Low neurotoxicity HSV vector

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 patent application claims rights under 35 U.S.C. § 119e of U.S. Provisional Patent Application No. 62 / 773,119 (filed November 29, 2018) (the said application is incorporated herein by reference in its entirety for all purposes). (Technical field) This invention relates to HSV vectors that generally have low neurotoxicity. [Background technology]

[0002] Oncolytic virus therapy is recognized as a promising and novel therapeutic approach for cancer treatment. This is because oncolytic viruses cause potent tumor lysis and induce systemic tumor-specific immunity, while producing significantly fewer side effects than chemotherapy or radiation therapy. Among the various types of vectors (OVs), those derived from herpes simplex virus type 1 ("HSV-1") are the most advanced. For example, herpesvirus OVs (T-Vec) are 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. Patents 7,223,593, 7,537,924, 7,063,835, 7,063,851, 7,118,755, 8,277,818, and 8,680,068. One challenge with oncolytic herpesvirus vectors is the neurotropic nature of HSV. Neurotoxicity is primarily mediated by the viral protein ICP34.5, which leads to the common strategy of deleting ICP34.5 from vectors used in oncolytic virus therapy. However, complete deletion of ICP34.5 reduces viral replication ability in a wide range of tissues by almost tenfold. This invention overcomes certain challenges with conventional HSV vectors and offers other relevant advantages. Not everything considered in the background art is necessarily prior art, nor should it be assumed to be prior art simply as a result of being considered in the prior art. Following these guidelines, any recognition of a prior art problem considered in the background art, or related to such content, should not be considered prior art unless it is explicitly stated to be prior art. Rather, any consideration of the content of the background art should be considered part of the inventor's approach to the present problem, and such consideration may also be novel in itself. [Overview of the project] [Problems that the invention aims to solve]

[0003] This invention overcomes certain challenges related to conventional HSV vectors and also provides other relevant advantages. [Means for solving the problem]

[0004] In short, this 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 having 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 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 more frequently found as the final product is indicated as miRNA, and the less frequently found partner is miRNA. * This is indicated. In other embodiments, the recombinant herpes simplex virus further comprises a modified ICP27 or ICP4 gene, the modification being the 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 the FGF gene.

[0005] In certain embodiments, the recombinant herpes simplex virus further comprises a gene sequence encoding at least one immunostimulator, a checkpoint-blocking peptide, or both. The disclosure also provides a method for treating cancer, comprising the step of 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 of the Invention] is provided to introduce certain concepts in a simplified form, which are described in more detail in the [Modes for Carrying Out the Invention] below. Unless otherwise stated, this brief [Summary of the Invention] is not intended to identify any important or essential features of the claims, nor is it intended to limit the scope of the claims. Details of one or more embodiments are described below. Features illustrated or described in relation to one exemplary embodiment may be combined with features of other embodiments. Other features, purposes, and advantages will be evident from this description, drawings, and claims. In addition, all patent and patent application disclosures referenced herein are incorporated herein by reference in their entirety. The exemplary features, nature, and various advantages of this disclosure will be evident from the accompanying drawings and the detailed description of the various embodiments below. Non-limiting and non-exclusive embodiments are described with reference to the accompanying drawings, where the same markings or reference numerals refer to the same parts throughout the various drawings unless otherwise specified. The size and relative position of elements in the drawings are not necessarily drawn to scale. For example, the shapes of various elements are selected, enlarged, and positioned to improve the readability of the drawings. The individual shapes of the elements depicted are selected to facilitate recognition of the drawings. One or more embodiments are described below with reference to the following accompanying drawings: [Brief explanation of the drawing]

[0006] [Figure 1] This is a schematic diagram of an exemplary HSV vector having three different miRNA targets in the 3' untranslated region of ICP34.5. [Figure 2] This is a schematic diagram of an exemplary HSV vector containing a modified γ34.5 gene and a modified ICP4 or ICP27 gene. [Figure 3] This graph shows the expression levels of ICP27, ICP4, and ICP47 in the brains of normal mice and mice with human brain tumors (U87). [Figure 4]Western blot showing the expression of ICP34.5 and β-actin in neurons and tumor cells (LNCaP and A549). [Figure 5] Schematic diagram of a transcription- and translation-dual regulated virus. [Figure 6] Showing various regulatory elements that can be used in the platform virus. [Figure 7] Photograph of a mouse brain section after intracranial injection of either CXCR4-TF-Fc-h1215 virus or CXCR4-TF-Fc-h1215-miR virus. The brain section was stained with rabbit polyclonal anti-HSV primary antibody and fluorescent rat anti-rabbit secondary antibody. [Figure 8A] Graph of cell survival after infecting cells with the virus at various MOIs. Figure 8A shows the cell survival of lung tumor cells A549 and normal lung cells BEAS-2b. [Figure 8B] Graph of cell survival after infecting cells with the virus at various MOIs. Figure 8B shows the cell survival of lung tumor cells A549 and normal lung cells HPL1D. [Figure 8C] Graph of cell survival after infecting cells with the virus at various MOIs. Figure 8C shows the cell survival of lung tumor cells A549, PC9, H460, H23S, H1975. [Figure 9] Graph showing the replication of VG182LF virus in A549 lung tumor cells and BEAS-2b normal lung cells. [Figure 10] Bar graph showing the increase (fold increase) of IL-12 in A549 lung tumor cells and LNCaP prostate tumor cells after hVG161 or hVG182LF infection. [Figure 11A] Showing the replication of VG182LF virus in various lung tumor cells. Figure 11A: H1975 cells. [Figure 11B] Showing the replication of VG182LF virus in various lung tumor cells. Figure 11B: H460 cells. [Figure 11C] Showing the replication of VG182LF virus in various lung tumor cells. Figure 11C: PC9 cells. [Figure 12] This graph shows tumor size in H1975 tumor-carrying nude mice one week after treatment with either the vehicle or VG182LF virus. [Figure 13A] A selection list of microRNAs in tumors is disclosed. These microRNAs can be found in PubMed at https: / / www.ncbi.nlm.nih.gov / pubmed and the microRNA database “mIBRASE” at http: / / www.mirbase.org / (both of which are included herein by reference in their entirety). [Figure 13B] Figure 13A continued. [Figure 14A] These graphs show the 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] Figure 14A continued. [Figure 14C] Figure 14B continued. [Figure 15] These are photographs showing HSV-1 immunostaining of mouse brain and spinal cord sections. Mice were injected subcutaneously with a 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), along with a fusion-inducible mutation (gB-876t) at the carboxyl terminus of gb. [Figure 16] The graph shows the survival curves of mice injected subcutaneously with one of the following: wild-type HSV-1, ICP34.5-deleted HSV-1 variant (VG161), or a variant (VG301) encoding binding sites for miR-143 and miR-124 in the 3'UTR of ICP34.5 along with a fusion-inducible mutation at the carboxyl terminus of gb (gB-876t). [Figure 17]This is a photograph showing the results of a fusion assay. In the assay, cells were fixed and Giemsa stained to visualize viral plaques and syncytia produced by virus-induced cell fusion. Recombinant oncolytic HSV-1 with or without a fusion-inducible mutation at the carboxyl terminus of gB (+gB-876t) was used to infect the cells. [Modes for carrying out the invention]

[0007] Detailed description of the invention The present invention can be more readily understood by referring to the following detailed description of preferred embodiments of the invention and the examples included herein. As used herein, the terms “microRNA” or “miRNA” refer to a family of short (typically 21-25 nucleotides) endogenous single-stranded RNAs expressed in a wide range of organisms, including animals and plants. In humans, more than 1000 unique miRNAs are expressed. miRNAs bind to specific target sequences found in messenger RNA (mRNA). Binding to complementary or partially complementary sequences (target sequences) in the mRNA molecule results in downregulation of gene expression through mRNA cleavage, increased degradation due to shortening of its poly(A) tail, and direct translational repression. A selection list of microRNAs in tumors (with related literature) is provided in Figures 13A and 13B (the 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 can replicate in tumor cells and kill them. In certain embodiments, the virus can be manipulated to more selectively target tumor cells. Representative examples of oncolytic herpesviruses are described in U.S. Patents 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 entirety). As used herein, the terms “to treat” and “treatment” mean an approach to obtain a beneficial or desired outcome, including clinical outcomes. Beneficial or desired clinical outcomes include, but are not limited to, the reduction or alleviation of one or more symptoms or conditions, whether detectable or undetectable; a reduction in the scope of the disease; a state of stabilization (i.e., no exacerbation) of the disease; prevention of disease progression; delay or slowing of disease progression; relief or temporary suppression of disease stages; a reduction in disease recurrences; and remission (whether partial or complete). The terms “to treat” and “treatment” may also mean an extension of survival compared to the survival expected if no treatment were received.

[0008] The most common 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, colorectal, CNS (e.g., acoustic neuroma, astrocytoma, craniopharyogioma, ependymoma, glioblastoma, hemangioblastoma, medulloblastoma, meningioma, 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. Cancer may include solid tumors (e.g., sarcomas, e.g., fibrosarcomas, myxosarcomas, liposarcomas, chondrosarcomas, and osteogenic sarcomas), diffuse tumors (e.g., leukemia), or any combination of the above (e.g., metastatic cancer having both solid tumors and disseminated or diffuse cancer cells). Cancers particularly favored for treatment include lung tumors, breast and prostate tumors, glioblastoma, tumors of the gastrointestinal tract (and related organs, e.g., esophageal, bile duct, anal, stomach, intestinal, pancreatic, colon, and liver), and all surface injectable tumors (e.g., melanoma). Benign tumors and other symptoms of undesirable cell proliferation can also be treated. For further understanding of the various embodiments described herein, the following sections describing various embodiments are provided: A. Oncolytic herpesvirus; B. MicroRNA; C. Therapeutic composition; and D. Administration.

[0009] A. Oncolytic herpesvirus 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 referred to as the 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 approximately 75 known open reading frames. The viral genome has been engineered to develop, for example, oncolytic viruses used in 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., are non-viral / 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 originate from either HSV-1 or HSV-2 (including any laboratory or clinical isolate). In some embodiments, oHSV may be one of the laboratory HSV-1 strain 17, HSV-1 strain F, or HSV-2 strain HG52, or may be derived from these strains. In other embodiments, oHSV may be a non-laboratory strain JS-1, or may be derived from this strain. 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 (Title of Invention: HSV Vectors with Enhanced Replication in Cancer Cells) and PCT / US2017 / 018539 (Title of Invention: Compositions and Methods of Using Stat1 / 3 Inhibitors with Oncolytic Herpes Virus) (both of which are incorporated herein by reference in their entirety). .

[0011] The oHSV vector has at least one γ34.5 gene modified with respect to a miRNA target sequence in its 3'UTR as disclosed herein, and no unmodified γ34.5 gene is present in the vector. In some embodiments, oHSV has two modified γ34.5 genes, and in other embodiments, 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 a viral gene. When the modified γ34.5 gene is a replacement for only one γ34.5 gene, the other γ34.5 is deleted. Either of the native γ34.5 genes can be deleted. In some embodiments, terminal repeats (including the γ34.5 gene and the ICP4 gene) are deleted. As discussed herein, the modified γ34.5 gene may include additional changes (e.g., having an exogenous promoter). oHSV may contain additional mutations, which may include disabling mutations (e.g., deletions, substitutions, insertions) that can affect the virulence or replication ability of the virus. For example, mutations may occur in any one or more of ICP6, ICPO, ICP4, ICP27, ICP47, ICP24, and ICP56. Preferably, a mutation in one of these genes (or, where appropriate, in both copies of the gene) 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 target cells, or that can be induced upon delivery of an inducible substance, or upon a cellular event or in a specific environment. In certain embodiments, ICP4 or ICP27 expression is controlled by exogenous promoters (e.g., tumor-specific promoters). Exemplary tumor-specific promoters include survivor, 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 exist. In some cases, enhancers (e.g., NFκB / oct4 / sox2 enhancers) are present. 5'UTRs (e.g., growth factor gene (e.g., FGF)-derived 5'UTRs) may also be exogenous. See Figure 2 for exemplary constructs.

[0012] oHSV can also have genes and nucleotide sequences of non-HSV origin. For example, sequences encoding prodrugs, cytokines or other immunostimulants, tumor-specific promoters, inducible promoters, enhancers, and sequences homologous to host cells may be present in the oHSV genome, among others. Exemplary sequences encode IL12, IL15, IL15 receptor alpha subunit, OX40L, PD-L1 blockers, or PD-1 blockers. For product-encoding sequences, they are ligated to the promoter sequence and other regulatory sequences required or desired for expression (e.g., enhancers, polyadenylation signal sequences) to function. The regulatory regions of viral genes can be modified to include response elements that affect expression. Exemplary response elements include NF-κB, Oct-3 / 4-SOX2, response elements for enhancers and silencers, cAMP response elements, CAAT enhancer-binding sequences, and insulators. Other response elements may also be included. Viral promoters can be replaced with different promoters. The choice of promoter will depend on numerous requirements, such as the recommended 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 the treatment of cancer, when promoters are replaced, they will generally be replaced with cell-specific, tissue-specific, or tumor-specific promoters. Tumor-specific, cell-specific, and tissue-specific promoters are well known in the art. Other gene elements can also be modified. For example, the 5'UTR of a viral gene can be replaced with an exogenous UTR.

[0013] B. MicroRNA As described above, the present invention provides oHSV having at least two miRNA target sequences. Briefly, the miRNA binds to a target sequence within mRNA (typically at the 3'-untranslated region (3'-UTR)). Binding may begin at or require a region located approximately 2-8 nucleotides from the 5' end of the miRNA, called the “seed region.” When partial complementarity is present, the 5' end tends to have greater identity with respect to the target sequence than the 3' end. Higher complementarity can enhance mRNA repression, particularly through mRNA cleavage. Individual miRNAs and miRNA groups may be expressed exclusively or preferentially in certain tissue types. MiRNAs that are abundant or exclusive in neuronal cells include: mIR-122, miR-124, and 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 strands more frequently found as the final product are indicated as miRNAs, while the less frequent partners are miRNAs. * This is shown as follows. A list of microRNAs in tumors is provided in Figures 13A and 13B (along with related literature) (the aforementioned list and related 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 that are inserted in tandem. There may be at least three, at least four, at least five, at least six, at least ten, and so on. In other embodiments, there may be 10, 20, 50, or fewer than 100 target sequences. The optimal number of target sequences can be determined by assaying the expression level of ICP34.5. The numerous miRNA target sequences may all bind to the same miRNA, or they may bind to different miRNAs. The target sequences may also be in a cluster state (e.g., Figure 1), in which, for example, there are at least two target sequences in tandem that bind to a first miRNA, followed by at least two more target sequences in tandem that bind to a second miRNA, followed by at least two more target sequences that bind to a third miRNA. Alternatively, the numerous miRNA target sequences that bind to different miRNAs do not have to be in a specific 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 yet another embodiment, there are 3–5 different miRNA target sequences, and 3–5 copies of each of these target sequences exist in a clustered state. See Figure 1 for illustrative constructs. Numerous miRNA target sequences may be adjacent without intervening sequences, or may have 1 to about 25, 1 to about 20, 1 to about 15, 1 to about 10, 1 to about 5, 3 to about 10, or 5 to about 10 intervening nucleotides. The intervening nucleotides can be selected to have a G+C content similar to the 3'UTR, and preferably to avoid the presence of polyadenylated signal sequences. Other considerations for selecting intervening nucleotides are known in the art.

[0015] In certain embodiments of the present invention, the oHSV described herein is constructed to utilize dual regulation of both transcription and translation (also referred to as “TTDR”). An exemplary illustration of such a vector is provided in Figure 5. Briefly, in certain preferred embodiments, translational regulation of the ICP34.5 gene is achieved by inserting five copies of the binding sites for miR-124 and miR-143 into the 3'-UTR of the ICP34.5 gene. A key element of the platform viral vector may also include transcriptional regulation of the ICP27 gene (a gene essential for viral replication) using a tumor-specific promoter. A wide variety of HSV-1 strains (including strains 17, KOS, F, and McKrae) can be used as a backbone for constructing oncolytic recombinant viruses. All viral mutation induction can be performed in Escherichia coli using standard lambda Red-mediated recombination techniques with HSV-1 genomes cloned on bacterial artificial chromosomes (BACs) (see below for general information: 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 following 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 the expression of cassettes encoding immunomodulatory factors IL12 / IL15 / IL15RA (which boost anti-tumor immunity). Immunomodulatory factor 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., between viral genes US1 / US2, UL3 / UL4, and / or UL50 / UL51). To facilitate in vivo testing in diverse mouse models, other recombinant viruses expressing mouse IL12 instead of human IL12 can be constructed. Human IL15 can be retained in mouse-specific oncolytic viruses due to its activity in mouse cells. The vector may include an expression cassette encoding a fusion of the env protein of gibbon leukemia virus (GALV) lacking the C-terminal R-peptide (enhancing the cytotoxicity of the virus). In other embodiments, the expression cassette may encode a fusion 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 negatively affect viral gene expression and replication (e.g., between viral genes US1 / US2, UL3 / UL4 and / or UL50 / UL51). BAC recombination requires the presence of exogenous BAC DNA within the viral genome to facilitate mutation induction in E. coli. The BAC sequence is most typically inserted between viral genes (e.g., US1 / US2, UL3 / UL4, and / or UL50 / UL51) or into thymidine kinase (TK) genes (which can disrupt native TK expression). TK-deficient viral vectors may contain an expression cassette for the HSV-1 thymidine kinase (TK) gene, which is 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 conventional treatment with guanosine analogs (e.g., ganciclovir and acyclovir).

[0017] In another embodiment, the initially destroyed TK can be restored instead of being replaced with another TK, or the TK gene can be destroyed, further reducing neurotoxicity without any replacement or restoration (TK null viruses cannot be reactivated from the incubation period). Even if the TK is destroyed, the virus will still be susceptible to treatment with drugs whose function is not dependent on the TK. For example, foscarnet and cidofovir inhibit viral DNA polymerase and are not TK-dependent. The promoter driving the expression of the important HSV-1 transcription regulator ICP27 can be replaced with a tumor-specific promoter (e.g., hCEA, hCXCR4, PSA, or provasine (ARR2PB)). The 3'UTR of the viral gene encoding the neurotoxic factor ICP34.5 can also be modified by inserting multiple copies of a microRNA recognition element to halt ICP34.5 production in tissues containing high levels of the corresponding microRNA. In an exemplary embodiment, five copies of the miR-124 and five copies of the miR-143 recognition elements can be inserted in tandem into the 3'UTR of ICP34.5. Complete deletion of terminal repeat regions in the viral genome can reduce the overall genome size, creating more space for transgene insertions, and the deleted TR can be manipulated to avoid disruption of the native promoter of the ICP47 gene (which is normally the terminal repeat region). Similar modifications can be carried out by deleting internal repeat regions instead of terminal repeat regions. Further details of the exemplary elements discussed herein are illustrated in Figure 6.

[0018] C. Therapeutic composition A therapeutic composition is provided that can be used to prevent, treat, or mitigate the effects of a disease (e.g., cancer). More specifically, a therapeutic composition comprising at least one oncolytic virus as described herein is provided. In certain embodiments, the composition may further include a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable carrier” is intended to encompass any carrier, diluent, or excipient that does not interfere with the efficacy of the biological activity of the oncolytic virus and is not toxic to the target animal to which the composition is administered (see below for general information: 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 Supplement)). In the cases of oncolytic viruses described herein, non-limiting examples of suitable pharmaceutically acceptable carriers include phosphate-buffered saline, water, emulsions (e.g., oil / water emulsions), various types of wetting agents, sterile solutions, and others. Additional pharmaceutically acceptable carriers include gels containing 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 ways and administered to the target animal 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 also herein may include, for example, mineral salts (e.g., hydrochloride, hydrobromide, phosphate, sulfate) and salts of organic acids (e.g., acetate, propionate, malonate, benzoate). Such pharmaceutically acceptable (pharmaceutical grade) carriers, diluents, and excipients that can be used to deliver oHSV to cancer cells are preferably not likely to induce an immune response in the individual (subject animal) given the composition (and preferably will be administered without excessive toxicity).

[0019] The compositions provided herein may be available in a variety of concentrations. For example, the dosage of oncolytic viruses is approximately 10 6 From about 10 9 It can be provided in the range of pfu. In yet another embodiment, the drug can be administered in approximately 10 6 From about 10 8 Within the range of pfu / mL, patients with large lesions (e.g., >5cm) may receive up to 4mL, while patients with small lesions (e.g., <0.5cm) may receive smaller doses (e.g., up to 0.1mL) every 2-3 weeks. In certain embodiments of the present invention, a lower-than-standard dosage can be used. Therefore, in certain embodiments, about 10 6 The patient may be administered doses of less than pfu / mL (maximum 4 mL injected into the patient every 2-3 weeks). The composition can be stored at temperatures that contribute to a stable shelf life, including room temperature (approximately 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 composition can be stored in a dry (e.g., freeze-dried) or liquid form.

[0020] D. Administration In addition to the compositions described herein, a variety of methods for using such compositions to alleviate cancer are provided, the methods comprising the step of administering an effective dose of the oHSV described herein to a target animal. The terms “effective dose” and “effective amount” refer to the amount of oncolytic virus that is sufficient to achieve the treatment of the target cancer, for example, the amount that is effective in reducing the target tumor size or tumor load, or otherwise in inhibiting the growth rate of the target tumor cells. More specifically, such terms refer to the amount of oncolytic virus that is effective in achieving the desired results with the required dosage and duration of treatment. For example, in relation to cancer treatment, the effective amount of the composition described herein is the amount that induces remission, reduces the tumor load, and / or prevents tumor spread or cancer growth. The effective amount may vary according to several requirements (e.g., disease stage, age, sex, and weight of the animal, as well as the prescription of the medicine, route of administration, etc.), but nevertheless, a person skilled in the art can routinely determine the amount. The therapeutic composition is administered to target animals diagnosed with or suspected of having cancer. The target animals may be humans or non-human animals.

[0021] This composition is used to treat cancer. As used herein, the terms “to treat” and “treatment” mean an approach to achieve beneficial or desired outcomes, including clinical outcomes. Beneficial or desired clinical outcomes include, but are not limited to, the reduction or alleviation of one or more symptoms or conditions, whether detectable or undetectable; a reduction in the extent of the disease; a state of stabilization (i.e., no worsening) of the disease; prevention of disease progression; delay or slowing of disease progression; mitigation or temporary suppression of disease stage; a reduction in disease recurrence; and remission (whether partial or complete). The terms “to treat” and “treatment” may also mean an extension of survival compared to the survival expected if no treatment is received.

[0022] The most common 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, colorectal, CNS (e.g., acoustic neuroma, astrocytoma, craniopharyogioma, ependymoma, glioblastoma, hemangioma, medulloblastoma, meningioma, 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., esophageal, gastric, and colon), and thyroid. Cancer may include solid tumors (e.g., sarcomas, e.g., fibrosarcomas, myxosarcomas, liposarcomas, chondrosarcomas, and osteogenic sarcomas), diffuse tumors (e.g., leukemia), or any combination of the above (e.g., metastatic cancer having both solid tumors and disseminated or diffuse cancer cells). Cancer may also be resistant to conventional treatments (e.g., conventional chemotherapy and / or radiotherapy). Cancers particularly favored for treatment include lung tumors, breast and prostate tumors, glioblastoma, tumors of the gastrointestinal tract (and related organs, e.g., esophageal, bile duct, anal, stomach, intestinal, pancreatic, colon, and liver), and injectable tumors of all surfaces (e.g., melanoma). Benign tumors and other symptoms of undesirable cell proliferation can also be treated. The recombinant herpes simplex viruses described herein can be administered, for example, orally, topically, parenterally, systemically, intravenously, intramuscularly, intraocularly, intrathecally, intratumorally, subcutaneously, or percutaneously. In certain embodiments, the oncolytic virus can be delivered by cannula, catheter, or direct injection. The administration site may be intratumorally or a site distant from the tumor. The route of administration will often depend on the type of cancer being targeted.

[0023] The optimal or appropriate drug regimen for oncolytic viruses is readily determined by the attending physician within the scope of the art of this field based on patient data, patient examination, and various clinical requirements (including, for example, the size, body surface area, age, sex, and specific oncolytic viruses administered, timing and route of administration, type of cancer being treated, the patient's overall health status, and other drug therapies the patient is receiving). In certain embodiments, treatment of animals using oncolytic viruses as described herein may be combined with additional types of treatment (e.g., chemotherapy using chemotherapeutic agents (e.g., etoposide, ifosfamide, adriamycin, vincristine, doxycycline, and others)). The recombinant herpes simplex viruses described herein can be formulated as pharmaceuticals and pharmaceutical compositions for clinical use and can be combined with pharmaceutically acceptable carriers, diluents, excipients, or adjuvants. The formulation will be at least partially dependent on the route of administration. A suitable formulation may contain the virus and inhibitors in a sterile medium. The formulation may be in liquid, gel, paste, or solid form. The formulation can be provided to the target animal or to a healthcare professional. Preferably, a therapeutically effective dose is administered. The dose is sufficient to provide a benefit to the animal under treatment. The actual dose administered and the time course of administration will depend, at least in part, on the nature of the cancer, the condition of the animal under treatment, the site of delivery, and other requirements. In yet another embodiment of the present invention, the oncolytic virus may be administered in a variety of ways, for example, intratumor, intravenously, or after surgical resection of the tumor. The present invention has been described extensively and comprehensively herein. Each of the narrower groups of species and subgenera included in this comprehensive disclosure also forms part of the present invention. This includes the comprehensive description of the present invention conditionally or with negative limitations that exclude any content derived from a genus (whether or not the content to be excluded is specifically enumerated herein).

[0024] The following are additional exemplary embodiments of the disclosure of the present invention: 1) 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. In a related embodiment, a recombinant herpes simplex virus comprising a modified oncolytic herpesvirus genome is provided, the modified herpesvirus genome comprising at least one miRNA target sequence ligated to a first or first and second copy of the ICP34.5 gene. 2) The recombinant herpes simplex virus according to Embodiment 1, wherein at least two of the miRNA target sequences are targets of the same miRNA. 3) Recombinant herpes simplex virus according to either Embodiment 1 or 2, wherein 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 strands more frequently found as the final product are indicated as miRNAs, while the less frequent partners are miRNAs. * This is shown as follows. In certain embodiments of the present invention, a recombinant herpes simplex virus as described in Embodiment 1, 2, or 3 is provided, wherein the miRNA target site comprises one, two, three, four, five, six, or more than six copies of the binding sites to 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 other embodiments, the ICP27 or ICP4 gene is modified by replacement of a natural promoter. In a particularly preferred embodiment of the present invention, ICP27 is modified by replacement of the natural promoter with the hCEA promoter or the hCXCR4 promoter. 5) 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 complete replacement of the promoter-regulatory region of ICP27. In the further embodiments described above, the herpes simplex virus is HSV-1. In any one further embodiment of Embodiments 1, 2, 3, or 4, the recombinant herpes simplex virus further comprises a fusion-inducible mutation in the gene encoding glycoprotein B(gB). In the relevant embodiments, 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 includes additional mutations or modifications to at least one viral gene selected from the group consisting of ICP6, ICP0, ICP4, ICP27, ICP47, ICP24, and ICP56. In certain preferred embodiments, the additional mutations or modifications are located in the non-coding region of the viral gene.

[0026] 6) Recombinant herpes simplex virus according to any one of Embodiments 1, 2, 3, 4, or 5, further comprising a gene sequence encoding at least one immunostimulator. Representative immunostimulators include IL12, IL15, IL15 receptor alpha subunit, OX40L, and PD-L1 blockers. 7) Recombinant herpes simplex virus according to any one of Embodiments 1, 2, 3, 4, 5, or 6, further comprising a gene sequence encoding an immunostimulator 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 immunostimulators, antibodies, and checkpoint-blocking peptides. In the relevant embodiments, the at least one nucleic acid is ligated to act as a tumor-specific promoter. 8) A method for treating cancer, comprising the step of administering a recombinant herpes simplex virus as described in any of Embodiments 1-7. Particularly preferred cancers to be treated include lung tumors, breast and prostate tumors, glioblastoma, tumors of the gastrointestinal tract (and related organs, e.g., esophageal, bile duct, anal, stomach, intestinal, pancreatic, colon and liver), and injectable tumors of all surfaces (e.g., melanoma).

[0027] The following is yet another embodiment of the present invention: 9) Recombinant herpes simplex virus comprising a modified oncolytic herpesvirus genome, wherein the modified herpesvirus genome comprises at least one miRNA target sequence ligated to the first or first and second copies of the 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) Recombinant herpes simplex virus according to Embodiment 9, wherein a second copy of the ICP34.5 gene contains an inactivating mutation. 11) Recombinant herpes simplex virus according to Embodiment 9, comprising two to ten miRNA target sequences ligated to act on the first or first and second copies of the ICP34.5 gene. 12) Recombinant herpes simplex virus according to Embodiment 10 or 11, comprising two miRNA target sequences ligated to act on the first or first and second copies of the ICP34.5 gene. 13) 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 first and second copies of the ICP34.5 gene. 14) Recombinant herpes simplex virus according to Embodiment 13, wherein the miRNA target sequence is inserted in tandem in the 3' untranslated region. 15) Recombinant herpes simplex virus according to embodiment 10 or 11, wherein miRNA target sequences 2 to 10 bind to a single miRNA. 16) Recombinant herpes simplex virus according to Embodiment 10 or 11, wherein 2 to 10 miRNA target sequences bind to at least two different miRNAs.

[0028] 17) Recombinant herpes simplex virus according to Embodiment 15 or 16, 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. By convention, the strand more frequently found as the final product is indicated as miRNA, and the less frequently found partner is miRNA. * This is indicated. 18) Recombinant herpes simplex virus according to Embodiment 17, wherein the miRNA target site includes five copies of binding sites for miR-124 and miR-143. 19) The recombinant herpes simplex virus according to Embodiment 9, wherein the oncolytic herpesvirus is HSV-1. 20) Recombinant herpes simplex virus according to Embodiment 9, wherein the modified herpesvirus genome includes additional mutations or modifications to 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) Recombinant herpes simplex virus according to Embodiment 20, wherein additional mutations or modifications affect the virulence or replication activity of the virus. 22) Recombinant herpes simplex virus according to Embodiment 20, wherein the mutated or modified viral gene is ICP4 and / or ICP27. 23) Recombinant herpes simplex virus according to Embodiment 22, wherein the mutation or modification involves a functional linkage of the ICP4 and ICP27 genes with an exogenous 5' untranslated region.

[0029] 24) The recombinant herpes simplex virus according to 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 according to Embodiment 22, further comprising a modified ICP27, wherein the modification is a complete replacement of the promoter-regulatory region of ICP27. In certain embodiments, the ICP27 promoter is replaced with an 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 according to Embodiment 25, further comprising at least one nucleic acid encoding a non-viral protein selected from the group consisting of immunostimulators, antibodies, and checkpoint-blocking peptides, wherein the at least one nucleic acid is linked to act as a tumor-specific promoter. 27) Recombinant herpes simplex virus according to Embodiment 26, wherein the nonviral protein is selected from the group consisting of IL12, IL15, IL15 receptor alpha subunit, OX40L, and PD-L1 blockers. 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 leukemia virus lacking a C-terminal R-peptide, wherein the nucleic acid optionally 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 shortened (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, wherein at least one internal or terminal repeat region of the viral genome is deleted. In some yet another embodiment, the recombinant herpes simplex virus according to any one of Embodiments 1 to 28 has 5x miR-124 and 5x miR-143 binding sites at the 3'UTR of ICP34.5, and the terminal repeat is deleted (the second copies of ICP0, ICP4, and ICP34.5 are also deleted). 30) A method for lysing tumor cells, comprising the step of providing a therapeutically effective amount of recombinant herpes simplex virus according to any of the embodiments 1 to 29 described above. 31) A therapeutic composition comprising a recombinant herpes simplex virus according to any of the embodiments 1 to 29 described above and a pharmaceutically acceptable carrier. 32) A method for treating cancer in a patient suffering from cancer, comprising the step of administering a therapeutically effective amount of the composition described in Embodiment 31. Particularly preferred cancers to be treated include lung tumors, breast and prostate tumors, glioblastoma, tumors of the gastrointestinal tract (and related organs, e.g., esophageal, bile duct, anal, stomach, intestinal, pancreatic, colon and liver), and injectable tumors of all surfaces (e.g., melanoma). [Examples]

[0030] HSV-1 pre-early gene expression in normal mouse brains and human brain tumor U87 In this study, we compared HSV-1 pre-early gene expression 24 hours after injection of microRNA-regulated virus in normal mouse brains and human brain tumors (U87). Five nude mice without tumors and five nude mice with human U87 brain tumors in the cranial cavity were subjected to a total of 1 x 10⁶ injections. 6 Either PFU / mouse CXCR4-miR virus or control CXCR4 virus was injected intracranially once. 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 a tumor-specific CXCR4 promoter. The construct also includes expression cassettes for secretible IL12 / IL15 / IL15RA and for secretible peptides that inhibit PD-1 binding with PD-L1. The CXCR4 virus contains wild-type ICP34.5 lacking a microRNA binding site, but is otherwise identical to the CXCR4-miR virus. The expression of the viral pre-initial genes ICP27, ICP4, and ICP47 in normal brain tissue and tumor tissue was measured 24 hours after injection using RT-qPCR. Changes in CXCR4-miR virus gene expression levels were determined by comparing them with those of CXCR4 virus. 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 exhibit a very significant (p<0.01) reduction in the expression of all tested viral genes in normal brain tissue, but maintain high levels of viral gene expression within tumors. These results suggest that miRNA virus-dependent downregulation of ICP34.5 gene expression reduces HSV-1 replication in normal brain tissue compared to brain tumor tissue. [Examples]

[0031] ICP34.5 expression in neurons and tumor cells This example demonstrates the expression of ICP34.5 protein in neurons and tumor cells after injection with either CXCR4-miR virus or control CXCR4 virus. Mouse neuronal cells (LNCap cells) and A549 cells were treated with either CXCR4-miR virus or CXCR4 virus. Cells were pelleted 16 hours post-injection, washed with Dulbecco's Phosphate Buffer Saline (PBS), and lysed by incubation on ice for 40 minutes in RIPA buffer containing 1 mM phenylmethylsulfonyl fluoride (PMSF) and a protease inhibitor cocktail (10 mM Tris-Cl (pH 8.0), 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 140 mM NaCl). The lysates were then centrifuged at 13,000 rpm at 4°C for 10 minutes, and the supernatant was collected. ICP34.5 protein levels in each sample were determined by Western blot analysis. Total protein concentration was measured using a BSA assay. Protein lysates (30-40 μg) were mixed with 4x SDS loading dye and subsequently heated at 95°C for 10 minutes. Samples were then loaded and subjected to electrophoresis on 10% SDS-PAGE, followed by transfer to a nitrocellulose membrane. The membrane was then blocked at room temperature for 1 hour in Tris-buffered saline + Tween 20 (TBST) containing 5% BSA. The blocked membrane was incubated overnight at 4°C with anti-ICP34.5 or β-actin antibody. Subsequently, the membrane was washed with TBST three times for 10 minutes and incubated at room temperature for 1 hour with the corresponding secondary antibody. After three 10-minute washes with TBST, the membrane was incubated with enhanced chemiluminescence (ECL) reagent for 1 minute and subsequently exposed to a BIO-RAD ChemiDoc XRS+ imaging system. Band intensity was quantified using ImageJ. Figure 4 shows the results of the Western blot. The column labeled "miRNA" indicates whether the cells were infected with a virus containing (+) or lacking (-) the miRNA binding element in the 3'UTR of the ICP34.5 gene. ICP34.5 expression was found to be low in neurons 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 embodiment provides a microRNA-based oncolytic virus platform having several exemplary manipulated viral genomes. This platform is referred to herein as “Transcriptional and Translational Dual Regulation” (TTDR). The basic platform HSV-1 vector is illustrated in Figure 5. A key feature of the platform HSV-1 virus is the translational regulation of the ICP34.5 gene by inserting five copies of the binding sites for miR-124 and miR-143 into the 3'UTR of the ICP34.5 gene. A key element of this platform vector may also include transcriptional regulation of the ICP27 gene (a gene essential for viral replication) using a tumor-specific promoter. A variety of HSV-1 strains can be used as a backbone for constructing recombinant oncolytic viruses, including strains 17, KOS, F, and McKrae. All viral mutation induction can be performed in E. coli using standard lambda Red-mediated recombination techniques with HSV-1 genomes cloned on bacterial artificial chromosomes (BACs) (see below for general information: 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 following 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 the expression of cassettes encoding immunomodulatory factors IL12 / IL15 / IL15RA (which boost anti-tumor immunity). Immunomodulatory factor 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., between viral genes US1 / US2, UL3 / UL4, and / or UL50 / UL51). To facilitate in vivo testing in diverse mouse models, other recombinant viruses expressing mouse IL12 instead of human IL12 can be constructed. Human IL15 can be retained in mouse-specific oncolytic viruses due to its activity in mouse cells. The vector may include an expression cassette encoding a fusion of the env protein of gibbon leukemia virus (GALV) lacking the C-terminal R-peptide (enhancing viral cytotoxicity). Alternatively, the expression cassette may encode a fusion of glycoprotein B (e.g., truncated gB876t). The cassette can be inserted into the viral genome at locations that do not negatively affect viral gene expression and replication (e.g., between viral genes US1 / US2, UL3 / UL4 and / or UL50 / UL51). The viral vector may also contain an expression cassette for the HSV-1 thymidine kinase (TK) gene, which is inserted between viral genes at US1 / US2, UL3 / UL4, and / or UL50 / UL51. The original native TK gene is disrupted if the BAC sequence is inserted into the viral genome to facilitate mutation induction in E. coli. The presence of the exogenous TK gene enhances viral safety by making the virus susceptible to conventional treatment with guanosine analogs (e.g., ganciclovir and acyclovir).

[0034] The promoter driving the expression of the important HSV-1 transcription regulator ICP27 can be replaced with a tumor-specific promoter (e.g., hCEA, hCXCR4, PSA, or provasine (ARR2PB)). The 3'UTR of the viral gene encoding the neurotoxic factor ICP34.5 can also be modified by inserting multiple copies of a microRNA recognition element to halt ICP34.5 production in tissues containing high levels of the corresponding microRNA. In an exemplary embodiment, five copies of the miR-124 and five copies of the miR-143 recognition elements can be inserted in tandem into the 3'UTR of ICP34.5. By completely deleting the terminal repeat region of the viral genome, the overall genome size can be reduced, creating more space for transgene insertions, and the deleted TR can be manipulated to avoid disruption of the native promoter of the ICP47 gene (which is the terminal repeat portion under normal conditions). Further details of the exemplary elements discussed herein are illustrated in Figure 6. The obtained recombinant virus can be isolated using the Qiagen HiSpeed ​​MidiPrep kit and transfected into Vero cells to recover the virus (e.g., lipofectamine 2000 is used). Genome integrity can be verified using targeted sequencing and restriction profiling of all modified regions. The stability of the final recombinant virus can be confirmed by serial passage and periodic verification of transgene expression by Western blotting and ELISA. Five exemplary embodiments of this platform are shown in the table below. Two viruses were manipulated for the treatment of lung cancer (or other cancers of epithelial origin, such as renal cancer and breast cancer), and three viruses were manipulated for the treatment of prostate cancer.

[0035] TIFF0007862491000001.tif76155 [Examples]

[0036] MicroRNA-mediated regulation of ICP34.5 expression results in reduced neurotoxicity in vivo. One dose (5x10) is administered into the skull of a mouse. 7Participants were injected with either the CXCR4-TF-Fc-h1215-miR virus (with five miR-124 and miR-143 elements inserted into the 3'UTR of the ICP34.5a gene) or a control CXCR4-TF-Fc-h1215 virus lacking the aforementioned insertions. Both viral constructs also included a terminal repeat region substituted with a cassette expressing the CXCR4 promoter-driven ICP27 gene, a TF+Fc PD-L1 blocker expression cassette inserted between UL3 and UL4, and human IL12, IL15, and IL15 receptor alpha subunit. Following infection, the extent of infection was visualized by staining of mouse brain sections with rabbit polyclonal anti-HSV primary antibody and AlexaFluor 488-conjugated rat anti-rabbit secondary antibody. As shown in Figure 7, mice infected with the virus containing miR-controlled ICP34.5 showed detectable virus only along the path of the injection needle, while the virus containing wild-type ICP34.5 was widely disseminated throughout the brain. [Examples]

[0037] The 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 for 72 hours while increasing the MOI. Following infection, cell viability was measured using the MTT assay. As shown in Figures 8A and 8B, VG182LF virus demonstrated a dose-dependent increase in lung cancer cell killing compared to normal lung cells. The table below provides the determined IC50 values ​​for each cell line, showing that normal lung cells HPL1D and BEAS-2b exhibit an increase of 6.54 times and 18.93 times, respectively, in IC50 compared to the lung cancer cell line (A549).

[0038] TIFF0007862491000002.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 the expression of ICP27 and IL12 / IL15 / IL15RA genes. This experiment was repeated using further lung cancer cell lines. As shown in Figure 8C, the VG182LF virus efficiently kills a wide variety of lung cancer cells available on the market. The calculated IC50 values ​​for each cell line are shown in the table below. TIFF0007862491000003.tif45153 [Examples]

[0039] VG182LF selectively replicates in in vitro lung cancer cells. Lung cancer cells (A549) and normal lung cells (BEAS-2b) were treated with VG182LF virus at a MOI of 0.1 for various time intervals. After infection, the virus was collected and titrated with Vero cells. As shown in Figure 9, VG182LF virus successfully replicated in lung cancer cells but not in normal lung cells. At 48 hours, 6 x 10⁻⁶ cells were observed. 6 Viral particle titers exceeding [a certain level] were obtained from A549 lung cancer cells, while no significant virus was obtained from BEAS-2b normal lung cells. This suggests that microRNA regulation of ICP34.5 and the use of tumor-specific promoters to drive the expression of ICP27 and IL12 / IL15 / IL15RA negatively affect viral replication in normal cells but promote viral replication in tumor cells. We investigated VG182LF virus replication in A549 lung tumor cells or LNCaP prostate tumor cells. In short, cells were infected with either VG161 (control) or VG182LF virus for 12 or 24 hours. Cells were then collected and intracellularly stained with anti-human IL-12p70 antibody. Human IL-12-positive cells were detected by flow cytometry, and the multiplier increase in human IL-12 expression was calculated. As shown in Figure 10, increased human IL-12 expression showed a positive correlation with enhanced viral replication. The replication capacity of VG182LF virus in various lung cancer cell lines was evaluated. Cells 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. The virus from each sample was titrated with Vero cells. The data from this experiment are shown in Figures 11A-C, where the titration values ​​represent the average of three biological replicas. These data indicate that the virus can replicate to significant levels in each lung cancer cell line 48 hours post-infection. [Examples]

[0040] In vivo antitumor efficacy of VG182LF in the H1975 lung cancer model Nude mice carrying tumors (H1975) were treated with VG182LF one week after transplantation. 5.65x10 7 PFU / mouse VG182LF was injected three times at 2-day intervals. Vehicle-treated mice reached the humane endpoint 12 days after the start of treatment 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 the start of treatment. [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 less important for replication in non-neuronal cultured cells (e.g., 293FT cells). In this study, we evaluated the ability of miR-143 to influence ICP34.5 expression in 293FT cells. Initially, cells were transfected with miR-143 on day 0. As a control, 293FT cells were either transfected with scrambled miR or left untransfected. Twenty hours after transfection, the cells were washed and subsequently infected with recombinant oncolytic HSV-1 (MOI=1). This recombinant virus, along with a fusion-inducible mutation at the carboxyl terminus of gB (gB-876t), encodes binding sites for miR-143 and miR-124 in the 3'UTR of ICP34.5. Cells were collected 6 hours post-infection for RNA isolation to measure gene expression and transfection efficiency, and cells were collected 0 and 24 hours post-infection for DNA isolation to measure viral replication. As shown in Figure 14A, high levels of miR-143 were detected by RT-qPCR 6 hours post-infection in cells transfected with miR-143, while untransfected cells 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 clearly showed a significant decrease in ICP34.5 expression in samples previously transfected with miR-143, whereas a similar decrease was not observed in another viral gene (ICP27) that does not contain the miR binding site. Viral replication was quantified by qPCR 24 hours post-infection to measure the number of ICP27 copies (each copy corresponds to a separate viral genome). As shown in Figure 14C, when comparing samples transfected with miR-143 or scrambled miR, there was no significant difference in viral replication levels, suggesting that the dramatic decrease in ICP34.5 expression observed in miR-143 transfected samples was not due to a decrease in viral copy number. [Examples]

[0042] ICP34.5 miR modification improves safety by preventing neurotoxicity. In this study, DBA / 2 mice (N=3 in each group) were subcutaneously injected with one of the following: vehicle control, wild-type HSV-1, VG161 viral variant lacking ICP34.5 and without fusion-inducible mutations, or VG301 viral variant encoding binding sites for miR-143 and miR-124 in the 3'UTR of ICP34.5 along with a fusion-inducible mutation at the carboxyl terminus of gB (gB-876t). Samples were collected 6 days post-infection for HSV-1 immunostaining. As shown in Figure 15, a vigorous viral replication pattern was observed in both the brain and spinal cord of mice injected with wild-type HSV-1. In contrast, no viral replication was observed in the neuronal tissue of mice injected with either the VG161 or VG301 variant, suggesting that miR modulation of ICP34.5 is just as effective as complete deletion of ICP34.5 in preventing neurotoxicity. The remaining wild-type HSV-1 injected mice rapidly developed neurological symptoms and had to be euthanized, while all other mice treated with either the VG161 or VG301 variant remained healthy throughout the experiment, as shown in Figure 16. These results provide further evidence supporting the safety and effectiveness of OV-induced neurotoxicity prevention utilizing miR modulation of ICP34.5. Furthermore, it can be concluded that fusion-induced mutations in the VG301 variant do not result in increased morbidity or mortality. [Examples]

[0043] Evaluation of fusion-inducible mutations in oncolytic HSV-1 In this example, A549wt and BPH1 cells were infected with recombinant oncolytic HSV-1 (the HSV-1 encoded a fusion-inducible mutation in the carboxyl terminus of gB (+gB-876t)). As a control, A549wt and BPH1 cells were infected with HSV-1 lacking the fusion-inducible mutation (-gB-876t). 48 hours after infection, the cells were fixed and Giemsa stained to visualize viral plaques and syncytia formed by virus-induced cell fusion. As shown in Figure 17, a large amount of cell-for-cell fusion was observed in cells infected with the virus carrying the fusion-inducible mutation, while minimal fusion was clearly observed in cells infected with the virus lacking the fusion-inducible mutation.

[0044] The present invention has been described extensively and comprehensively in this specification. Each of the narrower groups of species and subgenera included in this comprehensive disclosure also forms part of the present invention. This includes the comprehensive description of the present invention conditionally or with negative limitations that exclude any content derived from a genus (whether or not the content to be excluded is specifically enumerated herein). As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include multiple related words unless the context clearly indicates otherwise; the term “X and / or Y” means either “X” or “Y” or both “X” and “Y”; and the “s” following a noun indicates the plural and singular forms of that noun. In addition, where the features or characteristics of the present invention are described as a group of Markushes, the present invention is intended to encompass and be described as any individual member and any subgroup of such Markushes, and a person skilled in the art would recognize this. Furthermore, the applicant reserves the right to amend the application or claims to specifically refer to any individual member and any subgroup of such Markushes. It should be understood that the terminology used herein is intended solely to describe specific embodiments and is not intended to limit them. Furthermore, unless otherwise specified herein, the terminology used herein should be given the conventional meaning known in the relevant industry. Throughout this specification, the term “embodiment” and its variations mean that the specific features, structures, or characteristics described in relation to such embodiment are included in at least one embodiment. Therefore, the phrase “in one embodiment” appearing in various places throughout this specification does not necessarily refer to the same embodiment. Furthermore, such specific 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 plural (i.e., one or more) related terms unless the content and context clearly indicate otherwise. Furthermore, it should be noted that the conjunctions “and” and “or” are generally used in the broadest sense to include “and / or” unless the content and context clearly indicate inclusiveness or exclusion as there may be. Thus, the use of alternatives (e.g., “or”) should be understood to mean one, both, or any combination thereof of those alternatives. In addition, when listed herein as “and / or,” the compound “and” and “or” are intended to include embodiments that include all of the related items or ideas and one or more other alternative embodiments that include less than all of the related items or ideas. Unless the context requires otherwise, throughout this specification and the appended claims, “comprise” and its synonyms and variations thereof (e.g., “have” and “include”) should be understood to have an open, inclusive sense (e.g., “including, but not limited to)” as variations of “comprise” (e.g., “comprises” and “comprising”). The term “consisting essentially of” is used to limit the claims to those that do not substantially affect the specified materials or processes or the fundamental or novel features of the claimed invention. Any headings used in this document are provided solely to facilitate the reader's overview of the document and should not be construed as limiting the invention or claims in any way. Accordingly, such headings and the summaries provided herein are for convenience only and should not be construed as defining the scope or meaning of the embodiments.

[0046] Where a range of values ​​is provided herein, each intermediate value between the upper and lower limits of that range (up to 1 / 10 of the lower limit unit unless the context clearly indicates otherwise) and any other intermediate values ​​described or within the range described herein are included in the present invention. The upper and lower limits of smaller ranges that may be independently included within these smaller ranges are also included in the present invention, but subject to any designated exclusion limits within the range described herein. Where a described range includes one or both limits, the range that excludes one or both of those included limits is also included in the present 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 enumerated range, and, where appropriate, fractions thereof (e.g., 1 / 10 and 1 / 100 of an integer), unless otherwise specified. Furthermore, any range of numbers enumerated herein with respect to any physical feature (e.g., polymer subunit, size, or thickness) should be understood to include any integer within the enumerated range, unless otherwise specified. As used herein, the term “approximately” means ±20% of the specified range, value, or structure, unless otherwise specified.

[0047] Any U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications cited in and / or enumerated in the application data sheet are incorporated herein by reference in their entirety. Such documents may be included by reference for the purpose of describing and disclosing, for example, materials and methodologies (which may be used in connection with the invention described herein) described in the publications. The publications considered above and throughout this text provide solely their disclosures prior to the filing date of this application. Nothing in this should be interpreted as an acceptance that the inventors do not have priority over any of the referenced publications for prior invention. All patents, publications, chemical articles, websites, and other documents and materials referenced or described herein are indicators of the level of the art in the industry to which the present invention belongs, and each of such reference documents and materials is thus included herein as if by reference the whole or individually or in whole were described herein. The applicant reserves the right to physically incorporate any and all materials and information from such patents, publications, chemical articles, websites, electronically available information, and other reference materials or documents into this specification. Generally speaking, the terms used in the following claims should not be interpreted as limiting the claims to specific embodiments disclosed in the specification and claims, but rather as encompassing all possible embodiments in addition to the full scope of the equivalents for which such claims qualify. Therefore, the claims are not limited by the disclosure.

[0048] Furthermore, the written portion of the patent includes all of the claims. Moreover, all of the claims (including all original claims along with any and all claims derived from priority documents) are included in their entirety by reference in the written portion of the specification, and the applicant reserves the right to physically incorporate any and all such claims into the written portion or any other portion of the application. Therefore, under no circumstances should the patent be construed as not providing a written description of the claims on the grounds, for example, that the exact wording of the claims is not instead stated in the specification of the patent document. The claims shall be interpreted in accordance with the law. However, notwithstanding any claimed or recognized ease or difficulty of interpretation of any claim or part thereof, under no circumstances shall any adjustment or modification of the claims or any part thereof during the examination of the application be construed as a waiver of any rights of the Patent to any and all equivalents that do not form part of the prior art. Other non-limiting embodiments exist within the following claims. The patent should not be construed as being limited to specific examples or non-limiting embodiments or methods specifically and / or expressly disclosed herein. Under no circumstances should any statement made by the examiner or any other officer or employee of the Patent Office be construed as limiting the patent unless such statement is specifically, unconditionally, or expressly adopted in the applicant's response.

Claims

1. Recombinant herpes simplex virus comprising a modified oncolytic herpesvirus genome, wherein the modified herpesvirus genome includes: 1) at least two miRNA target sequences in the 3'UTR of the ICP34.5 gene in the genome, which are ligated to one or both of the two ICP34.5 genes in the genome, wherein the at least two miRNA target sequences include five copies of a binding site for miR-124 and five copies of a binding site for miR-143; 2) a mutation or modification in at least one viral gene selected from the group consisting of ICP27 and ICP47, wherein the mutation or modification includes substitution by the CEA promoter or CXCR4 promoter of the natural promoter; and 3) a nucleic acid encoding IL12, IL15, and the IL15 receptor alpha subunit, which are ligated to act on a tumor-specific promoter.

2. Recombinant herpes simplex virus according to claim 1, further comprising a target sequence for a miRNA selected from the group consisting of: miR-122, 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-145, miR-154, miR-184, miR-188, miR-204, miR216a, miR-299, miR-300-3p, m iR-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 according to claim 1, wherein the oncolytic herpesvirus is HSV-1.

4. The recombinant herpes simplex virus according to claim 3, wherein the genome further includes a mutation that induces cell-cell fusion in a gene encoding glycoprotein B, and the gene encoding glycoprotein B encodes a glycoprotein B variant that terminates after amino acid 876.

5. The recombinant herpes simplex virus according to claim 1, further comprising at least one nucleic acid encoding a nonviral protein selected from the group consisting of immunostimulators and checkpoint-blocking peptides, wherein the at least one nucleic acid is ligated to act as a tumor-specific promoter.

6. The recombinant herpes simplex virus according to claim 5, wherein the nonviral protein is selected from the group consisting of OX40L and PD-L1 blockers.

7. A therapeutic composition for lysing tumor cells, comprising a therapeutically effective amount of recombinant herpes simplex virus according to any one of claims 1 to 6.

8. A method for lysing tumor cells in vitro, comprising providing a therapeutically effective amount of recombinant herpes simplex virus according to any one of claims 1 to 6.

9. A therapeutic composition comprising the recombinant herpes simplex virus according to any one of claims 1 to 6.

10. A therapeutic composition according to claim 9 for treating cancer in a subject.

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