucine (I), methionine (M), phenylalanine (F), tyrosine (Y), or tryptophan (W). In some embodiments, the amino acid at position 419 is F. In some embodiments, the provided clone strain comprises a variant 059(E2L) ORF having an L419F mutation as compared to the 059(E2L) ORF set forth in SEQ ID NO:2. In some embodiments, the variant 059(E2L) ORF contains any of the above amino acid changes at position 419 and encodes an amino acid sequence having at least 96% sequence identity to SEQ ID NO:60. In some embodiments, the variant 059(E2L) ORF contains any of the above amino acid changes at position 66 and encodes an amino acid sequence having at least 97% sequence identity to SEQ ID NO:60. In some embodiments, the variant 059(E2L) ORF contains any of the above amino acid changes at position 419 and encodes an amino acid sequence having at least 98% sequence identity to SEQ ID NO:60. In some embodiments, the variant 059(E2L) ORF contains any of the above amino acid changes at position 66 and encodes an amino acid sequence having at least 99% sequence identity to SEQ ID NO:60. In some embodiments, the variant 059(E2L) ORF has the sequence set forth in SEQ ID NO:60. In some embodiments, such vaccinia virus clone strain has a nucleic acid genome having at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.99% sequence identity to SEQ ID NO:1.

[0179] In some embodiments, the provided clone strain comprises, or is characterized by, a variant 104 (H4L) ORF encoding an amino acid sequence having at least 95% sequence identity to SEQ ID NO:61 and containing an amino acid other than asparagine (N) at position 591. In some embodiments, the amino acid at position 591 is a negatively charged amino acid. In some embodiments, the amino acid at position 591 is aspartic acid (D) or glutamic acid (E). In some embodiments, the amino acid at position 591 is D. In some embodiments, the provided clone strain comprises a variant 104 (H4L) ORF having an N591D mutation as compared to the 104 (H4L) ORF set forth in SEQ ID NO:2. In some embodiments, the variant 104 (H4L) ORF contains any of the above amino acid changes at position 591 and encodes an amino acid sequence having at least 96% sequence identity to SEQ ID NO:61. In some embodiments, the variant 104 (H4L) ORF contains any of the above amino acid changes at position 591 and encodes an amino acid sequence having at least 97% sequence identity to SEQ ID NO:61. In some embodiments, the variant 104 (H4L) ORF contains any of the above amino acid changes at position 591 and encodes an amino acid sequence having at least 98% sequence identity to SEQ ID NO:61. In some embodiments, the variant 104 (H4L) ORF contains any of the above amino acid changes at position 591 and encodes an amino acid sequence having at least 99% sequence identity to SEQ ID NO:61. In some embodiments, the variant 104 (H4L) ORF has the sequence set forth in SEQ ID NO:61. In some embodiments, such vaccinia virus clone strain has a nucleic acid genome having at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.99% sequence identity to SEQ ID NO:1.

[0180] In some embodiments, the provided clone strain is a variant 182 (A56R) ORF having a nucleotide deletion that results in a frameshift mutation, comprising or characterized by a variant 182 (A56R) ORF in which the 182 (A56R) gene product is modified. In some embodiments, the nucleotide deletion is a deletion of two consecutive nucleotides corresponding to nucleotides after nucleotide position 165972 of SEQ ID NO:2. In some embodiments, the 182 (A56R) gene product is set forth in SEQ ID NO:63. In some embodiments, the variant 182 (A56R) ORF is set forth in SEQ ID NO:62. In some embodiments, such a vaccinia virus clone strain has a nucleic acid genome having at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.99% sequence identity to SEQ ID NO:1. In some embodiments, the variant 182 (A56R) ORF is characterized by being modified as compared to the nucleic acid set forth in SEQ ID NO:75, or the amino acid sequence set forth in SEQ ID NO:76.

[0181] In some embodiments, the provided clonal strain is characterized by a nucleic acid genome that includes at least one of any of the above mutations in the 017 ORF, 038 (K5L) ORF, 059 (E2L) ORF, 104 (H4L) ORF, and 182 (A56R) ORF. In some embodiments, the provided clonal strain is characterized by a nucleic acid genome that includes at least two of any of the above mutations in the 017 ORF, 038 (K5L) ORF, 059 (E2L) ORF, 104 (H4L) ORF, and 182 (A56R) ORF. In some embodiments, the provided clonal strain is characterized by a nucleic acid genome that includes at least three of any of the above mutations in the 017 ORF, 038 (K5L) ORF, 059 (E2L) ORF, 104 (H4L) ORF, and 182 (A56R) ORF. In some embodiments, the provided clonal strain is characterized by a nucleic acid genome that includes at least four of any of the above mutations in the 017 ORF, 038 (K5L) ORF, 059 (E2L) ORF, 104 (H4L) ORF, and 182 (A56R) ORF. In some embodiments, at least one of the mutations is in the 017 ORF. In some embodiments, at least one of the mutations is in the 038 (K5L) ORF. In some embodiments, at least one of the mutations is in the 059 (E2L) ORF. In some embodiments, at least one of the mutations is in the 104 (H4L) ORF. In some embodiments, at least one of the mutations is in the 182 (A56R) ORF. In some embodiments, such a vaccinia virus clonal strain has a nucleic acid genome having at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.99% sequence identity to SEQ ID NO:1.

[0182] In some embodiments, the provided clonal strain is characterized by a nucleic acid genome comprising each of the above mutations in the 017 ORF, 038 (K5L) ORF, 059 (E2L) ORF, 104 (H4L) ORF and 182 (A56R) ORF. In some embodiments, the provided clonal strain comprises a variant 017 ORF encoding the amino acid sequence set forth in SEQ ID NO:57, a variant 038 (K5L) ORF set forth in SEQ ID NO:58, a variant of 038 (K5L) encoding the amino acid sequence set forth in SEQ ID NO:59, a variant 059 (E2L) ORF encoding the amino acid sequence set forth in SEQ ID NO:60, a variant 104 (H4L) ORF encoding the amino acid sequence set forth in SEQ ID NO:61, a variant 182 (A56R) ORF set forth in SEQ ID NO:62, and a variant of 182 (A56R) encoding the amino acid sequence set forth in SEQ ID NO:63. In some embodiments, such a vaccinia virus clonal strain has a nucleic acid genome having at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.99% sequence identity to SEQ ID NO:1.

[0183] In some embodiments, the provided vaccinia virus clone strain has at least 95% sequence identity to SEQ ID NO:1 and has a nucleic acid genome characterized by one or more of the following: (i) guanine (G) at the position corresponding to position 7770 of SEQ ID NO:1; (ii) thymine (T) at the position corresponding to position 15261 of SEQ ID NO:1; (iii) G at the position corresponding to position 32136 of SEQ ID NO:1; (iv) G at the position corresponding to position 49455 of SEQ ID NO:1; (v) cytosine (C) at the position corresponding to position 92969 of SEQ ID NO:1; (vi) the continuous nucleotide sequence CACTTATATAT (as set forth in SEQ ID NO:77) at the position corresponding to positions 106870 - 106880 of SEQ ID NO:1; (vii) the nucleic acid sequence GTTTTCATTA (as set forth in SEQ ID NO:78) at the position corresponding to positions 111267 - 111276 of SEQ ID NO:1; (viii) adenine (A) at the position corresponding to position 162715 of SEQ ID NO:1; (ix) the nucleic acid sequence TACAGACACC (as set forth in SEQ ID NO:79) at the position corresponding to positions 165844 - 185853 of SEQ ID NO:1; and (x) C at the position corresponding to position 187805 of SEQ ID NO:1.

[0184] In some embodiments, the vaccinia virus clone strain provided herein includes those having a nucleotide sequence characterized by one point mutation, insertion, and / or deletion selected from any one of (i) - (x) above.

[0185] In some embodiments, the vaccinia virus clone strain provided herein includes those having a nucleotide sequence characterized by one point mutation, insertion, and / or deletion selected from any two of (i) - (x) above.

[0186] In some embodiments, the vaccinia virus clone strain provided herein includes those having a nucleotide sequence characterized by one point mutation, insertion, and / or deletion selected from any three of (i) - (x) above.

[0187] In some embodiments, the vaccinia virus clone strains provided herein include those having a nucleotide sequence characterized by one point mutation, insertion, and / or deletion selected from any four of the above (i)-(x).

[0188] In some embodiments, the vaccinia virus clone strains provided herein include those having a nucleotide sequence characterized by one point mutation, insertion, and / or deletion selected from any five of the above (i)-(x).

[0189] In some embodiments, the vaccinia virus clone strains provided herein include those having a nucleotide sequence characterized by one point mutation, insertion, and / or deletion selected from any six of the above (i)-(x).

[0190] In some embodiments, the vaccinia virus clone strains provided herein include those having a nucleotide sequence characterized by one point mutation, insertion, and / or deletion selected from any seven of the above (i)-(x).

[0191] In some embodiments, the vaccinia virus clone strains provided herein include those having a nucleotide sequence characterized by one point mutation, insertion, and / or deletion selected from any eight of the above (i)-(x).

[0192] In some embodiments, the vaccinia virus clone strains provided herein include those having a nucleotide sequence characterized by one point mutation, insertion, and / or deletion selected from any nine of the above (i)-(x).

[0193] In some embodiments, the vaccinia virus clone strains provided herein include those having a nucleotide sequence characterized by one point mutation, insertion, and / or deletion selected from each of the above (i)-(x).

[0194] A. Exemplary Features In some embodiments, IHD-J-derived clones exhibited good antitumorigenicity and low pathogenicity / toxicity compared to the starting virus preparation or mixture, or other reference or isolated strains including recombinant strains, in in vitro and / or in vivo assays. In some embodiments, IHD-J-derived clones exhibited good antitumorigenic properties compared to the starting virus preparation or mixture, or other reference or isolated strains including recombinant strains. In some embodiments, IHD-J-derived clones exhibited low toxicity compared to the starting virus preparation or mixture, or other reference or isolated strains including recombinant strains. In some embodiments, IHD-J-derived clones exhibited similar antitumorigenic properties and / or similar toxicity compared to the starting virus preparation or mixture, or other reference or isolated strains including recombinant strains.

[0195] Provided herein are IHD-J clone isolates that exhibited improved properties compared to the starting virus preparation or mixture, or other reference or isolated strains including recombinant strains, in the absence of inserted heterologous DNA. In some embodiments, the IHD-J clone isolates exhibited good antitumorigenicity and low toxicity compared to the starting virus preparation or mixture, or other reference or isolated strains including recombinant strains, in the absence of inserted heterologous DNA. In some embodiments, the IHD-J clone isolates exhibited improved or good antitumorigenic activity compared to the starting virus preparation or mixture, or other reference or isolated strains including recombinant strains, in the absence of inserted heterologous DNA. In some embodiments, the IHD-J clone isolates exhibited low toxicity compared to the starting virus preparation or mixture, or other reference or isolated strains including recombinant strains, in the absence of inserted heterologous DNA. In some embodiments, the IHD-J clone isolates exhibited similar toxicity and / or antitumorigenic activity compared to the starting virus preparation or mixture, or other reference or isolated strains including recombinant strains, in the absence of inserted heterologous DNA.

[0196] In some embodiments, the cloned isolates that exhibited improved or good antitumorigenic activity compared to the starting virus preparation or mixture, or other reference or isolated strains containing recombinant strains, showed 120% or more and 1000% or less of the antitumorigenic activity of the reference virus preparation (starting virus preparation or mixture, or other reference or isolated strains containing recombinant strains) in an assay or method for evaluating the parameters indicating antitumorigenicity, for example, at least 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 400%, 500%, 1000% or more. Antitumorigenicity can be determined using either in vitro or in vivo tests related to the parameters indicating antitumorigenicity described herein.

[0197] In some embodiments, the clonal isolates provided herein exhibited an increase in the production of extracellular enveloped virus (EEV) compared to the starting virus preparation or mixture, or other reference or isolate including a recombinant strain. Vaccinia virus replicates intracellularly and produces both intracellular virus (IMV, intracellular mature virus; IEV, intracellular enveloped virus) and extracellular virus (EEV, extracellular enveloped virus; CEV, cell-associated extracellular virus) (Smith et al. (1998) Adv Exp Med Biol. 440:395-414). IMV corresponds to approximately 99% of the viral yield after replication by wild-type vaccinia virus strains. The IMV viral form is relatively stable in the external environment and is mainly responsible for inter-individual spread, but the IMV virus does not spread efficiently within the infected host due to inefficient release from cells and sensitivity to complement and / or antibody neutralization. In contrast, the EEV form is released into the extracellular environment and typically corresponds to only about 1% of the viral yield (Smith et al. (1998) Adv Exp Med Biol. 440:395-414). EEV is responsible for viral spread within the infected host and is relatively easily degraded outside the host. In addition, the EEV form has developed several mechanisms to inhibit its neutralization in the bloodstream. EEV is relatively resistant to complement (Vanderplasschen et al. (1998) Proc Natl Acad Sci USA. 95(13):7544-9), which is because a host cell inhibitor of complement is incorporated into its outer membrane coat and vaccinia virus complement control protein (VCP) is secreted into the local extracellular environment. In addition, EEV is relatively resistant to the neutralizing antibody effect compared to IMV (Smith et al. (1997) Immunol Rev. 159:137-54; Vanderplasschen et al. (1997) J Gen Virol. 78(Pt 8):2041-8). EEV is released at an earlier time point after infection (e.g., 4-6 hours) compared to IMV (released only during / after cell death), so the spread of the EEV form is faster (Blasco et al. (1993) J Virol. 67(6):3319-25).

[0198] Since EEV is relatively resistant to complement effects and antibody-mediated neutralization, when grown within cell types from the same species, this viral form becomes more stable and retains activity in the blood for a longer time after intravenous administration (Smith et al. (1998) Adv Exp Med Biol. 440:395-414; Vanderplasschen et al., (1998) Proc Natl Acad Sci U S A. (13):7544-9). This is particularly important for repeated dosing as neutralizing antibody levels increase and anti-cancer therapies require repeated dosing. Thus, an increase in the EEV form of vaccinia and other poxviruses can result in enhanced systemic efficacy.

[0199] In some embodiments, the clonal isolates provided herein showed an increase in the production of extracellular enveloped virus (EEV) compared to other clonal isolates derived from the IDH-J or Copenhagen strains. In some embodiments, the clonal isolates provided herein showed an increase in the production of extracellular enveloped virus (EEV), for example, at least 120% to 1000% more of extracellular enveloped virus (EEV), compared to a starting virus preparation or mixture, or other reference or isolated strains including recombinant strains, for example, at least 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 400%, 500%, 1000% or more production.

[0200] In some embodiments, more than 5% or about more than 5%, more than 6% or about more than 6%, more than 7% or about more than 7%, more than 8% or about more than 8%, more than 9% or about more than 9%, more than 10% or about more than 10%, more than 11% or about more than 11%, more than 12% or about more than 12%, more than 13% or about more than 13%, more than 14% or about more than 14%, more than 15% or about more than 15%, more than 16% or about more than 16%, more than 17% or about more than 17%, more than 18% or about more than 18%, more than 19% or about more than 19%, more than 20% or about more than 20% of the infectious particles after cell infection are EEV. In some embodiments, more than 5% of the infectious particles after cell infection are EEV. In some embodiments, more than 10% of the infectious particles after cell infection are EEV. In some embodiments, more than 15% of the infectious particles after cell infection are EEV. In some embodiments, more than 20% of the infectious particles after cell infection are EEV.

[0201] In other embodiments, the clonal isolates provided herein showed a decrease in tumor and / or metastatic growth, or an increase in tumor and / or metastatic shrinkage, in in vitro or in vivo assays or models. Tumors can be removed from a subject, weighed, and compared in weight to tumors removed from tumor-bearing subjects infected with the virus from a starting virus preparation or mixture, or other reference strains or isolates containing recombinant strains. It is also possible to compare the weight of the tumor with tumors removed from control treatment subjects at the same time after infection. The weight can be expressed as tumor volume / weight, and / or the ratio of tumor volume / weight (tumor weight of control-treated animals / tumor weight of clonal isolate-treated subjects). For example, a ratio of tumor weight of 1.2 or 5 means that the virus results in a decrease in tumor / metastatic weight / growth, and / or an increase in tumor / metastatic shrinkage, as well as an antitumorigenic activity of 120% or 500% compared to a reference or control.

[0202] In some embodiments, the clonal isolates provided herein showed a decrease in tumor and / or metastatic growth, or an increase in tumor and / or metastatic shrinkage. In some embodiments, the tumor / metastasis volume / weight ratio is greater than 1.0, for example, greater than 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 or more. In some embodiments, the increase in tumor / metastatic shrinkage is at least 120% - 500%, for example, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500% or more.

[0203] In some embodiments, the clonal isolates provided herein show 70% - 120%, for example, at least 70%, 80%, 90%, 95%, 100%, 110%, 115%, or 120%, or about 70%, 80%, 90%, 95%, 100%, 110%, 115%, or 120%, or 70%, 80%, 90%, 95%, 100%, 110%, 115%, or 120% of the anti - tumorigenic activity of the parental virus preparation or mixture or other reference virus strain in an assay or method that evaluates parameters such as toxicity showing similar anti - tumorigenic activity compared to the starting virus preparation or mixture, or other reference strain or isolate containing a recombinant strain.

[0204] In some embodiments, the clonal isolates provided herein show a decrease in the volume, size or weight of tumors and / or metastases in in vitro or in vivo assays or models. In some embodiments, the clonal isolates provided herein show a decrease in the volume of tumors and / or metastases, for example, a decrease in the volume, size or weight of tumors and / or metastases of 0% or more and 99% or less, for example, 99%, 98%, 97%, 96%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% or less than that, compared to the starting virus preparation or mixture, or other reference strain or isolate containing a recombinant strain, in terms of toxicity or more.

[0205] Parameters indicating toxicity or pathogenicity include, without limitation, a decrease in the percentage of viable cells in 2D (two-dimensional) and 3D (three-dimensional) cell cultures, a decrease in the body weight of a subject, fever, rash or other allergies, the presence of fatigue or abdominal pain, the tissue distribution of a virus, a decrease in the survival rate of a subject, induction of an immune response in a subject, the amount of tumor antigen released, and a decrease in the plaque formation rate. Toxicity or pathogenicity can be determined using any in vitro or in vivo test well known to those skilled in the art.

[0206] In some embodiments, the clonal isolates provided herein exhibit lower toxicity compared to a starting virus preparation or mixture, or other reference strain or isolate containing a recombinant strain, e.g., in an assay or method for evaluating a parameter indicating toxicity, 0% or more and 99% or less, e.g., 99%, 98%, 97%, 96%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% or less than that of a starting virus preparation or mixture, or other reference strain or isolate containing a recombinant strain. In some embodiments, the IHD-J clonal isolates provided herein exhibit 0% or more and 99% or less, e.g., 99%, 98%, 97%, 96%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% or less than that of other clonal isolates derived from the IHD-J or Copenhagen strain. In some embodiments, the method for evaluating a parameter indicating toxicity includes the step of quantifying the percentage of viable cells in a cell culture. In some embodiments, the method for evaluating a parameter indicating toxicity includes the step of quantifying the percentage of viable cells in 2D (two-dimensional) and 3D (three-dimensional) cell cultures. (Should all cell types used in the examples be listed?)

[0207] In some embodiments, the cloned isolates provided herein exhibit similar toxicity and / or cytotoxicity compared to the starting virus preparation or mixture, or other reference or isolated strains comprising recombinant strains, e.g., in an assay or method that evaluates parameters indicative of toxicity, 70% to 120% of the starting virus preparation or mixture, or other reference or isolated strains comprising recombinant strains, e.g., at least 70%, 80%, 90%, 95%, 100%, 110%, 115%, or 120%, or about 70%, 80%, 90%, 95%, 100%, 110%, 115%, or 120%, or 70%, 80%, 90%, 95%, 100%, 110%, 115%, or 120% of the anti-tumorigenic activity.

[0208] In certain embodiments, the cloned isolates provided herein exhibit improved anti-tumorigenicity and are less toxic (i.e., less pathogenic) compared to the starting virus preparation or mixture, or other reference or isolated strains comprising recombinant strains. For example, when administered to a subject in an amount effective to induce anti-tumorigenic activity, the cloned strain is less toxic (i.e., less pathogenic). In the treatment of human subjects or other similarly sized subjects, exemplary therapeutic amounts of the cloned strain are about 1×10 6 ~1×10 14 pfu or 1×10 6 ~1×10 14 pfu, about 1×10 7 ~1×10 10 pfu or 1×10 7 ~1×10 10 pfu, e.g., 1×10 9 ~1×10 10 pfu, e.g., at least 1×10 6 、1×10 7 、1×10 8 、1×10 9 、2×10 9 、3×10 9 、4×10 9 、or 5×10 9 pfu, or about 1×10 6 、1×10 7 、1×10 8 、1×109 , 2×10 9 , 3×10 9 , 4×10 9 , or 5×10 9 pfu within the range. In the treatment of mice or other similarly sized subjects, an exemplary therapeutic dose of the cloned strain is about 1×10 3 ~1×10 9 pfu or 1×10 3 ~1×10 9 pfu, for example, 1×10 5 ~1×10 7 pfu, for example, at least 1×10 3 , 1×10 4 , 1×10 5 , 1×10 6 , 2×10 6 , 3×10 6 , 4×10 6 , or 5×10 6 pfu, or about 1×10 3 , 1×10 4 , 1×10 5 , 1×10 6 , 2×10 6 , 3×10 6 , 4×10 6 , or 5×10 6 pfu, or 1×10 3 , 1×10 4 , 1×10 5 , 1×10 6 , 2×10 6 , 3×10 6 , 4×10 6 , or 5×10 6It is within the range of pfu. Such an effective amount can be determined empirically by those skilled in the art and depends on various factors including the subject, the condition or disease being treated, the stage or progression of the disease, the type of cancer, tumor, metastasis or hyperplasia, and other factors. The dosing regimen can vary. In some embodiments, the clonal isolates provided herein show a 100% survival rate of the subject over the course of the treatment regimen and are not associated with causing weight loss of the subject over the course of the treatment. In one embodiment, the clonal strain provided herein shows an increased survival rate when administered to a subject compared to the survival rate of subjects administered other clonal isolates of the same or similar therapeutic amount. In some embodiments, the clonal isolates provided herein show 100% tumor growth inhibition over the course of the treatment regimen.

[0209] The isolated clonal virus provided herein can be derived from plaque isolation of the IHD-J strain propagated through serial passage in cell lines. In some embodiments, the clonal isolates provided herein can be obtained by passaging the virus in embryonated chicken egg cultures, chicken embryo fibroblasts (CEF), Hela S3 cells, confluent CV-1 cells, or BHK-21 cells. In some embodiments, the clonal isolates provided herein can be obtained by passaging the virus in confluent CV-1 grown in a 6-well plate infected with a series of dilutions of the vaccinia virus strain, in African green monkey kidney fibroblast cultures. The clonal isolates provided herein have a homogeneous sequence. The exemplary clonal virus provided herein is a clonal isolate that shows enhanced antitumorigenic properties and reduced toxicity.

[0210] II. Attenuated vaccinia virus strain Also provided herein are recombinant vaccinia viruses that exhibit one or more modifications for attenuating viral virulence as compared to a wild-type or parental strain of a virus, e.g., as compared to any of the isolated clonal virus strains described in Section I. In some embodiments, provided herein are recombinant vaccinia viruses that are attenuated as compared to the vaccinia virus strain VIP02, e.g., having reduced virulence. In some embodiments, provided herein are recombinant vaccinia viruses that are attenuated as compared to the vaccinia virus strain described in SEQ ID NO:1, e.g., having reduced virulence. In some embodiments, the attenuated virus is a virus having low toxicity to normal cells, e.g., low or reduced viral replication, cytolytic activity, or cytotoxicity to normal cells such as non-tumor cells.

[0211] In some embodiments, the attenuated virus is a recombinant oncolytic vaccinia virus comprising an inactivating mutation of B2R, a heterologous nucleic acid encoding interferon regulatory factor 3 (IRF3), and at least one heterologous nucleic acid encoding one or more cytokines and / or chemokines. In some embodiments, the at least one heterologous nucleic acid encoding one or more cytokines and / or chemokines comprises a heterologous nucleic acid encoding chemokine ligand 9 (CXCL9) and / or IL-12.

[0212] In some embodiments, the attenuated virus is a recombinant oncolytic virus comprising an inactivating mutation of at least one viral gene and at least one heterologous nucleic acid encoding one or more heterologous gene products, wherein the one or more heterologous gene products are immunomodulatory proteins, complement inhibitors, T cell evasion factors or NK cell evasion factors, anti-angiogenic proteins, interferon regulatory factors or apoptosis-inducing proteins, or any combination or inclusion thereof.

[0213] In some embodiments, any of the vaccinia viruses provided is vaccinia growth factor (VGF) (McCart et al. (2001) Cancer Research 61:8751); thymidine kinase (TK) gene (International Publication No. 2005 / 047458); hemagglutinin (HA) gene (International Publication No. 2005 / 047458, and Zhang et al. (2007) Cancer Research 67:10038); F3 gene (also called F14.5L; International Publication No. 2005 / 047458, and Zhang et al. (2007) Cancer Research 67:10038); ribonucleotide reductase (Gammon et al. (2010) PLoS Pathogens 6:e1000984); serine protease inhibitor (e.g., SPI-1, SPI-2) (Guo et al. (2005) Cancer Research 65:9991, and Yang et al. (2007) Gene Therapy 14:638); ribonucleotide reductase gene F4L or I4L (Child et al. (1990) Virology 174:625; Potts et al. (2017) EMBO Mol. Med. 9:638); B2R (Eaglesham et al. (2019) Nature 566:259-263); B18R (Symons et al. (1995) Cell 81:551; Kirn et al. (2007) PLoS Medicine 4:e353); A48R (Hughes et al. (1991) J. Biol. Chem. 266:20103); B8R (Verardi et al. (2001) J. Virol. 75:11); B15R (Spriggs et al. (1992) Cell 71:145); A41R (Ng et al. (2001) Journal of General Virology 82:2095); A52R (Bowie et al. (2000) Proc. Natl. Acad. Sci. USA 97:10162); F1L (Gerlic et al. (2013) Proc. Natl. Acad. Sci. USA 110:7808); E3L (Chang et al.(1992) Proc. Natl. Acad. Sci. USA 89:4825); A44R - A46R (Bowie et al. (2000) Proc. Natl. Acad. Sci. USA 97:10162); K1L (Bravo Cruz et al. (2017) Journal of Virology 91:e00524); A48R, B18R, C11R and TK (Mejias - Perez et al. (2017) Molecular Therapy: Oncolytics 8:27) can be attenuated by modifying vaccinia virus so that the functions are defective. In some embodiments, several non - essential genes such as J2R (thymidine kinase TK) (Buller et al. 1985), C11R (secreted epidermal growth factor - like) (Buller et al. 1988), A56R (hemagglutinin HA) (Shida et al. 1988), B8R (soluble interferon - gamma receptor - like) (Verardi et al. 2001) and F14.5L (WO 2005 / 047458, and Zhang et al. (2007) Cancer Research 67:10038) are known to result in a decrease in pathogenicity when deleted or disrupted.

[0214] In some embodiments, provided herein are recombinant vaccinia virus strains having a genome in which any of the above genes has an inactivating mutation that inactivates the gene and thereby attenuates the virus. In some embodiments, the viral gene is selected from the group consisting of hemagglutinin (HA), J2R (thymidine kinase), F14.5L, A56R (hemagglutinin), B2R, vaccinia growth factor (VGF), A35R, A49R, A55R, B14R, C4L, C6L, C16L, NIL / N2L, E2L / E3L, K1L / K2L, K7L, superoxide dismutase locus, 7.5K, C2L-F3L, C4L-F1L, C7-K1L, B13R + B14R, A26L and I4L. In some embodiments, the inactivating mutation is a deletion of some or all of the viral gene. In some embodiments, the inactivating mutation is a deletion of the entire ORF of the viral gene. In some embodiments, the inactivating mutation is a deletion of a portion of the ORF of the viral gene that renders the encoded gene product non-functional. In some embodiments, the portion of the ORF that is deleted is a contiguous sequence of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more nucleotides up to the entire sequence of the ORF of the viral gene.

[0215] In some embodiments, the gene region or the encoded gene product can be rendered non-functional by any of a variety of methods known to those of skill in the art. In some embodiments, the gene region or gene product can be rendered non-functional as a result of one or more mutations (e.g., substitutions), truncations or deletions of the gene region. In some embodiments, the gene region or gene product can be rendered non-functional as a result of a mutation, truncation or deletion of the promoter region that controls the expression of the gene region. In some embodiments, the gene region or gene product can be rendered non-functional by a mutation, truncation or deletion of the polyadenylation sequence such that translation of the polypeptide encoded by the gene region is reduced or eliminated.

[0216] In some embodiments, the attenuated recombinant vaccinia virus of the disclosure that is deficient in a given vaccinia virus gene exhibits a decrease in the production and / or activity of the gene product of the gene (e.g., mRNA gene product; polypeptide gene product). In some embodiments, the amount and / or activity of the gene product is less than 75%, less than 60%, less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5% or less than 1% of the amount and / or activity of the same gene product produced by the wild-type vaccinia virus or a control vaccinia virus that does not contain genetic modifications. For example, in some embodiments, the amount and / or activity of the gene product is less than 75%, less than 60%, less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5% or less than 1% of the amount and / or activity of the same gene product produced by VIP02 or a vaccinia virus having the nucleic acid genome set forth in SEQ ID NO:1. In some embodiments, the amount and / or activity of the gene product is less than 75%, less than 60%, less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5% or less than 1% of the amount and / or activity of the same gene product produced by the IHD-W1 strain or a vaccinia virus having the nucleic acid genome set forth in SEQ ID NO:2.

[0217] In some embodiments, the attenuated recombinant vaccinia virus of the disclosure lacking a viral gene may have a deletion in a region consisting of a designated gene region or in an adjacent gene region containing the designated gene region. By way of example, mutations and / or shortening and / or deletion of a promoter region that reduces transcription of a gene region can result in a deletion. The gene region can also be deleted by incorporation of a transcription termination element such that translation of the polypeptide encoded by the gene region is reduced or eliminated. The gene region can also be deleted by using a gene editing enzyme or gene editing complex to reduce or eliminate transcription of the gene region. The gene region can also be deleted by using competitive reverse promoter / polymerase occupancy to reduce or eliminate transcription of the gene region. The gene region can also be deleted by inserting a nucleic acid into the gene region, thereby knocking out the gene region. Optionally, a heterologous nucleic acid may be inserted into the viral gene as described for exemplary recombinant vaccinia virus strains in Section III.

[0218] In some embodiments, the OVV provided by the disclosure is deficient in vaccinia virus thymidine kinase (TK). Optionally, the OVV of the disclosure includes deletion of all or part of the vaccinia virus TK coding region such that the recombinant oncolytic vaccinia virus having replication ability is TK-deficient. For example, optionally, the OVV of the disclosure includes deletion of the J2R gene (i.e., the gene encoding viral thymidine kinase). See, e.g., Mejia-Perez et al. (2018) Mol. Ther. Oncolytics 8:27. Optionally, the OVV of the disclosure includes an insertion into the J2R region, thereby resulting in reduced expression or activity of vaccinia virus TK.

[0219] In some embodiments, any of the cloned vaccinia virus strains described in Section I, such as VIP02, or the vaccinia virus strain described in SEQ ID NO:1, may have their genomes further modified to attenuate the virus. In some embodiments, the vaccinia virus strain has one or more of the TK (J2R), hemagglutinin (HA), A35R or B2R genes modified. In some embodiments, the modification renders the gene product encoded by the locus non-functional or deletes it. In some embodiments, all or part of the ORF of TK, HA, A35R or B2R is deleted.

[0220] In some embodiments, the attenuated recombinant vaccinia virus provided herein has an inactivating mutation such as an insertion, mutation or deletion of the J2R gene (TK; SEQ ID NO:66) encoding thymidine kinase. In some embodiments, the TK locus is not essential for virus replication, and its modification has been reported to reduce virus pathogenicity, prevent virus replication in the brain or ovary, and retain the ability to preferentially replicate in tumor tissue (e.g., Buller et al. (1985) Nature, 317:813-815). In some embodiments, the nucleic acid genome of the recombinant vaccinia virus strain comprises a nucleotide sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the sequence set forth in SEQ ID NO:4. In some embodiments, the nucleic acid genome of the recombinant vaccinia virus strain has the nucleotide sequence set forth in SEQ ID NO:4. In some embodiments, the recombinant vaccinia virus is a vaccinia virus designated VIR13.

[0221] In some embodiments, the attenuated recombinant vaccinia virus provided herein has an inactivating mutation such as an insertion, mutation, or deletion in the B2R locus encoding cytoplasmic cGAMP nuclease (poxin) (SEQ ID NO: 54). In some embodiments, the B2R locus has been reported to effect vaccinia virus attenuation in a skin scarification model (Eaglesham et al. 2019, Nature 566:259-263). In some embodiments, the attenuated recombinant vaccinia virus provided herein has an inactivating mutation such as an insertion, mutation, or deletion in the B2R gene and an inactivating mutation such as an insertion, mutation, or deletion in the J2R gene. In some embodiments, the nucleic acid genome of the recombinant vaccinia virus strain comprises a nucleotide sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence set forth in SEQ ID NO: 48. In some embodiments, the nucleic acid genome of the recombinant vaccinia virus strain has the nucleotide sequence set forth in SEQ ID NO: 48. In some embodiments, the recombinant vaccinia virus is a vaccinia virus designated VIR94.

[0222] In some embodiments, the attenuated recombinant vaccinia virus provided herein has an inactivating mutation such as an insertion, mutation, or deletion at the A35R locus. A35R is a pathogenicity gene that regulates the adaptive immune response, and its inactivation by deletion or the like can lead to a decrease in virus replication ability and can reduce virus pathogenicity (Brennan et al. 2015, J. Virol., 89:9986 - 9997). In some embodiments, the nucleic acid genome of the recombinant vaccinia virus strain comprises a nucleotide sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence set forth in SEQ ID NO:3. In some embodiments, the nucleic acid genome of the recombinant vaccinia virus strain has the nucleotide sequence set forth in SEQ ID NO:3. In some embodiments, the recombinant vaccinia virus is a vaccinia virus designated VIR11. In some embodiments, the attenuated recombinant vaccinia virus provided herein has an inactivating mutation such as an insertion, mutation, or deletion in the A35R gene and an inactivating mutation such as an insertion, mutation, or deletion in the J2R gene. In some embodiments, the nucleic acid genome of the recombinant vaccinia virus strain comprises a nucleotide sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence set forth in SEQ ID NO:12. In some embodiments, the nucleic acid genome of the recombinant vaccinia virus strain has the nucleotide sequence set forth in SEQ ID NO:12. In some embodiments, the recombinant vaccinia virus is a vaccinia virus designated VIR52.

[0223] In some embodiments, the attenuated recombinant vaccinia virus provided herein has an inactivating mutation such as an insertion, mutation, or deletion of the A56R locus (HA; SEQ ID NO: 67) encoding hemagglutinin. In some embodiments, the HA locus is not essential for virus replication, and its modification has been reported to reduce viral pathogenicity, prevent the virus from replicating in the brain or ovaries, and retain the ability to preferentially replicate in tumor tissue (e.g., Shida et al. (1988) J. Virol., 62:4474-4480).

[0224] In some embodiments, the attenuated recombinant vaccinia virus provided herein has an inactivating mutation such as an insertion, mutation, or deletion of the F14.5L gene (SEQ ID NO: 65). In some embodiments, the attenuated recombinant vaccinia virus provided herein has an insertion, mutation, or deletion of the F3 gene product encoded by the F14.5L gene (SEQ ID NO: 64). In some embodiments, the F14.5L gene (also called F3) is not essential for virus replication, and its modification has been reported to reduce viral pathogenicity, prevent the virus from replicating in the brain or ovaries, and retain the ability to preferentially replicate in tumor tissue (e.g., U.S. Patent Application Publication No. 2005 / 0031643).

[0225] The level of attenuation of a virus can be evaluated or determined using a variety of methods. Such methods for measuring the level of attenuation can be performed in vitro or in vivo and can include the evaluation of changes in the following characteristics of the virus, namely, a) viral mRNA synthesis, b) viral protein expression, c) viral DNA replication, d) viral plaque size, e) viral titer, or f) in vivo toxicity. Methods for evaluating the level of attenuation of a virus by in vitro and in vivo methods are known in the art and include, without limitation, methods such as plaque assays and mouse models of viral pathogenicity. Exemplary methods for testing early, intermediate, and late transcription of vaccinia can be found in Broyles et al. Methods Mol Biol. (2004) 269:135-142 and Wright et al. Methods Mol. Biol. (2004) 269:143-150. Methods for assaying viral RNA transcripts and proteins include, without limitation, RNA hybridization and blotting techniques as well as techniques well known as immunohistochemical examinations.

[0226] III. Recombinant virus strains having heterologous nucleic acids Recombinant virus strains with modified genomic sequences are provided herein. In some embodiments, recombinant oncolytic viruses are provided herein that contain at least one heterologous nucleic acid encoding one or more heterologous gene products. The heterologous gene products are not particularly limited and, in some embodiments, can be complement inhibitors, T cell evasion factors or NK cell evasion factors, immunostimulatory proteins, anti-angiogenic proteins, interferon regulatory factors, apoptosis-inducing proteins, or any combination of the foregoing. Accordingly, in some embodiments, recombinant oncolytic viruses are also provided herein that contain at least one heterologous nucleic acid encoding one or more heterologous gene products, and the one or more heterologous gene products are complement inhibitors, T cell evasion factors or NK cell evasion factors, immunostimulatory proteins, anti-angiogenic proteins, interferon regulatory factors, apoptosis-inducing proteins, or any combination of the foregoing.

[0227] Recombinant oncolytic vaccinia viruses are provided herein that contain an inactivating mutation of B2R, a heterologous nucleic acid encoding interferon regulatory factor 3 (IRF3), and at least one heterologous nucleic acid encoding one or more cytokines and / or chemokines. In some embodiments, the at least one heterologous nucleic acid encoding one or more cytokines and / or chemokines contains a heterologous nucleic acid encoding chemokine ligand 9 (CXCL9) and / or IL-12.

[0228] A recombinant oncolytic virus is also provided herein, comprising an inactivating mutation of at least one viral gene and at least one heterologous nucleic acid encoding one or more heterologous gene products, wherein the one or more heterologous gene products are immunomodulatory proteins, complement inhibitors, T cell evasion factors or NK cell evasion factors, anti-angiogenic proteins, interferon regulatory factors or apoptosis-inducing proteins, or any combination thereof or including any of the foregoing.In some embodiments, at least one heterologous nucleic acid encoding one or more heterologous gene products comprises one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, such as one or more immunomodulatory proteins selected from the group consisting of LIGHT, IRF3, IL-2, IL-12, and CXCL9, and / or at least one heterologous nucleic acid encoding one or more heterologous gene products comprises one or more heterologous nucleic acids encoding apoptosis-inducing proteins, such as iDED, iFas, or iCas9, and / or at least one heterologous nucleic acid encoding one or more heterologous gene products comprises one or more heterologous nucleic acids encoding one or more T cell evasion factor proteins or NK cell evasion factor proteins, such as a set of proteins encoded by vaccinia virus ORFs 012, 203, and 018 (CPXV012-203-018), and / or at least one heterologous nucleic acid encoding one or more heterologous gene products comprises one or more heterologous nucleic acids encoding one or more complement inhibitors, such as CRASP-2, or miniFH, and / or one or more heterologous nucleic acids encoding one or more complement inhibitors are introduced into a viral membrane gene, optionally F14.5L, to produce a fusion gene encoding a fusion protein, and / or at least one heterologous nucleic acid encoding one or more heterologous gene products comprises one or more heterologous nucleic acids encoding one or more anti-angiogenic proteins, such as a VEGF inhibitor, an angiopoietin inhibitor, or versican, and / or at least one heterologous nucleic acid encoding one or more heterologous gene products comprises one or more heterologous nucleic acids encoding one or more therapeutic or diagnostic agents.

[0229] An inactivating mutation includes any of several methods that modify the expression and / or functionality of a gene product expressed by a viral gene inactivated by gene disruption or the like. Gene disruption can be achieved, for example, by gene deletion, nucleic acid insertion, nucleic acid mutation or substitution, knockout, premature stop codon, transcriptional promoter modification, RNAi, or gene editing, such as by CRISPR. In some embodiments, the inactivating mutation is by gene deletion and / or insertion (also referred to as introduction) of a heterologous nucleic acid encoding one or more gene products. In certain embodiments, the inactivating mutation combines a gene deletion with the insertion of a heterologous nucleic acid at such a locus. For example, in some methods that result in an inactivating mutation, such as by homologous recombination and other methods, the heterologous nucleic acid can be inserted within the region of the deleted gene. Thus, in some embodiments, it is understood that reference to the locus into which the heterologous nucleic acid is inserted is the deletion locus of the gene, inactivated by deletion of all or part of the gene. In some embodiments, the gene deletion removes the entire sequence of the gene. In other embodiments, the gene deletion is a partial deletion, i.e., one that removes a portion of the gene sequence. In one embodiment, the gene deletion is a partial deletion that removes at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% of the gene sequence. In one embodiment, the gene deletion is a partial deletion that removes at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of the protein-coding sequence of the gene. In other embodiments, the gene deletion removes 100% of the gene sequence. In still other embodiments, the gene deletion removes 100% of the protein-coding sequence of the gene. In one embodiment, the gene deletion removes at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900 or at least 1000 nucleotides of the gene sequence.In another aspect, the gene deletion is a partial deletion that removes at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900 or at least 1000 nucleotides of the gene sequence. In a specific aspect, the partial deletion of the gene results in a partial gene.

[0230] Recombinant oncolytic viruses comprising at least one heterologous nucleic acid encoding one or more heterologous gene products are also provided herein, wherein the one or more heterologous gene products are complement inhibitors, T cell evasion factors or NK cell evasion factors, immunomodulatory proteins, anti-angiogenic proteins, interferon regulatory factors, apoptosis-inducing proteins, or any combination or inclusion thereof.

[0231] Recombinant oncolytic viruses are also provided herein that have a nucleic acid genome having at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:1 and at least one heterologous nucleic acid encoding one or more heterologous gene products inserted into the genome.

[0232] Exemplary heterologous proteins are described in the following subsections. In addition to recombinant virus strains, any of the heterologous proteins described can also be incorporated into gene therapy vectors (e.g., AAV, lentivirus and retrovirus), or cell-based therapies (e.g., chimeric antigen receptor-expressing T cell (CAR-T) therapy, natural killer (NK) cell therapy or tumor-infiltrating lymphocyte (TIL) therapy).

[0233] Among the viral strains provided, there are recombinant viral strains containing at least one heterologous nucleic acid encoding one or more heterologous gene products. In some embodiments, the recombinant viruses include, without limitation, vaccinia virus, vesicular stomatitis virus (VSV), Maraba virus (MARAV), measles virus (MV), myxoma virus, orf virus, parvovirus, arctic foxpox virus, coxsackievirus, reovirus, Newcastle disease virus, Seneca Valley virus, Semliki Forest virus, influenza virus, echovirus, poliovirus (PV), adenovirus (e.g., mastadenovirus and aviadenovirus), herpesvirus (e.g., herpes simplex virus 1, herpes simplex virus 2, herpes simplex virus 5, herpes simplex virus 6, Epstein–Barr virus, HHV6–HHV8, and cytomegalovirus), levivirus (e.g., levivirus, gut microbiota MS2, allolevirus), poxvirus (e.g., codepoxvirus, parapoxvirus, avipoxvirus, capripoxvirus, lepripoxvirus, swipoxvirus, molluscipoxvirus, entomopoxvirus), papovavirus (e.g., polyomavirus and papillomavirus), paramyxovirus (e.g., paramyxovirus, parainfluenza virus 1 (e.g., measles rubella virus), rubulavirus (e.g., mumps virus), pneumovirus, (pneumovirus, (pneumovirus) human), human respiratory syncytial virus and metapneumovirus (e.g., avian pneumovirus and human metapneumovirus)), picornavirus (e.g., enterovirus, rhinovirus, hepatovirus (e.g., human hepatitis A virus), cardiovirus and aptovirus), reovirus (e.g., orthoreovirus, orbivirus, rotavirus, cytoreovirus, fijivirus, phytoreovirus and oryzavirus), retrovirus (e.g., mammalian type B retrovirus, mammalian type C retrovirus, group D retrovirus, BLV–HTLV retrovirus,Retroviruses (e.g., human immunodeficiency virus type 1 and human immunodeficiency virus type 2 (e.g., HIV gp 160), spumaviruses), flaviviruses (e.g., hepatitis C virus, dengue virus, West Nile virus), hepatadaviruses (e.g., hepatitis B virus), togaviruses (e.g., alphaviruses (e.g., sindbis virus) and rubiviruses (e.g., rubella virus)), rhabdoviruses (e.g., vesiculovirus, lyssavirus, ephemerovirus and cytoradovirus), arenaviruses (e.g., arenavirus, lymphocytic choriomeningitis virus, Ippy virus and Lassa virus) and coronaviruses (e.g., coronavirus and torovirus) are included.

[0234] In some embodiments, the recombinant virus includes an oncolytic virus. In some embodiments, the recombinant virus is a recombinant oncolytic virus. In some embodiments, the recombinant virus, e.g., the recombinant oncolytic virus, is vaccinia virus, herpes simplex virus, vesicular stomatitis virus (VSV), Maraba virus (MARAV), measles virus (MV), adenovirus, myxoma virus, orf virus, parvovirus, araigpox virus, coxsackievirus, reovirus, Newcastle disease virus, Seneca Valley virus, Semliki Forest virus, mumps virus, influenza virus, echovirus or poliovirus (PV). In some embodiments, the recombinant virus, e.g., the recombinant oncolytic virus, is vaccinia virus.

[0235] In some embodiments, the recombinant virus is a virus that is not an oncolytic virus. In some embodiments, the recombinant virus is a virus that is not vaccinia virus. In some embodiments, the recombinant virus includes vaccinia virus. In some embodiments, the recombinant virus is derived from the Copenhagen strain.

[0236] In certain embodiments, the recombinant virus is a virus derived from IHD-J. In some embodiments, the recombinant virus is a virus derived from VIP02. In some embodiments, provided herein are recombinant viruses that include one or more mutations, insertions, deletions, or substitutions (replacements) of nucleic acids, or other modifications of the viral genomic sequence, e.g., recombinant oncolytic viruses. In some embodiments, provided herein is a modified VIP02 strain in which the genomic sequence is modified as compared to the genomic sequence set forth in SEQ ID NO:1. In some embodiments, the recombinant virus is a virus derived from a virus having the nucleic acid genome set forth in SEQ ID NO:1, wherein the genome is modified by the insertion of a nucleic acid encoding a heterologous gene product.

[0237] Methods for generating recombinant viruses using recombinant DNA technology are well known in the art (see, e.g., U.S. Patent Nos. 4,769,330; 4,603,112; 4,722,848; 4,215,051; 5,110,587; 5,174,993; 5,922,576; 6,319,703; 5,719,054; 6,429,001; 6,589,531; 6,573,090; 6,800,288; 7,045,313; He et al. (1998) PNAS U S A. 95(5):2509-2514; Racaniello et al., (1981) Science 214:916-919). Methods for generating recombinant vaccinia viruses for the methods can also be found in the examples described herein.

[0238] In some embodiments, the recombinant virus has a large carrying capacity for foreign genes into which exogenous DNA fragments can be inserted. For example, the vaccinia virus genome has a large carrying capacity for foreign genes into which exogenous DNA fragments of up to 25 kb can be inserted. The genomes of several vaccinia strains have been completely sequenced, and many essential and non-essential genes have been identified. Due to the high sequence homology between different strains, genomic information from one vaccinia strain can be used to design and generate modified viruses in other strains. Finally, the technology for producing modified vaccinia strains by genetic manipulation is well established (Moss, Curr. Opin. Genet. Dev. 3:86-90 (1993); Broder and Earl, Mol. Biotechnol. 13:223-245 (1999); Timiryasova et al., Biotechniques 31:534-540 (2001)).

[0239] Insertion sites of heterologous nucleic acid molecules are known in the art and have been described for various viral vectors (see, for example, 5,166,057, 5,266,489, 6,338,846, 6,248,320, 6,221,646, 6,841,158, 7,101,685, 7,001,760, and references therein). Heterologous nucleic acid molecules are typically inserted into non-coding or coding regions of genes that are not essential for viral replication.For example, in the vaccinia virus, the insertion site of a heterologous DNA molecule can be, without limitation, the thymidine kinase (TK) gene, the hemagglutinin (HA) gene, F14.5L (see, for example, U.S. Patent Application Publication No. 2005-0031-643), the VGF gene (see, for example, U.S. Patent Application Publication No. 2003-0031681), Hind III F, F13L, or Hind III M (see, for example, U.S. Patent No. 6,548,068); the hemorrhagic region or the A-type inclusion body region (ATI) (see, for example, U.S. Patent No. 6,265,189 and U.S. Patent No. 6,596,279); the A33R gene, the A34R gene, the A36R gene or the B5R gene (see, for example, Katz et al., (2003) J. Virology 77:12266-12275); SalF7L (see, for example, Moore et al., (1992) EMBO J. 11:1973-1980); N1L (see, for example, Kotwal et al. (1989) Virology 171:579-587); M1 lambda (see, for example, Child et al. (1990) Virology. 174:625-629); HR, HindIIII-MK, HindIII-MKF, HindIII-CNM, RR, or BamF (see, for example, Lee et al. (1992) J Virol. 66:2617-2630); C21L (see, for example, Isaacs et al. (1992) Proc Natl Acad Sci USA. 89:628-632), the host range region genes K1L and C7L, A35R (see, for example, U.S. Patent No. 6,265,189, U.S. Patent No. 7,045,313; U.S. Patent Application Publication No. 2005-0244428, U.S. Patent Application Publication No. 2006-0159706; Coupar et al. J. Gen. Virol. (2000) 81:431-439; Smith et al. (1993) Vaccine 11(1):43-53), and can be within the intergenic regions, non-coding regions, and / or non-essential genes or non-essential gene regions. When multiple gene expression cassettes are inserted, the insertions can be at the same insertion site or at different insertion sites.Alternatively, the heterologous nucleic acid molecule can be inserted into an essential gene, and a cell line for packaging the virus can be used for virus production.

[0240] In some embodiments, at least one heterologous nucleic acid encoding one or more heterologous gene products is inserted into or instead of a non-essential gene or non-essential region within the viral genome. In some embodiments, at least one heterologous nucleic acid encoding one or more heterologous gene products is inserted into or instead of hemagglutinin (HA), J2R (thymidine kinase), F14.5L, A56R (hemagglutinin), B2R, vaccinia growth factor (VGF), A35R, A49R, A55R, B14R, C4L, C6L, C16L, NIL / N2L, E2L / E3L, K1L / K2L, K7L, superoxide dismutase locus, 7.5K, C2L-F3L, C4L-F1L, C7-K1L, B13R + B14R, A26L or I4L locus, or any combination thereof within the viral genome. In some embodiments, at least one heterologous nucleic acid encoding one or more heterologous gene products is inserted into or instead of the F14.5L locus. The F14.5 locus encodes a viral membrane protein. In some embodiments, at least one heterologous nucleic acid encoding one or more heterologous gene products is inserted into or instead of the A35R locus. In some embodiments, at least one heterologous nucleic acid encoding one or more heterologous gene products is inserted into or instead of the J2R locus. In some embodiments, the insertion into the locus is an insertion where the locus contains a partial deletion and the heterologous nucleic acid replaces the deleted portion. In some embodiments, the insertion into the locus is an insertion where a portion of the endogenous locus is not deleted. In some embodiments, the insertion in place of the locus is an insertion where the entire locus is deleted and replaced by the heterologous nucleic acid.

[0241] Mutations in non-essential vaccinia genes can also contribute to increased attenuation of the virus. Thus, insertion of heterologous expression cassettes into non-essential genes such as the TK gene can attenuate the virus in two scenarios: by genetic mutation and by additional transcriptional and / or translational burden. In the methods described herein, mutations in non-essential genes are not required, but one or more non-essential genes can be modified to enhance the attenuation effect of the gene expression cassette. Virus attenuation can be subsequently reduced by removing the expression cassette and replacing it with non-coding sequences such that the gene remains inactive (i.e., the virus shows increased replication). Thus, removal or replacement of the gene expression cassette reduces the transcriptional and / or translational burden on the virus and reduces virus attenuation.

[0242] In some embodiments, at least one heterologous nucleic acid encoding one or more heterologous gene products is fused to a gene encoding a viral membrane protein within the genome of the virus. In some embodiments, at least one heterologous nucleic acid encoding one or more heterologous gene products is fused to a gene encoding a viral membrane protein to produce a fusion protein. In some embodiments, at least one heterologous nucleic acid encoding one or more heterologous gene products is fused to a viral membrane protein to produce a fusion protein. In some embodiments, the gene encoding the viral membrane protein fused to at least one heterologous nucleic acid encoding one or more heterologous gene products is F14.5L. In some embodiments, the viral membrane protein is F14.5L. In some embodiments, the viral membrane protein is F14.5L and the fusion is at the C-terminus of F14.5L. In some embodiments, the fusion protein is incorporated into the outer membrane of intracellular mature virus (IMV), e.g., vaccinia virus. These fusion proteins containing the viral membrane protein F14.5L are predicted to be incorporated into the outer membrane of IMV virus particles, thereby providing them with resistance to inactivation by complement in the blood.

[0243] Modifications can include mutations, insertions, deletions or substitutions (replacements) of nucleic acids, or other modifications of the viral genomic sequence. For example, the viruses provided herein can be modified to contain one or more heterologous nucleic acid molecules that are inserted into or replaced into the viral genome. Viral genes can be replaced by homologous genes from another virus or by different genes. In one aspect, the modification includes the insertion or replacement of one or more nucleotides, for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 1000, 2000, 3000 or more nucleotides. In some aspects, the modification includes the deletion of one or more nucleotides, such as at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 1000, 2000, 3000 or more nucleotides. In some aspects, the modification includes the substitution of one or more nucleotides, for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 1000, 2000, 3000 or more nucleotides.

[0244] Modifications include the insertion and / or replacement (substitution) of nucleic acids, or other modifications of the viral genomic sequence by heterologous nucleic acids. Generally, a heterologous gene is a gene encoding a non-viral protein. For example, a heterologous nucleic acid molecule encoding a heterologous gene can be inserted. In some embodiments, the heterologous nucleic acid replaces all or part of a viral gene. In other embodiments, the viruses provided herein can be modified by the insertion of one or more heterologous nucleic acid molecules. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more heterologous nucleic acid molecules can be inserted. The heterologous nucleic acid molecule can contain an open reading frame or can be a non-coding sequence. Generally, the inserted heterologous nucleic acid contains an open reading frame and is a continuous nucleotide sequence corresponding to the coding region of a gene. The gene to be inserted or replaced can be transcribed and / or translated from the viral genome after infection of a host cell such as a tumor cell. As described below, the heterologous nucleic acid can contain regulatory sequences for controlling gene expression. For example, the heterologous nucleic acid can be operably linked to a promoter for the expression of the open reading frame. In some embodiments, the promoter has a sequence identity of 70, 80, 90, 100% identical to the sequence set forth in SEQ ID NO: 68, 69, 70, 71 or 72. In some embodiments, the promoter has the same sequence identity as the sequence set forth in SEQ ID NO: 68, 69, 70, 71 or 72.

[0245] The modifications to the viral genome provided herein can result in changes in the characteristics or properties of the virus. Exemplary changes include changes in parameters that exhibit antitumorigenicity and / or toxicity. For example, insertions, mutations or deletions can reduce the pathogenicity of the clonal strain, for example, reducing the infectivity, toxicity, replicative ability or number of non-tumor organs or non-tumor tissues in which the vaccinia virus can accumulate. Exemplary insertions, deletions, mutations and / or substitutions of nucleic acids result in a vaccinia virus having good antitumorigenic properties and low toxicity compared to a clonal strain that does not contain the modification, and / or other reference strains or isolates including the starting virus preparation or mixture, or recombinant strains. In some embodiments, the insertions, deletions, mutations and / or substitutions of nucleic acids result in a vaccinia virus having similar antitumorigenic properties and toxicity compared to a clonal strain that does not contain the modification, and / or other reference strains or isolates including the starting virus preparation or mixture, or recombinant strains. In some embodiments, the modification to the viral genome reduces toxicity compared to a clonal strain that does not contain the modification, and / or other reference strains or isolates including the starting virus preparation or mixture, or recombinant strains. In some embodiments, insertions, mutations or deletions include, but are not limited to, those that increase antitumorigenicity and reduce the toxicity of the virus compared to a clonal strain that does not contain the modification, and / or other reference strains or isolates including the starting virus preparation or mixture, or recombinant strains.

[0246] In some embodiments, the insertions, mutations or deletions include those that increase the ability of the cloned virus strain to evade the host immune system, without limitation, as compared to a cloned strain without modifications, and / or a starting virus preparation or mixture, or other reference or isolated strain including a recombinant strain. In some embodiments, the insertions, mutations or deletions include those that increase the ability of the cloned virus strain to stimulate the host immune system, without limitation, as compared to a cloned strain without modifications, and / or a starting virus preparation or mixture, or other reference or isolated strain including a recombinant strain. In some embodiments, the insertions, mutations or deletions include those that increase the host anti-angiogenic activity, without limitation, as compared to a cloned strain without modifications, and / or a starting virus preparation or mixture, or other reference or isolated strain including a recombinant strain. In some embodiments, the insertions, mutations or deletions include those that increase the host apoptotic activity, without limitation, as compared to a cloned strain without modifications, and / or a starting virus preparation or mixture, or other reference or isolated strain including a recombinant strain.

[0247] In some embodiments, one or more heterologous nucleic acid molecules can encode, for example, an anti-apoptosis gene product of a fragment thereof, for example, a gene product capable of modifying the host's apoptotic response; an angiogenesis gene product or a fragment thereof, for example, a gene product capable of modifying the host's angiogenesis response; an immune system gene product or a fragment thereof, for example, a gene product capable of modifying the host's immune response. In some embodiments, a gene product or a fragment thereof capable of modifying the host's immune response can increase the ability of the host immune system to escape complement-mediated inhibition compared to an unmodified clonal strain and / or other reference or isolated strains including the starting virus preparation or mixture, or recombinant strain. In some embodiments, a gene product capable of modifying the host's immune response increases the activity of the host immune system compared to an unmodified clonal strain and / or other reference or isolated strains including the starting virus preparation or mixture, or recombinant strain.

[0248] In some embodiments, the recombinant virus is a vaccinia virus with a modified genomic sequence as compared to the genomic sequence set forth in SEQ ID NO:1 or a sequence having at least 99% sequence identity to SEQ ID NO:1. In some embodiments, the recombinant virus is a vaccinia virus with a modified genomic sequence as compared to the genomic sequence set forth in SEQ ID NO:1. The large genomic size of the vaccinia virus provided herein allows for the insertion of large and / or multiple nucleotide sequences of heterologous DNA into the viral genome (Smith and Moss (1983) Gene 25(1):21-28). The viruses provided herein can be modified by the insertion or substitution of one or more nucleotides. In one embodiment, the modification includes the insertion or substitution of one or more nucleotides, for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 1000, 2000, 3000, 4000, 5000 or more nucleotides. In some embodiments, one or more heterologous DNA molecules are inserted at a locus of the viral genome such as any described herein. In some embodiments, one or more heterologous DNA molecules are inserted into a non-essential region of the viral genome. For example, the DNA molecule is inserted at a locus that is not essential for viral replication in proliferating cells such as tumor cells. Exemplary insertion sites are known in the art and provided herein. In some embodiments, the recombinant vaccinia virus provided herein can contain an inactivating mutation in a viral gene such as any described, such as a complete or partial gene deletion of the viral gene. In such embodiments, one or more heterologous nucleic acids can be inserted at or instead of such a locus. In some embodiments, the recombinant virus is a virus modified as compared to the genomic sequence set forth in SEQ ID NO:1, in which one or more heterologous nucleic acids are inserted and one or more viral loci are inactivated by, for example, gene deletion.The modified recombinant virus can be any of the viruses provided herein that have a genome described in SEQ ID NO:1, or a genome that is at least 99% identical to SEQ ID NO:1, generated by the introduction of the heterologous DNA described herein, or any other virus. In some embodiments, the recombinant virus has a modified genomic sequence as compared to the genomic sequence described in SEQ ID NO:1 and has an amino acid sequence that exhibits at least 85%, 90% or 95% sequence identity to the sequence described in SEQ ID NO:1. In some embodiments, the nucleic acid genome of the recombinant oncolytic vaccinia virus has at least 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence described in SEQ ID NO:1.

[0249] In some embodiments, the recombinant virus can be modified to express an exogenous or heterologous gene. Exemplary exogenous gene products include proteins involved in the regulation of apoptosis, angiogenesis, and / or the immune system. In some embodiments, the gene products include proteins that affect the host's apoptotic pathway, such as caspase-9, the DED (death effector domain) of FADD (Fas-associated death domain protein), and Fas. In some embodiments, the gene products include proteins that affect the host's angiogenesis pathway, such as vascular endothelial growth factor (VEGF) and versican (VK). In some embodiments, the gene products include proteins that affect the host's immune system, such as minimized complement regulatory factor H (miniFH), Borrelia burgdorferi complement regulatory acquired surface protein 2 (CRASP-2), vaccinia virus ORFs 012, 203 and 018 (CPXV012-203-018), and human LIGHT variant (hmLIGHT). The characteristics of such gene products are described herein and elsewhere.

[0250] In particular, the viruses provided herein can be modified to express genes in vivo and in vitro. In some embodiments, the virus can be modified to express two or more gene products, such as 2, 3, 4, 5, 6, 7, 8, 9, 10 or more gene products, and any combination of two or more gene products can be one or more detectable gene products. In one embodiment, the virus can be modified to express an apoptosis-related gene product. In another example, the virus can be modified to express two or more gene products for generating a fusion protein. In some examples, one or more proteins involved in angiogenesis can be co-expressed. When two or more heterologous genes are introduced, the genes can be regulated under the same or different regulatory sequences, and the genes can be inserted into the same or different regions of the viral genome in a single or multiple gene manipulation steps. In some embodiments, one gene can be under the control of a constitutive promoter and a second gene can be under the control of an inducible promoter. Methods for inserting two or more genes into a virus are known in the art and can be readily performed on a variety of viruses using a variety of exogenous genes, regulatory sequences and / or other nucleic acid sequences.

[0251] The viruses provided herein can be modified by the insertions, deletions, substitutions or mutations described herein. Standard methods for modifying viruses by nucleic acid insertions, deletions, substitutions and mutations are well known in the art. Such methods include in vitro recombination techniques, synthetic methods, direct cloning and in vivo recombination methods. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, cold Spring Harbor N.Y. (1989), and the examples disclosed herein. Techniques for generating recombinant viruses include nucleic acid transfer protocols, various nucleic acid manipulation techniques, nucleic acid amplification protocols, which typically involve the generation of gene cassettes or transfer vectors using standard techniques in molecular biology. See, for example, U.S. Patent No. 5,494,807 and U.S. Patent No. 5,185,146, which describe exemplary methods for generating recombinant vaccinia viruses and other molecular biology techniques known in the art. Methods for generating recombinant viruses using recombinant DNA techniques are well known in the art (see, for example, U.S. Patent No. 4,769,330, U.S. Patent No. 4,603,112, U.S. Patent No. 4,722,848, U.S. Patent No. 4,215,051, U.S. Patent No. 5,110,587, U.S. Patent No. 5,174,993, U.S. Patent No. 5,922,576, U.S. Patent No. 6,319,703, U.S. Patent No. 5,719,054, U.S. Patent No. 6,429,001, U.S. Patent No. 6,589,531, U.S. Patent No. 6,573,090, U.S. Patent No. 6,800,288, U.S. Patent No. 7,045,313; He et al. (1998) PNAS 95(5):2509-2514; Racaniello et al., (1981) Science 214:916-919; and Hruby et al., (1990) Clin Micro Rev. 3:153-170).Methods for generating recombinant vaccinia viruses are well known in the art (see, for example, Hruby et al., (1990) Clin Micro Rev. 3:153-170, U.S. Patent Application Publication No. 2005-0031643, now U.S. Patent No. 7,588,767, U.S. Patent No. 7,588,771, U.S. Patent No. 7,662,398 and U.S. Patent No. 7,045,313).

[0252] In some embodiments, homologous recombination can be used to introduce insertions or deletions of nucleic acid molecules into target sequences of interest. The use of nucleic acid tools such as vectors, plasmids, promoters, and other regulatory sequences for a wide variety of viruses and cellular organisms is well known in the art. Nucleic acid amplification protocols include, without limitation, polymerase chain reaction (PCR), or amplification via viruses or organisms such as yeast, bacteria, insects, or mammalian cells without limitation. Nucleic acid transfer protocols include electroporation, calcium chloride transformation / transfection, liposome-mediated nucleic acid transfer, and the like. A wide variety of tools for modifying nucleic acids are available from many different sources, including a variety of commercial sources. For example, point mutations or small insertions or deletions can be introduced into genes of interest by use of oligonucleotide-mediated site-directed mutagenesis. In another example, homologous recombination can be used to introduce mutations into nucleic acid sequences, or to insert or delete nucleic acid molecules into target sequences of interest. In some examples, mutations, insertions, or deletions of nucleic acids within a particular gene can be selected for use of positive or negative selection pressure. See, for example, Current Techniques in Molecular Biology, (Ed. Ausubel, et al.). One of ordinary skill in the art will be able to readily select the appropriate tools and methods for genetic modification of any particular virus, according to knowledge in the art and design choices. In some embodiments, plasmids are used for homologous recombination to construct recombinant viruses. In some embodiments, plasmids are constructed using gene splicing to ligate two fragments. In some embodiments, the primers used to amplify two fragments include 70%, 80%, 90%, or 100% of SEQ ID NO:14, 15, 16, 17, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 94, 95, 96, or 97.

[0253] Insertions, deletions, substitutions or mutations can be specifically directed to a particular sequence within the viral genome. Such sequences within the viral genome include, without limitation, intergenic sequences, regulatory sequences, sequences of unknown function, gene coding sequences, or non-essential regions of the viral genome. For many viruses, the regions of the viral genome available for modification are well known in the art.

[0254] In some embodiments, a recombinant virus, e.g., a recombinant oncolytic virus, contains an inactivating mutation in at least one viral gene. The inactivating mutation is not particularly limited and, in some embodiments, can be any mutation that results in a decrease or loss of function of the gene product of the viral gene as compared to the case where there is no inactivating mutation. In some embodiments, the inactivating mutation is a deletion of all or part of at least one viral gene. In some embodiments, the deletion of at least one viral gene is a deletion of the entire gene ORF of the viral gene. In some embodiments, the deletion of at least one viral gene is a deletion of a part of the ORF of the viral gene. In some embodiments, the deletion of at least one viral gene is a deletion of a part of the ORF of the viral gene sufficient to render the encoded gene product non-functional. In some embodiments, at least one viral gene is selected from the group consisting of hemagglutinin (HA), J2R (thymidine kinase), F14.5L, A56R (hemagglutinin), B2R, vaccinia growth factor (VGF), A35R, A49R, A55R, B14R, C4L, C6L, C16L, NIL / N2L, E2L / E3L, K1L / K2L, K7L, superoxide dismutase locus, 7.5K, C2L-F3L, C4L-F1L, C7-K1L, B13R+B14R, A26L, and I4L. In some embodiments, at least one viral gene comprises two or more viral genes selected from the group consisting of hemagglutinin (HA), J2R (thymidine kinase), F14.5L, A56R (hemagglutinin), B2R, vaccinia growth factor (VGF), A35R, A49R, A55R, B14R, C4L, C6L, C16L, NIL / N2L, E2L / E3L, K1L / K2L, K7L, superoxide dismutase locus, 7.5K, C2L-F3L, C4L-F1L, C7-K1L, B13R+B14R, A26L, and I4L. In some embodiments, at least one viral gene is A35R. In some embodiments, at least one viral gene is J2R. In some embodiments, at least one viral gene is B2R. In some embodiments, at least one viral gene is B2R.In some embodiments, at least one viral gene is B2R. In some embodiments, at least one viral gene includes A35R and J2R. In some embodiments, at least one viral gene is B2R. In some embodiments, at least one viral gene includes B2R and J2R.

[0255] Heterologous nucleic acid molecules are typically inserted into the viral genome within intergenic regions or at loci encoding non-essential viral gene products. Insertion of heterologous nucleic acids at such sites generally does not significantly affect viral infection or replication in the target tissue. Examples of insertion sites include, without limitation, J2R (thymidine kinase (TK)), A56R (hemagglutinin (HA)), F14.5L, vaccinia growth factor (VGF), A35R, N1L, E2L / E3L, K1L / K2L, superoxide dismutase locus, 7.5K, C7-K1L (host range gene region), B13R + B14R (hemorrhage region), A26L (type A inclusion body region (ATI)), or I4L (large subunit, ribonucleotide reductase) locus. Insertion sites provided herein also include sites corresponding to intragenic regions described for other poxviruses such as modified vaccinia Ankara (MVA) virus (exemplary sites described in U.S. Patent No. 7,550,147), NYVAC (exemplary sites described in U.S. Patent No. 5,762,938). In some embodiments, insertion, deletion, substitution, and / or mutation sites include J2R, F14.5L, and / or A35R.

[0256] For example, the generation of recombinant vaccinia viruses that express heterologous gene products typically involves the use of a recombinant plasmid containing a heterologous nucleic acid that is optionally operably linked to a promoter and has a vaccinia virus DNA sequence adjacent to the heterologous nucleic acid to facilitate homologous recombination and insertion of the gene into the viral genome. Generally, the viral DNA adjacent to the heterologous gene is complementary to a non-essential segment of the vaccinia virus DNA, and the gene is inserted at a non-essential position or any other position. The recombinant plasmid can be propagated in Escherichia coli, purified from E. coli, and introduced into suitable host cells, such as, without limitation, CV-1, BSC-40, BSC-1, and TK-143 cells. The transfected cells are then super-infected with vaccinia virus, thereby initiating the replication cycle. The heterologous DNA can be integrated into the vaccinia virus genome by homologous recombination and packaged into infectious progeny. The recombinant virus can be identified by methods known in the art, such as, for example, by detection of the expression of the heterologous gene product or by using a positive selection method or a negative selection method (U.S. Patent No. 7,045,313). In some embodiments, the recombinant virus is generated by homologous integration of a plasmid into the viral genomic region corresponding to the J2R gene. In some embodiments, the recombinant virus is generated by homologous integration of a plasmid into the viral genomic region corresponding to the A35R gene. In some embodiments, the recombinant virus is generated by homologous integration of a plasmid into the viral genomic region corresponding to the F14.5L gene. In some embodiments, the recombinant virus is generated by homologous integration of one plasmid into the viral genomic region corresponding to the J2R gene and homologous integration of another plasmid into the viral genomic region corresponding to the F14.5L gene. In some embodiments, the recombinant virus is generated by homologous integration of one plasmid into the viral genomic region corresponding to the J2R gene and homologous integration of another plasmid into the viral genomic region corresponding to the F14.5L gene.In some embodiments, the recombinant virus is generated by homologous integration of one plasmid into the viral genomic region corresponding to the J2R gene and homologous integration of another plasmid into the viral genomic region corresponding to the A35R gene. In some embodiments, the recombinant virus is generated by homologous integration of one plasmid into the viral genomic region corresponding to the F14.5L gene and homologous integration of another plasmid into the viral genomic region corresponding to the A35R gene. In some embodiments, the recombinant virus is generated by homologous integration of one plasmid into the viral genomic region corresponding to the J2R gene, homologous integration of another plasmid into the viral genomic region corresponding to the A35R gene, and homologous integration of another plasmid into the viral genomic region corresponding to the F14.5L gene.

[0257] In another example, a recombinant vaccinia virus expressing a heterologous gene product can be generated by direct cloning (see, e.g., U.S. Patent No. 6,265,183 and Scheiflinger et al. (1992) Proc. Natl. Acad. Sci. USA 89:9977-9981). In such a method, a heterologous nucleic acid optionally operably linked to a promoter is adjacent to a restriction endonuclease cleavage site for insertion into a unique restriction endonuclease site within the target virus. The viral DNA is purified using standard techniques and cleaved with a sequence-specific restriction endonuclease, and the sequence is a unique site within the viral genome. Any unique site within the viral genome can be used as long as modification at that site does not interfere with viral replication. Generally, the insertion is at a site located in a non-essential region of the viral genome. For example, an exemplary modification herein includes insertion of a foreign DNA sequence into NotI-digested viral DNA.

[0258] In some instances, the heterologous nucleic acid can also contain one or more regulatory sequences for regulating the expression of open reading frames encoding heterologous RNA and / or protein. For example, suitable regulatory sequences that are functional in mammalian host cells are well known in the art. Expression can also be affected by one or more proteins or RNA molecules expressed by the virus. Gene regulatory elements such as promoters and enhancers have cell type-specific activity and can be activated by specific inducers (e.g., hormones, growth factors, cytokines, cytostatic agents, irradiation, heat shock) via response elements. The controlled and restricted expression of these genes can be achieved using such regulatory elements as internal promoters to promote gene expression within viral vector constructs.

[0259] In some embodiments, a heterologous nucleic acid encoding one or more heterologous gene products is operably linked to a promoter. In some embodiments, one or more heterologous nucleic acids encoding one or more heterologous gene products are operably linked to a promoter for the expression of heterologous RNA and / or protein. For example, a heterologous nucleic acid operably linked to a promoter is also referred to as an expression cassette. Thus, the viruses provided herein can have the ability to express one or more heterologous genes. Gene expression can include the expression of a protein encoded by a gene and / or the expression of an RNA molecule encoded by a gene. In some embodiments, the viruses provided herein can express a heterologous gene at a level high enough to enable the harvesting of the product of the exogenous gene from a tumor. Expression of the heterologous gene can be controlled by a constitutive promoter or by an inducible promoter. In other examples, organ-specific or tissue-specific expression can be controlled by regulatory sequences. To achieve expression only within a target organ, e.g., within a tumor being treated, a foreign nucleotide sequence can be linked to a tissue-specific promoter and used in gene therapy. Such promoters are well known to those of skill in the art (see, for example, Zimmermann et al., Neuron 12:11-24 (1994); Vidal et al., EMBO J. 9:833-840 (1990); Mayford et al., Cell 81:891-904 (1995); and Pinkert et al., Genes & Dev. 1:268-76 (1987)).

[0260] Exemplary promoters for the expression of heterologous genes are known in the art. The heterologous nucleic acid can be operably linked to a native promoter or a heterologous promoter that is not native to the virus. Any suitable promoter can be used, including synthetic promoters, native promoters, and modified promoters. Exemplary promoters include synthetic promoters, including synthetic viral promoters and synthetic animal promoters. Native or heterologous promoters include, without limitation, viral promoters such as the vaccinia virus promoter and the adenovirus promoter.

[0261] In some embodiments, the promoter is a poxvirus promoter, such as a vaccinia virus promoter. Thus, in some embodiments, the promoter is a poxvirus promoter or a variant or derivative thereof, such as a vaccinia virus promoter. In some embodiments, the promoter is a vaccinia virus promoter. The vaccinia virus promoter for the expression of one or more heterologous genes can be a synthetic promoter or a native promoter and includes vaccinia early, intermediate, early / late, and late promoters. Exemplary vaccinia virus promoters for controlling the expression of heterologous genes include, but are not limited to, 7.5E, 7.5E / L, SSE, 11KL, SSL, SSEL, mH5, LEO, P7.5k, P11k, PSE, PSEL, PSL, H5R, TK, P28, C11R, G8R, F17R, I3L, I8R, A1L, A2L, A3L, H1L, H3L, H5L, H6R, H8R, D1R, D4R, D5R, D9R, D11L, D12L, D13L, M1L, N2L, P4b, or K1 promoter. Thus, in some embodiments, the nucleic acid encoding the heterologous gene product is operably linked to a promoter selected from the group consisting of 7.5E, 7.5E / L, SSE, 11KL, SSL, SSEL, mH5, LEO, P7.5k, P11k, PSE, PSEL, PSL, H5R, TK, P28, C11R, G8R, F17R, I3L, I8R, A1L, A2L, A3L, H1L, H3L, H5L, H6R, H8R, D1R, D4R, D5R, D9R, D11L, D12L, D13L, M1L, N2L, P4b, and K1 promoters. Other viral promoters include, but are not limited to, the adenovirus late promoter, the vaccinia ATI promoter, or the T7 promoter. A strong late promoter can be used to achieve high-level expression of heterologous genes. Early and intermediate stage promoters can also be used.In one example, the promoter is a modified H5 promoter, PmH5, that contains both early and late promoter elements, such as both the native early and late vaccinia promoter regions of vaccinia virus, the synthetic early / late vaccinia PSEL promoter, and the PSE synthetic early promoter (Hammond et al., Journal of Virological Methods 66:1, 135-138 (1997); Stritzker et al., Journal of Virology 88:19, 11556-11567 (2014); Kugler et al., Virol J. 16: 100 (2019)). In some embodiments, the promoter is a synthetic strong early promoter (SSE). In some embodiments, the promoter is a strong early / late promoter (SEL).

[0262] In some embodiments, the promoter is selected from the group consisting of 7.5E, 7.5E / L, SSE, 11KL, SSL, SSEL, mH5, and LEO. In some embodiments, the promoter has an amino acid sequence shown in any one of SEQ ID NO:29, 53, 55, 68, 69, 70, 71, or 72. In some embodiments, the promoter has an amino acid sequence shown in SEQ ID NO:29. In some embodiments, the promoter is a synthetic strong early promoter (SSE) and comprises the amino acid sequence shown in SEQ ID NO:29. In some embodiments, the promoter has an amino acid sequence shown in SEQ ID NO:55. In some embodiments, the promoter is a strong early / late promoter (SEL) and comprises the amino acid sequence shown in SEQ ID NO:55. In some embodiments, the promoter is a poxvirus promoter, and the poxvirus promoter is mH5. In some embodiments, the poxvirus promoter is mH5 and comprises the amino acid sequence shown in SEQ ID NO:53.

[0263] Combinations of different promoters can be used to express different gene products in the same virus or in two different viruses. The viruses provided herein can exhibit differences in characteristics such as attenuation as a result of using a stronger promoter relative to a weaker promoter. For example, in vaccinia, the synthetic early / late and late promoters are relatively strong promoters, while the vaccinia synthetic early promoter is a relatively weaker promoter (see, e.g., Chakrabarti et al. (1997) BioTechniques 23(6) 1094-1097).

[0264] As is known in the art, regulatory sequences can enable constitutive expression of an exogenous gene or can enable inducible expression of an exogenous gene. Further, regulatory sequences can enable control of the expression level of an exogenous gene. In some instances, such as the production and harvesting of gene products, regulatory sequences can result in constitutive high-level gene expression. In some instances, such as the harvesting of anti-(gene product) antibodies, regulatory sequences can result in constitutive lower-level gene expression. In the example of tumor treatment, the therapeutic protein can be under the control of an internal inducible promoter or an external inducible promoter.

[0265] Thus, the expression of a heterologous gene can be controlled by a constitutive promoter or by an inducible promoter. Inducible promoters can be used to provide tissue-specific expression of a heterologous gene or can be inducible by the addition of a regulatory molecule to provide temporal-specific induction of the promoter. In some instances, inducible expression can be under the control of a cellular or other factor present in tumor cells or in virus-infected tumor cells. In further instances, inducible expression can be under the control of an administrable substance, including IPTG, RU486, or other known inducible compounds. Additional regulatory sequences can be used to control the expression of one or more heterologous genes inserted into a virus. One of various regulatory sequences can be utilized by one of ordinary skill in the art according to known factors and design preferences.

[0266] In some embodiments, one or more heterologous gene products include a therapeutic or diagnostic agent. In some embodiments, one or more heterologous gene products, such as a therapeutic or diagnostic agent, are an anti-cancer agent, an anti-metastatic agent, an anti-angiogenic agent, an immunomodulatory molecule, an antigen, a cell matrix degrading gene, a gene for tissue regeneration and for reprogramming human somatic cells to pluripotency, an enzyme that modifies a substrate to produce a detectable product or signal or is detectable by an antibody, a protein that can bind to a contrast agent, a gene for optical imaging or detection, a gene for PET imaging, and a gene for MRI imaging. In some embodiments, one or more heterologous gene products, such as a therapeutic or diagnostic agent, include a therapeutic agent selected from the group consisting of a hormone, a growth factor, a cytokine, a chemokine, a costimulatory molecule, a ribozyme, a transporter protein, a single-chain antibody, an antisense RNA, a prodrug converting enzyme, siRNA, microRNA, a toxin, an anti-tumor oligopeptide, a mitosis-inhibiting protein, an anti-mitotic oligopeptide, an anti-cancer polypeptide antibiotic, an angiogenesis inhibitor, a tumor suppressor, a cytotoxic protein, a cell growth inhibitory protein, and a tissue factor.

[0267] In some of any such embodiments, a recombinant virus, such as a recombinant oncolytic virus, comprises a nucleic acid sequence comprising at least one heterologous nucleic acid encoding one or more heterologous gene products, such as any of the heterologous gene products described herein, for example, in Parts A, B, C, and D of Section III, which is selected from the group consisting of, for example, a complement inhibitor, a T cell evasion factor or an NK cell evasion factor, an immune stimulating protein, an anti-angiogenic protein, an interferon regulatory factor, an apoptosis-inducing protein, or any combination thereof, and optionally, at least one viral gene, for example, hemagglutinin (HA), J2R (thymidine kinase), F14.5L, A56R (hemagglutinin), B2R, vaccinia growth factor (VGF), A35R, A49R, A55R, B14R, C4L, C6L, C16L, NIL / N2L, E2L / E3L, K1L / K2L, K7L, superoxide dismutase locus, 7.5K, C2L-F3L, C4L-F1L, C7-K1L, B13R+B14R, A26L, or I4L locus in the genome of the virus, optionally, one or more viral genes are one or more of B2R, J2R, A35R, and A56R, and any combination thereof.

[0268] In some of any such embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises a nucleic acid sequence shown in any one of SEQ ID NO: 48, 80, 82, and 84 - 93, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence shown in any one of SEQ ID NO: 48, 80, 82, and 84 - 93. In some embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises a nucleic acid sequence shown in any one of SEQ ID NO: 85, 86, 88, and 90, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence shown in any one of SEQ ID NO: 85, 86, 88, and 90. In some embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises a nucleic acid sequence shown in SEQ ID NO: 85, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence shown in SEQ ID NO: 85. In some embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises a nucleic acid sequence shown in SEQ ID NO: 48, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence shown in SEQ ID NO: 48. In some embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises a nucleic acid sequence shown in SEQ ID NO: 80, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence shown in SEQ ID NO: 80. In some embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises a nucleic acid sequence shown in SEQ ID NO: 82, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence shown in SEQ ID NO: 82. In some embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises a nucleic acid sequence shown in SEQ ID NO: 84, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence shown in SEQ ID NO: 84.In some embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown in SEQ ID NO:86, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence shown in SEQ ID NO:86. In some embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown in SEQ ID NO:87, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence shown in SEQ ID NO:87. In some embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown in SEQ ID NO:88, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence shown in SEQ ID NO:88. In some embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown in SEQ ID NO:89, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence shown in SEQ ID NO:89. In some embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown in SEQ ID NO:90, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence shown in SEQ ID NO:90. In some embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown in SEQ ID NO:91, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence shown in SEQ ID NO:91. In some embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown in SEQ ID NO:92, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence shown in SEQ ID NO:92.In some embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence set forth in SEQ ID NO:93, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:93.

[0269] In some of any such embodiments, the recombinant oncolytic virus comprises an inactivating mutation in at least one viral gene; and at least one heterologous nucleic acid encoding one or more heterologous gene products, optionally, the one or more heterologous gene products are immunomodulatory proteins, complement inhibitors, T cell or NK cell evasion factors, anti-angiogenic proteins, interferon regulatory factors, or apoptosis-inducing proteins, or any combination of the foregoing or include them; and, the at least one viral gene is A35R or includes it, optionally, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence of SEQ ID NO:3, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:3.

[0270] In some of any such embodiments, the recombinant oncolytic virus comprises an inactivating mutation in at least one viral gene; and at least one heterologous nucleic acid encoding one or more heterologous gene products, optionally, the one or more heterologous gene products are immunomodulatory proteins, complement inhibitors, T cell or NK cell evasion factors, anti-angiogenic proteins, interferon regulatory factors, or apoptosis-inducing proteins, or any combination of the foregoing or include them; and, the at least one viral gene is A35R and J2R or includes them, optionally, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence of SEQ ID NO:12, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:12.

[0271] In some of any such embodiments, the recombinant oncolytic virus comprises an inactivating mutation of at least one viral gene; and at least one heterologous nucleic acid encoding one or more heterologous gene products, optionally, the one or more heterologous gene products are immunomodulatory proteins, complement inhibitors, T cell or NK cell evasion factors, anti-angiogenic proteins, interferon regulatory factors, or apoptosis-inducing proteins, or any combination or inclusion thereof; and, the at least one viral gene is J2R or includes it, and the inactivating mutation of J2R is by insertion of one or more heterologous nucleic acids each encoding one or more T cell or NK cell evasion factor proteins, optionally, the one or more T cell or NK cell evasion factor proteins include a set of proteins encoded by vaccinia virus ORFs 012, 203, and 018 (CPXV012-203-018), and, the at least one heterologous nucleic acid encoding the one or more heterologous gene products includes one or more heterologous nucleic acids each encoding one or more complement inhibitors, optionally, the viral membrane gene is F14.5L, optionally, the fusion is a fusion at the C-terminus of the F14.5L protein, and optionally, the nucleic acid genome of the recombinant oncolytic virus includes the nucleic acid sequence of SEQ ID NO:10, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence shown in SEQ ID NO:10.

[0272] In some of any such embodiments, the recombinant oncolytic virus comprises an inactivating mutation of at least one viral gene; and at least one heterologous nucleic acid encoding one or more heterologous gene products, optionally, the one or more heterologous gene products are immunomodulatory proteins, complement inhibitors, T cell or NK cell evasion factors, anti-angiogenic proteins, interferon regulatory factors, or apoptosis-inducing proteins, or any combination of the foregoing or include them; and, the at least one viral gene is J2R or includes it, optionally, the nucleic acid genome of the recombinant oncolytic virus comprises a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO:4, or the nucleic acid sequence shown in SEQ ID NO:4.

[0273] In some of any such embodiments, the recombinant oncolytic virus comprises an inactivating mutation of at least one viral gene; and at least one heterologous nucleic acid encoding one or more heterologous gene products, optionally, the one or more heterologous gene products are immunomodulatory proteins, complement inhibitors, T cell or NK cell evasion factors, anti-angiogenic proteins, interferon regulatory factors, or apoptosis-inducing proteins, or any combination of the foregoing or include them; and, the at least one viral gene is J2R and A35R or includes them, and the inactivating mutation of A35R is by insertion of one or more heterologous nucleic acids each encoding one or more immunomodulatory proteins, optionally, the one or more immunomodulatory proteins are selected from the group consisting of LIGHT, IRF3, IL-2, IL-12, and CXCL9, optionally, the one or more immunomodulatory proteins are LIGHT; and optionally, the nucleic acid genome of the recombinant oncolytic virus comprises a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO:11, or the nucleic acid sequence shown in SEQ ID NO:11.

[0274] In some of any such embodiments, the recombinant oncolytic virus comprises an inactivating mutation of at least one viral gene; and at least one heterologous nucleic acid encoding one or more heterologous gene products, optionally, the one or more heterologous gene products are immunomodulatory proteins, complement inhibitors, T cell or NK cell evasion factors, anti-angiogenic proteins, interferon regulatory factors, or apoptosis-inducing proteins, or any combination of the foregoing or include them; and the at least one viral gene is or includes J2R and A35R, and the inactivating mutation of J2R is by insertion of one or more heterologous nucleic acids encoding one or more anti-angiogenic proteins, optionally, the one or more anti-angiogenic proteins include inhibitors or inhibitors of VEGF and / or Ang2, optionally, the one or more anti-angiogenic proteins are bispecific anti-VEGF / anti-Ang2 antibodies; and optionally, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence of SEQ ID NO:13, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence shown in SEQ ID NO:13.

[0275] In some of any such embodiments, the recombinant oncolytic virus comprises an inactivating mutation of at least one viral gene; and at least one heterologous nucleic acid encoding one or more heterologous gene products, optionally, the one or more heterologous gene products are, or comprise, an immunomodulatory protein, a complement inhibitor, a T cell or NK cell evasion factor, an anti-angiogenic protein, an interferon regulatory factor, or an apoptosis-inducing protein, or any combination of the foregoing; and the at least one viral gene is, or comprises, J2R and A35R, and the inactivating mutation of A35R is by insertion of one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally, the one or more immunomodulatory proteins are selected from the group consisting of LIGHT, IRF3, IL-2, IL-12, and CXCL9, optionally, the one or more immunomodulatory proteins are LIGHT; and the inactivating mutation of J2R is by insertion of one or more heterologous nucleic acids encoding one or more anti-angiogenic proteins, optionally, the one or more anti-angiogenic proteins comprise an inhibitor or an inhibitor of VEGF and / or Ang2, optionally, the one or more anti-angiogenic proteins are a bispecific anti-VEGF / anti-Ang2 antibody; and optionally, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence of SEQ ID NO:47, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence shown in SEQ ID NO:47.

[0276] In some of any such embodiments, the recombinant oncolytic virus comprises an inactivating mutation of at least one viral gene; and at least one heterologous nucleic acid encoding one or more heterologous gene products, optionally, the one or more heterologous gene products are immunomodulatory proteins, complement inhibitors, T cell or NK cell evasion factors, anti-angiogenic proteins, interferon regulatory factors, or apoptosis-inducing proteins, or any combination of the foregoing or include them; and, the at least one viral gene is or includes J2R, and the inactivating mutation of J2R is by insertion of one or more heterologous nucleic acids each encoding an apoptosis-inducing protein, optionally, the apoptosis-inducing protein is inducible DED (iDED), inducible Fas (iFas), or inducible Cas9 (iCas9), optionally, the nucleic acid genome of the recombinant oncolytic virus comprises a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO:7, 8, or 9, or the nucleic acid sequence shown in SEQ ID NO:7, 8, or 9.

[0277] In some of any such embodiments, the recombinant oncolytic virus comprises an inactivating mutation of at least one viral gene; and at least one heterologous nucleic acid encoding one or more heterologous gene products, optionally, the one or more heterologous gene products are immunomodulatory proteins, complement inhibitors, T cell or NK cell evasion factors, anti-angiogenic proteins, interferon regulatory factors, or apoptosis-inducing proteins, or any combination of the foregoing or include them; and, the at least one viral gene is J2R or includes it, and the inactivating mutation of J2R is by insertion of one or more heterologous nucleic acids each encoding one or more immunomodulatory proteins, optionally, the one or more immunomodulatory proteins are selected from the group consisting of LIGHT, IRF3, IL-2, IL-12, and CXCL9, optionally, the one or more immunomodulatory proteins is IRF3; optionally, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence of SEQ ID NO:49, 50, or 93, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence shown in SEQ ID NO:49, 50, or 93.

[0278] In some of any such embodiments, the recombinant oncolytic virus comprises an inactivating mutation of at least one viral gene; and at least one heterologous nucleic acid encoding one or more heterologous gene products, optionally, the one or more heterologous gene products are immunomodulatory proteins, complement inhibitors, T cell or NK cell evasion factors, anti-angiogenic proteins, interferon regulatory factors, or apoptosis-inducing proteins, or any combination of the foregoing or include them; and, the at least one viral gene is J2R and B2R or includes them, optionally, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence of SEQ ID NO:48, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence shown in SEQ ID NO:48.

[0279] In some of any such embodiments, the recombinant oncolytic virus comprises an inactivating mutation of at least one viral gene; and at least one heterologous nucleic acid encoding one or more heterologous gene products, optionally, the one or more heterologous gene products are immunomodulatory proteins, complement inhibitors, T cell or NK cell evasion factors, anti-angiogenic proteins, interferon regulatory factors, or apoptosis-inducing proteins, or any combination of the foregoing or include them; and, the at least one viral gene is or includes J2R and B2R, and the inactivating mutation of J2R is by insertion of one or more heterologous nucleic acids each encoding one or more immunomodulatory proteins, optionally, the one or more immunomodulatory proteins are selected from the group consisting of LIGHT, IRF3, IL-2, IL-12, and CXCL9, optionally, the one or more immunomodulatory proteins is IRF3; optionally, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence of SEQ ID NO:80, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence shown in SEQ ID NO:80.

[0280] In some of any such embodiments, the recombinant oncolytic virus comprises an inactivating mutation of at least one viral gene; and at least one heterologous nucleic acid encoding one or more heterologous gene products, optionally, the one or more heterologous gene products are immune regulatory proteins, complement inhibitors, T cell or NK cell evasion factors, anti-angiogenic proteins, interferon regulatory factors, or apoptosis-inducing proteins, or any combination of the foregoing or include them; and the at least one viral gene is, or includes, J2R, B2R, and A35R; the inactivating mutation of J2R is by insertion of one or more heterologous nucleic acids encoding one or more anti-angiogenic proteins, optionally, the one or more anti-angiogenic proteins include inhibitors or inhibitors of VEGF and / or Ang2, optionally, the one or more anti-angiogenic proteins are bispecific anti-VEGF / anti-Ang2 antibodies; the inactivating mutation of B2R is by insertion of one or more heterologous nucleic acids each encoding one or more immune regulatory proteins, optionally, the one or more immune regulatory proteins are selected from the group consisting of LIGHT, IRF3, IL-2, IL-12, and CXCL9, optionally, the one or more immune regulatory proteins are IRF3; and the inactivating mutation of A35R is by insertion of one or more heterologous nucleic acids encoding one or more immune regulatory proteins, optionally, the one or more immune regulatory proteins are selected from the group consisting of LIGHT, IRF3, IL-2, IL-12, and CXCL9, optionally, the one or more immune regulatory proteins are LIGHT; and optionally, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence of SEQ ID NO:82, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence shown in SEQ ID NO:82.

[0281] In some of any such embodiments, the recombinant oncolytic virus comprises an inactivating mutation of at least one viral gene; and at least one heterologous nucleic acid encoding one or more heterologous gene products, optionally, the one or more heterologous gene products are immunomodulatory proteins, complement inhibitors, T cell or NK cell evasion factors, anti-angiogenic proteins, interferon regulatory factors, or apoptosis-inducing proteins, or combinations thereof or include them; and the at least one viral gene is or includes J2R, B2R, and A56R; the inactivating mutation of J2R is by insertion of one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally, the one or more immunomodulatory proteins are IRF3; the inactivating mutation of A56R is by insertion of one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally, the one or more immunomodulatory proteins are IL-2, optionally, IL-2 is an IL-2 supercytokine, optionally MDNA11; and optionally, the nucleic acid genome of the recombinant oncolytic virus comprises a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO:84, or the nucleic acid sequence shown in SEQ ID NO:84.

[0282] In some of any such embodiments, the recombinant oncolytic virus comprises an inactivating mutation of at least one viral gene; and at least one heterologous nucleic acid encoding one or more heterologous gene products, optionally, the one or more heterologous gene products are immunomodulatory proteins, complement inhibitors, T cell or NK cell evasion factors, anti-angiogenic proteins, interferon regulatory factors, or apoptosis-inducing proteins, or any combination or inclusion thereof; and the at least one viral gene is or comprises J2R, B2R, and A56R; the inactivating mutation of J2R is by insertion of one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally, the one or more immunomodulatory proteins are IRF3; the inactivating mutation of A56R is by insertion of one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally, the one or more immunomodulatory proteins include two or more immunomodulatory proteins selected from the group consisting of LIGHT, IRF3, IL-2, IL-12, and CXCL9, optionally, the two or more immunomodulatory proteins include IL-12 and CXCL9; and optionally, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence of SEQ ID NO:85, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence shown in SEQ ID NO:85.

[0283] In some of any of such embodiments, the recombinant oncolytic virus comprises an inactivating mutation of at least one viral gene; and at least one heterologous nucleic acid encoding one or more heterologous gene products, optionally, the one or more heterologous gene products are immunomodulatory proteins, complement inhibitors, T cell or NK cell evasion factors, anti-angiogenic proteins, interferon regulatory factors, or apoptosis-inducing proteins, or any combination of the foregoing or include them; and the at least one viral gene is or includes J2R, B2R, and A56R; the inactivating mutation of B2R is due to the insertion of one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally, the one or more immunomodulatory proteins are IRF3; the inactivating mutation of A56R is due to the insertion of one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally, the one or more immunomodulatory proteins include two or more immunomodulatory proteins selected from the group consisting of LIGHT, IRF3, IL-2, IL-12, and CXCL9, optionally, the two or more immunomodulatory proteins include IL-12 and CXCL9; the inactivating mutation of J2R is due to the insertion of one or more heterologous nucleic acids each encoding an apoptosis-inducing protein, optionally, the apoptosis-inducing protein is inducible DED (iDED); and optionally, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence of SEQ ID NO:86, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence shown in SEQ ID NO:86.

[0284] In some of any of such embodiments, the recombinant oncolytic virus comprises an inactivating mutation of at least one viral gene; and at least one heterologous nucleic acid encoding one or more heterologous gene products, optionally, the one or more heterologous gene products are immunomodulatory proteins, complement inhibitors, T cell or NK cell evasion factors, anti-angiogenic proteins, interferon regulatory factors, or apoptosis-inducing proteins, or any combination of the foregoing or include them; and the at least one viral gene is, or includes, J2R, B2R, A35R, and A56R; the inactivating mutation of J2R is by insertion of one or more heterologous nucleic acids encoding one or more anti-angiogenic proteins, optionally, the one or more anti-angiogenic proteins include inhibitors or inhibitors of VEGF and / or Ang2, optionally, the one or more anti-angiogenic proteins are bispecific anti-VEGF / anti-Ang2 antibodies; the inactivating mutation of B2R is by insertion of one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally, the one or more immunomodulatory proteins are IRF3; the inactivating mutation of A35R is by insertion of one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally, the one or more immunomodulatory proteins are selected from the group consisting of LIGHT, IRF3, IL-2, IL-12, and CXCL9, optionally, the one or more immunomodulatory proteins are LIGHT; the inactivating mutation of A56R is by insertion of one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, the one or more immunomodulatory proteins are the IL-2 supercytokine MDNA11; and optionally, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence of SEQ ID NO:87, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence shown in SEQ ID NO:87.

[0285] In some of any such embodiments, the recombinant oncolytic virus comprises an inactivating mutation of at least one viral gene; and at least one heterologous nucleic acid encoding one or more heterologous gene products, optionally, the one or more heterologous gene products are immunomodulatory proteins, complement inhibitors, T cell or NK cell evasion factors, anti-angiogenic proteins, interferon regulatory factors, or apoptosis-inducing proteins, or any combination of the foregoing or include them; and the at least one viral gene is or includes J2R, B2R, A35R, and A56R; the inactivating mutation of J2R is by insertion of one or more heterologous nucleic acids encoding one or more anti-angiogenic proteins, optionally, the one or more anti-angiogenic proteins include inhibitors or inhibitors of VEGF and / or Ang2, optionally, the one or more anti-angiogenic proteins are bispecific anti-VEGF / anti-Ang2 antibodies; the inactivating mutation of B2R is by insertion of one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally, the one or more immunomodulatory proteins are IRF3; the inactivating mutation of A35R is by insertion of one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally, the one or more immunomodulatory proteins are selected from the group consisting of LIGHT, IRF3, IL-2, IL-12, and CXCL9, optionally, the one or more immunomodulatory proteins are LIGHT; the inactivating mutation of A56R is by insertion of one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, the one or more immunomodulatory proteins are the IL-2 supercytokine MDNA11T, optionally, MDNA11T includes the amino acid sequence shown in SEQ ID NO:98; and optionally, the nucleic acid genome of the recombinant oncolytic virus includes the nucleic acid sequence of SEQ ID NO:88, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence shown in SEQ ID NO:88.

[0286] In some of any such embodiments, the recombinant oncolytic virus comprises an inactivating mutation of at least one viral gene; and at least one heterologous nucleic acid encoding one or more heterologous gene products, optionally, the one or more heterologous gene products are immunomodulatory proteins, complement inhibitors, T cell or NK cell evasion factors, anti-angiogenic proteins, interferon regulatory factors, or apoptosis-inducing proteins, or combinations or include any of the foregoing; and, the at least one viral gene is or includes J2R, B2R, and A56R; the inactivating mutation of J2R is by insertion of one or more heterologous nucleic acids each encoding one or more T cell or NK cell evasion factor proteins, optionally, the one or more T cell or NK cell evasion factor proteins include a set of proteins encoded by vaccinia virus OFR 012, 203, and 018 (CPXV012-203-018); the inactivating mutation of B2R is by insertion of one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally, the one or more immunomodulatory proteins are IRF3; the inactivating mutation of A56R is by insertion of one or more heterologous nucleic acids each encoding one or more immunomodulatory proteins, optionally, the one or more immunomodulatory proteins are selected from the group consisting of LIGHT, IRF3, IL-2, IL-12, and CXCL9, optionally, the one or more immunomodulatory proteins are IL-2 supercytokine, optionally MDNA11 or MDNA11T; the at least one heterologous nucleic acid encoding the one or more heterologous gene products is introduced into a viral membrane gene, optionally F14.5L, to produce a fusion gene encoding a fusion protein, and includes one or more heterologous nucleic acids each encoding one or more complement inhibitors, optionally CRASP-2, optionally, the fusion is F14.A fusion at the C-terminus of the 5L protein; and optionally, the nucleic acid genome of the recombinant oncolytic virus comprises a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO:89, or the nucleic acid sequence shown in SEQ ID NO:89.

[0287] In some of any such embodiments, the recombinant oncolytic virus comprises an inactivating mutation of at least one viral gene; and at least one heterologous nucleic acid encoding one or more heterologous gene products, optionally, the one or more heterologous gene products are immunomodulatory proteins, complement inhibitors, T cell or NK cell evasion factors, anti-angiogenic proteins, interferon regulatory factors, or apoptosis-inducing proteins, or any combination of the foregoing or include them; and, the at least one viral gene is, or includes, J2R, B2R, and A56R; the inactivating mutation of J2R is by insertion of one or more heterologous nucleic acids each encoding one or more T cell or NK cell evasion factor proteins, optionally, the one or more T cell or NK cell evasion factor proteins include a set of proteins encoded by vaccinia virus ORFs 012, 203, and 018 (CPXV012-203-018); the inactivating mutation of B2R is by insertion of one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally, the one or more immunomodulatory proteins are IRF3; the inactivating mutation of A56R is by insertion of one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally, the one or more immunomodulatory proteins include two or more immunomodulatory proteins selected from the group consisting of LIGHT, IRF3, IL-2, IL-12, and CXCL9, optionally, the two or more immunomodulatory proteins include IL-12 and CXCL9; the at least one heterologous nucleic acid encoding the one or more heterologous gene products includes one or more heterologous nucleic acids each encoding one or more complement inhibitors, optionally CRASP-2, introduced into the viral membrane gene, optionally F14.5L, to produce a fusion gene encoding a fusion protein, optionally, the fusion is F14.a fusion at the C-terminus of the 5L protein; and optionally, the nucleic acid genome of the recombinant oncolytic virus comprises a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO:90, or the nucleic acid sequence shown in SEQ ID NO:90.

[0288] In some of any such embodiments, the recombinant oncolytic virus comprises an inactivating mutation of at least one viral gene; and at least one heterologous nucleic acid encoding one or more heterologous gene products, optionally, the one or more heterologous gene products are immunomodulatory proteins, complement inhibitors, T cell or NK cell evasion factors, anti-angiogenic proteins, interferon regulatory factors, or apoptosis-inducing proteins, or any combination of the foregoing or comprising them; and the at least one viral gene is or comprises B2R and J2R, and the inactivating mutation of J2R is by insertion of one or more heterologous nucleic acids each encoding one or more immunomodulatory proteins, optionally, the one or more immunomodulatory proteins are selected from the group consisting of LIGHT, IRF3, IL-2, IL-12, and CXCL9, optionally, the one or more immunomodulatory proteins is IRF3; and optionally, the nucleic acid genome of the recombinant oncolytic virus comprises a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO:91, or the nucleic acid sequence shown in SEQ ID NO:91.

[0289] In some of any such embodiments, the recombinant oncolytic virus comprises an inactivating mutation of at least one viral gene; and at least one heterologous nucleic acid encoding one or more heterologous gene products, optionally, the one or more heterologous gene products are immunomodulatory proteins, complement inhibitors, T cell or NK cell evasion factors, anti-angiogenic proteins, interferon regulatory factors, or apoptosis-inducing proteins, or any combination or inclusion thereof; and the at least one viral gene is, or comprises, B2R, J2R, and A56R, and the inactivating mutation of J2R is by insertion of one or more heterologous nucleic acids each encoding one or more immunomodulatory proteins, optionally, the one or more immunomodulatory proteins are selected from the group consisting of LIGHT, IRF3, IL-2, IL-12, and CXCL9, optionally, the one or more immunomodulatory proteins are IRF3; and the inactivating mutation of A56R is by insertion of one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally, the one or more immunomodulatory proteins include two or more immunomodulatory proteins selected from the group consisting of LIGHT, IRF3, IL-2, IL-12, and CXCL9, optionally, the two or more immunomodulatory proteins include IL-12 and CXCL9; and optionally, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence of SEQ ID NO:92, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence shown in SEQ ID NO:92.

[0290] In some of any such embodiments, the recombinant oncolytic virus comprises an inactivating mutation of at least one viral gene; and at least one heterologous nucleic acid encoding one or more heterologous gene products, optionally, the one or more heterologous gene products are immunomodulatory proteins, complement inhibitors, T cell or NK cell evasion factors, anti-angiogenic proteins, interferon regulatory factors, or apoptosis-inducing proteins, or any combination of the foregoing or include them; and the at least one viral gene is J2R or includes it, and the inactivating mutation of J2R is by insertion of one or more heterologous nucleic acids each encoding one or more immunomodulatory proteins, optionally, the one or more immunomodulatory proteins are selected from the group consisting of LIGHT, IRF3, IL-2, IL-12, and CXCL9, optionally, the one or more immunomodulatory proteins are IRF3; and optionally, the nucleic acid genome of the recombinant oncolytic virus comprises a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO:93, or the nucleic acid sequence shown in SEQ ID NO:93.

[0291] In some of any of such embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises a nucleic acid sequence shown in any one of SEQ ID NO: 48, 80, 82, and 84-93, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence shown in any one of SEQ ID NO: 48, 80, 82, and 84-93; and (i) a variant 017 open reading frame (ORF) encoding an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 57 and having a polar uncharged amino acid at position 66, optionally threonine (T) at position 66; (ii) a variant 038(K5L) ORF comprising a nucleotide insertion to cause a frameshift mutation in which the 038(K5L) gene product is modified; (iii) a variant 059(E2L) ORF having at least 95% sequence identity to SEQ ID NO: 60 and encoding an amino acid sequence having a hydrophobic amino acid other than leucine at position 419, optionally phenylalanine (F) at position 419; (iv) a variant 104(H4L) ORF having at least 95% sequence identity to SEQ ID NO: 61 and encoding an amino acid sequence having a negatively charged amino acid at position 591, optionally aspartic acid (D) at position 591; and (v) a variant 182(A56R) ORF comprising a deletion of two nucleotides to cause a frameshift mutation in which the 182(A56R) ORF gene product is modified, characterized by one or more of them.

[0292] In some of any such embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises a nucleic acid sequence shown in any one of SEQ ID NO: 48, 80, 82, and 84 - 93, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence shown in any one of SEQ ID NO: 48, 80, 82, and 84 - 93; and one or more of (i) guanine (G) at the position corresponding to position 7770 of SEQ ID NO: 1; (ii) thymine (T) at the position corresponding to position 15261 of SEQ ID NO: 1; (iii) G at the position corresponding to position 32136 of SEQ ID NO: 1; (iv) G at the position corresponding to position 49455 of SEQ ID NO: 1; (v) cytosine (C) at the position corresponding to position 92969 of SEQ ID NO: 1; (vi) the nucleic acid sequence CACTTATATAT at the position corresponding to positions 106870 - 106880 of SEQ ID NO: 1; (vii) the nucleic acid sequence GTTTTCATTA at the position corresponding to positions 111267 - 111276 of SEQ ID NO: 1; (viii) adenine (A) at the position corresponding to position 162715 of SEQ ID NO: 1; (ix) the nucleic acid sequence TACAGACACC at the position corresponding to positions 165844 - 185853 of SEQ ID NO: 1; and (x) C at the position corresponding to position 187805 of SEQ ID NO: 1, are characteristic thereof.

[0293] A. Stealth Virus In various embodiments, recombinant viruses are provided herein that contain a heterologous nucleic acid encoding a "stealth protein" that can be stably and efficiently expressed in many types of virus-infected cells. Such stealth proteins can increase the ability of the virus to avoid attack by the host immune system, such as by T cells, e.g., cytotoxic T lymphocytes (CTLs) or natural killer (NK) cells. In some embodiments, such stealth proteins can increase the ability of the recombinant virus to avoid activation of the host complement cascade / system.

[0294] Thus, in some embodiments, provided herein is a recombinant oncolytic virus comprising at least one heterologous nucleic acid encoding one or more heterologous gene products, wherein the one or more heterologous gene products are, or comprise, a complement inhibitor, or a T cell or NK cell evasion factor (sometimes also referred to as a stealth protein).

[0295] In some embodiments, the recombinant virus comprises an oncolytic virus. In some embodiments, the recombinant virus does not comprise an oncolytic virus. In some embodiments, the recombinant virus comprises any virus described herein or incorporated herein by reference. In some embodiments, the recombinant virus comprises vaccinia virus. In some embodiments, the recombinant virus comprises a virus derived from VIP02.

[0296] Oncolytic viruses (OVs) can create a favorable microenvironment for the action of the immune system against specific cancer cell determinants; however, the antiviral immunity induced against the resulting viral antigens from the infection also plays a key role during OV-based therapies. Indeed, the induced antiviral immunity can be detrimental to cancer virotherapy, as activation of the immune system against the virus itself is expected to limit viral replication and spread, leading to a decrease in therapeutic efficacy. Lemos de Matos et al., Mol Ther Methods Clin Dev. 2020 Jun 12; 17: 349-358. The complement system is constantly vigilant against viruses. Its ability to recognize viruses and virus-infected cells and trigger an immune response results in the neutralization of viruses and the killing of infected cells. This selective pressure exerted by complement against viruses has evolved numerous countermeasures in viruses. Agrawal et al., Front Microbiol. 2017; 8: 1117.

[0297] In some embodiments, stealth proteins include, but are not limited to, Borrelia burgdorferi complement regulator-acquiring surface protein 2 (CRASP-2), minimized complement factor H (miniFH), and vaccinia virus ORFs 012, 203, and 018 (CPXV012-203-018). Further details about stealth proteins and mechanisms involved in evading the host immune system, such as avoiding host complement or cytotoxicity of NK cells or T cells, can be found in Monrat Chulanetra and Wanpen Chaicumpa, Front. Cell. Infect. Microbiol., 2021, Front. Cell. Infect. Microbiol. 11:702125, which is incorporated herein by reference for all purposes.

[0298] The complement system is an important component of innate immunity that helps eliminate pathogens, and thus, during evolution, pathogens have developed various strategies to avoid destruction by complement activation. One strategy is the ability of pathogens to acquire proteins that enable them to control steps involved in activating the host immune response during infection, hereinafter referred to as "stealth proteins". See Kraiczy et al., Infect Immun. 2001 Dec; 69(12): 7800-7809.

[0299] The complement system employs a complex cascade of proteolytic cleavage of more than 30 plasma and cell membrane proteins, resulting in the induction of inflammatory responses, chemotaxis of phagocytes and neutrophils, neutralization of pathogens and subsequent opsonization, and lysis of infected cells. Activation can be initiated via three independent pathways: (i) the classical pathway, which involves binding of C1q, the first component of the cascade, to antibody-antigen complexes; (ii) the alternative pathway, which involves spontaneous hydrolysis of the downstream complement component 3 (C3) convertase and its interaction with pathogen surfaces; and (iii) the mannose-binding lectin (MBL) pathway, which is triggered by binding of MBL to mannose residues on pathogen surfaces. All three pathways converge at the stage of cleavage of C3 into the anaphylatoxin C3a and the opsonin C3b, which binds to pathogens and marks them for destruction. Effector compounds generated in the complement cascade can be delivered to any surface, including host membranes, so intact host cells defend themselves by expressing multiple complement regulatory proteins. Janeway et al., Immunobiology: The Immune System in Health and Disease. 5th edition。

[0300] Inappropriate regulation of the complement system underlies or exacerbates many human diseases. The alternative pathway (AP) of complement has unique properties in that it is continuously and indiscriminately activated, albeit at low levels. In the AP, C3b propagates itself via a positive feedback amplification loop that requires very tight regulation mediated by two key soluble AP regulators, factor H (FH) and its splice product FH-like-1 (FHL-1). MiniFH, an engineered version of FH, contains only the N-terminal and C-terminal portions of FH linked by optimized peptides and exhibits in vitro potency to inhibit complement activation that is approximately 10-fold higher compared to FH. Markus J. Harder, author manuscript available at *J Immunol. PMC 2017 Jan 15*. J Immunol. 2016 Jan 15; 196(2): 866-876. and Christoph Q. Schmidt / author manuscript available at *J Immunol. PMC 2014 Jun 1*. J Immunol. 2013 Jun 1; 190(11): 10.4049 / jimmunol.1203548. Published online Apr 24, 2013. doi: 10.4049 / jimmunol.1203548.

[0301] Microorganisms that have evolved the ability to avoid the complement by producing stealth proteins are Borrelia burgdorferi, a spirochete that causes Lyme disease (LD), the most common vector-borne disease in the Northern Hemisphere transmitted by ticks. During tick feeding, the spirochete is exposed to the host's blood and thus to the first line of innate immunity that they must overcome to survive. The key evasion mechanism that B. burgdorferi has evolved is the production of complement-binding proteins or CRP-binding proteins, including CRASP, a stealth protein that can promote complement inactivation. See Yi-Pin Lin et al., Front Cell Infect Microbiol. 2020; 10: 1, US20120142023A1. CRASP-2 (also named CspZ) confers serum resistance in gain-of-function B. burgdorferi by binding to FH / FHL-1 and inhibiting complement activation on the spirochete surface. Infect Immun. 2001 Dec; 69(12): 7800-7809. Peter Kraiczy. US20200323972A1 Composition and method for generating immunity to borrelia burgdorferi.

[0302] Downregulation of MHC class I on the cell surface is an immune evasion mechanism shared by many DNA viruses, including vaccinia virus. CPXV is a member of the orthopoxvirus genus, which includes variola virus, camelpox virus, and simian pox virus, and encodes a sophisticated arsenal of immune evasion proteins. The ability of CPXV to infect a wide range of mammalian hosts is likely due to the fact that among orthopoxviruses, CPXV encodes the most complete set of open reading frames expected to encode immunomodulatory proteins. Among the proteins encoded are CPXV012 and CPXV203, which can block cytotoxic T cell recognition by interfering with MHC I-mediated antigen presentation. CPXV012 inhibits antigen peptide transport from the cytoplasm to the ER, whereas CPXV203 blocks MHC I transport to the cell surface. Dina Alzhanova and Klaus Fruh* Microbes Infect. 2010 Nov; 12(12-13): 900-909. McCoy et al., Molecular Immunology 55 (2013) 156-158. Furthermore, the Brighton Red strain produces OMCP (also named CPXV018), a 171-residue protein that is abundantly secreted from infected cells and can block NKG2D-mediated target cell killing by natural killer cells in vitro. Cell Host & Microbe Volume 6, Issue 5, 19 November 2009, Pages 422-432 / Cell Host & Microbe journal homepage / Two Mechanistically Distinct Immune Evasion Proteins of Cowpox Virus Combine to Avoid Antiviral CD8 T Cells.

[0303] In some embodiments, one or more heterologous gene products include a complement inhibitor. In some embodiments, the complement inhibitor is Borrelia burgdorferi complement regulator-acquiring surface protein 2 (CRASP-2) or minimized complement factor H (miniFH). In some embodiments, the complement inhibitor is the CRASP-2 gene product (UniProtKB-050665). The CRASP-2 protein can increase the ability of a recombinant virus to evade the host complement. Specifically, in some embodiments, the recombinant virus comprises an expression cassette containing a CRASP-2 cDNA fused to the F14.5L locus under the control of the vaccinia F14.5L gene promoter. In some embodiments, the CRASP-2 molecule comprises full-length CRASP-2. In some embodiments, the complement inhibitor is CRASP-2 and has an amino acid sequence that exhibits at least 85%, 90%, or 95% sequence identity to the sequence shown in SEQ ID NO:18. In some embodiments, the complement inhibitor has the sequence shown in SEQ ID NO:18.

[0304] In some embodiments, the recombinant virus comprises a heterologous nucleic acid encoding a CRASP-2 molecule comprising a CRASP-2 cDNA fused to the F14.5L locus, wherein CRASP-2 comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:18.

[0305] In some embodiments, the recombinant virus comprises an amino acid sequence having at least 70%, such as at least 75%, 80%, 85%, or 90% sequence identity to the amino acid sequence of SEQ ID NO:18. For example, in some embodiments, the recombinant virus comprises an amino acid sequence having at least 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% sequence identity to the amino acid sequence of SEQ ID NO:18, but less than 100% identical to the amino acid sequence of SEQ ID NO:18.

[0306] In some embodiments, the heterologous nucleic acid encoding the CRASP-2 gene product is operably linked to the F14.5L gene promoter. In some embodiments, the recombinant virus comprising the heterologous nucleic acid encoding the CRASP-2 gene product (e.g., comprising the amino acid sequence of SEQ ID NO:18) is derived from the clonal VIP02 strain (comprising the nucleic acid sequence of SEQ ID NO:1) and comprises a nucleotide sequence having at least 70%, such as at least 75%, 80%, 85%, or 90% sequence identity to SEQ ID NO:5 (also named VIR27). In some embodiments, the heterologous gene product is CRASP-2 and is operably linked to the F14.5L gene promoter in the genome of the virus. In some embodiments, the recombinant virus comprises a nucleic acid sequence having at least 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to the nucleic acid sequence of SEQ ID NO:5, but less than 100% identical to the nucleic acid sequence of SEQ ID NO:5. In some embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises a nucleotide sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence shown in SEQ ID NO:5. In some embodiments, the heterologous gene product is CRASP-2 and is operably linked to the F14.5L gene promoter in the genome of the virus, and the recombinant virus comprises a nucleic acid sequence having at least 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to the nucleic acid sequence of SEQ ID NO:5. In some embodiments, the heterologous gene product is CRASP-2 and is operably linked to the F14.5L gene promoter in the genome of the virus, and the recombinant virus comprises the nucleic acid sequence of SEQ ID NO:5.In some embodiments, a recombinant virus, e.g., a recombinant oncolytic virus, comprises the nucleic acid sequence of SEQ ID NO:5. The recombinant oncolytic virus comprising the nucleic acid sequence of SEQ ID NO:5 is also referred to herein as VIR27.

[0307] The recombinant viruses provided herein exhibit an increased ability to evade the host complement in various embodiments. In some embodiments, the recombinant viruses provided herein can escape complement inhibition in in vivo and in vitro systems. In certain embodiments, VIR27 (comprising the nucleic acid sequence of SEQ ID NO:5) can escape complement inhibition in an in vitro system of complement inhibition upon incubation of an effective dose of VIR27 (comprising the nucleic acid sequence of SEQ ID NO:5) with human and / or BABL / c mouse serum (Figure 6). In certain embodiments, administration of an effective dose of VIR27 (comprising the nucleic acid sequence of SEQ ID NO:5) to a subject inhibits the growth of tumors, hyperplasia, or metastases in an in vivo model (Figure 7).

[0308] In some embodiments, the complement inhibitor is a miniFH gene product. Specifically, in some embodiments, the recombinant virus comprises an expression cassette comprising a miniFH cDNA fused to the F14.5L locus under the control of the vaccinia F14.5L gene promoter. Further details regarding minFH can be found in Schmidt et al., J Immunol. 2013 Jun 1; 190(11): 10.4049 / jimmunol.1203548., which is incorporated herein by reference for all purposes. In some embodiments, the complement inhibitor is a miniFH gene product comprising an amino acid sequence having at least 85%, 90%, or 95% sequence identity to the amino acid sequence shown in SEQ ID NO:19. In some embodiments, the complement inhibitor is a miniFH gene product comprising the amino acid sequence shown in SEQ ID NO:19.

[0309] In some embodiments, the stealth protein comprises miniFH, an FH-based inhibitor. In some embodiments, the miniFH gene product can increase the ability of a recombinant virus to evade the host complement. In some embodiments, provided is a recombinant virus comprising a polynucleotide encoding a miniFH gene product comprising a miniFH cDNA fused to the F14.5L locus, wherein the miniFH polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:19. In some embodiments, the recombinant virus comprises a polypeptide having at least 70%, such as at least 75%, 80%, 85%, or 90% sequence identity to the amino acid sequence of SEQ ID NO:19. For example, in some embodiments, the recombinant virus comprises an amino acid sequence having at least 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to the amino acid sequence of SEQ ID NO:19, but less than 100% identical to the amino acid sequence of SEQ ID NO:19.

[0310] In some embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises a nucleotide sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:6. In some embodiments, the heterologous gene product is miniFH, is operably linked to the F14.5L gene promoter in the genome of the virus, and the recombinant virus comprises a nucleic acid sequence having at least 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to the nucleic acid sequence of SEQ ID NO:6. In some embodiments, the heterologous gene product is miniFH, is operably linked to the F14.5L gene promoter in the genome of the virus, and the recombinant virus comprises the nucleic acid sequence of SEQ ID NO:6. In some embodiments, the recombinant virus, e.g., the recombinant oncolytic virus, comprises the nucleic acid sequence of SEQ ID NO:6. The recombinant oncolytic virus comprising the nucleic acid sequence of SEQ ID NO:6 is also referred to herein as VIR37.

[0311] In some embodiments, the polynucleotide encoding the miniFH molecule is operably linked to the F14.5L gene promoter. In some embodiments, a recombinant virus comprising a polynucleotide encoding a miniFH molecule (e.g., comprising the amino acid sequence of SEQ ID NO:19) is derived from the clonal VIP02 strain (comprising the nucleic acid sequence of SEQ ID NO:1) and comprises a nucleic acid sequence having at least 70%, such as at least 75%, 80%, 85%, or 90% sequence identity to SEQ ID NO:6 (also named VIR37). In some embodiments, the recombinant virus comprises a nucleic acid sequence having at least 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to the nucleic acid sequence of SEQ ID NO:6, but less than 100% identical to SEQ ID NO:6. In some embodiments, the recombinant virus comprises a nucleic acid sequence comprising the nucleic acid sequence of SEQ ID NO:6. In some embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises a nucleotide sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence shown in SEQ ID NO:6. In some embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence of SEQ ID NO:6.

[0312] The recombinant viruses provided herein exhibit an increased ability to evade the host complement in various embodiments. In some embodiments, the recombinant viruses provided herein can escape complement inhibition in in vivo and in vitro systems. In certain embodiments, VIR37 (comprising the nucleic acid sequence of SEQ ID NO:6) can escape complement inhibition in an in vitro system of complement inhibition upon incubation of an effective dose of VIR37 (comprising the nucleic acid sequence of SEQ ID NO:6) with human and / or BABL / c mouse serum (Figure 6).

[0313] In some embodiments, one or more heterologous gene products are T cell evasion factors or NK cell evasion factors. Gene products of T cell evasion factors or NK cell evasion factors can increase the ability of a virus to evade attack by the host immune system by T cells, such as cytotoxic T lymphocytes (CTLs) or natural killer (NK) cells. In particular, gene products of such T cell evasion factors or NK cell evasion factors can increase the ability of a recombinant virus to evade activation of the host complement cascade / system.

[0314] In some embodiments, the T cell evasion factor or NK cell evasion factor is a set of proteins encoded by vaccinia virus ORFs 012, 203, and 018 (CPXV012-203-018). CPXV012-203-018 is a synthetic DNA fragment. ORFs 012, 203, and 018 are expressed separately under their own promoters within the CPXV012-203-018 synthetic DNA fragment that encodes the CPXV012, CPXV203, and CPXV018 proteins. Vaccinia virus evades CTLs by CPXV012 and CPXV203. CPXV012 inhibits antigen peptide transport from the cytoplasm to the endoplasmic reticulum (ER), whereas CPXV203 blocks MHC I transport to the cell surface by exploiting the KDEL-receptor recycling pathway. CPXV018 encodes a soluble NKG2D ligand known as the orthopoxvirus major histocompatibility complex (MHC) class I-like protein (OMCP), which can block NKG2D-mediated cytotoxicity.

[0315] Recombinant viruses expressing the CRASP-2 gene product (UniProtKB-050665) as well as vaccinia virus open reading frames (ORFs) 012, 203, and 018 (CPXV012-203-018) have been generated herein in various embodiments. Specifically, in some embodiments, the recombinant virus, e.g., recombinant oncolytic virus, contains an expression cassette containing a continuous polynucleotide sequence comprising a CRASP-2 cDNA fused to the F14.5L locus under the control of the vaccinia F14.5L gene promoter, as well as open reading frames (ORFs) 012, 203, and 018 (CPXV012-203-018) having their own promoters inserted into the J2R locus.

[0316] In some embodiments, the stealth protein comprises Borrelia burgdorferi complement regulator-acquiring surface protein 2 (CRASP-2). In some embodiments, the expression of the CRASP-2 protein can increase the ability of the recombinant virus to evade the host's complement. In some embodiments, there is provided a recombinant virus comprising a polynucleotide encoding a CRASP-2 molecule comprising a CRASP-2 cDNA fused to the F14.5L locus, wherein the CRASP-2 polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:18. In some embodiments, the stealth protein comprises vaccinia virus open reading frames (ORFs) 012, 203, and 018 (CPXV012-203-018) having their own promoters. In some embodiments, the expression of vaccinia virus open reading frames (ORFs) 012, 203, and 018 (CPXV012-203-018) can increase the ability of the recombinant virus to evade the host's T cells and NK cells. In some embodiments, the expression of the stealth protein comprising CRASP-2 as well as vaccinia virus open reading frames (ORFs) 012, 203, and 018 can increase the ability of the recombinant virus to evade the host's complement as well as T cells and NK cells.

[0317] In some embodiments, the T cell evasion factor or NK cell evasion factor is a set of proteins encoded by vaccinia virus ORFs 012, 203, and 018 (CPXV012-203-018) that includes the amino acid sequences of SEQ ID NO: 20, 21, and 22, or amino acid sequences having at least 70%, 80%, 85%, 90%, or 95% sequence identity to the amino acid sequences of SEQ ID NO: 20, 21, and 22. In some embodiments, the recombinant virus comprises a polypeptide encoding CPXV012 having an amino acid sequence having at least 70%, such as at least 75%, 80%, 85%, or 90% sequence identity to the amino acid sequence of SEQ ID NO: 20. For example, in some embodiments, the recombinant virus comprises an amino acid sequence having at least 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to the amino acid sequence of SEQ ID NO: 20, but less than 100% identical to the amino acid sequence of SEQ ID NO: 20. In some embodiments, the recombinant virus comprises the amino acid sequence of SEQ ID NO: 20. In some embodiments, the recombinant virus comprises a polypeptide encoding CPXV203 having an amino acid sequence having at least 70%, such as at least 75%, 80%, 85%, or 90% sequence identity to the amino acid sequence of SEQ ID NO: 21. For example, in some embodiments, the recombinant virus comprises an amino acid sequence having at least 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to the amino acid sequence of SEQ ID NO: 21, but less than 100% identical to the amino acid sequence of SEQ ID NO: 21. In some embodiments, the recombinant virus comprises the amino acid sequence of SEQ ID NO: 21.In some embodiments, the recombinant virus comprises a polypeptide encoding CPXV018 having an amino acid sequence with at least 70%, such as at least 75%, 80%, 85%, or 90% sequence identity to the amino acid sequence of SEQ ID NO:22. For example, in some embodiments, the recombinant virus comprises an amino acid sequence having at least 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to the amino acid sequence of SEQ ID NO:22, but less than 100% identical to the amino acid sequence of SEQ ID NO:22. In some embodiments, the recombinant virus comprises the amino acid sequence of SEQ ID NO:22.

[0318] In some embodiments, the T cell evasion factor or NK cell evasion factor is a set of proteins encoded by vaccinia virus ORFs 012, 203, and 018 (CPXV012-203-018), and the set of proteins encoded by CPXV012-203-018 comprises an amino acid sequence (CPXV012) having at least 85%, 90%, or 95% sequence identity to the sequence shown in SEQ ID NO:20, an amino acid sequence (CPXV0203) having at least 85%, 90%, or 95% sequence identity to the sequence shown in SEQ ID NO:21, and an amino acid sequence (CPXV018) having at least 85%, 90%, or 95% sequence identity to the sequence shown in SEQ ID NO:22. In some embodiments, the set of proteins encoded by CPXV012-203-018 comprises the amino acid sequences shown in SEQ ID NO:20, SEQ ID NO:21, and SEQ ID NO:22.

[0319] In some embodiments, the recombinant virus comprises a polypeptide encoding CRASP-2 having an amino acid sequence with at least 70%, such as at least 75%, 80%, 85%, or 90% sequence identity to SEQ ID NO:18. For example, in some embodiments, the recombinant virus comprises a nucleic acid sequence encoding a polypeptide having at least 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to the amino acid sequence of SEQ ID NO:18, but less than 100% identical to the amino acid sequence of SEQ ID NO:18. In some embodiments, the recombinant virus comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:18. In some embodiments, the polynucleotide encoding the CRASP-2 molecule is operably linked to the F14.5L gene promoter. In some embodiments, the nucleotide sequences encoding CPXV012, CPXV203, and CPXV018 are inserted into the J2R genomic region.

[0320] In some embodiments, a recombinant virus comprising nucleic acid sequences encoding CRASP-2 (e.g., SEQ ID NO:18), CPXV012 (e.g., SEQ ID NO:20), CPXV203 (e.g., SEQ ID NO:21), and CPXV018 (e.g., SEQ ID NO:22) is derived from the VIR27 strain (comprising the nucleic acid sequence of SEQ ID NO:5) and comprises a nucleotide sequence having at least 70%, such as at least 75%, 80%, 85%, or 90% sequence identity to SEQ ID NO:10 (also named VIR46). For example, in some embodiments, the recombinant virus has at least 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% sequence identity to the nucleic acid sequence of SEQ ID NO:10, but is less than 100% identical to the nucleic acid sequence of SEQ ID NO:10. In some embodiments, the recombinant virus comprises the nucleic acid sequence of SEQ ID NO:10. The recombinant oncolytic virus comprising the nucleic acid sequence of SEQ ID NO:10 is also referred to herein as VIR46.

[0321] The recombinant viruses provided herein exhibit an increased ability to evade the host complement in various embodiments. In some embodiments, the recombinant viruses provided herein can escape complement inhibition in in vivo and in vitro systems. In certain embodiments, administration of an effective dose of VIR46 (comprising the nucleic acid sequence of SEQ ID NO:10) to a subject inhibits tumor, hyperplasia, or metastasis growth in an in vivo model (Figure 8).

[0322] B. Immunomodulatory Viruses Recombinant viruses are provided herein that contain a heterologous nucleic acid encoding an immunomodulatory protein that can be stably and efficiently expressed in many types of virus-infected cells in various embodiments. In some embodiments, the immunomodulatory protein includes cytokines, chemokines, immune receptors, antigens against immune receptors, proteins in immune cell activation pathways, signal transduction proteins within immune cells that stimulate immune cell activation or cytokine secretion from immune cells, and antigens. In some embodiments, the immunomodulatory protein includes one or more cytokines and / or chemokines. In some embodiments, the one or more cytokines and / or chemokines include one or more of chemokine ligand 9 (CXCL9), IL-2, and IL-12. In some embodiments, the immunomodulatory protein is tumor necrosis factor superfamily member 14 (LIGHT). In some embodiments, the immunomodulatory protein is an interferon regulatory factor that activates the Toll-like receptor 3 (TLR3)-interferon regulatory factor 3 (IRF3) signaling pathway. In some embodiments, the immunomodulatory protein is interleukin 12 (IL-12). In some embodiments, the immunomodulatory protein is chemokine ligand 9 (CXCL9). In some embodiments, the immunomodulatory protein is IL-2 or an IL-2 supercytokine. In some embodiments, the immunomodulatory protein is an interleukin 2 (IL-2) supercytokine. In some embodiments, the immunomodulatory protein is MDNA11. In some embodiments, MDNA11 is mutated to increase the anti-tumor efficacy of the recombinant virus, and the immunomodulatory protein is MDNA11T. In some embodiments, the recombinant virus contains a heterologous nucleic acid encoding one or more of the following immunomodulatory proteins: LIGHT, IRF3, IL-12, CXCL9, MDNA11, MDNA11T, and other immunomodulatory proteins. In some embodiments, the one or more immunomodulatory proteins are immunostimulatory proteins such as LIGHT.

[0323] In some embodiments, at least one heterologous nucleic acid encoding one or more heterologous gene products comprises one or more heterologous nucleic acids each encoding one or more immunomo...

Claims

1. an inactivating mutation of B2R, a heterologous nucleic acid encoding interferon regulatory factor 3 (IRF3), and at least one heterologous nucleic acid encoding one or more cytokines and / or chemokines A recombinant oncolytic vaccinia virus comprising:

2. The recombinant oncolytic vaccinia virus according to claim 1, wherein the at least one heterologous nucleic acid encoding one or more cytokines and / or chemokines comprises a heterologous nucleic acid encoding chemokine ligand 9 (CXCL9) and / or IL-12.

3. The recombinant oncolytic vaccinia virus according to claim 1 or 2, wherein the at least one heterologous nucleic acid encoding one or more cytokines and / or chemokines comprises a heterologous nucleic acid encoding CXCL9 and a heterologous nucleic acid encoding IL-12.

4. The recombinant oncolytic vaccinia virus according to claim 1, wherein the at least one heterologous nucleic acid encoding one or more cytokines and / or chemokines comprises a heterologous nucleic acid encoding CXCL9 and a heterologous nucleic acid encoding IL-12.

5. CXCL9 is human CXCL9 and comprises the amino acid sequence set forth in SEQ ID NO:99, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:99, or CXCL9 is mouse CXCL9 and comprises the amino acid sequence set forth in SEQ ID NO:106, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:106, The recombinant oncolytic vaccinia virus according to any one of claims 1 to 4.

6. IL-12 is human single-chain IL-12 and comprises the amino acid sequence set forth in SEQ ID NO:103, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:103, or IL-12 is mouse single-chain IL-12 and comprises the amino acid sequence set forth in SEQ ID NO:102, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:102, The recombinant oncolytic vaccinia virus according to any one of claims 1 to 5.

7. The recombinant oncolytic vaccinia virus according to any one of claims 1 to 6, wherein at least one heterologous nucleic acid encoding the one or more cytokines and / or chemokines comprises a heterologous nucleic acid encoding IL-2.

8. The recombinant oncolytic vaccinia virus according to claim 7, wherein IL-2 comprises the amino acid sequence set forth in any one of SEQ ID NOs: 98, 100, 101, 104 and 105, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 98, 100, 101, 104 and 105.

9. The recombinant oncolytic vaccinia virus according to claim 7 or claim 8, wherein IL-2 is an IL-2 supercytokine.

10. The IL-2 supercytokine is H9, H9T, MDNA11 or MDNA11T, the H9 IL-2 supercytokine comprises the amino acid sequence of SEQ ID NO: 100, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 100, or the H9T IL-2 supercytokine comprises the amino acid sequence of SEQ ID NO: 104, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 104, or the MDNA11 IL-2 supercytokine comprises the amino acid sequence of SEQ ID NO: 101, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 101, or the MDNA11T IL-2 supercytokine comprises the amino acid sequence of SEQ ID NO: 98, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 98, The recombinant oncolytic vaccinia virus according to claim 9.

11. The IL-2 supercytokine is MDNA11T, and the MDNA11T comprises the amino acid sequence set forth in SEQ ID NO:98, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:98, the recombinant oncolytic vaccinia virus according to claim 6 or claim 7.

12. The recombinant oncolytic vaccinia virus according to any one of claims 1 to 11, further comprising one or more heterologous gene products selected from the group consisting of a complement inhibitor, a T cell evasion factor or an NK cell evasion factor, an immunostimulatory protein, an anti-angiogenic protein, an interferon regulatory factor, an apoptosis-inducing protein, or any combination of the foregoing.

13. The recombinant oncolytic vaccinia virus according to any one of claims 1 to 12, wherein the inactivating mutation of B2R is a deletion of all or part of the B2R locus.

14. The recombinant oncolytic vaccinia virus according to claim 13, wherein the deletion is sufficient to render the encoded B2R gene product non-functional.

15. The recombinant oncolytic virus according to any one of claims 1 to 14, wherein the inactivating mutation of B2R is characterized by the insertion of at least one of the heterologous nucleic acids encoding IRF3 into the B2R locus and / or at least one of the at least one heterologous nucleic acid encoding the one or more cytokines and / or chemokines.

16. The recombinant oncolytic virus according to any one of claims 1 to 15, wherein the inactivating mutation of B2R is characterized by the insertion of a heterologous nucleic acid encoding chemokine ligand 9 (CXCL9) and / or IL-12 into the B2R locus.

17. The heterologous nucleic acid encoding IRF3 is inserted into the hemagglutinin (HA), J2R (thymidine kinase), F14.5L, A56R (hemagglutinin), vaccinia growth factor (VGF), A35R, A49R, A55R, B14R, C4L, C6L, C16L, NIL / N2L, E2L / E3L, K1L / K2L, K7L, superoxide dismutase locus, 7.5K, C2L-F3L, C4L-F1L, C7-K1L, B13R + B14R, A26L or I4L locus in the genome of the virus, and / or At least one of the at least one heterologous nucleic acid encoding the one or more cytokines and / or chemokines is inserted into HA, J2R, F14.5L, A56R, vaccinia growth factor, A35R, A49R, A55R, B14R, C4L, C6L, C16L, NIL / N2L, E2L / E3L, K1L / K2L, K7L, superoxide dismutase locus, 7.5K, C2L-F3L, C4L-F1L, C7-K1L, B13R + B14R, A26L or I4L locus within the genome of the virus. The recombinant oncolytic vaccinia virus according to any one of claims 1 to 16.

18. The recombinant oncolytic vaccinia virus according to any one of claims 1 to 17, wherein the nucleic acid genome of the recombinant oncolytic vaccinia virus has at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:1 and is optionally modified from a parental vaccinia virus having the nucleic acid genome set forth in SEQ ID NO:

1.

19. The nucleic acid genome of the parental vaccinia virus is (i) an amino acid sequence having at least 95% sequence identity to SEQ ID NO:57, having a polar uncharged amino acid at position 66, optionally containing threonine (T) at position 66, encoding variant 017 open reading frame (ORF); (ii) variant 038 (K5L) ORF containing a nucleotide insertion to cause a frameshift mutation and modifying the 038 (K5L) gene product; (iii) having at least 95% sequence identity to SEQ ID NO:60, having a hydrophobic amino acid other than leucine at position 419, optionally containing phenylalanine (F) at position 419, encoding variant 059 (E2L) ORF; (iv) having at least 95% sequence identity to SEQ ID NO:61, having a negatively charged amino acid at position 591, optionally containing aspartic acid (D) at position 591, encoding variant 104 (H4L) ORF; and (v) variant 182 (A56R) ORF containing a deletion of two nucleotides to cause a frameshift mutation and modifying the 182 (A56R) ORF gene product The recombinant oncolytic vaccinia virus according to any one of claims 1 to 18, characterized by one or more of the following.

20. The nucleic acid genome of the parental virus is (i) guanine (G) at a position corresponding to position 7770 of SEQ ID NO:1; (ii) thymine (T) at a position corresponding to position 15261 of SEQ ID NO:1; (iii) G at a position corresponding to position 32136 of SEQ ID NO:1; (iv) G at a position corresponding to position 49455 of SEQ ID NO:1; (v) cytosine (C) at a position corresponding to position 92969 of SEQ ID NO:1; (vi) the nucleic acid sequence CACTTATATAT at a position corresponding to positions 106870 to 106880 of SEQ ID NO:1; (vii) the nucleic acid sequence GTTTTCATTA at a position corresponding to positions 111267 to 111276 of SEQ ID NO:1; (viii) adenine (A) at a position corresponding to position 162715 of SEQ ID NO:1; (ix) the nucleic acid sequence TACAGACACC at a position corresponding to positions 165844 to 185853 of SEQ ID NO:1; and (x) C at a position corresponding to position 187805 of SEQ ID NO:1 The recombinant oncolytic vaccinia virus according to any one of claims 1 to 19, characterized by one or more of the following.

21. The recombinant oncolytic vaccinia virus according to any one of claims 1 to 20, wherein the nucleic acid genome of the recombinant oncolytic vaccinia virus has at least 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:

1.

22. The heterologous nucleic acid encoding IRF3 is inserted into the J2R (thymidine kinase) locus within the genome of the virus, The at least one heterologous nucleic acid encoding the one or more cytokines and / or chemokines includes heterologous nucleic acids encoding CXCL9 and IL-12, and the heterologous nucleic acids encoding CXCL9 and IL-12 are inserted into the A56R locus within the genome of the virus. The recombinant oncolytic vaccinia virus according to any one of claims 1 to 14 and 17 to 21.

23. The nucleic acid genome of the recombinant oncolytic vaccinia virus comprises a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence of SEQ ID NO:85, or the nucleic acid sequence described in SEQ ID NO:

85. The recombinant oncolytic vaccinia virus according to any one of claims 1 to 14 and 17 to 22.

24. The heterologous nucleic acid encoding IRF3 is inserted into the B2R (viral cGAMP-specific nuclease) locus within the genome of the virus. The at least one heterologous nucleic acid encoding the one or more cytokines and / or chemokines comprises heterologous nucleic acids encoding CXCL9 and IL-12, and the heterologous nucleic acids encoding CXCL9 and IL-12 are inserted into the A56R locus within the genome of the virus. The recombinant oncolytic vaccinia virus according to any one of claims 1 to 21.

25. The recombinant oncolytic vaccinia virus according to any one of claims 1 to 24, further comprising a heterologous nucleic acid encoding an apoptosis-inducing protein.

26. The recombinant oncolytic vaccinia virus according to claim 25, wherein the apoptosis-inducing protein is an inducible death effector domain (iDED).

27. The recombinant oncolytic vaccinia virus according to claim 26, wherein the iDED comprises the amino acid sequence described in SEQ ID NO:27, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence described in SEQ ID NO:

27.

28. The recombinant oncolytic vaccinia virus according to claim 26 or claim 27, wherein the heterologous nucleic acid encoding the iDED is inserted into or instead of the J2R locus within the genome of the virus.

29. The nucleic acid genome of the recombinant oncolytic vaccinia virus comprises a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence of SEQ ID NO:86, or the nucleic acid sequence described in SEQ ID NO:

86. The recombinant oncolytic vaccinia virus according to any one of claims 1 to 21 and 24 to 28.

30. The recombinant oncolytic vaccinia virus according to any one of claims 1 to 29, further comprising a heterologous nucleic acid encoding one or more T cell evasion factor proteins or NK cell evasion factor proteins.

31. The recombinant oncolytic vaccinia virus according to claim 30, wherein the one or more T cell evasion factor proteins or NK cell evasion factor proteins comprise a set of proteins encoded by vaccinia virus ORFs 012, 203 and 018 (CPXV012-203-018).

32. The set of proteins encoded by CPXV012-203-018 is (i) the amino acid sequence set forth in SEQ ID NO:20 (CPXV012), or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:20, (ii) the amino acid sequence set forth in SEQ ID NO:21 (CPXV0203), or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:21, and (iii) the amino acid sequence set forth in SEQ ID NO:22 (CPXV018), or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:22 The recombinant oncolytic vaccinia virus according to claim 31, comprising.

33. The recombinant oncolytic vaccinia virus according to any one of claims 1 to 32, further comprising a heterologous nucleic acid encoding a complement inhibitor.

34. The recombinant oncolytic vaccinia virus according to claim 33, wherein the complement inhibitor is Borrelia burgdorferi complement regulator-acquiring surface protein 2 (CRASP-2).

35. The recombinant oncolytic vaccinia virus according to claim 34, wherein the heterologous nucleic acid encoding CRASP-2 produces a fusion gene encoding a fusion protein and is optionally fused to the viral membrane gene F14.5L.

36. The recombinant oncolytic vaccinia virus according to claim 35, wherein the fusion protein comprises CRASP-2 fused to a viral membrane protein encoded by the viral membrane gene.

37. The recombinant oncolytic virus according to claim 36, wherein the viral membrane protein is F14.5L, and optionally, the fusion is a fusion at the C-terminus of F14.5L.

38. The nucleic acid genome of the recombinant oncolytic vaccinia virus according to any one of claims 1 to 22, 24 to 28, and 30 to 37, comprising a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO:90 or the nucleic acid sequence described in SEQ ID NO:

90.

39. The heterologous nucleic acid encoding IRF3 is inserted into or instead of the B2R (viral cGAMP-specific nuclease) locus within the genome of the virus, The at least one heterologous nucleic acid encoding one or more cytokines and / or chemokines comprises a heterologous nucleic acid encoding IL-2, and the IL-2 is an IL-2 supercytokine that is MDNA11T. The recombinant oncolytic vaccinia virus according to any one of claims 1 to 21, 25 to 28, and 30 to 37.

40. The recombinant oncolytic vaccinia virus according to any one of claims 1 to 39, further comprising a heterologous nucleic acid encoding an immunostimulatory protein and / or a heterologous nucleic acid encoding one or more anti-angiogenic proteins.

41. The recombinant oncolytic vaccinia virus according to claim 40, wherein the immunostimulatory protein is recombinant LIGHT.

42. The recombinant oncolytic vaccinia virus according to claim 41, wherein the recombinant LIGHT comprises the amino acid sequence described in SEQ ID NO:30 or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence described in SEQ ID NO:

30.

43. The recombinant oncolytic vaccinia virus according to any one of claims 40 to 42, wherein the one or more anti-angiogenic proteins comprise a VEGF inhibitor, an angiopoietin inhibitor, versican, or a fusion protein of any two or more of the foregoing.

44. The recombinant oncolytic vaccinia virus according to claim 43, wherein the one or more anti-angiogenic proteins comprise an anti-VEGF antibody and / or an anti-Ang2 antibody.

45. The recombinant oncolytic vaccinia virus according to claim 43 or claim 44, wherein the one or more anti-angiogenic proteins are bispecific anti-VEGF / anti-Ang2 antibodies.

46. The recombinant oncolytic vaccinia virus according to claim 45, wherein the bispecific anti-VEGF / anti-Ang2 antibody comprises the amino acid sequence set forth in SEQ ID NO: 23, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO:

23.

47. The recombinant oncolytic vaccinia virus according to any one of claims 1 to 22, 24 to 28, 30 to 37 and 39 to 46, wherein the nucleic acid genome of the recombinant oncolytic vaccinia virus comprises the nucleic acid sequence of SEQ ID NO: 88, or a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:

88.

48. an inactivating mutation of at least one viral gene and at least one heterologous nucleic acid encoding one or more heterologous gene products, wherein the one or more heterologous gene products are immunomodulatory proteins, complement inhibitors, T cell evasion factors or NK cell evasion factors, anti-angiogenic proteins, interferon regulatory factors or apoptosis-inducing proteins, or any combination or inclusion thereof, and at least one heterologous nucleic acid A recombinant oncolytic virus comprising.

49. A recombinant oncolytic virus comprising at least one heterologous nucleic acid encoding one or more heterologous gene products, wherein the one or more heterologous gene products are a complement inhibitor, a T cell evasion factor or an NK cell evasion factor, an immunomodulatory protein, an anti-angiogenic protein, an interferon regulatory factor, an apoptosis-inducing protein, or any combination thereof or include them, said recombinant oncolytic virus.

50. The recombinant oncolytic virus according to claim 48 or claim 50, which is vaccinia virus, herpes simplex virus, vesicular stomatitis virus (VSV), Maraba virus (MARAV), measles virus (MV), adenovirus, myxoma virus, orf virus, parvovirus, araigpox virus, coxsackievirus, reovirus, Newcastle disease virus, Seneca Valley virus, Semliki Forest virus, mumps virus, influenza virus, echovirus, and poliovirus (PV).

51. The recombinant oncolytic virus according to any one of claims 48 to 50, which is vaccinia virus.

52. The recombinant oncolytic vaccinia virus, wherein the nucleic acid genome of the recombinant oncolytic vaccinia virus is modified from a parental vaccinia virus having a nucleic acid genome having at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:1, and optionally, the parental vaccinia virus has the nucleic acid genome set forth in SEQ ID NO:1, the recombinant oncolytic vaccinia virus according to any one of claims 48 to 51.

53. A recombinant oncolytic virus comprising a nucleic acid genome modified from a parental vaccinia virus genome having at least 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:1, and optionally, the parental vaccinia virus has the nucleic acid genome set forth in SEQ ID NO:1, and the nucleic acid genome of the recombinant oncolytic virus comprises at least one heterologous nucleic acid encoding one or more heterologous gene products inserted into the genome, said recombinant oncolytic virus.

54. The nucleic acid genome of the parental vaccinia virus is (i) having at least 95% sequence identity to SEQ ID NO:57 and having a polar uncharged amino acid at position 66, optionally including threonine (T) at position 66, an amino acid sequence Variant 017 open reading frame (ORF) encoding; (ii) Variant 038 (K5L) ORF, which includes a nucleotide insertion that results in a frameshift mutation and modifies the 038 (K5L) gene product; (iii) An amino acid sequence having at least 95% sequence identity to SEQ ID NO:60, with a hydrophobic amino acid other than leucine at position 419, optionally including phenylalanine (F) at position 419; Variant 059 (E2L) ORF encoding; (iv) An amino acid sequence having at least 95% sequence identity to SEQ ID NO:61, with a charged amino acid at position 591, optionally including aspartic acid (D) at position 591; Variant 104 (H4L) ORF encoding; and (v) Variant 182 (A56R) ORF, which includes a two-nucleotide deletion that results in a frameshift mutation and modifies the 182 (A56R) ORF gene product The recombinant oncolytic vaccinia virus according to claim 52 or claim 53, characterized by one or more of the above.

55. The parental vaccinia virus genome is (i) Guanine (G) at the position corresponding to position 7770 of SEQ ID NO:1; (ii) Thymine (T) at the position corresponding to position 15261 of SEQ ID NO:1; (iii) G at the position corresponding to position 32136 of SEQ ID NO:1; (iv) G at the position corresponding to position 49455 of SEQ ID NO:1; (v) Cytosine (C) at the position corresponding to position 92969 of SEQ ID NO:1; (vi) The nucleic acid sequence CACTTATATAT at the position corresponding to positions 106870 - 106880 of SEQ ID NO:1; (vii) The nucleic acid sequence GTTTTCATTA at the position corresponding to positions 111267 - 111276 of SEQ ID NO:1; (viii) Adenine (A) at the position corresponding to position 162715 of SEQ ID NO:1; (ix) The nucleic acid sequence TACAGACACC at the position corresponding to positions 165844 - 185853 of SEQ ID NO:1; and (x) C at the position corresponding to position 187805 of SEQ ID NO:1 The recombinant oncolytic virus according to any one of claims 52 - 54, characterized by one or more of the above.

56. The recombinant oncolytic vaccinia virus according to any one of claims 52 to 55, wherein the nucleic acid genome of the recombinant oncolytic vaccinia virus has at least 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:

1.

57. The recombinant oncolytic virus is a recombinant oncolytic vaccinia virus, and the nucleic acid genome of the recombinant oncolytic vaccinia virus is (i) an amino acid sequence having at least 95% sequence identity to SEQ ID NO:57, having a polar uncharged amino acid at position 66, optionally containing threonine (T) at position 66, a variant 017 open reading frame (ORF) encoding; (ii) a variant 038 (K5L) ORF containing a nucleotide insertion to cause a frameshift mutation and modifying the 038 (K5L) gene product; (iii) having at least 95% sequence identity to SEQ ID NO:60, having a hydrophobic amino acid other than leucine at position 419, optionally containing phenylalanine (F) at position 419, a variant 059 (E2L) ORF encoding; (iv) having at least 95% sequence identity to SEQ ID NO:61, having a charged amino acid at position 591, optionally containing aspartic acid (D) at position 591, a variant 104 (H4L) ORF encoding; and (v) a variant 182 (A56R) ORF containing a deletion of two nucleotides to cause a frameshift mutation and modifying the 182 (A56R) ORF gene product The recombinant oncolytic virus according to any one of claims 1 to 56, characterized by one or more of them.

58. The recombinant oncolytic virus is a recombinant oncolytic vaccinia virus, and the nucleic acid genome of the recombinant oncolytic vaccinia virus is (i) guanine (G) at the position corresponding to position 7770 of SEQ ID NO:1; (ii) thymine (T) at the position corresponding to position 15261 of SEQ ID NO:1; (iii) G at the position corresponding to position 32136 of SEQ ID NO:1; (iv) G at the position corresponding to position 49455 of SEQ ID NO:1; (v) cytosine (C) at the position corresponding to position 92969 of SEQ ID NO:1; (vi) The nucleic acid sequence CACTTATATAT at the position corresponding to positions 106870 to 106880 of SEQ ID NO:1; (vii) The nucleic acid sequence GTTTTCATTA at the position corresponding to positions 111267 to 111276 of SEQ ID NO:1; (viii) Adenine (A) at the position corresponding to position 162715 of SEQ ID NO:1; (ix) The nucleic acid sequence TACAGACACC at the positions corresponding to positions 165844 to 185853 of SEQ ID NO:1; and (x) C at the position corresponding to position 187805 of SEQ ID NO:1 The recombinant oncolytic virus according to any one of claims 1 to 57, characterized by one or more of the above.

59. The recombinant oncolytic virus according to any one of claims 48 to 58, wherein at least one of the at least one heterologous nucleic acid encoding the one or more heterologous gene products is inserted into a non-essential gene or non-essential region within the genome of the virus.

60. The recombinant oncolytic virus according to any one of claims 48 to 59, wherein at least one of the at least one heterologous nucleic acid encoding the one or more heterologous gene products is inserted into the hemagglutinin (HA), J2R (thymidine kinase), F14.5L, A56R (hemagglutinin), B2R, vaccinia growth factor (VGF), A35R, A49R, A55R, B14R, C4L, C6L, C16L, NIL / N2L, E2L / E3L, K1L / K2L, K7L, superoxide dismutase locus, 7.5K, C2L-F3L, C4L-F1L, C7-K1L, B13R + B14R, A26L or I4L locus within the genome of the virus.

61. Each of at least one heterologous nucleic acid encoding one or more heterologous gene products inserted into a non-essential gene or non-essential region within the genome of said virus is independently inserted into the hemagglutinin (HA), J2R (thymidine kinase), F14.5L, A56R (hemagglutinin), B2R, vaccinia growth factor (VGF), A35R, A49R, A55R, B14R, C4L, C6L, C16L, NIL / N2L, E2L / E3L, K1L / K2L, K7L, superoxide dismutase locus, 7.5K, C2L-F3L, C4L-F1L, C7-K1L, B13R + B14R, A26L or I4L locus within the genome of said virus. The recombinant oncolytic virus according to claim 59.

62. The at least one viral gene comprises one or more viral genes selected from the group consisting of hemagglutinin (HA), J2R (thymidine kinase), F14.5L, A56R (hemagglutinin), B2R, vaccinia growth factor (VGF), A35R, A49R, A55R, B14R, C4L, C6L, C16L, NIL / N2L, E2L / E3L, K1L / K2L, K7L, superoxide dismutase locus, 7.5K, C2L-F3L, C4L-F1L, C7-K1L, B13R + B14R, A26L and I4L, and any combination thereof. The recombinant oncolytic virus according to any one of claims 48, 50 to 52, and 54 to 59.

63. The at least one viral gene is (i) B2R; (ii) A35R; (iii) A35R and J2R; (iv) J2R; (v) B2R and J2R; (vi) A35R, B2R and J2R; (vii) B2R, J2R and A56R; or (viii) A35R, B2R, J2R and A56R or comprises them. The recombinant oncolytic virus according to any one of claims 48, 50 to 52, 54 to 59 and 62.

64. One or more inactivating mutations in one or more of the at least one viral gene are independently insertion of at least one of at least one heterologous nucleic acid encoding one or more heterologous gene products into a locus within the genome of said virus, deletion of all or part of the at least one viral gene, and / or One or more nucleic acid substitutions within said at least one viral gene The recombinant oncolytic virus according to any one of claims 48, 50 to 52, and 54 to 63, which is a mutation by **Claim 65** The recombinant oncolytic virus according to any one of claims 48, 50 to 52, and 54 to 64, wherein said inactivating mutation is a deletion of all or part of said at least one viral gene. **Claim 66** The recombinant oncolytic virus according to claim 64 or claim 65, wherein the deletion of said at least one viral gene is a deletion of the entire gene ORF of the viral gene. **Claim 67** The recombinant oncolytic virus according to any one of claims 64 to 66, wherein said deletion is sufficient to render the encoded viral gene product non-functional. **Claim 68** The recombinant oncolytic virus according to claim 64, wherein said one or more nucleic acid substitutions are sufficient to render the encoded viral gene product non-functional. **Claim 69** The recombinant oncolytic virus according to any one of claims 48, 50 to 52, 54 to 59, and 62 to 68, wherein said at least one viral gene is B2R or includes the same. **Claim 70** The recombinant oncolytic virus according to any one of claims 48, 50 to 52, and 54 to 68, wherein one or more inactivating mutations of said at least one viral gene are characterized by the insertion of at least one of at least one heterologous nucleic acid encoding one or more heterologous gene products into a viral locus. **Claim 71** The recombinant oncolytic virus according to claim 70, wherein said at least one viral gene includes B2R. **Claim 72** The recombinant oncolytic virus according to claim 70 or claim 71, wherein said at least one viral gene includes J2R. **Claim 73** The recombinant oncolytic virus according to any one of claims 70 to 72, wherein said at least one viral gene includes A35R. **Claim 74** The recombinant oncolytic virus according to any one of claims 70 to 73, wherein said at least one viral gene includes A56R. **Claim 75** The recombinant oncolytic virus according to any one of claims 70 to 74, wherein said at least one viral gene includes B2R, J2R, and A35R. **Claim 76** The recombinant oncolytic virus according to any one of claims 70 to 75, wherein the at least one viral gene comprises B2R, J2R, A35R and A56R.

77. The recombinant oncolytic virus according to any one of claims 70 to 76, wherein the at least one viral gene comprises B2R, J2R and A56R.

78. The recombinant oncolytic virus according to any one of claims 48 to 77, wherein at least one heterologous nucleic acid encoding the one or more heterologous gene products is inserted into or instead of F14.5L.

79. The recombinant oncolytic virus according to any one of claims 48 to 78, wherein at least one heterologous nucleic acid encoding the one or more heterologous gene products is inserted into or instead of A35R.

80. The recombinant oncolytic virus according to any one of claims 48 to 78, wherein at least one heterologous nucleic acid encoding the one or more heterologous gene products is inserted into or instead of J2R.

81. The recombinant oncolytic virus according to any one of claims 48 to 80, wherein at least one heterologous nucleic acid encoding the one or more heterologous gene products comprises one or more heterologous nucleic acids encoding one or more immunomodulatory proteins respectively.

82. The recombinant oncolytic virus according to any one of claims 48, 50 to 52, and 54 to 81, wherein one or more inactivating mutations in the at least one viral gene are inactivating mutations by insertion of one or more heterologous nucleic acids encoding one or more immunomodulatory proteins respectively.

83. The recombinant oncolytic virus according to claim 81 or claim 82, wherein the one or more immunomodulatory proteins comprise one or more immunostimulatory proteins.

84. The recombinant oncolytic virus according to any one of claims 81 to 83, wherein the one or more immunomodulatory proteins comprise one or more cytokines and / or chemokines.

85. The recombinant oncolytic virus according to any one of claims 81 to 84, wherein the one or more immunomodulatory proteins comprise one or more interferon regulatory factors, optionally including IRF3.

86. The recombinant oncolytic virus according to claim 85, wherein the one or more interferon regulatory factors is or comprises interferon regulatory factor 3 (IRF3).

87. The recombinant oncolytic virus according to any one of claims 81 to 86, wherein the one or more immunomodulatory proteins comprise interferon regulatory factor 3 (IRF3) and one or more cytokines and / or chemokines.

88. The recombinant oncolytic virus according to any one of claims 81 to 87, wherein the one or more immunomodulatory proteins comprise one or more immunomodulatory proteins selected from the group consisting of LIGHT, IRF3, IL-2, IL-12, and CXCL9.

89. The recombinant oncolytic virus according to claim 88, wherein CXCL9 is human CXCL9 and comprises the amino acid sequence set forth in SEQ ID NO: 99, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO:

99.

90. The recombinant oncolytic virus according to claim 88, wherein CXCL9 is mouse CXCL9 and comprises the amino acid sequence set forth in SEQ ID NO: 106, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO:

106.

91. The recombinant oncolytic virus according to claim 88, wherein IL-12 is human single-chain IL-12 and comprises the amino acid sequence set forth in SEQ ID NO: 103, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO:

103.

92. The recombinant oncolytic virus according to claim 88, wherein IL-12 is mouse single-chain IL-12 and comprises the amino acid sequence set forth in SEQ ID NO: 102, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO:

102.

93. The recombinant oncolytic virus according to any one of claims 81 to 92, wherein the one or more immunomodulatory proteins comprise IRF3.

94. The recombinant oncolytic virus according to claim 93, wherein IRF3 is human IRF3 (hIRF3).

95. The recombinant oncolytic virus according to claim 94, wherein hIRF3 comprises the amino acid sequence set forth in SEQ ID NO: 51, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:

51.

96. The recombinant oncolytic virus according to claim 93, wherein IRF3 is mouse IRF3 (mIRF3).

97. The recombinant oncolytic virus according to claim 96, wherein mIRF3 comprises the amino acid sequence set forth in SEQ ID NO: 52, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:

52.

98. The recombinant oncolytic virus according to any one of claims 81 to 97, wherein the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence set forth in any one of SEQ ID NOs: 49, 50, 80, 82, and 84 to 93, or a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence set forth in any one of SEQ ID NOs: 49, 50, 80, 82, and 84 to 93.

99. The recombinant oncolytic virus according to any one of claims 81 to 98, wherein the one or more immunomodulatory proteins comprise IRF3 and one or more immunomodulatory proteins selected from the group consisting of LIGHT, IL-2, IL-12 and CXCL9.

100. The recombinant oncolytic virus according to any one of claims 81 to 99, wherein the one or more immunomodulatory proteins comprise IL-2.

101. The recombinant oncolytic virus according to any one of claims 81 to 100, wherein the one or more immunomodulatory proteins comprise IL-12.

102. The recombinant oncolytic virus according to any one of claims 81 to 101, wherein the one or more immunomodulatory proteins comprise LIGHT.

103. The recombinant oncolytic virus according to any one of claims 81 to 102, wherein the one or more immunomodulatory proteins comprise CXCL9.

104. The one or more immunomodulatory proteins are (i) IRF3; (ii) LIGHT; (iii) IRF3 and LIGHT; (iv) IRF3 and IL-2; (v) IRF3, CXCL9 and IL-12; (vi) IRF3, LIGHT and IL-2; (vii) IRF3 and CXCL9; or (viii) IRF3, CXCL9 and IL-2 The recombinant oncolytic virus according to any one of claims 81 to 103, which is or contains any of them.

105. The recombinant oncolytic virus according to any one of claims 88 to 104, wherein IL-2 is human IL-2.

106. The recombinant oncolytic virus according to any one of claims 88 to 105, wherein IL-2 is an IL-2 supercytokine.

107. The recombinant oncolytic virus according to claim 106, wherein the IL-2 supercytokine is H9, H9T, MDNA11 or MDNA11T.

108. The H9 IL-2 supercytokine contains the amino acid sequence of SEQ ID NO: 100, or contains an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 100, or The H9T IL-2 supercytokine contains the amino acid sequence of SEQ ID NO: 104, or contains an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 104, or The MDNA11 IL-2 supercytokine contains the amino acid sequence of SEQ ID NO: 101, or contains an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 101, or The MDNA11T IL-2 supercytokine contains the amino acid sequence of SEQ ID NO: 98, or contains an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 98, The recombinant oncolytic virus according to claim 106.

109. The recombinant oncolytic vaccinia virus according to any one of claims 106 to 108, wherein the IL-2 supercytokine is MDNA11 or MDNA11T.

110. The IL-2 supercytokine is MDNA11T, The MDNA11T comprises the amino acid sequence set forth in SEQ ID NO:98, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:

98. The recombinant oncolytic vaccinia virus according to any one of claims 106 to 109. **Claim 111** The recombinant oncolytic virus according to any one of claims 88 to 110, wherein the LIGHT is a recombinant LIGHT. **Claim 112** The recombinant oncolytic virus according to claim 111, wherein the recombinant LIGHT is a human LIGHT protein or a variant thereof. **Claim 113** The recombinant oncolytic virus according to claim 111 or claim 112, wherein the recombinant LIGHT comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:

30. **Claim 114** The recombinant oncolytic virus according to any one of claims 111 to 113, wherein the recombinant LIGHT is a human LIGHT variant (hmLIGHT) that is a human LIGHT variant that binds to human and mouse LTβR and HVEM. **Claim 115** The recombinant oncolytic virus according to any one of claims 111 to 114, wherein the recombinant LIGHT comprises one or more mutations selected from the group consisting of threonine at position 138, glycine at position 160, glycine at position 221, and lysine at position 222. **Claim 116** The recombinant oncolytic virus according to any one of claims 111 to 115, wherein the recombinant LIGHT comprises the amino acid sequence set forth in SEQ ID NO:25, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:

25. **Claim 117** The recombinant oncolytic virus according to any one of claims 111 to 116, wherein the recombinant LIGHT comprises the sequence set forth in SEQ ID NO:

25. **Claim 118** The nucleic acid genome of the recombinant oncolytic virus comprises a nucleic acid sequence as set forth in any one of SEQ ID NOs: 11, 82, 87, and 88, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence as set forth in any one of SEQ ID NOs: 11, 82, 87, and 88. The recombinant oncolytic virus according to any one of claims 111 to 117.

119. The recombinant oncolytic virus according to any one of claims 88 to 118, wherein IL-12 is human IL-12.

120. The recombinant oncolytic virus according to claim 119, wherein human IL-12 is human single-chain IL-12 (hscIL-12).

121. The recombinant oncolytic virus according to claim 120, wherein hscIL-12 comprises an amino acid sequence as set forth in SEQ ID NO: 103, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:

103.

122. The recombinant oncolytic virus according to any one of claims 88 to 121, wherein CXCL9 is human CXCL9.

123. The recombinant oncolytic virus according to claim 122, wherein human CXCL9 comprises an amino acid sequence as set forth in SEQ ID NO: 99, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:

99.

124. The recombinant oncolytic virus according to any one of claims 48 to 123, wherein at least one heterologous nucleic acid encoding the one or more heterologous gene products comprises one or more heterologous nucleic acids each encoding an apoptosis-inducing protein.

125. The recombinant oncolytic virus according to any one of claims 48, 50 to 52, and 54 to 124, wherein one or more inactivating mutations of the one or more of the at least one viral gene are inactivating mutations by insertion of one or more heterologous nucleic acids each encoding an apoptosis-inducing protein.

126. The recombinant oncolytic virus according to claim 124 or claim 125, wherein the apoptosis-inducing protein comprises an apoptosis-promoting molecule fused to an FKBP variant capable of binding to a chemical dimerization inducer (CID).

127. The recombinant oncolytic virus according to claim 126, wherein the FKBP variant is FKBP-F36V, and optionally, the FKBP-F36V comprises the amino acid sequence set forth in SEQ ID NO:56, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:

56.

128. The recombinant oncolytic virus according to claim 126 or claim 127, wherein the chemical dimerization inducer is AP1903 (Rimiducid).

129. The recombinant oncolytic virus according to any one of claims 126 to 128, wherein the apoptosis-promoting molecule is, or comprises, Fas, the death effector domain (DED) of Fas-associated death domain-containing protein (FADD), or caspase, and optionally, the caspase is caspase 9.

130. The recombinant oncolytic virus according to any one of claims 124 to 129, wherein the apoptosis-inducing protein is inducible DED (iDED).

131. The recombinant oncolytic virus according to claim 130, wherein iDED comprises the amino acid sequence set forth in SEQ ID NO:27, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:

27.

132. The recombinant oncolytic virus according to any one of claims 124 to 131, wherein the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence set forth in SEQ ID NO:8 or 86, or a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:8 or 86.

133. The recombinant oncolytic virus according to any one of claims 124 to 129, wherein the apoptosis-inducing protein is inducible Fas (iFas).

134. The recombinant oncolytic virus according to claim 133, wherein iFas comprises the amino acid sequence set forth in SEQ ID NO:28, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:

28.

135. The recombinant oncolytic virus according to any one of claims 124 to 129, 133 and 134, wherein the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence set forth in SEQ ID NO:9, or a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:

9.

136. The recombinant oncolytic virus according to any one of claims 124 to 129, wherein the apoptosis-inducing protein is inducible caspase 9 (iCas9).

137. The recombinant oncolytic virus according to claim 136, wherein iCas9 comprises the amino acid sequence set forth in SEQ ID NO:26, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:

26.

138. The recombinant oncolytic virus according to any one of claims 124 to 129, 136 and 137, wherein the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence set forth in SEQ ID NO:7, or a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:

7.

139. The recombinant oncolytic virus according to any one of claims 48 to 138, wherein the at least one heterologous nucleic acid encoding the one or more heterologous gene products comprises one or more heterologous nucleic acids encoding one or more T cell evasion factor proteins or NK cell evasion factor proteins, respectively.

140. The recombinant oncolytic virus according to any one of claims 48, 50 to 52, and 54 to 139, wherein one or more inactivating mutations of the one or more viral genes are inactivating mutations by insertion of one or more heterologous nucleic acids encoding one or more T cell evasion factor proteins or NK cell evasion factor proteins, respectively.

141. The recombinant oncolytic vaccinia virus according to claim 139 or claim 140, wherein the one or more T cell evasion factor proteins or NK cell evasion factor proteins comprise a set of proteins encoded by vaccinia virus ORFs 012, 203 and 018 (CPXV012-203-018).

142. The recombinant oncolytic vaccinia virus according to any one of claims 139 to 141, wherein the one or more T cell evasion factor proteins or NK cell evasion factor proteins are or comprise a set of proteins comprising the CPXV012 protein, the CPXV203 protein and the CPXV018 protein.

143. The set of proteins encoded by CPXV012-203-018 is (i) the amino acid sequence set forth in SEQ ID NO:20 (CPXV012), or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:20, (ii) the amino acid sequence set forth in SEQ ID NO:21 (CPXV0203), or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:21, and (iii) the amino acid sequence set forth in SEQ ID NO:22 (CPXV018), or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:22 The recombinant oncolytic vaccinia virus according to claim 141 or claim 142, comprising.

144. The recombinant oncolytic vaccinia virus according to any one of claims 141 to 143, wherein the set of proteins encoded by CPXV012-203-018 comprises the amino acid sequences set forth in SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:

22.

145. The nucleic acid genome of the recombinant oncolytic virus comprises a nucleic acid sequence described in any one of SEQ ID NOs: 10, 89, and 90, or a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence described in any one of SEQ ID NOs: 10, 89, and 90, the recombinant oncolytic vaccinia virus according to any one of claims 139 to 144.

146. The recombinant oncolytic virus according to any one of claims 48 to 145, wherein at least one heterologous nucleic acid encoding the one or more heterologous gene products comprises one or more heterologous nucleic acids each encoding one or more complement inhibitors.

147. The recombinant oncolytic virus according to any one of claims 48, 50 to 52, and 54 to 146, wherein one or more inactivating mutations of the one or more viral genes are inactivating mutations by insertion of one or more heterologous nucleic acids each encoding one or more complement inhibitors.

148. The recombinant oncolytic virus according to claim 146 or claim 147, wherein the one or more complement inhibitors are Borrelia burgdorferi complement regulator-acquiring surface protein 2 (CRASP-2) and / or minimized complement factor H (miniFH).

149. The recombinant oncolytic virus according to claim 148, wherein the one or more complement inhibitors is or comprises CRASP-2.

150. The recombinant oncolytic virus according to claim 149, wherein CRASP-2 comprises the amino acid sequence described in SEQ ID NO: 18, or has an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence described in SEQ ID NO:

18.

151. The recombinant oncolytic virus according to any one of claims 148 to 150, wherein the one or more complement inhibitors is or comprises miniFH.

152. The recombinant oncolytic virus according to claim 151, wherein miniFH comprises the amino acid sequence described in SEQ ID NO: 19, or has an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence described in SEQ ID NO:

19.

153. The recombinant oncolytic virus according to any one of claims 146 to 152, wherein one or more heterologous nucleic acids encoding the one or more complement inhibitors are introduced into a viral membrane gene, optionally F14.5L, to produce a fusion gene encoding a fusion protein.

154. The recombinant oncolytic virus according to claim 153, wherein the fusion protein comprises the complement inhibitor fused to a viral membrane protein encoded by the viral membrane gene.

155. The recombinant oncolytic virus according to claim 153 or claim 154, wherein the viral membrane gene is F14.5L, and optionally, the fusion is a fusion at the C-terminus of the F14.5L protein.

156. The recombinant oncolytic virus according to any one of claims 153 to 155, wherein the fusion protein is incorporated into the outer membrane of intracellular mature virus (IMV).

157. The nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence set forth in SEQ ID NO:5, or a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:5, or The nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence set forth in SEQ ID NO:6, or a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:6, or The nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence set forth in SEQ ID NO:89, or a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:89, or The nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence set forth in SEQ ID NO:90, or a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:90, The recombinant oncolytic vaccinia virus according to any one of claims 153 to 156.

158. The recombinant oncolytic virus according to any one of claims 48 to 157, wherein at least one heterologous nucleic acid encoding the one or more heterologous gene products comprises one or more heterologous nucleic acids each encoding one or more anti-angiogenic proteins.

159. The recombinant oncolytic virus according to any one of claims 48, 50 to 52, and 54 to 157, wherein one or more inactivating mutations of the one or more viral genes are inactivating mutations by insertion of one or more heterologous nucleic acids each encoding one or more anti-angiogenic proteins.

160. The recombinant oncolytic virus according to claim 158 or claim 159, wherein the one or more anti-angiogenic proteins are a VEGF inhibitor, an angiopoietin inhibitor, versican, or a fusion protein of any two or more of the foregoing.

161. The recombinant oncolytic virus according to any one of claims 158 to 160, wherein the one or more anti-angiogenic proteins comprise a VEGF inhibitor and / or an angiopoietin inhibitor, optionally including an inhibitor of Ang2.

162. The recombinant oncolytic virus according to any one of claims 158 to 161, wherein the one or more anti-angiogenic proteins comprise an anti-VEGF antibody and / or an anti-Ang2 antibody.

163. The recombinant oncolytic virus according to any one of claims 160 to 162, wherein the VEGF inhibitor is an anti-VEGF antibody, optionally an anti-VEGF single-chain antibody (scAb).

164. The recombinant oncolytic virus according to any one of claims 160 to 163, wherein the angiopoietin inhibitor is an anti-angiopoietin-2 (Ang2) antibody, optionally an anti-Ang2 single-chain antibody (scAb).

165. The recombinant oncolytic virus according to any one of claims 158 to 164, wherein the one or more anti-angiogenic proteins are a bispecific anti-VEGF / anti-Ang2 antibody.

166. The recombinant oncolytic virus according to claim 165, wherein the bispecific anti-VEGF / anti-Ang2 antibody comprises the amino acid sequence set forth in SEQ ID NO:23, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO:

23.

167. The recombinant oncolytic virus according to any one of claims 158 to 166, wherein the one or more anti-angiogenic proteins comprise versican.

168. The recombinant oncolytic virus according to claim 167, wherein versican comprises the amino acid sequence set forth in SEQ ID NO:24 or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the sequence set forth in SEQ ID NO:

24.

169. The recombinant oncolytic virus according to any one of claims 158 to 168, wherein the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence set forth in any one of SEQ ID NOs: 13, 47, 82, 87 and 88, or a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence set forth in any one of SEQ ID NOs: 13, 47, 82, 87 and 88.

170. The recombinant oncolytic virus according to any one of claims 48 to 169, wherein the at least one heterologous nucleic acid encoding the one or more heterologous gene products comprises one or more heterologous nucleic acids encoding one or more therapeutic or diagnostic agents, respectively.

171. The recombinant oncolytic virus according to any one of claims 48, 50 to 52, and 54 to 170, wherein one or more inactivating mutations of the at least one viral gene are inactivating mutations by insertion of one or more heterologous nucleic acids encoding one or more therapeutic or diagnostic agents, respectively.

172. The one or more therapeutic or diagnostic agents are selected from the group consisting of anti-cancer agents, anti-metastasis agents, anti-angiogenic agents, immunomodulatory molecules, antigens, genes for cell matrix degradation, genes for tissue regeneration and for reprogramming human somatic cells to pluripotency, enzymes that modify a substrate to produce a detectable product or signal or are detectable by an antibody, proteins that can bind to a contrast agent, genes for optical imaging or detection, genes for PET imaging, and genes for MRI imaging, the recombinant oncolytic virus according to claim 170 or claim 171.

173. The recombinant oncolytic virus according to any one of claims 170 to 172, wherein the one or more therapeutic or diagnostic agents comprise a therapeutic agent selected from the group consisting of hormones, growth factors, cytokines, chemokines, costimulatory molecules, ribozymes, transporter proteins, single-chain antibodies, antisense RNAs, prodrug-converting enzymes, siRNAs, microRNAs, toxins, anti-tumor oligopeptides, mitosis-inhibiting proteins, anti-mitotic oligopeptides, anti-cancer polypeptide antibiotics, angiogenesis inhibitors, tumor suppressors, cytotoxic proteins, cell growth inhibitory proteins, and tissue factors.

174. (i) the at least one viral gene is or comprises A35R, and optionally, the nucleic acid genome of the recombinant oncolytic virus comprises a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence of SEQ ID NO:3, or the nucleic acid sequence described in SEQ ID NO:3, or (ii) the at least one viral gene is or comprises A35R and J2R, and optionally, the nucleic acid genome of the recombinant oncolytic virus comprises a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence of SEQ ID NO:12, or the nucleic acid sequence described in SEQ ID NO:12, or (iii) the at least one viral gene is or comprises J2R, and the inactivating mutation of J2R is an inactivating mutation by insertion of one or more heterologous nucleic acids each encoding one or more T cell evasion factor proteins or NK cell evasion factor proteins, and optionally, the one or more T cell evasion factor proteins or NK cell evasion factor proteins comprise a set of proteins encoded by vaccinia virus ORFs 012, 203 and 018 (CPXV012-203-018), and At least one heterologous nucleic acid encoding the one or more heterologous gene products comprises one or more heterologous nucleic acids each encoding one or more complement inhibitors to be introduced into a viral membrane gene to produce a fusion gene encoding a fusion protein, optionally wherein the viral membrane gene is F14.5L, optionally wherein the fusion is a fusion at the C-terminus of the F14.5L protein, and optionally wherein the nucleic acid genome of the recombinant oncolytic virus comprises a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence of SEQ ID NO:10, or the nucleic acid sequence set forth in SEQ ID NO:10, or (iv) the at least one viral gene is or comprises J2R, and optionally the nucleic acid genome of the recombinant oncolytic virus comprises a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence of SEQ ID NO:4, or the nucleic acid sequence set forth in SEQ ID NO:4, or (v) the at least one viral gene is or comprises J2R and A35R, and the inactivating mutation of A35R is an inactivating mutation by insertion of one or more heterologous nucleic acids each encoding one or more immunomodulatory proteins, optionally wherein the one or more immunomodulatory proteins are selected from the group consisting of LIGHT, IRF3, IL-2, IL-12 and CXCL9, optionally wherein the one or more immunomodulatory proteins is LIGHT, and optionally the nucleic acid genome of the recombinant oncolytic virus comprises a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence of SEQ ID NO:11, or the nucleic acid sequence set forth in SEQ ID NO:11, A recombinant oncolytic virus according to any one of claims 48, 50 to 52, and 54 to 173.

175. (i) The at least one viral gene is or includes J2R and A35R, and the inactivating mutation of J2R is an inactivating mutation by insertion of one or more heterologous nucleic acids encoding one or more anti-angiogenic proteins. Optionally, the one or more anti-angiogenic proteins include an inhibitor or inhibitors of VEGF and / or Ang2, and optionally, the one or more anti-angiogenic proteins are bispecific anti-VEGF / anti-Ang2 antibodies, and optionally, the nucleic acid genome of the recombinant oncolytic virus includes a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence of SEQ ID NO:13, or the nucleic acid sequence described in SEQ ID NO:13, or (ii) The at least one viral gene is or includes J2R and A35R, and the inactivating mutation of A35R is an inactivating mutation by insertion of one or more heterologous nucleic acids encoding one or more immunomodulatory proteins. Optionally, the one or more immunomodulatory proteins are selected from the group consisting of LIGHT, IRF3, IL-2, IL-12 and CXCL9, and optionally, the one or more immunomodulatory proteins are LIGHT, and the inactivating mutation of J2R is an inactivating mutation by insertion of one or more heterologous nucleic acids encoding one or more anti-angiogenic proteins. Optionally, the one or more anti-angiogenic proteins include an inhibitor or inhibitors of VEGF and / or Ang2, and optionally, the one or more anti-angiogenic proteins are bispecific anti-VEGF / anti-Ang2 antibodies, and optionally, the nucleic acid genome of the recombinant oncolytic virus includes a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence of SEQ ID NO:47, or the nucleic acid sequence described in SEQ ID NO:47, or (iii) the at least one viral gene is or includes J2R, and the inactivating mutation of J2R is an inactivating mutation by insertion of one or more heterologous nucleic acids each encoding an apoptosis-inducing protein, optionally, the apoptosis-inducing protein is inducible DED (iDED), inducible Fas (iFas) or inducible Cas9 (iCas9), optionally, the nucleic acid genome of the recombinant oncolytic virus comprises a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 7, 8 or 9, or the nucleic acid sequence described in SEQ ID NO: 7, 8 or 9, or (iv) the at least one viral gene is or includes J2R, and the inactivating mutation of J2R is an inactivating mutation by insertion of one or more heterologous nucleic acids each encoding one or more immunomodulatory proteins, optionally, the one or more immunomodulatory proteins are selected from the group consisting of LIGHT, IRF3, IL-2, IL-12 and CXCL9, optionally, the one or more immunomodulatory proteins is IRF3, optionally, the nucleic acid genome of the recombinant oncolytic virus comprises a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 49, 50 or 93, or the nucleic acid sequence described in SEQ ID NO: 49, 50 or 93, or (v) the at least one viral gene is or includes J2R and B2R, optionally, the nucleic acid genome of the recombinant oncolytic virus comprises a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 48, or the nucleic acid sequence described in SEQ ID NO: 48, The recombinant oncolytic virus according to any one of claims 48, 50 to 52, and 54 to 173.

176. The recombinant oncolytic virus according to any one of claims 48, 50 to 52, and 54 to 173, wherein the at least one viral gene is or includes J2R and B2R.

177. (i) The at least one viral gene is or includes J2R and B2R, the inactivating mutation of J2R is an inactivating mutation by insertion of one or more heterologous nucleic acids each encoding one or more immunomodulatory proteins, optionally, the one or more immunomodulatory proteins are selected from the group consisting of LIGHT, IRF3, IL-2, IL-12, and CXCL9, optionally, the one or more immunomodulatory proteins is IRF3, optionally, the nucleic acid genome of the recombinant oncolytic virus comprises a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO:80, or the nucleic acid sequence described in SEQ ID NO:80, or (ii) The at least one viral gene is or includes J2R, B2R, and A35R, the inactivating mutation of J2R is an inactivating mutation by insertion of one or more heterologous nucleic acids encoding one or more anti-angiogenic proteins, optionally, the one or more anti-angiogenic proteins include an inhibitor or an inhibitor of VEGF and / or Ang2, optionally, the one or more anti-angiogenic proteins is a bispecific anti-VEGF / anti-Ang2 antibody, the inactivating mutation of B2R is an inactivating mutation by insertion of one or more heterologous nucleic acids each encoding one or more immunomodulatory proteins, optionally, the one or more immunomodulatory proteins are selected from the group consisting of LIGHT, IRF3, IL-2, IL-12, and CXCL9, optionally, the one or more immunomodulatory proteins is IRF3, and the inactivating mutation of A35R is an inactivating mutation by insertion of one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally, the one or more immunomodulatory proteins are selected from the group consisting of LIGHT, IRF3, IL-2, IL-12, and CXCL9, optionally, the one or more immunomodulatory proteins is LIGHT, and Optionally, the nucleic acid genome of the recombinant oncolytic virus comprises a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence of SEQ ID NO:82, or the nucleic acid sequence described in SEQ ID NO:82, or (iii) said at least one viral gene is or comprises J2R, B2R and A56R, the inactivating mutation of J2R is an inactivating mutation by insertion of one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally, said one or more immunomodulatory proteins being IRF3, the inactivating mutation of A56R is an inactivating mutation by insertion of one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally, said one or more immunomodulatory proteins being IL-2, optionally, said IL-2 being an IL-2 supercytokine, optionally MDNA11, and Optionally, the nucleic acid genome of the recombinant oncolytic virus comprises a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence of SEQ ID NO:84, or the nucleic acid sequence described in SEQ ID NO:84, or (iv) said at least one viral gene is or comprises J2R, B2R and A56R, the inactivating mutation of J2R is an inactivating mutation by insertion of one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally, said one or more immunomodulatory proteins being IRF3, the inactivating mutation of A56R is an inactivating mutation by insertion of one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally, said one or more immunomodulatory proteins comprising two or more immunomodulatory proteins selected from the group consisting of LIGHT, IRF3, IL-2, IL-12 and CXCL9, optionally, said two or more immunomodulatory proteins comprising IL-12 and CXCL9, and Optionally, the nucleic acid genome of the recombinant oncolytic virus comprises a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence of SEQ ID NO:85, or the nucleic acid sequence described in SEQ ID NO:85, or (v) The at least one viral gene may be or include J2R, B2R, and A56R, the inactivating mutation of B2R is an inactivating mutation by insertion of one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, and optionally, the one or more immunomodulatory proteins are IRF3, the inactivating mutation of A56R is an inactivating mutation by insertion of one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, and optionally, the one or more immunomodulatory proteins include two or more immunomodulatory proteins selected from the group consisting of LIGHT, IRF3, IL-2, IL-12, and CXCL9, and optionally, the two or more immunomodulatory proteins include IL-12 and CXCL9, the inactivating mutation of J2R is an inactivating mutation by insertion of one or more heterologous nucleic acids encoding apoptosis-inducing proteins, and optionally, the apoptosis-inducing proteins are inducible DED (iDED), and optionally, the nucleic acid genome of the recombinant oncolytic virus includes a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO:86, or the nucleic acid sequence described in SEQ ID NO:86, or (vi) The at least one viral gene may be or include J2R, B2R, A35R, and A56R, the inactivating mutation of J2R is an inactivating mutation by insertion of one or more heterologous nucleic acids encoding one or more anti-angiogenic proteins, and optionally, the one or more anti-angiogenic proteins include an inhibitor or inhibitors of VEGF and / or Ang2, and optionally, the one or more anti-angiogenic proteins are bispecific anti-VEGF / anti-Ang2 antibodies, the inactivating mutation of B2R is an inactivating mutation by insertion of one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, and optionally, the one or more immunomodulatory proteins are IRF3, The inactivating mutation of A35R is an inactivating mutation by insertion of one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally, the one or more immunomodulatory proteins are selected from the group consisting of LIGHT, IRF3, IL-2, IL-12 and CXCL9, optionally, the one or more immunomodulatory proteins are LIGHT, The inactivating mutation of A56R is an inactivating mutation by insertion of one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, the one or more immunomodulatory proteins are IL-2 supercytokine MDNA11, and optionally, the nucleic acid genome of the recombinant oncolytic virus comprises a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence of SEQ ID NO:87, or the nucleic acid sequence described in SEQ ID NO:87, or (vii) the at least one viral gene is, or includes, J2R, B2R, A35R and A56R, The inactivating mutation of J2R is an inactivating mutation by insertion of one or more heterologous nucleic acids encoding one or more anti-angiogenic proteins, optionally, the one or more anti-angiogenic proteins include an inhibitor or an inhibitor of VEGF, and / or Ang2, optionally, the one or more anti-angiogenic proteins are a bispecific anti-VEGF / anti-Ang2 antibody, The inactivating mutation of B2R is an inactivating mutation by insertion of one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally, the one or more immunomodulatory proteins are IRF3, The inactivating mutation of A35R is an inactivating mutation by insertion of one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally, the one or more immunomodulatory proteins are selected from the group consisting of LIGHT, IRF3, IL-2, IL-12 and CXCL9, optionally, the one or more immunomodulatory proteins are LIGHT, The inactivating mutation of A56R is an inactivating mutation by insertion of one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, the one or more immunomodulatory proteins being the IL-2 supercytokine MDNA11T, and optionally, the MDNA11T comprising the amino acid sequence set forth in SEQ ID NO:98, and optionally, the nucleic acid genome of the recombinant oncolytic virus comprises a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence of SEQ ID NO:88, or the nucleic acid sequence set forth in SEQ ID NO:88, or (viii) the at least one viral gene is or comprises J2R, B2R and A56R, The inactivating mutation of J2R is an inactivating mutation by insertion of one or more heterologous nucleic acids encoding one or more T cell evasion factor proteins or NK cell evasion factor proteins, and optionally, the one or more T cell evasion factor proteins or NK cell evasion factor proteins comprise the set of proteins encoded by vaccinia virus ORFs 012, 203 and 018 (CPXV012-203-018), The inactivating mutation of B2R is an inactivating mutation by insertion of one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, and optionally, the one or more immunomodulatory proteins is IRF3, The inactivating mutation of A56R is an inactivating mutation by insertion of one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, and optionally, the one or more immunomodulatory proteins are selected from the group consisting of LIGHT, IRF3, IL-2, IL-12 and CXCL9, and optionally, the one or more immunomodulatory proteins are the IL-2 supercytokine, optionally MDNA11 or MDNA11T, at least one heterologous nucleic acid encoding the one or more heterologous gene products comprises one or more heterologous nucleic acids encoding one or more complement inhibitors, optionally CRASP-2, which are introduced into a viral membrane gene, optionally F14.5L, to produce a fusion gene encoding a fusion protein, and optionally, the fusion is a fusion at the C-terminus of the F14.5L protein, and Optionally, the nucleic acid genome of the recombinant oncolytic virus comprises a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence of SEQ ID NO:89, or the nucleic acid sequence described in SEQ ID NO:89, or (ix) the at least one viral gene is or includes J2R, B2R and A56R, the inactivating mutation of J2R is an inactivating mutation by insertion of one or more heterologous nucleic acids encoding one or more T cell evasion factor proteins or NK cell evasion factor proteins, optionally, the one or more T cell evasion factor proteins or NK cell evasion factor proteins include a set of proteins encoded by vaccinia virus ORF 012, 203 and 018 (CPXV012-203-018), the inactivating mutation of B2R is an inactivating mutation by insertion of one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally, the one or more immunomodulatory proteins is IRF3, the inactivating mutation of A56R is an inactivating mutation by insertion of one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally, the one or more immunomodulatory proteins include two or more immunomodulatory proteins selected from the group consisting of LIGHT, IRF3, IL-2, IL-12 and CXCL9, optionally, the two or more immunomodulatory proteins include IL-12 and CXCL9, at least one heterologous nucleic acid encoding the one or more heterologous gene products includes one or more heterologous nucleic acids encoding one or more complement inhibitors, optionally CRASP-2, introduced into a viral membrane gene, optionally F14.5L, to produce a fusion gene encoding a fusion protein, optionally, the fusion is a fusion at the C-terminus of the F14.5L protein, and Optionally, the nucleic acid genome of the recombinant oncolytic virus comprises a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence of SEQ ID NO:90, or the nucleic acid sequence described in SEQ ID NO:90, or (x) The at least one viral gene is or includes B2R and J2R, and the inactivating mutation of J2R is an inactivating mutation by insertion of one or more heterologous nucleic acids each encoding one or more immunomodulatory proteins. Optionally, the one or more immunomodulatory proteins are selected from the group consisting of LIGHT, IRF3, IL-2, IL-12, and CXCL9. Optionally, the one or more immunomodulatory proteins are IRF3, and Optionally, the nucleic acid genome of the recombinant oncolytic virus comprises a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO:91, or the nucleic acid sequence described in SEQ ID NO:91, or (xi) The at least one viral gene is or includes B2R, J2R, and A56R, The inactivating mutation of J2R is an inactivating mutation by insertion of one or more heterologous nucleic acids each encoding one or more immunomodulatory proteins. Optionally, the one or more immunomodulatory proteins are selected from the group consisting of LIGHT, IRF3, IL-2, IL-12, and CXCL9. Optionally, the one or more immunomodulatory proteins are IRF3, The inactivating mutation of A56R is an inactivating mutation by insertion of one or more heterologous nucleic acids encoding one or more immunomodulatory proteins. Optionally, the one or more immunomodulatory proteins include two or more immunomodulatory proteins selected from the group consisting of LIGHT, IRF3, IL-2, IL-12, and CXCL9. Optionally, the two or more immunomodulatory proteins include IL-12 and CXCL9, and Optionally, the nucleic acid genome of the recombinant oncolytic virus comprises a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO:92, or the nucleic acid sequence described in SEQ ID NO:92, or (xii) the at least one viral gene is or includes J2R, and the inactivating mutation of J2R is an inactivating mutation by insertion of one or more heterologous nucleic acids each encoding one or more immunomodulatory proteins, optionally, the one or more immunomodulatory proteins are selected from the group consisting of LIGHT, IRF3, IL-2, IL-12 and CXCL9, optionally, the one or more immunomodulatory proteins are IRF3, and, optionally, the nucleic acid genome of the recombinant oncolytic virus comprises a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence of SEQ ID NO:93, or the nucleic acid sequence described in SEQ ID NO:93, The recombinant oncolytic virus according to any one of claims 48, 50 to 52, and 54 to 173.

178. The nucleic acid genome of the recombinant oncolytic vaccinia virus according to any one of claims 48 to 173, comprising the nucleic acid sequence described in any one of SEQ ID NO:48, 80, 82, and 84 to 93, or a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence described in any one of SEQ ID NO:48, 80, 82, and 84 to 93.

179. The nucleic acid genome of the recombinant oncolytic vaccinia virus according to any one of claims 48 to 173, comprising the nucleic acid sequence described in any one of SEQ ID NO:85, 86, 88 and 90, or a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence described in any one of SEQ ID NO:85, 86, 88 and 90.

180. The nucleic acid genome of the recombinant oncolytic vaccinia virus according to any one of claims 48 to 173, comprising the nucleic acid sequence described in SEQ ID NO:85, or a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence described in SEQ ID NO:

85.

181. The recombinant oncolytic virus according to any one of claims 48 to 180, wherein one or more of the heterologous nucleic acids encoding the heterologous gene product are operably linked to a promoter.

182. The recombinant oncolytic virus according to claim 181, wherein each of the one or more heterologous nucleic acids encoding the heterologous gene product, which is operably linked to a promoter, is selected from the group consisting of 7.5E, 7.5E / L, SSE, 11KL, SSL, SSEL, mH5, and LEO.

183. The recombinant oncolytic virus according to any one of claims 48 to 182, wherein each heterologous nucleic acid encoding the heterologous gene product is independently operably linked to a promoter, and optionally, each heterologous nucleic acid encoding the heterologous gene product is independently operably linked to a promoter selected from the group consisting of 7.5E, 7.5E / L, SSE, 11KL, SSL, SSEL, mH5, and LEO.

184. The recombinant oncolytic virus according to claim 182 or claim 183, wherein the promoter is a poxvirus promoter or a variant or derivative thereof.

185. The recombinant oncolytic virus according to claim 182 or claim 183, wherein the promoter is a vaccinia virus promoter.

186. The recombinant oncolytic virus according to any one of claims 182 to 185, wherein the promoter is selected from the group consisting of 7.5E, 7.5E / L, SSE, 11KL, SSL, SSEL, mH5, and LEO.

187. The recombinant oncolytic virus according to any one of claims 182 to 186, wherein the promoter has the amino acid sequence set forth in any one of SEQ ID NO: 29, 53, 55, 68, 69, 70, 71, or 72.

188. The recombinant oncolytic virus according to any one of claims 182 to 187, wherein the promoter is a synthetic strong early promoter (SSE).

189. The recombinant VACV strain according to claim 188, wherein the SSE promoter comprises the sequence set forth in SEQ ID NO:

29.

190. The recombinant oncolytic virus according to any one of claims 182 to 189, wherein the promoter is a strong early / late promoter (SEL).

191. The recombinant oncolytic virus according to claim 190, wherein the SEL promoter comprises the sequence set forth in SEQ ID NO:

55. **Claim 192** The recombinant oncolytic virus according to any one of claims 182 to 191, wherein the promoter is mH5. **Claim 193** The recombinant oncolytic virus according to claim 192, wherein the mH5 promoter comprises the sequence set forth in SEQ ID NO:

53. **Claim 194** An isolated cloned vaccinia virus (VACV) strain comprising a nucleic acid genome having at least 95% sequence identity with the nucleic acid sequence set forth in SEQ ID NO:1, wherein the nucleic acid genome (i) has at least 95% sequence identity to SEQ ID NO:57 and encodes an amino acid sequence having a polar uncharged amino acid at position 66, optionally including threonine (T) at position 66 for the variant 017 open reading frame (ORF); (ii) a variant 038 (K5L) ORF comprising a nucleotide insertion to cause a frameshift mutation, wherein the 038 (K5L) gene product is modified; (iii) having at least 95% sequence identity to SEQ ID NO:60 and encoding an amino acid sequence having a hydrophobic amino acid other than leucine at position 419, optionally including phenylalanine (F) at position 419 for the variant 059 (E2L) ORF; (iv) having at least 95% sequence identity to SEQ ID NO:61 and encoding an amino acid sequence having a negatively charged amino acid at position 591, optionally including aspartic acid (D) at position 591 for the variant 104 (H4L) ORF; and (v) a variant 182 (A56R) ORF comprising a two-nucleotide deletion to cause a frameshift mutation, wherein the 182 (A56R) ORF gene product is modified characterized by one or more of the above, said isolated cloned vaccinia virus (VACV) strain. **Claim 195** The isolated cloned VACV strain according to claim 194, wherein the nucleic acid genome is characterized by (i), and the variant 017 ORF encodes an amino acid sequence having at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

57. **Claim 196** The isolated cloned VACV strain according to claim 194 or claim 195, wherein the nucleic acid genome is characterized by (i), and the variant 017 ORF encodes the amino acid sequence set forth in SEQ ID NO:

57.

197. The isolated cloned VACV strain according to any one of claims 194 to 196, wherein the nucleic acid genome is characterized by (ii), the nucleotide insertion is guanine (G) corresponding to the insertion after nucleotide position 32135 of SEQ ID NO: 1, and optionally, the variant 038 (K5L) ORF is as described in SEQ ID NO:

58.

198. The isolated cloned VACV strain according to any one of claims 194 to 197, wherein the nucleic acid genome is characterized by (ii), and the 038 (K5L) gene product is as described in SEQ ID NO:

59.

199. The isolated cloned VACV strain according to any one of claims 194 to 198, wherein the nucleic acid genome is characterized by (iii), and the variant 059 (E2L) ORF encodes an amino acid sequence having at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

60.

200. The isolated cloned VACV strain according to any one of claims 194 to 199, wherein the nucleic acid genome is characterized by (iii), and the variant 059 (E2L) ORF encodes the amino acid sequence set forth in SEQ ID NO:

60.

201. The isolated cloned VACV strain according to any one of claims 194 to 200, wherein the nucleic acid genome is characterized by (iv), and the 104 (H4L) ORF encodes an amino acid sequence having at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

61.

202. The isolated cloned VACV strain according to any one of claims 194 to 201, wherein the nucleic acid genome is characterized by (iv), and the variant 104 (H4L) ORF encodes the amino acid sequence set forth in SEQ ID NO:

61.

203. The isolated cloned VACV strain according to any one of claims 194 to 202, wherein the nucleic acid genome is characterized by (v), and the deletion of the two nucleotides is a deletion of two consecutive nucleotides corresponding to the nucleotides after nucleotide position 165972 of SEQ ID NO: 2, and optionally, the variant 182 (A56R) is as described in SEQ ID NO:

62.

204. The isolated cloned VACV strain according to any one of claims 194 to 203, wherein the nucleic acid genome is characterized by (v), and the VACV protein is as described in SEQ ID NO:

63.

205. The isolated cloned VACV strain according to any one of claims 194 to 204, wherein the nucleic acid genome is characterized by any two of (i) to (v).

206. The isolated cloned VACV strain according to any one of claims 194 to 204, wherein the nucleic acid genome is characterized by any three of (i) to (v).

207. The isolated cloned VACV strain according to any one of claims 194 to 204, wherein the nucleic acid genome is characterized by any four of (i) to (v).

208. The isolated cloned VACV strain according to any one of claims 194 to 204, wherein the nucleic acid genome is characterized by each of (i) to (v).

209. An isolated cloned vaccinia virus (VACV) strain comprising a nucleic acid genome having at least 95% sequence identity with the nucleic acid sequence set forth in SEQ ID NO: 1, wherein the nucleic acid genome is (i) guanine (G) at the position corresponding to position 7770 of SEQ ID NO: 1; (ii) thymine (T) at the position corresponding to position 15261 of SEQ ID NO: 1; (iii) G at the position corresponding to position 32136 of SEQ ID NO: 1; (iv) G at the position corresponding to position 49455 of SEQ ID NO: 1; (v) cytosine (C) at the position corresponding to position 92969 of SEQ ID NO: 1; (vi) the nucleic acid sequence CACTTATATAT at the position corresponding to positions 106870 to 106880 of SEQ ID NO: 1; (vii) the nucleic acid sequence GTTTTCATTA at the position corresponding to positions 111267 to 111276 of SEQ ID NO: 1; (viii) adenine (A) at the position corresponding to position 162715 of SEQ ID NO: 1; (ix) The nucleic acid sequence TACAGACACC at the position corresponding to positions 165844 to 185853 of SEQ ID NO:1; and (x) C at the position corresponding to position 187805 of SEQ ID NO:1 The isolated cloned vaccinia virus (VACV) strain, characterized by one or more of the above.

210. The isolated cloned VACV strain according to claim 209, wherein the nucleic acid genome is characterized by any two of (i) to (x).

211. The isolated cloned VACV strain according to claim 209, wherein the nucleic acid genome is characterized by any three of (i) to (x).

212. The isolated cloned VACV strain according to claim 209, wherein the nucleic acid genome is characterized by any four of (i) to (x).

213. The isolated cloned VACV strain according to claim 209, wherein the nucleic acid genome is characterized by any five of (i) to (x).

214. The isolated cloned VACV strain according to claim 209, wherein the nucleic acid genome is characterized by any six of (i) to (x).

215. The isolated cloned VACV strain according to claim 209, wherein the nucleic acid genome is characterized by any seven of (i) to (x).

216. The isolated cloned VACV strain according to claim 209, wherein the nucleic acid genome is characterized by any eight of (i) to (x).

217. The isolated cloned VACV strain according to claim 209, wherein the nucleic acid genome is characterized by any nine of (i) to (x).

218. The isolated cloned VACV strain according to claim 209, wherein the nucleic acid genome is characterized by each of (i) to (x).

219. The isolated cloned VACV strain according to any one of claims 209 to 218, wherein the nucleic acid genome has at least 96% sequence identity with the nucleotide sequence set forth in SEQ ID NO:

1.

220. The isolated cloned VACV strain according to any one of claims 209 to 219, wherein the nucleic acid genome has at least 97% sequence identity with the nucleotide sequence set forth in SEQ ID NO:

1.

221. The isolated cloned VACV strain according to any one of claims 209 to 220, wherein the nucleic acid genome has at least 98% sequence identity with the nucleotide sequence set forth in SEQ ID NO:

1.

222. An isolated cloned VACV strain according to any one of claims 209 to 221, wherein the nucleic acid genome has at least 99% sequence identity with the nucleotide sequence set forth in SEQ ID NO:

1.

223. An isolated cloned vaccinia virus (VACV) strain comprising a nucleic acid genome having at least 99% sequence identity with the nucleotide sequence set forth in SEQ ID NO:

1.

224. A recombinant oncolytic virus according to any one of claims 1 to 193, or an isolated cloned VACV strain according to any one of claims 194 to 223, wherein the nucleic acid genome has at least 99.5% sequence identity with the nucleotide sequence set forth in SEQ ID NO:

1.

225. A recombinant oncolytic virus according to any one of claims 1 to 193, or an isolated cloned VACV strain according to any one of claims 194 to 224, wherein the nucleic acid genome has at least 99.9% sequence identity with the nucleotide sequence set forth in SEQ ID NO:

1.

226. A recombinant oncolytic virus according to any one of claims 1 to 193, or an isolated cloned VACV strain according to any one of claims 194 to 225, wherein the nucleic acid genome has at least 99.95% sequence identity with the nucleotide sequence set forth in SEQ ID NO:

1.

227. A recombinant oncolytic virus according to any one of claims 1 to 193, or an isolated cloned VACV strain according to any one of claims 194 to 226, wherein the nucleic acid genome does not contain the nucleotide sequence set forth in SEQ ID NO:

2.

228. An isolated cloned VACV strain according to any one of claims 194 to 227, wherein the nucleic acid genome is not modified to contain a non-viral heterologous nucleic acid containing an open reading frame encoding a non-viral heterologous protein.

229. An isolated cloned VACV strain according to any one of claims 194 to 228, wherein the nucleic acid genome is as set forth in SEQ ID NO:

1.

230. The recombinant oncolytic virus or cloned VACV strain optionally exhibits enhanced production of extracellular enveloped virions (EEV) after cell infection, as determined by the percentage of EEV, and the percentage of the EEV is given by the formula: Viral titer in supernatant / (Viral titer in supernatant + Viral titer in cell lysate) * 100 The recombinant oncolytic virus according to any one of claims 1 to 193, or the isolated clone VACV strain according to any one of claims 194 to 229, as determined by .

231. The recombinant oncolytic virus according to claim 230, or the isolated clone VACV strain according to claim 244, wherein more than 5% of the infectious particles after cell infection are EEV.

232. The recombinant oncolytic virus according to claim 230, or the isolated clone VACV strain according to claim 244, wherein more than 10% of the infectious particles after cell infection are EEV.

233. The recombinant oncolytic virus according to claim 230, or the isolated clone VACV strain according to claim 244, wherein more than 15% of the infectious particles after cell infection are EEV.

234. The recombinant oncolytic virus according to any one of claims 230 to 233, or the isolated clone VACV strain according to any one of claims 230 to 233, as determined by the percentage of at least 5%, 10% or 15% of the infectious particles being EEV, showing enhanced production of extracellular enveloped virions (EEV) after cell infection.

235. The recombinant oncolytic virus according to any one of claims 1 to 193, 224 to 227, and 230 to 234, or the isolated clone VACV strain according to any one of claims 194 to 234, showing oncolytic activity for killing tumor cells.

236. A VACV preparation comprising the isolated clone VACV strain according to any one of claims 194 to 235.

237. A VACV preparation comprising the recombinant oncolytic virus according to any one of claims 1 to 193, 224 to 227, and 230 to 235, which is a recombinant oncolytic vaccinia virus.

238. A recombinant oncolytic virus preparation comprising the recombinant oncolytic virus according to any one of claims 1 to 193, 224 to 227, and 230 to 235, optionally wherein at least 70%, 80%, 90%, 95% or 98% of the virus particles in the preparation have the genomic sequence of the cloned oncolytic virus strain.

239. The VACV preparation according to claim 236 or claim 237, which is substantially homogeneous and wherein a plurality of virus particles in the preparation have the genomic sequence of the cloned VACV strain.

240. The VACV preparation according to any one of claims 236, 237, and 239, wherein at least 70% of the virus particles in the preparation have the genomic sequence of the cloned VACV strain.

241. The VACV preparation according to any one of claims 236, 237, and 239, wherein at least 80% of the virus particles in the preparation have the genomic sequence of the cloned VACV strain.

242. The VACV preparation according to any one of claims 236, 237, and 239, wherein at least 90% of the virus particles in the preparation have the genomic sequence of the cloned VACV strain.

243. The VACV preparation according to any one of claims 236, 237, and 239, wherein at least 95% of the virus particles in the preparation have the genomic sequence of the cloned VACV strain.

244. The VACV preparation according to any one of claims 236, 237, and 239, wherein at least 98% of the virus particles in the preparation have the genomic sequence of the cloned VACV strain.

245. A pharmaceutical composition comprising an isolated VACV cloned strain according to any one of claims 208 to 248.

246. A pharmaceutical composition comprising a VACV preparation according to any one of claims 194 to 234.

247. A pharmaceutical composition comprising a recombinant oncolytic virus according to any one of claims 1 to 193, 224 to 227, and 230 to 235.

248. A recombinant vaccinia virus (VACV) strain comprising a nucleic acid genome of a VACV cloned strain according to any one of claims 194 to 235, which contains an inactivating mutation in at least one viral gene.

249. The recombinant VACV strain according to claim 248, wherein the viral gene is selected from the group consisting of hemagglutinin (HA), J2R (thymidine kinase), F14.5L, A56R (hemagglutinin), B2R, vaccinia growth factor (VGF), A35R, A49R, A55R, B14R, C4L, C6L, C16L, NIL / N2L, E2L / E3L, K1L / K2L, K7L, superoxide dismutase locus, 7.5K, C2L-F3L, C4L-F1L, C7-K1L, B13R + B14R, A26L and I4L.

250. The recombinant VACV according to claim 248 or claim 249, wherein the inactivating mutation is a deletion of all or part of the at least one viral gene.

251. The recombinant VACV strain according to claim 250, wherein the deletion of the at least one viral gene is a deletion of the entire gene ORF of the viral gene.

252. The recombinant VACV strain according to claim 250, wherein the deletion of the at least one viral gene is a deletion of a part of the ORF of the viral gene, and the deletion is sufficient to render the encoded gene product non-functional.

253. The recombinant VACV strain according to any one of claims 248 to 252, wherein the at least one viral gene is A35R or includes the same.

254. The recombinant VACV strain according to claim 253, wherein the nucleic acid genome of the recombinant VACV strain comprises the nucleic acid sequence set forth in SEQ ID NO:3, or a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the sequence set forth in SEQ ID NO:

3.

255. The recombinant VACV strain according to any one of claims 248 to 254, wherein the at least one viral gene is J2R or includes the same.

256. The recombinant VACV strain according to claim 255, wherein the nucleic acid genome of the recombinant VACV strain comprises the nucleic acid sequence set forth in SEQ ID NO:4, or a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the sequence set forth in SEQ ID NO:

4.

257. The recombinant VACV strain according to any one of claims 248 to 256, wherein the at least one viral gene is B2R or comprises B2R.

258. The recombinant VACV strain according to any one of claims 248 to 257, wherein the at least one viral gene is A35R and J2R or comprises A35R and J2R.

259. The recombinant VACV strain according to claim 258, wherein the nucleic acid genome of the recombinant VACV strain comprises the nucleic acid sequence set forth in SEQ ID NO:12, or a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the sequence set forth in SEQ ID NO:

12.

260. The recombinant VACV strain according to any one of claims 248 to 259, wherein the at least one viral gene is B2R and J2R or comprises B2R and J2R.

261. The recombinant VACV strain according to claim 260, wherein the nucleic acid genome of the recombinant VACV strain comprises the nucleic acid sequence set forth in SEQ ID NO:48, or a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the sequence set forth in SEQ ID NO:

48.

262. The recombinant VACV strain according to any one of claims 248 to 261, wherein the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence set forth in any one of SEQ ID NO:48, 80, 82, and 84 - 93, or a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence set forth in any one of SEQ ID NO:48, 80, 82, and 84 - 93.

263. The recombinant VACV strain according to any one of claims 248 to 261, wherein the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence set forth in any one of SEQ ID NO:85, 86, 88 and 90, or a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence set forth in any one of SEQ ID NO:85, 86, 88 and 90.

264. The nucleic acid genome of the recombinant oncolytic virus comprises a nucleic acid sequence set forth in SEQ ID NO:85, or a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence set forth in SEQ ID NO:85, the recombinant VACV strain according to any one of claims 248 to 261.

265. A nucleic acid comprising the genome of a recombinant oncolytic virus according to any one of claims 1 to 193, 224 to 227, and 230 to 235, or an isolated VACV clone strain according to any one of claims 194 to 234.

266. A recombinant oncolytic virus comprising the nucleic acid according to claim 265.

267. The nucleic acid according to claim 265, wherein the recombinant oncolytic virus is a recombinant oncolytic vaccinia virus.

268. The recombinant oncolytic virus according to claim 266, which is a recombinant oncolytic vaccinia virus.

269. A pharmaceutical composition comprising a recombinant VACV strain according to any one of claims 248 to 264.

270. A pharmaceutical composition comprising a recombinant oncolytic virus according to any one of claims 1 to 193, 224 to 227, and 230 to 235, optionally wherein the recombinant oncolytic virus is a recombinant oncolytic vaccinia virus.

271. The pharmaceutical composition according to any one of claims 245 to 247, 269 and 270, further comprising a pharmaceutically acceptable carrier.

272. The pharmaceutical composition according to any one of claims 245 to 247 and 269 to 271, formulated for intravenous, intratumoral, intraperitoneal or intrathoracic administration.

273. The pharmaceutical composition according to any one of claims 245 to 247 and 269 to 272, formulated for intravenous administration.

274. The pharmaceutical composition according to any one of claims 245 to 247 and 269 to 273, which is a liquid composition.

275. The pharmaceutical composition according to any one of claims 245 to 247 and 269 to 273, which is lyophilized.

276. A method for treating a proliferative disorder in a subject, comprising administering to the subject a recombinant oncolytic virus according to any one of claims 1 to 193, 224 to 227, and 230 to 235, an isolated clone VACV strain according to any one of claims 194 to 235, a recombinant VACV strain according to any one of claims 248 to 264, or a pharmaceutical composition according to any one of claims 245 to 247 and 269 to 275.

277. The method according to claim 276, wherein the proliferative disorder is a tumor or metastasis.

278. The method according to claim 276 or claim 277, wherein the proliferative disorder is cancer.

279. The cancer is pancreatic cancer, ovarian cancer, lung cancer, colon cancer, prostate cancer, cervical cancer, breast cancer, rectal cancer, kidney (renal) cancer, gastric cancer, esophageal cancer, liver (hepatic) cancer, endometrial cancer, bladder cancer, brain cancer, head and neck cancer, oral cancer (e.g., oral cavity cancer), cervical cancer, uterine cancer, thyroid cancer, testicular cancer, prostate cancer, skin cancer, e.g., melanoma, e.g., malignant melanoma, cholangiocarcinoma (bile duct cancer), thymic epithelial cancer, e.g., thymoma, leukemia, lymphoma or multiple myeloma, according to the method of claim 278.

280. The method according to claim 278 or claim 279, wherein the cancer is microsatellite stable (MSS) colorectal cancer.

281. The method according to claim 280, wherein the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence set forth in SEQ ID NO:8, or a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the sequence set forth in SEQ ID NO:

8.

282. The recombinant oncolytic virus or isolated oncolytic virus is administered in an amount of 1×10 5 pfu to 1×10 14 pfu, according to the method of any one of claims 276 to 281.

283. The method according to any one of claims 276 to 282, further comprising administering a second therapeutic agent for treating the proliferative disorder.

284. The method according to any one of claims 276 to 283, further comprising another treatment selected from surgery, radiation therapy, immunosuppressive therapy, and administration of an anticancer agent.

285. The method according to claim 284, wherein said other treatment is administration of an anti-cancer agent selected from cytokines, chemokines, growth factors, photosensitizers, toxins, anti-cancer antibiotics, chemotherapy compounds, radionuclides, angiogenesis inhibitors, signal transduction modulators, antimetabolites, anti-cancer vaccines, anti-cancer oligopeptides, mitosis-inhibiting proteins, anti-mitotic oligopeptides, anti-cancer antibodies, anti-cancer antibiotics, immunotherapeutic agents, and combinations of any of the foregoing thereof.

286. The method according to any one of claims 276 to 285, wherein the recombinant oncolytic virus or the isolated oncolytic virus is administered intravenously.

287. The method according to any one of claims 276 to 286, further comprising the step of administering AP1903 (rimiducid) to the subject.

288. The method according to any one of claims 276 to 287, wherein the recombinant oncolytic virus to be administered to the subject contains a heterologous nucleic acid encoding an apoptosis-inducing protein.

289. The method according to claim 287 or claim 288, wherein the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence set forth in SEQ ID NO: 8, or a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the sequence set forth in SEQ ID NO:

8.

290. The method according to any one of claims 276 to 289, wherein the subject exhibits severe immunodeficiency and is susceptible to viral infection.

291. The method according to any one of claims 276 to 290, wherein the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence set forth in any one of SEQ ID NO: 48, 80, 82, and 84 to 93, or a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence set forth in any one of SEQ ID NO: 48, 80, 82, and 84 to 93.

292. A method for inhibiting virus replication, comprising the step of contacting cells infected with a recombinant oncolytic virus with AP1903 (rimiducid), wherein the recombinant oncolytic virus contains a heterologous nucleic acid encoding an apoptosis-inducing protein.

293. A method for inhibiting viral replication, comprising the step of contacting cells with AP1903 (rimiducid), and infecting the cells with a recombinant oncolytic virus according to any one of claims 1 to 193, 224 to 227, and 230 to 235, an isolated cloned VACV strain according to any one of claims 194 to 235, or a recombinant VACV strain according to any one of claims 248 to 264.

294. The method according to claim 292 or claim 293, wherein the contacting step is performed in vivo in a subject.

295. The method according to claim 292 or claim 294, wherein AP1903 (rimiducid) has been administered to a subject who has previously been administered the recombinant oncolytic virus containing the heterologous nucleic acid encoding the apoptosis-inducing protein.

296. The method according to claim 293 or claim 294, wherein AP1903 (rimiducid) has been administered to a subject who has previously been administered a recombinant oncolytic virus according to any one of claims 1 to 193, 224 to 227, and 230 to 235, an isolated cloned VACV strain according to any one of claims 194 to 235, or a recombinant VACV strain according to any one of claims 248 to 264.

297. A method for inhibiting viral replication in a subject, comprising the step of administering AP1903 (rimiducid) to the subject, wherein the subject has previously been administered a recombinant oncolytic virus containing a heterologous nucleic acid encoding an apoptosis-inducing protein.

298. A method for inhibiting viral replication in a subject, comprising the step of administering AP1903 (rimiducid) to the subject, wherein the subject has previously been administered a recombinant oncolytic virus according to any one of claims 1 to 193, 224 to 227, and 230 to 235, an isolated cloned VACV strain according to any one of claims 194 to 235, or a recombinant VACV strain according to any one of claims 248 to 264.

299. The method according to any one of claims 287 to 298, which preferentially inhibits viral replication in non-cancerous cells.

300. The method according to any one of claims 287 to 299, wherein the apoptosis-inducing protein is inducible DED (iDED).

301. The method according to claim 300, wherein iDED comprises the amino acid sequence set forth in SEQ ID NO: 27, or an amino acid sequence having at least 85%, 90% or 95% sequence identity to SEQ ID NO:

27.

302. The method according to claims 300 and 301, wherein the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence set forth in SEQ ID NO: 8, or a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the sequence set forth in SEQ ID NO:

8.

303. The method according to any one of claims 292 to 302, wherein the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence set forth in any one of SEQ ID NO: 48, 80, 82, and 84 to 93, or a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the nucleic acid sequence set forth in any one of SEQ ID NO: 48, 80, 82, and 84 to 93.

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