Hsv vector with reduced neurotoxicity

Abstract

To provide recombinant herpes simplex viruses having a modified oncolytic herpes virus genome, and also provide pharmaceutical compositions having such recombinant herpes simplex viruses, as well as methods of using such compositions in the treatment of subject animals having cancer.SOLUTION: In the present invention, the modified herpes virus genome has at least one miRNA target sequence operably linked to a first copy or to first and second copies of an ICP34.5 gene.SELECTED DRAWING: None

Description

[Technical field]

[0001] RELATED APPLICATIONS This application claims the benefit of 35 USC § 119e of U.S. Provisional Patent Application No. 62 / 773,119, filed November 29, 2018, which is incorporated herein by reference in its entirety for all purposes. (Technical field) The present invention relates generally to HSV vectors with reduced neurovirulence. [Background technology]

[0002] Oncolytic virus therapy has been recognized as a promising novel therapeutic approach for cancer treatment because oncolytic viruses cause potent tumor lysis and induce systemic tumor-specific immunity, while causing significantly fewer side effects than chemotherapy or radiation therapy. Among the various OVs, herpes simplex virus type 1 ("HSV-1")-based OVs are the most advanced. For example, herpes virus-based OV (T-Vec) has been approved by the U.S. Food and Drug Administration for the treatment of melanoma. Representative examples of HSV vectors include those described in U.S. Patent Nos. 7,223,593, 7,537,924, 7,063,835, 7,063,851, 7,118,755, 8,277,818, and 8,680,068. One of the challenges of oncolytic herpesvirus vectors is the neurotropic nature of HSV.Neuroinvasiveness is mainly mediated by viral protein ICP34.5, which leads to the general strategy of deleting ICP34.5 from vectors used in oncolytic virotherapy.However, complete deletion of ICP34.5 reduces viral replication capacity by approximately 10-fold in a wide range of tissues.The present invention overcomes certain challenges of traditional HSV vectors and also provides other related advantages. Not all of the material discussed in the Background Art is necessarily prior art, and should not be assumed to be prior art merely as a result of being discussed in the Prior Art. Along these lines, any recognition of a prior art problem discussed in the Background Art or related to such material should not be considered prior art unless expressly stated to be prior art. Rather, discussion of any material in the Background Art should be considered part of the inventor's approach to the problem at hand, which may also be innovative in and of itself. Summary of the Invention [Problem to be solved by the invention]

[0003] The present invention overcomes certain challenges associated with conventional HSV vectors, and further provides other related advantages. [Means for solving the problem]

[0004] Briefly, the present application relates to a recombinant herpes simplex virus (also referred to as an "oHSV vector"), the recombinant virus comprising at least one ICP34.5 gene with at least two miRNA target sequences in the 3' untranslated region of ICP34.5. In certain embodiments, the at least two miRNA target sequences are targets of the same miRNA. In other embodiments, the at least two miRNA target sequences are targets of a miRNA selected from the group consisting of: miR-122, miR-124, miR-124 * , miR-127, miR-128, miR-129, miR-129 * , miR-132, mIR-133a, mIR133b, miR-135b, miR-136, miR-136 *, miR-137, miR-139-5p, miR-143, mIR-145, miR-154, miR-184, miR-188, miR-204, mIR216a, miR-299, miR-300- 3p, miR-300-5p, miR-323, miR-329, miR-337, miR-335, miR-341, miR-369-3p, miR-369-5p, miR-376a, miR-376a * , miR-376b-3p, miR-376b-5p, miR-376c, miR-377, miR-379, miR-379 * , miR-382, miR-382 * , miR-409-5p, miR-410, miR-411, miR-431, miR-433, miR-434, miR-451, miR-466b, miR-485, miR-495, miR-539, miR-541, miR-543 * , miR-551b, miR-758, and miR-873. By convention, the strand that is more frequently found in the end product is designated the miRNA, and the rarer partner is designated the miRNA. * It is shown that: In other embodiments, the recombinant herpes simplex virus further comprises a modified ICP27 or ICP4 gene, wherein the modification is a replacement of the 5'UTR, the promoter-regulatory region, or both the 5'UTR and the promoter-regulatory region. In some embodiments, the 5'UTR is derived from an FGF gene.

[0005] In certain embodiments, the recombinant herpes simplex virus further comprises a genetic sequence encoding at least one immune stimulatory factor, a checkpoint blocking peptide, or both. The present disclosure also provides a method of treating cancer comprising administering a recombinant herpes simplex virus comprising at least one ICP34.5 gene having at least two miRNA target sequences in the 3' untranslated region of ICP34.5. This brief Summary is provided to introduce certain concepts in a simplified form that are described in more detail below in the Detailed Description. Unless otherwise 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 set forth in the following description. Features illustrated or described in connection with one exemplary embodiment may be combined with the features of other embodiments. Other features, objects, and advantages will be apparent from the description, drawings, and claims. Additionally, the disclosures of all patents and patent applications referenced herein are hereby incorporated by reference in their entirety. Exemplary features of the present disclosure, its nature and various advantages will be apparent from the accompanying drawings and the detailed description of various embodiments below. Non-limiting and non-exhaustive embodiments are described with reference to the accompanying drawings, in which the same labels or reference numbers refer to the same parts throughout the various drawings unless otherwise specified. The sizes and relative positions of elements in the drawings are not necessarily drawn to scale. For example, the shapes of the various elements have been selected, enlarged and positioned to improve the legibility of the drawings. The individual shapes of the depicted elements have been selected to facilitate recognition of the drawings. One or more embodiments are described below with reference to the following accompanying drawings: [Brief description of the drawings]

[0006] [Figure 1] FIG. 1 is a schematic diagram of an exemplary HSV vector carrying three different miRNA targets in the 3′ untranslated region of ICP34.5. [Diagram 2] FIG. 1 is a schematic diagram of an exemplary HSV vector carrying a modified γ34.5 gene and a modified ICP4 or ICP27 gene. [Diagram 3] 1 is a graph showing the expression levels of ICP27, ICP4, and ICP47 in the brains of normal mice and mice bearing human brain tumors (U87). [Figure 4]1 is a Western blot showing the expression of ICP34.5 and β-actin in neuronal and tumor cells (LNCaP and A549). [Diagram 5] FIG. 1 is a schematic diagram of a transcriptional and translational dual-regulated virus. [Figure 6] 1 shows various regulatory elements that can be used in the platform viruses. [Figure 7] Photographs of brain sections from mice after intracranial injection of either CXCR4-TF-Fc-h1215 or CXCR4-TF-Fc-h1215-miR viruses. Brain sections were stained with rabbit polyclonal anti-HSV primary antibody and fluorescent rat anti-rabbit secondary antibody. [Figure 8A] 8A shows graphs of cell survival after infection with viruses at various MOIs, and FIG. 8B shows cell survival of lung tumor cells A549 and normal lung cells BEAS-2b. [Figure 8B] Figure 8B is a graph of cell survival after infection with viruses at various MOIs, showing cell survival of lung tumor cells A549 and normal lung cells HPL1D. [Figure 8C] Figure 8C is a graph of cell survival after infection with viruses at various MOIs for lung tumor cells A549, PC9, H460, H23S, and H1975. [Figure 9] 1 is a graph showing VG182LF virus replication in A549 lung tumor cells and BEAS-2b normal lung cells. [Figure 10] 1 is a bar graph showing the increase (fold increase) in IL-12 in A549 lung tumor cells and LNCaP prostate tumor cells following infection with hVG161 or hVG182LF. [Figure 11A] Replication of VG182LF virus in various lung tumor cells is shown in Figure 11A: H1975 cells. [Figure 11B] Replication of VG182LF virus in various lung tumor cells is shown in Figure 11B: H460 cells. [Figure 11C] Replication of VG182LF virus in various lung tumor cells is shown in Fig. 11C: PC9 cells. [Figure 12] 1 is a graph showing tumor size in H1975 tumor-bearing nude mice one week after treatment with either vehicle or VG182LF virus. [Figure 13A] Disclosed is a select list of microRNAs in tumors that can be found in PubMed at https: / / www.ncbi.nlm.nih.gov / pubmed and in the microRNA database ("mIBRASE") at http: / / www.mirbase.org / , both of which are incorporated herein by reference in their entireties. [Figure 13B] Continued from Figure 13A. [Figure 14A] 13 is a graph showing miR-143 transfection efficiency 6 hours post-infection, viral gene expression 6 hours post-infection, and viral replication 24 hours post-infection in 293FT cells, respectively. [Figure 14B] Continued Figure 14A. [Figure 14C] Figure 14B continued. [Figure 15] Photographs showing HSV-1 immunostaining of mouse brain and spinal cord sections. Mice were subcutaneously injected with control vehicle, wild-type HSV-1, an ICP34.5 deleted HSV-1 variant (VG161), or a variant encoding binding sites for miR-143 and miR-124 in the 3'UTR of ICP34.5 (VG301) together with a fusogenic mutation in the carboxyl terminus of gb (gB-876t). [Figure 16] Graph showing survival curves for mice injected subcutaneously with either wild-type HSV-1, an ICP34.5-deleted HSV-1 variant (VG161), or a variant encoding binding sites for miR-143 and miR-124 in the 3'UTR of ICP34.5 together with a fusogenic mutation in the carboxyl terminus of gb (gB-876t) (VG301). [Figure 17]Photographs showing the results of a fusion assay, in which cells were fixed and stained with Giemsa to visualize viral plaques and syncytia resulting from virus-induced cell fusion. Cells were infected with recombinant oncolytic HSV-1 with (+gB-876t) or without (-gB-876t) a fusogenic mutation in the carboxyl terminus of gB. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0007] Detailed Description of the Invention The present invention may be understood more readily by reference to the following detailed description of the preferred invention embodiments and the examples included therein. The term "microRNA" or "miRNA" as used herein refers to a family of short (typically 21-25 nucleotides), endogenous, single-stranded RNAs expressed in a wide range of organisms, including animals and plants. Over 1000 unique miRNAs are expressed in humans. miRNAs bind to specific target sequences found in messenger RNA (mRNA). Binding to complementary or partially complementary sequences (target sequences) in mRNA molecules results in downregulation of gene expression by cleavage of the mRNA, its increased degradation due to shortening of its polyA tail, and direct translational repression. A selected list of microRNAs in tumors (along with related literature) is provided in Figures 13A and 13B (said list and related literature are incorporated herein by reference in their entirety). The term "oncolytic herpesvirus" or "oHSV" generally refers to a herpesvirus that is capable of replicating in and killing tumor cells. In certain embodiments, the virus can be engineered to more selectively target tumor cells. Representative examples of oncolytic herpesviruses are described in U.S. Patent 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 herein by reference in their entireties. As used herein, the terms "treat" and "treatment" refer to an approach for obtaining beneficial or desired results (including clinical results). Beneficial or desired clinical results include, but are not limited to, reduction or alleviation of one or more symptoms or symptoms, whether detectable or undetectable, reduction in the extent of disease, stabilization (i.e., not worsening) of disease, prevention of disease spread, slowing or slowing of disease progression, mitigation or palliative treatment of disease, reduction in recurrence of disease, and remission (whether partial or complete). The terms "treat" and "treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment.

[0008] Representative forms of cancer include carcinoma, leukemia, lymphoma, myeloma, and sarcoma. Further examples include, but are not limited to, cancers of the bile duct, brain (glioblastoma), breast, cervix, colorectum, CNS (e.g., acoustic neuroma, astrocytoma, craniopharyogioma, ependymoma, glioblastoma, hemangioblastoma, medulloblastoma, menangioma, neuroblastoma, oligodendroglioma, pinealoma, and retinoblastoma), 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), and thyroid. The cancer may include a solid tumor (e.g., a sarcoma, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, and osteogenic sarcoma), may be diffuse (e.g., a leukemia), or any combination of the above (e.g., a metastatic cancer having both a solid tumor and disseminated or diffuse cancer cells). Particularly preferred cancers for treatment include lung tumors, breast and prostate tumors, glioblastoma, tumors of the gastrointestinal tract (and associated organs, e.g., esophagus, cholangiocarcinoma, anus, stomach, intestine, pancreas, colon and liver), and all superficial injectable tumors (e.g., melanoma). Benign tumors and other conditions of unwanted cell proliferation may also be treated. For a further understanding of the various embodiments herein, the following sections describing the various embodiments are provided: A. Oncolytic Herpesviruses; B. MicroRNAs; C. Therapeutic Compositions; and D. Administration.

[0009] A. Oncolytic herpesviruses Herpes simplex virus (HSV) types 1 and 2 are members of the Herpesviridae family that infect humans. The HSV genome contains two unique regions, which are unique long (U L ) and unique short (U s ) regions. Each of these regions is flanked by a pair of inverted terminal repeat sequences. There are about 75 known open reading frames. The viral genome has been manipulated to develop oncolytic viruses for use in, for example, cancer therapy. Tumor-specific 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 ICP34.5 are known to lack neurovirulence (i.e., non-virulent / non-neurovirulent) and be oncolytic. Tumor-selective replication of HSV can also be conferred by controlling the expression of key viral genes (e.g., ICP27 and / or ICP4).

[0010] 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 or 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 or 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), G2O7 (Mineta et al. Nature Medicine. 1(9):938-943, 1995; Kooby et al. The FASEB Journal, 13(11):1325-1334, 1999); G47 delta (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 Gene 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 vectors described in the following 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] The oHSV vector has at least one γ34.5 gene modified with respect to the miRNA target sequence in its 3'UTR as disclosed herein, and there is no unmodified γ34.5 gene in the vector. In some embodiments, the oHSV has two modified γ34.5 genes, and 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. Either native γ34.5 gene can be deleted. In some embodiments, the terminal repeat (including the γ34.5 gene and the ICP4 gene) is deleted. As discussed herein, the modified γ34.5 gene can include additional changes (e.g., having an exogenous promoter). The oHSV may contain additional mutations, including disabling mutations (e.g., deletions, substitutions, insertions), that affect the virulence of the virus or its ability to replicate. For example, mutations may occur in any one or more of ICP6, ICPO, ICP4, ICP27, ICP47, ICP24, ICP56. Preferably, a mutation in one of these genes (possibly occurring in both copies of the gene, where appropriate) results in the inability (or reduced ability) of the HSV 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 that can be induced upon delivery of an inducing agent or that can be induced upon a cellular event or in a particular environment. In certain embodiments, 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 also be present. In some cases, enhancers are present (e.g., NFkB / oct4 / sox2 enhancers). 5'UTRs may also be exogenous (e.g., 5'UTRs from growth factor genes (e.g., FGF)). See FIG. 2 for exemplary constructs.

[0012] oHSVs can also have genes and nucleotide sequences that are non-HSV in origin. For example, sequences encoding prodrugs, cytokines or other immune stimulants, tumor-specific promoters, inducible promoters, sequences encoding enhancers, sequences homologous to the host cell, among others, can be present in the oHSV genome. Exemplary sequences encode IL12, IL15, IL15 receptor alpha subunit, OX40L, PD-L1 blockers or PD-1 blockers. For product-encoding sequences, they are operably linked to promoter sequences and other regulatory sequences necessary or desired for expression (e.g. enhancers, polyadenylation signal sequences). The regulatory region of the viral gene can be modified to include response elements that affect expression. Exemplary response elements include NF-κB, Oct-3 / 4-SOX2, enhancers, response elements for silencers, cAMP response elements, CAAT enhancer binding sequences, and insulators. Other response elements can also be included. The viral promoter can be replaced with a different promoter. The choice of promoter will depend on a number of factors, such as the proposed use of the HSV vector, the treatment of the patient, the disease status or symptoms, and the ease of application of the inducer (for inducible promoters). For cancer treatment, when the promoter is replaced, it will generally be replaced with 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 can also be modified. For example, the 5'UTR of the viral gene can be replaced with an exogenous UTR.

[0013] B. MicroRNA As mentioned above, the present invention provides an oHSV having at least two miRNA target sequences. Briefly, the miRNA binds to a target sequence within the mRNA, typically at the 3'-untranslated region (3'-UTR). Binding may initiate or require a region located approximately 2-8 nucleotides from the 5' end of the miRNA, called the "seed region". When partial complementarity exists, the 5' end tends to have greater identity to the target sequence than the 3' end. Higher complementarity can enhance the suppression of the mRNA, particularly through mRNA cleavage. Individual miRNAs and miRNA groups may be exclusively or preferentially expressed in certain tissue types. miRNAs that are abundant or exclusive in neuronal cells include: miR-122, miR-124, miR-124 * , miR-127, miR-128, miR-129, miR-129 * , miR-132, mIR-133a, mIR133b, miR-135b, miR-136, miR-136 *, miR-137, miR-139-5p, miR-143, mIR-145, miR-154, miR-184, miR-188, miR-204, mIR216a, miR-299, miR-300- 3p, miR-300-5p, miR-323, miR-329, miR-337, miR-335, miR-341, miR-369-3p, miR-369-5p, miR-376a, miR-376a * , miR-376b-3p, miR-376b-5p, miR-376c, miR-377, miR-379, miR-379 * , miR-382, miR-382 * , miR-409-5p, miR-410, miR-411, miR-431, miR-433, miR-434, miR-451, miR-466b, miR-485, miR-495, miR-539, miR-541, miR-543 * , miR-551b, miR-758, and miR-873. By convention, the strand that is more frequently found in the final product is designated as the miRNA, and the rarer partner is designated as the miRNA. * A selected list of microRNAs in tumors (along with associated literature) is provided in Figures 13A and 13B (said list and associated literature are incorporated herein by reference in their entirety).

[0014] The miRNA target sequence is inserted into the 3'UTR of the γ34.5 gene. There are at least two miRNA target sequences inserted in tandem. There may be at least three, at least four, at least five, at least six, at least ten, and so forth. In other embodiments, there are less than 10, 20, 50, or 100 target sequences. The optimal number of target sequences can be determined by assaying the expression level of ICP34.5. The multiple miRNA target sequences may all bind to the same miRNA or may bind to different miRNAs. The target sequences may be in a cluster (e.g., FIG. 1), where, for example, there are at least two target sequences in tandem that bind a first miRNA, which are followed by at least two target sequences in tandem that bind a second miRNA, which are followed by at least two target sequences that bind a third miRNA. Alternatively, the multiple miRNA target sequences that bind different miRNAs may not be in a particular order. Furthermore, only one copy of each miRNA target sequence may be present. In some embodiments, there are 3-5 different miRNA target sequences. In other embodiments, there are 3-5 copies of each target sequence. In other embodiments, there are 3-5 different miRNA target sequences, with 3-5 copies of each of these target sequences present in a cluster. See FIG. 1 for an exemplary construct. Multiple miRNA target sequences can be adjacent with no intervening sequences or have 1 to about 25, or 1 to about 20, or 1 to about 15, or 1 to about 10, or 1 to about 5, or 3 to about 10, or 5 to about 10 intervening nucleotides. The intervening nucleotides can be selected to have a similar G+C content as the 3'UTR and preferably do not include a polyadenylation signal sequence. Other considerations for selecting intervening nucleotides are known in the art.

[0015] In certain embodiments of the invention, the oHSVs described herein are constructed to take advantage of dual regulation of both transcription and translation (also referred to as "TTDR"). An exemplary diagram of such a vector is provided in FIG. 5. Briefly, in certain preferred embodiments, translational control of the ICP34.5 gene is achieved by inserting five copies of binding sites for miR-124 and miR-143 into the 3'-UTR of the ICP34.5 gene. An important element of the platform virus vector can also include transcriptional control of the ICP27 gene (a gene essential for viral replication) using a tumor-specific promoter. A wide variety of HSV-1 strains can be used as backbones for the construction of oncolytic recombinant viruses, including strain 17, strain KOS, strain F, and strain McKrae. All viral mutagenesis was performed in Escherichia coli using standard lambda Red-mediated recombination techniques performed on the HSV-1 genome cloned in a bacterial artificial chromosome (BAC) (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 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).

[0016] Tumor-specific promoters can also be used to drive expression of cassettes encoding immunomodulators IL12 / IL15 / IL15RA (boosting anti-tumor immunity). The immunomodulator expression cassettes can be controlled by hCEA, hCXCR4 or PSA promoters and can be inserted into the viral genome at locations that do not negatively affect viral gene expression and replication (e.g., viral intergenic US1 / US2, UL3 / UL4 and / or UL50 / UL51). Other recombinant viruses can be constructed that express mouse IL12 instead of human IL12 to facilitate in vivo testing in various mouse models. Human IL15 can be retained in mouse-specific oncolytic viruses for its activity in mouse cells. The vector can include an expression cassette encoding a fusion form of the env protein of Gibbon Ape Leukemia Virus (GALV) lacking the C-terminal R-peptide (enhancing viral cytotoxicity). In other embodiments, the expression cassette can encode a fusion form of HSV-1 glycoprotein B. In certain preferred embodiments, glycoprotein B can be truncated (e.g., by a deletion occurring after amino acid 876 of gB) ("gB-876"). The cassette can be inserted into the viral genome at a location that does not have a negative effect on viral gene expression and replication (e.g., viral intergenic US1 / US2, UL3 / UL4, and / or UL50 / UL51). BAC recombination engineering requires the presence of exogenous BAC DNA in the viral genome to facilitate mutagenesis in E. coli. BAC sequences are most commonly inserted between viral genes (e.g., US1 / US2, UL3 / UL4, and / or UL50 / UL51) or into the thymidine kinase (TK) gene (which can disrupt expression of native TK). TK-deficient viral vectors can contain an expression cassette for the HSV-1 thymidine kinase (TK) gene, under the control of a constitutive promoter inserted into a non-coding region of the viral genome. The presence of an exogenous TK gene enhances viral safety by making the virus susceptible to routine treatment with guanosine analogs (e.g., ganciclovir and acyclovir).

[0017] In another embodiment, the originally disrupted TK can be restored instead of inserting another TK, or the TK gene can be disrupted without replacement or restoration at all to further reduce neurovirulence (TK null viruses cannot reactivate from latency). Even if TK is disrupted, the virus will still be sensitive to treatment with drugs that do not depend on TK for their function. For example, foscarnet and cidofovir inhibit viral DNA polymerase and are not TK dependent. The promoter driving expression of the key HSV-1 transcriptional regulator ICP27 can be replaced with a tumor-specific promoter (e.g., hCEA, hCXCR4, PSA, or probasin (ARR2PB)). The 3'UTR of the viral gene encoding the neurovirulence factor ICP34.5 can also be modified by inserting multiple copies of a microRNA recognition element to silence ICP34.5 production in tissues that contain high levels of the corresponding microRNA. In an exemplary embodiment, five copies of miR-124 and five copies of miR-143 recognition elements can be inserted in tandem into the 3'UTR of ICP34.5. The terminal repeat regions of the viral genome can be deleted completely to reduce the overall genome size and create more room for transgene insertion, and the deleted TRs are engineered to avoid disrupting the native promoter of the ICP47 gene (which is normally part of the terminal repeat). Similar modifications can be made by deleting the internal repeat regions instead of the terminal repeat regions. Further details of the exemplary elements discussed herein are illustrated in FIG.

[0018] C. Therapeutic composition Therapeutic compositions are provided that can be used to prevent, treat, or ameliorate the effects of disease, such as cancer. More specifically, 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 "pharma-ceutically 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 the subject to which the composition is administered (see generally: Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21st ed. (May 1, 2005 and in The United States PharmacopE1A: The National Formulary (USP 40-NF 35 and supplements). In the case of the oncolytic viruses described herein, non-limiting examples of suitable pharma- ceutically acceptable carriers include phosphate buffered saline, water, emulsions (e.g., oil / water emulsions), various types of wetting agents, sterile solutions, and others. Additional pharma- ceutically acceptable carriers include gels containing the oncolytic viruses, bioabsorbable matrix materials, implant components, or any other suitable vehicle, delivery or distribution means or material. Such carriers can be formulated in conventional manner and administered to the subject in an effective dose. Additional pharma- ceutically acceptable excipients include, but are not limited to, water, saline, polyethylene glycol, hyaluronic acid, and ethanol. Pharmaceutically acceptable salts also include herein, for example, mineral acid salts (such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like), and salts of organic acids (such as acetates, propionates, malonates, benzoates, and the like). Such pharma- ceutically acceptable (pharmaceutical grade) carriers, diluents and excipients that can be used to deliver oHSV to cancer cells will preferably not induce an immune response in the individual (subject animal) receiving the composition (and will preferably be administered without undue toxicity).

[0019] The compositions provided herein can be provided in a variety of concentrations. For example, the dosage of the oncolytic virus is about 10 6 About 10 9 In yet another embodiment, the dosage may be provided in the range of about 10 pfu. 6 About 10 8 In the range of pfu / mL, up to 4 mL for patients with large lesions (e.g., >5 cm) and smaller amounts (e.g., up to 0.1 mL) for patients with small lesions (e.g., <0.5 cm) may be injected every 2-3 weeks for treatment. In certain embodiments of the present invention, lower than standard dosages can be used. Thus, in certain embodiments, about 10 6 Less than pfu / mL may be administered to patients (maximum of 4 mL injected into the patient every 2-3 weeks). The compositions can be stored at temperatures conducive to a stable shelf life, including room temperature (about 20° C.), 4° C., −20° C., −80° C., and liquid nitrogen. Compositions intended for in vivo use do not contain preservatives, so storage will generally be at lower temperatures. The compositions can be stored in dry (e.g., lyophilized) or liquid form.

[0020] D. Administration In addition to the compositions described herein, various methods of using such compositions to alleviate cancer are provided, the methods comprising administering to a subject an effective dose of an oHSV described herein. The terms "effective dose" and "effective amount" refer to an amount of oncolytic virus that is sufficient to achieve treatment of a target cancer, e.g., an amount that is effective to reduce the target tumor size or tumor load or otherwise impede the proliferation rate of target tumor cells. More specifically, such terms refer to an amount of oncolytic virus that is effective to achieve a desired result at the required dosage and treatment period. For example, in the context of cancer treatment, an effective amount of the compositions described herein is an amount that induces remission, reduces tumor burden, and / or prevents tumor spread or cancer growth. An effective amount may vary according to several factors (e.g., the stage, age, sex, and weight of the subject animal 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 non-human animal.

[0021] The composition is used to treat cancer. As used herein, the terms "treat" and "treatment" refer to an approach to achieve a beneficial or desired result, including a clinical result. Beneficial or desired clinical results include, but are not limited to, reduction or alleviation of one or more symptoms or symptoms, whether detectable or undetectable, reduction in the extent of disease, stabilization (i.e., not worsening) of disease, prevention of disease spread, delay or slowing of disease progression, mitigation or palliative treatment of disease stage, reduction in recurrence of disease, and remission (whether partial or complete). The terms "treat" and "treatment" can also refer to prolonging survival as compared to expected survival if not receiving treatment.

[0022] Representative forms of cancer include carcinoma, leukemia, lymphoma, myeloma and sarcoma. Further examples include (but are not limited to) cancers of the bile duct, brain (glioblastoma), breast, cervix, colorectum, CNS (e.g. acoustic neuroma, astrocytoma, craniopharyogioma, ependymoma, glioblastoma, hemangioblastoma, medulloblastoma, menangioma, neuroblastoma, oligodendroglioma, pinealoma and retinoblastoma), 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 a solid tumor (e.g., a sarcoma, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, and osteogenic sarcoma), may be diffuse (e.g., a leukemia), or any combination of the above (e.g., a metastatic cancer having both a solid tumor and disseminated or diffuse cancer cells). The cancer may also be resistant to conventional treatments (e.g., conventional chemotherapy and / or radiation therapy). Particularly preferred cancers for treatment include lung tumors, breast and prostate tumors, glioblastoma, tumors of the gastrointestinal tract (and associated organs, e.g., esophagus, bile duct cancer, anus, stomach, intestine, pancreas, colon and liver), and all superficial injectable tumors (e.g., melanoma). Benign tumors and other conditions of unwanted cell proliferation may also be treated. The recombinant herpes simplex viruses described herein can be administered, for example, by oral, topical, parenteral, systemic, intravenous, intramuscular, intraocular, intrathecal, intratumoral, subcutaneous, or transdermal routes. In certain embodiments, the oncolytic viruses can be delivered by cannula, catheter, or by direct injection. The site of administration can be within the tumor or at a site distant from the tumor. The route of administration will often depend on the type of cancer being targeted.

[0023] Optimal or appropriate dosing regimens for oncolytic viruses are readily determined within the skill of the art by the attending physician based on patient data, patient examination, and various clinical considerations, including, for example, the subject's size, body surface area, age, sex, and the specific oncolytic virus administered, the time and route of administration, the type of cancer being treated, the patient's general health, and other drug therapies the patient is receiving. According to certain embodiments, treatment of a subject with an oncolytic virus described herein may be combined with an additional type of treatment, such as chemotherapy using chemotherapeutic agents, such as etoposide, ifosfamide, adriamycin, vincristine, doxycycline, and others. The recombinant herpes simplex viruses described herein can be formulated as medicaments and pharmaceutical compositions 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 inhibitor in a sterile medium. The formulation can be in liquid, gel, paste, or solid form. The formulation can be provided to the subject or to a medical professional. Preferably, a therapeutically effective amount is administered, said amount being 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 considerations. In yet other embodiments of the invention, the oncolytic virus can be administered in a variety of ways, for example, intratumorally, intravenously, or following surgical resection of the tumor. The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with any condition or negative limitation that removes any generic content (whether or not the removed content is specifically recited herein).

[0024] The following are additional exemplary embodiments of the present disclosure: 1) A recombinant herpes simplex virus comprising at least one ICP34.5 gene with at least two miRNA target sequences in the 3' untranslated region of ICP34.5. In a related embodiment, a recombinant herpes simplex virus is provided comprising a modified oncolytic herpesvirus genome, the modified herpesvirus genome comprising at least one miRNA target sequence operably linked to a first or first and second copies of the ICP34.5 gene. 2) A recombinant herpes simplex virus as described in embodiment 1, wherein the at least two miRNA target sequences are targets of the same miRNA. 3) The recombinant herpes simplex virus according to any of the preceding claims 1 or 2, wherein the at least two miRNA target sequences are targets of miRNAs selected from the group consisting of: miR-122, miR-124, miR-124 * , miR-127, miR-128, miR-129, miR-129 * , miR-132, mIR-133a, mIR133b, miR-135b, miR-136, miR-136 * , miR-137, miR-139-5p, miR-143, mIR-145, miR-154, miR-184, miR-188, miR-204, mIR216a, miR-299, miR-300- 3p, miR-300-5p, miR-323, miR-329, miR-337, miR-335, miR-341, miR-369-3p, miR-369-5p, miR-376a, miR-376a *, miR-376b-3p, miR-376b-5p, miR-376c, miR-377, miR-379, miR-379 * , miR-382, miR-382 * , miR-409-5p, miR-410, miR-411, miR-431, miR-433, miR-434, miR-451, miR-466b, miR-485, miR-495, miR-539, miR-541, miR-543 * , miR-551b, miR-758, and miR-873. By convention, the strand that is more frequently found in the final product is designated as the miRNA, and the rarer partner is designated as the miRNA. * In certain embodiments of the invention, a recombinant herpes simplex virus according to embodiment 1, 2 or 3 is provided, wherein the miRNA target site comprises 1, 2, 3, 4, 5, 6 or more than 6 copies of the binding sites for miR-124 and miR-143.

[0025] 4) A recombinant herpes simplex virus according to any one of embodiments 1-3, further comprising a modified ICP27 or ICP4 gene, wherein the modification is a replacement of the 5'UTR. In another embodiment, the ICP27 or ICP4 gene is modified by replacement of the native promoter. In a particularly preferred embodiment of the invention, ICP27 is modified by replacement of the native promoter with the hCEA promoter or the hCXCR4 promoter. 5) A recombinant herpes simplex virus according to any one of embodiments 1, 2, 3 or 4, further comprising a modified ICP27, wherein the modification is a replacement of the complete promoter-regulatory region of ICP27. In a further embodiment of the above, the herpes simplex virus is HSV-1. In a further embodiment of any one of embodiments 1, 2, 3 or 4, the recombinant herpes simplex virus further comprises a fusogenic mutation in the gene encoding glycoprotein B (gB). In a related embodiment, the gene encoding glycoprotein B (gB) encodes a glycoprotein B variant that terminates after amino acid 876. In yet another embodiment, a recombinant herpes simplex virus according to any one of embodiments 1, 2, 3 or 4 is provided, wherein the genome further comprises a modified gene encoding glycoprotein B (gB), wherein the modified gene encodes a glycoprotein B variant that terminates after amino acid 876. In yet another embodiment, the recombinant herpes simplex virus comprises an additional mutation or modification in at least one viral gene selected from the group consisting of ICP6, ICP0, ICP4, ICP27, ICP47, ICP24, and ICP56, hi certain preferred embodiments, the additional mutation or modification is in a non-coding region of the viral gene.

[0026] 6) The recombinant herpes simplex virus of any one of embodiments 1, 2, 3, 4 or 5, further comprising a gene sequence encoding at least one immune stimulator. Exemplary immune stimulators include IL12, IL15, IL15 receptor alpha subunit, OX40L, and PD-L1 blockers. 7) The recombinant herpes simplex virus of any of embodiments 1, 2, 3, 4, 5, or 6, further comprising a genetic sequence encoding an immune stimulator or a checkpoint blocking peptide. In further features of embodiments 1, 2, 3, 4, or 5, the recombinant herpes simplex virus further comprises at least one nucleic acid encoding a non-viral protein selected from the group consisting of an immune stimulator, an antibody, and a checkpoint blocking peptide. In a related embodiment, the at least one nucleic acid is operably linked to a tumor-specific promoter. 8) A method for treating cancer, comprising administering a recombinant herpes simplex virus according to any of embodiments 1-7. Particularly preferred cancers to be treated include lung tumors, breast and prostate tumors, glioblastomas, tumors of the gastrointestinal tract (and associated organs, e.g. esophagus, cholangiocarcinoma, anus, stomach, intestine, pancreas, colon and liver), and all superficial injectable tumors (e.g. melanoma).

[0027] The following is yet another embodiment of the present invention: 9) A recombinant herpes simplex virus comprising a modified oncolytic herpes virus genome, the modified herpes virus genome comprising at least one miRNA target sequence operably linked to a first or first and second copies of an ICP34.5 gene. In a preferred embodiment, the herpes simplex virus produces significantly lower levels of functional ICP34.5 protein in non-transformed cells compared to tumor cells. 10) A recombinant herpes simplex virus according to embodiment 9, wherein the second copy of the ICP34.5 gene contains an inactivating mutation. 11) A recombinant herpes simplex virus according to embodiment 9, comprising two to ten miRNA target sequences operably linked to the first or the first and second copies of the ICP34.5 gene. 12) A recombinant herpes simplex virus according to embodiment 10 or 11, comprising two miRNA target sequences operably linked to the first or the first and second copies of the ICP34.5 gene. 13) A recombinant herpes simplex virus according to embodiment 10 or 11, wherein the miRNA target sequence is inserted into the 3' untranslated region of the first or the first and second copies of the ICP34.5 gene. 14) A recombinant herpes simplex virus according to embodiment 13, wherein the miRNA target sequences are inserted in tandem in the 3' untranslated region. 15) A recombinant herpes simplex virus described in embodiment 10 or 11, wherein 2 to 10 miRNA target sequences bind to a single miRNA. 16) A recombinant herpes simplex virus described in embodiment 10 or 11, wherein the 2 to 10 miRNA target sequences bind to at least two different miRNAs.

[0028] 17) A recombinant herpes simplex virus according to embodiment 15 or 16, in which the miRNA is selected from the group consisting of: mIR-122, miR-124, miR-124 * , miR-127, miR-128, miR-129, miR-129 * , miR-132, mIR-133a, mIR133b, miR-135b, miR-136, miR-136 * , miR-137, miR-139-5p, miR-143, mIR-145, miR-154, miR-184, miR-188, miR-204, mIR216a, miR-299, miR-300- 3p, miR-300-5p, miR-323, miR-329, miR-337, miR-335, miR-341, miR-369-3p, miR-369-5p, miR-376a, miR-376a * , miR-376b-3p, miR-376b-5p, miR-376c, miR-377, miR-379, miR-379 * , miR-382, miR-382 * , miR-409-5p, miR-410, miR-411, miR-431, miR-433, miR-434, miR-451, miR-466b, miR-485, miR-495, miR-539, miR-541, miR-543 * , miR-551b, miR-758, and miR-873. By convention, the strand that is more frequently found in the end product is designated the miRNA, and the rarer partner is designated the miRNA. * It is shown that: 18) A recombinant herpes simplex virus described in embodiment 17, wherein the miRNA target site contains 5 copies of binding sites for miR-124 and miR-143. 19) The recombinant herpes simplex virus described in embodiment 9, wherein the oncolytic herpes virus is HSV-1. 20) The recombinant herpes simplex virus according to embodiment 9, wherein the modified herpesvirus genome comprises additional mutations or modifications in at least one viral gene selected from the group consisting of ICP6, ICP0, ICP4, ICP27, ICP47, ICP24 and ICP56. In a preferred embodiment, the coding sequence remains intact and the viral gene is modified by replacing the native promoter with a tumor-specific promoter. 21) A recombinant herpes simplex virus according to embodiment 20, in which additional mutations or modifications affect the virulence of the virus or its replicative activity. 22) A recombinant herpes simplex virus according to embodiment 20, wherein the mutated or modified viral gene is ICP4 and / or ICP27. 23) A recombinant herpes simplex virus according to embodiment 22, wherein the mutation or modification comprises an operable linkage of the ICP4 and ICP27 genes with an exogenous 5' untranslated region.

[0029] 24) A recombinant herpes simplex virus as described in embodiment 23, further comprising a modified ICP27 or ICP4 gene, wherein the modification is a replacement of the 5'UTR. 25) The recombinant herpes simplex virus of embodiment 22, further comprising a modified ICP27, the modification being a replacement of the complete promoter-regulatory region of ICP27. In certain embodiments, the ICP27 promoter is replaced with the hCEA or hCXCR4 promoter. In certain embodiments, only a portion of the promoter region is replaced, and the native 5'UTR is retained. 26) The recombinant herpes simplex virus of embodiment 25, 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 blocking peptide, wherein the at least one nucleic acid is operably linked to a tumor-specific promoter. 27) The recombinant herpes simplex virus of embodiment 26, wherein the non-viral protein is selected from the group consisting of IL12, IL15, IL15 receptor alpha subunit, OX40L, and PD-L1 blocker. 28) A recombinant herpes simplex virus according to any one of embodiments 1 to 27, further comprising an expression cassette having a nucleic acid sequence encoding a fusion variant of the env protein of Gibbon Ape Leukemia Virus lacking the C-terminal R-peptide, optionally wherein the nucleic acid encodes HSV-1 thymidine kinase. In other embodiments, a recombinant herpes simplex virus according to any one of embodiments 1 to 27 is provided, comprising an expression cassette having a nucleic acid sequence encoding a fusion form of HSV-1 glycoprotein B. In certain preferred embodiments, glycoprotein B can be truncated (e.g., by a deletion occurring after amino acid 876 of gB). 29) A recombinant herpes simplex virus according to any one of embodiments 1 to 28, in which at least one internal or terminal repeat region of the viral genome is deleted. In certain further embodiments, the recombinant herpes simplex virus according to any one of embodiments 1 to 28 has 5x miR-124 and 5x miR-143 binding sites in the 3'UTR of ICP34.5, and the terminal repeat is deleted (ICP0, ICP4 and the second copy of ICP34.5 are also deleted). 30) A method for lysing tumor cells, comprising the step of providing a therapeutically effective amount of a recombinant herpes simplex virus according to any one of embodiments 1 to 29 above. 31) A therapeutic composition comprising a recombinant herpes simplex virus according to any one of embodiments 1 to 29 above and a pharma- ceutically acceptable carrier. 32) A method for treating cancer in a patient suffering from cancer, comprising administering a therapeutically effective amount of the composition according to embodiment 31. Particularly preferred cancers to be treated include lung tumors, breast and prostate tumors, glioblastoma, tumors of the gastrointestinal tract (and associated organs, e.g. esophagus, bile duct cancer, anus, stomach, intestine, pancreas, colon and liver), and all superficial injectable tumors (e.g. melanoma). EXAMPLES

[0030] HSV-1 immediate early gene expression in normal mouse brain and human brain tumor U87 In this example, we compared HSV-1 immediate early gene expression 24 hours after injection of a microRNA-regulated virus in normal mouse brain and human brain tumor U87. Five nude mice without tumors and five nude mice carrying human U87 brain tumors in the cranial cavity were injected with a total of 1x10 6 PFU / mouse were intracranially injected with either CXCR4-miR virus or control CXCR4 virus. The CXCR4-miR virus was engineered to insert five miR-124 / 143 binding sites in tandem into the 3'UTR of ICP34.5 and the viral ICP27 gene was modified to replace the native ICP27 promoter with the tumor-specific CXCR4 promoter. The construct also contains an expression cassette for secretable IL12 / IL15 / IL15RA and a secretable peptide that inhibits PD-1 binding to PD-L1. The CXCR4 virus contains wild-type ICP34.5 lacking the microRNA binding site but is otherwise identical to the CXCR4-miR virus. The expression of viral immediate early genes ICP27, ICP4, and ICP47 in normal brain and tumor tissues was measured 24 hours after injection using RT-qPCR. Changes in gene expression levels of CXCR4-miR viruses were determined by comparing them with gene expression of CXCR4 viruses. Actin expression was used for normalization. Adjusted p-values ​​were calculated using the Bonferroni-Sidak method. Figure 3 shows that CXCR4-miR virus-treated mice exhibited a highly significant (p<0.01) reduction in expression of all tested viral genes in normal brain tissue, but maintained high levels of viral gene expression in tumors. These results suggest that miRNA virus-dependent downregulation of ICP34.5 gene expression reduces HSV-1 replication in normal brain tissue versus brain tumor tissue. EXAMPLES

[0031] Expression of ICP34.5 in neurons and tumor cells This example shows the expression of ICP34.5 protein in neurons and tumor cells after injection with either CXCR4-miR or control CXCR4 viruses. Mouse neuronal cells (LNCap cells) and A549 cells were treated with either CXCR4-miR or CXCR4 viruses. 16 hours after injection, cells were pelleted, washed with Dulbecco's Phosphate Buffer Saline (PBS), and lysed by incubation on ice for 40 minutes in RIPA buffer (10 mM Tris-Cl (pH 8.0), 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 140 mM NaCl) containing 1 mM phenylmethylsulfonyl fluoride (PMSF) and protease inhibitor cocktail. The lysate was then centrifuged at 13,000 rpm at 4° C. for 10 minutes to collect the supernatant. ICP34.5 protein levels in each sample were determined by Western blot analysis. Total protein concentration was measured using the BSA assay. Protein lysates (30-40 μg) were mixed with 4x SDS loading dye and subsequently heated at 95°C for 10 min. Samples were then loaded and electrophoresed on a 10% SDS-PAGE followed by transfer to a nitrocellulose membrane. The membrane was then blocked in Tris-buffered saline + Tween 20 (TBST) containing 5% BSA for 1 h at room temperature. The blocked membrane was incubated with anti-ICP34.5 or β-actin antibodies overnight at 4°C. The membrane was subsequently washed 3x10 min with TBST and incubated with the corresponding secondary antibody for 1 h at room temperature. After three washes with TBST for 10 min, the membrane was incubated with enhanced chemiluminescence (ECL) reagent for 1 min followed by exposure to a BIO-RAD ChemiDoc XRS+ imaging system. Band intensity was quantified using ImageJ. The results of the Western blot are shown in Figure 4. The column marked "miRNA" indicates whether cells were infected with viruses containing (+) or lacking (-) a miRNA-binding element in the 3'UTR of the ICP34.5 gene. Expression of ICP34.5 was found to be low in neuronal cells infected with viruses containing the miRNA-binding element. In contrast, in tumor cells, expression was similar in cells infected with viral constructs containing or lacking the miRNA-binding element. EXAMPLES

[0032] MicroRNA-based oncolytic virus platform This example provides a microRNA-based oncolytic virus platform with several exemplary engineered viral genomes. This platform is referred to herein as "transcriptional and translational dual regulation" (TTDR). The basic platform HSV-1-based vector is illustrated in FIG. 5. A key feature of the platform HSV-1 virus is the translational control of the ICP34.5 gene by inserting five copies of binding sites for miR-124 and miR-143 into the 3'UTR of the ICP34.5 gene. An important element of this platform vector can also include transcriptional control (using a tumor-specific promoter) of the ICP27 gene, a gene essential for viral replication. A variety of HSV-1 strains can be used as backbones for constructing recombinant oncolytic viruses, including strain 17, strain KOS, strain F, strain McKrae, and others. All viral mutagenesis can be performed in E. coli using standard lambda Red-mediated recombination techniques performed on the HSV-1 genome cloned in a bacterial artificial chromosome (BAC) (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 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 Sceince+Business Media, LLC 2010).

[0033] Tumor-specific promoters can also be used to drive expression of cassettes encoding immunomodulators IL12 / IL15 / IL15RA, which boost anti-tumor immunity. The immunomodulator expression cassettes can be controlled by hCEA, hCXCR4 or PSA promoters and can be inserted into the viral genome at locations that do not negatively affect viral gene expression and replication (e.g., viral intergenic US1 / US2, UL3 / UL4 and / or UL50 / UL51). Other recombinant viruses can be constructed that express mouse IL12 instead of human IL12 to facilitate in vivo testing in various mouse models. Human IL15 can be retained in mouse-specific oncolytic viruses for its activity in mouse cells. The vector can contain an expression cassette encoding a fusion form of the env protein of Gibbon Ape Leukemia Virus (GALV) lacking the C-terminal R-peptide (enhancing viral cytotoxicity). Alternatively, the expression cassette can encode a fusion form of glycoprotein B (e.g., truncated gB876t). The cassette can be inserted into the viral genome at a location that does not have a negative effect on viral gene expression and replication (e.g., viral intergenic US1 / US2, UL3 / UL4, and / or UL50 / UL51). The viral vector can also contain an expression cassette for the HSV-1 thymidine kinase (TK) gene, which is inserted into the viral intergenic US1 / US2, UL3 / UL4 and / or UL50 / UL51. If a BAC sequence is inserted into the viral genome to facilitate mutagenesis in E. coli, the original native TK gene is disrupted. The presence of an exogenous TK gene enhances the safety of the virus by making it susceptible to routine treatment with guanosine analogues (e.g., ganciclovir and acyclovir).

[0034] The promoter driving expression of the key HSV-1 transcriptional regulator ICP27 can be replaced with a tumor-specific promoter (e.g., hCEA, hCXCR4, PSA, or probasin (ARR2PB)). The 3'UTR of the viral gene encoding the neurovirulence factor ICP34.5 can also be modified by inserting multiple copies of a microRNA recognition element to silence ICP34.5 production in tissues that contain high levels of the corresponding microRNA. In an exemplary embodiment, five copies of miR-124 and five copies of miR-143 recognition elements can be inserted in tandem into the 3'UTR of ICP34.5. The terminal repeat region of the viral genome can be completely deleted to reduce the overall genome size and create more room for transgene insertion, and the deleted TR is engineered to avoid disrupting the native promoter of the ICP47 gene (which is normally part of the terminal repeat). Further details of the exemplary elements discussed herein are illustrated in FIG. The resulting recombinant virus can be isolated using Qiagen HiSpeed ​​MidiPrep kit and transfected into Vero cells to recover the virus (e.g., using Lipofectamine 2000). Targeted sequencing and restriction profiling of all modified regions can be used to verify genomic integrity. Stability of the final recombinant virus can be confirmed by serial passaging and periodic verification of transgene expression by Western blot and ELISA. Five exemplary embodiments of this platform are shown in the table below: Two viruses have been engineered for the treatment of lung cancer (or other cancers of epithelial cell origin, such as renal and breast cancer) and three viruses for the treatment of prostate cancer.

[0035] TIFF2024178171000001.tif76155 EXAMPLES

[0036] MicroRNA-mediated regulation of ICP34.5 expression results in reduced neurovirulence in vivo Mice were given one dose (5x10 7The mice were injected with 1000 PFU / mL of either the CXCR4-TF-Fc-h1215-miR virus (containing five miR-124 and miR-143 elements inserted into the 3'UTR of the ICP34.5a gene) or the control CXCR4-TF-Fc-h1215 virus lacking said insertion. Both viral constructs also contain the CXCR4 promoter-driven ICP27 gene, a TF+Fc PD-L1 blocker expression cassette inserted between UL3 and UL4, and the terminal repeat region replaced with cassettes expressing human IL12, IL15, and the IL15 receptor alpha subunit. Following infection, the extent of infection was visualized by staining murine brain sections with rabbit polyclonal anti-HSV primary antibody and rat anti-rabbit secondary antibody conjugated to AlexaFluor 488. As shown in Figure 7, mice infected with virus containing miR-regulated ICP34.5 showed detectable virus only along the needle track, whereas virus containing wild-type ICP34.5 was widely disseminated throughout the brain. EXAMPLES

[0037] VG182LF virus selectively kills lung cancer cells in vitro Lung cancer cells (A549) or normal lung cells (BEAS-2b and HPL1D) were incubated with VG182LF virus at increasing MOIs for 72 hours. Following infection, cell viability was measured using an MTT assay. As shown in Figures 8A and 8B, VG182LF virus was found to exhibit increased killing of lung cancer cells compared to normal lung cells in a dose-dependent manner. The table below provides the IC50 values ​​determined for each cell line and shows that there is a 6.54-fold and 18.93-fold increase in IC50 for normal lung cells HPL1D and BEAS-2b, respectively, compared to the lung cancer cell line (A549).

[0038] TIFF2024178171000002.tif28153 These data indicate that increased tumor killing is associated with microRNA regulation of ICP34.5 gene expression and the use of tumor-specific promoters to drive expression of the ICP27 and IL12 / IL15 / IL15RA genes. This experiment was repeated using additional lung cancer cell lines. As shown in Figure 8C, VG182LF virus efficiently kills a wide variety of commercially available lung cancer cells. The calculated IC50 values ​​for each cell line are shown in the table below. TIFF2024178171000003.tif45153 EXAMPLES

[0039] VG182LF selectively replicates in lung cancer cells in vitro Lung cancer cells (A549) and normal lung cells (BEAS-2b) were treated with VG182LF virus at an MOI of 0.1 for various times. After infection, the virus was harvested and titrated on Vero cells. As shown in Figure 9, the VG182LF virus replicates successfully in lung cancer cells but not in normal lung cells. At 48 hours, 6x10 6 Viral particle titers exceeding 1000 μg / kg were obtained from A549 lung cancer cells, whereas no significant virus was obtained from BEAS-2b normal lung cells, suggesting that microRNA regulation of ICP34.5 and the use of tumor-specific promoters to drive expression of ICP27 and IL12 / IL15 / IL15RA negatively impacts viral replication in normal cells but promotes viral replication in tumor cells. Replication of VG182LF virus in A549 lung tumor cells or LNCaP prostate tumor cells was examined. Briefly, cells were infected with either VG161 (control) or VG182LF virus for 12 or 24 hours. Cells were then harvested and intracellularly stained with anti-human IL-12p70 antibody. Human IL-12 positive cells were detected by flow cytometry, and the fold increase in human IL-12 was calculated. As shown in Figure 10, increased expression of human IL-12 positively correlated with enhanced viral replication. The replication ability of VG182LF virus in various lung cancer cell lines was evaluated. Cells derived from lung cancer cell lines H1975, PC9, and H460 were treated with VG182LF virus at an MOI of 0.1, and supernatants were collected at 0, 6, 24, and 48 hours post-infection. Virus from each sample was titrated on Vero cells. Data from this experiment are shown in Figures 11A-C, and titration values ​​represent the average of three biological replicates. These data indicate that at 48 hours post-infection, the virus can replicate to significant levels in each lung cancer cell line. EXAMPLES

[0040] In vivo antitumor efficacy of VG182LF in H1975 lung cancer model H1975 tumor-bearing nude mice were treated with VG182LF one week after implantation. 7 VG182LF was injected three times at 2-day intervals at PFU / mouse. Vehicle-treated mice reached a humane endpoint 12 days after treatment initiation and were sacrificed. As shown in Figure 12, VG182LF virus-treated mice showed dramatically reduced tumor growth compared to vehicle-treated controls and were still alive 29 days after treatment initiation. EXAMPLES

[0041] miR-mediated regulation of ICP34.5 expression in cultured transfected cells The HSV-1 protein ICP34.5 is required for efficient viral replication in neurons but is dispensable for replication in non-neuronal cultured cells (e.g., 293FT cells). Here, we evaluated the ability of miR-143 to affect ICP34.5 expression in 293FT cells. Cells were first transfected with miR-143 on day 0. As a control, 293FT cells were transfected with scrambled miR or not transfected at all. 20 hours after transfection, cells were washed and subsequently infected with recombinant oncolytic HSV-1 (MOI=1). This recombinant virus encodes binding sites for miR-143 and miR-124 in the 3'UTR of ICP34.5, along with a fusogenic mutation in the carboxyl terminus of gB (gB-876t). Cells were harvested for RNA isolation 6 hours post-infection to measure gene expression and transfection efficiency, and for DNA isolation at 0 and 24 hours post-infection to measure viral replication. As shown in Figure 14A, high levels of miR-143 were detected by RT-qPCR in miR-143-transfected cells 6 hours post-infection, whereas non-transfected and scrambled miR-transfected cells showed negligible levels of miR-143. As shown in Figure 14B, viral gene expression determined by RT-qPCR 6 hours post-infection revealed a significant decrease in ICP34.5 expression in samples previously transfected with miR-143, whereas no similar decrease was observed for another viral gene (ICP27) that does not contain a miR binding site. Viral replication was quantified by qPCR 24 hours post-infection to measure copies of ICP27 (each copy corresponds to a separate viral genome). As shown in Figure 14C, viral replication levels were not significantly different when comparing samples transfected with miR-143 or scrambled miR, suggesting that the dramatic decrease in ICP34.5 expression observed in the miR-143-transfected samples was not due to a decrease in viral copy number. EXAMPLES

[0042] miR modulation of ICP34.5 improves safety by preventing neurotoxicity In this example, DBA / 2 mice (N=3 in each group) were subcutaneously injected with either vehicle control, wild-type HSV-1, VG161 virus variant lacking ICP34.5 and no fusogenic mutation, or VG301 virus variant encoding binding sites for miR-143 and miR-124 in the 3'UTR of ICP34.5 along with a fusogenic mutation in the carboxyl terminus of gB (gB-876t). Samples were collected for HSV-1 immunostaining 6 days after infection. As shown in FIG. 15, an intense viral replication pattern was observed in both the brain and spinal cord of wild-type HSV-1 injected mice. In contrast, no viral replication was observed in neuronal tissues of mice injected with either the VG161 or VG301 variants, suggesting that miR modulation of ICP34.5 is just as effective as complete deletion of ICP34.5 in preventing neurovirulence. The remaining wild-type HSV-1 injected mice rapidly developed neurological symptoms and had to be euthanized, whereas all remaining mice treated with either the VG161 or VG301 variants remained healthy for the entire duration of the experiment as shown in Figure 16. These results provide further evidence supporting the safety and efficacy of preventing OV-induced neurovirulence using miR regulation of ICP34.5. Furthermore, it can be concluded that the fusogenic mutations of the VG301 variant do not result in increased morbidity or mortality. EXAMPLES

[0043] Evaluation of fusogenic mutations in oncolytic HSV-1 In this example, A549wt and BPH1 cells were infected with recombinant oncolytic HSV-1, which encodes a fusogenic mutation in the carboxyl terminus of gB (+gB-876t). As a control, A549wt and BPH1 cells were infected with HSV-1 lacking the fusogenic mutation (-gB-876t). 48 hours post-infection, cells were fixed and stained with Giemsa to visualize viral plaques and syncytia resulting from virus-induced cell fusion. As shown in FIG. 17, extensive cell-cell fusion was observed in cells infected with viruses carrying the fusogenic mutation, whereas minimal fusion was evident in cells infected with viruses lacking the fusogenic mutation.

[0044] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with any condition or negative limitation that removes any generic content (whether or not the removed content is specifically recited herein). As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural related words unless the context clearly dictates otherwise, and the term "X and / or Y" means "X" or "Y," or both "X" and "Y," and it should also be understood that an "s" following a noun indicates the plural as well as the singular form of that noun. In addition, when a feature or characteristic of the invention is described as a Markush group, the invention is intended to encompass, and thereby be described as, any individual member and any member subgroup of that Markush group, and one of skill in the art would recognize that. Moreover, applicants reserve the right to amend the application or claims to specifically refer to any individual member and any member subgroup of that Markush group. It should be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to be limiting. Furthermore, it should also be understood that unless otherwise specified herein, the terms used herein should be given their customary meanings as known in the relevant industry. Throughout this specification, the term "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 phrase "in one embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0045] As used herein and in the appended claims, the singular forms "a," "an," and "the" include the plural (i.e., one or more) of the associated terms unless the content and context clearly dictate otherwise. Furthermore, it should be noted that the conjunctions "and" and "or" are generally used in their broadest sense to include "and / or," unless the content and context clearly dictate inclusiveness or exclusion, as the case may be. Thus, the use of alternative terms (e.g., "or") should be understood to mean one, both, or any combination thereof of the alternatives. In addition, when recited herein as "and / or," the compound words "and" and "or" are intended to encompass embodiments including all of the associated items or ideas and one or more other alternative embodiments including less than all of the associated items or ideas. Unless the context requires otherwise, throughout this specification and the following appended claims, the word "comprise" and synonyms and variations thereof (e.g., "have" and "include"), as well as variations of "comprise" (e.g., "comprises" and "comprising"), are to be construed in an open, inclusive sense (e.g., "including, but not limited to"). The term "consisting essentially of" limits the claim to specified materials or steps or those that do not materially affect the basic or novel characteristics of the claimed invention. Any headings used in this document are used merely to facilitate the reader's review of the document and should not be construed as limiting the scope of the invention or the claims in any manner. Thus, the headings and summaries provided herein are merely for convenience and should not be interpreted as dictating the scope or meaning of the embodiments.

[0046] Where a range of values ​​is provided herein, each intervening value between the upper and lower limits of that range (to 1 / 10 of the unit of the lower limit unless the context clearly dictates otherwise), as well as any other stated or intervening values ​​in that stated range, is encompassed within the invention. The upper and lower limits of smaller ranges that may be independently included in these smaller ranges are also encompassed within the invention, but subject to any stated excluded limits in that stated range. Where the stated range includes one or both limits, ranges excluding either or both of those included 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., 1 / 10 and 1 / 100 of an integer), unless otherwise specified. Furthermore, any numerical range recited herein for any physical feature (e.g., polymer subunits, size, or thickness) should be understood to include any integer within the recited range, unless otherwise specified. As used herein, the term "about" means ±20% of the specified range, value, or structure, unless otherwise specified.

[0047] All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications cited in this application and / or listed in the Application Data Sheet are hereby incorporated by reference in their entirety. Such documents may be included by reference for the purpose of describing and disclosing, for example, materials and methodology described in the publications, which 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 this application. Nothing herein should be construed as an admission that the inventors are not entitled to antedate any referenced publication by virtue of prior invention. All patents, publications, scientific articles, websites, and other documents and materials referenced or described herein are indicative of the level of skill in the art to which this invention pertains, and each such reference document and material is hereby incorporated by reference herein as if it were individually or in its entirety set forth herein. Applicant reserves the right to physically incorporate into this specification any and all materials and information from such patents, publications, scientific articles, websites, electronically available information, and other reference materials or documents. In general, the terms used in the following claims should not be construed to limit the scope of 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. Thus, the claims are not limited by the disclosure.

[0048] Furthermore, the written description portion of the patent includes all claims. Furthermore, all claims (including all original claims as well as any and all claims derived from priority documents) are incorporated by reference in their entirety into the written description portion of the specification, and applicant reserves the right to physically incorporate any and all such claims into the written description or any other part of the application. Thus, for example, under no circumstances should the patent be construed as not providing a written description of the claims based on a contention that the exact language of the claims is not instead set forth in the patent document specification. The claims will be interpreted in accordance with law, but further notwithstanding any alleged or perceived ease or difficulty of interpretation of any claim or portion thereof, under no circumstances shall any adjustment or modification of the claims or any portion thereof during the prosecution of the application be construed as a waiver of any and all rights of this patent to any and all equivalents that do not form part of the prior art. Other non-limiting embodiments are within the scope of the following claims. The patent should 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 should the patent be construed as being limited by any statement made by the examiner or any other official or employee of the Patent Office unless such statement is specifically and unrestrictedly or unconditionally clearly adopted in the applicant's reply.

Claims

1. A recombinant herpes simplex virus comprising a modified oncolytic herpesvirus genome, the modified herpesvirus genome comprising at least one miRNA target sequence operably linked to a first or first and second copies of an ICP34.5 gene.

2. The recombinant herpes simplex virus of claim 1, wherein the miRNA is selected from the group consisting of: mIR-122, miR-124, miR-124 * , miR-127, miR-128, miR-129, miR-129 * , miR-132, mIR-133a, mIR133b, miR-135b, miR-136, miR-136 * , miR-137, miR-139-5p, miR-143, mIR-145, miR-154, miR-184, miR-188, miR-204, mIR216a, miR-299, miR-300- 3p, miR-300-5p, miR-323, miR-329, miR-337, miR-335, miR-341, miR-369-3p, miR-369-5p, miR-376a, miR-376a * , miR-376b-3p, miR-376b-5p, miR-376c, miR-377, miR-379, miR-379 * , miR-382, miR-382 * , miR-409-5p, miR-410, miR-411, miR-431, miR-433, miR-434, miR-451, miR-466b, miR-485, miR-495, miR-539, miR-541, miR-543 * , miR-551b, miR-758, and miR-873.

3. The recombinant herpes simplex virus of claim 2, wherein the miRNA target site comprises five copies of binding sites for miR-124 and miR-143.

4. The recombinant herpes simplex virus of claim 1, wherein the oncolytic herpes virus is HSV-1.

5. 5. The recombinant herpes simplex virus of claim 4, wherein the genome further comprises a fusogenic mutation in the gene encoding glycoprotein B (gB).

6. 6. The recombinant herpes simplex virus of claim 5, wherein the glycoprotein B (gB) coding gene encodes a glycoprotein B variant that terminates after amino acid 876.

7. The recombinant herpes simplex virus of claim 1, wherein the modified oncolytic herpesvirus genome comprises an additional mutation or modification in at least one viral gene selected from the group consisting of ICP6, ICP0, ICP4, ICP27, ICP47, ICP24, and ICP56.

8. 8. The recombinant herpes simplex virus of claim 7, wherein at least one viral gene is modified by replacing the native promoter.

9. The recombinant herpes simplex virus of claim 1, further comprising at least one nucleic acid encoding a non-viral protein selected from the group consisting of immune stimulators and checkpoint blocking peptides, wherein the at least one nucleic acid is operably linked to a tumor-specific promoter.

10. The recombinant herpes simplex virus of claim 9, wherein the non-viral protein is selected from the group consisting of IL12, IL15, IL15 receptor alpha subunit, OX40L, and a PD-L1 blocker.

11. 10. A method for lysing tumor cells comprising the step of providing a therapeutically effective amount of a recombinant herpes simplex virus according to any one of claims 1 to 9.

12. 10. A therapeutic composition comprising a recombinant herpes simplex virus according to any one of claims 1 to 9 and a pharma- ceutically acceptable carrier.

13. 13. A method of treating cancer in a subject suffering from cancer, comprising administering a therapeutically effective amount of the composition of claim 12.

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