(12) Patent Application Publication (10) Pub. No.: US 2013/0039933 A1 BARBER (43) Pub

US 201300399.33A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2013/0039933 A1 BARBER (43) Pub. Date: Feb. 14, 2013

(54) STING (STIMULATOR OF INTERFERON Publication Classification GENES), A REGULATOR OF INNATE MMUNE RESPONSES (51) Int. Cl. A6139/00 (2006.01) (75) Inventor: GLEN N. BARBER, Palmetto Bay, FL A6IP3L/2 (2006.01) (US) A6IP37/06 (2006.01) A6IP3I/00 (2006.01) (73) Assignee: University of Miami, Miami, FL (US) CI2N5/071 (2010.01) A6IP37/04 (2006.01) (21) Appl. No.: 13/460,408 (52) U.S. Cl...... 424/185.1; 424/1841; 435/375 (22) Filed: Apr. 30, 2012 (57) ABSTRACT Related U.S. Application Data Novel molecules termed STING which include nucleic acids, (63) Continuation-in-part of application No. 13/057,662, polynucleotides, oligonucleotides, peptides, mutants, vari filed on Jun. 14, 2011, filed as application No. PCT/ ants and active fragments thereof, modulate innate and adap US2009/052767 on Aug. 4, 2009. tive immunity in a subject. STING compositions are useful (60) Provisional application No. 61/129.975, filed on Aug. for the treatment of an immune-related disorder, including 4, 2008. treating and preventing infection by modulating immunity. Patent Application Publication Feb. 14, 2013 Sheet 1 of 83 US 2013/00399.33 A1

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STING (STIMULATOR OF INTERFERON analysis of STING in HEK 293 cells. RNAi to STING (hST GENES), A REGULATOR OF INNATE ING) or control RNAi (NS) was used to confirm specificity of IMMUNE RESPONSES the antibody. FIG. 1E shows a confocal analysis of HEK 293 cells transfected with hSTING tagged at the carboxyl end RELATED APPLICATIONS with HA. Transfected cells were also analyzed using ER 0001. This application is a continuation in part of U.S. dsRed, Mitotracker or Golgi-dsRed. FIG.1F shows that frac application Ser. No. 13/057,662 filed Feb. 4, 2011 which is tionation experiments confirm that STING resides in the ER. the US National Stage Entry of PCT application no. PCT/ Control antibodies indicate accuracy of fractionation (Cal US2009/052767 filed Aug. 4, 2009 and claims priority under reticulin-ER, COX IV-mitochondria, beta actin-cytosol). 35 USC S 119 to U.S. Provisional Patent Application No. 0009 FIG. 2A: 293T cells were transfected with 50 ng 61/129.975 filed Aug. 4, 2008, the disclosures of which are p110-Luc plasmid and 10 ng pRL-TK normalization plasmid incorporated by reference in their entirety. with increasing amounts of (50 ng, 150 ng, 250 ng) human hSTING, murine mSTING or control ARIG-I. Luciferase FIELD OF THE INVENTION assays indicating IFNB promoteractivity were taken 36 hours post-transfection. 293T cells transfected as in (FIG. 2A) with 0002 Embodiments of the invention relate to composi either PRDIII-1-Luc (FIG. 2B), NF-kB-Luc (FIG. 2C) or tions and methods for modulating innate and adaptive immu ISRE-Luc (FIG. 2D) responsive plasmids were analyzed nity in a Subject and/or for the treatment of an immune-related similarly. FIG.2E: 293T cells were transfected with 250ng of disorder, cancer, autoimmunity, treating and preventing vector alone or STING, IPS-1 or TBK-1 expressing plasmids infections. for 24hrs and lysed cells analyzed by native gel electrophore sis and by Subsequent immunoblot using antibody to detect BACKGROUND IRF3 dimerization. FIG. 2F: MEF's were transfected with 0003 Cellular host defense responses to pathogen inva vector alone or hSTING or mSTING or IPS-1 expressing sion principally involves the detection of pathogen associated plasmid (500 ng) and mRNA retrieved after 24 hrs post molecular patterns (PAMPs) such as viral nucleic acid or transfection. IFNB mRNA was analyzed by qRT-PCR. FIG. bacterial cell wall components including lipopolysaccharide 2G: Medium from transfected MEFs was analyzed for IFNB or flagellar proteins that results in the induction of anti-patho protein by ELISA. FIG.2H:293T cells were transfected with gen genes. For example, viral RNA can be detected by mem 250 ng of control or hSTING expressing plasmid and mRNA brane bound Toll-like receptors (TLRs) present in the endo retrieved after 36 hrs for analysis by DNA microarray. FIG. plasmic reticulum (ER) and/or endosomes (e.g. TLR 3 and 2I: MEF's were transfected with vector alone or hSTING or 7/8) or by TLR-independent intracellular DExD/H box RNA mSTING or ARIG-I or IPS-1 expressing plasmid (500 ng) helicases referred to as retinoic acid inducible gene 1 (RIG-I) and after 36 hrs post-transfection were infected at an MOI of or melanoma differentiation associated antigen 5 (MDA5, 1 with VSV-GFP. FIG. 2J: Viral replication from experiment also referred to as IFIH1 and helicard). These events culmi (FIG.2H) was measured by plaque assay. FIG.2K: Normal or nate in the activation of downstream signaling events, much TBK-1 deficient MEFs were transfected with vector alone, of which remains unknown, leading to the transcription of hSTING, mSTING or TBK-1 expressing plasmids (500 ng) NF-kB and IRF3/7-dependent genes, including type IIFN. and 100 ng murine IFNB-Luc reporter plasmid with 10 ng PRL-TK for 24hrs and luciferase measured. FIG.2L: Normal SUMMARY or FADD MEFs were treated as in (FIG. 2K) and luciferase measured. FIG.2M. Schematic of hSTING variants. FIG.2N: 0004. This Summary is provided to present a summary of 293T cells were transfected as in (FIG. 2A) with hSTING the invention to briefly indicate the nature and substance of full-length or variants and luciferase measured. FIG. 20: the invention. It is submitted with the understanding that it 293T cells were transfected with 100 ng full length STING will not be used to interpret or limit the scope or meaning of and increasing amounts of hSTING-Full, hSTING-N or hST the claims. ING-C (0 ng, 150 ng, 250 ng) with luciferase plasmids as in 0005 STING molecules (Stimulator of Interferon Genes) (FIG. 2A). Luciferase was measured after 36 hrs. Asterisks modulate the immune system, in particular the innate immune indicate significant difference (P<0.05) as determined by Stu system. Compositions comprising STING and/or other dent's t-test. agents which modulate STING expression, activity and/or functions treat diseases Such as cancer, infections, autoim 0010 FIG.3A: MEFs (C57/BL6) were treated with RNAi mune diseases or disorders, inflammation and the like are to mSTING and knockdown confirmed after 72 hours by administered to patients at risk of developing or for the treat immunoblot using anti-STING rabbit antiserum. FIG. 3B: ment of patients afflicted with such diseases. Fluorescence microscopy (GFP) of MEF's treated with RNAi 0006 Embodiments of the invention are also directed to to mSTING following 24 hrs infection with VSV-GFP (MOI molecules and pathways which directly or indirectly interact 1). FIG. 3C: RNAi treated cells were infected with VSVGFP or associate with STING. (MOI 1) for 16 hrs and IFNB mRNA measured using quan titative RT-PCR. FIG. 3D: Viral titers taken from RNAi 0007. Other aspects of the invention are described infra. treated or untreated MEF's after 24 hours. FIG. 3E schematic diagram depicting targeted homologous recombination strat BRIEF DESCRIPTION OF THE DRAWINGS egy of STING in ES cells. FIG. 3F: quantitative RT-PCR 0008 FIG. 1A shows the amino acid sequence of human analysis of STING mRNA in STING or control litter mate and mouse STING (SEQID NOS: 1 and 2 respectively). FIG. MEFs. FIG. 3G: Immunoblot of STING cells or control 1B shows a schematic representation of hSTING indicating MEFs using antiserum as in (FIG. 3A). FIG. 3H: Fluores TM and leucine rich regions. FIG. 1C shows a Northern blot cence microscopy (GFP) of STING or control MEFs fol analysis of human STING. FIG. 1D shows an immunoblot lowing 12 hrs infection with VSV-GFP (MOI 0.1). FIG. 3I: US 2013/0039933 A1 Feb. 14, 2013

Viral titers taken from STING or control MEF's following as described in materials and methods. FIG. 4L: HEK 293 infection with VSV-GFP after 24 hours. FIG. 3J: Viral titers cells were transfected with HA-STING and GFPTBK-1 for taken from STING or control MEF's following infection with 36 hours. Co-immunoprecipitation experiments were done VSVAM after 24 hours. FIG. 3K: Endogenous IFNB levels using and HA-tagged antibody and immunoblot carried out measured from STING or control MEFs infected with using an antibody GFP. Asterisks indicate significant differ VSV-GFP (MOI 1) or Sendai Virus (SeV MOI 1) after 24 ence (P<0.05) as determined by Student's t-test. hours. FIG. 3L: STING MEF's or controls were treated (0012 FIGS. 5A-5D are graphs showing 293T cells were with transfected (Lipo 2000 3 ul/ml) poly dA-dT for 24 hrs co-transfected with STING or ARIG-I as in FIG. 2A using an and IFNB measured by ELISA. FIG.3M: Time course analy IFNB (p110), RB, E2F or p53 promoter driving luciferase sis of DNA transfected MEFs. FIG. 3N: BMDM were treated reporter. 36 hours later, luciferase activity was measured. with exogenous poly I:C (10 ug/ml) or LPS (10 ug/ml) or Asterisks indicate significant difference (P<0.05) as deter transfected poly dA-dT (as in FIG.3L. 10 ug/ml) and IFNB mined by Student's t-test. after 24 hours by ELISA. FIG.3O: GM-DC's were treated as 0013 FIG. 6A is a blot showing the confirmation of hST in FIG. 3N. Asterisks indicate significant difference (P<0.05) ING RNAi knockdown, as described in materials and meth as determined by Student's t-test. ods, in HEK 293 cells by immunoblot using anti-STING 0011 FIG. 4A: 293T cells were co-transfected with HA rabbit antiserum, after 72 hours. FIG. 6B: is a graph showing tagged STING and FLAG-tagged RIG-I or MDA5 for 24 RNAi treated cells, as in (FIG. 6A), which were infected for hours. Cells were infected with SeV (MOI 1) for 12 hours. 16 hrs with VSV-GFP (MOI 1) and IFNB mRNA measured Cells were lysed and co-immunoprecipitated with anti-FLAG using quantitative RT-PCR. Asterisks indicate significant dif antibody and after immunoblotting analyzed using antibody ference (P<0.05) as determined by Student's t-test. to HA. FIG. 4B: HUVECs were lysed and immunoprecipi 0014 FIG. 7A is a graph showing luciferase activity tated using an antibody to endogenous hSTING. Washed which was measured in STING or control MEF's following precipitates were immunoblotted using an antibody to endog infection with VSV-Luc after 24 hours. FIG. 7B is a graph enous RIG-I. FIG. 4C: 293T cells were co-transfected with showing the viral titers taken from STING or control MEFs HA-tagged STING and FLAG-tagged RIG-I, ARIG-I (aa1 following infection with VSV-Luc after 24 hours. FIG.7C is 284) or RIG-I-C (aa218-925) for 24 hours. Cells were lysed a scan of photographs showing that STING or control MEF's and co-immunoprecipitated with anti-FLAG antibody and were transfected with STING or STING carboxyl region after immunoblotting analyzed using antibody to HA. FIG. (aa173-378) for 24 hours. Cells were then infected with VSV 4D: 293T cells were co-transfected with control vector (-) or GFP (MOI0.1) for 24hours. Fluorescence was detected using increasing amounts of full-length, amino (aa1-230) or car microscopy as in FIG. 3B. FIG. 7D is an immunoblot con boxyl (aa173-379) STING (0, 150 ng and 250 ng) together firming expression of re-constituted STING using anti with 150 ng of ARIG-I. Luciferase was measured after 36 hrs. STING antibody. FIG.7E is a graph showing VSV-GFP titers FIG. 4E: 293T cells were co-transfected with HA-tagged of FIG.7C which were measured 24 hours post-infection by STING and FLAG-tagged RIG-I for 24 hours. Cells were plaque assay. fixed and stained with anti-FLAG and anti-HA antibody as 10015 FIG. 8A is a graph showing STING or control described in materials and methods. FIG. 4F: Control or MEFs which were transfected with poly I:C (1 lug/ml in Lipo STING MEFs were transfected with ARIG-I (aa1-284) or 2000-3 ul/ml). IFNB was measured using ELISA as described IPS-1 for 24 hours and endogenous IFNB measured by in materials and methods. FIG. 8B is a graph showing that ELISA. FIG. 4G: STING lacking the first 35 amino acids EMCV was used to inoculate STING or control MEF's. comprising the signal peptide (amino acids 36-369: BD-hST Virus replication was measured by plaque assay 24hours later INGASP) or the carboxyl region of STING (amino acids using BHK cells. 173-379; BD-hSTING-C) was fused to the binding domain of 0016 FIG. 9A is a graph showing HeLa cells which were yeast GAL.4 transcription factor and used to Screen a human treated with RNAi to STING for 72 hours. Quantitative-RT IFN-inducible fibroblast cDNA library fused to the activation PCR was used to confirm knockdown. FIG.9B is a graph domain of the yeast GAL.4 transcription factor as described in showing that after 72 hours of RNAi treatment, ISD (3 ug/ml materials and methods. BD-hSTING-C was found to interact with Lipo 2000 3 ul/ml) or poly dA-dT (3 ug/ml with Lipo with Ssr-2/TRAPB (AD-hTRAP3) but not control vector 20003 ul/ml) was used to treat cells for 6 hours. IFNBmRNA (AD) or negative control (AD vector containing). Efficiency production was measured using quantitative-RT-PCR. FIG. of yeast harboring both BD (-Trp) and AD (-Leu) plasmids is 9C showing: BMDM or GM-DC's from STING deficient shown on left panel (-Trp.-Leu) and high Stringency interac mice were analyzed by immunoblot using anti-STING anti tion on the right panel (-Trp. -Leu, -Ade -His). FIG.4H: HEK serum. FIG. 9D: is a graph showing BMDM from STING 293 cells were transfected with FLAG-tagged TRAPP (+) for deficient mice which were infected with VSV-GFP (MOI 1) 36 hours. Cell lysates were immunoprecipitated with anti for 24 hours. Virus replication was measured by plaque assay FLAG antibody and immunoblotted using antibody to endog as described in materials and methods. FIG. 9E is a graph enous STING or endogenous Sec61 B. FIG. 4I: HEK293 cells showing GM-DC's from STING deficient mice which were were transfected with FLAG-TRAPP and HA-STING for 36 infected with VSV-GFP (MOI 1) for 24 hours. Virus replica hours. Cells were lysed and immunoblotted using antibody to tion was measured by plaque assay as described in materials endogenous Sec61 B, or FLAG or HA. FIG. 4J: 293T cells and methods. Asterisks indicate significant difference (P<0. were treated for 48 hours with RNAi to TRAPB, SEC61 B or 05) as determined by Student's t-test. Sec5. Cells were then co-transfected with HA-STING and (0017 FIG. 10A is a photograph showing 293T cells which IFNB-Luc for 24 hours. Luciferase activity was measured as were co-transfected with FLAG-IPS-1 and HA-STING or described. FIG. 4K: 293T cells were co-transfected with HA control vector. After 24 hours, cells were fixed as described in tagged STING and FLAG-tagged TRAPB for 24 hours. Cells materials and methods and examined using confocal micros were fixed and stained with anti-FLAG and anti-HA antibody copy as described in materials and methods. FIG. 10B: is a US 2013/0039933 A1 Feb. 14, 2013

blot showing 293T cells which were co-transfected with IFN-Y production measured by ELISA. FIG. 15D: STING FLAG-IPS-1 and HA-STING or control vector (1 mg/ml). deficient animals () or controls ("") (n=4; approximately 8 After 24 hours, cells were lysed and immunoprecipitated weeks of age were infected with vaccinia expressing oval using HA-antibody. Washed immunoprecipitates were sepa bumin (VV-OVA; i.v., 5x10) and spleen anti-OVA-specific rated using SDS/PAGE and immunoblotted using anti-FLAG IFN-Y production measured by ELISA. Asterisks indicate antibody. significant difference (P<0.05) as determined by Students 0018 FIG. 11, is a graph showing RIG-I or IPS-1 deficient t-test. Error bars indicates.d. MEFs, or control MEFs which were co-transfected with 0023 FIGS. 16A-16I: STING translocates from the ER to mSTING (1 lug/ml) and IFNB-Luc (100 ng/ml) for 24 hours. Sec5 containing vesicles. FIG.16A: STING MEFs, stably Luciferase activity was measured as described in materials reconstituted with HA tagged STING (mSTING-HA) were and methods. stained using HA (green) and calreticulin (red) antibody. FIG. 0019 FIG. 12A is a graph showing 293T cells which were 16B: HA-STING MEFs was stained for HA-Sting (green), treated with RNAi to TRAPB or control RNAi for 72 hours. calreticulin (blue) or Mitotracker (red). FIG.16C: Immuno Confirmation of knockdown was carried out using quantita blot analysis of fractionation experiments of uninfected or tive-RT-PCR. FIG. 12B is a blot showing 293T cells were HSV infected (moi 10; 4 hours) HA-STING MEFs (HA for treated with RNAi to Sec61 B or control RNAi for 72 hours. STING, calreticulin, ER: Sigma 1 R, mitochondrial associ Confirmation of knockdown was carried out using an anti ated ER membranes MAMI; COXIV, mitochondria). FIG. body to Sec61B. FIG. 12C is a graph showing that after 72 16D: HA (green) or Calreticulin (red) staining of mSTING hours cell viability of the RNAi treated 293T cells was mea HA MEF's following treatment with transfected ISD (1 Sured using Trypan blue exclusion analysis. ug/ml), transfected single stranded DNA (ssDNA, 1 ug/ml) or 0020 FIGS. 13 A-13G show that STING is essential for HSV infection as in FIG.16C. FIG.16E: mSTING-HAMEFS intracellular DNA-mediated IFN production. FIG. 13A: were transfected with or without ISD and cells stained with Murine embryonic fibroblasts (MEFs) were transfected with HA (green) and TBK-1 (red) antibody. FIG. 16F: mSTING 1 g/ml of DNA ligands (with Lipofectamine 2000) for 16 HA MEFs were transfected as in FIG. 16E and stained with hours and IFNB or IL-6 measured. FIG. 13B: MEF's were HA (green) and Transferrin receptor (Tfr. red) antibody. FIG. transfected with ISD (interferon stimulatory DNA) for 4 16G: mSTING-HA MEFs were transfected as in FIG. 16E hours and Ifn or Ifna2 mRNA measured. FIG. 13C: MEFs and stained with HA (green) and Sec5 antibody (red). FIGS. treated as in FIG. 13B, was stained by antibody for IRF3 16H, 16I: MEF's were treated with RNAi to TRAPB or SEC5 translocation. FIG.13D: Bone-marrow derived macrophages for 72 hours and transfected with ISD. IFNs mRNA and were transfected with poly(dAT:dAT), poly(I:C) or ISD or protein was measured at 4 and 16 hours respectively. Aster infected with HSV (moi 10) or Listeria (moi10) for 16 hours isks indicate significant difference (P<0.05) as determined by and IFN3 measured. FIG. 13E: Macrophages was infected Student's t-test. Error bars indicates.d. with HSV-1 for 16 hours and IL-13 measured. FIG. 13F: (0024 FIG. 17: IL-6 production in the absence of STING is GM-CSF induced dendritic cells (GM-DC) treated as in FIG. defective in response to poly(dAT:dAT) and ISD, but not 13D, and IFNB or IFNC. measured after 16 hours. FIG. 13G: HSV-1 or CpG ODN. Macrophages were transfected with 1 Flt3 stimulated DC’s were treated as in FIG. 13F (exogenous ug/ml of DNA ligands or infected for 16 hours with HSV-1 CpG-ODN (1 lug/ml) was additionally used). Asterisks indi (moi 10). IL-6 was measured by ELISA. Asterisks indicate cate significant difference (P<0.05) as determined by Stu significant difference (P<0.05) as determined by Students dent's t-test. Error bars indicates.d. t-test. Error bars indicates.d. 0021 FIGS. 14A-14G show that STING is required for (0025 FIGS. 18A-18F: Defective type IIFN production in effective in vivo host defense. FIG. 14A. STING deficient viral-infected STING knockout mice. FIGS. 18A, 18B: animals () or litter mate controls ("") (n=7; approximately STING deficient animals (Sting) or litter mate controls 8 weeks of age) were infected intravenously (i.v.) with 1x107 ("") (n=3; approximately 8 weeks of age were infected intra HSV and survival monitored. FIG. 14B: Sting mice or con venously (i.v.) with 1x107 HSV and survival monitored. trols were infected with HSV-1 as in FIG. 14A and brain Serum from animals were analyzed for IFNB or IFNC. pro retrieved after 5 days for HSV-1 plaque assays. FIGS. 14C, duction after 24 hours). FIGS. 18C, 18D: STING deficient 14D: Serum from animals (n=3) infected with HSV (1x107 animals or controls (n-3) were infected i.v. with vesicular i.v.) were analyzed for IFNB or IFNC production after 6 stomatitis virus (VSV. 5x107) and IFNB or IFNC measured hours). FIG. 14E, 14F: Serum from animals infected as in after 12 hours). FIGS. 18E, 18F: STING deficient animals or FIG. 14C, was analyzed for RANTES and IL-6 production. controls (n=3) were infected intraperitoneally (i.p.) with FIG. 14G: Increasing amounts of Yellow Fever Virus (YFV) encephalomyocarditis virus (EMCV, 1x10). Serum from NS4b was co-transfected into 293T cells with STING or the animals was analyzed for IFNB or IFNC. production after 6 amino terminus of RIG-I (ARIG-1, 1-284) and transfected and 24 hours. Asterisks indicate significant difference (P<0. IFNB promoter driven luciferase (IFN-B-Luc) measured after 05) as determined by Student's t-test. Error bars indicates.d. 36 hours. Asterisks indicate significant difference (P<0.05) as 0026 FIGS. 19A-19C: Human STING (hSTING, amino determined by Student's t-test. Error bars indicates.d. acids 125-222) exhibits significant homology to flavivirus 0022 FIGS. 15A-15D: STING is required for effective NS4B proteins. FIG. 19A: Homology between Yellow Fever DNA-mediated adaptive immune responses. FIG. 15A: Virus (YFV), Dengue Virus (DV) and hepatitis C virus (HCV) STING deficient animals () or controls ("") (n=4; approxi NS4b are shown. FIG. 19B: Schematic of YFV NS4b homol mately 8 weeks of age were immunized twice (100 ug, ogy versus STING. FIG. 19C:293T cells were co-transfected intramuscularly i.m.) by electroporation with a DNA vac with FLAG-tagged STING and HA taggedYFV-NS4B for 36 cine encoding ovalbumin. Serum was measured for anti-OVA hours and observed by confocal microscopy. IgG. FIGS. 15B,15C: Mice were treated as in a and spleen 0027 FIG. 20: STING translocates to endosomes follow CD8"/IFN-y" cells measured by FACS and anti-OVA-specific ing exposure to ISD. STING MEFs, stably reconstituted US 2013/0039933 A1 Feb. 14, 2013

with HA tagged STING (mSTING-HA) were treated with or immobilized on Ambion BrightStar(R-Plus Membrane, without transfected ISD (4 hours) and stained using HA anti probed with 'P-labeled human-STING or B-actin DNA body (green) and either calreticulin, ER, mitotracker, mito probes, and finally, exposed for autoradiography. 7 human chondria; TfR, recycled endosome; EEA1, early endosome; Burkitt lymphoma lines (BL-5, BL-7, BL-8, SM1, peterson, TGN46, trans-Golgi, Giantin, cis-Golgi, LAMP1 lysosome BL-30 and canute) and 2 primary effusion lymphoma lines (red). (BC-1 and BC-3) were tested in this northern blot. Huvec cell 0028 FIGS. 21A, 21B: Confirmation of RNAi knock was used as a positive control. (+), EBV positive: (-), EBV down in MEFs. FIG. 21A: Immunoblot of RNAi (to Sec5) negative. treated cells indicating knockdown of Sec5. FIG. 21B: RT 0035 FIG. 28 is a Western blot of endogenous STING PCR measurements of RNAi (Trap?3) treated cells indicating expression in various types of normal human cells: 30 g of knockdown of Trap?3. whole cell extracts were resolved by 10% SDS-PAGE fol 0029 FIG. 22 is a graph showing that the intracellular lowed by western blot using either rabbit-anti-human-STING DNA pathway is defective in human cancer cells, but not antiserum or mouse-anti-beta-actin monoclonal antibody normal cells. Normal human cells (PASMC, NHDF-ad and (Sigma). Normal human cells were purchased from Lonza TFF) or human cancer cell lines (Hek293, HeLa, MCF7, and names of cells tested are labeled above the image. H1299 and 293T) were either mock transfected or transfected 0036 FIGS. 29 A-29B: STING is lost from numerous can with Lipofectamine and 5 g/ml of polyIC, poly(dG-dC), calf cer cells although reconstitution of STING can rescue the thymus DNA or E. coli DNA. Culture supernatants were intracellular DNA-mediated type I IFN induced pathway. collected 16 hours after transfection and endogenous IFN-B FIG. 29A is an immunoblot blot analysis of hSTING expres was analyzed by ELISA (Invitrogen). The data indicates that sion in normal human cells (PASMC and NHDF-ad) or dsRNA pathways (polyIC), but not intracellular DNA (poly human cancer cells (293T and MCF7). FIG. 29B: 293T cells dG-dC, calf thymus DNA or E. coli DNA) pathways which were transfected with an IFN-B-Luc (p110-Luc) plasmid and are regulated by STING are functional. increasing amounts of human STING (hSTING), murine 0030 FIG. 23 is a blot showing that STING is absent in STING (mSTING) or control ARIG-I, and analyzed for numerous cancer cell-lines but not normal cells. Western blot luciferase activity. MCF7 cells were transfected with increas of endogenous STING expression in normal human cells ing amount of hSTING and culture supernatants were col (PASMC, NHDF-ad) and cancer cells lines (293T-H1299): lected and analyzed for IFN-B protein by ELISA. 30 ug of whole cell extracts were resolved by 10% SDS 0037 FIGS. 30A-30K show that STING is required for PAGE followed by western blot using either rabbit-anti-hu intracellular DNA-mediated type IIFN type production. FIG. man-STING antiserum or mouse-anti-beta-actin monoclonal 30A: Sting" or MEFS were transfected with FLAG-IRF7 antibody (Sigma). Names of cells tested are labeled above the for 24 hours and immunostained using anti-FLAG antibody. image. Plasmid transfection stimulates the STING pathway, activat 0031 FIG. 24 is a Western blot of endogenous STING ing IRF7 and nuclear localization. This does not occur in the expression in various human cell lines: 30 g of whole cell absence of STING. FIG.30B: Sting" or MEFS were treated extracts were resolved by 10% SDS-PAGE followed by west with ISD for 16 hours and cell lysates analyzed for NF-KB ern blot using either rabbit-anti-human-STING antiserum or p65 activity using ELISA. FIG.30C: Sting" or macroph mouse-anti-beta-actin monoclonal antibody (Sigma). Names age were treated with ISD or poly (dAT:dAT) for 16 hours and of cell lines tested are labeled above the image. lysates analyzed for caspase-1 p10 fragment using antibody. 0032 FIG. 25 is a Northern blot of endogenous STING FIG. 30D: Macrophages were transfected with 1 g/ml of expression in various human cell lines: mRNAs were DNA ligands or infected for 16 hours with HSV-1 (moi 10). extracted using the MicroPoly(A)PuristTM Kit (Ambion). IL-6 was measured by ELISA. IL-6 production in the absence Northern blot was performed following the Northern Max(R)- of STING is defective in response to poly(dAT:dAT) and ISD, Gly Kit (Ambion) procedure. Briefly, 2 ug of each mRNA was but not HSV-1 or CpG ODN. FIG.30E: Sting MEFS were resolved by 1% agarose gel, transferred and immobilized on re-constituted with STING and IFNB production rescued as Ambion BrightStar R-Plus Membrane, probed with 32P-la measured by ELISA. FIGS. 30F and 30G: Sting" or MEFS beled human-STING or B-actin DNA probes, and finally, were infected (moi 10) with human cytomegalovirus exposed for autoradiography. Names of cell lines tested are (HCMV) or vaccinia virus (VVAE3L) or baculovirus and labeled above the image. IFNB production measured by ELISA after 16 hours. FIG. 0033 FIG. 26 is a Western blot of endogenous STING 30H is a scan of a photograph showing GM-DCs from expression in various human lymphoma cell lines: 30 ug of Sting" or without treatment or treated with FITC-ISD. FIG. whole cell extracts were resolved by 10% SDS-PAGE fol 30I: Sting" or MEFS were transfected with murine DAI for lowed by western blot using either rabbit-anti-human-STING 16 hours and treated with ISD for 16 hours, and endogenous antiserum or mouse-anti-beta-actin monoclonal antibody IFNB was measured. FIG. 30J: L929 cells were treated with (Sigma). 7 human Burkitt lymphoma lines (BL-5, BL-7, RNAi to STING for 3 days, and treated with poly(dA:dT). BL-8, SM1, peterson, BL-30 and canute) and 2 primary effu After 16 hours, IFNB production measured by ELISA. FIG. sion lymphoma lines (BC-1 and BC-3) were tested in this 30K: Knockdown of STING in (FIG.30I) was confirmed by western blot. Huvec cell was used as a positive control. (+), immunoblot using anti-STING antibody. Asterisks indicate EBV positive: (-), EBV negative. significant difference (P<0.05) as determined by Students 0034 FIG. 27 is a Northern blot of endogenous STING t-test. Error bars indicates.d. expression in various human lymphoma cell lines: mRNAS 0038 FIGS. 31A-31D: Normal Tand B cell repertoire in were extracted using the MicroPoly(A)PuristTM Kit (Am STING animals under non-stimulatory conditions. FIGS. bion). Northern blot was performed following the Northern 31A-31C: Splenocytes from STING"" or mice were ana Max(R)-Gly Kit (Ambion) procedure. Briefly, 2 ug of each lyzed by FACS using anti-CD3, anti-CD4 or CD8 (for T cell) mRNA was resolved by 1% agarose gel, transferred and or anti-CD19 (for B cells) under non-stimulated conditions. US 2013/0039933 A1 Feb. 14, 2013

FIG. 31 D: Ovalbumin (10 ug) in incomplete Freunds adju generate hybridomas, which were isolated to generate clones. vant (IFA) was inoculated i.p. into STING" or mice (n=4). Secreted antibody was screened using 293T cells as controls Animals were boosted after 14 days. Antibody production to or 293T cells expressing human STING, in immunofluores OVA was measured 14 days after the last boost. FIGS. 32A cence assays (FIG. 35A). Antibody fluorescing the STING 32I: Brefeldin A blocks STING trafficking. FIG.32A: Immu transfected cells but not controls was used to incubate immu noblot analysis of STING expression in Sting MEF's stably noblots of lysates expressing human STING or control vector. transfected with HA-STING, to demonstrate comparable Thus Mab AS-1 (for anti-STING 1 from clone 2g.9) recog amounts of STING in both cell types. FIG.32B: MEFs stably nizes STING in immunofluorescence studies and by immu transfected with HA-STING were treated with Brefeldin A noblot. (0.1 g/ml) or chloroquine (0.1 mM) for 1 hour prior to ISD 0042 FIGS. 36A-36G show STING dependent innate stimulation (3 hours 1 lug/ml in Lipofectamine 2000). immune signaling. FIG.36A: Human Telomerase Fibroblasts Green-anti-HA and red-anti-calreticulin. FIG. 32C: Wild (hTERT-BJ1) were transfected with various nucleotides (3 type MEFs were treated with Brefeldin A as above and IFNB mg/ml) for 16 h. Endogenous IFNB levels were measured. mRNA measured. FIGS. 32D, 32E: 293T cells were treated hTERT-BJ1 cells were transfected with FITC conjugated with various concentrations of Brefeldin A as above were dsDNA90 was examined by fluorescent microscopy to ensure co-transfected with STING, constitutively active IRF3 or efficient transfection. FIG. 36B: hTERT-BJ1 cells were trans IRF7 and IFN-luciferase reporter plasmid. Luciferase was fected with mock, random or two independent human STING measured after 24 hours. FIGS. 32F, 32G: Macrophages siRNAs (siRNA 3 or 4) for 3 days followed by dsDNA90 treated with various chloroquine amounts for one hour were transfection (3 ug/ml) for 16 hours. Endogenous IFNB levels treated with ISD (1 lug/ml), poly (dAT-dAT) 1 ug/ml) or LPS were measured. Silencing of hSTING protein was demon (1 lug/ml) and after 16 hours IFNB or IL6 measured by ELISA. strated by immunoblotting, with 3-actin serving as a control. FIG. 32H: STING MEFS were transfected with FIG. 36C: Primary STING"', STING', STAT1* or HA-STING or HA-STING ASP (that lacks the first 36 amino STAT1 MEFs were transfected with or without dsDNA90 acids representing a potential signal peptide motif) for 24 (3 g/ml) for 3 hours. Total RNA was purified and examined hours prior to treatment with ISD (1 lug/ml). After 3 hours for gene expression by Illumina Sentrix BeadChip Array STING localization was measured using immunofluores (Mouse WG6 version 2). FIG. 36D: hTERT-BJ1 cells were cence and an anti-HA antibody (green) or anti calreticulin treated NS or STING siRNA. After 3 days, cells were treated antibody (red). FIG.32I:293T cells were co-transfected with with dsDNA90, ssDNA90 or ssDNA45 (3 ug/ml). IFNB HA-STING or HA-STINGASP and an IFN-Luciferase plas mRNA levels were measured by real time RT-PCR after 16 mid and after 24 hours luciferase was measured. STING ASP hours. FIG. 36E: hTERT-BJ1 cells were treated NS or STING does not traffic in response to ISD and cannot activate type I siRNA. At 3 days, cells were treated with dsDNA90, IFN. Asterisks indicate significant difference (P<0.05) as ssDNA90 or ssDNA45 (3 g/ml). Endogenous IFNB levels determined by Student's t-test. Error bars indicates.d. were measured after 16 hours. FIG. 36F: Primary STING" 0039 FIGS.33A-33B: STING is essential for TBK-1 traf or STING MEFs were transfected with or without ficking in response to intracellular DNA. FIG. 33A: dsDNA90 (3 g/ml). After 3 h, the same as FIG. 36C. FIG. STING" or MEFs were treated with ISD (1 g/ml with 36G: hTERT-BJ1 cells were treated with or without Lipofactamine 2000) and after 3 hours endogenous TBK1 dsDNA90 (3 g/ml) for 3 hours and stained with anti-HA localization was analyzed using immunofluorescence using antibody and calreticulin as a ER marker. and anti-TBKantibody (green) or anti-calreticulin (red). FIG. 0043 FIGS. 37A-37J show that STING binds to DNA. 33B:TBK1* or MEFs were transfected with HA-STING FIG. 37A: 293T cells were transfected with indicated plas and after 24 hours cells were treated with ISD for 3 hours and mids. Cell lysates were precipitated with biotin-dsDNA90 STING localization analyzed by immunofluorescence using agarose and analyzed by immunoblotting using anti-HA anti an anti HA antibody (green) or anti-calreticulin (red). STING body. FIG. 37B: Schematic of STING mutants. FIG. 37C: localizes in the absence of TBK1. Same as FIG. 37A. FIG. 37D: Same as FIG. 37A. STING 0040 FIGS. 34A-34F: Sec5 is required for efficient intra variants unable to bind DNA are labeled in red. FIG. 37E: cellular DNA-mediated signaling. FIG. 34A: Immunoblot of hTERT-BJ1 cells were transfected biotin conjugated RNAi (to Sec5 or Sec61B) treated cells indicating knockdown dsDNA90 (B-dsDNA90; 3 g/ml) for 6 hand treated with of Sec5 and Sec61 B. FIG. 34B: RT-PCR measurements of DSS. Lysates were precipitated using streptavidin agarose RNAi (Trap?3) treated cells indicating knockdown of Trap?3. and analyzed by immunoblotting using anti-HA antibody. FIG. 34C. MEFS treated with RNAi to Sec5 or STING for 3 FIG. 37F: STING was expressed in 293T cells and after 36 days were infected with HSV1 (moi 1) for 24 hours and viral hours lysates were incubated with dsDNA90 agarose in the titers measured. FIG.34D: Same as in (FIG.34C) except that presence of competitor dsDNA90, Poly(I:C), B-DNA or endogenous IFNB was measured. FIG. 34E: MEF's treated ssDNA90 and analyzed by immunoblotting using anti-HA with RNAi to Trap?3, Sec61 B or Sec5 for 3 days were treated antibody. FIG. 37G: 293T cells were transfected with HA with ISD for 3 hours and Ifnb mRNA measured by RT-PCR. tagged STING, GFP or TREX1. Cells were lysed and biotin FIG. 34F: Same as in (FIG. 34E) except that endogenous labeled ssDNA or dsDNA added with streptavidin agarose IFNB was measured. beads. Precipitates were analyzed by immunoblotting using 004.1 FIGS. 35A, 35B show immunostaining (FIG.35A) anti-HA antibody. FIG. 37H: 293T cells were transfected and immunobloting (FIG.35B) of 293T cells transfected with with IFNB-Luciferase and STING variants and luciferase hSTING for 24 hours using anti-STING monoclonal anti activity measured. FIG. 37I: htERT-BJ1 cells were trans body AS-1. The monoclonal antibody (Mab) was generated fected with dsDNA90 and crosslinked with formaldehyde. by inoculating/immunizing mice (Balbfc) with plasmid DNA STING was precipitated and bound DNA detected by PCR (100 lug) expressing the human STING gene. Spleen cells using dsDNA90 specific primers. NC: negative control. PC: from the immunized animals were fused to myeloma cells to positive control, dsDNA90. FIG.37J: STING" or STING US 2013/0039933 A1 Feb. 14, 2013

MEFs were transfected with dsDNA90 and then same as FIG. ined by real time PCR for IFNB (FIG. 41A), IFIT1 (FIG. 37I. Error bars indicates S.d. Data are representative of at least 41 B), IFIT2 (FIG. 41C), IFIT3 (FIG. 41D), CXCL 10 (FIG. two independent experiments. 41E), GBP1 (FIG.41F), RSAD2 (FIG.41G) and CCL5 (FIG. 0044 FIGS. 38A-38H show that TREX1 is negative regu 41H). *P<0.05, Student's t-test. Error Bars indicated s.d. lator of STING signaling. FIG.38A: Immunoblot confirming Data are representative of at least two independent experi STING and/or TREX1 knockdown in siRNA treated hTERT mentS. BJ1 cells. FIG. 38B: siRNA treated hTERT-BJ1 cells were 0048 FIGS. 42A-42H show that cytoplasmic DNAS transfected with dsDNA90 (3 ug/ml). Endogenous IFNB lev induce STING-dependent genes in MEFs. In FIGS. 42A els were measured after 16 hours. P<0.05. FIG.38C: siRNA 42H, STING" or STING MEFs were treated with or treated hTERT-BJ1 cells were infected with HSV-1 (m.o.i=1) without dsDNA45, dsDNA90, ssDNA45 or ssDNA90 for 3 h. and virus titers measured. P-O.05. FIG.38D: siRNA treated Total RNAs were purified and examined by real time PCR for hTERT-BJ1 cells were infected with Y34.5 deleted-HSV and IFNB (FIG. 42A), IFIT1 (FIG.42B), IFIT2 (FIG. 42C), IFIT3 virus titers measured. P-O.05. FIG. 38E: Immunoblot of NS (FIG.42D), CCL5 (FIG. 42E), CXCL 10 (FIG.42F), RSAD2 or STING siRNA treated TREX1* or TREX1 MEFs, (FIG. 42G) or GBP1 (FIG. 42H). *P<0.05, Student's t-test. confirming STING knockdown. FIG. 38F: siRNA treated Error Bars indicated S.d. Data are representative of at least TREX1* or TREX1 MEFs were treated with dsDNA90 two independent experiments. and IFNB levels were measured after 16 hours. *P<0.05. FIG. 0049 FIGS. 43 A-43D show STING localization and 38G: siRNA treated TREX1''' or TREX1 MEFs were dimerization. FIG.43A: MEF's stably expressing STING-HA infected HSV-1 (m.o.i=1) and virus titers measured. *P<0.05. were treated with ssDNA45, dsDNA45, ssDNA90 or FIG. 38H: Immunofluorescence analysis using anti-TREX1 dsDNA90. After 3 h, cells were stained using anti-HA or oranti-STING antibody of hTERT-BJ1 cells transfected with anti-calreticulin antibody. FIG. 43B: 293T cells were trans or without dsDNA90. *P<0.05, Student's t-test. Error bars fected with STING-HA and Myc-STING. Lysates were pre indicated S.d. Data are representative of at least two indepen cipitated by anti-Myc antibody and analyzed by immunob dent experiments. lotting using anti-HA antibody. FIG. 43C. hTERT-BJ 1 cells 004.5 FIGS. 39A-39J: show TREX1 associates with oli were treated with or without the cross linker DSS. Cell lysates gosaccharyltransferase complex. FIG. 39A shows a sche were subjected to immunoblot using anti-STING antibody. matic of TREX1. Red indicates RPN1 binding site. FIG. 39B FIG. 43D: 293T cells were transfected with indicated plas shows a schematic of STING. Red indicates DAD1 binding mids and treated with DSS. Cell lysates were analyzed by site. FIG. 39C: RPN1 interacts with TREX1 in yeast two immunoblotting using anti-HA antibody. hybrid analysis (1 pCBKT7, 2-pCBKT7-NFARM9, 3 pC 0050 FIGS. 44A-44I show that DNA virus induces BKT7-TREX1, 4 pCBKT7-STING full length, 5.pGBKT7 STING-dependent genes in MEFs. FIG. 44A: MEFs were STING C-terminal). FIG. 39D: 293T cells were co-trans infected with Y34.5 deleted-HSV (m.o.i.–1) for 3 h. Total fected with TREX1-tGFP and RPN1-Myc. Lysates were RNA was purified and examined for gene expression using immunoprecipitated using anti-Myc antibody and analyzed Illumina Sentric Bead Chip array (Mouse WGS version2). by immunoblotting. FIG. 39H: hTERT-BJ1 cells were treated FIGS. 44 B-44I: STING"', STING or STAT/STING" with or without dsDNA90 (3 ug/ml). At 6 h after transfection, MEFs were treated with or without dsDNA90, HSV or Y34.5 cells were examined by immunofluorescence using anti deleted-HSV for 3 h. Total RNAs were purified and examined STING or anti-DAD1 antibody. FIG.39I: Immunoblot analy by real time PCR for IFNB (FIG. 44B), IFIT1 (FIG. 44C), sis of microsome fractions after Sucrose gradient centrifuga IFIT2 (FIG. 44D), IFIT3 (FIG. 44E), CCL5 (FIG. 44F), tion using indicated antibodies. I: input. FIG. 39J: hTERT CXCL 10 (FIG. 44G), RSAD2 (FIG. 44H) or GBP1 (FIG. BJ1 cells were treated with TREX1, Sec61A1, TRAPB, NS or 44I). Error Bars indicated S.d. STING siRNA. After 72 h, cells were treated with dsDNA90 0051 FIGS. 45A-45F show that STING interacts with (3 ug/ml) for 16 h and then endogenous IFNB levels were IRF3/7 and NFK-B. FIG. 45A: IRF7 binding sites in the measured. PK0.05, Student's t-test. Error bars indicated S.d. promoter regions of STING dependent dsDNA90 stimulatory Data are representative of at least two independent experi genes. FIGS. 45B-45D: Nudear extract was isolated from mentS. mock treated or dsDNA90 treated STING" or STING 0046 FIGS. 40A-40G show that cytoplasmic DNA MEFs and were examined for IRF3 (FIG. 45B), IRF7 (FIG. induces STING-dependent genes in hTERT-BJ1 cells. FIG. 45C) and NF-kB (FIG. 45D) activation following the manu 40A: hTERT-BJ1 cells were treated with STING siRNA. facturer's instruction. Nuclear extract kit, TransAM IRF3, After 3 days, cells were treated with or without dsDNA90 for TransAM IRF7 and TransAMNFkB family Elisa kits were 3 h. Total RNA was purified and examined for gene expres from Active Motif *P<0.05, Student's t-test. Error Bars indi sion using Human HT-12 V4 Bead Chip. FIGS. 40B-40G: cated S.d Data are representative of at least two independent hTERT-BJ 1 cells were treated as in FIG. 40A. Total RNAS experiments. FIG. 45E: STING" or STING MEFs were were examined by real time PCR for IFNB (FIG. 40B), treated with poly(I:C), dsDNA90 or HSV1 and cells were PMAIP1 (FIG. 40C), IFIT1 (FIG. 40D), IFIT2 (FIG. 40E), stained by anti-IRF3 antibody. FIG. 43F. STING" or IFIT3 (FIG. 40F) and PTGER4 (FIG. 40G). Real time PCR STING MEFs were treated dsDNA90 or HSV1 and cells was performed using TaqMan gene Expression Assay (Ap were stained by anti-p65 antibody. Loss of STING did not plied Biosystem). *P<0.05, Student's t-test. Error Bars indi affect poly(I:C) mediated innate immune signaling. cated S.d. Data are representative of at least two independent 0052 FIGS. 46A-46F show that STING binds DNA in experiments. vitro. FIG. 46A: In vitro translation products were precipi 0047 FIGS. 41A-41H show that STING-dependent genes tated with biotin conjugated dsDNA90 and immunoblotted are induced by cytoplasmic DNA in MEFs. Primary by anti-HA antibody. FIG. 46B: schematic of STING vari STAT1*.* or STAT1 MEFs were treated dsDNA90, IFNo. ants. FIGS. 46 C, 46D: Same as FIG. 46A. STING variants ordsDNA90 with IFNC. Total RNAs were purified and exam lacking aa 242-341 (red) failed to bind DNA. FIGS. 46E-46F: US 2013/0039933 A1 Feb. 14, 2013

In vitro translation products were precipitated with biotin 52H: Total RNAs were examined by RT-PCR for IFNB (FIG. conjugated ssDNA90 and immunoblotted by anti-HA anti 52B), IFIT1 (FIG.52C), IFIT2 (FIG.52D), IFIT3 (FIG.52E), body. CCL5 (FIG.52F), CXCL 10 (FIG.52G), RSAD2 (FIG.52H). 0053 FIGS. 47A-47G show that STING binds DNA in No significant difference in IFN induced genes was observed vivo and in vitro. hTERT BJ1 cells were transfected with in TREX1 lacking cells. *P<0.05, Student's t-test. Error bars biotin-dsDNA90 and crosslinked by UV. Cells were lysed and indicated S.d. Data are representative of at least two indepen precipitated by Streptavidin agarose and analyzed by immu dent experiments. noblotting. FIGS. 47B-47C hTERT-BJ1 cells were treated 0059 FIGS. 53A-53D show that TREX1 associates with with IFI 16 (FIG.47B) or STING (FIG.47C) siRNA and then oligosaccharyltransferase complex. An IFN-treated hTERT same as in FIG. 47A. FIG. 47D: STING" or STING cDNA library was used to develop a yeast two hybrid library MEFs were treated as in FIG. 47A. FIG. 47E: STING-Flag (AH109). Full length TREX 1 was used as bait to screen the expressing 293T cells were treated with or without biotin library. Approximately 5 million cDNA expressing yeast dsDNA90 and crosslinked by DSS. Lysates were precipitated were screened (Clontech). 44 clones were isolated from 3 and analyzed by immunoblotting. FIG. 47F: 293T cells were independent yeast mating procedures. RPN1 was isolated 8 transfected with dsDNA90 or ssDNA90 and crosslinked by times in total (three times in screen 1, twice in Screen 2 and UV or DSS and then precipitated and analyzed by immuno three times in screen 3). The majority of the clones, aSide blotting. FIG. 47G: STING-Flag expression 293T cells were from RPN1, failed to interact with IREX1 after re-testing. Of lysed and incubated with dsONA90 or Poly(I:C) and biotin these eight RPN1 isolated clones, four clones encoded aa dsDNA90 agarose and then same as FIGS. 47E. 258-397, two clones aa 220-390 and two clones aa 240-367. 0054 FIGS. 48A-48C show that STING binds viral DNA. TREX1 variants were generated and the interation between FIG. 48A: Oligonucleotide sequences of HSV, cytomegalovi TREX1 (aa.241-369) and RPN 1 (aa 256-397) was mapped. rus (CMV) or adenovirus (ADV). FIGS. 48B-48C:293T cells To isolate DAD1, the C-terminal region of STING (aa 173 were transfected with indicated plasmids. Cell-lysates were 379) was used to screen the same library. 24 isolated clones, precipitated with biolin-dsDNA90, biotin-HSV DNA 120 full length DAD I was found twice. The majority of the mer, biotin-ADV DNA 120 mer or biotin-CMV DNA 120 clones, aSide from DAD1, failed to interact with STING after meragarose and analyzed by immunoblotting using anti-HA re-comfirmation studies. Full length DAD1 was seen to asso antibody. ciate with region 242-310 of STING. FIG.53A: Schematic of 0055 FIGS. 49A-49C show that STING binds DNA. FIG. TREX1 mutants. FIG. 53B: GAL4 binding domain fused to 49A. Schematic of STING ELISA. FIG. 49B: Process of an TREX1 or TREX1-4 interact with RPN1 fused to the GAL4 embodiment of a STINGELISA. FIG.49C: Binding capacity activation domain in yeast two hybrid screening. FIG. 53C: of dsDNA90 was measured by ELISA. *P<0.05, Student’s Schematic of STING mutants. FIG. 53D. GAL4 binding t-test. Error Bars indicated S.d. Data are representative of at domain fused to STING-C-terminal or STING-C2 interact least two independent experiments. with DAD1 fused to the GAL.4 activation domain in yeast two 0056 FIGS. 50A-50D show that TREX1 is a negative hybrid Screening. regulator of STING signalling. FIGS.50A: hTERT-BJ1 cells 0060 FIGS.54A-54C show that TREX1 and STING asso were treated with TREX1, STING or STING and TREX1 ciate with oligosaccharyltransferase complex. FIG. 54A: siRNA. After 3 days, cells were infected with HSV-Luc (m.o. 293T cells were co-transfected with TREX1-tGFP and i=1) for 48 h. Lysates were measured luciferase activity. FIG. RPN1-Myc. Lysates were immunoprecipitated with anti 50B: Primary TREX1** or TREX MEFs were transfected tGFP antibody or IgG control and analyzed by immunoblot with NS or STING siRNA. After 3 days, cells were infected ting using indicated antibodies. FIG. 5B; 293T cells were with HSV-Luc (m.o.i.-1) for 48 h. Lysates were measured. co-transfected with Myc-STING or GFP-DAD1 and cells FIG. 50C: Primary TREX1* or TREX17 MEFs were were lysed. Lysates were immunoprecipitated with anti-Myc transfected with wild type TREX1. After 48 h, cells were antibody or IgG control and immunoblotted using anti-GFP infected with HSV and measured HSV replication. TREX1 antibody. FIG. 54C: 293T cells were co-transfected with expression was checked by immunoblotting. *P<0.05, Stu TREX1-to-FP and RPN1-Myc, GFP-DAD1 or STING-HA. dent's t-test. Error Bars indicated S.d. Data are representative Lysates were immunoprecipitated by anti-tGFP antibody or of at least two independent experiments. FIG.50D:STING" IgG control and analyzed by immunoblotting using anti or STING MEFs were treated with or without dsDNA90 tGFP, anti-Myc, anti-GFP or anti-HA antibodies. for 3 h. Total RNAs were purified and examined for gene 0061 FIG.55 shows that TREX1 localizes in the endo expression by Illumina Sentrix Bead Chip array (Mouse WG6 plasmic reticulum. hTERT-BJ1 cells were transfected with version2). RPN1-Myc. After 48 h, cells were examined by immunofluo 0057 FIG.51 shows that STING regulates ssDNA90-me rescence using anti-TREX1 antibody (red), anti-Myc anti diated IFNB production in TREX MEFs. siRNA treated body (green) or anti-calreticulin antibody (blue) as an endo TREX1** TREX1 MEFs were treated with ssDNA45 or plasmic reticulum marker. ssDNA90 and IFNB levels were measured after 16 hours. 0062 FIGS. 56A-56H show that exogenously expressed *P<0.05, Student's t-test. Error Bars indicated S.d. Data are STING in 293T cells reconstitutes dsDNA90 response. FIG. representative of at least two independent experiments. 56A: 293T cells were infected with control lentivirus or hST 0058 FIGS. 52A-52H show that TREX1 is not a negative ING lentivirus. 1 day after infection, cells were treated with regulator of STING-dependent genes. FIG. 52A: TREX1"" dsDNA90. After 6 h, cells were stained using anti-STING or or TREX1 MEFs were treated with HSV1, IFNC, anti-calreticulin antibody. FIG. 56B: Cell lysates (from FIG. dsDNA90, triphosphate RNA (TPRNA) and VSV. TPRNA 56A) were subjected to immunoblot using anti-STING anti and VSV weakly activated IFN induced genes. Total RNAs body. FIGS. 56C-56D: Lentivirus infected 293T cells were were purified and examined for gene expression by Illumina stimulated with dsDNA90 for 6 h. Total RNAs were purified Sentrix Bead Chip array (Mouse WG6 version2). FIGS. 52B and examined by real time PCR for IFNB (FIG.56C) or IFIT2 US 2013/0039933 A1 Feb. 14, 2013

(FIG. 56D). FIG. 56E: 293T cells stably transduced with by H&E staining on mock or DMBA treated skin/skin tumor control or hSTING lentivirus were subjected to Brefeldin A biopsies. Images were taken at 20x magnification. FIG. 64D: (BFA) experiment as shown in the flow chart. FIG. 56F: Cell Cytokine upregulation in STING expressing mice exposed to lysates (from FIG. 56E) were subjected to immunoblot using carcinogens. RNAs extracted from mock or DMBA treated anti-STING antibody. FIG.56G: Cell lysates (from FIG.56E) skin/skin tumor biopsies were analyzed by Illumina Sentrix were measured for IFNB-luciferase activity. FIG. 56H: Pri BeadChip Array (Mouse WG6 version 2) in duplicate. Total mary STING MEFs were stably transduced with controlor gene expression was analyzed. Most variable genes were mSTING. Cells were treated with dsDNA90 and endogenous selected. Rows represent individual genes; columns represent IFNB levels were measured by ELISA. *P<0.05, Student’s individual samples. Pseudo-colors indicate transcript levels t-test. Error bars indicated S.d. Data are representative of at below, equal to, or above the mean (green, black, and red, least two independent experiments. respectively). Gene expression; fold change log 10 scale 0063 FIG. 57 show that translocon members regulate ranges between -5 to 5. No cytokines were observed in the HSV1 replication. htERT-BJ1 cells were treated with skin of STING-deficient animals. TREX1, Sec61A1, TRAPB, NS or STING siRNA. 0064 FIGS. 58A-58C show that IFI16 is not required for DETAILED DESCRIPTION IFN-production by dsDNA90 inhTER-BJ1 cells. FIG. 58A: hTERT-BJt cells were treated with NS, IFI16, STING or 0071. The present invention is described with reference to TREX1 siRNA. After 3 days, cells were lysed and checked the attached figures, wherein like reference numerals are used expression levels by immunoblotting. FIG. 58B; siRNA throughout the figures to designate similar or equivalent ele treated hTERT-BJ1 cells were stimulated with dsDNA90 and ments. The figures are not drawn to scale and they are pro IFNB production was measured by ELISA. FIG.58C: siRNA vided merely to illustrate the instant invention. Several treated hTERT-BJ1 cells were infected with HSV-luciferase aspects of the invention are described below with reference to (m.o.i=0.1). At 2 days after infection, cells were lysed and example applications for illustration. It should be understood luciferase activity was measured. *P-0.05, Student's t-test. that numerous specific details, relationships, and methods are Error bars indicated S.d. Data are representative of at least two set forth to provide a full understanding of the invention. One independent experiments. having ordinary skill in the relevant art, however, will readily recognize that the invention can be practiced without one or 0065 FIG. 59 shows an embodiment of a STING cell more of the specific details or with other methods. The present based assay. invention is not limited by the illustrated ordering of acts or 0.066 FIG. 60 shows that Drug “A” induces STING traf events, as some acts may occur in different orders and/or ficking. concurrently with other acts or events. Furthermore, not all 0067 FIG. 61 shows that drug “X” inhibits IFNB mRNA illustrated acts or events are required to implement a meth production. odology in accordance with the present invention. 0068 FIG. 62 is a schematic showing that STING is phos 0072 All genes, gene names, and gene products disclosed phorylated in response to cytoplannic DNA. hTERT-BJ1 cells herein are intended to correspond to homologs from any were transfected with 4 lug/ml of ISD for 6 h. The cell lysates species for which the compositions and methods disclosed were prepared in TNE buffer and then subjected to immuno herein are applicable. Thus, the terms include, but are not precipitation with anti-STING antibody followed by SDS limited to genes and gene products from humans and mice. It PAGE. The gel was stained with CBB and then bands includ is understood that when a gene or gene product from a par ing STING were analyzed by mLC/MS/MS at the Harvard ticular species is disclosed, this disclosure is intended to be Mass Spectrometry and Proteomics Resource Laboratory. exemplary only, and is not to be interpreted as a limitation Alignment of STING amino acid sequence from different unless the context in which it appears clearly indicates. Thus, species and the phosphorylation sites identified by mass spec for example, for the genes disclosed herein, which in some trometry. Serine 345, 358, 366, and 379 were identified by embodiments relate to mammalian nucleic acid and amino mass spectrometry. Serine 358 and S366 are important for acid sequences are intended to encompass homologous and/ STING function. or orthologous genes and gene products from other animals 0069 FIG. 63 shows that Serine 366 (S366) of STING is including, but not limited to other mammals, fish, amphib important for IFNB production in cytoplasmic DNA pathway. ians, reptiles, and birds. In preferred embodiments, the genes 293T cells were transfected with plasmid encoding mutant or nucleic acid sequences are human. STING and reporter plasmid. After 36 hr, luciferase activity 0073. Unless otherwise defined, all terms (including tech was measured. STING MEF cells were reconstituted with nical and Scientific terms) used herein have the same meaning mutant STING and then the amount of IFNB in culture media as commonly understood by one of ordinary skill in the art to was measured by ELISA. S366 is important for IFN produc which this invention belongs. It will be further understood tion by STING and S358 is also likely to play an important that terms, such as those defined in commonly used dictio role. naries, should be interpreted as having a meaning that is 0070 FIGS. 64A-64D show that STING deficient mice consistent with their meaning in the context of the relevant art are resistant to DMBA induced inflammation and skin onco and will not be interpreted in an idealized or overly formal genesis: STING" and STING.' mice were either mocked sense unless expressly so defined herein. treated with acetone or treated with 10 ug of DMBA on the shaved dorsal weekly for 20 weeks. FIG. 64A: STING defi DEFINITIONS cient animals are resistant to DNA-damaging agants that cause skin cancer. Percentages of skin tumor-free mice were 0074 The terminology used herein is for the purpose of shown in the Kaplan-Meier curve. FIG. 64B: Pictures of describing particular embodiments only and is not intended to representative mice of each treatment groups were shown. be limiting of the invention. As used herein, the singular FIG. 64C. Histopathological examinations were performed forms “a”, “an and “the are intended to include the plural US 2013/0039933 A1 Feb. 14, 2013

forms as well, unless the context clearly indicates otherwise. 0081. The term “variant, when used in the context of a Furthermore, to the extent that the terms “including, polynucleotide sequence, may encompass a polynucleotide “includes”, “having”, “has”, “with', or variants thereof are sequence related to a wildtype gene. This definition may also used in either the detailed description and/or the claims, such include, for example, “allelic,” “splice.” “species,” or “poly terms are intended to be inclusive in a manner similar to the morphic' variants. A splice variant may have significant iden term "comprising.” tity to a reference molecule, but will generally have a greater 0075. The term “about” or “approximately” means within or lesser number of polynucleotides due to alternate splicing an acceptable error range for the particular value as deter of exons during mRNA processing. The corresponding mined by one of ordinary skill in the art, which will depend in polypeptide may possess additional functional domains oran part on how the value is measured or determined, i.e., the absence of domains. Species variants are polynucleotide limitations of the measurement system. For example, “about sequences that vary from one species to another. Of particular can mean within 1 or more than 1 Standard deviation, per the utility in the invention are variants of wildtype gene products. practice in the art. Alternatively, “about can mean a range of Variants may result from at least one mutation in the nucleic up to 20%, preferably up to 10%, more preferably up to 5%, acid sequence and may result in altered mRNAS or in and more preferably still up to 1% of a given value. Alterna polypeptides whose structure or function may or may not be tively, particularly with respect to biological systems or pro altered. Any given natural or recombinant gene may have cesses, the term can mean within an order of magnitude, none, one, or many allelic forms. Common mutational preferably within 5-fold, and more preferably within 2-fold, changes that give rise to variants are generally ascribed to of a value. Where particular values are described in the appli natural deletions, additions, or Substitutions of nucleotides. cation and claims, unless otherwise stated the term “about Each of these types of changes may occur alone, or in com meaning within an acceptable error range for the particular bination with the others, one or more times in a given value should be assumed. Sequence. 0076. The term “induces or enhances an immune I0082. The resulting polypeptides generally will have sig response' is meant causing a statistically measurable induc nificantamino acid identity relative to each other. A polymor tion or increase in an immune response over a control sample phic variant is a variation in the polynucleotide sequence of a to which the peptide, polypeptide or protein has not been particular gene between individuals of a given species. Poly administered. Preferably the induction or enhancement of the morphic variants also may encompass 'single nucleotide immune response results in a prophylactic or therapeutic polymorphisms” (SNPs) or single base mutations in which response in a subject. Examples of immune responses are the polynucleotide sequence varies by one base. The presence increased production of type I IFN, increased resistance to of SNPs may be indicative of, for example, a certain popula viral and other types of infection by alternate pathogens. The tion with a propensity for a disease state, that is Susceptibility enhancement of immune responses to tumors (anti-tumor Versus resistance. responses), or the development of vaccines to prevent tumors I0083) Derivative polynucleotides include nucleic acids or eliminate existing tumors. Subjected to chemical modification, for example, replace 0077. The term “STING” is meant to include, without ment of hydrogen by an alkyl, acyl, or amino group. Deriva limitation, nucleic acids, polynucleotides, oligonucleotides, tives, e.g., derivative oligonucleotides, may comprise non sense and antisense polynucleotide Strands, complementary naturally-occurring portions, such as altered Sugar moieties sequences, peptides, polypeptides, proteins, homologous orinter-Sugar linkages. Exemplary among these are phospho and/or orthologous STING molecules, isoforms, precursors, rothioate and other Sulfur containing species which are mutants, variants, derivatives, splice variants, alleles, differ known in the art. Derivative nucleic acids may also contain ent species, and active fragments thereof. labels, including radionucleotides, enzymes, fluorescent 0078. The term “lacks a functional STING gene' is meant agents, chemiluminescent agents, chromogenic agents, Sub that a transgenic animal lacks a gene that encodes STING, or strates, cofactors, inhibitors, magnetic particles, and the like. lacks other genetic components (e.g. promoters) required for I0084. A "derivative' polypeptide or peptide is one that is expression of STING. modified, for example, by glycosylation, pegylation, phos 007.9 The term “active fragment or variant' is meant a phorylation, Sulfation, reduction/alkylation, acylation, fragment that is at least 380 amino acid residues in length and chemical coupling, or mild formalin treatment. A derivative is 100% identical to a contiguous portion of the peptide, may also be modified to contain a detectable label, either polypeptide or protein, or a variant that is at least 90%, pref directly or indirectly, including, but not limited to, a radio erably 95% identical to a fragment up to and including the full isotope, fluorescent, and enzyme label. length peptide, polypeptide or protein. A variant, for example, I0085. The term “immunoregulatory” is meant a com may include conservative amino acid Substitutions, as defined pound, composition or Substance that is immunogenic (i.e. in the art, or nonconservative Substitutions, providing that at stimulates or increases an immune response) or immunosup least e.g. 10%, 25%, 50%, 75% or 90% of the activity of the pressive (i.e. reduces or suppresses an immune response). original peptide, polypeptide or protein is retained. Also I0086) “An antigen presenting cell (APC) is a cell that is included are STING molecules, fragments or variants having capable of activating T cells, and includes, but is not limited post-translational modifications such as Sumoylation, phos to, monocytes/macrophages, B cells and dendritic cells phorylation glycosylation, splice variants, and the like, all of (DCs). The term “dendritic cell” or “DC” refers to any mem which may effect the efficacy of STING function and/or ber of a diverse population of morphologically similar cell activity, both known and yet to be discovered. types found in lymphoid or non-lymphoid tissues. These cells 0080 Unless otherwise indicated, the terms “peptide', are characterized by their distinctive morphology, high levels "polypeptide' or “protein’ are used interchangeably herein, of surface MHC-class II expression. DCs can be isolated from although typically they refer to peptide sequences of varying a number of tissue sources. DCs have a high capacity for S17S. sensitizing MHC-restricted T cells and are very effective at US 2013/0039933 A1 Feb. 14, 2013 presenting antigens to T cells in situ. The antigens may be tive in the assay, are termed “true negatives.” The “specific self-antigens that are expressed during T cell development ity of a diagnostic assay is 1 minus the false positive rate, and tolerance, and foreign antigens that are present during where the “false positive' rate is defined as the proportion of normal immune processes. those without the disease who test positive. While a particular 0087. The term “expression vector” as used herein refers diagnostic method may not provide a definitive diagnosis of a to a vector containing a nucleic acid sequence coding for at condition, it suffices if the method provides a positive indi least part of a gene product capable of being transcribed. In cation that aids in diagnosis. Some cases, RNA molecules are then translated into a protein, (0093. “Treatment” is an intervention performed with the polypeptide, or peptide. In other cases, these sequences are intention of preventing the development or altering the not translated, for example, in the production of antisense pathology or symptoms of a disorder. Accordingly, “treat molecules, siRNA, ribozymes, and the like. Expression vec ment” refers to both therapeutic treatment and prophylactic or tors can contain a variety of control sequences, which refer to preventative measures. Those in need of treatment include nucleic acid sequences necessary for the transcription and those already with the disorder as well as those in which the possibly translation of an operatively linked coding sequence disorder is to be prevented. In tumor (e.g., cancer) treatment, in a particular host organism. In addition to control sequences a therapeutic agent may directly decrease the pathology of that govern transcription and translation, Vectors and expres tumor cells, or render the tumor cells more susceptible to sion vectors may contain nucleic acid sequences that serve treatment by other therapeutic agents, e.g., radiation and/or other functions as well. chemotherapy. As used herein, “ameliorated' or “treatment' I0088. By “encoding or “encoded”, “encodes', with refers to a symptom which is approaches a normalized value respect to a specified nucleic acid, is meant comprising the (for example a value obtained in a healthy patient or indi information for translation into the specified protein. A vidual), e.g., is less than 50% different from a normalized nucleic acid encoding a protein may comprise non-translated value, preferably is less than about 25% different from a sequences (e.g., introns) within translated regions of the normalized value, more preferably, is less than 10% different nucleic acid, or may lack Such intervening non-translated from a normalized value, and still more preferably, is not sequences (e.g., as in cDNA). The information by which a significantly different from a normalized value as determined protein is encoded is specified by the use of codons. Typically, using routine statistical tests. For example the term “treat' or the amino acid sequence is encoded by the nucleic acid using “treating with respect to tumor cells refers to stopping the the “universal genetic code. progression of said cells, slowing down growth, inducing I0089. As used herein, "heterologous” in reference to a regression, or amelioration of symptoms associated with the nucleic acid is a nucleic acid that originates from a foreign presence of said cells. Treatment of an individual Suffering species, or, if from the same species, is Substantially modified from an infectious disease organism refers to a decrease and from its native form in composition and/or genomic locus by elimination of the disease organism from an individual. For deliberate human intervention. For example, a promoteroper example, a decrease of viral particles as measured by plaque ably linked to a heterologous structural gene is from a species forming units or other automated diagnostic methods such as different from that from which the structural gene was ELISA etc. derived, or, if from the same species, one or both are Substan 0094. The “treatment of cancer', refers to one or more of tially modified from their original form. A heterologous pro the following effects: (1) inhibition, to some extent, of tumor tein may originate from a foreign species or, if from the same growth, including, (i) slowing down and (ii) complete growth species, is substantially modified from its original form by arrest; (2) reduction in the number of tumor cells; (3) main deliberate human intervention. taining tumor size; (4) reduction in tumor size; (5) inhibition, 0090 “Sample is used herein in its broadest sense. A including (i) reduction, (ii) slowing down or (iii) complete sample comprising polynucleotides, polypeptides, peptides, prevention, of tumor cell infiltration into peripheral organs; antibodies and the like may comprise a bodily fluid; a soluble (6) inhibition, including (i) reduction, (ii) slowing down or fraction of a cell preparation, or media in which cells were (iii) complete prevention, of metastasis; (7) enhancement of grown; a chromosome, an organelle, or membrane isolated or anti-tumor immune response, which may result in (i) main extracted from a cell; genomic DNA, RNA, or cDNA, taining tumor size, (ii) reducing tumor size, (iii) slowing the polypeptides, or peptides in Solution or bound to a Substrate; growth of a tumor, (iv) reducing, slowing or preventing inva a cell; a tissue; a tissue print; a fingerprint, skin or hair, and the sion and/or (8) relief, to some extent, of the severity or num like. ber of one or more symptoms associated with the disorder. 0091. The terms “patient”, “subject” or “individual” are 0095. As used herein, the term “safe and effective amount used interchangeably herein, and refers to a mammalian Sub refers to the quantity of a component which is sufficient to ject to be treated, with human patients being preferred. In yield a desired therapeutic response without undue adverse Some cases, the methods of the invention find use in experi side effects (such as toxicity, irritation, or allergic response) mental animals, in Veterinary application, and in the devel commensurate with a reasonable benefit/risk ratio when used opment of animal models for disease, including, but not lim in the manner of this invention. By “therapeutically effective ited to, rodents including mice, rats, and hamsters; and amount' is meant an amount of a compound of the present primates. invention effective to yield the desired therapeutic response. 0092 “Diagnostic' or “diagnosed’ means identifying the For example, an amount effective to delay the growth of or to presence or nature of a pathologic condition. Diagnostic cause a cancer, eithera sarcoma or lymphoma, or to shrink the methods differ in their sensitivity and specificity. The “sen cancer or prevent metastasis. The specific safe and effective sitivity of a diagnostic assay is the percentage of diseased amount or therapeutically effective amount will vary with individuals who test positive (percent of “true positives”). Such factors as the particular condition being treated, the Diseased individuals not detected by the assay are “false physical condition of the patient, the type of mammal or negatives. Subjects who are not diseased and who test nega animal being treated, the duration of the treatment, the nature US 2013/0039933 A1 Feb. 14, 2013 of concurrent therapy (if any), and the specific formulations vided are an agent reactive with the peptide, a pharmaceutical employed and the structure of the compounds or its deriva composition that includes the peptide, isolated nucleic acid tives. molecules, an isolated nucleic acid molecules encoding the 0096 “Cells of the immune system” or “immune cells', is peptides, recombinant nucleic acid constructs that include the meant to include any cells of the immune system that may be nucleic acid molecules, at least one host cell comprising the assayed, including, but not limited to, B lymphocytes, also recombinant nucleic acid constructs, and a method of produc called B cells, T lymphocytes, also called T cells, natural ing the peptide using the host cell. The present invention killer (NK) cells, natural killer T (NK) cells, lymphokine further provides a method for treating and/or preventing activated killer (LAK) cells, monocytes, macrophages, neu infection in a Subject by administering the peptide of the trophils, granulocytes, mast cells, platelets, Langerhans cells, invention to the Subject, thereby modulating innate immunity stem cells, dendritic cells, peripheral blood mononuclear in the Subject. Additionally, the present invention provides a cells, tumor-infiltrating (TIL) cells, gene modified immune method for identifying candidate therapeutic agents. cells including hybridomas, drug modified immune cells, and 0103) In the Examples section which follows, the data derivatives, precursors or progenitors of the above cell types. provide for the identification following expression cloning 0097. “Immune effector cells' refers to cells capable of and characterization of a novel molecule, STING (STimula binding an antigen and which mediate an immune response tor of INterferon Genes) that regulates innate immune signal selective for the antigen. These cells include, but are not ing processes. Both the nucleic acid and peptide sequences limited to, T cells (T lymphocytes), B cells (B lymphocytes), are identified in humans and mice. STING, comprising 5 monocytes, macrophages, natural killer (NK) cells and cyto putative transmembrane (TM) regions, predominantly toxic T lymphocytes (CTLs), for example CTL lines, CTL resides in the endoplasmic reticulum (ER) and is able to clones, and CTLs from tumor, inflammatory, or other infil activate both NF-kB and IRF3 transcription pathways to trates. induce type IIFN and to exert a potent anti-viral state follow 0098 “Immune related molecules” refers to any molecule ing expression. In contrast, loss of STING reduced the ability identified in any immune cell, whether in a resting (“non of polyIC to activate type IIFN and rendered murine embry stimulated') or activated State, and includes any receptor, onic fibroblasts lacking STING ( MEFs) generated by tar ligand, cell Surface molecules, nucleic acid molecules, geted homologous recombination, Susceptible to vesicular polypeptides, variants and fragments thereof. stomatitis virus (VSV) infection. DNA-mediated ability to 0099. “T cells” or “T lymphocytes' are a subset of lym stimulate type IIFN responses in the absence of STING, was phocytes originating in the thymus and having heterodimeric also greatly inhibited, indicating that STING may play an receptors associated with proteins of the CD3 complex (e.g., important role in recognizing DNA-virus and bacteria as well a rearranged T cell receptor, the heterodimeric protein on the as other pathogen infection. Yeast-two hybrid and co-immu T cell surfaces responsible for antigen/MHC specificity of the noprecipitation studies indicated that STING interacts with cells). T cell responses may be detected by assays for their RIG-I and with Ssr2/TRAPB, a member of the translocon effects on other cells (e.g., target cell killing, activation of associated protein (TRAP) complex required for protein other immune cells, such as B-cells) or for the cytokines they translocation across the ER membrane following translation. produce. RNAi ablation of TRAPB was subsequently found to inhibit 0100. The phrase “T cell response' means an immuno STING function as well as impede the production of type I logical response involving T cells. The T cells that are “acti IFN in response to polyIC. Thus, aSide from identifying a vated divide to produce antigen specific memory T cells or novel regulator of innate immune signaling, this data further antigen specific cytotoxic T cells. The cytotoxic T cells bind implicates, for the first time, a potential role for the translocon to and destroy cells recognized as containing the antigen. The in intracellular dsRNA/virus signaling and DNA/virus, plas memory T cells are activated by the antigen and thus provide mid DNA based pathways in mammalian cells which appears a response to an antigen already encountered. This overall central for the activation of primary innate immune response to the antigen is the antigen specific T cell response, responses, including the production of Type IIFN. e.g. tumor specific. 0104. In preferred embodiments, STING molecules and 0101. A “secondary immune response' or “adaptive the molecules STING regulates in the various pathways immune response' may be active or passive, and may be described herein, provide the design of new adjuvants, vac humoral (antibody based) or cellular that is established dur cines and therapies to regulate the immune system and other ing the life of an animal, is specific for an inducing antigen, systems. The molecule, and/or pathways it regulates, would and is marked by an enhanced immune response on repeated be utilized in vaccine and therapies to combat disease. Modu encounters with said antigen. A key feature of the T lympho lation of the immune system by STING provides for the cytes of the adaptive immune system is their ability to detect treatment of diseases, including diseases caused by foreign minute concentrations of pathogen-derived peptides pre agents. Exemplary infections by foreign agents which may be sented by MHC molecules on the cell surface. treated and/or prevented by the method of the present inven tion include an infection by a bacterium (e.g., a Gram-positive STING Compositions or Gram-negative bacterium), an infection by a fungus, an 0102 Embodiments of the invention are directed to infection by a parasite, and an infection by a virus. In one nucleic acid sequences and encoded products thereof, to a embodiment of the present invention, the infection is a bac molecule termed herein STING. The novel STING nucleic terial infection (e.g., infection by E. coli, Klebsiella pneumo acids, polynucleotides, oligonucleotides, peptides, mutants, niae, Pseudomonas aeruginosa, Salmonella spp., Staphyllo variants and active fragments thereof, can be used to modu coccus aureus, Streptococcus spp., or Vancomycin-resistant late innate and adaptive immunity in a subject and/or for the enterococcus). In another embodiment, the infection is a fun treatment of an immune-related disorder, including treating gal infection (e.g. infection by a mould, a yeast, or a higher and preventing infection by modulating immunity. Also pro fungus). In still another embodiment, the infection is a para US 2013/0039933 A1 Feb. 14, 2013 sitic infection (e.g., infection by a single-celled or multicel far. Thus suppression of the STING pathway may be an early, lular parasite, including Giardia duodenalis, Cryptospo critical and hitherto novel requirement for the cellular trans ridium parvum, Cyclospora Cayetanensis, and Toxoplasma formation process. The intracellular DNA pathway is defec gondiz). In yet another embodiment, the infection is a viral tive and that this event involves STING or STING interacting infection (e.g., infection by a virus associated with AIDS, molecules, or molecules upstream or downstream of STING, avian flu, chickenpox, cold Sores, common cold, gastroenteri including TRAP complexes, Vesicular pathways and endo tis, glandular fever, influenza, measles, mumps, pharyngitis, Some pathways. pneumonia, rubella, SARS, and lower or upper respiratory 0113. In a preferred embodiment, STING and/or STING tract infection (e.g., respiratory syncytial virus)). interacting molecules are administered to patients for the 0105 STING also associates with components of the pro treatment of cancer. STING can be administered as a poly teosome (PSMB1), which is essential for the elimination of nucleotide, polypeptide, peptides, antisense oligonucle misfolded proteins. Removal of misfolded proteins from the otides, vectors expressing STING, antibodies and the like. secretory pathway depends on their recognition in the endo The data herein, indicate that STING is absent in a variety of plasmic reticulum ER). STING resides in the ER and associ lymphoma Such as Burkett's lymphoma, breast cancer, other ates with PSMB1 to potentially play a role in ERAD-ER leukemia, lymphoma, melanoma and so on. The data herein associated protein degradation. A functional ubiquitin also show that there is defective intracellular DNA signaling proteosome system is essential and defects can lead to neu in cancer cells but not normal cells. Thus, reconstitution of rodegenerative diseases (Parkinson, Alzheimer's) and car STING and/or STING associated molecules are used intreat diac disorders. Up-regulation of proteosome activity can lead ments for cancer and other diseases or disorders. For to wasting conditions (sepsis, cachexia). Immunoproteo example, the data show that reconstitution of STING into Somes occurring in the brain and muscle reflect persistent cancer cell-lines previously shown to be defective in intrac inflammation and are downregulated in cancer cells. The ellular DNA signaling such as 293T or MCF7, rescues the induction of apoptosis by proteosome inhibitors is a treatment intracellular pathway. That is reconstitution of STING can for cancer (such as myeloma). Thus STING could play a role induce type IIFN and perhaps other signal transduction path in these pathways and provide a target for cancer therapy and ways. STING would thus be useful to reconstitute in cancer neurodegenerative disease. cells, perhaps to kill cancer but not normal cells. STING or 0106. In another embodiment, STING is associated with a other molecules associated directly or indirectly with STING molecule of interest (e.g. peptide, antigen, enzyme, etc) either in the intracellular DNA pathway may be useful targets for by binding, as a fusion moiety, or linked via a chemical therapeutic intervention. Without wishing to be bound by moiety to modulate the molecule of interests half-life, reten theory, it is also proposed that the STING pathway will be tion in an intracellular body, degradation, altering of intrac deregulated/suppressed in viral infected tumors such as HPV ellular or surface expression of the molecule of interest, etc. related cervical carcinoma, HCV or HBV-related hepatocel 0107. In a preferred embodiment, an isolated nucleic acid lular carcinoma, herpes virus associated cancers (EBV posi sequence encoding a protein or peptide comprises a sequence tive Burkett's lines—(see, for example, FIGS. 26, 27). as set forth in SEQID NOS: 1 or 2, or an active fragment or STING may also be suppressed in latently infected cells such variant thereofthat induces or enhances an immune response. as those mentioned above (EBV, HPV) as well as CMV, HIV 0108. In another preferred embodiment, an active frag etc. ment is of at least 380 consecutive amino acid residues in 0114. In another preferred embodiment, STING is admin length in humans and induces or enhances an immune istered to a patient to prevent or treat cancer. Cancer refers to response in Vivo or in vitro. all types of cancer or neoplasm or malignant tumors found in 0109. In another preferred embodiment, expression of mammals, including, but not limited to: leukemias, lympho SEQID NOS: 1 or 2 or active fragments thereof, induce type mas, melanomas, carcinomas and sarcomas. Examples of IIFN in a cell, tissue or organ, in vivo or in vitro. cancers are cancer of the brain, breast, pancreas, cervix, 0110. In another preferred embodiment, the expression of colon, head and neck, kidney, lung, non-Small cell lung, mela SEQID NOS: 1 or 2 in vivo or in vitro induces an antiviral noma, mesothelioma, ovary, sarcoma, stomach, uterus and immune response. Medulloblastoma. As used herein, the terms “cancer,” “neo 0111. In another preferred embodiment, STING induces plasm, and "tumor are used interchangeably and in either an anti-tumor response. Cancer cells may activate the STING the singular or plural form, refer to cells that have undergone pathway by numerous ways, for example following virus a malignant transformation that makes them pathological to infection (EBV, HPV, HBV ETC) of known or unknown the host organism. Primary cancer cells (that is, cells obtained viruses, by the re-activation of endogenous retroviruses from near the site of malignant transformation) can be readily (ERV), or other retroelements (retroelement-derived DNA, distinguished from non-cancerous cells by well-established retrotranscription) or proteins or nucleic acids which activate techniques, particularly histological examination. The defi the STING pathway (Endogenous retroviruses and cancer, nition of a cancer cell, as used herein, includes not only a Ruprecht Ketal., Cell Mol Life Sci. 2008 November; 65(21): primary cancer cell, but any cell derived from a cancer cell 3366-82) Gifford and Tristem Virus Genes 2003 26:3 291 ancestor. This includes metastasized cancer cells, and in vitro 315) (Dolei A. Perron H. Neurovirol. 2009 January; 15(1):4- cultures and cell lines derived from cancer cells. When refer 13)(Goodier J. L. Kazazian H H Jr Cell. 2008 Oct. 3: 135(1): ring to a type of cancer that normally manifests as a solid 23-35). tumor, a “clinically detectable” tumor is one that is detectable 0112. In another preferred embodiment, treatment of can on the basis of tumor mass; e.g., by procedures such as CAT cer comprises administration of STING or agents which Scan, MR imaging, X-ray, ultrasound or palpation, and/or induce STING and pathways thereof. The data shown herein, which is detectable because of the expression of one or more evidences that the intracellular DNA pathway that is regu cancer-specific antigens in a sample obtainable from a lated by STING is defective in all cancer cells examined so patient. US 2013/0039933 A1 Feb. 14, 2013

0115. In a preferred embodiment, administration of immune-mediated encephalomyelitis; inclusion body myosi STING prevents (neuroprotective) or treats neurological dis tis; incontinentia pigmenti; infantile phytanic acid storage orders. Neurological disorder refers to any disorder of the disease; infantile refsum disease; infantile spasms; inflamma nervous system and/or visual system. “Neurological disor tory myopathy; intracranial cyst; intracranial hypertension; ders' include disorders that involve the central nervous sys Joubert syndrome; Kearns-Sayre syndrome; Kennedy dis tem (brain, brainstem and cerebellum), the peripheral ner ease Kinsbourne syndrome; Klippel Feil syndrome; Krabbe Vous system (including cranial nerves), and the autonomic disease: Kugelberg-Welander disease; kuru; Lafora disease; nervous system (parts of which are located in both central and Lambert-Eaton myasthenic syndrome; Landau-Kleffner Syn peripheral nervous system). Neurodegenerative diseases, drome; lateral medullary (Wallenberg) syndrome; learning include, for example, Alzheimer's Disease, stroke, multiple disabilities; Leigh's disease; Lennox-Gustaut Syndrome; Sclerosis etc. Lesch-Nyhan syndrome; leukodystrophy; Lewy body 0116. The following is a list of several neurological disor dementia; Lissencephaly; locked-in syndrome; Lou Gehrig's ders, symptoms, signs and syndromes that can be treated disease (i.e., motor neuron disease or amyotrophic lateral using compositions and methods according to the present Sclerosis); lumbar disc disease; Lyme disease—neurological invention: acquired epileptiform aphasia; acute disseminated sequelae; Machado-Joseph disease; macrencephaly: mega encephalomyelitis; adrenoleukodystrophy; age-related lencephaly: Melkersson-Rosenthal syndrome: Menieres dis macular degeneration; agenesis of the corpus calloSum; agno ease; meningitis; Menkes disease; metachromatic leukodys sia; Aicardi syndrome; Alexander disease; Alpers disease; trophy; microcephaly; migraine; Miller Fisher syndrome; alternating hemiplegia; Alzheimer's disease; Vascular mini-strokes; mitochondrial myopathies; Mobius syndrome; dementia; amyotrophic lateral Sclerosis; anencephaly; Angel monomelic amyotrophy; motor neuron disease; Moyamoya man syndrome; angiomatosis; anoxia; aphasia; apraxia; disease; mucopolysaccharidoses; milti-infarct dementia; arachnoid cysts; arachnoiditis; Anronl-Chiari malformation; multifocal motor neuropathy; multiple Sclerosis and other arteriovenous malformation; Asperger syndrome; ataxia tel demyelinating disorders; multiple system atrophy with pos egiectasia; attention deficit hyperactivity disorder, autism; tural hypotension; p muscular dystrophy; myasthenia gravis; autonomic dysfunction; back pain; Batten disease; Behcet’s myelinoclastic diffuse Sclerosis; myoclonic encephalopathy disease; Bell's palsy, benign essential blepharospasm; of infants; myoclonus; myopathy; myotonia congenital; nar benign focal; amyotrophy; benign intracranial hypertension; colepsy; neurofibromatosis; neuroleptic malignant Syn Binswanger's disease; blepharospasm; Bloch Sulzberger drome; neurological manifestations of AIDS; neurological syndrome; brachial plexus injury; brain abscess; brain injury: sequelae of lupus; neuromyotonia; neuronal ceroid lipofus brain tumors (including glioblastoma multiforme); spinal cinosis; neuronal migration disorders; Niemann-Pick dis tumor, Brown-Sequard syndrome; Canavan disease; carpal ease; O'Sullivan-McLeod syndrome; occipital neuralgia; tunnel syndrome; causalgia; central pain syndrome; central occult spinal dysraphism sequence; Ohtahara syndrome; pontine myelinolysis; cephalic disorder; cerebral aneurysm; olivopontocerebellar atrophy; opSoclonus myoclonus; optic cerebral arteriosclerosis; cerebral atrophy; cerebral gigan neuritis; orthostatic hypotension; overuse syndrome; pares tism; cerebral palsy; Charcot-Marie-Tooth disease; chemo thesia; Parkinson's disease; paramyotonia congenital; para therapy-induced neuropathy and neuropathic pain; Chiari neoplastic diseases; paroxysmal attacks; Parry Romberg Syn malformation; chorea; chronic inflammatory demyelinating drome: Pelizaeus-Merzbacher disease; periodic paralyses; polyneuropathy; chronic pain; chronic regional pain Syn peripheral neuropathy; painful neuropathy and neuropathic drome; Coffin Lowry syndrome; coma, including persistent pain; persistent vegetative state; pervasive developmental dis Vegetative state; congenital facial diplegia; corticobasal orders; photic Sneeze reflex; phytanic acid storage disease; degeneration; cranial arteritis; craniosynostosis; Creutzfeldt Pick's disease; pinched nerve: pituitary tumors; polymyosi Jakob disease; cumulative trauma disorders; Cushing's Syn tis; porencephaly; post-polio syndrome; postherpetic neural drome; cytomegalic inclusion body disease; cytomegalovirus gia; postinfectious encephalomyelitis; postural hypotension; infection; dancing eyes-dancing feet syndrome; Dandy Prader-Willi syndrome; primary lateral sclerosis: prion dis Walker syndrome; Dawson disease; De Morsier's syndrome: eases; progressive hemifacial atrophy; progressive multifocal Dejerine-Klumke palsy, dementia; dermatomyositis; dia leukoencephalopathy; progressive Sclerosing poliodystro betic neuropathy; diffuse Sclerosis; dysautonomia; dys phy; progressive Supranuclear palsy; pseudotumor cerebri; graphia; dyslexia; dystonias; early infantile epileptic Ramsay-Hunt syndrome (types I and II); Rasmussen's encephalopathy; empty sella syndrome; encephalitis; encephalitis; reflex sympathetic dystrophy syndrome; Ref encephaloceles; encephalotrigeminal angiomatosis; epi Sum disease; repetitive motion disorders; repetitive stress lepsy; Erb's palsy; essential tremor; Fabry's disease; Fahr's injuries; restless legs syndrome; retrovirus-associated myel syndrome; fainting; familial spastic paralysis; febrile sei opathy: Rett syndrome; Reye's syndrome: Saint Vitus dance: Zures; Fisher syndrome; Friedreich's ataxia; fronto-temporal Sandhoff disease; Schilder's disease; schizencephaly; septo dementia and other “tauopathies'; Gaucher's disease; Gerst optic dysplasia; shaken baby syndrome; shingles; Shy mann's syndrome; giant cell arteritis; giant cell inclusion Drager syndrome; Sjögren's syndrome, sleep apnea; Soto's disease; globoid cell leukodystrophy: Guillain-Barre syn syndrome; spasticity; spina bifida; spinal cord injury; spinal drome; HTLV-1-associated myelopathy; Hallervorden-Spatz cord tumors; spinal muscular atrophy; Stiff-Person Syn disease; head injury; headache; hemifacial spasm; hereditary drome; stroke; Sturge-Weber syndrome; Subacute Sclerosing spastic paraplegia; heredopathia atactica polyneuritiformis; panencephalitis; Subcortical arteriosclerotic encephalopathy; herpes Zoster oticus; herpes Zoster, Hirayama syndrome; Sydenham chorea; syncope; syringomyelia; tardive dyskine HIV-associated dementia and neuropathy (also neurological sia; Tay-Sachs disease; temporal arteritis; tethered spinal cord manifestations of AIDS); holoprosencephaly: Huntington's syndrome; Thomsen disease; thoracic outlet syndrome; Tic disease and other polyglutamine repeat diseases; hydranen Douloureux; Todd's paralysis; Tourette syndrome; transient cephaly; hydrocephalus; hypercortisolism; hypoxia; ischemic attack; transmissible spongiform encephalopathies; US 2013/0039933 A1 Feb. 14, 2013 transverse myelitis; traumatic braininjury; tremor; trigeminal tially including constituents of cell debris (such as neuralgia; tropical spastic paraparesis; tuberous sclerosis: nucleosomes or cellular RNAs), STING may also play a role vascular dementia (multi-infarct dementia); vasculitis includ in the expression of a broader array of autoimmune and auto ing temporal arteritis; Von Hippel-Lindau disease; Wallen inflammatory conditions. It is thus notable that the STING berg's syndrome; Werdnig-Hoffman disease; West syn gene is located in humans at 5q31.2, at or immediately adja drome; whiplash: Williams syndrome; Wildon's disease; and cent to the IBD5 locus that has been linked to disease risk Zellweger syndrome. and/or to more severe disease in processes including Crohn's 0117. In another preferred embodiment, administration of disease, rheumatoid arthritis, diabetes mellitus type I, and STING prevents or treats patients suffering from autoimmune celiac disease. At least one report has also observed an asso diseases. Examples of autoimmune diseases comprise: rheu ciation between this region and risk for lupus (De Jager P L. matoid arthritis, systemic lupus erythematosus, inflammatory et al. Genes Immun. 2006 June; 7(4):327–34). Without wish bowel diseases (IBDs) comprising Crohn disease (CD) and ing to be bound by theory, it is hypothesized that increased ulcerative colitis (UC) which are chronic inflammatory con induction of STING-associated pathways increases pro-im ditions with polygenic Susceptibility. mune signaling, increasing the likelihood and severity of 0118. In another preferred embodiment, STING is a prog autoimmune diseases, including the tendency for anti-ds nostic marker for disease progression or outcome or response DNA-associated lupus to target major organs and to lead to or resistance to anti-cancer therapies. For example, loss of poorer outcomes. Other diseases regulated by the STING STING, change in expression and/or function of STING as pathway include multiple Sclerosis, the cause of which may compared to a normal cell, or detection of a mutant STING involve DNA based retroelements (Dolei A. Perron H.J Neu molecule would be an indication of a cell progressing towards rovirol. 2009 January; 15(1):4-13; Christensen T. Association being a tumor cell. of human endogenous retroviruses with multiple Sclerosis 0119. In another preferred embodiment, STING is prog and possible interactions with herpes viruses Rev Med. Virol. nostic of a disease state comprising cancer, autoimmunity, 2005 May-June; 15(3):179-211). infection by an agent and the like. (0.124 STING Interacting Molecules: 0120 In a preferred embodiment, a peptide, polypeptide 0.125 STING interacts or associates with a variety, includ or protein encoded by an isolated nucleic acid set forth as ing, as yet unknown molecules and pathways. SEQID NOS: 1 or 2, or an active fragment or variant thereof. I0126. In a preferred embodiment, STING, compositions In certain embodiments, the peptide is at least 380 consecu thereof, active fragment, variant, or mutant thereof modulates tive amino acid residues in length. expression and/or function of molecules which interact or 0121. In another preferred embodiment, a biomarker com associate with, directly or indirectly, with STING. This prises a nucleic acid sequence set forth as SEQID NOS: 1 or includes all pathways and molecules involved in those path 2, an active fragment, variant, or mutant thereof. ways, directly or indirectly, which STING may affect (di 0122. In another preferred embodiment, a biomarker com rectly or indirectly). prises a peptide, polypeptide or protein encoded by a nucleic I0127. For example, TRAP-B which binds to STING, inter acid sequence set forth as SEQ ID NOS: 1 or 2, an active acts with (identified via UniProtKB, MINT, and/or STRING), fragment, variant, or mutant thereof. for example: SSR2 (ENSP000003572953); SSR4 0123 Role in Autoimmunity: Antibodies to double NSP000003591033 STRING (score=0.996); SSRI stranded DNA are present in roughly half of patients with NSPOOOOO2447633 STRING (score=0.995); systemic lupus erythematosus, and are highly specific to the SR3ENSP000002650443 STRING (score=0.995); SRPR diagnosis of SLE (Simon JA, et al. Rheumatology (Oxford). NSPOOOOO328O233 STRING (score=0.96); TRAM1 2004 February; 43(2):220-4). In addition, these antibodies NSPOOOOO26221.33 STRING (score=0.955) DDOST have prominent prognostic importance: SLE patients with NSPOOOOO3641883 STRING (score=0.943); RPN2 anti-dsDNA antibodies have a much greater likelihood of NSPOOOOO3459,043 STRING (score=0.931); SEC61B developing severe manifestations of lupus including lupus NSPOOOOO2236413 STRING (score=0.925); SEC11A nephritis and vasculitis, and have a Substantially poorer long NSPOOOOO2682203 STRING (score=0.918); SRP19 term prognosis. Despite the long-standing awareness of the NSPOOOOO2829.993 STRING (score=0.917); SEC61G importance of dsDNA antibodies to lupus, the relevance of NSP000003415383 STRING (score=0.916); SRP9 these antibodies to disease pathogenesis has remained mys NSP000003052303 STRING (score=0.915); DAD1 terious. Isolated dsDNA specificities have been found in NSP000002504983 STRING (score=0.91); SRP68 Some patients that directly interact with target tissue antigens, NSP000003120663 STRING (score=0.91); SPCS2 but patients without identified cross-reactivities of this sort NSPOOOOO2636723 STRING (score=0.907); SEC11C still often target the kidneys with similarly poor prognoses. B NSPOOOOO2.997143 STRING (score=0.906); STT3B and T cell tolerance protocols toward dsDNA antigens in NSPOOOOO2957703 STRING (score=0.905); SEC61A1 patients or in animal models of lupus have had very limited NSPOOOOO243.2533 STRING (score=0.903); SEC61A2 success (RenaudineauY, et al. Arthritis Rheum. 2006 August; NSP000003.683193 STRING (score=0.903); SRP72 54(8):2523-32: Cardiel M. H. et al. Arthritis Rheum. 2008 NSP000003421813 STRING (score=0.902); ERO1L August; 58(8):2470-80: Kang H K, et al. J Immunol. 2007 NSP000003520443 STRING (score=0.899) INS Jun. 15; 178(12):7849-58.). Interest in the relevance of NSP000003707313 STRING (score=0.899); RPN1 nucleosomal DNA to induction of innate immunity and in NSP000002962553 STRING (score=0.899); SPCS1 particular severe dsDNA-associated lupus continues to exist, NSP000002330253 STRING (score=0.899); SPCS3 though, and non-TLR9 innate immune recognition pathways NSP000002645973 STRING (score=0.899); SRP14 for self DNA to induce immune reactivity have been identi NSP000002678843 STRING (score=0.899); SRP54 fied. Given the potential role that STING may play in induc NSP000002167743 STRING (score=0.899); SRPRB ing/amplifying innate immune responses to products poten NSPOOOOO2734O63 STRING (score=0.899); TRAM2 US 2013/0039933 A1 Feb. 14, 2013

ENSPOOOOO1825273 STRING (score=0.899); ants, mutants or active fragments thereof. For example, frag ENSP000003515683 STRING (score=0.905). ments comprising domains that bind DNA. 0128. In another example, SEC61B interacting proteins I0131 The vector can be administered to a patient wherein (P604681, 2 ENSP000002236413) via UniProtKB, MINT, expression of SEQID NOS: 1, 2, variants, mutants or active and/or STRING: Interactant Interaction Details GeneCard fragments thereof, induces a response comprising at least one External ID(s) comprise: SSRI P433072, of anti-viral activity, immune responses, immune signaling ENSP000002447633 MINT-4546723 STRING (score=0. or intracellular B-form DNA. 96); SEC61G ENSP000003415383 STRING (score=0.986); SEC61A1 ENSP000002432533 STRING (score=0.981); 0.132. A number of vectors are known to be capable of TRAM1 ENSP000002622133 STRING (score=0.964); mediating transfer of gene products to mammalian cells, as is SSR3ENSP000002650443 STRING (score=0.934); SSR4 known in the art and described herein. A “vector' (sometimes ENSPOOOOO359 1033 STRING (score=0.934): referred to as gene delivery or gene transfer “vehicle') refers SR2ENSP000003572953 STRING (score=0.925); RPN2 to a macromolecule or complex of molecules comprising a NSP000003459043 STRING (score=0.923); SRP9 polynucleotide to be delivered to a host cell, either in vitro or NSP000003052303 STRING (score=0.912); DAD1 in vivo. The polynucleotide to be delivered may comprise a NSP000002504983 STRING (score=0.911); SPCS2 coding sequence of interest in gene therapy. Vectors include, NSP000002636723 STRING (score=0.908) DDOST for example, viral vectors (such as adenoviruses (“Ad”), NSP000003641883 STRING (score=0.906); TRAM2 adeno-associated viruses (AAV), and vesicular stomatitis NSP000001825273 STRING (score=0.904); SRP68 virus (VSV) and retroviruses), liposomes and other lipid NSP000003120663 STRING (score=0.903); SEC61A2 containing complexes, and other macromolecular complexes NSP000003.683193 STRING (score=0.9); ERO1L capable of mediating delivery of a polynucleotide to a host NSP000003520443 STRING (score=0.899) INS cell. Vectors can also comprise other components or function NSP000003707313 STRING (score=0.899); RPN1 alities that further modulate gene delivery and/or gene NSP000002962553 STRING (score=0.899); SEC11A expression, or that otherwise provide beneficial properties to NSP000002682203 STRING (score=0.899); SEC11C the targeted cells. As described and illustrated in more detail NSP000002997143 STRING (score=0.899); SPCS1 below, such other components include, for example, compo NSP000002330253 STRING (score=0.899); SPCS3 nents that influence binding or targeting to cells (including NSP000002645973 STRING (score=0.899); SRP14 components that mediate cell-type or tissue-specific bind NSP000002678843 STRING (score=0.899); SRP19 ing); components that influence uptake of the vector nucleic NSP000002829993 STRING (score=0.899); SRP54 acid by the cell; components that influence localization of the NSP000002167743 STRING (score=0.899); SRP72 polynucleotide within the cell after uptake (Such as agents NSP000003421813 STRING (score=0.899); SRPR mediating nuclear localization); and components that influ NSP000003280233 STRING (score=0.899); SRPRB ence expression of the polynucleotide. Such components also NSP000002734063 STRING (score=0.899); STT3B might include markers, such as detectable and/or selectable NSP000002957703 STRING (score=0.899); DERL2 markers that can be used to detect or select for cells that have NSP000001587713 STRING (score=0.762); DERL3 taken up and are expressing the nucleic acid delivered by the NSP000003153033 STRING (score=0.762); EDEM1 vector. Such components can be provided as a natural feature NSP000002564973 STRING (score=0.746); SEC62 of the vector (such as the use of certain viral vectors which NSP000003376883 STRING (score=0.732): BCAP31 have components or functionalities mediating binding and P515721 EBI-1788.819, EBI-77683; ENSP000003515683 uptake), or vectors can be modified to provide Such function STRING (score=0.899). alities. Other vectors include those described by Chen et al; 0129. Examples of interacting proteins for EXOC2/Sec 5 BioTechniques, 34: 167-171 (2003). A large variety of such (Q96 KP11 ENSP000002304493) via UniProtKB, MINT, vectors are known in the art and are generally available. and/or STRING: Interactant Interaction Details GeneCard 0133. A “recombinant viral vector” refers to a viral vector External ID(s) comprise: SERGEF Q9UGK81, comprising one or more heterologous gene products or ENSP000002659653 EBI-465715, EBI-465368 STRING sequences. Since many viral vectors exhibit size-constraints (score=0.998); EXOC3 ENSP000003233773 STRING associated with packaging, the heterologous gene products or (score=0.997); EXOC8 ENSP000003535643 STRING sequences are typically introduced by replacing one or more (score=0.997); RALA ENSP000000052573 STRING portions of the viral genome. Such viruses may become rep (score=0.997); EXOC7 ENSP000003341003 STRING lication-defective, requiring the deleted function(s) to be pro (score=0.987); EXOC4 ENSP000002538613 STRING vided in trans during viral replication and encapsidation (by (score=0.973); EXOC5 ENSP000003421003 STRING using, e.g., a helper Virus or a packaging cell line carrying (score=0.973); RALB ENSP000003.653.983 STRING gene products necessary for replication and/or encapsida (score=0.972); EXOC6 ENSP000002607623 STRING tion). Modified viral vectors in which a polynucleotide to be (score=0.969); EXOC1 ENSP000003706953 STRING delivered is carried on the outside of the viral particle have (score=0.954); ARF6 ENSP000002983.163 STRING also been described (see, e.g., Curiel, DT, et al. PNAS 88: (score=0.935); DPH3 ENSP000002850823 STRING 8850-8854, 1991). (score=0.928); RHOQ ENSP000002387383 STRING I0134) Suitable nucleic acid delivery systems include viral (score=0.899); TRIP10 ENSP000003201173 STRING vector, typically sequence from at least one of an adenovirus, (score=0.899). adenovirus-associated virus (AAV), helper-dependent aden ovirus, retrovirus, or hemagglutinating virus of Japan-lipo Vectors Expressing STING: some (HVJ) complex. Preferably, the viral vector comprises a 0130. In another preferred embodiment, a vector com strong eukaryotic promoter operably linked to the polynucle prises a polynucleotide set forth as SEQID NOS: 1, 2, vari otide e.g., a cytomegalovirus (CMV) promoter. US 2013/0039933 A1 Feb. 14, 2013

0135 Additionally preferred vectors include viral vectors, 0.139. Another delivery method is to use single stranded fusion proteins and chemical conjugates. Retroviral vectors DNA producing vectors which can produce the STING intra include Moloney murine leukemia viruses and HIV-based cellularly. See for example, Chen et al. BioTechniques, 34: viruses. One preferred HIV-based viral vector comprises at 167-171 (2003), which is incorporated herein, by reference, least two vectors wherein the gag and pol genes are from an in its entirety. HIV genome and the env gene is from another virus. DNA 0140 Expression of STING may be controlled by any viral vectors are preferred. These vectors include pox vectors promoter/enhancer element known in the art, but these regu Such as Orthopox or avipox vectors, herpesvirus vectors such latory elements must be functional in the host selected for as a herpes simplex I virus (HSV) vector Geller, A.I. et al., expression. Promoters which may be used to control STING J. Neurochem, 64: 487 (1995); Lim, F., et al., in DNA Clon gene expression include, but are not limited to, cytomegalovi ing: Mammalian Systems, D. Glover, Ed. (Oxford Univ. rus (CMV) promoter (U.S. Pat. Nos. 5,385.839 and 5,168, Press, Oxford England) (1995); Geller, A.I. et al., Proc Natl. 062), the SV40 early promoter region (Benoist and Chambon, Acad. Sci. U.S.A. 90 7603 (1993); Geller, A.I., et al., Proc 1981, Nature 290:304-310), the promoter contained in the 3' Natl. Acad. Sci. USA: 87:1149 (1990), Adenovirus Vectors longterminal repeat of Rous sarcomavirus (Yamamoto, et al., LeGal LaSalle et al., Science, 259:988 (1993); Davidson, et Cell 22:787-797, 1980), the herpes thymidine kinase pro al., Nat. Genet. 3: 219 (1993); Yang, et al., J. Virol. 69: 2004 moter (Wagner et al., Proc. Natl. Acad. Sci. U.S.A. 78:1441 (1995) and Adeno-associated Virus Vectors Kaplitt, M. G., 1445, 1981), the regulatory sequences of the metallothionein et al., Nat. Genet. 8:148 (1994). gene (Brinster et al., Nature 296:39-42, 1982); prokaryotic 0.136 Pox viral vectors introduce the gene into the cells expression vectors such as the beta.-lactamase promoter cytoplasm. Avipox virus vectors result in only a short term (VIIIa-Kamaroff, et al., Proc. Natl. Acad. Sci. U.S.A. expression of the nucleic acid. Adenovirus vectors, adeno 75:3727-3731, 1978), or the tac promoter (DeBoer, et al., associated virus vectors and herpes simplex virus (HSV) vec Proc. Natl. Acad. Sci. U.S.A. 80:21-25, 1983); see also “Use tors may be an indication for Some invention embodiments. ful proteins from recombinant bacteria' in Scientific Ameri The adenovirus vector results in a shorter term expression can, 242:74–94, 1980; promoter elements from yeast or other (e.g., less than about a month) than adeno-associated virus, in fungi such as the Gal 4 promoter, the ADC (alcohol dehydro Some embodiments, may exhibit much longer expression. genase) promoter, PGK (phosphoglycerol kinase) promoter, The particular vector chosen will depend upon the target cell alkaline phosphatase promoter, and the animal transcrip and the condition being treated. The selection of appropriate tional control regions, which exhibit tissue specificity and promoters can readily be accomplished. Preferably, one have been utilized in transgenic animals: elastase I gene con would use a high expression promoter. An example of a trol region which is active in pancreatic acinar cells (Swift et suitable promoter is the 763-base-pair cytomegalovirus al., Cell 38:639-646, 1984; Ornitz et al., Cold Spring Harbor (CMV) promoter. The Rous sarcoma virus (RSV) (Davis, et Symp. Ouant. Biol. 50:399-409, 1986; MacDonald, Hepatol al., Hum Gene Ther 4:151 (1993)) and MMT promoters may ogy 7:425-515, 1987); insulin gene control region which is also be used. Certain proteins can expressed using their native active in pancreatic beta cells (Hanahan, Nature 315: 115 promoter. Other elements that can enhance expression can 122, 1985), immunoglobulin gene control region which is also be included such as an enhancer or a system that results active in lymphoid cells (Grosschedl et al., Cell 38:647-658, in high levels of expression Such as a tatgene and tar element. 1984; Adames et al., Nature 318:533-538, 1985; Alexander et This cassette can then be inserted into a vector, e.g., a plasmid al., Mol. Cell. Biol. 7:1436-1444, 1987), mouse mammary vector such as, puC19, puC118, pBR322, or other known tumor virus control region which is active intesticular, breast, plasmid vectors, that includes, for example, an E. coli origin lymphoid and mast cells (Leder et al., Cell 45:485-495, of replication. See, Sambrook, et al., Molecular Cloning: A 1986), albumin gene control region which is active in liver Laboratory Manual, Cold Spring Harbor Laboratory press, (Pinkert et al., Genes and Devel. 1:268-276, 1987), alpha (1989). The plasmid vector may also include a selectable fetoprotein gene control region which is active in liver marker Such as the B-lactamase gene for amplicillin resis (Krumlauf et al., Mol. Cell. Biol. 5:1639-1648, 1985; Ham tance, provided that the marker polypeptide does not meret al., Science 235:53-58, 1987), alpha 1-antitrypsin gene adversely effect the metabolism of the organism being control region which is active in the liver (Kelsey et al., Genes treated. The cassette can also be bound to a nucleic acid and Devel. 1: 161-171, 1987), beta-globin gene control binding moiety in a synthetic delivery system, such as the region which is active in myeloid cells (Mogram et al., Nature system disclosed in WO95/22618. 315:338-340, 1985; Kollias et al., Cell 46:89-94, 1986), myelin basic protein gene control region which is active in 0.137 If desired, the polynucleotides of the invention may oligodendrocyte cells in the brain (Readhead et al., Cell also be used with a microdelivery vehicle such as cationic 48:703-712, 1987), myosin light chain-2 gene control region liposomes and adenoviral vectors. For a review of the proce which is active in skeletal muscle (Sani, Nature 314:283-286, dures for liposome preparation, targeting and delivery of con 1985), and gonadotropic releasing hormone gene control tents, see Mannino and Gould-Fogerite, BioTechniques, region which is active in the hypothalamus (Mason et al., 6:682 (1988). See also, Felgner and Holm, Bethesda Res. Science 234:1372-1378, 1986). Lab. Focus, 11(2):21 (1989) and Maurer, R.A., Bethesda Res. 0.141. A wide variety of host/expression vector combina Lab. Focus, 11(2):25 (1989). tions may be employed in expressing the DNA sequences of 0138 Replication-defective recombinant adenoviral vec this invention. Useful expression vectors, for example, may tors, can be produced in accordance with known techniques. consist of segments of chromosomal, non-chromosomal and See, Quantin, et al., Proc. Natl. Acad. Sci. USA, 89:2581 synthetic DNA sequences. Suitable vectors include deriva 2584 (1992); Stratford-Perricadet, et al., J. Clin. Invest. tives of SV40 and known bacterial plasmids, e.g., E. coli 90:626-630 (1992); and Rosenfeld, et al., Cell, 68:143-155 plasmids col E1, pCR1, pBR322, pMal-C2, pET, pGEX (1992). (Smith et al., Gene 67:31-40, 1988), pMB9 and their deriva