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WO2025129693A1 - Virus de l'herpès oncolytique recombinant ayant un domaine de dégradation dépendant de l'oxygène - Google Patents

Virus de l'herpès oncolytique recombinant ayant un domaine de dégradation dépendant de l'oxygène Download PDF

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WO2025129693A1
WO2025129693A1 PCT/CN2023/141255 CN2023141255W WO2025129693A1 WO 2025129693 A1 WO2025129693 A1 WO 2025129693A1 CN 2023141255 W CN2023141255 W CN 2023141255W WO 2025129693 A1 WO2025129693 A1 WO 2025129693A1
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cancer
hsv
gene
odd
tumor
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Kuan Zhang
Xiaohu Liu
William Wei-Guo JIA
Zhenglong Wang
Dong ZUO
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Virogin Biotech Canada Ltd
Virogin Biotech Shanghai Ltd
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Virogin Biotech Canada Ltd
Virogin Biotech Shanghai Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A61K35/763—Herpes virus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
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    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
    • C12N2710/16621—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
    • C12N2710/16622—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
    • C12N2710/16632—Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
    • C12N2710/16641—Use of virus, viral particle or viral elements as a vector
    • C12N2710/16643—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention relates generally to compositions and methods for making and using recombinant oncolytic herpes viruses.
  • HSV-1 Oncolytic herpes simplex virus type-1 (oHSV-1) has been explored for more than a decade in laboratories and clinical trials for the treatment of many types of cancer (Ning and Wakimoto, 2014) .
  • herpes virus-based oncolytic virus (oHSV-1) T-VEC, an oncolytic HSV-1 carrying GM-CSF, became the first oncolytic virus (OV) to be approved by the FDA for treating melanoma (Ferrucci et al., 2021) .
  • Other well-known oncolytic HSV vectors include G207 (see Markert, J.M. et al. A phase 1 trial of oncolytic HSV-1, G207, given in combination with radiation for recurrent GBM demonstrates safety and radiographic responses. Mol. Ther. 22, 1048–1055 (2014) ) and G47 ⁇ (see Todo, T. et al. Intratumoral oncolytic herpes virus G47 ⁇ for residual or recurrent glioblastoma: a phase 2 trial. Nat. Med. 28, 1630–1639 (2022) ) .
  • HSV-based vectors A key characteristic of most HSV-based vectors is their ability to infect normal tissues, particularly neuronal cells, in addition to tumor cells.
  • Common approaches to making HSV-1 safer and more tumor specific include mutation or deletion of one or multiple viral genes, as well as the use of tumor specific promoters (Martuza et al., 1991) .
  • T- VEC was attenuated by deletion of the neurovirulence factor ICP34.5, which significantly compromised its ability to replicate in normal cells while enabling selective replication in tumor cells that lack protein kinase R (PKR) activity (Bommareddy et al., 2017) .
  • PTR protein kinase R
  • IFN interferon
  • ICP34.5 and ICP0 play important roles in counteracting the innate antiviral immune response, leading to severe defects in viral replication in vivo if the genes encoding either ICP34.5 or ICP0 are deleted (see E.A. Kurt-Jones, M.H. Orzalli, D.M. Knipe, Innate Immune Mechanisms and Herpes Simplex Virus Infection and Disease, Advances in anatomy, embryology, and cell biology, 223 (2017) 49-75; R. Manivanh, J.Mehrbach, D.M. Knipe, D.A.
  • the present invention provides recombinant oncolytic HSV vectors that exhibit enhanced stability in tumor microenvironments with low oxygen (hypoxic) conditions.
  • recombinant herpes simplex viruses comprising a modified oncolytic herpes virus genome, wherein the modified oncolytic herpes virus genome comprises an O 2 -dependent degradation domain (ODD) operably linked to an HSV gene selected from the group consisting of ICP0, ICP4, ICP27, and ICP34.5.
  • ODD O 2 -dependent degradation domain
  • the gene is ICP34.5.
  • the ICP34.5 gene is important to counteract IFN- ⁇ -related antiviral immunity.
  • the ICP34.5 gene is fused with an O2-dependent degradation domain (ODD) from hypoxia-inducible factor 1 alpha subunit (HIF-1 ⁇ ) .
  • ODD O2-dependent degradation domain
  • IPF-1 ⁇ hypoxia-inducible factor 1 alpha subunit
  • ICP34.5-ODD is unstable in normal tissues, resulting in rapid clearance of the virus by innate immunity.
  • the ICP34.5-ODD is protected from degradation in tumors and thereby supports oHSV replication in tumors, particularly in tumors that are hypoxic (such as solid tumors) .
  • the oHSV is constructed to express one or more desired proteins, including for example, immunomodulatory proteins such as cytokines and checkpoint inhibitors, bispecific T cell engagers (BiTEs) , multispecific immune cell engagers, bispecific adapter proteins, and tumor antigens.
  • immunomodulatory proteins such as cytokines and checkpoint inhibitors
  • BiTEs bispecific T cell engagers
  • multispecific immune cell engagers multispecific immune cell engagers
  • bispecific adapter proteins such as tumor antigens.
  • oHSVs are provided wherein the oHSV expresses a mutated or truncated US12 (tUS12) to reduce virus-induced immune suppression due to US12-mediated inhibition of antigen presentation in infected cells.
  • tUS12 mutated or truncated US12
  • oHSVs are provided wherein the oHSV expresses a mutated or truncated US9, and/or glycoprotein E (gE) , and/or glycoprotein I (gI) to reduce neurovirulence by impairing axonal trafficking of viral particles.
  • gE glycoprotein E
  • gI glycoprotein I
  • oHSVs are provided that express truncated gB (tgB) to enhance cell-to-cell spread of the virus within a tumor.
  • tgB truncated gB
  • oHSVs are provided that express mutated UL37 and/or mutated US9 to reduce the risk of neurovirulence.
  • FIG. 1A is a schematic illustration of two plasmids.
  • FIG. s 1B, 1C and 1D are graphs depicting SEAP activity in cells transfected with expression plasmids, SEAP activity in cells transfected with expression plasmids and subjected to CoCl 2 treatment, and rate of accumulation of SEAP protein in cells subjected to CoCl 2 treatment, respectively.
  • FIG. s 2A, 2B, 2C, 2D and 2E are graphs depicting SEAP activity in cells co-transfected with plasmids directing expression of various miRNAs under various conditions.
  • FIG. s 3A, 3B, 3C and 3D are diagrammatic illustrations of expression cassettes introduced into the genomes of various recombinant HSV-1 viruses.
  • FIG. s 4A and 4B are graphs depicting titers of HSV-1 viruses with and without ICP34.5 gene in the presence and absence of IFN ⁇ .
  • FIG. s 5A and 5B are graphs depicting the impact of ICP34.5-deletion, ICP34.5-ODD-mirT, or ICP0-ODD-mirT on HSV replication.
  • FIG. s 6A and 6B are graphs depicting the impact of ICP34.5-ODD-mirT on virus replication in the presence and absence of IFN ⁇ treatment.
  • FIG. s 7A and 7B are graphs depicting virus replication in an in vitro hypoxic environment.
  • the present invention relates generally to recombinant oncolytic herpes viruses having an oxygen-dependent domain.
  • the following definitions are provided:
  • ODD oxygen-dependent degradation domain from hypoxia-inducible factor 1 alpha subunit (HIF-1 ⁇ ) , which comprises approximately 200 amino acid residues located in the central region of HIF-1 ⁇ .
  • the ODD domain is responsible for stabilizing HIF-1 ⁇ in hypoxic conditions and causes HIF-1 degradation in normoxic conditions (see Huang, L.E., et al., Regulation of hypoxia-inducible factor 1 alpha is mediated by an O2-dependent degradation domain via the ubiquitin-proteasome pathway. Proc Natl Acad Sci U S A, 1998. 95 (14) : p. 7987-92) .
  • ODD comprises the minimal human ODD domain (SEQ ID NO: 13) , either with or without flanking sequences.
  • ODD comprises the homologous minimal ODD sequence from any land vertebrate, either with or without flanking sequences.
  • ODD comprises the consensus minimal ODD sequence as set forth in SEQ ID NO: 14, either with or without flanking sequences. Further embodiments of ODD domains are discussed in more detail below.
  • oncolytic herpes virus refers generally to a herpes virus capable of replicating in and killing tumor cells. Within certain embodiments the virus can be engineered to more selectively target tumor cells. Representative examples of oncolytic herpes viruses are described in US 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 by reference in their entirety.
  • Herpes Simplex Virus (HSV) 1 and 2 are members of the Herpesviridae family, which infects humans.
  • the HSV genome contains two unique regions, which are known as the unique long (U L ) and unique short (U S ) region. Each of these regions is flanked by a pair of identical inverted repeat sequences, with the repeats flanking the U L region designated as R L and the repeats flanking the U S region designated as R S .
  • R L unique long
  • R S unique short
  • the viral genome has been engineered to develop oncolytic viruses for use in e.g. cancer therapy. Tumor-selective replication of HSV may be conferred by mutation of the HSV ICP34.5 (also called ⁇ 34.5) gene.
  • HSV contains two copies of ICP34.5. Mutants inactivating one or both copies of the ICP34.5 gene are known to lack neurovirulence, i.e., be avirulent/non-neurovirulent and be oncolytic. Abrogation of neurovirulence may also be achieved by translational control of ICP34.5 using microRNA binding sites inserted into the 3’-untranslated region of ICP34.5 without inactivating both copies of the ICP34.5 gene, or after inactivating only one copy of the ICP34.5 gene and leaving one copy of ICP34.5 to be regulated via microRNA.
  • Neuropathic activity may also be inhibited by mutating other HSV genes such as the UL37 gene, the US9 gene, the gene encoding glycoprotein E, and/or the gene encoding glycoprotein I.
  • Tumor selective replication of HSV may also be conferred by controlling expression or degradation of ICP34.5 or of other key viral genes such as ICP27, ICP0, and/or ICP4.
  • Suitable oncolytic HSV may be derived from either HSV-1 or HSV-2, including any laboratory strain or clinical isolate.
  • the oHSV may be derived from one of laboratory strains HSV-1 strain 17, HSV-1 strain F, or HSV-2 strain HG52. In other embodiments, it may be derived from 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.
  • HSV vectors (and associated elements and accessory molecules) are also disclosed in U.S. Patent application entitled “Oncolytic viral vectors and uses thereof” (e.g., US2023/0241140) ; U.S. Patent entitled “Oncolytic viral delivery of therapeutic polypeptides: (U.S. Patent No. 11,078,280) ; U.S. Patent application entitled “HSV vector with Reduced Neurotoxicity” (U.S. Patent Application No. 2020/0171110) ; PCT application entitled “Targeting Moiety Decorated Oncolytic Viruses” (PCT Publication No.
  • the oHSV vector has at least one ⁇ 34.5 gene that is modified with miRNA target sequences in its 3’ UTR as disclosed herein.
  • the oHSV has two modified ⁇ 34.5 genes; in other embodiments, the oHSV has only one ⁇ 34.5 gene, and it is modified.
  • the modified ⁇ 34.5 gene (s) are constructed in vitro and inserted into the oHSV vector as replacements for the viral gene (s) .
  • the modified ⁇ 34.5 gene is a replacement of only one ⁇ 34.5 gene, the other ⁇ 34.5 is deleted. Either native ⁇ 34.5 gene can be deleted. Alternatively, both native ⁇ 34.5 genes can be deleted simultaneously.
  • the terminal repeat region which comprises ⁇ 34.5 gene, ICP0 gene, and ICP4 gene
  • the internal repeat region which is identical to the terminal repeat region in wild-type HSV and comprises a second identical copy of the ⁇ 34.5 gene, ICP0 gene, and ICP4 gene, is deleted instead of the terminal repeat region.
  • the modified ⁇ 34.5 gene may comprise additional changes, such as having an exogenous promoter and/or an ODD domain fusion.
  • the oHSV vector has at least one ICP0 gene that is modified with miRNA target sequences in its 3’ UTR as disclosed herein.
  • the oHSV has two modified ICP0 genes; in other embodiments, the oHSV has only one ICP0 gene, and it is modified.
  • the modified ICP0 gene (s) are constructed in vitro and inserted into the oHSV vector as replacements for the viral gene (s) .
  • the modified ICP0 gene is a replacement of only one ICP0 gene, the other ICP0 is deleted. Either native ICP0 gene can be deleted. Alternatively, both native ICP0 genes can be deleted simultaneously.
  • the terminal repeat region which comprises ⁇ 34.5 gene, ICP0 gene, and ICP4 gene
  • the internal repeat region which is identical to the terminal repeat region in wild-type HSV and comprises a second identical copy of the ⁇ 34.5 gene, ICP0 gene, and ICP4 gene, is deleted instead of the terminal repeat region.
  • the modified ICP0 gene may comprise additional changes, such as having an exogenous promoter and/or an ODD domain fusion.
  • the oHSV vector has at least one ICP27 gene that is modified with miRNA target sequences in its 3’ UTR as disclosed herein.
  • the oHSV has only one ICP27 gene and it is modified.
  • the modified ICP27 gene (s) are constructed in vitro and inserted into the oHSV vector as replacements for the viral gene (s) .
  • the modified ICP27 gene may comprise additional changes, such as having an exogenous promoter and/or an ODD domain fusion.
  • the oHSV may have additional mutations, which may include disabling mutations e.g., deletions, substitutions, insertions) , which may affect the virulence of the virus or its ability to replicate.
  • mutations may be made in any one or more of ICP6, ICP0, ICP4, ICP27, ICP47, ICP24, ICP56, ICP8, ICP22, UL5, UL8, UL9, UL30, UL37, UL39/40, UL42.
  • a mutation in one of these genes leads to an inability (or reduction of the ability) of the HSV to express the corresponding functional polypeptide.
  • the promoter of a viral gene may be substituted with a promoter that is selectively active in target cells or inducible upon delivery of an inducer or inducible upon a cellular event or particular environment.
  • the oHSV may have additional mutations, which may include disabling mutations, e.g., deletions, substitutions, insertions) , which may reduce the risk of recombination events in a repetitive and non-unique region of the viral genome.
  • mutations may be made in any one or more of R S region, R L region, US1 promoter, US12 promoter.
  • the promoter of a viral gene may be substituted with a promoter of a different viral gene that reduces the risk of recombination event in the repetitive and non-unique region of the native, or natural, promoter.
  • the expression of ICP4 or ICP27 is controlled by an exogenous (i.e., heterologous) promoter, e.g., a tumor-specific promoter.
  • a tumor-specific promoter e.g., a tumor-specific promoter.
  • exemplary tumor specific promoters include survivin, CEA, CXCR4, PSA, ARR2PB, or telomerase; other suitable tumor-specific promoters may be specific to a single tumor type and are known in the art. Other elements may be present. In some cases, an enhancer such as NFkB/oct4/sox2 enhancer is present.
  • the 5’UTR may be exogenous, such as a 5’UTR from growth factor genes such as FGF.
  • the oHSV may also have genes and nucleotide sequences that are non-HSV in origin.
  • a sequence that encodes a prodrug, a sequence that encodes a cytokine or other immune stimulating factor, a tumor-specific promoter, an inducible promoter, an enhancer, a sequence homologous to a host cell, among others may be in the oHSV genome.
  • Exemplary sequences encode IL12, IL15, IL15 receptor alpha subunit, IL-18, OX40L, CD40L, CXCL13, PD-L1 blocker, PD-1 blocker, or an immune cell engager (e.g., BiTE) .
  • sequences that encode a product they are operatively linked to a promoter sequence and other regulatory sequences (e.g., enhancer, polyadenylation signal sequence) necessary or desirable for expression.
  • the regulatory region of viral genes may be modified to comprise response elements that affect expression.
  • exemplary response elements include response elements for NF- ⁇ B, Oct-3/4-SOX2, enhancers, silencers, cAMP response elements, CAAT enhancer binding sequences, and insulators. Other response elements may also be included.
  • a viral promoter may be replaced with a different promoter. The choice of the promoter will depend upon a number of factors, such as the proposed use of the HSV vector, treatment of the patient, disease state or condition, and ease of applying an inducer (for an inducible promoter) . For treatment of cancer, generally when a promoter is replaced it will be 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 gene elements may be modified as well. For example, the 5’ UTR of the viral gene may be replaced with an exogenous UTR.
  • the oHSV constructs of the present invention may comprise many different elements, including for example: 1) Post Transcriptional Regulation; 2) Expression of ICP27; 3) Truncated Glycoprotein B; 4) Modified ICP47; and 5) Payload Expression.
  • oHSV are provided herein with the following specific elements: (i) both copies of ICP34.5 are regulated by fusion with ODD domain and optionally, multiple miR binding sites are inserted into the 3’-UTR of ICP34.5; (ii) an immunomodulatory payload comprising IL-12, IL-15, IL-15RA1 and/or IL-18 is inserted between HSV-1 genes UL3 and UL4; (iii) optionally, glycoprotein B (gB) is truncated to improve cell-to-cell spread; optionally, (iv) US12 is partially or entirely deleted to reduce virus-induced immune suppression; optionally, (v) US9 is partially or entirely deleted to reduce neurovirulence; optionally, (vi) glycoprotein E is partially or entirely deleted to reduce neurovirulence; optionally, (vii) glycoprotein I is partially or entirely deleted to reduce neurovirulence; optionally, (viii) UL37 is mutated or partially deleted to reduce neurovirulence.
  • an immunomodulatory payload comprising
  • both copies of ICP0 are regulated by fusion with ODD domain and insertion of multiple miR binding sites into the 3’-UTR of ICP0, optionally paired with a deletion of both copies of ICP34.5; an immunomodulatory payload comprising IL-12, IL-15, IL-15RA1 and/or IL-18 is inserted between HSV-1 genes UL3 and UL4; optionally, glycoprotein B (gB) is truncated to improve cell-to-cell spread; optionally, US12 is partially or entirely deleted to reduce virus-induced immune suppression; optionally, US9 is partially or entirely deleted to reduce neurovirulence; optionally, glycoprotein E is partially or entirely deleted to reduce neurovirulence; optionally, glycoprotein I is partially or entirely deleted to reduce neurovirulence; optionally, UL37 is mutated or partially deleted to reduce neurovirulence.
  • gB glycoprotein B
  • oHSV are provided wherein ICP34.5 expression or ICP0 expression is post-transcriptionally regulated.
  • ICP34.5 expression or ICP0 expression is post-transcriptionally regulated.
  • wild-type HSV-1 there are 2 copies of the ICP34.5 gene and 2 copies of the ICP0 gene.
  • one copy of ICP34.5 and/or one copy of ICP0 has been deleted.
  • neither copy of ICP34.5 and/or ICP0 has been deleted.
  • neither copy of ICP0 has been deleted but both copies of ICP34.5 have been deleted.
  • oHSV may be constructed wherein an ODD domain is fused to ICP34.5 and multiple copies of binding domains for miR-124 and miR-143 are inserted into the 3’-UTR region to regulate the expression of ICP34.5 post-transcriptionally.
  • oHSV may be constructed wherein an ODD domain is fused to ICP0 and multiple copies of binding domains for miR-125b-5p, miR-1226-3p, and miR-145-5p are inserted into the 3’-UTR region to regulate the expression of ICP0 post-transcriptionally.
  • Both ICP34.5 and ICP0 may be modified as described herein within the same construct. Within other embodiments of the invention, only one or both copies of the ICP34.5 gene may be modified as described herein, and the ICP0 gene remains unmodified. Within other embodiments of the invention, only one of both copies of the ICP0 gene may be modified as described herein, and the ICP34.5 gene remains unmodified. Alternatively, within yet other embodiments of the invention, one or both copies of the ICP0 gene may be modified as described herein, and the ICP34.5 gene may be deleted.
  • ICP34.5 is encoded by the HSV late gene g-34.5. It is well known for its function of suppressing anti-viral immunity in host cells, particularly neuronal cells, to cause neurotoxicity.
  • ICP0 is a viral E3 ubiquitin ligase that remodels the SUMO proteome to counteract the host immune defense to infection.
  • oHSVs can be constructed which use microRNAs as a post-transcriptional control to achieve differential expression of ICP34.5 or ICP0 in tumor cells.
  • miRNAs are ⁇ 22 nucleotides, noncoding small RNAs coded by miRNA genes, which are transcribed by RNA polymerase II to produce primary miRNA (pri-miRNA) .
  • Mature single-stranded (ss) miRNA forms the miRNA-associated RNA-induced silencing complex (miRISC) .
  • miRNA in miRISC may influence gene expression by binding to the 3′-untranslated region (3′-UTR) in the target mRNA. This region consists of sequences recognized by miRNA. If the complementarity of the miRNA: mRNA complex is perfect, the mRNA is degraded by Ago2, a protein belonging to the Argonaute family. However, if the complementarity is not perfect, the translation of the target mRNA while not fully degraded, is still suppressed.
  • miRNAs are expressed differentially in a tissue specific fashion.
  • miR-124 One example is miR-124. While the precursors of miR-124 from different species are different, the sequences of mature miR-124 in humans, mice, and rats are completely identical.
  • MiR-124 is the most abundantly expressed miRNA in neuronal cells and is highly expressed in the immune cells and organs (Qin et al., 2016, miRNA-124 in immune system and immune disorders. Frontiers in Immunology, 7 (OCT) , 1–8) .
  • Another example of differential expression of miRNA is miR-143 (Lagos-Quintana et al., 2002, Identification of tissue-specific MicroRNAs from mouse. Current Biology, 12 (9) , 735–739) .
  • MiR-143 is constitutively expressed in normal tissues but significantly downregulated in cancer cells (Michael et al., 2003, Reduced Accumulation of Specific MicroRNAs in Colorectal Neoplasia. Molecular Cancer Research, 1 (12) , 882–891) .
  • Results of RT-qPCR analysis have also shown that miR-125b is significantly suppressed in both HCC tissue and liver cancer-derived cell lines, including HepG2, SMMC-7721, and MHC97H when compared to a non-cancerous control cell line, HL-7702 (see, e.g., Zhao, L. and W. Wang. 2015. “miR-125b suppresses the proliferation of hepatocellular carcinoma cells by targeting Sirtuin7.
  • miR-125b has two subtypes, termed miR-125b-1 and miR-125b-2. They share the same miR-125b-5p sequence but contain different 3p sequences. Deep sequencing detects a high proportion of 5p sequences in both subtypes. Consequently, in certain embodiments, miR-125b-5p targeting sites are used for tumor-specific regulation of ICP0.
  • the 3’ UTR region of the ICP34.5 gene or of the ICP0 gene can be constructed to contain multiple copies of binding domains (also referred to as “miRNA target sequences” , “miRNA binding sequences” or “miRNA binding sites” ) that are completely complementary to the miRNAs of interest. Binding of miR-124 and miR-143 to the 3’-UTR of ICP34.5 mRNA and binding of miR-125b-5p, miR-1226-3p, and miR-145-5p to the 3’-UTR of ICP0 mRNA causes degradation of the mRNA; therefore the gene is post-transcriptionally downregulated in normal cells but not in tumor cells. This design allows differential expression of ICP34.5 or ICP0 in tumor cells.
  • identical or varying lengths of linker DNA can be inserted between different miRNA binding sites. Within other embodiments the linkers range from 1 to 50 base pairs. Within other embodiments the linkers separating different miRNA binding sites are less than 10 base pairs.
  • Another method of post-transcriptional regulation exploits the ODD domain of HIF-1 ⁇ to control the degradation of selected HSV-1 genes by stabilizing said genes in cancer tissues while promoting the degradation of said genes in healthy tissues.
  • hypoxia-inducible factor HIF
  • cancer entails a multifaceted regulatory mechanism that plays a pivotal role in tumor progression.
  • HIF hypoxia-inducible factor
  • PHDs prolyl hydroxylase enzymes
  • Tumor hypoxia presents a daunting barrier to many common antitumor therapeutics such as chemotherapy, radiotherapy, and immunotherapy (see Muz B, de la Puente P, Azab F, Azab AK. The role of hypoxia in cancer progression, angiogenesis, metastasis, and resistance to therapy.
  • Hypoxia (Auckl) . 2015 Dec 11; 3: 83-92 and Rakotomalala A, Escande A, Furlan A, Meignan S, Lartigau E.
  • Hypoxia in Solid Tumors How Low Oxygenation Impacts the “Six Rs” of Radiotherapy. Front Endocrinol (Lausanne) . 2021 Sep 2; 12: 742215) .
  • an ODD domain is fused to one or more functional viral genes that affect viral infection and/or replication in vivo, resulting in a virus with selective tumor tropism.
  • recombinant herpes simplex virus constructs comprising a modified oncolytic herpes virus genome, wherein the modified oncolytic herpes virus genome comprises an O 2 -dependent degradation domain (ODD) operably linked to an HSV gene selected from the group consisting of ICP0, ICP4, ICP27, and ICP34.5.
  • ODD O 2 -dependent degradation domain
  • ODD-regulated HSV-1 vectors are constructed as described herein wherein one or both copies of ICP34.5 are regulated by fusion with an ODD domain as described herein.
  • ODD domain is highly conserved among land vertebrates, with amino acid sequence similarity ranging between 78%and 98%when comparing the human ODD domain (SEQ ID NO: 13) from human HIF-1 ⁇ (SEQ ID NO: 12) to homologous regions in the genomes of rodents (SEQ ID NOs: 15 and 16) , bovines (SEQ ID NO: 17) , birds (SEQ ID NO: 18) , and non-human primates (SEQ ID NO: 19) .
  • ODD comprises the minimal human ODD domain (SEQ ID NO: 13) , either with or without flanking sequences.
  • ODD comprises the homologous minimal ODD sequence from any land vertebrate, either with or without flanking sequences.
  • SEQ ID NO: 14 is the minimal consensus amino acid sequence for Hypoxia-inducible factor 1 alpha subunit (HIF-1 ⁇ ) O2-dependent degradation domain (ODD domain) :
  • X1 is G or S
  • X2 is N, D, or no amino acid
  • X3 is D, E, N, or no amino acid
  • X4 is T, E, N, S, or no amino acid
  • X5 is E, S, D, or no amino acid
  • X6 is T, S, D, E, or no amino acid
  • X7 is D, E, or no amino acid
  • X8 is D, E, or no amino acid
  • X9 is L or C
  • X10 is E or D
  • X11 is E or D
  • X12 is L or F
  • X13 is P or S
  • X14 is N or S
  • X15 is Q or no amino acid
  • X16 is L or I
  • X17 is T, A, or S
  • X18 is A or S
  • X19 is P or T
  • X20 is S or N
  • X21 is Q or R
  • X22 is A or V
  • X23 is L or S
  • X24 is P or S
  • X25 is N or S
  • X26 is P or T
  • X27 is S or T
  • X28 is E or G
  • X29 is I or V
  • X30 is D or E
  • X31 is T or P
  • X32 is P, A, or T
  • X33 is G or A
  • X34 is R or S
  • X35 is R or no amino acid
  • X36 is L or no amino acid
  • X37 is L or no amino acid
  • X38 is Q or no amino acid
  • X39 is E or no amino acid
  • X40 is N or no amino acid
  • X41 is V or no amino acid
  • X42 is N or no amino acid
  • X43 is T or no amino acid
  • X44 is P or no amino acid
  • X45 is N or no amino acid
  • X46 is F or no amino acid
  • X47 is S or no amino acid
  • X48 is Q or no amino acid
  • X49 is N or S
  • X50 is S or N
  • X51 is E or D
  • X52 is Y or D
  • X53 is S, G, or N
  • X54 is V or A
  • X55 is E or V
  • X56 is E or I
  • X57 is T or A
  • X58 is D or E
  • X59 is S or N
  • X60 is S or N
  • X61 is A, T, P, S, or no amino acid
  • X62 is S, G, or no amino acid
  • X63 is P, S, G, or no amino acid
  • X64 is E, Q, P, S, G, or no amino acid
  • X65 is S, N, or no amino acid
  • X66 is A, M, V, Q, or no amino acid
  • X67 is S, A, N, or no amino acid
  • X68 is P, T, A, or no amino acid
  • X69 is Q, T, A, or no amino acid
  • X70 is S, T, A, or no amino acid
  • X71 is T or no amino acid
  • X72 is V, N, I, or no amino acid
  • X73 is V, G, or I
  • X74 is F or L
  • ODD domains can be utilized within the present invention, including for example, ODD proteins or derivatives thereof which are greater than 75%, 78%, 80%, 85%, 90%, 95%, 98%, or, 100%homologous to any of SEQ ID Nos. 13, 14, 15, 16, 17, 18 or 19.
  • ODD activity can be readily assessed utilizing standard assays in vitro by fusing different ODD domain variants of interest to a protein of interest such as ICP34.5 or to a reporter gene expressing an easily quantifiable protein such as GFP or luciferase, and then evaluating production of said fusion protein (s) under a range of normoxic and hypoxic conditions.
  • HSV-1 viral replication depends on a cascade of expression of viral genes, with immediate early gene products (particularly ICP4 and ICP27) controlling subsequent expression of viral early genes and late genes that govern the lytic replication cycle of the virus. Deletion of ICP4 or ICP27 results in complete abrogation of viral replication and a significant reduction in viral gene expression, which makes ICP4 and ICP27 excellent targets for tumor specific regulation in oncolytic HSV.
  • ICP27 is a multi-functional protein that regulates transcription of many virus genes. ICP27 functions in all stages of mRNA biogenesis from transcription, RNA processing and export, to translation. ICP27 has also been implicated in nuclear protein quality control, cell cycle control, activation of stress signaling pathways and prevention of apoptosis.
  • the native promoter of ICP27 is replaced with a promoter for human C-X-C motif chemokine receptor 4 (CXCR4) to enhance expression in liver tumors, particularly hepatocellular carcinoma (HCC) .
  • CXCR4 C-X-C motif chemokine receptor 4
  • HCC hepatocellular carcinoma
  • ICP27 expression may be rendered even more tumor specific via incorporation of microRNA binding sites within the 3’ untranslated region (3’ UTR) of ICP27, in which the binding sites include multiple copies of DNA sequences that are complementary to microRNAs which are present at relatively high concentrations in normal cells but are downregulated in cancer cells.
  • the oHSV includes 5 copies of miR-223 and 5 copies of miR-125b binding sites in the 3’ untranslated region (3’-UTR) of ICP27.
  • HSV-1 membrane fusion is a crucial step during infection. It is dependent on four essential viral glycoproteins (gB, gD, gH, and gL) , which mediate entry into host cells by merging the viral envelope with a host cell membrane.
  • the core fusion protein is glycoprotein B (gB) , a 904-residue glycosylated transmembrane protein encoded by the UL27 gene of HSV-1.
  • gB glycoprotein B
  • Multiple types of mutations within the cytoplasmic domain of gB can be used to yield a hyperfusogenic phenotype, increasing cell-cell fusion.
  • gB may be modified by truncating C-terminal amino acids 877 to 904 from the full-length protein.
  • the promoter controlling expression of the US12 gene which encodes ICP47
  • the promoter controlling expression of the US1 gene which is located approximately 13k base pairs from the US12 gene.
  • large regions of the native ICP47 promoter include repetitive sequences that may facilitate spurious homologous recombination events.
  • replacement of the native ICP47 promoter with a heterologous (i.e., exogenous) promoter is predicted to improve viral genomic stability.
  • the oHSV construct may, in some embodiments, replace the native ICP47 promoter with the native ICP27 promoter.
  • the native ICP27 promoter includes the entire sequence of DNA located between the coding regions of UL53 (gK) and UL54 (ICP27) .
  • the ICP27 promoter sequence may be 90%, 80%, 70%, 60%, or 50%identical to the ICP27 promoter sequence of any known human herpes virus type 1 strain or human herpes virus type 2 strain, e.g., human herpes virus type 1 strain 17 (NCBI reference sequence NC_001806.2) .
  • ICP47 acts to block the transporter associated with antigen presentation (TAP) , preventing antigenic peptides from being transported into the endoplasmic reticulum, which is the site of assembly of MHC class I molecules.
  • TEP antigen presentation
  • the oHSV expresses a truncated US12 gene (tUS12) with an N-terminal deletion of amino acids 2-36 of ICP47 in order to ameliorate virus-induced defects in MHC presentation.
  • oHSV contain genes and nucleotide sequences that are non-HSV in origin.
  • a sequence that encodes a therapeutic molecule, a prodrug, a sequence that encodes a cytokine or other immune stimulating factor, a tumor-specific promoter, an inducible promoter, an enhancer, a sequence homologous to a host cell, among others, may be included in the oHSV genome.
  • Exemplary sequences encode immune cell engagers (e.g., bispecific T cell engagers) , bispecific antibodies, tumor antigens, multivalent adapter proteins for immune cell retargeting, IL-12, IL-15, IL-15 receptor alpha subunit, IL-18, OX40L, CD40L, CXCL13, or checkpoint inhibitors such as a CTLA-4 blocker, a PD-L1 blocker, or a PD-1 blocker.
  • a promoter sequence e.g., CXCR4, CEA, EF1 ⁇ , CAG, or CMV
  • other regulatory sequences e.g., enhancer, polyadenylation signal sequence
  • a “therapeutic molecule” refers to a molecule that enhances the therapeutic efficacy of an oncolytic virus described herein.
  • the therapeutic molecules described herein are proteins, nucleic acids, or a combination thereof.
  • Exemplary therapeutic molecules include cytokines, chemokines, antibodies, or antigen binding fragments thereof, proteases, RNA polynucleotides, and DNA polynucleotides.
  • the oHSV provided herein co-express multiple cytokines (e.g., any combination of IL-12, IL-15, IL-15 receptor alpha subunit and IL-18) to further stimulate an immunomodulatory response.
  • cytokines e.g., any combination of IL-12, IL-15, IL-15 receptor alpha subunit and IL-18
  • Expression of IL-12 promotes polarization of antigen exposed T cells towards an inflammatory and anti-tumor T H 1 phenotype, while IL-15 activates NK cells to further increase tumor killing and activation of antigen presenting cells.
  • the oHSV encodes IL-15R ⁇ to further enhance immune stimulation.
  • cytokine combinations include IL-12 and IL-15, IL-12, IL-15 and IL-15 receptor alpha subunit; IL-15 and IL-15 receptor alpha subunit; and IL-12, IL-15, IL-15 receptor alpha subunit and IL-18) .
  • IL-12, IL-15, and IL-15R ⁇ can be driven by a single strong promoter (e.g., a viral promoter such as CMV, or other strong promoters such as EF-1alpha or CAG) and the polypeptides are linked with 2A self-cleaving peptides (see Z. Liu et al., 2017, Systematic comparison of 2A peptides for cloning multi-genes in a polycistronic vector. Scientific Reports, 7 (1) , 1–9) that generate the 3 individual proteins through a mechanism of ribosomal skipping during translation.
  • a single strong promoter e.g., a viral promoter such as CMV, or other strong promoters such as EF-1alpha or CAG
  • expression cassettes are provided for the expression of one or more proteins, such as, for example, immunomodulatory proteins.
  • expression cassette is meant to include any type of genetic construct containing a nucleic acid coding for a gene product in which part or all of the nucleic acid coding sequence is capable of being transcribed.
  • the expression cassette comprises a promoter which is operably linked to a nucleic acid sequence encoding the one or more immunomodulatory proteins.
  • proteins that can be expressed include, but are not limited to, tumor antigens and immunomodulatory proteins such as cytokines and checkpoint inhibitors.
  • the expression cassette may be used to express human papillomavirus antigens. In other embodiments, the expression cassette may be used to express prostate specific antigens.
  • Tumor antigen or “tumor antigens” as utilized herein refers to antigens that are presented by MHC class I or class II molecules on the surface of tumor cells. Antigens which are found only on tumor cells are referred to as “Tumor Specific Antigens” or “TSAs, ” while antigens that are presented by both tumor cells and normal cells are referred to as “Tumor Associated Antigens” or “TAAs.
  • tumor antigens include, but are not limited to AIM-2, AIM-3, ART1, ART4, B7-H3, B7-H6, BAGE, ⁇ 1, 6-N, ⁇ -catenin, B-cyclin, BMI1, BRAF, BRAP, C13orf24, C6orfl53, C9orfl12, CA-125, CABYR, CASP-8, cathepsin B, Cav-1, CD70, CD74, CDK-1, CEA, CEAmidkin, COX-2, CRISP3, CSAG2, CSPG4, CTAG2, CYNL2, DHFR, E-cadherin, EGFRvIII, EpCAM , EphA2/Eck, ESO-1, EZH2, FAP, FR ⁇ , Fra-1/Fosl 1, FTHL17, GAGE1, Ganglioside/GD2, GD3, GLEA2, Glil, GLUT1, GnT-V, GOLGA, gp75, gpl
  • Particularly preferred tumor antigens include glucose transporter 1 (GLUT1) , transferrin receptor 1 (TfR1) , CEA, CEACAM6, HER-2, EGFR, FAP, 5T4, PSMA, Mesothelin, CA9, MUC16, NY-ESO1, Grp94, and WT1.
  • immunomodulatory protein refers to a protein that is capable of altering or modulating the immune system of a subject.
  • Immunomodulatory proteins can be derived from naturally occurring proteins, such as cytokines, chemokines, and/or co-stimulatory molecules (e.g., recombinantly produced from sequences encoding intact molecules or active fragments thereof) .
  • immunomodulatory proteins include: a) cytokines (or active fragments thereof) , such as IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-15, IL-15 receptor alpha subunit, IL-18, GM-CSF and interferon gamma; b) chemokines (or active fragments thereof) , such as IL-8, SDF-1 ⁇ , MCP1, MCP2, MCP3 and MCP4 or MCP5, RANTES, MIP-5, MIP-3, eotaxin, MIP-1 ⁇ , MIP-1 ⁇ , CMDC, TARC, LARC, or SLC; and/or C) co-stimulatory molecules (or active fragments thereof) , such as CD80, CD86, ICAM-1, LFA-3, C3d, CD40-L or Flt 3L.
  • the immunomodulatory protein can be secreted or linked to the surface of the recombinant
  • the immunomodulatory protein is an immune checkpoint modulator (e.g., an agonist of an immune cell stimulating receptor, such as an agonist of BAFFR, BCMA, CD27, CD28, CD40, CD122, CD137, CD226, CRTAM, GITR, HVEM, ICOS, DR3, LTBR, TACI, and/or OX40, or an antagonist of an inhibitory signal of an immune cell, such as an antagonist of A2AR, BTLA, B7-H3, B7-H4, CTLA4, GAL9, IDO, KIR, LAG3, PD-1, TDO, TIGIT, 3, and/or VISTA (see, e.g., "Immune Checkpoint Inhibitors in Cancer” 2019 Elsevier inc., ISBN-13: 978-49032 486, which is incorporated by reference in its entirety) .
  • an immune checkpoint modulator e.g., an agonist of an immune cell stimulating receptor, such as an agonist of BAFFR, BCMA, CD27
  • compositions are provided that may be used to prevent, treat, or ameliorate the effects of a disease, such as, for example, cancer. More particularly, therapeutic compositions are provided comprising at least one oncolytic virus as described herein.
  • compositions will further comprise a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is meant 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 whom it is administered (see generally Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21st ed. (May 1, 2005 and in The United States Pharmacopeia: The National Formulary (USP 40 –NF 35 and Supplements) .
  • suitable pharmaceutical carriers include phosphate buffered saline solutions, water, emulsions (such as oil /water emulsions) , various types of wetting agents, sterile solutions, and others.
  • Additional pharmaceutically acceptable carriers include gels, bioabsorbable matrix materials, implantation elements containing the oncolytic virus, or any other suitable vehicle, delivery, or dispensing means or material (s) .
  • Such carriers can be formulated by conventional methods and can be administered to the subject at an effective dose.
  • Additional pharmaceutically acceptable excipients include, but are not limited to, water, saline, polyethylene glycol, hyaluronic acid, and ethanol.
  • Pharmaceutically acceptable salts can also be included therein, e.g., mineral acid salts (such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like) and the salts of organic acids (such as acetates, propionates, malonates, benzoates, and the like) .
  • mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like
  • organic acids such as acetates, propionates, malonates, benzoates, and the like
  • Such pharmaceutically acceptable (pharmaceutical-grade) carriers, diluents and excipients that may be used to deliver the oHSV to a cancer cell will preferably not induce an immune response in the individual (subject) receiving the composition (and will preferably be administered without undue toxicity) .
  • compositions provided herein can be provided at a variety of concentrations.
  • dosages of oncolytic virus can be provided which range from about 10 6 to about 10 9 PFU/mL.
  • the dosage can range from about 10 6 to about 10 8 PFU/mL, or from about 10 7 to about 10 9 PFU/mL, with up to 8 mLs per dose being injected for intravenous delivery or up to 4 mLs per dose being injected into a patient with large lesions (e.g., >5 cm) , and smaller amounts (e.g., up to 0.1mLs per dose, or up to 0.5 mLs per dose, or up to 1 mLs per dose, or up to 2 mLs per dose, or up to 3 mLs per dose, or less than 4 mLs per dose) in patients with smaller lesions (e.g., ⁇ 5 cm) , repeated every day, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, about every
  • lower dosages than standard or higher dosages than standard may be utilized.
  • less than about 10 6 PFU/ml or more than about 10 9 PFU/mL (with up to 8 mLs being injected into a patient every day, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, about every week, about every 2 weeks, about every 2 –3 weeks, about every 3-4 weeks, about every month, about every 2 months, about every 3 months, about every 4 months, about every 5 months, or about every 6 months) can be administered to a patient.
  • compositions may be stored at a temperature conducive to stable shelf-life, which includes room temperature (about 20°C) , 4°C, -20°C, -80°C, and in liquid N2. Because compositions intended for use in vivo generally do not have preservatives, storage will generally be at colder temperatures. Compositions may be stored dry (e.g., lyophilized) or in liquid form.
  • compositions described herein comprising the step of administering an effective dose or amount of oHSV as described herein to a subject.
  • an effective amount of the compositions described herein is an amount that induces remission, reduces tumor burden, and/or prevents tumor spread or growth of the cancer. Effective amounts may vary according to factors such as the subject’s disease state, age, gender, and weight, as well as the pharmaceutical formulation, the route of administration, and the like, but can nevertheless be routinely determined by one skilled in the art.
  • the therapeutic compositions are administered to a subject diagnosed with cancer or suspected of having a cancer.
  • Subjects may be human or non-human animals.
  • compositions are used to treat cancer.
  • beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, diminishment of the reoccurrence of disease, and remission (whether partial or total) , whether detectable or undetectable.
  • the terms “treating” , and “treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment.
  • cancers include carcinomas and sarcomas. Further examples include, but are not limited to cancer of the bile duct, brain (e.g., glioblastoma) , breast, cervix, colorectal, CNS (e.g., acoustic neuroma, astrocytoma, craniopharyogioma, ependymoma, glioblastoma, hemangioblastoma, medulloblastoma, menangioma, neuroblastoma, oligodendroglioma, pinealoma and retinoblastoma) , endometrial lining, kidney, bladder, larynx, lung, liver, oral cavity, ovaries, pancreas, prostate, skin (e.g., melanoma and squamous cell carcinoma) , GI tract (e.g., esophagus, stomach, and colon) and thyroid.
  • CNS
  • Cancers can comprise solid tumors (e.g., sarcomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma and osteogenic sarcoma) , or be some combination of solid and diffuse tumors (e.g., a metastatic cancer having both solid tumors and disseminated or diffuse cancer cells) . Cancers can also be resistant to conventional treatment (e.g., conventional chemotherapy and/or radiation therapy) . Benign tumors and other conditions of unwanted cell proliferation may also be treated.
  • Particularly preferred cancers to be treated include hypoxic solid tumors or any solid tumor with the potential to become hypoxic as the tumor progresses.
  • Representative examples include lung tumors, breast and prostate tumors, glioblastomas, tumors of the gastro-intestinal tract (and associated organs) e.g., esophagus, cholangiocarcinoma, anal, stomach, bladder, intestine, pancreatic, colon and liver, and all surface injectable tumors (e.g., melanomas) . All types of solid tumors, especially malignant solid tumors, can exhibit some degree of hypoxia.
  • hypoxic tumors are defined as tumors with median intratumoral oxygen tension (pO 2 ) below the pO 2 of their healthy tissue of origin.
  • Particularly preferred hypoxic tumors to be treated exhibit a median intratumoral oxygen tension (pO 2 ) below 30 mmHg, below 25 mmHg, below 20 mmHg, below 15 mmHg, below 10 mmHg, below 5 mmHg, below 3 mmHg, below 2.5 mmHg, below 2 mmHg, below 1.5 mmHg, or below 1 mmHg.
  • Particularly preferred patients to be treated have more than 19%of measured tumor samples with oxygen tension (pO 2 ) below 25 mmHg, below 20 mmHg, below 15 mmHg, below 10 mmHg, below 5 mmHg, below 3 mmHg, below 2.5 mmHg, below 2 mmHg, below 1.5 mmHg, or below 1 mmHg.
  • oxygen tension pO 2
  • Tumor hypoxia may be detected by invasive direct methods such as by insertion of oxygen electrodes; by non-invasive direct methods such as phosphorescence quenching, photoacoustic lifetime imaging, electron paramagnetic resonance oximetry, 19 F-MRI relaxometry, or Overhauser-enhanced MRI; by non-invasive indirect methods such as near-infrared spectroscopy, blood oxygen level-dependent MRI, dynamic contrast-enhanced MRI, or photoacoustic tomography; or by detection of endogenous protein markers associated with cellular responses to oxygen deprivation such as CAIX, HIF-1 ⁇ , GLUT-1, OPN, LOX, Ephrin A1, or Galectin 1.
  • invasive direct methods such as by insertion of oxygen electrodes
  • non-invasive direct methods such as phosphorescence quenching, photoacoustic lifetime imaging, electron paramagnetic resonance oximetry, 19 F-MRI relaxometry, or Overhauser-enhanced MRI
  • non-invasive indirect methods such
  • the oncolytic herpes viruses provided herein may be administered along with (e.g., prior to, at the same time or subsequently) a therapeutic agent that blocks, binds to, or otherwise inhibits an immune checkpoint inhibitor.
  • immune checkpoints are key regulators of the immune system that, when stimulated, can dampen the immune response to an immunological stimulus such as cancer.
  • immune checkpoint inhibitors work to block inhibitory checkpoints, restoring immune system function (e.g., immunological response to a cancer) .
  • Representative examples of immune checkpoint inhibitors bind to CTLA-4, PD-1, or ligands thereof.
  • CTLA-4 or "cytotoxic T-lymphocyte-associated protein 4" , and also known as CD152
  • PD-1 or “Programmed cell death protein 1” , and also known as CD279 are proteins on the surface of T and B cells that have a role in down regulating the immune system, thereby suppressing T cell inflammatory activity and preventing or decreasing the ability of the immune system to kill tumor cells.
  • CTLA-4 also has primary ligands: CD80 and CD86 on the surface of T cells.
  • CTLA-4 antagonists include peptides and antibodies that block, bind to or otherwise inhibits the binding of CTLA-4 or it's ligands in a statistically significant manner, thereby inhibiting or diminishing their activity.
  • Representative examples of CTLA-4 antagonists include: Ipilimumab (Yervoy -a fully human anti-CTLA-4 monoclonal antibody, IgGl isotype) ; and Tremelimumab (Imjudo -a fully human anti-CTLA-4 antibody, lgG2 isotype) .
  • patents that relate to anti-CTLA-4 antibodies and variants thereof include US 5,811,097, 5,855,887, 5,977,318, 6,051,227, 6,207,156, 6,682,736, 6,984,720, 7,109,003, 7,132,281 and 10,196,445, and PCT Publication Nos. WO 2001/014424, WO 2004/035607, 2005/0201994, all of which are incorporate by reference in their entirety.
  • PD-1 Similar to CTLA-4, PD-1 also has two ligands: PD-L1 and PD-L2.
  • Representative examples of PD-1, PD-L1 and PD-L2 antagonists include peptides and antibodies that block, bind to or otherwise inhibits the binding of PD-1 or it's ligands in a statistically significant manner, thereby inhibiting or diminishing their activity.
  • PD-L1 antagonists include: Atezolizumab (TECENTRIQ-ahumanized anti-PD-Ll antibody) ; Avelumab (BAVENCIO-afully human anti-PD-Ll antibody) ; Durvalumab (IMFINZI -a human anti-PD-Ll antibody) ; Durvalumab (IMFINZI -a fully human igGl antibody) ; KN035; Cosibelimab (CK-301 by Checkpoint Therapeutics; AUNP12 (apeptide PD-1/PD-L1 inhibitor by Aurigene and Laboratoires Pierre Fabre) ; CA-170; and BMC-986189 (a macrocyclic peptide) .
  • Atezolizumab TECENTRIQ-ahumanized anti-PD-Ll antibody
  • Avelumab BAVENCIO-afully human anti-PD-Ll antibody
  • Durvalumab IMFINZI -a human anti-PD-Ll antibody
  • PD-1 antagonists include the following representative anti-PD-1 antibodies: Nivolumab (OPDIVO -a human anti-PD-1 antibody) ; Pembrolizumab (KEYTRUDA -a humanized anti-PD-1 antibody) ; Cemiplimab (LIBTAYO -an anti-PD-1 antibody) ; Dostarlimab (JEMPERLI -an anti-PD-1 antibody; Vopratelimab (JTX-4014) by Jounce Therapeutics; Spartalizumab (PDR001) by Novartis; Camrelizumab (SHR1210) -an anti-PD-1 monoclonal antibody by Jiangsu HengRui Medicine Co., Ltd.; Sintilimab (IBI308) , a human anti-PD-1 antibody developed by Innovent and Eli Lilly; Tislelizumab (BGB-A317) -a humanized lgG4 anti-PD-1 monoclonal antibody; Toripalimab (JS 001
  • Representative patents and patent applications describing PD-1, PD-L1 andPD-L2 antagonists and methods for assessing their activity include, for example: US Patent Nos. 7,595,048, 7,943,743, 8,952,136, 8,217,149, 8,609,089, 8,735,553, 8,779,105, 8,779,108, 8993731, and 9,815,897; U.S. Publication Nos. 2010/0203056, 2010/0266617, 2011/0229461, 2013/0017199, 2014/341917, 2015/0203579, 2016/0311903, 2016/0376367, 2016/0311903, 2017/0044259 and2018/0346569; and PCT Publication No. WO2012145493; all of which are incorporated by reference in their entirety.
  • the recombinant herpes simplex viruses and immune checkpoint inhibitors described herein may be given by a route that is e.g., oral, topical, parenteral, systemic, intravenous, intramuscular, intraocular, intrathecal, intratumoral, subcutaneous, or transdermal.
  • the oncolytic virus may be delivered by a cannula, by a catheter, or by direct injection.
  • the site of administration may be directly into the tumor, adjacent to the tumor, or at a site distant from the tumor. The route of administration will often depend on the type of cancer being targeted.
  • the immune checkpoint inhibitor may be delivered by a method different from the recombinant herpes virus (e.g., intravenously) .
  • the optimal or appropriate dosage regimen of the oncolytic virus is readily determinable within the skill of the art, by the attending physician based on patient data, patient observations, and various clinical factors, including for example a subject’s size, body surface area, age, gender, and the particular oncolytic virus being administered, the time and route of administration, the type of cancer being treated, the general health of the patient, and other drug therapies to which the patient is being subjected.
  • treatment of a subject using the oncolytic virus described herein may be combined with additional types of therapy, such as administration of a different oncolytic virus, radiotherapy, administration of a checkpoint inhibitor, chemotherapy using, e.g., a chemotherapeutic agent such as etoposide, ifosfamide, adriamycin, vincristine, doxycycline, and others.
  • additional types of therapy such as administration of a different oncolytic virus, radiotherapy, administration of a checkpoint inhibitor, chemotherapy using, e.g., a chemotherapeutic agent such as etoposide, ifosfamide, adriamycin, vincristine, doxycycline, and others.
  • the optimal or appropriate dosage regimen of the immune checkpoint inhibitor is readily determinable within the skill of the art, by the attending physician based on patient data, patient observations, and various clinical factors, including for example a subject’s size, body surface area, age and gender.
  • immune checkpoint inhibitors that have already been approved by regulatory bodies, they can be, within certain embodiments, administered in accordance with regulatory guidelines.
  • Recombinant herpes simplex viruses described herein may be formulated as medicaments and pharmaceutical compositions for clinical use and may be combined with a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.
  • the formulation will depend, at least in part, on the route of administration. Suitable formulations may comprise the virus and inhibitor in a sterile medium.
  • the formulations can be fluid, gel, paste or solid forms. Formulations may be provided to a subject or medical professional.
  • a therapeutically effective amount of virus and/or immune checkpoint inhibitor is preferably administered. This is an amount that is 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, site of delivery, and other factors.
  • the oncolytic virus and/or immune checkpoint inhibitor can be administered along with a variety of other methods, including for example, after radiation, chemotherapy, and/or surgical resection of a tumor.
  • a recombinant herpes simplex virus comprising a modified oncolytic herpes virus genome, wherein the modified oncolytic herpes virus genome comprises an O 2 -dependent degradation domain (ODD) operably linked to an HSV gene selected from the group consisting of ICP0, ICP4, ICP27, and ICP34.5.
  • ODD O 2 -dependent degradation domain
  • the gene is ICP34.5.
  • modified oncolytic herpes virus genome further comprises at least one nucleic acid encoding a non-viral protein selected from the group consisting of tumor antigens and immunomodulatory proteins, wherein the at least one nucleic acid is operably linked to a promoter.
  • non-viral protein is selected from the group consisting of IL12, IL15, IL15 receptor alpha subunit, IL-18, OX40L, CD40L, CXCL13, PD-L1 blocker, PD-1 blocker, and immune cell engagers (e.g., BiTE) .
  • modified oncolytic herpes virus genome further comprises a fusogenic mutation in a gene encoding glycoprotein B (gB) .
  • glycoprotein B (gB) encodes a glycoproptein B variant that terminates after amino acid 876.
  • modified oncolytic herpes virus genome further comprises a mutation in a gene encoding US12, wherein the gene encoding US12 encodes a truncated US12.
  • modified oncolytic herpes virus genome further comprises a mutation in a gene encoding UL37 and/or US9 and/or glycoprotein E and/or glycoprotein I to reduce neurovirulence.
  • a method of treating cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a recombinant HSV of any one of embodiments 1 to 10.
  • the cancer comprises adrenocortical carcinoma, anal cancer, bladder cancer, brain tumor, brain stem glioma, brain tumor, cerebellar astrocytoma, cerebral astrocytoma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal, pineal tumors, hypothalamic glioma, breast cancer, carcinoid tumor, carcinoma, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, extrahepatic bile duct cancer, Ewing’s family of tumors (Pnet) , extracranial germ cell tumor, eye cancer, intraocular melanoma, gallbladder cancer, gastric cancer, germ cell tumor, extragonadal, gestational trophoblastic tumor, head and neck cancer, hypopharyngeal cancer, islet cell carcinoma, laryngeal cancer, leukemia, acute lymphoblastic leukemia, oral cavity cancer, liver cancer, lung
  • inventions 11, 2, 13, or 14 further comprising the step of administering an immune checkpoint inhibitor.
  • immune checkpoint inhibitors inhibit CTLA-4 and/or its ligands, or, PD-1 and/or its ligands.
  • a therapeutic composition comprising a recombinant herpes simplex virus of any one of embodiments 1 to 10 and a pharmaceutically acceptable carrier.
  • the terms “about” and “approximately” are used as equivalents. Any numerals used in this application with or without about/approximately are meant to cover any normal fluctuations appreciated by one of ordinary skill in the relevant art.
  • the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100%of a possible value) .
  • “Increase” refers to an increase in a particular value of at least 5%, for example, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99, 100, 200, 300, 400, 500%or more as compared to a reference value.
  • An increase in a particular value may also be represented as a fold-change in the value compared to a reference value, for example, at least 1-fold, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 1000-fold or more, increase as compared to the level of a reference value.
  • sequence identity refers to the percentage of bases or amino acids between two polynucleotide or polypeptide sequences that are the same, and in the same relative position. As such one polynucleotide or polypeptide sequence has a certain percentage of sequence identity compared to another polynucleotide or polypeptide sequence. For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared.
  • reference sequence refers to a molecule to which a test sequence is compared.
  • Base pairings may include both canonical Watson-Crick base pairing and non-Watson-Crick base pairing (e.g., Wobble base pairing and Hoogsteen base pairing) .
  • adenosine-type bases (A) are complementary to thymidine-type bases (T) or uracil-type bases (U)
  • cytosine-type bases (C) are complementary to guanosine-type bases (G)
  • universal bases such as such as 3-nitropyrrole or 5-nitroindole can hybridize to and are considered complementary to any A, C, U, or T.
  • Inosine (I) has also been considered in the art to be a universal base and is considered complementary to any A, C, U, or T. See Watkins and SantaLucia, Nucl. Acids Research, 2005; 33 (19) : 6258-6267.
  • “Operably linked” refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner.
  • a promoter is operably linked to a polynucleotide sequence if the promoter affects the transcription or expression of the polynucleotide sequence.
  • subject or "patient” , as used herein, is considered to be any mammalian subject to which a composition described herein is administered according to the methods described herein.
  • methods of disseminating the present are employed to treat a human subject.
  • the methods of the present disclosure can also be used to treat non-human primates (for example, monkeys, baboons and chimpanzees) , rats, mice, cattle, horses, cats, dogs, pigs, rabbits, goats, deer, sheep, ferrets, piglets guinea pigs, gerbils, hamsters, bats, birds (for example, chickens, turkeys and ducks) , fish and reptiles.
  • non-human primates for example, monkeys, baboons and chimpanzees
  • rats mice, cattle, horses, cats, dogs, pigs, rabbits, goats, deer, sheep, ferrets, piglets guinea pigs, gerbils,
  • Reduced viral replication refers to a level of viral replication that is lower in a first cell or first population of cells compared to a second cell or a second population of cells. In some embodiments, the level of viral replication in the first cell or first population of cells is reduced by at least 5%compared to the level of viral replication in the second cell or population of cells. In some embodiments, the level of viral replication in the first cell or first population of cells is reduced by at least 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%or more compared to the level of viral replication in the second cell or population of cells. In some embodiments, viral replication in the first cell or first population of cells is completely inhibited compared to the viral replication in the second cell or population of cells.
  • Reduced (or reduce) neurovirulence refers to a reduction in neurovirulence of the HSV vector in a patient who has received the vector.
  • reduced (or reduce) neurovirulence refers to reducing the incidence of neurotoxicity (e.g., herpes encephalitis) in patients treated with the virus.
  • the amount of latent viral genomes present in a population of neurons infected by the mutant HSV-1 is reduced by at least 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%or more compared to the amount of latent viral genomes present in a population of neurons infected by wild-type HSV-1.
  • the level of viral reactivation from latency observed in a population of neurons infected by the mutant HSV-1 is reduced by at least 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%or more compared to the level of viral reactivation from latency observed in a population of neurons infected by wild-type HSV-1.
  • the retrograde or anterograde axonal transport of mutant HSV-1 is impaired by at least 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%or more compared to the retrograde or anterograde axonal transport of wild-type HSV-1.
  • latent infection, reactivation from latency, retrograde axonal transport, or anterograde axonal transport is completely inhibited in mutant HSV-1 compared to wild-type HSV-1.
  • Assays for determining neurotoxicity and viral reactivation from latency are widely known to those skilled in the art.
  • Avoid (or avoidance of) virus-induced immune suppression refers to inhibition of antigen presentation in cells infected with wild-type HSV-1 compared to cells infected with mutant HSV-1 comprising an inactivating mutation or deletion in ICP47, which normally functions to impair the MHC-I antigen presentation pathway by binding to the human transporter associated with antigen presentation (TAP) to block its binding to antigenic peptides.
  • TAP antigen presentation
  • MHC-I antigen presentation in cells infected with wild-type HSV-1 is reduced by at least 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%or more compared to MHC-I antigen presentation in cells infected with mutant HSV-1. In some embodiments, MHC-I antigen presentation is completely inhibited in cells infected with wild-type HSV-1 compared to cells infected with mutant HSV-1.
  • ODD O2-dependent degradation domain
  • SEAP alkaline phosphatase
  • ODD O2-dependent degradation domain
  • plasmids L21P51 and L22F12 were constructed to express SEAP and SEAP-ODD, respectively (Fig 1A) . These plasmids were transfected into cells for expression of SEAP or SEAP-ODD protein. Secreted SEAP in cell supernatant was quantified using a colorimetric enzyme assay (InvivoGen, QUANTI-Blue TM ) (FIG. 1B and C) . The results demonstrate that SEAP protein level accumulated over time in L21P51-transfected cells. However, the level of SEAP protein in L22F12-transfected cells demonstrated minor increases up to 72h post-transfection, suggesting that fusion with the ODD induced degradation of SEAP in L22F12-transfected cells.
  • a suitable reporter protein may comprise the protein that will be regulated using an ODD domain in vivo such as the HSV-1 ICP34.5 protein or the HSV-1 ICP0 protein, or it may comprise a protein that is easily quantifiable such as alkaline phosphatase or a fluorescent protein (e.g., GFP, RFP, NeonGreen, or luciferase) .
  • alkaline phosphatase or a fluorescent protein (e.g., GFP, RFP, NeonGreen, or luciferase)
  • SEAP secreted alkaline phosphatase
  • ODDs O2-dependent degradation domains
  • plasmids are constructed to express SEAP alone and SEAP fused to each ODD variant being tested. These plasmids are transfected into cells for expression of SEAP or SEAP-ODD proteins under normoxic conditions. Secreted SEAP in the cell supernatant is quantified using a colorimetric enzyme assay, demonstrating reduced levels of SEAP protein in cells transfected with some SEAP-ODD variants compared to cells transfected with SEAP lacking the ODD domain.
  • SEAP-ODD variants exhibiting the highest levels of SEAP degradation under normoxic conditions are further tested under hypoxic conditions in tissue culture either by incubating transfected cells in oxygen-controlled incubators or by treating the transfected cells with a chemical inducer of hypoxia-inducible factor-1 such as CoCl 2 .
  • CoCl2 should be observed to reduce SEAP protein levels in cells transfected with SEAP constructs lacking the ODD domain when compared to cells transfected with SEAP-ODD variants.
  • a subset of SEAP-ODD variants exhibiting significantly reduced SEAP protein levels under normoxic conditions and significantly increased SEAP protein levels under hypoxic conditions is selected for further testing by fusing each promising ODD variant to a protein of interest such as ICP34.5 in the context of a recombinant oncolytic HSV-1, wherein the protein of interest may also incorporate one or more microRNA binding sites inserted into the 3’-untranslated region (3’-UTR) .
  • mirT miRNA-targeting sequences
  • Mucin 1 is a transmembrane that is expressed by normal epithelial cells but is overexpressed in the majority of carcinoma tissues.
  • the 3’UTR of MUC1 mRNA contains conserved motifs for binding miR1226, miR125, and miR145. In cancer cells, the levels of these miRNAs are reduced, leading to aberrant gene transcription of MUC1 in cancer tissue.
  • the binding motifs of miR1226, miR125, and miR145 were inserted in the 3’UTR of viral genes that are important for viral infection.
  • the reduction in gene expression induced by miR1226, miR125, and miR145 was confirmed using a SEAP gene that included a mirT in the 3’UTR.
  • the gene encoding SEAP was followed by mirT sequences that a targeted by the mature miRNA sequences of miR1226, miR125 and miR145.
  • the mirT sequence following the gene encoding SEAP was identical to the 3’UTR of the MUC1 gene, which is targeted by the seed sequences of miR1226, miR125, and miR145.
  • the SEAP protein level was observed to be suppressed in the presence of miR1226, miR125, and miR145 at different levels (see FIG. 2 A-B, in the Vero and HEK293T cells lines, respectively) .
  • the suppression was observed to be more effective with L22F23 than with L22F24 (see Fig 2 C-E, in the Vero cell line at 24, 48, or 72 hours post transfection, respectively) , indicating the mirT targeting by mature miRNA sequences were more effective than the mirT targeting by seed regions.
  • HSV1 The genes of HSV1 are classified as either essential genes or non-essential genes based on the requirement of the gene for lytic infection.
  • Essential genes include ICP27, ICP4, and gB and others.
  • Using tumor or tissue-specific regulatory elements to control expression of essential genes allows the virus to specifically replicate in the relevant tissue.
  • viral replication in manufacturing cell lines is limited due to the lack of the translational or transcriptional elements to support viral replication. Therefore, viral genes that are important for replication in vivo, but have little impact on virus production in cell culture are better choices for linkage to tumor or tissue-specific regulatory elements.
  • An example of such genes is ICP34.5. Viruses lacking the gene retain efficient replication in tissue culture but are severely restricted in in host infection [3, 5] .
  • ICP34.5 In the role of ICP34.5 in the ability of HSV1 to resist innate immunity the following experiment was undertaken. All virus construction was performed using standard Lambda Red-mediated BAC recombineering techniques in E. coli, followed by transfection and virus recovery in Vero cells. First, two copies of the ICP34.5 gene were deleted in the viral genome, resulting in the virus T22F05 (see FIG. s 3A and 3B) . Viral replication in tissue culture was tested in the presence and absence of IFN- ⁇ . In the presence of IFN- ⁇ , the titer of the wildtype virus, VG17, was reduced by 10-fold whereas the titer of the T22F05 virus was reduced by 10 ⁇ 4 fold (see FIG. s 4A and 4B) , indicating a major role for ICP34.5 in the mechanism of opposing antiviral immunity.
  • FIG. 3C and FIG. 3D Two viruses with tumor-specific expression elements were generated, as depicted in FIG. 3C and FIG. 3D.
  • the presence of genes in each of the viruses were tested with PCR (Fig 5A) .
  • the ICP34.5 gene was linked to ODD-mirT sequences at the 3’-terminus of the gene.
  • the mirT sequences were consecutive complementing sequences of miR143 and miR124.
  • ODD-mirT elements were fused to ICP0 gene.
  • the mirT sequences are consecutive targeting sequences of miR1226, miR125, and miR145.
  • T22F14 (as shown in FIG. 3C) was used to assess the impact of IFN- ⁇ treatment on viral yield, with and without hypoxic pressure.
  • the sensitivity of T22F14 in IFN- ⁇ treatment was tested in Vero (Fig 6A) . In the presence of IFN- ⁇ , virus replication was significantly inhibited. Comparing T22F14 with VG17 and T22F05, we observed that T22F14 exhibited a higher sensitivity to IFN- ⁇ than T22F05 but a lower sensitivity than VG17 (Fig 6B) .
  • CoCl 2 treatment was used to mimic hypoxic conditions in tissue culture. An optimal concentration of CoCl 2 was used to minimize reducing viral replication in the absence of IFN- ⁇ (see FIG. 7A) . IFN- ⁇ treatment was observed to suppress viral replication by over 100-fold under normoxic conditions (see FIG. 7C) . In the presence of IFN- ⁇ following exposure to CoCl 2 , the viral yield was observed to increase by over 10-fold (see FIG. 7C) . Overall, in the presence of IFN- ⁇ , the T22F14 virus was observed to replicate better under hypoxic conditions than under normoxic conditions.

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Abstract

L'invention concerne un virus de l'herpès simplex recombinant (VHS) ayant un génome de virus de l'herpès oncolytique modifié, le génome du virus de l'herpès oncolytique modifié comprenant un domaine de dégradation dépendant de O2 (ODD) lié de manière fonctionnelle à une copie d'un gène VHS.
PCT/CN2023/141255 2023-12-22 2023-12-22 Virus de l'herpès oncolytique recombinant ayant un domaine de dégradation dépendant de l'oxygène Pending WO2025129693A1 (fr)

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