[go: up one dir, main page]

WO2013166000A1 - Modulation de réponses immunitaires - Google Patents

Modulation de réponses immunitaires Download PDF

Info

Publication number
WO2013166000A1
WO2013166000A1 PCT/US2013/038840 US2013038840W WO2013166000A1 WO 2013166000 A1 WO2013166000 A1 WO 2013166000A1 US 2013038840 W US2013038840 W US 2013038840W WO 2013166000 A1 WO2013166000 A1 WO 2013166000A1
Authority
WO
WIPO (PCT)
Prior art keywords
sting
cells
dna
nucleic acid
ifn
Prior art date
Application number
PCT/US2013/038840
Other languages
English (en)
Inventor
Glen N. Barber
Original Assignee
Barber Glen N
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US13/460,408 external-priority patent/US20130039933A1/en
Application filed by Barber Glen N filed Critical Barber Glen N
Priority to AU2013256468A priority Critical patent/AU2013256468A1/en
Priority to KR20147033632A priority patent/KR20150004416A/ko
Priority to EP13784572.3A priority patent/EP2844756A4/fr
Priority to CN201380029115.7A priority patent/CN104540945A/zh
Priority to US15/120,694 priority patent/US20170037400A1/en
Priority to JP2015510379A priority patent/JP2015516989A/ja
Priority to CA2907616A priority patent/CA2907616A1/fr
Publication of WO2013166000A1 publication Critical patent/WO2013166000A1/fr
Priority to US16/717,325 priority patent/US20200392492A1/en
Priority to US17/357,822 priority patent/US20210324023A1/en

Links

Classifications

    • 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
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]

Definitions

  • Embodiments of the invention relate to compositions and methods for modulating innate and adaptive immunity in a subject and/or for the treatment of an immune-related disorder, cancer, autoimmunity, treating and preventing infections.
  • PAMPs pathogen associated molecular patterns
  • viral RNA can be detected by membrane bound Toll-like receptors (TLR's) present in the endoplasmic reticulum (ER) and/or endosomes (e.g.
  • TLR 3 and 7/8 TLR-independent intracellular DExD/H box RNA helicases referred to as retinoic acid inducible gene 1 (RIG-I) or melanoma differentiation associated antigen 5 (MDA5, also referred to as IFIH1 and helicard).
  • RIG-I retinoic acid inducible gene 1
  • MDA5 melanoma differentiation associated antigen 5
  • STING Stimulator of Interferon Genes
  • TM transmembrane
  • ER endoplasmic reticulum
  • Loss of STING reduced the ability of polylC to activate type I IFN and rendered murine embryonic fibroblasts lacking STING ( _ ⁇ MEFs) generated by targeted homologous recombination, susceptible to vesicular stomatitis virus (VSV) infection.
  • VSV vesicular stomatitis virus
  • DNA-mediated type I IFN responses were inhibited, indicating that STING may play an important role in recognizing DNA from viruses, bacteria, and other pathogens which can infect cells.
  • Yeast-two hybrid and co-immunoprecipitation studies indicated that STING interacts with RIG-I and with Ssr2/TRAPp, a member of the translocon-associated protein (TRAP) complex required for protein translocation across the ER membrane following translation. RNAi ablation of TRAP inhibited STING function and impeded the production of type I IFN in response to polylC.
  • TRAP translocon-associated protein
  • STING itself binds nucleic acids including single- and double-stranded DNA such as from pathogens and apoptotic DNA, and plays a central role in regulating proinflammatory gene expression in inflammatory conditions such as DNA-mediated arthritis and cancer.
  • nucleic acids including single- and double-stranded DNA
  • apoptotic DNA binds nucleic acids including single- and double-stranded DNA
  • plays a central role in regulating proinflammatory gene expression in inflammatory conditions such as DNA-mediated arthritis and cancer.
  • Various new methods of, and compositions for, upregulating STING expression or function are described herein along with further characterization of other cellular molecule which interact with STING.
  • Described herein are methods for modulating an immune response in a subject having a disease or disorder associated with aberrant STING function. These methods can include the step of administering to the subject an amount of a pharmaceutical composition including an agent which modulates STING function and a pharmaceutically acceptable carrier, wherein amount the pharmaceutical composition is effective to ameliorate the aberrant STING function in the subject.
  • the agent can be a small molecule that increases or decreases STING function, or a nucleic acid molecule that binds to STING under intracellular conditions.
  • the STING-binding nucleic acid molecule can be a single-stranded DNA between 40 and 150 base pairs in length or a double-stranded DNA between 40 and 150, 60 and 120, 80 and 100, or 85 and 95 base pairs in length or longer.
  • the STING-binding nucleic acid molecule can be nuclease -resistant, e.g., made up of nuclease-resistant nucleotides. It can also be associated with a molecule that facilitates transmembrane transport. In these methods, the disease or disorder can be a DNA-dependent inflammatory disease.
  • These methods can include the step of administering to the subject an amount of a pharmaceutical composition including an agent which downregulates STING function or expression and a pharmaceutically acceptable carrier, wherein amount the pharmaceutical composition is effective to reduce the number of inflammatory immune cells infiltrating the cancerous tumor by at least 50% (e.g., at least 50, 60, 70, 80, or 90%, or until reduction of inflammatory cell infilitration is detectably reduced by histology or scanning).
  • Figs. 1A-G show STING dependent innate immune signaling.
  • Fig. 1A Human Telomerase Fibroblasts (hTERT-BJl) were transfected with various nucleotides (3 ⁇ g/ml) for 16h. Endogenous IFN levels were measured.
  • hTERT-BJl cells were transfected with FITC conjugated dsDNA90 was examined by fluorescent microscopy to ensure efficient transfection.
  • Fig. IB hTERT-BJl cells were transfected with mock, random or two independent human STING siRNAs (siRNA 3 or 4) for 3 days followed by dsDNA90 transfection (3 ⁇ g/ml) for 16 hours.
  • Fig. 36C Primary STING + + , STING " ' " , STAT1 + + or STATl " ' " MEFs were transfected with or without dsDNA90 (3 ⁇ g/ml) for 3 hours. Total RNA was purified and examined for gene expression by Illumina Sentrix BeadChip Array (Mouse WG6 version 2).
  • Fig. ID hTERT- BJl cells were treated NS or STING siRNA. After 3days, cells were treated with dsDNA90, ssDNA90 or ssDNA45 (3 ⁇ g/ml).
  • IFN mRNA levels were measured by real time RT-PCR after 16 hours.
  • Fig. IE hTERT-BJl cells were treated NS or STING siRNA. At 3days, cells were treated with dsDNA90, ssDNA90 or ssDNA45 (3 ⁇ g/ml). Endogenous IFN levels were measured after 16 hours.
  • Fig. IF Primary STING + + or STING " ' " MEFs were transfected with or without dsDNA90 (3 ⁇ g/ml). After 3h, the same as Fig. 1C.
  • Fig. 1G hTERT-BJl cells were treated with or without dsDNA90 (3 ⁇ g/ml) for 3 hours and stained with anti-HA antibody and calreticulin as an ER marker.
  • Figs. 2A-J show that STING binds to DNA.
  • Fig. 2A 293T cells were transfected with indicated plasmids. Cell lysates were precipitated with biotin-dsDNA90 agarose and analyzed by immunoblotting using anti-HA antibody.
  • Fig. 2B Schematic of STING mutants.
  • Fig. 2C Same as Fig. 2A.
  • Fig. 2D Same as Fig. 2A. STING variants unable to bind DNA are labeled in red.
  • Fig. 2E hTERT-BJl cells were transfected biotin conjugated dsDNA90 (B-dsDNA90; 3 ⁇ g/ml) for 6h and treated with DSS.
  • Fig. 2F STING was expressed in 293T cells and after 36 hours lysates were incubated with dsDNA90 agarose in the presence of competitor dsDNA90, Poly(I:C), B-DNA or ssDNA90 and analyzed by immunoblotting using anti-HA antibody.
  • Fig. 2G 293T cells were transfected with HA-tagged STING, GFP or TREX1. Cells were lysed and biotin labeled ssDNA or dsDNA added with streptavidin agarose beads. Precipitates were analyzed by immunoblotting using anti-HA antibody.
  • Fig. 2F STING was expressed in 293T cells and after 36 hours lysates were incubated with dsDNA90 agarose in the presence of competitor dsDNA90, Poly(I:C), B-DNA or ssDNA90 and analyzed by immunoblotting using anti-HA antibody.
  • Fig. 2G 293T cells were transfected with HA-tagged STING, G
  • 2H 293T cells were transfected with ⁇ - Luciferase and STING variants and luciferase activity measured.
  • Fig. 21 hTERT-BJl cells were transfected with dsDNA90 and crosslinked with formaldehyde. STING was precipitated and bound DNA detected by PCR using dsDNA90 specific primers. NC: negative control. PC: positive control, dsDNA90.
  • Fig. 2J STING + + or STING " ' " MEFs were transfected with dsDNA90 and then same as Fig. 21. Error bars indicates s.d. Data are representative of at least two independent experiments.
  • Figs. 3A-3H show that TREXl is negative regulator of STING signaling.
  • Fig. 3A Immunoblot confirming STING and/or TREXl knockdown in siRNA treated hTERT- BJl cells.
  • Fig. 3B siRNA treated hTERT-BJl cells were transfected with dsDNA90 (3 ⁇ g/ml). Endogenous IFN levels were measured after 16 hours. *P ⁇ 0.05.
  • Fig. 3A-3H show that TREXl is negative regulator of STING signaling.
  • Fig. 3A Immunoblot confirming STING and/or TREXl knockdown in siRNA treated hTERT- BJl cells.
  • Fig. 3B siRNA treated hTERT-BJl cells were transfected with dsDNA
  • FIG. 38D siRNA treated hTERT-BJl cells were infected with ⁇ 34.5 deleted-HSV and virus titers measured. *P ⁇ 0.05.
  • Fig. 3E Immunoblot of NS or STING siRNA treated TREX1 + + or TREXl " ' " MEFs, confirming STING knockdown.
  • Fig. 3F siRNA treated TREX1 + + or TREXl " ' " MEFs were treated with dsDNA90 and IFN levels were measured after 16 hours. *P ⁇ 0.05.
  • Fig. 3H Immunofluorescence analysis using anti-TREXl or anti-STING antibody of hTERT-BJl cells transfected with or without dsDNA90. *P ⁇ 0.05, Student's t-test. Error bars indicated s.d. Data are representative of at least two independent experiments.
  • Figs. 4A-J show TREXl associayes with oligosaccharyltransf erase complex.
  • Fig. 4 A shows a schematic of TREXl. Red indicates RPN1 binding site.
  • Fig. 4B shows a schematic of STING. Red indicates DAD1 binding site.
  • Fig. 4C RPN1 interacts with TREXl in yeast two hybrid analysis (l.pGBKT7, 2.pGBKT7-NFAR M9, 3.pGBKT7- TREX1, 4.pGBKT7-STING full length, 5.pGBKT7-STING C-terminal).
  • Fig. 4D 293T cells were co-transfected with TREXl-tGFP and RPNl-Myc.
  • Fig. 4H hTERT-BJl cells were treated with or without dsDNA90 (3 ⁇ g/ml). At 6h after transfection, cells were examined by immunofluorescence using anti-STING or anti-DADl antibody.
  • Fig. 41 Immunoblot analysis of microsome fractions after sucrose gradient centrifugation using indicated antibodies. I: input.
  • Fig. 4J hTERT-BJl cells were treated with TREXl, Sec61Al, TRAP , NS or STING siRNA. After 72h, cells were treated with dsDNA90 (3 ⁇ ) for 16h and then endogenous IFN levels were measured. *P ⁇ 0.05, Student's i-test. Error bars indicated s.d. Data are representative of at least two independent experiments.
  • Figs. 5A-G show that cytoplasmic DNA induces STING- dependent genes in hTERT-BJl cells.
  • Fig. 5A hTERT-BJl cells were treated with STING siRNA. After 3 days, cells were treated with or without dsDNA90 for 3h. Total RNA was purified and examined for gene expression using Human HT-12_V4_Bead Chip.
  • Figs. 5B-G hTERT-BJ 1 cells were treated as in Fig. 5A. Total RNAs were examined by real time PCR for IFN (Fig. 5B), PMAIP1 (Fig. 5C), IFIT1 (Fig. 5D), IFIT2 (Fig. 5E), IFIT3 (Fig.
  • Figs. 6A-H show that STING-dependent genes are induced by cytoplasmic DNA in MEFs.
  • Primary STAT1 + + or STAT1 " ' " MEFs were treated dsDNA90, IFNa or dsDNA90 with IFNa.
  • Total RNAs were purified and examined by real time PCR for IFN (Fig. 6A), IFITl (Fig. 6B), IFIT2 (Fig. 6C), IFIT3 (Fig. 6D), CXCL 10 (Fig. 6E), GBP1 (Fig. 6F), RSAD2 (Fig. 6G) and CCL5 (Fig. 6H).
  • Figs. 7A-H show that cytoplasmic DNAs induce STING-dependent genes in MEFs.
  • STING + + or STING " ' " MEFs were treated with or without dsDNA45, dsDNA90, ssDNA45 or ssDNA90 for 3h.
  • Total RNAs were purified and examined by real time PCR for IFN (Fig. 7A), IFITl (Fig. 7B), IFIT2 (Fig. 7C), IFIT3 (Fig. 7D), CCL5 (Fig. 7E), CXCL 10 (Fig. 7F), RSAD2 (Fig. 7G) or GBP1 (Fig. 7H).
  • Figs. 8A-D show STING localization and dimerization.
  • Fig. 8A MEFs stably expressing STING-HA were treated with ssDNA45, dsDNA45, ssDNA90 or dsDNA90. After 3h, cells were stained using anti-HA or anti-calreticulin antibody.
  • Fig. 8B 293T cells were transfected with STING-HA and Myc-STING. Lysates were precipitated by anti-Myc antibody and analyzed by immunoblotting using anti-HA antibody.
  • Fig. 8C hTERT-BJ 1 cells were treated with or without the cross linker DSS. Cell lysates were subjected to immunoblot using anti-STING antibody.
  • Fig. 8D 293T cells were transfected with indicated plasmids and treated with DSS. Cell lysates were analyzed by immunoblotting using anti-HA antibody.
  • Figs. 9A-I show that DNA virus induces STING-dependent genes in MEFs.
  • Total RNA was purified and examined for gene expression using Illumina Sentric Bead Chip array (Mouse WGS version2).
  • Figs. 9B-I STING + + , STING " ' " or STAT ' /STING ⁇ MEFS were treated with or without dsDNA90, HSV or ⁇ 34.5 deleted-HSV for 3h.
  • Total RNAs were purified and examined by real time PCR for IFN (Fig. 9B), IFTT1 (Fig. 9C), IFIT2 (Fig. 9D), IFIT3 (Fig. 9E), CCL5 (Fig. 9F), CXCL 10 (Fig. 9G), RSAD2 (Fig. 9H) or GBP1 (Fig. 91). Error Bars indicate s.d.
  • Figs. 10A-F show that STING interacts with IRF3/7 and NFK-B.
  • Fig. 10A IRF7 binding sites in the promoter regions of STING dependent dsDNA90 stimulatory genes.
  • Fig.s 10B-D Nudear extract was isolated from mock treated or dsDNA90 treated STING + + or STING " ' " MEFs and were examined for IRF3 (Fig. 10B), IRF7 (Fig. IOC) and NF- ⁇ (Fig. 10D) activation following the manufacturer's instruction.
  • Nuclear extract kit, Trans AM IRF3, Trans AM IRF7 and Trans AMNFKB family Elisa kits were from Active Motif. *P ⁇ 0.05, Student' s i-test.
  • Fig. 10E STING + + or STING " ' " MEFs were treated with poly(I:C). dsDNA90 or HSV1 and cells were stained by anti-IRF3 antibody.
  • Fig. 10F STING + + or STING " ' " MEFs were treated dsDNA90 or HSV1 and cells were stained by anti- p65 antibody. Loss of STING did not affect poly(I:C) mediated innate immune signaling.
  • Figs. 11A-F show that STING binds DNA in vitro.
  • Fig. 11A In vitro translation products were precipitated with biotin conjugated dsDNA90 and immunoblotted by anti-HA antibody.
  • Fig. 11B schematic of STING variants.
  • Figs. 11C, 11D Same as Fig. 11A. STING variants lacking aa 242-341 (red) failed to bind DNA.
  • Figs. 11E-F In vitro translation products were precipitated with biotin conjugated ssDNA90 and immunoblotted by anti-HA antibody.
  • Figs. 12A-G show that STING binds DNA in vivo and in vitro.
  • hTERT BJl cells were transfected with biotin-dsDNA90 and crosslinked by UV. Cells were lysed and precipitated by streptavidin agarose and analyzed by immunoblotting.
  • Figs. 12B-C hTERT- BJ1 cells were treated with IFI16 (Fig. 12B) or STING (Fig. 12C) siRNA and then same as in Fig. 12A.
  • Fig. 12D STING + + or STING " ' " MEFs were treated as in Fig. 12A.
  • Fig. 12D STING + + or STING " ' " MEFs were treated as in Fig. 12A.
  • FIG. 12E STING-Flag expressing 293T cells were treated with or without biotin-dsDNA90 and crosslinked by DSS. Lysates were precipitated and analyzed by immunoblotting.
  • Fig. 12F 293T cells were transfected with dsDNA90 or ssDNA90 and crosslinked by UV or DSS and then precipitated and analyzed by immunoblotting.
  • Fig. 12G STING-Flag expression 293T cells were lysed and incubated with ds0NA90 or Poly(I:C) and biotin-dsDNA90 agarose and then same as Fig. 12E.
  • Figs. 13A-C show that STING binds viral DNA.
  • Fig. 13A Oligonucleotide sequences of HSV, cytomegalovirus (CMV) or adenovirus (ADV).
  • Figs. 13B-C 293T cells were transfected with indicated plasmids. Cell-lysates were precipitated with biolin- dsDNA90, biotin-HSV DNA 120 mer, biotin-ADV DNA 120 mer or biotin-CMV DNA 120 mer agarose and analyzed by immunoblotting using anti-HA antibody.
  • FIGs. 14A-C show that STING binds DNA.
  • Fig. 14A Schematic of STING ELISA.
  • Fig. 14B Process of an embodiment of a STING ELISA.
  • Fig. 14C Binding capacity of dsDNA90 was measured by ELISA. *P ⁇ 0.05, Student' s i-test. Error Bars indicated s.d. Data are representative of at least two independent experiments.
  • Figs. 15A-D show that TREX1 is a negative regulator of STING signalling.
  • Fig. 15B
  • RNAs were purified and examined for gene expression by Illumina Sentrix Bead Chip array (Mouse WG6 version2).
  • Fig. 16 shows that STING regulates ssDNA90-mediated IFN production in TREX _ " MEFs.
  • siRNA treated TREX1 + + or TREX1 _ " MEFs were treated with ssDNA45 or ssDNA90 and IFN levels were measured after 16 hours. *P ⁇ 0.05, Student' s t-test. Error Bars indicated s.d. Data are representative of atleast two independent experiments.
  • Figs. 17A-H show that TREX1 is not a negative regulator of STING-dependent genes.
  • Fig. 17A TREX1 + + or TREX1 _ " MEFs were treated wilh HSV1, IFNa, dsDNA90, triphosphate RNA (TPRNA) and VSV. TPRNA and VSV weakly activated IFN induced genes. Total RNAs were purified and examined for gene expression by Illumina Sentrix Bead Chip array (Mouse WG6 version2).
  • Figs. 17B-H Total RNAs were examined by RT- PCR for IFN (Fig. 17B), IFIT1 (Fig. 17C), IFIT2 (Fig. 17D), IFIT3 (Fig.
  • Figs. 18A-D show that TREX1 assoicates with oligosaccharyltransferase complex.
  • An IFN-treated hTERT cDNA library was used to develop a yeast two hybrid library (AH109). Full length TREX 1 was used as bait to screen the library. Approximately 5 million cDNA expressing yeast were screened (Clontech). 44 clones were isolated from 3 independent yeast mating procedures. RPN1 was isolated 8 times in total (three times in screen 1, twice in screen 2 and three times in screen 3). The majority of the clones, aside from RPN1, failed to interact with IREX1 after re-testing.
  • RPN1 isolated clones four clones encoded aa 258-397, two clones aa 220-390 and two clones aa 240-367.
  • TREX1 variants were generated and the interation between TREX1 (aa.241-369) and RPN 1 (aa 256-397) was mapped.
  • To isolate DADl the C-terminal region of STING (aa 173-379) was used to screen the same library.
  • 24 isolated clones, full length DAD I was found twice. The majority of the clones, aside from DADl, failed to interact with STING after re- comfirmation studies. Full length DADlwas seen to associate with region 242-310 of STING. Fig.
  • FIG. 18A Schematic of TREX1 mutants.
  • Fig. 18B GAL4 binding domain fused to TREX1 or TREX1-4 interact with RPN1 fused to the GAL4 activation domain in yeast two hybrid screening.
  • Fig. 18C Schematic of STING mutants.
  • Fig. 18D GAL4 binding domain fused to STING-C-terminal or STING-C2 interact with DADl fused to the GAL4 activation domain in yeast two hybrid screening.
  • Figs. 19A-C show that TREX1 and STING associate with oligosacchary transferase complex.
  • Fig. 19A 293T cells were co-transfected with TREX1- tGFP and RPNl-Myc. Lysates were immunoprecipitated with anti-tGFP antibody or IgG control and analyzed by immunoblotting using indicated antibodies.
  • Fig. 19B 293T cells were co-transfected with Myc-STING or GFP-DAD1 and cells were lysed. Lysates were immunoprecipitated with anti-Myc antibody or IgG control and immunoblotted using anti- GFP antibody.
  • Fig. 19A 293T cells were co-transfected with TREX1- tGFP and RPNl-Myc. Lysates were immunoprecipitated with anti-tGFP antibody or IgG control and analyzed by immunoblotting using indicated antibodies.
  • Fig. 19B 293T cells were co-transfected with My
  • 19C 293T cells were co-transfected with TREXl-tGFP and RPNl-Myc, GFP-DAD1 or STING-HA. Lysates were immunoprecipitated by anti-tGFP antibody or IgG control and analyzed by immunoblotting using anti-tGFP, anti-Myc, anti-GFP or anti-HA antibodies.
  • Fig. 20 shows that TREX1 localizes in the endoplasmic reticulum.
  • hTERT-BJl cells were transfected with RPNl-Myc. After 48h, cells were examined by immunofluorescence using anti-TREXl antibody (red), anti-Myc antibody (green) or anti- calreticulin antibody (blue) as an endoplasmic reticulum marker.
  • Figs. 21A-H show that exogenously expressed STING in 293T cells reconstitutes dsDNA90 response.
  • Fig. 21 A 293T cells were infected with control lentivirus or hSTING lentivirus. 1 day afer infection, cells were treated with dsDNA90. After 6h, cells were stained using anti-STING or anti-calreticulin antibody.
  • Fig. 21B Cell lysates (from Fig. 56A) were subjected to immunoblot using anti-STING antibody.
  • Figs. 21C-D Lentivirus infected 293T cells were stimulated with dsDNA90 for 6h. Total RNAs were purified and examined by real time PCR for IFN (Fig.
  • Fig. 21C IFIT2
  • Fig. 21D IFIT2
  • Fig. 21E 293T cells stably transduced with control or hSTING lentivirus were subjected to Brefeldin A (BFA) experiment as shown in the flow chart.
  • Fig. 21F Cell lysates (from Fig. 21E) were subjected to immunoblot using anti-STING antibody.
  • Fig. 21G Cell lysates (from Fig. 21E) were measured for IFN -luciferase activity.
  • Fig. 21H Primary STING " _ MEFs were stably transduced with control or mSTING. Cells were treated with dsDNA90 and endogenous IFN levels were measured by ELISA. *P ⁇ 0.05, Student' s i-test. Error bars indicated s.d. Data are representative of at least two independent experiments.
  • Fig. 22 shows that translocon members regulate HSV1 replication.
  • hTERT-BJl cells were treated withTREXl, Sec61Al, TRAPp, NS or STING siRNA.
  • Figs. 23A-C show that IFI16 is not required for IFN-production by dsDNA90 in hTER-BJl cells.
  • Fig. 23A hTERT-BJt cells were treated with NS, IFI16, STING or TREX1 siRNA. After 3 days, cells were lysed and checked expression levels by immunoblotting.
  • Fig. 23B siRNA treated hTERT-BJl cells were stimulated with dsDNA90 and IFN production was measured by ELISA.
  • Fig. 24 shows an embodiment of a STING cell based assay.
  • Fig. 25 shows that Drug "A” induces STING trafficking.
  • Fig. 26 shows that drug "X" inhibits IFN mRNA production.
  • Fig. 27 is a schematic showing that STING is phosphorylated in response to cytoplamic DNA.
  • hTERT-BJl cells were transfected with 4 ⁇ g/ml of ISD for 6 h.
  • the cell lysates were prepared in TNE buffer and then subjected to immunoprecipitation with anti- STING antibody followed by SDS-PAGE.
  • the gel was stained with CBB and then bands including STING were analyzed by mLC/MS/MS at the Harvard Mass Spectrometry and Proteomics Resource Laboratory. Alignment of STING amino acid sequence from different species and the phosphorylation sites identified by mass spectrometry.
  • Serine 345, 358, 366, and 379 were identified by mass spectrometry.
  • Serine 358 and S366 are important for STING function.
  • Fig. 28 shows that Serine 366 (S366) of STING is important for IFN production in cytoplasmic DNA pathway.
  • S366 Serine 366
  • 293T cells were transfected with plasmid encoding mutant STING and reporter plasmid. After 36 hr, luciferase activity was measured.
  • STING " ' " MEF cells were reconstituted with mutant STING and then the amount of IFN in culture media was measured by ELISA.
  • S366 is important for IFN production by STING and S358 is also likely to play an important role.
  • Figs. 29A-D show that STING deficient mice are resistant to DMBA induced inflammation and skin oncogenesis: STING + + and STING " ' " mice were either mocked treated with acetone or treated with 10 ⁇ g of DMBA on the shaved dorsal weekly for 20 weeks.
  • Fig. 29A STING deficient animals are resistant to DNA-damaging agants that cause skin cancer. Percentages of skin tumor-free mice were shown in the Kaplan-Meier curve.
  • Fig. 29B Pictures of representative mice of each treatment groups were shown.
  • Fig. 29C Histopathological examinations were performed by H&E staining on mock or DMBA treated skin/skin tumor biopsies.
  • Fig. 29D Cytokine upregulation in STING expressing mice exposed to carcinogens.
  • RNAs extracted from mock or DMBA treated skin/skin tumor biopsies were analyzed by Illumina Sentrix BeadChip Array (Mouse WG6 version 2) in duplicate. Total gene expression was analyzed. Most variable genes were selected. Rows represent individual genes; columns represent individual samples. Pseudo-colors indicate transcript levels below, equal to, or above the mean (green, black, and red, respectively). Gene expression; fold change loglO scale ranges between -5 to 5. No cytokines were observed in the skin of STING-deficient animals.
  • Methods and compositions for modulating an immune response in a subject e.g., a human being, dog, cat, horse, cow, goat, pig, etc.
  • a pharmaceutical composition including an agent which modulates STING function and a pharmaceutically acceptable carrier, wherein amount the pharmaceutical composition is effective to ameliorate the aberrant STING function in the subject.
  • Diseases or disorders associated with aberrant STING function can be any where cells having defective STING function or expression cause or exacerbate the physical symtoms of the disease or disorder.
  • diseases or disorders are mediated by immune system cells, e.g., an inflammatory condition, an autoimmune condition, cancer (e.g., breast, colorectal, prostate, ovarian, leukemia, lung, endometrial, or liver cancer), atherosclerosis, arthritis (e.g., osteoarthritis or rheumatoid arthritis), an inflammatory bowel disease (e.g., ulcerative colitis or Crohn's disease), a peripheral vascular disease, a cerebral vascular accident (stroke), one where chronic inflammation is present, one characterized by lesions having inflammatory cell infiltration, one where amyloid plaques are present in the brain (e.g., Alzheimer's disease), Aicardi-Goutieres syndrome, juvenile arthritis, osteoporosis, amyotrophic lateral sclerosis, or multiple s
  • inflammatory condition
  • the agent can be a small molecule (i.e., an organic or inorganic molecule having a molecular weight less than 500, 1000, or 2000 daltons) that increases or decreases STING function or expression or a nucleic acid molecule that binds to STING under intracellular conditions (i.e., under conditions inside a cell where STING is normally located).
  • the agent can also be a STING-binding nucleic acid molecule which can be a single-stranded (ss) or double- stranded (ds) RNA or DNA.
  • the nucleic acid is between 40 and 150, 60 and 120, 80 and 100, or 85 and 95 base pairs in length or longer.
  • the STING-binding nucleic acid molecule can be nuclease-resistant, e.g., made up of nuclease-resistant nucleotides or in cyclic dinucleotide form. It can also be associated with a molecule that facilitates transmembrane transport.
  • Methods and compositions for treating cancer in a subject having a cancerous tumor infiltrated with inflammatory immune cells involve a pharmaceutical composition including an agent which downregulates STING function or expression and a pharmaceutically acceptable carrier, wherein amount the pharmaceutical composition is effective to reduce the number of inflammatory immune cells infiltrating the cancerous tumor by at least 50% (e.g., at least 50, 60, 70, 80, or 90%, or until reduction of inflammatory cell infilitration is detectably reduced by histology or scanning).
  • compositions described herein might be included along with one or more pharmaceutically acceptable carriers or excipients to make pharmaceutical compositions which can be administered by a variety of routes including oral, rectal, vaginal, topical, transdermal, subcutaneous, intravenous, intramuscular, insufflation, intrathecal, and intranasal administration.
  • routes including oral, rectal, vaginal, topical, transdermal, subcutaneous, intravenous, intramuscular, insufflation, intrathecal, and intranasal administration.
  • Suitable formulations for use in the present invention are found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th ed. (1985).
  • the active ingredient(s) can be mixed with an excipient, diluted by an excipient, and/or enclosed within a carrier which can be in the form of a capsule, sachet, paper or other container.
  • a carrier which can be in the form of a capsule, sachet, paper or other container.
  • the excipient serves as a diluent, it can be a solid, semi-solid, or liquid material, which acts as a vehicle, carrier or medium for the active ingredient.
  • compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments, soft and hard gelatin capsules, suppositories, sterile injectable solutions, sterile liquids for intranasal administration (e.g., a spraying device), or sterile packaged powders.
  • the formulations can additionally include: lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preserving agents such as methyl- and propylhydroxy-benzoates; sweetening agents; and flavoring agents.
  • lubricating agents such as talc, magnesium stearate, and mineral oil
  • wetting agents such as talc, magnesium stearate, and mineral oil
  • emulsifying and suspending agents such as methyl- and propylhydroxy-benzoates
  • sweetening agents and flavoring agents.
  • the compositions of the invention can be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the patient by employing procedures known in the art.
  • the composition can be mixed with a pharmaceutical excipient to form a solid preformulation composition containing a homogeneous mixture of a compound.
  • Tablets or pills may be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action.
  • the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former.
  • the two components can be separated by an enteric layer which serves to resist disintegration in the stomach and permit the inner component to pass intact into the duodenum or to be delayed in release.
  • enteric layers or coatings such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate.
  • Liquid forms of the formulations include suspensions and emulsions.
  • the formulations may be encapsulated, introduced into the lumen of liposomes, prepared as a colloid, or incorporated in the layers of liposomes.
  • a variety of methods are available for preparing liposomes, as described in, e.g., Szoka, et al., U.S. Pat. Nos. 4,235,871, 4,501,728 and 4,837,028 each of which is incorporated herein by reference.
  • compositions are preferably formulated in a unit dosage form of the active ingredient(s).
  • the amount administered to the patient will vary depending upon what is being administered, the purpose of the administration, such as prophylaxis or therapy, the state of the patient, the manner of administration, and the like all of which are within the skill of qualified physicians and pharmacists.
  • compositions are administered to a patient already suffering from a disease in an amount sufficient to cure or at least partially arrest the symptoms of the disease and its complications. Amounts effective for this use will depend on the disease condition being treated as well as by the judgment of the attending clinician depending upon factors such as the severity of the symptoms, the age, weight and general condition of the patient, and the like.
  • STING Stimulator of Interferon Genes
  • dsDNA of approximately 90 base pairs (referred to herein as interferon stimulatory dsDNA90) were required to fully activate type I IFN.
  • STING is indeed essential for the production of type I IFN in hTERTs (Fig. IB).
  • cytoplasmic dsDNA can induce a wide array of innate immune genes, in addition to type I IFN, in hTERT cells (Figs. 5A-G).
  • cytoplasmic dsDNA induced a variety of innate immune genes in a STING-dependent manner was confirmed using STING + + or _ ⁇ murine embryonic fibroblasts (MEFs) (Fig. 1C).
  • ssDNA90 transfected ssDNA comprising 90 nucleotides
  • ssDNA90 transfected ssDNA comprising 90 nucleotides
  • Figs. ID, IE type I IFN in hTERT cells
  • cytoplasmic ssDNA or dsDNA which includes transfected plasmid DNA, can potently induce the transcription of a wide array of innate immune related genes that is dependent on STING.
  • RNAi knockdown of STING in hTERT cells eliminated the observed binding and STING-DNA complexes were also only observed in wild type MEFs ( + + ) but not MEFs lacking STING ( _ ⁇ ) (Figs. 12A-G). It was similarly confirmed that HSV1, cytomegalovirus (CMV) as well as adenovirus (ADV) related dsDNA Competition experiments suggested that STING also could bind to ssDNA (ssDNA90) as well as dsDNA, but not dsRNA (Fig. 2F). This was confirmed by expressing STING in vitro and observing association with ssDNA90 (Fig. 2G).
  • CMV cytomegalovirus
  • ADV adenovirus
  • dsDNA was also transfected into hTERT or MEFs cells and treated with formaldehyde to cross-link cellular proteins to the nucleic acid. Subsequent CHIP analysis following STING pull down, further confirmed that transfected DNA can directly associate with STING as determined using dsDNA90 specific primers (Figs. 21 and 2J). It was observed that STING could bind to biotin-labeled DNA in ELISA assays (Figs. 14A-C). The data indicated that ssDNA and dsDNA-mediated innate signaling events were dependent on STING and evidence that STING itself was able to complex to these nucleic acid structures to help trigger these events.
  • TREX1 a 3'->5' DNA exonuclease is also an ER associated molecule, and important for degrading checkpoint activated ssDNA species that could otherwise activate the immune system.
  • RNAi used to silence TREX1 in hTERT cells significantly increased STING-dependent, production of type I IFN by dsDNA90 (Figs. 3A and 3B).
  • the replication of the dsDNA virus HSV1 was greatly reduced in hTERT cells lacking TREX1, likely due to the elevated production of type I IFN and antiviral IFN stimulated genes (ISO's) (Fig 3C and D).
  • Luciferase expression from a recombinant HSV- expressing the luciferase gene was also significantly lower in hTERT infected cells treated with RNAi to silence TREX1 (Figs. 15A-D). These observations were extended by using TREX1 deficient MEFs, which similarly indicated that cytoplasmic dsDNA-dependent gene induction was greatly elevated in the absence of TREX1 and that HSV1 replication was significantly reduced (Figs. 3E-G).
  • STING was silenced in TREX1 lacking hTERTs or TREX1 _ ⁇ MEFs and treated these cells with cytoplasmic dsDNA or HSV1.
  • RNAi knockdown of STING in TREX _ ⁇ MEFs also eliminated ssDNA90- mediated type I IFN production and innate gene stimulation (Fig. 16).
  • dsDNA species complex with STING and accessory molecules to mediate trafficking and downstream signaling events that activate the transcription factors IRF3/7 and NF-KB, responsible for the induction of primary innate immune genes including TREX1.
  • STING-activated TREX1 resides in the ER region to degrade activator dsDNA and repress cytoplasmic dsDNA signaling in a negative-feedback manner.
  • TREX1 is a negative regulator of STING.
  • the translocon complex includes Sec61 ⁇ ⁇ and ⁇ coupled with TRAP ⁇ ⁇ , ⁇ and ⁇ , which can attach to ribosomes.
  • Secretory and membrane proteins are translocated into the ER for proper folding and glycosylation prior to being exported.
  • full length TREX1 was used as bait in a two hybrid yeast screen.
  • TREX1 recurrently interacted with a protein referred to as Ribophorin I (RPN1), a 68 kDa type I transmembrane protein and member of the oligosaccharyltransferase (OST) complex (Figs. 4A-E; Figs. 18A-D).
  • the OST complex catalyses the transfer of mannose oligosaccharides onto asparagines residues of nascent polypeptides as they enter the ER through the translocon.
  • At least seven proteins include the OST complex including RPN1, RPN2, OST48, OST4, STT3A/B, TUSC3 and DADl.
  • TREX1 and STING cofractionated with the ER markers RPN1 and RPN2, DADl and calreticulin, but not nuclear histone H3, confirming that their subcellular localization is indistinguishable from components of the translocon/OST complex (Fig. 41).
  • TREXl is targeted to the OST/translocon complex of the ER, that includes STING, and this association occurs through association with RPN1, although the TM region of TREXl was found to be involved in TREXl 's localization to the ER.
  • SRP signal recognition peptide
  • STING can complex with cytoplasmic intracellular ssDNA and dsDNA, which can include plasmid-based DNA and gene therapy vectors, can regulate the induction of a wide array of innate immune genes such as type I IFN, the IFIT family, and a variety of chemokines important for antiviral activity and for initiating adaptive immune responses.
  • STING activation facilitates the escort of TBK1 to clathrin covered endosomal compartments plausibly to activate IRF3/7 by mechanisms that remain to be fully clarified.
  • TREXl appears present in low levels in the cell and is itself inducible by STING.
  • TREXl localizes to the OST complex in close proximity to unactivated STING (which also resides in the OST/translocon complex) where presumably it degrades DNA species that can otherwise provoke STING action.
  • Components of the translocon/OST complex which now involve STING and TREXl, regulate cytoplasmic ssDNA and dsDNA- mediated innate immune signaling. Since loss of TREXl manifests autoimmune disorders through elevated type I IFN production, it is possible that these diseases are induced through STING activity.
  • Example 2 STING Modulators [0053] Drug libraries were screened to identify agents that modulate STING expression, function, activity, etc.
  • Fig. 24 shows the steps of a STING cell based assay.
  • EMD InhibitorSelectTM 96-Well
  • Doxazosin mesylate 1 (4-amino-6,7-dimethoxy-2- alphal adrenoceptor blocker quinazolinyl)-4-[4-(1 ,4-benzodioxan- 2-yl)carpiperazin-1 -yl)]-6,7- dimethoxyquinazoline mesylate
  • alkaloid isolated from the bark of the Cinchona family of South American trees
  • (+)-Quisqualic acid L(+)-alpha-Amino-3,5-dioxo-1 ,2,4- Active enantiomer of oxadiazolidine-2-propanoic acid quisqualic acid; excitatory amino acid at glutamate receptors; anthelmentic agent
  • Activators of STING included dihydroouabain and BNTX maleate salt hydrate.
  • Example 3 STING manifests Self DNA-Dependent Inflammatory Disease
  • BMDM Bone marrow derived macrophages
  • dsDNA90 90 base pair dsDNA
  • aDNA apoptotic DNA
  • Dex dexamethasone
  • DNA microarray experiments confirmed that aDNA triggered STING-dependent production of a wide array of innate immune and inflammatory related cytokines in BMDM such as IFN as well as TNFa (Table 3).
  • Table 3 shows the gene expression of higher expressed genes in BMDM treated with apoptic DNA (aDNA). ' fiis
  • DNase IT 1' embryos exhibited anemia, as described above, which was in significant contrast to Sting '1' DNase IT 1' DKO embryos or controls which noticeably lacked this phenotype.
  • Lethal anemia has been reported to be due to type I IFN inhibition of erythropoiesis during development. It was subsequently observed by hematoxylin and eosin staining that the livers of DNase IT 1' embryos contained numerous infiltrating macrophages full of engulfed apoptotic cells responsible for producing high levels of cytokines. In contrast to control mice, the livers of Sting '1' DNase IT 1' embryos exhibited a similar phenotype.
  • Sting " ' " DNase II " ' " macrophages similar to DNase II " ' “ macrophages, were not able to digest the engulfed nuclei from dexamethasone treated apoptotic thymocytes compared to control macrophages taken from wild type or Sting " ' " mice.
  • macrophages harvested from the livers of Sting " ' “ DNase II " ' “ embryonic mice similarly exhibit an inability to digest engulfed apoptotic cells, analogous to DNase II " ' " macrophages.
  • IFN interferon- stimulated genes
  • OAS 2'-5' oligoadenylate synthetases
  • IFITs interferon-induced proteins with tetratricopeptide repeats
  • IFI27 interferon-inducible protein 27
  • ISG15 ubiquitin-like modifier
  • Sting 1' DNase ⁇ macrophages similar to DNase ⁇ macrophages, were not able to digest the engulfed nuclei from apoptotic thymocytes (Dex + ) compared to control macrophages taken from wild type or Sting " ' " mice.
  • the accumulation of undigested DNA in DNase IT _ Sting " ' " macrophages was less pronounced when WT thymocytes were used as targets (Dex ).
  • BMDM derived from Sting 1' DNase II mice are also incapable of digesting DNA from apoptotic cells, although in contrast to DNase II BMDM do not produce inflammatory cytokine responses.
  • DNase II mediated embryonic lethality can be avoided by crossing DNase if ' mice with type I IFN defective Ifnarl " mice, the resultant progeny suffer from severe polyarthritis approximately 8 weeks after birth (arthritis score of 2) since undigested DNA activates innate immune signaling pathways and triggers the production of inflammatory cytokines such as TNFa.
  • Sting " ' " DNase IT _ mice did not manifest any signs of polyarthritis following birth. Arthritis scores remained at approximately zero (no score) in the Sting A DNase ⁇ , up to 12 months of age in contrast to reported DNase ⁇ IfnarV A mice which exhibited an arthritis score of up to 7 after a similar period.
  • STING is responsible for inflammatory disease such as for example, Aicardi- Goutieres syndrome (AGS).
  • AGS is genetically determined encephalopathy and is characterized by calcification of basal ganglia and white matter, demyelination. High levels of lymphocytes and type I IFN in cerebrospinal fluid. The features mimic chronic infection. Serum levels of type I IFN are also raised in autoimmune syndrome systemic lupus erythromatosis (SLE).
  • AGS is caused by mutations in 3 '-5' DNA exonuclease TREX1. Loss of TREX1 function- DNA species accumulates in the ER of cells and activates cytoplasmic DNA sensors (STING). TREXl digests this DNA source (housekeeping function) to prevent innate immune gene activation.
  • mice defective in apoptosis it was next evaluated whether other types of self-DNA triggered disease occurred through activation of the STING pathway.
  • patients defective in the 3 ' repair exonuclease 1 suffer from Aicardi-Goutieres Syndrome (AGS) which instigates lethal encephalitis characterized by high levels of type I IFN production being present in the cerebrospinal fluid.
  • AGS Aicardi-Goutieres Syndrome
  • Trexl -deficient mice exhibit a median life span of approximately 10 weeks since as yet uncharacterized self-DNA, presumably normally digested by Trexl, activates intracellular DNA sensors which triggers cytokine production and causes lethal inflammatory aggravated myocarditis.
  • Example 5 STING is responsible for inflammation-associated cancer
  • STING WT and STING _ animals were treated with DNA damaging agents and mice lacking STING were resistant to tumor formation. This is because infiltrating immune cells such as dendritic cells, macrophages etc eat the damaged cells that have undergone necrosis or apoptosis and the DNA or other ligands from such cells activate STING and the production of cytokines that promote tumor formation. STING may be involved in facilitating tumor progression in a wide variety of other cancers.
  • Figs. 29A-D show that STING deficient mice are resistant to DMBA induced inflammation and skin oncogenesis: STING + + and STING " ' " mice were either mocked treated with acetone or treated with 10 ⁇ g of DMBA on the shaved dorsal weekly for 20 weeks.
  • Fig. 29A STING deficient animals are resistant to DNA-damaging agants that cause skin cancer. Percentages of skin tumor-free mice were shown in the Kaplan-Meier curve.
  • Fig. 29B Pictures of representative mice of each treatment groups were shown.
  • Fig. 29C Histopathological examinations were performed by H&E staining on mock or DMBA treated skin/skin tumor biopsies.
  • Fig. 29D Cytokine upregulation in STING expressing mice exposed to carcinogens.
  • RNAs extracted from mock or DMBA treated skin/skin tumor biopsies were analyzed by Illumina Sentrix BeadChip Array (Mouse WG6 version 2) in duplicate. Total gene expression was analyzed. Most variable genes were selected. Rows represent individual genes; columns represent individual samples. Pseudo-colors indicate transcript levels below, equal to, or above the mean. Gene expression; fold change loglO scale ranges between -5 to 5. No cytokines were observed in the skin of STING-deficient animals.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Medicinal Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Organic Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Engineering & Computer Science (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Genetics & Genomics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Immunology (AREA)
  • Epidemiology (AREA)
  • Biotechnology (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Communicable Diseases (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Oncology (AREA)
  • Pain & Pain Management (AREA)
  • Rheumatology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne des modulateurs de STING qui sont aptes à réguler à la hausse ou à la baisse des réponses immunitaires. L'administration de tels modulateurs peut être utilisée pour traiter des maladies ou d'autres états indésirables chez un sujet de façon directe ou en combinaison avec d'autres agents.
PCT/US2013/038840 2008-08-04 2013-04-30 Modulation de réponses immunitaires WO2013166000A1 (fr)

Priority Applications (9)

Application Number Priority Date Filing Date Title
AU2013256468A AU2013256468A1 (en) 2012-04-30 2013-04-30 Modulating immune responses
KR20147033632A KR20150004416A (ko) 2012-04-30 2013-04-30 면역 반응의 조절
EP13784572.3A EP2844756A4 (fr) 2012-04-30 2013-04-30 Modulation de réponses immunitaires
CN201380029115.7A CN104540945A (zh) 2012-04-30 2013-04-30 调节免疫应答
US15/120,694 US20170037400A1 (en) 2008-08-04 2013-04-30 Modulating immune responses
JP2015510379A JP2015516989A (ja) 2012-04-30 2013-04-30 免疫反応の変調
CA2907616A CA2907616A1 (fr) 2012-04-30 2013-04-30 Modulation de reponses immunitaires
US16/717,325 US20200392492A1 (en) 2008-08-04 2019-12-17 Modulating Immune Responses
US17/357,822 US20210324023A1 (en) 2008-08-04 2021-06-24 Modulating immune responses

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US13/460,408 2012-04-30
US13/460,408 US20130039933A1 (en) 2008-08-04 2012-04-30 Sting (stimulator of interferon genes), a regulator of innate immune responses

Related Parent Applications (3)

Application Number Title Priority Date Filing Date
US13/057,662 Continuation-In-Part US20110262485A1 (en) 2008-08-04 2009-08-04 Sting (stimulator of interferon genes), a regulator of innate immune responses
PCT/US2009/052767 Continuation-In-Part WO2010017248A2 (fr) 2008-08-04 2009-08-04 Sting (stimulateur de gènes d’interféron), un régulateur de réponses immunitaires innées
US13/460,408 Continuation-In-Part US20130039933A1 (en) 2008-08-04 2012-04-30 Sting (stimulator of interferon genes), a regulator of innate immune responses

Related Child Applications (2)

Application Number Title Priority Date Filing Date
US15/120,694 A-371-Of-International US20170037400A1 (en) 2008-08-04 2013-04-30 Modulating immune responses
US16/717,325 Continuation US20200392492A1 (en) 2008-08-04 2019-12-17 Modulating Immune Responses

Publications (1)

Publication Number Publication Date
WO2013166000A1 true WO2013166000A1 (fr) 2013-11-07

Family

ID=49514802

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2013/038840 WO2013166000A1 (fr) 2008-08-04 2013-04-30 Modulation de réponses immunitaires

Country Status (7)

Country Link
EP (1) EP2844756A4 (fr)
JP (1) JP2015516989A (fr)
KR (1) KR20150004416A (fr)
CN (1) CN104540945A (fr)
AU (1) AU2013256468A1 (fr)
CA (1) CA2907616A1 (fr)
WO (1) WO2013166000A1 (fr)

Cited By (28)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9549944B2 (en) 2013-05-18 2017-01-24 Aduro Biotech, Inc. Compositions and methods for inhibiting “stimulator of interferon gene”—dependent signalling
WO2017093933A1 (fr) 2015-12-03 2017-06-08 Glaxosmithkline Intellectual Property Development Limited Dinucléotides cycliques de purine à titre de modulateurs du sting
US9695212B2 (en) 2012-12-13 2017-07-04 Aduro Biotech, Inc. Compositions comprising cyclic purine dinucleotides having defined stereochemistries and methods for their preparation and use
US9724408B2 (en) 2013-05-18 2017-08-08 Aduro Biotech, Inc. Compositions and methods for activating stimulator of interferon gene-dependent signalling
US9770467B2 (en) 2012-06-08 2017-09-26 Aduro Biotech, Inc. Compositions and methods for cancer immunotherapy
WO2017175147A1 (fr) 2016-04-07 2017-10-12 Glaxosmithkline Intellectual Property Development Limited Amides hétérocycliques utiles en tant que modulateurs de protéine
WO2017175156A1 (fr) 2016-04-07 2017-10-12 Glaxosmithkline Intellectual Property Development Limited Amides hétérocycliques utiles en tant que modulateurs de protéine
US9840533B2 (en) 2013-04-29 2017-12-12 Memorial Sloan Kettering Cancer Center Compositions and methods for altering second messenger signaling
US10047115B2 (en) 2015-01-29 2018-08-14 Glaxosmithkline Intellectual Property Development Limited Cyclic dinucleotides useful for the treatment of inter alia cancer
US10176292B2 (en) 2013-07-31 2019-01-08 Memorial Sloan-Kettering Cancer Center STING crystals and modulators
WO2019069269A1 (fr) 2017-10-05 2019-04-11 Glaxosmithkline Intellectual Property Development Limited Modulateurs de stimulateur des gènes (sting) d'interféron utiles dans le traitement du vih
WO2019069270A1 (fr) 2017-10-05 2019-04-11 Glaxosmithkline Intellectual Property Development Limited Modulateurs de stimulateur des gènes (sting) de l'interféron
WO2019069275A1 (fr) 2017-10-05 2019-04-11 Glaxosmithkline Intellectual Property Development Limited Méthodes d'administration d'agonistes de sting
US10336786B2 (en) 2012-12-19 2019-07-02 Board Of Regents, The University Of Texas System Pharmaceutical targeting of a mammalian cyclic di-nucleotide signaling pathway
US10450341B2 (en) 2014-06-04 2019-10-22 Glaxosmithkline Intellectual Property Development Limited Cyclic di-nucleotides as modulators of STING
US10449211B2 (en) 2015-03-10 2019-10-22 Aduro Biotech, Inc. Compositions and methods for activating “stimulator of interferon gene”—dependent signalling
WO2019219820A1 (fr) 2018-05-16 2019-11-21 Ctxt Pty Limited Thiophènes condensés substitués utilisés en tant que modulateurs de sting
US10519188B2 (en) 2016-03-18 2019-12-31 Immunesensor Therapeutics, Inc. Cyclic di-nucleotide compounds and methods of use
WO2020006432A1 (fr) 2018-06-28 2020-01-02 Eternity Bioscience Inc. Composés hétérocycliques tricycliques fusionnés et leurs utilisations thérapeutiques
WO2020202091A1 (fr) 2019-04-05 2020-10-08 Glaxosmithkline Intellectual Property Development Limited Composés chimiques
US10881730B2 (en) 2017-02-01 2021-01-05 Modernatx, Inc. Immunomodulatory therapeutic MRNA compositions encoding activating oncogene mutation peptides
WO2021009365A1 (fr) 2019-07-18 2021-01-21 Ctxt Pty Limited Dérivés de benzothiophène, de thiénopyridine et de thiénopyrimidine pour la modulation de sting
WO2021009362A1 (fr) 2019-07-18 2021-01-21 Ctxt Pty Limited Dérivés de benzothiophène, de thiénopyridine et de thiénopyrimidine permettant la modulation d'une piqûre
WO2021119753A1 (fr) 2019-12-18 2021-06-24 Ctxt Pty Limited Composés
US11098077B2 (en) 2016-07-05 2021-08-24 Chinook Therapeutics, Inc. Locked nucleic acid cyclic dinucleotide compounds and uses thereof
US11873319B2 (en) 2013-05-03 2024-01-16 The Regents Of The University Of California Cyclic di-nucleotide induction of type I interferon
US11897888B1 (en) 2020-04-30 2024-02-13 Stinginn Llc Small molecular inhibitors of sting signaling compositions and methods of use
US12134605B2 (en) 2019-12-18 2024-11-05 Stinginn, Llc Substituted 1,2,4-triazoles and methods of use

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106540260A (zh) * 2015-12-09 2017-03-29 聊城市奥润生物医药科技有限公司 干扰素基因刺激蛋白（sting）激动剂在抗阿尔兹海默症中的应用
CN106539757A (zh) * 2016-03-20 2017-03-29 聊城市奥润生物医药科技有限公司 环二核苷酸cGAMP-脂质体在抗肿瘤中的应用
CN106667914B (zh) * 2017-03-13 2022-02-01 杭州星鳌生物科技有限公司 靶向脂质体-环二核苷酸的组成、制备方法及其在抗肿瘤中的应用

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110262485A1 (en) * 2008-08-04 2011-10-27 University Of Miami Sting (stimulator of interferon genes), a regulator of innate immune responses

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110262485A1 (en) * 2008-08-04 2011-10-27 University Of Miami Sting (stimulator of interferon genes), a regulator of innate immune responses

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
GLEN N BARBER.: "Innate immune DNA sensing pathways: STING, AIMII and the regulation of interferon production and inflammatory responses.", CURRENT OPINION IN IMMUNOLOGY, vol. 23, 2011, pages 10 - 20, XP028371143 *
HIROKI ISHIKAWA ET AL.: "The STING pathway and regulation of innate immune signaling in response to DNA pathogens.", CELL. MOL. LIFE SCI., vol. 68, 2011, pages 1157 - 1165, XP019890447 *
See also references of EP2844756A4 *

Cited By (50)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9770467B2 (en) 2012-06-08 2017-09-26 Aduro Biotech, Inc. Compositions and methods for cancer immunotherapy
US10414789B2 (en) 2012-12-13 2019-09-17 Aduro Biotech, Inc. Compositions comprising cyclic purine dinucleotides having defined stereochemistries and methods for their preparation and use
US9695212B2 (en) 2012-12-13 2017-07-04 Aduro Biotech, Inc. Compositions comprising cyclic purine dinucleotides having defined stereochemistries and methods for their preparation and use
US10508129B2 (en) 2012-12-19 2019-12-17 The Board Of Regents Of The University Of Texas System Pharmaceutical targeting of a mammalian cyclic di-nucleotide signaling pathway
US10633411B2 (en) 2012-12-19 2020-04-28 The Board Of Regents Of The University Of Texas System Pharmaceutical targeting of a mammalian cyclic di-nucleotide signaling pathway
US11492368B2 (en) 2012-12-19 2022-11-08 The Board Of Regents Of The University Of Texas System Pharmaceutical targeting of a mammalian cyclic di-nucleotide signaling pathway
US10336786B2 (en) 2012-12-19 2019-07-02 Board Of Regents, The University Of Texas System Pharmaceutical targeting of a mammalian cyclic di-nucleotide signaling pathway
US10696710B2 (en) 2012-12-19 2020-06-30 The Board Of Regents Of The University Of Texas System Pharmaceutical targeting of a mammalian cyclic di-nucleotide signaling pathway
US10131686B2 (en) 2013-04-29 2018-11-20 Memorial Sloan Kettering Cancer Center Compositions and methods for altering second messenger signaling
US9840533B2 (en) 2013-04-29 2017-12-12 Memorial Sloan Kettering Cancer Center Compositions and methods for altering second messenger signaling
US10385091B2 (en) 2013-04-29 2019-08-20 Memorial Sloan Kettering Cancer Center Compositions and methods for altering second messenger signaling
US11014956B2 (en) 2013-04-29 2021-05-25 Memorial Sloan Kettering Cancer Center; The Rockefeller Compositions and methods for altering second messenger signaling
US11873319B2 (en) 2013-05-03 2024-01-16 The Regents Of The University Of California Cyclic di-nucleotide induction of type I interferon
US10653774B2 (en) 2013-05-18 2020-05-19 Aduro Biotech, Inc. Compositions and methods for activating “stimulator of interferon gene”-dependent signalling
US9724408B2 (en) 2013-05-18 2017-08-08 Aduro Biotech, Inc. Compositions and methods for activating stimulator of interferon gene-dependent signalling
US10189873B2 (en) 2013-05-18 2019-01-29 Aduro Biotech, Inc. Compositions and methods for inhibiting “stimulator of interferon gene”-dependent signalling
US9549944B2 (en) 2013-05-18 2017-01-24 Aduro Biotech, Inc. Compositions and methods for inhibiting “stimulator of interferon gene”—dependent signalling
US10176292B2 (en) 2013-07-31 2019-01-08 Memorial Sloan-Kettering Cancer Center STING crystals and modulators
US10450341B2 (en) 2014-06-04 2019-10-22 Glaxosmithkline Intellectual Property Development Limited Cyclic di-nucleotides as modulators of STING
US10047115B2 (en) 2015-01-29 2018-08-14 Glaxosmithkline Intellectual Property Development Limited Cyclic dinucleotides useful for the treatment of inter alia cancer
US10449211B2 (en) 2015-03-10 2019-10-22 Aduro Biotech, Inc. Compositions and methods for activating “stimulator of interferon gene”—dependent signalling
US11040053B2 (en) 2015-03-10 2021-06-22 Chinook Therapeutics, Inc. Compositions and methods for activating “stimulator of interferon gene”13 dependent signalling
US10730907B2 (en) 2015-12-03 2020-08-04 Glaxosmithkline Intellectual Property Development Limited Compounds
WO2017093933A1 (fr) 2015-12-03 2017-06-08 Glaxosmithkline Intellectual Property Development Limited Dinucléotides cycliques de purine à titre de modulateurs du sting
US9718848B2 (en) 2015-12-03 2017-08-01 Glaxosmithkline Intellectual Property Development Limited Compounds
US10364266B2 (en) 2015-12-03 2019-07-30 Glaxosmithkline Intellectual Property Development Limited Compounds
EP3366691A1 (fr) 2015-12-03 2018-08-29 GlaxoSmithKline Intellectual Property Development Limited Dinucléotides cycliques de purine utilisés comme modulateurs de sting
US9994607B2 (en) 2015-12-03 2018-06-12 Glaxosmithkline Intellectual Property Development Limited Compounds
US11299512B2 (en) 2016-03-18 2022-04-12 Immunesensor Therapeutics, Inc. Cyclic di-nucleotide compounds and methods of use
US10519188B2 (en) 2016-03-18 2019-12-31 Immunesensor Therapeutics, Inc. Cyclic di-nucleotide compounds and methods of use
EP4032885A1 (fr) 2016-04-07 2022-07-27 GlaxoSmithKline Intellectual Property Development Limited Amides hétérocycliques utiles en tant que modulateurs de protéine
US11970480B2 (en) 2016-04-07 2024-04-30 Glaxosmithkline Intellectual Property Development Limited Heterocyclic amides useful as protein modulators
WO2017175156A1 (fr) 2016-04-07 2017-10-12 Glaxosmithkline Intellectual Property Development Limited Amides hétérocycliques utiles en tant que modulateurs de protéine
US11365190B2 (en) 2016-04-07 2022-06-21 Glaxosmithkline Intellectual Property Development Limited Heterocyclic amides useful as protein modulators
WO2017175147A1 (fr) 2016-04-07 2017-10-12 Glaxosmithkline Intellectual Property Development Limited Amides hétérocycliques utiles en tant que modulateurs de protéine
US11098077B2 (en) 2016-07-05 2021-08-24 Chinook Therapeutics, Inc. Locked nucleic acid cyclic dinucleotide compounds and uses thereof
US10881730B2 (en) 2017-02-01 2021-01-05 Modernatx, Inc. Immunomodulatory therapeutic MRNA compositions encoding activating oncogene mutation peptides
WO2019069270A1 (fr) 2017-10-05 2019-04-11 Glaxosmithkline Intellectual Property Development Limited Modulateurs de stimulateur des gènes (sting) de l'interféron
WO2019069269A1 (fr) 2017-10-05 2019-04-11 Glaxosmithkline Intellectual Property Development Limited Modulateurs de stimulateur des gènes (sting) d'interféron utiles dans le traitement du vih
WO2019069275A1 (fr) 2017-10-05 2019-04-11 Glaxosmithkline Intellectual Property Development Limited Méthodes d'administration d'agonistes de sting
WO2019219820A1 (fr) 2018-05-16 2019-11-21 Ctxt Pty Limited Thiophènes condensés substitués utilisés en tant que modulateurs de sting
US11613525B2 (en) 2018-05-16 2023-03-28 Ctxt Pty Limited Substituted condensed thiophenes as modulators of sting
WO2020006432A1 (fr) 2018-06-28 2020-01-02 Eternity Bioscience Inc. Composés hétérocycliques tricycliques fusionnés et leurs utilisations thérapeutiques
US11535633B2 (en) 2018-06-28 2022-12-27 Jiangsu Hengrui Medicine Co., Ltd. Fused tricyclic heterocycle compounds and therapeutic uses thereof
WO2020202091A1 (fr) 2019-04-05 2020-10-08 Glaxosmithkline Intellectual Property Development Limited Composés chimiques
WO2021009362A1 (fr) 2019-07-18 2021-01-21 Ctxt Pty Limited Dérivés de benzothiophène, de thiénopyridine et de thiénopyrimidine permettant la modulation d'une piqûre
WO2021009365A1 (fr) 2019-07-18 2021-01-21 Ctxt Pty Limited Dérivés de benzothiophène, de thiénopyridine et de thiénopyrimidine pour la modulation de sting
WO2021119753A1 (fr) 2019-12-18 2021-06-24 Ctxt Pty Limited Composés
US12134605B2 (en) 2019-12-18 2024-11-05 Stinginn, Llc Substituted 1,2,4-triazoles and methods of use
US11897888B1 (en) 2020-04-30 2024-02-13 Stinginn Llc Small molecular inhibitors of sting signaling compositions and methods of use

Also Published As

Publication number Publication date
EP2844756A4 (fr) 2016-02-24
KR20150004416A (ko) 2015-01-12
CA2907616A1 (fr) 2013-11-07
JP2015516989A (ja) 2015-06-18
AU2013256468A1 (en) 2014-12-04
CN104540945A (zh) 2015-04-22
EP2844756A1 (fr) 2015-03-11

Similar Documents

Publication Publication Date Title
US20200392492A1 (en) Modulating Immune Responses
WO2013166000A1 (fr) Modulation de réponses immunitaires
Li et al. SARS-CoV-2 ORF10 suppresses the antiviral innate immune response by degrading MAVS through mitophagy
JP2013500265A (ja) 抗ウイルス剤として用いられるｍＴＯＲキナーゼ阻害剤
US20150301067A1 (en) Methods and assays for facioscapulohumeral muscular dystrophy
Accardi et al. Epstein-Barr virus transforming protein LMP-1 alters B cells gene expression by promoting accumulation of the oncoprotein ΔNp73α
Lin et al. Influenza a virus NS1 resembles a TRAF3-interacting motif to target the RNA sensing-TRAF3-type I IFN axis and impair antiviral innate immunity
US9814741B2 (en) Methods relating to DNA-sensing pathway related conditions
Furrer et al. Absent in Melanoma 2 (AIM2) limits pro-inflammatory cytokine transcription in cardiomyocytes by inhibiting STAT1 phosphorylation
Cai et al. G3BP1 inhibition alleviates intracellular nucleic acid–induced autoimmune responses
Doan et al. Virofree, an herbal medicine-based formula, interrupts the viral infection of delta and omicron variants of SARS-CoV-2
Guo et al. Inhibition of histone deacetylase 1 suppresses pseudorabies virus infection through cGAS-STING antiviral innate immunity
Basu et al. Identification of age-specific gene regulators of La Crosse virus neuroinvasion and pathogenesis
US20220088127A1 (en) Targeting the bag family for the treatment of neurodegenerative disease
Cai et al. FOSL1 inhibits type I interferon responses to malaria and viral infections by blocking TBK1 and TRAF3/TRIF interactions. mBio 8: e02161-16
US20190015473A1 (en) Targeting the hdac2-sp3 complex to enhance synaptic funcation
CN119082287A (zh) 去乙酰化酶sirt2的应用及调控rna病毒感染的药物
Liu et al. Forkhead box O1-mediated ubiquitination suppresses RIG-I-mediated antiviral immune responses via its DNA binding domain
Sposito Mechanisms of airway inflammation in health and disease
CN117794584A (zh) 包含msh2或msh6抑制剂作为有效成分的用于预防或治疗癌症的组合物
KR20250136253A (ko) Dusp22 억제제를 포함하는 근감소증의 치료 또는 예방용 약학 조성물

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 13784572

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2015510379

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

WWE Wipo information: entry into national phase

Ref document number: 2013784572

Country of ref document: EP

ENP Entry into the national phase

Ref document number: 20147033632

Country of ref document: KR

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 2013256468

Country of ref document: AU

Date of ref document: 20130430

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 2907616

Country of ref document: CA

WWE Wipo information: entry into national phase

Ref document number: 15120694

Country of ref document: US