Syncytial oncolytic herpes simplex variants as potential cancer therapeutics

Abstract

The present disclosure provides non-native herpes simplex viruses ("HSV"), compositions comprising, or alternatively consisting essentially of, or further consisting of HSV, and methods of making HSV or infecting cells with HSV. Also provided herein are methods of treating cancer or inhibiting the growth or metastasis of cancer cells in a subject in need thereof.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application Nos. 62 / 818,577 and 62 / 932,725, filed March 14, 2019 and November 8, 2019, respectively, the contents of each of which are incorporated herein by reference.

[0002] Government support statement This invention was made with Government support under Grant No. CA223104 awarded by the National Institutes of Health and National Cancer Institute (NIH / NCI). The Government has certain rights in this invention.

[0003] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on March 13, 2020, is named 106887-7660_ST25.txt and is 245,014 bytes in size. [Background technology]

[0004] background The antitumor effects of oncolytic herpes simplex viruses (oHSVs), such as Imlygic™, recently approved by the FDA to treat melanoma, are extremely promising. These vectors have two main mechanisms of action: (1) a lytic phase determined by direct infection and lysis of cells, and (2) an immune phase driven by stimulation of antitumor immunity. However, not all cancers respond similarly, as some cancers have an inherently slower spread of the virus. In culture, cells have different levels of growth permissiveness to the virus. In animals, variation in tumor stroma and immune cell composition leads to variation in the capacity for virus spread and immune response. Thus, there remains a need for strategies to improve the efficacy of the lytic phase to reach optimal therapeutic utility. The present disclosure meets these needs and provides related advantages as well. Summary of the Invention [Means for solving the problem]

[0005] overview The present disclosure provides a non-naturally occurring herpes simplex virus ("HSV"), wherein the virus comprises, consists essentially of, or consists of mutations in one or more of: (a) the gene encoding glycoprotein E ("gE"), (b) the gene encoding infected cell protein 0 ("ICP0"), (c) the gene encoding DNA packaging terminase subunit 1, (d) the gene encoding ICP8, or (e) the gene encoding ICP34.5.

[0006] In another embodiment, the disclosure provides a composition or pharmaceutical composition comprising, consisting essentially of, or consisting of a non-naturally occurring HSV of the disclosure. In another embodiment, the disclosure provides a method for infecting a cell, comprising, or alternatively consisting essentially of, or even further consisting of contacting a cell with a non-naturally occurring HSV or a composition or pharmaceutical composition containing a non-naturally occurring HSV.

[0007] In one aspect, the disclosure provides a method of preparing a non-naturally occurring HSV of the disclosure, comprising, or alternatively consisting essentially of, or even further consisting of mutating a gene in a non-naturally occurring HSV viral particle or introducing a transgene into the non-naturally occurring HSV. In another aspect, a method of making a non-naturally occurring HSV vector comprises, or alternatively consists essentially of, or even further consisting of (a), (b) and (c), (a) introducing a 17TermA HSV vector and a rRp450 HSV vector into a host cell; (b) growing the host cell for at least three passages; and (c) isolating the HSV particles produced by the host cell.

[0008] Also provided is a method for inhibiting the growth or metastasis of cancer cells or metastatic cancer cells, comprising, consisting essentially of, or consisting of contacting cells with an effective amount of a non-naturally occurring HSV vector or a composition or pharmaceutical composition containing a non-naturally occurring HSV vector as described herein. The contacting is in vitro or in vivo. In one embodiment, the contacting is in vivo by administering the non-naturally occurring HSV or the composition or pharmaceutical composition to a subject. In vitro, the method is carried out by contacting the non-naturally occurring HSV with cells. The in vitro method can be used as a personalized assay to test new therapies or to determine whether a therapy is suitable for the cancer to be treated. Additional cancer therapy can be combined with the disclosed method, which may be simultaneous or sequential.

[0009] The cancer cells to be treated may be solid tumors or blood cancers, such as carcinomas or sarcomas, non-limiting examples of which include pancreatic cancer, renal cancer, small cell lung cancer, brain cancer, neuroblastoma, neural cancer, bone cancer, lymphoma, myeloma, colon cancer, uterine cancer, breast cancer, leukemia, liver cancer, prostate cancer, skin cancer or melanoma. The cells may be of any species, such as mammalian and human, and when performed in vitro, the cells may be cultured cell lines or primary cells, such as from tissue biopsies. The cells may be adult or juvenile cells or cancer stem cells (i.e., cancer cells that have characteristics associated with normal stem cells, particularly the ability to give rise to all cell types found in a particular cancer sample) or cancer cells that lack such characteristics associated with normal stem cells. In one embodiment, the cells express the N-myc proto-oncogene protein (MYCN) and / or express MYCN at a higher level than non-cancer cells.

[0010] In another embodiment, the present disclosure also provides a method for treating cancer or metastatic cancer or inhibiting cancer cell growth or metastasis in a subject in need of such treatment, comprising, or alternatively consisting essentially of, or even consisting of administering to the subject an effective amount of a non-naturally occurring HSV, composition or pharmaceutical composition of the present disclosure.The subject to be treated can be any species, such as mammals and humans, such as dogs, horses, cows, cats, monkeys, rats or mice.Administration can be as a first line treatment, a second line treatment, a third line treatment, a fourth line treatment or a fifth line treatment.Additional cancer treatments can be combined with the disclosed methods, which can be simultaneous or sequential. The cancer to be treated may be a solid tumor or a blood cancer, such as a carcinoma or sarcoma, non-limiting examples of which include pancreatic cancer, renal cancer, small cell lung cancer, brain cancer, neuroblastoma, neural cancer, bone cancer, lymphoma, myeloma, colon cancer, uterine cancer, breast cancer, leukemia, liver cancer, prostate cancer, skin cancer or melanoma.

[0011] The methods of the present disclosure can be combined with appropriate diagnostics to monitor disease remission or progression. Several methods for such monitoring are known in the art.

[0012] In one embodiment, the present disclosure provides a method for inducing cell lysis, comprising, or alternatively consisting essentially of, or even consisting of contacting a cell with an effective amount of a non-naturally occurring HSV, composition and / or pharmaceutical composition of the present disclosure. The contacting is in vitro or in vivo. In one embodiment, the contacting is in vivo by administering the non-naturally occurring HSV or composition or pharmaceutical composition to a subject. In vitro, the method is carried out by contacting the non-naturally occurring HSV with a cell. The in vitro method can be used as an individualized assay to test new therapies or to determine whether a therapy is suitable for the subject to be treated. Additional cell lysis therapy can be combined with the disclosed method, which can be simultaneous or sequential.

[0013] The cells to be treated may be solid tumors or blood cancers, such as carcinomas or sarcomas, including, but not limited to, pancreatic cancer, renal cancer, small cell lung cancer, brain cancer, neuroblastoma, neural cancer, bone cancer, lymphoma, myeloma, colon cancer, uterine cancer, breast cancer, leukemia, liver cancer, prostate cancer, skin cancer or melanoma. The cells may be of any species, such as mammalian and human, and when performed in vitro, the cells may be cultured cell lines or primary cells, such as from tissue biopsies. The cells may be adult or juvenile cells, or cancer stem cells, or cancer cells that do not have characteristics associated with normal stem cells. The treatment may be combined with an appropriate assay to test for the effectiveness of the treatment, such as remission or progression of cancer.

[0014] In another embodiment, the present disclosure also provides kits comprising, or alternatively consisting essentially of, or even further consisting of the non-naturally occurring HSV, compositions and / or pharmaceutical compositions of the present disclosure. In certain embodiments, for example, the following are provided: (Item 1) A non-naturally occurring herpes simplex virus ("HSV"), said virus comprising: (a) a gene encoding glycoprotein E ("gE"); (b) a gene encoding infected cell protein 0 ("ICP0"); (c) a gene encoding DNA packaging terminase subunit 1; (d) a gene encoding ICP8; or (e) The gene encoding ICP34.5 A non-naturally occurring herpes simplex virus ("HSV") comprising one or more mutations in a virulence gene that is from the group consisting of: (Item 2) 2. The non-naturally occurring HSV of item 1, wherein the HSV further comprises a gene encoding a dysfunctional ICP34.5 protein and / or a gene encoding a dysfunctional ICP6 protein. (Item 3) 3. The non-naturally occurring HSV of item 2, wherein the gene encoding the dysfunctional ICP34.5 protein comprises a polynucleotide having a sequence selected from SEQ ID NO:5, SEQ ID NO:7, and a sequence at least 95% identical to SEQ ID NO:5 or SEQ ID NO:7, and maintaining the mutation at a nucleotide in any one of SEQ ID NO:5 or SEQ ID NO:7. (Item 4) 3. The non-naturally occurring HSV of item 2, wherein the gene encoding the dysfunctional ICP6 protein comprises a polynucleotide having a sequence selected from SEQ ID NO: 45, SEQ ID NO: 47, and a sequence at least 95% identical to SEQ ID NO: 45 or 47, and maintaining the mutation at a nucleotide in any one of SEQ ID NO: 45 or 47. (Item 5) 5. The non-naturally occurring HSV of any one of items 1 to 4, wherein the mutation in the virulence gene comprises an insertion, deletion, truncation, frameshift, substitution or point mutation. (Item 6) 6. The non-naturally occurring HSV of any one of items 1 to 5, wherein the HSV lacks a gene encoding a functional ICP34.5 protein and / or a functional ICP6 protein. (Item 7) 7. The non-naturally occurring HSV of any one of items 1 to 6, wherein the mutation is a non-synonymous mutation in the virulence gene. (Item 8) The mutation or equivalent thereof is (a) an alanine to threonine mutation at position 151 of the gE protein; (b) an arginine to histidine mutation at position 258 of the ICP0 protein; (c) an alanine to threonine mutation at position 376 of the DNA packaging terminase subunit 1 protein; (d) a threonine to methionine mutation at position 1155 of the ICP8 protein; or (e) a proline to histidine mutation at position 119 of the ICP34.5 protein; 8. The non-naturally occurring HSV or its equivalent according to any one of items 1 to 7, which encodes: (Item 9) The following: (a) a polynucleotide encoding an amino acid sequence selected from SEQ ID NOs: 2, 6, 8, 10, and 52, and / or a polynucleotide having a sequence selected from SEQ ID NOs: 1, 5, 7, 9, and 51; (b) a polypeptide having an amino acid sequence selected from SEQ ID NOs: 2, 6, 8, 10, and 52; (c) a polynucleotide encoding an amino acid sequence selected from SEQ ID NOs: 13, 15, 17, and 19, and / or a polynucleotide having a sequence selected from SEQ ID NOs: 12, 14, 16, and 18; (d) a polypeptide having an amino acid sequence selected from SEQ ID NOs: 13, 15, 17 and 19; (e) a polynucleotide encoding an amino acid sequence selected from SEQ ID NOs: 21, 23 and 26, and / or a polynucleotide having a sequence selected from SEQ ID NOs: 20, 22, 24, 25 and 53, or these sequences not containing one or two or more introns; (f) a polypeptide having an amino acid sequence selected from SEQ ID NOs: 21, 23 and 26; (g) a polynucleotide encoding an amino acid sequence selected from SEQ ID NOs: 28, 30, 32, and 34, and / or a polynucleotide having a sequence selected from SEQ ID NOs: 27, 29, 31, and 33; (h) a polypeptide having an amino acid sequence selected from SEQ ID NOs: 28, 30, 32 and 34; (i) a polynucleotide encoding an amino acid sequence selected from SEQ ID NOs: 36, 38, 40, and 42, and / or a polynucleotide having a sequence selected from SEQ ID NOs: 35, 37, 39, and 41; (j) a polypeptide having an amino acid sequence selected from SEQ ID NOs: 36, 38, 40 and 42; (k) a polynucleotide encoding an amino acid sequence selected from SEQ ID NOs: 44, 46, 48, and 50, and / or a polynucleotide having a sequence selected from SEQ ID NOs: 43, 45, 47, and 49; (l) a polypeptide having an amino acid sequence selected from SEQ ID NOs: 44, 46, 48 and 50; 9. The non-naturally occurring HSV according to any one of items 1 to 8, comprising one or more of the following: (Item 10) 10. The non-naturally occurring HSV of any one of items 1 to 9, further comprising a polynucleotide having a sequence identical to at least a fragment of a virulence gene from 17TermA HSV. (Item 11) 11. The non-naturally occurring HSV of any one of paragraphs 1 to 10, further comprising a polynucleotide having a sequence identical to at least a fragment of a virulence gene from rRp450 HSV. (Item 12) 12. The non-naturally occurring HSV of any one of items 1 to 11, wherein the HSV is derived from an HSV type 1 ("HSV-1") or HSV type 2 ("HSV-2") strain. (Item 13) 12. The non-naturally occurring HSV according to any one of items 1 to 11, wherein the HSV is derived from the HSV-1 KOS strain. (Item 14) 14. The non-naturally occurring HSV of any one of items 1 to 13, further comprising a transgene. (Item 15) 15. A composition comprising the non-naturally occurring HSV according to any one of items 1 to 14 and a carrier. (Item 16) 15. The composition according to item 14, wherein the carrier is a pharma- ceutically acceptable carrier. (Item 17) 17. The composition of claim 15 or 16, further comprising a cryopreservation agent that facilitates freezing and thawing of the non-native HSV without significant loss of pathogenicity. (Item 18) 15. A non-human mammal infected with a non-naturally occurring HSV according to any one of items 1 to 14. (Item 19) A cell, optionally a lymphocyte, infected with a non-naturally occurring HSV according to any one of items 1 to 14. (Item 20) 18. A method for infecting a cell, comprising contacting said cell with a non-naturally occurring HSV according to any one of items 1 to 14 or with a composition according to any one of items 15 to 17. (Item 21) 21. The method of item 20, wherein the cell is a lymphocyte. (Item 22) 22. The method of claim 20 or 21, wherein the cells are infected with Epstein-Barr Virus ("EBV"). (Item 23) 14. A method for preparing a viral vector, comprising introducing a transgene into the non-naturally occurring HSV according to any one of items 1 to 13. (Item 24) 18. A method for inhibiting the growth or metastasis of cancer cells, comprising contacting said cells with an effective amount of a non-naturally occurring HSV vector according to any one of items 1 to 14 or a composition according to any one of items 15 to 17. (Item 25) 25. The method of claim 24, wherein the contacting is in vitro or in vivo. (Item 26) 25. The method of claim 24, wherein the contacting is in vivo by administration of the non-natural product to a subject. (Item 27) 18. A method for treating cancer in a subject, comprising administering to the subject an effective amount of a non-naturally occurring HSV vector according to any one of items 1 to 14 or a composition according to any one of items 15 to 17. (Item 28) 28. The method according to any one of items 24 to 27, wherein the cancer is a cancer of the following types or the cancer cells are selected from the following types: pancreatic cancer, renal cancer, small cell lung cancer, brain cancer, neural cancer, bone cancer, lymphoma, myeloma, colon cancer, uterine cancer, breast cancer, leukemia, liver cancer, prostate cancer, skin cancer or melanoma cells. (Item 29) 29. The method according to item 27 or 28, wherein the HSV vector or the pharmaceutical composition is administered by injection, infusion, instillation and / or inhalation. (Item 30) 29. The method according to any one of items 27 to 28, wherein said administering comprises a treatment from the group of first line treatment, second line treatment, third line treatment, fourth line treatment or fifth line treatment. (Item 31) 28. The method of claim 27, further comprising administering to the subject an effective amount of an anti-cancer treatment. (Item 32) 32. The method according to any one of items 27 to 31, wherein the subject is a mammal. (Item 33) 33. The method according to item 32, wherein the mammal is from the group of humans, mice, rats, guinea pigs, non-human primates, dogs, cats, horses, cows, pigs, goats or sheep. (Item 34) 33. The method of claim 32, wherein the subject is a human. (Item 35) A method for producing the HSV vector according to any one of items 1 to 13, comprising: (a) introducing a 17TermA HSV vector and a rRp450 HSV vector into a host cell; (b) growing the host cells for at least three passages; (c) isolating HSV particles produced by the host cell; A method comprising: (Item 36) 36. The method of claim 35, wherein the HSV is introduced into the host cell by transfection, infection, transformation, electroporation, injection, microinjection, or a combination thereof. (Item 37) 37. The method of claim 35 or 36, wherein the host cells are grown for at least 3 passages, 10 passages, 20 passages, 30 passages, 40 passages or 50 passages. (Item 38) 38. The method of any one of items 35 to 37, wherein the host cell comprises complementary gene products to support replication of the introduced HSV vector. (Item 39) 39. The method according to item 38, wherein the complementary gene encodes an ICP6 protein and / or an ICP34.5 protein. (Item 40) 40. The method of any one of items 35 to 39, further comprising introducing a transgene into the HSV vector. (Item 41) 41. HSV particles produced by the method according to any one of items 35 to 40. (Item 42) 18. A method for inducing cell lysis, comprising contacting said cells with a non-naturally occurring HSV according to any one of items 1 to 14 or with a composition according to any one of items 15 to 17. (Item 43) 43. The method of claim 42, wherein the cell is a cancer cell. (Item 44) A kit comprising the HSV vector according to any one of items 1 to 14 or the composition according to any one of items 15 to 17, and instructions for use. (Item 45) A method for producing the HSV vector according to any one of items 1 to 13, comprising: (a) introducing into a host cell a polynucleotide encoding the viral genome of the HSV vector; (b) growing the host cell; (c) isolating HSV particles produced by the host cell; Includes, a method. (Item 46) 46. ​​The method of claim 45, wherein the polynucleotide is introduced into the host cell by transfection, infection, transformation, electroporation, injection, microinjection, or a combination thereof. (Item 47) 47. The method of claim 45 or 46, wherein the nucleic acid sequence encoding the viral genome is introduced into the host cell in a vector. (Item 48) 48. The method of claim 47, wherein the vector is HSV or a plasmid. (Item 49) 49. The method of any one of items 45 to 48, wherein the host cell comprises complementary gene products to support replication of the introduced HSV vector. (Item 50) 50. The method of any one of items 45 to 49, wherein the isolated HSV is substantially free of host cells, cell debris, culture medium or any other factors used in culturing the host cells. (Item 51) 51. The method according to any one of items 45 to 50, wherein the isolation is by centrifugation, filtration, chromatography or any combination thereof. [Brief description of the drawings]

[0015] [Figure 1A-1B]1A-1B show schematics of the generation of Mut-3 and Mut-3Δ34.5 (FIG. 1A) and Mut-3 SNP (FIG. 1B). HSV Mut-3 was isolated from serial passages mixing 17TermA and rRp450 in a non-permissive line ("directed evolution"), and the attenuated mutant Mut-3Δ34.5 was constructed through gene editing (denoted as the "CRISPR / Cas9" step) (FIG. 1A). FIG. 1B shows a sequence comparison of Mut-3 with its parent virus. Nonsynonymous mutations in Mut-3 that differ from either parent are marked with a downward-right slash, including UL15, UL29, US8, RL1, and RL2. Genomic sequences identical to 17TermA are shown as blanks in the lower panel, and genomic sequences identical to rRp450 are marked with a downward-left slash.

[0016] [Figure 2A-2B]Figures 2A-2C show the plaque size and receptor usage of Mut-3, Mut-3Δ34.5, rRp450 and 17TermA. Applicant performed plaque assays of the four viruses simultaneously, and after 3 days, scanned and analyzed the plaque images via a Keyence HS All-in-one Fluorescence Microscope BZ-II Analyzer. (Figure 2A) Raw images (top) and masked images (bottom, numbered plaques) of the four viruses. (Figure 2B) The average plaque size (left) and number of plaques (right) of the four viruses were calculated. The plaque size of Mut-3 and Mut-3Δ34.5 is significantly larger than either of the parent viruses rRp450 and 17TermA. (Figure 2C) In vitro cytotoxicity / MTS assay of a set of CHO cells. Applicants infected CHO-K1 (as marked), CHO-nectin-1 (as marked), CHO-nectin-2 (as marked) and CHO-HVEM (as marked) with the four viruses at different multiplicities of infection (MOI). Applicants measured cell viability colorimetric cell proliferation / MTS assays 3 days post virus infection (pvi) compared to untreated controls. Only CHO-nectin-1 and CHO-HVEM were sensitive to the treatment of the four viruses, but not CHO-K1 or CHO-nectin-2 (mainly for HSV-2 entry), suggesting that Mut-3 and Mut-3Δ34.5 do not bypass the receptor barrier and may still relay on the standard HSV entry receptor to infect cells. [Figure 2C]Figures 2A-2C show the plaque size and receptor usage of Mut-3, Mut-3Δ34.5, rRp450 and 17TermA. Applicant performed plaque assays of the four viruses simultaneously, and after 3 days, scanned and analyzed the plaque images via a Keyence HS All-in-one Fluorescence Microscope BZ-II Analyzer. (Figure 2A) Raw images (top) and masked images (bottom, numbered plaques) of the four viruses. (Figure 2B) The average plaque size (left) and number of plaques (right) of the four viruses were calculated. The plaque size of Mut-3 and Mut-3Δ34.5 is significantly larger than either of the parent viruses rRp450 and 17TermA. (Figure 2C) In vitro cytotoxicity / MTS assay of a set of CHO cells. Applicants infected CHO-K1 (as marked), CHO-nectin-1 (as marked), CHO-nectin-2 (as marked) and CHO-HVEM (as marked) with the four viruses at different multiplicities of infection (MOI). Applicants measured cell viability colorimetric cell proliferation / MTS assays 3 days post virus infection (pvi) compared to untreated controls. Only CHO-nectin-1 and CHO-HVEM were sensitive to the treatment of the four viruses, but not CHO-K1 or CHO-nectin-2 (mainly for HSV-2 entry), suggesting that Mut-3 and Mut-3Δ34.5 do not bypass the receptor barrier and may still relay on the standard HSV entry receptor to infect cells.

[0017] [Figure 3A]3A-C: Mut-3Δ34.5 is more potent than 17TermA (γ134.5-null) in killing human and mouse neuroblastoma cells, and this is not due to an increased yield of infectious viral particles in vitro. (FIG. 3A) In vitro cytotoxicity / MTS assays of neuroblastoma cell lines. Applicants infected both human (SK-N-AS) and mouse (975A2) neuroblastoma cells with Mut-3 (as marked), Mut-3Δ34.5 (as marked) or 17TermA (as marked) at different MOIs. Applicants measured cell survival by MTS assay 4 days after virus infection compared to untreated controls. N=6, error bars represent standard error of the mean. ****p<0.0001, 2-way ANOVA. (FIG. 3B) In vitro virus replication assays. Applicants infected neuroblastoma cell lines with 17TermA (as marked) or Mut-3Δ34.5 (as marked) at MOI=0.1 (upper panel, human line SK-N-AS) or MOI=0.5 (lower panel, mouse line 975A2) and washed cells with PBS 2 hours after virus infection. Applicants harvested cell lysates 2, 24, 48 and 72 hours after virus infection and determined virus yields by plaque assay. N=3. Error bars represent standard deviation. *p<0.05, **p<0.001, ****p<0.0001, 2-way ANOVA. (Figure 3C) Comparison of relative free released virions in neuroblastoma cultures infected with Mut-3Δ34.5 and 17TermA. Applicants performed a similar assay as described in (Figure 3B) by taking the supernatant and pellet and performing plaque assays over time on two separate portions instead of measuring the whole culture in Figure 3B. Applicants calculated the relative released virions as the ratio of infectious particles in the supernatant to the whole culture (supernatant + pellet). Mut-3Δ34.5 shows significantly more relative released virions than 17TermA at 48 hours. N=3. Error bars represent standard deviation. *p<0.05. [Figure 3B]3A-C: Mut-3Δ34.5 is more potent than 17TermA (γ134.5-null) in killing human and mouse neuroblastoma cells, and this is not due to an increased yield of infectious viral particles in vitro. (FIG. 3A) In vitro cytotoxicity / MTS assays of neuroblastoma cell lines. Applicants infected both human (SK-N-AS) and mouse (975A2) neuroblastoma cells with Mut-3 (as marked), Mut-3Δ34.5 (as marked) or 17TermA (as marked) at different MOIs. Applicants measured cell survival by MTS assay 4 days after virus infection compared to untreated controls. N=6, error bars represent standard error of the mean. ****p<0.0001, 2-way ANOVA. (FIG. 3B) In vitro virus replication assays. Applicants infected neuroblastoma cell lines with 17TermA (as marked) or Mut-3Δ34.5 (as marked) at MOI=0.1 (upper panel, human line SK-N-AS) or MOI=0.5 (lower panel, mouse line 975A2) and washed cells with PBS 2 hours after virus infection. Applicants harvested cell lysates 2, 24, 48 and 72 hours after virus infection and determined virus yields by plaque assay. N=3. Error bars represent standard deviation. *p<0.05, **p<0.001, ****p<0.0001, 2-way ANOVA. (Figure 3C) Comparison of relative free released virions in neuroblastoma cultures infected with Mut-3Δ34.5 and 17TermA. Applicants performed a similar assay as described in (Figure 3B) by taking the supernatant and pellet and performing plaque assays over time on two separate portions instead of measuring the whole culture in Figure 3B. Applicants calculated the relative released virions as the ratio of infectious particles in the supernatant to the whole culture (supernatant + pellet). Mut-3Δ34.5 shows significantly more relative released virions than 17TermA at 48 hours. N=3. Error bars represent standard deviation. *p<0.05. [Figure 3C]3A-C: Mut-3Δ34.5 is more potent than 17TermA (γ134.5-null) in killing human and mouse neuroblastoma cells, and this is not due to an increased yield of infectious viral particles in vitro. (FIG. 3A) In vitro cytotoxicity / MTS assays of neuroblastoma cell lines. Applicants infected both human (SK-N-AS) and mouse (975A2) neuroblastoma cells with Mut-3 (as marked), Mut-3Δ34.5 (as marked) or 17TermA (as marked) at different MOIs. Applicants measured cell survival by MTS assay 4 days after virus infection compared to untreated controls. N=6, error bars represent standard error of the mean. ****p<0.0001, 2-way ANOVA. (FIG. 3B) In vitro virus replication assays. Applicants infected neuroblastoma cell lines with 17TermA (as marked) or Mut-3Δ34.5 (as marked) at MOI=0.1 (upper panel, human line SK-N-AS) or MOI=0.5 (lower panel, mouse line 975A2) and washed cells with PBS 2 hours after virus infection. Applicants harvested cell lysates 2, 24, 48 and 72 hours after virus infection and determined virus yields by plaque assay. N=3. Error bars represent standard deviation. *p<0.05, **p<0.001, ****p<0.0001, 2-way ANOVA. (Figure 3C) Comparison of relative free released virions in neuroblastoma cultures infected with Mut-3Δ34.5 and 17TermA. Applicants performed a similar assay as described in (Figure 3B) by taking the supernatant and pellet and performing plaque assays over time on two separate portions instead of measuring the whole culture in Figure 3B. Applicants calculated the relative released virions as the ratio of infectious particles in the supernatant to the whole culture (supernatant + pellet). Mut-3Δ34.5 shows significantly more relative released virions than 17TermA at 48 hours. N=3. Error bars represent standard deviation. *p<0.05.

[0018] [Fig. 4A-4H]Figures 4A-4H: TEM analysis of Mut3Δ34.5 and 17TermA uptake in neuroblastoma cells. Neuroblastoma cells SK-N-AS and 975A2 were infected with Mut-3Δ34.5 or 17TermA at an MOI of 50 for 20 min at 37°C. (Figures 4A, 4B, 4E and 4F) Insets show that Mut-3Δ34.5 virions were found mainly in intracellular vesicles (arrows except the right arrow in Figure 4B) and few were found fused with the plasma membrane (arrows on the right side of Figure 4B). (Figures 4C, 4D, 4G and 4H) Insets show that 17TermA virions were found mainly in intracellular vesicles (arrows except the lower arrow in Figure 4G) and few were found fused with the plasma membrane (arrows on the lower side of Figure 4G). TEM analysis was performed via a Hitachi H-7650 TEM. N: nucleus. Scale bar: 500 nm.

[0019] [Figure 5A]Figures 5A-5C: Attenuated 17Δ34.5 virions showed comparable potency to 17TermA in neuroblastoma cells. Applicants generated 17Δ34.5 via CRISPR-Cas9 gene editing technology to replace the g134.5 gene in wild-type strain 17+ with an EGFP expression cassette. (Figure 5A) Plaque images of 17Δ34.5 clones on Vero cells. Both B4 and G1 clones produce 100% GFP-positive (left panel) non-syncytial (right panel, phase contrast) plaques after three rounds of plaque purification. Two days after viral infection. Images were captured via an EVOS® FL Imaging System. Scale bar: 400 μm. (Figure 5B) In vitro cytotoxicity / MTS assay of neuroblastoma cell lines. Applicants infected neuroblastoma cell lines SK-N-AS and 975A2 with wild-type strain 17+ (as marked), 17TermA (as marked), 17Δ34.5 clone B4 (as marked) or 17Δ34.5 clone G1 (as marked) at different MOIs. Applicants measured cell survival by MTS assay 3 days after virus infection compared to untreated control. The potency of both 17Δ34.5 clones was significantly lower than their wild-type counterpart strain 17+ but comparable to 17TermA. EGFP cassette and EGFP DNA sequence are shown in FIG. 5C (SEQ ID NO: 11). [Figure 5B]Figures 5A-5C: Attenuated 17Δ34.5 virions showed comparable potency to 17TermA in neuroblastoma cells. Applicants generated 17Δ34.5 via CRISPR-Cas9 gene editing technology to replace the g134.5 gene in wild-type strain 17+ with an EGFP expression cassette. (Figure 5A) Plaque images of 17Δ34.5 clones on Vero cells. Both B4 and G1 clones produce 100% GFP-positive (left panel) non-syncytial (right panel, phase contrast) plaques after three rounds of plaque purification. Two days after viral infection. Images were captured via an EVOS® FL Imaging System. Scale bar: 400 μm. (Figure 5B) In vitro cytotoxicity / MTS assay of neuroblastoma cell lines. Applicants infected neuroblastoma cell lines SK-N-AS and 975A2 with wild-type strain 17+ (as marked), 17TermA (as marked), 17Δ34.5 clone B4 (as marked) or 17Δ34.5 clone G1 (as marked) at different MOIs. Applicants measured cell survival by MTS assay 3 days after virus infection compared to untreated control. The potency of both 17Δ34.5 clones was significantly lower than their wild-type counterpart strain 17+ but comparable to 17TermA. EGFP cassette and EGFP DNA sequence are shown in FIG. 5C (SEQ ID NO: 11). [Figure 5C-1]Figures 5A-5C: Attenuated 17Δ34.5 virions showed comparable potency to 17TermA in neuroblastoma cells. Applicants generated 17Δ34.5 via CRISPR-Cas9 gene editing technology to replace the g134.5 gene in wild-type strain 17+ with an EGFP expression cassette. (Figure 5A) Plaque images of 17Δ34.5 clones on Vero cells. Both B4 and G1 clones produce 100% GFP-positive (left panel) non-syncytial (right panel, phase contrast) plaques after three rounds of plaque purification. Two days after viral infection. Images were captured via an EVOS® FL Imaging System. Scale bar: 400 μm. (Figure 5B) In vitro cytotoxicity / MTS assay of neuroblastoma cell lines. Applicants infected neuroblastoma cell lines SK-N-AS and 975A2 with wild-type strain 17+ (as marked), 17TermA (as marked), 17Δ34.5 clone B4 (as marked) or 17Δ34.5 clone G1 (as marked) at different MOIs. Applicants measured cell survival by MTS assay 3 days after virus infection compared to untreated control. The potency of both 17Δ34.5 clones was significantly lower than their wild-type counterpart strain 17+ but comparable to 17TermA. EGFP cassette and EGFP DNA sequence are shown in FIG. 5C (SEQ ID NO: 11). [Figure 5C-2]Figures 5A-5C: Attenuated 17Δ34.5 virions showed comparable potency to 17TermA in neuroblastoma cells. Applicants generated 17Δ34.5 via CRISPR-Cas9 gene editing technology to replace the g134.5 gene in wild-type strain 17+ with an EGFP expression cassette. (Figure 5A) Plaque images of 17Δ34.5 clones on Vero cells. Both B4 and G1 clones produce 100% GFP-positive (left panel) non-syncytial (right panel, phase contrast) plaques after three rounds of plaque purification. Two days after viral infection. Images were captured via an EVOS® FL Imaging System. Scale bar: 400 μm. (Figure 5B) In vitro cytotoxicity / MTS assay of neuroblastoma cell lines. Applicants infected neuroblastoma cell lines SK-N-AS and 975A2 with wild-type strain 17+ (as marked), 17TermA (as marked), 17Δ34.5 clone B4 (as marked) or 17Δ34.5 clone G1 (as marked) at different MOIs. Applicants measured cell survival by MTS assay 3 days after virus infection compared to untreated control. The potency of both 17Δ34.5 clones was significantly lower than their wild-type counterpart strain 17+ but comparable to 17TermA. EGFP cassette and EGFP DNA sequence are shown in FIG. 5C (SEQ ID NO: 11). [Figure 5C-3]Figures 5A-5C: Attenuated 17Δ34.5 virions showed comparable potency to 17TermA in neuroblastoma cells. Applicants generated 17Δ34.5 via CRISPR-Cas9 gene editing technology to replace the g134.5 gene in wild-type strain 17+ with an EGFP expression cassette. (Figure 5A) Plaque images of 17Δ34.5 clones on Vero cells. Both B4 and G1 clones produce 100% GFP-positive (left panel) non-syncytial (right panel, phase contrast) plaques after three rounds of plaque purification. Two days after viral infection. Images were captured via an EVOS® FL Imaging System. Scale bar: 400 μm. (Figure 5B) In vitro cytotoxicity / MTS assay of neuroblastoma cell lines. Applicants infected neuroblastoma cell lines SK-N-AS and 975A2 with wild-type strain 17+ (as marked), 17TermA (as marked), 17Δ34.5 clone B4 (as marked) or 17Δ34.5 clone G1 (as marked) at different MOIs. Applicants measured cell survival by MTS assay 3 days after virus infection compared to untreated control. The potency of both 17Δ34.5 clones was significantly lower than their wild-type counterpart strain 17+ but comparable to 17TermA. EGFP cassette and EGFP DNA sequence are shown in FIG. 5C (SEQ ID NO: 11).

[0020] [Figures 6A-6C] Figures 6A-6C: Mut-3Δ34.5 shows much faster viral gene transfer and cell killing compared to 17Δ34.5. We treated (Figure 6A) Vero (Figure 6B) SK-N-AS (Figure 6C) 975A2 cells with either mock CTL, Mut-3Δ34.5 or 17Δ34.5 at different MOIs and monitored GFP positive area (upper panel) and cell confluency (lower panel) over time using IncuCyte ZOOM live cells. N=6 wells per condition, n=2 measurements per well for each time point. Post viral infection (pvi). Mann-Whitney U test is used to compare time to maximum GFP area between viruses for (Figure 6A). Error bars represent standard deviation.

[0021] [Figure 7] Figure 7: Mut-3Δ34.5 is more effective than 17TermA to control human neuroblastoma growth in vivo. Athymic nude mice bearing sub-q SK-N-AS tumors were intratumorally injected with either phosphate-buffered saline (PBS) control (as indicated, n=8), three doses of 1e7 pfu of 17TermA (as indicated, n=8) or Mut-3Δ34.5 (as indicated, n=9). Kaplan-Meier survival curves were plotted. Statistical significance between 17TermA and Mut-3Δ34.5 was scored using the log-rank test. *p<0.05.

[0022] [Figure 8A-8C]Figure 8A-8C: Mut-3ΔICP6, an attenuated version of Mut-3, induces superior cytotoxicity in human neuroblastoma cell line CHP-134 compared to oncolytic herpesvirus rRp450. (Figure 8A) Map of Mut-3ΔICP6 constructed by CRISPR-Cas9 gene editing strategy, in which the UL39 gene encoding ICP-6 was replaced with a GFP reporter cassette driven by CMV. MTS cell viability assay comparing the cytotoxicity profile of Mut-3, rRp450 and Mut3-ΔICP6 / D7-1 in human neuroblastoma cell lines. Cells were seeded at 4000 cells / well in 96-well dishes, incubated overnight at 37 °C, and then infected with each of the listed viruses at a multiplicity of infection (MOI) of 0.001, 0.01, 0.1 and 1 infectious viral particle per cell. Assays were performed using the Cell Titer96 AQuesous Non-Radioactive Cell Proliferation Assay / MTS (Promega, Madison, WI) on days 3 or 4 post-infection according to the manufacturer's instructions. Each sample group was run in quadruplicate, and results were presented as percent cell survival compared to uninfected controls. Error bars represent standard deviation. Statistical significance was assessed using t-test. *p≦0.05, **p≦0.01, ***p≦0.001. Figure 8B SK-N-AS and Figure 8C CHP-134 show the results graphically.

[0023] [Figure 9A-9B]Figures 9A-9B: Mut-3ΔICP6 induces superior cytotoxicity in mouse neuroblastoma cell lines Neuro-2a and 975A2 compared to rRp450. Figures 9A-9B show the results of an MTS cell viability assay comparing the cytotoxicity profiles of Mut-3, rRp450 and Mut3-ΔICP6 / D7-1 in mouse neuroblastoma cell lines. Cells were seeded at 4000 cells / well in 96-well dishes, incubated overnight at 37°C, and then infected with each of the listed viruses at a multiplicity of infection (MOI) of 0.001, 0.01, 0.1 and 1 infectious viral particle per cell. Assays were performed on days 3 or 4 post-infection using a Cell Titer96 AQuesous Non-Radioactive Cell Proliferation Assay / MTS (Promega, Madison, WI) according to the manufacturer's instructions. Each sample group was run in quadruplicate and results were presented as percent cell survival compared to uninfected controls. Error bars represent standard deviation. Statistical significance was assessed using t-test. **p≦0.01, ***p≦0.001. Figure 9A: Neuro-2a and Figure 9B: 975A2.

[0024] [Figure 10A-10B]Figure 10A-B: Mut-3ΔICP6 results in significantly higher virus yields than rRp450 in mouse neuroblastoma cell line 975A2 over 48 and 72 hour infection periods. Mouse 975A2 (Figure 10A) and human SK-N-AS (Figure 10B) neuroblastoma cells were seeded at 2x105 cells / well in 12-well dishes, incubated overnight at 37°C, and infected with each virus in 200 μL of serum-free medium for 2 hours with gentle shaking every 20 minutes at an MOI of 0.01 for SK-N-AS cells (Figure 10B) or at an MOI of 0.5 for 975A2 cells (Figure 10A). Cells were washed once with PBS and covered with 1 mL of complete medium. Cells and supernatants were then harvested at 2, 24, 48 and 72 hours post-infection, frozen and thawed three times, serially diluted and titrated on Vero cells to determine infectious virus yields by standard plaque assay. Each sample was assayed in triplicate. Error bars represent standard deviation. Statistical significance was assessed using t-test. *p ≤ 0.05, **p ≤ 0.01.

[0025] [Figure 11A-11B]Figures 11A-11B: Mut-3Δ34.5 and Mut-3ΔICP6 are significantly less potent than Mut-3 virus against differentiated human keratinocytes. Human keratinocyte (HFK) cells were grown in EpiLife Media (Cascade Biologics) supplemented with human keratinocyte growth supplements according to the manufacturer's instructions. Undifferentiated HFKs (Figure 11A) were seeded at a density of 2000 cells / well in 96-well plates and cultured overnight. Cultures were then infected with Mut-3, Mut-3Δ34.5 / C8G5 or Mut-3ΔICP6 D7-1 at MOIs of 0.0004, 0.004, 0.04, 0.4 and 4. Three days after virus infection, cell survival was determined by MTS assay. 24 hours after seeding, differentiated HFKs were produced by adding 10% FBS and 1 mmol / l CaCl2 to the culture medium of HFKs and allowing the HFKs to incubate for an additional 48 hours before infection. Four days after virus infection, the survival of differentiated cells (FIG. 11B) was determined by MTS assay. Each sample was assayed in quadruplicate. Error bars represent standard deviation. Statistical significance was evaluated using t-test. *p≦0.05, **p≦0.01.

[0026] [Figure 12]Intravenous injection of 1e6 pfu of wild-type KOS strain virus is lethal to naive Blab / c mice. Description of Mut-3Δ34.5 and Mut-3ΔICP6 safety studies. Applicants initiated virus biodistribution studies by injecting 1e8 plaque-forming units (pfu) of each virus into naive non-tumor-bearing Balb / c mice (20 males, 20 females) via the tail vein. Applicants then sacrificed the animals on days 1, 14, 28, 56 and 85 (n=4 / time point) and collected their peripheral blood, testes (males), ovaries (females), spleen, lungs, kidneys, heart, lungs and brain for analysis. Applicants observed each mouse daily for the first two weeks after virus administration and twice weekly thereafter until the scheduled day of sacrifice. The weight of each mouse was recorded before virus challenge and then weekly. In parallel, as a positive (non-safety) control, a group of mice was administered wild-type KOS virus [dose range: 1e5-1e7 pfu / mouse (n=3)]. Applicants also performed pathological analysis of harvested organs and compared samples obtained from mice administered mutant viruses to those administered wild-type KOS virus. Applicants also utilized plaque assays to detect the replicative capacity of these viruses. Results show survival curves of mice receiving a single dose of 1e5, 1e6 or 1e7 pfu of wild-type KOS virus.

[0027] [Figure 13A-13B] Figures 13A-13B: Naïve Balb / c mice can tolerate intravenous injections of up to 1e8 pfu of Mut-3Δ34.5(C8G5) or Mut-3ΔICP6(D7-1). Applicants have completed safety and biodistribution studies and found that mice receiving up to 1e8 pfu of Mut-3Δ34.5(C8G5) or Mut-3ΔICP6(D7-1) remained healthy until the designated sacrifice date (up to day 85) by physical examination. In female mice, plaque assays revealed the presence of infectious virus in the heart, kidney, liver, ovaries and spleen only at 24 hours post-infection. Figures 13A and 13B graphically show the results obtained from female and male mice, respectively.

[0028] [Figure 14] Replicating HSV can be detected in the brain, kidney and ovaries of mice given 1e6 pfu or more of wild type KOS virus. Figure 14 is a table summarizing the results of plaque assays from tissues of mice injected with 1e6 or 1e7 pfu of wild type KOS virus. Mice were sacrificed 5-6 days post virus injection (pvi) after they began to show outward signs of morbidity (kyphosis, lethargic hind limb paralysis, etc.). "+" indicates detectable plaque(s) and "-" indicates the absence of detectable plaque(s). Grey shaded boxes indicate that these data were not available.

[0029] [Figure 15] Figure 15 shows that replicating HSV can be detected in almost all harvested tissues except lungs of mice given 1e8 pfu of C8G5 or D7-1 24 hours after viral infection. The table summarizes the results of plaque assays from tissues of mice injected with MutΔ34.5 or Mut3ΔICP6 24 hours after viral infection. "+" indicates detectable plaque(s) and "-" indicates the absence of detectable plaque(s). Grey shaded boxes indicate that these data are not available.

[0030] [Figure 16] Figure 16 shows that replicating HSV cannot be detected 14 days after viral infection in any of the tissues harvested from mice given 1e8 pfu of C8G5 or D7-1. The table summarizes the results of plaque assays from tissues of mice injected with MutΔ34.5 or Mut3ΔICP6 14 days after viral infection. "-" indicates the absence of detectable plaque(s).

[0031] [Figure 17]Figure 17 shows that replicating HSV cannot be detected 28 days after viral infection in any of the harvested tissues of mice given 1e8 pfu of C8G5 or D7-1. The table summarizes the results of plaque assays from tissues of mice injected with MutΔ34.5 or Mut3ΔICP6 28 days after viral infection. "-" indicates the absence of detectable plaque(s). Grey shaded boxes indicate that these data are not available.

[0032] [Figure 18] 18 is a table summarizing the inability to detect replicating HSV 56 days after viral infection in any of the tissues harvested from mice given 1e8 pfu of C8G5 or D7-1. The table summarizes the results of plaque assays from tissues of mice injected with MutΔ34.5 or Mut3ΔICP6 56 days after viral infection. "-" indicates the absence of detectable plaque(s).

[0033] [Figure 19] Figure 19 shows that replicating HSV cannot be detected 85 days after viral infection in any of the harvested tissues of mice given 1e8 pfu of C8G5 or D7-1. The table summarizes the qPCR and plaque assay results from tissues of mice injected with MutΔ34.5 or Mut3ΔICP6 85 days after viral infection. "-" indicates the absence of detectable plaque(s).

[0034] [Figures 20A-20D]Figures 20A-D show the results of the study showing that the plaque size of Mut-3ΔgE is much smaller compared to Mut-3 or Mut-3D34.5(C8G5). Figure 20A: Mut-3ΔgE was constructed by a CRISPR-Cas9 gene editing strategy in which the Us8 gene encoding glycoprotein gE was replaced with a GFP reporter cassette driven by CMV. Figure 20B: Non-syncytial GFP-positive plaque phenotype of Mut-3ΔgE clones 28D5-B4 and 28D5-H1-Vero cells were infected with serially diluted 28D5-B4 or 28D5-H1 for 2 h and overlaid with overlay medium. Photographs of plaques were taken 3 days after virus infection. Figure 20C: qPCR analysis of Mut-3ΔgE clones 28D5-B4 and 28D5-H1 reveals loss of Us8 / gE. Baby hamster kidney (BHK) cells were infected with Mut-3ΔgE clones 28D5-B4 or 28D5-H1, and their lysates were harvested when more than 50% cytopathic effect was observed. 50-100 ng of genomic DNA isolated from virus-infected BHK cell lysates were used in qPCR reactions. Results are shown as gE / Us8 or GFP fold relative to the Us8a control HSV gene. Figure 20D: Comparison of plaque sizes of Mut-3ΔgE clones 28D5-B4 and 28D5-H1 with Mut-3 or Mut-3Δ34.5 / C8G5 obtained from standard plaque assays performed in Vero cells. Plaque pictures of Mut-3, Mut-3Δ34.5, Mut-3ΔgE clones 28D5-B4 and clones 28D5-H1 were taken 3 days after virus infection.

[0035] [Figures 21A-21D]Figure 21A-D provide the results of the study showing that the plaque size of 17ΔgE is much smaller compared to 17syn+ or 17Δ34.5. Figure 21A: 17ΔgE was constructed by a CRISPR-Cas9 gene editing strategy in which the Us8 gene encoding glycoprotein gE was replaced with a GFP reporter cassette driven by CMV. Figure 21B: Non-syncytial and small GFP-positive plaque phenotype of 17ΔgE clone 12G5 - Vero cells were infected with serially diluted 17ΔgE 12G5 for 2 h and overlaid with overlay medium. Photographs of plaques were taken 3 days after viral infection. Figure 21C: qPCR analysis of 17ΔgE clone 12G5 reveals loss of Us8 / gE. Baby hamster kidney (BHK) cells were infected with 17ΔgE clone 12G5 and lysates were harvested at the time when more than 50% cytopathic effect was observed. 50-100 ng of genomic DNA isolated from virus-infected BHK cell lysates were used in the qPCR reactions. Results are shown as gE / Us8 or GFP fold relative to the ICP6 control HSV gene. Figure 21D: Comparison of plaque size between 17ΔgE clone 12G5 17syn+ and 17Δ34.5 clone B4 from a standard plaque assay performed in Vero cells. Photographs of plaques were taken 3 days after virus infection. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0036] Detailed Description The embodiments according to the present disclosure will be described more completely below. However, aspects of the present disclosure can be embodied in different forms and should not be construed as being limited to the embodiments described herein. Rather, these embodiments are provided so that the present disclosure will be thorough and complete, and will fully convey the scope of the present invention to those skilled in the art. The terms used in the description herein are merely intended to describe specific embodiments, and are not intended to be limiting.

[0037] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by those skilled in the art. Terms as defined in commonly used dictionaries should be interpreted to have a meaning consistent with their meaning in the context of this application and related art, and it is further understood that they should not be interpreted in an idealized or overly formal sense unless expressly defined as such herein. Although not expressly defined below, such terms should be interpreted according to their general meaning.

[0038] The terms used in the description herein are for the purpose of describing particular embodiments only and are not intended to limit the invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety.

[0039] Unless otherwise indicated, the practice of the present technique employs conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology, and recombinant DNA, which are within the skill of the art.

[0040] Unless the context dictates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination.Furthermore, the present disclosure also contemplates that in some embodiments, any feature or combination of features described herein can be excluded or omitted.To illustrate, if the specification states that a complex comprises components A, B, and C, it is specifically intended that any or any combination of A, B, or C can be omitted and can be excluded from the scope, either alone or in any combination.

[0041] Unless otherwise stated, all specified embodiments, features and terms are intended to include both the recited embodiment, feature or term and their biological equivalents.

[0042] All numerical designations, including ranges, such as pH, temperature, time, concentration and molecular weight, are approximate values ​​that are varied (+) or (-) by increments of 1.0 or 0.1, or by variations of + / -15%, or + / -10%, or + / -5%, or + / -2%, as appropriate. It should be understood, although not always explicitly stated, that all numerical designations are preceded by the term "about". It should also be understood, although not always explicitly stated, that the reagents described herein are merely exemplary, and that equivalent reagents to such are known in the art.

[0043] As used herein, the term "comparable" refers to having the same level as a reference level or being within a + / - 50%, or alternatively 45%, or alternatively 40%, or alternatively 35%, or alternatively 30%, or alternatively 25%, or alternatively 20%, or alternatively 15%, or alternatively 10%, or alternatively 5%, or alternatively 2% variation compared to the reference level.

[0044] Throughout this disclosure, various publications, patents and published patent specifications are referenced by an identifying citation or by Arabic numerals. Full citations for publications identified by Arabic numerals are set forth immediately before the claims. The disclosures of these publications, patents and published patent specifications are incorporated by reference in their entireties into this disclosure in order to more fully describe the state of the art to which this invention pertains.

[0045] definition Unless otherwise stated, the practice of this technology uses conventional techniques of organic chemistry, pharmacology, immunology, molecular biology, microbiology, cell biology and recombinant DNA that are within the skill of the art.See, for example, Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual, 2nd edition (1989); Current Protocols In Molecular Biology (FM Ausubel et al., eds., (1987)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (MJ MacPherson, BD Hames and GR Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, a Laboratory Manual, and Animal Cell Culture (RI Freshney, ed. (1987)).

[0046] As used in the description of this invention and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise.

[0047] As used herein, the term "comprising" is intended to mean that the compositions and methods include the recited elements but do not exclude other elements. As used herein, the transitional phrase (and grammatical variations) consisting essentially of should be interpreted to include the recited materials or steps, as well as materials or steps that do not materially affect the basic and novel characteristics of the recited embodiment. These characteristics are recited in the method embodiment. Thus, the term "consisting essentially of" as used herein should not be interpreted as equivalent to "comprising." "Consisting of" is intended to mean excluding more than trace amounts of other ingredients and substantial method steps for administering the compositions disclosed herein. The aspects defined by each of these transitional terms are within the scope of this disclosure.

[0048] The term "about" as used herein when referring to a measurable value, such as an amount or concentration, is intended to encompass a variation of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of the specified amount.

[0049] The terms "acceptable," "effective," or "sufficient" when used to describe the selection of any ingredient, range, dosage form, etc. disclosed herein intend that said ingredient, range, dosage form, etc. is appropriate for the purpose disclosed.

[0050] As used herein, "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 combinations when interpreted in the alternative ("or").

[0051] As used herein, the term "cell" can refer to either prokaryotic or eukaryotic cells, obtained from the subject or from a commercially available source, as appropriate.

[0052] "Eukaryotic Cell" All kingdoms of life except Monera. Eukaryotic cells can be easily identified through their membrane-bound nucleus. Animals, plants, fungi and protists are eukaryotes, i.e. organisms whose cells are organized into complex structures by internal membranes and cytoskeleton. The most characteristic membrane-bound structure is the nucleus. Unless specifically stated, the term "host" includes eukaryotic hosts, including, for example, yeast, higher plant, insect and mammalian cells. Non-limiting examples of eukaryotic cells or eukaryotic hosts include monkey, cow, pig, mouse, rat, bird, reptile and human, e.g., HEK293 and 293T cells.

[0053] "Prokaryotic cells", which usually lack a nucleus or any other membrane-bound organelles, are divided into two domains: bacteria and archaea. In addition to chromosomal DNA, these cells can also contain genetic information in circular loops called episomes. Bacterial cells are very small, roughly the size of an animal mitochondrion (approximately 1-2 μm in diameter and 10 μm in length). Prokaryotic cells are characterized by three main shapes: rod-shaped, spherical and spiral. Instead of undergoing an elaborate replication process like eukaryotes, bacterial cells divide by binary fission. Examples include, but are not limited to, the bacteria Bacillus, Escherichia coli and Salmonella.

[0054] The term "encoding" when applied to a nucleic acid sequence refers to a polynucleotide that is said to "encode" a polypeptide if, in its native state or when manipulated by methods well known to those of skill in the art, it can be transcribed and / or translated to produce an mRNA for a polypeptide and / or fragment thereof. The antisense strand is the complement of such a nucleic acid, and the coding sequence can be deduced from the antisense strand.

[0055] The terms "equivalent" or "biological equivalent" are used interchangeably when referring to a particular molecule, biological or cellular material, and are intended to have minimal homology while still maintaining a desired structure or function. Non-limiting examples of equivalent polypeptides include polypeptides or polypeptide sequences having at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95% identity, or polypeptides encoded by polynucleotides or their complements that hybridize to polynucleotides encoding such polypeptide sequences under high stringency conditions. High stringency conditions are described herein and are incorporated by reference. Alternatively, the equivalent is a polypeptide encoded by a polynucleotide or its complement that has at least 70%, or at least 75%, or 80%, or at least 85%, or at least 90%, or at least 95% identity, or at least 97% sequence identity to a reference polynucleotide, such as a wild-type polynucleotide. In one embodiment, an equivalent polypeptide or polynucleotide has the same or a substantially similar biological function as the reference polypeptide or polynucleotide, respectively, e.g., a cytolytic function, an anti-tumor function, an anti-metastatic function or an anti-cancer function as determined by a suitable cellular assay or animal model as described herein.

[0056] Non-limiting examples of equivalent polypeptides include polynucleotides having at least 60%, or at least 65%, or at least 70%, or at least 75%, or 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97% identity to a reference polynucleotide. Equivalent also contemplates polynucleotides or their complements that hybridize to a reference polynucleotide under conditions of high stringency.

[0057] A polynucleotide or polynucleotide region (or polypeptide or polypeptide region) having a certain percentage (e.g., 80%, 85%, 90% or 95%) of "sequence identity" to another sequence means that, when aligned, that percentage of bases (or amino acids) are identical when comparing the two sequences. Alignment and percent homology or sequence identity can be determined using software programs known in the art, such as those described in Current Protocols in Molecular Biology (Ausubel et al., eds. 1987) Supplement 30, section 7.7.18, Table 7.7.1. In certain embodiments, default parameters are used for alignment. A non-limiting exemplary alignment program is BLAST, using default parameters. In particular, exemplary programs include BLASTN and BLASTP, using the following default parameters: Genetic code = standard; filter = none; strand = both; cutoff = 60; expectation = 10; matrix = BLOSUM62; description = 50 sequences; sort = high score; database = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translation + SwissProtein + SPupdate + PIR. Details of these programs can be found at the following Internet address: ncbi.nlm.nih.gov / cgi-bin / BLAST. Sequence identity and percent identity can be determined by incorporating them into clustalW (available at the Web address: genome.jp / tools / clustalw / , last accessed on January 13, 2017).

[0058] "Homology" or "identity" or "similarity" refers to sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing a position in each sequence that can be aligned for comparison. When a position in the compared sequences is occupied by the same base or amino acid, the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An "unrelated" or "non-homologous" sequence shares less than 40% identity, or less than 25% identity, with one of the sequences of the present disclosure.

[0059] "Homology" or "identity" or "similarity" can also refer to two nucleic acid molecules that hybridize under stringent conditions.

[0060] "Hybridization" refers to a reaction in which one or more polynucleotides react to form a complex stabilized through hydrogen bonding between the bases of the nucleotide residues. Hydrogen bonding can occur by Watson-Crick base pairing, Hoogsteen binding, or in any other sequence-specific manner. The complex may comprise, or alternatively may consist essentially of, or even consist of, two strands forming a double-stranded structure, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination thereof. A hybridization reaction may constitute a step in a larger process, such as the initiation of a PCR reaction or the enzymatic cleavage of a polynucleotide by a ribozyme.

[0061] Examples of stringent hybridization conditions include an incubation temperature of about 25°C to about 37°C; a hybridization buffer concentration of about 6xSSC to about 10xSSC; a formamide concentration of about 0% to about 25%; and a wash solution of about 4xSSC to about 8xSSC. Examples of mild hybridization conditions include an incubation temperature of about 40°C to about 50°C; a buffer concentration of about 9xSSC to about 2xSSC; a formamide concentration of about 30% to about 50%; and a wash solution of about 5xSSC to about 2xSSC. Examples of high stringency conditions include an incubation temperature of about 55°C to about 68°C; a buffer concentration of about 1xSSC to about 0.1xSSC; a formamide concentration of about 55% to about 75%; and a wash solution of about 1xSSC, 0.1xSSC, or deionized water. Generally, hybridization incubation times are 5 minutes to 24 hours, with one, two or more washing steps, with wash incubation times of about 1, 2 or 15 minutes. SSC is a 0.15M NaCl and 15mM citrate buffer. It is understood that equivalents of SSC using other buffer systems can be used.

[0062] As used herein, "expression" refers to the process by which a polynucleotide is transcribed into mRNA and / or the transcribed mRNA is subsequently translated into a peptide, polypeptide or protein. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.

[0063] "Gene" refers to a polynucleotide containing at least one open reading frame (ORF) that can encode a particular polypeptide or protein after being transcribed and translated. "Gene product" or "gene expression product" refers to the amino acid (e.g., peptide or polypeptide) produced when a gene is transcribed and translated.

[0064] "Under transcriptional control" is a term well understood in the art and indicates that transcription of a polynucleotide sequence, typically a DNA sequence, is dependent on being operably linked to elements that contribute to the initiation of transcription or promote transcription. "Operably linked" refers to a polynucleotide positioned such that it can function within a cell. In one embodiment, the present invention provides a promoter operably linked to a downstream sequence, such as an HSV virulence gene or a variant thereof.

[0065] The term "encoding" as applied to a polynucleotide refers to a polynucleotide that is said to "encode" a polypeptide if, in its native state or when manipulated by methods well known to those of skill in the art, it can be transcribed and / or translated to produce an mRNA for a polypeptide and / or fragment thereof. The antisense strand is the complement of such a nucleic acid, and the coding sequence can be deduced from the antisense strand.

[0066] As used herein, the term "isolated" refers to a molecule or biological or cellular material that is substantially free of other materials.

[0067] As used herein, the term "functional" may be used to modify any molecule, biologic, or cellular material with the intent of achieving a particular, specified effect.

[0068] As used herein, the terms "nucleic acid sequence" and "polynucleotide" are used interchangeably to refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides.Thus, the term includes, but is not limited to, single-stranded, double-stranded or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids or polymers that contain, or alternatively consist essentially of, or even consist of, purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derivatized nucleotide bases.

[0069] The term "wild type" refers to a gene or gene product that has the characteristics of that gene or gene product when isolated from a naturally occurring source. In some embodiments, a wild type gene or gene product contains a slightly different sequence, even for one viral strain.

[0070] The term "mutant" refers to a gene or gene product that exhibits modified sequence and / or functional properties (i.e., altered characteristics) when compared to the wild-type gene or gene product or to a gene or gene product from another mutant strain(s). In one embodiment, other mutant strains include the 17TermA strain or the rR450 strain.

[0071] The term "mutation" refers to a variation in a DNA sequence from the wild type or other mutant strain(s). Mutations may or may not produce a functional characteristic in an organism. There are multiple types of mutations, including but not limited to insertions, deletions, truncations, frameshifts, substitutions or point mutations.

[0072] The term "point mutation" refers to a mutation involving the change, insertion or deletion of a single nucleotide base in the genetic material, DNA or RNA.

[0073] A "deletion" refers to a mutation in which a portion of a chromosome or sequence of DNA is missing.

[0074] "Frameshift" refers to a mutation caused by an indel (insertion or deletion) of several nucleotides in a DNA sequence that is not divisible by three.

[0075] A "substitution" refers to a mutation involving the replacement of one or several nucleotides in a gene.

[0076] "Truncation" refers to a mutation involving premature termination of a protein extension due to removal of the N- or C-terminal portions of a protein by proteolysis or manipulation of a structural gene, or due to the presence of a stop codon in that structural gene as a result of a nonsense mutation.

[0077] In some embodiments, the mutation is a non-synonymous mutation. The term "non-synonymous mutation" refers to a mutation that changes the amino acid sequence of a protein, and is contrasted with a synonymous mutation, which does not change the amino acid sequence.

[0078] As used herein, the term "promoter" refers to any sequence that controls the expression of a coding sequence, such as a gene. A promoter can be, for example, constitutive, inducible, repressible, or tissue-specific. A "promoter" is a regulatory sequence that is a region of a polynucleotide sequence at which the initiation and rate of transcription are regulated. A promoter can contain genetic elements to which regulatory proteins and molecules, such as RNA polymerase and other transcription factors, can bind. Non-limiting exemplary promoters include the Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter, the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the phosphoglycerol kinase (PGK) promoter, the U6 promoter, or the EF1 promoter. In some embodiments, the promoter is a chicken β-actin ("CBA") promoter.

[0079] Further non-limiting exemplary promoters with specific target specificity are described herein below, including, but not limited to, CMV, EF1a, SV40, PGK1 (human or mouse), P5, Ubc, human β-actin, CAG, TRE, UAS, Ac5, polyhedrin, CaMKIIa, Gal1, TEF1, GDS, ADH1, CaMV35S, Ubi, H1, U6 and α-1-antitrypsin. Synthetic derived promoters can be used for ubiquitous or tissue-specific expression. In addition, viral derived promoters, some of which are described above, such as CMV, HIV, adenovirus and AAV promoters, can be useful in the methods disclosed herein. In some embodiments, the promoter is coupled to an enhancer to increase transcription efficiency.

[0080] Enhancers are regulatory elements that increase the expression of a target sequence. A "promoter / enhancer" is a polynucleotide that contains a sequence that can provide both promoter and enhancer functions. For example, the long terminal repeats of retroviruses contain both promoter and enhancer functions. Enhancers / promoters can be "endogenous" or "exogenous" or "heterologous." An "endogenous" enhancer / promoter is one that is naturally linked to a particular gene in a genome. An "exogenous" or "heterologous" enhancer / promoter is one that has been juxtaposed to a gene using genetic engineering (i.e., molecular biology techniques) such that transcription of that gene is induced by the linked enhancer / promoter.

[0081] The term "tumor-specific promoter or tissue-specific promoter" as used herein means a promoter that allows the expression of the gene under the control of the promoter specifically in desired tumor cells or tissue.Non-limiting examples of tissue-specific promoters that can be used in the present invention include prostate-specific antigen (PSA) promoter, prostate-specific membrane antigen (PSMA) promoter, casein promoter, IgG promoter, chorionic embryonic antigen promoter, elastase promoter, porphobilinogen deaminase promoter, insulin promoter, growth hormone factor promoter, acetylcholine receptor promoter, alcohol dehydrogenase promoter and alpha or beta globin promoter.

[0082] Non-limiting examples of tumor-specific promoters to be used in the present invention include the telomerase reverse transcriptase promoter, the glial fibrillary acidic protein promoter, the E2F promoter, the survivin promoter, the COX-2 promoter, the EGD-2 promoter; the ELF-1 promoter; the hypoxia-specific promoter; the carcinoembryonic antigen promoter and the stromelysin 3 promoter.

[0083] The term "cryopreservative" refers to a compound or material that can protect one or more tissues, viruses or other biological agents from being damaged or impaired. Examples of cryopreservatives include, but are not limited to, chondroitin sulfate, glycosaminoglycan dimethylsulfoxide, cell-permeable organic solutes, polysaccharides, glycerol, Dulbecco's Minimum Essential Medium (DMEM), glutamine, D-glucose, sodium pyruvate, fetal bovine serum, papaverine, DMSO, glycerol, trehalose, KH2PO4, K2HPO4, KCl, mannitol, NaHCO3, sodium ascorbate, 1,2-propanediol, formamide, 2,3-butanediol, probucol, curcumin and mixtures thereof.

[0084] The terms "protein", "peptide" and "polypeptide" are used interchangeably and in their broadest sense refer to a compound of two or more subunits of amino acids, amino acid analogs or peptidomimetics. The subunits may be linked by peptide bonds. In another embodiment, the subunits may be linked by other bonds, such as esters, ethers, etc. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that may constitute, or alternatively essentially consist of, or even further consist of, the protein or peptide sequence. As used herein, the term "amino acid" refers to any of the natural and / or unnatural or synthetic amino acids, including glycine and both D and L optical isomers, amino acid analogs and peptidomimetics.

[0085] As used herein, the term "vector" refers to a non-chromosomal nucleic acid that comprises, or alternatively consists essentially of, or even consists of an intact replicon such that the vector can replicate when placed in a cell, for example, by a transformation process. Vectors can be viral or non-viral. Viral vectors include retroviruses, adenoviruses, herpes simplex viruses ("HSV"), baculoviruses, modified baculoviruses, papoviruses, or other modified naturally occurring viruses. Exemplary non-viral vectors for delivering nucleic acids include the use of naked DNA; DNA complexed with cationic lipids, alone or in combination with cationic polymers; anionic and cationic liposomes; particles comprising, or alternatively consists essentially of, or even consists of DNA-protein complexes and DNA condensed with heterologous polylysine, oligopeptides of defined length, and cationic polymers, such as polyethyleneimine, optionally contained in liposomes; and ternary complexes comprising, or alternatively consists essentially of, or even consists of viruses and polylysine-DNA.

[0086] "Viral vector" is defined as a recombinantly produced virus or virus particle that contains, or alternatively consists essentially of, or even consists of a polynucleotide to be delivered into a host cell, either in vivo, ex vivo or in vitro. Examples of viral vectors include retroviral vectors, HSV vectors, AAV vectors, lentiviral vectors, adenoviral vectors, alphaviral vectors, and the like. Alphaviral vectors, such as Semliki Forest virus-based vectors and Sindbis virus-based vectors, have also been developed for use in gene therapy and immunotherapy. See Schlesinger and Dubensky (1999) Curr. Opin. Biotechnol. 5:434-439 and Ying et al. (1999) Nat. Med. 5(7):823-827.

[0087] In another embodiment, expression of the HSV virulence protein (e.g., wild-type or mutant) is controlled by a promoter that is an inducible promoter. In certain related embodiments, the promoter is an inducible tetracycline promoter. The Tet-Off and Tet-On gene expression systems provide researchers with ready access to high-level, controlled gene expression systems described as Tet-Off and Tet-On. In the Tet-Off system, gene expression is turned on when tetracycline (Tc) or doxycycline (Dox; a Tc derivative) is removed from the medium. In contrast, in the Tet-On system, expression is turned on by adding Dox. Both systems allow for tight control of gene expression in response to changes in the concentration of Tc or Dox. The maximum expression levels in the Tet system are very high and compare favorably with the maximum levels obtained from strong constitutive mammalian promoters such as CMV. Unlike other inducible mammalian expression systems, gene regulation in the Tet system is highly specific, so interpretation of results is not complicated by pleiotropic effects or non-specific induction. In E. coli, the Tet repressor protein (TetR) negatively regulates the genes of the tetracycline resistance operon on the Tn10 transposon. TetR blocks transcription of these genes by binding to the tet operator sequence (tetO) in the absence of Tc. TetR and tetO provide the basis for regulation and induction for use in mammalian experimental systems. In the Tet-on system, the regulatory protein is based on the "reverse" Tet repressor (rTetR) created by four amino acid changes in TetR (Hillen and Berens, Mechanisms underlying expression of Tn10 encoded tetracycline resistance. Annu Rev Microbiol. 1994;48:345-69; Gossen et al., Transcriptional activation by tetracyclines in mammalian cells. Science. 1995 Jun 23;268(5218):1766-9).The resulting protein, rtTA (reverse tTA, also called tetracycline-activated protein), is encoded by the pTet-On regulatory plasmid.

[0088] In a related embodiment, the vector further comprises, consists essentially of, or consists of a nucleic acid encoding a tetracycline activating protein and a promoter regulating expression of the tetracycline activating protein.

[0089] Other inducible systems useful in the vectors, isolated cells, viral packaging systems and methods described herein include regulation by ecdysone, estrogen, progesterone, chemical inducers of dimerization and isopropyl-β-D1-thiogalactopyranoside (IPTG).

[0090] As used herein, the term "recombinant expression system" or "recombinant vector" refers to one or more genetic constructs for the expression of specific genetic material formed by recombination.

[0091] "Gene delivery vehicle" is defined as any molecule that can carry inserted polynucleotide into host cell.Examples of gene delivery vehicles are biocompatible polymers, including liposomes, micelles, natural and synthetic polymers; lipoproteins; polypeptides; polysaccharides; lipopolysaccharides; artificial virus envelopes; metal particles; and bacteria or viruses, such as baculovirus, adenovirus and retrovirus, bacteriophages, cosmids, plasmids, fungal vectors and other recombinant vehicles typically used in the art that have been described for expression in various eukaryotic and prokaryotic hosts and can be used for gene therapy, in addition to simple protein expression.

[0092] The polynucleotides disclosed herein can be delivered to cells or tissues using gene delivery vehicles. As used herein, "gene delivery", "gene transfer", "transduction" and the like are terms that refer to the introduction of an exogenous polynucleotide (sometimes referred to as a "transgene") into a host cell, regardless of the method used for the transfer. Such methods include various well-known techniques, such as vector-mediated gene transfer (e.g., by viral infection / transfection, or various other protein-based or lipid-based gene delivery complexes), as well as techniques that facilitate the delivery of "naked" polynucleotides (such as electroporation, "gene gun" delivery, and various other techniques used for the transfer of polynucleotides). The transferred polynucleotide can be stably or transiently maintained in the host cell. Stable maintenance typically requires that the transferred polynucleotide contains an origin of replication compatible with the host cell, or is integrated into a host cell replicon, such as an extrachromosomal replicon (e.g., a plasmid), or into the nuclear or mitochondrial chromosome. As known in the art and described herein, many vectors are known to be capable of mediating the transfer of genes into mammalian cells.

[0093] A "plasmid" is an extrachromosomal DNA molecule that can replicate independently of chromosomal DNA and is separate from chromosomal DNA. Plasmids are often circular and double stranded. Plasmids provide a mechanism for horizontal gene transfer within a population of microorganisms and usually provide a selective advantage under given environmental conditions. Plasmids may carry genes that confer resistance to naturally occurring antibiotics in competitive niches, or the proteins produced may act as toxins under similar circumstances.

[0094] "Plasmids" used in genetic engineering are called "plasmid vectors". Many plasmids are commercially available for such use. The gene to be replicated is inserted into a copy of the plasmid that contains a gene that makes the cell resistant to a particular antibiotic and a multiple cloning site (MCS, or polylinker), a short region that contains several commonly used restriction sites that make it easy to insert a DNA fragment into this location. Another major use of plasmids is to make large amounts of proteins. In this case, researchers grow bacteria that contain a plasmid with the gene of interest. Just as bacteria produce proteins that confer antibiotic resistance, they can also be induced to produce large amounts of proteins from an inserted gene.

[0095] The term "herpes simplex virus" or "HSV" as used herein refers to a herpes simplex virus that produces the effects of the present invention, including wild-type or mutant herpes simplex viruses. In one embodiment, the mutant non-native HSV is obtained by mutating or modifying any of the genes of wild-type HSV, or by inserting any of the exogenous genes. Serotypes of HSV include HSV type 1 (or HSV-1) or HSV type 2 (or HSV-2), or consist essentially of HSV type 1 (or HSV-1) or HSV type 2 (or HSV-2), or even consist of HSV type 1 (or HSV-1) or HSV type 2 (or HSV-2). HSV-1 is an enveloped double-stranded DNA virus. In one embodiment, HSV-1 can infect human cells. In another embodiment, a sequence, a gene, or multiple genes can be incorporated into HSV-1. The size of the integrated sequence can be approximately 1 base, 5 bases, 10 bases, 100 bases, 1 kb, 10 kb, 100 kb or 150 kb. HSV-1 can induce cell lysis at a relatively low multiplicity of infection (MOI), and its growth can be inhibited by antiviral drugs. In one embodiment, HSV viral DNA remains outside the chromosome without being integrated into the genome of the host cell. HSV-1 can include various strains (e.g., KOS and McKrae). See Wang et al. (2013) Virus Res.173(2):436-440. In one embodiment, HSV-1 is HSV-1 KOS strain. In another embodiment, HSV-1 is HSV-1 McKrae strain.

[0096] There are several HSV mutants, for example, 17TermA HSV and rRp450 HSV. The term "17TermA HSV" refers to a mutant HSV-1 virus that contains the entire ICP34.5 gene but has a stop codon inserted before 100 bp of the coding region, resulting in premature termination of protein expression and expression of a truncated protein of 30 amino acids. The 17TermA HSV mutant shows growth defects due to the truncated ICP34.5 protein. See Orvedahl et al., (2007) Cell Host & Microbe, 1:1, 23-25. The term "rRp450" refers to an attenuated herpes simplex 1 vector that is defective in virally encoded ribonucleotide reductase or ICP6. See Aghi M et al., (1999) Cancer Res., 59(16):3861-5.

[0097] The HSV genome encodes multiple virulence proteins, including, but not limited to, glycoprotein E ("gE"), infected cell protein 0 ("ICP0"), infected cell protein 6 ("ICP6"), DNA packaging terminase subunit 1, infected cell protein 8 ("ICP8") and infected cell protein 34.5 ("ICP34.5"). An exemplary HSV1 genome can be found in NCBI Reference Sequence: NC_001806.2, last accessed March 13, 2020.

[0098] The term "gE-encoding gene" refers to a gene or DNA fragment thereof that encodes the gE protein. An exemplary gE-encoding gene can be identified at positions 33-2555 of the HSV-1 genome sequence in the NCBI Reference Sequence: NC_001806.2. The term "ICP6 protein" refers to infected cell protein 6 encoded by the HSV genome. ICP6 is a subunit of ribonucleotide reductase ("RR"), a key enzyme for nucleotide metabolism and viral DNA synthesis in non-dividing cells.

[0099] A "dysfunctional" protein refers to a protein that has impaired or no function of the original protein. In one embodiment, the dysfunctional protein is caused by a deletion or substitution in the coding sequence. For example, HSV cannot replicate in normal non-dividing cells because the dysfunctional ICP6 gene is deleted or inactivated. However, in actively dividing cells with increased RR activity, the virus's missing enzymatic activity is compensated and the virus is able to replicate. The DNA and amino acid sequences of ICP34.5 are provided in SEQ ID NOs: 1, 2, and 5-10. The DNA and amino acid sequences of gE are provided in SEQ ID NOs: 12-19. The DNA and amino acid sequences of ICP0 are provided in SEQ ID NOs: 20-26. The DNA and amino acid sequences of DNA packaging terminase subunit 1 are provided in SEQ ID NOs: 35-42. The DNA and amino acid sequences of ICP8 are provided in SEQ ID NOs: 27-34. The DNA and amino acid sequences of ICP6 are provided in SEQ ID NOs: 43-50.

[0100] The term "gene encoding ICP0" refers to a gene or DNA fragment thereof that encodes an ICP0 protein. Exemplary DNA and amino acid sequences for ICP0 are provided in SEQ ID NOs:20-26. The term "gene encoding DNA packaging terminase subunit 1" refers to a gene or DNA fragment thereof that encodes a DNA packaging terminase subunit 1 protein or peptide. Exemplary DNA and amino acid sequences for DNA packaging terminase subunit 1 are provided in SEQ ID NOs:35-42. The term "gene encoding ICP8" refers to a gene or DNA fragment thereof that encodes an ICP8 protein. Exemplary DNA and amino acid sequences for ICP8 are provided in SEQ ID NOs:27-34. The term "gene encoding ICP34.5" refers to a gene or DNA fragment thereof that encodes an ICP34.5 protein. Exemplary DNA and amino acid sequences for ICP34.5 are provided in SEQ ID NOs:1, 2, and 5-10. The term "gene encoding glycoprotein E ("gE")" refers to a gene or DNA fragment thereof that encodes a gE protein. Exemplary DNA and amino acid sequences for gE are provided in SEQ ID NOs:12-19.

[0101] The term "deletion or inactivation of a gene" refers to the deletion of all or part of a gene, or the suppression of gene expression by partial base substitution, modification, insertion of unnecessary sequences, etc. The deletion or inactivation of an HSV gene (e.g., gE, ICP0, and ICP8) can be performed by a person skilled in the art using a known method or a method based thereon. For example, a method using homologous recombination can be used. For example, a DNA fragment containing a part of an HSV gene and a sequence unrelated to the HSV gene is cloned into an appropriate plasmid vector, which is then introduced into HSV to cause homologous recombination in a part of the HSV gene region, thereby splitting and inactivating the HSV gene. Alternatively, mutation or deletion of an HSV gene can be caused by natural mutation during virus passage.

[0102] In embodiments in which gene transfer is mediated by a DNA viral vector, such as herpes simplex virus, a vector construct refers to a polynucleotide that comprises, or alternatively consists essentially of, or even consists of the viral genome or a portion thereof and a transgene. Thus, in one embodiment, the non-naturally occurring HSV further comprises a transgene encoding a therapeutic polynucleotide or protein.

[0103] Vectors containing both a promoter and a cloning site into which a polynucleotide can be operably linked are well known in the art. Such vectors are capable of transcribing RNA in vitro or in vivo and are commercially available from sources such as Agilent Technologies (Santa Clara, Calif.) and Promega Biotech (Madison, Wis.). To optimize expression and / or in vitro transcription, it may be necessary to remove, add, or modify the 5' and / or 3' untranslated portions of the clone to remove extra potentially inappropriate alternative translation initiation codons or other sequences that may disrupt or reduce expression at either the transcription or translation level. Alternatively, a consensus ribosome binding site can be inserted immediately 5' of the initiation codon to enhance expression.

[0104] Gene delivery vehicles also include DNA / liposome complexes, micelles, and targeted viral protein-DNA complexes. Liposomes that also contain, or alternatively essentially consist of, or alternatively essentially consist of, or alternatively consist of targeting antibodies or fragments thereof can be used in the methods disclosed herein. In addition to the delivery of polynucleotides to cells or cell populations, the direct introduction of the proteins described herein into cells or cell populations can be performed by non-limiting techniques of protein transfection, or culture conditions that can enhance the expression and / or activity of the proteins disclosed herein are other non-limiting techniques.

[0105] As used herein, the term "signal peptide" or "signal polypeptide" refers to an amino acid sequence that is usually present at the N-terminal end of newly synthesized secretory or membrane polypeptides or proteins. "Signal peptide" or "signal polypeptide" acts to guide a polypeptide to a specific cellular location, for example, across the cell membrane, into the cell membrane, or into the nucleus. In some embodiments, the signal peptide is removed after localization. Examples of signal peptides are well known in the art. Non-limiting examples are those described in U.S. Patent Nos. 8,853,381, 5,958,736, and 8,795,965.

[0106] In one embodiment, the HSV is detectably labeled. As used herein, the term "label" refers to a directly or indirectly detectable compound or composition that is directly or indirectly conjugated to a composition to be detected, e.g., a protein such as a polynucleotide or an antibody, to generate a "labeled" composition. The term also includes sequences conjugated to a polynucleotide that provide a signal upon expression of the inserted sequence, such as green fluorescent protein (GFP). The label may be detectable alone (e.g., a radioisotope label or a fluorescent label) or, in the case of an enzyme label, may catalyze a chemical change in a substrate compound or composition that is detectable. The label may be suitable for small-scale detection or may be more suitable for high-throughput screening. Thus, suitable labels include, but are not limited to, radioisotopes, fluorescent dyes, chemiluminescent compounds, dyes, and proteins, including enzymes. The label may simply be detected or quantified. A response that is merely detected generally includes, or alternatively consists essentially of, or even consists of a response whose presence is merely confirmed, whereas a response that is quantified generally includes, or alternatively consists essentially of, or even consists of a response that has a quantifiable (e.g., numerically reportable) value, such as intensity, polarization and / or other property. In luminescent or fluorescent assays, the detectable response may be generated directly, using a luminophore or fluorophore associated with the assay component actually responsible for binding, or indirectly, using a luminophore or fluorophore associated with another (e.g., reporter or indicator) component.

[0107] Examples of luminescent labels that generate signals include, but are not limited to, bioluminescence and chemiluminescence.Detectable luminescent responses generally include, or alternatively consist essentially of, or even consist of, the change or generation of a luminescent signal.Appropriate methods and luminophores for luminescent labeling of assay components are known in the art and are described, for example, in Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6th ed.).Examples of luminescent probes include, but are not limited to, aequorin and luciferase.

[0108] Examples of suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosine, coumarin, methylcoumarin, pyrene, Malachite Green, stilbene, Lucifer Yellow, Cascade Blue™, and Texas Red. Other suitable optical dyes are described in Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6th ed.).

[0109] In another embodiment, the fluorescent label is functionalized to facilitate covalent binding to cellular components present in or on the surface of cells or tissues, such as cell surface markers.Suitable functional groups include, but are not limited to, isothiocyanate groups, amino groups, haloacetyl groups, maleimides, succinimidyl esters, and sulfonyl halides, all of which can be used to attach the fluorescent label to a second molecule.The choice of functional group of the fluorescent label depends on the site of attachment to either linker, drug, marker, or second labeling agent.

[0110] Fluorescent labels can be attached directly to the cellular component or compound or via a linker. Binding pairs suitable for use in indirectly linking fluorescent labels to intermediates include, but are not limited to, antigen / antibody, e.g., rhodamine / anti-rhodamine, biotin / avidin and biotin / streptavidin.

[0111] The term "solid support" refers to a non-aqueous surface such as a "culture plate", "gene chip" or "microarray". Such gene chips or microarrays can be used for diagnostic and therapeutic purposes by many techniques known to those skilled in the art. In one technique, oligonucleotides are attached and arranged on the gene chip to determine DNA sequences by hybridization approaches, such as those outlined in U.S. Pat. Nos. 6,025,136 and 6,018,041. The polynucleotides of the invention can be modified into probes, which can then be used to detect gene sequences. Such techniques are described, for example, in U.S. Pat. Nos. 5,968,740 and 5,858,659. Probes can also be attached or affixed to electrode surfaces for electrochemical detection of nucleic acid sequences, as described by Kayem et al., U.S. Pat. No. 5,952,172 and Kelley et al., (1999) Nucleic Acids Res. 27:4830-4837.

[0112] A "composition" is intended to mean a combination of an active polypeptide, polynucleotide or antibody with another compound or composition, inert (e.g., a detectable label) or active (e.g., a gene delivery vehicle).

[0113] A "pharmaceutical composition" is intended to include a combination of an active polynucleotide, polynucleotide or antibody with an inert or active carrier, such as a solid support, making the composition suitable for in vitro, in vivo or ex vivo diagnostic or therapeutic use.

[0114] As used herein, the term "pharmaceutically acceptable carrier" includes any of the standard pharmaceutical carriers, such as phosphate buffered saline solution, water, and emulsions, such as oil / water or water / oil emulsions, as well as various types of wetting agents. The composition may also include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see Martin (1975) Remington's Pharm.Sci., 15th Ed. (Mack Publ.Co., Easton).

[0115] The "subject" of diagnosis or treatment is a cell or an animal, such as a mammal, or a human. The subject is not limited to a particular species, and includes non-human animals that are the subject of diagnosis or treatment, those that are subject to infection, or animal models, such as monkeys, rats, mice, murines such as chinchillas, canines such as dogs, lagomorphs such as rabbits, farm animals, sport animals, and pets. Human patients are also included in this term.

[0116] The term "tissue" is used herein to refer to tissue of a living or dead organism, or any tissue derived from or designed to mimic a living or dead organism. Tissues may be healthy, diseased, and / or have genetic mutations. Biological tissues may include any single tissue (e.g., a collection of cells that may be interconnected), or a group of tissues that make up an organ or part or region of an organism's body. Tissues may include, or may consist essentially of, or even more homogeneous cellular material, may include, or may consist essentially of, or even more homogeneous cellular material, or may be composite structures, such as those found in regions of the body including the chest, which may include, for example, lung tissue, skeletal tissue, and / or muscle tissue. Exemplary tissues include, but are not limited to, tissues from the liver, lung, thyroid, skin, pancreas, blood vessels, bladder, kidney, brain, bile duct, duodenum, abdominal aorta, iliac vein, heart and intestine (including any combination thereof).

[0117] As used herein, "treating" a disease in a subject or "treatment" of a disease in a subject refers to (1) preventing symptoms or disease from occurring in a subject who is predisposed to or has not yet shown symptoms of the disease, (2) arresting or halting the development of the disease, or (3) alleviating a disease or symptoms of the disease or causing a regression of the disease or symptoms of the disease. As understood in the art, "treatment" is an approach to obtain beneficial or desired results, including clinical results. In the present technology, beneficial or desired results can include, but are not limited to, one or more of the following: alleviation or relief of one or more symptoms, reduction in the severity of a symptom (including a disease), a stabilized (i.e., non-worsening) state of a symptom (including a disease), a delay or slowdown of a symptom (including a disease), progression, alleviation or remission, state and remission (whether partial or complete) of a symptom (including a disease), whether detectable or undetectable.

[0118] As used herein, the term "effective amount" is intended to mean an amount sufficient to achieve a desired effect. In the context of therapeutic or prophylactic applications, the effective amount will depend on the type and severity of the condition in question, as well as the characteristics of the individual subject, such as general health, age, sex, weight, and tolerance to the pharmaceutical composition. In the context of gene therapy, in some embodiments, the effective amount is an amount sufficient to cause partial or complete functional restoration of a gene that is deficient in the subject. In one aspect, the effective amount is an amount that provides a multiplicity of infection (MOI) of infectious viral particles of 0.001 to 1 per cell, with a range therebetween. Non-limiting examples include a multiplicity of infection (MOI) of infectious viral particles of at least 0.001, or at least 0.01, or at least 0.1, or at least 1, or 0.01 to 1, or 0.1 to 1, or about 0.01 to 0.1, or less than 1, or less than 0.1, or less than 0.01. In other embodiments, the effective amount of HSV viral particles is an amount sufficient to cause cell lysis in the subject. In some embodiments, the effective amount is the amount required to increase galactose metabolism in a subject in need thereof. One of skill in the art will be able to determine the appropriate amount depending on these and other factors.

[0119] In some embodiments, the effective amount will depend on the size and nature of the application in question.The effective amount also depends on the nature and sensitivity of the intended subject and the method used.Those skilled in the art will be able to determine the effective amount based on these and other considerations.The effective amount can include one or more administrations of the composition, or alternatively can consist essentially of one or more administrations of the composition, or even consist of one or more administrations of the composition, depending on the embodiment.

[0120] As used herein, the term "administer" or "administration" is intended to mean the delivery of a substance to a subject, such as an animal or human. Administration can be effected continuously or intermittently throughout the course of treatment, for example, intratumorally or intravenously, in one dose. Methods for determining the most effective means and dosages of administration are known to those skilled in the art and vary with the composition used for treatment, the purpose of the treatment, and the age, health, or sex of the subject being treated. Single or multiple administrations can be performed, with the dose level and pattern being selected by the treating physician or, in the case of pets and animals, by the treating veterinarian. Appropriate dosage formulations and methods for administering drugs are known in the art. The route of administration can also be determined, and methods for determining the most effective route of administration are known to those skilled in the art and vary with the composition used for treatment, the purpose of the treatment, the health or disease stage of the subject being treated, and the target cell or tissue. Non-limiting examples of routes of administration include direct and systemic, e.g., intravenous, intraarterial, intramuscular, intracardiac, intrathecal, subventricular, epidural, intracerebral, intratumor, intracranial, intraventricular, subretinal, intravitreal, intraarticular, intraocular, intraperitoneal, intrauterine, intradermal, subcutaneous, transdermal, transmucosal, and inhalation.

[0121] Oncolytic herpes simplex virus (oHSV) The efficacy of oHSV against tumors derives from direct cell killing (lytic phase) and enhanced anticancer immunity (immune phase). These viruses have been constructed in various ways to selectively target cancer cells. To achieve cancer selectivity, the most common mutation is the deletion of the neurovirulence gene γ134.5 / RL1. Expression of ICP34.5, encoded by γ134.5 / RL1, is essential for HSV-1 to counteract the host cell's antiviral protein kinase RNA-activated (PKR) pathway, which normally phosphorylates e-IF2α and halts protein translation in response to viral infection. ICP34.5 redirects cellular protein phosphatase-1 (PP1) to dephosphorylate e-IF2α to allow productive viral replication. Many cancer cells are defective in the PKR response and therefore support the replication of HSV vectors containing γ134.5 null mutants. Some vectors are constructed by mutating important metabolic viral genes such as ribonucleotide reductase (RR, whose large subunit is encoded by the ICP6 / UL39 gene). Since many malignant cells have increased expression and activity of RR, ICP6 null mutants selectively replicate in highly proliferating cancer cells due to the large ribonucleotide pools present.

[0122] HSV entry, cell-cell spread and syncytium formation HSV-1 entry involves sequential steps in which viral glycoproteins interact with cellular surface molecules. First, glycoproteins B and C (gB and gC) attach to cellular heparan sulfate proteoglycans, followed by gD binding to viral entry receptors (i.e., nectin-1, herpesvirus entry mediator (HVEM) or 3-O-sulfated heparan sulfate (3-OS-HS)). This process further allows gH / gL to interact with gB and trigger the fusion of the viral envelope to the target cell membrane, resulting in the release of virion capsid and tegument proteins into the cell. Some reports suggest that membrane fusion is also important for the subsequent lateral spread of the virus between neighboring cells. Viral cell-cell spread occurs when the virus spreads from an infected cell to an adjacent non-infected cell through areas of cell contact, which can occur even in the presence of virus-neutralizing antibodies. The presence of gE / gI promotes cell-cell spread, whereas gE hypomorphic mutants reduce cell-cell fusion and plaque size. Syncytia are the result of the fusion of multiple adjacent cells into a multinucleated giant cell. Mutations that cause syncytia have been found in at least four HSV viral genes (i.e., gB, gK, UL20 and UL24).

[0123] The present disclosure provides a strategy to combine "directed evolution" with CRISPR / Cas9 technology for the development of clinically relevant viral vectors. This effective combination represents a substantial departure from prior art approaches. Without being bound by theory, it is anticipated that an unexpected mechanism may result in increased viral potency for killing tumors. It is anticipated that this will reveal previously unknown mutation(s) that result in a hyperfusogenic phenotype and increased potency, as well as a springboard for developing next-generation oHSVs. The oHSVs thus generated have improved lytic phases, longer persistence, and maximized therapeutic outcomes, which are consistent with the objectives of the National Cancer Institute's Clinical and Translational Exploratory / Developmental Studies.

[0124] Although the focus has been on the "lytic phase" of virotherapy, a longer sustained viral response may enhance the inflammatory response in vivo, benefiting the subsequent "immune phase" of virotherapy to reach optimal antitumor efficacy. Efficacy studies in pediatric cancer models could address the use of non-native HSV in addressing pediatric cancers. The resulting improved viruses are expected to be active across a range of adult cancers as well.

[0125] MODES FOR CARRYING OUT THE DISCLOSURE Although HSV can target or infect a wide range of cells and induce lysis, the viral infection itself causes cytotoxicity and other disorders, such as encephalitis, esophagitis, and interstitial pneumonia. A large number of HSV genes affect pathogenicity. For example, γ34.5 (RL1) can cause neuropathogenesis. ICP6 (UL39), ribonucleotide reductase, thymidine kinase (UL23), uracil DNA glycosylase (UL2), dUTPase (UL50), and DNA polymerase (UL30) are involved in the nucleotide metabolism and pathogenicity of HSV. Therefore, it is necessary to create attenuated but replication-competent HSV particles to carry out its tumor-inhibiting functions while minimizing its side effects.

[0126] Provided herein is a new virus called Mut-3 that exhibits a giant syncytial plaque phenotype. Applicants isolated Mut-3 from serial passages mixing 17TermA and rRp450 in a non-permissive system ("directed evolution") and constructed an attenuated mutant Mut-3Δ34.5 through gene editing ("CRISPR / Cas9" step) (Figure 1A). Whole genome sequence analysis revealed that Mut-3 had acquired complete copies of both γ134.5 / RL1 and UL39 (encoding ICP6), and thus its genotype is similar to that of the wild-type (WT) virus. Without wishing to be bound by theory, Applicants found that the lytic activity of Mut-3 was much greater than many WT viruses, suggesting that other genomic changes (in addition to its complete viral genome) may be responsible for its increased potency. Five nonsynonymous mutations in Mut-3 were found that differ from either parental virus, including an alanine to threonine mutation at position 151 (A151T) in the gene encoding gE. An attenuated version of the Mut-3 virus was provided in which γ134.5 / RL1 was replaced with green fluorescent protein (GFP) via CRISPR / Cas9 gene editing (Figure 1A, bottom, labeled "CRISPR / Cas9" step), designated Mut-3Δ34.5. See Figure 1B, which outlines the results of the sequence comparison of Mut-3 with its parental virus. Nonsynonymous mutations in Mut-3 that differ from either parent are marked with a downward slash, including UL15, UL29, US8, RL1, and RL2. Genomic sequences identical to 17TermA are shown as blanks, and genomic sequences identical to rRp450 are marked with a downward slash.

[0127] Accordingly, the present disclosure provides a non-naturally occurring herpes simplex virus ("HSV"), the virus comprising, consisting essentially of, or even consisting of a mutation in a virulence gene that is one or more from the group of: (a) a gene encoding glycoprotein E ("gE"), (b) a gene encoding infected cell protein 0 ("ICP0"), (c) a gene encoding DNA packaging terminase subunit 1, (d) a gene encoding ICP8, or (e) a gene encoding ICP34.5. In one embodiment, the HSV further comprises, consists essentially of, or even consists of a gene encoding a dysfunctional ICP34.5 protein and / or a gene encoding a dysfunctional ICP6 protein. In another embodiment, the gene encoding the dysfunctional ICP34.5 protein comprises, consists essentially of, or even further consists of a polynucleotide having a sequence at least 95% identical to SEQ ID NO: 1, 5, 7, 9 or 51 and its equivalents, provided that the equivalents maintain the mutated or altered amino acids or nucleotides. In another embodiment, the gene encoding the dysfunctional ICP6 protein comprises, consists essentially of, or even further consists of a polynucleotide having a sequence at least 95% identical to SEQ ID NO: 43, 45, 47 or 49 and its equivalents, provided that the equivalents maintain the mutated or altered amino acids or nucleotides compared to the wild type sequence.

[0128] In certain embodiments, the gene encoding gE of the non-naturally occurring herpes simplex virus comprises, consists essentially of, or even consists of a polynucleotide having a sequence selected from SEQ ID NO: 12, 14, 16, 18, a sequence at least 80%, 85%, 90%, 95%, 97% or 99% identical to any one of SEQ ID NO: 12, 14, 16 and 18, and equivalents thereof, provided that the equivalents maintain mutated or altered amino acids or nucleotides compared to the wild-type sequence. In further embodiments, HSV containing such equivalents enter cells and / or spread between cells and / or replicate DNA at a level similar to that of non-naturally occurring HSV containing a mutated gE having the amino acid sequence of SEQ ID NO: 13. Non-limiting examples of assessing HSV entry into cells, spread between cells and replicate DNA can be found in the Examples. In still further embodiments, the polynucleotide of the gene encoding gE encodes a polypeptide having an amino acid sequence selected from SEQ ID NO: 13, 15, 17 and 19.

[0129] In certain embodiments, the gene encoding ICP0 of the non-naturally occurring herpes simplex virus comprises, consists essentially of, or even consists of a polynucleotide having a sequence selected from SEQ ID NOs: 20, 22, 24, 25, and 53, the sequence of any one of SEQ ID NOs: 20, 22, 24, 25, and 53 without one or both of the introns, a sequence at least 80%, 85%, 90%, 95%, 97% or 99% identical to SEQ ID NOs: 20, 22, 24, 25, and 53, and equivalents thereof, provided that the equivalents maintain mutated or altered amino acids or nucleotides compared to the wild-type sequence. In one embodiment, the introns are set forth in the sequence listing and below: nucleotides (nt) 58 to nt 861 of SEQ ID NO:20, nt 1529 to nt 1663 of SEQ ID NO:20, nt 58 to nt 822 of SEQ ID NO:22, nt 1490 to nt 1625 of SEQ ID NO:22, nt 58 to nt 862 of SEQ ID NO:24, nt 1530 to nt 1668 of SEQ ID NO:24, nt 58 to nt 861 of SEQ ID NO:25, nt 1529 to nt 1663 of SEQ ID NO:25, nt 58 to nt 822 of SEQ ID NO:53, and nt 1490 to nt 1625 of SEQ ID NO:53. In further embodiments, the equivalents encode polypeptides of ICP0 that function at the same level as wild-type ICP0 or mutated ICP0 having the amino acid sequence of SEQ ID NO: 21, for example, to promote transcription from viral genes, to disrupt structures in the nucleus known as nuclear dots or promyelocytic leukemia (PML) nuclear structures, and to alter the expression of host and viral genes in combination with neuronal specific proteins. Examples of assessing such functions can be found in Lee HR, Kim DJ, Lee JM et al. (June 2004).“Ability of the human cytomegalovirus IE1 protein to modulate sumoylation of PML correlates with its functional activities in transcriptional regulation and infectivity in cultured fibroblast cells” J.Virol.78(12):6527-42;Gu H, Liang Y, Mandel G, Roizman B (May 2005). “Components of the REST / CoREST / histone deacetylase repressor complex are disrupted, modified, and translocated in HSV-1-infected cells” Proc. Natl. Acad. Sci. USA102(21):7571-6; and Pinnoji RC, Bedadala GR, George B, Holland TC, Hill JM, Hsia SC (2007). "Repressor element-1 silencing transcription factor / neuronal restrictive silencer factor (REST / NRSF) can regulate HSV-1 immediate-early transcription via histone modification" Virol. J. 4:56. Additionally or alternatively, HSV containing such equivalents enter cells and / or spread between cells and / or replicate DNA at levels comparable to non-native HSV containing a mutated ICP0 having the amino acid sequence of SEQ ID NO:21. Non-limiting examples of assessing HSV entry into cells, spread between cells and replicate DNA can be found in the Examples. In still further embodiments, the polynucleotide of the gene encoding ICP0 encodes a polypeptide having an amino acid sequence selected from SEQ ID NOs:21, 23 and 26.

[0130] In certain embodiments, the gene encoding ICP8 of the non-naturally occurring herpes simplex virus comprises, consists essentially of, or even consists of a polynucleotide having a sequence selected from SEQ ID NO:27, 29, 31, 33, a sequence at least 80%, 85%, 90%, 95%, 97% or 99% identical to any one of SEQ ID NO:27, 29, 31 and 33, and equivalents thereof, provided that the equivalents maintain mutated or altered amino acids or nucleotides compared to the wild-type sequence. In further embodiments, the equivalents encode a polypeptide of ICP8 that functions (e.g., annealing to single-stranded DNA (ssDNA), melting small pieces of double-stranded DNA, or destabilizing double-stranded DNA during replication initiation) at a level similar to that of wild-type ICP8 or a mutated ICP8 having the amino acid sequence of SEQ ID NO:28. Such function can be assessed via methods available in the art, for example, Boehmer, PE; Lehman, IR (1993) "Herpes simplex virus type 1 ICP8: Helix-destabilizing properties". Journal of Virology. 67(2):711-5. Additionally or alternatively, HSV containing such equivalents enter cells and / or spread between cells and / or replicate DNA at a level similar to that of non-native HSV containing a mutated ICP8 having the amino acid sequence of SEQ ID NO:28. Non-limiting examples of assessing HSV entry into cells, spread between cells and replicate DNA can be found in the Examples. In still further embodiments, the polynucleotide of the gene encoding ICP8 encodes a polypeptide having an amino acid sequence selected from SEQ ID NOs:28, 30, 32 and 34.

[0131] In certain embodiments, the gene encoding the DNA packaging terminase subunit 1 of the non-naturally occurring herpes simplex virus comprises, consists essentially of, or even consists of a polynucleotide having a sequence selected from SEQ ID NO: 35, 37, 39, 41, a sequence at least 80%, 85%, 90%, 95%, 97% or 99% identical to any one of SEQ ID NO: 35, 37, 39 and 41, and equivalents thereof, provided that the equivalents maintain mutated or altered amino acids or nucleotides compared to the wild-type sequence. In further embodiments, HSV containing such equivalents enter cells and / or spread between cells and / or replicate DNA at a level similar to a non-naturally occurring HSV containing a mutated DNA packaging terminase subunit 1 having the amino acid sequence of SEQ ID NO: 36. Non-limiting examples of assessing HSV entry into cells, spread between cells and replicate DNA can be found in the Examples. In a further embodiment, the polynucleotide of the gene encoding DNA packaging terminase subunit 1 encodes a polypeptide having an amino acid sequence selected from SEQ ID NOs:36, 38, 40 and 42.

[0132] In certain embodiments, the HSV comprises one or more of the polynucleotides having a sequence selected from SEQ ID NO: 12, 20, 27 and 35, or essentially consists of one or more of the polynucleotides having a sequence selected from SEQ ID NO: 12, 20, 27 and 35, or even further consists of one or more of the polynucleotides having a sequence selected from SEQ ID NO: 12, 20, 27 and 35. In further embodiments, the HSV does not have a functional ICP34.5 (i.e., encodes a dysfunctional ICP34.5 or does not encode ICP34.5). Additionally or alternatively, the HSV does not have a functional ICP6 (i.e., encodes a dysfunctional ICP6 or does not encode ICP6). In another embodiment, the mutation in the virulence gene comprises, or essentially consists of, for example, an insertion, deletion, truncation, frameshift, substitution or point mutation of the ICP34.5 gene and / or the ICP6 gene, or even further consists of, for example, an insertion, deletion, truncation, frameshift, substitution or point mutation of the ICP34.5 gene and / or the ICP6 gene.In another embodiment, the HSV lacks the gene encoding a functional ICP34.5 protein and / or a functional ICP6 protein.In another embodiment, the mutation is a non-synonymous mutation in the virulence gene.

[0133] In one embodiment, the mutation on a non-naturally occurring HSV of the disclosure encodes one or more of: (a) an alanine to threonine mutation at position 151 of the gE protein; (b) an arginine to histidine mutation at position 258 of the ICP0 protein; (c) an alanine to threonine mutation at position 376 of the DNA packaging terminase subunit 1 protein; (d) a threonine to methionine mutation at position 1155 of the ICP8 protein; or (e) a proline to histidine mutation at position 119 of the ICP34.5 protein. In another embodiment, the non-naturally occurring HSV comprises, or alternatively consists essentially of, or even consists of one or more of SEQ ID NO:2, SEQ ID NO:13, SEQ ID NO:21, SEQ ID NO:28, or SEQ ID NO:36, and their equivalents, but with the proviso that the equivalents maintain mutated or altered amino acids or nucleotides.

[0134] In certain embodiments, a non-naturally occurring herpes simplex virus ("HSV") is provided, wherein the virus comprises, or consists essentially of, a mutation in one or more of (a) gE, (b) ICP0, (c) DNA packaging terminase subunit 1, (d) ICP8, or (e) ICP34.5. In certain embodiments, the HSV does not comprise a functional ICP34.5 protein (e.g., ICP34.5 of a 17TermA or rRp450 strain). In a further embodiment, the HSV does not comprise an ICP34.5 protein. Additionally or alternatively, the HSV does not comprise a functional ICP6 protein (e.g., ICP6 of strain 17TermA or rRp450). In yet a further embodiment, the HSV does not comprise an ICP6 protein.

[0135] In one embodiment, the one or more mutations on a non-naturally occurring HSV of the disclosure are one or more of: (a) an alanine to threonine mutation at position 151 of the gE protein; (b) an arginine to histidine mutation at position 258 of the ICP0 protein; (c) an alanine to threonine mutation at position 376 of the DNA packaging terminase subunit 1 protein; (d) a threonine to methionine mutation at position 1155 of the ICP8 protein; or (e) a proline to histidine mutation at position 119 of the ICP34.5 protein.

[0136] In certain embodiments, the non-naturally occurring HSV gE comprises, or consists essentially of, or even further consists of an amino acid sequence selected from SEQ ID NOs: 13, 15, 17, and 19. In further embodiments, the non-naturally occurring HSV further comprises a polynucleotide encoding the amino acid sequence of gE, e.g., a polynucleotide having a sequence selected from SEQ ID NOs: 12, 14, 16, 18, and equivalents thereof. In certain embodiments, the non-naturally occurring HSV ICP0 comprises, or consists essentially of, or even further consists of an amino acid sequence selected from SEQ ID NOs: 21, 23, and 26, and equivalents thereof. In further embodiments, the non-naturally occurring HSV further comprises a polynucleotide encoding the amino acid sequence of ICP0, e.g., a polynucleotide having a sequence selected from SEQ ID NOs: 20, 22, 24, 25, 53, and equivalents thereof. In certain embodiments, the ICP8 of the non-naturally occurring HSV comprises, or consists essentially of, or even consists of an amino acid sequence selected from SEQ ID NOs: 28, 30, 32, and 34. In further embodiments, the non-naturally occurring HSV further comprises a polynucleotide encoding the amino acid sequence of ICP8, e.g., a polynucleotide having a sequence selected from SEQ ID NOs: 27, 29, 31, 33, and equivalents thereof. In certain embodiments, the DNA packaging terminase subunit 1 of the non-naturally occurring HSV comprises, or consists essentially of, or even consists of an amino acid sequence selected from SEQ ID NOs: 36, 38, 40, and 42.In a further embodiment, the non-naturally occurring HSV further comprises a polynucleotide encoding the amino acid sequence of DNA packaging terminase subunit 1, eg, a polynucleotide having a sequence selected from SEQ ID NO: 35, 37, 39, 41, or an equivalent thereof.

[0137] In certain embodiments, the non-naturally occurring HSV comprises one or more of a polypeptide having an amino acid sequence selected from SEQ ID NOs: 13, 21, 28, and 36, or alternatively consists essentially of one or more of a polypeptide having an amino acid sequence selected from SEQ ID NOs: 13, 21, 28, and 36, or even further consists of one or more of a polypeptide having an amino acid sequence selected from SEQ ID NOs: 13, 21, 28, and 36. In further embodiments, the non-naturally occurring HSV comprises one or more of a polypeptide encoding one or more of an amino acid sequence selected from SEQ ID NOs: 13, 21, 28, and 36, or alternatively consists essentially of one or more of a polynucleotide encoding one or more of an amino acid sequence selected from SEQ ID NOs: 13, 21, 28, and 36, or even further consists of one or more of a polynucleotide encoding one or more of an amino acid sequence selected from SEQ ID NOs: 13, 21, 28, and 36.

[0138] In certain embodiments, the non-naturally occurring HSV is: (a) a polynucleotide encoding an amino acid sequence selected from SEQ ID NOs: 2, 6, 8, 10, and 52, and / or a polynucleotide having a sequence selected from SEQ ID NOs: 1, 5, 7, 9, and 51; (b) a polypeptide having an amino acid sequence selected from SEQ ID NOs: 2, 6, 8, 10, and 52; (c) a polynucleotide encoding an amino acid sequence selected from SEQ ID NOs: 13, 15, 17, and 19, and / or a polynucleotide having a sequence selected from SEQ ID NOs: 12, 14, 16, and 18; (d) a polypeptide having an amino acid sequence selected from SEQ ID NOs: 13, 15, 17 and 19; (e) a polynucleotide encoding an amino acid sequence selected from SEQ ID NOs: 21, 23 and 26, and / or a polynucleotide having a sequence selected from SEQ ID NOs: 20, 22, 24, 25 and 53, or a sequence thereof not containing one or two or more introns; (f) a polypeptide having an amino acid sequence selected from SEQ ID NOs: 21, 23 and 26; (g) a polynucleotide encoding an amino acid sequence selected from SEQ ID NOs: 28, 30, 32 and 34, and / or a polynucleotide having a sequence selected from SEQ ID NOs: 27, 29, 31 and 33; (h) a polynucleotide encoding an amino acid sequence selected from SEQ ID NOs: 28, 30, 32 and 34. (i) a polynucleotide encoding an amino acid sequence selected from SEQ ID NOs: 36, 38, 40 and 42, and / or having a sequence selected from SEQ ID NOs: 35, 37, 39 and 41; (j) a polypeptide having an amino acid sequence selected from SEQ ID NOs: 36, 38, 40 and 42; (k) a polynucleotide encoding an amino acid sequence selected from SEQ ID NOs: 44, 46, 48 and 50, and / or having a sequence selected from SEQ ID NOs: 43, 45, 47 and 49; (l) a polypeptide having an amino acid sequence selected from SEQ ID NOs: 44, 46, 48 and 50.

[0139] In another embodiment, the non-naturally occurring HSV further comprises, or alternatively consists essentially of, or even further consists of a polynucleotide having a sequence identical to at least one fragment of a pathogenicity gene from 17TermA HSV and its equivalents. In another embodiment, the non-naturally occurring HSV further comprises, or alternatively consists essentially of, or even further consists of a polynucleotide having a sequence identical to at least one fragment of a pathogenicity gene from rRp450 HSV and its equivalents. The non-naturally occurring HSV is, in some embodiments, derived from an HSV type 1 ("HSV-1") or HSV type 2 ("HSV-2") strain. In one embodiment, the non-naturally occurring HSV is derived from the HSV-1 KOS strain. In another embodiment, the non-naturally occurring HSV further comprises, or alternatively consists essentially of, or even further consists of a transgene.

[0140] Since the HSV disclosed herein retains its lytic function, in another aspect, the disclosure provides a method of treating cancer or inhibiting the growth or metastasis of cancer cells in a subject in need thereof, comprising, consisting essentially of, or even consisting of administering to the subject an effective amount of a non-naturally occurring HSV, or an effective amount of a composition comprising, or alternatively consisting essentially of, or even consisting of a non-naturally occurring HSV. In one aspect, the cancer comprises pancreatic cancer, renal cancer, small cell lung cancer, brain cancer, neuronal cancer, neuroblastoma, bone cancer, lymphoma, myeloma, colon cancer, uterine cancer, breast cancer, leukemia, liver cancer, prostate cancer, skin cancer, or melanoma. The subject to be treated can be an adult or pediatric patient, such as a mammal or human patient. In another embodiment, the non-naturally occurring HSV vector or composition or pharmaceutical composition is administered locally or systemically by injection, infusion, instillation, and / or inhalation. In another embodiment, the subject is a mammal. In some embodiments, the mammal is a mouse, rat, guinea pig, non-human primate, dog, cat, horse, cow, pig, goat or sheep. In another embodiment, the subject is a human.

[0141] In another aspect, the present disclosure provides a method of inducing cell lysis, comprising, consisting essentially of, or even further consisting of contacting a cell with a non-naturally occurring HSV of the present disclosure, or with a composition comprising, or alternatively consisting essentially of, or even further consisting of a non-naturally occurring HSV. In one embodiment, the cell is a cancer cell. In a further aspect, the cell is a cultured cell (for use as a preclinical model or assay) or a cell isolated from a subject. The cell can be cultured or can be present in an isolated tissue. Non-limiting examples of such cells include cells from pancreatic cancer, kidney cancer, small cell lung cancer, brain cancer, neuroblastoma, neural cancer, bone cancer, lymphoma, myeloma, colon cancer, uterine cancer, breast cancer, leukemia, liver cancer, prostate cancer, skin cancer, or melanoma. The cell can be isolated from a mammal, for example, the human can be an adult or a juvenile (child).

[0142] In another aspect, the disclosure provides a method for infecting a cell, the method comprising, or alternatively consisting essentially of, or even consisting of contacting the cell with a non-native HSV. In one embodiment, the cell is a eukaryotic cell. In another embodiment, the cell is a lymphocyte. In one embodiment, the cell is a cancer cell, such as a blood cancer or a solid tumor cell, such as a carcinoma or sarcoma. In a further aspect, the cell is a cultured cell (for use as a preclinical model or preclinical assay) or a cell isolated from a subject. The cell can be cultured or can be present in an isolated tissue. Non-limiting examples of such cells include cells from pancreatic cancer, renal cancer, small cell lung cancer, brain cancer, neuroblastoma, neural cancer, bone cancer, lymphoma, myeloma, colon cancer, uterine cancer, breast cancer, leukemia, liver cancer, prostate cancer, skin cancer, or melanoma. The cell can be isolated from a mammal, for example, the human can be an adult or a juvenile (child).

[0143] Applicants have discovered that conventional populations of lymphocytes infected with Epstein-Barr Virus ("EBV") are resistant to conventional oncolytic HSV ("oHSV"). Without wishing to be bound by theory, the resistance is due, at least in part, to low levels of expression of HSV entry receptors on EBV-infected lymphocytes. Surprisingly, the HSV of the claimed invention was able to induce lysis in EBV-infected lymphocytes that are resistant to wild-type strain 17, KOS and McKrae HSV viruses. Thus, in one embodiment, the cells comprise, or alternatively consist essentially of, or even further consist of, EBV-infected or EBV-resistant cells. In another embodiment, the cell comprises, or alternatively consists essentially of, or even further consists of pathogenic elements of Epstein-Barr Virus ("EBV"). In another embodiment, the cell comprises, or alternatively consists essentially of, or even further consists of lymphocytes infected by EBV. In another embodiment, the HSV of the claimed invention has a higher infection rate for cells infected with EBV compared to the control. In one embodiment, the control comprises, or alternatively consists essentially of, or even further consists of conventional oHSV. In another embodiment, the conventional oHSV comprises wild-type strain 17 HSV, KOS HSV, or McKrae HSV.

[0144] Generation of HSV mutants or derivatives The creation of HSV mutants or derivatives involves alteration or mutation of genes or combinations of genes encoded by parent HSV strains. For example, derivatives may have the sequence of HSV-1 or HSV-2 genome modified by nucleotide substitutions, such as 1, 2 or 3 to 10, 25, 50 or 100 substitutions. Alternatively or additionally, HSV-1 or HSV-2 genome may be modified by one or more insertions and / or deletions and / or extensions at either or both ends. Genetic modification methods, such as CRISPR, recombination construction or point mutations, are known in the art. Those skilled in the art know how to create HSV mutants based on the needs.

[0145] In addition to targeted gene modification methods, HSV mutants can be produced spontaneously. For example, the culture of viruses such as HSV involves a technique known as serial passage. To grow and maintain the virus, the virus is infected into suitable cells, the virus replicates in the cells, and then the virus is harvested and then reinfected into fresh cells, this process constituting one cycle of serial passage. Each such cycle may take several days, for example, in the case of HSV. As mentioned above, such serial passage may result in changes in the properties or gene sequence of the virus strain, in that selection occurs for properties favorable for clinical application of HSV. For example, enhanced properties may include rapid replication, or the ability to migrate along axons to infect human cells. In addition, spontaneous mutations can be generated by infecting cells with one HSV or more than one HSV.

[0146] Thus, the present disclosure provides a method for preparing HSV or a mutant or derivative thereof by mutating a gene in HSV. In another embodiment, the method comprises, or alternatively consists essentially of, or even further consists of introducing a transgene into a non-naturally occurring HSV.

[0147] In another aspect, provided herein is a method for producing HSV viral particles, comprising, or alternatively consisting essentially of, or even consisting of, (a) introducing a 17TermA HSV vector and a rRp450 HSV vector into a host cell; (b) growing the host cell for at least three passages; and (c) isolating the HSV particles produced by the host cell. In one embodiment, the HSV is introduced into the cell by transfection, infection, transformation, electroporation, injection, microinjection, or a combination thereof. In another embodiment, the host cell is grown for at least 3 passages, 10 passages, 20 passages, 30 passages, 40 passages, or 50 passages. In some embodiments, the host cell comprises, or alternatively consists essentially of, or even consists of a complementary gene product for supporting the replication of the introduced HSV vector. In another embodiment, the complementation genes encode ICP6 and / or ICP34.5 proteins. In another embodiment, the HSV particles so produced comprise, or alternatively consist essentially of, or even further consist of an HSV vector of this disclosure.

[0148] In certain embodiments, provided herein is a method for producing a non-naturally occurring HSV viral particle of the present disclosure, the method comprising, or alternatively consisting essentially of, or even consisting of, (a), (b) and (c): (a) introducing a non-naturally occurring HSV vector into a host cell; (b) growing the host cell; and (c) isolating HSV particles produced by the host cell.

[0149] In certain embodiments, the present disclosure provides a method for making non-natural HSV viral particles, comprising, or alternatively consisting essentially of, or even consisting of, (a), (b) and (c): (a) introducing a polynucleotide encoding the viral genome of a non-natural HSV vector into a host cell; (b) growing the host cell; and (c) collecting and isolating the HSV particles produced by the host cell. In one embodiment, the nucleic acid sequence encoding the viral genome is introduced into the host cell by transfection, infection, transformation, electroporation, injection, microinjection, or a combination thereof. In one embodiment, the nucleic acid sequence encoding the viral genome is introduced into the host cell in a vector. In a further embodiment, the vector is a viral vector (such as HSV) or a non-viral vector (such as a plasmid or nanoparticle). In a further embodiment, the vector is HSV. In some embodiments, the host cell comprises, or alternatively consists essentially of, or even further consists of complementary gene products for supporting the replication of the introduced HSV vector. In one embodiment, such complementary gene products are provided in the host cell via a helper virus. In another embodiment, the complementary genes encode ICP6 and / or ICP34.5 proteins. In another embodiment, the HSV particles thus produced comprise, or alternatively consists essentially of, or even further consists of the HSV vector of the present disclosure.

[0150] In one embodiment, isolating refers to a process of substantially separating HSV from other materials, such as host cells, cell debris, culture medium, or any other agent used in the culture of host cells, for example, by centrifugation, filtration, chromatography, or any combination thereof. Non-limiting examples can be found in Sia et al., Optimal purification method for Herpes-based viral vectors that confers minimal cytotoxicity for systemic route of vector administration. J Virol Methods. 2007 Feb;139(2):166-74.

[0151] composition In another aspect, the present disclosure provides a composition comprising or consisting essentially of a non-naturally occurring HSV as described herein, or even further consisting of a non-naturally occurring HSV as described herein.Compositions such as pharmaceutical compositions comprising or consisting essentially of an agent or virus particle as described herein, or even further consisting of an agent or virus particle as described herein, can be produced by conventional mixing, dissolving, granulating, levigating, emulsifying, encapsulating, encapsulating or lyophilizing processes.Compositions can be formulated in a conventional manner using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries that facilitate the processing of the virus particles provided herein into a medicamentically usable preparation.

[0152] The agents and viral particles of the present technology can be administered parenterally (e.g., intramuscular, intraperitoneal, intravenous, intracerebroventricular ("ICV"), intracisternal injection or infusion, subcutaneous injection, or implant), orally, by inhalation spray, nasal, vaginal, rectal, sublingual, urethral (e.g., urethral suppository), or topical routes of administration (e.g., gels, ointments, creams, aerosols, etc.), and can be formulated, alone or together, in suitable dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants, excipients and vehicles appropriate for each route of administration.

[0153] In one embodiment, the disclosure relates to a composition comprising, consisting essentially of, or even further consisting of the non-naturally occurring HSV viral particles and carriers described herein.

[0154] In another embodiment, the disclosure relates to a pharmaceutical composition comprising, or alternatively consisting essentially of, the non-naturally occurring viral particles and a pharma- ceutically acceptable carrier as described herein, or even further consisting of the non-naturally occurring viral particles and a pharma- ceutically acceptable carrier as described herein. In another embodiment, the composition comprises, or alternatively consists essentially of, a cryopreservation agent that facilitates freezing and thawing of the non-naturally occurring HSV without significant loss of pathogenicity, or even further consisting of a cryopreservation agent that facilitates freezing and thawing of the non-naturally occurring HSV without significant loss of pathogenicity.

[0155] In another embodiment, the present disclosure relates to a pharmaceutical composition comprising, or alternatively consisting essentially of, or even further consisting of a therapeutically effective amount of the non-naturally occurring HSV viral particles described herein and a pharma- ceutically acceptable carrier.

[0156] The pharmaceutical composition for administering HSV viral particles can be conveniently provided in dosage unit form and can be prepared by any method well known in the field of pharmacy.The pharmaceutical composition can be prepared, for example, by uniformly and intimately mixing the HSV viral particles provided herein with liquid carriers, finely divided solid carriers, or both, and then, if necessary, shaping the product into desired formulations.In the pharmaceutical composition, the compound provided herein is included in an amount sufficient to produce the desired therapeutic effect.For example, the pharmaceutical composition of the present disclosure can be in a form suitable for virtually any mode of administration, including, for example, topical, ocular, oral, buccal, systemic, nasal, injection, infusion, transdermal, rectal and vaginal, or in a form suitable for administration by inhalation or insufflation.

[0157] For topical administration, the non-native HSV viral particles can be formulated as solutions, gels, ointments, creams, suspensions, etc., as is known in the art.

[0158] Systemic formulations include those designed for administration by injection (e.g., subcutaneous, intravenous, infusion, intramuscular, intrathecal or intraperitoneal injection), as well as those designed for transdermal, transmucosal, oral or pulmonary administration.

[0159] Useful injection preparations include sterile suspensions, solutions or emulsions of HSV virus particles provided herein in aqueous or oily vehicles.The composition may also contain compounding agents, such as suspending agents, stabilizing agents and / or dispersing agents.Injection preparations may be provided in unit dosage form, such as ampoules or in multi-dose containers, and may contain added preservatives.

[0160] Alternatively, the injectable preparation can be provided in powder form for reconstitution with suitable vehicle, including but not limited to sterile pyrogen-free water, buffer and dextrose solution, before use.To this end, the HSV virus particles provided herein can be dried by any art-known technique, such as lyophilization, and reconstituted before use.

[0161] For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are known in the art.

[0162] For oral administration, the pharmaceutical composition may take the form of lozenges, tablets or capsules prepared by conventional means using pharma- ceutically acceptable excipients such as, for example, binders (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropylmethylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). Tablets may be coated, for example, with sugar, film or enteric coating, by methods well known in the art.

[0163] Compositions intended for oral use can be prepared according to any method known in the art for the manufacture of pharmaceutical compositions, and such compositions may contain one or more agents selected from the group consisting of sweeteners, flavoring agents, coloring agents, and preservatives to provide pharma- ceutically elegant and palatable preparations. Tablets contain the HSV virus particles provided herein in admixture with non-toxic pharma- ceutically acceptable excipients suitable for the manufacture of tablets. These excipients can be, for example, inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents (e.g., corn starch or alginic acid); binding agents (e.g., starch, gelatin or gum arabic); and lubricants (e.g., magnesium stearate, stearic acid or talc). Tablets can be left uncoated, or they can be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract, thereby providing a sustained action over a longer period of time. For example, a time-delay material such as glyceryl monostearate or glyceryl distearate can be used. Tablets may also be coated by techniques well known to those skilled in the art.The pharmaceutical compositions of the present technology may be in the form of oil-in-water emulsions.

[0164] Liquid preparations for oral administration can take the form of, for example, elixirs, solutions, syrups or suspensions, or can be provided as dry products for constitution with water or other suitable vehicles before use.Such liquid preparations can be prepared by conventional means using pharma-ceutically acceptable additives, such as suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats); emulsifying agents (e.g., lecithin or gum arabic); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, Cremophor™, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl p-hydroxybenzoate or sorbic acid).Preparations can also contain buffer salts, preservatives, flavoring agents, coloring agents, and sweeteners, as appropriate.

[0165] In one embodiment, the disclosure provides a method for preparing a non-naturally occurring HSV of the disclosure, comprising, or alternatively consisting essentially of, or even further consisting of, mutating a gene in a non-naturally occurring HSV virus particle or introducing a transgene into the non-naturally occurring HSV. In another aspect, a method for producing a non-naturally occurring HSV vector comprises, or alternatively consists essentially of, or even further consisting of (a), (b) and (c), (a) introducing a 17TermA HSV vector and a rRp450 HSV vector into a host cell; (b) growing the host cell for at least three passages; and (c) isolating the HSV particles produced by the host cell.

[0166] Also provided is a method for inhibiting the growth or metastasis of cancer cells or metastatic cancer cells, comprising, consisting essentially of, or even consisting of contacting cells with an effective amount of a non-naturally occurring HSV vector or a composition or pharmaceutical composition containing a non-naturally occurring HSV vector as described herein. The contacting is in vitro or in vivo. In one embodiment, the contacting is in vivo by administering the non-naturally occurring HSV or the composition or pharmaceutical composition to a subject. In vitro, the method is carried out by contacting the non-naturally occurring HSV with cells. The in vitro method can be used as a personalized assay to test new therapies or to determine whether a therapy is suitable for the cancer to be treated. Additional cancer therapy can be combined with the disclosed method, which may be simultaneous or sequential.

[0167] The cancer cells to be treated may be solid tumors or blood cancers, such as carcinomas or sarcomas, non-limiting examples of which include pancreatic cancer, renal cancer, small cell lung cancer, brain cancer, neuroblastoma, neural cancer, bone cancer, lymphoma, myeloma, colon cancer, uterine cancer, breast cancer, leukemia, liver cancer, prostate cancer, skin cancer or melanoma. The cells may be of any species, such as mammalian and human, and when performed in vitro, the cells may be cultured cell lines or primary cells, such as from tissue biopsies. The cells may be adult or juvenile cells or cancer stem cells from tissue biopsies. The cells may be adult or juvenile cells or cancer stem cells (i.e., cancer cells that have characteristics associated with normal stem cells, particularly the ability to give rise to all cell types found in a particular cancer sample), or cancer cells that lack such characteristics associated with normal stem cells. In one embodiment, the cells express N-myc proto-oncogene protein (MYCN) and / or express MYCN at a higher level than non-cancer cells.

[0168] Further provided herein is a method of treating cancer or inhibiting the growth or metastasis of cancer cells in a subject in need thereof, comprising, or alternatively consisting essentially of, or even consisting of administering to the subject an effective amount of a non-naturally occurring HSV, composition, or pharmaceutical composition of the present disclosure. The subject to be treated can be any species, such as a mammal and human, such as a dog, horse, cow, cat, monkey, rat, or mouse. Administration can be as a first line, second line, third line, fourth line, or fifth line treatment. Additional cancer treatments can be combined with the disclosed method, which treatments can be simultaneous or sequential. The cancer to be treated can be a solid tumor or a hematological cancer, such as a carcinoma or sarcoma, non-limiting examples of which include pancreatic cancer, renal cancer, small cell lung cancer, brain cancer, neuroblastoma, neural cancer, bone cancer, lymphoma, myeloma, colon cancer, uterine cancer, breast cancer, leukemia, liver cancer, prostate cancer, skin cancer, or melanoma.

[0169] The methods of the present disclosure can be combined with appropriate diagnostics to monitor disease remission or progression. Several methods for such monitoring are known in the art.

[0170] Further provided is a method of inducing cell lysis, comprising, or alternatively consisting essentially of, or even consisting of contacting a cell with an effective amount of the non-naturally occurring HSV, composition and / or pharmaceutical composition of the present disclosure. The contacting is in vitro or in vivo. In one embodiment, the contacting is in vivo by administering the non-naturally occurring HSV or composition or pharmaceutical composition to a subject. In vitro, the method is carried out by contacting the non-naturally occurring HSV with a cell. The in vitro method can be used as an individualized assay to test new therapies or to determine whether a therapy is suitable for the subject to be treated. Additional cell lysis therapy can be combined with the disclosed method, which can be simultaneous or sequential.

[0171] The cells to be treated can be solid tumors or blood cancers, such as carcinomas or sarcomas, including, but not limited to, pancreatic cancer, renal cancer, small cell lung cancer, brain cancer, neuroblastoma, neural cancer, bone cancer, lymphoma, myeloma, colon cancer, uterine cancer, breast cancer, leukemia, liver cancer, prostate cancer, skin cancer or melanoma. The cells can be of any species, such as mammalian and human, and when performed in vitro, the cells can be cultured cell lines or primary cells, such as from tissue biopsies. The cells can be adult or juvenile cells or cancer stem cells or cancer cells that do not have characteristics associated with normal stem cells. The treatment can be combined with an appropriate assay to test for the effectiveness of the treatment, such as remission or progression of cancer.

[0172] Use of HSV virus particles for preparing a medicine The HSV and composition of the present invention are also useful in the preparation of medicaments for treating various pathologies described herein.The methods and techniques for preparing medicaments of compositions are known in the art.For the purpose of illustration only, pharmaceutical formulations and routes of delivery are described in detail herein.

[0173] Thus, those skilled in the art will readily understand that any one or more of the above compositions, including many specific embodiments, can be used by applying standard pharmaceutical manufacturing procedures to prepare medicaments for treating many of the disorders described herein. Such medicaments can be delivered to a subject by using delivery methods known in the pharmaceutical arts.

[0174] Administration of additional therapeutic agents The methods disclosed herein can further comprise, or alternatively consist essentially of, or even further comprise, administration of an effective amount of an additional therapeutic agent to increase or enhance the efficacy of the treatment of the disclosed methods. In one embodiment, the additional therapeutic agent comprises, or alternatively consists essentially of, or even further comprises, surgical resection of the tumor, an anti-tumor agent, such as a small molecule or immunotherapy or cytolytic therapy.

[0175] The administration of the therapeutic agent or substance of the present disclosure to patients will follow the general protocol for administration of that particular first or second line therapy, taking into account the toxicity (if any) of the treatment. It is expected that the treatment cycle will be repeated as necessary. It is also contemplated that various standard therapies and surgical interventions may be applied in combination with the described treatment.

[0176] As will be apparent to one of skill in the art, the combination therapy can take the form of a combination therapy for simultaneous or sequential administration.

[0177] kit The agents or non-naturally occurring HSV described herein may, in some embodiments, be assembled into pharmaceutical or diagnostic or research kits to facilitate their use in therapeutic, diagnostic or research applications. The kits may include one or more containers housing the components of the present invention and instructions for use. Specifically, such kits may include one or more agents described herein, together with instructions describing the intended use and proper use of these agents. In certain embodiments, the agents in the kits may be in pharmaceutical formulations and dosages suitable for a particular use and method of administration of the agents. Kits for research purposes may contain components in appropriate concentrations or amounts to carry out various experiments.

[0178] The kits can be designed to facilitate the use of the methods described herein and can take many forms. Each composition of the kit can be provided in liquid form (e.g., in solution) or solid form (e.g., dry powder), as applicable. In certain cases, some of the compositions can be configurable or otherwise processable (e.g., into an active form), for example, by the addition of a suitable solvent or other species (e.g., water or cell culture medium), which may or may not be provided with the kit. In some embodiments, the compositions can be provided in a preservation solution (e.g., a cryopreservation solution). Non-limiting examples of preservation solutions include DMSO, paraformaldehyde, and CryoStor® (Stem Cell Technologies, Vancouver, Canada). In some embodiments, the preservation solution contains an amount of a metalloprotease inhibitor.

[0179] As used herein, "instructions" can define instructional and / or encouraging elements, and typically include written instructions on or accompanying the packaging of the present invention. Instructions can also include any verbal or electronic instructions provided in any format that clearly identifies to the user that the instructions are to be associated with the kit, such as audiovisual (e.g., videotape, DVD, etc.), internet and / or web-based communication means, etc. Written instructions can be in a format prescribed by a government agency regulating the manufacture, use, or sale of pharmaceutical or biological products, and the instructions can also reflect agency approval of manufacture, use, or sale for animal administration.

[0180] The kit may contain any one or more of the components described herein in one or more containers. As an example, in one embodiment, the kit may include instructions for mixing one or more components of the kit and / or for isolating and mixing a sample and applying to a subject. The kit may include a container that contains the agent described herein. The HSV may be in the form of a liquid, gel, or solid (powder). The HSV may be aseptically prepared, packaged in a syringe, and shipped refrigerated. Alternatively, it may be contained in a vial or other container for storage. A second container may have other agents that are aseptically prepared. Alternatively, the kit may contain an active agent that is premixed and shipped in a syringe, vial, tube, or other container. The kit may have one or more or all of the components required to administer the agent to a subject, such as a syringe, topical application device, or IV needle tube and bag.

[0181] Screening assays The present disclosure also provides screening assays for identifying potential therapeutic agents of known and novel compounds and combinations.For example, those skilled in the art can also determine whether HSV provides therapeutic benefit in vitro by contacting HSV with the sample cell or tissue to be treated.Cells or tissues can be derived from any species, such as monkey, dog, cow, sheep, rat, mouse or human.

[0182] The contacting can also be performed in vivo in suitable animal models or human patients.When performed in vitro, HSV can be added directly to cell culture medium.When performed in vitro, the method can be used to screen for new combination therapies, formulations or treatment regimens before administration to animals or human patients.

[0183] In another embodiment, the assay requires contacting a first sample ("control sample") containing suitable cells or tissue with an effective amount of HSV disclosed herein, and contacting a second sample ("test sample") of suitable cells or tissue with the HSV, agent, or combination to be assayed. In one embodiment for cancer, inhibition of growth of the first and second cell samples is determined. If the inhibition of growth of the second sample is substantially the same as or greater than the first sample, the agent is a potential drug for treatment. In one embodiment, the substantially same or greater inhibition of cell growth is less than about 1%, or alternatively less than about 5%, or alternatively less than about 10%, or alternatively more than about 10%, or alternatively more than about 20%, or alternatively more than about 50%, or alternatively more than about 90% difference. The contacting can be in vitro or in vivo. Means for determining inhibition of cell growth are well known in the art.

[0184] In further embodiments, the test agent is contacted with a third sample of cells or tissue, which comprises cells or tissue of the control and normal counterparts of the test sample, and an agent that treats the second sample of cells or tissue but does not adversely affect the third sample is selected.For the assays described herein, suitable cells or tissues are described herein, such as cancer or other diseases described herein.Examples of such include, but are not limited to, cancer cells or tissues obtained by biopsy or from blood.

[0185] The effectiveness of a test composition is determined using methods known in the art, including, but not limited to, cell viability assays.

[0186] In a further embodiment, the assay requires at least two cell types, the first cell type being a suitable control cell.

[0187] The assay is also useful for predicting whether a subject is appropriately treated by the present disclosure, by delivering HSV to the sample containing the cells to be treated, and assaying for treatment that varies with pathology, or for screening for new drugs and combinations.In one embodiment, cells or tissue are obtained from a subject or patient by biopsy.The present disclosure also provides a kit for determining whether a diseased cell or patient is appropriately treated by this therapy, by providing at least one composition of the present disclosure and instructions for use.

[0188] Test cells can be grown in small multi-well plates and used to detect the biological activity of test compounds. In the present disclosure, successful HSV or other agents inhibit the growth or kill cancer cells while leaving control cell types intact.

[0189] The following examples are provided to illustrate, but not to limit, the present disclosure. EXAMPLES

[0190] Generation of HSV mutants A nonpermissive cell line was infected with 17TermA and rRp450 ("directed evolution") and cultured after serial passage to isolate HSV Mut-3 mutants containing a WT-like genotype. The HSV Mut-3 mutants were then used to construct an attenuated mutant Mut-3Δ34.5 via gene editing (designated "CRISPR / Cas9") (Figure 1A). A sequence comparison of Mut-3 with its parent virus is shown in Figure 1B. Nonsynonymous mutations in Mut-3 that differ from either parent are marked with a diagonal line to the lower right, including UL15, UL29, US8, RL1, and RL2. Genomic sequences identical to 17TermA are shown as blanks, and genomic sequences identical to rRp450 are marked with a diagonal line.

[0191] We isolated a potent oHSV mutant Mut-3 that contains a WT-like genotype. Both Mut-3 and the attenuated version Mut-3Δ34.5 rely on the canonical HSV entry protein nectin-1 or herpesvirus entry mediator (HVEM) to achieve successful infection (not shown). The kinetics of Mut-3Δ34.5 viral gene transfer, as measured by the expression of detectable GFP-positive regions, is much faster when compared to 17Δ34.5, a γ134.5 null virus derived from wild-type strain 17 via the same CRISPR / Cas9 gene editing strategy. Furthermore, Mut-3Δ34.5 infection results in more cell killing, as measured by its smaller cell-crowding region. However, Mut-3Δ34.5 appears to replicate less compared to 17Δ34.5 or compared to the Mut-3Δ34.5 null parent virus 17TermA. These results indicate that even in the attenuated Mut-3Δ34.5 version, the unique genomic alteration(s) result in a syncytial phenotype (not shown) and enhanced potency remains. Studies of epidermal growth factor receptor (EGFR)-retargeted HSV reported that introducing syncytial mutations does not impair the specificity of entry and spread. Without being bound by theory, the applicants believe that the underlying cause of the syncytial phenotype in Mut-3Δ34.5 may result in altered kinetics during virus fusion to the cell membrane that may affect entry and / or virus-mediated cell-cell fusion, leading to faster cell killing and reduced virus particle production.

[0192] No mutations were found in gB, gD, gH and gL, the canonical glycoproteins involved in HSV-1 entry. However, five genes contain nonsynonymous (NS) mutations in Mut-3 that are different from either parent: RL1, RL2, UL15, UL29 and Us8 / gE. Both Mut-3 and its attenuated version Mut-3Δ34.5 exhibit a membrane fusogenic phenotype, suggesting that this phenomenon is not RL1-related. Whole genome analysis also suggests that this phenotype is not related to previously reported mutations, since the amino acid sequence of Mut-3 is identical to either rRp450 and the reference strain 17 genome (not a syncytial virus) in gK / UL53 and UL20, or to 17TermA (not a syncytial virus) in gB / UL27 and UL24. The A151T mutation in gE / Us8 is the only glycoprotein (virion surface protein) in Mut-3 that has a NS change compared to its parent virus. Although gE is not involved in either viral attachment or entry, gE / gI dimerization mediates viral cell-to-cell spread, as mutations in gE are associated with smaller plaque sizes. ** Applicant hypothesizes that A151T of gE in Mut-3Δ34.5 may be the underlying cause of its syncytial and increased potency phenotypes. Based on the order of Applicant's research (top to bottom), in Table 1, Applicant lists all five NS mutations found in five genes in Mut-3, as well as the corresponding functions of each gene, their possible roles related to the increased potency phenotype of Mut-3, and Applicant's proposed research. The other three mutations (except RL1) can also be determined as was done for gE A151T. [Table 1]

[0193] Comparison of HSV mutants Plaque assays were performed simultaneously for the four viruses shown in Figure 1A, and plaque images were scanned and analyzed via a Keyence HS All-in-one Fluorescence Microscope BZ-II Analyzer after 3 days. As shown in Figures 1A and 1B, the plaque sizes of Mut-3 and Mut-3Δ34.5 were significantly larger than either of the parental viruses rRp450 and 17TermA. For in vitro cytotoxicity / MTS assays of a set of CHO cells, CHO-K1, CHO-nectin-1, CHO-nectin-2 and CHO-HVEM were infected with the four viruses at different multiplicities of infection (MOI). Three days after virus infection (pvi), cell viability colorimetric cell proliferation and MTS assays were measured compared to untreated controls. Only CHO-nectin-1 and CHO-HVEM, but not CHO-K1 or CHO-nectin-2 (mainly for HSV-2 entry), were sensitive to the treatment of the four viruses (Figure 2C). Without being bound by theory, the results suggested that Mut-3 and Mut-3Δ34.5 did not bypass the receptor barrier and still relied on the canonical HSV entry receptor to infect cells.

[0194] The increased potency of Mut-3Δ34.5 in killing human and mouse neuroblastoma cells compared with 17TermA was not due to an increased yield of infectious virus (Fig. 3). Transmission electron microscopy ("TEM") analysis also revealed that following infection of neuroblastoma cells, Mut-3Δ34.5 virions were found primarily in intracellular vesicles, whereas 17TermA virions were found primarily in intracellular vesicles (Fig. 4).

[0195] An attenuated 17Δ34.5 mutant was generated by CRISPR-Cas9 gene editing technology to replace the g134.5 gene of wild-type strain 17+ with an EGFP expression cassette. The 17Δ34.5 mutant had lower potency than its wild-type strain 17+ but was comparable to 17TermA (Figure 5). The attenuation of 17Δ34.5 was further confirmed when Mut-3Δ34.5 showed much faster viral gene transfer and cell killing compared to 17Δ34.5 (Figure 6). Furthermore, Mut-3Δ34.5 was more effective than 17TermA to control human neuroblastoma growth in vivo (Figure 7).

[0196] Us8 / gE revertants in Mut-3Δ34.5 for loss-of-function studies Because of the existence of multiple copies of genome during viral replication and because this gene editing cannot be accompanied by a selection marker, it is difficult to introduce a single nucleotide change into the HSV genome through CRISPR / Cas9 technology alone.One construct can be the result of a two-step process in combination with CRISPR / Cas9 technology: 1) replace the entire gE coding region with a reporter gene (such as mCherry or red fluorescent protein (RFP)) to construct gE-null Mut-3Δ34.5, and then 2) replace the reporter gene with WT gE coding region to obtain gE-WT Mut-3Δ34.5 revertant.The degree to which both the gE-null intermediate and this gE revertant Mut-3Δ34.5 lose phenotype can be observed as described above.The present disclosure also provides this construct.

[0197] Us8 / gE A151T mutation in Mut-3 parent 17TermA for gain of function As described above, gE mutant 17TermA can be constructed in two steps: 1) completely knock out the gE coding region and replace it with a reporter gene (e.g., GFP), and then 2) replace the reporter gene by knocking in the gE coding region containing the A151T mutation. The phenotype of Mut-3Δ34.5 is determined. The present disclosure also provides this construct.

[0198] In cases where gE A151T is only partially or not at all responsible for the observed phenotype, or even if it is, other mutations in Mut-3 are provided by the present disclosure. Mutants are constructed by systematically attempting similar gain-of-function and loss-of-function approaches, alone or in combination, as described herein and shown in Table 1. The safety profile and efficacy of attenuated Mut-3Δ34.5 can be tested in mouse models. Thus, the present disclosure provides animal models used to test mutants and methods therefor.

[0199] A potent and WT-like HSV mutant, Mut-3 strain, was constructed from the recombination of 17TermA and rRp450. To ensure safety for clinical use, an attenuated version, Mut-3Δ34.5, was generated through deletion of the viral virulence protein γ134.5 / RL1. In our preliminary toxicology studies, no adverse clinical signs or significant changes in body weight were observed in Balb / c mice over 85 days after intravenous administration of up to 1e8 plaque forming units (pfu) (data not shown). Furthermore, Mut-3Δ34.5 also exhibits antitumor effects in highly aggressive neuroblastoma models both in vitro (Figure 3A) and in vivo (Figure 7) compared to 17TermA.

[0200] Biodistribution profile of Mut-3Δ34.5 in naive non-tumor-bearing mice Applicant's own studies have shown that naive Balb / c mice can tolerate up to 1e8 pfu of intravenously (iv) delivered Mut-3Δ34.5 virus for more than 85 days without physical signs of disease. Biodistribution studies were performed starting with intravenous administration of the highest dose previously tested (1e8 pfu of Mut-3Δ34.5 virus per mouse) to naive non-tumor-bearing Balb / c mice of both sexes (30 mice per sex). Peripheral blood is collected and then mice are sacrificed. Testes, ovaries, spleen, lungs, kidneys, heart, lungs and brain are harvested (n=6 per time point) at 24 hours, 14 days, 28 days, 56 days and 85 days after viral infection. Half of the organs are embedded in formalin to preserve for pathology analysis and the other half is homogenized for qPCR analysis of HSV genome and plaque assay to obtain the viral load in each organ. Mice are observed daily for the first two weeks after virus administration, then twice weekly until the scheduled sacrifice date. Each mouse is weighed before virus administration and weekly thereafter. Mice are sacrificed for the presence or absence of adverse clinical signs or with more than 20% weight loss and analyzed for viral activity in organs as described above. In parallel, wild-type KOS virus [dose range: 1 × 10 5 ~1×10 7 pfu / mouse (n=3)] will be administered to a group of mice as a positive (non-safety) control. Applicants will administer 1×10 6We previously found that a dose of pfu of KOS virus was uniformly lethal within 2-3 days. qPCR is performed to analyze HSV genome copies and plaque assays are performed to evaluate viral activity and pathology analysis in organs of positive control mice showing signs of disease. These results can serve as positive indicators / thresholds to evaluate data collected from Mut-3Δ34.5 treated mice. Pathological changes in tissues / organs are further evaluated in the Mut-3Δ34.5 treated group to show comparable viral loads as the positive control. Six mice per group for each sex are used to evaluate biodistribution and safety and tolerability over different time periods. Descriptive statistics are used to measure the biodistribution of Mut-3Δ34.5 by the number of HSV genome copies per nanogram of genomic DNA in different organs over different time periods and compared to univariate analysis (where applicable).

[0201] Cytotoxicity of Mut-3Δ34.5 and other γ134.5-null viruses (17TermA and T-VEC) in various pediatric cancer cell lines in vitro Superior killing by Mut-3Δ34.5 was observed in human and mouse neuroblastoma cells compared to 17TermA (Figure 3A). Using the same MTS in vitro assay shown in Figure 3A, the same analysis is applied to other pediatric cancer cells such as sarcoma, malignant peripheral nerve sheath tumor (MPNST) and brain tumors to determine whether the increased efficacy phenotype is applicable across different tumor types (applicants have multiple models available). The most effective lines from each cell type are used to perform in vivo efficacy studies. Examine the efficacy of Mut-3Δ34.5 in human pediatric tumor models in comparison with other oHSV therapeutics, 17TermA and T-VEC.

[0202] Select one highly responsive model of each tumor type (three in total) to conduct efficacy studies in xenografts using 5-6 week old female athymic nude mice. Tumors should be between 150-300 mm 3Once the mice reach this number, they are pooled and randomly assigned to three groups (n=11 each): i) phosphate-buffered saline (PBS) control, ii) Mut-3Δ34.5 virus, or iii) 17TermA or T-VEC virus. For efficacy between viruses, a minimum of 11 mice per group are used to detect significant differences in survival and tumor growth (20% vs. 80% at day 20) with a minimum of 80% power. Based on applicant's previous work, a treatment regimen of three total injections is used, with 1×10 7 Each mouse is treated intratumorally every other day with either pfu of virus or PBS only (control). Mice are monitored for tumor volume (twice weekly) and body weight (weekly) for 80 days after virus injection. Endpoint criteria include tumor volume above 2500 mm 3 These include tumor volumes exceeding 100 μg / kg, tumor diameters reaching 2 cm, or body weight loss exceeding 20%. Animal survival can be displayed using Kaplan-Meier curves, and survival can be compared between groups by the log-rank test.

[0203] Efficacy of Mut-3Δ34.5 in a mouse pediatric tumor model compared to other oHSV therapeutics (17TermA and T-VEC) One highly responsive mouse tumor model of each tumor type (total of 2) will be selected to conduct Applicant's efficacy studies in (5-6) week old gender matched C57BL6 mice. Similarly, mice will be pooled and randomly assigned to three groups, i) PBS, ii) Mut-3Δ34, iii) 17TermA or T-VEC, n=11 each. As per Applicant's previous studies, each mouse will be treated intratumorally with either 1e8 pfu of virus in 100 μl PBS or PBS only every other day for a total of three injections. Endpoint criteria include 2500 mm 3 These included tumor volumes exceeding 100 μg / kg, tumor diameters reaching 2 cm, or body weight loss exceeding 20%. Animal survival was displayed using Kaplan-Meier curves, and survival was compared between groups by the log-rank test.

[0204] Summary of pathology analysis Tissues from four wild-type KOS-injected mice (1e6 and 1e7 pfu, 2 mice / dose) were subjected to pathology analysis by the OSU Comparative Pathology & Mouse Phenotyping Shared Resource. Tissues from Mut-3D34.5 / C8G5 and Mut-3DICP6 / D7-1-injected mice (24 hours and 14 days post-viral infection, 2 mice / time point) were subsequently subjected to pathology analysis.

[0205] Pathology report (female): KOS-injected mouse The lymphoplasmacytic encephalitis of the brain stem is consistent with published reports of CNS pathology in Balb / c mice injected with HSV-1. All mice examined in this submission had this lesion, but one submitted brain only involved a small segment of the brain stem, and therefore the lesion appeared milder than in the other three mice. Adrenal sections were present along with kidney sections from two of these mice. Both mice had prominent necrosis of both the adrenal cortex and medulla.

[0206] Pathology report (female): 24 hours and 14 days for both mice injected with Mut-3Δ34.5 / C8G5 and Mut-3ΔICP6 / D7-1 All samples from mice sacrificed 24 hours after HSV infection had the following lesions of moderate to marked severity: periportal to midzone hepatic necrosis including necrosis of individual hepatocytes and focally extensive hepatic cords; marked splenic red pulp necrosis and moderate white pulp necrosis with smaller than expected follicles. Several 24 hour mice had increased apoptosis / necrosis of cells in follicles and corpora lutea in ovarian tissue. This was not seen above expected amounts in control mice evaluated several months earlier and was above expected amounts in the mice noted here.

[0207] In the 14-day mice, oval cell hyperplasia is a general chronic response of the injured liver, possibly in response to infection. Some lung samples had necrotic and inflammatory lesions that were not seen in the 24-hour animals. Overall, the 14-day mice had much fewer and much milder lesions than the 24-hour animals.

[0208] The laboratory-developed mutant virus may have contributed to necrosis of various cell types, including liver cells, splenocytes in the red and white pulp, and cells in the ovaries, particularly at 24 hours.

[0209] Equivalent Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0210] The invention illustratively described herein may suitably be practiced in the absence of any element or elements, or limitation or limitations not specifically disclosed herein. Thus, for example, terms such as "comprising," "including," "containing," etc., should be read expansively and without limitation. Moreover, the terms and expressions used herein are used as terms of description, not terms of limitation, and in the use of such terms and expressions, there is no intention to exclude any equivalents of the shown and described features or portions thereof, and it is recognized that various modifications are possible within the scope of the claimed invention.

[0211] Thus, while the present invention has been specifically disclosed by preferred embodiments and optional features, it should be understood that modifications, improvements, and variations of the invention embodied therein and disclosed herein may be employed by those skilled in the art, and that such modifications, improvements, and variations are considered to be within the scope of the present disclosure. The materials, methods, and examples provided herein are representative of preferred embodiments and are illustrative and are not intended as limitations on the scope of the present disclosure.

[0212] The present invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of this disclosure. This includes the generic description of this disclosure with a provisos or negative limitation removing any subject matter from the genus, regardless of whether the deleted material is specifically recited herein.

[0213] All publications, patent applications, patents, and other references cited herein, including all formulas and figures, are expressly incorporated by reference in their entirety to the same extent as if each was individually incorporated by reference. In the case of conflict, the present specification, including definitions, will control.

[0214] Other embodiments are within the scope of the following claims. [ka] [ka] [ka] [ka] [ka] [ka]

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Claims

1. A non-native herpes simplex virus ("HSV") comprising: The non-naturally occurring HSV comprises a polynucleotide encoding an amino acid sequence set forth in SEQ ID NO:13, inhibits the growth or metastasis of cancer cells, and has improved lytic activity compared to an HSV comprising a polynucleotide encoding an amino acid sequence set forth in SEQ ID NO:

19.

2. 2. The non-naturally occurring HSV of claim 1, wherein the HSV further comprises a gene encoding a dysfunctional ICP34.5 protein and / or a gene encoding a dysfunctional ICP6 protein.

3. The gene encoding the dysfunctional ICP34.5 protein is 3. The non-naturally occurring HSV of claim 2, comprising a polynucleotide having a sequence selected from SEQ ID NO:5, SEQ ID NO:7, and a sequence at least 95% identical to SEQ ID NO:5 or SEQ ID NO:7, and maintaining a mutation in a nucleotide in any one of SEQ ID NO:5 or SEQ ID NO:

7.

4. The gene encoding the dysfunctional ICP6 protein is 3. The non-naturally occurring HSV of claim 2, comprising a polynucleotide having a sequence selected from SEQ ID NO:45, SEQ ID NO:47, and a sequence at least 95% identical to SEQ ID NO:45 or 47, and maintaining a mutation in a nucleotide in any one of SEQ ID NO:45 or 47.

5. 3. The non-naturally occurring HSV of claim 1 or 2, wherein the non-naturally occurring HSV lacks a gene encoding a functional ICP34.5 protein and / or a functional ICP6 protein.

6. A non-naturally occurring HSV according to any one of claims 1 to 5, comprising a polynucleotide having the sequence set forth in SEQ ID NO:

12.

7. and / or further comprising a polynucleotide having a sequence identical to at least a fragment of a virulence gene from 17TermA HSV; and / or and / or further comprising a polynucleotide having a sequence identical to at least a fragment of a virulence gene from rRp450 HSV; the HSV is from an HSV type 1 ("HSV-1") or HSV type 2 ("HSV-2") strain; and / or the HSV is from the HSV-1 KOS strain; and / or The non-naturally occurring HSV of any one of claims 1 to 5, further comprising a transgene.

8. A composition comprising the non-naturally occurring HSV of any one of claims 1 to 5 and a carrier.

9. A non-human mammal infected with a non-naturally occurring HSV according to any one of claims 1 to 7.

10. A cell infected with a non-naturally occurring HSV according to any one of claims 1 to 7.

11. 10. A composition comprising a non-naturally occurring HSV according to any one of claims 1 to 7 or the composition according to claim 8 for use in a method of infecting a cell or inducing cell lysis, said method comprising contacting said cell with said composition.

12. A method for preparing a non-naturally occurring HSV, comprising introducing a transgene into the non-naturally occurring HSV according to any one of claims 1 to 7.

13. A composition comprising a non-naturally occurring HSV according to any one of claims 1 to 7 or a composition according to claim 8 for use in a method for inhibiting the growth or metastasis of a cancer cell, the method comprising contacting the cell with the composition, The composition, wherein the cancer cells are of a type selected from pancreatic cancer, renal cancer, small cell lung cancer, brain cancer, neural cancer, bone cancer, lymphoma, myeloma, colon cancer, uterine cancer, breast cancer, leukemia, liver cancer, prostate cancer, skin cancer or melanoma.

14. 10. A composition comprising a non-naturally occurring HSV according to any one of claims 1 to 7 or the composition according to claim 8 for use in a method of treating cancer in a subject, the method comprising administering the composition to the subject.

15. A method for producing the HSV according to any one of claims 1 to 7, comprising the steps of: (a) introducing 17TermA HSV and rRp450 HSV into a host cell; (b) growing the host cells for at least three passages; (c) isolating the HSV particles produced by the host cell; A method comprising:

16. A method for producing the HSV according to any one of claims 1 to 7, comprising the steps of: (a) introducing into a host cell a polynucleotide encoding the viral genome of HSV; (b) growing the host cell; (c) isolating the HSV particles produced by the host cell; Includes, a method.

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