Methods and compositions for improving oncolytic virus infection for nonpermissive cancers

Abstract

To provide methods and compositions for improving oncolytic virus infection for nonpermissive cancers.SOLUTION: Disclosed herein are methods and compositions of treating cancer, comprising administrating to a subject with cancer a therapeutically effective amount of an oncolytic virus and a nucleocytoplasmic transport inhibitor. Further described herein are methods of converting a nonpermissive cancer cell to a permissive cancer cell and methods of killing a cancer cell by contacting the cancer cell with a therapeutically effective amount of an oncolytic virus and a nucleocytoplasmic transport inhibitor.SELECTED DRAWING: Figure 3A

Description

[Technical field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 62 / 913,667, filed October 10, 2019, and U.S. Provisional Patent Application No. 62 / 894,929, filed September 2, 2019, the entire contents of which are incorporated herein by reference.

[0002] The present disclosure relates to oncolytic viruses, and in particular to methods of using oncolytic viruses in combination with nucleocytoplasmic transport inhibitors.

[0003] Statement of Government Support This invention was made with Government support under Grant No. R01 AI080607 awarded by the National Institutes of Health. The Government has certain rights in this invention. [Background technology]

[0004] Oncolytic viruses, such as those from the Poxviridae family of viruses, are mammalian viruses that have been engineered and / or selected for their ability to selectively infect and kill transformed cancer cells and to activate the host's immune system not only against the virus but also against tumor antigens (see, e.g., Lichty BD, Breitbach CJ, Stojdl DF, Bell JC. 2014. Going viral with cancer immunotherapy. Nature Reviews Cancer 14:559-567). However, the use of oncolytic viruses may be limited in certain tumor cells, e.g., nonpermissive tumor cells. Thus, there remains a need to improve oncolytic virus-based therapies.

[0005] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. Summary of the Invention

[0006] Disclosed herein in some aspects are methods of treating cancer comprising administering to a subject a therapeutically effective amount of an oncolytic virus and a nucleocytoplasmic transport inhibitor.

[0007] In some embodiments, the oncolytic virus is from the Poxviridae family. In some embodiments, the oncolytic virus is from the Chordopoxvirinae or Entomopoxvirinae subfamily. In some embodiments, the oncolytic virus is from the Orthopoxvirus, Cervidpoxvirus, Parapoxvirus, Avipoxvirus, Capripoxvirus, Leporipoxvirus, Suipoxvirus, Molluscipoxvirus, Yatapoxvirus, Alphaentomopoxvirus, Betaentomopoxvirus, or Gammaentopoxvirus virus genus. In some embodiments, the oncolytic virus is from the Leporipoxvirus genus. In some embodiments, the oncolytic virus is Myxoma virus (MYXV). In some embodiments, the oncolytic virus is from the Orthopoxvirus genus. In some embodiments, the oncolytic virus is genetically engineered. In some embodiments, the nucleocytoplasmic transport inhibitor is selected from the group consisting of Leptomycin A, Leptomycin B, Ratjadone A, Ratjadone B, Ratjadone C, Ratjadone D, Anginomycin A, Goniothalamin, piperlongumine, plumbagin, curcumin, valtrate, acetoxychavicolacetate, prenylcoumarin osthol, KOS 2464, PKF050-638, CBS9106, and Selinexor.In some embodiments, the nucleocytoplasmic transport inhibitor is leptomycin B. In some embodiments, the nucleocytoplasmic transport inhibitor is selinexor. In some embodiments, the cancer is a solid tumor. In some embodiments, the solid tumor is a sarcoma or a carcinoma. In some embodiments, the solid tumor is a fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovium, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma. , sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical carcinoma, uterine carcinoma, testicular tumor, lung cancer, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma, meningioma, melanoma, neuroblastoma, or retinoblastoma. In some embodiments, the solid tumor is a colon adenocarcinoma, pancreatic carcinoma, or melanoma. In some embodiments, the oncolytic virus and the nucleocytoplasmic transport inhibitor are administered simultaneously. In some embodiments, the oncolytic virus and the nucleocytoplasmic transport inhibitor are administered sequentially. In some embodiments, the oncolytic virus is administered prior to administration of the nucleocytoplasmic transport inhibitor. In some embodiments, the oncolytic virus is administered after the administration of the nucleocytoplasmic transport inhibitor. In some embodiments, the method comprises administering the oncolytic virus and the nucleocytoplasmic transport inhibitor for a period of at least 1 day, at least 2 days, at least 3 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 3 months, at least 6 months, or at least 1 year. In some embodiments, the method comprises administering the oncolytic virus, the nucleocytoplasmic transport inhibitor, or both to the subject twice a week, once a week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once a month, or once every 2 months. In some embodiments, the oncolytic virus is administered locally to the cancer tissue to be treated.In some embodiments, the oncolytic virus is administered by intratumoral injection. In some embodiments, the multiplicity of infection (MOI) of the oncolytic virus to the tissue is about 0.01 to about 10. In some embodiments, the multiplicity of infection (MOI) of the oncolytic virus to the tissue is about 0.05 to about 5. In some embodiments, the concentration of the nucleocytoplasmic transport inhibitor in the tissue is about 0.0001 μM to about 100 μM. In some embodiments, the concentration of the nucleocytoplasmic transport inhibitor in the tissue is about 0.01 μM to about 1 μM.

[0008] Disclosed herein, in some embodiments, is a method for killing cancer cells, the method comprising: contacting a cancer cell with an oncolytic virus and a nucleocytoplasmic transport inhibitor, thereby killing the cancer cell.

[0009] Disclosed herein, in some embodiments, is a method for enhancing the susceptibility of a cancer cell to infection by an oncolytic virus, the method comprising contacting the cancer cell with an oncolytic virus and a nucleocytoplasmic transport inhibitor.

[0010] Disclosed herein, in some embodiments, is a method for inducing cancer cells to be susceptible to infection by an oncolytic virus, the method comprising contacting the cancer cells with an oncolytic virus and a nucleocytoplasmic transport inhibitor.

[0011] Disclosed herein, in some embodiments, is a method for converting a non-permissive cancer cell into a permissive cancer cell, the method comprising contacting the non-permissive cancer cell with an oncolytic virus and a nucleocytoplasmic transport inhibitor.

[0012] Disclosed herein, in some embodiments, is a method for increasing the proliferation of an oncolytic virus in a cancer cell that is not susceptible to infection by the oncolytic virus, the method comprising contacting the cancer cell with an oncolytic virus and a nucleocytoplasmic transport inhibitor.

[0013] In some embodiments, the oncolytic virus is a virus belonging to the Poxviridae family. In some embodiments, the oncolytic virus is a virus belonging to the subfamily Chordopoxvirinae or Entomopoxvirinae. In some embodiments, the oncolytic virus is from the following virus genus: Orthopoxvirus, Serbidopoxvirus, Parapoxvirus, Avipoxvirus, Capripoxvirus, Leporipoxvirus, Suipoxvirus, Morsipoxvirus, Yatapoxvirus, Alphaentomopoxvirus, Betaentomopoxvirus, or Gammaentomopoxvirus. In some embodiments, the oncolytic virus is from the Leporipoxvirus genus. In some embodiments, the oncolytic virus is Myxoma virus (MYXV). In some embodiments, the oncolytic virus is from the Orthopoxvirus genus. In some embodiments, the oncolytic virus is genetically modified. In some embodiments, the nucleocytoplasmic transport inhibitor is selected from the group consisting of leptomycin A, leptomycin B, ratjadon A, ratjadon B, ratjadon C, ratjadon D, anginomycin A, goniothalamin, piperlongumine, plumbagin, curcumin, valtrate, acetoxychavicol acetate, prenylcoumarin osthole, KOS 2464, PKF050-638, CBS9106, and selinexor. In some embodiments, the nucleocytoplasmic transport inhibitor is leptomycin B. In some embodiments, the nucleocytoplasmic transport inhibitor is selinexor. In some embodiments, the cancer cell is a colon cancer cell, a pancreatic cancer cell, or a melanoma cancer cell. In some embodiments, the cancer cell is a human cancer cell. In some embodiments, the cancer cell is contacted in vivo with the oncolytic virus and the nucleocytoplasmic transport inhibitor. In some embodiments, the cancer cells are contacted with the oncolytic virus and the nucleocytoplasmic transport inhibitor ex vivo. In some embodiments, the method comprises contacting the cancer cells with the oncolytic virus and the nucleocytoplasmic transport inhibitor simultaneously.In some embodiments, the method comprises contacting the cancer cells with an oncolytic virus and a nucleocytoplasmic transport inhibitor sequentially. In some embodiments, the cancer cells are contacted with the oncolytic virus before being contacted with the nucleocytoplasmic transport inhibitor. In some embodiments, the cancer cells are contacted with the nucleocytoplasmic transport inhibitor before being contacted with the oncolytic virus. In some embodiments, the cancer cells are contacted with the oncolytic virus at an MOI of about 0.01 to about 10. In some embodiments, the cancer cells are contacted with the oncolytic virus at an MOI of about 0.05 to about 5. In some embodiments, the method comprises incubating the cancer cells in a medium comprising a nucleocytoplasmic transport inhibitor at a concentration of about 0.0001 μM to about 100 μM. In some embodiments, the method comprises incubating the cancer cells in a medium comprising a nucleocytoplasmic transport inhibitor at a concentration of about 0.01 μM to about 1 μM. In some embodiments, the method includes contacting the cancer cells with a medium containing a nucleocytoplasmic transport inhibitor at a concentration of about 0.0001 μM to about 100 μM. In some embodiments, the method includes contacting the cancer cells with a medium containing a nucleocytoplasmic transport inhibitor at a concentration of about 0.01 μM to about 1 μM. In some embodiments, the method further includes contacting the oncolytic virus with autologous or heterologous cells ex vivo. In some embodiments, the autologous or heterologous cells are bone marrow cells or macrophage cells. In some embodiments, the method further includes injecting the autologous or heterologous cells into the subject.

[0014] Disclosed herein, in some embodiments, is a pharmaceutical composition comprising an oncolytic virus and a nucleocytoplasmic transport inhibitor.

[0015] In some embodiments, the pharmaceutical composition further comprises a pharma- ceutically acceptable excipient or carrier. In some embodiments, the oncolytic virus is from the Poxviridae family. In some embodiments, the oncolytic virus is from the Chordopoxvirinae or Entomopoxvirinae subfamily. In some embodiments, the oncolytic virus is from the Orthopoxvirus, Serbidopoxvirus, Parapoxvirus, Avipoxvirus, Capripoxvirus, Leporipoxvirus, Suipoxvirus, Morsipoxvirus, Yatapoxvirus, Alphaentomopoxvirus, Betaentomopoxvirus, or Gammaentomopoxvirus genus of viruses. In some embodiments, the oncolytic virus is from the Leporipoxvirus genus. In some embodiments, the oncolytic virus is Myxoma virus (MYXV). In some embodiments, the oncolytic virus is from the Orthopoxvirus genus. In some embodiments, the oncolytic virus is genetically modified. In some embodiments, the nucleocytoplasmic transport inhibitor is selected from the group consisting of leptomycin A, leptomycin B, ratjadon A, ratjadon B, ratjadon C, ratjadon D, anginomycin A, goniothalamin, piperlongumine, plumbagin, curcumin, valtrate, acetoxychavicol acetate, prenylcoumarin osthole, KOS 2464, PKF050-638, CBS9106, and selinexor. In some embodiments, the nucleocytoplasmic transport inhibitor is leptomycin B. In some embodiments, the nucleocytoplasmic transport inhibitor is selinexor. In some embodiments, the pharmaceutical composition is in a unit dose dosage form suitable for intratumoral or parenteral administration. In some embodiments, the unit dose is about 1x10 per mL. 3 Plaque forming units (PFU) to approximately 1x10 10 In some embodiments, the unit dose comprises up to about 1x10 PFU of oncolytic virus per mL. 5 Plaque forming units (PFU) to approximately 1x10 10In some embodiments, the weight ratio between the oncolytic virus and the nucleocytoplasmic transport inhibitor is about 1x10 9 From about 1x10 -9 That is until now.

[0016] Some embodiments relate to a method of treating cancer, comprising administering to a subject a therapeutically effective amount of an oncolytic virus and a nucleocytoplasmic transport inhibitor. In one aspect, a method of killing cancer cells is described herein, comprising contacting a cancer cell with a therapeutically effective amount of an oncolytic virus and a nucleocytoplasmic transport inhibitor, thereby killing said cancer cell. In one aspect, a method of enhancing the susceptibility of a cancer cell to infection by an oncolytic virus is described herein, comprising contacting a cancer cell with an oncolytic virus and a nucleocytoplasmic transport inhibitor. In one aspect, a method of rendering a cancer cell susceptible to infection by an oncolytic virus is described herein, comprising contacting a cancer cell with an oncolytic virus and a nucleocytoplasmic transport inhibitor. In one aspect, a method of converting a non-permissive cancer cell to a permissive cancer cell is described herein, comprising contacting a non-permissive cancer cell with said therapeutically effective amount of an oncolytic virus and a nucleocytoplasmic transport inhibitor. In one aspect, a method is described herein for increasing the growth of an oncolytic virus in a cancer cell that is not susceptible to infection by the oncolytic virus, the method comprising contacting the cancer cell with an oncolytic virus and a nucleocytoplasmic transport inhibitor. In some embodiments, the oncolytic virus is a virus belonging to the family Poxviridae. In some embodiments, the oncolytic virus is a virus belonging to the subfamily Chordopoxvirinae or Entomopoxvirinae. In some embodiments, the oncolytic virus is from the following virus genus: Orthopoxvirus, Serbidopoxvirus, Parapoxvirus, Avipoxvirus, Capripoxvirus, Leporipoxvirus, Suipoxvirus, Morsipoxvirus, Yatapoxvirus, Alphaentomopoxvirus, Betaentomopoxvirus, or Gammaentomopox. In some embodiments, the oncolytic virus is from the genus Leporipoxvirus. In some embodiments, the oncolytic virus is Myxoma virus (MYXV). In some embodiments, the oncolytic virus is from the genus Orthopoxvirus. In some embodiments, the oncolytic virus is genetically engineered.In some embodiments, the nucleocytoplasmic transport inhibitor is selected from the group consisting of leptomycin A, leptomycin B, ratjadon A, ratjadon B, ratjadon C, ratjadon D, anginomycin A, goniothalamin, piperlongumine, plumbagin, curcumin, valtrate, acetoxychavicol acetate, prenylcoumarin osthole, KOS 2464, PKF050-638, CBS9106, and selinexor. In some embodiments, the nucleocytoplasmic transport inhibitor is leptomycin B. In some embodiments, the nucleocytoplasmic transport inhibitor is selinexor. In some embodiments, the cell is a human A549 cell, a HeLa cell, a 239 cell, or a primate Vero cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is an in vivo cell, an in vitro cell, or an ex vivo cell. In some embodiments, the cancer is a solid tumor. In some embodiments, the solid tumor is a sarcoma or a carcinoma. In some embodiments, the solid tumor is a fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelioma, lymphangiosarcoma, lymphangioendothelioma, synovium, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, The solid tumor is medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical carcinoma, uterine carcinoma, testicular, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma, meningioma, melanoma, neuroblastoma, or retinoblastoma. In some embodiments, the solid tumor is a colon adenocarcinoma, pancreatic carcinoma, or melanoma. In some embodiments, the oncolytic virus and the nucleocytoplasmic transport inhibitor are administered simultaneously or sequentially. In some embodiments, the oncolytic virus is administered before the administration of the nucleocytoplasmic transport inhibitor. In some embodiments, the oncolytic virus is administered after the administration of the nucleocytoplasmic transport inhibitor.In some embodiments, the method comprises administering the oncolytic virus and the nucleocytoplasmic transport inhibitor for a period of at least 1 day, at least 2 days, at least 3 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 3 months, at least 6 months, or at least 1 year. In some embodiments, the method comprises administering the oncolytic virus, the nucleocytoplasmic transport inhibitor, or both to the subject twice a week, once a week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once a month, or once every 2 months. In some embodiments, the oncolytic virus is administered locally to the cancer tissue to be treated. In some embodiments, the oncolytic virus is administered by injection. In some embodiments, the multiplicity of infection (MOI) of the oncolytic virus for the tissue is from about 0.01 to about 10. In some embodiments, the multiplicity of infection (MOI) of the oncolytic virus for the tissue is from about 0.05 to about 5. In some embodiments, the concentration of the nucleocytoplasmic transport inhibitor in the tissue is from about 0.0001 μM to about 100 μM. In some embodiments, the concentration of the nucleocytoplasmic transport inhibitor in the tissue is from about 0.01 μM to about 1 μM. In some embodiments, the method comprises contacting the cancer cells with the oncolytic virus and the nucleocytoplasmic transport inhibitor simultaneously or sequentially. In some embodiments, the cancer cells are contacted with the oncolytic virus before being contacted with the nucleocytoplasmic transport inhibitor. In some embodiments, the cancer cells are contacted with the nucleocytoplasmic transport inhibitor before being contacted with the oncolytic virus. In some embodiments, the method comprises pretreating the cancer cells with the nucleocytoplasmic transport inhibitor before being contacted with the oncolytic virus. In some embodiments, the cancer cells are contacted with the oncolytic virus at an MOI of from about 0.01 to about 10. In some embodiments, the cancer cells are contacted with the oncolytic virus at an MOI of from about 0.05 to about 5.In some embodiments, the method comprises incubating the cancer cells in a medium comprising the nucleocytoplasmic transport inhibitor at a concentration of about 0.0001 μM to about 100 μM. In some embodiments, the method comprises incubating the cancer cells in a medium comprising the nucleocytoplasmic transport inhibitor at a concentration of about 0.01 μM to about 1 μM. In some embodiments, the method comprises contacting the cancer cells with a medium comprising the nucleocytoplasmic transport inhibitor at a concentration of about 0.0001 μM to about 100 μM. In some embodiments, the method comprises contacting the cancer cells with a medium comprising the nucleocytoplasmic transport inhibitor at a concentration of about 0.01 μM to about 1 μM. In some embodiments, the method comprises contacting an autologous or heterologous cell with the oncolytic virus ex vivo. In some embodiments, the autologous or heterologous cell is a bone marrow cell or a macrophage cell. In some embodiments, the method comprises injecting the autologous or heterologous cell into the subject.

[0017] In one aspect, a pharmaceutical composition is described herein, the pharmaceutical composition comprising an oncolytic virus and a nucleocytoplasmic transport inhibitor. In some embodiments, the pharmaceutical composition further comprises a pharma- ceutical acceptable excipient or carrier. In some embodiments, the pharmaceutical composition is in a unit dose form suitable for intratumoral or parenteral administration. In some embodiments, the unit dose is about 1x10 per mL. 3 Plaque forming units (PFU) to approximately 1x10 10 In some embodiments, the unit dose comprises up to about 1x10 PFU of oncolytic virus per mL. 5 Plaque forming units (PFU) to approximately 1x10 10 In some embodiments, the weight ratio between the oncolytic virus and the nucleocytoplasmic transport inhibitor is about 1x10 9 From about 1x10 -9 That is until now. [Brief description of the drawings]

[0018] [Figure 1A] Figure 1A and Figure 1B illustrate that treatment of the human colon adenocarcinoma cell line HT29 with the nucleocytoplasmic transport inhibitor leptomycin B enhanced MYXV early (GFP) and late (TdTomato) gene expression levels. HT29 cells were pretreated with various concentrations of leptomycin B for 1 h and infected with vMyx-GFP-Tdtomato at an MOI of 5.0 in the presence of the inhibitor, and fluorescent images were captured using a fluorescent microscope at 24 hours post infection (HPI). [Figure 1B] Figure 1A and Figure 1B illustrate that treatment of the human colon adenocarcinoma cell line HT29 with the nucleocytoplasmic transport inhibitor leptomycin B enhanced MYXV early (GFP) and late (TdTomato) gene expression levels. HT29 cells were pretreated with various concentrations of leptomycin B for 1 h and infected with vMyx-GFP-Tdtomato at an MOI of 0.5 in the presence of the inhibitor, and fluorescent images were captured using a fluorescent microscope at 48 h post infection (HPI). [Diagram 2] Figure 2 illustrates that the nucleocytoplasmic transport inhibitor leptomycin B enhanced MYXV replication in the human colon adenocarcinoma cell line HT29. HT29 cells were pretreated with various concentrations of leptomycin B for 1 h. Cells were then infected with vMyx-GFP-TdTomato at an MOI of 0.5 or 0.05 for 1 h. After 1 h, unadsorbed virus was removed, cells were washed, and incubated with medium in the presence of inhibitors. Infected cells were harvested 72 h after infection, and virus titers were determined after serial dilution into RK13 cells. [Figure 3A] Figures 3A and 3B illustrate that treatment of human pancreatic cancer cell line PANC-1 with the nucleocytoplasmic transport inhibitor leptomycin B enhanced MYXV gene expression and replication. Figure 3A shows that PANC-1 cells were pretreated with various concentrations of leptomycin B for 1 hour and infected with vMyx-GFP-Tdtomato at an MOI of 5.0 in the presence of the inhibitor, and fluorescent images were taken 48 hours after infection using a fluorescent microscope. [Figure 3B] Figures 3A and 3B illustrate that treatment of human pancreatic cancer cell line PANC-1 with the nucleocytoplasmic transport inhibitor leptomycin B enhanced MYXV gene expression and replication. Figure 3B shows that PANC-1 cells were pretreated with various concentrations of leptomycin B for 1 h, and then the cells were infected with vMyx-GFP-TdTomato at an MOI of 5.0 or 0.5 for 1 h. After 1 h, unadsorbed virus was removed, and then the cells were washed and incubated with medium in the presence of the inhibitor. Infected cells were harvested 48 or 72 h after infection, and virus titers were determined after serial dilution into RK13 cells. [Figure 4A] Figures 4A and 4B illustrate that treatment of human melanoma cell line MDA-MB-435 with the nucleocytoplasmic transport inhibitor leptomycin B enhanced MYXV gene expression and replication. Figure 4A shows that MDA-MB-435 cells were pretreated with various concentrations of leptomycin B for 1 hour and infected with vMyx-GFP-Tdtomato at an MOI of 5.0 in the presence of the inhibitor, and fluorescent images were taken 48 hours after infection using a fluorescent microscope. [Figure 4B] Figures 4A and 4B illustrate that treatment of human melanoma cell line MDA-MB-435 with the nucleocytoplasmic transport inhibitor leptomycin B enhanced MYXV gene expression and replication. Figure 4B shows that MDA-MB-435 cells were pretreated with various concentrations of leptomycin B for 1 h, and then the cells were infected with vMyx-GFP-TdTomato at an MOI of 5.0 or 0.5 for 1 h. After 1 h, unadsorbed virus was removed, and then the cells were washed and cultured with medium in the presence of the inhibitor. Infected cells were harvested 48 or 72 h after infection, and virus titers were determined after serial dilution into RK13 cells. [Figure 5A]Figures 5A and 5B illustrate that treatment of human cancer cell lines with nucleocytoplasmic transport inhibitors and subsequent infection with MYXV reduced cell viability. Figure 5A shows that HT29 cells (20,000 cells in 100 ul medium / well) were cultured overnight in opaque-walled 96-well plates. The cells were then pretreated with various concentrations of leptomycin B for 1 hour and infected with vMyx-GFP-Tdtomato at an MOI of 5.0 in the presence of the inhibitors. Cell viability was determined by measuring ATP levels 64 hours after infection. [Figure 5B] Figure 5A and Figure 5B illustrate that treatment of human cancer cell lines with nucleocytoplasmic transport inhibitors and subsequent infection with MYXV reduced cell viability. Figure 5B shows that PANC-1 cells (20,000 cells in 100 ul medium / well) were cultured overnight in opaque-walled 96-well plates. The cells were then pretreated with various concentrations of leptomycin B for 1 hour and infected with vMyx-GFP-Tdtomato at an MOI of 5.0 in the presence of the inhibitors. Cell viability was determined by measuring the levels of ATP 40 hours after infection. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] Viruses naturally utilize permissive cells for infection and replication, but not all tumor cells are permissive to infection with oncolytic viruses. The present disclosure involves the surprising discovery that treating non-permissive cancer cells with one or more agents, such as nucleocytoplasmic transport inhibitors, can convert the non-permissive cells into permissive cells and enhance the replication of oncolytic viruses in the cancer cells, thereby enhancing the killing of the cancer cells.

[0020] The use of nucleocytoplasmic transport inhibitors, as demonstrated herein, can support and enhance gene expression and replication of oncolytic viruses in cancer cells that are not normally susceptible to infection by oncolytic viruses (i.e., not susceptible to oncolytic virus infection in the absence of nucleocytoplasmic transport inhibitors). The addition of nucleocytoplasmic transport inhibitors can also enhance the production of oncolytic virus progeny or the production of oncolytic viruses in host cells that are not normally susceptible to infection. The surprising impact of nucleocytoplasmic transport inhibitors on non-permissive cancer cells is manifested as an enhanced cancer cell killing effect when nucleocytoplasmic transport inhibitors are used in combination with oncolytic viruses, thereby providing a more effective method for treating cancer.

[0021] The present disclosure provides a method for treating cancer, for example, cancer that is not susceptible or has low susceptibility to oncolytic virus infection, by administering an oncolytic virus and a nucleocytoplasmic transport inhibitor. Also provided herein is a method for converting non-permissive cancer cells into permissive cancer cells, and a method for killing cancer cells by contacting the cancer cells with an oncolytic virus and a nucleocytoplasmic transport inhibitor. Further provided herein is a pharmaceutical composition comprising an oncolytic virus and a nucleocytoplasmic transport inhibitor.

[0022] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. Similar or equivalent methods and materials to those described herein can be used to practice or test this disclosure, and suitable methods and materials are described below. In addition, the materials, methods, and examples are merely illustrative and are not intended to be limiting.

[0023] The following explanations of terms and methods are provided to better describe the compounds, compositions, and methods of the present invention and to guide those of ordinary skill in the art in the practice of the present disclosure. It is also to be understood that the terms used in this disclosure are for the purpose of describing specific embodiments and examples only, and are not intended to be limiting.

[0024] As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0025] As used herein, the term "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, as well as the absence of a combination when interpreted as the alternative ("or").

[0026] The term "about" or "approximately" can mean within an acceptable error range of a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, "about" can mean 1 or more than 1 standard deviation per practice in the art. Alternatively, "about" can mean within a range of up to 20%, up to 15%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, especially with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, more preferably within 2-fold of a value.

[0027] As used herein, "one or more" or at least one may mean 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or any number more.

[0028] As used herein, the term "comprises" or "comprising" means "includes." Thus, "comprising A or B" means including A, B, or A and B. "Comprise," and variations such as "comprising," "comprises," and "comprised," as used herein, mean that various additional elements or steps can be used in combination.

[0029] "Effective amount" refers to the amount of a compound or composition sufficient to produce a desired effect (e.g., an increase in poxvirus production). In this example, the effective amount will vary within the knowledge and expertise of one of ordinary skill in the art, depending on factors such as the type of cell and the specific agent administered, the duration of treatment, the nature of any co-therapy, and the like.

[0030] The term "permissive" as used herein refers to whether a cell is prone to infection with a virus described in this application. The term "susceptible to infection" as used herein refers to the ability of a cell to be infected by a virus or another organism within the cell. It encompasses "permissive" infection, but the term is not intended to be limited to encompass situations where a cell is infected with an organism, but the organism does not necessarily replicate and / or spread from the infected cell to other cells. The phrase "viral propagation" as used herein describes the spread or passage of an infectious virus from a permissive cell type to additional cells, either of a permissive or susceptible nature.

[0031] The term "host cell" as used herein should be understood to mean broadly, without any limitation to a specific tissue in a tissue, organ, or isolated cell. Such cells may be a unique type of cell, or a group of different types of cells, such as cultured cell lines, primary cells, dividing cells, etc. Examples of host cells include, but are not limited to, prokaryotic cells, lower eukaryotic cells such as yeast, and other eukaryotic cells, such as insect cells and mammalian cells (human or non-human), as well as cells capable of producing oncolytic viruses. Host cells may be, for example, cancer cells in vivo.

[0032] "Nucleocytoplasmic transport inhibitors" refer to drugs that inhibit the transport of molecules through the nucleocytoplasmic transport pathway. The term "nucleocytoplasmic transport inhibitors" is used interchangeably with "nuclear export inhibitors". Examples of nucleocytoplasmic transport inhibitors include, but are not limited to, leptomycin A, leptomycin B, ratjadon A, ratjadon B, ratjadon C, ratjadon D, anginomycin A, goniothalamin, piperlongumine, plumbagin, curcumin, valtrate, acetoxychavicol acetate, prenylcoumarin osthole, KOS 2464, PKF050-638, CBS9106, and selinexol.

[0033] A "virus" is a microscopic infectious organism that reproduces within living cells. A virus may consist essentially of a single nucleic acid nucleus surrounded by a protein membrane and has the ability to replicate only inside living cells. "Viral replication" is the production of additional viruses by the occurrence of at least one viral life cycle. Viruses may disrupt the normal function of a host cell, causing the cell to behave in a manner determined by the virus. For example, viral infection may cause a cell to produce or respond to cytokines that an uninfected cell would not normally do. As used herein, the term "replication-competent" refers to a virus that is capable of infecting and replicating within a particular host cell.

[0034] "Poxvirus" is a virus belonging to the Poxviridae family. Poxvirus is a double-stranded DNA virus that can infect both vertebrates and invertebrates. Poxvirus includes the species and genera of viruses classified as part of the subfamily Chordopoxvirinae, such as Orthomopoxvirus, Parapoxvirus, Avipoxvirus, Capripoxvirus, Leporipoxvirus, Suipoxvirus, Morsipoxvirus, and Yatapoxvirus, and the subfamily Entomopoxvirinae, including Alphaentomopoxvirus, Betaentomopoxvirus, and Gammaentomopoxvirus.

[0035] "Contacting" means placing in direct physical association. Contacting includes contact between molecules, such as an inhibitor and a cell, for example, by placing an agent in direct physical association with a cell, such as in a culture with the cells. In some embodiments, contacting or exposing an agent to a cell includes placing the agent in the growth medium of the cell.

[0036] "Inhibit" means, for example, to suppress transport in a nuclear export pathway to a measurable extent. An "inhibitor" is, for example, a substance that can inhibit transport in a nuclear export pathway to a measurable extent.

[0037] The terms "treat", "treated", "treating", "treatment" and the like are meant to encompass prevention (e.g., preventative measures in subjects at risk of having the pathological disease to be treated) and / or therapy (e.g., in subjects diagnosed with the pathological disease). The outcome of treatment is to delay, cure, reduce, control, or ameliorate the disorder and / or symptoms associated therewith. "Treating" encompasses the concept of "alleviating", which refers to reducing the frequency of occurrence or recurrence, or the severity, of any symptoms and disease effects associated with the disorder and / or side effects. The term "treating" also encompasses the concept of "managing", which refers to reducing the severity or delaying the recurrence of a particular disease or disorder in a patient, for example, extending the period of remission in a patient suffering from the disease. The term "treating" further encompasses the concepts of "prevent", "preventing", and "prevention", as previously mentioned. It is understood that treating a disorder or disease does not require, but does not preclude, the complete elimination of the disorder, disease, or symptoms associated therewith.

[0038] A "pharmaceutical acceptable excipient or diluent" refers to an excipient, carrier, or diluent that can be administered to a subject together with a drug and that does not destroy its pharmacological activity and is non-toxic when administered in a dosage sufficient to deliver a therapeutic amount of the drug.

[0039] Suitable methods and materials for the practice or testing of the present disclosure are described below. Such methods and materials are merely illustrative and are not intended to be limiting. Other methods and materials similar or equivalent to those described herein can be used. For example, conventional methods well known in the art to which this disclosure pertains are described in a variety of general and more specific references, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, 1989; Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Press, 2001; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates, 1992 (and Supplements to 2000); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, 4th ed., Wiley & Sons, 1999; Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1990, and Harlow and Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0040] Oncolytic viruses Oncolytic viruses as described herein include viruses that can selectively replicate in dividing cells (e.g., proliferating cells such as cancer cells), with the goal of slowing down the proliferation of dividing cells and / or lysing them, while showing no or minimal replication in non-dividing cells, either in vitro or in vivo.Typically, oncolytic viruses contain viral genomes packaged into viral particles or virions, and are infectious (i.e., can infect and enter into host cells or subjects).As used herein, this term includes DNA or RNA vectors (depending on the type of virus), as well as the viral particles produced therewith.

[0041] In some embodiments, oncolytic viruses are mammalian viruses that have been selected and / or engineered for their ability to selectively infect and kill transfected cancer cells and for their ability to activate the host immune system not only against the virus but also against tumor antigens.

[0042] In some embodiments, the oncolytic virus is a virus that belongs to or is derived from the Poxviridae family. Members of the Poxviridae family of viruses are a diverse group of large, complex, double-stranded DNA viruses that can replicate in the cytoplasm of infected cells. The genomes of most poxviruses are about 150,000-300,000 base pairs in length and encode approximately 150-300 proteins. Approximately half of these viral proteins are highly conserved among the various poxvirus members and perform essential functions such as cell binding and entry, genome replication, transcription, and virion assembly. Other viral proteins are involved in evading many host defense functions. Poxvirus genes can be expressed in different phases. For example, early gene products can include proteins essential for viral DNA replication and are expressed before DNA is replicated. Meanwhile, intermediate / late gene products, expressed during or after DNA replication, can include structural proteins required for virion maturation. All steps of this complex viral replication process, starting from decoding the genome, through early gene expression, DNA replication, late gene expression, and the even more complex process of virion maturation, can occur exclusively in the cytoplasm of infected cells.

[0043] Poxviruses are large DNA viruses that can replicate exclusively in the cytoplasm of infected cells. They can encode all the proteins necessary for DNA and mRNA synthesis. Apart from the viral proteins involved in poxvirus replication, about half of the poxvirus genome encodes proteins shown to be necessary for the inhibition or manipulation of diverse intracellular antiviral signaling pathways that function in the cytoplasm and nucleus. However, there is evidence that host cell proteins from the cytoplasmic and nuclear compartments are involved in at least some steps of poxvirus replication. Many diverse cellular proteins and signaling pathways have been intimately linked to defending cells against poxvirus infection and replication. This is why more than half of the genome-encoded poxvirus proteins are involved in the specific inhibition or regulation of these host antiviral pathways. In some cases, the virally encoded proteins function in a host-specific manner and determine whether a given poxvirus can successfully infect and replicate in a particular species of host. Members of the poxviruses, such as myxoma virus (MYXV), leporipoxvirus, and vaccinia virus (VACV), an orthopoxvirus, can be developed as oncolytic viruses for the treatment of human cancers.

[0044] In certain embodiments, the poxvirus of interest is genetically modified. For example, the poxvirus of interest can be modified to carry other genes, such as therapeutic genes, and / or to delete or disrupt one or more endophytic viral genes. In the case of viruses modified to treat cancer, the genes will typically be selected to enhance the anti-cancer effect of the treatment. Such genes may be genes involved in causing apoptosis or genes involved in targeting infected cells for immune destruction, including genes that repair deficiencies in the response to interferon, or genes that cause the expression of cell surface markers that stimulate an antibody production response, such as bacterial cell surface antigens. Viruses may also be modified to express genes involved in stopping the proliferation and growth of tumorigenic or cancerous cells, thereby preventing the cells from dividing. In addition, viruses may be modified to include therapeutic genes, such as genes involved in the synthesis of chemotherapeutic agents, or to increase the level of replication in the cells of the particular species from which the cells to be inhibited or killed are derived, such as human cells.

[0045] In some embodiments, the oncolytic virus is a virus belonging to the subfamily Chordopoxvirinae or Entomopoxvirinae, or is derived from the subfamily Chordopoxvirinae or Entomopoxvirinae. In some embodiments, the oncolytic virus is a virus of the genus Orthopoxvirus, cervidopoxvirus, parapoxvirus, avipoxvirus, capripoxvirus, leporipoxvirus, suipoxvirus, morsipoxvirus, yatapoxvirus, alphaentomopoxvirus, betaentomopoxvirus, or gammaentomopoxvirus. In some embodiments, the oncolytic virus is a virus of the genus Orthopoxvirus, cervidopoxvirus, parapoxvirus, avipoxvirus, capripoxvirus, leporipoxvirus, suipoxvirus, morsipoxvirus, yatapoxvirus, alphaentomopoxvirus, betaentomopoxvirus, or gammaentomopoxvirus. In some embodiments, the oncolytic virus is a virus belonging to the Orthopoxvirus genus or derived from the Orthopoxvirus genus. In some embodiments, the oncolytic virus is a Vaccinia virus or a virus derived from the Vaccinia virus. In some embodiments, the Vaccinia virus is a Vaccinia virus strain selected from the group consisting of Lister, Wyeth, Western Reserve, Modified Vaccinia virus Ankara, and LC16m series. In some embodiments, the oncolytic virus is a Raccoonpox virus or a virus derived from the Raccoonpox virus. In some embodiments, the oncolytic virus is a virus belonging to the Serbidopoxvirus genus or derived from the Serbidopoxvirus genus. In some embodiments, the oncolytic virus is a Sheeppox virus or a virus derived from the Sheeppox virus.In some embodiments, the oncolytic virus belongs to the genus Parapoxvirus or is derived from a virus of the genus Parapoxvirus. In some embodiments, the oncolytic virus belongs to the genus Avipoxvirus or is derived from a virus of the genus Avipoxvirus. In some embodiments, the oncolytic virus belongs to the genus Capripoxvirus or is derived from a virus of the genus Capripoxvirus. In some embodiments, the oncolytic virus belongs to the genus Suipoxvirus or is derived from a virus of the genus Suipoxvirus. In some embodiments, the oncolytic virus belongs to the genus Morsipoxvirus or is derived from a virus of the genus Morsipoxvirus. In some embodiments, the oncolytic virus belongs to the genus Yatapoxvirus or is derived from a virus of the genus Yatapoxpoxvirus. In some embodiments, the oncolytic virus belongs to the genus Alphaentomopoxvirus or is derived from a virus of the genus Alphaentomopoxvirus. In some embodiments, the oncolytic virus belongs to the genus Betaentomopoxvirus or is derived from a virus of the genus Betaentomopox. In some embodiments, the oncolytic virus belongs to or is derived from the genus Gamma-entomopoxvirus. In some embodiments, the oncolytic virus belongs to or is derived from the genus Leporipoxvirus. In some embodiments, the oncolytic virus is or is derived from the genus Myxoma virus (MYXV).

[0046] Due to its unique biology, MYXV is potentially well suited as a therapeutic virus against solid cancers such as osteosarcoma. MYXV is a member of the Poxviridae family and a member of the Leporipoxvirus genus. Thus, in an embodiment, the poxvirus of interest is a candidate oncolytic virus. In an embodiment, the poxvirus of interest is a Leporipoxvirus, such as Myxoma virus. In an embodiment, the poxvirus of interest is an Orthopoxvirus, such as Vaccinia virus, including various strains of Vaccinia virus, such as Lister, Wyeth, Western Reserve (WR), Modified Vaccinia virus Ankara (MVA), and LC16m series, and Raccoon poxvirus (RCNV). In an embodiment, the poxvirus of interest is a Yatapoxvirus, such as Tanapoxvirus (TPV) or Yaba-like disease virus (YLDV). In certain embodiments, the poxvirus of interest is a capripoxvirus, such as sheeppoxvirus.

[0047] MYXV is a rabbit-specific pathogen. MYXV can be any autonomous virus belonging to the Leporipoxvirus species of poxvirus. In some embodiments, MYXV is a wild-type strain of MYXV or is derived from a wild-type strain of MYXV. In some embodiments, MYXV is a recombinant strain of MYXV or is derived from a recombinant strain of MYXV. In some examples, MYXV is a Lausanne strain or is derived from a Lausanne strain. In some examples, MYXV is a South American MYXV strain circulating in forest rabbits (Sylvilagus brasiliensis) or is derived from a South American MYXV strain circulating in forest rabbits (Sylvilagus bachmani). In some examples, MYXV is a California MYXV strain circulating in brush rabbits (Sylvilagus bachmani) or is derived from a California MYXV strain circulating in brush rabbits. In some examples, the MYXV is or is derived from 6918, an attenuated Spanish field strain (GenBank Accession number EU552530, provided by GenBank on July 27, 2019 and incorporated by reference) that contains modifications in genes M009L, M036L, M135R, and M148R. In some examples, the MYXV is or is derived from 6918VP60-T2 (GenBank Accession number EU552531, provided by GenBank on July 27, 2019 and incorporated by reference). In some examples, the MYXV is or is derived from a strain designated Standard laboratory Strain (SLS).

[0048] In some examples, the MYXV comprises at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, such as between 95% and 98%, between 95% and 99%, including 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, to the sequence disclosed in Cameron, et al., "The complete DNA sequence of Myxoma Virus," Virology 264: 298-318 (1999), which is incorporated by reference in its entirety. In some instances, the MYXV comprises the sequence disclosed in Cameron, et al., "The complete DNA sequence of Myxoma Virus," Virology 264: 298-318 (1999).

[0049] In some embodiments, MYXV is non-pathogenic in humans (e.g., immunocompetent humans), but has the ability to infect and kill a wide range of human cancer cells originating from various tissues. In normal primary human cells, MYXV replication can be limited by multiple factors, such as cell binding determinants, intracellular antiviral signaling pathways, and type I IFN and / or other cytokine-mediated cellular antiviral state. In human cancer cells, these self-defense cellular pathways are commonly defective. MYXV replication in some human cancer cells may depend on cellular RNA helicase family proteins. Without wishing to be bound by theory, RNA helicases that shuttle between nuclear and cytoplasmic compartments of cells may affect MYXV replication in virus-infected cells. Besides RNA helicases, other nuclear proteins may contribute to the replication cycle of MYXV and other poxviruses. For example, nuclear proteins may affect the replication efficiency of poxviruses in transformed human host cell lines.

[0050] In some embodiments, the oncolytic virus belongs to the virus family consisting of Poxviridae, Herpesviridae, Reoviridae, Paramyxoviridae, Retroviridae, Adenoviridae, Rhabdoviridae, Picornaviridae, Parvoviridae, and Picornaviridae, or is derived from the virus family consisting of Poxviridae, Herpesviridae, Reoviridae, Paramyxoviridae, Retroviridae, Adenoviridae, Rhabdoviridae, Picornaviridae, Parvoviridae, and Picornaviridae. In some embodiments, the oncolytic virus belongs to the Herpesviridae family or is derived from the Herpesviridae family. In some embodiments, the oncolytic virus belongs to the Reoviridae family or is derived from the Reoviridae family. In some embodiments, the oncolytic virus belongs to the Paramyxoviridae family or is derived from the Paramyxoviridae family. In some embodiments, the oncolytic virus belongs to the Retroviridae family or is derived from the Retroviridae family. In some embodiments, the oncolytic virus belongs to the Adenoviridae family or is derived from the Adenoviridae family. In some embodiments, the oncolytic virus belongs to the Rhabdoviridae family or is derived from the Rhabdoviridae family. In some embodiments, the oncolytic virus belongs to the Picornaviridae family or is derived from the Picornaviridae family. In some embodiments, the oncolytic virus belongs to the Parvoviridae family or is derived from the Parvoviridae family. In some embodiments, the oncolytic virus belongs to the Picornaviridae family. In some embodiments, the oncolytic virus belongs to the Simplexvirus, Rubulavirus, or Senecavirus genus or is derived from the Simplexvirus, Rubulavirus, or Senecavirus genus. In some embodiments, the oncolytic virus belongs to the Simplexvirus genus or is derived from the Simplexvirus genus.In some embodiments, the oncolytic virus belongs to or is derived from the Rubulavirus genus. In some embodiments, the oncolytic virus belongs to or is derived from the Senecavirus genus. In some embodiments, the oncolytic virus belongs to or is derived from the following virus species: Measles, Fowlpox, and Vesicular Stomatitis virus, Mumps rubulavirus, Coxsackievirus, and Vaccinia. In some embodiments, the oncolytic virus is a measles virus or is derived from a measles virus. In some embodiments, the oncolytic virus is a fowlpox virus or is derived from a fowlpox virus. In some embodiments, the oncolytic virus is a vesicular stomatitis virus or is derived from a vesicular stomatitis virus. In some embodiments, the oncolytic virus is a mumps rubula virus or derived from a mumps rubula virus. In some embodiments, the oncolytic virus is a coxsackie virus or derived from a coxsackie virus. In some embodiments, the oncolytic virus is a vaccinia virus or derived from a vaccinia virus.

[0051] In some embodiments, the oncolytic virus is autonomous. In some embodiments, the oncolytic virus belongs to or is derived from a wild-type strain. In some embodiments, the oncolytic virus belongs to or is derived from a genetically modified strain. In some embodiments, the oncolytic virus is genetically modified. In some embodiments, the oncolytic virus is non-pathogenic in humans, but capable of infecting and killing a wide range of human cancer cells originating from various tissues. In some embodiments, the nucleic acid sequence of the oncolytic virus comprises at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence of the wild-type virus sequence, such as between 95% and 98%, between 95% and 99%, including 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%.

[0052] Nucleocytoplasmic transport inhibitors As disclosed herein, nucleocytoplasmic transport inhibitors can enhance viral replication and suppress cell viability for cancer cells, such as non-permissive cancer cells. In some embodiments, the nucleocytoplasmic transport inhibitors are selective inhibitors. In some embodiments, the nucleocytoplasmic transport inhibitors are not selective. In some embodiments, the described nucleocytoplasmic transport inhibitors are agents that can interfere with nucleocytoplasmic transport. In some embodiments, the agents inhibit nucleocytoplasmic transport by interfering with transport proteins. In some embodiments, the agent comprises trifluoperazine hydrochloride, W13, ETP-45648, Vinblastine, Akt inhibitor X, INCA, SMIP001 / 004, Resveratrol, Elliticine, WGA, cSN50 peptide, bimax1 / 2 peptide, leptomycin B, anginomycin, goniothalamin, latjadone, valtrate, acetoxychavicol acetate, 15d-PGJ2, Peumusolide A, PKF050-638, KOS 2464, CBS9106, or a combination thereof. In some embodiments, the nucleocytoplasmic transport inhibitor comprises a small molecule compound. In some embodiments, the nucleocytoplasmic transport inhibitor comprises a natural compound, such as latjadone, valtrate, and acetoxychavicol acetate. In some embodiments, the nucleocytoplasmic transport inhibitor comprises a selective inhibitor of nuclear export (SINE). In some embodiments, the nucleocytoplasmic transport inhibitor comprises a reversible nuclear export inhibitor, such as CBS9106.

[0053] In an embodiment, the nucleocytoplasmic transport inhibitor is leptomycin A, leptomycin B, ratjadone A, B, C, anginomycin A, goniothalamin, piperlongumine, plumbagin, curcumin, valtrate, acetoxychavicol acetate, prenylcoumarin osthole, or synthetic nucleocytoplasmic transport inhibitors such as KOS 2464, PKF050-638 (N-azolylacrylate analogue, CBS9106, selinexor, and the like, as described in Mathew and Ghildyal, CRM1 inhibitors for antiviral therapy, Frontiers in Microbiology 2017, Vol 8, article herein incorporated by reference. 1171. In some embodiments, the nucleocytoplasmic transport inhibitor comprises leptomycin A. In some embodiments, the nucleocytoplasmic transport inhibitor comprises leptomycin B. In some embodiments, the nucleocytoplasmic transport inhibitor comprises ratjadone A. In some embodiments, the nucleocytoplasmic transport inhibitor comprises ratjadone B. In some embodiments, the nucleocytoplasmic transport inhibitor comprises ratjadone C. In some embodiments, the nucleocytoplasmic transport inhibitor comprises ratjadone D. In some embodiments, the nucleocytoplasmic transport inhibitor comprises anginomycin A. In some embodiments, the nucleocytoplasmic transport inhibitor comprises leptomycin B. In some embodiments, the nucleocytoplasmic transport inhibitor comprises ratjadone C. In some embodiments, the nucleocytoplasmic transport inhibitor comprises ratjadone D. In some embodiments, the nucleocytoplasmic transport inhibitor comprises anginomycin A. In some embodiments, the nucleocytoplasmic transport inhibitor comprises goniothalamin. In some embodiments, the nucleocytoplasmic transport inhibitor comprises piperlongumine. In some embodiments, the nucleocytoplasmic transport inhibitor comprises plumbagin. In some embodiments, the nucleocytoplasmic transport inhibitor comprises curcumin. In some embodiments, the nucleocytoplasmic transport inhibitor comprises valtrate. In some embodiments, the nucleocytoplasmic transport inhibitor comprises acetoxychavicol acetate. In some embodiments, the nucleocytoplasmic transport inhibitor comprises the prenylcoumarin osthole. In some embodiments, the nucleocytoplasmic transport inhibitor comprises KOS 2464. In some embodiments, the nucleocytoplasmic transport inhibitor comprises PKF050-638. In some embodiments, the nucleocytoplasmic transport inhibitor comprises CBS9106. In some embodiments, the nucleocytoplasmic transport inhibitor comprises selinexol.In some embodiments, the nucleocytoplasmic transport inhibitor is leptomycin B.

[0054] Treatment Method The use of nucleocytoplasmic transport inhibitors significantly helps to convert cancer cells that are not susceptible to infection by oncolytic viruses (e.g., MYXV) into cancer cells that are susceptible to infection by oncolytic viruses, i.e., induces the susceptibility of cancer cells to infection by oncolytic viruses. The addition of nucleocytoplasmic transport inhibitors increases the susceptibility of cancer cells to infection by oncolytic viruses. Nucleocytoplasmic transport inhibitors can be used to transform cancer cells that have low susceptibility to infection by oncolytic viruses into cancer cells that have high susceptibility to infection by oncolytic viruses. Thus, to improve the effectiveness of oncolytic viruses in treating cancer and killing tumor cells, nucleocytoplasmic transport inhibitors can be administered in combination with oncolytic viruses.

[0055] Disclosed herein is a method of using an oncolytic virus and a composition comprising an oncolytic virus. Disclosed herein, in some embodiments, is a method of treating cancer by administering to a subject a therapeutically effective amount of an oncolytic virus (e.g., MYXV or VACV) and a nucleocytoplasmic transport inhibitor. In one aspect, disclosed herein is a method of converting a non-permissive cell into a permissive cell, comprising contacting a cancer cell that is non-permissive to an oncolytic virus with an oncolytic virus and a nucleocytoplasmic transport inhibitor, thereby converting said cancer cell. Disclosed herein in another aspect is a method of killing a cancer cell, comprising contacting a cancer cell with an oncolytic virus and a nucleocytoplasmic transport inhibitor, thereby killing said cancer cell. Accordingly, in some embodiments, the present disclosure provides a method of increasing virus replication in a non-permissive cancer cell by treating the non-permissive cell with a nucleocytoplasmic transport inhibitor.

[0056] Some embodiments relate to a method of enhancing the susceptibility of a cancer cell to infection by an oncolytic virus, the method comprising contacting the cancer cell with an oncolytic virus and a nucleocytoplasmic transport inhibitor.Some embodiments relate to a method of rendering a cancer cell susceptible to infection by an oncolytic virus, the method comprising contacting the cancer cell with an oncolytic virus and a nucleocytoplasmic transport inhibitor.

[0057] Some embodiments relate to a method of increasing the proliferation of an oncolytic virus in a cancer cell that is not susceptible to infection by the oncolytic virus, the method comprising contacting the cancer cell with an oncolytic virus and a nucleocytoplasmic transport inhibitor.

[0058] The oncolytic virus and the nucleocytoplasmic transport inhibitor may be administered together or separately. In some embodiments, the oncolytic virus and the nucleocytoplasmic transport inhibitor may be administered together. In some embodiments, the oncolytic virus and the nucleocytoplasmic transport inhibitor may be administered separately. When the oncolytic virus and the nucleocytoplasmic transport inhibitor are administered separately, the oncolytic virus may be administered prior to the nucleocytoplasmic transport inhibitor. When the oncolytic virus and the nucleocytoplasmic transport inhibitor are administered separately, the oncolytic virus may be administered after the nucleocytoplasmic transport inhibitor.

[0059] In some embodiments, the cancer cell, e.g., the non-permissive cancer cell, is an animal cell, such as a mammalian cell. In some embodiments, the cancer cell, e.g., the non-permissive cancer cell, is a human cell. In some embodiments, the cell is an immortalized human cell or a primate cell. In some embodiments, the cell belongs to a cancer cell line. In some embodiments, the cell is a human A549 cell, a HeLa cell, a 239 cell, or a primate Vero cell. The cancer cell can be an in vivo cell, an in vitro cell, or an ex vivo cell. In some embodiments, the cancer cell is a cell of the cancer tissue of the subject to be treated.

[0060] The cancer may be a solid tumor or hematoma. In some embodiments, the cancer is leukemia (e.g., acute leukemia, acute lymphocytic leukemia, acute myelogenous leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia), polycythemia vera, lymphoma (e.g., Hodgkin's disease, non-Hodgkin's disease), Waldenstrom's macroglobulinemia, heavy chain disease, or solid tumor. In some embodiments, the cancer is a solid tumor. In some embodiments, the solid tumor is a sarcoma or carcinoma. In some embodiments, the solid tumor is selected from the group consisting of fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelioma, lymphangiosarcoma, lymphangioendothelioma, synovium, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, The solid tumor is a medullary carcinoma, a bronchogenic carcinoma, a renal cell carcinoma, a hepatoma, a cholangiocarcinoma, a choriocarcinoma, a seminoma, an embryonal carcinoma, a Wilms' tumor, a cervical carcinoma, a uterine carcinoma, a testicular carcinoma, a lung carcinoma, a small cell lung carcinoma, a bladder carcinoma, an epithelial carcinoma, a glioma, an astrocytoma, a medulloblastoma, a craniopharyngioma, an ependymoma, a pinealoma, a hemangioblastoma, an acoustic neuroma, an oligodendroglioma, a schwannoma, a meningioma, a melanoma, a neuroblastoma, or a retinoblastoma. In some embodiments, the solid tumor is a colon adenocarcinoma, a pancreatic carcinoma, or a melanoma. In some embodiments, the solid tumor is a bone cancer, such as a chondrosarcoma, a Ewing's sarcoma, and an osteosarcoma. In some embodiments, the solid tumor is an osteosarcoma.

[0061] The oncolytic virus and the nucleocytoplasmic transport inhibitor can be administered simultaneously or sequentially to a subject with cancer. In some embodiments, the oncolytic virus and the nucleocytoplasmic transport inhibitor are administered simultaneously to the subject. In some embodiments, the oncolytic virus and the nucleocytoplasmic transport inhibitor are premixed prior to administration to the subject. In some embodiments, the oncolytic virus and the nucleocytoplasmic transport inhibitor are administered separately to the subject. In some embodiments, the oncolytic virus is administered prior to the nucleocytoplasmic transport inhibitor. In some embodiments, the oncolytic virus is administered after the nucleocytoplasmic transport inhibitor. In some embodiments, the method comprises contacting the cancer cells or tissue with the oncolytic virus and the nucleocytoplasmic transport inhibitor simultaneously or sequentially. In some embodiments, the cancer cells or tissue are contacted with the oncolytic virus prior to being contacted with the nucleocytoplasmic transport inhibitor. In some embodiments, the cancer cells or tissue are contacted with the nucleocytoplasmic transport inhibitor prior to being contacted with the oncolytic virus. In some embodiments, the method comprises contacting the cancer cells or tissue with the premixed oncolytic virus and the nucleocytoplasmic transport inhibitor. In some embodiments, the cancer cells or tissues are contacted separately with the oncolytic virus and the nucleocytoplasmic transport inhibitor.

[0062] In some embodiments, the method comprises pretreating cancer cells or cancer tissue with a nucleocytoplasmic transport inhibitor.In some embodiments, the cancer cells or cancer tissue is pretreated with a nucleocytoplasmic transport inhibitor for at least 1 minute, at least 2 minutes, at least 5 minutes, at least 10 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 6 hours, at least 12 hours, at least 24 hours, or at least 1 week before contacting the cells or tissue with an oncolytic virus.In some embodiments, the cancer cells or cancer tissue is pretreated with a nucleocytoplasmic transport inhibitor for up to 1 minute, up to 2 minutes, up to 5 minutes, up to 10 minutes, up to 30 minutes, up to 1 hour, up to 2 hours, up to 6 hours, up to 12 hours, up to 24 hours, or up to 1 week before contacting the cells or tissue with an oncolytic virus. In some embodiments, the cancer cells or tissues are pretreated with a nucleocytoplasmic transport inhibitor for a period of about 1 minute to about 1 day, about 5 minutes to about 12 hours, about 10 minutes to about 2 hours, or about 30 minutes to about 90 minutes before contacting the cells or tissues with an oncolytic virus. In some embodiments, the cancer cells or tissues are pretreated with a nucleocytoplasmic transport inhibitor for about 1 hour before contacting the cells or tissues with an oncolytic virus.

[0063] In one aspect, methods are described herein that include administering an oncolytic virus to cells that are autologous, allogeneic, or xenogeneic to a subject harboring cancer cells. The oncolytic virus can be administered in vivo or ex vivo. In some embodiments, the method includes administering the oncolytic virus to autologous cells ex vivo. In some embodiments, the method includes administering the oncolytic virus to allogeneic cells ex vivo. In some embodiments, the method includes administering the oncolytic virus to xenogeneic cells ex vivo. In some embodiments, the autologous, allogeneic, or xenogeneic cells are bone marrow cells or macrophage cells. In some embodiments, the autologous, allogeneic, or xenogeneic cells are stem cells. In some embodiments, the autologous, allogeneic, or xenogeneic cells are any cells that are non-permissive to oncolytic viruses. In some embodiments, the autologous, allogeneic, or xenogeneic cells are any cells that are permissive to oncolytic viruses. In some embodiments, the autologous, allogeneic, or xenogeneic cells are healthy cells. In some embodiments, the administering step comprises contacting an oncolytic virus with an autologous, allogeneic, or xenogeneic cell. In some embodiments, the method comprises administering said autologous, allogeneic, or xenogeneic cell to a subject. In some embodiments, the method comprises injecting said autologous, allogeneic, or xenogeneic cell to a subject. In some embodiments, the method comprises administering a nucleocytoplasmic transport inhibitor prior to administration of the autologous, allogeneic, or xenogeneic cell. In some embodiments, the method comprises administering a nucleocytoplasmic transport inhibitor after administration of the autologous, allogeneic, or xenogeneic cell.

[0064] In some embodiments, the method comprises administering to the subject an oncolytic virus, a nucleocytoplasmic transport inhibitor, or both for a period of time. In some embodiments, the period of time is at least 1 day, at least 2 days, at least 3 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 3 months, at least 6 months, or at least 1 year. In some embodiments, the period of time is up to 1 day, up to 2 days, up to 3 days, up to 1 week, up to 2 weeks, up to 3 weeks, up to 4 weeks, up to 1 month, up to 3 months, up to 6 months, up to 1 year, or up to 10 years. In some embodiments, the period of time is from about 1 day to about 1 year, from about 1 month to about 12 months, or from about 1 month to about 6 months. In some embodiments, the method comprises administering to the subject an oncolytic virus, a nucleocytoplasmic transport inhibitor, or both twice a week, once a week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once a month, or once every 2 months. In some embodiments, the oncolytic virus, the nucleocytoplasmic transport inhibitor, or both, are administered to the subject from 1 to 4 weeks apart, e.g., about 1 week apart, about 2 weeks apart, or about 3 weeks apart.

[0065] In some embodiments, the method includes administering the oncolytic virus, the nucleocytoplasmic transport inhibitor, or both according to an initial dose schedule and a subsequent dose schedule. In some embodiments, the initial dose schedule includes a different dose schedule than the subsequent dose schedule. In some embodiments, the initial dose schedule includes 1-10 treatments, such as 1-4 treatments or 2-3 treatments, of the oncolytic virus, the nucleocytoplasmic transport inhibitor, or both. In some embodiments, each treatment of the oncolytic virus, the nucleocytoplasmic transport inhibitor, or both is administered 1 week to about 6 weeks apart according to the initial dose schedule. In some embodiments, each treatment of the oncolytic virus, the nucleocytoplasmic transport inhibitor, or both is administered about 1 week apart, about 2 weeks apart, about 3 weeks apart, or about 4 weeks apart according to the initial dose schedule. In some embodiments, each treatment of the oncolytic virus, the nucleocytoplasmic transport inhibitor, or both is administered 1 week to about 6 weeks apart according to the subsequent dose schedule. In some embodiments, each treatment of oncolytic virus, nucleocytoplasmic transport inhibitor, or both, is administered about 1 week apart, about 2 weeks apart, about 3 weeks apart, or about 4 weeks apart according to the subsequent dosing schedule. In some embodiments, the oncolytic virus, nucleocytoplasmic transport inhibitor, or both, is administered 3 weeks apart in the initial dosing schedule and about 2 weeks apart in the subsequent dosing schedule.

[0066] In some embodiments, the method includes local administration to the cancer tissue to be treated. For example, in some embodiments, the oncolytic virus, the nucleocytoplasmic transport inhibitor, or both, are administered locally to the cancer tissue to be treated. In some embodiments, the oncolytic virus is administered locally and the nucleocytoplasmic transport inhibitor is not administered locally. In some embodiments, the oncolytic virus, the nucleocytoplasmic transport inhibitor, or both, are administered parenterally. In some embodiments, the nucleocytoplasmic transport inhibitor, or both, are administered by injection. In some embodiments, the oncolytic virus, the nucleocytoplasmic transport inhibitor, or both, are administered by injection into the skin, subcutaneous injection, or into a nodular lesion. In some embodiments, the oncolytic virus, the nucleocytoplasmic transport inhibitor, or both, are administered by injection into the cancer tissue or into a cancer organ containing the cancer tissue.

[0067] In some embodiments, the method comprises administering a therapeutically effective amount of a nucleocytoplasmic transport inhibitor to or near the cancer tissue. In some embodiments, the method comprises contacting the cancer cells or cancer tissue with a medium containing a therapeutically effective amount of a nucleocytoplasmic transport inhibitor. In some embodiments, the method comprises incubating the cancer cells or cancer tissue with a medium containing a therapeutically effective amount of a nucleocytoplasmic transport inhibitor.

[0068] In some embodiments, a therapeutically effective amount of a nucleocytoplasmic transport inhibitor is determined for a given cancer cell or cancer tissue (e.g., a given inhibitor, virus strain, and host cell line). The effective amount of a nucleocytoplasmic transport inhibitor can be determined, for example, by titrating the amount of inhibitor and quantifying viral progeny replication and / or cell viability as disclosed herein. In some embodiments, a therapeutically effective amount of a nucleocytoplasmic transport inhibitor comprises a concentration of about 0.000001 μM to about 100 μM, about 0.000001 μm to about 10 μm, about 0.00001 μm to about 1 μm, about 0.01 μm to about 1 μm, about 0.00001 μm to about 0.1 μm, about 0.00005 μm to about 0.05 μm, or about 0.00005 μm to about 0.005 μm. In some embodiments, a therapeutically effective amount of a nucleocytoplasmic transport inhibitor comprises a concentration of about 0.01 μM to about 1 μM. In some embodiments, a therapeutically effective amount of a nucleocytoplasmic transport inhibitor comprises a concentration of greater than about 0.000001 μm, greater than about 0.000005 μm, greater than about 0.00001 μm, greater than about 0.00005 μm, greater than about 0.0001, greater than 0.0005 μm, greater than about 0.001 μm, greater than about 0.005 μm, greater than about 0.01 μm, greater than about 0.05 μm, or greater than about 0.1 μm. In some embodiments, a therapeutically effective amount of a nucleocytoplasmic transport inhibitor is less than about 0.0005 μM, less than about 0.001 μM, less than about 0.0015 μM, less than about 0.002 μM, less than about 0.0025 μM, less than about 0.003 μM, less than about 0.0035 μM, less than about 0.004 μM, less than about 0.0045 μM, less than about 0.005 μM, less than about 0.01 μM, less than about 0.015 μM, less than about 0.02 μM, less than about 0.025 μM, less than about 0.03 μM, or less than about 0.035 μM. , less than about 0.04 μM, less than about 0.045 μM, less than about 0.05 μM, less than about 0.1 μM, less than about 0.2 μM, less than about 0.25 μM, less than about 0.3 μM, less than about 0.4 μM, less than about 0.5 μM, less than about 0.75 μM, less than about 0.9 μM, less than about 1 μM, less than about 1.25 μM, less than about 1.5 μM, less than about 1.75 μM, less than about 2 μM, less than about 3 μM, less than about 4 μM, less than about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, or less than about 10 μM.In some embodiments, the therapeutically effective amount of the nucleocytoplasmic transport inhibitor is between about 0.000001 μM and about 10 μM, about 0.0005 μM, about 0.001 μM, about 0.0015 μM, about 0.002 μM, about 0.0025 μM, about 0.003 μM, about 0.0035 μM, about 0.004 μM, about 0.0045 μM, about 0.005 μM, about 0.01 μM, about 0.015 μM, about 0.02 μM, about 0.025 μM, about 0.03 μM, about 0.035 μM, M, about 0.04 μM, about 0.045 μM, about 0.05 μM, about 0.1 μM, about 0.2 μM, about 0.3 μM, about 0.4 μM, about 0.5 μM, about 0.6 μM, about 0.7 μM, about 0.8 μM, about 0.9 μM, about 1.0 μM, about 1.1 μM, about 1.2 μM, about 1.3 μM, about 1.4 μM, about 1.5 μM, about 1.6 μM, about 1.7 μM, about 1.8 μM, about 1.9 μM, about 2.0 μM, about 3.0 μM, about 4.0 μM, and about 5.0 μM.

[0069] In some embodiments, the method includes administering a therapeutically effective amount of an oncolytic virus (e.g., MYXV or VACV) to or near the cancer tissue. In some embodiments, the method includes infecting a cancer cell or cancer tissue with a therapeutically effective amount of an oncolytic virus. In some embodiments, a therapeutically effective amount of an oncolytic virus is present when the cancer cell or cancer tissue is infected at an effective multiplicity of infection (MOI). In some embodiments, the method includes contacting a cancer cell or cancer tissue with an oncolytic virus at an MOI as described herein. The MOI can be determined for a given cell or tissue, for example, by titrating the virus to cell or tissue ratio and quantifying viral progeny replication and / or cellular viability as disclosed herein. In some embodiments, an effective MOI can minimize drug-specific cellular toxicity while enhancing viral replication and reducing attenuating cancer cell viability. In some embodiments, the cancer cells are incubated with an oncolytic virus (e.g., MYXV or VACV) for a period of time to allow the virus of interest to adsorb to the surface of the cells, the period being from about 20 minutes to about 5 hours, such as about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 65 minutes, about 70 minutes, about 75 minutes, about 80 minutes, about 85 minutes, about 90 minutes, about 95 minutes, about 100 minutes, about 105 minutes, about 110 minutes, about 115 minutes, about 2 hours, about 2.5 hours, about 3 hours, about 3.5 hours, about 4 hours, about 4.5 hours, about 5 hours, 12 hours, 18 hours, 20 hours, 24 hours, 30 hours, 36 hours, or more.

[0070] In certain embodiments, the MOI of the oncolytic virus for the cancer cells or tissue is between about 0.001 and 2.0, or between about 0.01 and 1.0, such as about 0.001, about 0.005, 0.01, about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, about 0.1, about 0.15, about Such as 0.2, about 0.25, about 0.3, about 0.35, about 0.4, about 0.45, about 0.5, about 0.55, about 0.6, about 0.65, about 0.7, about 0.75, about 0.8, about 0.85, about 0.9, about 0.95, about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, or about 2.0. In some embodiments, the MOI of the oncolytic virus against the cancer cells or tissue is about 0.01 to about 10, about 0.05 to about 5, and / or ranges therebetween. In some embodiments, the MOI of the oncolytic virus against the cancer cells or tissue is about 0.05, about 0.5, or about 5. In some embodiments, the MOI of the oncolytic virus to the cancer cell or tissue is at least about 0.001, at least about 0.005, at least about 0.01, at least about 0.02, at least about 0.03, at least about 0.04, at least about 0.05, at least about 0.06, at least about 0.07, at least about 0.08, at least about 0.09, at least about 0.1, at least about 0.15, at least about 0.2, at least about 0.25, at least about 0.3, at least about 0.35, at least about 0.4, at least about 0.45, at least about 0.5, at least about 0.55, at least about 0.6, at least about 0.65, at least about 0.7, at least about 0.75, at least about 0.8, at least about 0.85, at least about 0.9, at least about 0.95, at least about 1.0, at least about 1.1, at least about 1.2, at least about 1.3, at least about 1.4, at least about 1.5, at least about 1.6, at least about 1.7, at least about 1.8, at least about 1.9, at least about 2.0, at least about 2.5, at least about 3.0, at least about 4.0, at least about 5.0, at least about 6.0.In some embodiments, the MOI of the oncolytic virus for the cancer cells or tissue is up to 0.01, up to 0.02, up to 0.03, up to 0.04, up to 0.05, up to 0.06, up to 0.07, up to 0.08, up to 0.09, up to 0.1, up to 0.15, up to 0.2, up to 0.25, up to 0.3, up to 0.35, up to 0.4, up to 0.45, up to 0.5, up to 0.6, up to 0.7, up to 0.8, up to 0.9, up to 0.10, up to 0.15, up to 0.16, up to 0.17, up to 0.18, up to 0.19, up to 0.20, up to 0.25, up to 0.30, up to 0.35, up to 0.4, up to 0.45, up to 0.5, up to 0.60, up to 0.75, up to 0.80, up to 0.95, up to 0.90, up to 0.95, up to 0.100, up to 0.15, up to 0.16, up to 0.17, up to 0.18, up to 0.19, up to 0.20, up to 0.25, up to 0.30, up to 0.35, up to 0.4, up to 0.45, up to 0.5, up to 0.60, up to 0.75, up to 0.80, up to 0.95, up to 0.95, up to 0.100, up to 0.15, up to 0.15, up to 0.1 .55, max 0.6, max 0.65, max 0.7, max 0.75, max 0.8, max 0.85, max 0.9, max 0.95, max 1.0, max 1.1, max 1.2, max 1.3, max 1.4, max 1.5, max 1.6, max 1.7, max 1.8, max 1.9, max 2.0, max 2.5, max 3.0, max 4.0, max 5.0, max 6.0, or max 10.

[0071] In some embodiments, the use of a nucleocytoplasmic transport inhibitor increases the replication of an oncolytic virus (e.g., MYXV or VACV) in cancer cells or tissues. In some embodiments, the oncolytic virus replicates at a rate at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% faster than the replication rate in the absence of a nucleocytoplasmic transport inhibitor. In some embodiments, the oncolytic virus replicates at a rate at least 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold faster than the replication rate in the absence of a nucleocytoplasmic transport inhibitor. In some embodiments, the nucleocytoplasmic transport inhibitor increases the replication of an oncolytic virus in cancer cells or tissues by at least 30%, 50%, 70%, 90%, 2-fold, 3-fold, 5-fold, 7-fold, 9-fold, 10-fold, 12-fold, or 15-fold 24 hours after infection. In some embodiments, the nucleocytoplasmic transport inhibitor increases oncolytic viral replication in cancer cells or tissues by up to 5-fold, 10-fold, 12-fold, 15-fold, 20-fold, 30-fold, 50-fold, 70-fold, or 100-fold 24 hours after infection.

[0072] In some embodiments, the use of a nucleocytoplasmic transport inhibitor in combination with an oncolytic virus reduces the viability of cancer cells or tissues compared to the use of an oncolytic virus in the absence of a nucleocytoplasmic transport inhibitor. In some embodiments, the viability of the cells is reduced by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the cancer cells compared to the viability of the cells when an oncolytic virus is used in the absence of a nucleocytoplasmic transport inhibitor.

[0073] Further disclosed is a delivery strategy in which the therapeutic MYXV virus is first incubated with mixed leukocytes derived from either bone marrow or peripheral blood mononuclear cells ex vivo before introducing the cells into a cancer-bearing subject. In some embodiments, the leukocytes and MYXV are incubated ex vivo with a nucleocytoplasmic transport inhibitor. In this strategy, MYXV can be delivered to the cancer site via migration of leukocytes pre-infected with the virus ex vivo. This systemic delivery method may be referred to as "ex vivo virus therapy," or EVV (aka EV2), since the virus is first delivered to isolated leukocytes before infusion into the patient. The MYXV construct and this delivery strategy can significantly reduce systemic tumor burden and extend survival in subjects in need. In some embodiments, BM or PBMC cells are incubated with the MYXV construct ex vivo for 1 hour, and then the MYXV-bearing leukocytes are infused into the recipient. In some embodiments, incubation with a nucleocytoplasmic transport inhibitor increases the uptake of MYXV by leukocytes and / or increases the delivery of MYXV to tumor sites.

[0074] In certain embodiments, mononuclear peripheral blood cells and / or bone marrow cells are obtained from a subject, e.g., as autologous cells. In some embodiments, mononuclear peripheral blood cells and / or bone marrow cells are obtained from one or more allogeneic donors, e.g., the donor is matched to the recipient for at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or at least eight HLA alleles (e.g., HLA-A, one or both copies of HLA-B alleles, HLA-A, and / or HLA-DR alleles). HLA alleles can be typed, e.g., using DNA-based methods. In some embodiments, mononuclear peripheral blood cells and / or bone marrow cells are obtained from one or more xenogeneic donors.

[0075] Methods for increasing virus yield In one aspect, disclosed herein is a method for replicating oncolytic viruses such as poxviruses, comprising exposing a host cell to an effective amount of a nucleocytoplasmic transport inhibitor and an oncolytic virus under conditions that allow the oncolytic virus to be adsorbed to the surface of the host cell, and incubating the infected host cell in culture medium to allow the oncolytic virus to replicate.In some embodiments, the oncolytic virus is a poxvirus.In some embodiments, the oncolytic virus is MYXV.

[0076] In some embodiments, the method further comprises harvesting the poxvirus produced in the host cell. In some embodiments, the method further comprises contacting the host cell with a nucleocytoplasmic transport inhibitor prior to infecting the host cell with the poxvirus. In some embodiments, the method further comprises contacting the host cell with a nucleocytoplasmic transport inhibitor after infecting the host cell with the poxvirus. In some embodiments, the method further comprises exposing the host cell to the nucleocytoplasmic transport inhibitor and the poxvirus simultaneously. In some embodiments, the poxvirus replicates at a rate that is at least 30% faster than the replication rate in the absence of the nucleocytoplasmic transport inhibitor. In some embodiments, the nucleocytoplasmic transport inhibitor increases the replication of the poxvirus by at least 3-fold 24 hours after infection. In some embodiments, the host cell is contacted with the poxvirus at a multiplicity of infection (MOI) of less than 2.0. In some embodiments, the effective amount of the nucleocytoplasmic transport inhibitor is in the range of about 0.5 μM to about 0.0005 μM. In some embodiments, the culture medium comprises a nucleocytoplasmic transport inhibitor. In some embodiments, the host cell is an immortalized human cell or a primate cell. In some embodiments, the host cell belongs to a cell line used in Good Manufacturing Practice (GMP) for poxvirus production. In some embodiments, the host cell is a human A549 cell, a HeLa cell, a 239 cell, or a primate Vero cell. In some embodiments, the method further comprises removing unadsorbed poxvirus.

[0077] Disclosed herein in some aspects is a method for producing an oncolytic virus with increased growth rate and / or titer in a cell, the method comprising: contacting a host cell with an effective amount of a nucleocytoplasmic transport inhibitor; contacting a host cell with an oncolytic virus of interest under conditions that allow the oncolytic virus of interest to adsorb to the surface of the host cell; and culturing the host cell to produce a progeny of the oncolytic virus of interest. In some embodiments, the method further comprises harvesting the progeny of the oncolytic virus of interest. In some embodiments, the oncolytic virus is a poxvirus. In some embodiments, the oncolytic virus is MYXV.

[0078] In some embodiments, disclosed herein is a method for increasing or enhancing gene expression and progeny virus formation of oncolytic viruses using drug inhibitors of nucleocytoplasmic transport pathways.In some embodiments, the inhibition of nuclear export pathways in immortalized human cells commonly used for GMP production of oncolytic viruses can be used to increase virus yield from these cells.Thus, the present disclosure provides a robust method for producing oncolytic viruses with potential therapeutic effects, and in some embodiments, the oncolytic virus is MYXV or the like.

[0079] In some embodiments, this enhanced viral replication is most potent when cells are treated with an effective amount of inhibitor, for example, beginning just before the time of the first viral adsorption. In some embodiments, the effective amount of inhibitor is determined for a given cell culture system (e.g., a given inhibitor, viral strain, and host cell line). The effective amount of inhibitor can be determined, for example, by titrating the amount of inhibitor in a cell culture system and quantifying the yield of viral progeny, as disclosed herein. In some embodiments, an effective amount of inhibitor can minimize drug-specific cellular toxicity while increasing the yield of viral progeny. In some embodiments, the effective amount of nucleocytoplasmic transport inhibitor is about 0.000001 μM to about 1 μM, about 0.000001 μM to about 0.1 μM, about 0.00001 μM to about 0.1 μM, about 0.00005 μM to about 0.05 μM, or about 0.00005 μM to about 0.005 μM. In some embodiments, an effective amount of a nucleocytoplasmic transport inhibitor is greater than about 0.000001 μM, greater than about 0.000005 μM, greater than about 0.00001 μM, greater than about 0.00005 μM, about 0.0001, greater than about 0.0005 μM, greater than about 0.001 μM, greater than about 0.005 μM, greater than about 0.01 μM, greater than about 0.05 μM, or greater than about 0.1 μM. In some embodiments, an effective amount of a nucleocytoplasmic transport inhibitor is at most about 0.0005 μM, about 0.001 μM, about 0.0015 μM, about 0.002 μM, about 0.0025 μM, about 0.003 μM, about 0.0035 μM, about 0.004 μM, about 0.0045 μM, about 0.005 μM, about 0.01 μM, about 0.015 μM, about 0.02 μM, about 0.025 μM, about 0.03 μM, about 0.04 ... 0.035 μM, about 0.04 μM, about 0.045 μM, about 0.05 μM, about 0.1 μM, about 0.2 μM, about 0.25 μM, about 0.3 μM, about 0.4 μM, about 0.5 μM, about 0.75 μM, about 0.9 μM, about 1 μM, about 1.25 μM, about 1.5 μM, about 1.75 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, or about 10 μM.

[0080] In some embodiments, enhanced viral replication occurs when cells are infected at an effective multiplicity of infection (MOI) and then maintained in the continuous presence of, for example, a nucleocytoplasmic transport inhibitor. MOI can be determined for a given cell culture system (e.g., a given inhibitor, virus strain, and host cell line), for example, by titrating the ratio of virus to host cells in the cell culture system and quantifying the yield of viral progeny. In some embodiments, an effective MOI can minimize drug-specific cellular toxicity while enhancing viral replication and increasing the yield of viral progeny.

[0081] The host cells can be contacted with the poxvirus of interest (e.g., MYXV) under conditions that allow the poxvirus of interest to adsorb to the surface of the host cells. In certain embodiments, the cells are contacted with the poxvirus of interest at a multiplicity of infection (MOI) of between about 0.001 and 2.0, or between about 0.01 and 1.0, such as about 0.001 MOI, about 0.005 MOI, 0.01 MOI, about 0.02 MOI, about 0.03 MOI, about 0.04 MOI, about 0.05 MOI, about 0.06 MOI, about 0.07 MOI, about 0.08 MOI, about 0.09 MOI, about 0.1 MOI, about 0.15 MOI, about 0.2 MOI, about 0.25 MOI, about 0.3 MOI, about 0.4 MOI, about 0.5 MOI, about 0.6 MOI, about 0.7 MOI, about 0.8 MOI, about 0.9 MOI, about 0.1 MOI, about 0.2 ... The MOI is about 0.35 MOI, about 0.4 MOI, about 0.45 MOI, about 0.5 MOI, about 0.55 MOI, about 0.6 MOI, about 0.65 MOI, about 0.7 MOI, about 0.75 MOI, about 0.8 MOI, about 0.85 MOI, about 0.9 MOI, about 0.95 MOI, about 1.0 MOI, about 1.1 MOI, about 1.2 MOI, about 1.3 MOI, about 1.4 MOI, about 1.5 MOI, about 1.6 MOI, about 1.7 MOI, about 1.8 MOI, about 1.9 MOI, or about 2.0 MOI. In some embodiments, the non-adsorbed poxvirus of interest is removed before culturing the cells, for example, by washing the cells. After treatment with the nucleocytoplasmic transport inhibitor, the host cells are cultured in the presence of the drug to produce the progeny of the poxvirus of interest. Appropriate culture conditions may be selected based on the specific cell type. In some embodiments, the cells are incubated with the poxvirus of interest for a period of time, such as about 20 minutes to about 5 hours, for example, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 65 minutes, about 70 minutes, about 75 minutes, about 80 minutes, about 85 minutes, about 90 minutes, about 95 minutes, about 100 minutes, about 105 minutes, about 110 minutes, about 115 minutes, about 2 hours, about 2.5 hours, about 3 hours, about 3.5 hours, about 4 hours, about 4.5 hours, about 5 hours, 12 hours, 18 hours, 20 hours, 24 hours, 30 hours, 36 hours, or even longer, such that the poxvirus of interest adsorbs to the surface of the host cells. In certain embodiments, the host cells are cultured in the presence of an effective amount of a nucleocytoplasmic transport inhibitor, for example, at the concentrations described above.The host cells can be cultured for about one to several days, such as about three to five days, before the progeny virus is harvested. In some embodiments, the method includes a step of harvesting the progeny of the poxvirus of interest.

[0082] As disclosed herein, nucleocytoplasmic transport inhibitors can enhance viral replication and progeny virus yield in such cells. This drug enhancement method provides a practical method for increasing the yield of poxviruses such as MYXV and VACV, and can be utilized for large-scale GMP production and clinical use of these viruses. In some embodiments, the present disclosure demonstrates that nucleocytoplasmic transport inhibitors can be used to increase the yield of poxviruses, including oncolytic virus candidates such as vaccinia virus (VACV) and myxoma virus (MYXV), when applied to poxvirus GMP manufacturing in immortalized human or primate cells.

[0083] The present disclosure provides a method for increasing poxvirus production yields in transformed human and primate cells, such as those commonly used for GMP manufacturing of viruses, such as oncolytic viruses. Compared to other viruses proposed as oncolytic virus candidates, poxviruses, such as MYXV and VACV, present several unique challenges in large-scale manufacturing and purification. Due to their large size, vaccinia virus cannot be sterilized by filtration, and therefore any manufacturing process is typically enclosed. Throughout its life cycle, which occurs within the host cell cytoplasm, the majority of infectious particles are not released into the cellular medium, but are retained within the infected cell, and therefore purification requires lysis of the infected cell and purification of the viral particles from the cellular debris.

[0084] In one embodiment, disclosed herein is a method of treating immortalized human cell lines with drugs, such as small molecules, that inhibit the nuclear export pathway when added just before the time of virus adsorption. For example, culturing cells in the presence of the drug throughout the later infection cycle can increase the replication and final yield of infectious poxviruses overall. Thus, the disclosed method provides an elegant way to increase the overall yield of poxviruses, such as myxoma virus (MYXV) and vaccinia virus (VACV), in transformed human and primate cells used for virus GMP production.

[0085] The use of a nucleocytoplasmic transport inhibitor increases the replication of the poxvirus. In some embodiments, the poxvirus replicates at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% faster than in the absence of the nucleocytoplasmic transport inhibitor. In some embodiments, the poxvirus replicates at least 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9, or 10 times faster than in the absence of the nucleocytoplasmic transport inhibitor.

[0086] In some embodiments, the nucleocytoplasmic transport inhibitor increases poxvirus replication in cancer tissue by at least 30%, 50%, 70%, 90%, 2-fold, 3-fold, 5-fold, 7-fold, 9-fold, 10-fold, 12-fold, or 15-fold 24 hours after infection. In some embodiments, the nucleocytoplasmic transport inhibitor increases poxvirus replication in cancer tissue by up to 5-fold, 10-fold, 12-fold, 15-fold, 20-fold, 30-fold, 50-fold, 70-fold, or 100-fold 24 hours after infection.

[0087] Aspects of the present disclosure relate to methods of producing increased growth rate and / or titer of poxvirus, etc. in cells. In some embodiments, the methods include contacting a host cell with an effective amount of a nucleocytoplasmic transport inhibitor, which may also be referred to as a nuclear export pathway inhibitor. The concentration of the inhibitor may be selected to be below that which would normally cause either cytotoxicity or antiviral effects, yet sufficient to result in an increase in the production of the poxvirus of interest. In some embodiments, the therapeutically effective amount of the nucleocytoplasmic transport inhibitor is between about 0.000001 μM and about 0.1 μM, such as between about 0.000001 μM, about 0.00001 μM, about 0.0001 μM, about 0.0005 μM, and about 0.05 μM, such as between about 0.0005 μM, about 0.001 μM, about 0.0015 μM, about 0. and the like.

[0088] Pharmaceutical Compositions In one aspect, disclosed herein is a pharmaceutical composition comprising an oncolytic virus, a nucleocytoplasmic transport inhibitor, a pharma- ceutically acceptable excipient or carrier, or a combination thereof. When administered as a combination, the therapeutic agents (i.e., the oncolytic virus and the nucleocytoplasmic transport inhibitor) can be formulated as separate compositions given at the same time or at different times, or the therapeutic agents can be given as a single composition.

[0089] The composition can be administered once daily, twice daily, once every 2 days, once every 3 days, once every 4 days, once every 5 days, once every 6 days, once every 7 days, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 2 months, once every 6 months, or once a year. The administration interval can be adjusted according to the needs of individual subjects. In an embodiment, the disclosed therapeutic agent is administered for a period of more than 2 weeks, more than 3 weeks, more than 1 month, more than 2 months, more than 3 months, more than 4 months, more than 5 months, more than 6 months, more than 1 year, more than 2 years, more than 3 years, more than 4 years, or more than 5 years, more than 10 years, or more than 15 years, or for any range of days, months, or years, for example, any range of 14 days and 15 years at the lower end and any range of 15 days and 20 years at the upper end. In some cases, it may be advantageous to administer the therapeutic agent for the rest of the patient's life.In some embodiments, the patient is monitored to check the progression of disease or disorder, and the dosage is adjusted accordingly.In some embodiments, the treatment according to the present invention is effective for at least 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 3 years, 4 years, or 5 years, 10 years, 15 years, 20 years, or the rest of the subject's life.

[0090] To prepare a pharmaceutical composition according to the present disclosure, a therapeutically effective amount of one or more therapeutic agents can be intimately mixed with a pharma- ceutically acceptable carrier according to conventional pharmaceutical compounding techniques to produce a dosage amount. The carrier can take a variety of forms depending on the desired preparation for administration, such as ophthalmic, oral, topical or parenteral forms, including gels, creams, ointments, lotions, and time-release implantable formulations, among others. In some embodiments, the pharma- ceutically acceptable carrier is aqueous, i.e., contains water and, optionally, additional co-solvents. Exemplary pharma- ceutically acceptable carriers include water, buffer solutions in water, such as phosphate buffered saline (PBS), and sugar alcohols, such as sorbitol. Compositions suitable for parenteral administration can be packaged in ampoules, disposable syringes, or multiple dose vials made of glass or plastic. In some embodiments, formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions, which may contain antioxidants, buffers, bacteriostats and solutes that render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions, which may contain suspending agents and thickening agents. The formulations may be presented in single-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in powder form or freeze-dried (lyophilized) condition, requiring only the addition of a sterile liquid carrier, for example, water for injection, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets of the kind previously described.

[0091] In some embodiments, the nucleocytoplasmic transport inhibitor is administered as a pharmaceutically acceptable salt, complex, or prodrug. Pharmaceutically acceptable salt or complex refers to a suitable salt or complex of the active compound according to the present disclosure, which retains the desired biological activity of the parent compound and shows limited toxicological effects on normal cells. Non-limiting examples of such salts include (a) acid addition salts formed with inorganic acids (e.g., hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, etc.) and salts formed with organic acids such as acetic acid, oxalic acid, tartaric acid, succinic acid, malic acid, ascorbic acid, benzoic acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, etc.; (b) base addition salts formed with various metal cations such as zinc, calcium, sodium, potassium, etc.

[0092] In some embodiments, the pharmaceutical composition is in a unit dosage form. In some embodiments, the unit dosage form is suitable for administration intratumorally or parenterally, e.g., intravenously. The unit dosage formulation may be a formulation containing a daily dose or unit, a daily sub-dose, a weekly dose or unit, or an appropriate fraction thereof, of the administered component. In some embodiments, the unit dosage comprises a deliverable volume of about 0.1 mL to about 100 mL, and / or a range therebetween. In some embodiments, the unit dosage comprises a deliverable volume of about 0.5 mL to about 5 mL, about 0.75 mL to about 2.5 mL, about 1.1 mL to about 0.9 mL. In some embodiments, the unit dosage comprises a deliverable volume of about 0.5 mL, about 1 mL, about 1.5 mL, or 2 mL. In some embodiments, the unit dosage comprises a deliverable volume of about 1x10 per mL. 3 Plaque forming units (PFU) to approximately 1x10 10 In some embodiments, the unit dose comprises about 1x10 PFU of oncolytic virus per mL. 4 PFU to approx. 1x10 9 Up to PFU or about 1x10 6 PFU to approx. 1x10 8In some embodiments, the unit dose is about 1x10 PFU of oncolytic virus per mL. 5 PFU to approx. 1x10 10 Up to PFU, approximately 1x10 6 PFU to approx. 1x10 10 Up to PFU, approximately 1x10 5 PFU to approx. 1x10 11 Up to PFU, approximately 1x10 5 PFU to approx. 1x10 9 Up to PFU, approximately 1x10 6 PFU to approx. 1x10 9 Up to PFU or about 1x10 7 PFU to approx. 1x10 8 PFU and / or ranges therebetween, including oncolytic viruses.

[0093] In some embodiments, the unit dose comprises from about 1 picogram to about 1000 micrograms, and / or any range therebetween, of oncolytic virus. In some embodiments, the unit dose comprises from about 10 picograms to about 100 micrograms, and / or any range therebetween, of oncolytic virus. In some embodiments, the unit dose comprises from about 10 picograms to about 100 micrograms, and / or any range therebetween, of oncolytic virus. In some embodiments, the unit dose comprises about 10 picograms to about 10 micrograms, about 10 picograms to about 1 micrograms, about 10 picograms to about 100 micrograms, about 100 picograms to about 100 micrograms, about 1000 picograms to about 100 micrograms, about 10000 picograms to about 100 micrograms, about 100 picograms to about 10 micrograms, or about 100 picograms to about 1 micrograms, and / or ranges therebetween of oncolytic virus. In some embodiments, the unit dose comprises about 1 picogram, about 10 picograms, about 100 picograms, about 1 nanograms, about 10 nanograms, about 100 nanograms, about 1 micrograms, about 10 micrograms, about 100 micrograms, or about 1000 micrograms of oncolytic virus. In some embodiments, the unit dose contains about 0.01 picograms to about 500 micrograms of oncolytic agent, and / or a range therebetween. In some embodiments, the unit dose contains about 0.05 picograms to about 50 micrograms of nucleocytoplasmic transport inhibitor, and / or a range therebetween. In some embodiments, the unit dose contains about 0.1 picograms to about 1 picograms to about 10 picograms to about 100 picograms, or about 1 picograms to about 0.1 micrograms to about 1 micrograms to about 10 micrograms to about 50 micrograms, and / or a range therebetween.In some embodiments, the unit dose comprises about 10 picograms to about 10 micrograms, about 10 picograms to about 1 micrograms, about 10 picograms to about 1 micrograms, about 100 picograms to about 50 micrograms, about 1000 picograms to about 50 micrograms, about 10000 picograms to about 50 micrograms, about 100 picograms to about 5 micrograms, or about 100 picograms to about 1 microgram, and / or ranges therebetween, of the nucleocytoplasmic transport inhibitor. In some embodiments, the unit dose comprises about 0.05 picograms, about 0.5 picograms, about 5 picograms, about 50 picograms, about 500 picograms, about 5 nanograms, about 50 nanograms, about 500 nanograms, about 5 micrograms, about 50 micrograms, or about 500 micrograms of the oncolytic virus. In some embodiments, the unit dose comprises from about 0.05 picograms to about 50 micrograms, and / or any range therebetween, of the nucleocytoplasmic transport inhibitor. In some embodiments, the unit dose comprises from about 0.01 picograms to about 100 micrograms, and / or any range therebetween, of the nucleocytoplasmic transport inhibitor. In some embodiments, the unit dose comprises from about 0.05 picograms to about 100 micrograms, and / or any range therebetween, of the nucleocytoplasmic transport inhibitor. In some embodiments, the unit dose comprises from about 0.01 picograms to about 90 micrograms, from about 0.01 picograms to about 80 micrograms, from about 0.01 picograms to about 60 micrograms, from about 0.01 picograms to about 50 micrograms, from about 0.05 picograms to about 80 micrograms, from about 0.05 picograms to about 40 micrograms, from about 0.1 picograms to about 50 micrograms, or from about 0.1 picograms to about 30 micrograms, and / or ranges therebetween, of a nucleocytoplasmic transport inhibitor.In some embodiments, the unit dose contains about 0.01 picograms, about 0.05 picograms, about 0.075 picograms, about 0.1 picograms, about 0.5 picograms, about 1 picograms, about 5 picograms, about 10 picograms, about 100 picograms, about 500 picograms, about 1 nanograms, about 10 nanograms, about 100 nanograms, about 1 micrograms, up to about 10 micrograms, or about 100 micrograms of the nucleocytoplasmic transport inhibitor. In some embodiments, the unit dose contains about 0.01 picograms to about 500 micrograms, and / or ranges therebetween, of the nucleocytoplasmic transport inhibitor. In some embodiments, the unit dose contains about 0.05 picograms to about 50 micrograms, and / or ranges therebetween, of the nucleocytoplasmic transport inhibitor. In some embodiments, the unit dose comprises a nucleocytoplasmic transport inhibitor in the range of about 0.1 picograms, about 1 picograms, about 10 picograms, about 100 picograms, or about 1 picograms to about 0.1 micrograms, about 1 micrograms, about 10 micrograms, or about 50 micrograms, and / or ranges therebetween. In some embodiments, the unit dose comprises a nucleocytoplasmic transport inhibitor in the range of about 10 picograms to about 10 micrograms, about 10 picograms to about 1 micrograms, about 100 picograms to about 50 micrograms, about 1000 picograms to about 50 micrograms, about 10000 picograms to about 50 micrograms, about 100 picograms to about 5 micrograms, or about 100 picograms to about 1 microgram, and / or ranges therebetween. In some embodiments, the unit dose comprises about 0.05 picograms, about 0.5 picograms, about 5 picograms, about 50 picograms, about 500 picograms, about 5 nanograms, about 50 nanograms, about 500 nanograms, about 5 micrograms, about 50 micrograms, or about 500 micrograms of a nucleocytoplasmic transport inhibitor.

[0094] In some embodiments, the weight ratio of the nucleocytoplasmic transport inhibitor to the oncolytic virus is about 1x109 From about 1x10 -9 In some embodiments, the weight ratio of oncolytic virus to nucleocytoplasmic transport inhibitor is about 1x10 8 From about 1x10 -8 Up to about 1x10 7 From about 1x10 -7 Up to about 1x10 6 From about 1x10 -6 Up to about 1x10 5 From about 1x10 -5 Up to about 1x10 4 From about 1x10 -3 Up to about 1x10 2 From about 1x10 -2 or from about 10 to about 0.1. In some embodiments, the weight ratio of oncolytic virus to nucleocytoplasmic transport inhibitor is from about 1 to about 1x10 -9 From about 1 to about 1x10 -8 From about 1 to about 1x10 -7 From about 1 to about 1x10 -6 From about 1 to about 1x10 -5 From about 1 to about 1x10 -4 From about 1 to about 1x10 -3 From about 1 to about 1x10 -2 or from about 1 to about 0.1. In some embodiments, the weight ratio of oncolytic virus to nucleocytoplasmic transport inhibitor is about 1x10 -9 From about 1 to about 1x10 -8 From about 1 to about 1x10 -7 From about 1 to about 1x10 -6 From about 1 to about 1x10 -5 From about 1 to about 1x10 -4 From about 1 to about 1x10 -3 From about 1 to about 1x10 -2 to about 1, or from about 0.1 to about 1. EXAMPLES

[0095] Example 1: Nucleocytoplasmic transport inhibitors enhance MYXV gene expression in non-permissive human cancer cell lines In some human cancer cell lines, the expression of MYXV genes is restricted, especially in late gene expression. Inhibition of MYXV gene expression may contribute to the restriction of viral replication. However, it has not been clearly documented which pathways and cellular proteins restrict MYXV replication in these non-permissive human cancer cell lines. For example, we tested nucleocytoplasmic transport inhibitors in human cancer cell lines that generally restrict MYXV gene expression and replication, such as colon adenocarcinoma cell line HT29, pancreatic cancer cell line PANC-1, and melanoma cell line MDA-MB-435.

[0096] As a representative nucleocytoplasmic transport inhibitor, leptomycin B was tested. Human HT29 cells (Figure 1A and Figure 1B), PANC-1 cells (Figure 3A), and MDA-MB-435 cells (Figure 4A) were mock-treated or pretreated with various concentrations of leptomycin B for 1 h. The cells were then infected with vMyx-GFP-TdTomato (wild-type MYXV expressing GFP under the poxvirus synthetic early / late promoter and TdTomato under the poxvirus p11 late promoter) at a multiplicity of infection (MOI) of 5.0 or 0.5.

[0097] HT29 cells were pretreated with various concentrations of leptomycin B for 1 h, infected with vMyx-GFP-Tdtomato at an MOI of 5.0 in the presence of inhibitors, and fluorescent images were taken using a fluorescent microscope 24 h (Fig. 1A) or 48 h (Fig. 1B) postinfection.

[0098] PANC-1 cells were pretreated with various concentrations of leptomycin B for 1 h, infected with vMyx-GFP-Tdtomato at an MOI of 5.0 in the presence of inhibitors, and fluorescent images were taken 48 h postinfection (Figure 3A).

[0099] MDA-MB-435 cells were pretreated with various concentrations of leptomycin B for 1 h, infected with vMyx-GFP-Tdtomato at an MOI of 5.0 in the presence of inhibitors, and fluorescent images were taken 48 h postinfection (Figure 4A).

[0100] To monitor the expression of GFP and TdTomato, expressions that indicate the progression of viral infection, cells were observed under a fluorescent microscope. In the presence of leptomycin B (e.g., 1uM, 0.1uM, and 0.01uM), enhanced GFP expression (early / late) and TdTomato expression (late) were observed in all tested human cancer cell lines (Figure 1A, Figure 1B, Figure 3A, Figure 4A). Treatment with nucleocytoplasmic transport inhibitors also allowed the formation of small foci in these restricted human cancer cell lines.

[0101] These results indicate that treatment with nucleocytoplasmic transport inhibitors enhances myxoma virus gene expression in infected cells. These results also suggest that certain nuclear proteins directly or indirectly restrict MYXV replication in these human cancer cell lines.

[0102] Example 2: Treatment with nucleocytoplasmic transport inhibitor drugs enhances production of MYXV progeny virus in non-permissive human cancer cell lines. To quantitatively assess whether the observed effects of the nucleocytoplasmic transport inhibitor leptomycin B on viral gene expression could be reflected in progeny virus formation, quantitative virus titration analysis was performed. HT29 (Figure 2), PANC-1 (Figure 3B) and MDA-MB-435 (Figure 4B) human cancer cell lines were mock-treated or pretreated with different concentrations of leptomycin B.

[0103] HT29 cells were pretreated with various concentrations of leptomycin B for 1 h. Cells were then infected with vMyx-GFP-TdTomato at an MOI of 0.05 or 0.5 for 1 h. After 1 h, unadsorbed virus was removed, and cells were then washed and cultured with medium in the presence of inhibitors. Infected cells were harvested 72 h postinfection, and virus titers were determined after serial dilution into RK13 cells. Leptomycin B treatment enhanced virus replication (Figure 2).

[0104] PANC-1 cells were pretreated with various concentrations of leptomycin B for 1 h. Cells were then infected with vMyx-GFP-TdTomato at an MOI of 5.0 or 0.5 for 1 h. After 1 h, unadsorbed virus was removed, and cells were then washed and incubated with medium in the presence of inhibitors. Infected cells were harvested 48 or 72 h postinfection, and virus titers were determined after serial dilution into RK13 cells. Leptomycin B treatment enhanced virus replication by at least 10-fold in all conditions (Figure 3B).

[0105] MDA-MB-435 cells were pretreated with various concentrations of leptomycin B for 1 h, after which the cells were infected with vMyx-GFP-TdTomato at an MOI of 5.0 or 0.5 for 1 h. After 1 h, unadsorbed virus was removed, and then the cells were washed and cultured with medium in the presence of inhibitors. Infected cells were harvested 48 or 72 h postinfection, and virus titers were determined after serial dilution into RK13 cells. Leptomycin B treatment enhanced virus replication by at least 10-fold in all conditions (Figure 4B).

[0106] In all tested cell lines, leptomycin B treatment significantly enhanced progeny MYXV formation by at least 10- to 20-fold when infected at an MOI of 5.0 or 0.5 (Fig. 2, Fig. 3B, Fig. 4B). In contrast, in the permissive A549 cell line, leptomycin B increased viral replication only at an MOI of 0.1 or lower.

[0107] These results indicate that blocking the nuclear export pathway using nucleocytoplasmic transport inhibitors can significantly enhance MYXV replication in nonpermissive human cancer cell lines.

[0108] Example 3: Pretreatment of human cancer cell lines with nucleocytoplasmic transport inhibitors enhances MYXV-mediated cancer cell killing. To test whether nucleocytoplasmic transport inhibitor-enhanced MYXV replication also enhanced cancer cell killing, we tested the viability of infected cells by measuring ATP, in this case HT29 and PANC-1 cells.

[0109] HT29 and PANC-1 cells were seeded in opaque-walled 96-well plates with 20,000 cells in 100ul medium / well and cultured overnight. Cells were then pretreated with various concentrations of leptomycin B for 1 hour and infected with vMyx-GFP-Tdtomato at an MOI of 5.0 in the presence of inhibitors. Cell viability was then measured based on ATP content. ATP content was measured by adding 100ul CellTiter-Glo(r) reagent (Promega) 64 hours (HT-29 cells, Figure 5A) or 40 hours (PANC-1 cells, Figure 5B) after infection and recording luminescence using a plate reader. Data shown represent the mean + / - SD (n=3 or 4).

[0110] The results showed that at the indicated time points, both HT29 and PANC-1 cells were only sensitive to leptomycin B treatment. Increasing concentrations of leptomycin B decreased cell viability. At these time points (64 h postinfection for HT29 and 40 h postinfection for PANC-1), only MYXV also reduced cell viability by 40-50%. However, pretreatment of both HT29 and PANC-1 cells with leptomycin B further reduced the viability of these human cancer cell lines. Interestingly, the lowest concentration of leptomycin B that had minimal effect on cell viability in the HT29 cell line further enhanced the cell killing ability of MYXV after combination with MYXV. Taken together, these results indicate that nucleocytoplasmic transport inhibitors not only enhance MYXV replication but also synergistically enhance the cell killing ability of MYXV in nonpermissive human cancer cell lines.

[0111] Example 4: Oncolytic virotherapy with myxoma virus (MYXV) and nucleocytoplasmic transport inhibitors The subject is identified as having cancer (eg, a non-permissive hematological cancer or a solid tumor).

[0112] As disclosed herein, MYXV was administered to a subject (e.g., via injection or infusion) in combination with selinexor, a nucleocytoplasmic transport inhibitor approved for use in human cancer patients. MYXV infected cancer cells in the subject, resulting in cancer cell death and increased anti-tumor immune responses. Administering the nucleocytoplasmic transport inhibitor enhanced the cancer treatment activity of myxoma virus, resulting in reduced tumor burden and longer survival.

[0113] Although the present disclosure has been described with emphasis on certain embodiments, it will be apparent to one of ordinary skill in the art that variations of the specific embodiments may be used, and it is intended that the disclosure may be practiced other than as specifically described herein. Features, properties, compositions, or examples of the invention described in conjunction with a particular aspect, embodiment, or example should be understood to be applicable to other aspects, embodiments, or examples of the invention. Accordingly, the present disclosure includes all modifications encompassed within the spirit and scope of the disclosure as defined by the following claims. Accordingly, it is claimed that all inventions fall within the scope and spirit of these claims.

Claims

1. a therapeutically effective amount of an oncolytic virus, and A method of treating cancer comprising administering to a subject a nucleocytoplasmic transport inhibitor.

2. The method of claim 1 , wherein the oncolytic virus is from the Poxviridae family.

3. The method of claim 1 or 2, wherein the oncolytic virus is derived from the subfamily Chordopoxvirinae or Entomopoxvirinae.

4. 4. The method of claim 1, wherein the oncolytic virus is derived from the virus genus Orthopoxvirus, Serbidopoxvirus, Parapoxvirus, Avipoxvirus, Capripoxvirus, Leporipoxvirus, Suipoxvirus, Morsipoxvirus, Yatapoxvirus, Alphaentomopoxvirus, Betaentomopoxvirus, or Gammaentomopoxvirus.

5. The method of any one of claims 1 to 4, wherein the oncolytic virus is from the genus Leporipoxvirus.

6. 6. The method according to claim 1, wherein the oncolytic virus is myxoma virus (MYXV).

7. The method of any one of claims 1 to 4, wherein the oncolytic virus is from the Orthopoxvirus genus.

8. 8. The method according to claim 1, wherein the virus is genetically modified.

9. 9. The method of any one of claims 1 to 8, wherein the nucleocytoplasmic transport inhibitor is selected from the group consisting of leptomycin A, leptomycin B, latjadone A, latjadone B, latjadone C, latjadone D, anginomycin A, goniothalamin, piperlongumine, plumbagin, curcumin, valtrate, acetoxychavicol acetate, prenylcoumarin osthole, KOS 2464, PKF050-638, CBS9106, and selinexor.

10. 10. The method according to claim 1, wherein the nucleocytoplasmic transport inhibitor is leptomycin B.

11. 10. The method of claim 1, wherein the nucleocytoplasmic transport inhibitor is selinexor.

12. The method according to any one of claims 1 to 11, wherein the cancer is a solid tumor.

13. The method of claim 12, wherein the solid tumor is a sarcoma or a carcinoma.

14. The solid tumors include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelioma, lymphangiosarcoma, lymphangioendothelioma, synovium, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, and renal cell carcinoma. , liver cancer, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma, meningioma, melanoma, neuroblastoma, or retinoblastoma.

15. 13. The method of claim 12, wherein the solid tumor is a colon adenocarcinoma, a pancreatic carcinoma or a melanoma.

16. 16. The method of any one of claims 1 to 15, wherein the oncolytic virus and the nucleocytoplasmic transport inhibitor are administered simultaneously.

17. 16. The method of any one of claims 1 to 15, wherein the oncolytic virus and the nucleocytoplasmic transport inhibitor are administered sequentially.

18. 18. The method of claim 17, wherein the oncolytic virus is administered prior to administration of the nucleocytoplasmic transport inhibitor.

19. 18. The method of claim 17, wherein the oncolytic virus is administered after administration of the nucleocytoplasmic transport inhibitor.

20. 20. The method of any one of claims 1 to 19, wherein the method comprises administering the oncolytic virus and the nucleocytoplasmic transport inhibitor for a period of at least 1 day, at least 2 days, at least 3 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 3 months, at least 6 months, or at least 1 year.

21. 21. The method of any one of claims 1 to 20, wherein the method comprises administering to the subject the oncolytic virus, the nucleocytoplasmic transport inhibitor, or both, twice a week, once a week, once every two weeks, once every three weeks, once every four weeks, once a month, or once every two months.

22. 22. The method of any one of claims 1 to 21, wherein the oncolytic virus is administered locally to the cancer tissue to be treated.

23. 23. The method of claim 22, wherein the oncolytic virus is administered by intratumoral injection.

24. 24. The method of claim 22 or 23, wherein the multiplicity of infection (MOI) of the oncolytic virus for the tissue is from about 0.01 to about 10.

25. 25. The method of any one of claims 22 to 24, wherein the multiplicity of infection (MOI) of the oncolytic virus for the tissue is from about 0.05 to about 5.

26. 26. The method of any one of claims 22 to 25, wherein the concentration of the nucleocytoplasmic transport inhibitor in the tissue is from about 0.0001 μM to about 100 μM.

27. 27. The method of any one of claims 22 to 26, wherein the concentration of the nucleocytoplasmic transport inhibitor in the tissue is from about 0.01 μM to about 1 μM.

28. 1. A method of killing a cancer cell, the method comprising the step of contacting said cancer cell with an oncolytic virus and a nucleocytoplasmic transport inhibitor, thereby killing said cancer cell.

29. 1. A method of increasing the susceptibility of a cancer cell to infection by an oncolytic virus, the method comprising contacting the cancer cell with an oncolytic virus and a nucleocytoplasmic transport inhibitor.

30. A method for inducing a cancer cell to be susceptible to infection by an oncolytic virus, the method comprising contacting the cancer cell with the oncolytic virus and the nucleocytoplasmic transport inhibitor.

31. 2. A method of converting a non-permissive cancer cell into a permissive cancer cell, the method comprising the step of contacting said non-permissive cancer cell with an oncolytic virus and a nucleocytoplasmic transport inhibitor.

32. A method for increasing the proliferation of an oncolytic virus in a cancer cell that is not susceptible to infection by the oncolytic virus, the method comprising contacting the cancer cell with an oncolytic virus and a nucleocytoplasmic transport inhibitor.

33. 33. The method of any one of claims 28 to 32, wherein the oncolytic virus is a virus belonging to the Poxviridae family.

34. 34. The method of any one of claims 28 to 33, wherein the oncolytic virus is a virus belonging to the subfamily Chordopoxvirinae or Entomopoxvirinae.

35. 35. The method of any one of claims 28 to 34, wherein the oncolytic virus belongs to the following viral genus: Orthopoxvirus, Serbidopoxvirus, Parapoxvirus, Avipoxvirus, Capripoxvirus, Leporipoxvirus, Suipoxvirus, Morsipoxvirus, Yatapoxvirus, Alphaentomopoxvirus, Betaentomopoxvirus, or Gammaentomopoxvirus.

36. 36. The method of any one of claims 28 to 35, wherein the oncolytic virus belongs to the genus Leporipoxvirus.

37. 37. The method of any one of claims 28 to 36, characterized in that the oncolytic virus is myxoma virus (MYXV).

38. 36. The method according to any one of claims 28 to 35, characterized in that the oncolytic virus belongs to the Orthopoxvirus genus.

39. 37. The method of any one of claims 28 to 36, wherein the oncolytic virus is genetically modified.

40. 40. The method of any one of claims 28 to 39, characterized in that the nucleocytoplasmic transport inhibitor is selected from the group consisting of leptomycin A, leptomycin B, latojadon A, latojadon B, latojadon C, latojadon D, anginomycin A, goniothalamin, piperlongumine, plumbagin, curcumin, valtrate, acetoxychavicol acetate, prenylcoumarin osthole, KOS 2464, PKF050-638, CBS9106, and selinexor.

41. 41. The method of any one of claims 28 to 40, wherein the nucleocytoplasmic transport inhibitor is leptomycin B.

42. 41. The method of any one of claims 28 to 40, wherein the nucleocytoplasmic transport inhibitor is selinexor.

43. 43. The method according to any one of claims 28 to 42, characterized in that the cancer cells are colon cancer cells, pancreatic cancer cells, melanoma cancer cells.

44. 44. The method of any one of claims 28 to 43, wherein the cancer cells are human cancer cells.

45. 45. The method of any one of claims 28 to 44, wherein the cancer cells are contacted in vivo with an oncolytic virus and a nucleocytoplasmic transport inhibitor.

46. 45. The method of any one of claims 28 to 44, wherein the cancer cells are contacted with the oncolytic virus and the nucleocytoplasmic transport inhibitor ex vivo.

47. 47. The method of any one of claims 28 to 46, wherein the method comprises contacting the cancer cells with the oncolytic virus and the nucleocytoplasmic transport inhibitor simultaneously.

48. 47. The method of any one of claims 28 to 46, wherein the method comprises contacting the cancer cell sequentially with the oncolytic virus and the nucleocytoplasmic transport inhibitor.

49. 49. The method of claim 48, wherein the cancer cells are contacted with the oncolytic virus prior to being contacted with the nucleocytoplasmic transport inhibitor.

50. 49. The method of claim 48, wherein the cancer cells are contacted with the nucleocytoplasmic transport inhibitor prior to contacting with the oncolytic virus.

51. 51. The method of any one of claims 28 to 50, wherein the cancer cells are contacted with the oncolytic virus at an MOI of about 0.01 to about 10.

52. 52. The method of any one of claims 28 to 51, wherein the cancer cells are contacted with the oncolytic virus at an MOI of about 0.05 to about 5.

53. 53. The method of any one of claims 28 to 52, wherein the method comprises incubating the cancer cells in a medium containing the nucleocytoplasmic transport inhibitor at a concentration of from about 0.0001 μM to about 100 μM.

54. 54. The method of any one of claims 28 to 53, wherein the method comprises incubating the cancer cells in a medium containing the nucleocytoplasmic transport inhibitor at a concentration of from about 0.01 μM to about 1 μM.

55. 55. The method of any one of claims 28 to 54, wherein the method comprises contacting the cancer cells with a medium containing the nucleocytoplasmic transport inhibitor at a concentration of from about 0.0001 μM to about 100 μM.

56. 56. The method of any one of claims 28 to 55, wherein the method comprises contacting the cancer cells with a medium containing the nucleocytoplasmic transport inhibitor at a concentration of from about 0.01 μM to about 1 μM.

57. 57. The method of any one of claims 28 to 56, further comprising contacting autologous or xenogeneic cells with the oncolytic virus ex vivo.

58. 58. The method of claim 57, wherein the autologous or xenogeneic cells are bone marrow cells or macrophage cells.

59. 60. The method of claim 57 or 58, further comprising the step of injecting said autologous or xenogeneic cells into said subject.

60. a. an oncolytic virus, and b. A pharmaceutical composition comprising a nucleocytoplasmic transport inhibitor.

61. 61. The pharmaceutical composition of claim 60, further comprising a pharma- ceutically acceptable excipient or carrier.

62. 62. The pharmaceutical composition of claim 60 or 61, wherein the oncolytic virus is from the Poxviridae family.

63. 63. The pharmaceutical composition of any one of claims 60 to 62, wherein the oncolytic virus is derived from the subfamily Chordopoxvirinae or Entomopoxvirinae.

64. 64. The pharmaceutical composition of any one of claims 60 to 63, wherein the oncolytic virus is derived from the viral genus Orthopoxvirus, Serbidopoxvirus, Parapoxvirus, Avipoxvirus, Capripoxvirus, Leporipoxvirus, Suipoxvirus, Morsipoxvirus, Yatapoxvirus, Alphaentomopoxvirus, Betaentomopoxvirus, or Gammaentomopoxvirus.

65. 65. The pharmaceutical composition of any one of claims 60 to 64, wherein the oncolytic virus is from the genus Leporipoxvirus.

66. 66. The pharmaceutical composition according to any one of claims 60 to 65, characterized in that the oncolytic virus is myxoma virus (MYXV).

67. 65. The pharmaceutical composition of any one of claims 60 to 64, wherein the oncolytic virus is from the Orthopoxvirus genus.

68. 68. The pharmaceutical composition of any one of claims 60 to 67, wherein the oncolytic virus is genetically modified.

69. 69. The pharmaceutical composition of any one of claims 60 to 68, wherein the nucleocytoplasmic transport inhibitor is selected from the group consisting of leptomycin A, leptomycin B, latojadone A, latojadone B, latojadone C, latojadone D, anginomycin A, goniothalamin, piperlongumine, plumbagin, curcumin, valtrate, acetoxychavicol acetate, prenylcoumarin osthole, KOS 2464, PKF050-638, CBS9106, and selinexor.

70. 70. The pharmaceutical composition of any one of claims 60 to 69, wherein the nucleocytoplasmic transport inhibitor is leptomycin B.

71. 71. The pharmaceutical composition according to any one of claims 60 to 70, wherein the nucleocytoplasmic transport inhibitor is selinexor.

72. 72. The pharmaceutical composition according to any one of claims 60 to 71, characterized in that the pharmaceutical composition is in a unit dosage form suitable for intratumoral or parenteral administration.

73. The unit dose is about 1 x 10 per mL ３ Plaque forming units (PFU) to approximately 1 x 10 １０ 73. The pharmaceutical composition of claim 72, comprising up to PFU of the oncolytic virus.

74. The unit dose is about 1 x 10 per mL ５ Approximately 1 x 10 PFU １０ 73. The pharmaceutical composition of claim 72, comprising up to PFU of the oncolytic virus.

75. The weight ratio between the oncolytic virus and the nucleocytoplasmic transport inhibitor is about 1×10 ９ From about 1 x 10 －９ 75. The pharmaceutical composition according to any one of claims 60 to 74, characterized in that

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