Oncolytic herpes simplex virus vectors that are dually regulated in transcription and translation

Abstract

Herpes virus vectors are provided that have both transcriptional and translational control. In various embodiments, the herpes virus vectors are based on modified herpes viruses and have both ICP27 and ICP34.5 under the control of the CEA promoter and miRNA-124 / 143, respectively, as well as deletion of at least one copy of the long terminal repeat region, which increases safety without sacrificing efficacy. The herpes virus vectors can also incorporate virally expressed cytokine cassettes encoding IL-12, IL-15 / IL-15RA under the control of the CXCR4 promoter.

Description

[Technical field]

[0001] INCORPORATION BY REFERENCE TO ALL PRIORITY APPLICATIONS Any application to which a foreign or domestic priority claim is identified in the Application Data Sheet filed with this application is hereby incorporated by reference. The present invention relates generally to oncolytic herpes simplex virus (oHSV) vectors that express molecules that stimulate the immune system. Reference to a sequence listing, table, or computer program An official copy of the Sequence Listing is being submitted contemporaneously herewith via EFS-Web as an ASCII formatted text file with the filename "VIRO413_ST25.txt", a creation date of January 24, 2022, and a size of 17.7 KB. This Sequence Listing submitted via EFS-Web is a part of the present specification and is incorporated herein by reference in its entirety. [Background technology]

[0002] Malignant tumors are a serious threat to human life and health. Although there are various standard treatment options, such as surgery, radiotherapy, chemotherapy, targeted therapy, and immunotherapy (including immune checkpoint inhibitors), most patients with advanced tumors still have poor prognosis. Currently, tumor immunotherapy has made breakthrough progress in the treatment of tumors. Immune-targeted drug therapy (e.g., immune checkpoint suppression) and immune cell therapy (e.g., chimeric antigen receptor T cells (CAR-T)) have transformed the field of antitumor therapy. However, among the currently approved indications for checkpoint inhibitors, the single-agent efficacy rate is only about 30% (Jiang et al., 2020, Progress and Challenges in Precise Treatment of Tumors With PD-1 / L1 Blockade. Frontiers in Immunology, 11(March)); on the other hand, CAR-T therapy mainly targets only cluster of differentiation 19 (CD19) and B-cell maturation antigen (BCMA), which are highly expressed by B-cell tumors. Clinical efficacy in solid tumors remains to be seen (Long et al., 2018, CAR T Cell Therapy of Non-hematopoietic Malignancies: Detours on the Road to Clinical Success. Frontiers in Immunology, 9(December), 2740). Nevertheless, there are many malignancies for which there is clear, long-term evidence of the benefit of immunotherapy. There is no clinically effective treatment for malignant tumors that recur after or are ineffective against standard treatments, and patients with this condition are more likely to die early due to widespread tumor metastasis or invasion of vital organs. Thus, these patients have a high and unmet need for effective treatments, which translates into an urgent need to develop new therapies that control disease progression and extend patient survival.

[0003] The present invention overcomes the shortcomings of current cancer therapies, such as immunotherapy, and yet provides additional, unexpected benefits. Not all of the subject matter described in the Background section is necessarily prior art, and should not be assumed to be prior art merely as a result of its description in the Background section. In line with these considerations, any awareness of a problem in the prior art described in the Background section, or a problem related to such subject matter, should not be treated as prior art unless expressly stated to be prior art. Instead, the discussion of any subject matter in the Background section should be treated as part of the inventor's approach to a particular problem, and may itself be an invention. Summary of the Invention

[0004] Briefly, the present invention relates to compositions and methods for treating cancer with recombinant herpesvirus vectors. In preferred embodiments of the invention, the recombinant vectors are both transcriptionally and post-transcriptionally (translationally) regulated to provide more precise control of the oncolytic potential of the virus. In one embodiment of the present invention, a recombinant herpesvirus is provided that comprises a modified oncolytic herpesvirus genome, the modified herpesvirus genome comprises at least one miRNA target sequence operably linked to a first copy of the ICP34.5 gene, and the second copy of the ICP34.5 gene comprises an inactivating mutation. In various embodiments, the recombinant virus may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 miRNA target sequences operably linked to the first copy of the ICP34.5 gene. In a further embodiment, the miRNA target sequence is a sequence of at least two different miRNAs (e.g., miR-124, miR-124 * , and miR-143).

[0005] In still other embodiments, the recombinant herpes virus can further comprise at least one nucleic acid encoding a non-viral protein. Examples of non-viral proteins include immune stimulators, antibodies, and checkpoint inhibitor peptides, and the at least one nucleic acid is operably linked to a tumor-specific promoter. In certain preferred embodiments, the non-viral protein is one or all of IL12, IL15, and IL15 receptor alpha subunit. In still other embodiments, the recombinant herpes simplex virus further comprises a nucleic acid sequence encoding a glycoprotein with enhanced fusogenicity (compared to the comparable wild-type virus), including various transgenes (e.g., the fusogenic glycoprotein from the Gibbon Ape Leukemia Virus, "GALV"), and / or mutations that enhance HSV fusion, such as truncations or mutations in glycoprotein B, glycoprotein K, and / or UL20. Also provided are therapeutic compositions comprising the recombinant herpesviruses described herein, as well as methods of lysing tumor cells and methods of treating cancer in a subject comprising administering to the subject one of the recombinant herpesviruses described herein.

[0006] This brief summary is provided to introduce certain concepts in a simplified form that are described in more detail below in the detailed description. Except as expressly stated, this brief summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to limit the scope of the claimed subject matter. The details of one or more embodiments are described in the following description. Features illustrated or described in connection with one exemplary embodiment may be combined with features of other embodiments. Thus, any of the various embodiments described herein can be combined to provide further embodiments. Where necessary to employ concepts of the various patents, applications, and publications identified herein, aspects of the embodiments can be modified to provide further embodiments. Other features, objects, and advantages will become apparent from the description, drawings, and claims. The present invention may be understood more readily by reference to the following detailed description of preferred embodiments of the invention and the examples included therein. [Brief description of the drawings]

[0007] [Figure 1] FIG. 1 is a schematic representation of the overall structural organization of the double-stranded deoxyribonucleic acid (DNA) elements of VG2025. [Diagram 2] FIG. 1 is a schematic representation of the transcriptional and translational dual regulated (TTDR) system. [Diagram 3] 1 is an image showing the results of an experiment in which multinucleated fused membrane plaques are observed in VG2025-infected A549 (tumor) cells but not in MRC-5 (non-tumor) cells. [Figure 4A] The graph shows CEA expression in different tumor cell lines correlating with viral copy number after infection with VG2025. [Figure 4B] The graph shows CEA expression in different tumor cell lines correlating with viral copy number after infection with VG2025. [Diagram 5] The miR124 / 143 regulation of ICP34.5 is shown in the graph. [Figure 6A] FIG. 6A shows the antitumor activity of hVG2025, measured as cell viability in 11 human tumor cell lines cultured in vitro (FIG. 6A). [Figure 6B]FIG. 6B shows the antitumor activity of hVG2025, measured as cell viability in six mouse tumor cell lines cultured in vitro. [Figure 7A] The growth curves of the two viruses in two tumor cell lines (A549 and BxPC-3, respectively) are shown in the graph. [Figure 7B] The growth curves of the two viruses in two tumor cell lines (A549 and BxPC-3, respectively) are shown in the graph. [Figure 8A] Payload expression of IL-12 is shown in the graph. [Figure 8B] Payload expression of IL-15 is shown in the graph. [Figure 9A] The graph shows payload expression from cells infected with hVG2025 or VG1905 or cells not infected with virus. [Figure 9B] The graph shows payload expression from cells infected with hVG2025 or VG1905 or cells not infected with virus. [Figure 10] The graph shows payload bioactivity on human IFN-g production from PHA-stimulated lymphoblasts. [Figure 11A] The graph shows A549 tumor size after hVG2025 treatment. [Figure 11B] The graph shows A549 tumor size after hVG2025 treatment. [Figure 11C] The graph shows A549 tumor size after hVG2025 treatment. [Figure 11D] The graph shows A549 tumor size after hVG2025 treatment. [Figure 11E] The graph shows A549 tumor size after hVG2025 treatment. [Figure 11F] The graph shows A549 tumor size after hVG2025 treatment. [Figure 11G] The graph shows A549 tumor size after hVG2025 treatment. [Figure 12] The graph shows the change in body weight after hVG2025 treatment. [Figure 13A] The graph shows the size of BxPC3 tumors after hVG2025 treatment. [Figure 13B] The graph shows the size of BxPC3 tumors after hVG2025 treatment. [Figure 13C] The graph shows the size of BxPC3 tumors after hVG2025 treatment. [Figure 13D] The graph shows the size of BxPC3 tumors after hVG2025 treatment. [Figure 13E] The graph shows the size of BxPC3 tumors after hVG2025 treatment. [Figure 13F] The graph shows the size of BxPC3 tumors after hVG2025 treatment. [Figure 13G] The graph shows the size of BxPC3 tumors after hVG2025 treatment. [Figure 14] The graph shows the body weight of the BxPC3 tumor model after hVG2025 treatment. [Figure 15A] A comparison of tumor size between hVG2025 and 34.5(-)HSV-1 is shown in the graph. [Figure 15B] A comparison of tumor size between hVG2025 and 34.5(-)HSV-1 is shown in the graph. [Figure 15C] A comparison of tumor size between hVG2025 and 34.5(-)HSV-1 is shown in the graph. [Figure 15D] A comparison of tumor size between hVG2025 and 34.5(-)HSV-1 is shown in the graph. [Figure 15E] A comparison of tumor size between hVG2025 and 34.5(-)HSV-1 is shown in the graph. [Figure 15F] A comparison of tumor size between hVG2025 and 34.5(-)HSV-1 is shown in the graph. [Figure 16] The graph shows the body weight of DBA / 2 mice subcutaneously injected with hVG2025. [Figure 17] The graph shows the percent survival of DBA / 2 mice injected subcutaneously with hVG2025. [Figure 18]The graph shows the body weight of DBA / 2 mice intranasally inoculated with hVG2025. [Figure 19] The graph shows the percent survival of DBA / 2 mice inoculated intranasally with hVG2025. [Figure 20A] Photographs provide clinical insight into virus-induced symptoms. [Figure 20B] Survival curves are plotted. [Figure 21] The graph shows RNA-seq analysis of the olfactory bulb and trigeminal ganglion after corneal scarification. [Figure 22] The sensitivity of hVG2025 to ganciclovir is shown in the graph. [Figure 23] The graph shows the stability of hVG2025 at 4° C. and −80° C. for up to one month. [Figure 24A] FIG. 24A graphically depicts the changes in body weight in Hep 3B-luc-burdened BALB / c nude mice treated with VG2025. [Figure 24B] FIG. 24B shows the tumor bioluminescence traces after administration of VG2025. [Figure 24C] FIG. 24C is a graph of the survival curves of treated mice. [Figure 24D] FIG. 24D is a graph showing the metastasis rate. [Figure 25A] FIG. 25A graphically depicts the change in body weight in A20-luc B cell lymphoma-bearing BALB / c nude mice treated with VG2025. [Figure 25B] FIG. 25B is a graph showing tumor bioluminescence tracings after administration of mVG2025. [Figure 25C] FIG. 25C is a graph showing the metastasis rate. [Figure 26A] FIG. 26A graphically depicts weight changes in A20-luc B cell lymphoma-burdened BALB / c nude mice treated with mVG2025. [Figure 26B] FIG. 26B is a graph showing tumor bioluminescence tracings after administration of mVG2025. [Figure 26C] FIG. 26C is a graph showing the metastasis rate. [Figure 27A] The graph depicts the mean tumor volumes of treated and contralateral untreated tumors after treatment up to 9 days after initiation of treatment. [Figure 27B] The graph depicts the mean tumor volumes of treated and contralateral untreated tumors after treatment up to 9 days after initiation of treatment. [Figure 28A] Individual tumor sizes of treated and contralateral tumors are graphed. [Figure 28B] Individual tumor sizes of treated and contralateral tumors are graphed. [Figure 28C] Individual tumor sizes of treated and contralateral tumors are graphed. [Figure 28D] Individual tumor sizes of treated and contralateral tumors are graphed. [Figure 29] Survival curves are plotted. [Figure 30A] The graph depicts the mean tumor volumes of treated and contralateral untreated tumors after treatment up to 17 days after initiation of treatment. [Figure 30B] The graph depicts the mean tumor volumes of treated and contralateral untreated tumors after treatment up to 17 days after initiation of treatment. [Figure 31A] Individual tumor sizes before and after subcutaneous tumor reimplantation are graphed. [Figure 31B] Individual tumor sizes before and after subcutaneous tumor reimplantation are graphed. [Figure 31C] Individual tumor sizes before and after subcutaneous tumor reimplantation are graphed. [Figure 31D] Individual tumor sizes before and after subcutaneous tumor reimplantation are graphed. [Diagram 32] Survival curves are plotted. [Figure 33A] Graph showing plots of human IL-12p70 and IL-15 / IL15Ralpha complex in tumors. [Figure 33B] Graph showing plots of human IL-12p70 and IL-15 / IL15Ralpha complex in tumors. [Figure 34A]Graph showing plots of human IL-12p70 and IL-15 / IL15Ralpha complex in serum. [Figure 34B] Graph showing plots of human IL-12p70 and IL-15 / IL15Ralpha complex in serum. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] As discussed above, the present invention provides recombinant herpesvirus vectors that are both transcriptionally and post-transcriptionally (translationally) regulated to provide more precise control of the oncolytic potential of the virus. For a further understanding of the various embodiments herein, the following sections are provided which describe the various embodiments: A. Oncolytic Herpesvirus; B. Specific Herpesvirus Construct - VG2025; C. Therapeutic Compositions, and D. Administration.

[0009] A. Oncolytic herpesviruses Briefly, herpes simplex viruses (HSV) 1 and 2 are members of the Herpesviridae family that infect humans. The HSV genome contains a long unique L ) region and short unique (U S ) region. Each of these regions is flanked by a pair of inverted repeat sequences. There are about 75 known open reading frames. The viral genome has been engineered to generate oncolytic viruses for use in, for example, cancer therapy. Tumor-selective replication of HSV can be conferred by mutations in the HSV ICP34.5 (also called γ34.5) gene. HSV contains two copies of ICP34.5. Mutants that inactivate one or both copies of the ICP34.5 gene are known to lack neurovirulence, i.e., are non-toxic / non-neurotoxic, as well as oncolytic. Tumor-selective replication of HSV can also be conferred by controlling the expression of key viral genes, such as ICP27 and / or ICP4.

[0010] The term "oncolytic herpes virus" or "oHV" generally refers to a herpes virus that is capable of replicating in and killing tumor cells. The term "oncolytic herpes simplex virus" or "oHSV" refers to a herpes simplex virus that is capable of replicating in and killing tumor cells. Suitable oncolytic HSV may be derived from either HSV-1 or HSV-2, including any laboratory strain or clinical isolate. In some embodiments, the oHSV may be derived from one of the laboratory strains HSV-1 strain 17, HSV-1 strain F, or HSV-2 strain HG52. In other embodiments, the oHSV may be derived from the non-laboratory strain JS-1. Other suitable HSV-1 viruses include HrrR3 (Goldstein and Weller, J. Virol. 62, 196-205, 1988), G2O7Mineta et al. Nature Medicine. 1(9):938-943, 1995; Kooby et al. The FASEB Journal, 13(11):1325-1334, 1999); G47Delta (Todo et al. Proceedings of the National Academy of Sciences. 2001; 98(11):6396-6401); HSV 1716 (Mace et al. Head & Neck, 2008; 30(8):1045-1051; Harrow et al. Gene Therapy. 2004; 11(22):1648-1658); HF10 (Nakao et al. Cancer Genetics. 2006; 11(22):1648-1658); Therapy. 2011; 18(3):167-175);NV1020(Fong et al. Molecular Therapy, 2009; 17(2):389-394);T-VEC(Andtbacka et al. Journal of Clinical Oncology, 2015: 33(25):2780-8);J100(Gaston et al. PloS one, 2013; 8(11):e81768);M002(Parker et al. Proceedings of the National Academy of Sciences, 2000; 97(5):2208-2213);NV1042(Passer et al. Cancer Gene Therapy. 2013; 20(1):17-24);G2O7-IL2(Carew et al.Molecular Therapy, 2001; 4(3):250-256); rQNestin34.5 (Kambara et al. Cancer Research, 2005; 65(7):2832-2839); G47Δ-mIL-18 (Fukuhara et al. Cancer Research, 2005; 65(23):10663-10668); and the vectors disclosed in PCT applications PCT / US2017 / 030308 entitled "HSV Vectors with Enhanced Replication in Cancer Cells" and PCT / US2017 / 018539 entitled "Compositions and Methods of Using Stat1 / 3 Inhibitors with Oncolytic Herpes Virus," all of which are incorporated herein by reference in their entireties.

[0011] Other representative examples of oncolytic herpesviruses are described in U.S. Pat. Nos. 7,223,593, 7,537,924, 7,063,835, 7,063,851, 7,118,755, 8,216,564, 8,277,818, and 8,680,068, all of which are incorporated by reference in their entireties. The oHSV vector has at least one γ34.5 gene modified by a miRNA target sequence in its 3'UTR as disclosed herein; there is no unmodified γ34.5 gene in the vector. In some embodiments, the oHSV has two modified γ34.5 genes; in other embodiments, the oHSV has only one γ34.5 gene, which is modified. In some embodiments, the modified γ34.5 gene is constructed in vitro and inserted into the oHSV vector as a replacement for the viral gene. When the modified γ34.5 gene is a replacement for only one γ34.5 gene, the other γ34.5 is deleted. Any native γ34.5 gene can be deleted. In one embodiment, the terminal repeat region including the γ34.5 gene and the ICP4 gene is deleted. As described herein, the modified γ34.5 gene may include additional modifications, such as having an exogenous promoter.

[0012] The oHSV may have additional mutations, including nullifying mutations (e.g., deletions, substitutions, insertions), which may affect the virulence of the virus or its ability to replicate. For example, mutations may be made in any one or more of ICP6, ICPO, ICP4, ICP27, ICP47, ICP24, ICP56. Preferably, a mutation in one of these genes (or in both copies of the gene, if appropriate) renders the HSV unable (or less able) to express the corresponding functional polypeptide. In some embodiments, the promoter of the viral gene may be replaced with a promoter that is selectively active in the target cell or inducible for delivery of an inducer or inducible for a cellular event or a specific environment. In certain embodiments, the expression of ICP4 or ICP27 is controlled by an exogenous promoter, e.g., a tumor-specific promoter. Exemplary tumor-specific promoters include survivin, CEA, CXCR4, PSA, ARR2PB, or telomerase; other suitable tumor-specific promoters may be specific to a single tumor type and are known in the art. Other elements may be present. In some cases, enhancers are present, such as the NFkB / oct4 / sox2 enhancer. Additionally, the 5'UTR may be exogenous, e.g., a 5'UTR from a growth factor gene, such as FGF. See FIG. 2 for an exemplary construct.

[0013] oHSV may also have genes and nucleotide sequences of non-HSV origin. For example, sequences encoding prodrugs, sequences encoding cytokines or other immune stimulants, tumor-specific promoters, inducible promoters, enhancers, sequences homologous to the host cell, among others, may be present in the oHSV genome. Exemplary sequences encode IL12, IL15, IL15 receptor alpha subunit, OX40L, PD-L1 blockers, or PD-1 blockers. In the case of product-encoding sequences, they are operably linked to promoter sequences and other regulatory sequences necessary or desirable for expression (e.g., enhancers, polyadenylation signal sequences). The regulatory region of the viral gene may be modified to include a response element that affects expression. Exemplary response elements include response elements for NFκB, Oct-3 / 4-SOX2, enhancers, silencers, cAMP response elements, CAAT enhancer binding sequences, and insulators. Other response elements may also be included. The viral promoter may be replaced with a different promoter. The choice of promoter will depend on several factors, such as the proposed use of the HSV vector, the patient's treatment, disease state or condition, and the ease of application of the inducer (in the case of an inducible promoter). In the case of cancer treatment, generally, if the promoter is replaced, it will be by a cell-specific or tissue-specific or tumor-specific promoter. Tumor-specific, cell-specific, and tissue-specific promoters are known in the art. Other genetic elements may also be modified. For example, the 5'UTR of the viral genome may be replaced by an exogenous UTR.

[0014] B. Specific herpesvirus construct - VG2025 One preferred construct of the present invention is provided in Figure 1. Briefly, Figure 1 diagrammatically depicts the overall structural organization of the double-stranded deoxyribonucleic acid (DNA) elements of VG2025. "CEA" means carcinoembryonic antigen; "CXCR4" means CXC Motif Chemokine Receptor 4; "gB" means glycoprotein B; "ICP" means infected cell polypeptide; "IL" means interleukin; "R L " means long repeat region; "RNA" means ribonucleic acid; "miR" means microRNA; "R S " means short repeat region; "UL" means long unique region; "US" means short unique region.

[0015] VG2025 is a recombinant HSV-1 platform that utilizes dual regulation of both transcription and translation of key viral genes ("TTDR" - see Figure 2) to restrict viral replication to tumor cells and enhance tumor-specific virulence without compromising safety. In addition, VG2025 expresses a payload cassette composed of IL12, IL15, and IL15 receptor alpha subunit. This payload cassette is controlled by the CXCR4 promoter for tumor-specific immune stimulation. Finally, the viral glycoprotein B (gB) in VG2025 was truncated to facilitate viral dissemination in the tumor microenvironment through enhanced membrane fusogenicity.

[0016] 1. Post-transcriptional (translational) regulation In VG2025, ICP34.5 expression is post-transcriptionally (translationally) regulated. Briefly, in wild-type HSV-1, there are two copies of ICP34.5 gene. In VG2025, one copy of ICP34.5 is deleted. For the remaining ICP34.5 gene, VG2025 inserts multiple copies of binding domains for miR124 and miR143 in 3'UTR region to regulate its expression post-transcriptionally.

[0017] ICP34.5 is encoded by HSV late gene g-34.5. Its function of suppressing antiviral immunity of host cells, especially neuronal cells, is well known to cause neurotoxic effects. Instead of deleting the gene or using a specific promoter to control the expression of ICP34.5 to target gliomas, VG2025 uses microRNA as post-transcriptional regulation to achieve differential expression of ICP34.5 in tumor cells, while maintaining its activity in tumor cells for robust replication, and thus abolishing the function of ICP34.5 in neurons and other normal cells. Briefly, microRNA (also called "miRNA" or "miR") is a non-coding small RNA of about 22 nucleotides that is encoded by miRNA genes and is transcribed by RNA polymerase II to generate primary miRNA (pri-miRNA). Mature single-stranded (ss) miRNA forms miRNA-associated RNA-induced silencing complex (miRISC). miRNAs in miRISC can affect gene expression by binding to the 3' untranslated region (3'-UTR) in target mRNAs. This region consists of the sequence recognized by the miRNA. If the complementarity of the miRNA:mRNA complex is perfect, the mRNA is degraded by Ago2, a protein belonging to the Argonaute family. However, if the complementarity is not perfect, the translation of the target mRNA is suppressed, but not completely destroyed.

[0018] MiRNAs are differentially expressed in a tissue-specific manner. One example is miR124. Although the precursors of miR-124 from different species are different, the sequences of mature miR-124 in humans, mice, and rats are completely identical. MiR-124 is the most abundantly expressed miRNA in neural cells and is highly expressed in immune cells and organs (Qin et al., 2016, miRNA-124 in immune system and immune disorders. Frontiers in Immunology, 7(OCT), 1-8). Another example of differential expression of miRNA is miR143 (Lagos-Quintana et al., 2002, Identification of tissue-specific MicroRNAs from mouse. Current Biology, 12(9), 735-739). MiR-143 is constitutively expressed in normal tissues, but is significantly downregulated in cancer cells (Michael et al., 2003, Reduced Accumulation of Specific MicroRNAs in Colorectal Neoplasia. Molecular Cancer Research, 1(12), 882-892). A representative example of the nucleic acid sequence of miR-124 is as set forth in SEQ ID NO:8, and an example of the nucleic acid sequence of miR-143 is as set forth in SEQ ID NO:9. The 3'UTR region of the ICP34.5 gene in VG2025 contains multiple copies of binding domains (also called "miRNA target sequence", "miRNA binding sequence" or "miRNA binding site") that are perfectly complementary to miR124 and miR143. Binding of miR124 and miR143 to the 3'UTR of ICP34.5 mRNA causes degradation of the mRNA, resulting in the gene being post-transcriptionally downregulated in normal cells but not in tumor cells. This design allows for differential expression of ICP34.5 in tumor cells.

[0019] 2. Expression of ICP27 in VG2025 is transcriptionally regulated HSV-1 viral replication depends on the sequential occurrence of viral gene expression, with immediate early gene products (especially ICP4 and ICP27) controlling the subsequent expression of viral early and late genes that govern the viral lytic replication cycle. Deletion of ICP4 or ICP27 results in a complete abrogation of viral replication and a significant reduction in viral gene expression, making ICP4 and ICP27 excellent targets for tumor-specific modulation in oncolytic HSV. ICP4 is the master transcription factor regulating viral gene expression, whereas ICP27 is a multifunctional protein that regulates the transcription of many viral genes. ICP27 functions in all stages of mRNA biogenesis, from transcription, RNA processing, and export to translation. ICP27 is also involved in nuclear protein quality control, cell cycle regulation, activation of stress signaling pathways, and prevention of apoptosis.

[0020] In VG2025, the native promoter of ICP27 is replaced with the 432 bp promoter of human carcinoembryonic antigen (CEA) (Beauchemin and Arabzadeh, 2013, Carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) in cancer progression and metastasis. Cancer and Metastasis Reviews, 32(3-4), 643-671; Hammarstrom 1999, The carcinoembryonic antigent (CEA) family. Structures, suggested functions and expression in normal and malignant tissues. In Seminars in cancer biology 9 (2), pp. 67-81; Kodera et al. 1993, Expression of carcinoembryonic antigen (CEA) and nonspecific crossreacting antigen (NCA) in gastrointestinal cancer; the correction with degress of differentiation. In Br. J Cancer 68 (1), pp 130-136). CEA belongs to a subgroup of 12 genes called carcinoembryonic antigen cell adhesion molecule (CEACAM) as part of a family of 22 genes (Beauchemin & Arabzadeh, 2013). CEA plays an important role in cellular processes such as inhibition of differentiation programs, inhibition of anoikis and apoptosis, and disruption of cell polarization and tissue architecture (Beauchemin & Arabzadeh, 2013). For example, the nucleic acid sequence of the CEA promoter is as set forth in SEQ ID NO:1.

[0021] 3. VG2025 payload expression is enhanced in tumors VG2025 further stimulates immune-modulatory responses by co-expressing IL12, IL15, and the IL15 receptor alpha subunit. Expression of IL12 promotes pro-inflammatory and anti-tumor T H IL-15 promotes the polarization of antigen-exposed T cells toward the IL-1 phenotype, while IL-15 further increases tumor killing and activation of antigen-presenting cells by activating NK cells. In addition to IL15 expression, VG2025 further enhances immune stimulation by encoding IL15Rα. For example, human IL12 can include an amino acid sequence as set forth in SEQ ID NO:4; human IL15 can include an amino acid sequence as set forth in SEQ ID NO:5, and human IL15 receptor alpha subunit can include an amino acid sequence as set forth in SEQ ID NO:7.

[0022] Transcription of IL-12, IL-15, and IL-15Rα is driven by a single promoter (CXCR4), and the polypeptides are linked by a 2A self-cleaving peptide that upon translation generates three separate proteins by a ribosomal skipping mechanism (Z. Liu et al., 2017, Systematic comparison of 2A peptides for cloning multi-genes in polycistronic vector. Scientific Reports, 7(2), 1-9). For example, the 2A peptide may comprise the amino acid sequence as set forth in SEQ ID NO:6. The payload is intended to be expressed in the tumor, but unwanted expression may occur in normal tissues if the virus "leaks" into extratumoral areas or if the virus is delivered systemically. To mitigate the potential risk of IL12 / 15 expression outside the tumor bed, the expression cassette of the payload in VG2025 is driven by a single promoter of CXC chemokine receptor 4 (CXCR4) (Moriuchi et al., 1997, Cloning and analysis of the promoter region of CXCR4, a coreceptor for HIV-1 entry. Journal of Immunology (Baltimore, Md. : 1950), 159(9), 4322-429; Caruz et al., 1998, Genomic organization and promoter characterization of human CXCR4 gene. FEBS Letters, 426(2), 271-278). CXCR4 is a seven-transmembrane G protein-coupled receptor originally isolated from human blood monocytes as a cofactor for HIV viral fusion and entry in T cells (Moriuchi et al. 1997). For example, the nucleic acid sequence of the CXCR4 promoter is as set forth in SEQ ID NO:3. Representative examples of selected expression cassettes / vectors are also described in PCT application WO2018 / 026872, which is incorporated by reference in its entirety.

[0023] 4. Truncated glycoprotein B (GB) HSV-1 membrane fusion is a key step in infection. It depends on four essential viral glycoproteins (gB, gD, gH, and gL) and mediates entry into the host cell by fusing the viral envelope with the host cell membrane. The core fusion protein is glycoprotein B (gB), a glycosylated transmembrane protein of 904 residues encoded by the UL27 gene of HSV-1. Multiple types of mutations in the cytoplasmic domain of gB result in a fusogenic phenotype, increasing cell-cell fusion (Chowdary & Heldwein, 2010, Synctial Phenotype of C-Terminally Truncated Herpes Simplex Virus Type 1 gB is Associated with Diminished Membrane Interactions. Journal of Virology, 84(10), 4923-4935). In one embodiment, gB can be modified by truncating the C-terminal amino acids 887-904 from the full-length protein. For example, the amino acid sequence of truncated glycoprotein B is as set forth in SEQ ID NO:2.

[0024] 5. Summary VG2025 is an oncolytic viral product with ICP27 and ICP34.5 under the control of the CEA promoter and miRNA-124 / 143, respectively. hVG2025 also incorporates a virally expressed cytokine cassette encoding IL-12, IL-15 / IL-15RA under the control of the CXCR4 promoter. The expression control mechanism in VG2025 is designed to increase safety without compromising efficacy. Specific modifications of wild-type HSV-1 strain 17+ are listed in Table 1 below.

[0025] TIFF2024512053000002.tif156163

[0026] VG2025 is a conditionally replicating oncolytic HSV-1 virus. The genome of VG2025 contains the HSV-1 long terminal repeat (TR) domain, which contains ICP34.5, ICP0, and one copy of LAT. L ) sequence. The remaining copy of ICP34.5 has an insertion in its 3'UTR region that contains multiple copies of the binding domain for the miRNAs miR-124 and miR-143. miR-124 and miR-143 are highly expressed in neural and normal tissues but not in tumor tissues. This product is further modified by replacing the native viral promoter of the essential viral gene UL54, which encodes ICP27 (infected cell polypeptide 27), with a tumor-specific promoter derived from the human carcinoembryonic antigen (CEA) gene. VG2025 also expresses a potent immunomodulatory payload consisting of IL-12, IL-15, and IL-15Rα, which are controlled by the tumor-selective CXC motif chemokine receptor 4 (CXCR4) promoter. Finally, VG2025 has a glycoprotein B (gB) terminal truncation that enhances fusion activity, thereby facilitating viral spread within the tumor microenvironment. mVG2025 and hVG2025 are very similar, except that mVG2025 contains the murine form of IL-12, whereas hVG2025 contains the human form of IL-12. As described in more detail below, preclinical pharmacology studies of VG2025 demonstrated significant anti-cancer activity in BxPC3 pancreatic cancer, Hep 3B liver cancer, A20 B-cell lymphoma, CT26 colon cancer, and A549 NSCLC tumor-bearing mouse models.

[0027] C. Therapeutic composition Therapeutic compositions are provided that may be used to prevent, treat, or ameliorate the effects of diseases, such as cancer, etc. More particularly, therapeutic compositions are provided that include at least one oncolytic virus as described herein. In certain embodiments, the composition will further comprise a pharma- ceutically acceptable carrier. The phrase "pharmaceutically acceptable carrier" is intended to encompass any carrier, diluent, or excipient that does not interfere with the effectiveness of the biological activity of the oncolytic virus and that is not toxic to a subject to which it is administered (see generally, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21st ed. (May 1, 2005 and in The United States Pharmacopeia: The National Formulary (USP 40 - NF 35 and Supplements).

[0028] For the oncolytic viruses described herein, non-limiting examples of suitable pharmaceutical carriers include phosphate buffered saline, water, emulsions (e.g., oil / water emulsions, etc.), various types of wetting agents, sterile solutions, and others. Additional pharmaceutically acceptable carriers include gels, bioabsorbable matrix materials, implantable elements containing the oncolytic viruses, or any other suitable vehicle, delivery or distribution means or materials. Such carriers can be formulated by conventional methods and administered to a subject in an effective dose. Additional pharmaceutically acceptable excipients include, but are not limited to, water, saline, polyethylene glycol, hyaluronic acid, and ethanol. Pharmaceutically acceptable salts, such as mineral acid salts (e.g., hydrochloride, hydrobromide, phosphate, sulfate, and the like) and salts of organic acids (e.g., acetate, propionate, malonate, benzoate, and the like) may also be included. Pharmaceutically acceptable (pharmaceutical grade) carriers, diluents, and excipients that can be used to deliver oHSV to cancer cells will preferably not elicit an immune response in the individual (subject) receiving the composition (and will preferably be administered without undue toxicity).

[0029] The compositions provided herein can be provided in a variety of concentrations. For example, about 10 4 pfu~about 10 10 A dosage of oncolytic virus in the range of pfu can be provided. In a further embodiment, the dosage is about 10 6 pfu~about 10 7 pfu, or about 10 7 pfu~about 10 8 pfu, or about 10 8 pfu~10 9 pfu range and can be administered as a single dose or as multiple doses over a period of time. Doses can be administered daily, weekly, biweekly, monthly, or bimonthly, and the dosing frequency can be cyclic, with each cycle comprising a repeat dosing pattern (e.g., a dose administered once a week or once every two weeks for about 4 weeks, including one cycle, repeated for up to about 24 cycles). In another embodiment of the invention, the virus is administered in a dose range of about 5x10 for intravenous delivery in humans. 4 pfu / kg ~ approx. 2x10 9 For intratumoral injection, the preferred dosage is about 10 pfu / kg per dose. 6 pfu~about 10 9 pfu (with injectable volumes ranging from about 0.1 mL to about 5 mL).

[0030] In certain embodiments of the invention, lower or higher dosages than normal may be utilized. Thus, in certain embodiments, about 10 6 Less than or about 10 pfu 9 More than pfu can be administered to a patient. The compositions can be stored at temperatures that provide a stable shelf life, including room temperature (about 20° C.), 4° C., −20° C., −80° C., and in liquid N2. Compositions intended for in vivo use generally do not contain preservatives, so storage will generally be at lower temperatures. The compositions can be stored in dry form (e.g., lyophilized) or in liquid form.

[0031] D. Administration In addition to the compositions described herein, various methods of using such compositions to treat or ameliorate cancer are provided, the methods comprising administering to a subject an effective dose or amount of an oHSV described herein. The terms "effective dose" and "effective amount" refer to an amount of oncolytic virus sufficient to achieve treatment of a target cancer, e.g., an amount effective to reduce the tumor size or tumor burden of the target or otherwise impede the growth rate of tumor cells of the target. More specifically, such terms refer to an amount of oncolytic virus effective to achieve the desired result, at the required dosage and treatment period. For example, in the context of treating cancer, an effective amount of the compositions described herein is an amount that produces remission, reduces tumor burden, and / or prevents tumor expansion or cancer growth. An effective amount may vary according to factors such as, for example, the condition, age, sex, and weight of the subject, as well as the pharmaceutical formulation, route of administration, etc., but can nevertheless be routinely determined by one of skill in the art. The therapeutic composition is administered to a subject diagnosed with or suspected of having cancer. The subject may be a human or a non-human animal.

[0032] The composition is used to treat cancer. The term "treat" or "treating" or "treatment" as used herein means an approach to obtain beneficial or desired results, including clinical results. Beneficial or desired clinical results may include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, whether detectable or undetectable, reduction in the extent of disease, stabilized (i.e., non-worsening) state of disease, prevention of disease spread, delay or slowing of disease progression, improvement or relief of disease state, reduction in recurrence of disease, and remission (whether partial or complete). The terms "treating" and "treatment" may also mean prolonging survival compared to expected survival in the absence of treatment. Representative forms of cancer include carcinomas, leukemias, lymphomas, myelomas, and sarcomas. Representative forms of leukemias include acute myeloid leukemia (AML), and representative forms of lymphomas include B-cell lymphomas. Further examples include, but are not limited to, cancers of the biliary tract, brain (e.g., glioblastoma), breast, cervical, colorectal, CNS (e.g., acoustic neuroma, astrocytoma, cephalopharyngioma, ependymoma, glioblastoma, hemangioblastoma, medulloblastoma, meningioma, neuroblastoma, oligodendroglioma, pinealoma, and retinal germinomas), endometrium, hematopoietic cells (e.g., leukemia and lymphoma), kidney, larynx, lung, liver, oral cavity, ovary, pancreas, prostate, skin (e.g., melanoma and squamous cell carcinoma), GI (e.g., esophagus, stomach, and colon), and thyroid. The cancer may include solid tumors (e.g., sarcomas, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma; and osteogenic sarcoma), diffuse (e.g., leukemia-based), or any combination of these (e.g., metastatic cancers having both solid tumors and scattered or diffuse cancer cells).

[0033] In certain embodiments of the invention, the cancer may be resistant or refractory to conventional treatments (e.g., conventional chemotherapy and / or radiation therapy). Benign tumors and other conditions of unwanted cell proliferation may also be treated. Particularly preferred cancers to be treated include those with high levels of CEA expression.Representative examples include lung tumors, breast and prostate tumors, hematopoietic cell tumors (e.g., leukemia and lymphoma), glioblastoma, tumors of the gastrointestinal tract (and associated organs), such as esophageal, bile duct cancer, anal, stomach, intestinal tract, pancreatic, colon, and liver, and all superficially injectable tumors (e.g., melanoma). The recombinant herpes simplex viruses described herein can be given by routes such as, for example, oral, topical, parenteral, systemic, intravenous, intramuscular, intraocular, intrathecal, intratumoral, subcutaneous, or transdermal. In certain embodiments, the oncolytic virus can be delivered by cannula, by catheter, or by direct injection. The site of administration can be directly at the tumor, adjacent to the tumor, or at a site distant from the tumor. The route of administration will often depend on the type of cancer being targeted.

[0034] Optimal or appropriate dosing regimens of oncolytic viruses can be readily determined within the skill of the art by the attending physician based on patient data, patient observations, and a variety of clinical factors, such as the subject's size, body surface area, age, sex, and the particular oncolytic virus administered, the time and route of administration, the type of cancer being treated, the patient's overall health, and other medications the patient is receiving, etc. According to certain embodiments, treatment of a subject using an oncolytic virus described herein can be combined with additional types of therapy, such as administration of a different oncolytic virus, radiation therapy, administration of a checkpoint inhibitor, chemotherapy using, for example, chemotherapeutic agents such as etoposide, ifosfamide, adriamycin, vincristine, doxycycline, etc.

[0035] The recombinant herpes simplex virus described herein can be formulated as a medicament and pharmaceutical composition for clinical use and can be combined with a pharma- ceutically acceptable carrier, diluent, excipient, or adjuvant. The formulation will depend, at least in part, on the route of administration. A suitable formulation can include the virus and an inhibitor in a sterile medium. The formulation can be in liquid, gel, paste, or solid form. The formulation can be provided to a subject or a medical professional. Preferably, a therapeutically effective amount is administered, that is, an amount sufficient to show benefit to the subject. The actual amount administered, and the time-course of administration, will depend, at least in part, on the nature of the cancer, the condition of the subject, the site of delivery, and other factors. In yet other embodiments of the invention, the oncolytic virus can be administered by various methods, for example, intratumorally or intravenously, or following surgical resection of the tumor. EXAMPLES

[0036] Overview: All viral mutagenesis can be performed in Escherichia coli using standard lambda Red-mediated recombination techniques performed on viral genomes cloned into bacterial artificial chromosomes (BACs) (see generally: Tischer BK, Smith GA, Osterrieder N. Methods Mol Biol. 2010;634:421-30. doi: 10.1007 / 978-1-60761-652-8_30. PMID: 20677001; Tischer BK, von Einem J, Kaufer B, and Osterrieder N., BioTechniques 40:191-197, Feb. 2006 (including the Supplementary Material, doi: 10.2144 / 000112096; and Tischer BK, Smith, GA and Osterrieder N. Chapter 30, Jeff Braman (ed.), In Vitro Mutagenesis Protocols: Third Edition, Methods in Molecular Biology, vol. 634, doi: 10.1007 / 978-1-60761-652-8_30, Springer Science+Business Media, LLC 2010). The BAC recombination method requires the presence of exogenous BAC DNA in the viral genome to facilitate mutagenesis in E. coli. BAC sequences are most commonly inserted either between viral genes, such as HSV genes US1 / US2, UL3 / UL4, and / or UL50 / UL51, or in the thymidine kinase (TK) gene, which can disrupt the expression of native TK. TK-deficient viral vectors can contain an expression cassette for a copy of the native viral thymidine kinase (TK) gene under the control of a constitutive promoter inserted into a non-coding region of the viral genome. Alternatively, TK function can be restored by removing the exogenous BAC sequence and reconstituting the native TK gene sequence via homologous recombination. The presence of a functional TK gene enhances the safety of the virus by making it sensitive to common treatments with guanosine analogs, such as ganciclovir and acyclovir.

[0037] TIFF2024512053000003.tif225153

[0038] Example 1 Membrane fusion test of VG2025 Objective: VG2025 contains a deletion of all amino acids between aa876 and the stop codon in gB and incorporates fusion-enhancing mutations. This study was conducted to demonstrate the fusion-enhancing effect of the mutations. Procedure: VG2025 was used to infect A549 tumor cells and MRC-5 non-tumor cells at MOfI=0.1 and incubated for 48 hours post-infection prior to imaging. Results: As shown in FIG. 3, multinucleated membrane-fused plaques were observed in VG2025-infected A549 cells (tumor cells) but not in MRC-5 cells (non-tumor cells). CONCLUSIONS: VG2025 is tumor-selective and highly fusogenic.

[0039] Example 2 Correlation of CEA expression levels with ICP27 expression and viral replication efficiency Objective: This study was to evaluate the correlation between CEA expression levels in different tumor cell lines and ICP27 expression levels and viral replication efficiency of hVG2025. Procedure: The cell lines listed below in Table 2 were seeded into 12-well plates and incubated overnight in the appropriate cell culture medium suggested by the vendor.

[0040] TIFF2024512053000004.tif73141

[0041] VG2025 was diluted and added to cell cultures at MOI 0.1 or mock infected. After 2 h incubation, the medium was changed. Cells were harvested 6 and 18 h after infection for RNA and DNA extraction, followed by RT-qPCR to detect CEA and ICP27 mRNA expression. Some samples were harvested 48 h after infection and processed for PCR to measure the DNA copy number of ICP27. GAPDH was used to normalize each sample. Other samples of uninfected cells were used to measure CEA protein shedding in cell culture supernatants using an ELISA kit (Abcam, AB99992). Normalized Ct (ΔCt) values ​​of CEA and ICP27 mRNA were plotted. R and P values ​​were calculated by regression analysis using EXCEL.

[0042] Results: Different human tumor cells express different levels of CEA. The results of this experiment are shown in Figures 4A and 4B. Briefly, CEA and ICP27 mRNA levels are expressed and plotted as ΔCt values ​​by RT-qPCR. Regression analysis showed that the ΔCt values ​​of ICP27 mRNA were positively correlated to the ΔCt values ​​of CEA mRNA, R=0.747 at p=0.0082. Viral copy numbers in CEA-positive hVG2025-infected cells were measured as ΔCt values ​​by ELISA. A correlation between CEA protein shedding and viral copy numbers was also shown. Regression analysis showed R=0.820 at p=0.0126. Conclusion: Certain types of tumors, including pancreatic, lung, and gastrointestinal cancers, have high levels of CEA expression. Transcription levels of ICP27 from hVG2025 showed a moderate but significant positive correlation with the transcriptional activity of CEA in some tumor cells. Tumor cell CEA protein expression, measured by shedding in cell culture, correlated highly with viral copy number following infection with hVG2025.

[0043] Example 3 miR124 / 143 transcriptional regulation of ICP34.5 expression demonstrated by evaluation of ICP34.5 expression in miR124 / 143-transduced HEK-293 cells Objective: The aim of this study was to investigate the function of miRNA-binding elements present in hVG2025 in regulating the expression of ICP34.5. The virus of the present disclosure is designed to have miR binding elements / sequences present in the 3'UTR region of the ICP34.5 gene. In hVG2025, the targeting domains for miR regulation are miR124 and miR143. The former is highly expressed in all neural cells, and the latter is underexpressed in most tumor cells. This will allow the use of viruses with the complete ICP34.5 gene, but the expression of ICP34.5 is differentially controlled by post-transcriptional regulation. The expression of ICP34.5 will not be affected in cells and tissues that do not express miR (tumor cells), but the expression of ICP34.5 will be hindered in normal tissues that express high levels of miR (e.g., neural cells).

[0044] In this study, we used 293FT cells or the same cells transfected with miR124 and miR143 mimics or miR precursors with scrambled sequences. The cells were then superinfected with hVG2025. This would allow direct testing and comparison of target gene expression in the presence or absence of miR precursors. Furthermore, the sequences of the binding domains downstream of the ICP34.5 coding region in hVG2025 match perfectly with miR, so the binding of these domains by miR would result in the degradation of mRNA (ICP34.5 in this case). Therefore, we can use RT-qPCR to measure the levels of ICP34.5 mRNA to test miR-regulated ICP34.5 expression in hVG2025. Procedure: 293FT cell cultures were transfected with miR124 and miR143 using Lipofectamine™ RNAiMAX Transfection Reagent and then multiply infected with hVG2025. RT-qPCR was performed 24 hours after viral infection to quantify ICP27 and ICP34.5 expression and copy number.

[0045] Experimental design: 293FT cells were transfected in triplicate with either miR124 / miR143 precursors. Control cells were transfected with scrambled miR precursors (Thermo Fisher AM17110) or mock transfected. 24 hours after transfection, cells were infected with hVG2025 at an MOI of 1. Cells were incubated at 37° C., 5% CO2 for 6 hours. For further studies, cells were harvested 6 hours after viral infection, RNA was purified, and RT-qPCR was performed to measure the levels of ICP27, ICP34.5, miR124, miR143, and actin. Results: The results of this experiment are shown in Figure 5. Expression levels of ICP27 were comparable in 293FT cells transfected with either miR124 / 143, control / scrambled miR, or non-transfected cells, whereas levels of ICP34.5 were significantly lower in miR124 / 143 transfected cells (p-value=0.0002).

[0046] To verify the presence of miR124 and miR143 in transfected cells, RT-qPCR was also performed in transfected cells (infected and non-infected). High levels of miR124 and miR143 were detected in transfected cells. Moreover, it was observed that viral infection did not affect or reduce the levels of miRs in these cells.

[0047] TIFF2024512053000005.tif57146 Conclusion: The results show a significant decrease in the expression of ICP34.5 in the presence of miR124 / 143 (p=0.0002). Meanwhile, the expression level of ICP27 was not regulated by miRs and was the same in all groups, suggesting that the downregulation of ICP34.5 was miR124 / 143 specific. Furthermore, viral infection did not alter the levels of miRs in treated cells.

[0048] Example 4 Dose-dependent vector-induced tumor cytotoxicity in various tumor cell lines Objective: This study aimed to evaluate cell viability and 50% inhibitory concentration (IC 50 The antitumor activity of hVG2025 was tested, measured as the antitumor activity (IgA) of hVG2025. Procedure: The following cell lines were cultured at 5 × 10 3 Cells were seeded at 100 cells / well in well plates and incubated overnight in various suitable cell-specific culture media as suggested by the vendor: A549 (human NSCLC), BxPC3 (human pancreatic cancer), Panc01 (human pancreatic cancer), Capan-1 (human pancreatic cancer), SW620 (human colon cancer), LnCAP and PC-3 (human prostate tumor cells), U2OS (human tibial osteosarcoma), HepG2 (human hepatocellular carcinoma), Kato III (human gastric cancer), SH-SY5Y (human neuroblastoma), Panc02 (mouse pancreatic cancer), Cloudman S91 (mouse melanoma), MB49-Luc (mouse bladder cancer), CT26 (mouse colon cancer), A20 (mouse reticulum cell sarcoma), 4T1 (mouse breast cancer).

[0049] hVG2025 was diluted and added to the cell cultures at MOIs ranging from 5, 1, 0.2, 0.04, and 0 (MOI 0 is medium alone as vehicle control). Incubated for 3 days. Cell viability was assessed by MTT method under standard MTT assay. Cell viability was plotted against the MOI for each cell line. IC 50 was calculated by GraphPad Prism. IC of resistant cell lines 50 were recorded as not determinable (ND). Results: The results of this experiment demonstrate the antitumor activity of hVG2025, measured as cell viability in 11 human tumor cell lines (Figure 6A) and 6 murine tumor cell lines (Figure 6B) cultured in vitro. More specifically, HepG2, A549, LnCap, and BxPC3 were the most sensitive to hVG2025, with IC values ​​lower than MOI 1. 50 Capan-1, PC3, and SW620 were found to be resistant to hVG2025. Other human tumor cells were found to be moderately tolerant to hVG2025. All mouse tumor cells tested in this study were found to be resistant to hVG2025. Conclusion: hVG2025 at an MOI < 1 is cytotoxic to several cell lines, including pancreatic (BxPC3), lung (A549), prostate (LnCaP), and hepatocellular carcinoma (HepG2). Mouse tumor cell lines were not sensitive to hVG2025-induced cytotoxicity.

[0050] Example 5 Viral replication efficiency of VG2025 in tumor cells in comparison with ICP34.5-oHSV-1 Objective: This study aimed to compare the replication efficiency of hVG2025 virus and the ICP34.5(-) oHSV-1 strain (VG160) in A549 and BxPC3 cells. Study design: A549 and BxPC3 were prepared in 6-well plates. After overnight incubation, cells were infected with hVG2025 and ICP34.5-oHSV-1 at MOI=0.5. Two hours after infection, the medium was replaced with virus-free fresh medium. At 6, 24, and 48 hours after infection, cells and medium were harvested together and stored at -80°C, after which plaque assays were performed in Vero cells. Two samples / virus / cell line were harvested each time.

[0051] Measurements: All samples were prepared for plaque assay according to SOPs. Results: The titers of hVG2025 grown in A549 and BxPC3 cells are shown in Table 4. Growth curves of the two viruses in the two tumor cell lines are shown in Figures 7A and 7B.

[0052] TIFF2024512053000006.tif52144

[0053] Conclusion: The results showed that the replication of hVG2025 was approximately 10-fold higher than that of VG160 (ICP34.5-) in A549 and BXPC3 cells (both of which are CEA-high expressing cells at 48 hours post-infection). The main differences between the two viruses in the context of this assay are: a) the viral essential gene ICP27 is controlled by the CEA promoter in hVG2025, whereas in VG160, the same gene is controlled by its native viral promoter; b) the ICP34.5 gene is controlled by miR124 and miR143 in hVG2025, whereas it is deleted in VG160. Thus, the replication advantage shown by hVG2025 in these two tumor cell lines can be attributed to both increased ICP27 transcription and functional ICP34.5.

[0054] Example 6 Expression of human IL-12p70 and human IL-15 / IL-15Ra from HVG2025 virus infected tumor cells Objective: To measure the secretion of human IL-12p70 and human IL-15 / IL-15Ra payloads from the virus hVG2025 infected tumor cells. Procedure: A549 (NSCLC) and MRC-5 (fibroblast) cell lines were seeded in 12-well plates at 37°C overnight and then infected with hVG2025 virus at MOI=1 for 24 hours. Cell lines infected with VG1905 backbone virus (non-human IL-12p70 and human IL-15 / IL-15Ra) under the same conditions were used as negative control. 24 hours after virus infection, supernatants were collected from cells and human IL-12p70 and human IL-15 / IL-15Ra secretion was quantified by ELISA assay.

[0055] Results: 24 hours after hVG2025 virus infection, payload expression was observed in A549 and MRC-5 cells. However, A549 cells produced significantly higher human IL-12p70 and human IL-15 / IL-15Ra payloads than MRC-5 cells (3.6-fold higher human IL-12p70 and 14.6-fold higher human IL-15 / IL-15Ra, respectively) (Figures 8A and 8B and Table 5).

[0056] TIFF2024512053000007.tif41150 Conclusion: A549 cells infected with hVG2025 virus produce more human IL-12p70 and IL-15 / IL-15Ra payloads compared to infected MRC5 cells.

[0057] Example 7 hVG2025 payload: biological functions of human IL-12p70 and human IL-15 / IL-15Ra AIM:To investigate the biological function of human IL-12p70 payload with human IL-15 / IL-15Ra produced from cells infected with hVG2025 virus. Procedure: Human IL-12p70 and human IL-15 / IL-15Ra payload expressing hVG2025 or VG1905 backbone (without human IL-12p70 and human IL-15 / IL-15Ra) viruses were used to infect Vero cells at an MOI of 1. Supernatants of infected Vero cell cultures were harvested 48 hours post-infection and payload expression was measured by ELISA assay and subsequently used for the following procedures. Human PBMCs were pre-stimulated with 0.25 mg / mL of the mitogen phytohemagglutinin (PHA) for 24 hours in a 37°C incubator. The next day, PHA-stimulated human lymphoblasts were co-incubated with different volumes of virus-infected Vero supernatant for 48 hours. Supernatants were harvested from the co-incubation and human IFN-γ secreted from immune cells was quantified by human IFN-γ ELISA assay.

[0058] Results: Payload expression in virus-infected Vero supernatants was determined by ELISA, and then the bioactivity of the payload was examined. Only supernatants from hVG2025-infected cells produced human IL-12 and IL-15 / IL-15Ra, but not from VG1905-infected cells or from supernatants without virus infection (Figures 9A and 9B and Table 6). Next, the bioactivity of the payload was examined based on human IFN-γ secretion by PBMC. The results showed dose-dependent human IFN-γ production from PHA-stimulated lymphoblasts co-incubated with supernatants from hVG2025 virus-infected cells, whereas no IFN-γ secretion was detected from PHA-stimulated lymphoblasts exposed to supernatants from cells infected with hVG2025, VG1905, or uninfected cells (Figure 10 and Table 7).

[0059] TIFF2024512053000008.tif36143

[0060] TIFF2024512053000009.tif47156 Conclusion: Human IL-12p70 and human IL-15 / IL-15Ra payloads were secreted from hVG2025 virus-infected Vero cells. Supernatants containing the above payloads secreted from hVG2025-infected cells stimulated human PBMCs to produce IFN-γ.

[0061] Example 8 TCID50 of hVG2025 in A549 and MRC5 cells Objective: To determine the viral titer of hVG2025 in A549 and MRC5 cells by TCID50 assay, and to find the difference in infection and replication of hVG2025 in tumor and normal cell lines. Study design: Six replicate dilutions of virus as follows: in the first tube, a 100-fold dilution was made by adding 10 μl of virus hVG2025 from -80°C stock to 990 μl of DMEM medium, then in seven tubes, a 10-fold serial dilution was made by adding 100 μl of virus to 900 μl of DMEM medium, making a total of 48 tubes of virus dilution preparations. The same dilutions of virus were replicated by two individuals. 100 ul / well of diluted virus was added to A549 or MRC5 cells in a 96-well plate to infect each cell line in total of 96 wells. Each dilution group infected wells in 12 replicates. TCID 200 was visualized under a microscope 3-5 days after inoculation at 37°C and 5% CO2. 50 was calculated.

[0062] Measurement: Cytopathic effect (CPE) was visualized under an inverted microscope by observing wells inoculated with virus dilutions. TCID50 was calculated based on the method of Reed and Muench. Results: The results of this experiment are provided in Table 8 below.

[0063] TIFF2024512053000010.tif26145 Conclusion: The TCID50 of hVG2025 in non-tumor MRC5 cells was much higher than that in A549 cells with a difference of 76.43-fold, indicating that the replication efficiency of VG2025 in A549 tumor cells was significantly higher.

[0064] Example 9 Treatment of A549-bearing BALB / c nude mice with hVG2025 Objective: This study was to determine the dose-dependent antitumor efficacy and survival benefit of intratumorally delivered hVG2025 virus in an A549 lung cancer xenograft model in athymic mice. Study design: 29 SPF-grade female Balb / c nude mice were randomly assigned to receive 5 × 10 6A549 cells / mouse were injected subcutaneously and randomized into six groups with five mice in each virus treatment group (four mice in the vehicle group). Group 1 was the vehicle (PBS) control. Groups 2-5 were test groups and were administered 10 2 , 10 3 , 10 4 , 10 5 , 10 6 A single dose of hVG2025 was administered in the dose of PFU / mouse. All animals were properly identified by marking different body sites, housed and fed according to standard protocols. Measurements: All mice were observed at least twice daily after dosing for clinical observations. Body weight and tumor size were measured at baseline and then 2-3 times per week. Data are expressed as mean ± SEM. The tumor is 1500 mm 3 Animals were sacrificed when they grew to maturity (measured by caliper) or showed grade 5 clinical symptoms.

[0065] Results: Compared with the vehicle control group, 10 3 , 10 4 , 10 5 , 10 6 Tumor growth inhibition was observed following intratumoral treatment of PFU / mouse with hVG2025 (see Figures 11A-11G). Tumor size was measured up to 40 days after implantation, terminating in spontaneous tumor regression in some vehicle control animals. Mice were allowed to survive until day 54 and sacrificed according to schedule. No statistical analysis was performed on tumor size, as sample sizes were too small to determine whether they were normally distributed. Instead, the duration of tumor inhibition in each group was analyzed by Gehan-Breslow-Wilcoxon test. Table 8 provides a summary of tumor regression in each group, and Figure 12 graphically depicts the change in body weight following hVG2025 treatment.

[0066] TIFF2024512053000011.tif47132 Conclusion: hVG2025 demonstrated antitumor activity in a dose-dependent manner in a mouse model of human lung cancer xenografts. 3 Virus doses above PFU / mouse appear to be sufficient to show inhibitory effects, but at higher doses, many mice had complete tumor regression, and no toxic symptoms were observed in any group of mice.

[0067] Example 10 Treatment of BxPC3-burdened BALB / c nude mice with hVG2025 Objective: This study was to determine the antitumor efficacy and survival benefit of intratumorally injectable hVG2025 virus in a BxPC3 pancreatic cancer xenograft model in athymic mice. Study design: Thirty SPF-grade female Balb / c nude mice were randomly assigned to receive 5 × 10 6 BxPC3 cells / mouse were injected subcutaneously and randomized into six groups with five mice per group. Group 1 was the vehicle control and was injected intratumorally with PBS. Groups 2-5 were test groups and were administered 10 2 , 10 3 , 10 4 , 10 5 , 10 6 A single dose of hVG2025 was administered in PFU / mouse. All animals were properly identified by marking different body sites, housed and fed according to standard protocols. Measurements: All mice were observed at least twice daily after dosing for clinical symptoms. Body weight and tumor size were measured at baseline and then 2-3 times weekly. Data were expressed as individual tumor size. The tumor is 1500 mm 3 Animals were sacrificed when they grew to maturity (measured by caliper) or showed grade 5 clinical symptoms.

[0068] Results: The results are presented in Figures 13A-13G. Compared to the vehicle control group, 3 , 10 4 , 105 , 10 6 We observed BxPC3 tumor growth inhibition following intratumoral treatment with hVG2025 PFU / mouse. Mice were euthanized due to tumor burden alone, while the remainder survived until day 89 post-implantation. Tumors did not grow in 2 of 5 animals in the vehicle control group. Statistical analysis was not performed on tumor size because the sample size was too small to determine whether it was normally distributed. Instead, the duration of tumor inhibition in each group was analyzed by Gehan-Breslow-Wilcoxon test (Figure 14). There was no significant difference in mouse body weight among all groups. No clinical symptoms were observed. Conclusions: Antitumor activity of hVG2025 was evident in nude mice xenografted with human pancreatic cancer. Some animals receiving hVG2025 showed complete tumor regression. Larger sample sizes are needed to demonstrate significant efficacy due to the wide variability of tumor growth among animals. hVG2025 was safe in tumor-bearing nude mice.

[0069] Example 11 Comparison of the antitumor efficacy of hVG2025 and ICP34.5-deficient HSV-1 in a lung cancer (A549) model Objective: The experimental goal was to compare the antitumor efficacy of hVG2025 and ICP34.5(-) oHSV-1 in an athymic A549 xenograft mouse model. Study design: Thirty SPF-grade female athymic nude mice were inoculated with 2.5 × 10 6 A549 cells / mouse were injected subcutaneously and randomized into 5 groups with 6 mice per group. Group 1 was the vehicle control and was injected intratumorally with vehicle containing 1xDPBS+7.5% glycerin. Groups 2-5 were test groups; Groups 2-4 received 5x10 3 , 5x10 5 , and 5x10 7 Finally, group 5 received a single dose of hVG2025 at 5x10 PFU / mouse. 7A single dose of ICP34.5 gene deleted HSV-1 vector was administered in PFU / mouse. All test groups were administered by intratumoral injection. All animals were properly housed and fed according to standard protocols and properly identified by marking different body parts.

[0070] Measurements: All mice were observed at least twice daily after dosing for clinical findings. Body weight and tumor size were measured at baseline and then 2-3 times per week. Statistical analysis was performed with GraphPad Prism 7.03. Tumor volumes were measured using a vernier caliper (length x width x depth x 0.5236). Data presented are tumor volumes (mm 3 ) and values ​​are means ± SEM. Statistical significance of tumor regression (P < 0.05) was determined using a multiple t test. 3 Animals were sacrificed when they grew to adulthood or showed grade 5 clinical symptoms. Results: The results are provided in Figures 15A, 15B, 15C, 15D, 15E, and 15F. Briefly, compared to the vehicle control group, 5x10 5 and 5x10 7 After a single intratumoral treatment with PFU / mouse, a statistically significant tumor growth inhibition was observed 39 days after treatment. The results were 5x10 7 Compared to the tumor-inhibitory effect of the ICP34.5 deletion mutant at doses of PFU / mouse, hVG2025 demonstrated much better efficacy, even at doses 100-fold lower than the ICP34.5 mutant. Conclusion: The antitumor effect of hVG2025 was confirmed in a mouse model xenografted with human lung cancer in a dose-dependent manner. Tumor growth was 5x10 5 and 5x10 7 The dose of PFU / mouse was significantly controlled. Compared with the ICP34.5 gene-deleted HSV-1 vector, a significantly increased anti-cancer effect was observed with hVG2025.

[0071] Example 12 Survival of young DBA / 2 mice after subcutaneous inoculation with VG2025. Objective: This study aimed to determine the toxicity of hVG2025 following subcutaneous inoculation in young DBA / 2 mice. Study Design: Thirty SPF grade female DBA / 2 mice as young as 4 weeks old were randomized into six groups with five mice per group. Group 1 was the vehicle control and was injected subcutaneously with PBS. Groups 2-4 were the test groups and received a single dose or multiple doses administered for five consecutive days (Group 4 only) of the test substance at different doses by subcutaneous injection as shown in Table 9. Groups 5-6 were the positive groups that received a single dose of wild type 17+ HSV virus.

[0072] TIFF2024512053000012.tif41127

[0073] All animals were properly identified by marking different body sites, housed and fed according to standard protocols. Measurements: All mice were observed at least twice daily after dosing for clinical observations. Body weights were measured at baseline and then 2-3 times per week. Data were expressed as mean body weight. Survival curves for each group were shown. Animals were sacrificed when they lost 20% body weight or showed grade 5 clinical symptoms. Results: No abnormalities were observed in general behavioral activity throughout the experiment. Compared to the vehicle control group, there was no significant difference in the weight of the mice between the groups, as shown in Figure 16. 6 Only one mouse in the PFU / mouse treatment group showed weight loss and morbidity 5 days after inoculation, and the mouse died despite being given special care. Percent survival is shown in FIG.

[0074] Conclusion: 10 6Some toxicity was seen in the 17+ strain at PFU / mouse, resulting in the death of one animal. hVG2025 was not detected in all animals injected subcutaneously with the 17+ strain, even at titers 100-fold higher than wild type, and was not detected in all animals injected subcutaneously with the 17+ strain at PFU / mouse, resulting in the death of one animal. 8 No toxicity was observed in all animals injected with PFU once daily (Group 4). Thus, hVG2025 is safe in young DBA / 2 mice administered by the subcutaneous route.

[0075] Example 13 Neurovirulence assay of VG2025 by intranasal inoculation in young DBA / 2 mice Objective: This study was to determine the toxicity of hVG2025 in comparison to the wild-type parental strain 17+ and VG161, an oncolytic HSV-1, ICP34.5(-), by intranasal inoculation in young DBA / 2 mice. As the nasal site is innervated by both the trigeminal ganglion and the olfactory bulb, this model is highly sensitive to testing HSV neurovirulence. Study Design: Thirty SPF grade female DBA / 2 mice aged 4 weeks were randomized into six groups with five mice in each group. Group 1 was the vehicle control and was inoculated intranasally with PBS. Group 2 was the positive control and was inoculated with a lethal dose of the wild type ICP34.5 positive 17+ strain. Groups 4-6 were the test groups that received two levels of single doses of hVG2025 or VG161 via intranasal inoculation (Table 10).

[0076] TIFF2024512053000013.tif47144 All animals were properly identified by marking different body sites, housed and fed according to standard protocols. Measurements: All mice were observed at least twice daily after dosing for clinical observations. Body weights were measured at baseline and then 2-3 times per week. Data were expressed as mean body weight. Survival curves for each group are shown. Animals were sacrificed when they lost 20% body weight or showed grade 5 clinical symptoms. Results: No abnormalities in general behavioral activity were observed in the hVG2025 and VG161 treatment groups during the entire experiment. As shown in Figure 18, there was no significant difference in mouse weight between the VG161 and hVG2025 groups by comparing with the vehicle control group, and all mice at both dose levels survived. All mice in the 17+ treatment group showed morbidity and weight loss 3 days after inoculation and had to be sacrificed on day 6. The survival curves are shown in Figure 19.

[0077] Conclusion: 10 5 The 17+ strain at PFU / mouse was virulent for all mice 3 days after inoculation and had to be killed by day 6. 7 The hVG2025 and ICP34.5(-)VG161 groups, including the PFU / mouse treatment group, did not show any toxicity. Both hVG2025 and VG161 showed good safety in young DBA / 2 mice, regardless of the difference in ICP34.5 status. The safety of miR124 / 143-regulated ICP34.5 expression by hVG2025 in the nervous system is demonstrated.

[0078] Example 14 Survival and viral gene expression in the trigeminal ganglion of BALB / c mice exposed to VG2025 by corneal scarification to assess neurotoxicity. Objective: This study aimed to evaluate the neurotoxicity of hVG2025 using a corneal scarification model in normal BALB / c mice. Study Design: The viruses tested were a) hVG161, HSV-1 with both copies of the ICP34.5 gene deleted; b) hVG2025, HSV-1 with the CEA promoter driving ICP27 and regulation of ICP34.5 by miR124 / 143. Both viral strains also express IL12 / IL15; c) HSV-1 17+ wild type, hVG161 and the parental virus of hVG2025. A total of 140 6-week-old BALB / c mice were divided into 8 groups: 1) mock infected (n=10 mice), 2) 17+ (n=10 mice), 3) 17+ (n=10 mice), 4) 17+ (n=10 mice), 5) 17+ (n=10 mice), 6) 17+ (n=10 mice), 7) 17+ (n=10 mice), 8) 17+ (n=10 mice), 9) 17+ (n=10 mice), 10) 17+ (n=10 mice), 12) 17+ (n=10 mice), 13) 17+ (n=10 mice), 14) 17+ (n=10 mice), 15) 17+ (n=10 mice), 16) 17+ (n=10 mice), 18) 17+ (n=10 mice), 19) 18) 19 ... 5 pfu / eye, n=30 animals), 3) hVG161 105 pfu / eye (n=30 animals), 4) hVG161 10 6 pfu / eye (n=10 animals), 5) hVG161 10 7 pfu / eye (n=10 animals), 6) hVG2025 10 5 pfu / eye (n=30 animals), 7) hVG2025 10 6 pfu / eye (n=10 animals), 8) hVG2025 10 7 pfu / eye (n=10). All animals received 5ul of PBS or virus solution at the indicated doses via corneal scarification. Three animals from groups 1), 2), 3), and 6) were killed on day 5 post-infection and trigeminal ganglion (TG) and olfactory bulb (OB) were harvested for RNAseq analysis. The remaining animals were allowed to survive until day 28 and then sacrificed.

[0079] Results: Similar to mock-infected animals, both mice inoculated with hVG161 and hVG2025 showed no observable symptoms, whereas wild-type 17+ showed severe corneal lesions (representative photograph in Figure 20A) and approximately 75% mortality within 28 days post-infection (Figure 20B). No HSV-1 transcripts were detectable in the trigeminal ganglion and olfactory bulb at 5 days post-infection from either hVG161 or hVG2025 infected mice, whereas the 17+ strain expressed high levels of virtually all HSV-1 transcripts in the trigeminal ganglion and, to a lesser extent, in the olfactory bulb (Figure 21). Conclusion: 1. The wild type 17+ strain produced 10 5 The corneal scarification model was effective for testing HSV-1-induced neurovirulence because pfu virus infection caused severe corneal inflammation, tissue damage, and high mortality. 2. HSV-1 strains engineered with either an ICP34.5 deletion (hVG161) or dual regulation of transcription and translation (hVG2025) did not show any pathogenicity in both the eye and CNS, even at doses 100-fold higher than wild type.

[0080] 3. On day 5 after infection, 5Expression of all viral genes was readily detectable in the trigeminal ganglia of animals corneal-infected with wild-type HSV-1 17+ infected at pfu. Low levels of transcriptional activity (approximately 200-fold lower than in TG) could also be detected in the olfactory bulb at this early stage of infection, suggesting rapid and widespread dissemination of the virus in the brain. 4. Neither hVG161 nor hVG2025 infection in the cornea resulted in any detectable viral gene expression in the trigeminal ganglion or olfactory bulb, indicating that neither virus replicates in the CNS during the acute infection phase. 5. hVG2025, which has regulated ICP34.5 expression, is at least as safe in the nervous system as ICP34.5(-) hVG161.

[0081] Example 15 Susceptibility to ganciclovir Objective: The purpose of this study was to investigate the sensitivity of hVG2025 #9 to ganciclovir. Study Design: Stocks of hVG2025(tk+) and its parental strain VG1925#2-1(tk-) were used at 2000 pfu / ml, 400 pfu / ml, and 80 pfu / ml to infect Vero cells in the presence of different concentrations of ganciclovir (GCV). The number of plaques on the plates was counted as a measure of susceptibility to GCV.

[0082] Measurement: hVG2025 or VG1925#2-1 virus was diluted into the designated virus solution, and then infected into Vero cells in 12-well plates at a final concentration of 500 pfu / well, 100 pfu / well, or 20 pfu / well. After 1 h incubation at room temperature, different concentrations of GCV were added to the indicated wells, and the cells were incubated at 37°C 5% CO2. After 3 days of incubation, the plates were stained with 2% crystal violet, and the number of plaques was counted. The above experiment was repeated three times.

[0083] Results: The results are provided in Tables 11 and 12 below and in FIG.

[0084] TIFF2024512053000014.tif93148

[0085] TIFF2024512053000015.tif93156

[0086] Conclusion: TK(+) hVG2025 is highly sensitive to ganciclovir. IC of GCV inhibiting hVG2025 50 hVG2025 was <0.195 μg / ml, with <0.39 μg / ml GCV resulting in 100% viral inhibition. This susceptibility to GCV by hVG2025 is in contrast to its parental strain VG1925#2-1(TK-), where 1.563 μg / ml GCV (the highest concentration tested) was able to produce only about 20% inhibition in viral replication. Thus, the concentrations required to inhibit hVG2025 are much lower than the clinical dose of ganciclovir in humans (see, e.g., Toxicity et al., 2017, Ganciclovir Injection [package insert]. Lenoir: EXELA Pharma Sciences, NC; 2017; in this case, the clinical dose of ganciclovir provides a Cmax of 9 μg / ml in humans).

[0087] Example 16 Virus Stability Stability data was collected using pilot batches of VG2025 virus stored at 4°C and -80°C, respectively, for up to one month. The titers were compared to those before filling into vials. There was no significant loss of virus titer at both temperatures for up to one month (Figure 23).

[0088] (Example 17) Evaluation of the antitumor efficacy of hVG2025 virus in a liver cancer (Hep 3B-luc) model Objective: The aim of this study was to evaluate the antitumor efficacy of VG2025 administered intravenously (iv) in an orthotopic human liver cancer xenograft model of Hep 3B-luc in female BALB / c nude mice.

[0089] Study Design: The experimental design of the antitumor efficacy study of hVG2025 is summarized in Table 13.

[0090] JPEG2024512053000016.jpg72170 Results: Body weight changes following administration of VG2025 to female BALB / c nude mice bearing established orthotopic Hep 3B-luc tumors are shown in Figure 24A. Data points represent the mean body weight of the group. Error bars represent the standard error of the mean (SEM).

[0091] The mean bioluminescence over time in female BALB / c nude mice bearing orthotopic Hep 3B-luc xenografts administered VG2025 is shown in Table 14 and Figure 24B.

[0092] JPEG2024512053000017.jpg57170 Survival curves after administration of VG2025 to female BALB / c nude mice bearing established orthotopic Hep 3B-luc tumors are shown in Figure 24C.

[0093] The metastasis rates of these mice are shown in Table 15 and Figure 24D. Bioluminescence intensity was calculated based on the euthanized animals detected by the IVIS machine. No significant metastasis was detected in any group.

[0094] JPEG2024512053000018.jpg83170Conclusion: The antitumor effect of intravenously delivered VG2025 was confirmed in this mouse model xenografted with human liver cancer cells. Compared to the vehicle control group, VG2025 (2.4 × 10 6 PFU / 100 μL / mouse) and VG2025 (2.4 × 10 7Treatment with 100 μL / mouse (PFU) demonstrated a clear antitumor effect in this model of liver cancer. Notably, no significant metastasis was observed in any of the treatment groups.

[0095] (Example 18) Evaluation of mVG2025 antitumor efficacy in a B cell lymphoma (A20-luc) model Objective: The objective of this study was to evaluate the antitumor efficacy of VG2025 administered intravenously (iv) in an orthotopic human B-cell lymphoma xenograft model in female BALB / c nude mice. Study design: Mice (8 per group) were incubated for 10 min at 4 °C for 1 h before inoculation with lymphoma cells. 6 Mice were pre-immunized with two subcutaneous injections of mVG2025 at PFU / mouse. Mice were then inoculated intravenously with A20-Luc B cell lymphoma cells. Details of the experimental design are described in Table 16.

[0096] JPEG2024512053000019.jpg57170 Results: Body weight changes following administration of mVG2025 to female BALB / c mice bearing established orthotopic A20-luc tumors are shown in Figure 25A. Data points represent the mean body weight of the group. Error bars represent standard error of the mean (SEM).

[0097] The mean bioluminescence over time in female BALB / c mice bearing orthotopic A20-luc xenograft tumors administered mVG2025 is shown in Table 17 and FIG. 25B.

[0098] JPEG2024512053000020.jpg86170

[0099] The metastasis rates observed in this study are shown in Table 18 and Figure 25C. Bioluminescence intensity was calculated based on euthanized animals detected by IVIS machine. JPEG2024512053000021.jpg94170Conclusion: In this study, the therapeutic efficacy of mVG2025 was evaluated in an orthotopic A20-luc B-cell lymphoma xenograft model. Compared to the control (vehicle) group, mVG2025 (2.4 × 10 7 Treatment with 100 μL / mouse (pre-immune) demonstrated pronounced antitumor activity in this model of B-cell lymphoma.

[0100] (Example 19) Evaluation of the antitumor efficacy of low dose mVG2025 virus in a B cell lymphoma (A20-luc) model Objective: The objective of this study was to evaluate the antitumor efficacy of VG2025 administered intravenously (iv) in an orthotopic human B-cell lymphoma xenograft model in female BALB / c nude mice. Study design: Mice were cultured for 10 min prior to inoculation with lymphoma cells. 6 Mice were pre-immunized with two subcutaneous injections of mVG2025 at PFU / mouse. Mice were then inoculated intravenously with A20-Luc B cell lymphoma cells. Details of the experimental design are described in Table 19.

[0101] JPEG2024512053000022.jpg49170 Results: Body weight changes following administration of mVG2025 to female BALB / c mice bearing established orthotopic A20-luc tumors are shown in Figure 26A. Data points represent the mean body weight of the group. Error bars represent standard error of the mean (SEM).

[0102] The mean bioluminescence over time in female BALB / c mice bearing orthotopic A20-luc xenograft tumors administered mVG2025 is shown in Table 20 and FIG. 26B.

[0103] JPEG2024512053000023.jpg128170

[0104] Metastasis rates are shown in Table 21 and Figure 26C. JPEG2024512053000024.jpg87170Conclusion: In this study, the therapeutic efficacy of mVG2025 was evaluated in an orthotopic A20-luc B-cell lymphoma xenograft model. Compared to the control (vehicle) group, mVG2025 (2.4 × 10 6 PFU / 100 μL / mouse, preimmunization) and mVG2025 (2.4 × 10 5 Treatment with 100 μL PFU / mouse (preimmune) demonstrated pronounced antitumor activity in this model of B cell lymphoma.

[0105] (Example 20) Assessment of acute toxicity of hVG2025 in primates This study was conducted to evaluate the acute toxicity of hVG2025 following single-dose administration in rhesus monkeys by subcutaneous or intravenous injection. Male and female rhesus monkeys were given a single dose of 0 PFU / kg, 1.0 × 10 9 PFU / kg and 2.0 × 10 9 Six rhesus monkeys were randomly assigned to three groups (one animal / sex / group), the intravenous infusion rate was 2 mL / min, and the dose volumes were 5 mL / kg and 10 mL / kg for subcutaneous and intravenous administration, respectively. The following parameters and endpoints were assessed: morbidity and mortality; clinical findings; body weight; food consumption; temperature; electrocardiography; hematology and coagulation; clinical chemistry; immune function and macroscopic findings. All animals survived until the scheduled time of sacrifice. No test article-related effects were noted on clinical findings, body weight, food consumption, temperature, electrocardiography, hematology and coagulation, clinical chemistry, immune function, or gross findings.

[0106] Conclusion: Male and female rhesus monkeys received 0 PFU / kg, 1.0×10 9 PFU / kg, and 2.0 × 10 9PFU / kg. All animals survived until the scheduled time of sacrifice. No hVG2025-related abnormal changes in clinical findings, body weight, food consumption, temperature, electrocardiography, hematology, blood coagulation, clinical chemistry, immune function, or macroscopic findings were observed. Thus, the maximum tolerated dose (MTD) of hVG2025 in rhesus monkeys by subcutaneous injection is 1.0 × 10 9 The maximum tolerated dose (MTD) of hVG2025 in rhesus monkeys by intravenous injection was determined to be 2.0 × 10 9 The PFU / kg was determined.

[0107] Example 21 Abscopal antitumor effect of mVG2025 in a mouse colon cancer (CT26) model OBJECTIVE:The experimental goal was to determine the efficacy and safety of mVG2025, an alternative version of hVG2025 that expresses murine IL-12, in a dual CT26 syngeneic mouse colon cancer model incorporating both primary and secondary tumors inoculated into opposite flanks of mice. Study design: 24 female SPF-grade BALB / c mice were inoculated with 5 × 10 5 The mice were injected subcutaneously twice, once in each flank, with 1 × 10 CT26 cells and randomly divided into two groups with 12 mice in each group. Group 1 was the vehicle control and was injected intratumorally with PBS. Group 2 was the test group and was injected intratumorally with 1 × 10 CT26 cells for each dose. 8 Five doses of mVG2025 were administered in PFU / mouse on five consecutive days. Injections were performed into one tumor per mouse, and a second tumor on the opposite flank was left uninjected. All animals were properly identified by marking different body sites according to standard protocols, housed, and fed. Measurements: All mice were observed at least twice daily after dosing for clinical symptoms. Mice were weighed and tumor sizes were measured three times a week. Tumor volumes were measured using calipers (length x width x depth x 0.5236).

[0108] Results: After five consecutive intratumoral treatments with mVG2025 9 days after initiation of treatment, statistically significant tumor growth inhibition was observed compared to the vehicle control group (Figure 27A). Tumor growth inhibition relative to the contralateral abscopal untreated tumors did not reach statistical significance at the same time point, but mean tumor size was observed to trend toward lower in treated mice compared to the control group (Figure 27B). Thirty days after initiation of treatment, 7 of 12 mice in the mVG2025-treated group showed a complete response, as evidenced by complete regression of both the virus-treated tumors and the contralateral abscopal tumors. In contrast, 10 of 12 mice in the control group were euthanized due to tumor burden by 34 days after initiation of treatment (see Figures 28A, 28B, 28C, and 28D). Mice treated with mVG2025 showed a statistically significant increase in percent survival compared to mice treated with vehicle control. Specifically, 8 of 12 mice treated with mVG2025 survived until the end of the study (58 days after treatment initiation), whereas 10 of 12 mice in the control group reached the humane end point due to tumor burden before reaching the end of the study (Figure 29). Conclusion: The abscopal antitumor immune efficacy and survival benefit of treatment with mVG2025 was confirmed in a syngeneic double CT26 mouse colon cancer model, resulting in complete elimination of transplanted tumors from both the injected and non-injected sides in 7 of 12 mice. Moreover, no clinical signs of HSV-1-associated toxicity were observed.

[0109] Example 22 Abscopal antitumor efficacy of mVG2025 in a murine B-cell lymphoma (A20) mouse model OBJECTIVE:The experimental goal was to determine the efficacy and safety of mVG2025, an alternative version of hVG2025 expressing murine IL-12, in a dual A20 syngeneic murine B cell lymphoma model in which mice were harboring both primary and secondary tumors inoculated into opposite flanks. Study design: 19 SPF-grade BALB / c mice were treated with 2.5 × 10 6The mice were injected with 1 × 10 A20 cells subcutaneously twice, once in each flank, and randomized into two groups: Group 1 was a vehicle control consisting of nine mice injected intratumorally with PBS; Group 2 consisted of 1 × 10 mice injected with 1 × 10 A20 cells for each dose. 8 The test group consisted of 10 mice that received 5 doses of mVG2025 by intratumoral injection over 5 consecutive days, at PFU / mouse. Injections were performed into a single tumor per mouse, with a second tumor on the opposite flank left uninjected. All animals were properly identified by marking different body sites according to standard protocols, housed, and fed.

[0110] Measurements: All mice were observed at least twice daily after dosing for clinical symptoms. Mice were weighed and tumor sizes were measured three times a week. Tumor volumes were measured using calipers (length x width x depth x 0.5236). Results: After five consecutive intratumoral treatments with mVG2025 17 days after initiation of treatment, statistically significant tumor growth inhibition was observed for treated tumors compared to the vehicle control group (Figure 30A). Although tumor growth inhibition for contralateral untreated tumors did not reach statistical significance at the same time point (Figure 30B), by 75 days after initiation of treatment, 4 of 10 mice in the mVG2025-treated group showed a complete response, as evidenced by complete regression of both the virus-treated tumors and the contralateral abscopal tumor. In contrast, all control mice were euthanized due to tumor burden by 42 days after initiation of treatment (see Figures 31A, 31B, 31C, and 31D).

[0111] To further demonstrate that the four tumor-free mice treated with mVG2025 were capable of generating an anti-tumor immune response, these mice were rechallenged with A20 tumor cells 77 days after the initial treatment. The newly established tumors slowly regressed in three of the four mice by 107 days after the start of treatment without further mVG2025 treatment (Figure 32), suggesting the presence of an immune response against the A20 tumor cells. Four of 10 mice treated with mVG2025 survived to the study's endpoint (107 days after treatment initiation), whereas all control mice reached the humane endpoint due to tumor burden by 42 days after treatment initiation. Conclusion: The abscopal antitumor immune efficacy and survival benefit of treatment with mVG2025 was confirmed in a syngeneic double A20 murine B-cell lymphoma model, resulting in complete elimination of the implanted tumor from both the injected and non-injected sides in 4 of 10 mice. The presence of antitumor immune memory was further demonstrated in 4 mice with complete response by rechallenging them with A20 tumor cells, resulting in complete elimination of the tumor in 3 of 4 mice. Moreover, no clinical signs of HSV-1-associated toxicity were observed.

[0112] Example 23 Intratumoral and systemic detection of IL-12 and IL-15 payloads Objective: The experimental goal was to determine the payload levels of intratumorally delivered hVG2025 virus in a human lung cancer (A549) xenograft mouse model. Study design: Twenty-three female SPF-grade athymic nude mice were treated with 2.5 × 10 6 A549 cells / mouse were injected subcutaneously and randomized into two groups: Group 1 was a vehicle group consisting of three mice injected intratumorally with 7.5% glycerol in PBS; Group 2 was a group consisting of three mice injected intratumorally with 5×10 7 The study group consisted of 20 mice that received a single dose of hVG2025 at PFU / mouse. All vehicle-injected mice were euthanized 2 days after treatment, and 4 of the virus-injected mice were euthanized 1, 2, 3, 7, and 14 days after treatment. Tumors and serum were collected from each mouse and used for ELISA to detect IL-12 and IL-15 / IL-15RA.

[0113] Measurements: Mouse body weight and tumor size were measured three times a week. Tumor volumes were measured using calipers (length x width x depth x 0.5236). Entire tumors were harvested and immediately flash frozen after the animals were humanely euthanized. Blood samples were also taken to extract serum. Both tumors and serum were subjected to ELISA assays to determine the concentrations of IL-12 and IL-15 according to the protocol provided by the ELISA kit vendor. Results: A549 tumor and serum samples were collected from nude mice intratumorally injected with either hVG2025 or vehicle control. The kinetics of human IL-12p70 and human IL-15 / IL-15Ra complex products in tumor tissue (Figures 33A and 33B) and serum (Figures 34A and 34B) were analyzed using ELISA at 24 hours, 48 ​​hours, 72 hours, 7 days, and 15 days after injection. In tumor tissue, detection of human IL-12p70 and human IL-15 / IL-15Ra was maximal at 24 hours after hVG2025 injection and remained detectable up to 15 days after injection. Human IL-12p70 and human IL-15 / IL-15Ra were detectable in serum samples at the 24 hour time point, but at less than 1% of the concentrations detected in tumor tissue, and they rapidly declined to undetectable levels in serum at subsequent time points. Conclusions: Human IL-12p70 and human IL-15 / IL-15Rα were largely localized to the tumor with no evidence of systemic release, indicating that payload leakage from intratumorally injected hVG2025 is not a safety concern.

[0114] The invention has been described broadly and generically herein. Narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a provisos or negative limitation removing all subject matter from the genus, regardless of whether the excised material is expressly recited herein. It should also be understood that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise, and that the term "X and / or Y" means "X" or "Y" or both "X" and "Y," and that the letter "s" following a noun designates both the plural and the singular form of that noun. Furthermore, when features or aspects of the invention are described in terms of a Markush group, the invention is intended to embrace and be described in terms of any individual members and subgroups of members of the Markush group, as will be recognized by those of skill in the art, and applicants reserve the right to amend this application or claims to explicitly mean any individual member or any subgroup of members of the Markush group.

[0115] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. It is to be further understood that, unless expressly defined herein, the terminology used herein is intended to be given its conventional meaning as known in the relevant art. References throughout this specification to "one embodiment" or "an embodiment" and variations thereof mean that a particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification do not necessarily all refer to the same embodiment. Moreover, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0116] Below are some numbered exemplary embodiments of the present disclosure. 1. A recombinant herpesvirus comprising a modified oncolytic herpesvirus genome, the modified herpesvirus genome comprising at least one miRNA target sequence operably linked to a first copy of an ICP34.5 gene, and a second copy of the ICP34.5 gene comprising an inactivating mutation. In certain embodiments, the recombinant herpesvirus is a recombinant herpes simplex virus (such as HSV-1 or HSV-2).

[0117] 2. The mutation is in at least one long terminal repeat (R L ) region or deletion of the long terminal repeat region and short terminal repeat (R S ) region of the viral genome that encodes one copy of genes functionally equivalent to HSV-1 ICP0 and ICP34.5. L ) region or the long terminal repeat (R ) region of the viral genome that encodes one copy of a gene functionally equivalent to HSV-1 ICP4. L In other embodiments, the mutation may include a deletion of at least one of the long internal repeat regions, or a deletion of a long internal repeat region and a short internal repeat region. In other embodiments, the mutation may include a deletion of one long repeat (R L ) region or one long repeat (R L ) region and one short repeat (R S ) region. In a preferred embodiment of the invention, the deletion is only one long terminal repeat region, resulting in increased stability during passage compared to HSV with deletions in both the long and short terminal repeat regions. In an optional embodiment, the mutation is a deletion involving a second copy of the ICP34.5 gene.

[0118] 3. The recombinant herpes virus of embodiment 1 or 2, which is a herpes simplex virus, further comprising 2 to 10 miRNA target sequences operably linked to the first copy of the ICP34.5 gene. 4. The recombinant herpes simplex virus of any one of embodiments 1, 2, or 3, wherein the miRNA target sequence is inserted into the 3' untranslated region of the first copy of the ICP34.5 gene. In further embodiments, the miRNA target sequence is inserted in tandem into the 3' untranslated region. In various embodiments, linker DNA of the same or various lengths can be inserted between the different miRNA binding sites. In certain embodiments, the linker is in the range of 1-50 base pairs. In other embodiments, the linker is less than 10 base pairs. 5. The recombinant herpes simplex virus of any one of embodiments 1, 2, 3, or 4, wherein the 2-10 miRNA target sequences bind to at least two different miRNAs. 6. The miRNA target sequence is miR-124, miR-124 * 6. The recombinant herpes simplex virus of any one of embodiments 1, 2, 3, 4, or 5, which targets a miRNA selected from the group consisting of miR-143, miR-144, and miR-143.

[0119] 7. The recombinant herpesvirus of any one of embodiments 1, 2, 3, 4, 5, or 6, wherein the herpesvirus is a herpes simplex virus and the modified herpesvirus genome comprises additional mutations or modifications in the viral genes ICP4 and / or ICP27. 8. The recombinant herpesvirus of any one of embodiments 1, 2, 3, 4, 5, 6, or 7, wherein the modification comprises replacement of the native viral promoter with a tumor-specific promoter. 9. The recombinant herpesvirus according to any one of embodiments 1, 2, 3, 4, 5, 6, 7, or 8, wherein the modification is a replacement of the entire promoter-regulatory region of ICP4 or ICP27, optionally with a tumor-specific promoter. 10. The recombinant herpesvirus of any one of embodiments 1, 2, 3, 4, 5, 6, 7, 8, or 9, wherein the ICP27 promoter is replaced by the hCEA promoter.

[0120] 11. The recombinant herpesvirus according to any one of embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, further comprising at least one nucleic acid encoding a non-viral protein selected from the group consisting of an immune stimulator, an antibody, and a checkpoint inhibitor peptide, wherein the at least one nucleic acid is operably linked to a general promoter or a tumor-specific promoter. Examples of general promoters include constitutive promoters, such as SV40, CMV, UBC, EF1alpha, PGK, and CAG. 12. The recombinant herpesvirus of any one of embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9, or 11, wherein the non-viral protein is selected from the group consisting of IL12, IL15, IL15 receptor alpha subunit. 13. A recombinant herpesvirus according to embodiment 11 or 12, wherein the promoter is a tumor-specific CXCR4 promoter.

[0121] 14. A recombinant herpesvirus according to any one of embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13, having a nucleic acid sequence encoding a glycoprotein with enhanced fusogenicity (compared to a similar wild-type virus). Examples include various transgenes (e.g., the fusogenic glycoprotein from gibbon ape leukocyte virus "GALV") and / or mutations that enhance HSV fusion, such as truncations or mutations in glycoprotein B, glycoprotein K, and / or UL20. In a preferred embodiment, the nucleic acid sequence encodes a fusogenic form of glycoprotein B (e.g., glycoprotein B truncated after amino acid 876). 15. The recombinant herpes simplex virus of embodiment 14, wherein glycoprotein B is optionally truncated by a deletion after amino acid 876. 16. The recombinant herpesvirus of any one of embodiments 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, wherein the oncolytic herpesvirus is HSV-1.

[0122] In a particularly preferred embodiment of the invention, the recombinant herpesvirus comprises the oncolytic herpesvirus HSV-1, wherein a) a deletion of the long terminal repeat comprising the ICP0 and ICP34.5 genes ; b) replacement of the native ICP27 promoter with the CEA promoter; c) insertion of binding sites for miR-143 and miR-124 in the ICP34.5 3'UTR; d) deletion of a portion of the 3' end of the glycoprotein B coding region (e.g., an 84 bp deletion); and e) insertion of an expression cassette capable of expressing IL-12, IL-15, and IL-15Rα under the control of the CXCR4 promoter. 17. A method for inhibiting or lysing tumor cells, comprising the step of providing a therapeutically effective amount of a recombinant herpesvirus according to any one of embodiments 1 to 16. 18. A therapeutic composition comprising a recombinant herpesvirus according to any one of embodiments 1 to 16 and a pharma- ceutically acceptable carrier. In a preferred embodiment, the herpesvirus is VG2025, or more particularly hVG2025, as described in Table 1 above. 19. A method for treating cancer in a subject, comprising administering a therapeutically effective amount of the composition of embodiment 18.

[0123] 20. The method of embodiment 19, wherein the cancer expresses high levels of a biomarker, the promoter of which is used to drive the ICP4 and / or ICP27 genes as described in any one of embodiments 1-19. In other embodiments, the cancer expresses high levels of a biomarker, such as hCEA. In related embodiments, the subject is tested for expression of high levels of hCEA (e.g., greater than 2.5 ng / ml) prior to administration of the herpesvirus described herein. Levels of hCEA greater than 10 ng / ml or even greater than 20 ng / ml indicate significant progression of the cancer. In certain embodiments, the cancer is selected from the group consisting of cervical, esophageal, lung, colorectal, hepatic, gastric, cholangiocarcinoma, and pancreatic cancer. In other embodiments, the cancer is selected from the group consisting of breast and prostate tumors, and glioblastoma. In other embodiments, the cancer is a leukemia or lymphoma. In other embodiments, the cancer is an acute myeloid leukemia (AML) or B-cell lymphoma. In other embodiments, the cancer is a surface injectable tumor. In yet other embodiments, the cancer expresses high levels of CEA. 21. The method of embodiment 19, wherein the step of administering a therapeutically effective amount of the composition of embodiment 18 comprises intravenous or intratumoral administration. In a preferred embodiment, the composition is administered within about 10 4 pfu~about 10 10 In a further embodiment, the dosage is about 10 pfu. 6 pfu~about 10 7 pfu, or about 10 7 pfu~about 10 8 pfu, or about 10 8 pfu~about 10 9 The pfu range may be administered as a single dose or as multiple doses spread over time. The doses may be administered as described in more detail above.

[0124] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents, i.e., one or more objects, unless the content and context clearly dictate otherwise. It should also be noted that the conjunctions "and" and "or" are generally used in a broad sense, including "and / or," unless the content and context dictate otherwise, as the case may be, to be inclusive or exclusive. Thus, the use of an alternative (e.g., "or") should be understood to mean either one, both, or a combination thereof of the alternatives. Furthermore, the "and" and "or" compositions when recited herein as "and / or" are intended to encompass embodiments including all of the associated items or ideas as well as one or more other alternative embodiments including less than all of the associated items or ideas.

[0125] Unless the context clearly indicates, throughout this specification and the claims that follow, the term "comprise" and its synonyms and variations, such as "have" and "include," and variations thereof, such as "comprises" and "comprising," are to be interpreted in their open, inclusive sense, such as, for example, "including, but not limited to." The term "consisting essentially of" limits the scope of the claims to specified materials or steps or those that do not materially affect the basic novel characteristics of the claimed invention. All headings used within this document are utilized solely to facilitate the reader's review thereof and should not be construed as limiting the scope of the invention or the claims in any manner. Accordingly, the headings and abstracts of the disclosure provided herein are for convenience only and do not interpret the scope or meaning of the embodiments.

[0126] When a range of values ​​is provided herein, it is to be understood that each intervening value between the upper and lower limits of that range, to one-tenth of the unit of the lower limit unless otherwise indicated by the context, as well as any other stated value or intervening value in that stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, which are also included within the invention, subject to any specifically excluded ranges in the stated ranges. When a stated range includes one or both of the upper and lower limits, ranges excluding one or both of those included upper and lower limits are also included in the invention. For example, any concentration range, percentage range, ratio range, or integer range provided herein should be understood to include any integer value within the recited range, and fractions thereof, where appropriate (e.g., integer tenths and hundredths, etc.), unless otherwise indicated. Additionally, any numerical range recited herein relating to any physical characteristic, such as polymer subunits, size, or thickness, should also be understood to include any integer within the recited range, unless otherwise indicated. As used herein, the term "about" means ±20% of the indicated range, value, or structure, unless otherwise indicated.

[0127] All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications mentioned herein and / or listed in the Application Data Sheet are incorporated herein by reference in their entirety. Such publications are incorporated by reference, for example, for the purpose of describing and disclosing the materials and methodologies described in the publications that may be used in connection with the invention described herein. The publications discussed above and throughout the text are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the inventors are not entitled to antedate any referenced publication by prior invention. All patents, publications, scientific articles, websites, and other documents and materials referenced or mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains, and each such referenced document and material is incorporated herein by reference to the same extent as if it were individually incorporated herein by reference in its entirety or set forth in its entirety. Applicant reserves the right to physically incorporate into this specification any and all materials and information from any such patents, publications, scientific articles, websites, electronically available information, and other referenced materials or documents.

[0128] Generally, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited to the present disclosure. Moreover, any portion of the written description in this patent includes all claims. Moreover, all claims, including all original claims as well as all claims from any priority document, are hereby incorporated by reference in their entirety into the written description herein, and the applicant reserves the right to physically incorporate any such claims into the written description or any other part of this application. Thus, for example, under no circumstances should this patent be construed as not providing a description of a claim because the exact wording of the claim is not recited in that exact wording in any portion of the description herein.

[0129] The claims will be interpreted according to law. However, nevertheless, any alleged or perceived ease or difficulty in interpreting any claim or part thereof may not, under any circumstances, cause any adjustment or modification of the claim or any part thereof at the time of filing of this application(s) to be interpreted as a waiver by this patent of any right to any equivalent thereof that does not form part of the prior art. Other non-limiting embodiments are within the scope of the following claims. This patent may not be construed as being limited to the specific examples or non-limiting embodiments or methods specifically and / or explicitly disclosed herein. Under no circumstances may this patent be construed as being limited by any examiner or any other officer or employee of the Patent and Trademark Office unless such a claim is expressly adopted in the applicant's reply brief, specifically and without conditions or limitations.

Claims

[Claim 1] A recombinant herpesvirus comprising a modified oncolytic herpesvirus genome, said modified herpesvirus genome comprising at least one miRNA target sequence operably linked to a first copy of an ICP34.5 gene and a second copy of the ICP34.5 gene comprising an inactivating mutation, wherein 1) said mutation is a deletion of at least one long terminal repeat region of the viral genome, 2) said modified herpesvirus genome comprises additional mutations or modifications in the viral genes ICP4 and / or ICP27, 3) further comprising at least one nucleic acid encoding a non-viral protein selected from the group consisting of an immune stimulator, an antibody, and a checkpoint inhibitor peptide, said at least one nucleic acid being operably linked to a general promoter or a tumor-specific promoter, or 4) a glycoprotein. and further comprising a nucleic acid sequence encoding a fusogenic form of substrate B. The recombinant herpes virus.

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