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WO2019123414A1 - Virus modifiés exprimant la gmp-amp synthase cyclique - Google Patents

Virus modifiés exprimant la gmp-amp synthase cyclique Download PDF

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WO2019123414A1
WO2019123414A1 PCT/IB2018/060515 IB2018060515W WO2019123414A1 WO 2019123414 A1 WO2019123414 A1 WO 2019123414A1 IB 2018060515 W IB2018060515 W IB 2018060515W WO 2019123414 A1 WO2019123414 A1 WO 2019123414A1
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promoter
recombinant virus
cgas
virus
family
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Rohan KAMAT
Aditya Kulkarni
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Straximm LLP
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Straximm LLP
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A61K48/0058—Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0075—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the delivery route, e.g. oral, subcutaneous
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16033—Use of viral protein as therapeutic agent other than vaccine, e.g. apoptosis inducing or anti-inflammatory
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • 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
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14133—Use of viral protein as therapeutic agent other than vaccine, e.g. apoptosis inducing or anti-inflammatory
    • 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
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • cGAS AMP Synthase
  • Cyclic GMP-AMP Synthase is a cytosolic sensor recognizing double stranded DNA (dsDNA). cGAS binding of dsDNA catalyzes cyclic GMP-AMP (cGAMP) synthesis, and cGAMP activates the STING pathway, leading to production of type 1 interferons (IFN) (Sun et al., Science. 2013. 339:786-791).
  • IFN interferons
  • Defense mechanisms against infectious agents and tumors critically rely on type I IFN response and production of type 1 IFNs amplifies innate immune responses to infectious pathogens and activate adaptive immune cells, such as T cells. Therefore, cGAS can play an important role in the generation of immune responses to infectious pathogens as well as cancer cells.
  • the present disclosure provides viral vectors and compositions comprising viral vectors, wherein the viral vectors comprise polynucleotides encoding cGAS polypeptides.
  • Administration of such vectors and compositions to cells can therefore mediate expression and/or activation of cGAS in cells, thereby resulting in production of cGAMP, activation of the STING pathway, and induction of IFN responses.
  • the vectors and compositions may also be used therapeutically to treat cell proliferative diseases, inflammatory diseases, and/or infectious diseases and/or to induce anti-tumor immune responses in subjects in need thereof.
  • the present disclosure further provides methods of increasing the efficacy of an immune checkpoint inhibitor by administering the vectors or compositions to a subject in need thereof.
  • the present disclosure provides a recombinant virus comprising a polynucleotide encoding a cyclic GMP-AMP synthase (cGAS) polypeptide.
  • the virus is from the Adenoviridae family, the Arenaviridae family, the Astroviridae family, the Bornaviridae family, the Caliciviridae family, the Coronaviridae family, the Filoviridae family, the Flaviviridae family, the Hantaviridae family, the Hepadnaviridae family, the Herpesviridae family, the Orthomyxoviridae family, the Papillomaviridae family, the Paramyxoviridae family, the Parvoviridae family, the Phenuviridae family, the Picornaviridae family, the Polyomaviridae family, the Poxviridae family, the Rhabdoviridae family, the Reovirid
  • the virus is from the Retroviridae family and is an Alpharetrovirus, a Betaretrovirus, a Deltaretrovirus, an Epsilonretrovirus, a Gammaretrovirus, or a Lentivirus.
  • the virus is from the Herpesviridae family and is an Alphherpesvirus, a Betahepesvirus, or a Gammaherpesvirus.
  • the virus is from the Parvoviridae family and is an adeno-associated virus (AAV) selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9.
  • the virus is a lentivirus or an adeno-associated virus (AAV).
  • the virus is pseudotyped.
  • the pseudotyped virus is an AAV comprising capsid proteins derived from two or more AAV serotypes.
  • the capsid proteins are derived from eight AAV serotypes.
  • the pseudotyped virus is a lentivirus comprising an envelope glycoprotein G derived from vesicular stomatitis virus (VSV-G)
  • the polynucleotide encoding the cGAS polypeptide is operably linked to a promoter.
  • the promoter is a ubiquitous promoter, a tissue-specific promoter, or a tumor- specific promoter.
  • the ubiquitous promoter is selected from EFl-a, CMV, SV40, PGK1, Ubc, Beta actin, CAG, Polyhedrin, CaMV35S, Hl, U6, and Ubiquitin B.
  • the tissue-specific promoter is selected from the tyronsinase (Tyr) promoter, the B29 promoter, the CD 14 promoter, the CD43 promoter, the CD45 promoter, the CD68 promoter, the Desmin promoter, the Elastase-l promoter, the Endoglin promoter, the Fibronectin promoter, the Flt-l promoter, the GFAP promoter, the GPIIb promoter, the ICAM-2 promoter, the mIFN-b promoter, the Mb promoter, the Nphsl promoter, the OG-2 promoter, the SP-B promoter, the SYN1 promoter, the WASP promoter, and the CaMKII promoter.
  • Tyr tyronsinase
  • the tumor-specific promoter is selected from the alpha fetal protein (AFP) promoter, the cholecystokinin type-A receptor (CCKAR) promoter, the carcinoembryonic antigen (CEA) promoter, the c-ErbB2 promoter, the COX-2 promoter, the CXCR4 promoter, the E2F-1 promoter, the human epididymis protein 4 (HE4) promoter, the lipoprotein lipase (LP) promoter, the MUC1 promoter, the prostate- specific antigen (PSA) promoter, the Survivin (Sur) promoter, and the phosphoribosyl-anthranilate isomerase (TRP-l) promoter.
  • AFP alpha fetal protein
  • CCKAR cholecystokinin type-A receptor
  • CEA carcinoembryonic antigen
  • c-ErbB2 promoter the COX-2 promoter
  • CXCR4 promoter the CXCR4 promoter
  • the promoter is a constitutive promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, the promoter is a tetracycline inducible promoter and comprises one or more tetracycline response elements (TRE).
  • TRE tetracycline response elements
  • the present disclosure provides a recombinant virus comprising a polynucleotide encoding a cGAS polypeptide and further comprising a polynucleotide encoding a detectable label.
  • the detectable label is selected from a fluorescent protein.
  • the present disclosure provides a recombinant virus comprising a polynucleotide encoding a cGAS polypeptide and further comprising a polynucleotide encoding a payload polypeptide.
  • the payload polypeptide is selected from an immune checkpoint inhibitor (e.g ., a PD1 antagonist, a PDL1 antagonist, a CTLA4 antagonist), an AIM2 agonist, a TNF receptor agonist (e.g., a GITR agonist, an 0X40 agonist, a CD30 agonist, a TRAIL receptor agonist), a Fas receptor agonist, a pro-apoptotic protein (e.g., BAX, BAK1, BOK, BID), a caspase (e.g., caspase 9), and a cytokine (e.g., IL-2, IL-12, IL- 18, IL-21, IFNa, IFNft IFNy, TNF a).
  • an immune checkpoint inhibitor e.g a PD1 antagonist, a PDL1 antagonist, a CTLA4 antagonist
  • an AIM2 agonist e.g., a TNF receptor agonist (e.g., a GITR
  • the present disclosure provides a recombinant virus comprising a polynucleotide encoding a cGAS polypeptide, wherein the polynucleotide encoding the cGAS polypeptide comprises a nucleotide sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1.
  • the polynucleotide encoding the cGAS polypeptide consists of a nucleotide sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1.
  • the polynucleotide encoding the cGAS polypeptide comprises a nucleotide sequence is 100% identical to SEQ ID NO: 1. In some embodiments, the polynucleotide encoding the cGAS polypeptide consists of SEQ ID NO: 1.
  • the encoded cGAS polypeptide comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2. In some embodiments, the encoded cGAS polypeptide comprises an amino acid sequence that is 100% identical to SEQ ID NO: 2. In some embodiments, the encoded cGAS polypeptide consists of an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2. In some embodiments, the encoded cGAS polypeptide consists of SEQ ID NO: 2.
  • the recombinant viruses described herein comprise a nucleotide sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 3 or SEQ ID NO: 4. In some embodiments, the recombinant viruses described herein comprise a nucleotide sequence that is 100% identical to SEQ ID NO: 3 or SEQ ID NO: 4. In some embodiments, the recombinant viruses described herein consist of a nucleotide sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 3 or SEQ ID NO: 4. In some embodiments, the recombinant viruses described herein consist of a nucleotide sequence that is 100% identical to SEQ ID NO: 3 or SEQ ID NO: 4.
  • the recombinant virus described herein is pseudotyped.
  • the pseudotyped recombinant virus comprises one or more viral envelope proteins derived from a heterologous virus.
  • the present disclosure provides a composition comprising a recombinant virus described herein.
  • the composition further comprises a pharmaceutically acceptable carrier or excipient.
  • the present disclosure provides a method of expressing a cGAS polypeptide in a cell comprising administering a recombinant virus described herein or a composition thereof.
  • expression of the cGAS polypeptide induces production of cGAMP in the cell.
  • the method further comprises administering a cGAS activator to the cell.
  • administration of the cGAS activator induces production of cGAMP in the cell.
  • the present disclosure provides a method of activating the
  • Stimulator of IFN Gene (STING) pathway in a cell comprising administering a recombinant virus described herein or a composition thereof to the cell.
  • administration of the recombinant virus results in expression of cGAS and activation of the STING pathway in the cell.
  • the method further comprises administering a cGAS activator to the cell.
  • administration of the recombinant virus results in expression of cGAS in the cell and administration of the cGAS activator results in the activation of the STING pathway.
  • the present disclosure provides a method of inducing an interferon (IFN) response in a cell comprising administering a recombinant virus described herein or a composition thereof to the cell.
  • administration of the recombinant virus results in expression of cGAS and production of IFN in the cell.
  • the method further comprises administering a cGAS activator to the cell.
  • administration of the recombinant virus results in expression of cGAS in the cell and administration of the cGAS activator results in production of IFN in the cell.
  • the cGAS activator is selected from a double stranded DNA
  • the cell is in vitro or in vivo.
  • the cell is a tumor cell or an immune cell.
  • the cell is a human cell.
  • the present disclosure provides a method of treating a disease or disorder in a subject in need thereof, comprising administering a recombinant virus described herein or a composition thereof to the subject, and expressing a cGAS polypeptide in the subject.
  • the method further comprises administering a cGAS activator to the subject.
  • the recombinant virus is administered to the subject prior to the administration of the cGAS activator, or wherein the recombinant virus and cGAS activator are administered to the subject simultaneously or wherein the cGAS activator is administered to the subject prior to the administration of the recombinant virus.
  • the method described herein further comprise administering an immune checkpoint inhibitor to the subject.
  • the immune checkpoint inhibitor is an inhibitor of PD1, PDL1, CTLA4, A2AR, B7-H3, B7-H4, BTLA, IDO, LAG3, KIR, ⁇ M3, TIGIT, or VISTA.
  • the immune checkpoint inhibitor is administered prior to the recombinant virus and/or the cGAS activator, simultaneously with the recombinant virus and/or the cGAS activator, or after the recombinant virus and/or the cGAS activator.
  • the recombinant virus and/or the cGAS activator, and/or the immune checkpoint inhibitor are administered intravenously or intratumorally.
  • the disease is a cancer, an infection, or a disease caused by an infection.
  • the cancer is selected from colorectal, renal, brain/glioblastoma, prostate, and lung cancer.
  • the subject is a human.
  • the present disclosure provides a method of inducing anti tumor immunity in a subject in need thereof, comprising administering a recombinant virus described herein or a composition thereof to the subject, and expressing the cGAS polypeptide in the subject.
  • the method further comprises administering a cGAS activator to the subject.
  • the recombinant virus is administered to the subject prior to the administration of the cGAS activator, or the recombinant virus and cGAS activator are administered to the subject simultaneously or wherein the cGAS activator is administered to the subject prior to the administration of the recombinant virus.
  • the tumor is selected a colorectal tumor, a renal tumor, a brain tumor ( e.g ., a glioblastoma), a prostate tumor, or a lung tumor.
  • the present disclosure provides a method of increasing the efficacy of an immune checkpoint inhibitor comprising administering the immune checkpoint inhibitor to a subject suffering from a cancer; and administering a recombinant virus described herein or a composition thereof to the subject, thereby expressing the cGAS polypeptide in the subject, wherein the efficacy of the immune checkpoint inhibitor is increased by administration of the recombinant virus compared to the efficacy of the immune checkpoint inhibitor without administration of the recombinant virus.
  • the immune checkpoint inhibitor is administered prior to the recombinant virus, simultaneously with the recombinant virus, or after the recombinant virus.
  • the increased efficacy is measured by a decrease in tumor volume or number of tumors, an increase in tumor growth inhibition, an increase in survival of the subject, and/or an increase in length of remission time.
  • Fig. 1 shows a schematic of the STX-VP-001 lentiviral vector.
  • FIG. 2 shows a schematic of the STX- VP-002 AAV vector.
  • Fig. 3 shows STX-VP-001 induced IFN expression in THP-l luciferase reporter cells.
  • Fig. 4 A - Fig. 4B show schematics of dosing regimens for controls (Fig. 4A) and prophylactic and treatment (Fig. 4B) groups in a syngenic B16 murine model of melanoma.
  • Fig. 5A - Fig. 5B shows tumor volume over time (Fig. 5A) and % tumor growth inhibition (Fig. 5B) for treatment groups in the syngenic B16 murine model of melanoma.
  • Fig. 6A - Fig. 6B provide histological sections (Fig. 6A) and histological scores
  • FIG. 7 shows staining of histological sections for cGAS expression in B16F10
  • Fig. 8A - Fig. 8B show schematics of dosing regimens for control (Fig. 8A) and STX-VP-001 treatment (Fig. 8B) groups in a murine xenograft HCC model.
  • Fig. 9A - Fig. 9B shows tumor volume over time (Fig. 9A) and % tumor growth inhibition (Fig. 9B) for treatment groups in the murine xenograft HCC model.
  • Fig. 10 shows in vitro expression of cGAS from STX-VP-001 and STX- VP-002 vectors.
  • Fig. 11A - Fig. 11B show in vivo virus distribution and cGAS expression in the indicated tissue types.
  • Fig. 12A - Fig. 12B show schematics of dosing regimens for control and mono treatment groups (Fig. 12A) and combined treatment groups (Fig. 12B) groups in a syngenic B16 murine model of melanoma.
  • Fig. l3A - Fig. 13B show tumor volume (Fig. 13 A) and tumor growth inhibition
  • Fig. 14A - Fig. 14B provide histological sections (Fig. 14A) and histological scores
  • cGAS cyclic GMP-AMP synthase
  • cGAS is a nucleotidyltransferase and is part of the cGAS-STING innate immune pathway for sensing of intracellular DNA.
  • cGAS Upon cGAS binding of DNA in the cytoplasm, cGAS triggers a reaction of GTP and ATP to form the secondary messenger molecule, cyclic GMP-AMP (cGAMP).
  • cGAMP then binds to Stimulator of Interferon Genes (STING), located on the endoplasmic recticulum, and triggers phosphorylation of IRF3 via TBK1.
  • STING Stimulator of Interferon Genes
  • IRF3 is then translocated into the nucleus, where it activates transcription of type-I IFN genes.
  • cGAMP packaged in viral particles mediated the dispersion of innate immune response activation from infected cells to distant cells (Gentili et al., Science. 2015. 349:6253; 1232-1236) and various cancer types demonstrate decreased expression of cGAS (Xia et al., Cell Rep. 2016; 14(2): 282-297).
  • the recombinant viruses described herein therefore enable activation of innate immune signaling pathways (i.e., the cGAS-cGAMP-STING pathway) through targeted expression of cGAS and can be used in the treatment of a variety of cancers and infectious diseases.
  • 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).
  • sequence identity refers to the percentage of nucleotides 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 a reference polynucleotide sequence or a reference polypeptide sequence.
  • reference sequence refers to a molecule to which a test sequence is compared.
  • An“expression cassette” refers to a DNA polynucleotide operably linked to a promoter.“Operably linked” refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. For instance, a promoter is operably linked to a polynucleotide sequence if the promoter affects the transcription or expression of the linked polynucleotide sequence.
  • recombinant vector refers to a polynucleotide molecule capable of transferring or transporting another polynucleotide inserted into the recombinant vector.
  • the recombinant vector can be a recombinant viral vector.
  • the inserted polynucleotide may be an expression cassette.
  • sample refers to a biological composition (e.g ., a cell or a portion of a tissue) that is subjected to analysis and/or modification.
  • a sample is a “primary sample” in that it is obtained directly from a subject; in some embodiments, a“sample” is the result of processing of a primary sample, for example to remove certain components and/or to isolate or purify certain components of interest.
  • administering refers herein to introducing an agent or composition into a subject.
  • Treating refers to delivering an agent or composition to a subject to affect a physiologic outcome.
  • the term“effective amount” refers to the minimum amount of an agent or composition required to result in a particular physiological effect.
  • the effective amount of a particular agent may be represented in a variety of ways based on the nature of the agent, such as mass/volume, of cells/volume, particles/volume, (mass of the agent)/(mass of the subject), # of cells/(mass of subject), or particles/(mass of subject).
  • the effective amount of a particular agent may also be expressed as the half-maximal effective concentration (ECso), which refers to the concentration of an agent that results in a magnitude of a particular physiological response that is half-way between a reference level and a maximum response level.
  • phrases“pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • the present disclosure provides a recombinant virus comprising a polynucleotide encoding a cyclic GMP-AMP synthase (cGAS) polypeptide.
  • cGAS cyclic GMP-AMP synthase
  • the cGAS protein is encoded by the CGAS gene (NCBI Gene Id: 115004; NC_000006.12; NM 138441.1), also referred to as the MB21D1 or C6orfl50 gene.
  • the amino acid sequence of human cGAS is provided in SEQ ID NO: 2 (encoded by SEQ ID NO: 1).
  • the polynucleotide encoding the cGAS polypeptide comprises a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 1. In some embodiments, the polynucleotide encoding the cGAS polypeptide comprises a nucleic acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. In some embodiments, the polynucleotide encoding the cGAS polypeptide comprises a nucleic acid sequence that is 100% identical to the polynucleotide sequence of SEQ ID NO: 1.
  • the polynucleotide encoding the cGAS polypeptide consists of a nucleic acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. In some embodiments, the polynucleotide encoding the cGAS polypeptide consists of a nucleic acid sequence that is 100% identical to the polynucleotide sequence of SEQ ID NO: 1. [0051] In some embodiments, the cGAS polypeptide encoded by the recombinant viruses described herein comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 2.
  • the cGAS polypeptide encoded by the recombinant viruses described herein comprises an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2. In some embodiments, the cGAS polypeptide encoded by the recombinant viruses described herein comprises an amino acid sequence that is 100% identical to SEQ ID NO: 2. In some embodiments, the cGAS polypeptide encoded by the recombinant viruses described herein consists of an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2. In some embodiments, the cGAS polypeptide encoded by the recombinant viruses described herein consists of an amino acid sequence that is 100% identical to SEQ ID NO: 2
  • ETSA 85:2444 by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), by manual alignment and visual inspection (see, e.g, Brent et ah, (2003) Current Protocols in Molecular Biology), by use of algorithms known in the art including the BLAST and BLAST 2.0 algorithms, which are described in Altschul et ah, (1977) Nuc. Acids Res. 25:3389-3402; and Altschul et ah, (1990) J. Mol. Biol. 215:403-410, respectively.
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.
  • the polynucleotide encoding the cGAS polypeptide is operably linked to a promoter.
  • the promoter is a ubiquitously expressed promoter, such as a CAG promoter, a Ubc promoter, an EFla promoter, a PGK1 promoter, an SV40 promoter, a CMV promoter, or a human beta actin promoter.
  • expression of the cGAS polypeptide is not limited to a particular cell or tissue type
  • the promoter is a tissue or cell-type specific promoter such that the expression of the cGAS polypeptide is restricted to a particular tissue or cell type.
  • the promoter is a hepatocyte specific promoter such as the apolipoprotein A-II (APOA2) promoter, the SERPINA1 promoter (also referred to as an hAAT promoter), the CYP3A4 promoter, or the MTR122 promoter.
  • the promoter is a pancreas-specific promoter such as the insulin (INS) promoter, the insulin receptor substrate 2 (IRS2) promoter, the pancreatic and duodenal homeobox 1 (Pdxl) promoter, the aristaless-like homeobox 3 (Alx3) promoter, the Elastasel promoter, or the pancreatic polypeptide (Ppy) promoter.
  • INS insulin
  • IRS2 insulin receptor substrate 2
  • Pdxl pancreatic and duodenal homeobox 1
  • Alx3 aristaless-like homeobox 3
  • Elastasel the pancreatic polypeptide
  • the promoter is a cardiac tissue-specific promoter such as the myosin heavy chain 6 (Myh6) promoter, the myosin light chain 2 (MYL2) promoter, the troponin I type 3 (TNNI3) promoter, the natriuretic peptide precursor A (NPPA) promoter, or the solute carrier family 8 member 1 (Slc8al) promoter.
  • Myh6 myosin heavy chain 6
  • MYL2 myosin light chain 2
  • TNNI3 troponin I type 3
  • NPPA natriuretic peptide precursor A
  • Solute carrier family 8 member 1 solute carrier family 8 member 1
  • the promoter is a central nervous system (CNS) cell-specific promoter such as the synapsin 1 (Synl) promoter, the glial fibrillary acidic protein (GFAP) promoter, the internexin neuronal intermediate filament protein alpha (INA) promoter, the nestin (NES) promoter, the myelin-associated oligodendrocyte basic protein (MOBP) promoter, the tyrosine hydroxylase (TH) promoter, or the forkhead box A2 (FOXA2) promoter.
  • CNS central nervous system
  • the promoter is a urogenital cell-specific promoter such as the probasin (Pbsn) promoter, the uroplakin 2 (ETpk2) promoter, the spermine binding protein (Sbp), or the Fer-l-like 4 (Ferll4) promoter.
  • the promoter is a lung- specific promoter such as the SP-B promoter.
  • the promoter is a melanocyte specific promoter, such as the tyrosinase (Tyr) promoter, the melanocyte-specific MITF isoform (MITF-M) promoter, or the TRP-l promoter.
  • the promoter is an immune-cell specific promoter such as the B29 promoter (B cells), the CD 14 promoter (macrophages, dendritic cells, monocytes), the CD43 promoter, or the CD68 promoter (macrophages and monocytes).
  • the promoter is a hematopoietic cell-specific promoter such as the CD45 promoter or the WASP promoter.
  • the promoter is a muscle-specific promoter such as the desmin (DES) promoter or the myoglobulin (Mb) promoter.
  • the promoter is an endothelial cell-specific promoter such as the Endoglin promoter or the Fltl promoter.
  • the promoter is a podocyte-specific promoter such as the Nphsl promoter.
  • the promoter is a megakaryocyte-specific promoter such as the GPIIb promoter.
  • the promoter is an osteoblast-specific promoter such as OG-2.
  • the promoter is the Fibronectin promoter.
  • the promoter is a tumor specific promoter such that the expression of the cGAS polypeptide is restricted to tumor cells.
  • the promoter is the alpha fetal protein (AFP) promoter, the carcinoembryonic antigen (CEA) promoter, the CgA promoter, the cholecystokinin type-A receptor (CCKAR) promoter, the COX-2 promoter, the CXCR4 promoter, the E2F-1 promoter, the ERBB2 promoter, the GRP78 promoter, the H19 promoter, the human epididymis protein 4 (HE4) promoter, the IGF2-P4 promoter, the insulinoma-associated 1 (INSM1) promoter, the lipoprotein lipase (LP) promoter, the mesothelin promoter, the MUC1 promoter, the osteocalcin (OC) promoter, the uPAR promoter, the prostate-specific antigen (PSA) promoter, the AFP and fetal protein (AFP) promoter, the C
  • the ubiquitous promoter, the tissue-specific, and/or the tumor- specific promoter is a constitutive promoter.
  • transcription of the cGAS-encoding polynucleotide occurs in any cell that expresses the transcription factor(s) capable of binding to the constitutive promoter and driving transcription.
  • the ubiquitous promoter, the tissue-specific, and/or the tumor-specific promoter is an inducible promoter.
  • transcription of the cGAS-encoding polynucleotide requires the requisite transcription factors and further requires the presence (or absence) of a second factor or stimulus.
  • the promoter comprises a tetracycline response element (TRE) such that transcription of the cGAS-encoding polynucleotide is regulated by the presence or absence of tetracycline (or its derivatives, demeclocycline, doxycycline and minocycline).
  • TRE tetracycline response element
  • Expression of cGAS can be determined by a variety of means known in the art, for example by Western blot, immunohistochemistry, ELISA, flow cytometry.
  • the recombinant viruses described herein comprise a polynucleotide encoding a cGAS polypeptide and further comprise a polynucleotide encoding a detectable label.
  • the detectable label can be any molecule that allows for detection or selection of the virus during production of the viral vector or during use of the vector for treatment.
  • the detectable label is a fluorescent marker (e.g ., GFP, RFP, BFP, YFP, and the like).
  • the detectable label is an antibiotic resistance cassette (e.g., a puromycin resistance cassette).
  • the recombinant viruses described herein comprise a polynucleotide encoding a cGAS polypeptide and further comprise a polynucleotide encoding a payload protein.
  • the payload protein is a protein that enhances the therapeutic efficacy of the recombinant viral virus.
  • the payload protein enhances the activation of the immune response elicited by the recombinant virus (e.g ., enhanced cytokine expression, increased expression of pro-inflammatory genes, increased immune cell proliferation, increased immune cell infiltration to a tumor, etc.)
  • the payload protein enhances the anti-tumor immune response elicited by the recombinant virus (e.g., increased apoptosis of tumor cells and/or increased cytotoxicity against tumor cells).
  • the payload protein is an immune checkpoint inhibitor such as a PD1 antagonist, a PDL1 antagonist, or a CTLA4 antagonist.
  • the payload protein is a TNF receptor agonist, e.g., a CD30 agonist, a GITR agonist, an 0X40 agonist, a TRAIL receptor agonist (e.g. a TRAIL-R1, TRAIL-R2, TRAIL-R3, or TRAIL-R4), or a DR3 agonist.
  • the payload protein is a Fas receptor agonist.
  • the payload protein is a pro-apoptotic protein such as BAX, BAK1, BOK, or BID.
  • the payload protein is a pro-apoptotic caspase (e.g., caspase 9).
  • the payload protein is an agonist of an innate immune receptor such as an AIM2 agonist, a NLRP3 agonist, a TLR agonist (e.g., a TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, or TLR10 agonist), a NOD agonist, a C-type lectin receptor agonist, or a RIG-like receptor agonist.
  • the payload protein is a pro-inflammatory cytokine such as IL-la, IL-2, IL-12, IL-6, IL-18, IL-21, IFNa, IFNfr IFNy, or TNFa.
  • the recombinant virus is a virus from the Adenoviridae family (e.g. , an adenovirus), the Arenaviridae family, the Astroviridae family (e.g., an Avastrovirus or a Mamastrovirus ), the Bomaviridae family (e.g., a Carbovirus or an Orthobomavirus), the Bunyaviridae family, the Caliciviridae family, the Coronaviridae family (e.g, Alphacoronaviruses and Betacoronoaviruses), the Flaviviridae family (e.g., a Flavivirus or a Hepacivirus), the Filoviridae family, the Hantaviridae family, the Hepadnaviridae family (e.g., an orthohepadnavirus such as Hepatitis D virus), the Herpesviridae family (e.g., an equine herpes virus or herpe
  • the recombinant virus is a virus selected from an
  • Adenovirus an HSV-l virus, an influenza virus, an AAV (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9), a coxsackie virus, a polio virus, a Seneca valley virus, a vaccina virus, a myxoma virus, a retrovirus (e.g., a lentivirus), or a VSV virus.
  • AAV e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9
  • coxsackie virus e.g., polio virus, a Seneca valley virus, a vaccina virus, a myxoma virus, a retrovirus (e.g., a lentivirus), or a VSV virus.
  • the recombinant virus is a lentivirus.
  • the lentivirus is selected from mouse Mammary Tumor Virus (MMTV), Human T-Lymphotrophic virus (HTLV), Avian Sarcoma Leukosis Virus (ASLV), Human Immunodeficiency Virus (HIV), Simian Immunodeficiency Virus (SIV), Feline Immunodeficiency Virus (FIV), and Equine Infectious Anemia Virus (EIAV).
  • the recombinant virus is an AAV.
  • the AAV is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9.
  • the recombinant virus is a pseudotyped virus.
  • the recombinant virus refers to a virus comprising one or more heterologous capsid or envelope protein, or wherein one or more of the capsid or envelop proteins have been modified, such that the pseudotyped virus demonstrates altered tropism compared to the corresponding non- pseudotyped virus.
  • the recombinant virus is a lentivirus comprising an envelope protein derived from Vesicular stomatitis virus (VSV) (e.g., VSV glycoprotein G) (Akkina et al., J Virol. 1996 Apr; 70(4):258l-5; Naldini et al, Science.
  • VSV Vesicular stomatitis virus
  • the recombinant virus is a lentivirus and is pseudotyped with VSV glycoprotein G (VSV-G).
  • the recombinant virus is a pseudotyped AAV and comprises capsid proteins derived from two or more AAV serotypes (e.g ., two or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9).
  • the recombinant virus is an AAV and comprises capsid proteins derived from 3, 4, 5, 6, 7, 8, or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9.
  • the recombinant virus is an AAV and comprises capsid proteins derived from 8 different AAV serotypes.
  • the recombinant virus is an AAV and comprises capsid proteins derived from 8 different AAV serotypes and is an AAV DJ virus (Cell Biolabs, Inc.). In some embodiments, the recombinant virus is an AAV and comprises capsid proteins derived from 8 different AAV serotypes. In some embodiments, the recombinant virus is an AAV and comprises capsid proteins derived from 8 different AAV serotypes and is an AAV DJ/8 virus (Cell Biolabs, Inc.).
  • the recombinant virus is a lentivirus comprising the PGK1 promoter operably linked to a polynucleotide encoding a cGAS polypeptide.
  • the nucleic acid sequence of the recombinant virus is at least 90% identical to SEQ ID NO: 3. In some such embodiments, the nucleic acid sequence of the recombinant virus is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 3. In some such embodiments, the nucleic acid sequence of the recombinant virus is 100% identical to SEQ ID NO: 3.
  • the nucleic acid sequence of the recombinant virus consists of SEQ ID NO: 3.
  • the lentivirus is a pseudotyped lentivirus comprising VSV-G envelope proteins.
  • the recombinant virus is an AAV comprising the Tyr promoter operably linked to a polynucleotide encoding a cGAS polypeptide.
  • the nucleic acid sequence of the recombinant virus is at least 90% identical to SEQ ID NO: 4.
  • the nucleic acid sequence of the recombinant virus is at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 4.
  • the nucleic acid sequence of the recombinant virus is 100% identical to SEQ ID NO: 4. In some such embodiments, the nucleic acid sequence of the recombinant virus consists of SEQ ID NO: 4. In further aspects of this embodiment, the AAV is a pseudotyped AAV comprising capsid proteins derived from 8 AAV serotypes.
  • the present disclosure provides methods for expressing a cGAS polypeptide in a cell or a subject, comprising administering a cGAS-expressing virus or composition thereof described herein to the cell or the subject.
  • Expression of cGAS, production of cGAMP, and activation of the STING pathway lead to induction of a type I IFN response and provide anti-tumor responses and/or or anti-viral responses in subjects.
  • activation of innate immune responses by cGAS expression or cGAMP produced in virally infected cells and packaged in viral particles has been described (Gentili et al., Science. 2015. 349:6253; 1232-1236).
  • viral vectors expressing the cGAS polypeptide for use as a therapeutic have not been described and provide several advantages over the small molecules and cGAMP particles.
  • both the small molecules and cGAMP loaded viral-like particles activate the cGAS-cGAMP-STING pathway in all cell types, therefore leading to death of healthy cells as well as cancerous and/or infected cells.
  • the small molecules in most cases cyclic dinucleotides must be delivered directly to the tumor due to their poor bioavailability, therefore limiting the cancer and tumor types that can be treated with these small molecules.
  • the recombinant viruses described herein provide advantages over these previously described methods in one or more of the following ways: expression of cGAS in particular cell types through the use of tissue-specific or tumor-specific promoters; use of pseudotyped viruses to target the recombinant viruses to particular tissues; formulation of the recombinant viruses for systemic (e.g ., intravenous) delivery.
  • the expression of cGAS in a cell activates the STING pathway and induces a type I IFN response.
  • the present disclosure further provides methods for activating the STING pathway and/or inducing a type I IFN response in a cell or subject comprising administering a cGAS-expressing virus or composition thereof described herein to the cell or the subject.
  • Activation of the STING pathway can be measured by expression of IRF3 (e.g., by qPCR), production of type I IFN, and/or cell death (e.g., by MTT assay, Tunnel staining, or propidium idodide staining and assessment by flow cytometry).
  • the present disclosure provides methods for the treatment of a disease in a subject in need thereof, comprising administering a cGAS-expressing virus or composition thereof described herein to the subject.
  • the subject may be a neonate, a juvenile, or an adult.
  • Mammalian species that may be treated with the present methods include canines and felines; equines; bovines; ovines; etc. and primates, particularly humans.
  • Animal models, particularly small mammals e.g. mice, rats, guinea pigs, hamsters, rabbits, etc. may be used for experimental investigations.
  • administration route is local or systemic.
  • the administration route is intraarterial, intracranial, intradermal, intraduodenal, intrammamary, intrameningeal, intraperitoneal, intrathecal, intratumoral, intravenous, intravitreal, ophthalmic, parenteral, spinal, subcutaneous, ureteral, urethral, vaginal, or intrauterine.
  • the administration route is intratumoral or intravenous.
  • the administration route is by infusion (e.g., continuous or bolus).
  • infusion e.g., continuous or bolus
  • methods for local administration that is, delivery to the site of injury or disease, include through an Ommaya reservoir, e.g. for intrathecal delivery (See e.g., US Patent Nos. 5,222,982 and 5,385,582, incorporated herein by reference); by bolus injection, e.g. by a syringe, e.g. into a joint; by continuous infusion, e.g. by cannulation, such as with convection (See e.g., US Patent Application Publication No. 2007-0254842, incorporated herein by reference).
  • the administration route is by direct injection to a tumor.
  • the recombinant viruses described herein are administered to a subject in order to treat a disease.
  • the disease is a cancer or an infectious disease.
  • treatment comprises delivering an effective amount of the recombinant virus or composition thereof to a subject in need thereof.
  • treating refers to the treatment of a disease in a mammal, e.g., in a human, including (a) inhibiting the disease, i.e., arresting disease development or preventing disease progression; (b) relieving the disease, i.e., causing regression of the disease state or relieving one or more symptoms of the disease; and (c) curing the disease, i.e., remission of one or more disease symptoms.
  • treatment may refer to a short-term (e.g., temporary and/or acute) and/or a long term (e.g., sustained) reduction in one or more disease symptoms.
  • treatment results in an improvement or remediation of the symptoms of the disease.
  • the improvement is an observable or measurable improvement, or may be an improvement in the general feeling of well being of the subject.
  • the effective amount of the recombinant virus administered to a particular subject will depend on a variety of factors, several of which will differ from patient to patient including the disorder being treated and the severity of the disorder; activity of the specific agent(s) employed; the age, body weight, general health, sex and diet of the patient; the timing of administration, route of administration; the duration of the treatment; drugs used in combination; the judgment of the prescribing physician; and like factors known in the medical arts.
  • the effective amount of the recombinant virus may be the amount of virus required to result in at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more fold decrease in tumor mass or volume, decrease in the number of tumor cells, or decrease in the number of metastases.
  • the effective amount of the recombinant virus may be the amount of virus required to achieve an increase in life expectancy, an increase in progression-free or disease-free survival, or amelioration of various physiological symptoms associated with the disease being treated.
  • the effective amount of the recombinant virus is expressed in terms of transfection units (TU)/mL.
  • the effective amount of the recombinant virus is expressed in terms of viral genomes (VG)/mL. In some embodiments, the effective amount of the recombinant virus is at least 10 4 TU/mL, at least 10 5 TU/mL, at least 10 6 TU/mL, at least 10 7 TU/mL, at least 10 8 TU/mL, at least 10 9 TU/mL, at least 10 10 TU/mL, at least 10 11 TU/mL, or at least 10 12 TU/mL.
  • the effective amount of the recombinant virus is at about 10 4 TU/mL, about 10 5 TU/mL, about 10 6 TU/mL, about 10 7 TU/mL, about 10 8 TU/mL, about 10 9 TU/mL, about 10 10 TU/mL, about 10 11 TU/mL, or about 10 12 TU/mL.
  • the effective amount of the recombinant virus is at least 10 4 VG/mL, at least 10 5 VG/mL, at least 10 6 VG/mL, at least 10 7 VG/mL, at least 10 8 VG/mL, at least 10 9 VG/mL, at least 10 10 VG/mL, at least 10 11 VG/mL, or at least 10 12 VG/mL.
  • the effective amount of the recombinant virus is at about 10 4 VG/mL, about 10 5 VG/mL, about 10 6 VG/mL, about 10 7 VG/mL, about 10 8 VG/mL, about 10 9 VG/mL, about 10 10 VG/mL, about 10 11 VG/mL, or about 10 12 VG/mL.
  • the number of administrations of treatment to a subject may vary.
  • administering the recombinant virus to the subject may be a one-time event.
  • such treatment may require an on-going series of repeated treatments.
  • multiple administrations of the recombinant virus may be required before an effect is observed.
  • the exact protocols depend upon the disease or condition, the stage of the disease and parameters of the individual subject being treated.
  • the recombinant viruses described herein or compositions thereof are administered to a subject to treat a viral infection.
  • the virus is selected from one of adenoviruses, herpesviruses (including, for example, herpes simplex virus and Epstein Barr virus, and herpes zoster virus), poxviruses, papovaviruses, hepatitis viruses, (including, for example, hepatitis B virus and hepatitis C virus), papilloma viruses, orthomyxoviruses (including, for example, influenza A, influenza B, and influenza C), paramyxoviruses, coronaviruses, picornaviruses, reoviruses, togaviruses, flaviviruses, bunyaviridae, rhabdoviruses, rotavirus, respiratory syncitial virus, human immunodeficiency virus, or retroviruses.
  • herpesviruses including, for example, herpes simplex virus
  • the recombinant viruses described herein or compositions thereof are administered to a subject to treat a cancer.
  • Cancers that may be treated using the compositions and methods disclosed herein include cancers of the blood and solid tumors.
  • cancers that may be treated using the compositions and methods disclosed herein include, but are not limited to, adenoma, carcinoma, sarcoma, leukemia or lymphoma.
  • the cancer is chronic lymphocytic leukemia (CLL), B cell acute lymphocytic leukemia (B-ALL), acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), non- Hodgkin’s lymphoma (NHL), diffuse large cell lymphoma (DLCL), diffuse large B cell lymphoma (DLBCL), Hodgkin’s lymphoma, multiple myeloma, renal cell carcinoma (RCC), neuroblastoma, colorectal cancer, breast cancer, ovarian cancer, melanoma, sarcoma, prostate cancer, lung cancer, esophageal cancer, hepatocellular carcinoma (HCC), pancreatic cancer, astrocytoma, mesothelioma, head and neck cancer, and medulloblastoma, and liver cancer.
  • CLL chronic lymphocytic leukemia
  • B-ALL B cell acute lymphocytic leukemia
  • ALL acute lymphoblastic le
  • the present disclosure provides methods of increasing the efficacy of an immune checkpoint inhibitor comprising administering the immune checkpoint inhibitor to a subject suffering from a cancer; and administering a cGAS-expressing virus, or composition thereof to the subject, thereby expressing the cGAS polypeptide in the subject.
  • administration of the recombinant virus increases the efficacy of the immune checkpoint inhibitor compared to the efficacy observed without administration of the recombinant virus.
  • FDA-approved PD-L1 inhibitors include atezolizumab (Tecentriq ® , Genentech), avelumab (Bavencio®, Pfizer), and durvalumab (Imfinzi®, AstraZeneca); FDA-approved PD-l inhibitors include pembrolizumab (Keytruda ® , Merck), nivolumab (Opdivo ® , Bristol-Myers Squibb), and cemiplimab (Libtayo®, Regeneron); and FDA-approved CTLA4 inhibitors include ipilimumab (Yervoy®, Bristol-Myers Squibb).
  • Additional inhibitory immune checkpoint molecules that may be the target of future therapeutics include A2AR, B7-H3, B7-H4, BTLA, IDO, LAG3 (e.g, BMS-986016, under development by BMS), KIR (e.g., Lirilumab, under development by BMS), TIM3, HGIT, and VISTA.
  • administration of a recombinant virus described herein results in an enhanced therapeutic effect (e.g., a more significant reduction in tumor growth, an increase in tumor infiltration by lymphocytes, an increase in the length of progression free survival, etc.) than is observed after treatment with an immune checkpoint inhibitor.
  • the combined administration of a recombinant virus described herein and an immune checkpoint inhibitor results in an enhanced therapeutic effect (e.g., a more significant reduction in tumor growth, an increase in tumor infiltration by lymphocytes, an increase in the length of progression free survival, etc.) than is observed after treatment with either agent alone.
  • the combined administration of a recombinant virus described herein and an immune checkpoint inhibitor results in a synergistic therapeutic effect, that is, an effect that is greater than expected and/or that is greater than the sum of each of the individual treatments given separately.
  • the recombinant virus and immune checkpoint inhibitor may be administered at the same time.
  • the recombinant virus and immune checkpoint inhibitor may be administered at different times.
  • the recombinant virus may be administered before or after the immune checkpoint inhibitor.
  • oncologic indications are non-responsive (i.e., are insensitive) to treatment with immune checkpoint inhibitors. Further still, some oncologic indications that are initially responsive to treatment with immune checkpoint inhibitors develop an inhibitor-resistant phenotype during the course of treatment (i.e., are resistant). Therefore, in some embodiments, the recombinant virus described herein, or compositions thereof, are administered to treat a cancer that is resistant or insensitive to treatment with one or more immune checkpoint inhibitors.
  • administration of the recombinant virus or compositions thereof to a subject suffering from a cancer that is resistant or insensitive to treatment with the immune checkpoint inhibitor results in treatment of the cancer (e.g ., reduction in tumor growth, an increase in the length of progression free survival, etc.).
  • the cancer is resistant or insensitive to treatment with a PD1 inhibitor, such as an anti-PDl antibody.
  • the present disclosure provides methods of inducing an anti tumor immune response in a subject in need thereof.
  • An anti-tumor immune response can be measured by a variety of means known in the art, such as tumor growth inhibition (TGI), pro- inflammatory cytokine production (e.g., IFNa, PTMb, IFNy, TNFa, IL- 1 b, IL-12, IL-2, IL-17, CXCL8, and/or IL-6), immune cell infiltration into a tumor, and/or proliferation of tumor-specific T cells.
  • TGI tumor growth inhibition
  • pro- inflammatory cytokine production e.g., IFNa, PTMb, IFNy, TNFa, IL- 1 b, IL-12, IL-2, IL-17, CXCL8, and/or IL-6
  • the methods described herein further comprise administering a cGAS activation agent to the cell or subject.
  • A“cGAS” activation agent refers to an agent capable of inducing cGAS-mediated production of cGAMP.
  • Known cGAS activation agents include double stranded DNA molecules and chitosan.
  • cGAS is active as a homodimer, and therefore cGAS activation agents include agents capable of inducing cGAS dimerization.
  • the cGAS activation agent is administered to the cell or subject prior to the administration of a recombinant virus described herein.
  • the cGAS activation agent is administered to the cell or subject concurrent with the administration of a recombinant virus described herein. In some embodiments, the cGAS activation agent is administered to the cell or subject after the administration of a recombinant virus described herein. In some embodiments, the cGAS activation agent is administered to the cell or subject before the administration of a recombinant virus described herein. In some embodiments, the cGAS activation agents are encapsulated in a nanoparticle, liposome, exosome, or liposome for delivery to a subject.
  • the present disclosure relates to the use of cGAS expressing virus or composition thereof in the manufacture of a medicament.
  • Expression of cGAS, production of cGAMP, and activation of the STING pathway lead to induction of a type I IFN response and provide anti-tumor responses and/or or anti-viral responses in subjects.
  • the present disclosure relates to cGAS expressing virus or composition thereof for use as a medicament for treating a disease selected from cell proliferative diseases, inflammatory diseases, and infectious diseases, as well as to induce anti-tumor immune responses.
  • each step need not be executed by the same individual or at the same location.
  • the agents can be administered by different practitioners or by the same practitioner. Where necessary, each of the administrations can take place at a different location.
  • composition refers to a formulation of a cGAS- expressing virus described herein.
  • formulations include all physiologically acceptable compositions including derivatives and/or prodrugs, solvates, stereoisomers, racemates, or tautomers thereof.
  • the composition further comprises a physiologically acceptable carrier, diluent, and/or excipient.
  • A“therapeutic composition” or“pharmaceutical composition” refer to a composition of a cGAS-expressing virus described herein administered to a subject for the treatment of a particular disease or disorder.
  • pharmaceutically acceptable carrier, diluent or excipient includes without limitation any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye/colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, surfactant, and/or emulsifier which has been approved by the United States Food and Drug Administration as being acceptable for use in humans and/or domestic animals.
  • Exemplary pharmaceutically acceptable carriers include, but are not limited to, to sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; tragacanth; malt; gelatin; talc; cocoa butter, waxes, animal and vegetable fats, paraffins, silicones, bentonites, silicic acid, zinc oxide; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water
  • kits described herein further comprise one or more immune checkpoint inhibitors.
  • the kit comprises cGAS-expressing virus or composition thereof and a reagent for reconstituting and/or diluting the components.
  • a kit comprises a cGAS-expressing virus and further comprises one or more additional reagents, where such additional reagents can be selected from: a buffer for introducing the cGAS-expressing virus into a cell; a wash buffer; a control reagent; a control expression virus, and the like.
  • Components of a kit can be in separate containers or can be combined in a single container.
  • a kit further comprises instructions for using the components of the kit to practice the methods of the present disclosure.
  • the instructions for practicing the methods are generally recorded on a suitable recording medium.
  • the instructions may be printed on a substrate, such as paper or plastic, etc.
  • the instructions may be present in the kits as a package insert or in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or sub-packaging).
  • the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g. CD-ROM, diskette, flash drive, etc.
  • the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g. via the internet, are provided.
  • An example of this embodiment is a kit that includes a web address where the instructions can be viewed and/or from which the instructions can be downloaded.
  • the means for obtaining the instructions is recorded on a suitable substrate.
  • STX- VP- 001 is a 3rd generation lentiviral vector (LV) expressing cGAS under the human EFl-a promoter and the puromycin resistant gene under the PGK promoter.
  • STX-VP-001 is further pseudotyped using a VSV envelope glycoproteins (VSV-G) to expand the trophism of the LV.
  • VSV-G VSV envelope glycoproteins
  • the transducing vector (12 pg), psPAX (Gag-pol) (8.0 pg), Md2G
  • VSVGEnv (4.0 pg) (4.0 pg)
  • 20 pL of 2.5M CaCh was combined in 200 pL total volume of sterile water.
  • 200 pL of sterile BES buffer was added dropwise to the mixture and incubated at room temperature for 45 minutes before adding to cells.
  • 24 hrs post-transfection viral supernatants were collected and stored at 4°C and fresh DMEM was added to the cells.
  • 72 hrs post transfections viral supernatants were collected and pooled with the 24 hour supernatant.
  • Viral supernatants were centrifuged at 4°C at 2000 rpm for 10 minutes to pellet cell debris. Supernatants were filtered through a 0.45 pm filter using a syringe into a sterile 50 mL tube. Filtered supernatants were loaded into 100 kDa cut-off filter (Amicon) and spun at 1500g for 30 minutes to concentrate the virus. This step was repeated until the desired concentrated volume was achieved. The concentrated virus was is stored at -80°C for later use.
  • the virus Prior to use, the virus was titered by infecting HEK293 cells with various dilutions and calculating the transduction units per mL (TU/mL) by counting the number of colonies (in case of vectors with antibiotic resistance cassettes, e.g., Fig. 1) or fluorescent cells (in case of vectors with a fluorescent marker cassette, e-g Fig ⁇ 2).
  • STX-VP-002 is an adeno-associated viral vector expressing cGAS under the murine Tyrosinase (Tyr) promoter, which is exclusively expressed in melanocytes.
  • Tyr murine Tyrosinase
  • the same vector also expresses eGFP as a reporter gene under the ubiquitous human EFl-a promoter, which also shows activity in mouse cells.
  • the AAV vector for STX-VP-002 is AAV-DJ (Cell Biolabs Inc.) and has broad tropism due to its modified envelope. (Fig. 2)
  • the cells were then freeze-thawed four times by incubating alternately in a dry ice/ethanol bath and a water bath of 37° C. Cells were centrifuged at 10,000 g for 10 minutes and supernatants were collected as AAV crude lysate. The AAV crude lysates were then filtered using a 0.45 pm syringe filter and further concentrated to the desired volume using a 100 kDa cut-off filter and spinning at l500g for 30 min.
  • the virus was titered by infecting HEK293 cells with various dilutions and calculating the TU/mL by counting the number of colonies (in case of vectors with antibiotic resistance cassettes) or fluorescent cells (in case of vectors with a fluorescent marker cassette).
  • the THP-l Dual cells express the secreted Lucia luciferase under the ISG54 minimal promoter in conjunction with five IFN-stimulated response elements.
  • STING agonists like STX-VP-001
  • IFN Interferon pathway
  • Example 3 Anti-tumor efficacy of STX-VP-001 in a murine syngeneic melanoma model
  • the B16F10 mouse melanoma cancer cell line was purchased from ATCC and cultured according to manufacturer’s recommended protocols.
  • B16F10 cells were collected from culture during the exponential growth phase and approximately 1 x 10 6 cells in 100-200 pL of saline were subcutaneously (SC) injected on the right flank of each mouse (1 site/mouse) for implantation of tumors on Day 1.
  • SC subcutaneously
  • mice were randomized into one of five treatment groups, shown below in Table 1. Treatments were performed as described in Table 1 and as shown in Fig. 4A - Fig. 4B.
  • Body weights were recorded on the day of tumor implantation and treatment days. Tumor volumes were measured post implantation of cells and 3x/week for 4 weeks.
  • TGI Tumor growth inhibition
  • mice were sacrificed with CO2 in an appropriate exposure chamber. Approximately 20-30% of the tumor was taken from each mouse and snap frozen immediately in liquid nitrogen for later analysis. The remaining part of the tumors was excised from all the mice and embedded in in paraffin. 2 pm thick sections were then cut from each paraffin block and stained using hematoxylin (H) and eosin (E) stain for assessment of the following histological characteristics: immune cell infiltration, blood vessels, melanin, necrosis, and epithelium damage. Sections were also stained for cGAS expression using MB21D1 antibody (Sigma, USA).
  • H hematoxylin
  • E eosin
  • prophylactic STX-001 treatment and post-implantation STX- 001 treatment significantly reduced tumor growth compared to vehicle treated controls. Further, prophylactic STX-001 treatment and post-implantation STX-001 treatment also reduced tumor growth compared to the anti -PD 1 reference treatment group.
  • Fig. 6 and Fig. 7 provide the histological analyses of these experiments.
  • prophylactic STX-001 treatment (Pr) and post-implantation STX-001 treatment (Tr) reduced the melanin presence and vascularity scores compared to vehicle treated controls.
  • Representative immunohistochemistry images of these experiments are shown in Fig. 6A.
  • cGAS expression in tumors from each group is provided in Fig. 7.
  • cGAS expression was higher in the STX- VP-001 treated groups compared to untreated and PD1 antibody treated groups.
  • Example 4 Anti- tumor efficacy of STX- VP-001 in a murine hepatocellular carcinoma xenograft model
  • HCC HepG2 hepatocellular carcinoma
  • Table 4 Tumor volume measurements in HCC xenograft model
  • Example 5 Tissue distribution of STX- VP-001 and STX- VP-002 and cGAS expression
  • cGAS cGAS expression under control of the melanocyte/melanoma specific Tyr promoter
  • Ctl Virus AAV.GFP (does not express cGAS)
  • STX-VP-001 FV.EFl-a.cGAS (cGAS expression under control of the ubiquitous human EFl-a promoter).
  • FV.EFl-a.cGAS cGAS expression under control of the ubiquitous human EFl-a promoter
  • STX-VP-001 expressing Puromycin resistance gene under the PGK promoter
  • STX- VP-002 expressing eGFP under the EF1 alpha promoter
  • the target organs brain, liver, spleen, kidney, and lungs
  • Vector distribution in formalin-fixed tissues was analyzed by staining tissue sections with an anti-GFP antibody (murine anti-GFP, Sigma SAB2702197).
  • cGAS expression after vector administration was analyzed by IHC using an anti-cGAS antibody (Anti-Mb2lDl, Sigma, USA). Histological images were taken at 20x and lOOx magnification. Grading criteria for cGAS staining is as provided above in Table 2. Grading criteria for GFP staining is provided below in Table 6.
  • Tyr.cGAS.EFla.GFP was distributed throughout all tissues analyzed. As shown in Fig. 11B, cGAS expression for intratumoral administration of STX-VP-001 (LV + UP) and intravenous administration of STX- VP-002 (AAV+TSP) was comparable.
  • Example 6 Anti-tumor efficacy of STX- VP-002 in a murine syngeneic melanoma model
  • mice 1 x 10 6 cells in 100-200 pL of saline were subcutaneously (SC) injected on the right flank of each mouse (1 site/mouse) for implantation of tumors on Day 1.
  • SC subcutaneously
  • mice When tumor volume reached an average of 100 mm 3 , mice were randomized into one of seven treatment groups, shown below in Table 8. Treatments were performed as described in Table 8 and as shown in Fig. 12A - Fig. 12B. Body weights were recorded on the day of tumor implantation and treatment days. Tumor volumes were measured post-implantation of cells and 3x/week. Mice were sacrificed when tumor volumes reached ⁇ 6000 mm 3 in the vehicle control group ( ⁇ 2 weeks after I st administration of treatments).
  • TGI Tumor growth inhibition
  • FIG. 12A A schematic of the experimental protocol and treatment dosing regimens is provided in Fig. 12A - Fig. 12B. Details of the experimental design are provided below in Table 8 Table 8: Experimental Design of STX- VP-002 B16F10 Melanoma Mouse Study
  • Table 9 Tumor volume measurements for STX- VP-002 mono and combination therapy in
  • ⁇ 20-30% of tumors was taken from each mouse and fixed in 10% neutral buffered formalin. The remaining part of the tumors was excised from each mouse and stored in 10% neutral buffered formalin for histopathology. The tumors were embedded in paraffin and 2 pm thick sections were cut from each paraffin block and stained using hematoxylin (H) and eosin (E) stain. The following histological characteristics were assessed: immune cell infiltration, blood vessels, melanin, necrosis, and epithelium damage. The grading of each parameter was based on the following system: minimal (1/+), mild (2 /++), moderate (3/+++), marked (4/++++). Histological images for each group are shown in Fig. 14A and the results for tumor necrosis, melanin presence, and vascularity are quantified in Fig. 14B.

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Abstract

La présente invention concerne des vecteurs viraux comprenant des polynucléotides codant pour des polypeptides cGAS. L'invention concerne également des compositions comprenant les vecteurs viraux, des procédés d'expression et d'activation de cGAS dans des cellules, de production de cGAMP, d'activation de la voie STING, et d'induction de réponses IFN. Les vecteurs et les compositions peuvent être utilisés thérapeutiquement pour traiter des maladies associées à une prolifération cellulaire, des maladies inflammatoires et des maladies infectieuses, ainsi que pour induire des réponses immunitaires antitumorales chez des sujets le nécessitant. La présente invention concerne en outre des procédés d'augmentation de l'efficacité d'un inhibiteur de point de contrôle immunitaire par l'administration des vecteurs ou des compositions à un sujet le nécessitant.
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CN113384710A (zh) * 2020-03-14 2021-09-14 杭州星鳌生物科技有限公司 新型免疫激动剂复合物的组成及其在抗多种疾病药物中的应用
US12071633B2 (en) 2020-10-13 2024-08-27 Kriya Therapeutics, Inc. Viral vector constructs for delivery of nucleic acids encoding cytokines and uses thereof for treating cancer

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113384710A (zh) * 2020-03-14 2021-09-14 杭州星鳌生物科技有限公司 新型免疫激动剂复合物的组成及其在抗多种疾病药物中的应用
US12071633B2 (en) 2020-10-13 2024-08-27 Kriya Therapeutics, Inc. Viral vector constructs for delivery of nucleic acids encoding cytokines and uses thereof for treating cancer

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