Chimeric pox virus compositions and uses thereof

Abstract

To provide compositions taking advantage of the beneficial features of oncolytic viruses, while maximizing safety and clinical outcomes.SOLUTION: Disclosed herein is a chimeric poxvirus comprising a nucleic acid sequence having a sequence identity of at least 70% to two specific sequences, where the nucleic acid sequence includes (i) nucleic acid fragments from at least two poxvirus strains selected from the group including cowpox virus strain Brighton, raccoonpox virus strain Herman, rabbitpox virus strain Utrecht, vaccinia virus strain WR, vaccinia virus strain IHD, vaccinia virus strain Elstree, vaccinia virus strain CL, vaccinia virus strain Lederle-Chorioallantoic, vaccinia virus strain AS, orf virus strain NZ2 and pseudocowpox virus strain TJS (ii) one or more anticancer nucleic acid sequences or (iii) a nucleic acid sequence encoding a detectable moiety.SELECTED DRAWING: None

Description

[Technical field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 372,408, filed August 9, 2016, and U.S. Provisional Patent Application No. 62 / 519,010, filed June 13, 2017, the entire contents of which are incorporated by reference herein for all purposes.

[0002] The sequence listing set forth in file 48440-606001WO_ST25 (created on August 8, 2017, 696,239 bytes, instrument format: IBM-PC, operating system: MS-Windows) is incorporated herein by reference. [Background technology]

[0003] Cancer is the second leading cause of death in the United States. In recent years, there have been many advances in cancer immunotherapy, including immune checkpoint inhibitors, T cells with chimeric antigen receptors, and oncolytic viruses. Oncolytic viruses are natural or genetically modified viruses that infect, replicate in, and ultimately kill cancer cells while sparing healthy cells. However, the clinical benefits of using oncolytic viruses as a monotherapy remain limited. Summary of the Invention [Problem to be solved by the invention]

[0004] There is a need in the art for novel compositions that leverage the advantageous characteristics of oncolytic viruses while maximizing safety and clinical outcomes. Disclosed herein are, among other things, solutions to these and other problems in the art. [Means for solving the problem]

[0005] Summary of the Invention In one aspect, a chimeric poxvirus is provided comprising a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:1 or SEQ ID NO:2, said nucleic acid sequence comprising nucleic acid fragments from at least two poxvirus strains selected from the group consisting of: Bovine poxvirus strain Brighton, Raccoon poxvirus strain Herman, Rabbit poxvirus strain Utrecht, Vaccinia virus strain WR, Vaccinia virus strain IHD, Vaccinia virus strain Elstree, Vaccinia virus strain CL, Vaccinia virus strain Lederle-Chorioallantoic, Vaccinia virus strain AS, Orf virus strain NZ2 and Pseudobovine poxvirus strain TJS.

[0006] In one aspect, an isolated nucleic acid encoding a chimeric poxvirus described herein is provided.

[0007] In one aspect, a pharmaceutical composition is provided that comprises a therapeutically effective amount of a chimeric poxvirus described herein.

[0008] In another aspect, a method of treating cancer in a subject in need thereof is provided, the method comprising administering to the subject a therapeutically effective amount of a chimeric poxvirus described herein, thereby treating the cancer in the subject. In embodiments, the cancer is breast cancer, colon cancer, kidney cancer, leukemia, lung cancer, melanoma, ovarian cancer, prostate cancer, pancreatic cancer, brain cancer, liver cancer, gastric cancer, or sarcoma.

[0009] In another aspect, a method of forming a chimeric poxvirus is provided, the method comprising infecting a cell with at least two poxvirus strains selected from the group comprising: Brighton strain of bovine poxvirus, Herman strain of raccoon poxvirus, Utrecht strain of rabbit poxvirus, WR strain of vaccinia virus, IHD strain of vaccinia virus, Elstree strain of vaccinia virus, CL strain of vaccinia virus, Lederle-Chorioallantoic strain of vaccinia virus, AS strain of vaccinia virus, NZ2 strain of orf virus and TJS strain of pseudobovine poxvirus, and allowing the at least two poxvirus strains to replicate, thereby forming a chimeric poxvirus.

[0010] In one aspect, a method of inhibiting cell proliferation in a cell is provided, the method comprising contacting a cell with a chimeric poxvirus described herein.

[0011] In one aspect, a chimeric poxvirus is provided comprising a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:1 or SEQ ID NO:2, the nucleic acid sequence comprising: (i) a nucleic acid fragment from at least two poxvirus strains selected from the group consisting of: Bovine poxvirus strain Brighton, Raccoon poxvirus strain Herman, Rabbit poxvirus strain Utrecht, Vaccinia virus strain WR, Vaccinia virus strain IHD, Vaccinia virus strain Elstree, Vaccinia virus strain CL, Vaccinia virus strain Lederle-Chorioallantoic, Vaccinia virus strain AS, Orf virus strain NZ2, and Pseudobovine poxvirus strain TJS, (ii) one or more anti-cancer nucleic acid sequences, or (iii) a nucleic acid sequence encoding a detectable moiety.

[0012] In another aspect, a chimeric poxvirus is provided comprising a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:1, the nucleic acid sequence comprising: (i) a nucleic acid fragment from bovine poxvirus strain Brighton, raccoon poxvirus strain Herman, rabbit poxvirus strain Utrecht, vaccinia virus strain WR, vaccinia virus strain IHD, vaccinia virus strain Elstree, vaccinia virus strain CL, vaccinia virus strain Lederle-Chorioallantoic, and vaccinia virus strain AS, (ii) one or more anti-cancer nucleic acid sequences, or (iii) a nucleic acid sequence encoding a detectable moiety.

[0013] In another aspect, a chimeric poxvirus is provided comprising a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:2, the nucleic acid sequence comprising: (i) a nucleic acid fragment derived from orf virus strain NZ2 and pseudobovine poxvirus strain TJS; (ii) one or more anti-cancer nucleic acid sequences; or (iii) a nucleic acid sequence encoding a detectable moiety.

[0014] In another aspect, a chimeric poxvirus is provided comprising a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:3, said nucleic acid sequence comprising: (i) a nucleic acid fragment from bovine poxvirus strain Brighton, raccoon poxvirus strain Herman, rabbit poxvirus strain Utrecht, vaccinia virus strain WR, vaccinia virus strain IHD, vaccinia virus strain Elstree, vaccinia virus strain CL, vaccinia virus strain Lederle-Chorioallantoic, and vaccinia virus strain AS; (ii) one or more anti-cancer nucleic acid sequences; or (iii) a nucleic acid sequence encoding a detectable moiety.

[0015] In one aspect, a chimeric poxvirus is provided comprising a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:1 or SEQ ID NO:2, the nucleic acid sequence comprising: (i) a nucleic acid fragment from at least two poxvirus strains selected from the group consisting of: Bovine poxvirus strain Brighton, Raccoon poxvirus strain Herman, Rabbit poxvirus strain Utrecht, Vaccinia virus strain WR, Vaccinia virus strain IHD, Vaccinia virus strain Elstree, Vaccinia virus strain CL, Vaccinia virus strain Lederle-Chorioallantoic, Vaccinia virus strain AS, Orf virus strain NZ2, and Pseudobovine poxvirus strain TJS, (ii) one or more anti-cancer nucleic acid sequences, (iii) one or more nucleic acid binding sequences, or (iv) a nucleic acid sequence encoding a detectable moiety.

[0016] In another aspect, a chimeric poxvirus is provided comprising a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:1, the nucleic acid sequence comprising: (i) a nucleic acid fragment from Bovine poxvirus strain Brighton, Raccoon poxvirus strain Herman, Rabbit poxvirus strain Utrecht, Vaccinia virus strain WR, Vaccinia virus strain IHD, Vaccinia virus strain Elstree, Vaccinia virus strain CL, Vaccinia virus strain Lederle-Chorioallantoic, and Vaccinia virus strain AS, (ii) one or more anti-cancer nucleic acid sequences, (iii) one or more nucleic acid binding sequences, or (iv) a nucleic acid sequence encoding a detectable moiety.

[0017] In another aspect, a chimeric poxvirus is provided comprising a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:2, the nucleic acid sequence comprising: (i) orf virus strain NZ2 and pseudobovine poxvirus strain TJS; (ii) one or more anti-cancer nucleic acid sequences; (iii) one or more nucleic acid binding sequences; or (iv) a nucleic acid sequence encoding a detectable moiety.

[0018] In another aspect, a chimeric poxvirus is provided comprising a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:3, the nucleic acid sequence comprising: (i) a nucleic acid fragment from Bovine poxvirus strain Brighton, Raccoon poxvirus strain Herman, Rabbit poxvirus strain Utrecht, Vaccinia virus strain WR, Vaccinia virus strain IHD, Vaccinia virus strain Elstree, Vaccinia virus strain CL, Vaccinia virus strain Lederle-Chorioallantoic, and Vaccinia virus strain AS, (ii) one or more anti-cancer nucleic acid sequences, (iii) one or more nucleic acid binding sequences, or (iv) a nucleic acid sequence encoding a detectable moiety. [Brief description of the drawings]

[0019] [Figure 1] The novel chimeric orthopoxvirus isolates #33 (SEQ ID NO: 1) and #17 (SEQ ID NO: 3) show superior cancer cell killing ability compared to their parental wild-type virus strains, as well as the control viruses GLV-1h68 and OncoVEX GFP. [Diagram 2] A novel chimeric parapoxvirus isolate #189 (SEQ ID NO:2) exhibits superior cancer cell killing ability compared to its parent wild-type virus strain and the control viruses GLV-1h68 and OncoVEX GFP. [Diagram 3] The novel chimeric orthopoxvirus isolates #17 (SEQ ID NO: 3) and #33 (SEQ ID NO: 1) show potent cancer cell killing ability against pancreatic cancer cell lines compared to their parental virus strains and the control viruses GLV-1h68 and OncoVEX GFP. [Figure 4] A novel chimeric parapoxvirus isolate #189 (SEQ ID NO:2) exhibits potent cancer cell killing ability against pancreatic cancer cell lines compared to its parental virus strain and the control viruses GLV-1h68 and OncoVEX GFP. [Figure 5A]Figures 5A-5C show the excellent cell killing activity of novel chimeric virus isolates #33 (SEQ ID NO: 1) and #189 (SEQ ID NO: 2) against gastric cancer cell lines. Cancer cells were infected with each virus at MOI of 0.01, 0.1 and 1.0. Cell viability at 96 hours post-infection was plotted against MOI in gastric cancer cell lines MKN-45 (Figure 5A), OCUM-2M (Figure 5B) and KATO-3 (Figure 5C). [Figure 5B] Figures 5A-5C show the excellent cell killing activity of novel chimeric virus isolates #33 (SEQ ID NO: 1) and #189 (SEQ ID NO: 2) against gastric cancer cell lines. Cancer cells were infected with each virus at MOI of 0.01, 0.1 and 1.0. Cell viability at 96 hours post-infection was plotted against MOI in gastric cancer cell lines MKN-45 (Figure 5A), OCUM-2M (Figure 5B) and KATO-3 (Figure 5C). [Figure 5C] Figures 5A-5C show the excellent cell killing activity of novel chimeric virus isolates #33 (SEQ ID NO: 1) and #189 (SEQ ID NO: 2) against gastric cancer cell lines. Cancer cells were infected with each virus at MOI of 0.01, 0.1 and 1.0. Cell viability at 96 hours post-infection was plotted against MOI in gastric cancer cell lines MKN-45 (Figure 5A), OCUM-2M (Figure 5B) and KATO-3 (Figure 5C). [Figure 6A] Figures 6A-6D show that the in vitro cytotoxic effect of HOV-189 (SEQ ID NO: 2) is time- and dose-dependent in triple-negative breast cancer cell lines: (Figure 6A) Hs578T. LD50, MOI 0.396 (SD 0.113); (Figure 6B) BT549. LD50, MOI 1.636 (SD 0.539); (Figure 6C) MDA-MB-468. LD50, MOI 0.185 (SD 0.071); (Figure 6D) MDA-MB-231. LD50, MOI 1.712 (SD 1.263). LD50 (96 hours), median lethal dose, MOI: multiplicity of infection, SD: standard deviation. [Figure 6B]Figures 6A-6D show that the in vitro cytotoxic effect of HOV-189 (SEQ ID NO: 2) is time- and dose-dependent in triple-negative breast cancer cell lines: (Figure 6A) Hs578T. LD50, MOI 0.396 (SD 0.113); (Figure 6B) BT549. LD50, MOI 1.636 (SD 0.539); (Figure 6C) MDA-MB-468. LD50, MOI 0.185 (SD 0.071); (Figure 6D) MDA-MB-231. LD50, MOI 1.712 (SD 1.263). LD50 (96 hours), median lethal dose, MOI: multiplicity of infection, SD: standard deviation. [Figure 6C] Figures 6A-6D show that the in vitro cytotoxic effect of HOV-189 (SEQ ID NO: 2) is time- and dose-dependent in triple-negative breast cancer cell lines: (Figure 6A) Hs578T. LD50, MOI 0.396 (SD 0.113); (Figure 6B) BT549. LD50, MOI 1.636 (SD 0.539); (Figure 6C) MDA-MB-468. LD50, MOI 0.185 (SD 0.071); (Figure 6D) MDA-MB-231. LD50, MOI 1.712 (SD 1.263). LD50 (96 hours), median lethal dose, MOI: multiplicity of infection, SD: standard deviation. [Figure 6D] Figures 6A-6D show that the in vitro cytotoxic effect of HOV-189 (SEQ ID NO: 2) is time- and dose-dependent in triple-negative breast cancer cell lines: (Figure 6A) Hs578T. LD50, MOI 0.396 (SD 0.113); (Figure 6B) BT549. LD50, MOI 1.636 (SD 0.539); (Figure 6C) MDA-MB-468. LD50, MOI 0.185 (SD 0.071); (Figure 6D) MDA-MB-231. LD50, MOI 1.712 (SD 1.263). LD50 (96 hours), median lethal dose, MOI: multiplicity of infection, SD: standard deviation. [Figure 7]Replication of HOV-189 (SEQ ID NO: 2) in triple-negative breast cancer cell lines. Effective viral replication in vitro occurred in BT549, Hs578T and MDA-MB-231 cell lines at a low multiplicity of infection (MOI 0.01). HOV-189 replication in MDA-MB-468 was low at MOI 0.01. HOV-189 replication in MDA-MB-468 remained approximately log lower than the other three cell lines at MOI 10. [Figure 8] Intratumoral injection of HOV-189 (SEQ ID NO: 2) into MDA-MB-468 xenografts effectively reduces relative tumor size at concentrations as low as 103 PFU per tumor compared to controls. Tumors were injected with PBS (control), 103 PFU per tumor, 104 PFU per tumor, or 105 PFU per tumor with initial tumor volumes of approximately 100-150 mm3. Tumor sizes were measured approximately every 3 days, and the therapeutic effect was sustained for 6 weeks after injection. [Figure 9] No significant relative weight loss was observed in nude mice injected intratumorally with HOV-189 (SEQ ID NO: 2) compared to PBS-injected controls. Body weights were measured approximately every 3 days. [Figure 10A] In Figures 10A-10C, HOV-189 (SEQ ID NO:2) is infected by MDA-MB-468 tumors in vivo. Immunofluorescence detection of polyclonal antibodies against ORF virus shows virus infection in MDA-MB-468 xenograft tumor tissues harvested one week after intratumoral HOV-189 injection. (Figure 10A) Control tumor, 10x; (Figure 10B) 105 PFU-treated tumor, 10x; (Figure 10C) 105 PFU-treated tumor, 60x (ORF and DAPI counterstain). [Figure 10B]In Figures 10A-10C, HOV-189 (SEQ ID NO:2) is infected by MDA-MB-468 tumors in vivo. Immunofluorescence detection of polyclonal antibodies against ORF virus shows virus infection in MDA-MB-468 xenograft tumor tissues harvested one week after intratumoral HOV-189 injection. (Figure 10A) Control tumor, 10x; (Figure 10B) 105 PFU-treated tumor, 10x; (Figure 10C) 105 PFU-treated tumor, 60x (ORF and DAPI counterstain). [Figure 10C] In Figures 10A-10C, HOV-189 (SEQ ID NO:2) is infected by MDA-MB-468 tumors in vivo. Immunofluorescence detection of polyclonal antibodies against ORF virus shows virus infection in MDA-MB-468 xenograft tumor tissues harvested one week after intratumoral HOV-189 injection. (Figure 10A) Control tumor, 10x; (Figure 10B) 105 PFU-treated tumor, 10x; (Figure 10C) 105 PFU-treated tumor, 60x (ORF and DAPI counterstain). [Figure 11] Intratumoral HOV-189 (SEQ ID NO: 2) injection produces tumor silencing effects on distant non-injected tumors. A second breast tumor produced by MDA-MB-468 xenografting was treated with a single intratumoral injection of HOV-189 at 105 PFU, while a fourth breast tumor was not injected. Control tumors were injected with PBS. Tumor sizes were measured approximately every 3 days. [Figure 12A]In Figures 12A-12L, cytotoxicity assays were performed for PANC-1, MiaPaCa-2, BxPC-3, SU.86.86, Capan-1, and AsPC-1 cancer cell lines by plating 3x103 cancer cells per well in 100μL RPMI containing 5% FBS, 1% antibiotic (antimycotic) solution for 24 hours. 20μL of the indicated viruses were then added at multiplicities of infection (MOI) of 1, 0.1, and 0.01. Daily cell viability assays were performed by adding 20μL of CellTiter 96 Aqueous One Solution Cell Proliferation Assay to all wells and colorimetrically analyzing after 1 hour of incubation. Experimental results were normalized with respect to media only and zero MOI controls. Each experiment was repeated in triplicate. Graphs showing cell viability (%) over time in PANC-1 (FIG. 12A), MiaPaCa-2 (FIG. 12C), BxPC-3 (FIG. 12E), SU.86.86 (FIG. 12G), Capan-1 (FIG. 12I), and AsPC-1 (FIG. 12K) treated with #33 at MOIs of 1, 0.1, or 0.01. Also shown are bar graphs comparing cell viability (%) after 120 hours in cancer cells PANC-1 (FIG. 12B), MiaPaCa-2 (FIG. 12D), BxPC-3 (FIG. 12F), SU.86.86 (FIG. 12H), Capan-1 (FIG. 12J), and AsPC-1 (FIG. 12L) treated with the indicated viruses. Statistical analysis was performed at each time point using one-way ANOVA comparing #33 with the other experimental groups indicated. For SU.86.86 (FIG. 12H), statistical analysis was performed using unpaired t-tests at each MOI. [Figure 12B]In Figures 12A-12L, cytotoxicity assays were performed for PANC-1, MiaPaCa-2, BxPC-3, SU.86.86, Capan-1, and AsPC-1 cancer cell lines by plating 3x103 cancer cells per well in 100μL RPMI containing 5% FBS, 1% antibiotic (antimycotic) solution for 24 hours. 20μL of the indicated viruses were then added at multiplicities of infection (MOI) of 1, 0.1, and 0.01. Daily cell viability assays were performed by adding 20μL of CellTiter 96 Aqueous One Solution Cell Proliferation Assay to all wells and colorimetrically analyzing after 1 hour of incubation. Experimental results were normalized with respect to media only and zero MOI controls. Each experiment was repeated in triplicate. Graphs showing cell viability (%) over time in PANC-1 (FIG. 12A), MiaPaCa-2 (FIG. 12C), BxPC-3 (FIG. 12E), SU.86.86 (FIG. 12G), Capan-1 (FIG. 12I), and AsPC-1 (FIG. 12K) treated with #33 at MOIs of 1, 0.1, or 0.01. Also shown are bar graphs comparing cell viability (%) after 120 hours in cancer cells PANC-1 (FIG. 12B), MiaPaCa-2 (FIG. 12D), BxPC-3 (FIG. 12F), SU.86.86 (FIG. 12H), Capan-1 (FIG. 12J), and AsPC-1 (FIG. 12L) treated with the indicated viruses. Statistical analysis was performed at each time point using one-way ANOVA comparing #33 with the other experimental groups indicated. For SU.86.86 (FIG. 12H), statistical analysis was performed using unpaired t-tests at each MOI. [Figure 12C]In Figures 12A-12L, cytotoxicity assays were performed for PANC-1, MiaPaCa-2, BxPC-3, SU.86.86, Capan-1, and AsPC-1 cancer cell lines by plating 3x103 cancer cells per well in 100μL RPMI containing 5% FBS, 1% antibiotic (antimycotic) solution for 24 hours. 20μL of the indicated viruses were then added at multiplicities of infection (MOI) of 1, 0.1, and 0.01. Daily cell viability assays were performed by adding 20μL of CellTiter 96 Aqueous One Solution Cell Proliferation Assay to all wells and colorimetrically analyzing after 1 hour of incubation. Experimental results were normalized with respect to media only and zero MOI controls. Each experiment was repeated in triplicate. Graphs showing cell viability (%) over time in PANC-1 (FIG. 12A), MiaPaCa-2 (FIG. 12C), BxPC-3 (FIG. 12E), SU.86.86 (FIG. 12G), Capan-1 (FIG. 12I), and AsPC-1 (FIG. 12K) treated with #33 at MOIs of 1, 0.1, or 0.01. Also shown are bar graphs comparing cell viability (%) after 120 hours in cancer cells PANC-1 (FIG. 12B), MiaPaCa-2 (FIG. 12D), BxPC-3 (FIG. 12F), SU.86.86 (FIG. 12H), Capan-1 (FIG. 12J), and AsPC-1 (FIG. 12L) treated with the indicated viruses. Statistical analysis was performed at each time point using one-way ANOVA comparing #33 with the other experimental groups indicated. For SU.86.86 (FIG. 12H), statistical analysis was performed using unpaired t-tests at each MOI. [Figure 12D]In Figures 12A-12L, cytotoxicity assays were performed for PANC-1, MiaPaCa-2, BxPC-3, SU.86.86, Capan-1, and AsPC-1 cancer cell lines by plating 3x103 cancer cells per well in 100μL RPMI containing 5% FBS, 1% antibiotic (antimycotic) solution for 24 hours. 20μL of the indicated viruses were then added at multiplicities of infection (MOI) of 1, 0.1, and 0.01. Daily cell viability assays were performed by adding 20μL of CellTiter 96 Aqueous One Solution Cell Proliferation Assay to all wells and colorimetrically analyzing after 1 hour of incubation. Experimental results were normalized with respect to media only and zero MOI controls. Each experiment was repeated in triplicate. Graphs showing cell viability (%) over time in PANC-1 (FIG. 12A), MiaPaCa-2 (FIG. 12C), BxPC-3 (FIG. 12E), SU.86.86 (FIG. 12G), Capan-1 (FIG. 12I), and AsPC-1 (FIG. 12K) treated with #33 at MOIs of 1, 0.1, or 0.01. Also shown are bar graphs comparing cell viability (%) after 120 hours in cancer cells PANC-1 (FIG. 12B), MiaPaCa-2 (FIG. 12D), BxPC-3 (FIG. 12F), SU.86.86 (FIG. 12H), Capan-1 (FIG. 12J), and AsPC-1 (FIG. 12L) treated with the indicated viruses. Statistical analysis was performed at each time point using one-way ANOVA comparing #33 with the other experimental groups indicated. For SU.86.86 (FIG. 12H), statistical analysis was performed using unpaired t-tests at each MOI. [Figure 12E]In Figures 12A-12L, cytotoxicity assays were performed for PANC-1, MiaPaCa-2, BxPC-3, SU.86.86, Capan-1, and AsPC-1 cancer cell lines by plating 3x103 cancer cells per well in 100μL RPMI containing 5% FBS, 1% antibiotic (antimycotic) solution for 24 hours. 20μL of the indicated viruses were then added at multiplicities of infection (MOI) of 1, 0.1, and 0.01. Daily cell viability assays were performed by adding 20μL of CellTiter 96 Aqueous One Solution Cell Proliferation Assay to all wells and colorimetrically analyzing after 1 hour of incubation. Experimental results were normalized with respect to media only and zero MOI controls. Each experiment was repeated in triplicate. Graphs showing cell viability (%) over time in PANC-1 (FIG. 12A), MiaPaCa-2 (FIG. 12C), BxPC-3 (FIG. 12E), SU.86.86 (FIG. 12G), Capan-1 (FIG. 12I), and AsPC-1 (FIG. 12K) treated with #33 at MOIs of 1, 0.1, or 0.01. Also shown are bar graphs comparing cell viability (%) after 120 hours in cancer cells PANC-1 (FIG. 12B), MiaPaCa-2 (FIG. 12D), BxPC-3 (FIG. 12F), SU.86.86 (FIG. 12H), Capan-1 (FIG. 12J), and AsPC-1 (FIG. 12L) treated with the indicated viruses. Statistical analysis was performed at each time point using one-way ANOVA comparing #33 with the other experimental groups indicated. For SU.86.86 (FIG. 12H), statistical analysis was performed using unpaired t-tests at each MOI. [Figure 12F]In Figures 12A-12L, cytotoxicity assays were performed for PANC-1, MiaPaCa-2, BxPC-3, SU.86.86, Capan-1, and AsPC-1 cancer cell lines by plating 3x103 cancer cells per well in 100μL RPMI containing 5% FBS, 1% antibiotic (antimycotic) solution for 24 hours. 20μL of the indicated viruses were then added at multiplicities of infection (MOI) of 1, 0.1, and 0.01. Daily cell viability assays were performed by adding 20μL of CellTiter 96 Aqueous One Solution Cell Proliferation Assay to all wells and colorimetrically analyzing after 1 hour of incubation. Experimental results were normalized with respect to media only and zero MOI controls. Each experiment was repeated in triplicate. Graphs showing cell viability (%) over time in PANC-1 (FIG. 12A), MiaPaCa-2 (FIG. 12C), BxPC-3 (FIG. 12E), SU.86.86 (FIG. 12G), Capan-1 (FIG. 12I), and AsPC-1 (FIG. 12K) treated with #33 at MOIs of 1, 0.1, or 0.01. Also shown are bar graphs comparing cell viability (%) after 120 hours in cancer cells PANC-1 (FIG. 12B), MiaPaCa-2 (FIG. 12D), BxPC-3 (FIG. 12F), SU.86.86 (FIG. 12H), Capan-1 (FIG. 12J), and AsPC-1 (FIG. 12L) treated with the indicated viruses. Statistical analysis was performed at each time point using one-way ANOVA comparing #33 with the other experimental groups indicated. For SU.86.86 (FIG. 12H), statistical analysis was performed using unpaired t-tests at each MOI. [Figure 12G]In Figures 12A-12L, cytotoxicity assays were performed for PANC-1, MiaPaCa-2, BxPC-3, SU.86.86, Capan-1, and AsPC-1 cancer cell lines by plating 3x103 cancer cells per well in 100μL RPMI containing 5% FBS, 1% antibiotic (antimycotic) solution for 24 hours. 20μL of the indicated viruses were then added at multiplicities of infection (MOI) of 1, 0.1, and 0.01. Daily cell viability assays were performed by adding 20μL of CellTiter 96 Aqueous One Solution Cell Proliferation Assay to all wells and colorimetrically analyzing after 1 hour of incubation. Experimental results were normalized with respect to media only and zero MOI controls. Each experiment was repeated in triplicate. Graphs showing cell viability (%) over time in PANC-1 (FIG. 12A), MiaPaCa-2 (FIG. 12C), BxPC-3 (FIG. 12E), SU.86.86 (FIG. 12G), Capan-1 (FIG. 12I), and AsPC-1 (FIG. 12K) treated with #33 at MOIs of 1, 0.1, or 0.01. Also shown are bar graphs comparing cell viability (%) after 120 hours in cancer cells PANC-1 (FIG. 12B), MiaPaCa-2 (FIG. 12D), BxPC-3 (FIG. 12F), SU.86.86 (FIG. 12H), Capan-1 (FIG. 12J), and AsPC-1 (FIG. 12L) treated with the indicated viruses. Statistical analysis was performed at each time point using one-way ANOVA comparing #33 with the other experimental groups indicated. For SU.86.86 (FIG. 12H), statistical analysis was performed using unpaired t-tests at each MOI. [Figure 12H]In Figures 12A-12L, cytotoxicity assays were performed for PANC-1, MiaPaCa-2, BxPC-3, SU.86.86, Capan-1, and AsPC-1 cancer cell lines by plating 3x103 cancer cells per well in 100μL RPMI containing 5% FBS, 1% antibiotic (antimycotic) solution for 24 hours. 20μL of the indicated viruses were then added at multiplicities of infection (MOI) of 1, 0.1, and 0.01. Daily cell viability assays were performed by adding 20μL of CellTiter 96 Aqueous One Solution Cell Proliferation Assay to all wells and colorimetrically analyzing after 1 hour of incubation. Experimental results were normalized with respect to media only and zero MOI controls. Each experiment was repeated in triplicate. Graphs showing cell viability (%) over time in PANC-1 (FIG. 12A), MiaPaCa-2 (FIG. 12C), BxPC-3 (FIG. 12E), SU.86.86 (FIG. 12G), Capan-1 (FIG. 12I), and AsPC-1 (FIG. 12K) treated with #33 at MOIs of 1, 0.1, or 0.01. Also shown are bar graphs comparing cell viability (%) after 120 hours in cancer cells PANC-1 (FIG. 12B), MiaPaCa-2 (FIG. 12D), BxPC-3 (FIG. 12F), SU.86.86 (FIG. 12H), Capan-1 (FIG. 12J), and AsPC-1 (FIG. 12L) treated with the indicated viruses. Statistical analysis was performed at each time point using one-way ANOVA comparing #33 with the other experimental groups indicated. For SU.86.86 (FIG. 12H), statistical analysis was performed using unpaired t-tests at each MOI. [Figure 12I]In Figures 12A-12L, cytotoxicity assays were performed for PANC-1, MiaPaCa-2, BxPC-3, SU.86.86, Capan-1, and AsPC-1 cancer cell lines by plating 3x103 cancer cells per well in 100μL RPMI containing 5% FBS, 1% antibiotic (antimycotic) solution for 24 hours. 20μL of the indicated viruses were then added at multiplicities of infection (MOI) of 1, 0.1, and 0.01. Daily cell viability assays were performed by adding 20μL of CellTiter 96 Aqueous One Solution Cell Proliferation Assay to all wells and colorimetrically analyzing after 1 hour of incubation. Experimental results were normalized with respect to media only and zero MOI controls. Each experiment was repeated in triplicate. Graphs showing cell viability (%) over time in PANC-1 (FIG. 12A), MiaPaCa-2 (FIG. 12C), BxPC-3 (FIG. 12E), SU.86.86 (FIG. 12G), Capan-1 (FIG. 12I), and AsPC-1 (FIG. 12K) treated with #33 at MOIs of 1, 0.1, or 0.01. Also shown are bar graphs comparing cell viability (%) after 120 hours in cancer cells PANC-1 (FIG. 12B), MiaPaCa-2 (FIG. 12D), BxPC-3 (FIG. 12F), SU.86.86 (FIG. 12H), Capan-1 (FIG. 12J), and AsPC-1 (FIG. 12L) treated with the indicated viruses. Statistical analysis was performed at each time point using one-way ANOVA comparing #33 with the other experimental groups indicated. For SU.86.86 (FIG. 12H), statistical analysis was performed using unpaired t-tests at each MOI. [Figure 12J]In Figures 12A-12L, cytotoxicity assays were performed for PANC-1, MiaPaCa-2, BxPC-3, SU.86.86, Capan-1, and AsPC-1 cancer cell lines by plating 3x103 cancer cells per well in 100μL RPMI containing 5% FBS, 1% antibiotic (antimycotic) solution for 24 hours. 20μL of the indicated viruses were then added at multiplicities of infection (MOI) of 1, 0.1, and 0.01. Daily cell viability assays were performed by adding 20μL of CellTiter 96 Aqueous One Solution Cell Proliferation Assay to all wells and colorimetrically analyzing after 1 hour of incubation. Experimental results were normalized with respect to media only and zero MOI controls. Each experiment was repeated in triplicate. Graphs showing cell viability (%) over time in PANC-1 (FIG. 12A), MiaPaCa-2 (FIG. 12C), BxPC-3 (FIG. 12E), SU.86.86 (FIG. 12G), Capan-1 (FIG. 12I), and AsPC-1 (FIG. 12K) treated with #33 at MOIs of 1, 0.1, or 0.01. Also shown are bar graphs comparing cell viability (%) after 120 hours in cancer cells PANC-1 (FIG. 12B), MiaPaCa-2 (FIG. 12D), BxPC-3 (FIG. 12F), SU.86.86 (FIG. 12H), Capan-1 (FIG. 12J), and AsPC-1 (FIG. 12L) treated with the indicated viruses. Statistical analysis was performed at each time point using one-way ANOVA comparing #33 with the other experimental groups indicated. For SU.86.86 (FIG. 12H), statistical analysis was performed using unpaired t-tests at each MOI. [Figure 12K]In Figures 12A-12L, cytotoxicity assays were performed for PANC-1, MiaPaCa-2, BxPC-3, SU.86.86, Capan-1, and AsPC-1 cancer cell lines by plating 3x103 cancer cells per well in 100μL RPMI containing 5% FBS, 1% antibiotic (antimycotic) solution for 24 hours. 20μL of the indicated viruses were then added at multiplicities of infection (MOI) of 1, 0.1, and 0.01. Daily cell viability assays were performed by adding 20μL of CellTiter 96 Aqueous One Solution Cell Proliferation Assay to all wells and colorimetrically analyzing after 1 hour of incubation. Experimental results were normalized with respect to media only and zero MOI controls. Each experiment was repeated in triplicate. Graphs showing cell viability (%) over time in PANC-1 (FIG. 12A), MiaPaCa-2 (FIG. 12C), BxPC-3 (FIG. 12E), SU.86.86 (FIG. 12G), Capan-1 (FIG. 12I), and AsPC-1 (FIG. 12K) treated with #33 at MOIs of 1, 0.1, or 0.01. Also shown are bar graphs comparing cell viability (%) after 120 hours in cancer cells PANC-1 (FIG. 12B), MiaPaCa-2 (FIG. 12D), BxPC-3 (FIG. 12F), SU.86.86 (FIG. 12H), Capan-1 (FIG. 12J), and AsPC-1 (FIG. 12L) treated with the indicated viruses. Statistical analysis was performed at each time point using one-way ANOVA comparing #33 with the other experimental groups indicated. For SU.86.86 (FIG. 12H), statistical analysis was performed using unpaired t-tests at each MOI. [Figure 12L]In Figures 12A-12L, cytotoxicity assays were performed for PANC-1, MiaPaCa-2, BxPC-3, SU.86.86, Capan-1, and AsPC-1 cancer cell lines by plating 3x103 cancer cells per well in 100μL RPMI containing 5% FBS, 1% antibiotic (antimycotic) solution for 24 hours. 20μL of the indicated viruses were then added at multiplicities of infection (MOI) of 1, 0.1, and 0.01. Daily cell viability assays were performed by adding 20μL of CellTiter 96 Aqueous One Solution Cell Proliferation Assay to all wells and colorimetrically analyzing after 1 hour of incubation. Experimental results were normalized with respect to media only and zero MOI controls. Each experiment was repeated in triplicate. Graphs showing cell viability (%) over time in PANC-1 (FIG. 12A), MiaPaCa-2 (FIG. 12C), BxPC-3 (FIG. 12E), SU.86.86 (FIG. 12G), Capan-1 (FIG. 12I), and AsPC-1 (FIG. 12K) treated with #33 at MOIs of 1, 0.1, or 0.01. Also shown are bar graphs comparing cell viability (%) after 120 hours in cancer cells PANC-1 (FIG. 12B), MiaPaCa-2 (FIG. 12D), BxPC-3 (FIG. 12F), SU.86.86 (FIG. 12H), Capan-1 (FIG. 12J), and AsPC-1 (FIG. 12L) treated with the indicated viruses. Statistical analysis was performed at each time point using one-way ANOVA comparing #33 with the other experimental groups indicated. For SU.86.86 (FIG. 12H), statistical analysis was performed using unpaired t-tests at each MOI. [Figure 13A]In Figures 13A-13L, for the PANC-1, MiaPaCa-2, BxPC-3, SU.86.86, Capan-1 and AsPC-1 cancer cell lines, 5x105 cancer cells were plated per well in 2mL of RPMI containing 10% FBS, 1% antibiotic (antifungal) solution, and viral growth curves were generated in triplicate over 24 hours. The medium was then aspirated, and #33, OncoVEX GFP, GLV-1h68 or #189 was added at a multiplicity of infection (MOI) of 0.01 in 500μL of RPMI containing 2.5% FBS, 1% antibiotic (antifungal) solution, and left for 1 hour with shaking every 20 minutes. At 1 hour, the medium was aspirated, and 1.5mL of RPMI containing 2.5% FBS, 1% antibiotic (antifungal) solution was added. Cells and supernatants were harvested at 24, 48 and 72 hours and serial dilutions were performed in duplicate after three freeze-thaw cycles. This experiment was repeated in duplicate. Graphs showing PFU / million cells over time in cancer cells PANC-1 (FIG. 13A), MiaPaCa-2 (FIG. 13C), BxPC-3 (FIG. 13E), SU.86.86 (FIG. 13G), Capan-1 (FIG. 13I) and AsPC-1 (FIG. 13K) treated with the indicated viruses. Also shown are bar graphs comparing PFU / million cells at each time point in cancer cells PANC-1 (FIG. 13B), MiaPaCa-2 (FIG. 13D), BxPC-3 (FIG. 13F), SU.86.86 (FIG. 13H), Capan-1 (FIG. 13J) and AsPC-1 (FIG. 13L) treated with each virus. At each time point, statistical analysis was performed using one-way ANOVA comparing #33 with other experimental groups. [Figure 13B]In Figures 13A-13L, for the PANC-1, MiaPaCa-2, BxPC-3, SU.86.86, Capan-1 and AsPC-1 cancer cell lines, 5x105 cancer cells were plated per well in 2mL RPMI containing 10% FBS, 1% antibiotic (antifungal) solution, and viral growth curves were performed in triplicate over 24 hours. The medium was then aspirated, and #33, OncoVEX GFP, GLV-1h68 or #189 was added at a multiplicity of infection (MOI) of 0.01 in 500μL RPMI containing 2.5% FBS, 1% antibiotic (antifungal) solution, and left for 1 hour with shaking every 20 minutes. At 1 hour, the medium was aspirated, and 1.5mL RPMI containing 2.5% FBS, 1% antibiotic (antifungal) solution was added. Cells and supernatants were harvested at 24, 48 and 72 hours and serial dilutions were performed in duplicate after three freeze-thaw cycles. The experiment was repeated in duplicate. Graphs showing PFU / million cells over time in cancer cells PANC-1 (FIG. 13A), MiaPaCa-2 (FIG. 13C), BxPC-3 (FIG. 13E), SU.86.86 (FIG. 13G), Capan-1 (FIG. 13I) and AsPC-1 (FIG. 13K) treated with the indicated viruses. Also shown are bar graphs comparing PFU / million cells at each time point in cancer cells PANC-1 (FIG. 13B), MiaPaCa-2 (FIG. 13D), BxPC-3 (FIG. 13F), SU.86.86 (FIG. 13H), Capan-1 (FIG. 13J) and AsPC-1 (FIG. 13L) treated with each virus. At each time point, statistical analysis was performed using one-way ANOVA comparing #33 with other experimental groups. [Figure 13C]In Figures 13A-13L, for the PANC-1, MiaPaCa-2, BxPC-3, SU.86.86, Capan-1 and AsPC-1 cancer cell lines, 5x105 cancer cells were plated per well in 2mL RPMI containing 10% FBS, 1% antibiotic (antifungal) solution, and viral growth curves were performed in triplicate over 24 hours. The medium was then aspirated, and #33, OncoVEX GFP, GLV-1h68 or #189 was added at a multiplicity of infection (MOI) of 0.01 in 500μL RPMI containing 2.5% FBS, 1% antibiotic (antifungal) solution, and left for 1 hour with shaking every 20 minutes. At 1 hour, the medium was aspirated, and 1.5mL RPMI containing 2.5% FBS, 1% antibiotic (antifungal) solution was added. Cells and supernatants were harvested at 24, 48 and 72 hours and serial dilutions were performed in duplicate after three freeze-thaw cycles. This experiment was repeated in duplicate. Graphs showing PFU / million cells over time in cancer cells PANC-1 (FIG. 13A), MiaPaCa-2 (FIG. 13C), BxPC-3 (FIG. 13E), SU.86.86 (FIG. 13G), Capan-1 (FIG. 13I) and AsPC-1 (FIG. 13K) treated with the indicated viruses. Also shown are bar graphs comparing PFU / million cells at each time point in cancer cells PANC-1 (FIG. 13B), MiaPaCa-2 (FIG. 13D), BxPC-3 (FIG. 13F), SU.86.86 (FIG. 13H), Capan-1 (FIG. 13J) and AsPC-1 (FIG. 13L) treated with each virus. At each time point, statistical analysis was performed using one-way ANOVA comparing #33 with other experimental groups. [Figure 13D]In Figures 13A-13L, for the PANC-1, MiaPaCa-2, BxPC-3, SU.86.86, Capan-1 and AsPC-1 cancer cell lines, 5x105 cancer cells were plated per well in 2mL RPMI containing 10% FBS, 1% antibiotic (antifungal) solution, and viral growth curves were performed in triplicate over 24 hours. The medium was then aspirated, and #33, OncoVEX GFP, GLV-1h68 or #189 was added at a multiplicity of infection (MOI) of 0.01 in 500μL RPMI containing 2.5% FBS, 1% antibiotic (antifungal) solution, and left for 1 hour with shaking every 20 minutes. At 1 hour, the medium was aspirated, and 1.5mL RPMI containing 2.5% FBS, 1% antibiotic (antifungal) solution was added. Cells and supernatants were harvested at 24, 48 and 72 hours and serial dilutions were performed in duplicate after three freeze-thaw cycles. The experiment was repeated in duplicate. Graphs showing PFU / million cells over time in cancer cells PANC-1 (FIG. 13A), MiaPaCa-2 (FIG. 13C), BxPC-3 (FIG. 13E), SU.86.86 (FIG. 13G), Capan-1 (FIG. 13I) and AsPC-1 (FIG. 13K) treated with the indicated viruses. Also shown are bar graphs comparing PFU / million cells at each time point in cancer cells PANC-1 (FIG. 13B), MiaPaCa-2 (FIG. 13D), BxPC-3 (FIG. 13F), SU.86.86 (FIG. 13H), Capan-1 (FIG. 13J) and AsPC-1 (FIG. 13L) treated with each virus. At each time point, statistical analysis was performed using one-way ANOVA comparing #33 with other experimental groups. [Figure 13E]In Figures 13A-13L, for the PANC-1, MiaPaCa-2, BxPC-3, SU.86.86, Capan-1 and AsPC-1 cancer cell lines, 5x105 cancer cells were plated per well in 2mL RPMI containing 10% FBS, 1% antibiotic (antifungal) solution, and viral growth curves were performed in triplicate over 24 hours. The medium was then aspirated, and #33, OncoVEX GFP, GLV-1h68 or #189 was added at a multiplicity of infection (MOI) of 0.01 in 500μL RPMI containing 2.5% FBS, 1% antibiotic (antifungal) solution, and left for 1 hour with shaking every 20 minutes. At 1 hour, the medium was aspirated, and 1.5mL RPMI containing 2.5% FBS, 1% antibiotic (antifungal) solution was added. Cells and supernatants were harvested at 24, 48 and 72 hours and serial dilutions were performed in duplicate after three freeze-thaw cycles. The experiment was repeated in duplicate. Graphs showing PFU / million cells over time in cancer cells PANC-1 (FIG. 13A), MiaPaCa-2 (FIG. 13C), BxPC-3 (FIG. 13E), SU.86.86 (FIG. 13G), Capan-1 (FIG. 13I) and AsPC-1 (FIG. 13K) treated with the indicated viruses. Also shown are bar graphs comparing PFU / million cells at each time point in cancer cells PANC-1 (FIG. 13B), MiaPaCa-2 (FIG. 13D), BxPC-3 (FIG. 13F), SU.86.86 (FIG. 13H), Capan-1 (FIG. 13J) and AsPC-1 (FIG. 13L) treated with each virus. At each time point, statistical analysis was performed using one-way ANOVA comparing #33 with other experimental groups. [Figure 13F]In Figures 13A-13L, for the PANC-1, MiaPaCa-2, BxPC-3, SU.86.86, Capan-1 and AsPC-1 cancer cell lines, 5x105 cancer cells were plated per well in 2mL RPMI containing 10% FBS, 1% antibiotic (antifungal) solution, and viral growth curves were performed in triplicate over 24 hours. The medium was then aspirated, and #33, OncoVEX GFP, GLV-1h68 or #189 was added at a multiplicity of infection (MOI) of 0.01 in 500μL RPMI containing 2.5% FBS, 1% antibiotic (antifungal) solution, and left for 1 hour with shaking every 20 minutes. At 1 hour, the medium was aspirated, and 1.5mL RPMI containing 2.5% FBS, 1% antibiotic (antifungal) solution was added. Cells and supernatants were harvested at 24, 48 and 72 hours and serial dilutions were performed in duplicate after three freeze-thaw cycles. This experiment was repeated in duplicate. Graphs showing PFU / million cells over time in cancer cells PANC-1 (FIG. 13A), MiaPaCa-2 (FIG. 13C), BxPC-3 (FIG. 13E), SU.86.86 (FIG. 13G), Capan-1 (FIG. 13I) and AsPC-1 (FIG. 13K) treated with the indicated viruses. Also shown are bar graphs comparing PFU / million cells at each time point in cancer cells PANC-1 (FIG. 13B), MiaPaCa-2 (FIG. 13D), BxPC-3 (FIG. 13F), SU.86.86 (FIG. 13H), Capan-1 (FIG. 13J) and AsPC-1 (FIG. 13L) treated with each virus. At each time point, statistical analysis was performed using one-way ANOVA comparing #33 with other experimental groups. [Figure 13G]In Figures 13A-13L, for the PANC-1, MiaPaCa-2, BxPC-3, SU.86.86, Capan-1 and AsPC-1 cancer cell lines, 5x105 cancer cells were plated per well in 2mL RPMI containing 10% FBS, 1% antibiotic (antifungal) solution, and viral growth curves were performed in triplicate over 24 hours. The medium was then aspirated, and #33, OncoVEX GFP, GLV-1h68 or #189 was added at a multiplicity of infection (MOI) of 0.01 in 500μL RPMI containing 2.5% FBS, 1% antibiotic (antifungal) solution, and left for 1 hour with shaking every 20 minutes. At 1 hour, the medium was aspirated, and 1.5mL RPMI containing 2.5% FBS, 1% antibiotic (antifungal) solution was added. Cells and supernatants were harvested at 24, 48 and 72 hours and serial dilutions were performed in duplicate after three freeze-thaw cycles. This experiment was repeated in duplicate. Graphs showing PFU / million cells over time in cancer cells PANC-1 (FIG. 13A), MiaPaCa-2 (FIG. 13C), BxPC-3 (FIG. 13E), SU.86.86 (FIG. 13G), Capan-1 (FIG. 13I) and AsPC-1 (FIG. 13K) treated with the indicated viruses. Also shown are bar graphs comparing PFU / million cells at each time point in cancer cells PANC-1 (FIG. 13B), MiaPaCa-2 (FIG. 13D), BxPC-3 (FIG. 13F), SU.86.86 (FIG. 13H), Capan-1 (FIG. 13J) and AsPC-1 (FIG. 13L) treated with each virus. At each time point, statistical analysis was performed using one-way ANOVA comparing #33 with other experimental groups. [Figure 13H]In Figures 13A-13L, for the PANC-1, MiaPaCa-2, BxPC-3, SU.86.86, Capan-1 and AsPC-1 cancer cell lines, 5x105 cancer cells were plated per well in 2mL RPMI containing 10% FBS, 1% antibiotic (antifungal) solution, and viral growth curves were performed in triplicate over 24 hours. The medium was then aspirated, and #33, OncoVEX GFP, GLV-1h68 or #189 was added at a multiplicity of infection (MOI) of 0.01 in 500μL RPMI containing 2.5% FBS, 1% antibiotic (antifungal) solution, and left for 1 hour with shaking every 20 minutes. At 1 hour, the medium was aspirated, and 1.5mL RPMI containing 2.5% FBS, 1% antibiotic (antifungal) solution was added. Cells and supernatants were harvested at 24, 48 and 72 hours and serial dilutions were performed in duplicate after three freeze-thaw cycles. This experiment was repeated in duplicate. Graphs showing PFU / million cells over time in cancer cells PANC-1 (FIG. 13A), MiaPaCa-2 (FIG. 13C), BxPC-3 (FIG. 13E), SU.86.86 (FIG. 13G), Capan-1 (FIG. 13I) and AsPC-1 (FIG. 13K) treated with the indicated viruses. Also shown are bar graphs comparing PFU / million cells at each time point in cancer cells PANC-1 (FIG. 13B), MiaPaCa-2 (FIG. 13D), BxPC-3 (FIG. 13F), SU.86.86 (FIG. 13H), Capan-1 (FIG. 13J) and AsPC-1 (FIG. 13L) treated with each virus. At each time point, statistical analysis was performed using one-way ANOVA comparing #33 with other experimental groups. [Figure 13I]In Figures 13A-13L, for the PANC-1, MiaPaCa-2, BxPC-3, SU.86.86, Capan-1 and AsPC-1 cancer cell lines, 5x105 cancer cells were plated per well in 2mL RPMI containing 10% FBS, 1% antibiotic (antifungal) solution, and viral growth curves were performed in triplicate over 24 hours. The medium was then aspirated, and #33, OncoVEX GFP, GLV-1h68 or #189 was added at a multiplicity of infection (MOI) of 0.01 in 500μL RPMI containing 2.5% FBS, 1% antibiotic (antifungal) solution, and left for 1 hour with shaking every 20 minutes. At 1 hour, the medium was aspirated, and 1.5mL RPMI containing 2.5% FBS, 1% antibiotic (antifungal) solution was added. Cells and supernatants were harvested at 24, 48 and 72 hours and serial dilutions were performed in duplicate after three freeze-thaw cycles. The experiment was repeated in duplicate. Graphs showing PFU / million cells over time in cancer cells PANC-1 (FIG. 13A), MiaPaCa-2 (FIG. 13C), BxPC-3 (FIG. 13E), SU.86.86 (FIG. 13G), Capan-1 (FIG. 13I) and AsPC-1 (FIG. 13K) treated with the indicated viruses. Also shown are bar graphs comparing PFU / million cells at each time point in cancer cells PANC-1 (FIG. 13B), MiaPaCa-2 (FIG. 13D), BxPC-3 (FIG. 13F), SU.86.86 (FIG. 13H), Capan-1 (FIG. 13J) and AsPC-1 (FIG. 13L) treated with each virus. At each time point, statistical analysis was performed using one-way ANOVA comparing #33 with other experimental groups. [Figure 13J]In Figures 13A-13L, for the PANC-1, MiaPaCa-2, BxPC-3, SU.86.86, Capan-1 and AsPC-1 cancer cell lines, 5x105 cancer cells were plated per well in 2mL RPMI containing 10% FBS, 1% antibiotic (antifungal) solution, and viral growth curves were performed in triplicate over 24 hours. The medium was then aspirated, and #33, OncoVEX GFP, GLV-1h68 or #189 was added at a multiplicity of infection (MOI) of 0.01 in 500μL RPMI containing 2.5% FBS, 1% antibiotic (antifungal) solution, and left for 1 hour with shaking every 20 minutes. At 1 hour, the medium was aspirated, and 1.5mL RPMI containing 2.5% FBS, 1% antibiotic (antifungal) solution was added. Cells and supernatants were harvested at 24, 48 and 72 hours and serial dilutions were performed in duplicate after three freeze-thaw cycles. The experiment was repeated in duplicate. Graphs showing PFU / million cells over time in cancer cells PANC-1 (FIG. 13A), MiaPaCa-2 (FIG. 13C), BxPC-3 (FIG. 13E), SU.86.86 (FIG. 13G), Capan-1 (FIG. 13I) and AsPC-1 (FIG. 13K) treated with the indicated viruses. Also shown are bar graphs comparing PFU / million cells at each time point in cancer cells PANC-1 (FIG. 13B), MiaPaCa-2 (FIG. 13D), BxPC-3 (FIG. 13F), SU.86.86 (FIG. 13H), Capan-1 (FIG. 13J) and AsPC-1 (FIG. 13L) treated with each virus. At each time point, statistical analysis was performed using one-way ANOVA comparing #33 with other experimental groups. [Figure 13K]In Figures 13A-13L, for the PANC-1, MiaPaCa-2, BxPC-3, SU.86.86, Capan-1 and AsPC-1 cancer cell lines, 5x105 cancer cells were plated per well in 2mL RPMI containing 10% FBS, 1% antibiotic (antifungal) solution, and viral growth curves were performed in triplicate over 24 hours. The medium was then aspirated, and #33, OncoVEX GFP, GLV-1h68 or #189 was added at a multiplicity of infection (MOI) of 0.01 in 500μL RPMI containing 2.5% FBS, 1% antibiotic (antifungal) solution, and left for 1 hour with shaking every 20 minutes. At 1 hour, the medium was aspirated, and 1.5mL RPMI containing 2.5% FBS, 1% antibiotic (antifungal) solution was added. Cells and supernatants were harvested at 24, 48 and 72 hours and serial dilutions were performed in duplicate after three freeze-thaw cycles. The experiment was repeated in duplicate. Graphs showing PFU / million cells over time in cancer cells PANC-1 (FIG. 13A), MiaPaCa-2 (FIG. 13C), BxPC-3 (FIG. 13E), SU.86.86 (FIG. 13G), Capan-1 (FIG. 13I) and AsPC-1 (FIG. 13K) treated with the indicated viruses. Also shown are bar graphs comparing PFU / million cells at each time point in cancer cells PANC-1 (FIG. 13B), MiaPaCa-2 (FIG. 13D), BxPC-3 (FIG. 13F), SU.86.86 (FIG. 13H), Capan-1 (FIG. 13J) and AsPC-1 (FIG. 13L) treated with each virus. At each time point, statistical analysis was performed using one-way ANOVA comparing #33 with other experimental groups. [Figure 13L]In Figures 13A-13L, for the PANC-1, MiaPaCa-2, BxPC-3, SU.86.86, Capan-1 and AsPC-1 cancer cell lines, 5x105 cancer cells were plated per well in 2mL RPMI containing 10% FBS, 1% antibiotic (antifungal) solution, and viral growth curves were performed in triplicate over 24 hours. The medium was then aspirated, and #33, OncoVEX GFP, GLV-1h68 or #189 was added at a multiplicity of infection (MOI) of 0.01 in 500μL RPMI containing 2.5% FBS, 1% antibiotic (antifungal) solution, and left for 1 hour with shaking every 20 minutes. At 1 hour, the medium was aspirated, and 1.5mL RPMI containing 2.5% FBS, 1% antibiotic (antifungal) solution was added. Cells and supernatants were harvested at 24, 48 and 72 hours and serial dilutions were performed in duplicate after three freeze-thaw cycles. This experiment was repeated in duplicate. Graphs showing PFU / million cells over time in cancer cells PANC-1 (FIG. 13A), MiaPaCa-2 (FIG. 13C), BxPC-3 (FIG. 13E), SU.86.86 (FIG. 13G), Capan-1 (FIG. 13I) and AsPC-1 (FIG. 13K) treated with the indicated viruses. Also shown are bar graphs comparing PFU / million cells at each time point in cancer cells PANC-1 (FIG. 13B), MiaPaCa-2 (FIG. 13D), BxPC-3 (FIG. 13F), SU.86.86 (FIG. 13H), Capan-1 (FIG. 13J) and AsPC-1 (FIG. 13L) treated with each virus. At each time point, statistical analysis was performed using one-way ANOVA comparing #33 with other experimental groups. [Figure 14A]In Figures 14A-14C, 18 female athymic Nude-Foxn1nu nude mice (Envigo, Indianapolis, IN) were implanted with 2x106 tumors of MiaPaCa-2 in both flanks. When tumor size reached 400mm3, the tumors on the left side were injected with 50μL of PBS (3 mice), #33-(SE)hNIS or #33-(SE)hNIS-E9LmiR100t (5 mice) at approximately 1x105 PFU / dose. Net body weight % change (Figure 14A), and injected tumor % change (Figure 14B), and non-injected tumor % change (Figure 14C) were recorded twice weekly for 43 days. [Figure 14B] In Figures 14A-14C, 18 female athymic Nude-Foxn1nu nude mice (Envigo, Indianapolis, IN) were implanted with 2x106 tumors of MiaPaCa-2 in both flanks. When tumor size reached 400mm3, the tumors on the left side were injected with 50μL of PBS (3 mice), #33-(SE)hNIS or #33-(SE)hNIS-E9LmiR100t (5 mice) at approximately 1x105 PFU / dose. Net body weight % change (Figure 14A), and injected tumor % change (Figure 14B), and non-injected tumor % change (Figure 14C) were recorded twice weekly for 43 days. [Figure 14C] In Figures 14A-14C, 18 female athymic Nude-Foxn1nu nude mice (Envigo, Indianapolis, IN) were implanted with 2x106 tumors of MiaPaCa-2 in both flanks. When tumor size reached 400mm3, the tumors on the left side were injected with 50μL of PBS (3 mice), #33-(SE)hNIS or #33-(SE)hNIS-E9LmiR100t (5 mice) at approximately 1x105 PFU / dose. Net body weight % change (Figure 14A), and injected tumor % change (Figure 14B), and non-injected tumor % change (Figure 14C) were recorded twice weekly for 43 days. [Figure 15A]In Figures 15A-15C, 26 female athymic Nude-Foxn1nu nude mice (Envigo, Indianapolis, IN) were implanted with tumors PANC-1 at 1.25 x 106 cells in both flanks. When tumor size reached approximately 250 mm3, the tumors on the left side were injected with 50 μL of PBS (4 mice), #33 (6 mice), #33-(SE)hNIS (6 mice), #33-(SE)hNIS-E9LmiR100t (5 mice) or #33-(H5)Fluc2 at approximately 1 x 103 PFU / dose. Net body weight % change (Figure 15A), and injected tumor % change (Figure 15B), and non-injected tumor % change (Figure 15C) were recorded twice weekly for 43 days. [Figure 15B] In Figures 15A-15C, 26 female athymic Nude-Foxn1nu nude mice (Envigo, Indianapolis, IN) were implanted with tumors PANC-1 at 1.25x106 cells in both flanks. When tumor size reached approximately 250mm3, the tumors on the left side were injected with 50μL of PBS (4 mice), #33 (6 mice), #33-(SE)hNIS (6 mice), #33-(SE)hNIS-E9LmiR100t (5 mice) or #33-(H5)Fluc2 at approximately 1x103 PFU / dose. Net body weight % change (Figure 15A), and injected tumor % change (Figure 15B), and non-injected tumor % change (Figure 15C) were recorded twice weekly for 43 days. [Figure 15C] In Figures 15A-15C, 26 female athymic Nude-Foxn1nu nude mice (Envigo, Indianapolis, IN) were implanted with tumors PANC-1 at 1.25x106 cells in both flanks. When tumor size reached approximately 250mm3, the tumors on the left side were injected with 50μL of PBS (4 mice), #33 (6 mice), #33-(SE)hNIS (6 mice), #33-(SE)hNIS-E9LmiR100t (5 mice) or #33-(H5)Fluc2 at approximately 1x103 PFU / dose. Net body weight % change (Figure 15A), and injected tumor % change (Figure 15B), and non-injected tumor % change (Figure 15C) were recorded twice weekly for 43 days. [Figure 16]Twice a week, one PBS control mouse and three #33-(H5)Fluc2-injected mice were intraperitoneally injected with 4.28 mg luciferin / 150 μL PBS. After 7 min, luciferase imaging was obtained at standard exposure. Relative units were recorded at each time point and analyzed relative to the PBS control mice as background. [Figure 17A] In Figures 17A-17D, HT-29 and HCT-116 cancer cell lines were plated at 3 x 103 cells per well in 100 μL of McCoy's 5A medium containing 5% FBS, 1% antibiotic (antimycotic) solution, and cytotoxicity assays were performed for 24 hours. Viruses #33, #33-(SE)hNIS, #33-(H5)Emerald, OncoVEXGFP, GLV-1h68, or #189 were then added at 20 μL, multiplicity of infection (MOI) of 1, 0.1, and 0.01, respectively. Daily cell viability assays were performed by adding 20 μL of CellTiter 96 Aqueous One Solution Cell Proliferation Assay to all wells and colorimetrically analyzing after 1 hour of incubation. Experimental results were normalized with respect to media only and zero MOI controls. Each experiment was repeated in triplicate. Graphs of cell viability (%) over time are shown for HT-29 (FIG. 17A) and HCT-116 (FIG. 17C) treated with #33 at MOI of 1, 0.1, or 0.01. Also shown are bar graphs comparing cell viability (%) after 120 hours for HT-29 (FIG. 17B) and HCT-116 (FIG. 17D) cancer cells treated with the indicated viruses. Statistical analysis was performed using one-way ANOVA comparing #33 to other experimental groups at each time point. [Figure 17B]In Figures 17A-17D, HT-29 and HCT-116 cancer cell lines were plated at 3 x 103 cells per well in 100 μL of McCoy's 5A medium containing 5% FBS, 1% antibiotic (antimycotic) solution, and cytotoxicity assays were performed for 24 hours. Viruses #33, #33-(SE)hNIS, #33-(H5)Emerald, OncoVEXGFP, GLV-1h68, or #189 were then added at 20 μL, multiplicity of infection (MOI) of 1, 0.1, and 0.01, respectively. Daily cell viability assays were performed by adding 20 μL of CellTiter 96 Aqueous One Solution Cell Proliferation Assay to all wells and colorimetrically analyzing after 1 hour of incubation. Experimental results were normalized with respect to media only and zero MOI controls. Each experiment was repeated in triplicate. Graphs of cell viability (%) over time are shown for HT-29 (FIG. 17A) and HCT-116 (FIG. 17C) treated with #33 at MOI of 1, 0.1, or 0.01. Also shown are bar graphs comparing cell viability (%) after 120 hours for HT-29 (FIG. 17B) and HCT-116 (FIG. 17D) cancer cells treated with the indicated viruses. Statistical analysis was performed using one-way ANOVA comparing #33 to other experimental groups at each time point. [Figure 17C]In Figures 17A-17D, HT-29 and HCT-116 cancer cell lines were plated at 3 x 103 cells per well in 100 μL of McCoy's 5A medium containing 5% FBS, 1% antibiotic (antimycotic) solution, and cytotoxicity assays were performed for 24 hours. Viruses #33, #33-(SE)hNIS, #33-(H5)Emerald, OncoVEXGFP, GLV-1h68, or #189 were then added at 20 μL, multiplicity of infection (MOI) of 1, 0.1, and 0.01, respectively. Daily cell viability assays were performed by adding 20 μL of CellTiter 96 Aqueous One Solution Cell Proliferation Assay to all wells and colorimetrically analyzing after 1 hour of incubation. Experimental results were normalized with respect to media only and zero MOI controls. Each experiment was repeated in triplicate. Graphs of cell viability (%) over time are shown for HT-29 (FIG. 17A) and HCT-116 (FIG. 17C) treated with #33 at MOI of 1, 0.1, or 0.01. Also shown are bar graphs comparing cell viability (%) after 120 hours for HT-29 (FIG. 17B) and HCT-116 (FIG. 17D) cancer cells treated with the indicated viruses. Statistical analysis was performed using one-way ANOVA comparing #33 to other experimental groups at each time point. [Figure 17D]In Figures 17A-17D, HT-29 and HCT-116 cancer cell lines were plated at 3 x 103 cells per well in 100 μL of McCoy's 5A medium containing 5% FBS, 1% antibiotic (antimycotic) solution, and cytotoxicity assays were performed for 24 hours. Viruses #33, #33-(SE)hNIS, #33-(H5)Emerald, OncoVEXGFP, GLV-1h68, or #189 were then added at 20 μL, multiplicity of infection (MOI) of 1, 0.1, and 0.01, respectively. Daily cell viability assays were performed by adding 20 μL of CellTiter 96 Aqueous One Solution Cell Proliferation Assay to all wells and colorimetrically analyzing after 1 hour of incubation. Experimental results were normalized with respect to media only and zero MOI controls. Each experiment was repeated in triplicate. Graphs of cell viability (%) over time are shown for HT-29 (FIG. 17A) and HCT-116 (FIG. 17C) treated with #33 at MOI of 1, 0.1, or 0.01. Also shown are bar graphs comparing cell viability (%) after 120 hours for HT-29 (FIG. 17B) and HCT-116 (FIG. 17D) cancer cells treated with the indicated viruses. Statistical analysis was performed using one-way ANOVA comparing #33 to other experimental groups at each time point. [Figure 18A]In Figures 18A-18F, cancer cell lines SW620, SW480 and COLO 320DM were plated at 3x103 cells per well in 100μL of RPMI containing 5% FBS, 1% antibiotic (antimycotic) solution and cytotoxicity assays were performed for 24 hours. Viruses #33, #33-(SE)hNIS, #33-(H5)Emerald, OncoVEXGFP, GLV-1h68 or #189 were then added at 20μL, multiplicity of infection (MOI) of 1, 0.1 and 0.01, respectively. Daily cell viability assays were performed by adding 20μL of CellTiter 96 Aqueous One Solution Cell Proliferation Assay to all wells and colorimetrically analyzing after 1 hour of incubation. Experimental results were normalized with respect to media only and zero MOI controls. Each experiment was repeated in triplicate. Graphs of cell viability (%) over time are shown for SW620 (FIG. 18A), SW480 (FIG. 18C), and COLO 320DM (FIG. 18E) treated with #33 at MOIs of 1, 0.1, or 0.01. Also shown are bar graphs comparing cell viability (%) after 120 hours for SW620 (FIG. 18B), SW480 (FIG. 18D), and COLO 320DM (FIG. 18F) cancer cells treated with the indicated viruses. Statistical analysis was performed using one-way ANOVA to compare #33 with other experimental groups at each time point. "NS" above the comparison bars means "not significant." [Figure 18B]In Figures 18A-18F, cancer cell lines SW620, SW480 and COLO 320DM were plated at 3x103 cells per well in 100μL of RPMI containing 5% FBS, 1% antibiotic (antimycotic) solution and cytotoxicity assays were performed for 24 hours. Viruses #33, #33-(SE)hNIS, #33-(H5)Emerald, OncoVEXGFP, GLV-1h68 or #189 were then added at 20μL, multiplicity of infection (MOI) of 1, 0.1 and 0.01, respectively. Daily cell viability assays were performed by adding 20μL of CellTiter 96 Aqueous One Solution Cell Proliferation Assay to all wells and colorimetrically analyzing after 1 hour of incubation. Experimental results were normalized with respect to media only and zero MOI controls. Each experiment was repeated in triplicate. Graphs of cell viability (%) over time are shown for SW620 (FIG. 18A), SW480 (FIG. 18C), and COLO 320DM (FIG. 18E) treated with #33 at MOIs of 1, 0.1, or 0.01. Also shown are bar graphs comparing cell viability (%) after 120 hours for SW620 (FIG. 18B), SW480 (FIG. 18D), and COLO 320DM (FIG. 18F) cancer cells treated with the indicated viruses. Statistical analysis was performed using one-way ANOVA to compare #33 with other experimental groups at each time point. "NS" above the comparison bars means "not significant." [Figure 18C]In Figures 18A-18F, cancer cell lines SW620, SW480 and COLO 320DM were plated at 3x103 cells per well in 100μL of RPMI containing 5% FBS, 1% antibiotic (antimycotic) solution and cytotoxicity assays were performed for 24 hours. Viruses #33, #33-(SE)hNIS, #33-(H5)Emerald, OncoVEXGFP, GLV-1h68 or #189 were then added at 20μL, multiplicity of infection (MOI) of 1, 0.1 and 0.01, respectively. Daily cell viability assays were performed by adding 20μL of CellTiter 96 Aqueous One Solution Cell Proliferation Assay to all wells and colorimetrically analyzing after 1 hour of incubation. Experimental results were normalized with respect to media only and zero MOI controls. Each experiment was repeated in triplicate. Graphs of cell viability (%) over time are shown for SW620 (FIG. 18A), SW480 (FIG. 18C), and COLO 320DM (FIG. 18E) treated with #33 at MOIs of 1, 0.1, or 0.01. Also shown are bar graphs comparing cell viability (%) after 120 hours for SW620 (FIG. 18B), SW480 (FIG. 18D), and COLO 320DM (FIG. 18F) cancer cells treated with the indicated viruses. Statistical analysis was performed using one-way ANOVA to compare #33 with other experimental groups at each time point. "NS" above the comparison bars means "not significant." [Figure 18D]In Figures 18A-18F, cancer cell lines SW620, SW480 and COLO 320DM were plated at 3x103 cells per well in 100μL of RPMI containing 5% FBS, 1% antibiotic (antimycotic) solution and cytotoxicity assays were performed for 24 hours. Viruses #33, #33-(SE)hNIS, #33-(H5)Emerald, OncoVEXGFP, GLV-1h68 or #189 were then added at 20μL, multiplicity of infection (MOI) of 1, 0.1 and 0.01, respectively. Daily cell viability assays were performed by adding 20μL of CellTiter 96 Aqueous One Solution Cell Proliferation Assay to all wells and colorimetrically analyzing after 1 hour of incubation. Experimental results were normalized with respect to media only and zero MOI controls. Each experiment was repeated in triplicate. Graphs of cell viability (%) over time are shown for SW620 (FIG. 18A), SW480 (FIG. 18C), and COLO 320DM (FIG. 18E) treated with #33 at MOIs of 1, 0.1, or 0.01. Also shown are bar graphs comparing cell viability (%) after 120 hours for SW620 (FIG. 18B), SW480 (FIG. 18D), and COLO 320DM (FIG. 18F) cancer cells treated with the indicated viruses. Statistical analysis was performed using one-way ANOVA to compare #33 with other experimental groups at each time point. "NS" above the comparison bars means "not significant." [Figure 18E]In Figures 18A-18F, cancer cell lines SW620, SW480 and COLO 320DM were plated at 3x103 cells per well in 100μL of RPMI containing 5% FBS, 1% antibiotic (antimycotic) solution and cytotoxicity assays were performed for 24 hours. Viruses #33, #33-(SE)hNIS, #33-(H5)Emerald, OncoVEXGFP, GLV-1h68 or #189 were then added at 20μL, multiplicity of infection (MOI) of 1, 0.1 and 0.01, respectively. Daily cell viability assays were performed by adding 20μL of CellTiter 96 Aqueous One Solution Cell Proliferation Assay to all wells and colorimetrically analyzing after 1 hour of incubation. Experimental results were normalized with respect to media only and zero MOI controls. Each experiment was repeated in triplicate. Graphs of cell viability (%) over time are shown for SW620 (FIG. 18A), SW480 (FIG. 18C), and COLO 320DM (FIG. 18E) treated with #33 at MOIs of 1, 0.1, or 0.01. Also shown are bar graphs comparing cell viability (%) after 120 hours for SW620 (FIG. 18B), SW480 (FIG. 18D), and COLO 320DM (FIG. 18F) cancer cells treated with the indicated viruses. Statistical analysis was performed using one-way ANOVA to compare #33 with other experimental groups at each time point. "NS" above the comparison bars means "not significant." [Figure 18F]In Figures 18A-18F, cancer cell lines SW620, SW480 and COLO 320DM were plated at 3x103 cells per well in 100μL of RPMI containing 5% FBS, 1% antibiotic (antimycotic) solution and cytotoxicity assays were performed for 24 hours. Viruses #33, #33-(SE)hNIS, #33-(H5)Emerald, OncoVEXGFP, GLV-1h68 or #189 were then added at 20μL, multiplicity of infection (MOI) of 1, 0.1 and 0.01, respectively. Daily cell viability assays were performed by adding 20μL of CellTiter 96 Aqueous One Solution Cell Proliferation Assay to all wells and colorimetrically analyzing after 1 hour of incubation. Experimental results were normalized with respect to media only and zero MOI controls. Each experiment was repeated in triplicate. Graphs of cell viability (%) over time are shown for SW620 (FIG. 18A), SW480 (FIG. 18C), and COLO 320DM (FIG. 18E) treated with #33 at MOIs of 1, 0.1, or 0.01. Also shown are bar graphs comparing cell viability (%) after 120 hours for SW620 (FIG. 18B), SW480 (FIG. 18D), and COLO 320DM (FIG. 18F) cancer cells treated with the indicated viruses. Statistical analysis was performed using one-way ANOVA to compare #33 with other experimental groups at each time point. "NS" above the comparison bars means "not significant." [Figure 19A]In Figures 19A-19B, LoVo cancer cell line was plated at 3 x 103 cells per well in 100 μL of F-12K medium containing 5% FBS, 1% antibiotic (antimycotic) solution, and cytotoxicity assays were performed for 24 hours. Viruses #33, #33-(SE)hNIS, #33-(H5)Emerald, OncoVEXGFP, GLV-1h68, or #189 were then added at 20 μL, 1, 0.1, and 0.01 multiplicity of infection (MOI), respectively. Daily cell viability assays were performed by adding 20 μL of CellTiter 96 Aqueous One Solution Cell Proliferation Assay to all wells and colorimetrically analyzing after 1 hour of incubation. Experimental results were normalized with respect to media only and zero MOI controls. Each experiment was repeated in triplicate. Figure 19A shows the percentage of cell viability over time in LoVo cancer cells treated with #33 at an MOI of 1, 0.1, or 0.01. Figure 19B shows a bar graph comparing the percentage of cell viability after 120 hours in LoVo cancer cells treated with the indicated viruses. Statistical analysis was performed using one-way ANOVA comparing #33 to other experimental groups at each time point. "NS" above the comparison bars means "not significant." [Figure 19B]In Figures 19A-19B, LoVo cancer cell line was plated at 3 x 103 cells per well in 100 μL of F-12K medium containing 5% FBS, 1% antibiotic (antimycotic) solution, and cytotoxicity assays were performed for 24 hours. Viruses #33, #33-(SE)hNIS, #33-(H5)Emerald, OncoVEXGFP, GLV-1h68, or #189 were then added at 20 μL, 1, 0.1, and 0.01 multiplicity of infection (MOI), respectively. Daily cell viability assays were performed by adding 20 μL of CellTiter 96 Aqueous One Solution Cell Proliferation Assay to all wells and colorimetrically analyzing after 1 hour of incubation. Experimental results were normalized with respect to media only and zero MOI controls. Each experiment was repeated in triplicate. Figure 19A shows the percentage of cell viability over time in LoVo cancer cells treated with #33 at an MOI of 1, 0.1, or 0.01. Figure 19B shows a bar graph comparing the percentage of cell viability after 120 hours in LoVo cancer cells treated with the indicated viruses. Statistical analysis was performed using one-way ANOVA comparing #33 to other experimental groups at each time point. "NS" above the comparison bars means "not significant." [Figure 20A]In Figures 20A-20D, HT-29 and HCT-116 cancer cell lines were plated at 5x105 cells per well in 2mL of McCoy's 5A medium containing 10% FBS, 1% antibiotic (antimycotic) solution, and viral growth curves were generated in triplicate over 24 hours. The medium was then aspirated and #33, #33-(SE)hNIS, #33-(H5)Emerald, OncoVEXGFP, GLV-1h68, or #189 was added at a multiplicity of infection (MOI) of 0.01 in 500μL of McCoy's 5A medium containing 2.5% FBS, 1% antibiotic (antimycotic) solution for 1 hour with shaking every 20 minutes. After 1 hour, the medium was aspirated and 1.5mL of McCoy's 5A medium containing 2.5% FBS, 1% antibiotic (antimycotic) solution was added. Cells and supernatants were harvested at 24, 48, and 72 hours, and serial dilutions were performed in duplicate after three freeze-thaw cycles. The experiment was repeated in duplicate. Graphs showing PFU / million cells over time in HT-29 (FIG. 20A) and HCT-116 (FIG. 20C). Also, bar graphs comparing PFU / million cells at each time point in cancer cells HT-29 (FIG. 20B) and HCT-116 (FIG. 20D) treated with each virus. Statistical analysis was performed using one-way ANOVA to compare #33 with other experimental groups at each time point. [Figure 20B]In Figures 20A-20D, HT-29 and HCT-116 cancer cell lines were plated at 5x105 cells per well in 2mL of McCoy's 5A medium containing 10% FBS, 1% antibiotic (antimycotic) solution, and viral growth curves were performed in triplicate over 24 hours. The medium was then aspirated and #33, #33-(SE)hNIS, #33-(H5)Emerald, OncoVEXGFP, GLV-1h68, or #189 was added at a multiplicity of infection (MOI) of 0.01 in 500μL of McCoy's 5A medium containing 2.5% FBS, 1% antibiotic (antimycotic) solution for 1 hour with shaking every 20 minutes. After 1 hour, the medium was aspirated and 1.5mL of McCoy's 5A medium containing 2.5% FBS, 1% antibiotic (antimycotic) solution was added. Cells and supernatants were harvested at 24, 48, and 72 hours, and serial dilutions were performed in duplicate after three freeze-thaw cycles. The experiment was repeated in duplicate. Graphs showing PFU / million cells over time in HT-29 (FIG. 20A) and HCT-116 (FIG. 20C). Also, bar graphs comparing PFU / million cells at each time point in cancer cells HT-29 (FIG. 20B) and HCT-116 (FIG. 20D) treated with each virus. Statistical analysis was performed using one-way ANOVA to compare #33 with other experimental groups at each time point. [Figure 20C]In Figures 20A-20D, HT-29 and HCT-116 cancer cell lines were plated at 5x105 cells per well in 2mL of McCoy's 5A medium containing 10% FBS, 1% antibiotic (antimycotic) solution, and viral growth curves were performed in triplicate over 24 hours. The medium was then aspirated and #33, #33-(SE)hNIS, #33-(H5)Emerald, OncoVEXGFP, GLV-1h68, or #189 was added at a multiplicity of infection (MOI) of 0.01 in 500μL of McCoy's 5A medium containing 2.5% FBS, 1% antibiotic (antimycotic) solution for 1 hour with shaking every 20 minutes. After 1 hour, the medium was aspirated and 1.5mL of McCoy's 5A medium containing 2.5% FBS, 1% antibiotic (antimycotic) solution was added. Cells and supernatants were harvested at 24, 48, and 72 hours, and serial dilutions were performed in duplicate after three freeze-thaw cycles. The experiment was repeated in duplicate. Graphs showing PFU / million cells over time in HT-29 (FIG. 20A) and HCT-116 (FIG. 20C). Also, bar graphs comparing PFU / million cells at each time point in cancer cells HT-29 (FIG. 20B) and HCT-116 (FIG. 20D) treated with each virus. Statistical analysis was performed using one-way ANOVA to compare #33 with other experimental groups at each time point. [Figure 20D]In Figures 20A-20D, HT-29 and HCT-116 cancer cell lines were plated at 5x105 cells per well in 2mL of McCoy's 5A medium containing 10% FBS, 1% antibiotic (antimycotic) solution, and viral growth curves were performed in triplicate over 24 hours. The medium was then aspirated and #33, #33-(SE)hNIS, #33-(H5)Emerald, OncoVEXGFP, GLV-1h68, or #189 was added at a multiplicity of infection (MOI) of 0.01 in 500μL of McCoy's 5A medium containing 2.5% FBS, 1% antibiotic (antimycotic) solution for 1 hour with shaking every 20 minutes. After 1 hour, the medium was aspirated and 1.5mL of McCoy's 5A medium containing 2.5% FBS, 1% antibiotic (antimycotic) solution was added. Cells and supernatants were harvested at 24, 48, and 72 hours, and serial dilutions were performed in duplicate after three freeze-thaw cycles. The experiment was repeated in duplicate. Graphs showing PFU / million cells over time in HT-29 (FIG. 20A) and HCT-116 (FIG. 20C). Also, bar graphs comparing PFU / million cells at each time point in cancer cells HT-29 (FIG. 20B) and HCT-116 (FIG. 20D) treated with each virus. Statistical analysis was performed using one-way ANOVA to compare #33 with other experimental groups at each time point. [Figure 21A]In Figures 21A-21D, SW620 and SW480 cancer cell lines were plated at 5x105 cells per well in 2mL RPMI containing 10% FBS, 1% antibiotic (antimycotic) solution, and viral growth curves were generated in triplicate over 24 hours. The medium was then aspirated, and #33, #33-(SE)hNIS, #33-(H5)Emerald, OncoVEXGFP, GLV-1h68, or #189 was added at a multiplicity of infection (MOI) of 0.01 in 500μL RPMI containing 2.5% FBS, 1% antibiotic (antimycotic) solution, and left for 1 hour with shaking every 20 minutes. After 1 hour, the medium was aspirated, and 1.5mL RPMI containing 2.5% FBS, 1% antibiotic (antimycotic) solution was added. Cells and supernatants were harvested at 24, 48, and 72 hours, and serial dilutions were performed in duplicate after three freeze-thaw cycles. The experiment was repeated in duplicate. Graphs showing PFU / million cells over time in SW620 (FIG. 21A) and SW480 (FIG. 21C). Also, bar graphs comparing PFU / million cells at each time point in cancer cells SW620 (FIG. 21B) and SW480 (FIG. 21D) treated with each virus. Statistical analysis was performed using one-way ANOVA to compare #33 with other experimental groups at each time point. [Figure 21B]In Figures 21A-21D, SW620 and SW480 cancer cell lines were plated at 5x105 cells per well in 2mL RPMI containing 10% FBS, 1% antibiotic (antimycotic) solution, and viral growth curves were generated in triplicate over 24 hours. The medium was then aspirated, and #33, #33-(SE)hNIS, #33-(H5)Emerald, OncoVEXGFP, GLV-1h68, or #189 was added at a multiplicity of infection (MOI) of 0.01 in 500μL RPMI containing 2.5% FBS, 1% antibiotic (antimycotic) solution, and left for 1 hour with shaking every 20 minutes. After 1 hour, the medium was aspirated, and 1.5mL RPMI containing 2.5% FBS, 1% antibiotic (antimycotic) solution was added. Cells and supernatants were harvested at 24, 48, and 72 hours, and serial dilutions were performed in duplicate after three freeze-thaw cycles. The experiment was repeated in duplicate. Graphs showing PFU / million cells over time in SW620 (FIG. 21A) and SW480 (FIG. 21C). Also, bar graphs comparing PFU / million cells at each time point in cancer cells SW620 (FIG. 21B) and SW480 (FIG. 21D) treated with each virus. Statistical analysis was performed using one-way ANOVA to compare #33 with other experimental groups at each time point. [Figure 21C]In Figures 21A-21D, SW620 and SW480 cancer cell lines were plated at 5x105 cells per well in 2mL RPMI containing 10% FBS, 1% antibiotic (antimycotic) solution, and viral growth curves were generated in triplicate over 24 hours. The medium was then aspirated, and #33, #33-(SE)hNIS, #33-(H5)Emerald, OncoVEXGFP, GLV-1h68, or #189 was added at a multiplicity of infection (MOI) of 0.01 in 500μL RPMI containing 2.5% FBS, 1% antibiotic (antimycotic) solution, and left for 1 hour with shaking every 20 minutes. After 1 hour, the medium was aspirated, and 1.5mL RPMI containing 2.5% FBS, 1% antibiotic (antimycotic) solution was added. Cells and supernatants were harvested at 24, 48, and 72 hours, and serial dilutions were performed in duplicate after three freeze-thaw cycles. The experiment was repeated in duplicate. Graphs showing PFU / million cells over time in SW620 (FIG. 21A) and SW480 (FIG. 21C). Also, bar graphs comparing PFU / million cells at each time point in cancer cells SW620 (FIG. 21B) and SW480 (FIG. 21D) treated with each virus. Statistical analysis was performed using one-way ANOVA to compare #33 with other experimental groups at each time point. [Figure 21D]In Figures 21A-21D, SW620 and SW480 cancer cell lines were plated at 5x105 cells per well in 2mL RPMI containing 10% FBS, 1% antibiotic (antimycotic) solution, and viral growth curves were generated in triplicate over 24 hours. The medium was then aspirated, and #33, #33-(SE)hNIS, #33-(H5)Emerald, OncoVEXGFP, GLV-1h68, or #189 was added at a multiplicity of infection (MOI) of 0.01 in 500μL RPMI containing 2.5% FBS, 1% antibiotic (antimycotic) solution, and left for 1 hour with shaking every 20 minutes. After 1 hour, the medium was aspirated, and 1.5mL RPMI containing 2.5% FBS, 1% antibiotic (antimycotic) solution was added. Cells and supernatants were harvested at 24, 48, and 72 hours, and serial dilutions were performed in duplicate after three freeze-thaw cycles. The experiment was repeated in duplicate. Graphs showing PFU / million cells over time in SW620 (FIG. 21A) and SW480 (FIG. 21C). Also, bar graphs comparing PFU / million cells at each time point in cancer cells SW620 (FIG. 21B) and SW480 (FIG. 21D) treated with each virus. Statistical analysis was performed using one-way ANOVA to compare #33 with other experimental groups at each time point. [Figure 22] Immunohistochemical analysis of HCT-116 cancer cells infected with virus #33 or #33-(SE)hNIS. Images were acquired 24 hours post-infection with an MOI of 0.01. [Diagram 23] Immunohistochemical analysis of HT-29 cancer cells infected with virus #33 or #33-(SE)hNIS. Images were acquired 24 hours post-infection with an MOI of 0.01. [Figure 24]Tumor HT-29 was implanted at 5x106 cells in both flanks of 14 female athymic Nude-Foxn1nu nude mice (Envigo, Indianapolis, IN). When tumor size reached approximately 200 mm3, tumors were injected bilaterally with 50 μL of PBS (4 mice), #33 (5 mice) or #33-(H5)Fluc2 (5 mice) at approximately 1x105 PFU / dose. Net weight percent change and tumor percent change were recorded twice weekly for 42 days. Figure 24 shows the change (%) of tumor HT-29 over time. Significant differences in tumor volume percent change (p=0.02 and p=0.03, respectively) are highlighted when comparing PBS control with #33 (3 mice) and #33-(H5)Fluc2. [Diagram 25] Twice a week, one PBS control mouse and three #33-(H5)Fluc2-injected mice were intraperitoneally injected with 4.28 mg luciferin / 150 μL PBS. After 7 min, luciferase imaging was obtained at standard exposure. Relative units were recorded at each time point and analyzed relative to the PBS control mice as background. [Figure 26] Nineteen female athymic Nude-Foxn1nu nude mice (Envigo, Indianapolis, IN) were implanted with tumor HCT-116 at 5x106 cells in both flanks. When tumor size reached approximately 200 mm3, tumors were injected bilaterally with 50 μL of PBS (2 mice), #33-(SE)hNIS or #33-(H5)Fluc2 at approximately 1x105 PFU / dose (3 mice). Net weight % change and tumor % change were recorded twice weekly for 42 days. Figure 25 shows the % change of HCT-116 tumor over time. Significant differences in % tumor volume change when comparing PBS control with #33 (3 mice), #33-(SE)hNIS and #33-(H5)Fluc2 (p=0.0002, p=0.0001 and p=0.0002, respectively) are highlighted. [Figure 27]Twice a week, one PBS control mouse and three #33-(H5)Fluc2-injected mice were intraperitoneally injected with 4.28 mg luciferin / 150 μL PBS. After 7 min, luciferase imaging was obtained at standard exposure. Relative units were recorded at each time point and analyzed relative to the PBS control mice as background. [Figure 28A] Figures 28A-28C show oncolytic virus-mediated cytotoxicity to lung cancer and lung fibroblast cells at 72 hours post-infection. Five thousand A549, H2199 or HF1 fibroblast cells were plated in each well of a 96-well plate. The next day, cells were infected with various viruses (#33, #33-(H5)Emerald, #189, GLV-1h68, OncoVEXGFP) at the indicated multiplicity of infection (MOI, 0, 0.001, 0.01, 0.1, 1 MOI) or mock-infected. Cell viability was measured at 72 hours post-infection using CellTiter 96 AQueous One Solution (Promega, Cat#G3581). The viability of infected A549 cells (FIG. 28A), H2199 cells (FIG. 28B) or HF1 fibroblasts (FIG. 28C) was calculated in comparison to the viability of mock-infected cells. [Figure 28B] Figures 28A-28C show oncolytic virus-mediated cytotoxicity to lung cancer and lung fibroblast cells at 72 hours post-infection. Five thousand A549, H2199 or HF1 fibroblast cells were plated in each well of a 96-well plate. The next day, cells were infected with various viruses (#33, #33-(H5)Emerald, #189, GLV-1h68, OncoVEXGFP) at the indicated multiplicity of infection (MOI, 0, 0.001, 0.01, 0.1, 1 MOI) or mock-infected. Cell viability was measured at 72 hours post-infection using CellTiter 96 AQueous One Solution (Promega, Cat#G3581). The viability of infected A549 cells (FIG. 28A), H2199 cells (FIG. 28B) or HF1 fibroblasts (FIG. 28C) was calculated in comparison to the viability of mock-infected cells. [Figure 28C]Figures 28A-28C show oncolytic virus-mediated cytotoxicity to lung cancer and lung fibroblast cells at 72 hours post-infection. Five thousand A549, H2199 or HF1 fibroblast cells were plated in each well of a 96-well plate. The next day, cells were infected with various viruses (#33, #33-(H5)Emerald, #189, GLV-1h68, OncoVEXGFP) at the indicated multiplicity of infection (MOI, 0, 0.001, 0.01, 0.1, 1 MOI) or mock-infected. Cell viability was measured at 72 hours post-infection using CellTiter 96 AQueous One Solution (Promega, Cat#G3581). The viability of infected A549 cells (FIG. 28A), H2199 cells (FIG. 28B) or HF1 fibroblasts (FIG. 28C) was calculated in comparison to the viability of mock-infected cells. [Figure 29-1] GFP images of A549 xenografts are shown over several days following a single dose of 1000 PFU into the right tumor with the indicated virus (#33-(H5)Emerald, GLV-1h68 or OncoVEXGFP, intratumoral). [Figure 29-2] GFP images of A549 xenografts are shown over several days following a single dose of 1000 PFU into the right tumor with the indicated virus (#33-(H5)Emerald, GLV-1h68 or OncoVEXGFP, intratumoral). [Figure 30-1] Mouse weights over days in the A549 xenograft model are shown. Three weeks after injection of A549 tumor cells, mice were divided into different treatment groups (n=4 or 5) to achieve similar mean tumor volumes (approximately 200 mm3) and injected with the indicated viruses (#33, #33-(H5)Emerald, GLV-1h68, OncoVEXGFP, T-VECTM, #189, PBS control) into the right tumor or #33-(H5)Emerald intraperitoneally (ip) at 103 PFU. Mice were weighed twice weekly and their % weight change is shown. Each line represents the weight of an individual mouse. [Figure 30-2]Mouse weights over days in the A549 xenograft model are shown. Three weeks after injection of A549 tumor cells, mice were divided into different treatment groups (n=4 or 5) to achieve similar mean tumor volumes (approximately 200 mm3) and injected with the indicated viruses (#33, #33-(H5)Emerald, GLV-1h68, OncoVEXGFP, T-VECTM, #189, PBS control) into the right tumor or #33-(H5)Emerald intraperitoneally (ip) at 103 PFU. Mice were weighed twice weekly and their % weight change is shown. Each line represents the weight of an individual mouse. [Figure 31A-1] Figures 31A-31B show tumor regression in the A549 xenograft model. Three weeks after injection of A549 tumor cells, mice were divided into various treatment groups (n=4 or 5) to achieve similar mean tumor volumes (approximately 200 mm3) and injected with the indicated viruses (#33, #33-(H5)Emerald, GLV-1h68, OncoVEXGFP, T-VECTM, #189, PBS control) into the right tumor or #33-(H5)Emerald intraperitoneally (ip) at 103 PFU. Tumor volumes in the injected (Figure 31A) and non-injected (Figure 31B) groups were measured twice weekly using digital calipers. Each line represents the tumor volume of an individual mouse. [Figure 31A-2] Figures 31A-31B show tumor regression in the A549 xenograft model. Three weeks after injection of A549 tumor cells, mice were divided into various treatment groups (n=4 or 5) to achieve similar mean tumor volumes (approximately 200 mm3) and injected with the indicated viruses (#33, #33-(H5)Emerald, GLV-1h68, OncoVEXGFP, T-VECTM, #189, PBS control) into the right tumor or #33-(H5)Emerald intraperitoneally (ip) at 103 PFU. Tumor volumes in the injected (Figure 31A) and non-injected (Figure 31B) groups were measured twice weekly using digital calipers. Each line represents the tumor volume of an individual mouse. [Figure 31B-1]Figures 31A-31B show tumor regression in the A549 xenograft model. Three weeks after injection of A549 tumor cells, mice were divided into various treatment groups (n=4 or 5) to achieve similar mean tumor volumes (approximately 200 mm3) and injected with the indicated viruses (#33, #33-(H5)Emerald, GLV-1h68, OncoVEXGFP, T-VECTM, #189, PBS control) into the right tumor or #33-(H5)Emerald intraperitoneally (ip) at 103 PFU. Tumor volumes in the injected (Figure 31A) and non-injected (Figure 31B) groups were measured twice weekly using digital calipers. Each line represents the tumor volume of an individual mouse. [Figure 31B-2] Figures 31A-31B show tumor regression in the A549 xenograft model. Three weeks after injection of A549 tumor cells, mice were divided into various treatment groups (n=4 or 5) to achieve similar mean tumor volumes (approximately 200 mm3) and injected with the indicated viruses (#33, #33-(H5)Emerald, GLV-1h68, OncoVEXGFP, T-VECTM, #189, PBS control) into the right tumor or #33-(H5)Emerald intraperitoneally (ip) at 103 PFU. Tumor volumes in the injected (Figure 31A) and non-injected (Figure 31B) groups were measured twice weekly using digital calipers. Each line represents the tumor volume of an individual mouse. [Diagram 32] Figure 1 shows the volume of virus-injected tumors in the A549 xenograft model. Three weeks after injection of A549 tumor cells, mice were divided into various treatment groups (n=4 or 5) to achieve similar mean tumor volumes (approximately 200 mm3) and injected with the indicated viruses (#33, #33-(H5)Emerald, GLV-1h68, OncoVEXGFP, T-VECTM, #189, PBS control) into the right intratumoral space or #33-(H5)Emerald intraperitoneally (ip) at 103 PFU. Tumor volumes were measured twice weekly using digital calipers. Each line represents the mean volume with standard deviation for each treatment group that received tumor injections. Statistical analysis: one-way ANOVA at day 24 (*=p<0.05). [Diagram 33]Volume of uninjected tumors in A549 xenograft model. Three weeks after injection of A549 tumor cells, mice were divided into different treatment groups (n=4 or 5) to achieve similar mean tumor volumes (approximately 200 mm3) and injected with the indicated viruses (#33, #33-(H5)Emerald, GLV-1h68, OncoVEXGFP, T-VECTM, #189, PBS control) into the right intratumoral space or #33-(H5)Emerald intraperitoneally (ip) at 103 PFU. Tumor volumes of uninjected tumors were measured twice weekly using digital caliper splints. Each line represents the mean volume with standard deviation of each tumor-injected treatment group. Statistical analysis: One-way ANOVA at day 24 (*=p<0.05). [Figure 34A] Figures 34A-34B show the fold change in tumor volume. Three weeks after injection of A549 tumor cells, mice were divided into various treatment groups (n=4 or 5) to achieve similar mean tumor volumes (approximately 200 mm3) and each mouse was injected with the indicated viruses (#33, #33-(H5)Emerald, GLV-1h68, OncoVEXGFP, T-VECTM, #189, PBS control) either intratumorally on the right side or intraperitoneally (ip) with #33-(H5)Emerald at 103 PFU. Tumor volumes were measured twice weekly using digital caliper splints. Fold change in tumor volume was calculated for injected (Figure 34A) and non-injected (Figure 34B) tumors by normalizing tumor volumes at various time points to the time of virus injection (i.e., day 0). In Figures 34A-34B, each line represents the mean tumor volume with standard deviation for each treatment group. Statistical analysis: One-way ANOVA on day 24 (*=p<0.05). [Figure 34B]Figures 34A-34B show the fold change in tumor volume. Three weeks after injection of A549 tumor cells, mice were divided into various treatment groups (n=4 or 5) to achieve similar mean tumor volumes (approximately 200 mm3) and each mouse was injected with the indicated viruses (#33, #33-(H5)Emerald, GLV-1h68, OncoVEXGFP, T-VECTM, #189, PBS control) either intratumorally on the right side or intraperitoneally (ip) with #33-(H5)Emerald at 103 PFU. Tumor volumes were measured twice weekly using digital caliper splints. Fold change in tumor volume was calculated for injected (Figure 34A) and non-injected (Figure 34B) tumors by normalizing tumor volumes at various time points to the time of virus injection (i.e., day 0). In Figures 34A-34B, each line represents the mean tumor volume with standard deviation for each treatment group. Statistical analysis: One-way ANOVA on day 24 (*=p<0.05). [Figure 35A] Figures 35A-35B show the biodistribution of virus in injected and non-injected tumors (A549 model). Three weeks after injection of A549 tumor cells, mice were divided into different treatment groups (n=3) with similar mean tumor volumes (approximately 200 mm3) and the indicated viruses (#33, #33-(H5)Emerald, GLV-1h68, OncoVEXGFP) were injected at 103 PFU into the right tumor only of each mouse. Six days after virus injection, tumors and healthy organs were harvested. The harvested tissues were weighed, chopped into small pieces, and homogenized in 1 ml of PBS using a Bullet Blender Gold homogenizer. The homogenate was subjected to three freeze-thaw cycles and sonicated for 1 min. The homogenate was spun down at 1000 rpm for 3 min and the supernatant was collected. The supernatants were serially diluted and viral titers were measured using standard plaque assays. Figure 35A shows the PFU / g tumor of each virus in injected tumors. Figure 35B shows the PFU / g tumor of each virus in non-injected tumors. [Figure 35B]Figures 35A-35B show the biodistribution of virus in injected and non-injected tumors (A549 model). Three weeks after injection of A549 tumor cells, mice were divided into different treatment groups (n=3) with similar mean tumor volumes (approximately 200 mm3) and the indicated viruses (#33, #33-(H5)Emerald, GLV-1h68, OncoVEXGFP) were injected at 103 PFU into the right tumor only of each mouse. Six days after virus injection, tumors and healthy organs were harvested. The harvested tissues were weighed, chopped into small pieces, and homogenized in 1 ml of PBS using a Bullet Blender Gold homogenizer. The homogenate was subjected to three freeze-thaw cycles and sonicated for 1 min. The homogenate was spun down at 1000 rpm for 3 min and the supernatant was collected. The supernatants were serially diluted and viral titers were measured using standard plaque assays. Figure 35A shows the PFU / g tumor of each virus in injected tumors. Figure 35B shows the PFU / g tumor of each virus in non-injected tumors. [Diagram 36] Viral titers in the ovaries of mice (A549 model). Three weeks after injection of A549 tumor cells, mice were divided into different treatment groups (n=3) to achieve similar mean tumor volumes (approximately 200 mm3) and the indicated viruses (#33, #33-(H5)Emerald, GLV-1h68, OncoVEXGFP, T-VECTM) were injected at 103 PFU into the right tumor only of each mouse. Six days after virus injection, tumors and healthy organs were harvested. The harvested tissues were weighed, chopped into small pieces, and homogenized in 1 ml of PBS using a Bullet Blender Gold homogenizer. The homogenate was subjected to three freeze-thaw cycles and sonicated for 1 min. The homogenate was spun down at 1000 rpm for 3 min and the supernatant was collected. The supernatant was serially diluted and viral titers were measured using a standard plaque assay. Figure 36 shows the PFU / g tissue (ovary) of each virus. Not detected (ND). [Figure 37]Virus titers in blood 20 days after virus injection are shown. Three weeks after injection of A549 tumor cells, mice were divided into different treatment groups (n=3) to achieve similar mean tumor volumes (approximately 200 mm3) and the indicated viruses (#33, #33-(H5)Emerald, GLV-1h68, OncoVEXGFP, T-VECTM) were injected at 103 PFU into the right tumor only of each mouse. Blood was collected from mice (n=3) by facial vein puncture. After three freeze-thaw cycles, blood was serially diluted and virus titers were measured using a standard plaque assay. Figure 37 shows the PFU / mL in blood injected with each virus. Not detected (ND). [Figure 38] Chimeric virus #33 has stronger killing ability on lung cancer cells (A549) than the parental virus. Cytotoxicity assay: 5000 cells were plated in each well of a 96-well plate. The next day, cells were infected with chimeric virus #33 or parental virus at the indicated multiplicity of infection (MOI), or mock-infected. Cell viability was measured 72 hours after infection using CellTiter 96 AQueous One Solution (Promega, Cat#G3581). The viability of infected cells was calculated by comparison with that of mock-infected cells. [Figure 39-1] The weight change after treatment is shown. A549 (human lung cancer cells) were cultured, trypsinized, washed with PBS, and resuspended in PBS and Matrigel = 1:1 to prepare 5 x 106 cells per 100 μL. 100 μL of the cell suspension was subcutaneously injected into both upper flanks of thymectomized nude mice, resulting in the development of two tumors per mouse. Three weeks after tumor cell injection, the mice were divided into various treatment groups (n = 4 or 5) such that each group had a similar average tumor volume (approximately 200 mm3). After division, the indicated viruses were injected intratumorally at 103 PFU only into the right tumor of each mouse. Mice were weighed twice a week, and their weight change % was plotted. Each line represents the weight of an individual mouse. [Figure 39-2]The weight change after treatment is shown. A549 (human lung cancer cells) were cultured, trypsinized, washed with PBS, and resuspended in PBS and Matrigel = 1:1 to prepare 5 x 106 cells per 100 μL. 100 μL of the cell suspension was subcutaneously injected into both upper flanks of thymectomized nude mice, resulting in the development of two tumors per mouse. Three weeks after tumor cell injection, the mice were divided into various treatment groups (n = 4 or 5) such that each group had a similar average tumor volume (approximately 200 mm3). After division, the indicated viruses were injected intratumorally at 103 PFU only into the right tumor of each mouse. Mice were weighed twice a week, and their weight change % was plotted. Each line represents the weight of an individual mouse. [Figure 40-1] Tumor regression is shown. A549 (human lung cancer cells) were cultured, trypsinized, washed with PBS, and resuspended in PBS and Matrigel = 1:1 to 5 x 106 cells per 100 μL. 100 μL of the cell suspension was injected subcutaneously into both upper flanks of thymectomized nude mice, resulting in the development of two tumors per mouse. Three weeks after tumor cell injection, mice were divided into various treatment groups (n = 4 or 5) such that each group had a similar average tumor volume (approximately 200 mm3). After splitting, the indicated viruses were injected intratumorally at 103 PFU only into the right tumor of each mouse. Tumor volumes (injected and uninjected) were measured twice weekly using digital calipers (volume = {(length)2 x width / 2}. Each line represents the tumor volume of an individual mouse. [Figure 40-2] Tumor regression is shown. A549 (human lung cancer cells) were cultured, trypsinized, washed with PBS, and resuspended in PBS and Matrigel = 1:1 to 5 x 106 cells per 100 μL. 100 μL of the cell suspension was injected subcutaneously into both upper flanks of thymectomized nude mice, resulting in the development of two tumors per mouse. Three weeks after tumor cell injection, mice were divided into various treatment groups (n = 4 or 5) such that each group had a similar average tumor volume (approximately 200 mm3). After splitting, the indicated viruses were injected intratumorally at 103 PFU only into the right tumor of each mouse. Tumor volumes (injected and uninjected) were measured twice weekly using digital calipers (volume = {(length)2 x width / 2}. Each line represents the tumor volume of an individual mouse. [Figure 40-3] Tumor regression is shown. A549 (human lung cancer cells) were cultured, trypsinized, washed with PBS, and resuspended in PBS and Matrigel = 1:1 to 5 x 106 cells per 100 μL. 100 μL of the cell suspension was injected subcutaneously into both upper flanks of thymectomized nude mice, resulting in the development of two tumors per mouse. Three weeks after tumor cell injection, mice were divided into various treatment groups (n = 4 or 5) such that each group had a similar average tumor volume (approximately 200 mm3). After splitting, the indicated viruses were injected intratumorally at 103 PFU only into the right tumor of each mouse. Tumor volumes (injected and uninjected) were measured twice weekly using digital calipers (volume = {(length)2 x width / 2}. Each line represents the tumor volume of an individual mouse. [Figure 40-4] Tumor regression is shown. A549 (human lung cancer cells) were cultured, trypsinized, washed with PBS, and resuspended in PBS and Matrigel = 1:1 to 5 x 106 cells per 100 μL. 100 μL of the cell suspension was injected subcutaneously into both upper flanks of thymectomized nude mice, resulting in the development of two tumors per mouse. Three weeks after tumor cell injection, mice were divided into various treatment groups (n = 4 or 5) such that each group had a similar average tumor volume (approximately 200 mm3). After splitting, the indicated viruses were injected intratumorally at 103 PFU only into the right tumor of each mouse. Tumor volumes (injected and uninjected) were measured twice weekly using digital calipers (volume = {(length)2 x width / 2}. Each line represents the tumor volume of an individual mouse. [Diagram 41]Virus titers of injected and non-injected tumors 7 days after infection are shown. A549 (human lung cancer cells) were cultured, trypsinized, washed with PBS, and resuspended in PBS and Matrigel = 1:1 to 5 x 106 cells per 100 μL. 100 μL of cell suspension was injected subcutaneously into both upper flanks of thymectomized nude mice, resulting in the development of two tumors per mouse. Three weeks after tumor cell injection, mice were divided into different treatment groups (n = 3) such that each group had a similar average tumor volume (approximately 200 mm3). After splitting, the indicated viruses were injected intratumorally at 103 PFU only into the right tumor of each mouse. Tumors and healthy organs were harvested 6 days after virus injection. The harvested tissues were weighed, chopped into small pieces, and homogenized in 1 ml of PBS using a Bullet Blender Gold homogenizer. The homogenate was subjected to three freeze-thaw cycles and sonicated for 1 min. The homogenate was spun down at 1000 rpm for 3 min and the supernatant was collected. The supernatant was serially diluted and virus titer was determined using a standard plaque assay. [Figure 42-1] Virus biodistribution is shown. A549 (human lung cancer cells) were cultured, trypsinized, washed with PBS, and resuspended in PBS and Matrigel = 1:1 to 5 x 106 cells per 100 μL. 100 μL of the cell suspension was injected subcutaneously into both upper flanks of thymectomized nude mice, resulting in the development of two tumors per mouse. Three weeks after tumor cell injection, mice were divided into different treatment groups (n = 3) such that each group had a similar average tumor volume (approximately 200 mm3). After splitting, the indicated viruses were injected intratumorally at 103 PFU only into the right tumor of each mouse. Tumors and healthy organs were harvested 6 days after virus injection. The harvested tissues were weighed, chopped into small pieces, and homogenized in 1 ml of PBS using a Bullet Blender Gold homogenizer. The homogenate was subjected to three freeze-thaw cycles and sonicated for 1 min. The homogenate was spun down at 1000 rpm for 3 min and the supernatant was collected. The supernatant was serially diluted and virus titer was determined using a standard plaque assay. [Figure 42-2]Virus biodistribution is shown. A549 (human lung cancer cells) were cultured, trypsinized, washed with PBS, and resuspended in PBS and Matrigel = 1:1 to 5 x 106 cells per 100 μL. 100 μL of the cell suspension was injected subcutaneously into both upper flanks of thymectomized nude mice, resulting in the development of two tumors per mouse. Three weeks after tumor cell injection, mice were divided into different treatment groups (n = 3) such that each group had a similar average tumor volume (approximately 200 mm3). After splitting, the indicated viruses were injected intratumorally at 103 PFU only into the right tumor of each mouse. Tumors and healthy organs were harvested 6 days after virus injection. The harvested tissues were weighed, chopped into small pieces, and homogenized in 1 ml of PBS using a Bullet Blender Gold homogenizer. The homogenate was subjected to three freeze-thaw cycles and sonicated for 1 min. The homogenate was spun down at 1000 rpm for 3 min and the supernatant was collected. The supernatant was serially diluted and virus titer was determined using a standard plaque assay. [Figure 43-1] Figure 1 shows virus titers in the blood of injected mice. Blood was collected from the facial vein of A549 tumor-bearing mice at various time points after intratumoral injection of 1000 pfu of the indicated viruses. Viral titers in blood samples were measured using standard plaque assay methods. No virus was detectable in urine up to 10 days after injection. [Figure 43-2] Figure 1 shows virus titers in the blood of injected mice. Blood was collected from the facial vein of A549 tumor-bearing mice at various time points after intratumoral injection of 1000 pfu of the indicated viruses. Viral titers in blood samples were measured using standard plaque assay methods. No virus was detectable in urine up to 10 days after injection. [Diagram 44]Survival of mice after virus injection is shown. A549 (human lung cancer cells) were cultured, trypsinized, washed with PBS, and resuspended in PBS and Matrigel = 1:1 to 5 x 106 cells per 100 μL. 100 μL of cell suspension was injected subcutaneously into both upper flanks of thymectomized nude mice, resulting in the development of two tumors per mouse. Three weeks after tumor cell injection, mice were divided into different treatment groups (n = 3) such that each group had a similar average tumor volume (approximately 200 mm3). After splitting, each mouse was injected intratumorally with the indicated virus at 103 PFU only into the right tumor. Tumor volumes were measured twice weekly using digital calipers, and mice were euthanized when one of the bilateral tumors exceeded a certain tumor burden (3000 mm3) or when the mice became ill due to virus treatment (>20% weight loss). [Figure 45A] Figures 45A-45C show a comparison of the cytotoxicity potential of chimeric #33 and parental poxviruses in A549. In Figure 45A, the MOI of virus required to kill 50% of A549 cells (LD50) was calculated for all viruses and compared. In Figure 45B, cells were infected with #33 or parental virus at an MOI of 0.03 pfu, and the fold increase in virus titer relative to the amount of virus added at 24 hours post-infection was measured and compared between viruses. In Figure 45C, A549 cells were infected with viruses as in Figure 45B, and supernatants were collected from infected wells at 12 hours post-infection and 18 hours post-infection. Viral titers of the supernatants were measured by plaque assay and compared between viruses. [Figure 45B]Figures 45A-45C show a comparison of the cytotoxicity potential of chimeric #33 and parental poxviruses in A549. In Figure 45A, the MOI of virus required to kill 50% of A549 cells (LD50) was calculated for all viruses and compared. In Figure 45B, cells were infected with #33 or parental virus at an MOI of 0.03 pfu, and the fold increase in virus titer relative to the amount of virus added at 24 hours post-infection was measured and compared between viruses. In Figure 45C, A549 cells were infected with viruses as in Figure 45B, and supernatants were collected from infected wells at 12 hours post-infection and 18 hours post-infection. Viral titers of the supernatants were measured by plaque assay and compared between viruses. [Figure 45C] Figures 45A-45C show a comparison of the cytotoxicity potential of chimeric #33 and parental poxviruses in A549. In Figure 45A, the MOI of virus required to kill 50% of A549 cells (LD50) was calculated for all viruses and compared. In Figure 45B, cells were infected with #33 or parental virus at an MOI of 0.03 pfu, and the fold increase in virus titer relative to the amount of virus added at 24 hours post-infection was measured and compared between viruses. In Figure 45C, A549 cells were infected with viruses as in Figure 45B, and supernatants were collected from infected wells at 12 hours post-infection and 18 hours post-infection. Viral titers of the supernatants were measured by plaque assay and compared between viruses. [Figure 46A]In Figures 46A-46B, A549 cells were infected at various MOIs with #33 or #33-(H5)Emerald, which has the J2R(TK) gene replaced with an Emerald(green) expression cassette. In Figure 46A, 5000 cells were plated in each well of a 96-well plate. The next day, cells were infected at various MOIs with chimeric viruses #33 or #33-(H5)Emerald, which has the J2R(TK) gene replaced with an Emerald(green) expression cassette. Cell viability was measured 72 hours after infection using CellTiter 96 AQueous One Solution (Promega, Cat#G3581). The viability of infected cells was calculated by comparison with that of mock-infected cells. In FIG. 46B, A549 cells were infected with #33 or #33-(H5) at an MOI of 0.03 pfu, and the fold increase in viral titer relative to the viral infectivity was determined at the indicated time points. [Figure 46B] In Figures 46A-46B, A549 cells were infected at various MOIs with #33 or #33-(H5)Emerald, which has the J2R(TK) gene replaced with an Emerald(green) expression cassette. In Figure 46A, 5000 cells were plated in each well of a 96-well plate. The next day, cells were infected at various MOIs with chimeric viruses #33 or #33-(H5)Emerald, which has the J2R(TK) gene replaced with an Emerald(green) expression cassette. Cell viability was measured 72 hours after infection using CellTiter 96 AQueous One Solution (Promega, Cat#G3581). The viability of infected cells was calculated by comparison with that of mock-infected cells. In FIG. 46B, A549 cells were infected with #33 or #33-(H5) at an MOI of 0.03 pfu, and the fold increase in viral titer relative to the viral infectivity was determined at the indicated time points. [Figure 47A]Imaging is shown. A549 (human lung cancer cells) were cultured, trypsinized, washed with PBS, and resuspended in PBS and Matrigel = 1:1 to prepare 5 x 106 cells per 100 μL. 100 μL of the cell suspension was subcutaneously injected into both upper flanks of thymectomized nude mice, and two tumors were generated per mouse. Three weeks after tumor cell injection, mice were divided into various treatment groups (n = 5) so that each group had a similar average tumor volume (approximately 200 mm3). After division, #33-(H5) Emerald was injected intratumorally at 103 plaque forming units (PFU) or PBS only in the right tumor of each mouse. Mice were imaged twice a week for green fluorescence (excitation: 465 and emission: 530 nm) using a small animal imaging device (LagoX imaging system), and image processing was performed using AMIview image processing software. [Figure 47B] Using AMIview image processing software, the mean fluorescence intensity (MFI) of Emerald was calculated for each tumor at various time points. The mean MFI of tumors with and without injection (n=5 mice / group) was compared. [Figure 48A] A549 (human lung cancer cells) were cultured, trypsinized, washed with PBS, and resuspended in PBS and Matrigel = 1:1 to 5 x 106 cells per 100 μL. 100 μL of the cell suspension was injected subcutaneously into both upper flanks of thymectomized nude mice, resulting in the development of two tumors per mouse. Three weeks after tumor cell injection, mice were divided into various treatment groups (n = 7) such that each group had a similar average tumor volume (approximately 200 mm3). After division, #33-(H5)Emerald was injected intratumorally at 103 PFU only into the right tumor of each mouse. Mice were weighed twice weekly, and their % weight change was plotted. Each line represents the weight of an individual mouse. [Figure 48B] Tumor volumes were measured twice weekly using digital calipers (volume={(length)2×width / 2}. Each line represents the mean volume with SD of injected tumors in an individual treatment group. Statistics: Unpaired T-test, ****=p<0.0001. **33-GFP refers to animals treated with #33-(H5)Emerald. [Figure 48C] Tumor volumes for individual mice in each treatment group were plotted. [Figure 48D] Mice were euthanized when one of the bilateral tumors exceeded a certain tumor burden (3000 mm3) and survival curves of virus-treated groups were compared with survival curves of PBS-treated groups. Statistical methods: Log-rank (Mantel Cox) test, ****=p<0.0001. [Figure 49A-1] A549 (human lung cancer cells) were cultured, trypsinized, washed with PBS, and resuspended in PBS and Matrigel = 1:1 to prepare 5 x 106 cells per 100 μL. 100 μL of the cell suspension was injected subcutaneously into both upper flanks of thymectomized nude mice, resulting in the development of two tumors per mouse. Three weeks after tumor cell injection, mice were divided into various treatment groups (n = 4 or 5) such that each group had a similar mean tumor volume (approximately 200 mm3). After division, the indicated viruses were injected intratumorally at 103 PFU only into the right tumor of each mouse. At 7 and 56 days after virus injection, three mice in the virus treatment group were euthanized and their organs and tumors were harvested. Viral titers in the harvested organs were measured by plaque assay and compared between tumors and organs. Statistical method: One-way ANOVA; *** = p < 0.0001. ND = not detectable. [Figure 49A-2] A549 (human lung cancer cells) were cultured, trypsinized, washed with PBS, and resuspended in PBS and Matrigel = 1:1 to prepare 5 x 106 cells per 100 μL. 100 μL of the cell suspension was injected subcutaneously into both upper flanks of thymectomized nude mice, resulting in the development of two tumors per mouse. Three weeks after tumor cell injection, mice were divided into various treatment groups (n = 4 or 5) such that each group had a similar mean tumor volume (approximately 200 mm3). After division, the indicated viruses were injected intratumorally at 103 PFU only into the right tumor of each mouse. At 7 and 56 days after virus injection, three mice in the virus treatment group were euthanized and their organs and tumors were harvested. Viral titers in the harvested organs were measured by plaque assay and compared between tumors and organs. Statistical method: One-way ANOVA; *** = p < 0.0001. ND = not detectable. [Figure 49B-1]Tumor sections (7 days after virus injection) were stained for vaccinia virus. Dark staining represents virus-infected areas in the tumor section. Each section is from a separate mouse. [Figure 49B-2] Tumor sections (7 days after virus injection) were stained for vaccinia virus. Dark staining represents virus-infected areas in the tumor section. Each section is from a separate mouse. [Figure 49C] Figure 49D. Tumor sections obtained 7 days after virus injection were stained for apoptotic cells using In Situ Cell Death Detection Fluorescein (Roche). As a "positive control", tumor sections were treated with recombinant Dnase I (300 U / ml) for 10 min at room temperature. Grey signals represent apoptotic cells. [Figure 50] In vitro cytotoxicity of OVCAR8 cells (72 hours post-infection) is shown. Five thousand OVCAR8 (human ovarian cancer) cells were plated in each well of a 96-well plate. The next day, cells were infected with chimeric virus #33, or #33 with TK deleted (#33 / TK), or #33 virus with miR100 and Let-7c target sequences inserted into essential viral genes E9L or D4R. Infection was performed at the indicated multiplicity of infection (MOI). CellTiter 96 AQueous One Solution (Promega, Cat#G3581) was used to measure cell viability at 72 hours post-infection. The viability of infected cells was calculated by comparison with that of mock-infected cells. [Figure 51] Figure 1 shows the viral growth kinetics of OVCAR8 cells. OVCAR8 cells were infected with the indicated viruses at an MOI of 0.03 pfu in 6-well plates. Cell lysates were harvested from infected wells at 24, 48, and 72 hours post-infection. Viral titers in the cell lysates were measured by plaque assay, and the fold increase in viral titer was plotted against the amount of virus added. [Figure 52-1]The % weight change of mice is shown. OVCAR8 (human ovarian cancer cells) were cultured, trypsinized, washed with PBS, and resuspended in PBS and Matrigel = 1:1 to prepare 5 x 106 cells per 100 μL. 100 μL of the cell suspension was injected subcutaneously into both upper flanks of thymectomized nude mice, resulting in the development of two tumors per mouse. Three weeks after tumor cell injection, mice were divided into various treatment groups (n = 8 for PBS, and n = 5 for all other groups) such that each group had a similar mean tumor volume (approximately 200 mm3). After division, each mouse was injected intratumorally with the indicated virus at 105 PFU or with PBS only in the right tumor. Mice were weighed twice weekly, and their % weight change was plotted. Each line represents the weight of an individual mouse. [Figure 52-2] The % weight change of mice is shown. OVCAR8 (human ovarian cancer cells) were cultured, trypsinized, washed with PBS, and resuspended in PBS and Matrigel = 1:1 to prepare 5 x 106 cells per 100 μL. 100 μL of the cell suspension was injected subcutaneously into both upper flanks of thymectomized nude mice, resulting in the development of two tumors per mouse. Three weeks after tumor cell injection, mice were divided into various treatment groups (n = 8 for PBS, and n = 5 for all other groups) such that each group had a similar mean tumor volume (approximately 200 mm3). After division, each mouse was injected intratumorally with the indicated virus at 105 PFU or with PBS only in the right tumor. Mice were weighed twice weekly, and their % weight change was plotted. Each line represents the weight of an individual mouse. [Figure 53-1]Tumor volumes are shown. OVCAR8 (human ovarian cancer cells) were cultured, trypsinized, washed with PBS, and resuspended in PBS and Matrigel = 1:1 to 5 x 106 cells per 100 μL. 100 μL of cell suspension was injected subcutaneously into both upper flanks of thymectomized nude mice, resulting in the development of two tumors per mouse. Three weeks after tumor cell injection, mice were divided into various treatment groups (n = 8 for PBS, and n = 5 for all other groups) such that each group had a similar mean tumor volume (approximately 200 mm3). After splitting, each mouse was injected intratumorally with the indicated virus at 105 PFU or with PBS, only in the right tumor. Tumor volumes were measured twice weekly using digital calipers (volume = {(length)2 x width / 2}. Volumes of virus-injected and non-injected tumors were plotted for individual mice in each treatment group. [Figure 53-2] Tumor volumes are shown. OVCAR8 (human ovarian cancer cells) were cultured, trypsinized, washed with PBS, and resuspended in PBS and Matrigel = 1:1 to 5 x 106 cells per 100 μL. 100 μL of cell suspension was injected subcutaneously into both upper flanks of thymectomized nude mice to develop two tumors per mouse. Three weeks after tumor cell injection, mice were divided into various treatment groups (n = 8 for PBS, and n = 5 for all other groups) such that each group had a similar mean tumor volume (approximately 200 mm3). After splitting, each mouse was injected intratumorally with the indicated virus at 105 PFU or with PBS only in the right tumor. Tumor volumes were measured twice weekly using digital calipers (volume = {(length)2 x width / 2}). Volumes of virus-injected and non-injected tumors for individual mice in each treatment group were plotted. [Figure 53-3]Tumor volumes are shown. OVCAR8 (human ovarian cancer cells) were cultured, trypsinized, washed with PBS, and resuspended in PBS and Matrigel = 1:1 to 5 x 106 cells per 100 μL. 100 μL of cell suspension was injected subcutaneously into both upper flanks of thymectomized nude mice, resulting in the development of two tumors per mouse. Three weeks after tumor cell injection, mice were divided into various treatment groups (n = 8 for PBS, and n = 5 for all other groups) such that each group had a similar mean tumor volume (approximately 200 mm3). After splitting, each mouse was injected intratumorally with the indicated virus at 105 PFU or with PBS, only in the right tumor. Tumor volumes were measured twice weekly using digital calipers (volume = {(length)2 x width / 2}. Volumes of virus-injected and non-injected tumors were plotted for individual mice in each treatment group. [Figure 53-4] Tumor volumes are shown. OVCAR8 (human ovarian cancer cells) were cultured, trypsinized, washed with PBS, and resuspended in PBS and Matrigel = 1:1 to 5 x 106 cells per 100 μL. 100 μL of cell suspension was injected subcutaneously into both upper flanks of thymectomized nude mice, resulting in the development of two tumors per mouse. Three weeks after tumor cell injection, mice were divided into various treatment groups (n = 8 for PBS, and n = 5 for all other groups) such that each group had a similar mean tumor volume (approximately 200 mm3). After splitting, each mouse was injected intratumorally with the indicated virus at 105 PFU or with PBS, only in the right tumor. Tumor volumes were measured twice weekly using digital calipers (volume = {(length)2 x width / 2}. Volumes of virus-injected and non-injected tumors were plotted for individual mice in each treatment group. [Figure 53-5]Tumor volumes are shown. OVCAR8 (human ovarian cancer cells) were cultured, trypsinized, washed with PBS, and resuspended in PBS and Matrigel = 1:1 to 5 x 106 cells per 100 μL. 100 μL of cell suspension was injected subcutaneously into both upper flanks of thymectomized nude mice, resulting in the development of two tumors per mouse. Three weeks after tumor cell injection, mice were divided into various treatment groups (n = 8 for PBS, and n = 5 for all other groups) such that each group had a similar mean tumor volume (approximately 200 mm3). After splitting, each mouse was injected intratumorally with the indicated virus at 105 PFU or with PBS, only in the right tumor. Tumor volumes were measured twice weekly using digital calipers (volume = {(length)2 x width / 2}. Volumes of virus-injected and non-injected tumors were plotted for individual mice in each treatment group. [Figure 54A] Figures 54A-54B show the average tumor volume of injected and non-injected tumors. OVCAR8 (human ovarian cancer cells) were cultured, trypsinized, washed with PBS, and resuspended in PBS and Matrigel = 1:1 at 5 x 106 cells per 100 μL. 100 μL of the cell suspension was injected subcutaneously into both upper flanks of thymectomized nude mice, resulting in the development of two tumors per mouse. Three weeks after tumor cell injection, mice were divided into various treatment groups (n = 8 for PBS, and n = 5 for all other groups) such that each group had a similar average tumor volume (approximately 200 mm3). After division, only the right tumor of each mouse was injected intratumorally with the indicated virus at 105 PFU or with PBS. Tumor volumes were measured twice weekly using digital calipers (volume = {(length)2 x width / 2}. Mean tumor volumes for each treatment group were plotted with SD. Figures 54A and 54B show the mean tumor volumes for injected and non-injected tumors, respectively. [Figure 54B]Figures 54A-54B show the average tumor volume of injected and non-injected tumors. OVCAR8 (human ovarian cancer cells) were cultured, trypsinized, washed with PBS, and resuspended in PBS and Matrigel = 1:1 at 5 x 106 cells per 100 μL. 100 μL of the cell suspension was injected subcutaneously into both upper flanks of thymectomized nude mice, resulting in the development of two tumors per mouse. Three weeks after tumor cell injection, mice were divided into various treatment groups (n = 8 for PBS, and n = 5 for all other groups) such that each group had a similar average tumor volume (approximately 200 mm3). After division, only the right tumor of each mouse was injected intratumorally with the indicated virus at 105 PFU or with PBS. Tumor volumes were measured twice weekly using digital calipers (volume = {(length)2 x width / 2}. Mean tumor volumes for each treatment group were plotted with SD. Figures 54A and 54B show the mean tumor volumes for injected and non-injected tumors, respectively. [Figure 55] Viral titers in organs 7 days after infection are shown. OVCAR8 (human ovarian cancer cells) were cultured, trypsinized, washed with PBS, and resuspended in PBS and Matrigel = 1:1 to 5 x 106 cells per 100 μL. 100 μL of cell suspension was subcutaneously injected into both upper flanks of thymectomized nude mice, resulting in the development of two tumors per mouse. Three weeks after tumor cell injection, mice were divided into different treatment groups (n = 3) such that each group had a similar average tumor volume (approximately 200 mm3). After division, the indicated viruses were injected intratumorally at 105 PFU only into the right tumor of each mouse. Seven days after virus injection, mice were euthanized and their organs and tumors were harvested. Viral titers in the harvested organs were measured by plaque assay and compared between tumors and organs. Note: Virus was not detected in healthy organs (lung, liver, ovaries, kidneys, spleen and brain) and in non-injected tumors. [Figure 56]Figure 1 shows miR100 in OVCAR8 tumors and mouse organs. Thymectomized nude mice bearing OVCAR8 xenografts (n=3) were euthanized and their organs and tumors were harvested. Harvested tissues were homogenized and total RNA was isolated using miRNeasy mini kit (Qiagen). Real-time PCR was performed to measure the levels of miR-100 in the lysates. [Figure 57] Figure 1 shows Let-7c in OVCAR8 tumors and mouse organs. Thymectomized nude mice bearing OVCAR8 xenografts (n=3) were euthanized and their organs and tumors were harvested. Harvested tissues were homogenized and total RNA was isolated using miRNeasy mini kit (Qiagen). Real-time PCR was performed to measure the levels of Let-7c in the lysates. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] Herein, we describe a chimeric poxvirus composition that combines favorable characteristics from different virus species to produce a novel composite chimeric poxvirus that is superior to the individual wild-type viruses. Applicant has generated chimeric poxviruses from different genera. The chimeric orthopoxvirus and parapoxvirus isolates showed superior killing ability in a panel of NCI60 cancer cell lines compared to their parental individual wild-type viruses. Furthermore, based on the favorable fact that members from different genera of the poxviridae family are antigenic, the potent chimeric orthopoxvirus and potent chimeric parapoxvirus generated in this study can potentially be combined in the same treatment to achieve maximum therapeutic effect.

[0021] I. Definition Although various embodiments and aspects of the present invention are described herein, it will be apparent to those skilled in the art that such embodiments and aspects are provided by way of example only. Numerous variations, changes and substitutions will occur to those skilled in the art without departing from the present invention. It should be understood that various alternative forms of the embodiments of the present invention described herein can be applied in the practice of the present invention.

[0022] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described. All documents or portions of documents cited in this application, including but not limited to patents, patent applications, articles, books, manuals, and papers, are expressly incorporated herein by reference in their entirety for any purpose.

[0023] Unless otherwise defined, technical and scientific terms used herein generally have the same meaning as understood by those skilled in the art.See, for example, Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY 2nd ed., J.Wiley&Sons (New York, NY 1994); Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989). Any methods, devices and materials similar or equivalent to those described herein can be used in the practice of the present invention. The following definitions are provided to facilitate understanding of certain terms frequently used herein and are not intended to limit the scope of the present disclosure.

[0024] The terms "isolate" or "isolated" when applied to a nucleic acid, virus, or protein, mean that the nucleic acid, virus, or protein is essentially free from other cellular components with which it is naturally associated. It can be, for example, in a homogeneous state, and can be in a dry or aqueous solution. Purity and homogeneity are typically measured using analytical chemistry methods such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein that is abundant in a preparation is substantially purified.

[0025] As used herein, "nucleic acid" or "oligonucleotide" or "polynucleotide" or grammatical equivalents means at least two nucleotides covalently linked together. The term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides in single- or double-stranded form, and polymers thereof or their complements. The term "polynucleotide" refers to a linear sequence of nucleotides. The term "nucleotide" typically refers to a single unit (i.e., monomer) that constitutes a polynucleotide. Nucleotides can be ribonucleotides, deoxyribonucleotides, or modified versions thereof. Examples of polynucleotides contemplated herein include single- and double-stranded DNA, single- and double-stranded RNA (including siRNA), and hybrid molecules having mixtures of single- and double-stranded DNA and RNA. The term also encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, natural, and non-natural, which have binding properties comparable to the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotide. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral methyl phosphonates, and 2-O-methyl ribonucleotides.

[0026] Nucleic acids may contain non-specific sequences. As used herein, the term "non-specific sequence" refers to a nucleic acid sequence that contains a series of residues that are not designed to be complementary to any other nucleic acid sequence or that are only partially complementary. For example, a non-specific nucleic acid sequence is a series of nucleic acid residues that does not function as an inhibitory nucleic acid when contacted with a cell or organism. An "inhibitory nucleic acid" is a nucleic acid (e.g., a polymer of DNA, RNA, nucleotide analogs) that can bind to a target nucleic acid (e.g., an mRNA that can be translated into a protein) and reduce the transcription of the target nucleic acid (e.g., from DNA to mRNA), or reduce the translation of the target nucleic acid (e.g., an mRNA), or change post-transcriptional splicing (e.g., a single-stranded morpholino oligo).

[0027] A "labeled nucleic acid or oligonucleotide" is one that is attached, either covalently via a linker or chemical bond, or non-covalently via ionic, van der Waals, electrostatic, or hydrogen bonds, to a label whose presence can be detected by detecting the presence or absence of a detectable label attached to the nucleic acid. Alternatively, methods using high affinity interactions can also achieve the same result, in which one of a pair of binding partners binds to the other (e.g., biotin, streptavidin). In embodiments, the phosphorothioate nucleic acid or phosphorothioate polymer backbone comprises a detectable label as disclosed herein and generally known in the art.

[0028] The term "complementary" or "complementarity" refers to the ability of a nucleic acid in a polynucleotide to base pair with another nucleic acid in a second polynucleotide. For example, the sequence AGT is complementary to the sequence TCA. Complementarity may be partial, where only a portion of the nucleic acid is matched according to base pairing, or may be complete, where all of the nucleic acid is matched according to base pairing.

[0029] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein involved in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to promote translation. "Operably linked" broadly means that the DNA sequences being linked are located near each other, and, in the case of a secretory leader, are contiguous and in reading phase. Enhancers, however, need not be contiguous. Linking is accomplished by ligation at convenient restriction enzyme sites. If no such sites exist, synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

[0030] The term "gene" refers to a segment of DNA involved in the production of a protein, including the regions preceding and following the coding region (leader and trailer), as well as the intervening sequences (introns) between individual coding segments (exons). Leaders, trailers, and introns contain the control elements required for transcription and translation of a gene. Furthermore, a "protein gene product" is a protein expressed from a particular gene.

[0031] The term "expression" or "expressed" as used herein with respect to a gene refers to the transcription and / or translation product of that gene. The expression level of a DNA molecule in a cell can be measured based on the amount of corresponding mRNA present in the cell or the amount of protein encoded by the DNA produced by the cell. The expression level of a non-coding nucleic acid molecule (e.g., siRNA) can be detected by standard PCR or Northern blot techniques well known in the art. See Sambrook et al., 1989 Molecular Cloning: A Laboratory Manual, 18.1-18.88.

[0032] As used herein, "siRNA", "small interfering RNA", "small RNA" or "RNAi" refers to a nucleic acid that forms a double-stranded RNA, which has the ability to reduce or inhibit expression of a gene or target gene when expressed in the same cell as the gene or target gene. The complementary portions of the nucleic acid that hybridize to form the double-stranded molecule typically have substantial or complete identity. In one embodiment, siRNA or RNAi refers to a nucleic acid that has substantial or complete identity with a target gene and forms a double-stranded siRNA. In an embodiment, the siRNA inhibits gene expression by interacting with a complementary mRNA in the cell, thereby interfering with the expression of the complementary mRNA. Typically, the nucleic acid is at least about 15-50 nucleotides in length (e.g., each complementary sequence of a double-stranded siRNA is 15-50 nucleotides in length, and the double-stranded siRNA is about 15-50 base pairs in length). In other embodiments, the length is 20 to 30 nucleotides, preferably about 20 to 25, or about 24 to 29 nucleotides, for example, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. Non-limiting examples of siRNA include ribozymes, RNA decoys, short hairpin RNAs (shRNAs), microRNAs (miRNAs), and small nucleolar RNAs (snoRNAs).

[0033] The term "recombinant" when used with respect to, for example, a cell or a nucleic acid, protein, or vector, refers to the cell, nucleic acid, protein, or vector being modified by the introduction of a heterologous nucleic acid or protein, or the alteration of a naturally occurring nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, a recombinant cell expresses genes that are not present in the native (non-recombinant) form of the cell, or expresses naturally occurring genes that are aberrantly expressed, under-expressed, or not expressed at all in the absence of the recombinant cell. Transgenic cells and plants are those that express heterologous genes or coding sequences, typically as a result of recombinant techniques.

[0034] The term "heterologous" when used in reference to a portion of a nucleic acid refers to the fact that the nucleic acid comprises two or more subsequences in a relationship that is not found in the same relationship to each other in nature. For example, a nucleic acid is typically produced recombinantly, with two or more sequences from unrelated genes (e.g., a promoter from one source and a coding region from another source) arranged to create a novel functional nucleic acid. Similarly, a heterologous protein refers to the fact that the protein comprises two or more subsequences in a relationship that is not found in the same relationship to each other in nature (e.g., a fusion protein).

[0035] The term "exogenous" refers to a molecule or substance (e.g., a compound, nucleic acid, or protein) that originates from outside a given cell or organism. For example, an "exogenous promoter" as referred to herein is a promoter that is not native to the cell or organism in which it is expressed. Conversely, the term "endogenous" or "endogenous promoter" refers to a molecule or substance that is naturally present in or derived from a given cell or organism.

[0036] The term "isolated," when applied to a nucleic acid or protein, means that the nucleic acid or protein is essentially free from other intracellular components with which it is naturally associated. It can be, for example, in a homogeneous state, and can be in a dry or aqueous solution. Purity and homogeneity are typically measured using analytical chemistry methods such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The predominant type of protein present in a preparation is substantially purified.

[0037] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues, which may, in embodiments, be conjugated to a moiety that is not composed of amino acids. The terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of the corresponding naturally occurring amino acids, as well as to naturally occurring and non-natural amino acid polymers. A "fusion protein" refers to a chimeric protein encoding two or more separate protein sequences that are recombinantly expressed as a single moiety.

[0038] The terms "peptidyl" and "peptidyl moiety" refer to a monovalent peptide.

[0039] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that have been subsequently modified, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. "Amino acid analogs" refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an α-carbon with a hydrogen, a carboxyl group, an amino group, and an R group attached thereto, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. An amino acid mimetic refers to a chemical compound that has a structure that is different from the general chemical structure of an amino acid, but functions in a manner similar to a naturally occurring amino acid. The terms "non-naturally occurring amino acid" and "unnatural amino acid" refer to amino acid analogs, synthetic amino acids, and non-naturally occurring amino acid mimetics.

[0040] Amino acids may be represented herein by their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Similarly, nucleotides may be represented by their commonly accepted one-letter symbols.

[0041] "Conservatively modified variants" applies to both amino acid and nucleic acid sequences. "Conservatively modified variants" with respect to a particular nucleic acid sequence refers to nucleic acids that encode identical or essentially identical amino acid sequences. Due to the degeneracy of the genetic code, several nucleic acid sequences encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at any position where alanine is specified by a codon, the codon can be altered to any of the corresponding codons listed above without altering the encoded polypeptide.

[0042] Such nucleic acid mutations are "silent mutations", which are a type of conservatively modified mutation. For any nucleic acid sequence that encodes a polypeptide, all possible silent mutations of the nucleic acid are intended to be described herein. Those skilled in the art will recognize that each nucleic acid codon (except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan) can be modified to obtain a functionally identical molecule. Therefore, each silent mutation of a nucleic acid that encodes a polypeptide is intended to be included in each described sequence.

[0043] With respect to amino acid sequences, one of skill in the art will recognize that individual substitutions, deletions, or additions to nucleic acid, peptide, polypeptide, or protein sequences that substitute, add, or delete a single amino acid or a small percentage of amino acids in the encoded sequence are "conservatively modified variants" that result in the substitution of amino acids with chemically similar amino acids. Tables compiling conservative substitutions that provide functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to, but do not exclude, polymorphic variants, interspecies homologs, and alleles of the invention.

[0044] The following eight groups each contain amino acids that are conservative substitutions for one another: 1) alanine (A), glycine (G), 2) aspartic acid (D), glutamic acid (E), 3) asparagine (N), glutamine (Q), 4) arginine (R), lysine (K), 5) isoleucine (I), leucine (L), methionine (M), valine (V), 6) phenylalanine (F), tyrosine (Y), tryptophan (W), 7) serine (S), threonine (T), and 8) cysteine ​​(C), methionine (M) (see Creighton, Proteins (1984)).

[0045] The term "identical" or percent "identity", in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that are identical or have a predetermined percentage of identical amino acid residues or nucleotides in a given region (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity), as measured in a comparison window or a given region, using the BLAST or BLAST 2.0 sequence comparison algorithm with default parameters as described below to align sequences for best fit, or by manual alignment and visual assessment (see, e.g., the NCBI website http: / / www.ncbi.nlm.nih.gov / BLAST / ). Such sequences are said to be "substantially identical". This definition may also refer to or be applied to the complement of a test sequence. The definition also encompasses sequences that have deletions and / or additions, as well as sequences that have substitutions. Gaps and the like can be provided by a suitable algorithm, as described below. Preferably, the identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is 50-100 amino acids or nucleotides in length.

[0046] As used herein, the terms "thymidine kinase gene," "TK gene," "TK," "J2R gene," or "J2R" refer to a recombinant or naturally occurring form of a thymidine kinase gene or a mutant or homolog thereof that encodes a thymidine kinase polypeptide that can retain an activity of a thymidine kinase polypeptide (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the activity of a thymidine kinase polypeptide). In some embodiments, the mutant or homolog has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity over the entire sequence or a portion of the sequence (e.g., 50, 100, 150, or 200 contiguous nucleic acid portions) over the entire sequence or a portion of the sequence ... In an embodiment, the thymidine kinase gene is substantially identical to the nucleic acid sequence corresponding to positions 83422 to 83955 of the nucleic acid sequence identified by Accession No. DQ121394, or a mutant or homolog having substantial identity thereto. In an embodiment, the thymidine kinase gene comprises the nucleic acid sequence of SEQ ID NO: 4. In an embodiment, the thymidine kinase gene is the nucleic acid sequence of SEQ ID NO: 4. In an embodiment, the thymidine kinase gene has a mutation. In an embodiment, the thymidine kinase gene is partially deleted. In an embodiment, the thymidine kinase gene comprises the nucleic acid sequence of SEQ ID NO: 5. In an embodiment, the thymidine kinase gene comprises the nucleic acid sequence of SEQ ID NO: 5.

[0047] As used herein, the terms "F14.5L gene", "F14.5L sequence", "F14.5L" and the like refer to a recombinant or native F14.5L gene or a mutant or homolog thereof that encodes an F14.5L polypeptide that can maintain the activity of the F14.5L polypeptide (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the F14.5L polypeptide). In some embodiments, the mutant or homolog has at least 90%, 95%, 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity over the entire sequence or a portion of the sequence (e.g., 50, 100, 150 or 200 contiguous nucleic acid portions) compared to the native F14.5L gene. In an embodiment, the F14.5L gene is substantially identical to the nucleic acid sequence corresponding to positions 44428-44279 of the nucleic acid sequence identified by Accession No. KX781953, or a mutant or homolog having substantial identity thereto. In an embodiment, the F14.5L gene comprises the nucleic acid sequence of SEQ ID NO: 6. In an embodiment, the F14.5L gene is the nucleic acid sequence of SEQ ID NO: 6. In an embodiment, the F14.5L gene has a mutation. In an embodiment, the F14.5L gene is partially deleted. In an embodiment, the F14.5L gene comprises the nucleic acid sequence of SEQ ID NO: 7. In an embodiment, the F14.5L gene comprises the nucleic acid sequence of SEQ ID NO: 7.

[0048] As used herein, the terms "D4R gene," "uracil DNA glycosylase gene," and the like refer to any recombinant or native uracil DNA glycosylase gene, or mutant or homolog thereof, that encodes a uracil DNA glycosylase polypeptide that can retain the activity of the uracil DNA glycosylase polypeptide (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the activity of the uracil DNA glycosylase polypeptide). In some embodiments, the mutant or homolog has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity over the entire sequence or a portion of the sequence (e.g., 50, 100, 150, or 200 contiguous nucleic acid portions) compared to the native uracil DNA glycosylase gene. In an embodiment, the uracil DNA glycosidase gene is substantially identical to the nucleic acid sequence corresponding to positions 102720 to 103376 of the nucleic acid sequence identified by Accession No. DQ439815, or a variant or homolog having substantial identity thereto. In an embodiment, the uracil DNA glycosidase gene comprises the nucleic acid sequence of SEQ ID NO: 8. In an embodiment, the uracil DNA glycosidase gene is the nucleic acid sequence of SEQ ID NO: 8.

[0049] As used herein, the terms "E9L gene," "DNA polymerase gene," and the like refer to any recombinant or native DNA polymerase gene, or mutant or homolog thereof, that encodes a DNA polymerase polypeptide that can maintain the activity of the DNA polymerase polypeptide (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the activity of the DNA polymerase polypeptide). In some embodiments, the mutant or homolog has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity over the entire sequence or a portion of the sequence (e.g., 50, 100, 150, or 200 contiguous nucleic acid portions) over the native DNA polymerase gene. In an embodiment, the DNA polymerase gene is substantially identical to the nucleic acid sequence corresponding to positions 56656 to 53636 of the nucleic acid sequence identified by Accession No. AY243312, or a variant or homolog having substantial identity thereto. In an embodiment, the DNA polymerase gene comprises the nucleic acid sequence of SEQ ID NO: 12. In an embodiment, the DNA polymerase gene is the nucleic acid sequence of SEQ ID NO: 12.

[0050] As used herein, the terms "human sodium and iodine symporter gene," "hNIS gene," "NIS gene," and the like refer to any recombinant or native human sodium and iodine symporter gene, or variants or homologs thereof, that encode a human sodium and iodine symporter polypeptide that can retain the activity of a human sodium and iodine symporter polypeptide (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the activity of a human sodium and iodine symporter polypeptide). In some embodiments, the variant or homolog has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity over the entire sequence or a portion of the sequence (e.g., 50, 100, 150, or 200 contiguous nucleic acid portions) over the native human sodium and iodine symporter gene. In an embodiment, the human sodium and iodine symporter gene is substantially identical to the nucleic acid sequence identified by Accession No. NM_000453, or a variant or homolog having substantial identity thereto. In an embodiment, the human sodium and iodine symporter gene comprises the nucleic acid sequence of SEQ ID NO: 13. In an embodiment, the human sodium and iodine symporter gene is the nucleic acid sequence of SEQ ID NO: 13.

[0051] As used herein, the terms "Emerald gene" and "Emerald sequence" refer to a genetically engineered gene encoding an Emerald polypeptide that can maintain an activity of an Emerald polypeptide (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the activity of an Emerald polypeptide), or a variant thereof. In some embodiments, the variant or homolog has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity to an Emerald sequence over the entire sequence or a portion of the sequence (e.g., over 50, 100, 150, or 200 contiguous nucleic acid portions). In an embodiment, Emerald is substantially identical to a nucleic acid sequence corresponding to positions 3215-3931 of the nucleic acid sequence identified by Accession No. KF293661, or a variant or homolog having substantial identity thereto. In an embodiment, the Emerald gene comprises the nucleic acid sequence of SEQ ID NO: 14. In an embodiment, the Emerald gene is the nucleic acid sequence of SEQ ID NO:14.

[0052] As used herein, the term "firefly luciferase gene" or "firefly luciferase sequence" refers to a recombinant or native firefly luciferase gene, or a mutant or homolog thereof, that encodes a firefly luciferase polypeptide that can retain the activity of a firefly luciferase polypeptide (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the activity of a firefly luciferase polypeptide). In some embodiments, the mutant or homolog has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity over the entire sequence or a portion of the sequence (e.g., 50, 100, 150, or 200 contiguous nucleic acid portions) compared to a native firefly luciferase gene. In an embodiment, the firefly luciferase gene is substantially identical to the nucleic acid sequence corresponding to positions 3129 to 4781 of the nucleic acid sequence identified by Accession No. KF990214, or a mutant or homolog having substantial identity thereto. In an embodiment, the firefly luciferase gene comprises the nucleic acid sequence of SEQ ID NO: 15. In an embodiment, the firefly luciferase gene is the nucleic acid sequence of SEQ ID NO: 15.

[0053] As used herein, the term "mCherry gene" or "mCherry sequence" refers to a recombinant or naturally occurring gene encoding an mCherry polypeptide that maintains the activity of the mCherry polypeptide (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the activity of the mCherry polypeptide), or a variant or homolog thereof. In some embodiments, the variant or homolog has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity over the entire sequence or a portion of the sequence (e.g., over 50, 100, 150, or 200 contiguous nucleic acid portions) compared to a naturally occurring mCherry gene. In embodiments, the mCherry gene is substantially identical to a nucleic acid sequence corresponding to positions 1073-1783 of the nucleic acid sequence identified by Accession No. KX446949, or a variant or homolog having substantial identity thereto. In an embodiment, the mCherry gene comprises the nucleic acid sequence of SEQ ID NO: 16. In an embodiment, the mCherry gene is the nucleic acid sequence of SEQ ID NO: 16.

[0054] As used herein, the terms "H5 promoter", "H5" and the like refer to a recombinant or native H5 promoter, or a variant or homologue thereof, that maintains the activity of the H5 promoter (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the H5 promoter). In some embodiments, the variant or homologue has at least 90%, 95%, 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity over the entire sequence or a portion of the sequence (e.g., 50, 100, 150 or 200 contiguous nucleic acid portions) compared to the native H5 promoter. In an embodiment, the H5 promoter is substantially identical to a nucleic acid sequence corresponding to positions 7 to 76 of the nucleic acid sequence identified by Accession No. FJ386852, or a variant or homologue having substantial identity thereto. In an embodiment, the H5 promoter comprises the nucleic acid sequence of SEQ ID NO: 18. In an embodiment, the H5 promoter is the nucleic acid sequence of SEQ ID NO:18.

[0055] As used herein, the terms "SE promoter", "SE" and the like refer to a recombinant or native SE promoter, or a variant or homologue thereof, that maintains the activity of the SE promoter (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the SE promoter). In some embodiments, the variant or homologue has at least 90%, 95%, 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity compared to the native SE promoter over the entire sequence or a portion of the sequence (e.g., 50, 100, 150 or 200 contiguous nucleic acid portions). In an embodiment, the SE promoter comprises the nucleic acid sequence of SEQ ID NO:19. In an embodiment, the SE promoter comprises the nucleic acid sequence of SEQ ID NO:19.

[0056] As used herein, the terms "11K promoter", "11K" and the like refer to a recombinant or native 11K promoter that maintains the activity of the 11K promoter (e.g., at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to the 11K promoter), or a mutant or homolog thereof. In some embodiments, the mutant or homolog has at least 90%, 95%, 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity over the entire sequence or a portion of the sequence (e.g., 50, 100, 150 or 200 consecutive nucleic acid portions) compared to the native 11K promoter. In an embodiment, the 11K promoter is substantially identical to a nucleic acid sequence corresponding to positions 40734 to 40771 of the nucleic acid sequence identified by Accession No. KF179385, or a mutant or homolog having substantial identity thereto. In an embodiment, the 11K promoter comprises the nucleic acid sequence of SEQ ID NO: 20. In an embodiment, the 11K promoter is the nucleic acid sequence of SEQ ID NO: 20.

[0057] Antibodies are large (approximately 150,000 molecular weight or approximately 1320 amino acids), composite molecules with complex internal structures. Natural antibody molecules contain two pairs of identical polypeptide chains (each pair with one light and one heavy chain). Each light and heavy chain consists of two contiguous regions: a variable ("V") region, which is responsible for binding to the target antigen, and a constant ("C") region, which interacts with other components of the immune system. The light and heavy chain variable regions come together in three-dimensional space to form the variable region that binds to the antigen (e.g., a receptor on the surface of a cell). Within each light or heavy chain variable region, there are three short segments (average 10 amino acids long) called complementarity determining regions ("CDRs"). The six CDRs of an antibody variable domain (three from the light chain and three from the heavy chain) fold together in three-dimensional space to form the actual antibody binding site that docks onto the target antigen. The positions and lengths of the CDRs are precisely defined by Kabat, E. et al. (Sequences of Proteins of Immunological Interest, USDepartment of Health and Human Services, 1983, 1987). The parts of the variable regions not included in the CDRs are called the framework ("FR"), which form the environment for the CDRs.

[0058] The term "antibody" is used according to its commonly known meaning in the art. Antibodies exist, for example, as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases. Thus, for example, digestion of an antibody with pepsin below the disulfide bonds in the hinge region produces F(ab)'2, which is itself a V fragment separated by disulfide bonds. H -C H1The F(ab)'2 is reduced under mild conditions to cleave the disulfide bond in the hinge region and convert the F(ab)'2 dimer into a Fab' monomer. The Fab' monomer is essentially a Fab with a portion of the hinge region (see Fundamental Immunology, ed. Paul, 3rd ed., 1993). While various antibody fragments are defined as digests of intact antibodies, one skilled in the art will appreciate that such fragments can be synthesized de novo either chemically or using recombinant DNA methodology. Thus, the term "antibody" as used herein includes antibody fragments produced by the modification of full-length antibodies or those synthesized de novo using recombinant DNA methodology (e.g., single chain Fv) or those identified using a phage display library (see, e.g., McCafferty et al., Nature 348:552-554 (1990)).

[0059] An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer consists of two pairs of identical polypeptide chains, each pair having one "light" chain (about 25 kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100-110 or more amino acids that are primarily responsible for antigen recognition. The terms "variable light chain" (VL) and "variable heavy chain" (VH) refer to these light and heavy chains, respectively. The Fc (or fragment crystallizable region) is the "base" or "tail" of the immunoglobulin and typically consists of two heavy chains that form two or three constant domains, depending on the type of antibody. The Fc region ensures that each antibody mounts an appropriate immune response to a given antigen by binding to specific proteins. The Fc region also binds to various cellular receptors, such as Fc receptors, and other immune molecules, such as complement proteins.

[0060] The term "antigen" as provided herein refers to a molecule that can bind to the binding domain of an antibody provided herein. The "antigen-binding domain" as provided herein is a region in an antibody that binds to an antigen (epitope). As described above, the antigen-binding domain broadly consists of one constant domain and one variable domain (VL, VH, CL and CH1, respectively) of each heavy and light chain. The paratope or antigen-binding site is formed on the N-terminus of the antigen-binding domain. The two variable domains of the antigen-binding domain typically bind to an epitope on an antigen.

[0061] Antibodies exist, for example, as intact immunoglobulins or as several well-characterized fragments produced by digestion with various peptidases. Thus, for example, digestion of an antibody downstream of the disulfide bond in the hinge region with pepsin produces a dimer of F(ab)'2, a light chain Fab that is itself linked to VH-CH1 by a disulfide bond. F(ab)'2 is reduced under mild conditions to cleave the disulfide bond in the hinge region and convert the F(ab)'2 dimer into a Fab' monomer. The Fab' monomer is essentially an antigen binding site with part of the hinge region (see Fundamental Immunology, ed. Paul, 3rd ed., 1993). While various antibody fragments are defined as digests of intact antibodies, one of skill in the art will understand that such fragments can be synthesized de novo either chemically or using recombinant DNA methodology. Thus, the term "antibody," as used herein, includes antibody fragments produced by the modification of full-length antibodies or those synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv) or those identified using phage display libraries (see, e.g., McCafferty et al., Nature 348:552-554 (1990)).

[0062] Single chain variable fragments (scFv) are typically fusion proteins of the variable regions of immunoglobulin heavy (VH) and light (VL) chains, linked by a short linker peptide of 10 to about 25 amino acids. The linker is usually glycine-rich for flexibility and may be serine- or threonine-rich for solubility. The linker may link the N-terminus of VH to the C-terminus of VL, or vice versa.

[0063] The epitope of an antibody is the region of the antigen to which the antibody binds. Two antibodies bind to the same or overlapping epitopes if each competitively inhibits (blocks) the binding of the other antigen. That is, a 1x, 5x, 10x, 20x, or 100x excess of one antibody inhibits the binding of the other antibody by at least 30%, preferably 50%, 75%, 90% or 99%, as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res. 50:1495, 1990). Alternatively, two antibodies have the same epitope if substantially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody also reduce or eliminate binding of the other antibody. Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other antibody.

[0064] For the preparation of suitable antibodies of the invention and for use according to the invention (e.g., recombinant, monoclonal or polyclonal antibodies), many techniques known in the art can be used (see, e.g., Kohler and Milstein, Nature 256:495-497, 1975; Kozbor et al., Immunology Today 4:72 (1983); Cole et al., Monoclonal Antibodies and Cancer Therapy, pp.77-96, Alan R. Liss Inc. (1985); Coligan, Current Protocols in Immunology (1991); Harlow and Lane, Antibodies, A Laboratory Manual (1988); and Goding, Monoclonal Antibodies: Principles and Practice (2nd ed., 1986)). Genes encoding the heavy and light chains of the antibody of interest can be cloned from cells, e.g., genes encoding a monoclonal antibody can be cloned from a hybridoma and used to produce recombinant monoclonal antibodies. Libraries of genes encoding heavy and light chains of monoclonal antibodies can also be produced from hybridomas or plasma cells. Random combination of heavy and light chain gene products can generate a large pool of antibodies with different antigen specificities (see, for example, Kuby, Immunology (3rd ed., 1997)). The technology of single chain antibody or recombinant antibody production can optimize the production of antibodies against the polypeptides of the present invention (U.S. Pat. Nos. 4,946,778 and 4,816,567).Also, transgenic mice or other organisms such as other mammals can be used to express humanized or human antibodies (see, e.g., U.S. Pat. Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, and 5,661,016; Marks et al., Bio / Technology 10:779-783 (1992); Lonberg et al., Nature 368:856-859 (1994); Morrison, Nature 368:812-13 (1994); Fishwild et al., Nature Biotechnology 14:845-51 (1996); Neuberger, Nature Biotechnology 14:845-51 (1996)). 14:826 (1996); and Lonberg and Huszar (Intern. Rev) Immunol. 13:65-93 (1995). Alternatively, phage display techniques can be used to identify antibodies and heteromeric Fab fragments that specifically bind to a selected antigen (see, e.g., McCafferty et al., Nature 348:552-554 (1990); Marks et al., Biotechnology 10:779-783 (1992)). Bispecific antibodies can also be generated, i.e., capable of recognizing two different antigens (see, e.g., WO 93 / 08829; Traunecker et al., EMBO J. 10:3655-3659, 1991; and Suresh et al., Methods in Enzymology 121:210 (1986)). The antibody may also be a heteroconjugate (e.g., two covalently linked antibodies or immunotoxin conjugates) (see, e.g., U.S. Pat. No. 4,676,980, WO 91 / 00360, WO 92 / 200373, and EP 03089).

[0065] The phrases "specifically (or selectively) bind to an antibody" or "specifically (or selectively) immunoreactive" when referring to a protein or peptide often refer to a binding reaction that is determinative for the presence of that protein in a heterogeneous population of proteins and other biological substances. That is, under given immunological assay conditions, a particular antibody binds to a particular protein at least twice background, more typically 10-100 times background or more. Specific binding to an antibody under such conditions typically requires that the antibody be selected based on its specificity for a particular protein. For example, polyclonal antibodies can be selected to obtain only a subset of antibodies that are specifically immunoreactive with a selected antigen and not with other proteins. This selection is made possible by excluding antibodies that cross-react with other molecules. A variety of immunological assay formats may be used to select antibodies that are specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies having specific immunoreactivity with a protein (see, e.g., Harlow and Lane, Using Antibodies, A Laboratory Manual (1998), for a description of immunoassay formats and conditions that can be used to measure specific immunoreactivity).

[0066] "Contacting" is used according to its plain and ordinary meaning to refer to the process of bringing at least two different species (e.g., chemicals, including biological molecules or cells) into sufficient proximity to react, interact, or physically touch. However, it should be understood that reaction products may result directly from the reaction between the added reagents or may result from intermediates that may be generated in the reaction mixture from the added reagent or reagents.

[0067] The term "contacting" may include reacting, interacting, or physically contacting two species, where the two species may be, for example, an antibody domain and an antibody binding domain as described herein. In embodiments, the contacting includes, for example, interacting an antibody domain as described herein with an antibody binding domain.

[0068] "Patient" or "subject in need thereof" refers to an organism suffering from or prone to a disease or condition that can be treated by administration of a composition or pharmaceutical composition provided herein. Non-limiting examples include humans, other mammals, cows, rats, mice, dogs, monkeys, goats, sheep, cattle, deer, and other non-mammals. In some embodiments, the patient is a human.

[0069] The term "disease" or "condition" as used herein refers to a condition or health state of a patient or subject that may be treated with the compounds or methods provided. The disease may be cancer. In some further examples, "cancer" refers to human cancer and carcinoma, sarcoma, adenocarcinoma, lymphoma, leukemia, including solid and lymphatic cancer, kidney cancer, breast cancer, lung cancer, bladder cancer, colon cancer, ovarian cancer, prostate cancer, pancreatic cancer, gastric cancer, brain cancer, head and neck cancer, skin cancer, uterine cancer, testicular cancer, glioma, esophageal cancer, and liver cancer, such as liver cancer, lymphoma, such as B acute lymphoblastic lymphoma, non-Hodgkin's lymphoma (e.g., Burkitt's, small cell and large cell lymphoma), Hodgkin's lymphoma, leukemia (including AML, ALL and CML) or multiple myeloma.

[0070] As used herein, the term "cancer" refers to any type of cancer, neoplasm, or malignant tumor found in a mammal (e.g., human), including leukemia, carcinoma, and sarcoma. Examples of cancers that may be treated with the compounds or methods provided herein include breast cancer, colon cancer, kidney cancer, leukemia, lung cancer, melanoma, ovarian cancer, prostate cancer, pancreatic cancer, brain cancer, liver cancer, stomach cancer, or sarcoma.

[0071] The term "leukemia" broadly refers to progressive, malignant diseases of the blood-forming organs and is primarily characterized by the abnormal proliferation and development of white blood cells and their precursors in the blood and bone marrow. Leukemias are usually classified clinically on the basis of (1) the duration and nature of the disease (acute or chronic), (2) the type of cells involved (myeloid (myeloid), lymphocytic (lymphoid), or monocytic), and (3) the abnormal increase or absence of an increase in the number of cells in the blood (leukocytosis or aleukemic (subleukemic)). Examples of leukemias that may be treated with the compounds or methods provided herein include, for example, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, leukocytosis leukemia, basophilic leukemia, blast cell leukemia, bovine leukemia, chronic myeloid leukemia, leukemia cutis, embryonic cell leukemia, eosinophilic leukemia, gross leukemia, hairy cell leukemia, hemoblastic leukemia, hemoblastic leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphatic leukemia, leukemia), lymphoblastic leukemia, lymphocytic leukemia, lymphoid leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia, monocytic leukemia, myeloblastic leukemia, myelogenous leukemia, myelogenous granulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasma cell leukemia, multiple myeloma, plasma cell leukemia, promyelocytic leukemia, Rieder cell leukemia, Schilling's leukemia, stem cell leukemia, subleukemia or anaplastic cell leukemia.

[0072] The term "sarcoma" generally refers to a tumor formed from a material like embryonic connective tissue and is usually composed of tightly packed cells embedded in a fibrous or homogeneous substance. Sarcomas that may be treated with the compounds or methods provided herein include chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abemethy's sarcoma, liposarcoma (adipose tissue), and sarcoma of the ... sarcoma), liposarcoma, cellulitic soft tissue sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, choriocarcinoma, embryonal sarcoma, Wilms' tumor sarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple chromatohemorrhagic sarcoma, immunoblastic sarcoma of B cell, lymphoma, immunoblastic sarcoma of T cell, Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukosarcoma, malignant mesenchymal sarcoma, parosteal osteosarcoma, reticulocytic sarcoma, Rous sarcoma, serum cystic sarcoma, synovial sarcoma, or telangiectatic sarcoma.

[0073] The term "melanoma" is intended to mean a tumor arising from the pigment cell system of the skin and other organs. For example, melanomas that may be treated with the compounds or methods provided herein include acral lentigo melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma, Harding-Passey melanoma, juvenile melanoma, lentigo maligna melanoma, malignant melanoma, nodular melanoma, subungual melanoma, or superficial spreading melanoma.

[0074] The term "cancer" refers to a malignant new growth composed of epithelial cells that tend to invade surrounding tissues and give rise to metastases. For example, examples of cancers that can be treated with the compounds or methods provided herein include medullary thyroid carcinoma, familial medullary thyroid carcinoma, lobular carcinoma, lobular carcinoma, adenoid cystic carcinoma, adenomatous carcinoma, adrenal cortical carcinoma, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, basaloid carcinoma, basal squamous cell carcinoma, bronchiolocarcinoma, bronchiolar carcinoma, bronchial carcinoma, cerebriform carcinoma, cholangiocarcinoma, choriocarcinoma, colloid carcinoma, comedocarcinoma, corpus carcinoma, cribriform carcinoma, armor carcinoma, skin carcinoma, cylindrical carcinoma, cylindrical cell carcinoma, ductal carcinoma, compact carcinoma, embryonal carcinoma, cerebriform carcinoma, epidermoid carcinoma, adenoid epithelial carcinoma, exophytic carcinoma, ulcer carcinoma, fibrous carcinoma, colloid carcinoma, cerebriform carcinoma, epidermoid carcinoma, adenoid epithelial carcinoma, exophytic carcinoma, ulcer carcinoma, fibrous carcinoma, geratiniform carcinoma, cerebriform carcinoma, epidermoid carcinoma, epithelial ... colloid carcinoma, giant cell carcinoma, giant cell carcinoma, adenocarcinoma, granulosa cell carcinoma, hair matrix carcinoma, hematoid carcinoma, liver cancer, Hurthle cell carcinoma, vitreous carcinoma, supernumerary renal carcinoma, infantile embryonic carcinoma, carcinoma in situ, intraepithelial carcinoma, intraepithelial carcinoma, Krompecher's carcinoma, Kulchitzky cell carcinoma, large cell carcinoma, lenticular carcinoma carcinoma lenticulare, lipomatous carcinoma, lymphoepithelial carcinoma, medullary carcinoma, medullary carcinoma, melanoma, soft carcinoma, mucinous carcinoma, carcinoma muciparum, carcinoma mucocellulare, mucinous adenocarcinoma, mucinous epidermoid carcinoma, carcinoma mucosum, mucinous carcinoma, myxomatous carcinoma, nasopharyngeal carcinoma, oat cell carcinoma, ossifying carcinoma, osteoid carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, squamous cell carcinoma, medullary carcinoma, renal cell carcinoma, reserve cell carcinoma, sarcomatoid carcinoma, Schneider's carcinoma, scirrhous carcinoma, scrotal carcinoma, signet ring cell carcinoma, simplex carcinoma, small cell carcinoma, potato carcinoma, spindle cell carcinoma, spindle cell carcinoma, cavernous carcinoma, squamous cell carcinoma, fasciate carcinoma, carcinoma telangiectaticum, carcinoma telangiectodes, transitional cell carcinoma, carcinoma tuberosum, nodular carcinoma, verrucous carcinoma or choriocarcinoma.

[0075] The terms "associated with" or "associated with," in the context of a substance or an activity or function of a substance associated with a disease (such as, for example, cancer, asthma, ulcerative colitis, irritable bowel syndrome, arthritis, uveitis, pyoderma gangrenosum, or erythema nodosum), refer to the substance or the activity or function of the substance causing (in whole or in part) the symptom or causing (in whole or in part) the manifestation of the disease.

[0076] As used herein, the terms "immune checkpoint", "immune checkpoint protein" or "checkpoint protein" are used interchangeably and are defined to refer to compositions (molecules) that can regulate the duration and extent of a physiological immune response (e.g., attenuate and / or eliminate persistent immune cell activation, i.e., control normal immune homeostasis). Immune checkpoint proteins can stimulate (enhance) an immune response. In embodiments, checkpoint proteins are cellular receptors. Examples of stimulatory checkpoint molecules include, but are not limited to, members of the tumor necrosis factor (TNF) receptor superfamily (e.g., CD27, CD40, OX40, glucocorticoid-induced TNFR family related gene (GITR) and CD137), members of the B7-CD28 superfamily (e.g., CD28 itself and inducible T cell co-stimulator (ICOS)). Alternatively, immune checkpoint proteins may inhibit (reduce) an immune response. Examples of inhibitory checkpoint molecules include, but are not limited to, adenosine A2A receptor (A2AR), B7-H3, B7-H4, BTLA, CTLA-4, indoleamine 2,3-dioxygenase (IDO), killer immunoglobulin-like receptor (KIR), LAG3, PD-1, TIM-3, and V-domain immunoglobulin suppressor of T cell activation (VISTA) protein.

[0077] Similarly, an "immune checkpoint inhibitor" or "checkpoint inhibitor" as provided herein refers to a substance (e.g., an antibody or fragment thereof, a small molecule, or the like) that inhibits and negatively affects (e.g., decreases) the activity or function of a checkpoint protein (e.g., decreases expression or decreases activity of the checkpoint protein) compared to the activity or function of the checkpoint protein in the absence of the inhibitor. A checkpoint inhibitor at least partially, in part or in whole, blocks stimulation, reduces, prevents or delays activation, or inactivates, desensitizes or downregulates signaling or enzymatic activity, or decreases the amount of a checkpoint protein. A checkpoint inhibitor can inhibit a checkpoint protein, for example, by binding to the checkpoint protein, blocking in part or in whole, desensitizing, inhibiting, delaying, inactivating, desensitizing or downregulating activity. In an embodiment, the checkpoint inhibitor is an antibody. In an embodiment, the checkpoint inhibitor is an antibody fragment. In an embodiment, the checkpoint inhibitor is an antibody variant. In an embodiment, the checkpoint inhibitor is an scFv. In embodiments, the checkpoint inhibitor is an anti-CTLA-4 antibody. In embodiments, the checkpoint inhibitor is an anti-PD1 antibody. In embodiments, the checkpoint inhibitor is an anti-PD-L1 antibody. In embodiments, the checkpoint inhibitor is an anti-LAG-3 antibody. In embodiments, the checkpoint inhibitor is an anti-IgG1k antibody. In embodiments, the checkpoint inhibitor is an anti-CD25 antibody. In embodiments, the checkpoint inhibitor is an anti-IL2R antibody. In embodiments, the checkpoint inhibitor forms part of an oncolytic virus. Non-limiting examples of checkpoint inhibitors include ipilimumab, pembrolizumab, nivolumab, talimogene, laherparepvec, durvalumab, daclizumab, avelumab, and atezolizumab.

[0078] The term "abnormal" as used herein refers to something that is different from the norm. Abnormal, when used in reference to enzyme activity, refers to a stronger or weaker activity than that of a normal control or the average of normal non-affected control samples. Abnormal activity refers to a degree of activity that results in disease, where restoring the abnormal activity to a normal or non-disease-related degree (e.g., using the methods described herein) results in a reduction in the disease or one or more symptoms.

[0079] "Control" or "standard control" refers to a sample, measurement or value as a reference (usually a known reference) for comparison of a test sample, measurement or value. For example, a test sample can be taken from a patient suspected of a given disease (e.g., cancer) and compared to a known healthy (unaffected) individual (e.g., a standard control subject). A standard control can also represent the average of measurements or values ​​collected from a population of similar individuals (e.g., standard control subjects) who do not have a given disease, such as healthy individuals with a similar medical background, the same age, comparable weight, etc. (i.e., a standard control population). A standard control value can also be obtained from a sample previously obtained from the same individual, e.g., a patient before the onset of the disease. For example, a control can be used to compare therapeutic benefits based on pharmacological data (e.g., half-life) or therapeutic measures (e.g., comparison of side effects). Controls are also important for determining the significance of data. For example, if a given parameter value varies widely in the control, the variation in the test sample is not considered significant. One of skill in the art will recognize that standard controls can be designed for evaluation of any numerical parameter (e.g., RNA levels, protein levels, specific cell types, specific body fluids, specific tissues, synovial cells, synovial fluid, synovial tissue, fibroblast-like synovial cells, macrophage-like synovial cells, etc.).

[0080] Those skilled in the art can understand which standard control is optimal in a given situation and can analyze data based on comparison with the standard control value. Standard control is also important for determining the significance of data (e.g., statistical significance). For example, if a given parameter value varies widely in the standard control, the variation of the test sample is not considered to be significant.

[0081] The term "diagnosis" refers to the relative probability that a disease (e.g., cancer) exists in a subject. Similarly, the term "prognosis" relates to the relative probability that a particular future outcome may occur in a subject with respect to a disease state. For example, in the context of the present invention, prognosis relates to the likelihood that an individual will develop a disease (e.g., cancer) or the expected severity of a disease (e.g., duration of the disease). As will be appreciated by those skilled in the art of medical diagnostics, this term does not have an absolute meaning.

[0082] "Biological sample" or "sample" refers to material obtained from or derived from a subject or patient. Biological samples include tissue portions, such as biopsy and autopsy samples, and frozen sections taken for histological purposes. Such samples include bodily fluids, such as blood and blood fractions or preparations (e.g., serum, plasma, platelets, red blood cells, etc.), sputum, tissues, cultured cells (e.g., primary cultures, grafts, and transformed cells), stool, urine, synovial fluid, articular tissue, synovial tissue, synovial cells, fibroblast-like synoviocytes, macrophage-like synoviocytes, immune cells, hematopoietic cells, fibroblasts, macrophages, T cells, etc. Biological samples are typically obtained from eukaryotic organisms, such as mammals, such as primates (e.g., chimpanzees or humans), cows, dogs, cats, rodents (e.g., guinea pigs, rats, mice), rabbits, or birds, reptiles, or fish.

[0083] As used herein, a "cell" refers to a cell that is performing sufficient metabolic or other functions to preserve or replicate its genomic DNA. Cells can be identified by methods well known in the art, including, for example, the presence of an intact cell membrane, staining with a particular dye, the ability to produce progeny cells, or (in the case of a reproductive cell) the ability to combine with a second reproductive cell to produce viable progeny. Cells can include prokaryotic and eukaryotic cells. Prokaryotic cells include, but are not limited to, bacteria. Eukaryotic cells include, but are not limited to, yeast cells, and cells derived from plants and animals, such as mammalian, insect (e.g., Spodoptera) and human cells. Cells can be used if they are not normally adhesive, or if they have been treated (e.g., trypsinized) to prevent them from adhering onto a surface.

[0084] The term "replication" is used according to its clear and ordinary meaning, i.e., refers to the ability of a cell or virus to produce progeny. As will be clear to those skilled in the art, the term "replication", when used in relation to DNA, refers to the biological process of producing two identical DNA replicas from one original DNA molecule. Thus, the term "replication" includes inheritance and reinfection of progeny cells. In an embodiment, the chimeric poxvirus provided herein has high oncolytic activity compared to its parent virus. In an embodiment, the oncolytic activity (ability to induce cell death in infected cells) is 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 100, 10000, 10000 times or more higher than the oncolytic activity of the parent virus (one of the viruses used to generate the chimeric virus provided herein).

[0085] As used herein, a "synergistic amount" refers to a synergistic effect (i.e., an effect greater than additive) that is achieved by adding together a first amount (e.g., an amount of a first chimeric poxvirus) and a second amount (e.g., an amount of a second chimeric poxvirus). Thus, the terms "synergistic effect," "synergistic action," "synergistic," "combined synergistic amount," and "synergistic therapeutic effect" are used interchangeably herein and refer to a measured effect of co-administration of chimeric poxviruses that is greater than the sum of the individual effects of each chimeric poxvirus administered alone as a single agent.

[0086] In embodiments, the synergistic amount is about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0 .6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 4 The amount of the first chimeric poxvirus may be 6, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%.In embodiments, the synergistic amount is about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0 .6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9 ... .2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, The amount of the second chimeric poxvirus may be 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%.

[0087] The terms "virus" or "virus particle" are used according to their plain and ordinary meaning in virology and refer to a viral particle comprising the viral genome (e.g., DNA, RNA, single-stranded, double-stranded), viral capsid and associated proteins, and, in the case of enveloped viruses (e.g., herpesviruses, poxviruses), the envelope comprising lipids, any host cell membrane components, and / or viral proteins.

[0088] The term "poxvirus" is used according to its plain and ordinary meaning in virology to refer to any member of the poxvirus family that has the ability to infect vertebrates and invertebrates and replicate in the cytoplasm of these hosts. In an embodiment, a poxvirus virus particle has a size of about 200 nm in diameter and about 300 nm in length, and has a genome in the form of a single, linear, double-stranded DNA segment, typically of 130-375 kb. The term poxvirus includes all viruses of the genus poxviridae (e.g., betaentomo poxvirus, yata poxvirus, cervid poxvirus, gammaentomo poxvirus, lepori poxvirus, suipoxvirus, mollusci poxvirus, crocodylid poxvirus, alphaentomo poxvirus, capripoxvirus, orthopoxvirus, avipoxvirus, and parapoxvirus), but is not limited thereto. In embodiments, the poxvirus is an orthopoxvirus (e.g., smallpox virus, vaccinia virus, bovine pox virus, monkey pox virus), a parapoxvirus (e.g., ovine thrush virus, pseudobovine pox virus, bovine papular stomatitis virus), a yatapoxvirus (e.g., tanapox virus, yaba monkey tumor virus), or a molluscpoxvirus (e.g., molluscum contagiosum virus). In embodiments, the poxvirus is an orthopoxvirus (e.g., bovine poxvirus Brighton strain, raccoon poxvirus Herman strain, rabbit poxvirus Utrecht strain, vaccinia virus WR strain, vaccinia virus IHD strain, vaccinia virus Elstree strain, vaccinia virus CL strain, vaccinia virus Lederle-Chorioallantoic strain, or vaccinia virus AS strain). In an embodiment, the poxvirus is a parapoxvirus (eg, orf virus strain NZ2 or pseudobovine poxvirus strain TJS).

[0089] The term "chimera" as used in the context of chimeric poxviruses is used according to its clear and ordinary meaning in virology to refer to a hybrid microorganism (e.g., a chimeric poxvirus) created by joining nucleic acid fragments from two or more different microorganisms (e.g., two viruses from the same subfamily, two viruses from different subfamilies). In an embodiment, the nucleic acid fragments from at least two poxvirus strains that are combined comprise essential genes required for replication. In an embodiment, the nucleic acid fragments from one of the at least two poxvirus strains comprise essential genes required for replication. The chimeric poxviruses according to these embodiments provided herein may comprise one or more transgenes (i.e., nucleic acid sequences that are not present in the native viral genome). For example, the chimeric poxviruses according to these embodiments provided herein may comprise an anti-cancer nucleic acid sequence, a nucleic acid binding sequence, a nucleic acid sequence encoding a detectable moiety, or any combination thereof. In an embodiment, the chimeric poxviruses comprise a nucleic acid sequence comprising an anti-cancer nucleic acid sequence, a nucleic acid binding sequence, and a nucleic acid sequence encoding a detectable moiety. In an embodiment, the chimeric poxviruses comprise a nucleic acid sequence comprising an anti-cancer nucleic acid sequence and a nucleic acid sequence encoding a detectable moiety. In an embodiment, the chimeric poxvirus comprises a nucleic acid sequence comprising a nucleic acid binding sequence and a nucleic acid sequence encoding a detectable moiety, In an embodiment, the chimeric poxvirus comprises a nucleic acid sequence comprising an anti-cancer nucleic acid sequence and a nucleic acid binding sequence.

[0090] The term "plaque-forming unit" is used according to its normal and obvious meaning in virology and refers to the amount of plaques that can be formed on a cell monolayer per volume of viral particles. In some embodiments, the unit is based on the number of plaques that can be formed when a monolayer of susceptible cells is infected. For example, in embodiments, 1,000 PFU / μl refers to 1 μl of a solution containing viral particles containing enough viral particles to produce 1000 infectious plaques on a cell monolayer. In embodiments, plaque-forming unit is abbreviated as "PFU".

[0091] The term "multiple of infection" or "MOI" is used according to its clear and ordinary meaning in virology and refers to the ratio of infectious agent (e.g., poxvirus) to target (e.g., cells) in a given area or volume, which in embodiments is considered to be homogenous.

[0092] The term "bovine poxvirus Brighton strain" is used according to its common and usual meaning and refers to the same or similarly named virus strains, as well as functional fragments and homologues thereof. The term encompasses recombinant or native bovine poxvirus Brighton strain, or variants thereof that maintain the activity of bovine poxvirus Brighton strain (e.g., within at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%). The term encompasses recombinant or native bovine poxvirus Brighton strain, or variants thereof that have sequence identity to the genome of bovine poxvirus Brighton strain (e.g., about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity to the genome of bovine poxvirus Brighton strain). The Brighton bovine poxvirus may also refer to a variant in which an amino acid residue has been mutated, thereby modulating (e.g., increasing or decreasing) the activity, expression, cell targeting, or infectivity of the Brighton bovine poxvirus. The Brighton bovine poxvirus may be modified as described herein. In an embodiment, the Brighton bovine poxvirus is designated by the American Type Culture Collection (ATCC) reference number ATCC VR-302. TM In an embodiment, bovine poxvirus Brighton strain refers to the virus strain identified by Taxonomy reference number 265872, a mutant or homolog thereof.

[0093] The term "raccoon poxvirus Herman strain" is used according to its common and usual meaning and refers to the same or similarly named virus strain, as well as functional fragments and homologues thereof. The term encompasses recombinant or native raccoon poxvirus Herman strain, or variants thereof that maintain the activity of the raccoon poxvirus Herman strain (e.g., within at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%). The term encompasses recombinant or native raccoon poxvirus Herman strain, or variants thereof that have sequence identity to the genome of the raccoon poxvirus Herman strain (e.g., about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to the genome of the raccoon poxvirus Herman strain). The raccoon poxvirus strain Herman may also refer to a variant in which an amino acid residue has been mutated, thereby modulating (e.g., increasing or decreasing) the activity, expression, cell targeting, or infectivity of the raccoon poxvirus strain Herman. The raccoon poxvirus strain Herman can be modified as described herein. In an embodiment, the raccoon poxvirus strain Herman is identified by ATCC reference number ATCC VR-838. TM In an embodiment, the raccoon poxvirus Herman strain refers to the viral strain encoded by the nucleic acid sequence with reference number NC_027213.

[0094] The term "rabbit poxvirus Utrecht strain" is used according to its common and usual meaning and refers to the same or similarly named virus strain, as well as functional fragments and homologues thereof. The term encompasses recombinant or native rabbit poxvirus Utrecht strain, or variants thereof that maintain the activity of rabbit poxvirus Utrecht strain (e.g., within at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100%). The term encompasses recombinant or native rabbit poxvirus Utrecht strain, or variants thereof that have sequence identity to the genome of rabbit poxvirus Utrecht strain (e.g., about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity to the genome of rabbit poxvirus Utrecht strain). The rabbit poxvirus Utrecht strain may also refer to a mutant in which an amino acid residue has been mutated, thereby modulating (e.g., increasing or decreasing) the activity, expression, cell targeting, or infectivity of the rabbit poxvirus Utrecht strain compared to the rabbit poxvirus Utrecht strain. The rabbit poxvirus Utrecht strain can be modified as described herein. In an embodiment, the rabbit poxvirus Utrecht strain is designated by ATCC reference number ATCC VR-1591. TM In an embodiment, rabbit poxvirus Utrecht strain refers to the virus strain identified by Taxonomy reference number 45417, a mutant or homolog thereof.

[0095] The term "vaccinia virus strain WR" is used according to its common and usual meaning and refers to the same or similarly named virus strain, as well as functional fragments and homologues thereof. The term encompasses recombinant or native vaccinia virus strain WR, or variants thereof that maintain the activity of the vaccinia virus strain WR (e.g., within at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%). The term encompasses recombinant or native vaccinia virus strain WR, or variants thereof that have sequence identity to the genome of the vaccinia virus strain WR (e.g., about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to the genome of the vaccinia virus strain WR). The WR vaccinia virus strain may also refer to a mutant in which an amino acid residue has been mutated, thereby modulating (e.g., increasing or decreasing) the activity, expression, cell targeting, or infectivity of the WR vaccinia virus strain. The WR vaccinia virus strain can be modified as described herein. In an embodiment, the WR vaccinia virus strain is designated ATCC Reference No. ATCC VR-1354. TM In an embodiment, vaccinia virus strain WR refers to the virus strain identified by Taxonomy reference number 10254, a mutant or homolog thereof.

[0096] The term "vaccinia virus IHD strain" is used according to its common and usual meaning and refers to the same or similarly named virus strain, as well as functional fragments and homologues thereof. The term encompasses recombinant or naturally occurring vaccinia virus IHD strains, or variants thereof that maintain the activity of vaccinia virus IHD strains (e.g., within at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%). The term encompasses recombinant or naturally occurring vaccinia virus IHD strains, or variants thereof that have sequence identity to the genome of vaccinia virus IHD strains (e.g., about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to the genome of vaccinia virus IHD strains). Vaccinia virus strain IHD may also refer to a mutant in which an amino acid residue has been mutated, thereby modulating (e.g., increasing or decreasing) the activity, expression, cell targeting, or infectivity of the vaccinia virus strain IHD. The vaccinia virus strain IHD can be modified as described herein. In an embodiment, the vaccinia virus strain IHD is designated ATCC Reference No. ATCC VR-156. TM In an embodiment, vaccinia virus IHD strain refers to the virus strain identified by Taxonomy reference number 10251, its mutants or homologs thereof.

[0097] The term "Vaccinia virus strain Elstree" is used according to its common and usual meaning and refers to the same or similarly named virus strain, as well as functional fragments and homologues thereof. The term encompasses recombinant or native vaccinia virus strain Elstree, or variants thereof that maintain the activity of the vaccinia virus strain Elstree (e.g., within at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%). The term encompasses recombinant or native vaccinia virus strain Elstree, or variants thereof that have sequence identity to the genome of the vaccinia virus strain Elstree (e.g., about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to the genome of the vaccinia virus strain Elstree). The Elstree vaccinia virus strain may also refer to a mutant in which an amino acid residue has been mutated, thereby modulating (e.g., increasing or decreasing) the activity, expression, cell targeting, or infectivity of the Elstree vaccinia virus strain. The Elstree vaccinia virus strain can be modified as described herein. In an embodiment, the Elstree vaccinia virus strain is designated ATCC Reference No. ATCC VR-1549. TM It refers to a virus strain identified by a mutant or its homologue.

[0098] The term "vaccinia virus CL strain" is used according to its common and usual meaning and refers to the same or similarly named virus strain, as well as functional fragments and homologues thereof. The term encompasses recombinant or naturally occurring vaccinia virus CL strains, or variants thereof that maintain the activity of the vaccinia virus CL strain (e.g., within at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100%). The term encompasses recombinant or naturally occurring vaccinia virus CL strains, or variants thereof that have sequence identity to the genome of the vaccinia virus CL strain (e.g., about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity to the genome of the vaccinia virus CL strain). Vaccinia virus strain CL may also refer to a mutant in which an amino acid residue has been mutated, thereby modulating (e.g., increasing or decreasing) the activity, expression, cell targeting, or infectivity of the vaccinia virus strain CL. The vaccinia virus strain CL can be modified as described herein. In an embodiment, the vaccinia virus strain CL is designated ATCC Reference No. ATCC VR-1774. TM " refers to a virus strain identified by, or a mutant or homologue thereof.

[0099] The term "Lederle-Chorioallantoic vaccinia virus strain" is used according to its common and usual meaning and refers to the same or similarly named virus strains, as well as functional fragments and homologues thereof. The term encompasses recombinant or naturally occurring Lederle-Chorioallantoic vaccinia virus strains, or variants thereof that retain the activity of the Lederle-Chorioallantoic vaccinia virus strain (e.g., within at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100%). The term encompasses recombinant or naturally occurring vaccinia virus Lederle-Chorioallantoic strain or mutants having sequence identity to the genome of the vaccinia virus Lederle-Chorioallantoic strain (e.g., about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity to the genome of the vaccinia virus Lederle-Chorioallantoic strain). The vaccinia virus Lederle-Chorioallantoic strain may also refer to mutants with mutated amino acid residues, which modulate (e.g., increase or decrease) the activity, expression, cell targeting or infectivity of the vaccinia virus Lederle-Chorioallantoic strain. The vaccinia virus Lederle-Chorioallantoic strain can be modified as described herein. In an embodiment, the vaccinia virus Lederle-Chorioallantoic strain is designated ATCC reference number ATCC VR-118 TM " refers to a virus strain identified by, or a mutant or homologue thereof.

[0100] The term "vaccinia virus AS strain" is used according to its common and usual meaning and refers to the same or similarly named virus strain, as well as functional fragments and homologues thereof. The term encompasses recombinant or naturally occurring vaccinia virus AS strains, or mutants thereof that maintain the activity of the vaccinia virus AS strain (e.g., within at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100%). The term encompasses recombinant or naturally occurring vaccinia virus AS strains, or mutants thereof that have sequence identity to the genome of the vaccinia virus AS strain (e.g., about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity to the genome of the vaccinia virus AS strain). Vaccinia virus strain AS may also refer to a mutant in which an amino acid residue has been mutated, thereby modulating (e.g., increasing or decreasing) the activity, expression, cell targeting, or infectivity of the vaccinia virus strain AS compared to the vaccinia virus strain AS. The vaccinia virus strain AS can be modified as described herein. In an embodiment, the vaccinia virus strain AS is designated by ATCC reference number ATCC VR-2010. TM " refers to a virus strain identified by, or a mutant or homologue thereof.

[0101] The term "orf virus strain NZ2" is used according to its common and usual meaning and refers to the same or similarly named virus strains, as well as functional fragments and homologues thereof. The term encompasses recombinant or native orf virus strain NZ2, or variants thereof that maintain the activity of orf virus strain NZ2 (e.g., within at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%). The term encompasses recombinant or native orf virus strain NZ2, or variants thereof that have sequence identity to the genome of orf virus strain NZ2 (e.g., about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to the genome of orf virus strain NZ2). Orf virus strain NZ2 may also refer to a variant in which an amino acid residue has been mutated, thereby modulating (e.g., increasing or decreasing) the activity, expression, cell targeting, or infectivity of the orf virus strain NZ2. The orf virus strain NZ2 may be modified as described herein. In an embodiment, the orf virus strain NZ2 is designated by ATCC reference number ATCC VR-1548. TM Orf virus strain NZ2 refers to a virus strain identified by Taxonomy reference number 10259, a mutant or homolog thereof.

[0102] The term "pseudo bovine poxvirus TJS strain" is used according to its common and usual meaning and refers to the same or similarly named virus strain, as well as functional fragments and homologues thereof. The term encompasses recombinant or naturally occurring pseudo bovine poxvirus TJS strain, or variants thereof that maintain the activity of the pseudo bovine poxvirus TJS strain (e.g., within at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100%). The term encompasses recombinant or naturally occurring pseudo bovine poxvirus TJS strain, or variants thereof that have sequence identity to the genome of the pseudo bovine poxvirus TJS strain (e.g., about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% identity to the genome of the pseudo bovine poxvirus TJS strain). The pseudobovine poxvirus TJS strain may also refer to a mutant in which an amino acid residue has been mutated, thereby modulating (e.g., increasing or decreasing) the activity, expression, cell targeting, or infectivity of the pseudobovine poxvirus TJS strain compared to the pseudobovine poxvirus TJS strain. The pseudobovine poxvirus TJS strain can be modified as described herein. In an embodiment, the vaccinia virus strain WR is designated ATCC Reference No. ATCC VR-634. TM It refers to a virus strain identified by a mutant or its homologue.

[0103] In an embodiment, the bovine poxvirus Brighton strain is bovine poxvirus Brighton strain ATCC VR-302. TM In an embodiment, the raccoon poxvirus Herman strain is raccoon poxvirus Herman strain ATCC VR-838 TM In an embodiment, the rabbit poxvirus Utrecht strain is rabbit poxvirus Utrecht strain ATCC VR-1591. TM In an embodiment, the vaccinia virus strain WR is vaccinia virus strain WR ATCC VR-1354. TM In an embodiment, the vaccinia virus IHD strain is vaccinia virus IHD strain ATCC VR-156 TMIn an embodiment, the vaccinia virus Elstree strain is vaccinia virus Elstree strain ATCC VR-1549. TM In an embodiment, the vaccinia virus CL strain is vaccinia virus CL strain ATCC VR-1774. TM In an embodiment, the vaccinia virus Lederle-Chorioallantoic strain is vaccinia virus Lederle-Chorioallantoic strain ATCC VR-118. TM In an embodiment, the vaccinia virus strain AS is vaccinia virus strain AS ATCC VR-2010 TM In an embodiment, the orf virus NZ2 strain is orf virus NZ2 strain ATCC VR-1548 TM In an embodiment, the pseudobovine poxvirus TJS strain is the pseudobovine poxvirus TJS strain ATCC VR-634. TM In an embodiment, bovine poxvirus Brighton strain refers to the virus strain identified by Taxonomy reference number 265872, a mutant or homologue thereof.

[0104] In this disclosure, "comprises," "comprising," "containing," "having," and the like, have the meanings given to them in U.S. Patent Law and can mean "includes," "including," and the like. "Consisting essentially of" or "consists essentially of" likewise have the meanings given to them in U.S. Patent Law and are open-ended phrases permitting the presence of other elements than those recited, but excluding prior art embodiments, so long as the basic or novel characteristics of what is recited are not altered by the presence of other elements than those recited.

[0105] II. Viral Compositions In one aspect, a chimeric poxvirus is provided comprising a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:1 or SEQ ID NO:2, said nucleic acid sequence comprising nucleic acid fragments from at least two poxvirus strains selected from the group consisting of: Bovine poxvirus strain Brighton, Raccoon poxvirus strain Herman, Rabbit poxvirus strain Utrecht, Vaccinia virus strain WR, Vaccinia virus strain IHD, Vaccinia virus strain Elstree, Vaccinia virus strain CL, Vaccinia virus strain Lederle-Chorioallantoic, Vaccinia virus strain AS, Orf virus strain NZ2 and Pseudobovine poxvirus strain TJS.

[0106] The chimeric poxviruses described herein may comprise a transgene. As used herein, a "transgene" refers to a nucleic acid sequence derived from outside a given cell, organism, or virus. Thus, the transgenes provided herein are not naturally occurring or derived from inside the poxvirus. The transgenes provided herein may be protein-encoding or non-encoding nucleic acid sequences. The transgenes provided herein may comprise anti-cancer nucleic acid sequences (e.g., nucleic acid binding sequences and nucleic acid sequences encoding polypeptides useful for the treatment of cancer), or nucleic acid sequences encoding detectable moieties. Thus, in embodiments, the chimeric poxviruses described herein comprise one or more anti-cancer nucleic acid sequences or nucleic acid sequences encoding detectable moieties. In embodiments, the chimeric poxviruses described herein comprise one or more anti-cancer nucleic acid sequences and nucleic acid sequences encoding detectable moieties.

[0107] In an embodiment, the nucleic acid sequence has at least 71% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has at least 72% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has at least 73% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has at least 74% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has at least 75% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has at least 76% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has at least 77% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has at least 78% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has at least 79% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has at least 80% sequence identity to SEQ ID NO:1.

[0108] In an embodiment, the nucleic acid sequence has at least 81% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has at least 82% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has at least 83% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has at least 84% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has at least 85% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has at least 86% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has at least 87% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has at least 88% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has at least 89% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has at least 90% sequence identity to SEQ ID NO:1.

[0109] In an embodiment, the nucleic acid sequence has at least 91% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has at least 92% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has at least 93% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has at least 94% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has at least 95% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has at least 96% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has at least 97% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has at least 98% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has at least 99% sequence identity to SEQ ID NO:1.

[0110] In an embodiment, the nucleic acid sequence has at least 71% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has at least 72% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has at least 73% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has at least 74% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has at least 75% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has at least 76% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has at least 77% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has at least 78% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has at least 79% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has at least 80% sequence identity to SEQ ID NO:2.

[0111] In an embodiment, the nucleic acid sequence has at least 81% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has at least 82% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has at least 83% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has at least 84% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has at least 85% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has at least 86% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has at least 87% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has at least 88% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has at least 89% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has at least 90% sequence identity to SEQ ID NO:2.

[0112] In an embodiment, the nucleic acid sequence has at least 91% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has at least 92% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has at least 93% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has at least 94% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has at least 95% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has at least 96% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has at least 97% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has at least 98% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has at least 99% sequence identity to SEQ ID NO:2.

[0113] In an embodiment, the nucleic acid sequence has 71% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has 72% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has 73% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has 74% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has 75% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has 76% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has 77% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has 78% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has 79% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has 80% sequence identity to SEQ ID NO:1.

[0114] In an embodiment, the nucleic acid sequence has 81% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has 82% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has 83% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has 84% ​​sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has 85% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has 86% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has 87% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has 88% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has 89% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has 90% sequence identity to SEQ ID NO:1.

[0115] In an embodiment, the nucleic acid sequence has 91% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has 92% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has 93% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has 94% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has 95% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has 96% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has 97% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has 98% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has 99% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence is the sequence of SEQ ID NO:1.

[0116] In an embodiment, the nucleic acid sequence has 71% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has 72% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has 73% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has 74% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has 75% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has 76% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has 77% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has 78% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has 79% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has 80% sequence identity to SEQ ID NO:2.

[0117] In an embodiment, the nucleic acid sequence has 81% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has 82% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has 83% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has 84% ​​sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has 85% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has 86% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has 87% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has 88% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has 89% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has 90% sequence identity to SEQ ID NO:2.

[0118] In an embodiment, the nucleic acid sequence has 91% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has 92% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has 93% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has 94% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has 95% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has 96% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has 97% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has 98% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has 99% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence is the sequence of SEQ ID NO:1.

[0119] In an embodiment, the nucleic acid sequence has about 71% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has about 72% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has about 73% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has about 74% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has about 75% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has about 76% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has about 77% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has about 78% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has about 79% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has about 80% sequence identity to SEQ ID NO:1.

[0120] In an embodiment, the nucleic acid sequence has about 81% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has about 82% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has about 83% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has about 84% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has about 85% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has about 86% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has about 87% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has about 88% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has about 89% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has about 90% sequence identity to SEQ ID NO:1.

[0121] In an embodiment, the nucleic acid sequence has about 91% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has about 92% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has about 93% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has about 94% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has about 95% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has about 96% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has about 97% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has about 98% sequence identity to SEQ ID NO:1. In an embodiment, the nucleic acid sequence has about 99% sequence identity to SEQ ID NO:1.

[0122] In an embodiment, the nucleic acid sequence has about 71% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has about 72% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has about 73% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has about 74% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has about 75% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has about 76% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has about 77% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has about 78% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has about 79% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has about 80% sequence identity to SEQ ID NO:2.

[0123] In an embodiment, the nucleic acid sequence has about 81% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has about 82% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has about 83% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has about 84% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has about 85% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has about 86% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has about 87% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has about 88% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has about 89% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has about 90% sequence identity to SEQ ID NO:2.

[0124] In an embodiment, the nucleic acid sequence has about 91% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has about 92% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has about 93% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has about 94% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has about 95% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has about 96% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has about 97% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has about 98% sequence identity to SEQ ID NO:2. In an embodiment, the nucleic acid sequence has about 99% sequence identity to SEQ ID NO:2.

[0125] The nucleic acid sequence has at least 70% sequence identity, and the nucleic acid sequence having at least 70% sequence identity may be contiguous. In an embodiment, the nucleic acid sequence has at least 70% sequence identity, and the nucleic acid sequence having at least 70% sequence identity may be a non-contiguous sequence. As provided herein, a "non-contiguous sequence" refers to a sequence that includes one or more sequence fragments that do not have sequence identity to SEQ ID NO:1 or SEQ ID NO:2. In an embodiment, the non-contiguous sequence is a sequence that includes a first sequence fragment that has at least 70% sequence identity to SEQ ID NO:1 or SEQ ID NO:2, and a second sequence fragment that has at least 70% sequence identity to SEQ ID NO:1 or SEQ ID NO:2, linked via a sequence fragment that does not have sequence identity to SEQ ID NO:1 or SEQ ID NO:2. In an embodiment, the non-contiguous sequence is a sequence that includes multiple sequence fragments that have at least 70% sequence identity to SEQ ID NO:1 or SEQ ID NO:2, linked by multiple sequence fragments that do not have sequence identity to SEQ ID NO:1 or SEQ ID NO:2. In an embodiment, the chimeric poxvirus further comprises a nucleotide insertion, deletion or mutation.

[0126] In embodiments, the nucleic acid fragment is derived from the bovine poxvirus strain Brighton, the raccoon poxvirus strain Herman, the rabbit poxvirus strain Utrecht, the vaccinia virus strain WR, the vaccinia virus strain IHD, the vaccinia virus strain Elstree, the vaccinia virus strain CL, the vaccinia virus strain Lederle-Chorioallantoic, and the vaccinia virus strain AS.

[0127] In an embodiment, the nucleic acid sequence comprises a nucleic acid fragment from the Brighton strain of bovine poxvirus and the Herman strain of raccoon poxvirus. In an embodiment, the nucleic acid sequence comprises a nucleic acid fragment from the Brighton strain of bovine poxvirus and the Utrecht strain of rabbit poxvirus. In an embodiment, the nucleic acid sequence comprises a nucleic acid fragment from the Brighton strain of bovine poxvirus and the WR strain of vaccinia virus. In an embodiment, the nucleic acid sequence comprises a nucleic acid fragment from the Brighton strain of bovine poxvirus and the IHD strain of vaccinia virus. In an embodiment, the nucleic acid sequence comprises a nucleic acid fragment from the Brighton strain of bovine poxvirus and the Elstree strain of vaccinia virus. In an embodiment, the nucleic acid sequence comprises a nucleic acid fragment from the Brighton strain of bovine poxvirus and the CL strain of vaccinia virus. In an embodiment, the nucleic acid sequence comprises a nucleic acid fragment from the Brighton strain of bovine poxvirus and the Lederle-Chorioallantoic strain of vaccinia virus. In an embodiment, the nucleic acid sequence comprises a nucleic acid fragment from the Brighton strain of bovine poxvirus and the AS strain of vaccinia virus. In an embodiment, the nucleic acid sequence comprises a nucleic acid fragment from the bovine poxvirus strain Brighton and the orf virus strain NZ2.In an embodiment, the nucleic acid sequence comprises a nucleic acid fragment from the bovine poxvirus strain Brighton and the pseudobovine poxvirus strain TJS.

[0128] In an embodiment, the nucleic acid sequence comprises a nucleic acid fragment from the rabbit poxvirus Utrecht strain and the vaccinia virus WR strain. In an embodiment, the nucleic acid sequence comprises a nucleic acid fragment from the rabbit poxvirus Utrecht strain and the vaccinia virus IHD strain. In an embodiment, the nucleic acid sequence comprises a nucleic acid fragment from the rabbit poxvirus Utrecht strain and the vaccinia virus Elstree strain. In an embodiment, the nucleic acid sequence comprises a nucleic acid fragment from the rabbit poxvirus Utrecht strain and the vaccinia virus CL strain. In an embodiment, the nucleic acid sequence comprises a nucleic acid fragment from the rabbit poxvirus Utrecht strain and the vaccinia virus Lederle-Chorioallantoic strain. In an embodiment, the nucleic acid sequence comprises a nucleic acid fragment from the rabbit poxvirus Utrecht strain and the vaccinia virus AS strain. In an embodiment, the nucleic acid sequence comprises a nucleic acid fragment from the rabbit poxvirus Utrecht strain and the vaccinia virus NZ2 strain. In an embodiment, the nucleic acid sequence comprises a nucleic acid fragment from the rabbit poxvirus Utrecht strain and the vaccinia virus TJS strain.

[0129] In an embodiment, the nucleic acid sequence comprises a nucleic acid fragment from the WR strain of vaccinia virus and the IHD strain of vaccinia virus. In an embodiment, the nucleic acid sequence comprises a nucleic acid fragment from the WR strain of vaccinia virus and the Elstree strain of vaccinia virus. In an embodiment, the nucleic acid sequence comprises a nucleic acid fragment from the WR strain of vaccinia virus and the CL strain of vaccinia virus. In an embodiment, the nucleic acid sequence comprises a nucleic acid fragment from the WR strain of vaccinia virus and the Lederle-Chorioallantoic strain of vaccinia virus. In an embodiment, the nucleic acid sequence comprises a nucleic acid fragment from the WR strain of vaccinia virus and the AS strain of vaccinia virus. In an embodiment, the nucleic acid sequence comprises a nucleic acid fragment from the WR strain of vaccinia virus and the NZ2 strain of orf virus. In an embodiment, the nucleic acid sequence comprises a nucleic acid fragment from the WR strain of vaccinia virus and the TJS strain of pseudobovine poxvirus.

[0130] In an embodiment, the nucleic acid sequence comprises a nucleic acid fragment from the vaccinia virus IHD strain and the vaccinia virus Elstree strain. In an embodiment, the nucleic acid sequence comprises a nucleic acid fragment from the vaccinia virus IHD strain and the vaccinia virus CL strain. In an embodiment, the nucleic acid sequence comprises a nucleic acid fragment from the vaccinia virus IHD strain and the vaccinia virus Lederle-Chorioallantoic strain. In an embodiment, the nucleic acid sequence comprises a nucleic acid fragment from the vaccinia virus IHD strain and the vaccinia virus AS strain. In an embodiment, the nucleic acid sequence comprises a nucleic acid fragment from the vaccinia virus IHD strain and the orf virus NZ2 strain. In an embodiment, the nucleic acid sequence comprises a nucleic acid fragment from the vaccinia virus IHD strain and the pseudobovine poxvirus TJS strain.

[0131] In an embodiment, the nucleic acid sequence comprises a nucleic acid fragment from the Elstree strain of vaccinia virus and the CL strain of vaccinia virus. In an embodiment, the nucleic acid sequence comprises a nucleic acid fragment from the Elstree strain of vaccinia virus and the Lederle-Chorioallantoic strain of vaccinia virus. In an embodiment, the nucleic acid sequence comprises a nucleic acid fragment from the Elstree strain of vaccinia virus and the AS strain of vaccinia virus. In an embodiment, the nucleic acid sequence comprises a nucleic acid fragment from the Elstree strain of vaccinia virus and the NZ2 strain of orf virus. In an embodiment, the nucleic acid sequence comprises a nucleic acid fragment from the Elstree strain of vaccinia virus and the TJS strain of pseudobovine poxvirus.

[0132] In an embodiment, the nucleic acid sequence comprises a nucleic acid fragment from the CL strain of vaccinia virus and the Lederle-Chorioallantoic strain of vaccinia virus. In an embodiment, the nucleic acid sequence comprises a nucleic acid fragment from the CL strain of vaccinia virus and the AS strain of vaccinia virus. In an embodiment, the nucleic acid sequence comprises a nucleic acid fragment from the CL strain of vaccinia virus and the NZ2 strain of orf virus. In an embodiment, the nucleic acid sequence comprises a nucleic acid fragment from the CL strain of vaccinia virus and the TJS strain of pseudobovine poxvirus.

[0133] In an embodiment, the nucleic acid sequence comprises a nucleic acid fragment from the Lederle-Chorioallantoic vaccinia virus strain and the AS vaccinia virus strain. In an embodiment, the nucleic acid sequence comprises a nucleic acid fragment from the Lederle-Chorioallantoic vaccinia virus strain and the NZ2 orf virus strain. In an embodiment, the nucleic acid sequence comprises a nucleic acid fragment from the Lederle-Chorioallantoic vaccinia virus strain and the TJS pseudobovine poxvirus strain.

[0134] In an embodiment, the nucleic acid sequence comprises a nucleic acid fragment from the vaccinia virus strain AS and the orf virus strain NZ2. In an embodiment, the nucleic acid sequence comprises a nucleic acid fragment from the vaccinia virus strain AS and the pseudobovine poxvirus strain TJS. In an embodiment, the nucleic acid sequence comprises a nucleic acid fragment from the orf virus strain NZ2 and the pseudobovine poxvirus strain TJS.

[0135] In an embodiment, the chimeric poxvirus comprises a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO: 1, the nucleic acid sequence comprising a nucleic acid fragment from bovine poxvirus strain Brighton, raccoon poxvirus strain Herman, rabbit poxvirus strain Utrecht, vaccinia virus strain WR, vaccinia virus strain IHD, vaccinia virus strain Elstree, vaccinia virus strain CL, vaccinia virus strain Lederle-Chorioallantoic, and vaccinia virus strain AS. In an embodiment, the chimeric poxvirus comprises a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO: 1, the nucleic acid sequence comprising a nucleic acid fragment from bovine poxvirus strain Brighton, raccoon poxvirus strain Herman, rabbit poxvirus strain Utrecht, vaccinia virus strain WR, vaccinia virus strain IHD, vaccinia virus strain Elstree, vaccinia virus strain CL, vaccinia virus strain Lederle-Chorioallantoic, or vaccinia virus strain AS.

[0136] In an embodiment, the chimeric poxvirus comprises a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:1, the nucleic acid sequence comprises a nucleic acid fragment from the bovine poxvirus Brighton strain. In an embodiment, the chimeric poxvirus comprises a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:1, the nucleic acid sequence comprises a nucleic acid fragment from the raccoon poxvirus Herman strain. In an embodiment, the chimeric poxvirus comprises a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:1, the nucleic acid sequence comprises a nucleic acid fragment from the rabbit poxvirus Utrecht strain. In an embodiment, the chimeric poxvirus comprises a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:1, the nucleic acid sequence comprises a nucleic acid fragment from the vaccinia virus WR strain. In an embodiment, the chimeric poxvirus comprises a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:1, the nucleic acid sequence comprises a nucleic acid fragment from the vaccinia virus IHD strain. In an embodiment, the chimeric poxvirus comprises a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:1, wherein the nucleic acid sequence comprises a nucleic acid fragment derived from the Elstree strain of vaccinia virus. In an embodiment, the chimeric poxvirus comprises a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:1, wherein the nucleic acid sequence comprises a nucleic acid fragment derived from the CL strain of vaccinia virus. In an embodiment, the chimeric poxvirus comprises a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:1, wherein the nucleic acid sequence comprises a nucleic acid fragment derived from the Lederle-Chorioallantoic strain of vaccinia virus or the AS strain of vaccinia virus.

[0137] In an embodiment, the chimeric poxvirus comprises a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:2, the nucleic acid sequence comprising a nucleic acid fragment from the orf virus strain NZ2 and the pseudobovine poxvirus strain TJS. In an embodiment, the chimeric poxvirus comprises a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:2, the nucleic acid sequence comprising a nucleic acid fragment from the orf virus strain NZ2. In an embodiment, the chimeric poxvirus comprises a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:2, the nucleic acid sequence comprising a nucleic acid fragment from the pseudobovine poxvirus strain TJS.

[0138] In an embodiment, the chimeric poxvirus comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 1, the nucleic acid sequence comprising a nucleic acid fragment from bovine poxvirus strain Brighton, raccoon poxvirus strain Herman, rabbit poxvirus strain Utrecht, vaccinia virus strain WR, vaccinia virus strain IHD, vaccinia virus strain Elstree, vaccinia virus strain CL, vaccinia virus strain Lederle-Chorioallantoic, and vaccinia virus strain AS. In an embodiment, the chimeric poxvirus comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 1, the nucleic acid sequence comprising a nucleic acid fragment from bovine poxvirus strain Brighton, raccoon poxvirus strain Herman, rabbit poxvirus strain Utrecht, vaccinia virus strain WR, vaccinia virus strain IHD, vaccinia virus strain Elstree, vaccinia virus strain CL, vaccinia virus strain Lederle-Chorioallantoic, or vaccinia virus strain AS.

[0139] In an embodiment, the chimeric poxvirus comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO:1, the nucleic acid sequence comprises a nucleic acid fragment from the bovine poxvirus Brighton strain. In an embodiment, the chimeric poxvirus comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO:1, the nucleic acid sequence comprises a nucleic acid fragment from the raccoon poxvirus Herman strain. In an embodiment, the chimeric poxvirus comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO:1, the nucleic acid sequence comprises a nucleic acid fragment from the rabbit poxvirus Utrecht strain. In an embodiment, the chimeric poxvirus comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO:1, the nucleic acid sequence comprises a nucleic acid fragment from the vaccinia virus WR strain. In an embodiment, the chimeric poxvirus comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO:1, the nucleic acid sequence comprises a nucleic acid fragment from the vaccinia virus IHD strain. In an embodiment, the chimeric poxvirus comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO:1, wherein the nucleic acid sequence comprises a nucleic acid fragment derived from the Elstree strain of vaccinia virus. In an embodiment, the chimeric poxvirus comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO:1, wherein the nucleic acid sequence comprises a nucleic acid fragment derived from the CL strain of vaccinia virus. In an embodiment, the chimeric poxvirus comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO:1, wherein the nucleic acid sequence comprises a nucleic acid fragment derived from the Lederle-Chorioallantoic strain of vaccinia virus or the AS strain of vaccinia virus.

[0140] In an embodiment, the chimeric poxvirus comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO:2, the nucleic acid sequence comprising a nucleic acid fragment from the orf virus strain NZ2 and the pseudobovine poxvirus strain TJS. In an embodiment, the chimeric poxvirus comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO:2, the nucleic acid sequence comprising a nucleic acid fragment from the orf virus strain NZ2. In an embodiment, the chimeric poxvirus comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO:2, the nucleic acid sequence comprising a nucleic acid fragment from the pseudobovine poxvirus strain TJS.

[0141] In an embodiment, the chimeric poxvirus comprises a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 1, the nucleic acid sequence comprising a nucleic acid fragment from bovine poxvirus strain Brighton, raccoon poxvirus strain Herman, rabbit poxvirus strain Utrecht, vaccinia virus strain WR, vaccinia virus strain IHD, vaccinia virus strain Elstree, vaccinia virus strain CL, vaccinia virus strain Lederle-Chorioallantoic, and vaccinia virus strain AS. In an embodiment, the chimeric poxvirus comprises a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 1, the nucleic acid sequence comprising a nucleic acid fragment from bovine poxvirus strain Brighton, raccoon poxvirus strain Herman, rabbit poxvirus strain Utrecht, vaccinia virus strain WR, vaccinia virus strain IHD, vaccinia virus strain Elstree, vaccinia virus strain CL, vaccinia virus strain Lederle-Chorioallantoic, or vaccinia virus strain AS.

[0142] In an embodiment, the chimeric poxvirus comprises a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO:1, the nucleic acid sequence comprises a nucleic acid fragment from the bovine poxvirus Brighton strain. In an embodiment, the chimeric poxvirus comprises a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO:1, the nucleic acid sequence comprises a nucleic acid fragment from the raccoon poxvirus Herman strain. In an embodiment, the chimeric poxvirus comprises a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO:1, the nucleic acid sequence comprises a nucleic acid fragment from the rabbit poxvirus Utrecht strain. In an embodiment, the chimeric poxvirus comprises a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO:1, the nucleic acid sequence comprises a nucleic acid fragment from the vaccinia virus WR strain. In an embodiment, the chimeric poxvirus comprises a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO:1, the nucleic acid sequence comprises a nucleic acid fragment from the vaccinia virus IHD strain. In an embodiment, the chimeric poxvirus comprises a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO:1, wherein the nucleic acid sequence comprises a nucleic acid fragment derived from the Elstree strain of vaccinia virus. In an embodiment, the chimeric poxvirus comprises a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO:1, wherein the nucleic acid sequence comprises a nucleic acid fragment derived from the CL strain of vaccinia virus. In an embodiment, the chimeric poxvirus comprises a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO:1, wherein the nucleic acid sequence comprises a nucleic acid fragment derived from the Lederle-Chorioallantoic strain of vaccinia virus or the AS strain of vaccinia virus.

[0143] In an embodiment, the chimeric poxvirus comprises a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO:2, the nucleic acid sequence comprising a nucleic acid fragment from the orf virus strain NZ2 and the pseudobovine poxvirus strain TJS. In an embodiment, the chimeric poxvirus comprises a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO:2, the nucleic acid sequence comprising a nucleic acid fragment from the orf virus strain NZ2. In an embodiment, the chimeric poxvirus comprises a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO:2, the nucleic acid sequence comprising a nucleic acid fragment from the pseudobovine poxvirus strain TJS.

[0144] In an embodiment, the chimeric poxvirus comprises a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 1, the nucleic acid sequence comprises a nucleic acid fragment from bovine poxvirus strain Brighton, raccoon poxvirus strain Herman, rabbit poxvirus strain Utrecht, vaccinia virus strain WR, vaccinia virus strain IHD, vaccinia virus strain Elstree, vaccinia virus strain CL, vaccinia virus strain Lederle-Chorioallantoic, and vaccinia virus strain AS. In an embodiment, the chimeric poxvirus comprises a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 1, the nucleic acid sequence comprises a nucleic acid fragment from bovine poxvirus strain Brighton, raccoon poxvirus strain Herman, rabbit poxvirus strain Utrecht, vaccinia virus strain WR, vaccinia virus strain IHD, vaccinia virus strain Elstree, vaccinia virus strain CL, vaccinia virus strain Lederle-Chorioallantoic, or vaccinia virus strain AS.

[0145] In an embodiment, the chimeric poxvirus comprises a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 1, the nucleic acid sequence comprising a nucleic acid fragment from the bovine poxvirus Brighton strain, the raccoon poxvirus Herman strain, the rabbit poxvirus Utrecht strain, the vaccinia virus WR strain, the vaccinia virus IHD strain, the vaccinia virus Elstree strain, the vaccinia virus CL strain, the vaccinia virus Lederle-Chorioallantoic strain, or the vaccinia virus AS strain. In an embodiment, the chimeric poxvirus comprises a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 1, the nucleic acid sequence comprising a nucleic acid fragment from the bovine poxvirus Brighton strain, the raccoon poxvirus Herman strain, the rabbit poxvirus Utrecht strain, the vaccinia virus WR strain, the vaccinia virus IHD strain, the vaccinia virus Elstree strain, the vaccinia virus CL strain, the vaccinia virus Lederle-Chorioallantoic strain, or the vaccinia virus AS strain.

[0146] In an embodiment, the chimeric poxvirus comprises a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO:1, the nucleic acid sequence comprises a nucleic acid fragment from the bovine poxvirus Brighton strain. In an embodiment, the chimeric poxvirus comprises a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO:1, the nucleic acid sequence comprises a nucleic acid fragment from the raccoon poxvirus Herman strain. In an embodiment, the chimeric poxvirus comprises a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO:1, the nucleic acid sequence comprises a nucleic acid fragment from the rabbit poxvirus Utrecht strain. In an embodiment, the chimeric poxvirus comprises a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO:1, the nucleic acid sequence comprises a nucleic acid fragment from the vaccinia virus WR strain. In an embodiment, the chimeric poxvirus comprises a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO:1, the nucleic acid sequence comprises a nucleic acid fragment from the vaccinia virus IHD strain. In an embodiment, the chimeric poxvirus comprises a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO:1, wherein the nucleic acid sequence comprises a nucleic acid fragment derived from the Elstree strain of vaccinia virus. In an embodiment, the chimeric poxvirus comprises a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO:1, wherein the nucleic acid sequence comprises a nucleic acid fragment derived from the CL strain of vaccinia virus. In an embodiment, the chimeric poxvirus comprises a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO:1, wherein the nucleic acid sequence comprises a nucleic acid fragment derived from the Lederle-Chorioallantoic strain of vaccinia virus or the AS strain of vaccinia virus.

[0147] In an embodiment, the chimeric poxvirus comprises a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO:2, the nucleic acid sequence comprising a nucleic acid fragment from the orf virus strain NZ2 and the pseudobovine poxvirus strain TJS. In an embodiment, the chimeric poxvirus comprises a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO:2, the nucleic acid sequence comprising a nucleic acid fragment from the orf virus strain NZ2. In an embodiment, the chimeric poxvirus comprises a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO:2, the nucleic acid sequence comprising a nucleic acid fragment from the pseudobovine poxvirus strain TJS.

[0148] In an embodiment, the chimeric poxvirus comprises a nucleic acid sequence having at least 98% sequence identity to SEQ ID NO: 1, the nucleic acid sequence comprising a nucleic acid fragment from bovine poxvirus strain Brighton, raccoon poxvirus strain Herman, rabbit poxvirus strain Utrecht, vaccinia virus strain WR, vaccinia virus strain IHD, vaccinia virus strain Elstree, vaccinia virus strain CL, vaccinia virus strain Lederle-Chorioallantoic, and vaccinia virus strain AS. In an embodiment, the chimeric poxvirus comprises a nucleic acid sequence having at least 98% sequence identity to SEQ ID NO: 1, the nucleic acid sequence comprising a nucleic acid fragment from bovine poxvirus strain Brighton, raccoon poxvirus strain Herman, rabbit poxvirus strain Utrecht, vaccinia virus strain WR, vaccinia virus strain IHD, vaccinia virus strain Elstree, vaccinia virus strain CL, vaccinia virus strain Lederle-Chorioallantoic, or vaccinia virus strain AS.

[0149] In an embodiment, the chimeric poxvirus comprises a nucleic acid sequence having at least 98% sequence identity to SEQ ID NO:1, the nucleic acid sequence comprises a nucleic acid fragment from the bovine poxvirus Brighton strain. In an embodiment, the chimeric poxvirus comprises a nucleic acid sequence having at least 98% sequence identity to SEQ ID NO:1, the nucleic acid sequence comprises a nucleic acid fragment from the raccoon poxvirus Herman strain. In an embodiment, the chimeric poxvirus comprises a nucleic acid sequence having at least 98% sequence identity to SEQ ID NO:1, the nucleic acid sequence comprises a nucleic acid fragment from the rabbit poxvirus Utrecht strain. In an embodiment, the chimeric poxvirus comprises a nucleic acid sequence having at least 98% sequence identity to SEQ ID NO:1, the nucleic acid sequence comprises a nucleic acid fragment from the vaccinia virus WR strain. In an embodiment, the chimeric poxvirus comprises a nucleic acid sequence having at least 98% sequence identity to SEQ ID NO:1, the nucleic acid sequence comprises a nucleic acid fragment from the vaccinia virus IHD strain. In an embodiment, the chimeric poxvirus comprises a nucleic acid sequence having at least 98% sequence identity to SEQ ID NO:1, wherein the nucleic acid sequence comprises a nucleic acid fragment derived from the Elstree strain of vaccinia virus. In an embodiment, the chimeric poxvirus comprises a nucleic acid sequence having at least 98% sequence identity to SEQ ID NO:1, wherein the nucleic acid sequence comprises a nucleic acid fragment derived from the CL strain of vaccinia virus. In an embodiment, the chimeric poxvirus comprises a nucleic acid sequence having at least 98% sequence identity to SEQ ID NO:1, wherein the nucleic acid sequence comprises a nucleic acid fragment derived from the Lederle-Chorioallantoic strain of vaccinia virus or the AS strain of vaccinia virus.

[0150] In an embodiment, the chimeric poxvirus comprises a nucleic acid sequence having at least 98% sequence identity to SEQ ID NO:2, the nucleic acid sequence comprising a nucleic acid fragment from the orf virus strain NZ2 and the pseudobovine poxvirus strain TJS. In an embodiment, the chimeric poxvirus comprises a nucleic acid sequence having at least 98% sequence identity to SEQ ID NO:2, the nucleic acid sequence comprising a nucleic acid fragment from the orf virus strain NZ2. In an embodiment, the chimeric poxvirus comprises a nucleic acid sequence having at least 98% sequence identity to SEQ ID NO:2, the nucleic acid sequence comprising a nucleic acid fragment from the pseudobovine poxvirus strain TJS.

[0151] In an embodiment, the chimeric poxvirus is an oncolytic virus. An oncolytic virus as used herein is a virus that can target and eliminate cancer cells. In an embodiment, the oncolytic virus targets lung cancer cells. In an embodiment, the oncolytic virus targets ovarian cancer cells. In an embodiment, the oncolytic virus targets pancreatic cancer cells. In an embodiment, the oncolytic virus preferentially targets cancer cells relative to non-cancerous cells.

[0152] In an embodiment, the miRNA binding sequence forms part of the DNA polymerase gene of the chimeric poxvirus. In an embodiment, the poxvirus comprises a miRNA binding sequence. In an embodiment, the poxvirus comprises a plurality of miRNA binding sequences. In an embodiment, the plurality of miRNA binding sequences are independently different. In an embodiment, the plurality of miRNA binding sequences are identical. In an embodiment, the miRNA binding sequence is about 22 nucleotides in length. In an embodiment, the miRNA binding sequence is at least 22 nucleotides in length. In an embodiment, the miRNA binding sequence is about 22 nucleotides in length. In an embodiment, the plurality of miRNA binding sequences are each at least 22 nucleotides in length. In an embodiment, the plurality of miRNA binding sequences are each about 22 nucleotides in length. In an embodiment, the plurality of miRNA binding sequences are each 22 nucleotides in length.

[0153] In one aspect, an isolated nucleic acid is provided that encodes the chimeric poxvirus described herein.In an embodiment, the isolated nucleic acid is the nucleic acid sequence of SEQ ID NO:1.In an embodiment, the isolated nucleic acid is the nucleic acid sequence of SEQ ID NO:2.

[0154] III. Viral Compositions Containing Transgenes The chimeric poxvirus provided herein may, in its embodiment, comprise a transgene. The transgene contained in the chimeric poxvirus provided herein can enhance the oncolytic activity of the chimeric poxvirus compared to the chimeric poxvirus lacking the transgene. The transgene can further enhance the ability of the chimeric poxvirus to differentially express / replicate in cancer cells compared to healthy (non-cancerous) cells. When the chimeric poxvirus comprises a transgene, the nucleic acid of the chimeric poxvirus comprises an anti-cancer nucleic acid sequence, a nucleic acid binding sequence, a nucleic acid sequence encoding a detectable moiety, or any combination thereof. Thus, in one aspect, there is provided a chimeric poxvirus comprising a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:1 or SEQ ID NO:2, said nucleic acid sequence comprising: (i) a nucleic acid fragment from at least two poxvirus strains selected from the group consisting of: Bovine poxvirus strain Brighton, Raccoon poxvirus strain Herman, Rabbit poxvirus strain Utrecht, Vaccinia virus strain WR, Vaccinia virus strain IHD, Vaccinia virus strain Elstree, Vaccinia virus strain CL, Vaccinia virus strain Lederle-Chorioallantoic, Vaccinia virus strain AS, Orf virus strain NZ2 and Pseudo bovine poxvirus strain TJS, (ii) one or more anti-cancer nucleic acid sequences, or (iii) a nucleic acid sequence encoding a detectable moiety.

[0155] In one aspect, a chimeric poxvirus is provided comprising a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:1, the nucleic acid sequence comprising: (i) a nucleic acid fragment from Bovine poxvirus strain Brighton, Raccoon poxvirus strain Herman, Rabbit poxvirus strain Utrecht, Vaccinia virus strain WR, Vaccinia virus strain IHD, Vaccinia virus strain Elstree, Vaccinia virus strain CL, Vaccinia virus strain Lederle-Chorioallantoic, and Vaccinia virus strain AS, (ii) one or more anti-cancer nucleic acid sequences, or (iii) a nucleic acid sequence encoding a detectable moiety.

[0156] In one aspect, a chimeric poxvirus is provided comprising a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:2, the nucleic acid sequence comprising: (i) a nucleic acid fragment derived from the orf virus strain NZ2 and the pseudobovine poxvirus strain TJS; (ii) one or more anti-cancer nucleic acid sequences; or (iii) a nucleic acid sequence encoding a detectable moiety.

[0157] In one aspect, a chimeric poxvirus is provided comprising a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:3, the nucleic acid sequence comprising: (i) a nucleic acid fragment from bovine poxvirus strain Brighton, raccoon poxvirus strain Herman, rabbit poxvirus strain Utrecht, vaccinia virus strain WR, vaccinia virus strain IHD, vaccinia virus strain Elstree, vaccinia virus strain CL, vaccinia virus strain Lederle-Chorioallantoic, and vaccinia virus strain AS, (ii) one or more anti-cancer nucleic acid sequences, or (iii) a nucleic acid sequence encoding a detectable moiety.

[0158] As used herein, the term "anti-cancer nucleic acid sequence" or "anti-cancer nucleic acid sequence" refers to a nucleic acid sequence having anti-neoplastic properties and / or the ability to inhibit the growth or proliferation of cancer cells and / or to selectively express the chimeric poxvirus according to embodiments provided herein in cancer cells compared to healthy cells. The anti-cancer nucleic acid sequence may inhibit the progression of cancer or slow down the progression temporarily or permanently. Examples of anti-cancer nucleic acid sequences include sequences that encode proteins whose expression inhibits cancer cell proliferation directly or indirectly. For example, the anti-cancer nucleic acid sequence provided herein may encode a protein (e.g., a sodium iodide transporter) that is highly expressed in cancer cells compared to healthy cells. In another non-limiting example, the anti-cancer nucleic acid sequence may encode a polypeptide (antibody) that can de-inhibit the anti-tumor immune response (e.g., an anti-PD-L1 antibody or a fragment thereof). In an embodiment, the anti-cancer nucleic acid sequence comprises a nucleic acid sequence that can enhance the expression / replication of the chimeric poxvirus in cancer cells compared to healthy cells. Thus, in an embodiment, the expression (e.g., transcription, translation) rate of the chimeric poxvirus comprising the anti-cancer nucleic acid sequence is lower in healthy cells compared to cancer cells. In an embodiment, the chimeric poxvirus comprising the anti-cancer nucleic acid sequence is not expressed in detectable amounts in healthy cells. In an embodiment, the anti-cancer nucleic acid sequence is a nucleic acid binding sequence. In an embodiment, the anti-cancer nucleic acid sequence comprises a nucleic acid binding sequence.

[0159] As used herein, the term "nucleic acid binding sequence" refers to a nucleic acid sequence that can at least partially bind (hybridize) with a complementary nucleic acid (e.g., DNA, RNA, miRNA) in a cell, and the amount of nucleic acid in the cell is higher in healthy cells than in cancer cells. The nucleic acid binding sequence provided herein may be a part of the nucleic acid carried by the chimeric poxvirus, or may be operably linked to a gene of the chimeric poxvirus. Binding of the nucleic acid in the cell to the nucleic acid binding sequence targets the chimeric poxvirus gene for degradation (hydrolysis), thereby decreasing the expression / replication of the chimeric poxvirus. In an embodiment, the nucleic acid binding sequence is a DNA binding sequence. In an embodiment, the nucleic acid binding sequence is an RNA binding sequence. In an embodiment, the nucleic acid binding sequence is an miRNA binding sequence. Thus, in an embodiment, the anti-cancer nucleic acid sequence is a nucleic acid binding sequence. In an embodiment, the anti-cancer nucleic acid sequence is a DNA binding sequence. In an embodiment, the anti-cancer nucleic acid sequence is an RNA binding sequence. In an embodiment, the anti-cancer nucleic acid sequence is an miRNA binding sequence.

[0160] As used herein, a "nucleic acid sequence encoding a detectable moiety" refers to a nucleic acid sequence that encodes a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. The nucleic acid sequence encoding the detectable moiety may also encode a fluorescent moiety. Non-limiting examples of fluorescent moieties are mCherry, Emerald, and firefly luciferase.

[0161] In one aspect, a chimeric poxvirus is provided comprising a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:1 or SEQ ID NO:2, the nucleic acid sequence comprising: (i) a nucleic acid fragment from at least two poxvirus strains selected from the group consisting of: Bovine poxvirus strain Brighton, Raccoon poxvirus strain Herman, Rabbit poxvirus strain Utrecht, Vaccinia virus strain WR, Vaccinia virus strain IHD, Vaccinia virus strain Elstree, Vaccinia virus strain CL, Vaccinia virus strain Lederle-Chorioallantoic, Vaccinia virus strain AS, Orf virus strain NZ2, and Pseudobovine poxvirus strain TJS, (ii) one or more anti-cancer nucleic acid sequences, (iii) one or more nucleic acid binding sequences, or (iv) a nucleic acid sequence encoding a detectable moiety.

[0162] In another aspect, a chimeric poxvirus is provided comprising a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:1, the nucleic acid sequence comprising: (i) a nucleic acid fragment from bovine poxvirus strain Brighton, raccoon poxvirus strain Herman, rabbit poxvirus strain Utrecht, vaccinia virus strain WR, vaccinia virus strain IHD, vaccinia virus strain Elstree, vaccinia virus strain CL, vaccinia virus strain Lederle-Chorioallantoic, and vaccinia virus strain AS, (ii) one or more anti-cancer nucleic acid sequences, (iii) one or more nucleic acid binding sequences, or (iv) a nucleic acid sequence encoding a detectable moiety.

[0163] In another aspect, a chimeric poxvirus is provided comprising a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:2, the nucleic acid sequence comprising: (i) orf virus strain NZ2 and pseudobovine poxvirus strain TJS; (ii) one or more anti-cancer nucleic acid sequences; (iii) one or more nucleic acid binding sequences; or (iv) a nucleic acid sequence encoding a detectable moiety.

[0164] In another aspect, a chimeric poxvirus is provided comprising a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:3, the nucleic acid sequence comprising: (i) a nucleic acid fragment from Bovine poxvirus strain Brighton, Raccoon poxvirus strain Herman, Rabbit poxvirus strain Utrecht, Vaccinia virus strain WR, Vaccinia virus strain IHD, Vaccinia virus strain Elstree, Vaccinia virus strain CL, Vaccinia virus strain Lederle-Chorioallantoic, and Vaccinia virus strain AS, (ii) one or more anti-cancer nucleic acid sequences, (iii) one or more nucleic acid binding sequences, or (iv) a nucleic acid sequence encoding a detectable moiety.

[0165] In an embodiment, the nucleic acid sequence comprises: (i) a nucleic acid fragment from at least two poxvirus strains selected from the group consisting of: Brighton bovine poxvirus, Herman raccoon poxvirus, Utrecht rabbit poxvirus, WR vaccinia virus, IHD vaccinia virus, Elstree vaccinia virus, CL vaccinia virus, Lederle-Chorioallantoic vaccinia virus, AS vaccinia virus, NZ2 orf virus, and TJS pseudo bovine poxvirus; and (ii) one or more anti-cancer nucleic acid sequences. In an embodiment, the nucleic acid fragment is from Brighton bovine poxvirus, Herman raccoon poxvirus, Utrecht rabbit poxvirus, WR vaccinia virus, IHD vaccinia virus, Elstree vaccinia virus, CL vaccinia virus, Lederle-Chorioallantoic vaccinia virus, and AS vaccinia virus. In an embodiment, the nucleic acid fragment is a nucleic acid fragment derived from the orf virus strain NZ2 and the pseudobovine poxvirus strain TJS.

[0166] In an embodiment, the nucleic acid sequence comprises: (i) a nucleic acid fragment from at least two poxvirus strains selected from the group consisting of the bovine poxvirus strain Brighton, the raccoon poxvirus strain Herman, the rabbit poxvirus strain Utrecht, the vaccinia virus strain WR, the vaccinia virus strain IHD, the vaccinia virus strain Elstree, the vaccinia virus strain CL, the vaccinia virus strain Lederle-Chorioallantoic, the vaccinia virus strain AS, the orf virus strain NZ2, and the pseudo bovine poxvirus strain TJS, and (ii) one or more nucleic acid binding sequences. In an embodiment, the nucleic acid fragment is from the bovine poxvirus strain Brighton, the raccoon poxvirus strain Herman, the rabbit poxvirus strain Utrecht, the vaccinia virus strain WR, the vaccinia virus strain IHD, the vaccinia virus strain Elstree, the vaccinia virus strain CL, the vaccinia virus strain Lederle-Chorioallantoic, and the vaccinia virus strain AS. In an embodiment, the nucleic acid fragment is a nucleic acid fragment derived from the orf virus strain NZ2 and the pseudobovine poxvirus strain TJS.

[0167] In an embodiment, the nucleic acid sequence comprises: (i) a nucleic acid fragment from at least two poxvirus strains selected from the group consisting of: bovine poxvirus strain Brighton, raccoon poxvirus strain Herman, rabbit poxvirus strain Utrecht, vaccinia virus strain WR, vaccinia virus strain IHD, vaccinia virus strain Elstree, vaccinia virus strain CL, vaccinia virus strain Lederle-Chorioallantoic, vaccinia virus strain AS, orf virus strain NZ2, and pseudo bovine poxvirus strain TJS, and (ii) a nucleic acid sequence encoding a detectable moiety. In an embodiment, the nucleic acid fragment is from bovine poxvirus strain Brighton, raccoon poxvirus strain Herman, rabbit poxvirus strain Utrecht, vaccinia virus strain WR, vaccinia virus strain IHD, vaccinia virus strain Elstree, vaccinia virus strain CL, vaccinia virus strain Lederle-Chorioallantoic, and vaccinia virus strain AS. In an embodiment, the nucleic acid fragment is a nucleic acid fragment derived from the orf virus strain NZ2 and the pseudobovine poxvirus strain TJS.

[0168] In an embodiment, the nucleic acid sequence comprises: (i) a nucleic acid fragment from at least two poxvirus strains selected from the group consisting of bovine poxvirus strain Brighton, raccoon poxvirus strain Herman, rabbit poxvirus strain Utrecht, vaccinia virus strain WR, vaccinia virus strain IHD, vaccinia virus strain Elstree, vaccinia virus strain CL, vaccinia virus strain Lederle-Chorioallantoic, vaccinia virus strain AS, orf virus strain NZ2, and pseudobovine poxvirus strain TJS, (ii) one or more anti-cancer nucleic acid sequences, and (iii) a nucleic acid sequence encoding a detectable moiety. In an embodiment, the nucleic acid fragment is derived from the Brighton strain of bovine poxvirus, the Herman strain of raccoon poxvirus, the Utrecht strain of rabbit poxvirus, the WR strain of vaccinia virus, the IHD strain of vaccinia virus, the Elstree strain of vaccinia virus, the CL strain of vaccinia virus, the Lederle-Chorioallantoic strain of vaccinia virus, and the AS strain of vaccinia virus. In an embodiment, the nucleic acid fragment is derived from the NZ2 strain of orf virus and the TJS strain of pseudobovine poxvirus.

[0169] In an embodiment, the nucleic acid sequence comprises: (i) nucleic acid fragments from at least two poxvirus strains selected from the group consisting of bovine poxvirus strain Brighton, raccoon poxvirus strain Herman, rabbit poxvirus strain Utrecht, vaccinia virus strain WR, vaccinia virus strain IHD, vaccinia virus strain Elstree, vaccinia virus strain CL, vaccinia virus strain Lederle-Chorioallantoic, vaccinia virus strain AS, orf virus strain NZ2, and pseudobovine poxvirus strain TJS, (ii) one or more anti-cancer nucleic acid sequences, and (iii) one or more nucleic acid binding sequences. In an embodiment, the nucleic acid fragment is derived from the Brighton strain of bovine poxvirus, the Herman strain of raccoon poxvirus, the Utrecht strain of rabbit poxvirus, the WR strain of vaccinia virus, the IHD strain of vaccinia virus, the Elstree strain of vaccinia virus, the CL strain of vaccinia virus, the Lederle-Chorioallantoic strain of vaccinia virus, and the AS strain of vaccinia virus. In an embodiment, the nucleic acid fragment is derived from the NZ2 strain of orf virus and the TJS strain of pseudobovine poxvirus.

[0170] The anti-cancer nucleic acid sequence (transgene) may form part of the genome of the chimeric poxvirus according to embodiments provided herein. The chimeric poxvirus genome comprises genes required for expression and replication of the poxvirus. Genes required for expression and replication of the chimeric poxvirus are referred to herein as "essential genes". Genes not required for expression and replication of the chimeric poxvirus are referred to herein as "non-essential genes". The anti-cancer nucleic acid sequence may be incorporated into the chimeric poxvirus genome by insertion into a gene or may be operably linked to a gene. Insertion of the anti-cancer nucleic acid sequence into a chimeric poxvirus gene may result in deletion of a gene (e.g., a non-essential gene) or a portion thereof. In embodiments, the one or more anti-cancer nucleic acid sequences form part of a non-essential gene of the chimeric poxvirus. In embodiments, the one or more anti-cancer nucleic acid sequences are inserted into a non-essential gene of the chimeric poxvirus. In embodiments, the non-essential gene is a thymidine kinase gene. In embodiments, the non-essential gene is a J2R gene. In an embodiment, the non-essential gene is the F14.5L gene.

[0171] As described above, the anti-cancer nucleic acid sequence may encode a polypeptide useful for the treatment of cancer. In embodiments, the one or more anti-cancer nucleic acid sequences independently encode a PD-L1 inhibitor or a sodium iodide symporter. In embodiments, the PD-L1 inhibitor is an anti-PD-L1 scFv. In embodiments, the anti-PD-L1 scFv comprises the sequence of SEQ ID NO: 17. In embodiments, the anti-PD-L1 scFv is the sequence of SEQ ID NO: 17. In embodiments, the sodium iodide symporter comprises the sequence of SEQ ID NO: 13. In embodiments, the sodium iodide symporter is the sequence of SEQ ID NO: 13.

[0172] As used herein, "PD-L1" or "PD-L1 protein" includes recombinant or native programmed cell death ligand 1 (PD-L1), also known as Cluster of Differentiation 274 (CD274), or variants or homologs thereof that maintain PD-L1 activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the activity of PD-L1). In some embodiments, the variant or homolog has at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the native PD-L1 promoter over the entire sequence or a portion of the sequence (e.g., a portion of 50, 100, 150 or 200 contiguous amino acids). In an embodiment, the PD-L1 protein is substantially identical to a protein identified by UniProt reference number Q9NZQ7, or a variant or homologue having substantial identity thereto.

[0173] As used herein, the term "PD-L1 inhibitor" refers to an agent (e.g., an antibody, an antibody fragment, a single chain variable fragment (scFv)) that can detectably reduce the expression of PD-L1 or its activity level compared to a control. Inhibition of PD-L1 expression or activity can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more inhibition relative to a control. In particular examples, the inhibition is 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold or more inhibition compared to a control. A PD-L1 inhibitor inhibits PD-L1 by, for example, at least partially, partially or totally, blocking stimulation, reducing, preventing or delaying activation, inactivating or desensitizing signaling, or downregulating the activity or amount of PD-L1 compared to the absence of the PD-L1 inhibitor.

[0174] As used herein, the term "sodium iodide symporter," "NIS," or "hNIS" includes recombinant or native sodium iodide symporter, or a variant or homolog thereof that maintains sodium iodide symporter activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the activity of the sodium iodide symporter). In some embodiments, the variant or homolog has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a portion of 50, 100, 150, or 200 contiguous amino acids) over the native sodium iodide symporter. In an embodiment, the sodium iodide symporter is substantially identical to the protein identified by UniProt reference number Q92911, or a variant or homologue thereof having substantial identity thereto. In an embodiment, the sodium iodide symporter comprises the sequence of SEQ ID NO: 13. In an embodiment, the sodium iodide symporter is the sequence of SEQ ID NO: 13.

[0175] The expression of the anti-cancer nucleic acid sequences provided herein may be controlled by a promoter. Thus, in an embodiment, one or more anti-cancer nucleic acid sequences are each operably linked to a promoter. In an embodiment, the promoter is a vaccinia virus early promoter. In an embodiment, the promoter is a synthetic early promoter. In an embodiment, the synthetic early promoter comprises the sequence of SEQ ID NO: 19. In an embodiment, the synthetic early promoter is the sequence of SEQ ID NO: 19. In an embodiment, the promoter is a vaccinia virus late promoter. In an embodiment, the promoter is an H5 promoter or an 11K promoter. In an embodiment, the H5 promoter comprises the sequence of SEQ ID NO: 18. In an embodiment, the H5 promoter is the sequence of SEQ ID NO: 18. In an embodiment, the 11K promoter comprises the sequence of SEQ ID NO: 20. In an embodiment, the 11K promoter is the sequence of SEQ ID NO: 20.

[0176] The anti-cancer nucleic acid sequences (nucleic acid binding sequences) provided herein may be incorporated into the chimeric poxvirus genome so as to be in a functional (operably linked) relationship with a particular poxvirus gene. For example, the anti-cancer nucleic acid sequence (nucleic acid binding sequence) may be operably linked to a poxvirus gene if it affects the transcription or translation of the poxvirus gene. Typically, the anti-cancer nucleic acid sequence (nucleic acid binding sequence) and the poxvirus gene are operably linked when they are contiguous and / or in reading phase. In an embodiment, one or more anti-cancer nucleic acid sequences (one or more nucleic acid binding sequences) are operably linked to an essential gene of the chimeric poxvirus. In an embodiment, one or more anti-cancer nucleic acid sequences (one or more nucleic acid binding sequences) are operably linked to a DNA polymerase gene of the chimeric poxvirus. In an embodiment, one or more anti-cancer nucleic acid sequences (one or more nucleic acid binding sequences) are operably linked to the 3' end of the DNA polymerase gene of the chimeric poxvirus. In an embodiment, the one or more anti-cancer nucleic acid sequences (one or more nucleic acid binding sequences) are operably linked to the uracil DNA glycosylase gene. In an embodiment, the one or more anti-cancer nucleic acid sequences (one or more nucleic acid binding sequences) are operably linked to the 3' end of the uracil DNA glycosylase gene.

[0177] In an embodiment, the one or more anti-cancer nucleic acid sequences (one or more nucleic acid binding sequences) independently encode a miRNA binding sequence. In an embodiment, the miRNA binding sequence is a miR100 binding sequence or a let7c binding sequence. In an embodiment, the miRNA binding sequence is a miR100 binding sequence. In an embodiment, the miR100 binding sequence comprises the sequence of SEQ ID NO: 9. In an embodiment, the miR100 binding sequence is the sequence of SEQ ID NO: 9. In an embodiment, the miR100 binding sequence comprises the sequence of SEQ ID NO: 10. In an embodiment, the miR100 binding sequence is the sequence of SEQ ID NO: 10. In an embodiment, the miRNA binding sequence is a let7c binding sequence. In an embodiment, the let7c binding sequence comprises the sequence of SEQ ID NO: 11. In an embodiment, the let7c binding sequence is the sequence of SEQ ID NO: 11.

[0178] In an embodiment, the one or more anti-cancer nucleic acid sequences are a first anti-cancer nucleic acid sequence and a second anti-cancer nucleic acid sequence. As provided herein, the first anti-cancer nucleic acid sequence can be a first nucleic acid binding sequence, and the second anti-cancer nucleic acid sequence can be a second nucleic acid binding sequence.

[0179] In an embodiment, the first anti-cancer nucleic acid sequence encodes a sodium iodide symporter and said second anti-cancer nucleic acid sequence (second nucleic acid binding sequence) encodes a miRNA binding sequence. In an embodiment, the first anti-cancer nucleic acid sequence forms part of a thymidine kinase gene and the second anti-cancer nucleic acid sequence (second nucleic acid binding sequence) is operably linked to a uracil DNA glycosylase gene. In an embodiment, the first anti-cancer nucleic acid sequence forms part of a thymidine kinase gene and the second anti-cancer nucleic acid sequence (second nucleic acid binding sequence) is operably linked to a DNA polymerase gene.

[0180] In an embodiment, the first anti-cancer nucleic acid sequence encodes a sodium iodide symporter and the second anti-cancer nucleic acid sequence encodes a PD-L1 inhibitor. In an embodiment, the first anti-cancer nucleic acid sequence forms part of a thymidine kinase gene and the second anti-cancer nucleic acid sequence forms part of an F14.5L gene.

[0181] In an embodiment, the nucleic acid sequence comprises: (i) a nucleic acid fragment from at least two poxvirus strains selected from the group consisting of: bovine poxvirus strain Brighton, raccoon poxvirus strain Herman, rabbit poxvirus strain Utrecht, vaccinia virus strain WR, vaccinia virus strain IHD, vaccinia virus strain Elstree, vaccinia virus strain CL, vaccinia virus strain Lederle-Chorioallantoic, vaccinia virus strain AS, orf virus strain NZ2, and pseudo bovine poxvirus strain TJS; and (ii) a nucleic acid sequence encoding said detectable moiety. In an embodiment, the nucleic acid sequence encoding the detectable moiety encodes a fluorescent moiety. In an embodiment, the nucleic acid sequence encoding the detectable moiety forms part of a non-essential gene of the chimeric poxvirus. In an embodiment, the non-essential gene is a thymidine kinase gene. In an embodiment, a part of the non-essential gene is deleted.

[0182] In an embodiment, the nucleic acid sequence encoding the detectable moiety is operably linked to a promoter. In an embodiment, the promoter is a vaccinia virus early promoter. In an embodiment, the promoter is a synthetic early promoter. In an embodiment, the synthetic early promoter comprises the sequence of SEQ ID NO: 19. In an embodiment, the synthetic early promoter is the sequence of SEQ ID NO: 19. In an embodiment, the promoter is a vaccinia virus late promoter. In an embodiment, the promoter is an H5 promoter or an 11K promoter. In an embodiment, the H5 promoter comprises the sequence of SEQ ID NO: 18. In an embodiment, the H5 promoter is the sequence of SEQ ID NO: 18. In an embodiment, the 11K promoter comprises the sequence of SEQ ID NO: 20. In an embodiment, the 11K promoter is the sequence of SEQ ID NO: 20.

[0183] In one embodiment, the anti-cancer nucleic acid sequence (nucleic acid binding sequence) encodes an miRNA binding sequence having the sequence of SEQ ID NO: 10 and is operably linked to the 3' end of a uracil DNA glycosylase gene.

[0184] In one embodiment, the anti-cancer nucleic acid sequence (nucleic acid binding sequence) encodes an miRNA binding sequence having the sequence of SEQ ID NO: 9 and is operably linked to the 3' end of a uracil DNA glycosylase gene.

[0185] In one embodiment, the anti-cancer nucleic acid sequence (nucleic acid binding sequence) encodes an miRNA binding sequence having the sequence of SEQ ID NO: 11 and is operably linked to the 3' end of a uracil DNA glycosylase gene.

[0186] In one embodiment, the anti-cancer nucleic acid sequence (nucleic acid binding sequence) encodes an miRNA binding sequence having the sequence of SEQ ID NO: 9 and is operably linked to the 3' end of a DNA polymerase gene.

[0187] In one embodiment, the anti-cancer nucleic acid sequence (nucleic acid binding sequence) encodes an miRNA binding sequence having the sequence of SEQ ID NO: 11 and is operably linked to the 3' end of a DNA polymerase gene.

[0188] In one embodiment, the thymidine kinase gene of the chimeric poxvirus has the sequence of SEQ ID NO:5.

[0189] In one embodiment, the anti-cancer nucleic acid sequence encodes a sodium iodide symporter and is operably linked to a synthetic early promoter that is part of a thymidine kinase gene, the sodium iodide symporter having the sequence of SEQ ID NO: 13 and the synthetic early promoter having the sequence of SEQ ID NO: 19.

[0190] In one embodiment, the nucleic acid sequence encoding the detectable moiety encodes a fluorescent moiety having the sequence of SEQ ID NO: 14 and is operably linked to an H5 promoter and forms part of the thymidine kinase gene, the H5 promoter having the sequence of SEQ ID NO: 18.

[0191] In one embodiment, the nucleic acid sequence encoding the detectable moiety encodes a fluorescent moiety having the sequence of SEQ ID NO:15 and is operably linked to a synthetic early promoter and forms part of the thymidine kinase gene, the synthetic early promoter having the sequence of SEQ ID NO:19.

[0192] In one embodiment, the nucleic acid sequence encoding the detectable moiety encodes a fluorescent moiety having the sequence of SEQ ID NO: 15 and is operably linked to an H5 promoter and forms part of the thymidine kinase gene, the H5 promoter having the sequence of SEQ ID NO: 18.

[0193] In one embodiment, the nucleic acid sequence encoding the detectable moiety encodes a fluorescent moiety having the sequence of SEQ ID NO: 15, is operably linked to an 11K promoter and forms part of the thymidine kinase gene, the 11K promoter having the sequence of SEQ ID NO: 20.

[0194] In one embodiment, the nucleic acid sequence encoding the detectable moiety encodes a fluorescent moiety having the sequence of SEQ ID NO: 16 and is operably linked to an H5 promoter and forms part of the thymidine kinase gene, the H5 promoter having the sequence of SEQ ID NO: 18.

[0195] In one embodiment, the nucleic acid sequence encoding the detectable moiety encodes a fluorescent moiety having the sequence of SEQ ID NO: 16, is operably linked to an 11K promoter and forms part of the thymidine kinase gene, the 11K promoter having the sequence of SEQ ID NO: 20.

[0196] In one embodiment, a first anti-cancer nucleic acid sequence encodes a sodium iodide symporter and is operably linked to a synthetic early promoter and forms part of a thymidine kinase gene, a second anti-cancer nucleic acid sequence (nucleic acid binding sequence) encodes a miRNA binding sequence having the sequence of SEQ ID NO: 10 and is operably linked to the 3' end of a uracil DNA glycosylase gene, the sodium iodide symporter has the sequence of SEQ ID NO: 13 and the synthetic early promoter has the sequence of SEQ ID NO: 19.

[0197] In one embodiment, a first anti-cancer nucleic acid sequence encodes a sodium iodide symporter and is operably linked to a synthetic early promoter and forms part of a thymidine kinase gene, a second anti-cancer nucleic acid sequence (nucleic acid binding sequence) encodes a miRNA binding sequence having the sequence of SEQ ID NO: 9 and is operably linked to the 3' end of a uracil DNA glycosylase gene, the sodium iodide symporter has the sequence of SEQ ID NO: 13 and the synthetic early promoter has the sequence of SEQ ID NO: 19.

[0198] In one embodiment, a first anti-cancer nucleic acid sequence encodes a sodium iodide symporter and is operably linked to a synthetic early promoter and forms part of a thymidine kinase gene, a second anti-cancer nucleic acid sequence (nucleic acid binding sequence) encodes a miRNA binding sequence having the sequence of SEQ ID NO: 11 and is operably linked to the 3' end of a uracil DNA glycosylase gene, the sodium iodide symporter has the sequence of SEQ ID NO: 13 and the synthetic early promoter has the sequence of SEQ ID NO: 19.

[0199] In one embodiment, a first anti-cancer nucleic acid sequence encodes a sodium iodide symporter and is operably linked to a synthetic early promoter and forms part of a thymidine kinase gene, a second anti-cancer nucleic acid sequence (nucleic acid binding sequence) encodes a miRNA binding sequence having a sequence of SEQ ID NO: 9 and is operably linked to the 3' end of a DNA polymerase gene, the sodium iodide symporter has a sequence of SEQ ID NO: 13 and the synthetic early promoter has a sequence of SEQ ID NO: 19.

[0200] In one embodiment, a first anti-cancer nucleic acid sequence encodes a sodium iodide symporter and is operably linked to a synthetic early promoter and forms part of a thymidine kinase gene, a second anti-cancer nucleic acid sequence (nucleic acid binding sequence) encodes a miRNA binding sequence having the sequence of SEQ ID NO: 11 and is operably linked to the 3' end of a DNA polymerase gene, the sodium iodide symporter has the sequence of SEQ ID NO: 13 and the synthetic early promoter has the sequence of SEQ ID NO: 19.

[0201] In one embodiment, the F14.5L gene of the chimeric poxvirus has the sequence of SEQ ID NO:7.

[0202] In one embodiment, the anti-cancer nucleic acid sequence encodes an anti-PD-L1 scFv and is operably linked to the H5 promoter and forms part of the F14.5L gene, wherein the anti-PD-L1 scFv has the sequence of SEQ ID NO:17 and the H5 promoter has the sequence of SEQ ID NO:18.

[0203] In one embodiment, the anti-cancer nucleic acid sequence encodes a sodium iodide symporter and is operably linked to a synthetic early promoter and forms part of a thymidine kinase gene, the F14.5L gene having the sequence of SEQ ID NO:7, the sodium iodide symporter having the sequence of SEQ ID NO:13, and the synthetic early promoter having the sequence of SEQ ID NO:19.

[0204] In one embodiment, a first anti-cancer nucleic acid sequence encodes a sodium iodide symporter and is operably linked to a synthetic early promoter and forms part of a thymidine kinase gene, and a second anti-cancer nucleic acid sequence encodes an anti-PD-L1 scFv and is operably linked to the H5 promoter and forms part of the F14.5L gene, wherein the sodium iodide symporter has a sequence of SEQ ID NO: 13, the synthetic early promoter has a sequence of SEQ ID NO: 19, the anti-PD-L1 scFv has a sequence of SEQ ID NO: 17, and the H5 promoter has a sequence of SEQ ID NO: 18.

[0205] IV. Methods for generating chimeric poxviruses In another aspect, a method for forming a chimeric poxvirus is provided, the method comprising infecting a cell with at least two poxvirus strains selected from the group comprising: bovine poxvirus strain Brighton, raccoon poxvirus strain Herman, rabbit poxvirus strain Utrecht, vaccinia virus strain WR, vaccinia virus strain IHD, vaccinia virus strain Elstree, vaccinia virus strain CL, vaccinia virus strain Lederle-Chorioallantoic, vaccinia virus strain AS, orf virus strain NZ2 and pseudobovine poxvirus strain TJS, and allowing the at least two poxvirus strains to replicate, thereby forming a chimeric poxvirus.

[0206] The methods for forming chimeric poxviruses provided herein can be used to form chimeric poxviruses that contain a transgene described herein (e.g., an anti-cancer nucleic acid sequence, a nucleic acid binding sequence, a nucleic acid sequence encoding a detectable moiety).

[0207] In an embodiment, the at least two poxvirus strains are each present at a multiplicity of infectivity of less than about 1.0. In an embodiment, the at least two poxvirus strains are each present at a multiplicity of infectivity of less than about 0.5. In an embodiment, the at least two poxvirus strains are each present at a multiplicity of infectivity of less than about 0.1. In an embodiment, the at least two poxvirus strains are each present at a multiplicity of infectivity of less than about 0.05. In an embodiment, the at least two poxvirus strains are each present at a multiplicity of infectivity of less than about 0.01.

[0208] In an embodiment, the chimeric poxvirus is formed by a method comprising infecting a cell with at least two poxviruses selected from the group comprising: cowpoxvirus strain Brighton, raccoon poxvirus strain Herman, rabbit poxvirus strain Utrecht, vaccinia virus strain WR, vaccinia virus strain IHD, vaccinia virus strain Elstree, vaccinia virus strain CL, vaccinia virus strain Lederle-Chorioallantoic, vaccinia virus strain AS, orf virus strain NZ2 and pseudobovine poxvirus strain TJS, and allowing the at least two poxvirus strains to replicate, thereby forming the chimeric poxvirus.

[0209] In an embodiment, the cells are infected with the bovine poxvirus Brighton strain, the raccoon poxvirus Herman strain, the rabbit poxvirus Utrecht strain, the vaccinia virus WR strain, the vaccinia virus IHD strain, the vaccinia virus Elstree strain, the vaccinia virus CL strain, the vaccinia virus Lederle-Chorioallantoic strain, and the vaccinia virus AS strain.

[0210] In embodiments, the nucleic acid fragment is derived from the bovine poxvirus strain Brighton, the raccoon poxvirus strain Herman, the rabbit poxvirus strain Utrecht, the vaccinia virus strain WR, the vaccinia virus strain IHD, the vaccinia virus strain Elstree, the vaccinia virus strain CL, the vaccinia virus strain Lederle-Chorioallantoic, and the vaccinia virus strain AS.

[0211] In an embodiment, the cells are infected with the bovine poxvirus strain Brighton and the raccoon poxvirus strain Herman. In an embodiment, the cells are infected with the bovine poxvirus strain Brighton and the rabbit poxvirus strain Utrecht. In an embodiment, the cells are infected with the bovine poxvirus strain Brighton and the vaccinia virus strain WR. In an embodiment, the cells are infected with the bovine poxvirus strain Brighton and the vaccinia virus strain IHD. In an embodiment, the cells are infected with the bovine poxvirus strain Brighton and the vaccinia virus strain Elstree. In an embodiment, the cells are infected with the bovine poxvirus strain Brighton and the vaccinia virus strain CL. In an embodiment, the cells are infected with the bovine poxvirus strain Brighton and the vaccinia virus strain Lederle-Chorioallantoic. In an embodiment, the cells are infected with the bovine poxvirus strain Brighton and the vaccinia virus strain AS. In an embodiment, the cells are infected with the Brighton strain of bovine poxvirus and the NZ2 strain of orf virus.In an embodiment, the cells are infected with the Brighton strain of bovine poxvirus and the TJS strain of pseudobovine poxvirus.

[0212] In an embodiment, the cells are infected with rabbit pox virus Utrecht strain and vaccinia virus WR strain. In an embodiment, the cells are infected with rabbit pox virus Utrecht strain and vaccinia virus IHD strain. In an embodiment, the cells are infected with rabbit pox virus Utrecht strain and vaccinia virus Elstree strain. In an embodiment, the cells are infected with rabbit pox virus Utrecht strain and vaccinia virus CL strain. In an embodiment, the cells are infected with rabbit pox virus Utrecht strain and vaccinia virus Lederle-Chorioallantoic strain. In an embodiment, the cells are infected with rabbit pox virus Utrecht strain and vaccinia virus AS strain. In an embodiment, the cells are infected with rabbit pox virus Utrecht strain and orf virus NZ2 strain. In an embodiment, the cells are infected with rabbit pox virus Utrecht strain and pseudobovine pox virus TJS strain.

[0213] In an embodiment, the cells are infected with vaccinia virus strain WR and vaccinia virus strain IHD. In an embodiment, the cells are infected with vaccinia virus strain WR and vaccinia virus strain Elstree. In an embodiment, the cells are infected with vaccinia virus strain WR and vaccinia virus strain CL. In an embodiment, the cells are infected with vaccinia virus strain WR and vaccinia virus strain Lederle-Chorioallantoic. In an embodiment, the cells are infected with vaccinia virus strain WR and vaccinia virus strain AS. In an embodiment, the cells are infected with vaccinia virus strain WR and orf virus strain NZ2. In an embodiment, the cells are infected with vaccinia virus strain WR and pseudobovine pox virus strain TJS.

[0214] In an embodiment, the cells are infected with vaccinia virus IHD strain and vaccinia virus Elstree strain. In an embodiment, the cells are infected with vaccinia virus IHD strain and vaccinia virus CL strain. In an embodiment, the cells are infected with vaccinia virus IHD strain and vaccinia virus Lederle-Chorioallantoic strain. In an embodiment, the cells are infected with vaccinia virus IHD strain and vaccinia virus AS strain. In an embodiment, the cells are infected with vaccinia virus IHD strain and orf virus NZ2 strain. In an embodiment, the cells are infected with vaccinia virus IHD strain and pseudobovine pox virus TJS strain.

[0215] In an embodiment, the cells are infected with the Elstree and CL strains of vaccinia virus. In an embodiment, the cells are infected with the Elstree and Lederle-Chorioallantoic strains of vaccinia virus. In an embodiment, the cells are infected with the Elstree and AS strains of vaccinia virus. In an embodiment, the cells are infected with the Elstree and NZ2 strains of vaccinia virus. In an embodiment, the cells are infected with the Elstree and TJS strains of pseudobovine pox virus.

[0216] In an embodiment, the cells are infected with vaccinia virus strain CL and vaccinia virus strain Lederle-Chorioallantoic. In an embodiment, the cells are infected with vaccinia virus strain CL and vaccinia virus strain AS. In an embodiment, the cells are infected with vaccinia virus strain CL and orf virus strain NZ2. In an embodiment, the cells are infected with vaccinia virus strain CL and pseudobovine poxvirus strain TJS.

[0217] In an embodiment, the cells are infected with the Lederle-Chorioallantoic vaccinia virus and the AS vaccinia virus strain. In an embodiment, the cells are infected with the Lederle-Chorioallantoic vaccinia virus and the NZ2 orf virus strain. In an embodiment, the cells are infected with the Lederle-Chorioallantoic vaccinia virus and the TJS pseudobovine poxvirus strain.

[0218] In an embodiment, the cells are infected with vaccinia virus strain AS and orf virus strain NZ2. In an embodiment, the cells are infected with vaccinia virus strain AS and pseudobovine pox virus strain TJS. In an embodiment, the cells are infected with orf virus strain NZ2 and pseudobovine pox virus strain TJS.

[0219] In an embodiment, the cell is a kidney fibroblast cell. In an embodiment, the cell is an epithelial cell. In an embodiment, the cell is a CV-1 cell. In an embodiment, the cell is a bovine kidney epithelial cell.

[0220] V. Pharmaceutical Compositions In one aspect, a pharmaceutical composition is provided comprising a therapeutically effective amount of a chimeric poxvirus as described herein, including embodiments thereof, in which the chimeric poxvirus comprises a transgene, such as an anti-cancer nucleic acid sequence, a nucleic acid binding sequence, a nucleic acid sequence encoding a detectable moiety, or any combination thereof.

[0221] "Pharmaceutically acceptable excipient" and "pharmaceutically acceptable carrier" refer to substances that aid in the administration and absorption of active agents to a subject and can be included in the compositions of the present invention without causing significant toxic side effects to the patient. Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, saline, lactated Ringer's solution, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavorings, salt solutions (e.g., Ringer's solution), alcohol, fats and oils, gelatin, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidine, and dyes. Such preparations can be sterilized and, if necessary, mixed with auxiliary agents (e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts that affect osmotic pressure, buffers, dyes, and / or flavorings that do not adversely react with the compounds of the present invention, etc.). Those skilled in the art will recognize that other pharmaceutical excipients are useful in the present invention.

[0222] The chimeric poxvirus compositions according to the embodiments provided herein may be administered orally, gastrointestinally, or rectally. Administration may be in the form of a single bolus dose or may be continuous, for example, by a perfusion pump. In an embodiment, the chimeric poxviruses provided herein are combined with one or more excipients, such as disintegrants, fillers, glidants, or preservatives. In an embodiment, the chimeric poxviruses provided herein form part of a capsule. Suitable capsules include hard-shell capsules or soft-shell capsules. Any lipid-based or polymer-based colloid may be used to form the capsule. Exemplary polymers useful for colloidal preparations include gelatins, such as carrageenans and modified starches and modified celluloses, such as hypromellose, vegetable polysaccharides, or derivatives thereof. Optionally, other ingredients may be added to the gelling agent solution, such as plasticizers, such as glycerin and / or sorbitol, for example to reduce the hardness of the capsule, colorants, preservatives, disintegrants, lubricants, and surface treatments.

[0223] The chimeric poxvirus compositions can be formulated in unit dosage forms, each containing, for example, about 0.005 mg to about 2000 mg of a given chimeric poxvirus per dose, with minimal urease activity. The term "unit dosage form" refers to a physically separate unit suitable for unitary administration to a human subject or other mammal, each unit containing a predetermined amount of active ingredient calculated to produce a desired therapeutic effect together with a suitable pharmaceutical carrier. To prepare solid compositions such as tablets, the main active ingredient...

Claims

Claim 1: A chimeric poxvirus comprising a nucleic acid sequence having at least 95% sequence identity to positions 5,014 to 185,366 of SEQ ID NO: 1, The nucleic acid sequence i) one or more anti-cancer nucleic acid sequences, or ii) a nucleic acid sequence encoding a detectable moiety A chimeric poxvirus further comprising:

2. A chimeric poxvirus comprising a nucleic acid sequence containing an essential gene of SEQ ID NO: 1 for poxvirus replication, The nucleic acid sequence i) one or more anti-cancer nucleic acid sequences, or ii) a nucleic acid sequence encoding a detectable moiety A chimeric poxvirus further comprising:

3. The chimeric poxvirus described in claim 2, wherein the nucleic acid sequence has at least 95% sequence identity to positions 5,014 to 185,366 of SEQ ID NO:

1.

4. A chimeric poxvirus described in any one of claims 1 to 3, wherein the nucleic acid sequence further comprises one or more anti-cancer nucleic acid sequences.

5. A chimeric poxvirus as described in claim 4, wherein the one or more anti-cancer nucleic acid sequences are inserted into a non-essential gene of the chimeric poxvirus.

6. The chimeric poxvirus described in claim 5, wherein the non-essential gene is a thymidine kinase gene.

7. The chimeric poxvirus described in claim 5, wherein the non-essential gene is the F14.5L gene.

8. The chimeric poxvirus described in claim 4, wherein the one or more anti-cancer nucleic acid sequences independently encode a PD-L1 inhibitor or a sodium iodide symporter.

9. The chimeric poxvirus described in claim 8, wherein the PD-L1 inhibitor is an anti-PD-L1 scFv.

10. A chimeric poxvirus as described in claim 5, in which a portion of the non-essential gene is deleted.

11. A chimeric poxvirus as described in claim 4, wherein the one or more anti-cancer nucleic acid sequences are each operably linked to a promoter.

12. The chimeric poxvirus described in claim 11, wherein the promoter is a vaccinia virus early promoter.

13. A chimeric poxvirus as described in claim 11, wherein the promoter is a synthetic early promoter.

14. The chimeric poxvirus described in claim 11, wherein the promoter is a vaccinia virus late promoter.

15. The chimeric poxvirus described in claim 11, wherein the promoter is an H5 promoter or an 11K promoter.

16. A chimeric poxvirus as described in claim 4, wherein the one or more anti-cancer nucleic acid sequences are operably linked to an essential gene of the chimeric poxvirus.

17. A chimeric poxvirus as described in claim 4, wherein the one or more anti-cancer nucleic acid sequences are operably linked to a DNA polymerase gene of the chimeric poxvirus.

18. A chimeric poxvirus as described in claim 4, wherein the one or more anti-cancer nucleic acid sequences are operably linked to the 3' end of the DNA polymerase gene of the chimeric poxvirus.

19. The chimeric poxvirus described in claim 4, wherein the one or more anti-cancer nucleic acid sequences are operably linked to a uracil DNA glycosylase gene.

20. The chimeric poxvirus described in claim 1, wherein the one or more anti-cancer nucleic acid sequences are operably linked to the 3' end of a uracil DNA glycosylase gene.

21. The chimeric poxvirus described in claim 4, wherein the one or more anti-cancer nucleic acid sequences independently encode miRNA binding sequences.

22. The chimeric poxvirus described in claim 21, wherein the miRNA binding sequence is a miR100 binding sequence or a let7c binding sequence.

23. The chimeric poxvirus described in claim 4, wherein the one or more anti-cancer nucleic acid sequences are a first anti-cancer nucleic acid sequence and a second anti-cancer nucleic acid sequence.

24. The chimeric poxvirus described in claim 23, wherein the first anti-cancer nucleic acid sequence encodes a sodium iodide symporter and the second anti-cancer nucleic acid sequence encodes a miRNA binding sequence.

25. A chimeric poxvirus as described in claim 23, wherein the first anti-cancer nucleic acid sequence forms part of a thymidine kinase gene and the second anti-cancer nucleic acid sequence is operably linked to a uracil DNA glycosylase gene.

26. A chimeric poxvirus as described in claim 23, wherein the first anti-cancer nucleic acid sequence forms part of a thymidine kinase gene and the second anti-cancer nucleic acid sequence is operably linked to a DNA polymerase gene.

27. ​​The chimeric poxvirus described in claim 23, wherein the first anti-cancer nucleic acid sequence encodes a sodium iodide symporter and the second anti-cancer nucleic acid sequence encodes a PD-L1 inhibitor.

28. A chimeric poxvirus as described in claim 23, wherein the first anti-cancer nucleic acid sequence forms part of a thymidine kinase gene and the second anti-cancer nucleic acid sequence forms part of an F14.5L gene.

29. A chimeric poxvirus described in any one of claims 1 to 3, wherein the nucleic acid sequence further comprises a nucleic acid sequence encoding the detectable portion.

30. The chimeric poxvirus of claim 29, wherein the nucleic acid sequence encoding the detectable moiety encodes a fluorescent moiety.

31. A chimeric poxvirus as described in claim 29, wherein the nucleic acid sequence encoding the detectable portion is attached to a non-essential gene of the chimeric poxvirus.

32. The chimeric poxvirus described in claim 31, wherein the non-essential gene is a thymidine kinase gene.

33. A chimeric poxvirus as described in claim 31, in which a portion of the non-essential gene is deleted.

34. The chimeric poxvirus of claim 29, wherein the nucleic acid sequence encoding the detectable portion is operably linked to a promoter.

35. The chimeric poxvirus described in claim 34, wherein the promoter is a vaccinia virus early promoter.

36. A chimeric poxvirus as described in claim 34, wherein the promoter is a synthetic early promoter.

37. The chimeric poxvirus described in claim 34, wherein the promoter is a vaccinia virus late promoter.

38. The chimeric poxvirus described in claim 34, wherein the promoter is an H5 promoter or an 11K promoter.

39. (i) infecting cells with at least two poxvirus strains selected from the group consisting of cowpox virus strain Brighton, raccoon pox virus strain Herman, rabbit pox virus strain Utrecht, vaccinia virus strain WR, vaccinia virus strain IHD, vaccinia virus strain Elstree, vaccinia virus strain CL, vaccinia virus strain Lederle-Chorioallantoic, and vaccinia virus strain AS; and (ii) allowing said at least two poxvirus strains to replicate, thereby forming a chimeric poxvirus.

4. The chimeric poxvirus of claim 1, formed by a method comprising:

40. The chimeric poxvirus according to any one of claims 1 to 3, which is an oncolytic virus.

41. A chimeric poxvirus described in any one of claims 1 to 3, wherein the poxvirus comprises a miRNA binding sequence.

42. A chimeric poxvirus as described in claim 41, wherein the miRNA binding sequence forms part of the DNA polymerase gene of the chimeric poxvirus.

43. An isolated nucleic acid encoding the chimeric poxvirus of any one of claims 1 to 3.

44. A pharmaceutical composition comprising a therapeutically effective amount of the chimeric poxvirus of any one of claims 1 to 3.

45. 10. A method for treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a chimeric poxvirus of any one of claims 1 to 3, thereby treating the cancer in the subject.

46. The method described in claim 45, wherein the cancer is breast cancer, colon cancer, kidney cancer, leukemia, lung cancer, melanoma, ovarian cancer, prostate cancer, pancreatic cancer, brain cancer, liver cancer, gastric cancer or sarcoma.

47. The method described in claim 45, wherein the cancer is triple-negative breast cancer.

48. The method of claim 45, wherein the administration comprises administering a first chimeric poxvirus and a second chimeric poxvirus.

49. The method of claim 48, wherein the second chimeric poxvirus comprises a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO:2, and the nucleic acid sequence comprises nucleic acid fragments derived from the orf virus strain NZ2 and the pseudobovine poxvirus strain TJS.

50. The method of claim 48, wherein the first chimeric poxvirus and the second chimeric poxvirus are administered in a combined synergistic amount.

51. The method described in claim 48, wherein the first chimeric poxvirus and the second chimeric poxvirus are administered simultaneously.

52. The method described in claim 48, wherein the first chimeric poxvirus and the second chimeric poxvirus are administered sequentially.

53. The poxvirus, ３ 46. ​​The method of claim 45, administered in plaque forming units (Pfu) / kg.

54. The poxvirus, wherein the poxvirus is about 10 ３ 46. ​​The method of claim 45, administered in plaque forming units (Pfu) / kg.

55. The poxvirus is at least 10 ４ 54. The method of claim 53, administered in plaque forming units (Pfu) / kg.

56. The poxvirus is about 4 x 10 ４ 56. The method of claim 55, administered in plaque forming units (Pfu) / kg.

57. The poxvirus is about 5 x 10 ４ 57. The method of claim 56, administered in plaque forming units (Pfu) / kg.

58. Poxviruses are at least 10 ６ 58. The method of claim 57, administered in plaque forming units (Pfu) / kg.

59. The poxvirus, wherein the poxvirus is about 10 ８ 59. The method of claim 58, administered in plaque forming units (Pfu) / kg.

60. A method for forming the chimeric poxvirus of any one of claims 1 to 3, comprising: (i) infecting cells with at least two poxvirus strains selected from the group consisting of cowpox virus strain Brighton, raccoon pox virus strain Herman, rabbit pox virus strain Utrecht, vaccinia virus strain WR, vaccinia virus strain IHD, vaccinia virus strain Elstree, vaccinia virus strain CL, vaccinia virus strain Lederle-Chorioallantoic, and vaccinia virus strain AS; and (ii) allowing said at least two poxvirus strains to replicate, thereby forming a chimeric poxvirus. A method comprising:

61. The method described in claim 60, wherein the at least two poxvirus strains are each present at a multiplicity of infection of less than about 1.

62. The method of claim 61, wherein the at least two poxvirus strains are each present at a multiplicity of infection of less than about 0.

1.

63. The method of claim 62, wherein the at least two poxvirus strains are each present at a multiplicity of infection of about 0.

01.

64. The method described in claim 62, wherein the chimeric poxvirus is an oncolytic virus.

65. The method described in claim 60, wherein the poxvirus comprises a miRNA binding sequence.

66. A method for inhibiting cell proliferation in a cell, the method comprising contacting the cell with the chimeric poxvirus of any one of claims 1 to 3.

67. The method described in claim 66, wherein the cells are cancer cells.

68. The method described in claim 67, wherein the cancer cells are breast cancer cells, colon cancer cells, kidney cancer cells, leukemia cells, lung cancer cells, melanoma cells, ovarian cancer cells, prostate cancer cells, pancreatic cancer cells, brain cancer cells, liver cancer cells, gastric cancer cells or sarcoma cells.

69. The method described in claim 67 or 68, wherein the cancer cells are triple-negative breast cancer cells.

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