Oncolytic vaccinia viruses and recombinant viruses and methods of use thereof

Abstract

Provided herein are cloned vaccinia virus strains exhibiting enhanced anti-tumor properties and / or reduced immunogenicity, as well as recombinant vaccinia viruses derived therefrom. Also provided herein are recombinant oncolytic virus strains comprising inactivated mutations in one or more viral genes, and / or one or more heterologous nucleic acids each encoding one or more heterologous gene products. The viruses provided herein (e.g., vaccinia viruses, including recombinant vaccinia viruses) are useful as oncolytic viral therapies (e.g., oncolytic vaccinia viral therapies) for the treatment of cancer. Also provided herein are pharmaceutical compositions of viruses, such as vaccinia viruses, and methods and uses of viruses, such as vaccinia viruses, for the treatment of cancer, as well as nucleic acids encoding the viruses.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] 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,” which is incorporated herein by reference in its entirety for all purposes. Incorporation by reference into the sequence listing

[0002] This application is filed with an accompanying electronic Sequence Listing. The Sequence Listing is provided as a file named 773192000140SeqList.xml, created on July 6, 2023, and 6,262,578 bytes in size. The information in the electronic Sequence Listing is incorporated herein by reference in its entirety. Technical Field

[0003] The present disclosure provides clonal strains of vaccinia viruses that exhibit enhanced anti-tumor properties and / or reduced immunogenicity, as well as recombinant vaccinia viruses derived therefrom. The vaccinia viruses of the present disclosure, including recombinant vaccinia viruses, can be used as oncolytic vaccinia virus therapies for treating cancer. The present disclosure also provides pharmaceutical compositions, methods, and uses of vaccinia viruses for treating cancer. Background Art

[0004] Vaccinia is an oncolytic virus and accumulates in tumors. In some cases, oncolytic virus (OV) refers to a virus that replicates selectively or more efficiently in cancer cells than in non-cancerous cells. Oncolytic vaccinia virus includes recombinant virus, which is engineered to engineer natural virus by gene disruption or gene addition to improve its anti-tumor properties, such as tumor selectivity or preferential replication in tumor cells, host tropism, surface attachment, lysis and diffusion. Such recombinant vaccinia virus includes attenuated virus, one or more of which viral genes are modified to cause loss of expression of viral genes or reduction or inactivation of viral proteins. However, the effectiveness of oncolytic virus is hindered by the strong immune response induced by the virus. Immune factors (such as antibodies) neutralize the virus by directly binding to the virus and prevent the virus from successfully infecting cells, or by marking the virus so that complement or other immune cells destroy it. Therefore, there is still a need for improved oncolytic vaccinia virus, which has the ability to induce antiviral defense reduced by the virus, while having enhanced anti-tumor activity. Summary of the Invention

[0005] 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: the CXCL9 is human CXCL9. In some embodiments, the 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 the CXCL9 is set forth in SEQ ID NO:99. In some of any embodiments, the CXCL9 is mouse CXCL9. In some embodiments, the 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 the CXCL9 is set forth in SEQ ID NO:106.

[0008] In some of any embodiments: the IL-12 is a human single-chain IL-12. In some embodiments, the single-chain IL-12 consists of human IL-12A (p35) and human IL-12B (p40) subunits, optionally separated by a linker. In some embodiments, the single-chain IL-12 comprises the amino acid sequence shown 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 shown in SEQ ID NO: 103. In some of any embodiments, the IL-12 is a mouse single-chain IL-12. In some embodiments, the single-chain IL-12 consists of mouse IL-12A (p35) and mouse IL-12B (p40) subunits, optionally separated by a linker. In some embodiments, the single-chain IL-12 comprises the amino acid sequence shown 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 shown in SEQ ID NO: 102.

[0009] In some of any embodiments, 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, the 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 set forth in any one of SEQ ID NOs: 98, 100, 101, 104, and 105. In some embodiments, the IL-2 is set forth in SEQ ID NO: 105. In some embodiments, the IL-2 is a superkine having 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 of any embodiments, the IL-2 is an IL-2 superkine. In some embodiments, the IL-2 superkine is H9, H9T, MDNA11, or MDNA11T. In some of any embodiments, the H9 IL-2 superkine comprises the amino acid sequence shown in SEQ ID NO: 100, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 100. In some of any embodiments, the H9T IL-2 superkine comprises the amino acid sequence shown in SEQ ID NO: 104, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 104. In some of any embodiments, the MDNA11 IL-2 superkine comprises the amino acid sequence set forth in 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. In some of any embodiments, the MDNA11T IL-2 superkine comprises the amino acid sequence set forth in 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. In some of any embodiments, the IL-2 superkine is MDNA11T, and the MDNA11T comprises the amino acid sequence set forth in 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 set forth in SEQ ID NO: 98.

[0011] In some of any embodiments, the recombinant oncolytic virus further comprises one or more heterologous gene products, wherein the heterologous gene products are selected from the group consisting of complement inhibitors, T cell or NK cell escape products, immunostimulatory proteins, anti-angiogenic proteins, interferon regulatory factors, apoptosis-inducing proteins, or any combination of the foregoing.

[0012] In some of any embodiments, the inactivating mutation of B2R is a deletion of all or part of the B2R locus. In some of any embodiments, the deletion is sufficient to render the encoded B2R gene product inoperative. In some embodiments, the inactivating mutation of B2R is one or more amino acid substitutions in the encoded gene product. In some of any embodiments, the inactivating mutation of B2R is characterized by the insertion of a heterologous nucleic acid into the B2R locus, e.g., replacing the deletion of all or part of the B2R locus. In some embodiments, the heterologous nucleic acid encodes IRF3 or a cytokine and / or a chemokine. In some of any embodiments, the inactivating mutation of B2R is by the insertion of a heterologous nucleic acid encoding IRF3 into the B2R locus and / or at least one of the heterologous nucleic acids encoding one or more cytokines and / or chemokines. In some of any embodiments, 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 embodiments, the heterologous nucleic acid encoding IRF3 is inserted into the viral genome at 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 some of any embodiments, at least one of the at least one heterologous nucleic acid encoding one or more cytokines and / or chemokines is inserted into the viral genome at 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. In some of any such embodiments, the insertion replaces a deletion of all or part of the corresponding locus.

[0014] In some of any embodiments, the nucleic acid genome of the recombinant oncolytic vaccinia virus is modified from a parental vaccinia virus, and the nucleic acid genome of the parental vaccinia virus has at least 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 1. In some embodiments, the nucleic acid genome of the recombinant oncolytic vaccinia virus is modified from a parental vaccinia virus, and the parental vaccinia virus has the nucleic acid genome shown in SEQ ID NO: 1.

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

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

[0017] In some of any embodiments, 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 shown in SEQ ID NO: 1. In some of any 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 shown in SEQ ID NO: 1. In some of any embodiments, the nucleic acid genome of the recombinant oncolytic vaccinia virus has at least 95% sequence identity to the nucleic acid sequence shown in SEQ ID NO: 1. In some of any embodiments, the nucleic acid genome of the recombinant oncolytic vaccinia virus has at least 96% sequence identity to the nucleic acid sequence shown in SEQ ID NO: 1. In some of any embodiments, the nucleic acid genome of the recombinant oncolytic vaccinia virus has at least 97% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 1. In some of any embodiments, the nucleic acid genome of the recombinant oncolytic vaccinia virus has at least 98% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 1. In some of any embodiments, the nucleic acid genome of the recombinant oncolytic vaccinia virus has at least 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 1.

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

[0019] In some of any embodiments, the nucleic acid genome of the recombinant oncolytic vaccinia 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. In some embodiments, the nucleic acid genome of the recombinant oncolytic vaccinia virus is shown 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 in the viral genome; and the at least one heterologous nucleic acid encoding one or more cytokines and / or chemokines comprises heterologous nucleic acids encoding CXCL9 and IL-12, wherein the heterologous nucleic acids encoding CXCL9 and IL-12 are inserted into the A56R locus in the viral genome.

[0021] In some 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 the amino acid sequence shown 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 shown in SEQ ID NO: 27. In some embodiments, the iDED is shown in SEQ ID NO: 27. In some embodiments, the heterologous nucleic acid encoding iDED is inserted into the J2R locus in the viral genome or replaces the J2R locus in the viral genome.

[0022] In some of any embodiments, the nucleic acid genome of the recombinant oncolytic vaccinia 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. In some embodiments, the nucleic acid genome of the recombinant oncolytic vaccinia virus is shown in SEQ ID NO: 86.

[0023] In some of any embodiments, the recombinant oncolytic vaccinia virus further comprises a heterologous nucleic acid encoding one or more T cell or NK cell escape proteins. In some embodiments, the one or more T cell or NK cell escape proteins comprise a set of proteins encoded by vaccinia virus ORFs 012, 203, and 018 (CPXV012-203-018). In some embodiments, a set of proteins encoded by CPXV012-203-018 comprises: (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; (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: The amino acid sequence shown in NO:22 has an amino acid sequence with at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

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

[0025] In some of any embodiments, the nucleic acid genome of the recombinant oncolytic vaccinia 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 shown in SEQ ID NO: 90. In some embodiments, the nucleic acid genome of the recombinant oncolytic vaccinia virus is shown in SEQ ID NO: 90.

[0026] In some of any embodiments, the heterologous nucleic acid encoding IRF3 is inserted into the B2R (viral cGAMP-specific nuclease) locus in the viral genome or replaces the B2R locus in 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, wherein IL-2 is an IL-2 superkine, which is MDNA11T.

[0027] In some of any embodiments, 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 embodiments, the immunostimulatory protein is recombinant LIGHT. In some embodiments, the recombinant LIGHT comprises the amino acid sequence shown 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 shown in SEQ ID NO: 30. In some embodiments, the recombinant LIGHT has the sequence shown in SEQ ID NO: 30.

[0028] In some embodiments, the one or more anti-angiogenic proteins comprise a VEGF inhibitor, an angiogenin inhibitor, a versikine, or a fusion protein of any two or more of the foregoing. In some embodiments, the one or more anti-angiogenic proteins comprise an anti-VEGF antibody and / or an anti-Ang2 antibody. In some embodiments, the one or more anti-angiogenic proteins are bispecific anti-VEGF / anti-Ang2 antibodies. In some embodiments, 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, the bispecific anti-VEGF / anti-Ang2 antibody has the sequence set forth in SEQ ID NO: 23.

[0029] In some of any embodiments, 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 shown in SEQ ID NO: 88. In some embodiments, the nucleic acid genome of the recombinant oncolytic vaccinia virus is shown in SEQ ID NO: 88.

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

[0031] Also provided herein 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 or comprise immunomodulatory proteins, complement inhibitors, T cell or NK cell escape proteins, anti-angiogenic proteins, interferon regulatory factors, apoptosis-inducing proteins, or any combination of the foregoing.

[0032] In some of any embodiments, 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, raccoonpox 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 embodiments, the oncolytic virus is a vaccinia virus. In some of any embodiments, the nucleic acid genome of the recombinant oncolytic vaccinia virus is modified from a parental vaccinia virus, the nucleic acid genome of the parental vaccinia virus having at least 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 1. In some of any embodiments, the nucleic acid genome of the recombinant oncolytic vaccinia virus is modified from a parental vaccinia virus, the parental vaccinia virus having a nucleic acid genome having the nucleic acid genome shown in SEQ ID NO: 1.

[0034] Also 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 complement inhibitors, T cell or NK cell escape products, immunomodulatory proteins, anti-angiogenic proteins, interferon regulatory factors, apoptosis-inducing proteins, or any combination thereof.

[0035] Also provided herein is 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 shown in SEQ ID NO: 1; and at least one heterologous nucleic acid encoding one or more heterologous gene products inserted into the genome. Also provided herein is a recombinant oncolytic virus comprising: a nucleic acid genome modified from a parental vaccinia virus genome having the nucleic acid sequence shown in SEQ ID NO: 1; and at least one heterologous nucleic acid encoding one or more heterologous gene products inserted into the genome.

[0036] In some of any embodiments, the nucleic acid genome of the parental vaccinia virus is characterized by one or more of the following: (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 comprising a polar uncharged amino acid at position 66, optionally comprising a threonine (T) at position 66; (ii) a variant 038 (K5L) ORF comprising a nucleotide insertion to create a frameshift mutation, wherein the 038 (K5L) gene product is altered; (iii) a variant 059 (E2L) ORF encoding an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 57 and comprising a polar uncharged amino acid at position 66, optionally comprising a threonine (T) at position 66; NO:60 has at least 95% sequence identity and comprises a hydrophobic amino acid other than leucine at position 419, optionally comprising an amino acid sequence of phenylalanine (F) at position 419; (iv) a variant 104(H4L) ORF, said variant 104(H4L) ORF encoding an amino acid sequence having at least 95% sequence identity to SEQ ID NO:61 and comprising a negatively charged amino acid at position 591, optionally comprising aspartic acid (D) at position 591; and (v) a variant 182(A56R) ORF, said variant 182(A56R) ORF comprising a deletion of two nucleotides to cause a frameshift mutation, wherein the 182(A56R) ORF gene product is altered.

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

[0038] In some of any embodiments, 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 shown in SEQ ID NO: 1. In some of any 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 shown in SEQ ID NO: 1. In some of any embodiments, the nucleic acid genome of the recombinant oncolytic vaccinia virus has at least 95% sequence identity to the nucleic acid sequence shown in SEQ ID NO: 1. In some of any embodiments, the nucleic acid genome of the recombinant oncolytic vaccinia virus has at least 96% sequence identity to the nucleic acid sequence shown in SEQ ID NO: 1. In some of any embodiments, the nucleic acid genome of the recombinant oncolytic vaccinia virus has at least 97% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 1. In some of any embodiments, the nucleic acid genome of the recombinant oncolytic vaccinia virus has at least 98% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 1. In some of any embodiments, the nucleic acid genome of the recombinant oncolytic vaccinia virus has at least 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 1.

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

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

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

[0042] In some of any embodiments, at least one of the at least one heterologous nucleic acid encoding one or more heterologous gene products is inserted into the genome of the virus at a 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 some of any of the embodiments, at least one heterologous nucleic acid encoding one or more heterologous gene products inserted into a non-essential gene or region of the viral genome is each 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 viral genome. In some of any embodiments, 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. In some of any such embodiments, the insertion replaces a deletion of all or part of the corresponding locus.

[0043] In some of any embodiments, the at least one viral locus into which at least one of the at least one heterologous nucleic acid is inserted is or comprises: (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.

[0044] In some of any embodiments, one or more inactivating mutations of at least one viral gene are independently achieved by: 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 substitution of one or more nucleic acids in at least one viral gene. In some of any embodiments, one or more inactivating mutations of at least one viral gene are achieved by 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, wherein the insertion replaces the deletion of all or part of the viral gene.

[0045] 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 viral gene ORF. In some embodiments, the deletion is sufficient to render the encoded viral gene product inoperative. In some embodiments, one or more inactivating mutations of at least one viral gene are characterized by insertion of at least one heterologous nucleic acid encoding one or more heterologous gene products into the viral locus. In some embodiments, the at least one viral gene comprises B2R. In some embodiments, the at least one viral gene comprises J2R. In some embodiments, the at least one viral gene comprises A35R. In some embodiments, the at least one viral gene comprises A56R. In some embodiments, the at least one viral gene comprises B2R, J2R, and A35R. In some embodiments, the at least one viral gene comprises B2R, J2R, A35R, and A56R. In some embodiments, the at least one viral gene comprises B2R, J2R, and A56R.

[0046] In some of any of the embodiments: at least one heterologous nucleic acid encoding one or more heterologous gene products is inserted into F14.5L or replaces F14.5L; and / or at least one heterologous nucleic acid encoding one or more heterologous gene products is inserted into A35R or replaces A35R; and / or at least one heterologous nucleic acid encoding one or more heterologous gene products is inserted into J2R or replaces J2R.

[0047] In some of any embodiments, at least one heterologous nucleic acid encoding one or more heterologous gene products includes one or more heterologous nucleic acids encoding one or more immunomodulatory proteins. In some of any embodiments, one or more inactivating mutations in at least one viral gene are by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins. In some of any embodiments, one or more immunomodulatory proteins include one or more immunostimulatory proteins. In some of any embodiments, one or more immunomodulatory proteins include one or more cytokines and / or chemokines. In some of any embodiments, one or more immunomodulatory proteins include one or more interferon regulatory factors. In some of any embodiments, interferon regulatory factor is IRF3. In some of any embodiments, one or more interferon regulatory factors are or include interferon regulatory factor 3 (IRF3). In some of any embodiments, one or more immunomodulatory proteins include interferon regulatory factor 3 (IRF3) and one or more cytokines and / or chemokines.

[0048] In some of any embodiments, 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. In some of any embodiments: CXCL9 is human CXCL9. In some of any embodiments: 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 of any embodiments, CXCL9 is mouse CXCL9. In some of any embodiments, 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 of any embodiments: IL-12 is a human single-chain IL-12. In some of any embodiments: IL-12 is a 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 of any embodiments, IL-12 is a mouse single-chain IL-12. In some of any embodiments, IL-12 is a 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 of any embodiments, the one or more immunomodulatory proteins comprise IRF3. In some of any embodiments, the IRF3 is human IRF3 (hIRF3). In some of any embodiments, the 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 of SEQ ID NO:51. In some of any embodiments, the IRF3 is mouse IRF3 (mIRF3). In some of any embodiments, the 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 of SEQ ID NO:52.

[0051] In some of any embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown in any one of SEQ ID NOs: 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 shown in any one of SEQ ID NOs: 49, 50, 80, 82, and 84-93.

[0052] In some of any embodiments, one or more immunomodulatory proteins include IRF3 and one or more immunomodulatory proteins selected from the group consisting of LIGHT, IL-2, IL-12, and CXCL9. In some of any embodiments, one or more immunomodulatory proteins include IL-2. In some of any embodiments, one or more immunomodulatory proteins include IL-12. In some of any embodiments, one or more immunomodulatory proteins include LIGHT. In some of any embodiments, one or more immunomodulatory proteins include CXCL9. In some of any embodiments, one or more immunomodulatory proteins are or include: (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 embodiments, the IL-2 is human IL-2. In some of any embodiments, the IL-2 is an IL-2 superkine. In some of any embodiments, the IL-2 superkine is H9, H9T, MDNA11, or MDNA11T. In some embodiments, the H9 IL-2 superkine 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. In some of any embodiments, the H9T IL-2 superkine 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. In some of any embodiments, the MDNA11 IL-2 superkine 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. In some of any embodiments, the MDNA11T IL-2 superkine 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. In some of any embodiments, the IL-2 superkine is MDNA11 or MDNA11T. In some of any embodiments, the IL-2 superkine is MDNA11T, and MDNA11T comprises the amino acid sequence shown 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 shown in SEQ ID NO: 98.

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

[0055] In some of any embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown 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 shown in any one of SEQ ID NOs: 11, 82, 87, and 88.

[0056] In some of any embodiments, IL-12 is human IL-12. In some embodiments, human IL-12 is human single-chain IL-12 (hscIL-12). In some embodiments, hscIL-12 comprises the amino acid sequence shown 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 shown in SEQ ID NO: 103.

[0057] In some of any embodiments, CXCL9 is human CXCL9. In some of any 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 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 apoptosis-inducing protein. In some of any embodiments, one or more inactivating mutations in at least one viral gene are by inserting one or more heterologous nucleic acids each encoding apoptosis-inducing protein. In some of any embodiments, the apoptosis-inducing protein comprises a pro-apoptotic molecule fused to an FKBP variant capable of binding a chemical inducer (CID) of dimerization. In some of any embodiments, the FKBP variant is FKBP-F36V. In some of any embodiments, FKBP-F36V comprises the amino acid sequence shown 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 shown in SEQ ID NO: 56.

[0059] In some of any embodiments, the chemical inducer of dimerization is AP1903 (Rimiducid). In some of any embodiments, the pro-apoptotic molecule is or comprises Fas, a death effector domain (DED) containing a Fas-associated death domain protein (FADD), or a caspase, optionally wherein the caspase is caspase 9. In some of any embodiments, the apoptosis-inducing protein is an inducible DED (iDED). In some of any embodiments, iDED comprises the amino acid sequence shown 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 shown in SEQ ID NO: 27. In some of any embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown 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 shown in SEQ ID NO: 8 or 86.

[0060] In some of any embodiments, the apoptosis-inducing protein is an inducible Fas (iFas). In some of any embodiments, iFas comprises the amino acid sequence shown 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 shown in SEQ ID NO: 28. In some of any embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown 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 shown in SEQ ID NO: 9.

[0061] In some of any embodiments, the apoptosis-inducing protein is an inducible caspase 9 (iCas9). In some of any embodiments, iCas9 comprises the amino acid sequence shown 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 shown in SEQ ID NO: 26. In some of any embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown 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 shown in SEQ ID NO: 7.

[0062] 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 T cell or NK cell escape proteins. In some of any embodiments, one or more inactivating mutations of at least one viral genotype are by insertion of one or more heterologous nucleic acids each encoding one or more T cell or NK cell escape proteins.

[0063] In some of any embodiments, the one or more T cell or NK cell escape proteins comprise a set of proteins encoded by vaccinia virus ORFs 012, 203, and 018 (CPXV012-203-018). In some of any embodiments, the one or more T cell or NK cell escape proteins comprise a set of proteins that are or comprise CPXV012, CPXV203, and CPXV018 proteins. In some of any of the embodiments, a set of proteins encoded by CPXV012-203-018 comprises: (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; (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: NO: 22 has an amino acid sequence with at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity. In some of any embodiments, a group 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. In some of any embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown 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 shown in any one of SEQ ID NOs: 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 in at least one viral gene are achieved by insertion of one or more heterologous nucleic acids each encoding one or more complement inhibitors.

[0065] In some of any embodiments, the one or more complement inhibitors are Borrelia burgdorferi complement regulatory acquisition surface protein-2 (CRASP-2) and / or minimized complement regulatory factor H (miniFH). In some of any embodiments, the one or more complement inhibitors are or comprise CRASP-2. In some of any embodiments, CRASP-2 comprises the amino acid sequence set forth in SEQ ID NO: 18, 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: 18. In some of any embodiments, the one or more complement inhibitors are or comprise miniFH. In some of any embodiments, miniFH comprises the amino acid sequence set forth in SEQ ID NO: 19, 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: 19.

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

[0067] 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 anti-angiogenic proteins. In some embodiments, one or more inactivating mutations in at least one viral gene are introduced by insertion of one or more heterologous nucleic acids each encoding one or more anti-angiogenic proteins. In some embodiments, the one or more anti-angiogenic proteins are VEGF inhibitors, angiopoietin inhibitors, versikine, or a fusion protein of any two or more of the foregoing. In some embodiments, the one or more anti-angiogenic proteins comprise a VEGF inhibitor and / or angiopoietin inhibitor, optionally an Ang2 inhibitor. In some embodiments, the one or more anti-angiogenic proteins comprise an anti-VEGF antibody and / or an anti-Ang2 antibody. In some embodiments, the VEGF inhibitor is an anti-VEGF antibody, optionally an anti-VEGF single-chain antibody (scAb). In some embodiments, the angiopoietin inhibitor is an anti-angiopoietin-2 (Ang2) antibody, optionally an anti-Ang2 single-chain antibody (scAb). In some embodiments, the one or more anti-angiogenic proteins are bispecific anti-VEGF / anti-Ang2 antibodies. In some of any embodiments, 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 of any embodiments, the one or more anti-angiogenic proteins comprise versikine. In some of any embodiments, versikine 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 of any embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown 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 shown in any one of SEQ ID NOs: 13, 47, 82, 87, and 88.

[0068] 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 therapeutic or diagnostic agents. In some of any embodiments, one or more of the at least one viral genes is inactivated by insertion of one or more heterologous nucleic acids each encoding one or more therapeutic or diagnostic agents.

[0069] In some of any embodiments, the one or more therapeutic or diagnostic agents are selected from anticancer agents, anti-metastatic agents, anti-angiogenic agents, immunomodulatory molecules, antigens, cell matrix degradation genes, genes for tissue regeneration and reprogramming human cells to pluripotency, enzymes that modify substrates to produce a detectable product or signal or can be detected by antibodies, proteins that can bind contrast agents, genes for optical imaging or detection, genes for PET imaging, and genes for MRI imaging. In some of any embodiments, the one or more therapeutic or diagnostic agents comprise a therapeutic agent selected from hormones, growth factors, cytokines, chemokines, co-stimulatory molecules, ribozymes, transporters, single-chain antibodies, antisense RNA, prodrug converting enzymes, siRNA, microRNA, toxins, antitumor oligopeptides, mitotic inhibitory proteins, antimitotic oligopeptides, anticancer polypeptide antibiotics, angiogenesis inhibitors, tumor suppressors, cytotoxic proteins, cytostatic proteins, and tissue factors.

[0070] In some of any embodiments: at least one viral gene is or comprises A35R, optionally wherein 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 shown in SEQ ID NO:3.

[0071] In some of any embodiments: at least one viral gene is or comprises A35R and J2R, optionally wherein 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 shown in SEQ ID NO: 12.

[0072] In some of any embodiments: at least one viral gene is or comprises J2R, and the inactivating mutation of J2R is by inserting one or more heterologous nucleic acids each encoding one or more T cell or NK cell escape proteins, optionally wherein one or more T cell or NK cell escape proteins comprise a set of proteins encoded by vaccinia virus ORFs 012, 203, and 018 (CPXV012-203-018), and wherein 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, and the heterologous nucleic acids are introduced into the viral membrane gene to produce a fusion gene encoding a fusion protein. In some embodiments, the viral membrane gene is F14.5L. 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: 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.

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

[0074] In some of any embodiments: at least one viral gene is or comprises J2R and A35R, and the inactivating mutation of A35R is by inserting one or more heterologous nucleic acids each encoding one or more immunomodulatory proteins. In some embodiments, the one or more immunomodulatory proteins are selected from LIGHT, IRF3, IL-2, IL-12 and CXCL9. In some embodiments, the one or more immunomodulatory proteins are LIGHT. In some embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence of SEQ ID NO: 11, 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: 11.

[0075] In some of any embodiments: at least one viral gene is or comprises J2R and A35R, and the inactivating mutation of J2R is by inserting one or more heterologous nucleic acids encoding one or more anti-angiogenic proteins. In some embodiments, the one or more anti-angiogenic proteins comprise a VEGF inhibitor and / or an Ang2 inhibitor. In some embodiments, the 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: 13, or a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 13.

[0076] In some of any embodiments: at least one viral gene is or comprises J2R and A35R, and the inactivating mutation of A35R is by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, and the inactivating mutation of J2R is by inserting one or more heterologous nucleic acids encoding one or more anti-angiogenic proteins. In some embodiments, the one or more immunomodulatory proteins are selected from LIGHT, IRF3, IL-2, IL-12 and CXCL9. In some embodiments, the one or more immunomodulatory proteins are LIGHT. In some embodiments, the one or more anti-angiogenic proteins comprise a VEGF inhibitor and / or an inhibitor of Ang2. In some embodiments, the 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 shown in SEQ ID NO: 47.

[0077] In some of any embodiments: at least one viral gene is or comprises J2R, and the inactivating mutation of J2R is by inserting one or more heterologous nucleic acids encoding each apoptosis-inducing protein. In some embodiments, the apoptosis-inducing protein is an inducible DED (iDED), an inducible Fas (iFas) or an inducible Cas9 (iCas9). In some embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown in SEQ ID NO: 7, 8 or 9, 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: 7, 8 or 9. In some embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence of SEQ ID NO: 7. In some embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence of SEQ ID NO: 8. In some embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence of SEQ ID NO: 9.

[0078] In some of any embodiments: at least one viral gene is or comprises J2R, and the inactivating mutation of J2R is by inserting one or more heterologous nucleic acids each encoding one or more immunomodulatory proteins. In some embodiments, the one or more immunomodulatory proteins are selected from LIGHT, IRF3, IL-2, IL-12 and CXCL9. In some embodiments, the one or more immunomodulatory proteins are IRF3. In some embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises a 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.

[0079] In some of any embodiments: at least one viral gene is or comprises J2R and B2R. In some embodiments, 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 with the nucleic acid sequence shown in SEQ ID NO: 48.

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

[0081] In some of any embodiments: at least one viral gene is or comprises J2R and B2R, and the inactivating mutation of J2R is by inserting one or more heterologous nucleic acids each encoding one or more immunomodulatory proteins. In some embodiments, the one or more immunomodulatory proteins are selected from LIGHT, IRF3, IL-2, IL-12 and CXCL9. In some embodiments, the 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: 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.

[0082] In some of any embodiments: at least one viral gene is or comprises J2R, B2R, and A35R; wherein: 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 heterologous nucleic acids encoding one or more immunomodulatory proteins, and the inactivating mutation of A35R is by insertion of heterologous nucleic acids encoding one or more immunomodulatory proteins. In some embodiments, the one or more anti-angiogenic proteins comprise an inhibitor of VEGF and / or an inhibitor of Ang2. In some embodiments, the 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 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, the 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, the one or more immunomodulatory proteins are LIGHT. In some embodiments, 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.

[0083] In some of any embodiments: at least one viral gene is or comprises J2R, B2R and A56R; wherein: the inactivating mutation of J2R is by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally wherein one or more immunomodulatory proteins are IRF3; the inactivating mutation of A56R is by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally wherein one or more immunomodulatory proteins are IL-2. In some embodiments, IL-2 is an IL-2 super factor. In some embodiments, the IL-2 super factor is MDNA11. In some embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence of 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.

[0084] In some of any embodiments: at least one viral gene is or comprises J2R, B2R and A56R; wherein: the inactivating mutation of J2R is by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally wherein one or more immunomodulatory proteins are IRF3; the inactivating mutation of A56R is by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally wherein one or more immunomodulatory proteins comprise two or more immunomodulatory proteins selected from LIGHT, IRF3, IL-2, IL-12 and CXCL9. In some embodiments, the two or more immunomodulatory proteins comprise 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: 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.

[0085] In some of any embodiments: at least one viral gene is or comprises J2R, B2R and A56R; wherein: the inactivating mutation of B2R is by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally wherein one or more immunomodulatory proteins are IRF3; the inactivating mutation of A56R is by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally wherein one or more immunomodulatory proteins comprise two or more immunomodulatory proteins selected from LIGHT, IRF3, IL-2, IL-12 and CXCL9, and the inactivating mutation of J2R is by inserting one or more heterologous nucleic acids each encoding a cell apoptosis inducing protein. In some embodiments, the two or more immunomodulatory proteins comprise IL-12 and CXCL9. In some embodiments, the apoptosis inducing protein is an 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 shown in SEQ ID NO:86.

[0086] In some of any embodiments: at least one viral gene is or comprises J2R, B2R, A35R and A56R; wherein: the inactivating mutation of J2R is by inserting one or more heterologous nucleic acids encoding one or more anti-angiogenic proteins, optionally wherein the one or more anti-angiogenic proteins comprise an inhibitor of VEGF and / or an inhibitor of Ang2, optionally wherein the one or more anti-angiogenic proteins are bispecific anti-VEGF / anti-Ang2 antibodies; the inactivating mutation of B2R is by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally wherein the one or more immunomodulatory proteins are IRF3; the inactivating mutation of A35R is by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally wherein the one or more immunomodulatory proteins are selected from LIGHT, IRF3, IL-2, IL-12 and CXCL9, optionally wherein the one or more immunomodulatory proteins are LIGHT; the inactivating mutation of A56R is by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, wherein the one or more immunomodulatory proteins are IL-2 super factor 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 with the nucleic acid sequence shown in SEQ ID NO:87.

[0087] In some of any embodiments: at least one viral gene is or comprises J2R, B2R, A35R, and A56R; wherein: the inactivating mutation of J2R is by inserting one or more heterologous nucleic acids encoding one or more anti-angiogenic proteins, optionally wherein the one or more anti-angiogenic proteins comprise an inhibitor of VEGF and / or an inhibitor of Ang2, optionally wherein the one or more anti-angiogenic proteins are bispecific anti-VEGF / anti-Ang2 antibodies; the inactivating mutation of B2R is by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally wherein the one or more immunomodulatory proteins are IRF3; the inactivating mutation of A35R is by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally wherein the one or more immunomodulatory proteins are selected from LIGHT, IRF3, IL-2, IL-12, and CXCL9, optionally wherein the one or more immunomodulatory proteins are LIGHT; the inactivating mutation of A56R is by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, wherein the one or more immunomodulatory proteins are IL-2 super factor MDNA11T. In some embodiments, MDNA11T comprises the amino acid sequence shown in SEQ ID NO:98. In some embodiments, the nucleic acid genome of the recombinant oncolytic 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 with the nucleic acid sequence shown in SEQ ID NO:88.

[0088] In some of any embodiments: at least one viral gene is or comprises J2R, B2R and A56R; wherein: the inactivating mutation of J2R is by inserting one or more heterologous nucleic acids each encoding one or more T cell or NK cell escape proteins, optionally wherein the one or more T cell or NK cell escape proteins comprise a set of vaccinia virus ORFs 012, 203 and 018 (CPXV012-203-018) encoded proteins; the inactivating mutation of B2R is by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally wherein the one or more immunomodulatory proteins are IRF3; the inactivating mutation of A56R is by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally wherein the one or more immunomodulatory proteins are selected from LIGHT, IRF3, IL-2, IL-12 and CXCL9, optionally wherein the one or more immunomodulatory proteins are IL-2 super factors, optionally MDNA11 or MDNA11T; 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 (optionally CRASP-2), which are introduced into the viral membrane gene, optionally in 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:89, or a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO:89.

[0089] In some of any embodiments: at least one viral gene is or comprises J2R, B2R and A56R; wherein: the inactivating mutation of J2R is by inserting one or more heterologous nucleic acids each encoding one or more T cell or NK cell escape proteins, optionally wherein the one or more T cell or NK cell escape proteins comprise a set of vaccinia virus ORFs 012, 203 and 018 (CPXV012-203-018) encoded proteins; the inactivating mutation of B2R is by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally wherein the one or more immunomodulatory proteins are IRF3; the inactivating mutation of A56R is by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally wherein the one or more immunomodulatory proteins include two or more immunomodulatory proteins selected from LIGHT, IRF3, IL-2, IL-12 and CXCL9, optionally wherein the two or more immunomodulatory proteins include IL-12 and CXCL9; at least one heterologous nucleic acid encoding one or more heterologous gene products includes one or more heterologous nucleic acids encoding one or more complement inhibitors (optionally CRASP-2), the heterologous nucleic acids are introduced into the viral membrane gene, optionally in 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 with the nucleic acid sequence shown in SEQ ID NO:90.

[0090] In some of any embodiments: at least one viral gene is or comprises B2R and J2R, and the inactivating mutation of J2R is by inserting one or more heterologous nucleic acids each encoding one or more immunomodulatory proteins, optionally wherein the one or more immunomodulatory proteins are selected from LIGHT, IRF3, IL-2, IL-12 and CXCL9, optionally wherein the one or more immunomodulatory proteins are IRF3. In some embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises a 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 shown in SEQ ID NO: 91.

[0091] In some of any embodiments: at least one viral gene is or comprises B2R, J2R and A56R, and the inactivating mutation of J2R is by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally wherein the one or more immunomodulatory proteins are selected from LIGHT, IRF3, IL-2, IL-12 and CXCL9, optionally wherein the one or more immunomodulatory proteins are IRF3; the inactivating mutation of A56R is by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally wherein the one or more immunomodulatory proteins comprise two or more immunomodulatory proteins selected from LIGHT, IRF3, IL-2, IL-12 and CXCL9, optionally wherein the two or more immunomodulatory proteins comprise 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 shown in SEQ ID NO: 92.

[0092] In some of any embodiments: at least one viral gene is or comprises J2R, and the inactivating mutation of J2R is by inserting one or more heterologous nucleic acids each encoding one or more immunomodulatory proteins, optionally wherein the one or more immunomodulatory proteins are selected from LIGHT, IRF3, IL-2, IL-12 and CXCL9, optionally wherein the one or more immunomodulatory proteins are IRF3. In some embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises a 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 shown in SEQ ID NO: 93.

[0093] In some of any embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown 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 shown in any one of SEQ ID NOs 48, 80, 82, and 84-93. In some of any embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown 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 shown in any one of SEQ ID NOs 85, 86, 88, and 90. In some of any embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises the 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.

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

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

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

[0097] In some of any embodiments of any recombinant oncolytic virus or any isolated clonal 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 of any embodiments of any recombinant oncolytic virus or any isolated clonal VACV strain, the nucleic acid genome is characterized by (ii), and the nucleotide insertion is a guanine (G) inserted after nucleotide position 32135 corresponding to SEQ ID NO: 1, optionally wherein the variant 038 (K5L) ORF is shown in SEQ ID NO: 58. In some of any embodiments of any recombinant oncolytic virus or any isolated clonal VACV strain, the nucleic acid genome is characterized by (ii), and the 038 (K5L) gene product is shown in SEQ ID NO: 59. In some of any embodiments of any recombinant oncolytic virus or any isolated clonal 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 of any embodiments of any recombinant oncolytic virus or any isolated clonal VACV strain, the nucleic acid genome is characterized by (iii), and the variant 059 (E2L) ORF encodes the amino acid sequence shown in SEQ ID NO: 60. In some of any embodiments of any recombinant oncolytic virus or any isolated clonal 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 embodiments of any recombinant oncolytic virus or any isolated clonal VACV strain, the nucleic acid genome is characterized by (iv), and wherein the variant 104 (H4L) ORF encodes the amino acid sequence shown in SEQ ID NO: 61. In some of any embodiments of any recombinant oncolytic virus or any isolated clonal VACV strain, the nucleic acid genome is characterized by (v), and the deletion of two nucleotides is a deletion of two consecutive nucleotides corresponding to the nucleotide after nucleotide position 165972 of SEQ ID NO: 2, optionally wherein variant 182 (A56R) is shown in SEQ ID NO: 62. In some of any embodiments of any recombinant oncolytic virus or any isolated clonal VACV strain, the nucleic acid genome is characterized by (v), and the VACV protein is shown in SEQ ID NO: 63.

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

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

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

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

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

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

[0104] In some of any embodiments of any recombinant oncolytic virus or any isolated clonal VACV strain, the clonal VACV strain exhibits enhanced production of extracellular enveloped virus (EEV) after infection of cells, optionally as determined by the percentage of EEV, wherein the percentage of EEV is determined by the following formula: virus titer in supernatant / (virus titer in supernatant+virus titer in cell lysate)*100. In some of any embodiments of any recombinant oncolytic virus or any isolated clonal VACV strain, greater than 5% of the infectious particles after infection of cells are EEV. In some of any embodiments of any recombinant oncolytic virus or any isolated clonal VACV strain, greater than 10% of the infectious particles after infection of cells are EEV. In some of any embodiments of any recombinant oncolytic virus or any isolated clonal VACV strain, greater than 15% of the infectious particles after infection of cells are EEV. In some of any of the embodiments, the recombinant oncolytic virus or clonal VACV strain exhibits enhanced production of extracellular enveloped virus (EEV) upon infection of cells, as determined by having a percentage of infectious particles that are EEV of at least 5%, 10%, or 15%.

[0105] In some of any of the embodiments of any recombinant oncolytic virus or any isolated clonal VACV strain, the virus exhibits oncolytic activity to kill tumor cells.

[0106] Also provided herein is a VACV preparation comprising an isolated clonal VACV strain of any of the isolated clonal VACV strains provided herein.

[0107] Also provided herein is a VACV formulation comprising any of the recombinant oncolytic vaccinia viruses 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 the cloned recombinant oncolytic virus.

[0109] In some of any of the embodiments, the VACV preparation is substantially homogeneous, wherein the plurality of viral particles in the preparation have the genomic sequence of a clonal VACV strain.

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

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

[0112] Also provided herein is a pharmaceutical composition comprising any of the VACVs 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 clonal strains provided herein, wherein the nucleic acid genome comprises an inactivating mutation in at least one viral gene.

[0115] In some of any 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 of any embodiments, the inactivating mutation is a deletion of all or part of at least one viral gene. In some of any embodiments, the deletion of at least one viral gene is a deletion of the entire gene ORF of the viral gene. In some of any of the embodiments, the deletion of at least one viral gene is a deletion of a portion of the ORF of the viral gene, and wherein the deletion is sufficient to render the encoded gene product nonfunctional.

[0116] In some of any of the embodiments, the at least one viral gene is or comprises A35R.

[0117] In some of any embodiments, the nucleic acid genome of the recombinant VACV strain comprises the nucleic acid sequence shown 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 shown in SEQ ID NO:3.

[0118] In some of any of the embodiments, the at least one viral gene is or comprises J2R.

[0119] In some of any embodiments, the nucleic acid genome of the recombinant VACV strain comprises the nucleic acid sequence shown 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 shown in SEQ ID NO:4.

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

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

[0122] In some of any embodiments, the nucleic acid genome of the recombinant VACV strain comprises the nucleic acid sequence shown 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 shown in SEQ ID NO:12.

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

[0124] In some of any embodiments, the nucleic acid genome of the recombinant VACV strain comprises the nucleic acid sequence shown 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 shown in SEQ ID NO:48.

[0125] In some of any embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown 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 shown in any one of SEQ ID NOs 48, 80, 82, and 84-93. In some of any embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown 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 shown in any one of SEQ ID NOs 85, 86, 88, and 90. In some of any embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises the 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 with the nucleic acid sequence shown in SEQ ID NO 85.

[0126] Also provided herein is a nucleic acid comprising the genome of any recombinant oncolytic virus or any isolated VACV clonal strain provided herein.

[0127] Also provided herein is a recombinant oncolytic virus comprising the nucleic acid of any of the recombinant oncolytic viruses provided herein.

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

[0129] Also provided herein is a pharmaceutical composition comprising any one of the recombinant VACV strains provided herein.

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

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

[0132] In some of any embodiments, the pharmaceutical composition is formulated for intravenous administration, intratumoral administration, intraperitoneal administration, or intrapleural administration. In some of any embodiments, the pharmaceutical composition is formulated for intravenous administration. In some of any embodiments, the pharmaceutical composition is a liquid composition. In some of any embodiments, 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 of any embodiments, the proliferative disorder is cancer. In some of any embodiments, the cancer is pancreatic cancer, ovarian cancer, lung cancer, colon cancer, prostate cancer, cervical cancer, breast cancer, rectal cancer, kidney (kidney) cancer, stomach cancer, esophageal cancer, liver (liver) cancer, endometrial cancer, bladder cancer, brain cancer, head and neck cancer, mouth cancer (such as oral cancer), cervical cancer, uterine cancer, thyroid cancer, testicular cancer, prostate cancer, skin cancer (such as melanoma, such as malignant melanoma), cholangiocarcinoma (bile duct cancer), thymic epithelial cancer (such as thymoma), leukemia, lymphoma or multiple myeloma. In some of any embodiments, the cancer is microsatellite stable (MSS) colorectal cancer.

[0135] In some of any embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown 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 with the sequence shown in SEQ ID NO:8.

[0136] In some of any embodiments, the recombinant oncolytic virus or isolated oncolytic virus is at 1×10 5 pfu to 1x10 14 pfu were administered.

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

[0138] In some of any 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, chemotherapeutic compounds, radionuclides, angiogenesis inhibitors, signal transduction regulators, antimetabolites, anticancer vaccines, anticancer oligopeptides, mitotic inhibitory proteins, anti-mitotic oligopeptides, anticancer antibodies, anticancer antibiotics, immunotherapeutic agents, and any combination thereof.

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

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

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

[0142] In some of any embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown 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 with the sequence shown in SEQ ID NO:8.

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

[0144] In some of any embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown 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 shown in any one of SEQ ID NOs: 48, 80, 82, and 84-93.

[0145] Also provided herein is a method for inhibiting viral replication, comprising 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 contacting a cell with AP1903 (Rimiducid), wherein the cell is infected 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 (e.g., clonal VACV strains).

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

[0148] Also provided herein is a method of inhibiting viral replication in a subject, the method comprising administering AP1903 (Rimiducid) to the subject, wherein the subject has previously been 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 administering AP1903 (Rimiducid) to the subject, wherein the subject has previously been administered any of the recombinant oncolytic viruses provided herein or any of the isolated oncolytic viruses provided herein.

[0150] In some of any embodiments, the method preferentially inhibits viral replication in non-cancerous cells. In some of any embodiments, the apoptosis-inducing protein is an inducible death effector domain (iDED). In some of any embodiments, the iDED comprises the amino acid sequence shown 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 of any embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown 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 with the sequence shown in SEQ ID NO:8.

[0152] In some of any embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown 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 shown in any one of SEQ ID NOs: 48, 80, 82, and 84-93. BRIEF DESCRIPTION OF THE DRAWINGS

[0153] Figure 1 Depicted are the percentage of cell survival of BT-549, A549, LOX-IMVI, HCC-2998, and COLO-205 cells following infection with VIP01-06VACV clonal isolates. For each of VIP01, VIP02, VIP03, VIP04, VIP05, and VIP06, the bars correspond from left to right to BT-549, A549, LOX-IMVI, HCC-2998, and COLO-205 cells.

[0154] Figure 2 Shown are the percentages of extracellular enveloped virus (EEV) production by VCP02 and VIP02 in 4T1 and B16-F10 infected cells.

[0155] Figure 3 Depicted are changes in tumor volume in the 4T1 mouse mammary carcinoma model following infection with a single intravenous delivery of VCP02 (squares), VIP01 (triangles), VIP02 (diamonds), and vehicle (circles).

[0156] FIG4 depicts 2-D ( Figure 4A ) and 3-D( Figure 4B ) Percentage of cell survival in cell culture.

[0157] Figure 5 Depicted are a series of schematic diagrams representing the genomic structures of cryptic recombinant clones VIR27, VIR37, and VIR46 derived from parental VIP02.

[0158] Figure 6 Depicted are the percentage of host complement inhibition in human and mouse sera following incubation with the stealth oncolytic virus clones VIR27, VIR37, and VIP02.

[0159] Figure 7 Depicted are tumor volumes in the 4T1 mouse mammary cancer model following infection with a single intravenous delivery of VIP02, VIR27, and vehicle.

[0160] Figure 8 Depicted are tumor volumes in the 4T1 mouse mammary carcinoma model following infection with a single intravenous delivery of VIR46, VIR52, and vehicle.

[0161] Figure 9 Depicted are a series of schematic diagrams representing the genomic structures of the immunostimulatory oncolytic viruses VIR49 and VIR52.

[0162] Figure 10 Depicted are tumor volumes in the 4T1 mouse mammary cancer model following infection with a single intravenous delivery of VIR49 and VIR52.

[0163] Figure 11 Depicted are a series of schematic diagrams representing the genomic structure of the anti-angiogenic oncolytic virus VIR71.

[0164] Figure 12A and 12B Depicted is the administration of VIR71, VIR52, and vehicle in a single intravenous dose. Figure 12A ) and VIR13, VIR86 and vehicle ( Figure 12B ) in a 4T1 mouse breast cancer model after infection.

[0165] Figure 13 Depicted are a series of schematic diagrams representing the genomic structures of apoptosis-inducing oncolytic viruses VIR40, VIR41, VIR42, and a control virus VIR13.

[0166] Figure 14 Depicted are a series of graphs quantifying viral replication in primary healthy HBE, HME and MME of apoptosis-inducing viral clones VIR13, VIR40, VIR41 and VIR42 initially infected at an MOI of 0.01 and / or 10 in the presence of Rimiducid or DMSO as a control.

[0167] Figure 15 depicts a series of graphs quantifying the apoptosis-inducing viral clones VIR13, VIR40, VIR41 and VIR42 in BT-549 breast cancer cells ( Figure 15A ), Hs578T breast cancer cells ( Figure 15B ), MCF-7 and 4T1 breast cancer cells ( Figure 15C ), A549 and M14 lung cancer and melanoma cells ( Figure 15D ), HCT-15MSI colon cancer cells ( Figure 15E ), HCT-116MSI colon cancer cells ( Figure 15F) and KM12 MSI colon cancer cells ( Figure 15G ) in the virus replication.

[0168] FIG16 depicts a series of graphs quantifying the effect of Rimiducid on the proliferation of COLO205 cancer cells initially infected at an MOI of 0.01 and / or 10 in the presence of Rimiducid or DMSO as a control. Figure 16A )、HCC-2998 cancer cells ( Figure 16B ) and HT-29 cancer cells ( Figure 16C ) in the virus replication.

[0169] Figure 17 depicts a series of graphs quantifying the apoptosis-inducing viral clones VIR13, VIR40, VIR41 and VIR42 in primary human bronchial / tracheal epithelial cells (HBE, Figure 17A ), human primary mammary epithelial cells (HME, Figure 17B ), mouse primary mammary epithelial cells (MME), and human primary colon epithelial cells (HCE, Figure 17C ) in the cytotoxicity.

[0170] Figure 18 depicts a series of graphs quantifying the apoptosis-inducing viral clones VIR13, VIR40, VIR41 and VIR42 in BT-549 breast cancer cells ( Figure 18A ), Hs578T breast cancer cells ( Figure 18B ), 4T1 breast cancer cells ( Figure 18C ), DU-145 prostate cancer cells ( Figure 18D ), PC-3 prostate cancer cells ( Figure 18E ), A549 lung cancer and melanoma cells ( Figure 18F ), M14 lung cancer and melanoma cells ( Figure 18G ), COLO 320DM and HCT-15MSI colon cancer cells ( Figure 18H ), HCT-116 and KM12 MSI colon cancer cells ( Figure 18I )、KM12 MSI colon cancer cells ( Figure 18J ) and SW48MSI colon cancer cells ( Figure 18K ) in the cytotoxicity.

[0171] FIG19 depicts a series of graphs quantifying the apoptosis-inducing viral clones VIR13, VIR40, VIR41 and VIR42 in COLO205 MSS colon cancer cells ( Figure 19A )、HCC-2998 colon cancer cells ( Figure 19B ), HT-29 cells ( Figure 19C ), LS123 cells ( Figure 19D ), LS174T cells ( Figure 19E ), SW620 cells ( Figure 19F ) and WiDR cells ( Figure 19G ) in the cytotoxicity.

[0172] Figure 20 demonstrated complete inhibition of tumor growth in the SL-4 mouse colon adenocarcinoma model following infection with a single intravenous injection of VIR13.

[0173] Figure 21 AE depicts a series of graphs showing the effects of VIR13, VIR41 or control ( Figure 21 A); VIR13, VIR86 or control ( Figure 21 B); VIR13, VIR93 or control ( Figure 21 C); VIR13, VIR94 or control ( Figure 21 D); VIR13, VIR96 or control ( Figure 21 E) Tumor size in mice over time (days post-treatment). Figure 21 F depicts a graph of body weight (g) over time (days post-treatment) of mice administered VIR13, VIR41, VIR86, VIR93, VIR94, VIR96, or control.

[0174] Figure 22 AH depicts a series of graphs showing the effects of VIR94, VIR100, or control ( Figure 22 A); VIR94, VIR103 or control ( Figure 22 B); VIR94, VIR105 or control ( Figure 22 C); VIR94, VIR106 or control ( Figure 22 D); VIR94, VIR109 or control ( Figure 22 E); VIR94, VIR113 or control ( Figure 22 F); VIR94, VIR114 or control ( Figure 22 G); VIR94, VIR115 or control ( Figure 22H) Tumor volume of mice over time (days after treatment). *=p≤0.05; **=p≤0.01; ***=p≤0.001.

[0175] Figure 23 A is a graph depicting tumor size over time (days post-treatment) in mice after administration of VIR103, VIR111, or VIR113. Figure 23 B shows a schematic diagram of how MDNA11 and MDNA11T are generated from wild-type human interleukin-2 (wt hIL-2). * = p ≤ 0.05; ** = p ≤ 0.01.

[0176] Figure 24 A depicts a graph showing tumor volume in mice over time (days post-treatment) after administration of VIR106 or control. Figure 24 B depicts a graph showing body weight (g) of mice over time (days post-treatment) after administration of VIR106 or control. ***=p≤0.001. Figure 24 C shows images of tumor sites of mice harvested on day 8 after administration of VIR106 or control, showing detectable tumors in control mice but no detectable tumors in VIR106-treated mice. Figure 24 DE depicts tumor volume over time (days after treatment) in mice administered VIR113 or control. Figure 24 D) and body weight (g)( Figure 24 E) diagram. Figure 24 FG depicts tumor volume over time (days after treatment) in mice administered VIR115 or control. Figure 24 F) and body weight (g)( Figure 24 G) chart.

[0177] Figure 25 AF depicts a series of graphs showing the effects of VIR106 or control ( Figure 25 A and B), VIR113 or control ( Figure 25 C and D), VIR115 or control ( Figure 25 E and F), tumor volume and body weight (g) in mice over time (days after treatment). *=p≤0.05; **=p≤0.01; ***=p≤0.001.

[0178] Figure 26 AB depicts Western blot analysis showing the expression of VIR13 in B16-F10 cells infected with mock, iVIR13, VIR13, VIR93, VIR94, VIR100, VIR106, VIR113, VIR115, VIR123, or VIR127 ( Figure 26A) and Hela S3 cells ( Figure 26 In B), expression of human phospho-IRF3, mouse phospho-IRF3, human IRF3, mouse IRF3, and β-actin. Detailed Description of the Invention

[0179] Provided herein are isolated clonal strains that exhibit superior anti-tumor activity and enhanced evasion of the host immune system compared to other vaccinia viruses. Specifically, the clonal strains provided herein are from the American Type Culture Collection. Catalog number VR-156 TM clonal isolates from the parental IHD-J. Also provided herein are preparations produced by propagation of such isolated clonal strains. Also provided herein are recombinant vaccinia viruses derived from isolated clonal strains, which are attenuated by modification to delete or reduce the expression of viral genes or to inactivate viral proteins. Furthermore, provided herein are recombinant viruses that have been further improved to escape host antiviral defenses or to have further enhanced anti-tumor activity. For example, such recombinant viruses contain heterologous nucleic acids encoding proteins to escape inhibition by the complement system, to escape attack by natural killer (NK) cells or T cells, to incorporate immune checkpoint molecules to enhance immunostimulatory activity, or to provide anti-angiogenic activity. The recombinant viruses provided herein also include those equipped with viral induction systems to inhibit viral replication as a safety strategy (e.g., by mediating apoptosis in certain unwanted infected cells (e.g., 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, 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. A recombinant oncolytic virus is also specifically 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 or comprise immunomodulatory proteins, complement inhibitors, T cell or NK cell escape products, anti-angiogenic proteins, interferon regulatory factors, apoptosis-inducing proteins, or any combination thereof.

[0180] Oncolytic viruses (OVs) are viruses that replicate selectively or more efficiently in cancer cells than in non-cancerous cells. In some cases, the ability to selectively infect, replicate, and destroy cancer cells in cancer cells, and at many times without harming healthy cells at the same time, is due to the ability to exploit the biochemical differences between healthy cells and transformed cells during infection. Cancer cells are characterized by destroyed apoptosis pathways, the acquisition of new abilities to escape the immune system, and the ability to proliferate indefinitely, all of which are conducive to viral replication. Since one of the main challenges of cancer therapy is to minimize toxic effects while killing malignant cells, OVs have become an attractive option because they rarely cause off-target toxicity.

[0181] Oncolytic viruses can be divided into three major categories: (1) viruses that have a natural tendency to replicate preferentially in cancer cells while being non-pathogenic to 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 in vitro for safe use in humans. The last category includes oncolytic viruses derived from vaccinia virus, which are highly favored due to 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 efficient in replication and spread than adenovirus vectors.

[0182] Vaccinia virus (VV), a typical member of the Orthopoxvirus genus, replicates in the cytoplasm of the host cell. VV is a large, complex enveloped virus with a linear double-stranded DNA genome of approximately 190,000 base pairs in length, consisting of a single continuous polynucleotide chain encoding approximately 250 genes that can potentially express more than 200 proteins. See, for example, McCraith et al., (1982) PNAS, 97(9): 4879-4884. Typically, non-segmented, non-infectious genomes are arranged in such a way that genes located in the center are essential for viral replication (and therefore conserved), while genes near the two ends affect more peripheral functions such as host range and virulence. Vaccinia virus differentially expresses genes by utilizing grouped open reading frames (ORFs), which generally do not overlap. See, for example, Traktman, P., Chapter 27, Poxvirus DNA Replication, pp.775-798, in DNA Replication in Eukaryotic Cells, Cold Spring Harbor Laboratory Press (1996). The rapid replication ability of VV leads to the effective lysis of infected cells and spreads to other tumor cells during several consecutive rounds of replication, resulting in strong local destruction of the tumor. The VV genome encodes about 250 genes and can accept up to 20kb of external DNA, making it an ideal gene delivery vehicle. The recombinant VV vector being developed is intended to deliver eukaryotic genes (such as tumor-associated antigens) into tumors, thereby promoting the induction of the host immune system for killing cancer cells. However, a limiting factor for using VV as a cancer treatment delivery vector is the strong neutralizing antibody response induced by injecting VV into the blood, which limits the continued existence and spread of the virus and hinders the re-administration of the vector. In some cases, neutralizing antibodies recognize and bind to viral glycoproteins with high affinity and prevent the virus from interacting with host cell receptors (resulting in viral neutralization).

[0183] Vaccinia virus replicates in the cytoplasm of infected cells, where the assembly of progeny viruses begins in a special area called a virus factory. During the replication process, three morphologically and antigenically different viral forms are produced: intracellular mature virions (IMV), intracellular enveloped virions (IEV), and extracellular virions. A subset of IMV, the first infectious progeny produced, is transported to the trans-Golgi network (TGN), where they are enveloped by another two membranes to produce IEV. IEV is transported to the cell periphery through the cytoplasm, where the outermost membrane fuses with the plasma membrane to release the virus in the form of a double membrane, referred to as EV. The EV retained on the cell surface is referred to as cell-associated enveloped virions (CEV), while the EV that is no longer attached to the cell surface is referred to as extracellular enveloped virions (EEV). IMV is the most abundant infectious form and is believed to be responsible for the spread between hosts; CEV is believed to play a role in the spread of cells to cells; and EEV is believed to be important for the long-distance spread of viruses in host organisms. Specifically, EEV is believed to be related to the long-distance spread of viruses in vivo. See, for example, Blasco et al., (1993) Journal of Virology, 67(6):3319-3325. The outer protein of EEV can induce protective immunity against the virus (Blaso and Moss (1992) J. Virol., 66:4170-4179). However, the amount of EEV produced by vaccinia virus strains is highly variable.

[0184] Attenuated vaccinia virus strains have been developed for therapeutic and diagnostic applications. For example, the attenuated virus includes a recombinant virus modified in one or more viral genes, and the modification results in loss or reduction of viral gene expression, or inactivation of viral proteins. Nevertheless, although vaccinia virus is a well-studied attenuated virus with anti-tumor properties, many vaccinia virus strains (including recombinant strains) show differences in virulence and safety, which makes many strains unsuitable for clinical applications. Therefore, there is a need for improved vaccinia virus strains with enhanced anti-tumor properties and low cytotoxicity, because these characteristics are very necessary for effective oncolytic therapy. The oncolytic viruses and methods described herein meet this demand.

[0185] Various approaches have been investigated to improve OV antitumor activity, primarily focusing on viral replication and spread, as viral replication is generally associated with cancer cell killing efficacy. However, other aspects of viral infection (such as enhancement of host antitumor immune responses, induction of apoptosis, and control of tumor angiogenesis) are also important aspects of cancer virotherapy (Davola, ME and KLMossman (2019) Oncoimmunology 8(6):e1581528).

[0186] Provided herein are isolated clonal viruses derived from a strain of the human idiopathic hive known as IHD-J ( Catalog Number: VR-156 TM IHD-J is a vaccinia virus strain that is closely related to the Western Reserve (WR) strain but produces 10 to 40 times more EEV than the WR strain and spreads more efficiently to distant cells (Blasco and Moss, 1992). However, strains that exhibit more distant spread may not exhibit sufficient antitumor activity for oncolytic virotherapy.

[0187] The embodiments provided herein are based on the identification of a specific clonal isolate of IHD-J (designated VIP02) that not only exhibits a high percentage of EEV but also exhibits the highest antitumor activity among other clonal isolates from the same strain. Furthermore, results demonstrate that a single low-dose intravenous delivery of this clonal isolate significantly inhibits tumor growth in a syngeneic mouse tumor model and exhibits potent tumor cell killing against a variety of tumor cells in both 2-D and 3-D cultures in vitro. Also provided herein are vaccinia virus strains that have sequence characteristics of the VIP02 clonal isolate.

[0188] Embodiments provided herein also relate to recombinant viruses in which heterologous nucleic acid can be introduced into an isolated clonal virus having enhanced anti-tumor properties to further enhance the anti-tumor properties of the isolated clonal virus while minimizing cytotoxicity to healthy cells.

[0189] In some embodiments, the selected clonal strains and recombinantly derived strains thereof are oncolytic virus candidates for tumor diagnosis and therapy. In some embodiments, isolated clonal strains of vaccinia and recombinantly derived strains thereof can be used as therapeutic viruses for the treatment of proliferative disorders, including cancer, hyperplasia, metastasis and tumors, as well as for other therapeutic and / or diagnostic methods described herein. In some other embodiments, the clonal strains can be used in vaccination methods. In other embodiments, isolated clonal strains and recombinantly derived strains thereof can be used as parental vaccinia viruses to produce recombinant oncolytic viruses.

[0190] All publications, including patent documents, scientific articles, and databases, cited in this application are incorporated herein by reference in their entirety for the same purpose as if each individual publication were individually incorporated. If a definition set forth herein conflicts or is otherwise inconsistent with a definition set forth in patents, applications, published applications, and other publications incorporated herein by reference, the definition set forth in this document controls and does not control.

[0191] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. I. Isolated Clonal Virus Strains and Their Attenuated Strains

[0192] This article provides vaccinia virus strain IHD-J ( Catalog Number: VR-156 TM ) or a strain that exhibits characteristics of a clonal vaccinia virus strain isolated therefrom. The parental IHD-J strain is heterologous in sequence. It has been discovered herein that certain vaccinia virus clones with enhanced anti-tumor properties can be isolated from an IHD-J parental vaccinia virus preparation or mixture.

[0193] In some embodiments, the clonal strains provided herein are present in a viral preparation propagated from IHD-J. For example, a clonal strain or preparation thereof can be obtained by isolating an IHD-J-derived clonal isolate from a cell culture in which a parental IHD-J or variant thereof has been propagated. The clonal isolates provided herein are obtained by passage of IHD-J virus in confluent CV-1 cells (derived from African green monkey kidney fibroblast cultures) grown in 6-well plates and infected with a dilution series of vaccinia virus strains.

[0194] In some embodiments, the clonal 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 clonal strain can be used as a parental sequence for generating a recombinant virus that is modified with a heterologous nucleic acid encoding a non-viral heterologous protein.

[0195] In some embodiments, the IHD-J clonal line provided herein is named VIP02 and has the nucleotide sequence set forth in SEQ ID NO:1.

[0196] In some embodiments, the present invention provides 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, 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.

[0197] In some embodiments, the present invention also provides 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 or comprise immunomodulatory proteins, complement inhibitors, T cell or NK cell escape proteins, anti-angiogenic proteins, interferon regulatory factors, cell apoptosis inducing proteins, or any combination thereof.

[0198] In some embodiments, vaccinia virus clonal lines are provided that have a nucleic acid genome that has at least 95% sequence identity to SEQ ID NO: 1. In some embodiments, vaccinia virus clonal lines are provided that have a nucleic acid genome that has at least 96% sequence identity to SEQ ID NO: 1. In some embodiments, vaccinia virus clonal lines are provided that have a nucleic acid genome that has at least 97% sequence identity to SEQ ID NO: 1. In some embodiments, vaccinia virus clonal lines are provided that have a nucleic acid genome that has at least 98% sequence identity to SEQ ID NO: 1. In some embodiments, vaccinia virus clonal lines are provided that have a nucleic acid genome that has at least 99% sequence identity to SEQ ID NO: 1. In some embodiments, vaccinia virus clonal lines are provided that have a nucleic acid genome that has at least 99.1% sequence identity to SEQ ID NO: 1. In some embodiments, vaccinia virus clonal lines are provided that have a nucleic acid genome that has at least 99.2% sequence identity to SEQ ID NO: 1. In some embodiments, vaccinia virus clonal lines are provided that have a nucleic acid genome that has at least 99.3% sequence identity to SEQ ID NO: 1. In some embodiments, vaccinia virus clonal lines are provided that have a nucleic acid genome that has at least 99.4% sequence identity to SEQ ID NO: 1. In some embodiments, vaccinia virus clonal lines are provided that have a nucleic acid genome that has at least 99.5% sequence identity to SEQ ID NO: 1. In some embodiments, vaccinia virus clonal lines are provided that have a nucleic acid genome that has at least 99.6% sequence identity to SEQ ID NO: 1. In some embodiments, vaccinia virus clonal lines are provided that have a nucleic acid genome that has at least 99.7% sequence identity to SEQ ID NO: 1. In some embodiments, vaccinia virus clonal lines are provided that have a nucleic acid genome that has at least 99.8% sequence identity to SEQ ID NO: 1. In some embodiments, vaccinia virus clonal lines are provided that have a nucleic acid genome that has at least 99.9% sequence identity to SEQ ID NO: 1.

[0199] In some of any such embodiments, provided vaccinia virus clonal lines do not have a nucleic acid genome comprising the amino acid sequence set forth in SEQ ID NO: 2 (IHD-W1). In some embodiments, provided clonal lines have a nucleotide sequence that has 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, provided clonal lines have 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 clonal 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 clonal 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 clonal virus strains provided herein may include nucleotide deletions or mutations in any one or more nucleotides in any ORF compared to SEQ ID NO: 2, or may include additions or insertions of viral DNA compared to SEQ ID NO: 2.

[0200] In some embodiments, vaccinia virus clonal strains are provided having a nucleic acid genome having at least 95% sequence identity to SEQ ID NO: 1 and exhibiting sequence characteristics of SEQ ID NO: 1. For example, as described in Table E1 herein, the exemplary VIP02 clonal isolate is characterized by one or more nucleotide deletions or mutations compared to SEQ ID NO: 2, including one or more mutations in the ORF of SEQ ID NO: 2. With respect to ORFs, ORFs are numbered consecutively starting from 001. In other embodiments, vaccinia virus open reading frames can also be named by using a capital letter to represent the HindIII restriction endonuclease fragment, a number to represent the position within the HindIII fragment, and a letter (L or R) to represent the direction of transcription, e.g., K5L. The corresponding protein is designated by a capital letter and a number, e.g., K5. In some embodiments, the nucleotide changes are in a non-ORF region of the sequence.

[0201] In some embodiments, provided vaccinia virus clonal lines comprise or are characterized by a variant 017 open reading frame (ORF) encoding an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 57 and 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, provided clonal lines include a variant 017 ORF having an A66T mutation compared to the 017 ORF shown in SEQ ID NO: 2. In some embodiments, the variant 017 ORF encodes an amino acid sequence comprising any of the above-described amino acid changes at position 66 and having at least 96% sequence identity to SEQ ID NO: 57. In some embodiments, the variant 017 ORF encodes an amino acid sequence comprising any of the above-described amino acid changes at position 66 and having at least 97% sequence identity to SEQ ID NO: 57. In some embodiments, the variant 017 ORF encodes an amino acid sequence comprising any of the above-described amino acid changes at position 66 and having at least 98% sequence identity to SEQ ID NO: 57. In some embodiments, the variant 017 ORF encodes an amino acid sequence comprising any of the above-described amino acid changes at position 66 and 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 vaccinia virus clonal strains have a nucleic acid genome having at least 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.99% sequence identity to SEQ ID NO: 1.

[0202] In some embodiments, provided clonal strains comprise or are characterized by a variant 038(K5L) ORF having a nucleotide insertion resulting in a frameshift mutation, wherein the 038(K5L) gene product is altered. In some embodiments, the nucleotide insertion is a guanine (G) insertion at a position corresponding to 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 vaccinia virus clonal strains have a nucleic acid genome having at least 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.99% sequence identity to SEQ ID NO: 1. In some embodiments, the variant 038(K5L) ORF is characterized by an alteration compared to the nucleic acid sequence set forth in SEQ ID NO: 73 or the amino acid sequence set forth in SEQ ID NO: 74.

[0203] In some embodiments, provided clonal lines include or are characterized by variant 059(E2L), which encodes 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, provided clonal lines include a variant 059(E2L) ORF having an L419F mutation compared to the 059(E2L) ORF shown in SEQ ID NO:2. In some embodiments, the variant 059(E2L) ORF encodes an amino acid sequence comprising any of the above-described amino acid changes at position 419 and having at least 96% sequence identity to SEQ ID NO:60. In some embodiments, the variant 059(E2L) ORF encodes an amino acid sequence comprising any of the above-described amino acid changes at position 419 and having at least 97% sequence identity to SEQ ID NO: 60. In some embodiments, the variant 059(E2L) ORF encodes an amino acid sequence comprising any of the above-described amino acid changes at position 419 and having at least 98% sequence identity to SEQ ID NO: 60. In some embodiments, the variant 059(E2L) ORF encodes an amino acid sequence comprising any of the above-described amino acid changes at position 419 and having at least 99% sequence identity to SEQ ID NO: 60. In some embodiments, the variant 059(E2L) ORF has the sequence shown in SEQ ID NO: 60. In some embodiments, such vaccinia virus clonal strains have a nucleic acid genome having at least 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.99% sequence identity to SEQ ID NO: 1.

[0204] In some embodiments, provided clonal lines include or are characterized by a variant 104(H4L) ORF that encodes 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, provided clonal lines include a variant 104(H4L) ORF having an N591D mutation compared to the 104(H4L) ORF shown in SEQ ID NO: 2. In some embodiments, the variant 104(H4L) ORF encodes an amino acid sequence comprising any of the above-described amino acid changes at position 591 and having at least 96% sequence identity to SEQ ID NO: 61. In some embodiments, the variant 104(H4L) ORF encodes an amino acid sequence comprising any of the above-described amino acid changes at position 591 and having at least 97% sequence identity to SEQ ID NO: 61. In some embodiments, the variant 104(H4L) ORF encodes an amino acid sequence comprising any of the above-described amino acid changes at position 591 and having at least 98% sequence identity to SEQ ID NO: 61. In some embodiments, the variant 104(H4L) ORF encodes an amino acid sequence comprising any of the above-described amino acid changes at position 591 and having at least 99% sequence identity to SEQ ID NO: 61. In some embodiments, the variant 104(H4L) ORF has the sequence shown in SEQ ID NO: 61. In some embodiments, such vaccinia virus clonal strains have a nucleic acid genome having at least 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.99% sequence identity to SEQ ID NO: 1.

[0205] In some embodiments, provided clonal strains comprise or are characterized by a variant 182(A56R) ORF having a nucleotide deletion resulting in a frameshift mutation, wherein the 182(A56R) gene product is altered. In some embodiments, the nucleotide deletion is a deletion of two consecutive nucleotides corresponding to the nucleotide following 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 vaccinia virus clonal strains have a nucleic acid genome having at least 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.99% sequence identity to SEQ ID NO:1. In some embodiments, the variant 182(A56R) ORF is characterized by an alteration compared to the nucleic acid sequence set forth in SEQ ID NO:75 or the amino acid sequence set forth in SEQ ID NO:76.

[0206] In some embodiments, provided clonal lines are characterized by a nucleic acid genome comprising at least one of any of the aforementioned mutations in 017 ORF, 038 (K5L) ORF, 059 (E2L) ORF, 104 (H4L) ORF, and 182 (A56R) ORF. In some embodiments, provided clonal lines are characterized by a nucleic acid genome comprising at least two of any of the aforementioned mutations in 017 ORF, 038 (K5L) ORF, 059 (E2L) ORF, 104 (H4L) ORF, and 182 (A56R) ORF. In some embodiments, provided clonal lines are characterized by a nucleic acid genome comprising at least three of any of the aforementioned mutations in 017 ORF, 038 (K5L) ORF, 059 (E2L) ORF, 104 (H4L) ORF, and 182 (A56R) ORF. In some embodiments, provided clonal strains are characterized by a nucleic acid genome comprising at least four of any of the aforementioned 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 vaccinia virus clonal strains have a nucleic acid genome that has at least 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.99% sequence identity to SEQ ID NO: 1.

[0207] In some embodiments, provided clonal lines are characterized by a nucleic acid genome comprising each of the above mutations in 017 ORF, 038 (K5L) ORF, 059 (E2L) ORF, 104 (H4L) ORF, and 182 (A56R) ORF. In some embodiments, provided clonal lines are characterized by a nucleic acid genome comprising 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 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 182 (A56R) encoding the amino acid sequence set forth in SEQ ID NO: 63. In some embodiments, such vaccinia virus clonal strains have a nucleic acid genome that has at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.99% sequence identity to SEQ ID NO:1.

[0208] In some embodiments, a vaccinia virus clonal strain is provided having a nucleic acid genome having at least 95% sequence identity to SEQ ID NO: 1 and characterized by one or more of the following: (i) a guanine (G) at a position corresponding to position 7770 of SEQ ID NO: 1; (ii) a thymine (T) at a position corresponding to position 15261 of SEQ ID NO: 1; (iii) a G at a position corresponding to position 32136 of SEQ ID NO: 1; (iv) a G at a position corresponding to position 49455 of SEQ ID NO: 1; (v) a cytosine (C) at a position corresponding to position 92969 of SEQ ID NO: 1; (vi) the nucleic acid sequence CACTTATAT at positions corresponding to positions 106870 to 106880 of SEQ ID NO: 1 (shown in SEQ ID NO: 77); (vii) the nucleic acid sequence GTTTTCATTA at positions corresponding to positions 111267 to 111276 of SEQ ID NO: 1 NO:78); (viii) an adenine (A) at a position corresponding to position 162715 of SEQ ID NO:1; (ix) the nucleic acid sequence TACAGACACC (shown in SEQ ID NO:79) at positions corresponding to positions 165844 to 185853 of SEQ ID NO:1; and (x) a C at a position corresponding to position 187805 of SEQ ID NO:1.

[0209] In some embodiments, the vaccinia virus clonal strains provided herein include those having a nucleotide sequence characterized by a single point mutation, insertion, and / or deletion selected from any one of (i)-(x) above.

[0210] In some embodiments, the vaccinia virus clonal strains provided herein include those having a nucleotide sequence characterized by a single point mutation, insertion, and / or deletion selected from any two of (i)-(x) above.

[0211] In some embodiments, the vaccinia virus clonal strains provided herein include those having a nucleotide sequence characterized by a single point mutation, insertion, and / or deletion selected from any three of (i)-(x) above.

[0212] In some embodiments, the vaccinia virus clonal strains provided herein include those having a nucleotide sequence characterized by a single point mutation, insertion, and / or deletion selected from any four of (i)-(x) above.

[0213] In some embodiments, the vaccinia virus clonal strains provided herein include those having a nucleotide sequence characterized by a single point mutation, insertion, and / or deletion selected from any five of (i)-(x) above.

[0214] In some embodiments, the vaccinia virus clonal strains provided herein include those having a nucleotide sequence characterized by a single point mutation, insertion, and / or deletion selected from any six of (i)-(x) above.

[0215] In some embodiments, the vaccinia virus clonal strains provided herein include those having a nucleotide sequence characterized by a single point mutation, insertion, and / or deletion selected from any seven of (i)-(x) above.

[0216] In some embodiments, the vaccinia virus clonal strains provided herein include those having a nucleotide sequence characterized by a single point mutation, insertion, and / or deletion selected from any eight of (i)-(x) above.

[0217] In some embodiments, the vaccinia virus clonal strains provided herein include those having a nucleotide sequence characterized by a single point mutation, insertion, and / or deletion selected from any nine of (i)-(x) above.

[0218] In some embodiments, the vaccinia virus clonal strains provided herein include those having a nucleotide sequence characterized by a single point mutation, insertion, and / or deletion selected from each of (i)-(x) above. A. Exemplary Features

[0219] In some embodiments, the clonal strains derived from IHD-J exhibit better anti-tumor properties and lower pathogenicity / toxicity in in vitro and / or in vivo assays compared to the starting viral preparation or mixture or other reference strains or isolates (including recombinant strains). In some embodiments, the clonal strains derived from IHD-J exhibit better anti-tumor properties compared to the starting viral preparation or mixture or other reference strains or isolates (including recombinant strains). In some embodiments, the clonal strains derived from IHD-J exhibit lower toxicity compared to the starting viral preparation or mixture or other reference strains or isolates (including recombinant strains). In some embodiments, the clonal strains derived from IHD-J exhibit similar anti-tumor properties and / or similar toxicity compared to the starting viral preparation or mixture or other reference strains or isolates (including recombinant strains).

[0220] Provided herein are IHD-J clonal isolates that exhibit improved properties compared to a starting viral preparation or mixture or other reference strains or isolates, including recombinant strains lacking inserted heterologous DNA. In some embodiments, the IHD-J clonal isolate exhibits better anti-tumor activity and lower toxicity compared to a starting viral preparation or mixture or other reference strains or isolates, including recombinant strains lacking inserted heterologous DNA. In some embodiments, the IHD-J clonal isolate exhibits improved or better anti-tumor activity compared to a starting viral preparation or mixture or other reference strains or isolates, including recombinant strains lacking inserted heterologous DNA. In some embodiments, the IHD-J clonal isolate exhibits lower toxicity compared to a starting viral preparation or mixture or other reference strains or isolates, including recombinant strains lacking inserted heterologous DNA. In some embodiments, the IHD-J clonal isolate exhibits similar toxicity and / or anti-tumor activity compared to a starting viral preparation or mixture or other reference strains or isolates, including recombinant strains lacking inserted heterologous DNA.

[0221] In some embodiments, the cloned isolated strain that shows improved or better anti-tumor activity compared to the starting viral preparation or mixture or other reference strain or isolate (including recombinant strain) shows an anti-tumor activity of 120% to 1000% of the reference viral preparation (starting viral preparation or mixture or other reference strain or isolate, including recombinant strain) in an assay or method for assessing parameters indicating anti-tumor activity, for example, at least 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 400%, 500%, 1000% or more anti-tumor activity. Anti-tumor activity can be determined using any in vitro or in vivo test for parameters indicating anti-tumor activity as described herein.

[0222] In some embodiments, the clonal isolates provided herein exhibit increased production of extracellular enveloped virus (EEV) compared to the starting viral preparation or mixture or other reference strains or isolates (including recombinant strains). Vaccinia viruses replicate in cells and produce intracellular viruses (IMV, intracellular mature viruses; IEV, intracellular enveloped viruses) and extracellular viruses (EEV, extracellular enveloped viruses; CEV, cell-associated extracellular viruses) (Smith et al. (1998) Adv Exp Med Biol. 440: 395-414). After replication of wild-type vaccinia virus strains, IMV accounts for approximately 99% of viral production. The IMV virus form is relatively stable in the external environment and is primarily responsible for spread between individuals; however, due to low release efficiency in cells and sensitivity to complement and / or antibody neutralization, the IMV virus does not spread effectively within the infected host. In contrast, EEV forms are released into the extracellular environment and typically account for only about 1% of viral production (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, EEV forms have developed multiple mechanisms to inhibit their neutralization in the blood. EEV is relatively resistant to complement due to the incorporation of host cell complement inhibitors into its outer membrane envelope and the secretion of vaccinia virus complement control protein (VCP) into the local extracellular environment (Vanderplasschen et al. (1998) Proc Natl Acad Sci USA. 95(13):7544-9). In addition, EEV is relatively resistant to the effects of neutralizing antibodies compared to IMV (Smith et al. (1997) Immunol Rev. 159: 137-54; Vanderplasschen et al. (1997) J Gen Virol. 78 (Pt 8): 2041-8). Compared to IMV (which is released only during or after cell death), EEV is released at an earlier time point after infection (e.g., 4-6 hours), and therefore, the EEV form spreads more rapidly (Blasco et al. (1993) J Virol. 67 (6): 3319-25).

[0223] Because EEV is relatively resistant to complement and antibody-mediated neutralization, this viral form has enhanced stability in the blood after intravenous administration and remains active for a longer period of time when grown in cell types of the same species (Smith et al. (1998) Adv Exp Med Biol. 440:395-414; Vanderplaschen et al., (1998) Proc Natl Acad Sci USA (13):7544-9). This is particularly important for repeated administration after neutralizing antibody levels rise, as is often required for cancer therapy. Therefore, the addition of EEV forms of vaccinia virus and other vaccinia viruses could lead to enhanced systemic efficacy.

[0224] In some embodiments, the clonal isolates provided herein exhibit increased production of extracellular enveloped viruses (EEVs) compared to other clonal isolates derived from IDH-J or Copenhagen strains. In some embodiments, the clonal isolates provided herein exhibit increased production of extracellular enveloped viruses (EEVs), such as at or between 120% and 1000%, for example, at least 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 400%, 500%, 1000% or more extracellular enveloped viruses (EEVs) compared to a starting viral preparation or mixture or other reference strains or isolates (including recombinant strains).

[0225] In some embodiments, after infection of the cells, greater than or about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20% of the infectious particles are EEV. In some embodiments, after infection of the cells, greater than 5% of the infectious particles are EEV. In some embodiments, after infection of the cells, greater than 10% of the infectious particles are EEV. In some embodiments, after infection of the cells, greater than 15% of the infectious particles are EEV. In some embodiments, after infection of the cells, greater than 20% of the infectious particles are EEV.

[0226] In other embodiments, the clone isolates provided herein show reduced tumor and / or metastasis growth or increased tumor and / or metastasis atrophy in in vitro or in vivo assays or models. Tumors can be harvested from a subject, weighed, and compared with the tumor weight of a subject harvested from a tumor starting from infection with a viral preparation or mixture or other reference strains or isolates (including recombinant strains). Tumor weight can also be compared with the tumor weight of a subject harvested from a control treatment at the same time after infection. Weight can be expressed as the ratio of tumor volume / weight and / or tumor volume / weight (tumor weight of a subject treated with a control animal / tumor weight of a clone isolate treated with a control). It should be understood that, for example, a tumor weight ratio of 1.2 or 5 means that compared with a reference or control, the virus causes reduced tumor / metastasis body weight / growth or increased tumor / metastasis atrophy, and 120% or 500% antitumor activity.

[0227] In some embodiments, the clone isolates provided herein show reduced tumor and / or metastasis growth or increased tumor and / or metastasis 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 higher. In some embodiments, the increased tumor / metastasis shrinkage is at least 120% to 500%, for example, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500% or higher.

[0228] In some embodiments, the clonal isolates provided herein exhibit similar anti-tumor activity in an assay or method for assessing parameters indicative of toxicity compared to the starting viral preparation or mixture or other reference strains or isolates (including recombinant strains), for example, between 70% and 120% of the anti-tumor activity of the parental viral preparation, mixture or other reference viral strain, for example, at least or about or 70%, 80%, 90%, 95%, 100%, 110%, 115% or 120% of the anti-tumor activity of the parental viral preparation, mixture or other reference viral strain.

[0229] In some embodiments, provided herein are cloned isolates that show reduction in vitro or in vivo assays or models and / or metastasis volume, size or weight. In some embodiments, compared with starting viral preparations or mixtures or other reference strains or isolates (including recombinant strains), provided herein are cloned isolates that show reduction in tumor and / or metastasis volume, such as 0% to 99% toxicity or tumor and / or metastasis volume, size or weight, such as less than 99%, 98%, 97%, 96%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% or lower toxicity or more tumor and / or metastasis volume, size or weight.

[0230] Parameters indicative of toxicity or potency include, but are not limited to, reduced percentage of cell survival in 2-D (two dimensions) and 3-D (three dimensions) cell culture, decreased subject weight, fever, the onset of rash or other allergic symptoms, fatigue or abdominal pain, tissue distribution of the virus, reduced or decreased subject survival, induction of an immune response in the subject, the amount of tumor antigen released, and a reduced rate of acne formation. Toxicity or potency can be determined using any in vitro or in vivo test known to those skilled in the art.

[0231] In some embodiments, the clonal isolates provided herein exhibit lower toxicity compared to a starting viral preparation or mixture or other reference strain or isolate (including recombinant strains), including recombinant strains, for example, 0% to 99% of the toxicity of the starting viral preparation or mixture or other reference strain or isolate (including recombinant strains), such as recombinant strains that are less than 99%, 98%, 97%, 96%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% or less of the toxicity of the starting viral preparation or mixture or other reference strain or isolate (including recombinant strains), including recombinant strains in assays or methods for assessing parameters indicative of toxicity. In some embodiments, the IHD-J clonal isolates provided herein exhibit toxicity of between 0% and 99%, such as less than 99%, 98%, 97%, 96%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% or less, compared to other clonal isolates derived from the IHD-J or Copenhagen strains. In some embodiments, the method of assessing a parameter indicative of toxicity comprises quantifying the percentage of cell survival in cell culture. In some embodiments, the method of assessing a parameter indicative of toxicity comprises quantifying the percentage of cell survival in 2-D (two-dimensional) and 3-D (three-dimensional) cell culture.

[0232] In some embodiments, the clonal isolates provided herein exhibit similar toxicity and / or cytotoxicity compared to the starting viral preparation or mixture or other reference strains or isolates (including recombinant strains), such as recombinant strains having an anti-tumor activity of between 70% and 120%, for example, at least or about or 70%, 80%, 90%, 95%, 100%, 110%, 115% or 120% of the starting viral preparation or mixture or other reference strains or isolates (including recombinant strains), including recombinant strains in assays or methods for assessing parameters indicative of toxicity, for example.

[0233] In certain embodiments, the clonal isolates provided herein exhibit improved anti-tumor properties and lower toxicity (i.e., lower potency) compared to the starting viral preparation or mixture or other reference strains or isolates (including recombinant strains). For example, when administered to a subject in an amount effective to induce anti-tumor activity, the clonal strains exhibit lower toxicity (i.e., lower potency). For treatment of human subjects or other subjects of similar size, exemplary therapeutic amounts of the clonal strains range from about or between 1×10 6 to 1×10 14 pfu, for example, about or between 1×10 7 to 1×10 10 pfu, such as approximately or between 1×10 9 to 1×10 10 pfu, for example at least or about 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 For treatment of mice or other subjects of similar size, exemplary therapeutic amounts of clonal strains range from about or between 1×10 3 to 1×10 9 pfu, for example, about or between 1×10 5 to 1×10 7 pfu, for example, at least or about 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 6pfu. These effective amounts can be determined empirically by those skilled in the art and depend on a variety of 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. Dosage regimens may vary. In some embodiments, the clonal isolates provided herein show 100% survival of the subject throughout the treatment regimen and do not cause weight loss or reduction in the subject during the treatment. In one embodiment, the clonal strains provided herein show increased survival when administered to the subject when compared to subjects administered the same or similar therapeutic doses of other clonal isolates. In some embodiments, the clonal isolates provided herein show 100% tumor growth inhibition throughout the treatment regimen.

[0234] The isolated clonal viruses provided herein can be obtained by plaque isolation of the IHD-J strain, which is propagated by repeated passage in a cell line. In some embodiments, the clonal isolates provided herein can be obtained by passage of the virus in chicken embryo yolk sac culture, 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 passage of the virus in confluent CV-1 (African green monkey kidney fibroblast culture), which are grown in 6-well plates and infected with a series of dilutions of the vaccinia virus strain. The clonal isolates provided herein are homogeneous in sequence. The exemplary clonal viruses provided herein are clonal isolates that exhibit enhanced anti-tumor properties and reduced toxicity. III. Attenuated Vaccinia Virus Strains

[0235] Also provided herein are recombinant vaccinia viruses that exhibit one or more modifications to attenuate viral toxicity compared to a wild-type or parental strain of the virus (e.g., compared to any of the isolated clonal virus strains described in Section I herein). In some embodiments, provided herein is a recombinant vaccinia virus that is attenuated (e.g., has reduced toxicity) compared to vaccinia virus strain VIP02. In some embodiments, provided herein is a recombinant vaccinia virus that is attenuated (e.g., has reduced toxicity) compared to the vaccinia virus strain set forth in SEQ ID NO: 1. In some embodiments, the attenuated virus is a virus that has low toxicity to normal cells, e.g., has low or reduced viral replication, cytolytic activity, or cytotoxicity to normal cells (e.g., non-tumor cells).

[0236] 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, 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.

[0237] In some embodiments, the attenuated virus is a recombinant oncolytic virus comprising: an inactivating mutation in 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 or comprise immunomodulatory proteins, complement inhibitors, T cell or NK cell escape proteins, anti-angiogenic proteins, interferon regulatory factors, or apoptosis-inducing proteins, or any combination thereof.

[0238] In some embodiments, any of the vaccinia viruses provided herein can be modified to express vaccinia growth factor (VGF) (McCart et al. (2001) Cancer Research 61:8751), thymidine kinase (TK) gene (WO 2005 / 047458), hemagglutinin (HA) gene (WO 2005 / 047458; and Zhang et al. (2007) Cancer Research 67:10038), F3 gene (also known as F14.5L; WO 2005 / 047458; Zhang et al. (2007) Cancer Research 67:10038), ribonucleotide reductase (Gammon et al. (2010) PLoS Pathogens 6:e1000984), serine protease inhibitors (e.g., SPI-1, SPI-2) (Guo et al. (2005) Cancer Research 65:9991; 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. (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. (2019) Nature 566:259-263), B18R (Symons et al. (1995) Cell 81:551; Kirn et al. (2007) PLoS Medicine 4:e353), 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) have functional defects and are attenuated. In some embodiments, some 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-γ receptor-like) (Verardi et al. 2001), and F14.5L (WO 2005 / 047458; Zhang et al. (2007) Cancer Research 67:10038), are known to result in reduced virulence when deleted or disrupted.

[0239] In some embodiments, a recombinant vaccinia virus strain is provided herein, wherein any of the above genes in its genome have a mutation that inactivates the gene, thereby attenuating the virus. In some embodiments, the viral gene is selected from 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 all or part of a viral gene. In some embodiments, the inactivating mutation is a deletion of the entire ORF of a viral gene. In some embodiments, the inactivating mutation is a deletion of a portion of the ORF of the viral gene, which renders the encoded gene product nonfunctional. In some embodiments, the deleted ORF portion 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.

[0240] In some embodiments, a gene region or a gene product that is encoded can be functionally defective by any of a variety of methods known to those skilled in the art. In some embodiments, a gene region or a gene product may be functionally defective due to one or more mutations (e.g., replacements), truncation, or deletions in the gene region. In some embodiments, a gene region or a gene product may be functionally defective due to a mutation, truncation, or deletion in a promoter region that controls expression of the gene region. In some embodiments, a gene region or a gene product may be functionally defective due to a mutation, truncation, or deletion in a polyadenylation sequence, thereby reducing or eliminating translation of a polypeptide encoded by the gene region.

[0241] In some embodiments, the attenuated recombinant vaccinia viruses disclosed herein that are defective in a given vaccinia virus gene exhibit reduced production and / or activity of a gene product (e.g., an mRNA gene product; a polypeptide gene product) of the gene. 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 a wild-type vaccinia virus or a control vaccinia virus that does not contain the gene alteration. 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 a nucleic acid genome as 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.

[0242] In some embodiments, the attenuated recombinant vaccinia virus disclosed herein that has defects in viral genes may have a deletion in a region consisting of a specified gene region, or a deletion in an adjacent gene region comprising a specified gene region. For example, mutations and / or truncations and / or deletions in a promoter region result in reduced transcription of the gene region, which may result in defects. The gene region is made defective by incorporating transcription termination elements so that the translation of the polypeptide encoded by the gene region is reduced or eliminated. Gene regions may also be made defective using gene editing enzymes or gene editing complexes to reduce or eliminate transcription of the gene region. Gene regions may also be made defective by utilizing competitive reverse promoter / polymerase occupancy to reduce or eliminate transcription of the gene region. Gene regions may also be made defective by inserting nucleic acids into the gene region, thereby knocking out the gene region. In some cases, heterologous nucleic acids may be inserted into viral genes, as described in the exemplary recombinant vaccinia virus strains in III herein.

[0243] In some embodiments, the OVV provided by the present disclosure is thymidine kinase (TK) defective. In some cases, the OVV of the present disclosure comprises a deletion of all or part of the vaccinia virus TK coding region, so that the recombinant oncolytic vaccinia virus with replication ability is TK defective. For example, in some cases, the OVV of the present disclosure comprises a deletion in the J2R gene (i.e., a gene encoding viral thymidine kinase). See, for example, Mejia-Perez et al. (2018) Mol. Ther. Oncolytics 8: 27. In some cases, the OVV of the present disclosure comprises an insertion in the J2R region, resulting in reduced vaccinia virus TK expression or activity.

[0244] In some embodiments, any of the clonal vaccinia virus strains described in Section 1 herein, such as VIP02 or the vaccinia virus strain set forth in SEQ ID NO: 1, can be further modified in its genome to attenuate. In some embodiments, the vaccinia virus strain is modified in one or more of the TK (J2R), hemagglutinin (HA), A35R, or B2R genes. In some embodiments, the modification renders the gene product encoded by the locus nonfunctional or defective. In some embodiments, all or part of the TK, HA, A35R, or B2R ORF is deleted.

[0245] 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 encoding thymidine kinase (TK; SEQ ID NO: 66). In some embodiments, it has been reported that the TK locus is not essential for viral replication, and thus its modification can reduce viral virulence, resulting in the inability of the virus to replicate in the brain or ovary, while retaining 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 with the sequence shown in SEQ ID NO: 4. In some embodiments, the nucleic acid genome of the recombinant vaccinia virus strain has the nucleotide sequence shown in SEQ ID NO: 4. In some embodiments, the recombinant vaccinia virus is a vaccinia virus designated VIR13.

[0246] In some embodiments, the attenuated recombinant vaccinia virus provided herein has an inactivating mutation, such as an insertion, mutation, or deletion, of the B2R locus encoding the cytoplasmic cGAMP nuclease (poxin) (SEQ ID NO: 54). In some embodiments, the B2R locus has been reported to cause vaccinia virus attenuation in a skin scratch model (E 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, of the B2R gene, and an inactivating mutation, such as an insertion, mutation, or deletion, of 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 with the sequence shown in SEQ ID NO: 48. In some embodiments, the nucleic acid genome of the recombinant vaccinia virus strain has the nucleotide sequence shown in SEQ ID NO: 48. In some embodiments, the recombinant vaccinia virus is a vaccinia virus designated VIR94.

[0247] In some embodiments, the attenuated recombinant vaccinia virus provided herein has an inactivating mutation of the A35R locus, such as an insertion, mutation, or deletion. A35R is a virulence gene that regulates the adaptive immune response, and its inactivation (such as by deletion) can lead to a decrease in viral replication ability and reduced viral virulence (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 with the sequence shown in SEQ ID NO: 3. In some embodiments, the nucleic acid genome of the recombinant vaccinia virus strain has the nucleotide sequence shown 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 of the A35R gene, such as an insertion, mutation, or deletion, and an inactivating mutation of the J2R gene, such as an insertion, mutation, or deletion. 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 shown in SEQ ID NO: 12. In some embodiments, the nucleic acid genome of the recombinant vaccinia virus strain has the nucleotide sequence shown in SEQ ID NO: 12. In some embodiments, the recombinant vaccinia virus is a vaccinia virus designated VIR52.

[0248] 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 encoding hemagglutinin (HA; SEQ ID NO: 67). In some embodiments, it has been reported that the HA locus is not essential for viral replication, such that modification thereof can reduce viral virulence, resulting in the inability of the virus to replicate in the brain or ovary, while retaining the ability to preferentially replicate in tumor tissues (e.g., Shida et al. (1988) J. Virol., 62: 4474-4480).

[0249] In some embodiments, the attenuated recombinant vaccinia viruses provided herein have 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 viruses provided herein have an insertion, mutation, or deletion of the F3 gene product encoded by the F14.5L gene (SEQ ID NO: 64). In some embodiments, it has been reported that the F14.5L gene (also referred to as F3) is not essential for viral replication, such that modification thereof can reduce viral virulence, resulting in the inability of the virus to replicate in the brain or ovary, while retaining the ability to preferentially replicate in tumor tissue (e.g., U.S. Patent Publication No. US2005 / 0031643).

[0250] A variety of methods can be used to assess or determine the level of attenuation of a virus. These methods for measuring the level of attenuation can be performed in vitro or in vivo and can include assessing changes in any or all of the following properties of the virus: 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 assessing the level of viral attenuation by in vitro and in vivo methods are known in the art, including but not limited to methods such as plaque assays and mouse models of viral pathogenicity. Exemplary methods for studying early, middle, and late transcription of vaccinia virus 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 measuring viral RNA transcripts and proteins include but are not limited to well-known techniques such as Northern hybridization and blotting techniques and immunohistochemistry. IV. Recombinant Virus Strains with Heterologous Nucleic Acid

[0251] Provided herein are recombinant virus strains modified in genomic sequences. In some embodiments, a recombinant oncolytic virus is provided herein, comprising at least one heterologous nucleic acid encoding one or more heterologous gene products. The heterologous gene product is not particularly limited, and in some embodiments, it may be a complement inhibitor, a T cell or NK cell escape product, an immunostimulatory protein, an anti-angiogenic protein, an interferon regulatory factor, an apoptosis-inducing protein, or any combination thereof. Therefore, in some embodiments, a recombinant oncolytic virus is provided herein, 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 cells or NK cell escape products, immunostimulatory proteins, anti-angiogenic proteins, interferon regulatory factors, apoptosis-inducing proteins, or any combination thereof.

[0252] 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, 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.

[0253] Also provided herein 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 or comprise immunomodulatory proteins, complement inhibitors, T cell or NK cell escape products, anti-angiogenic proteins, interferon regulatory factors, apoptosis-inducing proteins, or any combination thereof. 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 immunomodulatory proteins, such as one or more immunomodulatory proteins selected from LIGHT, IRF3, IL-2, IL-12, and CXCL9; and / or the at least one heterologous nucleic acid encoding one or more heterologous gene products comprises one or more heterologous nucleic acids each encoding a cell apoptosis-inducing protein, such as iDED, iFas, or iCas9; and / or the 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 T cell or NK cell escape proteins, such as a group consisting of vaccinia virus ORF012, 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 each 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 the 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, such as VEGF inhibitors, angiogenin inhibitors or Versikine; and / or the 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 therapeutic or diagnostic agents.

[0254] Inactivation mutations include changing the expression and / or function of the gene product expressed by the inactivated viral gene in a variety of ways, such as gene disruption. Gene disruption can be achieved by, for example, gene deletion, nucleic acid insertion, nucleic acid mutation or substitution, gene knockout, premature termination codon, transcription promoter modification, RNAi or gene editing (such as CRISPR). In some embodiments, inactivation mutations are gene deletions and / or insertions (also referred to as introductions) of heterologous nucleic acids encoding one or more gene products. In specific embodiments, inactivation mutations combine gene deletions with insertions of heterologous nucleic acids into the locus. For example, in some methods for achieving inactivation mutations (such as by homologous recombination and other methods), heterologous nucleic acids can be inserted into the deleted gene region. Therefore, it is understood that in some embodiments, the locus mentioned for inserting heterologous nucleic acids refers to the locus of the deletion of a gene inactivated by the total or partial deletion of a gene. In some embodiments, gene deletion removes the entire sequence of a gene. In other embodiments, gene deletion is a partial deletion, i.e., a portion of a gene sequence is removed. In one embodiment, gene deletion is partial deletion, i.e., remove 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, gene deletion is partial deletion, i.e., remove 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, gene deletion removes 100% of the gene sequence. In another embodiment, gene deletion removes 100% of the gene protein coding sequence. In one embodiment, 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 embodiment, the gene deletion is a partial deletion, i.e., removal of 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 embodiment, the partial deletion in the gene results in a partial gene.

[0255] Also 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, a T cell or NK cell escape product, an immunomodulatory protein, an anti-angiogenic protein, an interferon regulatory factor, a cellular apoptosis-inducing protein, or any combination thereof.

[0256] Also provided herein is a recombinant oncolytic virus comprising: a nucleic acid genome having at least 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 1, and at least one heterologous nucleic acid encoding one or more heterologous gene products inserted into the genome.

[0257] The following subsections describe exemplary heterologous proteins. In addition to recombinant viral strains, any heterologous protein described herein can also be incorporated into gene therapy vectors (such as AAV, lentivirus, and retrovirus) or cell-based therapies (such as chimeric antigen receptor-expressing T cells (CAR-T), natural killer (NK) cells, or tumor infiltrating lymphocytes (TIL) therapy).

[0258] The virus strains provided herein include recombinant virus strains comprising at least one heterologous nucleic acid encoding one or more heterologous gene products. In some embodiments, the recombinant viruses include, but are not limited to, vaccinia virus, vesicular stomatitis virus (VSV), Maraba virus (MARAV), measles virus (MV), myxoma virus, stomatitis virus, parvovirus, raccoon pox virus, coxsackie virus, reovirus, Newcastle disease virus, Seneca Valley virus, Semliki Forest virus, influenza virus, echovirus, poliovirus (PV), adenovirus (such as mammalian adenovirus and avian adenovirus), herpes virus (such as herpes simplex virus type 1, herpes simplex virus type 2, herpes simplex virus type 5, herpes simplex virus type 6), herpes simplex virus (such as herpes simplex virus type 1, herpes simplex virus type 2, herpes simplex virus type 5, herpes simplex virus type 6), and herpes simplex virus (such as herpes simplex virus type 6). Herpes simplex virus, Epstein-Barr virus, HHV6-HHV8 and cytomegalovirus), levivirus (such as levivirus, enterobacteria phage MS2, allovirus), poxvirus (such as chordopoxvirinae, parapoxvirus, avipox virus, capripoxvirus, rabbitpox virus, swinepox virus, molluscumpox virus, entomopoxvirus), papovavirus (such as polyomavirus and papillomavirus), paramyxovirus (such as paramyxovirus, parainfluenza virus type 1 (such as measles-rubella virus), rubella virus (such as mumps virus), pneumovirus (such as pneumonia virus), viruses (human), human respiratory syncytial virus and metapneumoviruses (such as avian pneumonia virus and human metapneumovirus)), picornaviruses (such as enteroviruses, rhinoviruses, hepatoviruses (such as human hepatitis A virus), cardioviruses and aphthous viruses), reoviruses (such as orthoreoviruses, orbiviruses, rotaviruses, cytoplasmic polyhedrosis viruses, Fijiviruses, plant reoviruses and rice viruses), retroviruses (such as mammalian retrovirus type B, mammalian retrovirus type C, type D retrovirus group, BLV-HTLV retroviruses), lentiviruses (such as type 1 Human immunodeficiency virus and human immunodeficiency virus type 2 (such as HIV gp160), foamy viruses), flaviviruses (such as hepatitis C virus, dengue virus, West Nile virus), hepadnaviruses (such as hepatitis B virus), togaviruses (such as alphaviruses (such as Sindbis virus) and rubella viruses (such as rubella virus)), rhabdoviruses (such as vesiculovirus, lyssa virus, ephemeral fever virus and cytarabhavirus), arenaviruses (such as arenavirus, lymphocytic choriomeningitis virus, ephemeral fever virus and Lassa virus) and coronaviruses (such as coronavirus and torovirus).

[0259] In some embodiments, the recombinant virus comprises an oncolytic virus. In some embodiments, the recombinant virus is a recombinant oncolytic virus. In some embodiments, the recombinant virus (such as a recombinant oncolytic virus) is a vaccinia virus, herpes simplex virus, vesicular stomatitis virus (VSV), Maraba virus (MARAV), measles virus (MV), adenovirus, myxoma virus, sheep sore virus, parvovirus, raccoon pox virus, coxsackie virus, reovirus, Newcastle disease virus, Seneca Valley virus, Semliki Forest virus, mumps virus, influenza virus, Echo virus or poliovirus (PV). In some embodiments, the recombinant virus, such as a recombinant oncolytic virus, is a vaccinia virus.

[0260] In some embodiments, the recombinant virus is a non-oncolytic virus. In some embodiments, the recombinant virus is a non-vaccinia virus. In some embodiments, the recombinant virus comprises a vaccinia virus. In some embodiments, the recombinant virus is derived from the Copenhagen strain.

[0261] 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 (such as recombinant oncolytic viruses) comprising mutations, insertions, deletions, or substitutions (substitutions) of one or more nucleic acids, or other modifications of viral genomic sequences. In some embodiments, provided herein are modified VIP02 strains that have been modified in their genomic sequences compared to the genomic sequence shown in SEQ ID NO: 1. In some embodiments, the recombinant virus is a virus derived from a nucleic acid genome shown in SEQ ID NO: 1, whose genome has been modified by inserting a nucleic acid encoding a heterologous gene product.

[0262] Methods for producing 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 US A. 95(5):2509-2514. Racaniello et al., (1981) Science 214:916-919). Methods for producing recombinant vaccinia viruses can also be found in the Examples described herein.

[0263] In some embodiments, the recombinant virus has a large carrying capacity for insertable exogenous genes. For example, the vaccinia virus genome has a large carrying capacity for exogenous genes and can insert exogenous DNA fragments up to 25 kb. The genomes of several vaccinia virus strains have been completely sequenced, and many essential and non-essential genes have been identified. Due to the high degree of sequence homology between different strains, genomic information from one vaccinia virus strain can be used to design and produce modified viruses of other strains. Finally, the technology for producing modified vaccinia virus strains by genetic engineering is mature (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)).

[0264] Sites for inserting heterologous nucleic acid molecules are known in the art and have been described for a variety of viral vectors (see, e.g., 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 regions or into the coding region of genes that are not essential for viral replication. For example, in vaccinia virus, the site of insertion of the heterologous DNA molecule can be in an intergenic region, a noncoding region, and / or a non-essential gene or gene region, including, but not limited to, the thymidine kinase (TK) gene, the hemagglutinin (HA) gene, F14.5L (see, e.g., U.S. Patent Publication No. 2005-0031643), the VGF gene (see, e.g., U.S. Patent Publication No. 2003-0031681), Hind III F, F13L, or Hind III M (see, e.g., U.S. Patent No. 6,548,068); the hemorrhagic region or type A inclusion region (ATI) (see, e.g., U.S. Patent Nos. 6,265,189 and 6,596,279); the A33R, A34R, A36R, or B5R gene (see, e.g., Katz et al., (2003) J. Virology 77:12266-12275); the SalF7L (see, e.g., Moore et al., (2003) J. Virology 77:12266-12275); al., (1992) EMBO J. 11: 1973-1980); N1L (see, e.g., Kotwal et al. (1989) Virology 171: 579-587); M1λ (see, e.g., Child et al. (1990) Virology. 174: 625-629); HR, HindIII-MK, HindIII-MKF, HindIII-CNM, RR, or BamF (see, e.g., Lee et al. (1992) J Virol. 66: 2617-2630); C21L (see, e.g., Isaacs et al. (1992) Proc Natl Acad Sci USA.89:628-632), host range region genes K1L and C7L, A35R (see, e.g., U.S. Patent Nos. 6,265,189; 7,045,313; U.S. Patent Publication Nos. 2005-0244428; 2006-0159706; Coupar et al. J. Gen. Virol. (2000) 81:431-439; Smith et al. (1993) Vaccine 11(1):43-53). If more than one gene expression cassette is inserted, these insertions can be made at the same insertion site or at different insertion sites.Alternatively, the heterologous nucleic acid molecule can be inserted into an essential gene, and the virus can be produced using a cell line designed for virus packaging.

[0265] In some embodiments, at least one heterologous nucleic acid encoding one or more heterologous gene products is inserted into or replaces a non-essential gene or region in the viral genome. In some embodiments, at least one heterologous nucleic acid encoding one or more heterologous gene products is inserted into or replaces a 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 viral genome, or any combination thereof. In some embodiments, at least one heterologous nucleic acid encoding one or more heterologous gene products is inserted into or replaces 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 replaces the A35R locus. In some embodiments, at least one heterologous nucleic acid encoding one or more heterologous gene products is inserted into or replaces the J2R locus. In some embodiments, insertion into the locus refers to an insertion in which the locus comprises a partial deletion, and the heterologous nucleic acid replaces the deletion. In some embodiments, insertion into the locus refers to an insertion into the locus, but no part of the endogenous locus is deleted. In some embodiments, insertion into a replacement locus refers to an insertion in which the entirety of the locus is deleted and replaced by a heterologous nucleic acid.

[0266] Mutations in non-essential vaccinia virus genes also contribute to increasing the attenuation of the virus. Therefore, inserting a heterologous expression cassette into a non-essential gene (such as a TK gene) can attenuate the virus in two aspects: through gene mutation and increased transcription and / or translation load. For the methods described herein, mutations in non-essential genes are not required; however, one or more non-essential genes can be modified to enhance the attenuation effect of the gene expression cassette. Subsequently, the attenuation of the virus can be reduced by removing the expression cassette and replacing it with a non-coding sequence (i.e., the virus exhibits increased replication), thereby keeping the gene inactive. Therefore, removing or replacing the gene expression cassette reduces the transcription and / or translation load of the virus, resulting in a reduced degree of viral attenuation.

[0267] In some embodiments, at least one heterologous nucleic acid encoding one or more heterologous gene products is fused with a gene encoding a viral membrane protein in the viral genome. In some embodiments, at least one heterologous nucleic acid encoding one or more heterologous gene products is fused with 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 with a viral membrane protein to produce a fusion protein. In some embodiments, the gene encoding a viral membrane protein fused with 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 is fused at the C-terminus of F14.5L. In some embodiments, the fusion protein is incorporated into the outer membrane of an intracellular mature virus (IMV) (e.g., an IMV of vaccinia virus). These fusion proteins comprising the viral membrane protein F14.5L are expected to be incorporated into the outer membrane of the IMV virus particle, which can give it the ability to resist complement inactivation in the blood.

[0268] The modification can comprise mutation, insertion, deletion or substitution (replacement) of nucleic acid, or other modifications of viral genome sequence. For example, provided herein is a virus that can be modified to contain one or more heterologous nucleic acid molecules inserted into the viral genome or to substitute in the viral genome. Viral genes can be substituted by homologous genes or different genes from another virus. In one embodiment, modification comprises 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 or more nucleotides. In some embodiments, the modification comprises deleting one or more nucleotides, e.g., 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 embodiments, the modification comprises replacing one or more nucleotides, e.g., 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.

[0269] Modification comprises the insertion and / or replacement (replacement) of nucleic acid or the modification of viral genomic sequence with heterologous nucleic acid. Generally, heterologous gene is a gene encoding non-viral protein. For example, heterologous nucleic acid molecules encoding heterologous genes can be inserted. In some embodiments, heterologous nucleic acid replaces all or part of viral genes. In other embodiments, the virus provided herein can be modified by the insertion of one or more heterologous nucleic acids. 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 include an open reading frame, or can be a non-coding sequence. Generally, the heterologous nucleic acid inserted is a continuous nucleotide sequence including an open reading frame and corresponding to a gene coding region. The inserted or replaced gene can be transcribed and / or translated from the viral genome after infecting a host cell (such as a tumor cell). As described below, heterologous nucleic acid can include a regulatory sequence for controlling gene expression. For example, heterologous nucleic acid can be operably connected to a promoter to express an open reading frame. In some embodiments, the promoter has 70%, 80%, 90%, 100% sequence identity to the sequence shown in SEQ ID NO: 68, 69, 70, 71, or 72. In some embodiments, the promoter has the same sequence identity to the sequence shown in SEQ ID NO: 68, 69, 70, 71, or 72.

[0270] Modifications to the viral genomes provided herein can result in changes in viral characteristics or properties. Exemplary changes include changes in parameters that indicate anti-tumor properties and / or toxicity. For example, insertions, mutations, or deletions can reduce the pathogenicity of a clonal strain, such as reducing the infectivity, toxicity, or replication capacity of a vaccinia virus, or reducing the amount of accumulation thereof in non-tumor organs or tissues. Exemplary nucleic acid insertions, deletions, mutations, and / or substitutions are those that give the vaccinia virus better anti-tumor properties and lower toxicity relative to a clonal strain and / or starting virus preparation or mixture or other reference strain or isolate (including recombinant strain) that does not contain the modification. In some embodiments, the insertions, deletions, mutations, and / or substitutions of nucleic acids are those that give the vaccinia virus similar anti-tumor properties and toxicity relative to a clonal strain and / or starting virus preparation or mixture or other reference strain or isolate (including recombinant strain) that does not contain the modification. In some embodiments, the modification of the viral genome reduces toxicity relative to a clonal strain and / or starting virus preparation or mixture or other reference strain or isolate (including recombinant strain) that does not contain the modification. In some embodiments, insertions, mutations or deletions include, but are not limited to, those that increase the anti-tumor properties of the virus and reduce its toxicity relative to a clonal strain and / or starting virus preparation or mixture or other reference strain or isolate (including recombinant strain) that does not contain the modification.

[0271] In some embodiments, insertions, mutations or deletions include but are not limited to those that increase the ability of the clonal virus strain to escape the host's immune system relative to a clonal strain and / or starting virus preparation or mixture or other reference strain or isolate (including recombinant strain) that does not contain the modification. In some embodiments, insertions, mutations or deletions include but are not limited to those that increase the ability of the clonal virus strain to stimulate the host's immune system relative to a clonal strain and / or starting virus preparation or mixture or other reference strain or isolate (including recombinant strain) that does not contain the modification. In some embodiments, insertions, mutations or deletions include but are not limited to those that increase the host's anti-angiogenic activity relative to a clonal strain and / or starting virus preparation or mixture or other reference strain or isolate (including recombinant strain). In some embodiments, insertions, mutations or deletions include but are not limited to those that increase the host's apoptotic activity relative to a clonal strain and / or starting virus preparation or mixture or other reference strain or isolate (including recombinant strain) that does not contain the modification.

[0272] In some embodiments, one or more heterologous nucleic acid molecules may encode, for example, an anti-apoptotic gene product or fragment thereof, such as a gene product that can modulate the host's apoptotic response; an angiogenic gene product or fragment thereof, such as a gene product that can modulate the host's angiogenic response; an immune system gene product or fragment thereof, such as a gene product that can modulate the host's immune response. In some embodiments, the gene product or fragment thereof that can modulate the host's immune response can increase the host's immune system's ability to escape complement inhibition relative to a clonal strain and / or starting viral preparation or mixture or other reference strain or isolate (including recombinant strain) that does not contain the modification. In some embodiments, the gene product that can modulate the host's immune response can increase the activity of the host's immune system relative to a clonal strain and / or starting viral preparation or mixture or other reference strain or isolate (including recombinant strain) that does not contain the modification.

[0273] In some embodiments, the recombinant virus is a vaccinia virus that has been modified in its genomic sequence compared to the genomic sequence shown 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 that has been modified in its genomic sequence compared to the genomic sequence shown in SEQ ID NO: 1. The large genome size of the vaccinia viruses provided herein allows for the insertion of large and / or multiple heterologous DNA nucleotide sequences into the viral genome (Smith and Moss (1983) Gene 25(1): 21-28). The viruses provided herein can be modified by insertion or substitution of one or more nucleotides. In one embodiment, the modification comprises inserting or replacing one or more nucleotides, such as inserting or replacing 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 into a locus of the viral genome, such as any locus described herein. In some embodiments, one or more heterologous DNA molecules are inserted into a non-essential region of the viral genome; for example, a DNA molecule is inserted into a locus that is non-essential for viral replication in a proliferating cell (such as a tumor cell). Exemplary insertion sites are known in the art and are provided herein. In some embodiments, the recombinant vaccinia virus provided herein may comprise an inactivating mutation of a viral gene, such as any inactivating mutation described herein, such as a gene deletion of all or part of a viral gene. In such embodiments, one or more heterologous nucleic acids may be inserted into or replace such loci. In some embodiments, the recombinant virus is a virus that has been modified compared to the genomic sequence shown in SEQ ID NO: 1, wherein one or more heterologous nucleic acids are inserted and one or more viral loci are inactivated, such as by gene deletion. The modified recombinant virus can be any virus provided herein having a genome shown in SEQ ID NO: 1, or a genome at least 99% identical to SEQ ID NO: 1, or any other virus produced by introducing heterologous DNA as described herein. In some embodiments, the recombinant virus has been modified in its genomic sequence compared to the genomic sequence shown in SEQ ID NO: 1, and its amino acid sequence exhibits at least 85%, 90% or 95% sequence identity to the sequence shown 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 shown in SEQ ID NO: 1.

[0274] In some embodiments, the recombinant virus can be modified to express an exogenous or heterologous gene. Exemplary exogenous gene products include proteins involved in apoptosis, angiogenesis and / or immune system regulation. In some embodiments, the gene product includes proteins that affect the host's apoptosis pathway, such as caspase-9, the death effector domain (DED) of Fas-associated death domain protein (FADD) and Fas. In some embodiments, the gene product includes proteins that affect the host's angiogenesis pathway, such as vascular endothelial growth factor (VEGF) and Versikine (VK). In some embodiments, the gene product includes proteins that affect the host's immune system, such as minimized complement regulatory factor H (miniFH), Borrelia burgdorferi complement regulatory acquisition surface protein-2 (CRASP-2), vaccinia virus ORF 012, 203 and 018 (CPXV012-203-018) and human LIGHT variant (hmLIGHT). The characteristics of such gene products are described herein and elsewhere.

[0275] Specifically, 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, wherein 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 a gene product associated with apoptosis. In another example, the virus can be modified to express two or more gene products for the production of a fusion protein. In some examples, one or more proteins involved in angiogenesis can be expressed together. When two or more heterologous genes are introduced, these genes can be regulated under the same or different regulatory sequences, and these genes can be inserted into the same or different regions of the viral genome. This can be done through single or multiple genetic manipulation steps. In some embodiments, one gene can be under the control of a constitutive promoter, while the second gene can be under the control of an inducible promoter. Methods for inserting two or more genes into viruses are known in the art and can be easily applied to a variety of viruses using a variety of exogenous genes, regulatory sequences, and / or other nucleic acid sequences.

[0276] The viruses provided herein can be modified by insertion, deletion, substitution or mutation as described herein. Standard methods for modifying viruses by insertion, deletion, substitution and mutation of nucleic acids 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 NY (1989), and the Examples disclosed herein. The technology for producing recombinant viruses includes nucleic acid transfer protocols, various nucleic acid manipulation techniques, nucleic acid amplification protocols, and generally involves the use of standard molecular biology techniques to produce gene cassettes or transfer vectors. See, for example, U.S. Patent No. 5,494,807 and U.S. Patent No. 5,185,146, which describe exemplary methods for producing recombinant vaccinia viruses and other molecular biology techniques known in the art. Methods for producing 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 95(5):2509-2514; Racaniello et al., (1981) Science 214:916-919; and Hruby et al., (1990) Clin Microbiol. Methods for producing recombinant vaccinia viruses are known in the art (see, for example, Hruby et al., (1990) Clin Micro Rev. 3: 153-170; U.S. Patent Publication No. 2005-0031643, now U.S. Patent Nos. 7,588,767, 7,588,771, 7,662,398, and 7,045,313).

[0277] In some embodiments, homologous recombination can be used to introduce the insertion or deletion of a nucleic acid molecule into a target sequence of interest. For a variety of viruses and cell organisms, methods using nucleic acid tools (such as vectors, plasmids, promoters and other regulatory sequences) are well known in the art. Nucleic acid amplification protocols include, but are not limited to, polymerase chain reaction (PCR), or amplification by viruses or organisms (such as, but not limited to, yeast, bacteria, insects, or mammalian cells). Nucleic acid transfer protocols include electroporation, calcium chloride transformation / transfection, liposome-mediated nucleic acid transfer or other. A variety of tools for modifying nucleic acids can be obtained from many different sources, including various commercial sources. For example, point mutations or small insertions or deletions can be introduced into a gene of interest by using oligonucleotide-mediated site-directed mutagenesis. In another example, homologous recombination can be used to introduce mutations into a nucleic acid sequence, or insert or delete a nucleic acid molecule into a target sequence of interest. In some instances, positive or negative selection pressures can be used to select nucleic acid mutations, insertions, or deletions in a specific gene. See, for example, "Contemporary Molecular Biology Techniques" (Ausubel et al., ed.). Those skilled in the art will be able to readily select the appropriate tools and methods for genetic modification for any particular virus based on knowledge and design choices in the art. In some embodiments, a plasmid is used for homologous recombination to construct a recombinant virus. In some embodiments, a plasmid is constructed by connecting two fragments using gene splicing. In some embodiments, the primers used to amplify the two fragments comprise 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.

[0278] Insertions, deletions, substitutions or mutations can be specifically directed to specific sequences in the viral genome. Such sequences in the viral genome include, but are not limited to, intergenic sequences, regulatory sequences, sequences of unknown function, gene coding sequences, or non-essential regions of the viral genome. For many viruses, regions of the viral genome that can be used for modification are known in the art.

[0279] In some embodiments, the recombinant virus (such as a recombinant oncolytic virus) comprises an inactivating mutation of at least one viral gene. Inactivating mutations are not particularly limited. In some embodiments, they can be any mutation that causes the gene product function of the viral gene to be reduced or lost compared to when there is no inactivating mutation. In some embodiments, the inactivating mutation is a complete or partial deletion of at least one viral gene. In some embodiments, the deletion of at least one viral gene refers to the deletion of the entire 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, which is sufficient to make the encoded gene product lose its function. In some embodiments, the 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, the 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, the at least one viral gene is A35R. In some embodiments, the at least one viral gene is J2R. In some embodiments, the at least one viral gene is B2R. In some embodiments, the at least one viral gene is B2R. In some embodiments, the at least one viral gene comprises A35R and J2R. In some embodiments, the at least one viral gene is B2R. In some embodiments, the at least one viral gene comprises B2R and J2R.

[0280] Heterologous nucleic acid molecules are typically inserted into the intergenic regions of the viral genome or into loci encoding non-essential viral gene products. Insertion of heterologous nucleic acids at these sites generally does not significantly affect viral infection or replication in the target tissue. Examples of insertion sites include, but are not limited to, J2R (thymidine kinase (TK)), A56R (hemagglutinin (HA)), F14.5L, vaccinia growth factor (VGF), A35R, N1L, E2L / E3L, K1L / K2L, superoxide dismutase loci, 7.5K, C7-K1L (host range gene region), B13R+B14R (hemorrhagic region), A26L (A-type inclusion region (ATI)), or I4L (ribonucleotide reductase large subunit) loci. The insertion sites of the viruses provided herein also include sites corresponding to the intragenic regions described in other vaccinia viruses (e.g., modified vaccinia virus Ankara (MVA), exemplary sites shown in U.S. Patent No. 7,550,147), NYVAC (exemplary sites shown in U.S. Patent No. 5,762,938). In some embodiments, the insertion, deletion, substitution and / or mutation site comprises J2R, F14.5L and / or A35R.

[0281] For example, the production of a recombinant vaccinia virus expressing a heterologous gene product generally includes the use of a recombinant plasmid comprising a heterologous nucleic acid (optionally operably linked to a promoter) and carrying a vaccinia virus DNA sequence on both sides of the heterologous nucleic acid to promote homologous recombination and insert the gene into the viral genome. Typically, the viral DNA on both sides of the heterologous gene is complementary to the non-essential fragments of the vaccinia virus DNA so that the gene is inserted into a non-essential position or any other position. The recombinant plasmid can be grown and purified in Escherichia coli and then introduced into a suitable host cell, such as, but not limited to, CV-1, BSC-40, BSC-1, and TK-143 cells. The transfected cells are then superinfected with the vaccinia virus, which initiates the replication cycle. The heterologous DNA can be incorporated into the vaccinia virus genome by homologous recombination and packaged into the infected progeny. The recombinant virus can be identified by methods known in the art, such as detecting the expression of the heterologous gene product, or using positive or negative selection methods (U.S. Patent number 7,045,313). In some embodiments, recombinant viruses are produced by homologous integration of a plasmid into the viral genome corresponding to the region of the J2R gene. In some embodiments, recombinant viruses are produced by homologous integration of a plasmid into the viral genome corresponding to the region of the A35R gene. In some embodiments, recombinant viruses are produced by homologous integration of a plasmid into the viral genome corresponding to the region of the F14.5L gene. In some embodiments, recombinant viruses are produced by homologous integration of a plasmid into the viral genome corresponding to the region of the J2R gene, and another plasmid into the viral genome corresponding to the region of the F14.5L gene. In some embodiments, recombinant viruses are produced by homologous integration of a plasmid into the viral genome corresponding to the region of the J2R gene, and another plasmid into the viral genome corresponding to the region of the A35R gene. In some embodiments, recombinant viruses are produced by homologous integration of a plasmid into the viral genome corresponding to the region of the F14.5L gene, and another plasmid into the viral genome corresponding to the region of the A35R gene. In some embodiments, the recombinant virus is produced by homologously integrating one plasmid into the region of the viral genome corresponding to the J2R gene, and another plasmid into the region of the viral genome corresponding to the A35R gene, and another plasmid into the region of the viral genome corresponding to the F14.5L gene.

[0282] In another example, recombinant vaccinia viruses expressing heterologous gene products can be produced 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 this method, the heterologous nucleic acid (optionally operably linked to a promoter) is flanked by restriction endonuclease cleavage sites for insertion into the unique restriction endonuclease sites of the target virus. Viral DNA is purified using standard techniques and cleaved with sequence-specific restriction endonucleases that are unique sites in the viral genome. Any unique site in the viral genome can be used as long as the modification of the site does not interfere with viral replication. Typically, the insertion is into a site located in a non-essential region of the viral genome. For example, exemplary modifications herein include inserting an exogenous DNA sequence into viral DNA that has been cleaved by NotI.

[0283] In some instances, heterologous nucleic acid can also include one or more regulatory sequences to regulate the expression of open reading frames encoding heterologous RNA and / or protein. Regulatory sequences suitable for, for example, functioning in mammalian host cells are known in the art. Expression can also be affected by one or more proteins or RNA molecules expressed by viruses. Gene regulatory elements (such as promoters and enhancers) have cell type specific activity and can be activated by certain induction factors (such as hormones, growth factors, cytokines, cytostatics, radiation, heat shock) through response elements. Using such regulatory elements as internal promoters to drive gene expression in viral vector constructs can achieve controlled and restricted expression of these genes.

[0284] In some embodiments, the heterologous nucleic acid encoding one or more heterologous gene products is operably connected to a promoter. In some embodiments, the heterologous nucleic acid encoding one or more heterologous gene products is operably connected to a promoter to express heterologous RNA and / or protein. For example, a heterologous nucleic acid operably connected to a promoter is also referred to as an expression cassette. Therefore, provided herein is a virus that can have the ability to express one or more heterologous genes. Gene expression can include expressing a protein encoded by a gene and / or expressing an RNA molecule encoded by a gene. In some embodiments, provided herein is a virus that can express exogenous genes at sufficiently high levels, thereby allowing the product of the exogenous gene to be harvested from a tumor. The expression of the heterologous gene can be controlled by a constitutive promoter, or by an inducible promoter. In other examples, organ or tissue-specific expression can be controlled by a regulatory sequence. In order to achieve expression only in a target organ (e.g., a tumor to be treated), an exogenous nucleotide sequence can be connected to a tissue-specific promoter and used for gene therapy. Such promoters are well known to those skilled in the art (see, e.g., 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)).

[0285] Exemplary promoters for expressing heterologous genes are known in the art. The heterologous nucleic acid can be operably linked to a native promoter or a non-viral native heterologous promoter. Any suitable promoter can be used, including synthetic promoters, naturally occurring promoters, and modified promoters. Exemplary promoters include synthetic promoters, including synthetic viral promoters and animal promoters. Native promoters or heterologous promoters include, but are not limited to, viral promoters, such as vaccinia virus promoters and adenovirus promoters.

[0286] In some embodiments, the promoter is a poxvirus promoter, for example, 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. Vaccinia virus promoters used to express one or more heterologous genes can be synthetic promoters or natural promoters and include vaccinia virus early, middle, early / late, and late promoters. Exemplary poxvirus promoters for controlling heterologous gene expression include, but are not limited to, the 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, A1 L, 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. Strong late promoters can be used to achieve high levels of expression of heterologous genes. Early and intermediate promoters can also be used. In one example, the promoter comprises early and late promoter elements, e.g., a modified H5 promoter, PmH5, which comprises native early and late vaccinia promoter regions, a synthetic early / late vaccinia PSEL promoter, and a 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).

[0287] In some embodiments, the promoter is selected from 7.5E, 7.5E / L, SSE, 11KL, SSL, SSEL, mH5, and LEO. In some embodiments, the promoter has the amino acid sequence set forth in any one of SEQ ID NOs: 29, 53, 55, 68, 69, 70, 71, or 72. In some embodiments, the promoter has the amino acid sequence set forth in SEQ ID NO: 29. In some embodiments, the promoter is a synthetic strong early promoter (SSE) and comprises the amino acid sequence set forth in SEQ ID NO: 29. In some embodiments, the promoter has the amino acid sequence set forth in SEQ ID NO: 55. In some embodiments, the promoter is a strong early / late promoter (SEL) and comprises the amino acid sequence set forth 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 set forth in SEQ ID NO: 53.

[0288] 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 may exhibit differences in properties, such as the degree of attenuation, due to the use of stronger versus weaker promoters. For example, in vaccinia virus, the synthetic early / late and late promoters are relatively strong promoters, while the vaccinia virus synthetic early promoter is a relatively weak promoter (see, e.g., Chakrabarti et al. (1997) BioTechniques 23(6): 1094-1097).

[0289] As known in the art, regulatory sequences can allow constitutive expression of exogenous genes, or can allow inducible expression of exogenous genes. In addition, regulatory sequences can control the expression level of exogenous genes. In some instances, as in gene product manufacturing and results, regulatory sequences can cause constitutive, high-level expression of genes. In some instances, as in anti-(gene product) antibody results, regulatory sequences can cause constitutive, low-level expression of genes. In the example of tumor therapy, therapeutic proteins can be under the control of internal inducible promoters or external inducible promoters.

[0290] In some embodiments, the expression of heterologous genes can be controlled by constitutive promoters or inducible promoters. Inducible promoters can be used to provide tissue-specific expression of heterologous genes, or can be induced by adding regulatory molecules to provide the time-specific induction of promoters. In some instances, inducible expression can be under the control of cells or other factors present in tumor cells or virally infected tumor cells. In other instances, inducible expression can be under the control of administerable substances (including IPTG, RU486 or other known inducing compounds). Extra regulatory sequences can be used to control the expression of one or more heterologous genes inserted into the virus. According to known factors and design preferences, those skilled in the art can use any of a variety of regulatory sequences.

[0291] In some embodiments, one or more heterologous gene products include therapeutic agents or diagnostic agents. In some embodiments, one or more heterologous gene products (such as therapeutic agents or diagnostic agents) are selected from anticancer agents, anti-metastatic agents, anti-angiogenic agents, immune regulatory molecules, antigens, cell matrix degradation genes, genes for tissue regeneration and reprogramming human cells to pluripotency, enzymes that modify substrates to produce detectable products or signals or can be detected by antibodies, proteins that can bind contrast agents, genes for optical imaging or detection, genes for positron emission tomography (PET) imaging, and genes for MRI. In some embodiments, one or more heterologous gene products (such as therapeutic agents or diagnostic agents) include therapeutic agents selected from hormones, growth factors, cytokines, chemokines, costimulatory molecules, ribozymes, transporters, single-chain antibodies, antisense RNA, prodrug converting enzymes, siRNA, microRNA (microRNA), toxins, antitumor oligopeptides, mitosis inhibitor proteins, antimitotic oligopeptides, anticancer polypeptide antibiotics, angiogenesis inhibitors, tumor suppressors, cytotoxic proteins, cell growth inhibitory proteins, and tissue factors.

[0292] In some of any such embodiments, the recombinant virus (e.g., a recombinant oncolytic virus) comprises a nucleic acid sequence comprising at least one heterologous nucleic acid encoding one or more heterologous gene products, e.g., any heterologous gene product described herein (e.g., Section III A, B, C, and D), comprising, for example, one or more heterologous gene products selected from complement inhibitors, T cell escape proteins or NK cell escape proteins, immunostimulatory proteins, anti-angiogenic proteins, interferon regulatory factors, cell apoptosis inducing proteins, or any combination thereof, and optionally comprises an inactivating mutation of at least one viral gene, e.g., hemagglutinin (HA), in the viral genome. One or more of 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 loci, optionally wherein the one or more viral genes are one or more of B2R, J2R, A35R, and A56R, and any combination thereof.

[0293] In some of any such embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown 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 shown in any one of SEQ ID NOs 48, 80, 82, and 84-93. In some embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown 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 shown in any one of SEQ ID NOs 85, 86, 88, and 90. In some embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises the 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 the 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 the 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 the 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 the 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 shown 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 shown in SEQ ID NO 93.

[0294] 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 wherein the one or more heterologous gene products are or comprise an immunomodulatory protein, a complement inhibitor, a T cell or NK cell escape product, an anti-angiogenic protein, an interferon regulatory factor, an apoptosis-inducing protein, or any combination thereof; and wherein: the at least one viral gene is or comprises A35R, optionally wherein the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown in 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 shown in SEQ ID NO: 3.

[0295] 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 wherein the one or more heterologous gene products are or comprise an immunomodulatory protein, a complement inhibitor, a T cell or NK cell escape product, an anti-angiogenic protein, an interferon regulatory factor, an apoptosis-inducing protein, or any combination thereof; and wherein: the at least one viral gene is or comprises A35R and J2R, optionally wherein the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown in 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 shown in SEQ ID NO: 12.

[0296] 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 wherein the one or more heterologous gene products are or comprise immunomodulatory proteins, complement inhibitors, T cell or NK cell escape products, anti-angiogenic proteins, interferon regulatory factors, apoptosis-inducing proteins, or any combination thereof; and wherein: the at least one viral gene is or comprises J2R, and the inactivating mutation of J2R is achieved by inserting one or more heterologous nucleic acids, each encoding one or more T cell or NK cell escape proteins, optionally wherein the one or more T cell or NK cell escape proteins comprise a set of proteins encoded by vaccinia virus ORF012, 203 and 018 (CPXV012-203-018), and wherein 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, and the heterologous nucleic acids are introduced into the 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 at the C-terminus of the F14.5L protein, and optionally wherein the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown in 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.

[0297] 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 wherein the one or more heterologous gene products are or comprise an immunomodulatory protein, a complement inhibitor, a T cell or NK cell escape product, an anti-angiogenic protein, an interferon regulatory factor, an apoptosis-inducing protein, or any combination thereof; and wherein: the at least one viral gene is or comprises J2R, optionally wherein the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown in SEQ ID NO:4, or a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO:4.

[0298] 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 wherein the one or more heterologous gene products are or comprise an immunomodulatory protein, a complement inhibitor, a T cell or NK cell escape product, an anti-angiogenic protein, an interferon regulatory factor, an apoptosis-inducing protein, or any combination thereof; and wherein: 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 each encoding one or more immunomodulatory proteins, optionally wherein the one or more immunomodulatory proteins are selected from LIGHT, IRF3, IL-2, IL-12 and CXCL9, optionally wherein the one or more immunomodulatory proteins is LIGHT; and optionally wherein the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown in SEQ ID NO: 11, 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: 11.

[0299] 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 wherein the one or more heterologous gene products are or comprise an immunomodulatory protein, a complement inhibitor, a T cell or NK cell escape protein, an anti-angiogenic protein, an interferon regulatory factor, an apoptosis-inducing protein, or any combination thereof; and wherein: the at least one viral gene is or comprises 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 wherein the one or more anti-angiogenic proteins comprise an inhibitor of VEGF and / or an inhibitor of Ang2, optionally wherein the one or more anti-angiogenic proteins are bispecific anti-VEGF / anti-Ang2 antibodies; and optionally wherein the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown in 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.

[0300] 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 wherein the one or more heterologous gene products are or comprise an immunomodulatory protein, a complement inhibitor, a T cell or NK cell escape protein, an anti-angiogenic protein, an interferon regulatory factor, an apoptosis-inducing protein, or any combination thereof; and wherein: the at least one viral gene is or comprises J2R and A35R, and the inactivating mutation of A35R is by insertion of a heterologous nucleic acid encoding one or more immunomodulatory proteins, optionally wherein the one or more immunomodulatory proteins are selected from LIGHT, IRF3, IL-2, IL-12 and CXCL9, optionally wherein 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 wherein the one or more anti-angiogenic proteins comprise an inhibitor of VEGF and / or an inhibitor of Ang2, optionally wherein the one or more anti-angiogenic proteins are bispecific anti-VEGF / anti-Ang2 antibodies; and optionally wherein the nucleic acid genome of the recombinant oncolytic virus comprises SEQ ID The nucleic acid sequence shown in 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.

[0301] 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 wherein the one or more heterologous gene products are or comprise an immunomodulatory protein, a complement inhibitor, a T cell or NK cell escape protein, an anti-angiogenic protein, an interferon regulatory factor, an apoptosis-inducing protein, or any combination thereof; and wherein: the 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 a cell apoptosis-inducing protein, optionally wherein the cell apoptosis-inducing protein is inducible DED (iDED), inducible Fas (iFas) or inducible Cas9 (iCas9), optionally wherein the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown in SEQ ID NO: 7, 8 or 9, 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: 7, 8 or 9.

[0302] 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 wherein the one or more heterologous gene products are or comprise an immunomodulatory protein, a complement inhibitor, a T cell or NK cell escape protein, an anti-angiogenic protein, an interferon regulatory factor, an apoptosis-inducing protein, or any combination thereof; and wherein: the 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 immunomodulatory proteins, optionally wherein the one or more immunomodulatory proteins are selected from LIGHT, IRF3, IL-2, IL-12 and CXCL9, optionally wherein the one or more immunomodulatory proteins are IRF3; optionally wherein the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown in 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.

[0303] 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 wherein the one or more heterologous gene products are or comprise an immunomodulatory protein, a complement inhibitor, a T cell or NK cell escape protein, an anti-angiogenic protein, an interferon regulatory factor, an apoptosis-inducing protein, or any combination thereof; and wherein: the at least one viral gene is or comprises J2R and B2R, optionally wherein the nucleic acid genome of the recombinant oncolytic virus comprises the 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.

[0304] 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 wherein the one or more heterologous gene products are or comprise immunomodulatory proteins, complement inhibitors, T cell or NK cell escape proteins, anti-angiogenic proteins, interferon regulatory factors, apoptosis-inducing proteins, or any combination thereof; and wherein: the at least one viral gene is or comprises 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 wherein the one or more immunomodulatory proteins are selected from LIGHT, IRF3, IL-2, IL-12 and CXCL9, optionally wherein the one or more immunomodulatory proteins are IRF3; optionally wherein the nucleic acid genome of the recombinant oncolytic virus comprises the 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.

[0305] 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 wherein the one or more heterologous gene products are or comprise immunomodulatory proteins, complement inhibitors, T cell or NK cell escape proteins, anti-angiogenic proteins, interferon regulatory factors, apoptosis-inducing proteins, or any combination thereof; and wherein: the at least one viral gene is or comprises J2R, B2R and A35R; wherein: the inactivating mutation of J2R is by insertion of one or more heterologous nucleic acids encoding one or more anti-angiogenic proteins, optionally wherein the one or more anti-angiogenic proteins comprise an inhibitor of VEGF and / or an inhibitor of Ang2, optionally wherein the one or more anti-angiogenic proteins comprise an inhibitor of VEGF and / or an inhibitor of Ang2, optionally wherein the one or more anti-angiogenic proteins comprise an inhibitor of VEGF and / or an inhibitor of Ang2, The angiogenic protein is a bispecific anti-VEGF / anti-Ang2 antibody; the inactivating mutation of B2R is by inserting one or more heterologous nucleic acids each encoding one or more immunomodulatory proteins, optionally wherein the one or more immunomodulatory proteins are selected from LIGHT, IRF3, IL-2, IL-12 and CXCL9, optionally wherein the one or more immunomodulatory proteins are IRF3; and the inactivating mutation of A35R is by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally wherein the one or more immunomodulatory proteins are selected from LIGHT, IRF3, IL-2, IL-12 and CXCL9, optionally wherein the one or more immunomodulatory proteins are LIGHT; and optionally wherein the nucleic acid genome of the recombinant oncolytic virus comprises the 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 with the nucleic acid sequence shown in SEQ ID NO:82.

[0306] 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 wherein the one or more heterologous gene products are or comprise immunomodulatory proteins, complement inhibitors, T cell or NK cell escape proteins, anti-angiogenic proteins, interferon regulatory factors, apoptosis-inducing proteins, or any combination thereof; and wherein: the at least one viral gene is or comprises J2R, B2R and A56R; wherein: the inactivating mutation of J2R is by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally wherein the one or more immunomodulatory proteins are IRF3; the inactivating mutation of A56R is by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally wherein the one or more immunomodulatory proteins are IL-2, optionally wherein IL-2 is IL-2 super factor, optionally MDNA11; and optionally wherein the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown in SEQ ID NO: 84, or the nucleic acid sequence of SEQ ID NO: 84. The nucleic acid sequence shown in NO:84 has a nucleic acid sequence with at least 95%, 96%, 97%, 98% or 99% sequence identity.

[0307] 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 wherein the one or more heterologous gene products are or comprise immunomodulatory proteins, complement inhibitors, T cell or NK cell escape proteins, anti-angiogenic proteins, interferon regulatory factors, apoptosis-inducing proteins, or any combination thereof; and wherein: the at least one viral gene is or comprises J2R, B2R, and A56R; wherein: the inactivating mutation of J2R is by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally wherein the one or more immunomodulatory proteins are IRF3; the inactivating mutation of A56R is by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally wherein the one or more immunomodulatory proteins comprise two or more immunomodulatory proteins selected from LIGHT, IRF3, IL-2, IL-12, and CXCL9, optionally wherein the two or more immunomodulatory proteins comprise IL-12 and CXCL9; and optionally wherein the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown in SEQ ID NO: 85, or the nucleic acid sequence of SEQ ID NO: 86. The nucleic acid sequence shown in NO:85 has a nucleic acid sequence with at least 95%, 96%, 97%, 98% or 99% sequence identity.

[0308] 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 wherein the one or more heterologous gene products are or comprise immunomodulatory proteins, complement inhibitors, T cell or NK cell escape proteins, anti-angiogenic proteins, interferon regulatory factors, apoptosis-inducing proteins, or any combination thereof; and wherein: the at least one viral gene is or comprises J2R, B2R and A56R; wherein: the inactivating mutation of B2R is by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally wherein the one or more immunomodulatory proteins are The inactivating mutation of A56R is by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally wherein the one or more immunomodulatory proteins comprise two or more immunomodulatory proteins selected from LIGHT, IRF3, IL-2, IL-12 and CXCL9, optionally wherein the two or more immunomodulatory proteins comprise IL-12 and CXCL9; the inactivating mutation of J2R is by inserting one or more heterologous nucleic acids each encoding a cell apoptosis-inducing protein, optionally wherein the cell apoptosis-inducing protein is an inducible DED (iDED); and optionally wherein 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 with the nucleic acid sequence shown in SEQ ID NO:86.

[0309] 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 wherein the one or more heterologous gene products are or comprise an immunomodulatory protein, a complement inhibitor, a T cell or NK cell escape protein, an anti-angiogenic protein, an interferon regulatory factor, an apoptosis-inducing protein, or any combination thereof; and wherein: the at least one viral gene is or comprises J2R, B2R, A35R and A56R; wherein: the inactivating mutation of J2R is by insertion of one or more heterologous nucleic acids encoding one or more anti-angiogenic proteins, optionally wherein the one or more anti-angiogenic proteins comprise an inhibitor of VEGF and / or an inhibitor of Ang2, optionally wherein the one or more anti-angiogenic proteins are Bispecific anti-VEGF / anti-Ang2 antibody; the inactivating mutation of B2R is achieved by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally wherein the one or more immunomodulatory proteins are IRF3; the inactivating mutation of A35R is achieved by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally wherein the one or more immunomodulatory proteins are selected from LIGHT, IRF3, IL-2, IL-12 and CXCL9, optionally wherein the one or more immunomodulatory proteins are LIGHT; the inactivating mutation of A56R is achieved by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, wherein the one or more immunomodulatory proteins are IL-2 super factor MDNA11; and optionally wherein 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 with the nucleic acid sequence shown in SEQ ID NO:87.

[0310] 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 wherein the one or more heterologous gene products are or comprise immunomodulatory proteins, complement inhibitors, T cell or NK cell escape proteins, anti-angiogenic proteins, interferon regulatory factors, apoptosis-inducing proteins, or any combination thereof; and wherein: the at least one viral gene is or comprises J2R, B2R, A35R and A56R; wherein: the inactivating mutation of J2R is by inserting one or more heterologous nucleic acids encoding one or more anti-angiogenic proteins, optionally wherein the one or more anti-angiogenic proteins comprise an inhibitor of VEGF and / or an inhibitor of Ang2, optionally wherein the one or more anti-angiogenic The protein is a bispecific anti-VEGF / anti-Ang2 antibody; the inactivating mutation of B2R is achieved by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally wherein the one or more immunomodulatory proteins are IRF3; the inactivating mutation of A35R is achieved by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally wherein the one or more immunomodulatory proteins are selected from LIGHT, IRF3, IL-2, IL-12 and CXCL9, optionally wherein the one or more immunomodulatory proteins are LIGHT; the inactivating mutation of A56R is achieved by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, wherein the one or more immunomodulatory proteins are IL-2 super factor MDNA11T, optionally wherein MDNA11T comprises the amino acid sequence shown in SEQ ID NO: 98; and optionally wherein 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 with the nucleic acid sequence shown in SEQ ID NO: 88.

[0311] 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 wherein the one or more heterologous gene products are or comprise immunomodulatory proteins, complement inhibitors, T cell or NK cell escape proteins, anti-angiogenic proteins, interferon regulatory factors, apoptosis-inducing proteins, or any combination thereof; and wherein: the at least one viral gene is or comprises J2R, B2R and A56R; wherein: the inactivating mutation of J2R is by inserting one or more heterologous nucleic acids each encoding one or more T cell or NK cell escape proteins, optionally wherein the one or more T cell or NK cell escape proteins comprise a group of proteins encoded by vaccinia virus ORF012, 203 and 018 (CPXV012-203-018); the inactivating mutation of B2R is by inserting one or more encoding one or more A heterologous nucleic acid encoding an immunomodulatory protein, optionally wherein the one or more immunomodulatory proteins are IRF3; the inactivating mutation of A56R is achieved by inserting one or more heterologous nucleic acids each encoding one or more immunomodulatory proteins, optionally wherein the one or more immunomodulatory proteins are selected from LIGHT, IRF3, IL-2, IL-12 and CXCL9, optionally wherein the one or more immunomodulatory proteins are IL-2 super factors, optionally MDNA11 or MDNA11T; the 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 (optionally CRASP-2), the heterologous nucleic acid being introduced into a viral membrane gene (optionally F14.5L) to produce a fusion gene encoding a fusion protein, optionally wherein the fusion is at the C-terminus of the F14.5L protein; and optionally wherein 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.

[0312] 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 wherein the one or more heterologous gene products are or comprise immunomodulatory proteins, complement inhibitors, T cell or NK cell escape proteins, anti-angiogenic proteins, interferon regulatory factors, apoptosis-inducing proteins, or any combination thereof; and wherein: the at least one viral gene is or comprises J2R, B2R and A56R; wherein: the inactivating mutation of J2R is by inserting one or more heterologous nucleic acids each encoding one or more T cell or NK cell escape proteins, optionally wherein the one or more T cell or NK cell escape proteins comprise a group of proteins encoded by vaccinia virus ORF012, 203 and 018 (CPXV012-203-018); the inactivating mutation of B2R is by inserting one or more encoding one or more A heterologous nucleic acid encoding an immunomodulatory protein, optionally wherein the one or more immunomodulatory proteins are IRF3; the inactivating mutation of A56R is achieved by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally wherein the one or more immunomodulatory proteins comprise two or more immunomodulatory proteins selected from LIGHT, IRF3, IL-2, IL-12 and CXCL9, optionally wherein the two or more immunomodulatory proteins comprise IL-12 and CXCL9; the 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 (optionally CRASP-2), the heterologous nucleic acid being introduced into a viral membrane gene (optionally F14.5L) to produce a fusion gene encoding a fusion protein, optionally wherein the fusion is at the C-terminus of the F14.5L protein; and optionally wherein 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.

[0313] 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 wherein the one or more heterologous gene products are or comprise an immunomodulatory protein, a complement inhibitor, a T cell or NK cell escape protein, an anti-angiogenic protein, an interferon regulatory factor, an apoptosis-inducing protein, or any combination thereof; and wherein: 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 wherein the one or more immunomodulatory proteins are selected from LIGHT, IRF3, IL-2, IL-12 and CXCL9, optionally wherein the one or more immunomodulatory proteins are IRF3; and optionally wherein 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.

[0314] 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 wherein the one or more heterologous gene products are or comprise immunomodulatory proteins, complement inhibitors, T cell or NK cell escape proteins, anti-angiogenic proteins, interferon regulatory factors, apoptosis-inducing proteins, or any combination thereof; and wherein: the at least one viral gene is or comprises B2R, J2R and A56R, and the inactivating mutation of J2R is achieved by inserting one or more heterologous nucleic acids, each encoding one or more immunomodulatory proteins, optionally wherein wherein the one or more immunomodulatory proteins are selected from LIGHT, IRF3, IL-2, IL-12 and CXCL9, optionally wherein the one or more immunomodulatory proteins are IRF3; and the inactivating mutation of A56R is by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally wherein the one or more immunomodulatory proteins comprise two or more immunomodulatory proteins selected from LIGHT, IRF3, IL-2, IL-12 and CXCL9, optionally wherein the two or more immunomodulatory proteins comprise IL-12 and CXCL9; and optionally wherein 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 with the nucleic acid sequence shown in SEQ ID NO:92.

[0315] 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 wherein the one or more heterologous gene products are or comprise an immunomodulatory protein, a complement inhibitor, a T cell or NK cell escape protein, an anti-angiogenic protein, an interferon regulatory factor, an apoptosis-inducing protein, or any combination thereof; and wherein: the at least one viral gene is or comprises J2R, and the inactivating mutation of J2R is by one or more heterologous nucleic acids each encoding one or more immunomodulatory proteins, optionally wherein the one or more immunomodulatory proteins are selected from LIGHT, IRF3, IL-2, IL-12 and CXCL9, optionally wherein the one or more immunomodulatory proteins are IRF3; and optionally wherein the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown 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 shown in SEQ ID NO: 93.

[0316] In some of any such embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown 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 shown in any one of SEQ ID NOs: 48, 80, 82, and 84-93; and is characterized by one or more of the following: (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 comprising a polar uncharged amino acid at position 66, optionally a threonine (T) at position 66; (ii) a variant 038 (K5L) ORF comprising a nucleotide insertion to cause a frameshift mutation, wherein the 038 (K5L) gene product is altered; (iii) a variant 059 (E2L) ORF encoding an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 57 NO:60 has at least 95% sequence identity and comprises a hydrophobic amino acid other than leucine at position 419, optionally an amino acid sequence of phenylalanine (F) at position 419; (iv) variant 104 (H4L) ORF, which encodes an amino acid sequence having at least 95% sequence identity to SEQ ID NO:61 and comprising a negatively charged amino acid at position 591, optionally an aspartic acid (D) at position 591; and (v) variant 182 (A56R) ORF, which comprises a deletion of two nucleotides to cause a frameshift mutation, wherein the 182 (A56R) ORF gene product is altered.

[0317] In some of any such embodiments, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown 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 shown in any one of SEQ ID NOs: 48, 80, 82, and 84-93; and is characterized by one or more of the following: (i) a guanine (G) at a position corresponding to position 7770 of SEQ ID NO: 1; (ii) a thymine (T) at a position corresponding to position 15261 of SEQ ID NO: 1; (iii) a G at a position corresponding to position 32136 of SEQ ID NO: 1; (iv) a G at a position corresponding to position 49455 of SEQ ID NO: 1; (v) a cytosine (C) at a position corresponding to position 92969 of SEQ ID NO: 1; (vi) a thymine (T) at a position corresponding to position 15261 of SEQ ID NO: 1; NO:1, (i) the nucleic acid sequence CACTTATAT at positions corresponding to positions 106870 to 106880; (vii) the nucleic acid sequence GTTTTCATTA at positions corresponding to positions 111267 to 111276 of SEQ ID NO:1; (viii) adenine (A) at position corresponding to position 162715 of SEQ ID NO:1; (ix) the nucleic acid sequence TACAGACACC at positions corresponding to positions 165844 to 185853 of SEQ ID NO:1; and (x) C at position corresponding to position 187805 of SEQ ID NO:1. A. Recessive virus

[0318] In a number of embodiments, provided herein are recombinant viruses comprising heterologous nucleic acids encoding "cryptic proteins" that can be stably and efficiently expressed in cells infected by various types of viruses. Such cryptic proteins can enhance the ability of viruses to escape the immune system attack of the host, such as escaping T cells (such as cytotoxic T lymphocytes (CTL)) or natural killer (NK) cell attacks. In some embodiments, such cryptic proteins can enhance the ability of recombinant viruses to escape the complement cascade / system activation of the host.

[0319] 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 escape protein (sometimes also referred to as a cryptic protein).

[0320] 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 a vaccinia virus. In some embodiments, the recombinant virus comprises a virus derived from VIP02.

[0321] Oncolytic viruses (OVs) can create a favorable microenvironment for the immune system to target unique cancer cell determinants; however, antiviral immune responses against viral infection are also a key factor in OV-based therapies. Indeed, induced antiviral immunity can be detrimental to cancer virotherapy, as immune activation against the virus itself is expected to limit viral replication and spread, leading to reduced therapeutic efficacy (Lemos de Matos et al., Mol Ther Methods Clin Dev. 2020 Jun 12;17:349–358). The complement system continuously monitors viruses. Its ability to recognize viruses and virus-infected cells and trigger an immune response leads to viral neutralization and killing of infected cells. This selective pressure exerted by complement on viruses has led to the evolution of diverse coping strategies (Agrawal et al., Front Microbiol. 2017;8:1117).

[0322] In some embodiments, the cryptic proteins include, but are not limited to, burgdorferi complement regulatory acquisition surface protein-2 (CRASP-2), minimized complement regulatory factor H (miniFH), and vaccinia virus ORF012, 203, and 018 (CPXV012-203-018). More detailed information about cryptic proteins and mechanisms involved in escaping the host's immune system (such as escaping the host's complement or NK or T cell cytotoxicity) 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 in its entirety.

[0323] The complement system is an important component of innate immunity that helps eliminate pathogens. Therefore, during evolution, pathogens have developed a variety of strategies to avoid being destroyed by complement activation. One of these strategies is to acquire proteins that enable pathogens to control the steps involved in activating the host immune response during infection, hereinafter referred to as "cryptic proteins" (see Kraiczy et al., Infect Immun. 2001 Dec; 69(12): 7800–7809).

[0324] The complement system triggers inflammation, chemotaxis of phagocytes and neutrophils, pathogen neutralization, and subsequent opsonization, as well as lysis of infected cells through a complex proteolytic cascade targeting more than 30 plasma and cell membrane proteins. Complement activation can be initiated through three independent pathways: (i) the classical pathway, in which the first component of the complement cascade, C1q, binds to the antibody-antigen complex; (ii) the alternative pathway, in which the downstream complement component 3 (C3) convertase spontaneously hydrolyzes and interacts with the pathogen surface; and (iii) the mannose-binding lectin (MBL) pathway, which is triggered by the binding of MBL to mannose residues on the pathogen surface. All three pathways converge at the stage where C3 is cleaved into the antimicrobial peptide C3a and the opsonin C3b, which binds to the pathogen and marks it for degradation. Because effector compounds produced in the complement cascade can be delivered to any surface, including host cell membranes, intact host cells protect themselves by expressing a variety of complement regulatory proteins (Janeway et al., Immunobiology: The Immune System in Health and Disease. 5th edition).

[0325] Inadequate control of the complement system underlies or exacerbates numerous human diseases. The alternative pathway (AP) of complement has the unique property of being persistently and indiscriminately activated, albeit at low levels. In the AP, C3b propagates itself through a positive feedback amplification loop that requires tight regulation by two key soluble AP regulators, factor H (FH) and its spliced ​​product, FH-like-1 (FHL-1). The hidden protein miniFH is an engineered version of FH that contains only the N-terminal and C-terminal parts of FH, connected by an optimized peptide. Compared with FH, its potency in inhibiting complement activation in vitro is about 10-fold higher (FH.Markus J.Harder,*J Immunol.Author manuscript; available in PMC 2017Jan 15.J Immunol.2016Jan 15;196(2):866–876.&Christoph Q.Schmidt / J Immunol.Author manuscript; available in PMC 2014Jun 1.J Immunol.2013Jun 1;190(11):10.4049 / jimmunol.1203548.Publishedonline 2013Apr 24.doi:10.4049 / jimmunol.1203548).

[0326] A microorganism that has evolved to evade complement by producing cryptic proteins is Borrelia burgdorferi, a spirochete that causes Lyme disease (LD), the most common tick-borne disease in the Northern Hemisphere. During a blood meal, the spirochetes are exposed to the host's blood and are therefore faced with the first line of defense of innate immunity, which they must overcome in order to survive. A key escape mechanism developed by Borrelia burgdorferi is the production of complement or CRP-binding proteins, including CRASP, a cryptic protein that promotes complement inactivation (see Yi-Pin Lin et al., Front Cell Infect Microbiol. 2020; 10:1.; US20120142023A1). CRASP-2 (also known as CspZ) binds to FH / FHL-1 and confers serum resistance to Borrelia burgdorferi by inhibiting complement activation on the spirochete surface (Infect Immun. 2001 Dec; 69(12): 7800–7809. Peter Kraiczy. US20200323972A1 Compositions and methods for generating immunity against Borrelia burgdorferi).

[0327] Downregulating the expression of MHC class I molecules on the cell surface is an immune escape mechanism shared by many DNA viruses, including vaccinia virus. CPXV is a member of the genus Orthopoxvirus, which includes smallpox virus, camelpox virus, and monkeypox virus, and encodes a complex series of immune escape proteins. The ability of CPXV to infect a variety of mammalian hosts may be because among orthopoxviruses, CPXV encodes the most complete set of open reading frames that are expected to encode immunomodulatory proteins. The proteins it encodes include CPXV012 and CPXV203, which can prevent the recognition of cytotoxic T cells by interfering with antigen presentation mediated by MHC class I molecules. However, CPXV012 inhibits the transport of antigenic peptides from the cytoplasm to the ER, and CPXV203 blocks the transport of MHC class I molecules to the cell surface (Dina Alzhanova and Klaus Früh*Microbes Infect. 2010 Nov; 12(12-13): 900–909. McCoy et al., Molecular Immunology 55(2013) 156–158). In addition, the Brighton Red strain produces OMCP (also known as CPXV018), a 171-amino acid residue protein that is secreted in large quantities 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 / Journal home page for Cell Host & Microbe / Two Mechanistically Distinct Immune Evasion Proteins of Cowpox Virus Combine to Avoid Antiviral CD8 T Cells).

[0328] In some embodiments, one or more heterologous gene products comprise a complement inhibitor. In some embodiments, the complement inhibitor is Borrelia burgdorferi complement regulatory acquisition surface protein-2 (CRASP-2) or minimized complement regulatory factor H (miniFH). In some embodiments, the complement inhibitor is the CRASP-2 gene product (UniProtKB-050665). The CRASP-2 protein can enhance the ability of the recombinant virus to escape the host's complement. Specifically, in some embodiments, the recombinant virus comprises an expression cassette containing CRASP-2 cDNA fused to the F14.5L locus under the control of the vaccinia virus 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 having 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.

[0329] 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.

[0330] In some embodiments, the recombinant virus comprises an amino acid sequence that has at least 70% (e.g., 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 that 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%, 99.9% sequence identity to the amino acid sequence of SEQ ID NO: 18, but is less than 100% identical to the amino acid sequence of SEQ ID NO: 18.

[0331] 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 clone VIP02 (comprising the nucleic acid sequence of SEQ ID NO: 1) strain and comprises a nucleotide sequence having at least 70% (e.g., at least 75%, 80%, 85%, or 90%) sequence identity to SEQ ID NO: 5 (also known as VIR27). In some embodiments, the heterologous gene product is CRASP-2 and is operably linked to the F14.5L gene promoter in the viral genome. 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 viral genome, and the recombinant virus comprises a nucleic acid sequence with 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 viral genome, and the recombinant virus comprises a nucleic acid sequence with SEQ ID NO:5. In some embodiments, the recombinant virus (e.g., a recombinant oncolytic virus) comprises a nucleic acid sequence of SEQ ID NO:5. The recombinant oncolytic virus comprising a SEQ ID NO:5 nucleic acid sequence is also referred to herein as VIR27.

[0332] In various embodiments, the recombinant viruses provided herein exhibit increased ability to escape the host's immune system. In some embodiments, the recombinant viruses provided herein can escape complement inhibition in vivo and in vitro systems. In specific embodiments, when incubated with an effective dose of VIR27 (comprising the nucleic acid sequence of SEQ ID NO: 5), VIR27 (comprising the nucleic acid sequence of SEQ ID NO: 5) can escape complement inhibition in an in vitro complement inhibition system ( Figure 6 In certain embodiments, administering an effective dose of VIR27 (comprising the nucleic acid sequence of SEQ ID NO: 5) to a subject can inhibit tumor, hyperplasia, or metastasis growth in an in vivo model ( Figure 7 ).

[0333] In some embodiments, the complement inhibitor is a miniFH gene product. Specifically, in some embodiments, the recombinant virus comprises an expression cassette containing a miniFH cDNA fused to the F14.5L locus under the control of the vaccinia virus F14.5L gene promoter. For more detailed information about miniFH, see Schmidt et al., J Immunol. 2013 Jun 1; 190(11): 10.4049 / jimmunol.1203548., which is incorporated herein by reference in its entirety. 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.

[0334] In some embodiments, the stealth protein comprises an FH-based inhibitor, miniFH. In some embodiments, the miniFH gene product can enhance the ability of the recombinant virus to escape the host's complement. In some embodiments, a recombinant virus is provided, 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% (e.g., 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 that 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 amino acid sequence of SEQ ID NO: 19, but is less than 100% identical to the amino acid sequence of SEQ ID NO: 19.

[0335] 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 shown in SEQ ID NO: 6. In some embodiments, the heterologous gene product is miniFH and is operably linked to the F14.5L gene promoter in the viral genome, 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, but less than 100% identical to the nucleic acid sequence of SEQ ID NO: 6. In some embodiments, the heterologous gene product is miniFH and is operably linked to the F14.5L gene promoter in the viral genome, and the recombinant virus comprises the nucleic acid sequence of SEQ ID NO: 6. In some embodiments, the recombinant virus (e.g., a 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.

[0336] In some embodiments, the polynucleotide encoding the miniFH molecule is operably linked to the F14.5L gene promoter. In some embodiments, the recombinant virus comprising a polynucleotide encoding the miniFH molecule (e.g., comprising the amino acid sequence of SEQ ID NO: 19) is derived from the clone VIP02 (comprising the nucleic acid sequence of SEQ ID NO: 1) strain and comprises a nucleic acid sequence having at least 70% (e.g., at least 75%, 80%, 85%, or 90%) sequence identity to SEQ ID NO: 6 (also known as 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.

[0337] In various embodiments, the recombinant viruses provided herein exhibit increased ability to escape host complement. In some embodiments, the recombinant viruses provided herein can escape complement inhibition in vivo and in vitro systems. In specific embodiments, when an effective dose of VIR37 (comprising the nucleic acid sequence of SEQ ID NO: 6) is incubated with human serum and / or BABL / c mouse serum, VIR37 (comprising the nucleic acid sequence of SEQ ID NO: 6) can escape complement inhibition in an in vitro complement inhibition system ( Figure 6 ).

[0338] In some embodiments, one or more heterologous gene products are T cell escape proteins or NK cell escape proteins. T cell escape proteins or NK cell escape protein gene products can enhance the ability of the virus to escape the immune system attack of the host, such as the attack of escape T cells (such as cytotoxic T lymphocytes (CTL)) or natural killer (NK) cells. Specifically, such T cell escape proteins or NK cell escape protein gene products can enhance the ability of the recombinant virus to escape the complement cascade / system activation of the host.

[0339] In some embodiments, the T cell escape protein or NK cell escape protein is a group of proteins encoded by vaccinia virus ORFs 012, 203, and 018 (CPXV012-203-018). CPXV012-203-018 is a synthetic DNA fragment. In the CPXV012-203-018 synthetic DNA fragment, ORFs 012, 203, and 018 are expressed separately under their respective promoters to encode CPXV012, CPXV203, and CPXV018 proteins. Vaccinia virus escapes CTLs through CPXV012 and CPXV203. CPXV012 inhibits the transport of antigenic peptides from the cytoplasm to the endoplasmic reticulum (ER), while CPXV203 blocks the transport of MHC class I molecules to the cell surface by utilizing the KDEL receptor recycling pathway. CPXV018 encodes a soluble NKG2D ligand, called orthopoxvirus major histocompatibility complex (MHC) class I-like protein (OMCP), which can block NKG2D-mediated cytotoxicity.

[0340] In various embodiments, recombinant viruses expressing the CRASP-2 gene product (UniProtKB-050665) and vaccinia virus open reading frames (ORFs) 012, 203, and 018 (CPXV012-203-018) have been generated herein. Specifically, in some embodiments, the recombinant virus (e.g., a recombinant oncolytic virus) comprises an expression cassette containing CRASP-2 cDNA fused to the F14.5L locus under the control of the vaccinia virus F14.5L gene promoter and a continuous polynucleotide sequence comprising open reading frames (ORFs) 012, 203, and 018 (CPXV012-203-018) with their respective promoters and inserted into the J2R locus.

[0341] In some embodiments, the latent protein comprises the Borrelia burgdorferi complement regulatory acquisition surface protein-2 (CRASP-2). In some embodiments, expression of the CRASP-2 protein can enhance the ability of the recombinant virus to escape the host's complement. In some embodiments, a recombinant virus is provided, comprising a polynucleotide encoding a CRASP-2 molecule, the polynucleotide 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 latent protein comprises vaccinia virus open reading frames (ORFs) 012, 203, and 018 (CPXV012-203-018) with their respective promoters. In some embodiments, expression of vaccinia virus open reading frames (ORFs) 012, 203, and 018 (CPXV012-203-018) can enhance the ability of the recombinant virus to escape the host's T cells and NK cells. In some embodiments, expression of cryptic proteins including CRASP-2 and vaccinia virus open reading frames (ORFs) 012, 203, and 018 can enhance the ability of the recombinant virus to evade the host's complement, T cells, and NK cells.

[0342] In some embodiments, the T cell escape protein or NK cell escape protein is a group of proteins encoded by vaccinia virus ORFs 012, 203, and 018 (CPXV012-203-018), comprising the amino acid sequences set forth in SEQ ID NOs: 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 NOs: 20, 21, and 22. In some embodiments, the recombinant virus comprises a polypeptide encoding CPXV012 having an amino acid sequence having at least 70% (e.g., 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 that 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 amino acid sequence of SEQ ID NO: 20, but is 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 that has at least 70% (e.g., 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 that 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 amino acid sequence of SEQ ID NO: 21, but is 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 sequence encoding CPXV018 having an amino acid sequence that has at least 70% (e.g., 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 that 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 amino acid sequence of SEQ ID NO: 22, but is 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.

[0343] In some embodiments, the T cell escape protein or NK cell escape protein is a group of proteins encoded by vaccinia virus ORFs 012, 203, and 018 (CPXV012-203-018), and the group of proteins encoded by CPXV012-203-018 comprises an amino acid sequence that exhibits at least 85%, 90%, or 95% sequence identity to the sequence shown in SEQ ID NO: 20 (CPXV012), an amino acid sequence that exhibits at least 85%, 90%, or 95% sequence identity to the sequence shown in SEQ ID NO: 21 (CPXV0203), and an amino acid sequence that exhibits at least 85%, 90%, or 95% sequence identity to the sequence shown in SEQ ID NO: 22 (CPXV018). In some embodiments, the group 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.

[0344] In some embodiments, the recombinant virus comprises a polypeptide encoding CRASP-2 having an amino acid sequence that has at least 70% (e.g., 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 that 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 amino acid sequence of SEQ ID NO: 18, but is 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 that encodes 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.

[0345] In some embodiments, the recombinant virus comprising a nucleic acid sequence 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 (comprising the nucleic acid sequence of SEQ ID NO: 5) strain and comprises a nucleotide sequence having at least 70% (e.g., at least 75%, 80%, 85%, or 90%) sequence identity to SEQ ID NO: 10 (also known as VIR46). For example, in some embodiments, the recombinant virus comprises a nucleic acid sequence that 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.

[0346] In various embodiments, the recombinant viruses provided herein exhibit increased ability to escape the host's complement. In some embodiments, the recombinant viruses provided herein can escape complement inhibition in vivo and in vitro systems. In certain embodiments, administering an effective dose of VIR46 (comprising the nucleic acid sequence of SEQ ID NO: 10) to a subject can inhibit tumor, hyperplasia, or metastasis growth in an in vivo model ( Figure 8 ). B. Immunomodulatory viruses

[0347] In multiple embodiments, provided herein are recombinant viruses comprising heterologous nucleic acids encoding immunomodulatory proteins that can be stably and efficiently expressed in cells infected by various types of viruses. In some embodiments, immunomodulatory proteins include cytokines, chemokines, immune receptors, antigens of immune receptors, proteins in immune cell activation pathways, signaling proteins that stimulate immune cell activation or cytokine secretion in immune cells, and antigens. In some embodiments, the immunomodulatory protein comprises one or more cytokines and / or chemokines. In some embodiments, the one or more cytokines and / or chemokines comprise 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 super factor. In some embodiments, the immunomodulatory protein is an IL-2 super factor. In some embodiments, the immunomodulatory protein is MDNA11. In some embodiments, MDNA11 has been mutated to enhance the anti-tumor efficacy of the recombinant virus, and the immunomodulatory protein is MDNA11T. In some embodiments, the recombinant virus comprises 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, one or more immunomodulatory proteins are immunostimulatory proteins, such as LIGHT.

[0348] 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 immunomodulatory proteins. In some embodiments, the one or more immunomodulatory proteins are or comprise one or more cytokines and / or chemokines. In some embodiments, the one or more immunomodulatory proteins are or comprise one or more interferon regulatory factors, such as IRF3. In some embodiments,...

Claims

1. A recombinant oncolytic vaccinia virus, comprising: Inactivating mutations 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.

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 claim 2, wherein the at least one heterologous nucleic acid encoding one or more cytokines and / or chemokines comprises heterologous nucleic acids encoding CXCL9 and IL-12.

4. The recombinant oncolytic vaccinia virus of 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. The recombinant oncolytic vaccinia virus according to any one of claims 1 to 4, wherein: The 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 The CXCL9 is mouse CXCL9 and comprises the amino acid sequence shown 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.

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

102.

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

8. The recombinant oncolytic vaccinia virus of claim 7, wherein the IL-2 comprises the amino acid sequence shown 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 shown in 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 the IL-2 is an IL-2 superkine.

10. The recombinant oncolytic vaccinia virus of claim 9, wherein the IL-2 super factor is H9, H9T, MDNA11 or MDNA11T, and wherein: The H9 IL-2 super factor 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 super factor 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 super factor 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 super factor 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.

11. The recombinant oncolytic vaccinia virus of claim 6 or claim 7, wherein the IL-2 super factor is MDNA11T, and the MDNA11T comprises the amino acid sequence shown in SEQ ID NO: 98, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:

98.

12. The recombinant oncolytic vaccinia virus according to any one of claims 1 to 11, further comprising one or more heterologous gene products, wherein the heterologous gene products are selected from the group consisting of complement inhibitors, T cell or NK cell escape products, immunostimulatory proteins, anti-angiogenic proteins, interferon regulatory factors, apoptosis-inducing proteins, or any combination thereof.

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 of claim 13, wherein the deletion is sufficient to render the encoded B2R gene product nonfunctional.

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

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

17. The recombinant oncolytic vaccinia virus according to any one of claims 1 to 16, wherein: The heterologous nucleic acid encoding IRF3 is inserted into the genome of the virus 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; and / or The at least one heterologous nucleic acid encoding one or more cytokines and / or chemokines is inserted into the genome of the virus 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.

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 is modified from a parental vaccinia virus, and the nucleic acid genome of the parental vaccinia virus has at least 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 1, optionally the nucleic acid genome shown in SEQ ID NO:

1.

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

20. The recombinant oncolytic vaccinia virus according to any one of claims 1 to 19, wherein the nucleic acid genome of the parent 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 a position corresponding to position 92969 of SEQ ID NO: 1; (vi) the nucleic acid sequence CACTTATAT at positions corresponding to positions 106870 to 106880 of SEQ ID NO: 1; (vii) the nucleic acid sequence GTTTTCATTA at positions 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 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.

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 with the nucleic acid sequence shown in SEQ ID NO:

1.

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

23. The recombinant oncolytic vaccinia virus according to any one of claims 1-14 and 17-22, wherein the nucleic acid genome of the recombinant oncolytic vaccinia 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 with the nucleic acid sequence shown in SEQ ID NO:

85.

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

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 of claim 25, wherein the apoptosis-inducing protein is an inducible death effector domain (iDED).

27. The recombinant oncolytic vaccinia virus of claim 26, wherein the iDED comprises the amino acid sequence shown 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 shown in SEQ ID NO:

27.

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

29. The recombinant oncolytic vaccinia virus according to any one of claims 1-21 and 24-28, wherein the nucleic acid genome of the recombinant oncolytic vaccinia 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 with the nucleic acid sequence shown in SEQ ID NO:

86.

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

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

32. The recombinant oncolytic vaccinia virus according to claim 31, wherein the set of proteins encoded by CPXV012-203-018 comprises: (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.

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

34. The recombinant oncolytic vaccinia virus of claim 33, wherein the complement inhibitor is Borrelia burgdorferi complement regulatory acquisition surface protein-2 (CRASP-2).

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

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

38. The recombinant oncolytic vaccinia virus of any one of claims 1-22, 24-28, and 30-37, wherein the nucleic acid genome of the recombinant oncolytic vaccinia 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 shown in SEQ ID NO:

90.

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

40. The recombinant oncolytic vaccinia virus of any one of claims 1-39, 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 of claim 40, wherein the immunostimulatory protein is recombinant LIGHT.

42. The recombinant oncolytic vaccinia virus of claim 41, wherein the recombinant LIGHT comprises the amino acid sequence shown 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 shown in SEQ ID NO:

30.

43. The recombinant oncolytic vaccinia virus of any one of claims 40-42, wherein the 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.

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

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

46. ​​The recombinant oncolytic vaccinia virus of claim 45, wherein the bispecific anti-VEGF / anti-Ang2 antibody comprises the amino acid sequence shown 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 shown in SEQ ID NO:

23.

47. The recombinant oncolytic vaccinia virus of any one of claims 1-22, 24-28, 30-37, and 39-46, 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 shown in SEQ ID NO:

88.

48. A recombinant oncolytic virus, comprising: an inactivating mutation in 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 or comprise an immunomodulatory protein, a complement inhibitor, a T cell or NK cell escape product, an anti-angiogenic protein, an interferon regulatory factor, or an apoptosis-inducing protein, or any combination of the foregoing.

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 or comprise complement inhibitors, T cell or NK cell escape products, immunomodulatory proteins, anti-angiogenic proteins, interferon regulatory factors, apoptosis-inducing proteins, or any combination of the foregoing.

50. The recombinant oncolytic virus of claim 48 or claim 50, wherein the oncolytic virus is vaccinia virus, herpes simplex virus, vesicular stomatitis virus (VSV), Maraba virus (MARAV), measles virus (MV), adenovirus, myxoma virus, rubella virus, parvovirus, raccoon pox virus, coxsackie virus, reovirus, Newcastle disease virus, Seneca Valley virus, Semliki Forest virus, mumps virus, influenza virus, echovirus, and poliovirus (PV).

51. The recombinant oncolytic virus of any one of claims 48-50, wherein the oncolytic virus is a vaccinia virus.

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

1.

53. A recombinant oncolytic virus, comprising a nucleic acid genome modified from a parental vaccinia virus genome, wherein the parental vaccinia virus genome has at least 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 1, optionally wherein the parental vaccinia virus has the nucleic acid genome shown in SEQ ID NO: 1, wherein 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.

54. The recombinant oncolytic vaccinia virus of claim 52 or claim 53, wherein the nucleic acid genome of the parent vaccinia virus is characterized by one or more of the following: (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 comprising a polar uncharged amino acid at position 66, optionally comprising a threonine (T) at position 66; (ii) a variant 038(K5L) ORF comprising a nucleotide insertion resulting in a frameshift mutation, wherein the 038(K5L) gene product is altered; (iii) a variant 059(E2L) ORF encoding an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 60 and comprising a hydrophobic amino acid other than leucine at position 419, optionally comprising phenylalanine (F) at position 419; (iv) a variant 104(H4L) ORF encoding an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 61 and comprising a negatively charged amino acid at position 591, optionally comprising aspartic acid (D) at position 591; and (v) A variant 182(A56R) ORF comprising a deletion of two nucleotides resulting in a frameshift mutation, wherein the 182(A56R) ORF gene product is altered.

55. The recombinant oncolytic virus of any one of claims 52-54, wherein the parental vaccinia virus genome 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 a position corresponding to position 92969 of SEQ ID NO: 1; (vi) the nucleic acid sequence CACTTATAT at positions corresponding to positions 106870 to 106880 of SEQ ID NO: 1; (vii) the nucleic acid sequence GTTTTCATTA at positions 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 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.

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

1.

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

58. The recombinant oncolytic virus according to any one of claims 1 to 57, wherein the recombinant oncolytic virus is a recombinant oncolytic vaccinia virus, and wherein the nucleic acid genome of the recombinant oncolytic vaccinia 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 a position corresponding to position 92969 of SEQ ID NO: 1; (vi) the nucleic acid sequence CACTTATAT at positions corresponding to positions 106870 to 106880 of SEQ ID NO: 1; (vii) the nucleic acid sequence GTTTTCATTA at positions 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 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.

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

60. The recombinant oncolytic virus of any one of claims 48-59, wherein the at least one heterologous nucleic acid encoding one or more heterologous gene products is inserted into the genome of the virus comprising 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.

61. The recombinant oncolytic virus of claim 59, wherein the at least one heterologous nucleic acid encoding one or more heterologous gene products of non-essential genes or regions inserted into the viral genome is each independently inserted into the viral genome 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.

62. The recombinant oncolytic virus of any one of claims 48, 50-52, and 54-59, wherein 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.

63. The recombinant oncolytic virus according to any one of claims 48, 50-52, 54-59 and 62, wherein the at least one viral gene is or comprises: (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.

64. The recombinant oncolytic virus according to any one of claims 48, 50-52 and 54-63, wherein the inactivating mutation of one or more of the at least one viral gene is independently performed by: causing the at least one heterologous nucleic acid encoding one or more heterologous gene products to be inserted into a locus in the genome of the virus; All or part of the at least one viral gene is deleted; and / or One or more nucleic acid substitutions in said at least one viral gene.

65. The recombinant oncolytic virus of any one of claims 48, 50-52, and 54-64, wherein the inactivating mutation is a complete or partial deletion of at least one viral gene.

66. The recombinant oncolytic virus according to claim 64 or claim 65, wherein: The deletion of the at least one viral gene is a deletion of the entire gene ORF of the viral gene.

67. The recombinant oncolytic virus according to any one of claims 64-66, wherein: The deletion is sufficient to render the encoded viral gene product nonfunctional.

68. The recombinant oncolytic virus of claim 64, wherein the one or more nucleic acid substitutions are sufficient to render the encoded viral gene product nonfunctional.

69. The recombinant oncolytic virus of any one of claims 48, 50-52, 54-59, and 62-68, wherein the at least one viral gene is or comprises B2R.

70. A recombinant oncolytic virus according to any one of claims 48, 50-52 and 54-68, wherein the inactivating mutation(s) in at least one of the at least one viral genes is characterized by insertion of at least one of at least one heterologous nucleic acid encoding one or more heterologous gene products into the viral locus.

71. The recombinant oncolytic virus of claim 70, wherein the at least one viral gene comprises B2R.

72. The recombinant oncolytic virus of claim 70 or claim 71, wherein the at least one viral gene comprises J2R.

73. The recombinant oncolytic virus of any one of claims 70-72, wherein the at least one viral gene comprises A35R.

74. The recombinant oncolytic virus of any one of claims 70-73, wherein the at least one viral gene comprises A56R.

75. The recombinant oncolytic virus of any one of claims 70-74, wherein the at least one viral gene comprises B2R, J2R, and A35R.

76. The recombinant oncolytic virus of any one of claims 70-75, wherein the at least one viral gene comprises B2R, J2R, A35R, and A56R.

77. The recombinant oncolytic virus of any one of claims 70-76, 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 one or more heterologous gene products is inserted into or replaces F14.5L.

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

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

81. The recombinant oncolytic virus of any one of claims 48-80, wherein 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.

82. The recombinant oncolytic virus of any one of claims 48, 50-52, and 54-81, wherein the inactivating mutation of one or more of the at least one viral gene is achieved by inserting one or more heterologous nucleic acids each encoding one or more immunomodulatory proteins.

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

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

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

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

87. The recombinant oncolytic virus of any one of claims 81-86, 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 of any one of claims 81-87, 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 of claim 88, wherein the CXCL9 is human CXCL9 and comprises the amino acid sequence shown in SEQ ID NO: 99, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence of SEQ ID NO:

99.

90. The recombinant oncolytic virus of claim 88, wherein the CXCL9 is mouse CXCL9 and comprises the amino acid sequence shown 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 of claim 88, wherein the IL-12 is a human single-chain IL-12 and comprises the amino acid sequence shown in SEQ ID NO: 103, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence of SEQ ID NO:

103.

92. The recombinant oncolytic virus of claim 88, wherein the IL-12 is a mouse single-chain IL-12 and comprises the amino acid sequence shown 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 of any one of claims 81-92, wherein the one or more immunomodulatory proteins comprise IRF3.

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

95. The recombinant oncolytic virus of claim 94, wherein the hIRF3 comprises the amino acid sequence shown 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 shown in SEQ ID NO:

51.

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

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

52.

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

99. The recombinant oncolytic virus of any one of claims 81-98, 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 of any one of claims 81-99, wherein the one or more immunomodulatory proteins comprise IL-2.

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

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

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

104. The recombinant oncolytic virus according to any one of claims 81 to 103, wherein the 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.

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

106. The recombinant oncolytic virus of any one of claims 88-105, wherein the IL-2 is an IL-2 super factor.

107. The recombinant oncolytic virus of claim 106, wherein the IL-2 super factor is H9, H9T, MDNA11 or MDNA11T.

108. The recombinant oncolytic virus according to claim 106, wherein: The H9 IL-2 super factor 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 super factor 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 super factor 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 super factor 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.

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

110. The recombinant oncolytic vaccinia virus according to any one of claims 106 to 109, wherein the IL-2 super factor is MDNA11T, The MDNA11T comprises the amino acid sequence shown in SEQ ID NO: 98, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:

98.

111. The recombinant oncolytic virus of any one of claims 88-110, wherein the LIGHT is recombinant LIGHT.

112. The recombinant oncolytic virus according to claim 111, wherein the recombinant LIGHT is a human LIGHT protein or a mutant thereof.

113. The recombinant oncolytic virus of 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 with the amino acid sequence shown in SEQ ID NO:

30.

114. The recombinant oncolytic virus of any one of claims 111-113, wherein the recombinant LIGHT is a human LIGHT mutant (hmLIGHT), which is a human LIGHT mutant that binds to human and mouse LTβR and HVEM.

115. The recombinant oncolytic virus according to any one of claims 111-114, 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.

116. The recombinant oncolytic virus according to any one of claims 111-115, wherein the recombinant LIGHT comprises the amino acid sequence shown in SEQ ID NO: 25, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:

25.

117. The recombinant oncolytic virus of any one of claims 111-116, wherein the recombinant LIGHT comprises the sequence shown in SEQ ID NO:

25.

118. The recombinant oncolytic virus according to any one of claims 111-117, wherein the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown in any one of SEQ ID NO: 11, 82, 87 and 88, or a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the nucleic acid sequence shown in any one of SEQ ID NO: 11, 82, 87 and 88.

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

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

121. The recombinant oncolytic virus of claim 120, wherein the hscIL-12 comprises the amino acid sequence shown 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 shown in SEQ ID NO:

103.

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

123. The recombinant oncolytic virus of claim 122, wherein the human CXCL9 comprises the amino acid sequence shown 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 shown in SEQ ID NO:

99.

124. The recombinant oncolytic virus of any one of claims 48-123, wherein the 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.

125. The recombinant oncolytic virus of any one of claims 48, 50-52, and 54-124, wherein the inactivating mutation(s) in at least one of the at least one viral genes is achieved by inserting one or more heterologous nucleic acids each encoding an apoptosis-inducing protein.

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

127. The recombinant oncolytic virus of claim 126, wherein the FKBP variant is FKBP-F36V, optionally wherein the FKBP-F36V comprises the amino acid sequence shown 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 shown in SEQ ID NO:

56.

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

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

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

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

27.

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

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

134. The recombinant oncolytic virus of claim 133, wherein the iFas comprises the amino acid sequence shown 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 shown in SEQ ID NO:

28.

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

9.

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

137. The recombinant oncolytic virus of claim 136, wherein the iCas9 comprises the amino acid sequence shown 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 shown in SEQ ID NO:

26.

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

7.

139. The recombinant oncolytic virus of any one of claims 48-138, 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 T cell or NK cell escape proteins.

140. The recombinant oncolytic virus of any one of claims 48, 50-52, and 54-139, wherein the inactivating mutation(s) in at least one of the at least one viral genes is achieved by inserting one or more heterologous nucleic acids each encoding one or more T cell or NK cell escape proteins.

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

142. The recombinant oncolytic vaccinia virus of any one of claims 139-141, wherein the one or more T cell or NK cell escape proteins comprise a group of proteins that are or comprise CPXV012, CPXV203, and CPXV018 proteins.

143. The recombinant oncolytic vaccinia virus of claim 141 or claim 142, wherein the set of proteins encoded by CPXV012-203-018 comprises: (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.

144. The recombinant oncolytic vaccinia virus of any one of claims 141-143, wherein 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.

145. The recombinant oncolytic vaccinia virus according to any one of claims 139-144, wherein the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown 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 with the nucleic acid sequence shown in any one of SEQ ID NOs: 10, 89 and 90.

146. The recombinant oncolytic virus of any one of claims 48-145, wherein the 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.

147. The recombinant oncolytic virus of any one of claims 48, 50-52, and 54-146, wherein the inactivating mutation(s) in at least one of the at least one viral genes is achieved by inserting one or more heterologous nucleic acids each encoding one or more complement inhibitors.

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

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

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

18.

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

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

19.

153. The recombinant oncolytic virus of any one of claims 146-152, wherein the 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.

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

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

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

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

90.

158. The recombinant oncolytic virus of any one of claims 48-157, wherein the 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.

159. The recombinant oncolytic virus of any one of claims 48, 50-52, and 54-157, wherein the inactivating mutation of one or more of the at least one viral genes is achieved by inserting one or more heterologous nucleic acids each encoding one or more anti-angiogenic proteins.

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

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

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

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

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

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

166. The recombinant oncolytic virus of claim 165, wherein the bispecific anti-VEGF / anti-Ang2 antibody comprises the amino acid sequence shown 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 shown in SEQ ID NO:

23.

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

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

24.

169. A recombinant oncolytic virus according to any one of claims 158-168, wherein the nucleic acid genome of the recombinant oncolytic virus comprises a nucleic acid sequence as shown in any one of SEQ ID NO: 13, 47, 82, 87 and 88, or a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the nucleic acid sequence as shown in any one of SEQ ID NO: 13, 47, 82, 87 and 88.

170. The recombinant oncolytic virus of any one of claims 48-169, wherein the 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 therapeutic or diagnostic agents.

171. The recombinant oncolytic virus of any one of claims 48, 50-52, and 54-170, wherein the inactivating mutation of one or more of the at least one viral gene is achieved by inserting one or more heterologous nucleic acids each encoding one or more therapeutic or diagnostic agents.

172. The recombinant oncolytic virus of claim 170 or claim 171, wherein the one or more therapeutic or diagnostic agents are selected from anticancer agents, anti-metastatic agents, anti-angiogenic agents, immunomodulatory molecules, antigens, cell matrix degradation genes, genes for tissue regeneration and reprogramming of human cells to pluripotency, enzymes that modify substrates to produce detectable products or signals or that can be detected by antibodies, proteins that can bind contrast agents, genes for optical imaging or detection, genes for PET imaging, and genes for MRI imaging.

173. The recombinant oncolytic virus of any one of claims 170-172, wherein the one or more therapeutic agents or diagnostic agents comprise a therapeutic agent selected from hormones, growth factors, cytokines, chemokines, co-stimulatory molecules, ribozymes, transporters, single-chain antibodies, antisense RNA, prodrug converting enzymes, siRNA, microRNA, toxins, antitumor oligopeptides, mitotic inhibitory proteins, anti-mitotic oligopeptides, anticancer polypeptide antibiotics, angiogenesis inhibitors, tumor suppressors, cytotoxic proteins, cell inhibitory proteins, and tissue factors.

174. The recombinant oncolytic virus according to any one of claims 48, 50-52 and 54-173, wherein: (i) the at least one viral gene is or comprises A35R, optionally wherein 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 shown in SEQ ID NO: 3; or (ii) the at least one viral gene is or comprises A35R and J2R, optionally wherein 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 shown in SEQ ID NO: 12; or (iii) the at least one viral gene is or comprises J2R, and the inactivating mutation of J2R is achieved by inserting one or more heterologous nucleic acids each encoding one or more T cell or NK cell escape proteins, optionally wherein the one or more T cell or NK cell escape proteins comprise a set of proteins encoded by vaccinia virus ORFs 012, 203, and 018 (CPXV012-203-018), and wherein the 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, and the heterologous nucleic acids are 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 at the C-terminus of the F14.5L protein, and optionally wherein 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 shown in SEQ ID NO: 10; or (iv) the at least one viral gene is or comprises J2R, optionally wherein the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence of SEQ ID NO: 4, or a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the nucleic acid sequence shown 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 achieved 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 the nucleic acid sequence of SEQ ID NO: 11, or a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO:

11.

175. The recombinant oncolytic virus according to any one of claims 48, 50-52 and 54-173, wherein: (i) the at least one viral gene is or comprises J2R and A35R, and the inactivating mutation of J2R is achieved by inserting one or more heterologous nucleic acids each encoding one or more anti-angiogenic proteins, optionally wherein the one or more anti-angiogenic proteins comprise an inhibitor of VEGF and / or an inhibitor of Ang2, optionally wherein 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 with the nucleic acid sequence shown in SEQ ID NO: 13; or (ii) the at least one viral gene is or comprises J2R and A35R, and the inactivating mutation of A35R is achieved 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 The inactivating mutation of J2R is achieved by inserting one or more heterologous nucleic acids each encoding one or more anti-angiogenic proteins, optionally wherein the one or more anti-angiogenic proteins comprise an inhibitor of VEGF and / or an inhibitor of Ang2, optionally wherein 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: 47, or a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 47; or (iii) the at least one viral gene is or comprises J2R, and the inactivating mutation of J2R is achieved by inserting one or more heterologous nucleic acids of the respective apoptosis-inducing proteins, optionally wherein the apoptosis-inducing protein is inducible DED (iDED), inducible Fas (iFas) or inducible Cas9 (iCas9), optionally wherein the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence of SEQ ID NO: 7, 8 or 9, or a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 7, 8 or 9; or (iv) the at least one viral gene is or comprises J2R, and the inactivating mutation of the J2R is achieved by inserting 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 IRF3; optionally wherein 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 with the nucleic acid sequence shown in SEQ ID NO: 49, 50 or 93; or (v) the at least one viral gene is or comprises J2R and B2R, optionally wherein 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 with the nucleic acid sequence shown in SEQ ID NO:

48.

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

177. The recombinant oncolytic virus according to any one of claims 48, 50-52 and 54-173, wherein: (i) the at least one viral gene is or comprises J2R and B2R, and the inactivating mutation of the J2R is achieved by inserting 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 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 with the nucleic acid sequence shown in SEQ ID NO: 80; or (ii) the at least one viral gene is or comprises J2R, B2R and A35R; wherein: The inactivating mutation of J2R is achieved by inserting one or more heterologous nucleic acids encoding one or more anti-angiogenic proteins, optionally wherein the one or more anti-angiogenic proteins comprise a VEGF inhibitor and / or an Ang2 inhibitor, optionally wherein the one or more anti-angiogenic proteins are bispecific anti-VEGF / anti-Ang2 antibodies; The inactivating mutation of the B2R is achieved by inserting 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 are IRF3; and The inactivating mutation of A35R is achieved by inserting one or more heterologous nucleic acids 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 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; or (iii) the at least one viral gene is or comprises J2R, B2R and A56R; wherein: The inactivating mutation of J2R is achieved by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally wherein the one or more immunomodulatory proteins are IRF3; The inactivating mutation of A56R is achieved by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally wherein the one or more immunomodulatory proteins are IL-2, optionally wherein the IL-2 is an IL-2 superkine, optionally MDNA11; and Optionally, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence of 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; or (iv) the at least one viral gene is or comprises J2R, B2R and A56R; wherein: The inactivating mutation of J2R is achieved by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally wherein the one or more immunomodulatory proteins are IRF3; The inactivating mutation of A56R is achieved by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally wherein the 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 wherein the two or more immunomodulatory proteins comprise 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; or (v) the at least one viral gene is or comprises J2R, B2R and A56R; wherein: The inactivating mutation of B2R is achieved by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally wherein the one or more immunomodulatory proteins are IRF3; The inactivating mutation of A56R is achieved by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally wherein the 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 wherein the two or more immunomodulatory proteins comprise IL-12 and CXCL9; The inactivating mutation of J2R is achieved by inserting one or more heterologous nucleic acids each encoding an apoptosis-inducing protein, optionally wherein the apoptosis-inducing protein is an 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 with the nucleic acid sequence shown in SEQ ID NO: 86; or (vi) the at least one viral gene is or comprises J2R, B2R, A35R and A56R; wherein: The inactivating mutation of J2R is achieved by inserting one or more heterologous nucleic acids encoding one or more anti-angiogenic proteins, optionally wherein the one or more anti-angiogenic proteins comprise an inhibitor of VEGF and / or an inhibitor of Ang2, optionally wherein the one or more anti-angiogenic proteins are bispecific anti-VEGF / anti-Ang2 antibodies; The inactivating mutation of B2R is achieved by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally wherein the one or more immunomodulatory proteins are IRF3; The inactivating mutation of A35R is achieved by inserting one or more heterologous nucleic acids 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 are LIGHT; The inactivating mutation of A56R is achieved by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, wherein the one or more immunomodulatory proteins is IL-2 super factor 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 with the nucleic acid sequence shown in SEQ ID NO: 87; or (vii) the at least one viral gene is or comprises J2R, B2R, A35R and A56R; wherein: The inactivating mutation of J2R is achieved by inserting one or more heterologous nucleic acids encoding one or more anti-angiogenic proteins, optionally wherein the one or more anti-angiogenic proteins comprise an inhibitor of VEGF and / or an inhibitor of Ang2, optionally wherein the one or more anti-angiogenic proteins are bispecific anti-VEGF / anti-Ang2 antibodies; The inactivating mutation of B2R is by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally wherein the one or more immunomodulatory proteins are IRF3; The inactivating mutation of A35R is achieved by inserting one or more heterologous nucleic acids 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 are LIGHT; The inactivating mutation of A56R is by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, wherein the one or more immunomodulatory proteins are IL-2 super factor MDNA11T, optionally wherein the MDNA11T comprises the amino acid sequence shown in SEQ ID NO: 98; and Optionally, the nucleic acid genome of the recombinant oncolytic 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 with the nucleic acid sequence shown in SEQ ID NO: 88; or (viii) the at least one viral gene is or comprises J2R, B2R and A56R; wherein: The inactivating mutation of J2R is achieved by inserting one or more heterologous nucleic acids encoding one or more T cell or NK cell inducing proteins, wherein the one or more T cell or NK cell inducing proteins comprise a group of proteins encoded by vaccinia virus ORF012, 203 and 018 (CPXV012-203-018); The inactivating mutation of B2R is by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally wherein the one or more immunomodulatory proteins are IRF3; The inactivating mutation of A56R is by inserting 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 are IL-2 superkine, optionally MDNA11 or MDNA11T; the 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, optionally CRASP-2, which are introduced into a viral membrane gene, optionally F14.5L, to produce a fusion gene encoding a fusion protein, optionally wherein the fusion is at the C-terminus of the F14.5L protein; and Optionally, the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence of SEQ ID NO: 89, or a nucleic acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 89; or (ix) at least one viral gene is or comprises J2R, B2R and A56R; wherein The inactivating mutation of J2R is by inserting one or more heterologous nucleic acids each encoding one or more T cell or NK cell inducing proteins, optionally wherein the one or more T cell or NK cell inducing proteins comprise a group of proteins encoded by vaccinia virus ORFs 012, 203 and 018 (CPXV012-203-018); The inactivating mutation of B2R is by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally wherein the one or more immunomodulatory proteins are IRF3; The inactivating mutation of A56R is by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally wherein the 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 wherein the two or more immunomodulatory proteins comprise IL-12 and CXCL9; The 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, optionally CRASP-2, which are introduced into a viral membrane gene, optionally F14.5L, to produce a fusion gene encoding a fusion protein, optionally wherein the fusion is at the C-terminus of the F14.5L protein; and Optionally, 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 with the nucleic acid sequence shown in SEQ ID NO: 90; or (x) 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 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 IRF3; and Optionally, 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 with the nucleic acid sequence shown in SEQ ID NO: 91; or (xi) the at least one viral gene is or comprises B2R, J2R and A56R, and The inactivating mutation of J2R is by inserting 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 IRF3; and The inactivating mutation of A56R is by inserting one or more heterologous nucleic acids encoding one or more immunomodulatory proteins, optionally wherein the 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 wherein the two or more immunomodulatory proteins comprise 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 with the nucleic acid sequence shown in SEQ ID NO: 92; or (xii) the 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 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 IRF3; and Optionally, 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 with the nucleic acid sequence shown in SEQ ID NO:

93.

178. A recombinant oncolytic vaccinia virus according to any one of claims 48-173, wherein the nucleic acid genome of the recombinant oncolytic virus comprises a nucleic acid sequence as shown 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 with a nucleic acid sequence as shown in any one of SEQ ID NOs: 48, 80, 82 and 84-93.

179. The recombinant oncolytic vaccinia virus according to any one of claims 48 to 173, wherein the nucleic acid genome of the recombinant oncolytic virus comprises a nucleic acid sequence as shown 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 with a nucleic acid sequence as shown in any one of SEQ ID NOs: 85, 86, 88, and 90.

180. The recombinant oncolytic vaccinia virus according to any one of claims 48-173, wherein the nucleic acid genome of the recombinant oncolytic virus comprises the 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 with the nucleic acid sequence shown in SEQ ID NO 85.

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

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

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

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

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

186. The recombinant oncolytic virus of any one of claims 182-185, 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 of any one of claims 182-186, wherein the promoter has an amino acid sequence as shown in any one of SEQ ID NO: 29, 53, 55, 68, 69, 70, 71 or 72.

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

189. The recombinant VACV strain of claim 188, wherein the SSE promoter comprises the sequence shown in SEQ ID NO:

29.

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

191. The recombinant oncolytic virus of claim 190, wherein the SEL promoter comprises the sequence shown in SEQ ID NO:

55.

192. The recombinant oncolytic virus of any one of claims 182-191, wherein the promoter is mH5.

193. The recombinant oncolytic virus of claim 192, wherein the mH5 promoter comprises the sequence shown in SEQ ID NO:

53.

194. An isolated clonal vaccinia virus (VACV) strain comprising a nucleic acid genome having at least 95% sequence identity to the nucleic acid sequence shown in SEQ ID NO: 1, wherein the nucleic acid genome is characterized by one or more of the following: (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 comprising a polar uncharged amino acid at position 66, optionally comprising a threonine (T) at position 66; (ii) a variant 038(K5L) ORF comprising a nucleotide insertion resulting in a frameshift mutation, wherein the 038(K5L) gene product is altered; (iii) a variant 059(E2L) ORF encoding an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 60 and comprising a hydrophobic amino acid other than leucine at position 419, optionally comprising phenylalanine (F) at position 419; (iv) a variant 104(H4L) ORF encoding an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 61 and comprising a negatively charged amino acid at position 591, optionally comprising aspartic acid (D) at position 591; and (v) A variant 182(A56R) ORF comprising a deletion of two nucleotides resulting in a frameshift mutation, wherein the 182(A56R) ORF gene product is altered.

195. The isolated clonal VACV strain of 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.

196. The isolated clonal VACV strain of claim 194 or claim 195, wherein the nucleic acid genome is characterized by (i) and the variant 017 ORF encodes the amino acid sequence shown in SEQ ID NO:

57.

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

58.

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

59.

199. The isolated clonal VACV strain of any one of claims 194-198, 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 clonal VACV strain of any one of claims 194-199, wherein the nucleic acid genome is characterized by (iii) and the variant 059(E2L) ORF encodes the amino acid sequence shown in SEQ ID NO:

60.

201. The isolated clonal VACV strain of any one of claims 194-200, 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 clonal VACV strain of any one of claims 194-201, wherein the nucleic acid genome is characterized by (iv), and wherein the variant 104(H4L) ORF encodes the amino acid sequence shown in SEQ ID NO:

61.

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

62.

204. The isolated clonal VACV strain of any one of claims 194-203, wherein the nucleic acid genome is characterized by (v) and the VACV protein is shown in SEQ ID NO:

63.

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

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

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

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

209. An isolated clonal vaccinia virus (VACV) strain comprising a nucleic acid genome having at least 95% sequence identity to the nucleic acid sequence shown in SEQ ID NO: 1, wherein the nucleic acid genome 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 a position corresponding to position 92969 of SEQ ID NO: 1; (vi) the nucleic acid sequence CACTTATAT at positions corresponding to positions 106870 to 106880 of SEQ ID NO: 1; (vii) the nucleic acid sequence GTTTTCATTA at positions 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 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.

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

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

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

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

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

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

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

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

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

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

1.

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

1.

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

1.

222. The isolated clonal VACV strain of any one of claims 209-221, wherein the nucleic acid genome has at least 99% sequence identity to the nucleotide sequence shown in SEQ ID NO:

1.

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

1.

224. The recombinant oncolytic virus of any one of claims 1-193, or the isolated clonal VACV strain of any one of claims 194-223, wherein the nucleic acid genome has at least 99.5% sequence identity with the nucleotide sequence shown in SEQ ID NO:

1.

225. The recombinant oncolytic virus of any one of claims 1-193, or the isolated clonal VACV strain of any one of claims 194-224, wherein the nucleic acid genome has at least 99.9% sequence identity with the nucleotide sequence shown in SEQ ID NO:

1.

226. The recombinant oncolytic virus of any one of claims 1-193, or the isolated clonal VACV strain of any one of claims 194-225, wherein the nucleic acid genome has at least 99.95% sequence identity with the nucleotide sequence shown in SEQ ID NO:

1.

227. The recombinant oncolytic virus of any one of claims 1-193, or the isolated clonal VACV strain of any one of claims 194-226, wherein the nucleic acid genome does not comprise the nucleotide sequence shown in SEQ ID NO:

2.

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

229. The isolated clonal VACV strain of any one of claims 194-228, wherein the nucleic acid genome is shown in SEQ ID NO:

1.

230. The recombinant oncolytic virus of any one of claims 1-193, or the isolated clonal VACV strain of any one of claims 194-229, wherein the recombinant oncolytic virus or the clonal VACV strain exhibits enhanced production of extracellular enveloped virus (EEV) upon cell infection, optionally as determined by the percentage of EEV, wherein the percentage of EEV is determined by the following formula: virus titer in supernatant / (virus titer in supernatant + virus titer in cell lysate)*100.

231. The recombinant oncolytic virus of claim 230 or the isolated clonal VACV strain of claim 244, wherein greater than 5% of the infectious particles after infection of cells are EEV.

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

233. The recombinant oncolytic virus of claim 230 or the isolated clonal VACV strain of claim 244, wherein greater than 15% of the infectious particles after infection of cells are EEV.

234. The recombinant oncolytic virus of any one of claims 230-233, or the isolated clonal VACV strain of any one of claims 230-233, wherein the recombinant oncolytic virus or the clonal VACV strain exhibits enhanced production of extracellular enveloped virus (EEV) upon infection of cells, as determined by having a percentage of infectious particles that are EEV of at least 5%, 10% or 15%.

235. The recombinant oncolytic virus of any one of claims 1-193, 224-227, and 230-234, or the isolated clonal VACV strain of any one of claims 194-234, which exhibits oncolytic activity to kill tumor cells.

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

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

238. A recombinant oncolytic virus preparation comprising a recombinant oncolytic virus according to any one of claims 1-193, 224-227 and 230-235, 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 of claim 236 or 237, which is substantially homogeneous, wherein a plurality of viral particles in the preparation have the genomic sequence of the clonal VACV strain.

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

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

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

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

244. The VACV preparation of any one of claims 236, 237, and 239, wherein at least 98% of the viral particles in the preparation have the genomic sequence of a clonal VACV strain.

245. A pharmaceutical composition comprising the isolated VACV clonal strain according to any one of claims 208-248.

246. A pharmaceutical composition comprising the VACV formulation of any one of claims 194-234.

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

248. A recombinant vaccinia virus (VACV) strain comprising the nucleic acid genome of the VACV clonal strain of any one of claims 194-235, wherein the nucleic acid genome comprises an inactivating mutation in at least one viral gene.

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

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

252. The recombinant VACV strain of claim 250, wherein the deletion of at least one viral gene is a deletion of a portion of the viral gene ORF, and the deletion is sufficient to render the encoded gene product nonfunctional.

253. The recombinant VACV strain of any one of claims 248-252, wherein the at least one viral gene is or comprises A35R.

254. The recombinant VACV strain of claim 253, wherein the nucleic acid genome of the recombinant VACV strain comprises the nucleic acid sequence shown 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 shown in SEQ ID NO:

3.

255. The recombinant VACV strain of any one of claims 248-254, wherein the at least one viral gene is or comprises J2R.

256. The recombinant VACV strain of claim 255, wherein the nucleic acid genome of the recombinant VACV strain comprises the nucleic acid sequence shown 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 shown in SEQ ID NO:

4.

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

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

259. The recombinant VACV strain of claim 258, wherein the nucleic acid genome of the recombinant VACV strain comprises the nucleic acid sequence shown 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 shown in SEQ ID NO:

12.

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

261. A recombinant VACV strain according to claim 260, wherein the nucleic acid genome of the recombinant VACV strain comprises the nucleic acid sequence shown 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 with the sequence shown in SEQ ID NO:

48.

262. A recombinant VACV strain according to any one of claims 248-261, wherein the nucleic acid genome of the recombinant oncolytic virus comprises a nucleic acid sequence shown 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 with the nucleic acid sequence shown in any one of SEQ ID NOs: 48, 80, 82 and 84-93.

263. A recombinant VACV strain according to any one of claims 248-261, wherein the nucleic acid genome of the recombinant oncolytic virus comprises a nucleic acid sequence shown 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 with the nucleic acid sequence shown in any one of SEQ ID NOs: 85, 86, 88 and 90.

264. A recombinant VACV strain according to any one of claims 248-261, wherein the nucleic acid genome of the recombinant oncolytic virus comprises the 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 with the nucleic acid sequence shown in SEQ ID NO 85.

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

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

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

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

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

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

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

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

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

274. The pharmaceutical composition of any one of claims 245-247 and 269-273, wherein the pharmaceutical composition is a liquid composition.

275. The pharmaceutical composition of any one of claims 245-247 and 269-273, wherein the pharmaceutical composition is lyophilized.

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

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

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

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

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

281. The method of claim 280, wherein the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown 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 with the sequence shown in SEQ ID NO:

8.

282. The method according to any one of claims 276-281, wherein the recombinant oncolytic virus or the isolated oncolytic virus is 1×10 5 pfu to 1x10 14 pfu were administered.

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

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

285. The method of claim 284, wherein the additional treatment is the administration of an anticancer agent selected from the group consisting of cytokines, chemokines, growth factors, photosensitizers, toxins, anticancer antibiotics, chemotherapeutic compounds, radionuclides, angiogenesis inhibitors, signal transduction regulators, antimetabolites, anticancer vaccines, anticancer oligopeptides, mitotic inhibitory proteins, anti-mitotic oligopeptides, anticancer antibodies, anticancer antibiotics, immunotherapeutic agents, and any combination thereof.

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

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

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

289. The method of claim 287 or claim 288, wherein the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown 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 with the sequence shown in SEQ ID NO:

8.

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

291. The method of any one of claims 276-290, wherein the nucleic acid genome of the recombinant oncolytic virus comprises a nucleic acid sequence as shown 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 with a nucleic acid sequence as shown in any one of SEQ ID NOs: 48, 80, 82, and 84-93.

292. A method for inhibiting viral replication, the method comprising 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.

293. A method of inhibiting viral replication, the method comprising contacting a cell with AP1903 (Rimiducid), wherein the cell is infected with a recombinant oncolytic virus according to any one of claims 1-193, 224-227 and 230-235, an isolated cloned VACV strain according to any one of claims 194-235, or a recombinant VACV strain according to any one of claims 248-264.

294. The method of claim 292 or 293, wherein the contacting occurs in a subject.

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

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

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

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

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

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

301. The method of claim 300, wherein the iDED comprises the amino acid sequence shown 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 of any one of claims 300 and 301, wherein the nucleic acid genome of the recombinant oncolytic virus comprises the nucleic acid sequence shown 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 with the sequence shown in SEQ ID NO:

8.

303. The method of any one of claims 292-302, wherein the nucleic acid genome of the recombinant oncolytic virus comprises a nucleic acid sequence as shown 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 a nucleic acid sequence as shown in any one of SEQ ID NOs 48, 80, 82, and 84-93.

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