Inhibitors of ETS2 activating molecules for the treatment of inflammatory diseases

Abstract

The present invention provides inhibitors of proteins that activate the erythroblast transformation-specific proto-oncogene (2)(ETS2), and their use in the treatment or prevention of inflammatory and / or autoimmune diseases. Inhibitors are generally MEK inhibitors, HSP90 inhibitors, RAF inhibitors, SRC inhibitors, and / or ERK inhibitors. The present invention also encompasses diagnostic methods and methods for screening for agents that reduce macrophage activation.

Description

[Technical Field]

[0001] The present invention relates to the treatment and / or prevention of inflammatory and / or autoimmune diseases or disorders using erythrocyte transformation-specific proto-oncogene 2 (ETS2) inhibitors and / or inhibitors of the enhancer region in chr21q22. The present invention also includes diagnostic methods and methods for screening agents that reduce macrophage activation. [Background technology]

[0002] The global incidence of autoimmunity is steadily increasing, with nearly 5% of the world's population suffering from one or more autoimmune / inflammatory diseases. These heterogeneous conditions, ranging from inflammatory bowel disease (IBD) to systemic lupus erythematosus and multiple sclerosis, share an urgent need for better treatments, yet only about 10% of drugs that enter clinical development become approved therapies.[1] This high failure rate is mainly due to a lack of efficacy[2] and is increasing the urgent need to better understand the disease mechanisms. Genetics offers a unique opportunity to do this, with hundreds of regions of the human genome now directly linked to the development of at least one autoimmune disease.[3] In fact, drugs that target pathways identified by genetics are far more likely to constitute effective therapies.[4, 5]

[0003] However, to fully realize the potential of genetics, knowledge of where disease-risk variants exist must first be translated into an understanding of how they contribute to the disease[3]. This is a significant challenge because most disease-associated gene variants are not located in coding DNA where their impact on protein sequences / structures can be easily determined, but rather in the enigmatic non-coding genome where the same DNA sequence can produce different biological outcomes depending on cell type and / or external stimuli[3]. Most risk variants are thought to affect gene regulation[6], but the need to identify causative genes that may be located up to 1 million base pairs apart, and causative cell types that may express the causative gene only under specific conditions, has so far hindered attempts to discover disease mechanisms. For example, the genome-wide association study (GWAS) for IBD7 has reported more than 240 risk loci, but fewer than 10 have been mechanically resolved, and none have led to new therapies to date. [Overview of the Initiative]

[0004] Here, we demonstrate that the intergenetic "gene desert" on chr21q22, which predisposes individuals to several chronic inflammatory diseases, is a monocyte / macrophage-specific distal enhancer of ETS2, and that risk haplotypes describe a gain-of-function molecular mechanism by which ETS2 expression is altered. Furthermore, by combining a CRISPR-based loss-of-function technique in primary human macrophages cultured under chronic inflammatory conditions, targeting both the intergenetic region and ETS2, with a method for delivering dose-dependent overexpression, we show that ETS2, the gene primarily studied for its role in cancer, is a central regulator of the macrophage inflammatory response.

[0005] In line with this, the inventors identified that the transcriptional footprint of ETS2 is detectable in diseased tissues of multiple inflammatory conditions and is more rich in IBD-risk variants than most previously described disease processes, including autophagy. By screening a database of over 30,000 cell signatures, the inventors discovered potential means of therapeutically targeting this pathway and validated a class of approved small molecules using both in vitro and in vivo models, thereby illustrating the potential of genetics to reveal novel disease biology.

[0006] The present invention provides a method for treating or preventing a disease in a subject, comprising administering an inhibitor of a protein that activates erythroblast transformation-specific proto-oncogene 2 (ETS2) to the subject, wherein the protein that activates erythroblast transformation-specific proto-oncogene 2 (ETS2) is selected from the list consisting of MEK, HSP90, RAF, SRC, and ERK, and the disease is an inflammatory and / or autoimmune disease. The present invention provides a method for treating or preventing a disease in a subject, comprising administering an erythroblast transformation-specific proto-oncogene 2 (ETS2) inhibitor to the subject. The present invention provides a method for treating or preventing a disease in a subject, comprising administering an inhibitor of the chr21q22 enhancer (SEQ ID NO: 1) to the subject. The present invention provides a method for treating or preventing a disease in a subject, comprising modifying or deleting a portion of the chr21q22 enhancer (SEQ ID NO: 1) to the subject, optionally deleting the entire chr21q22 enhancer (SEQ ID NO: 1). The present invention provides a method for reducing macrophage activation by contacting the macrophages with an inhibitor of erythrocyte transformation-specific proto-oncogene 2 (ETS2). The present invention provides a method for reducing macrophage activation by contacting the macrophages with an inhibitor of chr21q22 enhancer (SEQ ID NO: 1). The present invention provides a method for reducing macrophage activation by modifying or deleting a portion of chr21q22 enhancer (SEQ ID NO: 1), wherein the entire chr21q22 enhancer (SEQ ID NO: 1) is optionally deleted. The present invention provides a method for reducing pro-inflammatory cytokine production by contacting macrophages with an inhibitor of erythrocyte transformation-specific proto-oncogene 2 (ETS2). The present invention provides a method for reducing pro-inflammatory cytokine production by contacting macrophages with an inhibitor of chr21q22 enhancer (SEQ ID NO: 1).The present invention provides a method for reducing pro-inflammatory cytokine production by modifying or deleting a portion of the chr21q22 enhancer (SEQ ID NO: 1), wherein the entire chr21q22 enhancer (SEQ ID NO: 1) is optionally deleted. The present invention provides a method for reducing reactive oxygen species (ROS) production by contacting macrophages with an inhibitor of erythrocyte transformation-specific proto-oncogene 2 (ETS2). The present invention provides a method for reducing reactive oxygen species (ROS) production by contacting macrophages with an inhibitor of the chr21q22 enhancer (SEQ ID NO: 1). The present invention provides a method for reducing reactive oxygen species (ROS) production by modifying or deleting a portion of the chr21q22 enhancer (SEQ ID NO: 1), wherein the entire chr21q22 enhancer (SEQ ID NO: 1) is optionally deleted. The present invention provides a method for reducing macrophage phagocytosis by contacting said macrophages with an inhibitor of erythrocyte transformation-specific proto-oncogene 2 (ETS2).

[0007] The present invention provides a method for reducing macrophage phagocytosis by contacting the macrophages with an inhibitor of the chr21q22 enhancer (sequence number 1). The present invention provides a method for reducing macrophage phagocytosis by modifying or deleting a portion of the chr21q22 enhancer (sequence number 1), wherein the entire chr21q22 enhancer (sequence number 1) is optionally deleted. The present invention provides a method for reducing macrophage migration by contacting the macrophages with an inhibitor of erythrocyte transformation-specific proto-oncogene 2 (ETS2). The present invention provides a method for reducing macrophage migration by contacting the macrophages with an inhibitor of the chr21q22 enhancer (sequence number 1). The present invention provides a method for reducing macrophage migration by modifying or deleting a portion of the chr21q22 enhancer (sequence number 1), wherein the entire chr21q22 enhancer (sequence number 1) is optionally deleted.

[0008] In some embodiments, reduced macrophage activation is indicated by one or more of the following: reduced cell activation, reduced pro-inflammatory cytokine production, metabolic reprogramming, reduced reactive oxygen species production, and / or reduced macrophage migration. In some embodiments, reduced pro-inflammatory cytokine production includes a reduction in the level of one or more of TNFα, IL-1, IL-6, IL-8, IL-12, IL-23, and / or IL-18. In some embodiments, reduced reactive oxygen species (ROS) production includes a reduction in the level of one or more of hydrogen peroxide, hydroxyl radicals, superoxide anions, and / or singlet oxygen. In some embodiments, reduced macrophage phagocytosis is indicated by a reduction in macrophage uptake of particulate matter (e.g., bacteria), quantified by an in vitro or in vivo assay. In some embodiments, reduced macrophage migration is indicated by in vivo measurement of macrophage motility (e.g., quantified by a chemotaxis assay) and / or a reduction in the total number of macrophages accumulating at the site of inflammation in vivo.

[0009] The present invention provides a method for treating a disease in a subject requiring treatment, comprising administering an erythroblast transformation-specific proto-oncogene 2 (ETS2) inhibitor to the subject, thereby reducing macrophage activation in the subject. The present invention provides a method for treating a disease in a subject requiring treatment, comprising administering a chr21q22 enhancer (SEQ ID NO: 1) inhibitor to the subject, thereby reducing macrophage activation in the subject. The present invention provides a method for treating a disease in a subject requiring treatment, comprising modifying or deleting a portion of the chr21q22 enhancer (SEQ ID NO: 1), thereby reducing macrophage activation in the subject.

[0010] In some embodiments, the subjects have inflammatory diseases and / or autoimmune diseases. In some embodiments, the inflammatory disease is selected from the list consisting of Crohn's disease, ulcerative colitis, inflammatory bowel disease (Crohn's disease and / or ulcerative colitis), ankylosing spondylitis, primary sclerosing cholangitis, Takayasu's arteritis, and rheumatoid arthritis. In some embodiments, the disease is an autoinflammatory disease. In some embodiments, the disease is a CHR21Q22-related disease. In some embodiments, the disease is selected from the group consisting of Crohn's disease, ulcerative colitis, primary sclerosing cholangitis, ankylosing spondylitis, and Takayasu's arteritis.

[0011] The present invention provides an inhibitor of a protein that activates erythroblast transformation-specific proto-oncogene 2 (ETS2) for use in a method of treating or preventing a disease in a subject, wherein the method comprises administering the inhibitor of the protein that activates erythroblast transformation-specific proto-oncogene 2 (ETS2) to a subject, the protein that activates erythroblast transformation-specific proto-oncogene 2 (ETS2) being selected from a list consisting of MEK, HSP90, RAF, SRC, and ERK, and the disease being an inflammatory and / or autoimmune disease. The present invention provides an erythroblast transformation-specific proto-oncogene 2 (ETS2) inhibitor for use in a method of treating or preventing a disease in a subject. The present invention provides an inhibitor of the chr21q22 enhancer (SEQ ID NO: 1) for use in a method of treating or preventing a disease in a subject. The present invention provides an erythroblast transformation-specific proto-oncogene 2 (ETS2) inhibitor for use in a method of reducing macrophage activation by contacting the macrophages with the erythroblast transformation-specific proto-oncogene 2 (ETS2) inhibitor. The present invention provides a chr21q22 enhancer (SEQ ID NO: 1) inhibitor for use in a method of reducing macrophage activation by contacting the macrophages with the chr21q22 enhancer (SEQ ID NO: 1) inhibitor. The present invention provides an erythrocyte transformation-specific proto-oncogene 2 (ETS2) inhibitor for use in a method of reducing pro-inflammatory cytokine production by contacting macrophages with the erythrocyte transformation-specific proto-oncogene 2 (ETS2) inhibitor. The present invention provides a chr21q22 enhancer (SEQ ID NO: 1) inhibitor for use in a method of reducing pro-inflammatory cytokine production by contacting macrophages with the chr21q22 enhancer (SEQ ID NO: 1) inhibitor. The present invention provides an erythrocyte transformation-specific proto-oncogene 2 (ETS2) inhibitor for use in a method of reducing reactive oxygen species (ROS) production by contacting macrophages with the erythrocyte transformation-specific proto-oncogene 2 (ETS2) inhibitor.The present invention provides a chr21q22 enhancer (SEQ ID NO: 1) inhibitor for use in a method of reducing reactive oxygen species (ROS) production by contacting macrophages with the inhibitor of the chr21q22 enhancer (SEQ ID NO: 1). The present invention provides an erythroblast transformation-specific proto-oncogene 2 (ETS2) inhibitor for use in a method of reducing macrophage phagocytosis by contacting said macrophages with the inhibitor of the erythroblast transformation-specific proto-oncogene 2 (ETS2). The present invention provides a chr21q22 enhancer (SEQ ID NO: 1) inhibitor for use in a method of reducing macrophage phagocytosis by contacting said macrophages with the inhibitor of the chr21q22 enhancer (SEQ ID NO: 1). The present invention provides an erythroblast transformation-specific proto-oncogene 2 (ETS2) inhibitor for use in a method of reducing macrophage migration by contacting said macrophages with the inhibitor of the erythroblast transformation-specific proto-oncogene 2 (ETS2). The present invention provides an inhibitor of CHR21Q22 enhancer (SEQ ID NO: 1) for use in a method of reducing macrophage migration by contacting the macrophages with an inhibitor of CHR21Q22 enhancer (SEQ ID NO: 1).

[0012] In some embodiments, reduced macrophage activation is indicated by one or more of the following: reduced cell activation, reduced pro-inflammatory cytokine production, metabolic reprogramming, reduced reactive oxygen species production, and / or reduced macrophage migration. In some embodiments, reduced pro-inflammatory cytokine production includes a reduction in the level of one or more of the following: TNFα, IL-1, IL-6, IL-8, IL-12, IL-23, and / or IL-18. In some embodiments, reduced reactive oxygen species (ROS) production includes a reduction in the level of one or more of the following: hydrogen peroxide, hydroxyl radical, superoxide anion, and / or singlet oxygen. In some embodiments, reduced macrophage phagocytosis is indicated by a reduction in macrophage uptake of particulate matter (e.g., bacteria), quantified by an in vitro or in vivo assay. In some embodiments, reduced macrophage migration is indicated by in vivo measurement of macrophage motility (e.g., quantified by a chemotaxis assay) and / or a reduction in the total number of macrophages accumulating at the site of inflammation in vivo.

[0013] The present invention provides an erythroblast transformation-specific proto-oncogene 2 (ETS2) inhibitor for use in a method of treating a disease in a subject requiring treatment for the disease, wherein the method comprises administering the erythroblast transformation-specific proto-oncogene 2 (ETS2) inhibitor to the subject, thereby reducing macrophage activation in the subject. The present invention also provides a chr21q22 enhancer (SEQ ID NO: 1) inhibitor for use in a method of treating a disease in a subject requiring treatment for the disease, wherein the method comprises administering the chr21q22 enhancer (SEQ ID NO: 1) inhibitor to the subject, thereby reducing macrophage activation in the subject.

[0014] In some embodiments, the subjects have inflammatory diseases and / or autoimmune diseases. In some embodiments, the inflammatory disease is selected from the list consisting of Crohn's disease, ulcerative colitis, inflammatory bowel disease (Crohn's disease and / or ulcerative colitis), ankylosing spondylitis, primary sclerosing cholangitis, Takayasu's arteritis, and rheumatoid arthritis. In some embodiments, the disease is an autoinflammatory disease. In some embodiments, the disease is a CHR21Q22-related disease. In some embodiments, the disease is selected from the group consisting of Crohn's disease, ulcerative colitis, primary sclerosing cholangitis, ankylosing spondylitis, and Takayasu's arteritis.

[0015] In some embodiments, the ETS2 inhibitor or chr21q22 enhancer inhibitor comprises an antisense nucleic acid molecule, a small interfering RNA (siRNA), or a short hairpin RNA (shRNA). In some embodiments, the ETS2 inhibitor or chr21q22 enhancer inhibitor comprises a small molecule, an inhibitory peptide, an antibody, or a nanobody. In some embodiments, the antibody is a monoclonal antibody that targets a surface marker specific to ETS2-positive macrophages to inhibit and / or induce disruption of effector function. In some embodiments, the nanobody targets a surface marker specific to ETS2-positive macrophages to inhibit and / or induce disruption of effector function.

[0016] In some embodiments, the ETS2 inhibitor or chr21q22 enhancer inhibitor includes a proteolytically targeted chimera (PROTAC) that targets ETS2 for degradation, a molecular adhesive that targets ETS2 for degradation, an antibody-drug conjugate, an antibody-RNA conjugate, an esterase-sensitive motif conjugate drug, or a nanobody-drug conjugate. In some embodiments, the antibody-drug conjugate includes an anti-CD163 antigen-binding molecule, and optionally, the antigen-binding molecule is an antibody. In some embodiments, the antibody-drug conjugate includes a MEK inhibitor, and optionally, the MEK inhibitor is selected from the list consisting of selumetinib, trametinib, and cobimetinib, preferably, the MEK inhibitor is selumetinib.

[0017] In some embodiments, the antibody-drug conjugate includes a cleavable linker, which is optionally glucuronide-based (e.g., GlyPro). In some embodiments, the antibody-drug conjugate includes a non-cleavable linker, which is optionally maleimide-PEG3-based.

[0018] In some embodiments, the esterase-sensitive motif conjugate drug comprises cyclopentyl L-leucinate or cyclopentyl(S)-2-amino-2-cyclohexyl acetate. In some embodiments, the esterase-sensitive motif conjugate drug comprises a MEK inhibitor, optionally selected from the list consisting of selumetinib, trametinib, and cobimetinib. In some embodiments, the esterase-sensitive motif conjugate drug comprises a linker. In some embodiments, the linker is one or two carbons long.

[0019] In some embodiments, the ETS2 inhibitor comprises a CRISPR nuclease system including a CRISPR-related protein (Cas protein) and a guide RNA (gRNA) that specifically binds to the ETS2 genome nucleic acid sequence, optionally comprising the sequence of Sequence ID No. 5. In some embodiments, the chr21q22 enhancer inhibitor comprises a CRISPR nuclease system including a CRISPR-related protein (Cas protein) and a guide RNA (gRNA) that specifically binds to the chr21q22 enhancer nucleic acid sequence, optionally comprising the sequence of Sequence ID No. 1.

[0020] Sequence ID 5 (chr21:40176507-40197479) is shown below. GTGATTGTTTCAGCCGTATAGATTCTGACTGTGACTCATGCCACGGTGATACCCGGAGAC CTTCGGGGACCCAGAAACATTAAGGTGCACCGATATCTCAATTATCCGGATAACTCTAGA CCTAGAGTCACCATGGAAACCTAGAACCCTGAGTCACAGAGAAGCTGGGCCATTCCTCTC CCTTCCCGGTGGTGCCGCAGTTCTTGAGATGTGAGCAGCTTTCAGCCCCGTGGCCTCGCC TAAGCTGGGGTTTCCTATTTCATCCACAACTTCAGGAATCTAAACTTTCCTGCAAAATAT TGAGCATGTGACCTGAGTCCAAACAGCCATGGACTCAAGCACCTTCTGATAAAAACGGAA ACCACAGTGTAGGGGGTGGTTTTGACCAGGGTTTCGGAGTCAGATAAACTCAGGTTTCCA CCTTGAATTTCTCATCAATCTGACGTGGCAGGTTATTCATCCTAAGGTTCAGTTCCCACC TGTGTAAAGTGGGAGCCGCAAGTCCTCCGAGAGTGACGATGATGTGCGTGGAGTGCCCAG CCCAGATTGAAGCGCGGCCAAGGCGGGTCGCTATCTGGGCACCGCTCAGCTCCAGAGGGC GCCACTCCCGCGGAGCCTGCGGGATCGGGGCTTCCCGGGAGCAGCGCGATCAGCACCACG ACTCGGGGACACAGCCAGGGCCCGGTTTCTACAGGAAGCGCCTCATTTGGAGCCTTTTTG TGATAGAATGATCATTAGTCCTAAGCCCATTCAGAGGTTCAAGAATGGGGTCGGCTCAAT TTCAGGGCCTTATTACCCAAGCCCGGCTGCCCTTCGGTGCCACCAGCACCACTGCTCCGT CGCTGCGGAATTCCAAAGGCAGGTTTGGCGTTAGGGCCTTGGCCCCAGAGAGGACGCCGA GCGCTCCACGGAAAGTCTCCGCCCGGCTCCCAGGGCGCACACTCGCGCGCACGTGGGGCC GAGGCCCTGCTCCCGGGGCCTCAGGGCCAGCCGGCGAGGGACCCAGCCGAGTGACAGCAG GAGGCGGAGGGAAGGTTGGGCCGGAAGGTGTCAGCCCCGCCCCGCGCTCCCTCGCCGCCT CCGCCCTCCTCTTCTCTCCCCTCGTGCGTTCCCTCTCCTCTCCCTCCGCTCCCCCAACCC TCCTGCTGCCCCCTTCCCTCTCCTCCCGCTTCTCCCCATCCTGCCTACCTCCCTTCCCCT CCTCTTCTCTCTCCTCCCCTTCCCTCCCCTCTCTCTTCTCTCCTCCCTCGTTTCCTCCCC TCCCCTCCACTCGGCCGTCCCTCCTTCCTCCTCCCTCCTCCCTCCTCCTCCCGCTCCTGA AGAGCGCGCCGCGTGGGGGACGGCCCGGTTACTTCCTCCAGAGACTGACGAGTGCGGTGT CGCTCCAGCTCAGAGCTCCCGGAGCCGCCCGGCCAGCGTCCGGCCTCCCTGATCGTCTCT GGCCGGCGCCCTCGCCCTCGCCCGGCGCGCACCGAGCAGCCGCGGGCGCCGAGCAGCCAC CGTCCCGACCAAGCGCCGGCCCTGCCCGCAGCGGCAGGGTAAGAGCTGGGCCCGCAGAGA GCGCCCGGCGCGCGGCTCCAGTCCCATGGAGGGTCACCCGGGGCCTGGGCGGGGGTCGCG GGGGGCACTGACACGCAGATCTCGGGGCGCTGCCGGGGGTGCAGGTGGGGGTGGCGGCTG CTGCGAGGACTCTAGGGGCGCGCGTCTGAGTTCCGCGCCGGCTCGTTTTCCGGTTATGGA GTGGCCTCCGGGGCTGGCGGGGTCGGCCGGGGGGTTCCTGCGTGCTAGGGCCGCTGTCTT CGGGGTCGCCTAGCGGCGGGCGCGGCCAGGGCGCGCTGGCTTGTTTCGCTCGCTTTTGTT TTTAAAAGGAAACGCAGGCCTGGTAGGGGGTCCTGCCCAGTGGATGTCCCGGCGAACATG ATTTCGCGAACGGGAGTGGGGGCACAGGAGAGCGTGTCCGAGGTGGCCTGGCGCCCCGGC TTTGAGGGTGACTTCCTGGAGCGGCGCCGGGCCCGGAGGATCTGGGGCGCCCAAGACACC TGAAGGCTGCGGCACCGCGGGAACCTGCGGGGCGCGGGGTGCCATGGTCACCTGCTCGCC GCGTCCAGGGCCCGGGCTGGGGACCCCTCGGTCGTGCGAGGAGAGCGTGGGGAACCTGTC GGAAATGAGATCTGGTTGCGCTGGGCTGCCTTTATTTTCTCGTTCCTACAGCATTTGAAT GAAGAGGTAACTGAGTGTTTGCTTGTGTGTGTTTGGGTTGCGTGTGTGTTATCCTATTTT ATTTTTTACGGCAGGAGACCTTTTATGTTAGCCTGTACACAATTTCAGGGTAGCCTAAAA CAGTAGTTGACGCGCTAGTTATTTAGAAAGTAAAGAAATGAGCAGTCCCCTTTGGAGACA GGAGTAATTTATTTTAATTCGCCAGTAAGAGATTATATTTGTTCCATACAATGGAAGCGC CTGTGCGTTATCTGCCTGCCCACACCACCACCAATCAGAGAGATAAATCTCCACCGTTAAG CTTTATTAAAATTCTGAGTAGTAGCGCAGCACAGTAAATGTTAGCACCGACCATCCGCAG GGAAATGCTCAGATACTCATTTCTGGGATTAGTCATTCATCTCTCTAAACTGATCGCTTT CAAAGCGAAAAAAGCAAACTACCTTCAAATGTGTGTTACAAGGTTGTGTTGTTTCTTTTT TTTGTTTGTTTTCAAATTTATCTAAATAATCTTACATAAGTGATTACTTGTTCCAATAAG GGTACTGTTGTCAGGCAAATTCTCCTTGTTTTTAAATGTAACCATTCTCAGCTGTTACAT TTCTGCTCTTTATCCTTTTTTTTTTTTTTTTTTTTGGTTTAAGCAGCGATCCATCAGCAA CACCAAACTTGAAATTGATTTATGTGGAAAAACTTGGCTTGTCTGCCATCTAACAAGCCC TGTTGAGTAAAATAAGCAAGCTTAAATTTGATTAGTTGTGTGTCTGCCTGAAACCATTTA GTAGAGCACTTTAATTCTGCATGGTTTTTACAAAACTCATTAAAAGCTGGACAACAAAAG AATTCTATTCTGTAGCTAGTAATTACAGCTCTTTATGTGGGAGTGGTAGGCTGCCTTTTC TCCTGGTATTTGTACACAAAAGCTGGGGAGAGCTTTTCCACTGCCTTCCACATTCATTCT CTGTCCATCTCAGAGAAGCTGAAAAACAGAATCAAGTCAGCAAGCAGTCTGCAAGCCTAA GGAAGAAAAAGAGCTGACCCATCCGGCTCTGGATGGATAACTGGCAACACCCAAGATGTC TATGATTTTGGCCTTCTGTTTGAGGCTTGTTTATTTCTTTAATCAGCAGCTACCAGGACT TATTTCATGCAGAGAAATGACATAAAGCTCTAGAGGAAGGAGCATTTCACATTGGAAGGA GTAGTGAGAAGGGCCTGATCTGATAATATCAGGATGGGTGGGAAGGGTCCATTCCTCATC ATTCATGTTCTCCCGGCACAGAGGACTGTGTGGAGGACTCAGGATTTAGGACCATACTCA ACTGGGGTTTGATCTCCACTTTGCCCTGCGGTGGCCCCTCTGGTAGCTATGTGGATCCTG GGCAGATTGTCCATGTTGCTGGGCCTCCGTTGCTTCACTGTATGACGTCTGCCTCTCAGG GATGCAGGAAGATTCAATTAGGTAGGAGTGGATGGTCCCCCAGCACGGGGCTTAGCTCTT AGTAACTAGGCATTCTGGCAATGGCAGCCATCAATAGGAGAGACTATTGATTGTTGAGTG AATATAAGTGGCATAATCCACCAAGTGGAGATTCTGAGTGAATGTGGCCACTTTGGACCC AAGACTGCTGCCCCTCTCTGTCCTCTCATAGGACTATTATTTATTGCCAGTGCTGACCAG CCTTTGTTGGCTACCATGATCATTACCTGCACTCCGAGTGTTTTAACAGAATTTTCTGAT GCCTCTGGCCTCACCTGCTGCCTTTTAGCCAAGAGGGGTGTGTGTGTGTATGTGTGTGTGTG TGTGTGTGTGTGTGTTGGGTGTGTGTTGGGTAGCAGGGTTCTTGGAAGGTCTGGTTTAGC CATAATGTTGATCATAAAGAGAAAACAAGAAAATAAATGCAAATTAAATAGTTCGGTGAG CCTTTGTTATTTCGCCACATCCAGTAAGTTCTATCAGTTGAATGTTGTCTGAATTACTTA AAAAGTTCTCAGTTGTAAACAGGTAAGAGCACCCAGTGGAGGCCATGGAGCTAGGCTTAG AGCTTTGCCACCAGCCTTCAGTTCCATAGCCATGAACCTGGGAGGGCAAGGAATCACCCT AGAGGTGAAGGCCATTCATCGGTGCTAACCCAAGGTCTCTGAAAATGAGTTTTAGTTACT CTATTATCTAAGTATCTTTCTTACTGATATGTAACTTACCTGCTGAACAATTACATTTT AGAGCTCCAAATAAATTTTCAGCCTATTATGTCATTGTCCAATTACAAGCCTCATTGTAA TAGCCATGTTAAAAACAATAAAGCCGGTGAAATTGATTTCAGTGTATTTCAAATTAACCT AATATATTCGAAATATTATGTATGTATAAAAATTATTGAGCTTTTTTCTTTTTTCCACTA AGTCTTGAGAGCTAGTGTGTATGTGTGGATTTGTACTAGCCACTGGTTGAGAGCTCTGT AGCCACACATGGCAAGTGGGTACCATATTGCTTAGCCCAGGCCTAGTAGAAGATTAAGAC TCAGTCAGAGGAAGTAAGGCAGATCCGTGCTTGGAATCAGTTCCTCTTGAAGAAGTGCAC AGCCAAGCAACACACAAAGCAGAAATATTTTGAGCTCTGTATTCAAAGCAGGAAGATCTG GGACCATGTAAGGGGGTTTGGTCTTCAGTTCTCCCTTTGATCCCTTCCCTGGGTGTGTGC TTTCTTACAGCCTGGAGAGTGGAAGGGAAGAGAGGGGGCTTCCTGGACTCCTTTCCTGTC TCTAGAAATGACCCCTGCAGCTGTCCTGAAGCTCTCAGGTTAAGCTGATTGTGTCCAGAG CAGACAGGAGGCGGCCCACTGCCCCACTGTACCCACCCAAGCACTCACCTAAGCTGGCCA TGGAAATGTGAATGAAAAGCCTGACCCAGGCCCTCTTGCCCTGCTAACCCAGCAAGGAGG CTCTGACCAGCCAGCCAGCAAGTGACTTTTTCTGTGACATCTAAGTGGAATTCAGTGAGG AATCAGCATTATTATTCAATCACTGAAACAGCATATCCCATTGTGATACTGAGAAATGCT TTCCTCTTGAGACCTTTGGGAGGACAGCCATTTAAACAAGAATTCCCTGCATTCACTTTT GAATTACCTGTTAGCTGTAGAACTCAGCCAGGATCAAGTCTTGATGCCACCAATGATGAG TTTAGTGTACTTAATCTTTTGCCTCTTTGACTTTTTTTTTTTCTTTTTTAAGATGAATGA TTTCGGAATCAAGAATATGGACCAGGTAGCCCCTGTGGCTAACAGTTACAGAGGGACACT CAAGGTGAGTGGGCAAGTCTTAATTTTTTTTTTTAATTGGAAAACTCGATCTCTAGGAGG AAAGAAAAAAAAGGCCTGGGTCCCAGAAAACTGGTTGTAGCCCTAGCTTCTTAATTTATT GGCCATGTGACTATAGACTATTTGCTTACTTGCTCTGGTCCTCAGTTTTCTGGTCTGCAG AATGGGTATCTTGATGTGTGTTGTTCTTCTCCTCCACGATATGAAACCAAAGGCCTTTAT AGCTTGTTTTCTGAGGCTCCTTCCAGGTGAAAATCCTATAACTCCCAATAAAACAGCTGG AGAGGCTGTCTCTACTGCCTTTTCTGTCTACCCAGGAGTTTCCTTGGTTGATGGGAAGCT GTTTAAGAACTGAAATGAGAGAGAAGAGGCAGACAGACCCAACCGCTACAGCTCCTATCA GAAAGGGCAGCGCCCGTGGAATTGTAGGTAGCCTGTTACTGGCTCAGAGAACAAGCAGGC CTACTTATGTTAAACCAAAAACCAGATGAAAAATGAAAGCGCCCTCTCTGCTGCATAAAC ATTTTATGTAGCTTCTATCATCATTCCTTTTGGGAACATTCTTTAGTAGCACAAACTGGT AATCTCCCACTTGTCTTCTCCAAATATGACAGCAAGATTTGATTGTTTTCAGTATTATGT AGTAAACATGTTTCAGAAGCATGATTTTAAAATTGGCCTCCTCAAAGTTTAGCGTCTTGC ATAATGATGATGTACGTCTCTGGCATATTACATTTTCCTTTGTATATCATTATTGAGGTT ATTTGTCTGATATGACCCAAAGAGGCAAAACTCAGCACAGTCCTTTCTGCAGTATTCTAA AGGTCATCAAACTTCAGCCTAGTGAGTCTGCTTGTTTGATTTGGCCGGACATTTTAAGCA TGGCAGAAGTGGTACAAGAAATCATGGTATTAAGTTGAAACCACACCCCTTAGAAAAATC CTTCTATTAATTCAAATAATTTGACGATGCTTATGCGGTTTCTGAAAAGAAGCAGTCGTT GCTGAAATTGATGTGTTGAAATAGGAAGCACAGATTTGTATTGTCTTTTGGCTTCCTGGC TTTAAAAAAAAAAAAAAAGAATTTACGGTTAGGAAAGGCATTTCTTAGTCACCTGGGCAA TGCTTGTGGGAACTTTAAAAGTTTATTGGAGGTTTCACCAAAATTAGCTCTGCTCTAAAG TAGTTTGTAAATCCAGATGAGTAATTTGCACTTGCCTGATTGATCGGTTTTCCTCGTAGA TCTCAGGCAGTTGGTCATCATTGACTTGGATTTTTTTGGGCTGGGCTCCAGCCTCTGCCT GCCCTCTTTTGTAATGCGTCATTCATTGTTCTGCATTTCTAAAAAGAAAGCGGGTAACCG ATTTTATTTGTAAATGTGTTACACATTTTCTTATTGATGACTCAACAGATGGGTCATTGT ATTACACATAACATGGGATTTCCAGCTTATGTTGGAAAAATATAGTCTTTCTTCACGTGT TCCTCAACAATTTAGGCGATGATGAATCCAGATATTATCGGTTACTTTCAAATGAGTCTT ACATTAAAAGGTATTTCTTTCATAAATGTTATGTAAGATGCTCAGAAATCGAGTTAGTGT AAGGTTTTATTTTCTGTGTGTCTCTTCTTTTCTGCAGATAGTTTAGTTTAACTTTAAAC CTATTTCCTTTGGAAATCTTTCCCCTTAGTAGGTGATTTGGGTTTTTTTTGTTTTGTTTT ATTGTTTTTTCGTTTTTGGTTTTAAGCAACAGGGTCCTTCTCTGTTGCCCAGGCGGGAG TACAGTGGCTTGATCGTAGTTCACCGCAGACTTGAACTCCTGGGCTCAAGTGATCCTCTG GCCTCAACCTCATAAAGCACTGGGGTTACAGGCATAAGCCACCCCGCCTGGCCTGGGGGC GGGGAGGGGTTCATTTTTAAGACACTTTACTTGTTATAAAATGAAACGTTAAGAATTT TGTCATGTATTATTATTTTCCAAACCATTGGAATTGATGTGGAAAAGCATCTCTAGTTTC AGTTAAATTGTTTCATGGCAAACATTTCAAAATAAATGCTTCCAAAGATAAAGTAGTGTA TTTCATTGTTTGATTTTTCAGTGTTTATCGGGTCATTTGCCTTTTAACTGCATTGTGCTC AGACGGCCTTGTCAAGGGTCATTTTTCTTACACACACAGCCTAGTTGAATTTTTCTCAAT GTAACAGGAGGGTCAGGTGTTTGGTAAAGGGGTCTCTGAGGCTCCTCTCGACACCATTGT AAGATTCTGTTATTTTCAGATTTGTGTATTTAATATATTAACTTAAGTGGATTAGGCCAC TAAAACAAATTCCCAGTCACTCCGACTCTGTGAGGTTACTTAAATAAATCCACAGTGGCC GGGCACCGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCAAGGCGGGTGGATTAC ACGAGGTCAGGAGTTTGAGACCAGCCTGGCCAACATGGTGAAACCCTGTCTGTACTAAAA ATGCAAAAATTATCCAGGGCGTGGTGGTGGGCGCCTGTAGTCCCAGCTACTCGGGAGGCTG AGGCAGGAGAATCGCTTGAACCCAGGAGGGGGAGGTTGCAGTGAGCCAAGATTGCACCAC TGCACTCTGGCCTGTGTGACAGAGTAAGACCTGTCTCAAAAATAATAATAATAATCCA CAGTAAAATACAATCATTTCAGTCACTTTGACCATTTTACGAGTCTGTTTTAAGGACTGA CTGGTTGATTCCATGTTTGCTTCAAAATTGCCAGTTTAATCAGGGACTATTGGTAGGATT GCCCACAGACCACTTTTACAACAGGTAATTCAATCAGTTTAAATATTGTATTTCCATTTT TTTTCCATCTGTTTTTGCAGCGCCAGCCAGCCTTTGACACCTTTGATGGGTCCCTGTTTG CTGTTTTTCCTTCTCTAAATGAAGAGCAAACACTGCAAGAAGTGCCAACAGGCTTGGATT CCATTTCTCATGGTAATTGGTTCCTCAGACTTGACAAATTGTGCATGATTTTCCTAAGTA GTTCAGTTAATAAAGAGATGACAGTTCCTAAGTGAGAAATGTTGTATCTGGAATCTTTG ATATGAATAGCCAGAAGTTCTGGACCTGGGAGAAGGCAGTTTTTCATTTGTATCAACTTG GTGATTCTGATGTGGATTCTGATGTCTTGAGTGCCAGACGCCCCCCAGTGAGCCGCTGTT AGGAAACTGCAGGGGAACCCCTGAGCTGCCCCTCCTGGATGTTTTCAACATTGGCAAGCA GGTCCTCATCTCCCTGCTCTGCCCTGGAAAACAGACAGAAGAGTAGACATTAAAACACAC CCAAACTTGTAATTTGGGTTTGCATCATTAGTTAAATCATCTCCCCTGCCCTGAGTTTTG GGGCAACAAATTGTTCTTTGTAATCCTTACCCATCGGTAGTTCTTTCTGTAGTTCTTCGT GTCATTGTTTATTCTTCTTAAAGGAATAGCACGTCAGATAATGTTCATACAATTACTGTG ACAGAGCTATTAGTTGGAAACATGCCAGAGATGACTGTGTCATGTGAAGTAGGTGCTGAG AATACAGGCCAGAAAATTAATTAAAACGCATACACTGGCGTAAGCATAACCCTGCATGGC AGCCATTTATCATTCTTGAAATTGCTGAGGGTGTCTGGAGAAGATTGTTTTTCAATAAAA ATGGAAGGTGTGGTTCCATTACCGTGCTAAGTGCGGCACATTCATATCACACTCACTTCT CAGCTCTACGCCTAGTTTAATGGAAGTGACTTCCAAATGATACAGTCCCTTCAGCCTTTT GACCAGAAACCATGCAACTGTTTTCATTGCCTACAATTTTAAAATAGACTTAGCAGTTTT CCCTACGCACAACTGCTTTGTGACGCTTTGCAAAATAGAATCAAATCTGAGTTTTATTTT AGATTTTTTTAATAAGTATAGTTCTGAGATATATATTTTCTCAATTATAGTCAGAAAAGT TTTTTGTTAATAGTTACACTGTTTTAAGGAATCATGCCAAGGTTTGAGATCAAAATTGTT CTTTTCCAAAAACTAAGATGTCTCTCCTAAATCTCCACCTGATATCACCAACTTGAAGTC CTAATGTCCCCATGGGGGGTTTCCTTCCAGACTCCGCCAACTGTGAATTGCCTTTGTTAA CCCCGTGCAGCAAGGCTGTGATGAGTCAAGCCTTAAAAGCTACCTTCAGTGGCTTCAAAA AGGAACAGCGGCGCCTGGGCATTCCAAAGAGTAAGTACTGCTTTCCTGAGCCTGCACTGG GTGAGAAGAACCAACTTCAGTGCAGTTGTTTGATCTTGACTTGTTTTATTAAGCTTTTGCT TGGGGTATTCTGCAAAGAGTAGCATGGATGTCGTTAACCTGAGCCAGTTTCTGTTTCACC CCAATCAACCCAAGACTTGATCCAGAACTCATTAAATATGTAGTAGAAGGACGCATTACT GGTGTCTTGAAATGTGCCTGGGTCGTGTCAGAATGGTGACGTGTCATCATGGTATCTTGC TCATTCGTGGGTTCTGGTGTATGTCGGTACTTGGTGCATAAATTAGGGATGACAGTGGTC TCACTACCTTTCCACTGTTTTTACCTCATGTGTTCCATTTTTTCTTTCTCTCCCTGGTAGAC CCCTGGCTGTGGAGTGAGCAACAGGTATGCCAGTGGCTTCTCTGGGCCACCAATGAGTTC AGTCTGGTGAACGTGAATCTGCAGAGGTTCGGCATGAATGGCCAGATGCTGTGTAACCTT GGCAAGGAACGCTTTCTGGAGCTGGCACCTGACTTTGTGGGTGACATTCTCTGGGAACAT CTGGAGCAAATGATCAAAGGTACCAGCTGAACGTCTTACTTCTCCTTGTCCAGGATGAGC TGTGGCCGGGAAGACTGATTGGGAAAGTCACGTGGGTGTTCTTCAACCTTAGGGTTGCCA CTTGAAATGACATAGAGTACCTTGCCTCAAAATGCCACTCAAGTGAGTCAGATATATGGC AGTGATTATAGATTTTTATCCCACTTTATGTGAGTGTGTGTGTGTGTGTGTGTGTGTGTG TATAGCATCAAGTATAGCCACAAGGTAGTAGCCTTAGTCACTAAATTGTTTGCTATTGCT GGCTGTATTCATCACGGGAGTCCATGTTGACTCAGATATGTAGGACAGCAAGAATTCCCA CGTCTTCTGAGCTCACCTTACAGAGCTAAGAGATATGGCAGCCTTAATGAAAGGGGGGAG CCTTTACTCACATAAACCCTGTGAATTCTACTGTAAATTTTCCTACATCCAAATGGAATG TATTTTGGATGTTGAGGATTGTTGAGGGGGCGGGGTCTGTTTTTAAACAAGTGAGAAATT GATATATATTTACACGCTATCATATTGTACAGAAACTGGCCATTGATCAGTAGAAATCTC ACCAAATACAGAGAAAGTCTTGATTCAAAACTTAAAATAGCAGGGAGACCAATGTTTAAA ACATGTTGAGTTAAAAAAGAAAAGAGGGCCAGGTGCGGTGGCTCACACCTGTAATCCCAG CACTTTGGGAGGCGGAGGCGGGCAGATTACTTGAGGTTAGGAGTTCAAGACCAGCCTAGC CAATATGGTGAAACCCTGTCTCTACTAAAAATACAAAAAATAAAAATATTAGCTGGGCAT AGTGGCACATGCCTGTAATCCCAGATACTCGGAAGCCTGAGGCAGGAGAATCCCTTGAGC CCGGGAGGCAGAGGTTGCAGTGAGTCGAGATCACACCACTGCACTCCAGCCTGTGGGAGT GAGACTCTGTTGAAAGGAAGGAAGGAAGGAAGGAAGGAAGGAAGGAAGGAAGGAAGGAAG GAAGGAAGGAAGGAAGGAAGGAAGGAAGGAGGGAGGGAGGGAAGGAAGGAAGGAAAGAAA GAGGGAGGGAGGGAGGGGAGGGAGGGAGGGAGGGAGGGAGGGGAGGGAGGGAGGGAGGGA GGGAGGGAAAGAAATAAAATACAGTAGGGATTATTTAGGTTAAAAAAATATAAGAAAATA AGCATGTTTATAGAATTCAGGGAACTAGTCCTCACCTGACTGATTTTAAACACACTGAAT TTCAGCATCAACTGTAGACTCAGAATCTCGAAAACACCTAGGATTCAACAGCTAACTTTT TTTAGTTACGGCGTCTGAGGGCCAGGGAAACTCATTTTTAAAGACCCTCATTTCTTCTGA ACTTGGACAGGGCTTTTCTACCAAGTTTCTGGTGATCTGTTTATTGGCAAATTGGGCCTG TTTTACCATTCCTCGGGCCTGTGAGTTTGTGTGTATTTGGGTTTCCGGGTGGACGTGCAC TTTGTAATATATTCGTAGTAAGTATGCATACACAGTGCACAAACCTTTTCCCTTTCAAAG TTCTTGGGCACATGTGTTCCCCATCCCAGACTCCCACAGCCCTGGAGACTTCCCACACCC TGGGGAAGCGTGCAGGTTTGGAAGCAGTGGAACTGTAAGCTTCACGGGGGCAGACACCAT CCATTTTATTAATTTCCTGTATGCAGCCTCTGGTACAGATGGCATTTCATAAATACTTAA CTGTTTCTTGAATGCATGAATGAATTCAATACACCATCTGGACCTTTTGCGATAGAAAAT TTAGTGTTGCCAATGACATCATTCATTTTGGAAAATTTATTTTAGTAATTGGAAAAATGG GTTGCTTTCTCTCGTTTTTTGAAAAAGACAGGCAGGGACGTGGAGCACCATGAGATTAAA TGTGAAGCACTCCGTGCATCTCATCATTCTAAAATTATCATCTCACAGGAATTCAGAAAG TCAATTTGTTCTGTGTAGTTTTCACCTACATCTGCCATCTTACGTCTCCTGTGTCTGCTT TTCAGAAAACCAAGAAAAGACAGAAGATCAATATGAAGAAAATTCACACCTCACCTCCGT TCCTCATTGGATTAACAGCAATACATTAGGTCAGTCCGATTGATTCTGCCCTTAAGAACT TTGTCTTCAGTCTTCCCAGTAGACTTGGAATCTCTCTACTGTAGGCTCCTAAGGGGCCAC CTAGACCTGTGTGTCTCTTATGGTGGCCACACGTGGGTGTTGAGCTATAAGCCATTGAAA TGTGACTGGTCCACATCGAGATGTGTAGGAAGAGTATAGTCATGCACACCAGATTTGGAA GTCTTAGTATGAAACAGAGAATATAAAATGTCTCCCTGATAATTTTTAATGTATTGATTA CATATTGAAATGATAATTGCTATATGAGGCCAAGTAAAATAAGAAAATTAACTTCACCTG TTTCTTTTTGCTTTTTTAATATGGCCACTAGAACTTTCGCAGTTGCTTAAGTGGCTCACA CGATCTTCCATAGGTCAGCACTGTTAGTGCTAGTCCAGGCAATTCTTAGAGCAAGGAGCA GAACGAAGTGAGCTGATTACCGCAGTCACCAGTAATGTTGGTTTAGTGTAGCAAACAAGA GGCGGCAGGCACAGAGTGCGTGAAAAGCAATTTGTAAACTGGCTCTGAACTTGGGAGCTT AAAGAGCTGTCATGGAATCTGGGTCCTTTCTTCTGGTTGCTGATACTGGGGAGGGCACAG GGTCTGGCCAGGCCAGCGAGGGAGGGGTCCTCGGAACAGCAGGCTTGGGTGGTGTTGGTC CGGCCCAGTGTTTTCTCTCTGGCCCCAGCTCCACCATTCAGACCCGGTGAAGCCCACCTC TCTGCCCCACTGGGGGCTTCCTGCTCAGGAGGTCTTTCTGAACATGACAGTGGGACACCC TGTGCCTGCCTGACCTGAATCAGAGTCAGAGAGCTTCCTTCCCTCTCCAGAAAATAAAT TATTCTGCAATGAACAGAGACAGGAGACGGTGTGATGAAGGAGCAGAAGCAGAGTGGGCT GTGCCTCAGCACACAGGGTTCATGGCCTGGTCTGCCCTTATGAATCACACAGTACATGA CACGTCACTTGGTCTGTCTGAGCCTTACTCTCCTCGGAAGCTAAAAGAGCCAGTGAGCCC TGTCTTCCTGAAGGTCCCATGAAAGCACTAGGGTTGTAGAATAGGAACATTGAGTCCTG CCTGCAGAGGCTGCAGTCATTTTGGCAGAGGGCTGCTGGTTGAGAAGCTGAATGGTTTGA GAAGAAGGCCTGGTGACAGACCCCACCAAAGCCCGGGTCCCACTGAGGTTCTGCAGAG GGCTGGAGTGTGCGGAGTGCTCACCTGTTCATTGCTCGTAGGGGTTAGTTACTGGGGTAA CACTGACTTTAAGAGCTCTGCCGTCCGATTGTTCTGTTCCAGGTTTGGCACAGAGCAGGG CGCCCTATGGAATGCAGACACAGAATTACCCCAAAGGCGGCCTCCTGGACAGCATGTGTC CGGCCTCCACACCCAGCGTACTCAGCTCTGAGCAGGAGTTTCAGATGTTCCCCAAGTCTC GGCTCAGCTCCGTCAGCGTCACCTACTGCTCTGTCAGTCAGGACTTCCCAGGCAGCAACT TGAATTTGCTCACCAACAATTCTGGTAAGATTGGAAGCATCTTTCAACAAGGCTGTTGCT TTGATTCTGAGAACCCCAGAGCCATAATGAACCTCTTAATAAATACTTCCTGGATTCAGC CATTAGAGAAGGGGGTCAAAGCCCATGTTCTAAGTGGATTTCCAACAAGCATACCCCTAA TATGTTTCAGGCACTGGCCCAGGTACTGCAGGCATTGAGTAAAAAACAGTCCCTGCTC TTACAGCCAGATTTCTCCACACTTTGGGATATTTATGGAGGCGATGCAGGAATGTGACAT TTCTTCTTATGGAGATGTAACATAAATACAGTGCCAAAGTCTCAGGTCATGTGGCTGGTT GGATTTGTGTATGTATGTATACCCATGTAATAACCATCCAGATGGAGATAGAGAAGTTTC CAGTAGCCTGGAAGATTCCTTCGTACCCCTTCCCATCAATAATGTCCCCCCGACAGAAGG GAACGATTCTGACTTTTATCATTACGGATTAGGTTTTGATTTTCTTGAACTTTACATAGA TGGAATCATAAGGCATGTTTTCTTTTGTGTTTGTCTTTTACTCATCATGTCTGGGATTCA TGCATGTTGTTGGATGTAGCAGCCATGTGTTCTTTTCATCGCTGTATAGTATTCCATTA TAAGAATTTTATTCATTCTACTGTTGATGGAAGATAGGATTTGTGTAGTATAGTTACCCT AAGGAATAAGCACCTGAGTAGAGAGCATTTTGAGAAGTCTGAGTGGTTCTACACCAGCAG GTGCATGATTGCACTGGTAGTGGTTGGTGATGCTATGGTCGTGCCCAAGACCAACTGTGT CCTGGAGGAATCAAAGTATGTGTTTGGTTGTCTTTGCCAGGGACTCCCAAAGACCACGAC TCCCCTGAGAACGGTGCGGACAGCTTCGAGAGCTCAGACTCCCTCCTCCAGTCCTGGAAC AGCCAGTCGTCCTTGCTGGATGTGCAACGGGTTCCTTCCTTCGAGAGCTTCGAAGATGAC TGCAGCCAGTCTCTCTGCCTCAATAAGCCAACCATGTCTTTCAAGGATTACATCCAAGAG AGGAGTGACCCAGTGGAGCAAGGCAAACCAGTTATACCTGCAGCTGTGCTGGCCGGCTTC ACAGGTGTGTGTGGAACTCCGAGAGCCTGGCCGCCCAGTCTCCTGGGTCCTGTCCCTTGC TTCCTTTCGAGCCACAGTACCACATTCACCGAGGGTGTTTCTAAGCTAGGTACACCAGTG CTCTACACTCCATGTTTTATGCGTGGCTTGCTGTATCTCTGAATTCGACAAAAGCCACAC TTGGAGGAATTTTCATGTACACAGAGCTCCATGGAATGAGTGAAAATTGACAGGCCACTC CCTCCACCTGGGACGTTCTGAGGAATAGTTGGAGCAGGGGACATGGCCTGTGCAGTCTTC AGCCCTGACTCACCCACTCCAAGAATCTCAAAGAATGTAAACTGGCTTAGGGAGGGGCAG CAGAGCCAGTCTCAAATAATATGGGCACTGTTTACCTTTTCTTGCTTCTAAAATCTATCT GCTCTTATTTTTGAGGTTTGGGTGAGAGCTATTCTAGGCAGTGGACAAATATAAAGCTGT TTTTAGAAACAGGTATTTCCAGTACTTTAGCGAAAATACTGGGGCTTTGTTTTCACTGCT TCGTGTATCCTTAAATGCGTTTTTGTTTTTGTTTTTTTAACCATATGGAGCCACTGTGGA TTGAAATTACCTAACTTAAAAAAATCTGATAAGTTTAAAATAAGGATACTGAGGCATTCA TTCTTTTTCTGTCATCTAAGAACAATCAGCCACGTAAACGTATTAAAATACACGGAAAGA GTGTGTGAAAGCCATTATCGGTTTCATTATTGGGAGTTTAGGGCCTGAGAAGAATAAACA GCCATAACGTTAGTGTGGTTCATTTTAATAGTAACATAAGTTTCTGTTTATTGACTGCCA ACTCTGTAGTTAAGTAGGCATACAGTCTGTAGGACAGCCTGGCATGGGGGTGGTATGAGG CCATTTTACAGATGGGGAAATTGGCTCACAGGCAGACAGCTATCGGTGCTCACACAGCTC TGGTTCTAGAATGCCTGTCCCGTGCTTTACAGCAAAGCTTTATTGATAACGGCCATTGCG TTTCTGAGGAAGAGCTATTTGGTAGAAAGAGGTTGGGCTTAGGGTCGGAAGATCTAGGCT CTTCTCCTGTTTGCTGTGATGGGCGTGTGATCCAGGACAAGGCACCTGGCCTCTGAGCCG CATCTTATCCCTCCTCTTTAGAACAGAACCACACTGACACCTGCTTTGCAAGGTGGTAGT TCTAAATGTCAATCGAGGACAGGTGGACGAGAAGCTCTTGGCAAAGCATGCTTCACCAGA CAGAGGCTGTGAGGCAACGGGTGTGACCCCGCGTGGCTATTGAGTAAACCGGGCTCACAT TAAGCAATTTACACATGGTCATTTGGCAGGAAGGTGGCATGGGATCCCACGTATGACACG CCTCATTCTGTGATGAAACACCCCCTCAGCCACCCCCATCTCTGCCCCACTGGGTGTTTC TAGAAAAAAACCTTGAAGGTTTTTACATAAGGAACAATTAAACCAGATGGGGAATTTTAA TGCCAAGAGTTGGTGGCCCTAAACTTTCTTCCAGAAAGAACAAACCTAGCAATCAAGGGG AAGAGTGTCTCGCCTAGTGACCTTATTTTCTAGGAGTAGGCAGTGTGGAGCAGGAACTCA CATTTGGTGCCCCGCCCCATCCCGTTAAAGCACTTAGTACTGTCACCAACACCTTGAGTG CCACCCTGAGTGTAACATCGGAACCCCATTCAGAGAGTTGGGTCTGCATTCCTAAATCAG CATGTACAATTAGGATGGTTAAAGACTTGCTGTATTTTACATCTGTGAAAGGGTATGATC CGTCTCCCTCCCTCTCCCCGCAGGAAGTGGACCTATTCAGCTGTGGCAGTTTCTCCTGGA GCTGCTATCAGACAAATCCTGCCAGTCATTCATCAGCTGGACTGGAGACGGATGGGAGTT TAAGCTCGCCGACCCCGATGAGGTATGGCCAGAGCCCTGGGAAATCTCTGGGCTTGAAAA CCTGATTTCCTGCTTGCATTCAAAAACTCAGTTCTTTGGGCACAAAAAAGGGTTCACCAG TACTGCTGAGAATCTTTCCACGTGAGGCATCCTTGGCTGTTGGGAAAATGGAAGTGGAGT CATTGCTTTGTTGATAAACGTGTACAGTGTTTTCTGGGTATGCTTCATACAAGGGCGTTG CACAGATTTCACTGTCTTGATCAGTTGTCTGATCAAGAGGCCCAAGCTGCAAACTGAGGT TCTCTGCTGACCATCTGAAAATGCCTTAGGAAGGCTGCCCTTGTCCATCGGGGGAACAAG CCTCATGTGGCCCCGAGCCAGCCTTCTGTCCACTCCTTAGCTGAGCCCAGCAGTGTTGAC CCCAGCCCTGCAGAGTTAGAACCAGGAGCCCTGTCTAAAGGAGAAGGCCAAGGGCAGGTG GGATACCAACCCCGCCAGAAAAGCGTTAGAGAGAAGCCCTTGGTCTCTGGCACTGGGGAG CGTAATCTGTCACCATTCCCACCCCGCCTCCAGTTCCCTTGGGAGACAAGCATTGAGGAA TGAGTATATCACACAGCTCAGAATCCCACTCGGCACCTGTTAACTTCAGTAGTTGGAATA TCCAGCCTGGAGGGTGGGTGACACCACCTTTCCCTGGTCTCAGGACCCTCCTGGCTCTGA ACCCTTGGCTCCCAGGAGGTTTCACTGAGCTGGGGCCAGGGAGCAGGGACCTCATTCCCC AGTGGTTCTGCCCCTTGGGGACACAGTGCCCCTACCATAGGTACTCAAAGGTACTCAGAG GTACTCAAAAGGTCCTCCTGCGGACCTTGTGTAGGTGTCAAGTTCTCTCTAGAGTGAACA TGCCTCAGAATCATAATCAGGGAGGAATGTCATTCACTTTTTCTTCATTGACAAATTGAG TTTAACTCTTTTCCATCCATGTTCACCAAAGGTGGCCCGCCGGTGGGGAAAGAGGAAAAA TAAGCCCAAGATGAACTACGAGAAGCTGAGCCGGGGCTTACGCTACTATTACGACAAGAA CATCATCCACAAGACGTCGGGGAAGCGCTACGTGTACCGCTTCGTGTGCGACCTCCAGAA CTTGCTGGGGTTCACGCCCGAGGAACTGCACGCCATCCTGGGCGTCCAGCCCGACACGGA GGACTGAGGTCGCCGGGACCACCCTGAGCCGGCCCCAGGCTCGTGGACTGAGTGGGAAGC CCATCCTGACCAGCTGCTCCGAGGACCCAGGAAAGGCAGGATTGAAAATGTCCAGGAAAG TGGCCAAGAAGCAGTGGCCTTATTGCATCCCAAACCACGCCTCTTGACCAGGCTGCCTCC CTTGTGGCAGCAACGGCACAGCTAATTCTACTCACAGTGCTTTTAAGTGAAAATGGTCGA GAAAGAGGCACCAGGAAGCCGTCCTGGCGCCTGGCAGTCCGTGGGACGGGATGGTTCTGG CTGTTTGAGATTCTCAAAGGAGCGAGCATGTCGTGGACACACACAGACTATTTTTAGATT TTCTTTTGCCTTTTGCAACCAGGAACAGCAAATGCAAAAACTCTTTGAGAGGGTAGGAGG GTGGGAAGGAAACAACCATGTCATTTCAGAAGTTAGTTTGTATATATTATTATAATCTTA TAATTGTTCTCAGAATCCCTTAACAGTTGTATTTAACAGAAATTGTATATTGTAATTTAA AATAATTATATAACTGTATTTGAAATAAGAATTCAGACATCTGAGGTTTTATTTCATTTT TCAATAGCACATATGGAATTTTGCAAAGATTTAATCTGCCAAGGGCCGACTAAGAGAAGT TGTAAAGTATGTATTATTTACATTTAATAGACTTACAGGGATAAGGCCTGTGGGGGGTAA TCCCTGCTTTTTGTGTTTTTTTGTTTGTTTGTTTGTTTGTTTTTGGGGGGTTTTCTTGCC TTGGTTGTCTGGCAAGGACTTTGTACATTTGGGAGTTTTTATGAGAAACTTAAATGTTAT TATCTGGGCTTATATCTGGCCTCTGCTTTCTCCTTTAATTGTAAAGTAAAAGCTATAAAG CAGTATTTTTCTTGACAAATGGCATATGTTTTCCACTTCTTTGCATGCGTTTAAGTCAGT TTATACACAAAATGGATTTTATTTTTTAGTTTAACTGTGTTTCTCCGACAGCTCACCTCT CTCTGACCACCCAGCCATTTCCTTCCTGTGCTCCACGTTCTTCTGTGTGATTAAAATAAG AATATTATTTTTGGAAATATGCAACTCCTTTTCAGAGATCAGGAGGGATTTATGTAGCAG CTATTTTTACTGCAAAAGTAATTCACTGGAAAAAAAATGTAATTTGTAAGAAAGCTTTAT TTTTATCTCAGCTCTATGTAAAGTTAAAGTTACTGTACAGAGCTGAAGGACGGGGGGCGG TAGGGGTCTTGATGAAACCTCTTGAACGAAGCACAGTTTGTCCCATCTTTGTTCACTCGT GTGTCTCAACCATCTTAATAGCATGCTGCTCCTTTTTGCTCAGTGTCCACAGCAAGATGA CGTGATTCTTATTTTCTTGGACACAGACTATTCTGAGGCACAGAGCGGGGACTTAAGATG GGAAAGAGAAAGCATCGGAGCCATTCATTCGGAGAAAACGTTTTGATCAAAATGGAGACT TTTGTAGTCGTTTCAAAAGAGCACCTGAGTCATGTGTATTCCCGGCCTTTATAAATGACC CGGTCAAGTTGGTTTCAAAGTCCGACAGGCTTGTCTGTTTACTAGCTGCGTGGCCTTGGA CGGGTGGCTGACATCTGTAAAGAATCCTCCTGTGATGAAACTGAGGAATCGGGTGGCCGG GCAAGCTGGGAAGAGCAAAGCCAGAGCTGCGCTGCCTCAATACCCACAAAAGACCATTCC CAGTATACATAAGCACAGGATGTTTTTCTCAAGAGGGATGTATTTATCACTTGGACATCT GTTTATAATATAAACAGACATGTGACTGGGAACATCTTGCTGCCAAAAGAATCCTAGGCA GTGGCTCATTGTATGTGAGGTTGAACCACGTGAAATTGCCAATATTAGGCTGGCTTTTAT CTACAAAGAAGGAGTTTCATGGGGTTCAGCCTAACAGTTATGGAAACTACAGTCCTTATA AACCATTGGCATGGTAATAAACAGATCTTAAGTATAAAAATTTTGTAATTGGGCCTTTAC TCTCTCAATAATAAAGTATTTTGTTTATATAAATTCTTTGTGATAGTCCTCGTTCTTCCT CTCCACACCCAGCATGAAGGAGTTGGAGGAAGGATGTTAACCCCAGATCCATTCTCTACT CAAAACATTCCATCATCAAGCGGCAAGTCTCTGTTTAACTGGTTTATACACAAGTCACTT AGAAACCACAACCCAAATTGGAATCAACTTTGAGCCCTTCCTAAAAGAATTCCCAAAAAG TGCTCTCTTTCAAAACAAAAAATTCTTTTAAGAAAGTGTTATAATAGAAAGATTCAAATG TCTTTCTTTGCCAGAAGCTTGGCAGAGATAACAGAGGAGAGATTCTGGAATGTTTATATT [[ID=?]]AAGTACATTAGAATTTGAGGTTTAAGTACTTTTGGAACTGAGGCCACAACACTCTGTCCC CTCAGTGGAGTCTGACATTGGTAAGGTGATGGTGGTCTTAGGACACCTGTTTTCAAATTT GTTCTCTTTACATGCTCCAAAATTAGTGAGTATGCCAAAAAGCTTATATTCATTGGTGCG CTAGAGCCAGCTCACTGTGTTCTCCCGAGAGCTACATGTCCCTTCCCATCATCGCATCAG TAATGGTAGCTTGAGATGGGGCGTGATGAGAGT (SEQ ID NO: 5)

[0021] It should be noted that there seems to be a problem with the "配列番号5" in the original text. I've translated it as "SEQ ID NO: 5" according to common practice for sequence number translation in patent texts. Also, there might be a mislabeling in your provided content where the "ID=?" should probably be "ID=12" for proper numbering continuity. Please double-check the original content for accuracy.In some embodiments, the ETS2 inhibitor comprises a small interfering RNA (siRNA) molecule including a sense strand. In some embodiments, the sense strand consists of 15 to 30 linked nucleosides. In some embodiments, the sense strand contains a sequence having at least 95% identity with the isolength portion of the pregenomic RNA and / or the mRNA encoding erythroblast-transforming proto-oncogene 2 (ETS2). In some embodiments, the sense strand contains a sequence having 100% identity with the isolength portion of the pregenomic RNA and / or the mRNA encoding erythroblast-transforming proto-oncogene 2 (ETS2). In some embodiments, the small interfering RNA (siRNA) molecule includes an antisense strand. In some embodiments, the antisense strand is at least 80%, at least 90%, at least 95%, or at least 99% complementary to the sense strand. In some embodiments, the antisense strand is fully complementary to the sense strand.

[0022] In some embodiments, ETS2 inhibitors can inhibit ETS2 expression in vitro by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%. In some embodiments, chr21q22 enhancer inhibitors can inhibit ETS2 expression in vitro by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%.

[0023] In some embodiments, the ETS2 inhibitor comprises an antisense nucleic acid molecule. In some embodiments, the antisense compound is an antisense oligonucleotide. In some embodiments, the antisense compound specifically binds to an ETS2 mRNA sequence containing SEQ ID NO: 2 or SEQ ID NO: 3, or to an ETS2 genomic DNA sequence containing SEQ ID NO: 5. In some embodiments, the antisense compound specifically binds to an ETS2 mRNA sequence containing SEQ ID NO: 2.

[0024] In some embodiments, the antisense oligonucleotide includes at least one modified nucleoside bond. In some embodiments, the modified nucleoside bond is a phosphorothioate bond. In some embodiments, the antisense oligonucleotide includes at least one modified sugar moiety. In some embodiments, the modified sugar moiety is a 2'-O-methoxyethyl sugar moiety. In some embodiments, the antisense oligonucleotide includes at least one modified nucleic acid base. In some embodiments, the modified nucleic acid base is 5-methylcytosine. In some embodiments, the antisense oligonucleotide is a chimeric oligonucleotide.

[0025] In some embodiments, the ETS2 inhibitor comprises a compound containing an ETS2 inhibitor and a conjugate group. In some embodiments, the chr21q22 enhancer inhibitor comprises a compound containing a chr21q22 enhancer inhibitor and a conjugate group. In some embodiments, the conjugate group comprises one or more antibodies or their antigen-binding moieties, e.g., Fab fragments. In some embodiments, the conjugate group comprises one or more carbohydrates. In some embodiments, the conjugate group comprises one or more galactose moieties, one or more lactose moieties, one or more N-acetyl-galactosamine moieties, and / or one or more mannose moieties. In some embodiments, the conjugate group comprises one or more N-acetyl-galactosamine moieties.

[0026] In some embodiments, the conjugate group comprises nanoparticles for delivering ETS2 or chr21q22 enhancer inhibitors to macrophages. In some embodiments, the conjugate group comprises a lipid carrier. In some embodiments, the lipid carrier comprises one or more components selected from the list consisting of poly(lactic acid) (PLA) nanoparticles, poly(D,L-lactic acid-coglycolic acid) (PLGA) nanoparticles, liposomes, lipid nanoparticles, micelles, reverse micelles, lipid crypts, and lipid microtubules.

[0027] The present invention provides a method for diagnosing or determining the prognosis of inflammatory or autoimmune diseases in a subject based on the expression status of erythrocyte transformation-specific proto-oncogene 2 (ETS2). The present invention also provides a method for diagnosing or determining the prognosis of inflammatory or autoimmune diseases in a subject based on the expression status of chr21q22 enhancer (SEQ ID NO: 1).

[0028] In some embodiments, determining the expression status of ETS2 or the chr21q22 enhancer involves determining one or more states in a biological sample obtained from a subject, selected from a list consisting of ETS2 mRNA levels, ETS2 protein levels, ETS2 DNA methylation status, ETS2 epigenetic status (such as histone modifications, RNA changes, or conformational changes), chr21q22 enhancer DNA methylation status, and chr21q22 enhancer epigenetic status (such as histone modifications, RNA changes, or conformational changes). In some embodiments, determining the expression status of ETS2 involves determining the ETS2 mRNA level or the ETS2 protein level. In some embodiments, determining the expression status of ETS2 involves a step of quantifying the expression status of an RNA transcript or cDNA molecule, the RNA or cDNA expression status being quantified using one or more of the following: microarray analysis, real-time quantitative PCR, DNA sequencing, RNA sequencing, Northern blot analysis, in situ hybridization, and / or detection and quantification of a specific binding molecule (e.g., an antibody). In some embodiments, the diagnostic method of the present invention further includes the step of comparing or normalizing the expression status of ETS2 with the expression status of a reference gene.

[0029] The present invention provides a method for treating or preventing a disease in a subject, comprising administering an inhibitor of a protein that activates erythroblast transformation-specific proto-oncogene 2 (ETS2) to the subject. In some embodiments, the protein that activates erythroblast transformation-specific proto-oncogene 2 (ETS2) is selected from a list consisting of MEK, HSP90, RAF, SRC, and ERK. In some embodiments, the MEK inhibitor is selected from a list consisting of selumetinib, trametinib, and cobimetinib, preferably the MEK inhibitor being selumetinib. In some embodiments, the inhibitor of the protein that activates erythroblast transformation-specific proto-oncogene 2 (ETS2) is an antibody-drug conjugate, an antibody-RNA conjugate, an esterase-sensitive motif conjugate drug, or a nanobody-drug conjugate. In some embodiments, the antibody-drug conjugate comprises an anti-CD163 antigen-binding molecule, and optionally, the antigen-binding molecule is an antibody. In some embodiments, the antibody-drug conjugate includes a cleavable linker, which is optionally glucuronide-based (e.g., GlyPro). In some embodiments, the antibody-drug conjugate includes a non-cleavable linker, which is optionally maleimide-PEG3-based. In some embodiments, the esterase-sensitive motif conjugate drug includes cyclopentyl L-leucinate or cyclopentyl(S)-2-amino-2-cyclohexyl acetate. In some embodiments, the esterase-sensitive motif conjugate drug includes a linker. In some embodiments, the linker is one or two carbons long. In some embodiments, the subject has an inflammatory disease and / or autoimmune disease. In some embodiments, the inflammatory disease is selected from the list consisting of Crohn's disease, ulcerative colitis, inflammatory bowel disease (Crohn's disease and / or ulcerative colitis), ankylosing spondylitis, primary sclerosing cholangitis, Takayasu's arteritis, and rheumatoid arthritis.

[0030] The present invention provides an inhibitor of a protein that activates erythroblast transformation-specific proto-oncogene 2 (ETS2) for use in a method of treating or preventing a disease in a subject, wherein the method comprises administering the inhibitor of the protein that activates erythroblast transformation-specific proto-oncogene 2 (ETS2) to the subject. In some embodiments, the protein that activates erythroblast transformation-specific proto-oncogene 2 (ETS2) is selected from a list consisting of MEK, HSP90, RAF, SRC, and ERK. In some embodiments, the MEK inhibitor is selected from a list consisting of selumetinib, trametinib, and cobimetinib, preferably the MEK inhibitor is selumetinib. In some embodiments, the inhibitor of the protein that activates erythroblast transformation-specific proto-oncogene 2 (ETS2) is an antibody-drug conjugate, an antibody-RNA conjugate, an esterase-sensitive motif conjugate drug, or a nanobody-drug conjugate. In some embodiments, the antibody-drug conjugate comprises an anti-CD163 antigen-binding molecule, and optionally, the antigen-binding molecule is an antibody. In some embodiments, the antibody-drug conjugate includes a cleavable linker, which is optionally glucuronide-based (e.g., GlyPro). In some embodiments, the antibody-drug conjugate includes a non-cleavable linker, which is optionally maleimide-PEG3-based. In some embodiments, the esterase-sensitive motif conjugate drug includes cyclopentyl L-leucinate or cyclopentyl(S)-2-amino-2-cyclohexyl acetate. In some embodiments, the esterase-sensitive motif conjugate drug includes a linker. In some embodiments, the linker is one or two carbons long. In some embodiments, the subject has an inflammatory disease and / or autoimmune disease. In some embodiments, the inflammatory disease is selected from the list consisting of Crohn's disease, ulcerative colitis, inflammatory bowel disease (Crohn's disease and / or ulcerative colitis), ankylosing spondylitis, primary sclerosing cholangitis, Takayasu's arteritis, and rheumatoid arthritis.

[0031] The present invention provides a method for reducing macrophage activation by contacting macrophages with an inhibitor of a protein that activates erythroid transformation-specific proto-oncogene 2 (ETS2). The present invention provides a method for reducing pro-inflammatory cytokine production by contacting macrophages with an inhibitor of a protein that activates erythroid transformation-specific proto-oncogene 2 (ETS2). The present invention provides a method for reducing reactive oxygen species (ROS) production by contacting macrophages with an inhibitor of a protein that activates erythroid transformation-specific proto-oncogene 2 (ETS2). The present invention provides a method for reducing macrophage phagocytosis by contacting macrophages with an inhibitor of a protein that activates erythroid transformation-specific proto-oncogene 2 (ETS2). The present invention provides a method for reducing macrophage migration by contacting macrophages with an inhibitor of a protein that activates erythroid transformation-specific proto-oncogene 2 (ETS2). The present invention provides an inhibitor of a protein that activates erythroid transformation-specific proto-oncogene 2 (ETS2) for use in a method of reducing macrophage activation by contacting the macrophages with an inhibitor of the protein that activates erythroid transformation-specific proto-oncogene 2 (ETS2). The present invention provides an inhibitor of a protein that activates erythroid transformation-specific proto-oncogene 2 (ETS2) for use in a method of reducing pro-inflammatory cytokine production by contacting macrophages with an inhibitor of the protein that activates erythroid transformation-specific proto-oncogene 2 (ETS2). The present invention provides an inhibitor of a protein that activates erythroid transformation-specific proto-oncogene 2 (ETS2) for use in a method of reducing reactive oxygen species (ROS) production by contacting macrophages with an inhibitor of the protein that activates erythroid transformation-specific proto-oncogene 2 (ETS2).The present invention provides an inhibitor of a protein that activates erythroid transformation-specific proto-oncogene 2 (ETS2) for use in a method of reducing macrophage phagocytosis by contacting the macrophages with an inhibitor of a protein that activates erythroid transformation-specific proto-oncogene 2 (ETS2). The present invention provides an inhibitor of a protein that activates erythroid transformation-specific proto-oncogene 2 (ETS2) for use in a method of reducing macrophage migration by contacting the macrophages with an inhibitor of a protein that activates erythroid transformation-specific proto-oncogene 2 (ETS2).

[0032] In some embodiments, the protein that activates erythroblast transformation-specific proto-oncogene 2 (ETS2) is selected from the list consisting of MEK, HSP90, RAF, SRC, and ERK. In some embodiments, the MEK inhibitor is selected from the list consisting of selumetinib, trametinib, and cobimetinib, and preferably the MEK inhibitor is selumetinib.

[0033] The present invention relates to a method for screening drugs that reduce macrophage activation, (a) bringing macrophages into contact with the candidate drug, (b) Determining the expression status of ETS2, This invention provides a method for identifying drugs that reduce ETS2 expression as drugs that reduce macrophage activation.

[0034] In some embodiments, determining the expression status of ETS2 includes determining the ETS2 mRNA level or the ETS2 protein level.

[0035] The present invention relates to a method for screening candidate genes involved in macrophage activation, (a) Introducing a CRISPR nuclease system containing a CRISPR-related protein (Cas protein) and a guide RNA (gRNA) that specifically binds to the ETS2 genome nucleic acid sequence into macrophages, (b) Measuring the expression status of one or more candidate genes, (c) A method is provided for identifying a gene as being associated with macrophage activation by comparing its expression status with the reference expression status of the same gene from control cells that do not contain a CRISPR nuclease system, wherein a gene having increased or decreased expression relative to the reference expression status is identified as being associated with macrophage activation.

[0036] In some embodiments, the ETS2 genome nucleic acid sequence includes the sequence of SEQ ID NO: 5.

[0037] The present invention relates to a method for screening candidate genes involved in macrophage activation, (a) Introducing a CRISPR nuclease system containing a CRISPR-related protein (Cas protein) and a guide RNA (gRNA) that specifically binds to the chr21q22 enhancer nucleic acid sequence into macrophages, (b) Measuring the expression status of one or more candidate genes, (c) A method is provided for identifying a gene as being associated with macrophage activation by comparing its expression status with the reference expression status of the same gene from control cells that do not contain a CRISPR nuclease system, wherein a gene having increased or decreased expression relative to the reference expression status is identified as being associated with macrophage activation.

[0038] In some embodiments, the chr21q22 enhancer nucleic acid sequence includes the sequence of sequence number 1.

[0039] The present invention relates to a method for screening candidate genes involved in macrophage activation, (a) Introducing a CRISPR nuclease system containing a CRISPR-related protein (Cas protein) and a guide RNA (gRNA) that specifically binds to candidate genes in the genome nucleic acid sequence into macrophages, (b) Measuring the expression status of ETS2, (c) A method is provided for identifying a gene associated with macrophage activation by comparing the expression status of ETS2 with the reference expression status of ETS2 from control cells that do not contain a CRISPR nuclease system, thereby identifying a candidate gene that regulates ETS2 expression status and is associated with macrophage activation.

[0040] In some embodiments, the expression status of one or more candidate genes is quantified using one or more of the following techniques: microarray analysis, real-time quantitative PCR, DNA sequencing, RNA sequencing, Northern blot analysis, in situ hybridization, and / or detection and quantification of specific binding molecules (e.g., antibodies).

[0041] In some embodiments, the antibody-drug conjugate includes an antigen-binding molecule that specifically binds to a macrophage marker, and optionally the antigen-binding molecule is an antibody. In some embodiments, the antibody-drug conjugate includes an anti-CD209 (DC-SIGN) or anti-CD206 (MRC1) antigen-binding molecule, and optionally the antigen-binding molecule is an antibody. In some embodiments, the antibody-drug conjugate has an antibody-to-drug ratio of at least 1:2, at least 1:4, at least 1:6, at least 1:8, or at least 1:10. In some embodiments, the ETS2 inhibitor or chr21q22 enhancer inhibitor includes a drug that inhibits the function of a co-transcription activator (e.g., BRD4 or CDK). In some embodiments, the ETS2 inhibitor or chr21q22 enhancer inhibitor includes a drug that targets chromatin regulators (e.g., readers, writers, and erasers of chromatin modifications). In some embodiments, the ETS2 inhibitor or chr21q22 enhancer inhibitor comprises a drug that targets non-coding RNA at the ETS2 locus or the chr21q22 enhancer locus. In some embodiments, the chr21q22 enhancer inhibitor comprises a gene editing technique selected from a list consisting of gene / base / prime editing strategies (e.g., CRISPR-based genome targeting tools), gene therapies (e.g., adeno-associated viruses (AAV), retroviral vectors, lentiviral vectors), and RNA therapies (including, but not limited to, antisense oligonucleotides). [Brief explanation of the drawing]

[0042] [Figure 1]Elucidation of the molecular mechanisms in chr21q22. A. Annotation of the disease-associated chr21q22 locus depicting IBD gene-related, physical interactions of disease-associated haplotypes in macrophages (promoter capture Hi-C data), and H3K27ac ChIP-seq data from various immune cell types. B. Schematic diagram of experiments to determine the function of the chr21q22 locus in monocyte-derived macrophages polarized under chronic inflammatory ("TPP") conditions. C. Histogram depicting the expression of ETS2, BRWD1, and PSMG1 in polarized inflammatory macrophages, measured using PrimeFlow RNA assay and directly quantified by flow cytometry. Data represent one of four donors. D. Relative ETS2, BRWD1, and PSMG1 expression in chr21q22-edited inflammatory macrophages (relative to untargeted control cells; NTCs). The plot shows the log2 factor change of mean fluorescence intensity (n=4, data represent mean= / - SEM, bidirectional ANOVA). E. SuSiE fine mapping posterior probability of IBD-related SNPs at the chr21q22 locus (99% confidence set). F. MPRA at the chr21q22 locus depicting oligonucleotide coverage (top), enhancer activity (analyzed using sliding window analysis of tiling oligos; middle), and the regulatory effect of candidate SNP expression within identified enhancer sites (bottom) in inflammatory macrophages (n=8). Shaded areas in the enhancer activity plot depict areas of significant enhancer activity. G. PU.1 ChIP-seq peaks at the chr21q22 locus in macrophages. H. BaalChIP analysis of allele-specific PU.1 binding in rs2836882 in two heterozygous macrophage datasets (data represent the 95% posterior distribution of allele binding ratios). I. H3K27ac ChIP-seq data from major (top) or minor (bottom) allele homozygotes at the chr21q22 locus. Data show two of four donors. [Figure 2]ETS2 is required for the inflammatory response of macrophages. A. Schematic diagram of an experiment in which primary monocytes were CRISPR-edited under chronically inflammatory ("TPP") conditions to differentiate monocyte-derived macrophages. B. Macrophage cytokine secretion after ETS2 disruption. The heatmap shows the log2 factor change in cytokine concentration in the supernatant of ETS2-edited macrophages compared to unedited macrophages transfected with untargeted control gRNA (NTC). n=8, Wilcoxon paired test, two-tailed. C. Histogram (left) depicting phagocytosis of fluorescently labeled dimosan particles by ETS2-edited and unedited macrophages. Data representing one of seven donors. Phagocytosis index (calculated as the product of the percentage of phagocytic cells and the mean fluorescence intensity) in ETS2-edited and unedited macrophages; right. The plot shows the log2 factor change in the phagocytic index of ETS2-edited macrophages compared to unedited macrophages (Wilcoxon signed-rank test, two-tailed; data represent mean + / - SEM). D. ROS production by ETS2-edited and unedited inflammatory macrophages (measured in relative light units; left). Data representing one of six donors. Western blot of gp91phox, p22phox, and EROS expression in ETS2-edited and unedited inflammatory macrophages (right). Data representing one of three donors. E. Volcano plot of differentially expressed genes in ETS2-edited vs. unedited (NTC) inflammatory macrophages (limma with voom transformation, n=8). F. Gene set enrichment analysis (fGSEA) of differentially expressed genes between ETS2-edited and unedited inflammatory macrophages. Results for selected gene ontology biological pathways are shown. The dot size represents the P-value, and the color indicates the normalized enrichment score (NES). Differential enrichment of expressed genes after deletion of disease-related chr21q22 locus (upwardly regulated gene, top; downwardly regulated gene, bottom) in G.ETS2 edited vs. unedited macrophages. *P<0.05, **P<0.01. [Figure 3]ETS2 modulates the macrophage inflammatory response. A. Schematic diagram of the ETS2 overexpression experiment. Resting (M0) human macrophages were transfected with a predefined amount of in vitro transcribed ETS2 mRNA or control mRNA (ETS2 reverse complement), activated with low-dose LPS (1 ng / ml), and collected. B. ETS2 mRNA levels in macrophages transfected with ETS2 or control mRNA, or untransfected (from another experiment). C. Cytokine secretion after ETS2 overexpression. The plot shows the log2 factor change in cytokine concentration in macrophage supernatant (ETS2 compared to control) after transfection with 500 ng of mRNA. D. Gene set enrichment analysis (fGSEA) of differentially expressed genes between ETS2-overexpressing macrophages and control macrophages. Results are shown for the same gene ontology biological pathways that were negatively enriched after ETS2 editing. Dot size represents P-value, color represents normalized enrichment score (NES), and border color indicates mRNA dose. E. Enrichment of disease-associated inflammatory macrophage gene sets derived from single-cell RNA-seq of Crohn's disease intestinal biopsy in ETS2-overexpressing macrophages (compared to control; top). Heatmap of leading genes showing log2 factor changes in gene expression in ETS2-overexpressing macrophages compared to control (500 ng mRNA; bottom). F. SNPsea analysis of enrichment of 241 IBD-associated gene loci within ETS2 regulatory genes (red) and pathways previously linked to IBD development (black). Pathways significantly enriched by § (Bonferroni-corrected substitution P<0.05). *P<0.05, **P<0.01. [Figure 4]ETS2 directs macrophage responses through transcriptional and metabolic effects. A. Genes co-expressed with ETS2 in a dataset of 64 monocyte-derived macrophages. Dotted line corresponds to FDR P<0.05. B. Effect of ETS2 disruption on glucose metabolism. Color indicates the median log2 factor change in label incorporation from 13C-glucose in ETS2-edited cells compared to unedited cells. Bold black border indicates P<0.05 (Wilcoxon corresponding pair, two-sided, n=6). C. Gene set enrichment analysis (fGSEA) of differentially expressed genes between ETS2-edited and unedited TPP macrophages treated with either roxadustat or vehicle. Results of selected gene ontology biological pathways are shown. D. Enrichment heatmap of ETS2 CUT&RUN peaks (IDR cutoff 0.01, n=2) in accessible chromatin from TPP macrophages (4-kb region centered on ATAC-seq peak). E. Characteristics of ETS2 binding sites (based on gene coordinates in TPP macrophages and H3K27ac ChIP-seq). F. Enrichment of ETS2 binding motifs at ETS2 cut & run peaks (hypergeometric p-values). G. ETS2 binding, chromatin accessibility (ATAC-seq), and enhancer activity (H3K27ac) at selected loci. H. Upset plot of crossovers between ETS2 gene lists, including genes with ETS2 peaks in core promoters or cis-regulatory elements following ETS2 editing (KO) or overexpression (OE), and genes that are significantly upregulated (Up) or downregulated (Dn). Vertical bars indicate shared genes between lists, shown as connected points in the lower panel. Horizontal bars indicate the proportion of gene lists within the crossover. I. ETS2 binding, chromatin accessibility (ATAC-seq), and enhancer activity (H3K27ac) at the disease-related chr21q22 locus. [Figure 5]ETS2-induced inflammation is evident in the disease and can be therapeutically targeted. A. Enrichment of chr21q22 regulatory genes in IBD intestinal macrophages (top), ankylosing spondylitis synovium (center), and primary sclerosing cholangitis liver (bottom). All compared to unaffected control tissues from the same dataset. The gene sets included genes that were significantly downregulated after chr21q22 deletion. B. Candidate drug classes that replicate the transcriptional outcomes of ETS2 disruption (from the NIH LINCS database). C. Volcano plot of fGSEA results for NIH LINCS drug signatures (FDR P;NES, normalized enrichment score estimated using an adaptive multilevel split Monte Carlo scheme). Set of significantly enriched MEK inhibitor genes colored by molecule. D. Schematic diagram of known upstream signaling pathways modulating ETS family transcription factors. E. Schematic diagram of experiments for an in vitro MEK inhibitor (PD-0325901) trial in inflammatory macrophages (TPP). F. Heatmap showing relative expression (log2 factor change) of the chr21q22 regulatory gene in inflammatory macrophages after MEK inhibitor treatment (compared to vehicle control, n=3). G. Gene set enrichment analysis (fGSEA) of differentially expressed genes between MEK inhibitor-treated macrophages and control inflammatory macrophages. Results are shown for the same gene ontology biological pathway that was negatively enriched after ETS2 editing. Dot size represents P value, and color indicates NES. H. Cytokine secretion from IBD mucosal biopsies cultured with vehicle control, PD-0325901, or infliximab I. I. Estimated plot of GSVA enrichment score of the chr21q22 downregulatory gene in IBD intestinal biopsies after MEK inhibition (MEKi). Error bars indicate 95% CI. J. GSVA enrichment score of biopsy-derived molecular inflammation score (bMIS). Data for h and i represent mean + / - SEM. Wilcoxon paired tests, two-tailed, n=10(h), n=9(j). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 6]Disease-associated mutations at the chr21q22 locus correlate with levels of ETS2 expression in monocytes. A. Risk haplotypes on chr21q22 are eQTLs of ETS2 expression in quiescent and activated monocytes (data from Fairfax et al, 2014). B. Manhattan plots depicting the association of chr21q22 with IBD (top panel), eQTLs in quiescent monocytes (center panel), and eQTLs in LPS-stimulated monocytes (bottom panel). C. IBD chr21q22 association co-localizes with eQTL signaling in quiescent but inactive monocytes. PP3, Probability that GWAS and eQTL signaling are independent. PP4, Probability that a shared variant is responsible for both associations. [Figure 7] CRISPR / Cas9 editing of the chr21q22 locus and ETS2 in monocytes. A. Cas9 gRNA was designed to be adjacent to the enhancer region within the chr21q22 locus at the indicated site. Enhancer activity shown from H3K27ac ChIP sequencing. B. Representative bioanalyzer traces of PCR amplifiers from the target region in macrophages after transfection with an equimolar mixture of Cas9 RNPs containing either 5' or 3' gRNA. Method for calculating the editing efficiency shown in the exemplary calculation. C. Editing efficiency at the chr21q22 locus. Mean deletion rate: 42.4%. D. Location and sequence of gRNA used to edit ETS2. E. Editing efficiency at the ETS2 locus. Mean indel rate: gRNA1, 89.7%; gRNA2, 78.6%. F. Survival rate of macrophages after nucleofection and differentiation with Cas9 RNP. Mean survival rates: NTC, 97.9%; gRNA1: 98.3%; gRNA2, 98.6%. Expression of myeloid markers after G.ETS2 editing and TPP differentiation. Data represent mean + / - SEM. [Figure 8] TPP macrophages replicate the transcriptional signatures of monocytes and macrophages in IBD. [Figure 9]MPRA in primary human macrophages. A. Schematic diagram of MPRA. A library of oligonucleotides (each containing a genome sequence and its own barcode) is cloned into a pGL4.10M cloning vector, and then promoter and reporter genes are inserted using directional cloning. The resulting plasmid is transfected into TPP macrophages, and RNA is extracted after 24 hours. After high-throughput sequencing, the number of mRNA barcodes is normalized to the corresponding number in the input DNA library to evaluate the expression regulatory activity. B. Identification of suitable promoters for MPRA in TPP macrophages. TPP macrophages were transfected with reporter vectors with GFP expression under the control of different promoters. GFP expression was quantified by flow cytometry after 24 hours. C. A suitable MPRA vector for use with primary human macrophages containing the RSV promoter. D. Principal component analysis of element count (total number of barcodes tagging the same genome sequence) in mRNA from TPP macrophages from eight donors (red) and DNA vectors (black). E. Heatmap showing pairwise correlations of expression regulatory activity of all constructs between donors. [Figure 10]Functional consequences of allele mutations in rs2836882. A. Schematic diagram of a PU.1 ChIP genotyping assay to evaluate allele-specific PU.1 binding in rs2836882. B. Schematic diagram of optimal approximation lines for various ratios of risk and non-risk-containing DNA sequences. C. Standard curves generated using different allele ratios of 200-nt DNA gene blocks centered on either the major (risk) or minor (non-risk) allele. D. Allele-specific PU.1 binding in rs2836882 in TPP macrophages (one-sample t-test, two-tailed) demonstrating increased PU.1 binding in the presence of the risk allele. Data are expressed in 95% confidence intervals. E. Rank-ordering analysis of superenhancers (ROSE) from H3K27ac ChIP sequencing data from TPP macrophages homozygous for major (left) and minor (right) alleles, demonstrating that the disease-related chr21q22 locus meets the criteria for superenhancer activity in homozygous major (risk) alleles but not in homozygous minor alleles. [Figure 11]Deficiency of the chr21q22 disease-associated enhancer replicates ETS2 disruption. A. Cytokine secretion from TPP macrophages after editing at the chr21q22 locus. The heatmap shows the log2-fold change in cytokine concentration in the supernatant of chr21q22-edited cells compared to untargeted control (NTC) cells (n=7, Wilcoxon paired signed-rank test, one-sided). B. Extracellular ROS production by NTC, chr21q22-edited, and ETS2 g1-edited TPP macrophages, quantified by chemiluminescence assay. The points represent the log2-fold change in area under the curve (AUC) for edited cells versus NTC (Wilcoxon signed-rank test). C. Representative histogram demonstrating phagocytosis of fluorescently labeled dimosan particles by NTC and chr21q22-edited TPP macrophages. D. Phagocytosis index of NTC and chr21q22-edited TPP macrophages. The phagocytosis index is calculated by multiplying the percentage of positive cells by the average fluorescence intensity (488 nm channel) of the positive cells. The plot shows the log2 factor change between chr21q22-edited cells and NTC cells (Wilcoxon signed-rank test). Data represent mean + / - SEM. *p<0.05. [Figure 12] Optimization of mRNA-based overexpression in primary human macrophages. Primary human macrophages (M0) were transfected with different amounts of GFP mRNA containing modified nucleotides using Lipofectamine MessengerMAX. GFP expression was quantified by flow cytometry 18 hours post-transfection. [Figure 13]Metabolic effects of ETS2 disruption. A and B. Changes in total metabolite abundance (A) and percentage of label incorporation from 13C-glucose (B) after ETS2 editing in TPP macrophages. Color indicates log2 ratio changes in abundance or incorporation in ETS2¬g1-edited macrophages compared to macrophages transfected with untargeted control (NTC) RNP. Bold black borders indicate significant changes (p<0.05, Wilcoxon paired signed-rank test, two-tailed). C. Heatmap summarizing metabolic changes after ETS2 disruption. Color indicates log2 ratio changes in metabolite abundance or label incorporation from 13C-glucose in ETS2 g1-edited cells compared to NTC cells (Wilcoxon paired signed-rank test, two-tailed). *p<0.05. [Figure 14] Functional studies in roxadustat-treated macrophages. A. Phagocytosis index in ETS2-edited or untargeted control (NTC) TPP macrophages treated with roxadustat or vehicle. The phagocytosis index is calculated by multiplying the percentage of positive cells by the mean fluorescence intensity (488 nm channel) of positive cells. The plot shows the log2-fold change in edited vs. NTC cells. B. Extracellular ROS production by ETS2-edited or NTC TPP macrophages treated with roxadustat or vehicle and quantified by chemiluminescence assay. The points represent the log2-fold change in area under the curve (AUC) of edited vs. NTC cells. Data represent mean + / - SEM. [Figure 15] Exemplary ETS2 nucleic acid and amino acid sequences. Sequence ID 1 - Exemplary chr21q22 enhancer sequence, hg19:chr21:40466236-40466677; Sequence ID 2 - Exemplary ETS2 mRNA sequence (NM005239.6); and Sequence ID 4 - Exemplary ETS2 amino acid sequence (Uniprot:P15036). [Figure 16]The transcriptional signature of ETS2 is detectable in affected tissue from chr21q22-linked diseases. A. Gene set enrichment analysis (fGSEA) of ETS2 regulatory genes in ankylosing synovial spondylitis (compared to control synovium). B. fGSEA of ETS2 regulatory genes in liver biopsies of primary sclerosing cholangitis (compared to control liver biopsies). C. fGSEA of ETS2 regulatory genes in intestinal macrophages isolated from patients with active inflammatory bowel disease (compared to healthy control intestinal macrophages). ETS2 regulatory genes in A, B, and C were defined as significantly downregulated after ETS2 editing using gRNA1-containing RNPs (upper panel) or gRNA2-containing RNPs (lower panel). [Figure 17] Effect of MEK1 / 2 inhibition on ETS2 regulatory genes. A, B, and C. Gene set enrichment analysis (fGSEA) of gene sets upregulated (upper panel) or downregulated (lower panel) after ETS2 or chr21q22 editing within a ranked list of genes dysregulated after MEK1 / 2 inhibitor treatment (MEKi-treated inflammatory macrophages vs. vehicle controls). MEK1 / 2 was inhibited using PD-0325901, 0.5 μM. Gene sets were derived from differential gene expression analysis (limma using voom conversion) after gRNA1 (A), ETS2 disruption by gRNA2 (B), or chr21q22 deletion (C). [Figure 18] Enrichment of macrophage signatures from patients with the indicated diseases (color-coded by category) in ETS2-overexpressing macrophages (compared to controls). Numbers represent normalized enrichment scores (NES), and dashed lines indicate FDR P0.05. [Figure 19](A) Spatial transcriptomics of PSCs and healthy liver (n=4). Images show representative fields with cell segmentation and semi-supervised clustering results (InsituType). Legend indicates InsituType cell type: Hep., hepatocyte; LSEC, hepatic sinusoidal endothelial cell. (B) Mean number of macrophages within a defined radius of cholangiocytes. (C) Distance from cholangiocytes to the nearest macrophage. Data are shown as Tukey box-and-whisker plots. Mann-Whitney test, two-tailed. Data in (B) and (C) represent 10,532 PSCs and 13,322 control cholangiocytes. (D) Scaled expression of ETS2 regulatory genes at a defined distance from cholangiocytes in 21,067 PSC macrophages (excluding genes used to define macrophage subsets). Data represent mean and 95% CI. [Modes for carrying out the invention]

[0043] The following provides definitions of certain terms, technical means, and embodiments used herein.

[0044] As used herein, the term “administration” refers to the administration of a composition to a subject. Administration to an animal subject (e.g., human) may be by any suitable route. For example, in some embodiments, administration may be by the bronchus (including by bronchial infusion), buccal, enteral, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, intra-organ or tissue (e.g., intrahepatic, intratumoral, peritumoral, etc.), mucosa, nose, oral, rectal, subcutaneous, sublingual, topical, trachea (including by intratracheal infusion), percutaneous, vaginal, and intravitreous. Administration may include intermittent dosing. Alternatively, administration may include continuous dosing (e.g., perfusion) for at least a selected period.

[0045] As used herein, the terms “erythroblast transformation-specific proto-oncogene 2” or “ETS2” refer to the amino acid sequence encoded by the ETS2 gene (HGNC reference 3489). The gene is also known as ETS2IT1. The gene is encoded by the genomic sequence identified by Ensembl reference ENSG00000157557. The transcripts ENST00000360938.8, ENST00000360214.8, and ENST00000667466.1 can also be used to define the ETS2 sequence. In some embodiments, ETS2 may comprise the amino acid sequence of SEQ ID NO: 4 (Uniprot reference: P15036). In some embodiments, ETS2 may consist of the amino acid sequence of SEQ ID NO: 4 (Uniprot reference: P15036). In some embodiments, ETS2 may be encoded by an mRNA sequence containing a sequence selected from either SEQ ID NO: 2 or SEQ ID NO: 3 (NCBI references: NM_005239.6 and NM_001256295.2). In some embodiments, ETS2 may be encoded by an mRNA sequence containing the SEQ ID NO: 2 sequence (NCBI reference: NM_005239.6). In some embodiments, ETS2 may be an mRNA sequence consisting of the SEQ ID NO: 2 sequence (NCBI reference: NM_005239.6).

[0046] As used herein, the terms “erythroblast transformation-specific proto-oncogene 2 inhibitor” or “ETS2 inhibitor” refer to any agent that reduces the expression or activity of erythroblast transformation-specific proto-oncogene 2 (ETS2) polypeptide or protein. The reduction in expression or activity can be measured by any standard means known in the art, including measuring the reduction in the levels of polynucleotides encoding ETS2 (such as ETS2 mRNA) and / or ETS2 polypeptide or protein. Methods for quantifying the amount of one or more ETS2 polynucleotides include fluorescence in situ hybridization (FISH), spectroscopy, quantitative sequencing, quantitative PCR (qPCR), RT-PCR, ddPCR, Southern blotting, or Northern blotting. Methods for quantifying the amount of one or more ETS2 polypeptides or proteins include immunohistochemistry (IHC), spectroscopy, colorimetric detection, Western blotting, mass spectrometry, or ELISA. The level of ETS2 inhibition induced by an ETS2 inhibitor may be at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of ETS2 expression or activity when measured in the absence of the ETS2 inhibitor. Ideally, levels of ETS2 activity can be compared within the same model system (e.g., cell line or tissue) in the presence and absence of an ETS2 inhibitor.

[0047] As used herein, the terms “chr21q22 enhancer inhibitor” or “chr21q22 enhancer inhibitor” refer to any agent that reduces the expression or activity of the erythroblast transformation-specific proto-oncogene 2 (ETS2) polypeptide or protein via binding to the chr21q22 enhancer (SEQ ID NO: 1). The reduction in ETS2 expression or activity can be measured by any standard means known in the art, including measuring the reduction in the levels of the polynucleotide encoding ETS2 (such as ETS2 mRNA) and / or the ETS2 polypeptide or protein. Methods for quantifying the amount of one or more ETS2 polynucleotides include fluorescence in situ hybridization (FISH), spectroscopy, quantitative sequencing, quantitative PCR (qPCR), RT-PCR, ddPCR, Southern blotting, or Northern blotting. Methods for quantifying the amount of one or more ETS2 polypeptides or proteins include immunohistochemistry (IHC), spectroscopy, colorimetric detection, Western blotting, mass spectrometry, or ELISA. The level of ETS2 inhibition induced by a chr21q22 enhancer inhibitor may be at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of ETS2 expression or activity when measured in the absence of the chr21q22 enhancer inhibitor. Ideally, levels of ETS2 activity can be compared within the same model system (e.g., cell line or tissue) in the presence and absence of the chr21q22 enhancer inhibitor.

[0048] As used herein, the terms “approximately” or “about” refer to values ​​similar to the stated reference values ​​when applied to one or more target values. In some embodiments, unless otherwise specified or evident from the context, the terms “approximately” or “about” refer to a range of values ​​that fall within or less than 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% in either direction (greater than or less than) the stated reference values ​​(except where such numbers exceed 100% of the possible values).

[0049] As used herein, the term “prevention” means delaying or preventing the onset, development, or progression of a disease, disorder, or condition for a period ranging from a few minutes to an indefinite period. “Prevention” also includes reducing the risk of developing a disease, disorder, or condition. “Prevention” includes, but is not required, the complete avoidance of a disease condition.

[0050] As used herein, the term “treatment” (and also “to treat” or “to treat”) refers to any administration of a therapeutic agent in accordance with a treatment regimen that achieves a desired effect in that it partially or completely alleviates, improves, reduces, decreases the severity of, and / or reduces the incidence of one or more symptoms or characteristics of a particular disease, disorder, and / or condition. In some embodiments, the administration of a therapeutic agent in accordance with a treatment regimen correlates with the achievement of the desired effect. Such treatment may be for subjects who do not show signs of the disease, disorder, and / or condition in question, and / or for subjects who show only initial signs of the disease, disorder, and / or condition. Alternatively or additionally, such treatment may be for subjects who show one or more established signs of the disease, disorder, and / or condition in question. In some embodiments, treatment may be for subjects who have been diagnosed with the disease, disorder, and / or condition in question. In some embodiments, treatment may be for subjects who are known to have one or more susceptibility factors that are statistically correlated with an increased risk of developing the disease, disorder, and / or condition in question.

[0051] As used herein, the term “equivalent” means a system, set of conditions, effect, or result that is sufficiently similar to a test system, set of conditions, effect, or result to enable a scientifically valid comparison. A person skilled in the art will recognize and understand which system, set of conditions, effect, or result is sufficiently similar to any particular test system, set of conditions, effect, or result described herein to be “equivalent.”

[0052] The term “correlated” as used herein has its usual meaning of “showing correlation with.” Those skilled in the art will understand that two features, items, or values ​​are correlated with each other if they tend to appear and / or change together. In some embodiments, the correlation is statistically significant when its p-value is less than 0.05, and in some embodiments, the correlation is statistically significant when its p-value is less than 0.01. In some embodiments, the correlation is assessed by regression analysis. In some embodiments, the correlation is the correlation coefficient.

[0053] As used herein, the terms “improve,” “increase,” or “decrease,” or their grammatical synonyms, refer to values ​​relative to reference (e.g., baseline) measurements, such as measurements taken in a control individual (or a group of control individuals) under comparable conditions (e.g., measurements taken in the same individual before the initiation of the treatment described herein, or in the absence of the treatment described herein).

[0054] As used herein, “polypeptide” is a chain of at least two amino acids linked to one another by peptide bonds. In some embodiments, a polypeptide may contain at least three to five amino acids, each of which is linked to another amino acid by at least one peptide bond. Those skilled in the art will understand that a polypeptide may optionally contain “unnatural” amino acids or other entities that can still be incorporated into the polypeptide chain.

[0055] As used herein, the term “protein” refers to a molecule comprising a polypeptide (i.e., a sequence of at least two amino acids linked to one another by peptide bonds). Proteins may contain non-amino acid sites (e.g., glycoproteins, proteoglycans, etc.) and / or may be otherwise processed or modified. Those skilled in the art will understand that a “protein” may be a complete polypeptide chain (with or without a signal sequence) as produced by a cell, or a characteristic portion thereof. Those skilled in the art will understand that a protein may comprise two or more polypeptide chains linked, for example, by one or more disulfide bonds, or associated by other means. Polypeptides may contain L-amino acids, D-amino acids, or both, and may contain any of the various amino acid modifiers or analogues known in the art. Useful modifications include, for example, terminal acetylation, amidation, and methylation. In some embodiments, proteins may comprise native amino acids, non-native amino acids, synthetic amino acids, and combinations thereof. The term "peptide" is generally used to refer to polypeptides having a length of less than approximately 100 amino acids, less than approximately 50 amino acids, less than 20 amino acids, or less than 10 amino acids.

[0056] As used herein, the terms “subject,” “individual,” or “patient” refer to any organism in which embodiments of the present invention may be used or administered, for example, for experimental, diagnostic, preventive, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans, insects, parasites, etc.). In preferred embodiments of the present invention, the subject is human.

[0057] As used herein, the term “therapeutic regimen” means any method used to partially or completely alleviate, improve, reduce, inhibit, prevent, delay the onset, reduce the severity, and / or reduce the incidence of one or more symptoms or characteristics of a particular disease, disorder, and / or condition. It may include the administration of one or more doses separated by regular or varying time intervals, at any choice. In some embodiments, a therapeutic regimen is designed so that its implementation achieves a particular effect, e.g., reduction or elimination of an adverse condition or disease, and / or correlates with the achievement of such a particular effect (e.g., across a population of relevant cells, tissues, or organisms). In some embodiments, the treatment includes administering one or more therapeutic agents simultaneously, sequentially, or at different times, at the same time or over different times. In some embodiments, a “therapeutic regimen” includes genetic methods such as gene therapy, gene ablation, or other methods known to induce or reduce expression (e.g., transcription, processing, and / or translation of a particular gene product, such as primary transcript or mRNA).

[0058] As used herein, the term “therapeutic dose” refers to the amount of a therapeutic agent that produces a therapeutic effect on a subject being treated in a reasonable benefit-to-risk ratio applicable to any medical treatment. Such a therapeutic effect may be objective (i.e., measurable by some test or marker) or subjective (i.e., the subject shows signs of or feels an effect). In some embodiments, “therapeutic dose” refers to the amount of a therapeutic agent or composition that is effective in treating, improving, or preventing (e.g., delaying the onset) an associated disease or condition, and / or exhibiting a detectable therapeutic or preventive effect, by improving symptoms associated with the disease, preventing or delaying the onset of the disease, and / or reducing the severity or frequency of symptoms of the disease. The therapeutic dose is usually administered in a dosing regimen that may contain multiple unit doses. For any particular therapeutic agent, the therapeutic dose (and / or an appropriate unit dose in an effective dosing regimen) may vary depending, for example, on the route of administration or in combination with other therapeutic agents. Alternatively or additionally, a specific therapeutically effective dose (and / or unit dose) for any particular patient may depend on a variety of factors, including the activity of the particular therapeutic agent used; the specific composition used; the patient's age, weight, general health, sex, and diet; the timing of administration, route of administration, and / or excretion or metabolic rate of the particular therapeutic agent used; the duration of treatment; and similar factors well known in the medical field.

[0059] As used herein, “part” means a defined number of consecutive (i.e., linked) nucleic acid bases of a nucleic acid. In some embodiments, a part is a defined number of consecutive nucleic acid bases of a target nucleic acid. In some embodiments, a part is a defined number of consecutive nucleic acid bases of an antisense compound. In some embodiments, a part may refer to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more consecutive nucleic acid bases. In some embodiments, a part may refer to at least 10, at least 20, at least 30, at least 40, or at least 50 consecutive nucleic acid bases. In some embodiments, a part may refer to fewer than 50 consecutive nucleic acid bases, for example, fewer than 40, fewer than 30, fewer than 20, or fewer than 10.

[0060] As used herein, "nucleoside" means a compound containing a nucleic acid base moiety and a sugar moiety. Nucleosides include, but are not limited to, naturally occurring nucleosides (found in DNA and RNA) and modified nucleosides. Nucleosides may be linked to a phosphate moiety.

[0061] As used herein, “nucleotide” means a nucleoside further containing phosphate linking groups. As used herein, “linked nucleoside” may or may not be linked by phosphate bonds and therefore includes, but is not limited to, “linked nucleotides.” As used herein, “linked nucleoside” is a nucleoside linked in a continuous sequence (i.e., there are no additional nucleosides between the linked nucleosides).

[0062] As used herein, “nucleic acid base” means a group of atoms capable of linking to a sugar moiety to form a nucleoside that can be incorporated into an oligonucleotide, and which can be bound to naturally occurring complementary nucleic acid bases of another oligonucleotide or nucleic acid. Nucleic acid bases may be naturally occurring or may be modified.

[0063] As used herein, the terms “unmodified nucleic acid bases” or “naturally occurring nucleic acid bases” mean the naturally occurring heterocyclic nucleic acid bases of RNA or DNA, namely the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) (including 5-methyl C), and uracil (U).

[0064] As used herein, “modified nucleic acid base” means any nucleic acid base that is not naturally occurring. As used herein, “modified nucleoside” means a nucleoside that contains at least one chemical modification compared to a naturally occurring RNA or DNA nucleoside. Modified nucleosides may contain modified sugar sites and / or modified nucleic acid bases.

[0065] As used herein, “oligonucleotide” means a compound comprising multiple linked nucleosides. In some embodiments, the oligonucleotide comprises one or more unmodified ribonucleosides (RNA) and / or unmodified deoxyribonucleosides (DNA) and / or one or more modified nucleosides.

[0066] As used herein, “conjugate” or “conjugate group” means an atom, group of atoms, or carrier bound to the inhibitory agent of the present invention. Generally, conjugate groups can modify one or more properties of the agent to which they are bound, including but not limited to pharmacodynamic, pharmacokinetic, binding, absorption, cell distribution, cell uptake, charge, and / or clearance properties.

[0067] As used herein, “nucleic acid base complementarity” or “complementarity” means, with respect to nucleic acid bases, a nucleic acid base that can base-pair with another nucleic acid base. For example, in DNA, adenine (A) is complementary to thymine (T). For example, in RNA, adenine (A) is complementary to uracil (U). In both DNA and RNA, guanine (G) is complementary to cytosine (C). In some embodiments, complementary nucleic acid bases mean nucleic acid bases of an oligomeric compound that can base-pair with nucleic acid bases of a target sequence. For example, if a nucleic acid base at a particular position in an oligomeric compound can hydrogen-bond with a nucleic acid base at a particular position in a target sequence, the positions of the hydrogen bonds between the oligomeric compound and the target sequence are considered complementary in that nucleic acid base pair. Nucleic acid base complementarity is still possible because nucleic acid bases with certain modifications can retain their ability to pair with the corresponding nucleic acid bases.

[0068] As used herein, “fully complementary” with respect to an oligomeric compound or its region means that each nucleic acid base of the oligomeric compound or its region can pair with a nucleic acid base of a complementary nucleic acid target sequence or a self-complementary region of the oligomeric compound. Therefore, a fully complementary oligomeric compound or its region does not contain mismatched or non-hybridized nucleic acid bases with respect to its target sequence or the self-complementary region of the oligomeric compound.

[0069] As used herein, “complementarity percentage” refers to the proportion of nucleic acid bases in the oligomer compound that are complementary to the isolength portion of the target nucleic acid. The complementarity percentage is calculated by dividing the number of nucleic acid bases in the oligomer compound that are complementary to the nucleic acid bases at the corresponding positions in the target nucleic acid by the total length of the oligomer compound.

[0070] As used herein, “identity percentage” means the number of nucleic acid bases in the first nucleic acid that are of the same type (regardless of chemical modification) as the nucleic acid base at the corresponding position in the second nucleic acid, divided by the total number of nucleic acid bases in the first nucleic acid.

[0071] As used herein, “modulation” means a change in the quantity or quality of a molecule, function, or activity compared to the quantity or quality of the molecule, function, or activity before modification. For example, modification includes any change that increases (stimulates or induces) or decreases (inhibits or reduces) gene expression.

[0072] As used herein, “activate” or “activating protein” refers to a process that turns on or upregulates gene expression, resulting in increased production and / or activity of its corresponding protein or RNA product. This process is controlled by a variety of molecular mechanisms, including transcription factors, epigenetic modifications, and signaling pathways. In gene activation, specific regulatory sequences within a DNA sequence, such as promoters and enhancers, are recognized and bound by transcription factors, which then replenish RNA polymerase to initiate gene transcription. This produces messenger RNA (mRNA), which is then translated into protein by ribosomes. Genetic activation can be influenced by a variety of factors, including environmental cues, developmental stages, and physiological conditions.

[0073] As used herein, "PROTAC" (PROteolysis TArgeting Chimeras) refers to a type of small molecule therapeutic agent that utilizes the ubiquitin-proteasome system to selectively degrade target proteins within cells. A PROTAC molecule consists of two functional domains: a ligand that binds to the target protein and a ligand that recruits the E3 ubiquitin ligase enzyme. When a PROTAC molecule binds to the target protein and the E3 ubiquitin ligase, they protrude into proximity, resulting in the transfer of ubiquitin molecules to the target protein. The ubiquitinated protein is then recognized and degraded by the proteasome, leading to a decrease in the target protein's level. Compared to conventional small molecule inhibitors, PROTACs offer several potential advantages, including the ability to target previously untreatable proteins, higher selectivity, and the potential for longer-lasting effects. They have shown promise as therapeutic strategies for a wide range of diseases, including cancer, neurodegenerative diseases, and viral infections.

[0074] As used herein, “molecular adhesives” refer to a class of small molecules capable of enhancing intracellular protein-protein interactions. These molecules function as “adhesives” by simultaneously binding to two or more protein surfaces, thereby stabilizing interactions that would otherwise be transient or weak. In the context of drug discovery, molecular adhesive molecules can be used to create novel therapeutic strategies by targeting proteins that cause diseases previously considered “untreatable” with conventional small molecules. By stabilizing protein-protein interactions essential for disease development, molecular adhesive molecules can inhibit or enhance protein function in ways that cannot be achieved with conventional small molecule inhibitors. Molecular adhesives have shown promise as therapeutic approaches for a variety of diseases, including cancer, neurodegenerative diseases, and viral infections. However, significant challenges remain to be overcome in the design and development of effective molecular adhesive molecules, including issues related to selectivity and off-target effects.

[0075] As used herein, “cotranscription activator disrupting agent” refers to a molecule or compound that interferes with the activity of a protein or complex involved in the process of cotranscriptional activation of gene expression. Cotranscription activators are proteins or complexes that interact with RNA polymerase during transcription, promoting the recruitment and binding of other proteins necessary for proper gene expression. Disrupting the function of cotranscription activators can affect a cell’s ability to produce functional RNA molecules, leading to alterations in gene expression and potentially contributing to disease. Some examples of cotranscription activators include chromatin remodelers, histone acetyltransferases, and transcription elongation factors. Cotranscription activator disrupting agents can act in various ways, such as inhibiting their enzymatic activity, preventing their binding to other proteins or RNA molecules, or inducing their degradation. These agents may be used as potential therapeutic agents for treating diseases caused by dysregulation of gene expression.

[0076] As used herein, “chromatin regulator-targeted agents” refers to molecules or compounds that interact with proteins involved in the modification, remodeling, or packaging of chromatin, the DNA-protein complex that constitutes chromosomes in the nucleus of cells. Chromatin regulators play a crucial role in regulating gene expression by modulating DNA accessibility to transcription factors and other regulatory proteins. Chromatin regulator-targeted agents can act in various ways, such as inhibiting their enzymatic activity, preventing them from binding to other proteins or DNA, or inducing their degradation. These agents can be used as potential therapeutic agents for diseases caused by dysregulation of gene expression. Examples of chromatin regulators that can be targeted by such agents include histone deacetylases (HDACs), histone methyltransferases (HMTs), histone demethylases (HDMs), and chromatin remodelers.

[0077] As used herein, “non-coding RNA-targeted drugs” refers to molecules or compounds that interact with non-coding RNA (ncRNA), also known as non-coding RNA. Non-coding RNA molecules play a crucial role in regulating gene expression, cell signaling, and other cellular processes and are increasingly recognized as important therapeutic targets for a wide range of diseases. Non-coding RNA-targeted drugs can act in various ways, such as blocking their interactions with other proteins or RNA molecules, promoting their degradation, or modulating their function. These drugs can be used as potential therapeutic agents to treat diseases caused by dysregulation of non-coding RNA. Examples of non-coding RNA molecules that can be targeted by such drugs include microRNA (miRNA), long non-coding RNA (lncRNA), and circular RNA (circRNA).

[0078] As used herein, “GlyPro” or “GlyPro linker” refers to a type of flexible amino acid linker commonly used in the design of protein therapeutics, particularly antibody-drug conjugates (ADCs). GlyPro linkers are composed of glycine and proline residues in a repeating pattern, which provides flexibility to the linker while minimizing the potential for immunogenicity and other adverse effects. In the context of ADCs, GlyPro linkers are used to connect the antibody portion of a molecule to a cytotoxic drug or other therapeutic payload. The linker plays a crucial role in determining the pharmacokinetics and pharmacodynamics of the ADC, including stability, solubility, and the ability to target specific cells or tissues. GlyPro is selectively cleaved by β-glucuronidase, thereby facilitating selective release in lysosomes. Additionally, this linker possesses favorable hydrophilic properties, which should reduce aggregation and promote solubility. Exemplary GlyPro linkers and related methods can be found in US2008 / 241128, which is incorporated herein by reference in its entirety.

[0079] As used herein, “esterase-sensitive motif” refers to a molecular structure or sequence that is cleaved by an esterase, a class of enzymes that hydrolyze ester bonds. In the context of drug delivery or controlled release, esterase-sensitive motifs can be used to create prodrugs or polymer-based systems that are activated by esterases in specific tissues or cell types. One common example of an esterase-sensitive motif is a carboxylic acid ester bond that can be hydrolyzed by an esterase to release an active drug molecule. In prodrug systems, the drug molecule is chemically linked to a carrier molecule via an esterase-sensitive motif, thereby enabling targeted delivery and activation of the drug. In polymer-based systems, the polymer backbone contains an esterase-sensitive motif that, in response to esterase activity, enables controlled release of the encapsulated drug. Esterase-sensitive motifs have been used in a variety of drug delivery and controlled release systems, including micelles, liposomes, and nanoparticles. They offer several advantages over other types of activation mechanisms, including high specificity, rapid kinetics, and compatibility with a wide range of drugs and polymers. Unlike most human carboxylesterases, which are ubiquitously expressed, human carboxylesterase-1 (hCE-1) is primarily expressed in monocyte-derived cells (e.g., monocytes and macrophages). ESM technology uses esters that are selectively hydrolyzed by hCE-1, and unlike esters, the acid cannot easily diffuse, thus leading to the intracellular accumulation of pharmacologically active acids (drugs) in hCE-1 expressing cells. This method can promote a 1000-fold increase in titer compared to non-conjugate inhibitors. Exemplary esterase-sensitive motifs and related methods can be found in WO06 / 117567, which is incorporated herein by reference in its entirety.

[0080] As used herein, “Crohn’s disease” refers to a chronic inflammatory bowel disease (IBD) that affects the gastrointestinal tract. It can occur anywhere along the digestive tract, from mouth to anus, but most commonly affects the small intestine and the beginning of the large intestine (colon). The exact cause of Crohn’s disease is unknown, but it is thought to be an autoimmune condition in which the body’s immune system attacks healthy tissue in the digestive tract. Genetics, environmental factors, and abnormal immune responses can all play a role in the development of the disease. The symptoms of Crohn’s disease are very diverse and can include abdominal pain, diarrhea, rectal bleeding, weight loss, and fatigue. Other complications can occur, including malnutrition, bowel obstruction, and abscesses.

[0081] As used herein, “ulcerative colitis” refers to a chronic inflammatory bowel disease (IBD) affecting the colon and rectum. It is characterized by inflammation and ulcers (sores) that develop in the lining of the colon, which can lead to symptoms such as diarrhea, rectal bleeding, abdominal pain, and weight loss. The exact cause of ulcerative colitis is unknown, but it is thought to be an autoimmune disease in which the immune system mistakenly attacks healthy tissue in the colon and rectum. Genetic and environmental factors may also play a role in the development of the disease. Ulcerative colitis is typically diagnosed through a combination of physical examination, medical history, blood tests, stool tests, and colonoscopy (examination of the colon using a flexible tube). Treatment for ulcerative colitis depends on the severity of the disease and may include medications to control inflammation, antibiotics to treat infections, and surgery to remove the affected parts of the colon and rectum. Lifestyle changes such as dietary changes, stress management, and regular exercise can also help manage symptoms and improve quality of life.

[0082] As used herein, “ankylosing spondylitis” (AS) refers to a type of chronic inflammatory arthritis that primarily affects the spine but can also involve other joints in the body. It is a form of spondyloarthritis and a group of inflammatory diseases that cause arthritis and inflammation in the spine and other joints. AS causes inflammation of the joints between the vertebrae of the spine, which can cause pain and stiffness in the lower back and buttocks. Over time, the inflammation can lead to vertebral fusion, which can cause the spine to become rigid and lose flexibility. AS can also affect other joints in the body, such as the hip, knee, and shoulder joints. The exact cause of AS is unknown, but it is thought to be an autoimmune disease in which the immune system mistakenly attacks the body's healthy tissues. Genetic and environmental factors may also play a role in the development of the disease. Diagnosis of AS involves a combination of physical examination, medical history, blood tests, imaging tests (such as X-rays and MRI), and assessment of symptoms. Treatment of AS may include medication to reduce inflammation and pain, physical therapy to maintain mobility and flexibility, and lifestyle changes such as regular exercise and proper posture. In severe cases, surgery may be necessary to correct the deformity or replace the damaged joint.

[0083] As used herein, “primary sclerosing cholangitis” (PSC) refers to a chronic liver disease affecting the bile ducts, which are the tubes that carry bile from the liver to the small intestine. It is characterized by inflammation and scarring of the bile ducts, which can lead to obstruction and damage to the liver. The exact cause of PSC is unknown, but it is thought to be an autoimmune disease in which the immune system mistakenly attacks the bile ducts. Genetic and environmental factors may also play a role in the development of the disease. Symptoms of PSC may include fatigue, itching, jaundice (yellowing of the skin and eyes), abdominal pain, and weight loss. Over time, PSC can lead to complications such as cirrhosis (scarring of the liver), liver failure, and an increased risk of liver cancer. Diagnosis of PSC typically involves blood tests, imaging tests (such as ultrasound, MRI, or CT scans), and a procedure called ERCP (endoscopic retrograde cholangiopancreatography), which examines the bile ducts using a flexible tube with a camera. Treatment of PSC involves managing symptoms and preventing complications. Medications may be used to reduce inflammation and control symptoms such as itching. In severe cases, a liver transplant may be necessary. Regular monitoring and follow-up with healthcare providers are important for people with PSC.

[0084] As used herein, “Takayas arteritis” (TA) refers to a rare type of chronic inflammatory disease affecting the body’s major arteries, particularly the aorta and its branches. It is also known as “pulseless disease” because the pulse in the arms and legs may be weak or absent. TA is thought to be an autoimmune disease in which the immune system mistakenly attacks healthy arterial tissue, causing inflammation and damage. The exact cause of TA is not fully understood, but genetic and environmental factors may also play a role in the development of the disease. Symptoms of TA may include fatigue, fever, weight loss, muscle aches, and joint pain. As the disease progresses, it can lead to narrowing or blockage of the arteries, potentially resulting in various complications such as hypertension, heart failure, stroke, and aneurysms (weak areas of the arterial wall that can rupture). Diagnosis of TA involves a combination of physical examination, medical history, blood tests, imaging tests (such as MRI or CT scans), and symptom assessment. Treatment of TA involves medications to reduce inflammation and prevent further damage to the arteries. In some cases, surgery may be necessary to repair or bypass the blocked artery.

[0085] As used herein, “rheumatoid arthritis” (RA) is a chronic autoimmune disease that primarily affects the joints. It is characterized by inflammation of the synovial membrane, the inner lining of the joint capsule, causing joint pain, swelling, stiffness, and injury. While RA can affect any joint in the body, it is most commonly seen in the hands, wrists, and feet. In addition to joint symptoms, RA can also cause fatigue, fever, and general malaise. The exact cause of RA is unknown, but it is thought to involve a combination of genetic and environmental factors that cause the immune system to attack the synovial membrane. Smoking and obesity are two examples of environmental factors associated with an increased risk of developing RA. Diagnosis of RA involves a combination of physical examination, medical history, blood tests (including tests for rheumatoid factor and anti-CCP antibodies), and imaging studies (such as X-rays and MRI). Early diagnosis and treatment of RA are important to prevent joint damage and improve long-term outcomes.

[0086] As used herein, “reactive oxygen species” (ROS) refer to oxygen-containing chemically reactive molecules produced as byproducts of normal cellular metabolism. They include, among others, superoxide anions, hydrogen peroxide, and hydroxyl radicals. ROS are important signaling molecules involved in many cellular processes, including cell proliferation, apoptosis, and inflammation. However, excessive levels of ROS can be harmful to cells and tissues, as they can cause oxidative damage to DNA, proteins, and lipids. This damage can lead to mutations, cellular dysfunction, and cell death, and is involved in the development of various diseases, including cancer, neurodegenerative diseases, and cardiovascular diseases. The body has several mechanisms to neutralize ROS, including antioxidant enzymes such as superoxide dismutase, catalase, and glutathione peroxidase, as well as small molecules such as vitamin C, vitamin E, and glutathione. However, when these defense mechanisms are overwhelmed, oxidative stress occurs, which can lead to cell and tissue damage.

[0087] As used herein, “macrophage phagocytosis” refers to the process by which macrophages, a type of immune cell, engulf and internalize foreign particles such as bacteria, viruses, and debris from dead cells. Phagocytosis is a crucial mechanism of the innate immune response, enabling macrophages to identify and remove pathogens and other harmful substances from the body. The process of phagocytosis involves several steps. First, macrophages use receptors on their cell surface to recognize and bind to foreign particles. Next, macrophages extend pseudopods (projections on the cell membrane) around the particle to form a phagosome. The phagosome then fuses with a lysosome containing enzymes that break down the contents of the phagosome. The resulting material is then released from the macrophage or presented to other immune cells for further processing.

[0088] As used herein, “macrophage migration” refers to the process by which macrophages, a type of immune cell, move from one location to another within the body. This migration is essential for macrophages to perform their functions in immune surveillance, tissue repair, and inflammation. Macrophage migration occurs in response to a variety of stimuli, including chemokines, growth factors, and microbial products. These stimuli activate signaling pathways within macrophages that induce changes in cytoskeletal structure and cell polarity, allowing macrophages to migrate towards the source of the stimulus. Macrophage migration is a complex process involving several steps, including adhesion to the extracellular matrix, cell polarization, and extension of pseudopods to enable migration. During migration, macrophages can interact with other immune cells, such as T cells and B cells, as well as other cell types in the tissue microenvironment, including fibroblasts and endothelial cells.

[0089] As used herein, macrophage activation refers to the process by which macrophages, a type of immune cell, are activated in response to stimuli such as infection or tissue injury. Activated macrophages undergo a series of changes in gene expression, morphology, and function that enable them to perform their roles in the immune response more effectively. Macrophage activation can occur through two distinct pathways: classical activation and alternative activation. Classical activation, also known as M1 activation, occurs in response to pro-inflammatory signals such as interferon-gamma (IFN-γ) and lipopolysaccharide (LPS) produced during infection or inflammation. M1 macrophages are characterized by increased production of pro-inflammatory cytokines, enhanced phagocytic activity, and increased expression of surface molecules involved in antigen presentation and T cell activation. Alternative activation, also known as M2 activation, occurs in response to anti-inflammatory signals such as interleukin-4 (IL-4) and interleukin-13 (IL-13) produced during tissue repair and wound healing. M2 macrophages are characterized by reduced production of pro-inflammatory cytokines and increased production of anti-inflammatory cytokines and growth factors. They also exhibit enhanced phagocytosis of apoptotic cells and debris, playing a role in tissue remodeling and angiogenesis.

[0090] As used herein, “pro-inflammatory cytokines” refer to a group of signaling proteins involved in the inflammatory response of the immune system. These cytokines are typically produced by immune cells such as macrophages, T cells, and mast cells in response to various stimuli, including infection, injury, or stress. Examples of pro-inflammatory cytokines include interleukin-1 (IL-1), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), interferon-gamma (IFN-γ), and interleukin-17 (IL-17), granulocyte-macrophage colony-stimulating factor (GM-CSF), or other interleukins such as IL-8, IL-12, IL-18, and IL-23. These cytokines can activate immune cells and cause inflammation characterized by redness, swelling, heat, and pain. Pro-inflammatory cytokines play a crucial role in the immune response to infection and tissue damage. They recruit immune cells to the site of infection or injury, promote the destruction of pathogens, and help initiate tissue repair. However, excessive or prolonged production of inflammatory cytokines can lead to chronic inflammation, tissue damage, and autoimmune diseases.

[0091] As used herein, the term “specifically binds” means that an antibody recognizes and binds to a specific target molecule, such as a protein or small molecule, with high affinity and selectivity. This is also known as immunospecific binding. The specificity of an antibody is determined by the unique three-dimensional structure of its variable region that forms the antigen-binding site that interacts with the target molecule. The specificity of an antibody to its target molecule is usually determined experimentally, for example, by measuring its binding affinity or by testing its ability to selectively recognize the target molecule in a complex mixture of other molecules. The binding specificity or immunospecific binding of an antibody can be assayed by any method known in the art. Immunoassays that can be used include, but are not limited to, competitive and non-competitive assay systems using techniques such as Western blotting, radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), “sandwich” immunoassay, immunoprecipitation assay, precipitation reaction, gel diffusion precipitation reaction, immunodiffusion assay, agglutination assay, complement fixation assay, immunoradiometry assay, fluorescence immunoassay, or protein A immunoassay.

[0092] As used herein, the term “antibody-drug ratio” (ADR) is used in the context of antibody-drug conjugates (ADCs), a type of targeted therapy that combines the specificity of an antibody with the cytotoxicity of a drug. ADR refers to the number of drug molecules conjugated to each antibody molecule within an ADC. It is typically expressed as a ratio, such as 2:1 or 4:1, indicating the number of drug molecules per antibody molecule. ADR can vary depending on the design and synthesis of the ADC and is a critical parameter that can affect the efficacy and safety of the therapy. The optimal ADR for an ADC depends on several factors, including the potency of the drug, the binding affinity of the antibody, and the expression level of the target antigen. A higher ADR can increase the potency of the therapy by delivering more drug molecules to target cells, but it can also increase the risk of toxicity by increasing the nonspecific binding of the ADC to healthy cells. A lower ADR can reduce the risk of toxicity, but it can also reduce the efficacy of the therapy. ADR is a critical parameter evaluated during the preclinical and clinical development of ADCs to optimize their therapeutic properties.

[0093] As used herein, the term "CHR21Q22-related disease" refers to a disease or disorder linked to an intergenic region on CHR21Q22. In particular, CHR21Q22-related diseases include Crohn's disease, ulcerative colitis, primary sclerosing cholangitis, ankylosing spondylitis, and Takayasu's arteritis.

[0094] The term "autoinflammatory disease" refers to a disease or disorder that involves episodes of inflammation caused by the innate immune system.

[0095] The term “comprising” is used herein to mean including the specified method steps or elements, but not to include an exclusive list, and therefore additional steps or elements may exist.

[0096] Furthermore, insofar as the term “includes” is used in either the detailed description or the claims, such term is intended to be inclusive in the same manner as the term “comprising” is used as a transitional term in the claims.

[0097] Pharmaceutical composition of drugs As used herein, “pharmaceutical composition” means a mixture of substances suitable for administration to an individual. For example, a pharmaceutical composition may comprise one or more active agents and a sterile aqueous solution.

[0098] As used herein, “pharmaceutically acceptable salt” means a physiologically and pharmaceutically acceptable salt of an antisense compound, i.e., a salt that retains the desired biological activity of the parent oligonucleotide and does not impart any undesirable toxicological effects thereto.

[0099] Other aspects of the present invention also relate to pharmaceuticals or diagnostic aids comprising an inhibitor according to the present invention, or a nucleic acid according to the present invention, or a cell according to the present invention, and, where appropriate, suitable excipients and additives such as physiological saline, stabilizers, or protease inhibitors.

[0100] kit Any pharmaceutical composition described herein may be provided in a kit. In some cases, the kit includes (a) a container containing the pharmaceutical composition described herein, and optionally (b) informational material. The informational material may be, for example, descriptive, instructional, marketing, or other material relating to the use of the methods and / or agents described herein for therapeutic benefit.

[0101] The informational materials in the kit are not limited to their form. In some cases, the informational materials may include information regarding the production of the drug of the present invention, such as concentration, expiration date, batch or production site information. In other cases, the informational materials may relate to a method of administering the drug of the present invention, for example, in a preferred amount, form, or method of administration (e.g., the dose, dosage form, or method of administration described herein).

[0102] In some cases, informational materials, such as instructions, may be provided in printed form, such as printed text, drawings, and / or photographs, such as labels or printed sheets. Informational materials may also be provided in other formats, such as Braille, computer-readable materials, video recordings, or audio recordings. In other cases, the informational materials in a kit may include contact information, such as a physical address, email address, website, or telephone number, which the user of the kit can use to obtain substantial information regarding the therapeutic agents and / or their use in the methods described herein. Informational materials may also be provided in any combination of formats.

[0103] In addition to the pharmaceutical composition of the present invention, the kit may contain other components such as solvents or buffers, stabilizers, or preservatives. The kit may also contain further agents, e.g., second or third agents, e.g., other therapeutic agents or other therapeutic compounds or compositions. Components may be provided in any form, e.g., liquid, dry, or lyophilized. Components may be substantially pure (they may be combined together or delivered separately) and / or sterile. If components are provided in a liquid solution, the liquid solution may be an aqueous solution, such as a sterile aqueous solution. If components are provided in a dry form, reconstitution is generally carried out by adding a suitable solvent. Solvents, e.g., sterile water or buffer solutions, may be provided in the kit at the option of their own.

[0104] The kit may include one or more containers for a pharmaceutical composition or other drug. In some cases, the kit may include separate containers, dividers, or compartments for the therapeutic agent and informational material. For example, the therapeutic agent may be contained in a bottle, vial, or syringe, and the informational material may be contained in a plastic sleeve or packet. In other situations, the separate elements of the kit are contained within a single, undivided container. In some cases, the kit may include a plurality (e.g., packs) of individual containers, each containing one or more unit dosage forms (e.g., dosage forms described herein) of the drug of the present invention. The containers may contain unit doses, for example, units containing the therapeutic agent. For example, the kit may include a plurality of syringes, ampoules, foil packets, blister packs, or medical devices, each containing a unit dose. The containers of the kit may be airtight, waterproof (e.g., impermeable to changes in moisture or evaporation), and / or lightly airtight.

[0105] The kit may optionally include a device suitable for administering the drug of the present invention, for example, a syringe or other suitable delivery device. The device may be provided pre-loaded with the drug of the present invention in a unit dose, or it may be empty but suitable for loading.

[0106] CRISPR As used herein, “CRISPR nuclease system” refers collectively to transcripts and other elements involved in directing the expression or activity of the Cas gene, including sequences encoding CRISPR-related ("Cas") genes, guide sequences (also referred to as “spacers” in the context of endogenous CRISPR systems), or other sequences and transcripts from the CRISPR locus.

[0107] In some embodiments, one or more elements of the CRISPR system are derived from type I, type II, or type III CRISPR systems. In some embodiments, one or more elements of the CRISPR system are derived from specific organisms that include an endogenous CRISPR system, such as Streptococcus pyogenes. The CRISPR system is characterized by elements that promote the formation of the CRISPR complex at a site of a target sequence.

[0108] In the context of CRISPR complex formation, the “target sequence” refers to a sequence designed to be complementary to the guide sequence, and hybridization between the target sequence and the guide sequence facilitates CRISPR complex formation. Complete complementarity is not necessarily required, as long as there is sufficient complementarity to induce hybridization and facilitate CRISPR complex formation. The target sequence may include any polynucleotide, such as DNA or RNA polynucleotides. In some embodiments, the target sequence is located in the nucleus or cytoplasm of a cell. In some embodiments, the target sequence may be located within an organelle of a eukaryotic cell, for example, in a mitochondria or chloroplast.

[0109] When using multiple different guide sequences, a single expression construct may be used to target CRISPR activity to multiple different corresponding target sequences within the cell. For example, a single vector may contain about one, two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty, or more guide sequences. In some embodiments, a vector containing about one, two, three, four, five, six, seven, eight, nine, ten, or more such guide sequences may be provided and optionally delivered to cells. In some embodiments, the vector includes a regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme, such as a Cas protein, also called a Cas enzyme.

[0110] Non-exclusive examples of Cas proteins (or Cas enzymes) include Cas1, Cas1.13, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csx12), Cas10, Csy1, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Cs Examples include m2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Crnr6, Csbl, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, their homologs, or modified forms thereof.

[0111] These enzymes are known; for example, the amino acid sequence of the S. pyogenes Cas9 protein can be found in the SwissProt database under accession number Q99ZW2. In some embodiments, the CRISPR enzyme, e.g., Cas9, has DNA cleavage activity. In some embodiments, the CRISPR enzyme is Cas9, which may be Cas9 derived from S. pyogenes or S. pneumoniae.

[0112] In some embodiments, the CRISPR enzyme induces a cleavage of one or both strands at a location within the target sequence, such as within the target sequence and / or within a complementary sequence of the target sequence. In some embodiments, the CRISPR enzyme induces a cleavage of one or both strands within approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of the target sequence.

[0113] In some embodiments, the CRISPR enzyme is instructed to cleave a nucleic acid sequence encoding ETS2 or a nucleic acid sequence encoding the enhancer region at chr21q22.

[0114] Alternative modalities In some embodiments, the ETS2 or chr21q22 enhancer inhibitor comprises a small molecule. In some embodiments, the ETS2 or chr21q22 enhancer inhibitor comprises an inhibitory peptide. In some embodiments, the ETS2 or chr21q22 enhancer inhibitor is an inhibitory nucleic acid molecule. In some embodiments, the ETS2 or chr21q22 enhancer inhibitor comprises an antibody.

[0115] Combination therapy In some embodiments, the present invention is characterized by a composition (e.g., one or more compositions, formulations, or drug formulations) or a pharmaceutical combination comprising an inhibitory agent and a second therapeutic agent according to the present invention.

[0116] In some embodiments, the composition includes a pharmaceutically acceptable carrier. In some embodiments, the inhibitor and the second agent according to the present invention may be present in a single composition or as two or more different compositions. The inhibitor and the second agent according to the present invention may be administered via the same route of administration or via different routes of administration. The inhibitor and the second agent according to the present invention may be administered simultaneously or sequentially. In some embodiments, the pharmaceutically acceptable combination includes the inhibitor and the second agent according to the present invention separately or together. [Examples]

[0117] Example 1 - Elucidation of the molecular mechanism in the multidirectional chr21q22 gene locus. Several genetic variants predispose individuals to two or more diseases, highlighting both their biological significance and the opportunity to discover common disease mechanisms. One notable example is the intergenetic haplotype on chr21q22 independently associated with Crohn's disease[7], ulcerative colitis[7] (collectively known as IBD), ankylosing spondylitis[8], primary sclerosing cholangitis[9], and Takayasu's arteritis

[10] . The associated locus contains no genes, but several nearby candidates, including PSMG1, BRWD1, and ETS2 (Figure 1a), have all been nominated as potentially causally related in different studies[7, 8, 9, 10, 11]. However, the underlying biological mechanisms remain unknown. We hypothesized that this intergenetic locus must be a distal enhancer, and since the associated diseases are all immune-mediated despite affecting different organs, we searched for features of enhancer activity in disease-associated immune cell types. Using H3K27ac ChIP-seq data marking active enhancers / promoters, we found that the chr21q22 locus contains monocyte / macrophage-specific enhancers (Figure 1a). Monocytes and monocyte-derived inflammatory macrophages play a central role in the development of many autoimmune and inflammatory diseases and produce cytokines that are often targeted by the most effective therapies

[12] . To identify the causative gene at this locus, we first examined publicly available data, including promoter-captured Hi-C

[13] and eQTL datasets

[14] from human monocytes. We found that the disease-associated locus physically interacts with the ETS2 promoter located approximately 290kb away, and that risk haplotypes correlate with higher ETS2 expression, particularly in post-activated monocytes (Figure 6). Interestingly, however, eQTL signaling in activated monocytes did not statistically co-localize with disease relevance, but co-localization with weaker eQTLs in quiescent monocytes was observed (Figure 6).To more directly confirm the identity of the target gene, CRISPR guide (g)RNAs were designed to be adjacent to the 1.85kb enhancer region and delivered to primary human monocytes as Cas9-gRNA-ribonucleoprotein (RNP) complexes (Figure 1b, Figure 7). Deletion of the entire enhancer was achieved in an average of 44% of cells (Figure 7). The transfected cells were then cultured in the presence of several inflammatory mediators, including TNFα (a pro-inflammatory cytokine), prostaglandin E2 (an inflammatory lipopeptide), and Pam3CSK4 (a TLR1 / 2 agonist). This model, called "TPP," is designed to mimic a chronic inflammatory environment

[15] and better reproduces the state of patient-derived monocytes / macrophages than classical M1 or M2 models (

[16] and Figure 7). Since flow cytometry antibodies were not available for any of the chr21q22 genes, we used PrimeFlow to measure the dynamics of RNA transcription after CRISPR editing of disease-related loci. In unedited cells, expression of all three candidate genes (ETS2, BRWD1, and PSMG1) increased after exposure to inflammatory stimuli (Figure 1c). Deletion of the chr21q22 enhancer did not affect the upregulation of BRWD1 and PSMG1, but ETS2 expression was significantly reduced (Figure 1d)—confirming that this multidirectional locus is a monocyte / macrophage-specific distal enhancer of ETS2.

[0118] Next, we attempted to identify the causative variant in chr21q22, where the biological effects were responsible for the risk of inflammatory disease. Unfortunately, statistical fine mapping of the largest IBD GWAS to date [7] was unable to reduce the number of candidate variants due to very high correlations (binding imbalance; LD) between them (Methods, Figure 1e). Therefore, we used a functional approach (large-scale parallel reporter assay; MPRA) to first identify the active enhancer sequence within the locus and then determine whether any candidate variant within this regulatory DNA could alter enhancer activity. MPRA is a high-throughput method that can simultaneously characterize enhancer activity in thousands of short DNA sequences by binding each short DNA sequence to a reporter gene uniquely barcoded within an expression vector

[17] . By normalizing the number of barcodes in mRNA extracted from transfected cells to an equivalent number in the input DNA library, the DNA sequences that regulate gene expression can be identified. Previously, MPRA was adapted for use in primary CD4+ T cells

[18] , and in this specification, the same principle was applied to adapt it to primary macrophages, thereby ensuring that an appropriate repertoire of transcription factors was present and interacted with the chr21q22 genomic sequence (Figure 9). Based on the sequences of 2-kb regions containing all candidate gene variants, duplicate oligonucleotides (oligos; 114 bp genomic sequences tiled at 50 bp intervals) were synthesized, and for each variant, additional oligos containing either a risk allele or a non-risk allele were included. After cloning, the resulting vector libraries were transfected into inflammatory macrophages from multiple independent donors, and barcode abundances were quantified using high-throughput RNA and DNA sequencing (Methods). Using sliding window analysis, we summarized the expression regulatory effects of the tiling sequence and identified a single 442 bp focus of enhancer activity (chr21:40466236-40466677, hg19, Figure 1f), which included three (seven) candidate mutants.Two of these mutants were transcriptionally inactive, but a third mutant (rs2836882) exhibited the strongest regulatory effect of any candidate mutant at the locus, with the risk allele (G) significantly increasing transcription, consistent with the reported direction of eQTLs (Figure 1f). Further investigation of rs2836882 revealed that this mutant is located within an experimentally confirmed PU.1 ChIP-seq peak in inflammatory macrophages (Figure 1g). PU.1 is an important myeloid pioneer factor that binds to heterochromatin, initiates nucleosome modification, and thus allows other transcription factors to bind and activate transcription [20, 21]

[19] . To determine whether the rs2836882 genotype may affect PU.1 binding, we identified two publicly available macrophage PU.1 ChIP-seq datasets from heterozygous individuals and assessed allelic imbalances in PU.1 binding using BaalChIP

[22] . Despite not being present within the normal PU.1 binding motif, significant allele-specific PU.1 binding was observed in rs2836882, accompanied by more than four-fold binding to the risk allele in both datasets (Figure 1h). This result was replicated by immunoprecipitation of PU.1 in TPP macrophages from five rs2836882 heterozygotes and genotyping of the binding DNA (Figure 10). This suggests that the rs2836882 risk allele should confer greater enhancer activity, consistent with MPRA and eQTL results. To test whether allele-specific differences in enhancer activity reside at an endogenous locus, H3K27ac ChIP-seq was performed in inflammatory macrophages from two major allele homozygotes and two minor allele homozygotes in rs2836882. While several nearby enhancer peaks were similar among these donors, enhancer activity located on rs2836882 was considerably stronger in the major (risk) allele homozygote (Figure 1i) and contributed to an approximately 2.5-fold increase in enhancer activity across the extended chr21q22 locus (Figure 10).Overall, these data identify the presumed causative variant in chr21q22 through its functional consequences in primary macrophages, revealing a genetic mechanism that enhances pioneer transcription factor binding and increases the activity of the long-range ETS2 enhancer.

[0119] Example 2 - ETS2 is required for the inflammatory response of macrophages. After identifying a plausible mechanism by which the chr21q22 risk haplotype increases ETS2 expression in monocytes / macrophages, we then sought to better understand the role of ETS2 in these cells. ETS2 is a member of the ETS family of transcription factors, which have been primarily studied as a proto-oncogene in cancer

[23] . In contrast, the role of ETS2 in primary human macrophages is not well defined, and previous studies have used either cell lines or compound mouse models and focused primarily on a single downstream molecule [24, 25, 26, 27, 28]. This has led to conflicting reports, with ETS2 being described as both required and surplus for macrophage development [29, 30] and both pro-inflammatory and anti-inflammatory [24, 25, 26, 27, 28]. To elucidate the specific role of ETS2 in inflammatory human macrophages and determine how dysregulated ETS2 expression contributes to disease, we first used a CRISPR-Cas9-based loss-of-function technique (Figure 2a). To minimize the chance that any desired effect might be due to off-target editing, two gRNAs targeting different ETS2 exons were designed and validated, and individually incorporated into Cas9 RNPs for transfection into primary monocytes, resulting in average on-target editing in 90% and 79% of cells, respectively (Figure 7). No difference in cell viability or macrophage marker expression was observed with either gRNA, suggesting that ETS2 is not required for inflammatory macrophage differentiation (Figure 7). In contrast, the production of pro-inflammatory cytokines, including IL-6, IL-8, and IL-1β, was significantly reduced after ETS2 deletion (Figure 2b), while the effect of the anti-inflammatory cytokine IL-10 was less pronounced. TNFα could not be evaluated as it was exogenously added. Next, we investigated whether ETS2 is also required for other macrophage effector functions. First, we examined phagocytosis using a fluorescently labeled substrate (dimosan particles) detectable by flow cytometry. Similar to pro-inflammatory cytokine production, phagocytosis was significantly impaired after ETS2 editing (Figure 2c).Next, we measured extracellular reactive oxygen species (ROS) production, a key effector response contributing to tissue damage in inflammatory diseases

[31] . We found that disrupting ETS2 significantly reduced the oxidative burst after macrophage activation, an effect that we thought was due to a reduction in the expression of major components of NADPH oxidase (Figure 2d, Figure 11). Overall, this suggests that ETS2 is required for multiple effector functions in inflammatory macrophages.

[0120] To better understand the molecular basis for these distinct functional effects, whole transcriptome RNA sequencing (RNA-seq) was performed on ETS2-edited and controlled inflammatory macrophages from multiple independent donors. Disruption of ETS2 resulted in widespread transcriptional changes and markedly reduced expression of many inflammatory genes, including several major initiators and amplification factors of inflammation (Figure 2e). The affected gene classes included cytokines (e.g., TNFSF10 / TRAIL, TNFSF13, IL1B), chemokines (e.g., CXCL1, CXCL3, CXCL5, CCL2, CCL5), secreted effector molecules (e.g., S100A8, S100A9, MMP14, MMP9), cell surface receptors (e.g., FCGR2A, FCGR2C, TREM1), pattern recognition receptors (e.g., TLR2, TLR6, NOD2), and signaling molecules (e.g., MAP2K, GPR84, NLRP3). To better characterize the pathways affected by ETS2 deletion, we performed gene set enrichment analysis (GSEA) using the Gene Ontology Biological Pathway Dataset. This supports the observed functional effects (Figure 2f), with the most negatively enriched pathways (downregulated after ETS2 disruption) being those involved in macrophage activation, pro-inflammatory cytokine production, phagocytosis, and ROS production. Genes involved in macrophage migration were also significantly downregulated, but gene sets related to monocyte-to-macrophage differentiation were not significantly affected—this is consistent with ETS2 directing the macrophage inflammatory response but not affecting monocyte-to-macrophage development. Few genes were upregulated after ETS2 deletion (Figure 2e), but two notably positively enriched pathways related to the metabolic processes of aerobic respiration and oxidative phosphorylation (OXPHOS; Figure 2f), which have been previously associated with anti-inflammatory macrophage behavior

[32] . In summary, these data identify the essential role of ETS2 in various macrophage effector functions, which can explain why dysregulation of ETS2 expression contributes to multiple inflammatory diseases. Indeed, deletion of the disease-related chr21q22 enhancer replicated both the functional and transcriptional consequences of ETS2 disruption (Figure 2g, Figure 11).

[0121] Example 3 - ETS2 controls the macrophage inflammatory response. The finding that ETS2 is required for effector function in monocyte-derived macrophages raised the question of whether it is sufficient to drive them, as one might expect from a central regulator of the inflammatory response. This is particularly important because loss-of-function techniques are useful for identifying the biological role of genes, but the chr21q22 risk haplotype increases ETS2 expression. To address this, we first optimized a method for inducing controlled overexpression of a specific gene in quiescent, inactive (M0) macrophages by transfecting them with a predefined amount of in vitro transcribed mRNA (Figure 3a, Figure 12). To minimize nonspecific activation associated with the transfected RNA, in vitro transcription was performed using co-transcriptional capping (to minimize uncapped products) and incorporating modified minimal immunogenic nucleotides (uridine replaced with N1-methyl-psoidouridine and cytidine replaced with methylcytidine). Control cells were transfected with an equal amount of mRNA encoding the reverse complement of ETS2, thereby controlling the amount, length, and purine / pyrimidine composition of the transfected RNA, but using untranslated transcripts (Figure 3b). ETS2 expression in these control cells was comparable to that of untransfected cells cultured under similar conditions, indicating that this method did not reduce ETS2 mRNA levels. Following RNA transfection, low-dose lipopolysaccharide exposure for 6 hours induced a low-grade inflammatory response (Figure 3a). Cytokine secretion in the cell culture supernatant was initially measured, and it was found that ETS2 overexpression was sufficient to increase the production of several pro-inflammatory cytokines, but again, the effect of IL-10 was minimal (Figure 3c). To better characterize the results of ETS2 overexpression, RNA-seq was performed to specifically investigate macrophage activation pathways that previously showed the required ETS2.Surprisingly, not only were multiple inflammatory pathways, including macrophage activation, pro-inflammatory cytokine production, ROS production, phagocytosis, and migration, induced by ETS2 overexpression, but this induction was also shown to be dose-dependent, with higher enrichment of all pathways as more ETS2 mRNA was transfected (Figure 3d). This demonstrates that ETS2 is both necessary and sufficient for the inflammatory response in primary human macrophages, revealing it to be a central regulator of bone marrow effector function, where dysregulation is directly linked to human disease.

[0122] Example 4 - In IBD, the ETS2 regulatory gene plays a central role. To understand whether ETS2 directly contributes to the macrophage phenotype observed in inflammatory diseases, the transcriptional outcomes of ETS2 overexpression were compared to a gene signature derived from intestinal macrophages in Crohn's disease, one of the chr21q22-associated states. Single-cell RNA-seq analysis has previously shown that active Crohn's disease is characterized by an expanded population of inflammatory monocyte-derived macrophages that contribute to anti-TNFα resistance

[33] . Using the Crohn's disease macrophage signature as a gene set, overexpression of ETS2 in quiescent macrophages induced a transcriptional state very similar to that of inflammatory disease macrophages, and core ("leading edge") enrichment of more than half of the genes in the signature, including many targets of approved therapies, was found (Figure 3e).

[0123] Based on the importance of ETS2 in the macrophage inflammatory response and the fact that ETS2 overexpression replicated disease-associated inflammatory states, we hypothesized that other genetic associations could also influence this previously uncharacterized pathway. A key goal of the GWAS was to identify a central disease pathway [3], which has proven challenging due to a lack of confidently identified causative genes and limited understanding of how these are affected by genetic mutations [3]. However, there have been some notable successes, including the discovery that autophagy is involved in Crohn's disease susceptibility through genetic associations of several autophagy genes

[34] . To better characterize the genetic risk attributable to the macrophage ETS2 pathway, we focused on IBD, as it has far more genetic associations than any other chr21q22-associated disease. Examining a list of genes that are generally downregulated after ETS2 editing (Padj<0.05 for both gRNAs), we identified more than 20 IBD risk genes, many of which have been proposed to be causally related at their respective loci [7, 35] (Table Sx). These included several genes thought to influence macrophage biology (e.g., SP140, LACC1 / FAMIN, CCL2, CARD9, CXCL5, TLR4, SLAMF8), as well as several genes highly expressed in macrophages but not previously associated with specific pathways (e.g., ADCY7, PTPRC, TAGAP, PTAFR, PDLIM5, DOK2). To more formally assess the extent to which ETS2-driven inflammation is related to previously identified disease pathways by IBD genetics, we used SNPsea

[36] , an algorithm designed to identify pathways affected by disease loci. 241 IBD-related loci were tested to enrich 7,658 gene ontology biological pathways and 7,660 pathways, including two duplicate lists of ETS2 regulatory genes (either downregulated after ETS2 editing or upregulated after ETS2 overexpression).The significance of enrichment was empirically calculated using a set of 5 million corresponding null SNPs, and pathways previously associated by IBD genetics were extracted for comparison. Surprisingly, ETS2 target genes, by any definition, were more strongly enriched for IBD-related loci than almost all previously associated pathways, and no set of null SNPs showed greater enrichment than any of the ETS2 regulatory genelists. After applying rigorous Bonferroni multiplexing correction, only ETS2 regulatory genes, as well as IBD pathways associated with T cell activation, T-helper 17 cells, autophagy, and IL-10 signaling, showed significant enrichment (Figure 3f). This suggests that ETS2 signaling in inflammatory macrophages plays a central role in IBD development and has higher gene enrichment than many previously involved pathways.

[0124] Example 5 - ETS2 modulates macrophage inflammatory responses through transcriptional and metabolic effects. Next, we sought to understand how ETS2 controls such diverse effector functions in macrophages. Studying ETS2 biology is challenging because ChIP-seq grade antibodies are not available, hindering the direct identification of its transcriptional targets. Even the ENCODE project, which performed ChIP-seq on 181 transcription factors, was unable to directly immunoprecipitate ETS2

[37] . Therefore, we first used a “guilt-by-association” technique to identify genes highly co-expressed with ETS2 across 64 different macrophage polarization conditions.15 This identified PFKFB3, which encodes a rate-limiting enzyme for glycolysis, as the most strongly co-expressed gene, with HIF1A also highly co-expressed (Figure 4a). Together, these genes are known to promote the “glycolysis switch” essential for myeloid inflammatory responses

[38] . Therefore, we hypothesized that ETS2 may regulate the inflammatory response through metabolic reprogramming—this idea was supported by the negative correlation of the OXPHOS gene with ETS2 (Figure 4a) and its upregulation after ETS2 deletion (Figure 2f). To characterize the metabolic consequences of disrupting ETS2, we used gas chromatography-mass spectrometry (GC-MS) to quantify the incorporation of labeled 13C-glucose in CRISPR-edited and unedited inflammatory macrophages. Following ETS2 disruption, we detected broad but slight decreases in both unlabeled and labeled glucose metabolites (Figure 4b, Figure 13). This affected both glycolysis and TCA cycle metabolites, including a marked decrease in intracellular and secretory lactates, characteristic of anaerobic glycolysis, as well as succinates, an important inflammatory signaling metabolite

[39] . These results are consistent with the reported inhibitory effect on glycolysis, and the decrease in TCA metabolites is attributed to uptake by mitochondrial OXPHOS [40, 41]. To determine whether these metabolic effects are responsible for the ETS2-mediated inflammatory effects, ETS2-editing inflammatory macrophages were treated with roxadustat, an HIF1α stabilizer that can promote glycolysis via HIF1α-mediated metabolic reprogramming.This had the predicted effects on the expression of genes involved in glycolysis and oxphos, but did not restore the effects of ETS2 disruption, either transcriptionally or functionally (Figure 4c, Figure 13). Therefore, while disrupting ETS2 appears to alter glucometabolism, these effects are not solely responsible for the observed differences in inflammation.

[0125] Therefore, we re-examined whether we could identify ETS2 target genes and sought to understand how ETS2 can control such diverse inflammatory effects. Using various anti-ETS2 antibodies, we confirmed that none functioned against ChIP (data not shown) and investigated whether any might function against Cleavage-Under-Targets-and-Release-Using-Nuclease (CUT&RUN), which does not require formaldehyde fixation. One of these antibodies identified several significantly enriched genomic regions (peaks) with 6,560 reproducibly detected across two biological replicas (non-reproducible detection rate <0.01) with an acceptable quality metric

[42] (mean FRiP score 0.23) (Figure 4d). These peaks were primarily located in the regulatory activity regions (90% of promoters or activity enhancers, Figure 4e), were highly enriched compared to the canonical ETS2 position-weight matrix (3.98-fold enrichment compared to the overall control, P=4.16e-126, Figure 4f), and coincided with ETS2 binding sites. After combining biological replicas to improve peak detection, ETS2 binding peaks were detected in the promoters of several major inflammatory genes, indicating that ETS2 likely directly modulates multiple macrophage effector responses (Figure 4g). Supporting this, 48.3% of genes dysregulated after ETS2 editing and 50.3% of genes dysregulated after ETS2 overexpression contained ETS2 binding peaks within their core promoters or their putative cis-regulatory elements (Figure 4h). These transcriptional targets include HIF1A, PFKFB3, and other glycolysis genes (e.g., GPI, HK2, and HK3), suggesting that the observed metabolic changes may also be directly induced by ETS2, not solely due to differences in inflammation. Notably, we also detected ETS2 binding at chr21q22 with its own enhancer (Figure 4i). This is consistent with previous reports that PU.1 and ETS2 can interact synergistically

[43] , suggesting a feedforward mechanism at disease-related loci in which increased ETS2 expression enhances ETS2 enhancer activity.In summary, these data suggest that ETS2 can be a master regulator of the monocyte / macrophage response in chronic inflammation, directing multifaceted transcriptional programs and contributing to a metabolic environment that tolerates inflammation.

[0126] Example 6 - ETS2-driven inflammation is detectable in diseased tissue and can be pharmacologically targeted. The potent enrichment of IBD GWAS hits in ETS2 regulatory genes led to the hypothesis that the transcriptional footprint of this pathway may be commonly detectable in affected organs of chr21q22-related diseases, which could have important therapeutic implications. Using publicly available gene expression data from chr21q22-related diseases (intestinal macrophages from IBD, synovium from ankylosing spondylitis, and liver from PSCs), we confirmed that diseased tissues were significantly enriched for genes regulated by ETS2 (Figure 5a, Figure 16). Therefore, we investigated whether this pathway could be pharmacologically targeted. No specific ETS2 inhibitors exist, and structural analysis indicates the absence of readily targetable allosteric inhibitory mechanisms

[44] . Therefore, we used the NIH LINCS database to identify drugs that may modulate ETS2 activity

[45] . This repository contains a list of more than 23,000 differentially expressed genes from cell lines exposed to more than 6,000 small molecules. Using GSEA, 906 drug signatures were found to mimic transcriptional effects that disrupt ETS2 in inflammatory macrophages (Padj<0.05), including several drugs already approved for the treatment of IBD and AS (e.g., JAK inhibitors). Of these candidate therapies, the most common class was MEK inhibitors, which are currently licensed for non-inflammatory human diseases (e.g., neurofibromatosis) (Figure 5b). This result was not due to a single compound, but rather a class effect in which multiple MEK1 / 2 inhibitors could downregulate ETS2 target genes (Figure 5c). This made biological sense, as MEK1 and MEK2, along with several other targets identified, are known upstream regulators of ETS family transcription factors (Figure 5d). Indeed, some of these drug classes, including MEK and HSP90 inhibitors, have been reported to be beneficial in mouse colitis models, but this is often an unreliable indicator of clinical efficacy, as some approved IBD treatments are ineffective in mice, and many drugs that improve mouse models are ineffective in human IBD

[46] .To determine whether MEK inhibition suppresses the inflammatory response in primary human macrophages, monocytes were differentiated under chronically inflammatory conditions and treated with selective non-ATP competitive MEK inhibitors (PD-0325901; Figure 5e). We observed potent anti-inflammatory activity that replicated the effects of ETS2 disruption or chr21q22 enhancer deletion, accompanied by downregulation of multiple inflammatory pathways (including several approved drug targets; Figure 5g) (Figure 5f, Figure 17). Considering the limitations of animal colitis models

[46] , we further investigated the therapeutic potential of targeting ETS2 signaling using a human intestinal explant model. To do this, intestinal mucosal biopsies were taken from active IBD patients who were not receiving immunosuppression or biological therapy and cultured for 18 hours on Transwell inserts

[47] with either a MEK inhibitor or a negative or positive control (Methods). The release of multiple IBD-related inflammatory cytokines was significantly reduced by MEK inhibition, to a level comparable to that observed with infliximab (a widely used anti-TNFα monoclonal antibody for IBD, Figure 5h). Furthermore, we confirmed reduced expression of ETS2 regulatory genes (Figure 5i) and significant improvement in the validated transcriptional inflammation score

[48] , which reflects IBD-related inflammation and has been shown to decrease with effective therapy (Figure 5j). Together, this suggests that targeting upstream regulators of ETS2 may be therapeutically useful in suppressing pathological inflammation in chr21q22-related diseases.

[0127] Example 7 - Antibody-drug conjugate (ADC) experiment Due to its limited but ETS2-dependent expression in inflammatory macrophages, its scavenger function, and previous literature in ADCs, we selected CD163 as the optimal target for ADC-mediated drug delivery.

[0128] We test cleavable and non-cleavable linkers via the maleimide conjugation method used in most approved ADCs to generate different drug:antibody ratios (DAR4, 6, and 8). This optimizes the trade-off between hydrophobicity / aggregation and therapeutic efficacy.

[0129] The selected cleavable linker is glucuronide-based (GlyPro). It is stable in circulation and selectively cleaved by β-glucuronidase, thereby promoting selective release in lysosomes. Additionally, this linker possesses favorable hydrophilic properties, which should reduce aggregation and promote solubility. Such linkers have been successfully used in several prodrugs. The selected non-cleavable linker is maleimide-PEG3-based. These linkers perform remarkably well in in vivo studies due to their low risk of premature drug release in non-target cells and lower toxicity. This should improve stability, tolerability, and therapeutic window, with drug release occurring only following antibody / linker degradation in CD163-expressing cells' lysosomes.

[0130] Example 8 - Experiment with esterase-sensitive motif (ESM) conjugate MEK inhibitors Unlike most human carboxylesterases, which are ubiquitously expressed, human carboxylesterase-1 (hCE-1) is primarily expressed in monocyte-derived cells (e.g., monocytes and macrophages). ESM technology utilizes esters selectively hydrolyzed by hCE-1, and unlike esters, the acid cannot easily diffuse, resulting in intracellular accumulation of pharmacologically active acids (drugs) in hCE-1 expressing cells. This technique can promote a 1000-fold increase in titer compared to non-conjugate inhibitors. hCE-1 is also expressed in the liver, and therefore requires intermediate-rate hydrolysis, which can be regulated by altering the ESM and inhibitor to ensure sufficient ester avoids first-pass metabolism. ESM-MEK inhibitor conjugates are synthesized with various ESMs, linker lengths, and MEK inhibitors to identify optimal combinations for downstream biological testing. Corresponding acid products and non-hydrolyzable ester controls are synthesized as controls.

[0131] Specifically, the following will be tested: - Three MEK inhibitors (to identify the most suitable candidate based on the pharmacological activity and synthetic traction of the acid-drug). - Two linker lengths (these link to the amino acid group and are typically one or two carbon atoms. This is important to ensure the molecule extends through the esterase active site). -Two ESMs (two validated ESMs listed in the literature: cyclopentyl L-leucinate and cyclopentyl(S)-2-amino-2-cyclohexyl acetate).

[0132] Example 9 - Biochemical and functional verification of MEKi conjugate molecules On-target binding: Competitive binding assays test whether ADCs bind to CD163 (using fluorescently tagged ADCs and measuring binding in the presence of various concentrations of non-conjugated antibodies). Other macrophage markers such as CD209 (DC-SIGN) and CD206 (MRC1) are also tested.

[0133] Internalization: Internalization of fluorescently tagged ADCs in inflammatory macrophages is examined by confocal microscopy.

[0134] ESM-MEKi-selective hydrolysis: Conjugates are incubated with peripheral blood mononuclear cells before individual cell populations (e.g., T cells, B cells, NK cells, and monocytes) are purified, and mass spectrometry (HPLC-MS / MS) is performed to determine the intracellular abundance of parent esters and hydrolyzable acids. Hydrolysis rates are quantified by incubation of ESM-MEKi conjugates with primary monocytes (hCE-1 positive) and hCE-1 deficient control cells.

[0135] Pharmacological activity (biochemical): The MEK inhibitory IC50 of each ADC and cleavage drug, or each ESM-MEKi and acid-drug, is calculated using a radiofilter-coupled assay with 33P ATP.

[0136] Pharmacological activity (physiological): ERK1 / 2 phosphorylation (Thr202 / Tyr204) is quantified by flow cytometry in individual leukocyte populations after culturing PBMCs with various concentrations of conjugates or cleavage agents.

[0137] Example 10 - Materials and Methods Analysis of existing data related to chr21q22 Using IBD GWAS summary statistics [7], multiple causal variant fine mappings were performed using susiE

[49] with reference minor alleles and LD information calculated from 503 European samples from 1000 genome phase 3. All R analyses were performed using v.4.2.1. Palindromic SNPs (A / T or C / G) and any SNPs that did not match by location or allele were pruned before imputation using the ssimp equation reimplemented in R. This did not affect any candidate SNPs in chr21q22. SuSiE fine mapping results for ETS2 (with identifiers ENSG00000157557 or ILMN_1720158) in monocyte datasets were obtained from the eQTL catalog. Colocalization analysis was performed using coloc v5.2.0

[50] , and colocalization was determined using the posterior probability of H4 (PP.H4.abf) > 0.5.

[0138] Raw H3K27ac ChIP-seq data from primary human immune cells were downloaded from the Gene Expression Omnibus (GEO series GSE18927 and GSE96014) and processed as described above

[51] . Processed promoter capture Hi-C data from 17 primary immune cell types were downloaded from OSF (https: / / osf.io / u8tzp)

[13] .

[0139] Monocyte purification and macrophage differentiation Leukocyte cones from healthy donors were obtained from NHS Blood and Transplant (Cambridge Blood Donor Centre, Colindale Blood Centre, or Tooting Blood Donor Centre). Peripheral blood mononuclear cells (PBMCs) were isolated by density centrifugation (Histopaque1077, Sigma), and monocytes were positively selected using CD14 microbeads (Miltenyi Biotec). Macrophage differentiation was performed using conditions that model chronic inflammation (TPP)

[15] : 3 days of GM-CSF (50 ng / ml, Peprotech), followed by 3 days of GM-CSF, TNFα (50 ng / ml, Peprotech), PGE2 (1 μg / ml, Sigma Aldrich), and Pam3CSK4 (1 μg / ml, Invivogen). All cultures were incubated in antibiotic-free RPMI1640 medium containing 10% FBS, GlutaMAX, and MEM non-essential amino acids (all ThermoFisher) at 37°C and 5% CO2. Cells were isolated using Accutase (Biolegend).

[0140] Identification of a model of chronic inflammatory macrophages Human monocyte-derived macrophage gene expression data files (n=299) associated with 64 different polarization conditions were downloaded from Gene Expression Omnibus (GSE47189) and quantile-normalized. Data from biological replicas were summarized to the median for each gene. Gene set variant analysis

[52] was performed (using the GSVA package in R) to identify the polarization conditions closest to CD14+ monocytes / macrophages from active IBD, using a disease-associated differentially expressed list

[53] .

[0141] CRISPR-Cas9 editing of the first human monocytes gRNA sequences were designed using CRISPick (formerly GPP sgRNA Designer) and synthesized by IDT. gRNA sequences: chr21q22 5'gRNA, CCUGGCUGCCUCGCGUUUCC; chr21q22 3'gRNA, CCUCGUCCAACAGAGAGCAA; ETS2 gRNA1, CAGACACAGAAUUACCCCAA; ETS2 gRNA2, UUGCUGCACGGGGUUAACAA. Alt-R CRISPR-Cas9 negative control crRNA#1 (IDT) was used as a non-targeting control. Cas9-gRNA ribonucleic acid protein was constructed as described above

[51] and synthesized using Nucleofector2b (Lonza, program Y-001) in 100 μL of nucleofection buffer (Human Monocyte Nucleofection Kit, Lonza) at a rate of 5 × 10⁶ 6 Nucleofection was performed on individual monocytes. After nucleofection, the monocytes were immediately transferred to 5 mL of preheated medium in a 6-well flat-bottom plate and differentiated into macrophages under TPP conditions. Editing efficiency was quantified by PCR amplification of the target region in the extracted DNA (chr21q22_Fw primer, GGTGGGGAGAGTTCCAAAGG;chr21q22_Rv, TCACCCTTCACCTCTTTGCT;ETS2_g1_Fw, TCCTGAAGGTCCCATGAAAG;ETS2_g1_Rv, TCATTATGGCTCTGGGGTTC;ETS2_g2_Fw, GCGGCACATTCATATCACAC;ETS2_g2_Rv, GCAGAATACCCCAAGCAAAA). Editing efficiency at the chr21q22 locus was measured via quantification of the amplified fragment (2100 Bioanalyzer, Agilent) as described above

[51] . The editing efficiency of individual gRNAs was evaluated using inference with the CRISPR Edits tool

[54] (ICE, Synthego).

[0142] PrimeFlow RNA assay RNA abundance was quantified by PrimeFlow (ThermoFisher) in chr21q22-edited and unedited (NTC) cells at days 0, 3, 4, 5, and 6 of TPP differentiation. Target probes specific to ETS2 (Alexa Fluor647), BRWD1 (Alexa Fluor568), and PSMG1 (Alexa Fluor568) were used according to the manufacturer's instructions. Data were analyzed using FlowJo v10 (BD Biosciences).

[0143] MPRA Duplicate oligonucleotides containing 114nt genomic sequences were designed to tile regions containing chr21q22 candidate SNPs (a 99% confidence set) at 50bp intervals. Six technical replicas were designed for each genomic sequence, each tagged with a unique 11nt barcode. The regulatory effects of all candidate SNPs within the 99% confidence set were tested, including additional oligonucleotides. Allelic constructs were designed as described above

[51] and tagged with 30 unique 11nt barcodes. Positive and negative controls were included as described above

[51] . 170nt oligonucleotides were synthesized as part of a larger MPRA pool (Twist Biosciences) containing the 16nt universal primer site ACTGGCCGCTTCACTG, the 114nt variable genomic sequence, KpnI and XbaI restriction sites (TGGACCTCTAGA), the 11nt barcode, and the 17nt universal primer site AGATCGGAAGAGCGTCG. Cloning into MPRA vectors was performed as described above

[51] . Suitable promoters for the MPRA vector (RSV) were identified by testing promoter activity in TPP macrophages. The MPRA vector library was nucleofected into TPP macrophages (5 μg of vector to 5 × 10⁶ cells) in 100 μl of nucleofection buffer (Human Macrophage Nucleofection Kit, Lonza) using Nucleofector2b (program Y-011). A minimum of 2 × 10⁶ cells were used for each donor (n=8) to ensure proper barcode representation. 7Nucleofection was performed on individual cells. After 24 hours, RNA was extracted and sequencing libraries were prepared from mRNA or DNA input vectors as described above

[51] . Library pools (each containing 6 samples) were sequenced on an Illumina HiSeq2500 high-power flow cell (50 bp, single-ended reads), and the data were preprocessed as described above

[51] . To identify regions of enhancer activity, paired t-tests were performed to identify transcription-enhancing genomic sequences. Then, sliding window analysis (300 bp window) was performed across all tiling sequences using the les package in R. Regulatory expression variants were identified using QuASAR-MPRA

[55] as described above

[51] .

[0144] BaalChIP We downloaded the publicly available PU.1 ChIP-seq dataset from human macrophages from GEO and examined the BAM files (using the IGV Genome Browser) to identify the rs2836882 heterozygote (i.e., the file containing both A and G allele reads for chr21:40,466,570; hg19). We identified two suitable samples (GSM1681423 and GSM1681429) and used them for Bayesian analysis of allele imbalance in PU.1 binding to correct for bias introduced by overdispersion and bias towards the reference allele, which is implemented in the BaalChIP package in R

[22] .

[0145] Allele-specific PU.1 ChIP genotyping 100 ml blood samples were collected from five healthy individuals heterozygous for rs2836882 (Taqman genotyping, evaluated via ThermoFisher). All participants provided written informed consent. Ethical approval was provided by the London-Brent Regional Ethics Committee (REC:21 / LO / 0682). Monocytes were isolated from PBMCs using CD14 microbeads (Miltenyi Biotec) and differentiated into inflammatory macrophages using TPP conditions

[15] . After differentiation, macrophages were isolated using Accutase and crosslinked in fresh medium containing 1% formaldehyde for 10 minutes. Crosslinking was quenched with glycine for 5 minutes (final concentration 0.125 M). Nuclear preparation and shearing were performed as described above using 10 cycles of sonication (30 sec ON / 30 sec OFF, Bioruptor Pico, Diagenode)

[51] . PU.1 was immunoprecipitated overnight at 4°C using a polyclonal anti-PU.1 antibody (1:25; Cell Signaling) with the SimpleCHIP Plus kit (Cell Signaling). The ratio of the rs2836882 allele in PU.1-binding DNA was dually quantified by TaqMan genotyping (Assay C__2601507_20). A standard curve was generated using a fixed-ratio gene block (200nt genome sequence centered on rs2836882; Genewiz) containing either the risk or non-risk allele.

[0146] PU.1 MPRA-ChIP-seq MPRA vector libraries were transfected into TPP macrophages from six healthy donors. PU.1 binding to SNP alleles was evaluated as described above

[51] , and minimal sonication was performed (to remove contaminants while minimizing chromatin shear). Immunoprecipitation was performed overnight at 4°C using the SimpleCHIP Plus kit (Cell Signaling) with polyclonal anti-PU.1 antibody (1:25; Cell Signaling). Similar to MPRA, sequencing libraries were prepared from isolated plasmids and sequenced on MiSeq (50 bp, single-ended reads).

[0147] H3K27ac ChIP-seq TPP macrophages from two rs2836882 major allele homozygotes and two minor allele homozygotes were collected, crosslinked, and quenched as described above. Donors were identified through NIHR BioResource. H3K27ac ChIP-seq was performed as described above using anti-H3K27ac antibody (1:250, Abcam) or isotype control (1:500, rabbit IgG, Abcam)

[51] . Libraries were sequenced with HiSeq4000 (50 bp, single-ended reads). Raw data were processed, QC'd, and analyzed as described above

[51] .

[0148] Assay of macrophage effector function Flow cytometry The expression of bone marrow markers was evaluated by flow cytometry (BD LSRFortessaTM X-20). Panel: CD11b PE / Dazzle594 (BioLegend), CD14 evolve605 (ThermoFisher), CD16 PerCP (BioLegend), CD68 FITC (BioLegend), Live / Dead Fixable Aqua Dead Cell Stain (ThermoFisher), and Fc receptor blocking reagent (Miltenyi). Data were analyzed using FlowJo v10 (BD Biosciences).

[0149] Cytokine quantification The supernatant was collected on day 6 of TPP macrophage culture and frozen. The cytokine concentration was quantified in duplicate via electrochemiluminescence using the U-PLEX assay (Meso Scale Diagnostics).

[0150] Phagocytosis Phagocytosis was evaluated using fluorescently labeled Zymosan particles (Green Zymosan, Abcam) according to the manufacturer's instructions. Cells were seeded at 10 5 cells / well in a 96-well round-bottom plate. Cytochalasin D (10 μg / ml, ThermoFisher), an inhibitor of cytoskeletal rearrangement, was used as a negative control. Phagocytosis was quantified via flow cytometry, and the phagocytosis index (the percentage of positive cells multiplied by their mean fluorescence intensity) was calculated.

[0151] Extracellular ROS production According to the manufacturer's protocol, extracellular ROS production was quantified using the Diogenes Enhanced Superoxide Detection Kit (National Diagnostics). Cells were seeded at a density of 10 5 cells / well and pre-stimulated with PMA (200 ng / ml, Sigma Aldrich).

[0152] Western blotting Western blotting was performed as described above using the following primary antibodies: rabbit anti-gp91phox, rabbit anti-p22phox (both Santa Cruz), rabbit anti-C17ORF62 / EROS (Atlas), and rabbit anti-actin (Abcam)

[56] . The secondary antibody was anti-rabbit IgG-horseradish peroxidase (Cell Signaling). Chemiluminescence was recorded on a ChemiDoc Touch imager (Bio-Rad) after incubating the membrane with ECL (ThermoFisher) or SuperSignal West Pico PLUS (ThermoFisher) reagent.

[0153] RNA sequencing RNA was isolated from macrophage lysates (AllPrep DNA / RNA Micro Kit, Qiagen), and a sequencing library was prepared from 10 ng of RNA using the SMARTer Stranded Total RNA-Seq Kit v2-Pico Input Mammalian (Takara) according to the manufacturer's instructions. The library was sequenced using NextSeq2000 (50 bp, PE reads: CRISPR-based loss of function, roxadustat, and PD-0325901 experiments) or NovaSeq6000 (100 bp, PE reads: overexpression experiments). Reads were trimmed using Trim Galore (Phred score 24), filtered to remove reads <20 bp, and ribosome reads were removed using human ribosomal DNA complete repeat units (GenBank: U13369.1) with the BBSplit function of BBMap (BBMap, sourceforge.net / projects / bbmap / ). Reads were aligned to the human genome (hg38) using HISAT2

[57] , converted to BAM files, and sorted and indexed using SAMtools

[58] . Gene read counts were obtained from Rsubread using the featureCounts program

[59] with GTF annotation files from the human genome build GRCh38 (version 102). Differential expression analysis was performed in R using the limma package

[60] with voom transformation and including donors as covariates.

[0154] Gene set enrichment analysis GSEA was performed using the fGSEA

[61] package in R. Gene sets were either obtained from gene ontology biological pathways (downloaded from MSigDB), experimentally derived based on differential expression analysis, or procured from published literature.7 The pathways shown in Figures 2–5 are GO:0002274, GO:0042116, GO:0097529, GO:0006909, GO:0071706, GO:0032732, GO:0032755, GO:0032757, GO:2000379, GO:0009060, GO:0006119, and GO:0045649. Statistical significance was calculated using adaptive multilevel partitioned Monte Carlo method.

[0155] In vitro transfer The cDNA sequence of ETS2 (NM005329.5) preceded by the Kozak sequence was synthesized and cloned into a TOPO vector. This was linearized, and the T7 promoter and AG start sequence (Phusion, NEB) were added to generate PCR amplifiers of the ETS2 gene. (Fw primer: GCTATACGACTCACTATAAGGACAGGCCACCATGAATGATTTCGGAATC, Rv primer: TCAGTCCTCCGTGTCGG). Reverse complement (control) amplifiers were also generated. (Fw primer: GCTATACGACTCACTATAAGGACAGGCCACCTCAGTCCTCCGTGTCGG, Rv primer: GCCACCATGAATGATTTCGGAATC). These amplifiers were used as templates for in vitro transcription using the CleanCap® Reagent AG Kit (NEB) with the HiScribe T7 mRNA Kit, following the manufacturer's instructions. However, N1-methyl-psoidouridine was replaced with uridine and methylcytidine with cytidine (both Stratech) to minimize nonspecific cell activation by the transfected mRNA. The mRNA was purified using the MEGAclear Transcription Clean-Up Kit (ThermoFisher) and polyadenylated using E. coli Poly(A) polymerase (NEB) before further cleanup (MEGAclear), product size quantification, and analysis (NorthernMax®-Gly gel, ThermoFisher). GFP mRNA was produced using the same method to optimize overexpression conditions. Fw primer (GCTAATACGACTCACTATAAGGACAGGCCACCATGGTGAGCAAGGGCGAG), Rv primer (TTACTTGTACAGCTCGTCCATGC).

[0156] mRNA overexpression Lipofectamine MessengerMAX (ThermoFisher) was diluted with Opti-MEM (1:75 v / v), vortexed, and incubated at room temperature for 10 minutes. Next, IVT mRNA was diluted with a fixed volume of Opti-MEM (112.5 μl per transfection), mixed with an equal volume of diluted Lipofectamine MessengerMAX, and incubated at room temperature for a further 5 minutes. The transfection mixture was then 2.5 × 10⁻⁶. 6 M0 macrophages were treated with the reagent (pre-cultured for 6 days in 6-well plates of antibiotic-free RPMI1640 macrophage medium containing M-CSF (50 ng / ml, Peprotech), with the medium changed on day 3). For GFP overexpression, cells were isolated using Accutase 18 hours after transfection, and GFP expression was measured by flow cytometry. For ETS2 / control overexpression, either 250 ng or 500 ng of mRNA was transfected, low-dose LPS (0.5 ng / ml) was added 18 hours after transfection, and cells were isolated using Accutase 6 hours later (n=8 donors). Representative ETS2 expression in untransfected macrophages obtained from previous data (GSE193336).

[0157] SNPsea Pathway analysis of 241 IBD-related GWAS hits [7] was performed using SNPsea

[36] . In summary, a junction interval was defined for each read SNP based on the most correlated SNPs (r2 > 0.5, EUR population in 1000 genomes) and extended to the nearest recombination hotspot with a recombination rate > 3 cM / Mb. If no gene was present in this region, the junction interval was extended 500 kb upstream and downstream. Genes within the junction interval were tested for enrichment within 7,660 pathways, including 7,658 gene ontology biological pathways and two lists of ETS2 regulatory genes (either significantly downregulated after ETS2 disruption with gRNA1 or significantly upregulated after ETS2 overexpression, based on a consensus list obtained from differential expression analysis using donor and mRNA levels as covariates, including all samples). The analysis was performed using single-score mode: it was assumed that only one gene per junction interval was associated with the pathway. A null distribution of scores for each pathway was performed by sampling a random set of SNPs corresponding to the number of linked genes (5,000,000 repeats). Substitution p-values ​​were calculated by comparing the enrichment of the IBD-related gene list with the null distribution. The following gene sets associated with IBD-related pathways were extracted for comparison: NOD2 signaling (GO:0032495), integrin signaling (GO:0033627, GO:0033622), TNFα signaling (GO:0033209, GO:0034612), intestinal epithelium (GO:0060729, GO:0030277), Th17 cells (GO :0072539, GO:0072538, GO:2000318), T cell activation (GO:0046631, GO:0002827), IL-10 signaling (GO:0032613, GO:0032733), and autophagy (GO:0061919, GO:0010506, GO:0010508, GO:1905037, GO:0010507).

[0158] ETS2 co-expression Genes co-expressed with ETS2 across 64 human monocyte-derived macrophage polarization conditions (normalized data from GSE47189) were identified using the rcorr function in the R Hmisc package.

[0159] 13 C-glucose GC-MS ETS2-edited or unedited TPP macrophages were generated from each donor in three replication cycles. On day 6, the culture medium was removed, the cells were washed with PBS, and fresh medium containing labeled glucose was added. Labeled media: RPMI1640 medium, glucose-free (ThermoFisher); 10% FBS (ThermoFisher); GlutaMAX (ThermoFisher); 13 14C-labeled glucose (Cambridge Isotype Laboratories). After 24 hours—a time point selected from the time course to establish steady-state conditions—the supernatant was rapidly frozen and macrophages were separated by scraping. Macrophages were washed three times with ice-cold PBS, counted, resuspended in 600 μl of ice-cold chloroform:methanol (2:1, v / v), and sonicated in a water bath (3 × 8 min). All extraction steps were performed at 4°C as previously described

[62] . Samples were analyzed using an Agilent 7890B-7000C GC-MS system. In electron ionization mode, spitless injection (injection temperature 270°C) into DB-5MS (Agilent) was used with helium as the carrier gas. The initial oven temperature was 70°C (2 min), followed by a temperature gradient of 12.5°C per min to 295°C and 25°C per min to 320°C (held for 3 min). The scan range was m / z 50–550. Data analysis was performed using the in-house software MANIC (version 3.0) based on the software package GAVIN

[63] . Label incorporation was calculated by subtracting the natural abundance of stable isotopes from the observed amounts. Total metabolite abundances were normalized to an internal standard (Aubronositol

[62] ).

[0160] Roxadustat ETS2-edited or unedited TPP macrophages were generated as described above. On day 5 of culture, cells were detached (Accutase) and reseeded in 96-well round-bottom plates in TPP medium containing roxadustat (FG-4592, 30 μM) at a density of 10 5 cells / well. After 12 hours, cells were harvested for functional assays and RNA-seq as described.

[0161] CUT&RUN Pre-cultured TPP macrophages were immediately harvested and processed using the CUT&RUN Assay Kit (Cell Signaling) according to the manufacturer's instructions, omitting the use of ConA-coated beads. Briefly, 5×10 5 cells per reaction were pelleted, washed, and resuspended in antibody binding buffer. Cells were incubated with anti-ETS2 (1:100, ThermoFisher) or IgG control (1:20, Cell Signaling) for 2 hours at 4°C. After washing with digitonin buffer, cells were incubated with pA / G-MNase for 1 hour at 4°C. Cells were washed twice in digitonin buffer, resuspended in the same buffer, and cooled on ice for 5 minutes. Calcium chloride was added to activate pA / G-MNase digestion (30 minutes, 4°C), after which the reaction was stopped and cells were incubated at 37°C for 10 minutes to release fragmented chromatin. The supernatant was collected by centrifugation and DNA was extracted using a spin column (Cell Signaling). Library preparation was performed using the protocol.

[0162] The IO protocol (dx.doi.org / 10.17504 / protocols.io.bagaibse) was followed using the NEBNext Ultra II DNA Library Prep Kit. Size selection was performed using AMPure XP beads (Beckman Coulter), and fragment sizes were assessed using the Agilent 2100 Bioanalyzer (High Sensitivity DNA Kit). Ecomoleric pools of the indexed libraries were sequenced using NovaSeq 6000 (100 bp PE reads). Raw data were analyzed using guidelines from the Henikoff lab

[64] . Briefly, paired-end reads were trimmed using Trim Galore and aligned to the human genome (GRCh37 / hg19) using Bowtie2. BAM files were sorted, merged (technical and, where indicated, biological copies), resorted, and indexed using SAMtools. Unmapped reads and SAMtools were marked using Picard, these reads were removed, and the files were resorted and reindexed. Bigwig files were constructed using the deepTools bamCoverage function. MACS2 was used to invoke peaks. Non-reproducible discovery rates were calculated over replicates (MACS2 peaks invoked using FDRq<0.05) using a 0.01 cutoff with the idr package in R. Enrichment of ETS2-binding motifs in consensus IDR peaks was calculated using TFmotifView

[65] with a global genome control. Overlap between consensus IDR peaks and putative cis-regulatory elements of core promoters (-250bp to +35bp from TSS) and / or ETS2 regulatory genes was assessed using a list of differentially expressed genes after ETS2 disruption or ETS2 overexpression with gRNA1 (based on consensus across mRNA doses, as previously stated). Putative cis-regulatory elements were defined as covalent interactions (CHiCAGO score > 5) in monocyte, M0, and M1 macrophage samples from publicly available promoter capture Hi-C data

[13] .

[0163] ATAC-seq ATAC-seq in TPP macrophages was performed using the Omni-ATAC protocol

[66] with the following modifications: increasing the cell count to 75,000 cells and increasing the cell lysis time to 5 minutes; doubling the volume of Tn5 transposase in the transposition mixture; and extending the duration of the transposition step to 40 minutes. The amplified libraries were purified using AMPure XP beads (Beckman Coulter) and sequenced with NovaSeq6000 (100 bp PE reads). The data were processed as described above

[67] .

[0164] chr21q22 disease dataset Publicly available raw RNA-seq data from affected tissues (and controls from the same experiments) of chr21q22-related diseases were downloaded from GEO: IBD macrophages (GSE123141), primary sclerosing cholangitis liver (GSE159676), and ankylosing spondylitis synovium (GSE41038). Reads were trimmed, filtered, and aligned as previously described. For each disease dataset, a ranked list of genes was obtained by differential expression analysis between cases and controls using limma with voom transformation. For IBD macrophages, only IBD samples with active disease were included. fGSEA using the ETS2 regulatory gene list was performed as described.

[0165] LINCS Signature A list of 31,027 downregulatory genes after cell line exposure to small molecules was obtained from the NIH LINCS database (downloaded January 2021). These were used as gene sets in fGSEA (as described) using a ranked list of genes obtained by differential expression analysis between ETS2-edited TPP macrophages and unedited TPP macrophages (gRNA1), with limma and donor with voom conversion as covariates. Drug classes of gene sets with FDR P < 0.05 were manually assigned based on known mechanisms of action.

[0166] PD-0325901 TPP macrophages were generated as described above. On day 4 of culture, PD-0325901 (0.5 μM, Sigma) or vehicle (DMSO) was added. Cells were harvested on day 6, RNA was extracted, and sequencing was performed as described.

[0167] Colon biopsy During colonoscopy, intestinal mucosal biopsies (6 per donor) were taken from 10 IBD patients (7 ulcerative colitis, 3 Crohn's disease). All had endoscopically active disease and were not receiving immunosuppression or biologic therapy. All biopsies were collected from a single site of inflammation. All patients provided written informed consent. Ethical approval was provided by the London-Brent Regional Ethics Committee (REC: 21 / LO / 0682). Biopsies were collected into Opti-MEM, weighed within 1 hour, and placed in pairs on Transwell inserts (ThermoFisher) designed to create an air-liquid interface 70 in 24-well plates. Each well contained 1 ml of medium supplemented with either DMSO (vehicle control), PD-0325901 (0.5 μM), or infliximab (10 μg / ml; MSD). Medium: Opti-MEM I (Gibco); GlutaMAX (ThermoFisher); 10% FBS (ThermoFisher); MEM non-essential amino acids (ThermoFisher); 1% sodium pyruvate (ThermoFisher); 1% penicillin / streptomycin (ThermoFisher); 50 μg / ml gentamicin (Merck). After 18 hours, the supernatant and biopsies were snap-frozen. Supernatant cytokine concentrations were quantified using the LEGENDplex Human Inflammation Panel (Biolegend). RNA was extracted from biopsies and libraries prepared as described above (n = 9, RNA from one donor was too degraded). Sequencing was performed on the NovaSeq 6000 (100 bp, PE reads). Data were processed as described above and GSVA was performed for ETS2 regulatory genes and biopsy-derived signatures of IBD-related inflammation

[48] .

[0168] Statistical methodology The statistical methods used in MPRA analysis, fGSEA, and SNPsea are described above. For other analyses, comparisons of continuous variables between paired samples in two groups were performed using the Wilcoxon paired tests for non-parametric data or paired t-tests for parametric data. Comparisons with assumed values ​​were performed using the Wilcoxon signed-rank tests for non-parametric data or one-sample t-tests for parametric data. Normality was confirmed using the Shapiro-Wilk test. Two-tailed tests were used as standard unless a specific hypothesis had been tested. Sample sizes are provided in their respective sections.

[0169] Example 11 - ETS2-driven inflammatory macrophages are present in all chr21q22-related autoinflammatory diseases. The inventors demonstrated that overexpression of ETS2 in quiescent macrophages results in a transcriptional state closely resembling the phenotype of intestinal macrophages in Crohn's disease. The inventors also expanded this analysis to show that it also applies to macrophages from other chr21q22-related diseases. To do this, they identified publicly reported macrophage signatures from various different diseases. Importantly, these represented true disease signatures derived from human macrophages from patients with each condition (rather than in response to in vivo stimulation). The signatures are shown in Table 1. TIFF2026513303000001.tif227170

[0170] These signatures were used as a gene set in fGSEA to evaluate whether ETS2 overexpression induced similar enrichment across all diseases. The results are shown in Figure 18. They demonstrate that ETS2 overexpression does not simply cause baseline activation, but rather induces genes that are characteristic of chronic inflammatory diseases and, to a lesser extent, responses to bacterial infections. In contrast, no significant enrichment of tumor-associated macrophage signatures or macrophages was observed during certain viral infections (e.g., influenza A) (Figure 18).

[0171] Due to the lack of a suitable dataset, one chr21q22-related disease missing from this analysis was primary sclerosing cholangitis (PSC). To address this, we performed spatial transcriptomics on fixed liver tissue from PSCs and revealed an increase in the number of inflammatory macrophages in the liver of PSCs that were close to cholangiocytes, the primary target of pathology, compared to unaffected controls (Figure 18). Furthermore, the ETS2 regulatory gene was most highly expressed in macrophages closest to PSC cholangiocytes, consistent with its role in disease development (Figure 19).

[0172] Together, the data demonstrate that ETS2-driven inflammatory macrophages are present in all chr21q22-related autoinflammatory diseases, and therefore demonstrate that this offers a novel therapeutic target.

[0173] method Spatial transcriptomics: 5 μm FFPE sections were cut from two PSC liver explants and two controls (healthy livers adjacent to tumor metastases), calcined overnight at 60°C, and prepared for CosMx according to manufacturer's instructions using 15 minutes of targeted retrieval and 30 minutes of protease digestion. Tissue samples were obtained via Tissue Access for Patient Benefit (TAP-B, part of the UCL-RFH Biobank) with research ethics approval. 16 / WA / 0289 (Wales Research Ethics Committee 4). One case and one control were included on each slide. The Human Universal Cell Characterization core panel (960 genes) was used, supplemented with eight additional genes to improve the identification of target cells: CD1D, EREG, ETS2, FCN1, G0S2, LYVE1, MAP2K1, MT1G. Segmentation was performed using the CosMx Human Universal Cell Segmentation Kit (RNA), Human IO PanCK / CD45 Kit (RNA), and Human CD68 Marker, Ch5 (RNA). FOV was tiled across all available regions (221 controls, 378 PSCs), and cyclic fluorescence in situ hybridization (FISH) was performed using CosMx SMI (Nanostring). Data were preprocessed using the AtoMx Spatial Informatics Platform, images were segmented to obtain cell boundaries, transcripts were assigned to single cells, and transcripts were obtained with a cell number matrix. Expression matrix, transcript coordinates, polygon coordinates, FOV coordinates, and cell metadata were exported, and QC, normalization, and cell type determination were performed using InsituType, an R package developed to extract all available information from the expression profile of any cell. Cell phenotypes were determined by incorporating a Liver Human Cell Atlas reference matrix using a semi-supervised strategy. Spatial analysis of macrophage phenotypes was performed according to their proximity to bile duct cells (anchor cell type).Using PhenoptR (https: / / akoyabio.github.io / phenoptr / ), radius and nearest neighbor analyses were performed on macrophage distributions from bile duct cells binned in 100 μm increments up to a maximum of 500 μm. Nearest neighbor analyses were performed to determine the distance from bile duct cells to the nearest inflammatory and non-inflammatory macrophages, and vice versa.

[0174] Gene Set Enrichment Analysis (GSEA): GSEA was performed using fGSEA in R with differentially expressed gene lists ranked by t-statistics. Gene sets were obtained from the Gene Ontology Biological Pathway (MSigDB), experimentally derived based on differential expression analysis, or sourced from published literature. Specific details of disease macrophage signatures are listed in Table 1. TIFF2026513303000002.tif183170

[0175] Equivalents and range Those skilled in the art will understand that the present invention is defined by the appended claims and not by the examples contained herein or by other descriptions of certain embodiments.

[0176] Similarly, the singular forms "a," "an," and "the" refer to multiple objects unless the context explicitly states otherwise.

[0177] Unless otherwise defined above, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Any methods and materials similar to or equivalent to those described herein may also be used in carrying out or testing the present invention. Generally, the terminology and techniques used herein in relation to cell and tissue culture, molecular biology, immunology, genetics, and protein and nucleic acid chemistry are well known and commonly used in the art, or are in accordance with the manufacturer's specifications.

[0178] All publications, patents, and patent applications referenced herein are incorporated herein by reference in whole to the same extent as each individual publication, patent, or patent application is specifically and individually indicated as being incorporated herein by reference. In addition, any citation or identification of references in this application should not be construed as an admission that such references are available as prior art of the present invention. Section headings should not necessarily be construed as restrictive to the extent in which they are used.

[0179] The present invention is also described in the following numbered embodiments. 1. A method for treating or preventing a disease in a subject, comprising administering an erythroblast transformation-specific proto-oncogene 2 (ETS2) inhibitor to the subject. 2. A method for treating or preventing a disease in a subject, comprising administering an inhibitor of a chr21q22 enhancer (SEQ ID NO: 1) to the subject. 3. A method for treating or preventing a disease in a subject, wherein the method comprises modifying or deleting a portion of the chr21q22 enhancer (sequence number 1) in the subject, and optionally deleting the entire chr21q22 enhancer (sequence number 1). 4. A method for reducing macrophage activation by exposing the macrophage to an inhibitor of erythrocyte transformation-specific proto-oncogene 2 (ETS2). 5. A method for reducing macrophage activation by contacting the macrophage with an inhibitor of the chr21q22 enhancer (SEQ ID NO: 1). 6. A method for reducing macrophage activation by modifying or deleting a portion of the chr21q22 enhancer (sequence number 1), wherein the entire chr21q22 enhancer (sequence number 1) is optionally deleted. 7. A method for reducing pro-inflammatory cytokine production by exposing macrophages to an inhibitor of erythrocyte transformation-specific proto-oncogene 2 (ETS2). 8. A method for reducing pro-inflammatory cytokine production by contacting macrophages with an inhibitor of the CHR21Q22 enhancer (SEQ ID NO: 1). 9. A method for reducing pro-inflammatory cytokine production by modifying or deleting a portion of the chr21q22 enhancer (sequence number 1), wherein the entire chr21q22 enhancer (sequence number 1) is optionally deleted. 10. A method for reducing reactive oxygen species (ROS) production by contacting macrophages with an inhibitor of erythrocyte transformation-specific proto-oncogene 2 (ETS2). 11. A method for reducing the production of reactive oxygen species (ROS) by contacting macrophages with an inhibitor of the chr21q22 enhancer (SEQ ID NO: 1). 12. A method for reducing reactive oxygen species (ROS) production by modifying or deleting a portion of the chr21q22 enhancer (sequence number 1), wherein the entire chr21q22 enhancer (sequence number 1) is optionally deleted. 13. A method for reducing macrophage phagocytosis by contacting the macrophage with an inhibitor of erythrocyte transformation-specific proto-oncogene 2 (ETS2). 14. A method for reducing macrophage phagocytosis by contacting the macrophage with an inhibitor of the chr21q22 enhancer (SEQ ID NO: 1). 15. A method for reducing macrophage phagocytosis by modifying or deleting a portion of the chr21q22 enhancer (sequence number 1), wherein the entire chr21q22 enhancer (sequence number 1) is optionally deleted. 16. A method for reducing macrophage migration by exposing the macrophages to an inhibitor of erythrocyte transformation-specific proto-oncogene 2 (ETS2). 17. A method for reducing macrophage migration by bringing the macrophages into contact with an inhibitor of the chr21q22 enhancer (SEQ ID NO: 1). 18. A method for reducing macrophage migration by modifying or deleting a portion of the chr21q22 enhancer (sequence number 1), wherein the entire chr21q22 enhancer (sequence number 1) is optionally deleted. 19. The method according to any one of Embodiments 4 to 6, wherein the reduction in macrophage activation is indicated by one or more of the following: reduction in cell activation, reduction in pro-inflammatory cytokine production, metabolic reprogramming, reduction in reactive oxygen species production, and / or reduction in macrophage migration. 20. The method according to any one of Embodiments 7 to 9, wherein the reduction in pro-inflammatory cytokine production includes a reduction in the level of one or more of TNFα, IL-1, IL-6, IL-8, IL-12, IL-23, and / or IL-18. 21. The method according to any one of Embodiments 10 to 12, wherein the reduction of reactive oxygen species (ROS) production includes a reduction in the level of one or more of hydrogen peroxide, hydroxyl radicals, superoxide anions, and / or singlet oxygen. 22. The method according to any one of Embodiments 13 to 15, wherein the reduction in macrophage phagocytosis is indicated by a reduction in the uptake of particulate matter (e.g., bacteria) by macrophages, which is quantified by an in vitro or in vivo assay. 23. The method according to any one of Embodiments 16 to 18, wherein the reduction in macrophage migration is indicated by in vivo measurement of macrophage motility (e.g., quantified by a chemotaxis assay) and / or a reduction in the total number of macrophages accumulating at the site of inflammation in vivo. 24. A method for treating a disease in a subject requiring treatment of the disease, comprising administering an erythroblast transformation-specific proto-oncogene 2 (ETS2) inhibitor to the subject, thereby reducing macrophage activation in the subject. 25. A method for treating a disease in a subject requiring treatment of the disease, comprising administering a CHR21Q22 enhancer (SEQ ID NO: 1) inhibitor to the subject to reduce macrophage activation in the subject. 26. A method for treating a disease in a subject requiring treatment, comprising modifying or deleting a portion of the chr21q22 enhancer (SEQ ID NO: 1) to thereby reduce macrophage activation in the subject. 27. A method for treating a disease in a subject according to any one of Embodiments 1-3 or 24-26, wherein the subject has an inflammatory disease and / or an autoimmune disease. 28. A method for treating a disease in the subject according to Embodiment 27, wherein the inflammatory disease is selected from the list consisting of Crohn's disease, ulcerative colitis, inflammatory bowel disease (Crohn's disease and / or ulcerative colitis), ankylosing spondylitis, primary sclerosing cholangitis, Takayasu's arteritis, and rheumatoid arthritis. 29. Erythroblast transformation-specific proto-oncogene 2 (ETS2) inhibitors for use in methods of treating or preventing diseases in the subject. 30. Inhibitors of the chr21q22 enhancer (SEQ ID NO: 1) for use in methods of treating or preventing diseases in the target population. 31. An erythroblast transformation-specific proto-oncogene 2 (ETS2) inhibitor for use in a method of reducing macrophage activation by contacting the macrophage with an erythroblast transformation-specific proto-oncogene 2 (ETS2) inhibitor. 32. An inhibitor of CHR21Q22 enhancer (SEQ ID NO: 1) for use in a manner that reduces macrophage activation by contacting the macrophage with an inhibitor of CHR21Q22 enhancer (SEQ ID NO: 1). 33. An erythroblast transformation-specific proto-oncogene 2 (ETS2) inhibitor for use in a method of reducing pro-inflammatory cytokine production by contacting macrophages with an erythroblast transformation-specific proto-oncogene 2 (ETS2) inhibitor. 34. An inhibitor of the CHR21Q22 enhancer (SEQ ID NO: 1) for use in a method of reducing pro-inflammatory cytokine production by contacting macrophages with an inhibitor of the CHR21Q22 enhancer (SEQ ID NO: 1). 35. An erythroblast transformation-specific proto-oncogene 2 (ETS2) inhibitor for use in a method of reducing reactive oxygen species (ROS) production by contacting macrophages with an erythroblast transformation-specific proto-oncogene 2 (ETS2) inhibitor. 36. An inhibitor of chr21q22 enhancer (SEQ ID NO: 1) for use in a method of reducing reactive oxygen species (ROS) production by contacting macrophages with an inhibitor of chr21q22 enhancer (SEQ ID NO: 1). 37. An erythroblast transformation-specific proto-oncogene 2 (ETS2) inhibitor for use in a method of reducing macrophage phagocytosis by contacting the macrophage with an erythroblast transformation-specific proto-oncogene 2 (ETS2) inhibitor. 38. An inhibitor of CHR21Q22 enhancer (SEQ ID NO: 1) for use in a manner that reduces macrophage phagocytosis by contacting the macrophage with an inhibitor of CHR21Q22 enhancer (SEQ ID NO: 1). 39. An erythroblast transformation-specific proto-oncogene 2 (ETS2) inhibitor for use in a method of reducing macrophage migration by contacting the macrophages with an erythroblast transformation-specific proto-oncogene 2 (ETS2) inhibitor. 40. An inhibitor of CHR21Q22 enhancer (SEQ ID NO: 1) for use in a method of reducing macrophage migration by contacting the macrophages with an inhibitor of CHR21Q22 enhancer (SEQ ID NO: 1). 41. A erythroblast transformation-specific proto-oncogene 2 (ETS2) or chr21q22 enhancer inhibitor for use according to Embodiment 31 or 32, wherein the reduction of macrophage activation is indicated by one or more of the following: reduction of cell activation, reduction of pro-inflammatory cytokine production, metabolic reprogramming, reduction of reactive oxygen species production, and / or reduction of macrophage migration. 42. A erythroblast transformation-specific proto-oncogene 2 (ETS2) or chr21q22 enhancer inhibitor for use according to Embodiment 33 or 34, wherein the reduction in pro-inflammatory cytokine production includes a reduction in the level of one or more of TNFα, IL-1, IL-6, IL-8, IL-12, IL-23, and / or IL-18. 43. A erythroblast transformation-specific proto-oncogene 2 (ETS2) or chr21q22 enhancer inhibitor for use according to Embodiment 35 or 36, wherein the reduction of reactive oxygen species (ROS) production includes a reduction in the level of one or more of hydrogen peroxide, hydroxyl radicals, superoxide anions, and / or singlet oxygen. 44. A erythroblast transformation-specific proto-oncogene 2 (ETS2) or chr21q22 enhancer inhibitor for use according to Embodiment 37 or 38, wherein the reduction in macrophage phagocytosis is indicated by a reduction in macrophage uptake of particulate matter (e.g., bacteria), which is quantified by an in vitro or in vivo assay. 45. A erythroblast transformation-specific proto-oncogene 2 (ETS2) or chr21q22 enhancer inhibitor for use according to Embodiment 39 or 40, wherein the reduction in macrophage migration is indicated by in vivo measurement of macrophage motility (e.g., quantified by a chemotaxis assay) and / or a reduction in the total number of macrophages accumulating at the site of inflammation in vivo. 46. ​​An erythroid transformation-specific proto-oncogene 2 (ETS2) inhibitor for use in a method of treating a disease in a subject requiring treatment of the disease, wherein the method comprises administering the erythroid transformation-specific proto-oncogene 2 (ETS2) inhibitor to the subject, thereby reducing macrophage activation in the subject. 47. An inhibitor of a chr21q22 enhancer (SEQ ID NO: 1) for use in a method of treating a disease in a subject requiring treatment of the disease, wherein the method comprises administering the chr21q22 enhancer (SEQ ID NO: 1) inhibitor to a subject, thereby reducing macrophage activation in the subject. 48. A erythroblast transformation-specific proto-oncogene 2 (ETS2) or chr21q22 enhancer inhibitor for use according to any one of embodiments 29 to 47, for subjects having inflammatory diseases and / or autoimmune diseases. 49. Erythroblast transformation-specific proto-oncogene 2 (ETS2) or chr21q22 enhancer inhibitor for use according to Embodiment 48, wherein the inflammatory disease is selected from the list consisting of Crohn's disease, ulcerative colitis, inflammatory bowel disease (Crohn's disease and / or ulcerative colitis), ankylosing spondylitis, primary sclerosing cholangitis, Takayasu's arteritis, and rheumatoid arthritis. 50. The method according to any one of Embodiments 1 to 49, wherein the ETS2 inhibitor or chr21q22 enhancer inhibitor comprises an antisense nucleic acid molecule, a small interfering RNA (siRNA), or a short hairpin RNA (shRNA), for use as an erythroblast transformation-specific proto-oncogene 2 (ETS2) or chr21q22 enhancer inhibitor. 51. An ETS2 inhibitor or chr21q22 enhancer inhibitor comprising a small molecule, inhibitory peptide, antibody, or nanobody, according to any one of Embodiments 1 to 49, for use as an erythroblast transformation-specific proto-oncogene 2 (ETS2) or chr21q22 enhancer inhibitor. 52. The method according to Embodiment 52, wherein the antibody is a monoclonal antibody that targets a surface marker specific to ETS2-positive macrophages and inhibits and / or induces the destruction of effector function, an erythroblast transformation-specific proto-oncogene 2 (ETS2) or chr21q22 enhancer inhibitor for use. 53. The method according to Embodiment 52, in which nanobodies target a surface marker specific to ETS2-positive macrophages and induce inhibition and / or disruption of effector function, an erythroblast transformation-specific proto-oncogene 2 (ETS2) or chr21q22 enhancer inhibitor for use. 54. The method according to any one of Embodiments 1 to 49, wherein the ETS2 inhibitor or chr21q22 enhancer inhibitor comprises a proteolytically targeted chimera (PROTAC) that targets ETS2 for degradation, a molecular adhesive that targets ETS2 for degradation, an antibody-drug conjugate, an antibody-RNA conjugate, an esterase-sensitive motif conjugate drug, or a nanobody-drug conjugate, for use by erythroblast transformation-specific proto-oncogene 2 (ETS2) or chr21q22 enhancer inhibitor. 55. The method according to Embodiment 54, wherein the antibody-drug conjugate comprises an antigen-binding molecule that specifically binds to a macrophage marker, and optionally the antigen-binding molecule is an antibody, for use with erythroblast transformation-specific proto-oncogene 2 (ETS2) or chr21q22 enhancer inhibitor. 56. The method according to Embodiment 54, wherein the antibody-drug conjugate comprises an anti-CD163 antigen-binding molecule, and optionally the antigen-binding molecule is an antibody, for use with erythroblast transformation-specific proto-oncogene 2 (ETS2) or chr21q22 enhancer inhibitor. 57. The method according to Embodiment 54, wherein the antibody-drug conjugate comprises an anti-CD209 (DC-SIGN) or anti-CD206 (MRC1) antigen-binding molecule, and optionally the antigen-binding molecule is an antibody, for use by an erythroblast transformation-specific proto-oncogene 2 (ETS2) or chr21q22 enhancer inhibitor. 58. The method according to Embodiments 54-57, wherein the antibody-drug conjugate comprises a MEK inhibitor, and optionally the MEK inhibitor is selected from the list consisting of selumetinib, trametinib, and cobimetinib, preferably the MEK inhibitor is selumetinib, an erythroblast transformation-specific proto-oncogene 2 (ETS2) or chr21q22 enhancer inhibitor for use. 59. The method according to any one of Embodiments 54 to 59, wherein the antibody-drug conjugate comprises a cleavable linker, and optionally the cleavable linker is glucuronide-based (e.g., GlyPro), for use with erythroblast transformation-specific proto-oncogene 2 (ETS2) or chr21q22 enhancer inhibitors. 60. The method according to any one of Embodiments 54 to 59, wherein the antibody-drug conjugate comprises a non-cleavable linker, and optionally the non-cleavable linker is maleimide-PEG3 based, for use by an erythroblast transformation-specific proto-oncogene 2 (ETS2) or chr21q22 enhancer inhibitor. 61. The method according to any one of Embodiments 54 to 60, wherein the antibody-drug conjugate has an antibody:drug ratio of at least 1:2, at least 1:4, at least 1:6, at least 1:8, or at least 1:10, for use by an erythroblast transformation-specific proto-oncogene 2 (ETS2) or chr21q22 enhancer inhibitor. 62. The method according to Embodiment 54, wherein the esterase-sensitive motif conjugate drug comprises cyclopentyl L-leucinate or cyclopentyl(S)-2-amino-2-cyclohexyl acetate, for use as an erythroblast transformation-specific proto-oncogene 2 (ETS2) or chr21q22 enhancer inhibitor. 63. The method according to Embodiment 54 or 62, wherein the esterase-sensitive motif conjugate drug comprises a MEK inhibitor, and optionally, the MEK inhibitor is selected from the list consisting of selumetinib, trametinib, and cobimetinib, an erythroblast transformation-specific proto-oncogene 2 (ETS2) or chr21q22 enhancer inhibitor for use. 64. An esterase-sensitive motif conjugate drug comprising a linker, according to the method of Embodiment 54, 62, or 63, is a erythroblast transformation-specific proto-oncogene 2 (ETS2) or chr21q22 enhancer inhibitor for use. 65. The method according to Embodiment 64, wherein the linker is one or two carbon lengths, for use with erythroblast transformation-specific proto-oncogene 2 (ETS2) or chr21q22 enhancer inhibitors. 66. The method according to any one of Embodiments 1 to 49, wherein the ETS2 inhibitor or chr21q22 enhancer inhibitor is an agent that disrupts the function of a co-transcription activator (e.g., BRD4 or CDK), for use as an erythroblast transformation-specific proto-oncogene 2 (ETS2) or chr21q22 enhancer inhibitor. 67. The method according to any one of Embodiments 1 to 49, wherein the ETS2 inhibitor or chr21q22 enhancer inhibitor is a drug that targets chromatin regulators (e.g., readers, writers, and erasers of chromatin modifications), an erythroblast transformation-specific proto-oncogene 2 (ETS2) or chr21q22 enhancer inhibitor for use. 68. The method according to any one of Embodiments 1 to 49, comprising an ETS2 inhibitor or chr21q22 enhancer inhibitor, wherein the ETS2 inhibitor or chr21q22 enhancer inhibitor is a drug that targets non-coding RNA at the ETS2 locus or chr21q22 enhancer locus, for use as an erythroblast transformation-specific proto-oncogene 2 (ETS2) or chr21q22 enhancer inhibitor. 69. Erythroblast transformation-specific proto-oncogene 2 (ETS2) for use by any one of Embodiments 1 to 49, wherein the ETS2 inhibitor comprises a CRISPR nuclease system including a CRISPR-related protein (Cas protein) and a guide RNA (gRNA) that specifically binds to the ETS2 genomic nucleic acid sequence, and optionally the ETS2 genomic nucleic acid sequence comprises the sequence of Sequence ID No. 5. 70. Erythroblast transformation-specific proto-oncogene 2 (ETS2) for use by any one of Embodiments 1 to 49 or 69, comprising a CRISPR nuclease system in which the ETS2 inhibitor comprises a CRISPR-related protein (Cas protein) and a guide RNA (gRNA) containing a sequence selected from SEQ ID NO: 8 and SEQ ID NO: 9. 71. A chr21q22 enhancer inhibitor for use according to any one of Embodiments 1 to 49, comprising a CRISPR nuclease system containing a CRISPR-related protein (Cas protein) and a guide RNA (gRNA) that specifically binds to a chr21q22 enhancer nucleic acid sequence, wherein the chr21q22 enhancer nucleic acid sequence optionally comprises the sequence of Sequence ID No. 1. 72. A chr21q22 enhancer inhibitor for use according to any one of Embodiments 1 to 49 or 71, comprising a CRISPR nuclease system, the chr21q22 enhancer inhibitor comprising a CRISPR-related protein (Cas protein) and a guide RNA (gRNA) containing a sequence selected from SEQ ID NO: 6 and SEQ ID NO: 7. 73. A chr21q22 enhancer inhibitor for use by any one of Embodiments 1 to 49, comprising a gene editing technique selected from the list of gene / base / prime editing strategies (e.g., CRISPR-based genome targeting tools), gene therapy (e.g., adeno-associated virus (AAV), retroviral vectors, lentiviral vectors), and RNA therapy (including, but not limited to, antisense oligonucleotides). 74. An erythroblast transformation-specific proto-oncogene 2 (ETS2) inhibitor for use according to or in the method of Embodiment 50, comprising a small interfering RNA (siRNA) molecule including a sense strand. 75. An erythroblast transformation-specific proto-oncogene 2 (ETS2) inhibitor for use according to the method in Embodiment 74, wherein the sense strand consists of 15 to 30 linked nucleosides. 76. An erythroblast transformation-specific proto-oncogene 2 (ETS2) inhibitor for use according to the method of Embodiment 74 or 75, wherein the sense strand comprises a sequence having at least 95% identity with an isolength portion of the mRNA encoding erythroblast transformation proto-oncogene 2 (ETS2). 77. An erythroblast transformation-specific proto-oncogene 2 (ETS2) inhibitor for use by any one of Embodiments 74 to 76, wherein the sense strand comprises a sequence having 100% identity with the isolength portion of the pregenomic RNA and / or mRNA encoding erythroblast transformation proto-oncogene 2 (ETS2). 78. A erythroblast transformation-specific proto-oncogene 2 (ETS2) inhibitor for use by any one of embodiments 74-77, wherein the small interfering RNA (siRNA) molecule comprises an antisense strand. 79. An erythroblast transformation-specific proto-oncogene 2 (ETS2) inhibitor for use by any one of Embodiments 74-78, wherein the antisense strand is at least 80%, at least 90%, at least 95%, or at least 99% complementary to the sense strand. 80. An erythroblast transformation-specific proto-oncogene 2 (ETS2) inhibitor for use by any one of embodiments 74-79, wherein the antisense strand is fully complementary to the sense strand. 81. An erythroblast transformation-specific proto-oncogene 2 (ETS2) inhibitor for use by the method described in any one of the prior embodiments, wherein the ETS2 inhibitor can inhibit ETS2 expression in vitro by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%. 82. A chr21q22 enhancer inhibitor for use according to any one of Embodiments 1 to 71, wherein the chr21q22 enhancer inhibitor can inhibit ETS2 expression in vitro by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%. 83. An erythroblast transformation-specific proto-oncogene 2 (ETS2) inhibitor for use according to or in the method of Embodiment 50, comprising an antisense nucleic acid molecule. 84. An erythroblast transformation-specific proto-oncogene 2 (ETS2) inhibitor for use according to or in the method of Embodiment 83, wherein the antisense compound is an antisense oligonucleotide. 85. An erythroblast transformation-specific proto-oncogene 2 (ETS2) inhibitor for use according to or in Embodiment 83 or 84, wherein the antisense compound specifically binds to an ETS2 mRNA sequence containing SEQ ID NO: 2 or SEQ ID NO: 3, or to an ETS2 genomic DNA sequence containing SEQ ID NO: 5. 86. An erythroblast transformation-specific proto-oncogene 2 (ETS2) inhibitor for the method or use of any one of embodiments 83 to 85, wherein the antisense compound specifically binds to an ETS2 mRNA sequence containing SEQ ID NO: 2. 87. An erythroblast transformation-specific proto-oncogene 2 (ETS2) inhibitor for use according to any one of embodiments 83 to 86, wherein the antisense oligonucleotide comprises at least one modified nucleoside bond. 88. An erythroblast transformation-specific proto-oncogene 2 (ETS2) inhibitor for use according to Embodiment 87, wherein the modified nucleoside bond is a phosphorothioate bond. 89. An erythroblast transformation-specific proto-oncogene 2 (ETS2) inhibitor for use by any one of embodiments 83 to 88, wherein the antisense oligonucleotide comprises at least one modified sugar site. 90. An erythroblast transformation-specific proto-oncogene 2 (ETS2) inhibitor for use according to the method described in Embodiment 89, wherein the modified sugar site is a 2'-O-methoxyethyl sugar site. 91. An erythroblast transformation-specific proto-oncogene 2 (ETS2) inhibitor for use according to any one of embodiments 83 to 90, wherein the antisense oligonucleotide comprises at least one modified nucleic acid base. 92. An erythroblast transformation-specific proto-oncogene 2 (ETS2) inhibitor for use according to the method of Embodiment 91, wherein the modified nucleic acid base is 5-methylcytosine. 93. An erythroblast transformation-specific proto-oncogene 2 (ETS2) inhibitor for the method or use according to any one of embodiments 83 to 92, wherein the antisense oligonucleotide is a chimeric oligonucleotide. 94. An erythroblast transformation-specific proto-oncogene 2 (ETS2) inhibitor for use by the method or use described in any one of the prior embodiments, comprising an ETS2 inhibitor and a compound containing a conjugate group. 95. A chr21q22 enhancer inhibitor for use according to any one of Embodiments 1 to 71, 83, or 94, wherein the chr21q22 enhancer inhibitor comprises a compound containing a chr21q22 enhancer inhibitor and a conjugate group. 96. The method according to Embodiment 94 or 95, wherein the conjugate group comprises one or more antibodies or their antigen-binding portions, e.g., Fab fragments, an erythroblast transformation-specific proto-oncogene 2 (ETS2) or chr21q22 enhancer inhibitor for use. 97. The method according to Embodiment 94 or 95, wherein the conjugate group comprises one or more carbohydrates, an erythroblast transformation-specific proto-oncogene 2 (ETS2) or chr21q22 enhancer inhibitor for use. 98. The method according to any one of Embodiments 94 to 97, wherein the conjugate group comprises one or more galactose moieties, one or more lactose moieties, one or more N-acetyl-galactosamine moieties, and / or one or more mannose moieties, an erythroblast transformation-specific proto-oncogene 2 (ETS2) or chr21q22 enhancer inhibitor for use. 99. An erythroblast transformation-specific proto-oncogene 2 (ETS2) inhibitor for use according to any one of embodiments 94 to 98, wherein the conjugate group comprises one or more N-acetyl-galactosamine moieties. 100. The method according to Embodiment 94 or 95, wherein the conjugate group comprises nanoparticles for delivering ETS2 or chr21q22 enhancer inhibitor to macrophages, for use with erythroblast transformation-specific proto-oncogene 2 (ETS2) or chr21q22 enhancer inhibitor. 101. The method according to any one of Embodiments 94 to 100, wherein the conjugate group comprises a lipid carrier, for use as an erythroblast transformation-specific proto-oncogene 2 (ETS2) or chr21q22 enhancer inhibitor. 102. The method according to Embodiment 101, wherein the lipid carrier comprises one or more components selected from the list consisting of poly(lactic acid) (PLA) microparticles, poly(D,L-lactic acid-coglycolic acid) (PLGA) microparticles, liposomes, lipid nanoparticles, micelles, reverse micelles, lipid cacleates, and lipid microtubules, for use as an erythroblast transformation-specific proto-oncogene 2 (ETS2) or chr21q22 enhancer inhibitor. 103. A method for providing diagnosis or prognosis of inflammatory or autoimmune diseases in a subject based on the expression status of erythroblast transformation-specific proto-oncogene 2 (ETS2). A method for providing a diagnosis or prognosis for inflammatory or autoimmune diseases in a subject based on the expression status of 104.chr21q22 enhancer (SEQ ID NO: 1). 105. A method for providing a diagnosis or prognosis according to Embodiment 103 or 104, comprising determining the expression status of ETS2 or chr21q22 enhancer, which is selected from a list consisting of ETS2 mRNA level, ETS2 protein level, ETS2 DNA methylation status, ETS2 epigenetic status (such as histone modification, RNA change, or conformational change), chr21q22 enhancer DNA methylation status, and chr21q22 enhancer epigenetic status (such as histone modification, RNA change, or conformational change). 106. A method for providing a diagnosis or prognosis according to any one of embodiments 103 to 105, wherein determining the expression status of ETS2 includes determining the ETS2 mRNA level or the ETS2 protein level. 107. A method for providing a diagnosis or prognosis according to any one of Embodiments 103 to 106, comprising the step of determining the expression status of ETS2, wherein the expression status of the RNA transcript or cDNA molecule is quantified using one or more of the following: microarray analysis, real-time quantitative PCR, DNA sequencing, RNA sequencing, Northern blot analysis, in situ hybridization, and / or detection and quantification of a specific binding molecule (e.g., antibody). 108. A method for providing a diagnosis or prognosis according to any one of embodiments 103 to 107, further comprising the step of comparing or normalizing the expression status of ETS2 with the expression status of a reference gene. 109. A method for treating or preventing a disease in a subject, comprising administering an inhibitor of a protein that activates erythroblast transformation-specific proto-oncogene 2 (ETS2) to the subject. 110. A method for treating or preventing a disease in a subject according to Embodiment 109, wherein the protein that activates erythrocyte transformation-specific proto-oncogene 2 (ETS2) is selected from the list consisting of MEK, HSP90, RAF, SRC, and ERK. 111. A method for treating or preventing a disease in the subject according to Embodiment 110, wherein the MEK inhibitor is selected from the list consisting of selumetinib, trametinib, and cobimetinib, and preferably the MEK inhibitor is selumetinib. 112. A method for treating a disease in a subject according to any one of Embodiments 109 to 111, wherein the inhibitor of a protein that activates erythroblast transformation-specific proto-oncogene 2 (ETS2) is an antibody-drug conjugate, an antibody-RNA conjugate, an esterase-sensitive motif conjugate drug, or a nanobody-drug conjugate. 113. A method for treating a disease in a subject according to Embodiment 112, wherein the antibody-drug conjugate comprises an anti-CD163 antigen-binding molecule, and optionally the antigen-binding molecule is an antibody. 114. A method for treating a disease in a subject according to Embodiment 112 or 113, wherein the antibody-drug conjugate comprises a cleavable linker, and optionally the cleavable linker is glucuronide-based (e.g., GlyPro). 115. A method for treating a disease in a subject according to Embodiment 112 or 113, wherein the antibody-drug conjugate comprises a non-cleavable linker, optionally the non-cleavable linker being maleimide-PEG3 based. 116. A method for treating a disease in a subject according to Embodiment 112, wherein the esterase-sensitive motif conjugate drug comprises cyclopentyl L-leucinate or cyclopentyl(S)-2-amino-2-cyclohexyl acetate. 117. A method for treating a disease in an object according to Embodiment 112 or 116, wherein an esterase-sensitive motif conjugate drug comprises a linker. 118. A method for treating a disease in an object according to Embodiment 117, wherein the linker is the length of one or two carbon atoms. 119. A method for treating a disease in a subject according to any one of embodiments 109 to 118, wherein the subject has an inflammatory disease and / or an autoimmune disease. 120. A method for treating a disease in the subject according to Embodiment 119, wherein the inflammatory disease is selected from the list consisting of Crohn's disease, ulcerative colitis, inflammatory bowel disease (Crohn's disease and / or ulcerative colitis), ankylosing spondylitis, primary sclerosing cholangitis, Takayasu's arteritis, and rheumatoid arthritis. 121. An inhibitor of a protein that activates erythroblast transformation-specific proto-oncogene 2 (ETS2) for use in a method for treating or preventing a disease in a subject, wherein the method comprises administering the inhibitor of the protein that activates erythroblast transformation-specific proto-oncogene 2 (ETS2) to a subject. 122. An inhibitor of a protein that activates erythroblast transformation-specific proto-oncogene 2 (ETS2) for use according to Embodiment 121, wherein the protein that activates erythroblast transformation-specific proto-oncogene 2 (ETS2) is selected from the list consisting of MEK, HSP90, RAF, SRC, and ERK. 123. An inhibitor of a protein that activates erythroblast transformation-specific proto-oncogene 2 (ETS2) for use according to Embodiment 122, wherein the MEK inhibitor is selected from the list consisting of selumetinib, trametinib, and cobimetinib, and preferably the MEK inhibitor is selumetinib. 124. An inhibitor of a protein that activates erythroblast transformation-specific proto-oncogene 2 (ETS2) for use according to any one of Embodiments 121 to 123, wherein the inhibitor of the protein that activates erythroblast transformation-specific proto-oncogene 2 (ETS2) is an antibody-drug conjugate, an antibody-RNA conjugate, an esterase-sensitive motif conjugate drug, or a nanobody-drug conjugate. 125. An inhibitor of a protein that activates erythroblast transformation-specific proto-oncogene 2 (ETS2) for use according to Embodiment 124, wherein the antibody-drug conjugate comprises an anti-CD163 antigen-binding molecule, and optionally the antigen-binding molecule is an antibody. 126. An inhibitor of a protein that activates erythroblast transformation-specific proto-oncogene 2 (ETS2) for use according to Embodiment 124 or 125, wherein the antibody-drug conjugate comprises a cleavable linker, and optionally the cleavable linker is glucuronide-based (e.g., GlyPro). 127. An inhibitor of a protein that activates erythroblast transformation-specific proto-oncogene 2 (ETS2) for use according to Embodiment 124 or 125, wherein the antibody-drug conjugate comprises a non-cleavable linker, and optionally the non-cleavable linker is maleimide-PEG3 based. 128. An inhibitor of a protein that activates erythroblast transformation-specific proto-oncogene 2 (ETS2) for use according to Embodiment 124, wherein the esterase-sensitive motif conjugate drug comprises cyclopentyl L-leucinate or cyclopentyl(S)-2-amino-2-cyclohexyl acetate. 129. An inhibitor of a protein that activates erythroblast transformation-specific proto-oncogene 2 (ETS2), comprising an esterase-sensitive motif conjugate drug, for use according to Embodiment 124 or 128. 130. An inhibitor of a protein that activates erythroblast transformation-specific proto-oncogene 2 (ETS2) for use according to Embodiment 129, wherein the linker is one or two carbon lengths. 131. An inhibitor of a protein that activates erythroblast transformation-specific proto-oncogene 2 (ETS2) for use according to any one of embodiments 121 to 130, for subjects having inflammatory diseases and / or autoimmune diseases. 132. An inhibitor of a protein that activates erythroblast transformation-specific proto-oncogene 2 (ETS2) for use according to Embodiment 131, wherein the inflammatory disease is selected from the list consisting of Crohn's disease, ulcerative colitis, inflammatory bowel disease (Crohn's disease and / or ulcerative colitis), ankylosing spondylitis, primary sclerosing cholangitis, Takayasu's arteritis, and rheumatoid arthritis. 133. A method for reducing macrophage activation by exposing the macrophage to an inhibitor of a protein that activates erythrocyte transformation-specific proto-oncogene 2 (ETS2). 134. A method for reducing pro-inflammatory cytokine production by contacting macrophages with an inhibitor of a protein that activates erythrocyte transformation-specific proto-oncogene 2 (ETS2). 135. A method for reducing reactive oxygen species (ROS) production by contacting macrophages with an inhibitor of a protein that activates erythrocyte transformation-specific proto-oncogene 2 (ETS2). 136. A method for reducing macrophage phagocytosis by contacting the macrophages with an inhibitor of a protein that activates erythroblast transformation-specific proto-oncogene 2 (ETS2). 137. A method for reducing macrophage migration by exposing the macrophages to an inhibitor of a protein that activates erythrocyte transformation-specific proto-oncogene 2 (ETS2). 138. An inhibitor of a protein that activates erythroblast transformation-specific proto-oncogene 2 (ETS2) for use in a method of reducing macrophage activation by contacting the macrophage with an inhibitor of a protein that activates erythroblast transformation-specific proto-oncogene 2 (ETS2). 139. An inhibitor of a protein that activates erythroblast transformation-specific proto-oncogene 2 (ETS2) for use in a method of reducing pro-inflammatory cytokine production by contacting macrophages with an inhibitor of a protein that activates erythroblast transformation-specific proto-oncogene 2 (ETS2). 140. An inhibitor of a protein that activates erythroblast transformation-specific proto-oncogene 2 (ETS2) for use in a method of reducing reactive oxygen species (ROS) production by contacting macrophages with an inhibitor of a protein that activates erythroblast transformation-specific proto-oncogene 2 (ETS2). 141. An inhibitor of a protein that activates erythroblast transformation-specific proto-oncogene 2 (ETS2), for use in a method of reducing macrophage phagocytosis by contacting the macrophages with an inhibitor of a protein that activates erythroblast transformation-specific proto-oncogene 2 (ETS2). 142. An inhibitor of a protein that activates erythroblast transformation-specific proto-oncogene 2 (ETS2), for use in a method of reducing macrophage migration by contacting the macrophages with an inhibitor of a protein that activates erythroblast transformation-specific proto-oncogene 2 (ETS2). 143. An inhibitor of the protein that activates erythroblast transformation-specific proto-oncogene 2 (ETS2) according to any one of embodiments 133 to 142, wherein the protein that activates erythroblast transformation-specific proto-oncogene 2 (ETS2) is selected from the list consisting of MEK, HSP90, RAF, SRC, and ERK. 144. An inhibitor of a protein that activates erythroblast transformation-specific proto-oncogene 2 (ETS2), for use according to or in the method of Embodiment 143, wherein the MEK inhibitor is selected from the list consisting of selumetinib, trametinib, and cobimetinib, and preferably the MEK inhibitor is selumetinib. 145. A screening method for drugs that reduce macrophage activation, wherein the method is a. Bringing macrophages into contact with the candidate drug, b. Determining the expression status of ETS2, A drug screening method in which drugs that reduce ETS2 expression are identified as drugs that reduce macrophage activation. 146. A drug screening method according to Embodiment 145, wherein determining the expression status of ETS2 includes determining the ETS2 mRNA level or the ETS2 protein level. 147. A method for screening candidate genes involved in macrophage activation, a. Introducing a CRISPR nuclease system containing CRISPR-related proteins (Cas proteins) and guide RNA (gRNA) that specifically binds to the ETS2 genome nucleic acid sequence into macrophages, b. Measuring the expression status of one or more candidate genes, c. A method for screening candidate genes involved in macrophage activation, comprising identifying a gene as being associated with macrophage activation by comparing its expression status with the reference expression status of the same gene from control cells that do not contain a CRISPR nuclease system, wherein a gene having increased or decreased expression relative to the reference expression status is identified as being associated with macrophage activation. 148. A method for screening candidate genes involved in macrophage activation according to Embodiment 147, wherein the ETS2 genome nucleic acid sequence includes the sequence of Sequence ID No. 5. 149. A method for screening candidate genes involved in macrophage activation, a. Introducing a CRISPR nuclease system containing a CRISPR-related protein (Cas protein) and a guide RNA (gRNA) that specifically binds to the chr21q22 enhancer nucleic acid sequence into macrophages, b. Measuring the expression status of one or more candidate genes, c. A method for screening candidate genes involved in macrophage activation, comprising identifying a gene as being associated with macrophage activation by comparing its expression status with the reference expression status of the same gene from control cells that do not contain a CRISPR nuclease system, wherein a gene having increased or decreased expression relative to the reference expression status is identified as being associated with macrophage activation. A method for screening candidate genes involved in macrophage activation according to Embodiment 148, wherein the 150.chr21q22 enhancer genome nucleic acid sequence includes the sequence of Sequence ID No. 1. 151. A method for screening candidate genes involved in macrophage activation, a. Introducing a CRISPR nuclease system containing CRISPR-related proteins (Cas proteins) and guide RNA (gRNA) that specifically binds to candidate genes in genomic nucleic acid sequences into macrophages, b. Measuring the expression status of ETS2, c. A method for screening candidate genes involved in macrophage activation, comprising identifying a gene as being associated with macrophage activation by comparing the expression status of ETS2 with the reference expression status of ETS2 from control cells that do not contain the CRISPR nuclease system, and identifying candidate genes that regulate ETS2 expression status as being associated with macrophage activation. 152. A method for screening candidate genes involved in macrophage activation according to any one of Embodiments 147 to 151, wherein the expression status of one or more candidate genes is quantified using one or more of the following techniques: microarray analysis, real-time quantitative PCR, DNA sequencing, RNA sequencing, Northern blot analysis, in situ hybridization, and / or detection and quantification of specific binding molecules (e.g., antibodies). 153. An inhibitor of a protein that activates erythroblast transformation-specific proto-oncogene 2 (ETS2) for use in a method of treating or preventing a disease in a subject, wherein the method comprises administering an inhibitor of a protein that activates erythroblast transformation-specific proto-oncogene 2 (ETS2) to a subject, the protein that activates erythroblast transformation-specific proto-oncogene 2 (ETS2) being selected from the list consisting of MEK, HSP90, RAF, SRC, and ERK, and the disease being an inflammatory and / or autoimmune disease. 154. An inhibitor of a protein that activates erythroblast transformation-specific proto-oncogene 2 (ETS2) for use in a method of treating or preventing a disease in a subject, wherein the method comprises administering an inhibitor of a protein that activates erythroblast transformation-specific proto-oncogene 2 (ETS2) to a subject, the protein that activates erythroblast transformation-specific proto-oncogene 2 (ETS2) being selected from the list consisting of MEK, HSP90, RAF, SRC, and ERK, and the disease being an autoinflammatory disease. 155. An inhibitor of a protein that activates erythroblast transformation-specific proto-oncogene 2 (ETS2) for use in a method for treating or preventing a disease in a subject, wherein the method comprises administering an inhibitor of a protein that activates erythroblast transformation-specific proto-oncogene 2 (ETS2) to a subject, the protein that activates erythroblast transformation-specific proto-oncogene 2 (ETS2) being selected from a list consisting of MEK, HSP90, RAF, SRC, and ERK, and the disease being chr21q22-related disease. 156. An inhibitor of a protein that activates erythroblast transformation-specific proto-oncogene 2 (ETS2) for use as described in Clause 153, for use in which the disease is an autoinflammatory disease. 157. An inhibitor of a protein that activates erythroblast transformation-specific proto-oncogene 2 (ETS2) for use as described in Clause 153, wherein the disease is a chr21q22-related disease. 158. An inhibitor of a protein that activates erythroblast transformation-specific proto-oncogene 2 (ETS2) for use as described in any one of clauses 153-157, wherein the disease is selected from the list consisting of Crohn's disease, ulcerative colitis, primary sclerosing cholangitis, ankylosing spondylitis, and Takayasu's arteritis.

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Claims

1. An inhibitor of a protein that activates erythroblast transformation-specific proto-oncogene 2 (ETS2), for use in a method of treating or preventing a disease in a subject, wherein the method comprises administering the inhibitor of the protein that activates erythroblast transformation-specific proto-oncogene 2 (ETS2) to the subject, the protein that activates erythroblast transformation-specific proto-oncogene 2 (ETS2) being selected from a list consisting of MEK, HSP90, RAF, SRC, and ERK, and the disease being an inflammatory and / or autoimmune disease.

2. An inhibitor of a protein that activates erythroblast transformation-specific proto-oncogene 2 (ETS2) for use according to claim 1, wherein the MEK inhibitor is selected from the list consisting of selumetinib, trametinib, and cobimetinib.

3. An inhibitor of a protein that activates erythroblast transformation-specific proto-oncogene 2 (ETS2) for use according to claim 1 or 2, wherein the inhibitor of the protein that activates erythroblast transformation-specific proto-oncogene 2 (ETS2) is an antibody-drug conjugate, an antibody-RNA conjugate, an esterase-sensitive motif conjugate drug, or a nanobody-drug conjugate.

4. An inhibitor of a protein that activates erythroblast transformation-specific proto-oncogene 2 (ETS2) for use according to claim 3, wherein the antibody-drug conjugate comprises an anti-CD163 antigen-binding molecule, and optionally the antigen-binding molecule is an antibody.

5. An inhibitor of a protein that activates erythroblast transformation-specific proto-oncogene 2 (ETS2) for use according to claim 3 or 4, wherein the antibody-drug conjugate comprises a cleavable linker, optionally the cleavable linker being glucuronide-based (e.g., GlyPro).

6. An inhibitor of a protein that activates erythroblast transformation-specific proto-oncogene 2 (ETS2) for use according to claim 3 or 4, wherein the antibody-drug conjugate comprises a non-cleavable linker, optionally the non-cleavable linker being maleimide-PEG3 based.

7. An inhibitor of a protein that activates erythroblast transformation-specific proto-oncogene 2 (ETS2) for use according to claim 3, wherein the esterase-sensitive motif conjugate drug comprises cyclopentyl L-leucinate or cyclopentyl (S)-2-amino-2-cyclohexyl acetate.

8. An inhibitor of a protein that activates erythroblast transformation-specific proto-oncogene 2 (ETS2) for use according to claim 3 or 7, wherein the esterase-sensitive motif conjugate drug comprises a linker.

9. An inhibitor of a protein that activates erythroblast transformation-specific proto-oncogene 2 (ETS2) for use according to claim 8, wherein the linker has a length of one or two carbon atoms.

10. An inhibitor of a protein that activates erythroblast transformation-specific proto-oncogene 2 (ETS2) for use according to claim 1, wherein the disease is an autoinflammatory disease.

11. An inhibitor of a protein that activates erythroblast transformation-specific proto-oncogene 2 (ETS2) for use according to claim 1, wherein the disease is a chr21q22-related disease.

12. An inhibitor of a protein that activates erythroblast transformation-specific proto-oncogene 2 (ETS2) for use according to claim 1, wherein the inflammatory disease is selected from the list consisting of Crohn's disease, ulcerative colitis, inflammatory bowel disease (Crohn's disease and / or ulcerative colitis), ankylosing spondylitis, primary sclerosing cholangitis, Takayasu's arteritis, and rheumatoid arthritis.

13. An inhibitor of a protein that activates erythroblast transformation-specific proto-oncogene 2 (ETS2) for use according to claim 1, wherein the disease is selected from the list consisting of Crohn's disease, ulcerative colitis, primary sclerosing cholangitis, ankylosing spondylitis, and Takayasu's arteritis.

14. A screening method for drugs that reduce macrophage activation, (a) bringing macrophages into contact with the candidate drug, (b) Determining the expression status of ETS2, A drug screening method in which drugs that reduce ETS2 expression are identified as drugs that reduce macrophage activation.

15. The drug screening method according to claim 15, wherein determining the expression status of ETS2 includes determining the ETS2 mRNA level or the ETS2 protein level.

16. A method for screening candidate genes involved in macrophage activation, (a) Introducing a CRISPR nuclease system containing a CRISPR-related protein (Cas protein) and a guide RNA (gRNA) that specifically binds to candidate genome nucleic acid sequence genes into macrophages, (b) Measuring the expression status of ETS2, (c) A method for screening candidate genes involved in macrophage activation, comprising: identifying a gene as being associated with macrophage activation by comparing the expression state of ETS2 with the reference expression state of ETS2 from control cells that do not contain the CRISPR nuclease system, and identifying a candidate gene that regulates the ETS2 expression state as being associated with macrophage activation.

17. A method for screening candidate genes involved in macrophage activation according to claim 16, wherein the expression status of one or more candidate genes is quantified using one or more of the following techniques: microarray analysis, real-time quantitative PCR, DNA sequencing, RNA sequencing, Northern blot analysis, in situ hybridization, and / or detection and quantification of specific binding molecules (e.g., antibodies).

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