Inflammatory bowel diseases

Abstract

The invention relates to DNA methylation and gene expression signatures for assessing the severity of IBD in a patient, monitoring the progression of IBD in a patient, and / or determining the efficacy of a therapeutic agent for treating IBD in a patient. The DNA methylation and gene expression signatures of the invention can also be used for the differential diagnosis of Crohn's Disease or Ulcerative Colitis in a patient having or suspected of having IBD. The invention further relates to intestinal epithelial organoids (IEOs) for predicting the efficacy of therapeutic agents and for screening agents for use in treating IBD. The invention further relates to therapeutic agents for use in treating IBD.

Description

[0001] INFLAMMATORY BOWEL DISEASES

[0002] TECHNICAL FIELD OF THE INVENTION

[0003] The invention relates to DNA methylation and gene expression signatures for assessing the severity of IBD in a patient, monitoring the progression of IBD in a patient, and / or determining the efficacy of a therapeutic agent for treating IBD in a patient. The DNA methylation and gene expression signatures of the invention can also be used for the differential diagnosis of Crohn's Disease or Ulcerative Colitis in a patient having or suspected of having IBD. The invention further relates to intestinal epithelial organoids (lEOs) for predicting the efficacy of therapeutic agents and for screening agents for use in treating IBD. The invention further relates to therapeutic agents for use in treating IBD.

[0004] BACKGROUND OF THE INVENTION

[0005] Inflammatory Bowel Diseases (IBD) are chronic conditions causing inflammation of the intestine. The two main entities are Crohn's Disease (CD) and Ulcerative Colitis (UC). Whilst CD can affect any part of the intestinal tract, in UC, inflammation is restricted to the large bowel. The incidence and prevalence of IBD has been rising over recent decades with an increasing proportion of patients being diagnosed in childhood or early adulthood.

[0006] Although genetic predisposition, altered gut microbiota, and exposure to environmental factors have been identified as key factors, understanding of disease pathogenesis remains incomplete. A hallmark of IBD, in particular CD, is the persistent chronic relapsing mucosal inflammation that favours specific gut segments such as the distal small intestine ( / .e. terminal ileum (Tl)). Furthermore, despite successful resolution of mucosal inflammation in response to medical treatments, relapsing inflammation tends to recur in the same anatomical location. This phenomenon suggests the presence of stable molecular changes leading to altered function in local tissue specific, resident cell types.

[0007] While genetic predisposition, altered gut microbiota, and exposure to environmental factors have all been identified as key contributory factors, fundamental drivers and reliable molecular markers of severe CD remain unclear. Altered function of the intestinal epithelium has long been considered a key factor in IBD pathogenesis. However, whilst major attention has been placed on potential mechanisms leading to impaired barrier function, the potential role of the intestinal epithelium as an antigen presenting and processing cell type has largely been neglected, and the underlying mechanisms remain ill defined. In particular, the mechanisms contributing towards lifelong relapsing mucosal inflammation remain unknown. One of the main obstacles to improving understanding of intestinal epithelial cell (IEC) specific mechanisms operative in human CD has been the lack of suitable, patient derived experimental models. Therapeutic interventions for IBD are typically effective in only 40-60% of patients, and current clinical practice relies on trial and error to find an effective treatment strategy. This approach is time consuming and can lead to prolonged suffering for patients.

[0008] Previous studies to determine whether gene expression plays a role in IBD have focussed on the analysis of whole gut tissue and immune compartments. For example, Venkateswaran et al. (Clinical Epigenetics 15:50 (2023)) mentions longitudinal DNA methylation profiling of the rectal mucosa using whole tissue samples from the rectum of UC patients. Venkateswaran et al. does not mention DNA methylation profiling of the terminal ileum or samples derived from patients with Crohn's disease. Similarly, Mo et a / . (The American Journal of Human Genetics 108, 1765-1779 (2021)) analyses whole tissue sections from colonic UC patients and provides no data on the DNA methylation of particular cell types. Data derived from whole tissue samples cannot be used to readily attribute particular gene expression levels to specific cell types, and so the clinical implications of these studies are limited.

[0009] There is an urgent and unmet need for methods of assessing the severity of IBD in patients. There is also an urgent and unmet need for methods of differentially diagnosing CD and UC in patients having or suspected of having IBD. In addition, there is an urgent and unmet need for methods of determining or predicting the efficacy of therapeutic agents for use in treating IBD. There is also an urgent and unmet need for further therapeutic agents for use in treating IBD.

[0010] SUMMARY OF THE INVENTION

[0011] The present invention is based upon the surprising discovery of distinct DNA methylation and gene expression signatures that are associated with both the presence and the severity of IBD. Advantageously, the DNA methylation and gene expression signatures of the invention can be used to assess the severity of IBD in patients. In addition, the DNA methylation and gene expression signatures of the invention can be used to differentially diagnose CD and UC in patients having or suspected of having IBD. The ability to stratify IBD patients based on disease severity and to differentially diagnose UC and CD advantageously allows therapeutic interventions to be optimised for particular patients. Optimising therapeutic intervention increases the likelihood that patients will receive treatments which are appropriate to their disease severity and type. For example, by ensuring that patients suffering from more severe IBD receive rapid and often more aggressive treatments; and by ensuring that patients suffering from milder IBD are not subjected to unnecessarily aggressive treatments and their associated side-effects. Uncovering the role of MHC-I DNA methylation and gene expression in the intestinal epithelium in IBD represents a paradigm-shifting discovery that, to the Inventors knowledge, has not previously been described. The present invention is also based on the surprising discovery that the DNA methylation and gene expression signatures described herein are highly stable in patient derived intestinal epithelial organoids (lEOs). The lEOs of the invention provide powerful translational research tools which can be used to predict the effectiveness of therapeutic agents for use in treating IBD.

[0012] The invention provides a method of assessing the severity of IBD in a patient, the method comprising: (a) comparing the level of DNA methylation of one or more MHC-I related gene(s) and / or the level of expression of one or more MHC-I related gene(s) in a biological sample obtained from the patient to the level of DNA methylation of said one or more MHC-I related gene(s) and / or the level of expression of said one or more MHC-I related gene(s) in a control, wherein the control is associated with a known severity of IBD; and (b) determining the severity of IBD based on the comparison performed in step (a).

[0013] The invention also provides a method for monitoring the progression of IBD in a patient, the method comprising: (a) comparing: (i) the level of DNA methylation of one or more MHC-I related gene(s) and / or the level of expression of one or more MHC-I related gene(s) in a biological sample obtained from the patient at a first time point with (ii) the level of DNA methylation of said one or more MHC-I related gene(s) and / or the level of expression of said one or more MHC-I related gene(s) in a biological sample obtained from the patient at a second subsequent time point; and (b) determining whether the IBD has progressed between the first and second time point based on the comparison performed in step (a).

[0014] The invention also provides a method for determining the efficacy of a therapeutic agent in a patient having IBD, the method comprising: (a) comparing: (i) the level of DNA methylation of one or more MHC-I related gene(s) and / or the level of expression of one or more MHC-I related gene(s) in a biological sample obtained from the patient prior to administration of the therapeutic agent with (ii) the level of DNA methylation of said one or more MHC-I related gene(s) and / or the level of expression of said one or more MHC-I related gene(s) in a biological sample obtained from the patient after administration of the therapeutic agent; and (b) determining the efficacy of the therapeutic agent based on the comparison performed in step (a).

[0015] The invention also provides a method of differentially diagnosing Crohn's Disease or Ulcerative Colitis in a patient having or suspected of having IBD, the method comprising: (a) comparing the level of DNA methylation of one or more MHC-I related gene(s) and / or the level of expression of one or more MHC- I related gene(s) in a biological sample obtained from the patient to the level of DNA methylation of said one or more MHC-I related gene(s) and / or the level of expression of said one or more MHC-I related gene(s) in a control; and (b) determining if the patient has Crohn's Disease or Ulcerative Colitis based on the comparison performed in step (a).

[0016] In some embodiments, the IBD is selected from Crohn's Disease and Ulcerative Colitis.

[0017] In some embodiments, the one or more MHC-I related gene(s) are selected from NLRC5, B2M, TAPI, TAP2, HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, HLA-DPA1, PSMB8, PSMB9, IFIT1, and IRF1.

[0018] In some embodiments, the one or more MHC-I related gene(s) comprise NLRC5.

[0019] In some embodiments, the method further comprises determining the level of DNA methylation of one or more MHC-I related gene(s) and / or the level of expression of one or more MHC-I related gene(s) in the biological sample obtained from the patient.

[0020] In some embodiments, the method comprises determining the level of DNA methylation at one or more CpG site(s) of one or more MHC-I related gene(s); optionally wherein the one or more CpG site(s) are selected from cg27587780, cgl5375424, cgl7394978, cgll666365, cgll086820, cg20287640, cgl3907973, cg25410123, cg05941961, cgl8890864, cg21428887, cg05915281, cg06942904, cg08977266, cg00483888, cgll971752, cgl5421363, cg00578828, cg26382286, cg21138405, cg00255919, cgl5018934, cg08698936, cgll201654, cg22298860, cgll768167, cg04186657, cgl4738290, cg03278703, cgll576659, cg02688650, cgl4402472, cg24703717, cg05237001, cg03851143, cgllll6776, cg23912231, cg09805507, cgl6728223, cg05603292, cgl7625506, cg20891813, cgl3924755, cgl9348622, cgl8676033, cgl0262357, cg26069562, cgll423684, cg07016276, cg20927242, cgl2588917, cg23892836, cg24351901, cgl5331332, cg00504902, cgll617938, cg01405582, cgll587584, cg05358170, cgl4140375, cgl0601943, cg22310628, cgl3040666, cg02945359, cg06036836, cg04892672, cg25197880, cg01393604, cg24952408, cg26987604, cg25252977, cgl6650906, cg23148731, cgl2550837, cgl6891968, cgl7264941, cg03176423, cgl8371410, cg21632181, cgl4317321, cg24177217, cgl5180617, cg08179037, cg08755130, cgl6289618, cgl5628633, cgl4574237, cg23611220, cg21727129, cg08708750, cg09430946, cg03432955, cg09865095, cg00126638, cg03521696, cg21529533, cgl2287358, cg03997176, cgl9141489, cg22257941, cgll363230, cgl5174396, cgl4546076, cgl3130352, cg26511972, cg23110482, cg03298800, cgl0807461, cg01818016, cgll682706, cg21855392, cgl5595495, cg05523662, cgl5319255, cgl7678719, cg23489273, cgll808100, cg05839762, cgl4772439, cgl4018363, cg08039587, cgl6742075, cg20408505, cg25637655, cgl7608381, cgll946459, cgl6367518, cgl6507955, cg21297314, cg08416870, cgl0794086, cg09275023, cgll861940, cg04807188, cgl0124767, cg02942965, cg20644330, cgl3223754, cg04305858, cgl7475918, cg26175526, cg01462744, cg22952566, cg20534075, cg20662397, cg25293221, cg08899786, cgl4849431, cgl5118824, cg02751503, cg01365762, cg21292025, cgl0457005, cg08180063, cg25664704, cg05384135, cgl5816267, cg09609649, cgl8183435, cg09620840, cg21176130, cg09326440, cgl2700271, cgl3007871, cg00340855, cgll594821, cg21758773, cg05201185, cg02188225, cg20652371, cg21366673, cg23235965, cg27486585, cgl7615629, cg20105257, cg02678305, cgl9109457, cg09569347, cgl3036546, cgl6535080, cgll019014, cg24710520, cgll247343, cgl3872627, cg01064373, cgl8511546, cg26392102, cgl7096289, cgl2965927, cgl8020334, cgl8747378, cg24877963, cgl7974398, cg05619024, cgl3155295, cg01006124, cg25355501, cg06659144, cg02827714, cgl8785300, cg27358585, cg08309069, cg05030953, cg23533285, cg06422189, cg23923934, cg27529346, cg05139028, cglll87245, cg01517384, cgl3902357, cg05932159, cg09588770, cgl2730088, cgl7731992, cg06646969, cg01035015, cg26706521, cgl0542672, cg04981410, cg22016094, cgll239749, cg09889987, cg21454965, cg02010152, cg03477130, cgl0294956, cgll469594, cg08905753, cg00409309, cg27246453, cg04576021, cg01530082, cgl7369196, cg01493678, cgl7103109, cg01200893, cgl9811863, cg00411901, cgl9121661, cgl2333338, cg06707784, cg21021279, cg25798341, cg22619784, cg05592483, cg02633767, cg25823314, cgl8243910, cg08985301, cgl3991568, cg27533873, cg26187287, cg00167916, cg08327277, cgl8050892, cg06121167, cg09408973, cgl4571125, cg00340328, cg26070383, cg07023208, cgl9887824, cg25744682, cgl0924050, cg27176262, cg09871008, cgl8511153, cgl3347198, cg02151752, cg01448551, cg02670545, cg09351471, cgll709793, cg07353306, cg01283574, cgll752893, cgl2121080, cgl3600652, cg00720839, cgl6026549, cgl2761728, cg07015256, cg24813704, cg21359558, cg09187413, cg24469596, cgl8045172, cg08513422, cgl4997446, cg20397692, cg25139471, cgl5785389, cg08358188, cgl2671948, cg08354908, cg00386460, cgl0451089, cg20224850, cgl8266257, cgl0894807, cgl0627913, cgl7390106, cg27618777, cg04255770, cgl5672065, cgl2854186, cg22940798, cg08998192, cgl3563634, cg23560159, cgl0088320, cg22230640, cgll668794, cg20724405, cg05729731, cg26260794, cg03026929, cg20898522, cgl9221044, cgll439192, cg02779707, cg07546106, cgl7253307, cg21780812, cgl2180249, cg00857968, cgl8607757, cgl9655544, cg03544736, cgl7670640, cg25728415, cg24364491, cgl3477582, cgl4867383, cg08557029, cg20558646, cgl6855458, cg05420994, cg23059310, cgl6951654, cg22798247, cgl4493899, cgl3871037, cg23750151, cg25693349, cgl2048225, cgl2094903, cgl8505752, cg08331398, cg21621645, cgll954557, cgl9646975, cgl2718404, cg22510079, cg08992729, cg26988373, cg22158175, cgl6009764, cg20060630, cg00971309, cg03078854, cg24031377, cg21593554, cgll453837, cg24898914, cg00533183, cg08675092, cg08099136, cg01309328, cgl4022881, cgll990058, cgl8696632, cg00777968, cg27232057, cg24893844, cg24625115, cg05115162, cg24767041, cg22007922, cg09006592, cg05545172, cg05872513, cg24326130, cg26829379, cg20848216, cg00889031, cg05242261, cg21322855, cg05135714, cg21568368, cgl0128166, cg07456831, cgl3853192, cg05629721, cgl8963307, cg01295600, cgl7392730, cg08772265, cg03995852, cgl5451100, cgl3603062, cg21535207, cg01702338, cgl0087598, cg20630683, cg06531943, cg03166550, cg06870868, cgl0192196, cglll41874, cg05697726, cgl6483840, cg02657012, cgl6890093, cgll706729, cg07218288, cgl9136673, cg05693489, cgl2253437, cgl3165140, cg09354037, cg25384897, cg02756056, cg09154880, cg02561720, cgll313335, cg05412855, cg05480623, cgl2499157, cg07072394, cg20880499, cg02351231, cgll969117, cg08351785, cg03807983, cg00240875, cg01255458, cg20357228, cg23132351, cg01979171, cg08151204, cgl7973694, cgl4293027, cg05262533, cg26033526, cg01673307, cg24111025, cg25042789, cg02181920, cg06473288, cg26234900, cgl0666909, cg08818207, cg04773990, cg24154161, cg23880953, cg26700949, cgl5926590, cgl8436324, cg05412930, cg06634056, cgl3403689, cg25110530, cg05826626, cg08880054, cg25524784, cgl4286550, cgl7378691, cg09968749, cg01170201, cg08466770, cgl4925994, cgl6501436, cgl9776032, cg01897517, cg01547742, cg09374375, cg27652200, cgll012835, cg23922920, cg27572016, cg05246277, cg25110511, cgl7199468, cgl8903583, cg20194454, cg23806084, cg06852255, cg03299066, cg00704844, cgl6494397, cg07504780, cg02439889, cgl4272068, cgl2641838, cgl2862002, cgl3274644, cg01692674, cg24718356, cg20729846, cg02551145, cg08766503, cg20813491, cg07250080, cg03735531, cgl2762680, cg02567488, cg07156249, cg03465320, cgl6853860, cg06791592, cg21826978, cgl0817441, cg04908668, cg21764921, cgl6229954, cgl9412007, cgl9760441, cg26265820, cg26811065, cg08209711, cg02878284, cgl9994157, cg00045690, cg04913118, cg06043617, cg09322555, cgl3869180, cg24461669, cgl8696027, cgl8555073, cg21445676, cgl0081723, cg07192821, cg08459087, cg00079638, cg08350173, cg24134304, cgl3964393, cg03355298, cg00837838, cgl9404757, cgl2468675, cgl9721944, cg08188779, cg27467953, cg03425812, cgl2828896, cg27537252, cg05475649, cgl6039157, cg02988397, cg21979287, cg22138564, cg07839457, cgl6411857, cg06862692, cg06754565, cg03056682, cg06181531, cg00881185, cg26963976, cg02493440, cg02169333, cg08558449, cg04097610, cg07862320, cg05723552, cgl2256630, cg24341550, cg03089589, cg07223648, cg21333585, cg02644232, cgl6677488, cg05757530, cg04601693, cgl0561843, cg20740356, eg 18440314, cg26661738, cgl6007266, cgl6362955, cgl7047468, cg24661931, cgl4196251, cgll345288, cg04799664, cgl7625032, cg09561419, cg02705619, cgll987321, cg02610906, cg06265737, cg09174467, cg05154222, cgl0854209, cg03754428, cg27462748, cgl7340138, cgl8398267, cg09624807, cg23314414, cg00125947, cg01834111, cg06600851, cgl0553082, cg23820818, cg07990412, cgl9840565, cgl6213529, cg07054754, cgl5875239, cg04344414, cg01517277, cgl8258710, cg00589550, cgl2439595, cgl2928479, cglll01747, Cg23848181, cgl8060687, cg24399801, cg09714724, cg23079860, cg04972244, and cgl4946319.

[0021] In some embodiments, the one or more CpG site(s) are selected from cglll87245, cg00340855, cg05475649, cg24341550, cgl2256630, cgl0601943, cgl2761728, cgl0817441, cgl5375424, cg23923934, cgl4140375, cg01405582, cg02181920, cgll617938, cgl8511546, cgl8696027, cg26033526, cg05697726, cg25384897, cg22310628, cg05358170, cgl7615629, cg07862320, cgll594821, cg07218288, cg24111025, cg07839457, and cgl3007871.

[0022] In some embodiments, the one or more CpG site(s) are selected from cg27529346, cglll87245, cgl6890093, cg05201185, cgll706729, cg09354037, cg00340855, cg06473288, cg05475649, cg24341550, cg02756056, cgl2256630, cgl0601943, cgl0817441, cgl5375424, cg05693489, cg23923934, cg04576021, cg27176262, cg01673307, cgl4140375, cg27537252, cg26234900, cg01405582, cg02181920, cgl9136673, cg02988397, cg25042789, cgl8511546, cg06791592, cg26033526, cg05697726, cg25384897, cg05412855, cg27486585, cgl8243910, cg06422189, cg24898914, cgll453837, cgl2253437, cg23235965, cg09187413, cg00533183, cgl7615629, cg22310628, cg05358170, cg07862320, cgll594821, cg07218288, cg24111025, cgll587584, cg07839457, and cgl3007871.

[0023] In some embodiments, the method further comprises obtaining the biological sample from the patient.

[0024] In some embodiments, the biological sample is obtained from an intestinal biopsy and / or a faecal sample.

[0025] In some embodiments, the biological sample comprises intestinal epithelial cells or intestinal epithelial organoids (lEOs).

[0026] In some embodiments, the level of DNA methylation is determined by sodium bisulphite conversion assay, next generation sequencing (NGS), differential enzymatic cleavage of DNA, a methylated DNA immunoprecipitation assay, methylation-specific PCR, methylation-sensitive melting assay, and / or a bisulphite-free detection method.

[0027] In some embodiments, the level of expression is determined by RNAseq, microarray, and / or quantitative PCR (qPCR).

[0028] In some embodiments, (a) if the patient is identified as having Crohn's Disease, the method further comprises treating said patient for Crohn's Disease; or (b) if the patient is identified as having Ulcerative Colitis, the method further comprises treating said patient for Ulcerative Colitis. In some embodiments, if the patient is identified as having moderate or severe IBD, the method further comprises treating said patient for IBD.

[0029] In some embodiments, if the patient is identified as having severe IBD, the method further comprises treating said patient for IBD.

[0030] In some embodiments, said treating comprises administering to the patient one or more of an aminosalicylate (5-ASA, e.g. mesalazine, sulfasalazine, olsalazine and balsalazide), a corticosteroid (e.g. prednisolone or budesonide), an immunosuppressant (e.g. azathioprine, mercaptopurine or methotrexate), cyclosporine, a biologic (e.g. adalimumab, infliximab, vedolizumab or ustekinumab), a JAK inhibitor (e.g. tofacitinib or filgotinib), and ozanimod.

[0031] In some embodiments, said treating comprises surgery, optionally resection or colectomy.

[0032] In some embodiments, said treating comprises administering to the patient a therapeutic agent which reduces or inhibits the activity of one or more proteins encoded by one or more MHC-I related gene(s).

[0033] In some embodiments, the therapeutic agent comprises one or more of a 5-ASA (e.g. mesalazine, sulfasalazine, olsalazine and balsalazide), a corticosteroid (e.g. prednisolone or budesonide), an immunosuppressant (e.g. azathioprine, mercaptopurine or methotrexate), cyclosporine, a biologic (e.g. adalimumab, infliximab, vedolizumab or ustekinumab), a JAK inhibitor (e.g. tofacitinib or filgotinib), and ozanimod.

[0034] The invention also provides a method of treating a patient identified as having Crohn's Disease or Ulcerative Colitis by a method of the invention, wherein the treating comprises: (i) administering to the patient one or more of a 5-ASA (e.g. mesalazine, sulfasalazine, olsalazine and balsalazide), a corticosteroid (e.g. prednisolone or budesonide), an immunosuppressant (e.g. azathioprine, mercaptopurine or methotrexate), cyclosporine, a biologic (e.g. adalimumab, infliximab, vedolizumab or ustekinumab), a JAK inhibitor (e.g. tofacitinib or filgotinib), and ozanimod; and / or (ii) surgery, optionally resection or colectomy.

[0035] The invention also provides a method of treating a patient identified as having moderate or severe IBD by a method of the invention, wherein the treating comprises: (i) administering to the patient one or more of a 5-ASA (e.g. mesalazine, sulfasalazine, olsalazine or balsalazide), a corticosteroid (e.g. prednisolone or budesonide), an immunosuppressant (e.g. azathioprine, mercaptopurine or methotrexate), cyclosporine, a biologic (e.g. adalimumab, infliximab, vedolizumab or ustekinumab), a JAK inhibitor (e.g. tofacitinib or filgotinib), and ozanimod; and / or (ii) surgery, optionally resection or colectomy. The invention also provides a method of treating a patient identified as having severe IBD by a method of the invention, wherein the treating comprises: (i) administering to the patient one or more of cyclosporine, a biologic, a JAK inhibitor, and / or ozanimod; and / or (ii) surgery, optionally resection or colectomy.

[0036] In some embodiments, the therapeutic agent reduces or inhibits the activity of one or more proteins encoded by one or more MHC-I related gene(s). In some embodiments, the therapeutic agent is an oligonucleotide that represses expression of one or more MHC-I related gene(s), e.g. by binding to corresponding DNA or mRNA. In some embodiments, the therapeutic agent is an antisense oligonucleotide, shRNA, siRNA, microRNA, or an aptamer. Aptamers are generally nucleic acid molecules that bind a specific target molecule. Aptamers can be engineered in vitro, are readily produced by chemical synthesis, possess desirable storage properties, and elicit little or no immunogenicity in therapeutic applications. As used herein, "aptamer" refers in general to a single or double stranded oligonucleotide or a mixture of such oligonucleotides, wherein the oligonucleotide or mixture is capable of binding specifically to a target. In some embodiments, the therapeutic agent is an antibody which binds with specificity to a protein, or a fragment thereof, encoded by one or more MHC-I related gene(s). As used herein, the term antibody encompasses the use of a monoclonal antibody or polyclonal antibody, as well as the antigen-binding fragments of a monoclonal or polyclonal antibody, or a peptide which binds with specificity to a protein encoded by one or more MHC-I related gene(s). The antibody may be a Fab, F(ab')2, Fv, scFv, Fd or dAb.

[0037] The invention also provides a method of treating IBD in a patient, wherein the method comprises: (i) administering to the patient a therapeutic agent which reduces or inhibits the activity of one or more proteins encoded by one or more MHC-I related gene(s) ; and / or (ii) surgery, optionally resection or colectomy.

[0038] The invention also provides a method of treating a patient identified as having Crohn's Disease or Ulcerative Colitis by a method of the invention, wherein the treating comprises: (i) administering to the patient a therapeutic agent which reduces or inhibits the activity of one or more proteins encoded by one or more MHC-I related gene(s); and / or (ii) surgery, optionally resection or colectomy.

[0039] The invention also provides use of the level of DNA methylation of one or more MHC-I related gene(s) and / or the level of expression of one or more MHC-I related gene(s) for assessing the severity of IBD.

[0040] The invention also provides use of the level of DNA methylation of one or more MHC-I related gene(s) and / or the level of expression of one or more MHC-I related gene(s) for monitoring the progression of IBD. The invention also provides use of the level of DNA methylation of one or more MHC-I related gene(s) and / or the level of expression of one or more MHC-I related gene(s) for determining the efficacy of a therapeutic agent in a patient having IBD.

[0041] The invention also provides use of the level of DNA methylation of one or more MHC-I related gene(s) and / or the level of expression of one or more MHC-I related gene(s) for the differential diagnosis of Crohn's Disease or Ulcerative Colitis.

[0042] The invention also provides a kit comprising (a) reagents for determining the level of DNA methylation of one or more MHC-I related gene(s) and / or the level of expression of one or more MHC-I related gene(s); and (b) instructions for use in: (i) assessing the severity of IBD; (ii) monitoring the progression of IBD; (iii) determining the efficacy of a therapeutic agent in a patient having IBD; and / or (iv) differential diagnosis of Crohn's Disease or Ulcerative Colitis.

[0043] In some embodiments, the reagents comprise primer(s) and / or probe(s) for detecting the level of methylation of one or more MHC-I related gene(s).

[0044] In some embodiments, the kit comprises: (i) one or more primer(s) specific to a methylated sequence; and / or (ii) one or more primer(s) specific to an unmethylated sequence.

[0045] In some embodiments, the one or more MHC-I related gene(s) are selected from NLRC5, B2M, TAPI, TAP2, HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, HLA-DPA1, PSMB8, PSMB9, IFIT1, and IRF1.

[0046] The invention also provides a method for predicting the likelihood that a patient will respond to therapy with a therapeutic agent, the method comprising: (a) exposing intestinal epithelial organoids (lEOs) derived from the patient to the therapeutic agent; and (b) quantifying the level of expression of MHC-I and / or the level of expression of one or more MHC-I related gene(s) in the lEOs; wherein reduced expression of MHC-I and / or reduced expression of said one or more MHC-I related gene(s) as compared to the level of expression of MHC-I and / or the level of expression of said one or more MHC- I related gene(s) in the absence of the therapeutic agent indicates an increased likelihood that the patient will respond to therapy.

[0047] The invention also provides a method for screening an agent for therapeutic activity against IBD, the method comprising: (a) exposing intestinal epithelial organoids (lEOs) derived from at least one patient having IBD to the agent; and (b) quantifying the level of expression of MHC-I and / or the level of expression of one or more MHC-I related gene(s) in the lEOs; wherein reduced expression of MHC- I and / or reduced expression of said one or more MHC-I related gene(s) as compared to the level of expression of MHC-I and / or the level of expression of said one or more MHC-I related gene(s) in the absence of the agent is indicative of therapeutic activity against IBD. In some embodiments, the method further comprises exposing the lEOs to IFN-y.

[0048] In some embodiments, the lEOs are derived from mucosal stem cells obtained from an intestinal biopsy.

[0049] In some embodiments, the one or more MHC-I related gene(s) are selected from NLRC5, B2M, TAPI, TAP2, HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, HLA-DPA1, PSMB8, PSMB9, IFIT1, and IRF1.

[0050] DESCRIPTION OF THE FIGURES

[0051] Figure 1. Stable loss of MHC-I gene DNAm in intestinal epithelial organoids derived from Crohn's Disease (CD) patients. (A) i) Overview of experimental setup and sample generation, i) Representative brightfield images of lEOs from duodenum (DUO), terminal ileum (Tl) and sigmoid colon (SC) from non-IBD controls, Ulcerative Colitis (UC) and Crohn's Disease (CD) patients. Images were taken by EVOS FL system (Life Technologies). Scale bars: 300 pm. (B) i) Correlation heat map of 31 comethylated CpG modules identified by WGCNA in Tl lEOs and CD diagnosis. The first principal component of Module 17 (ME17) demonstrates hypomethylation and the strongest association with CD diagnosis (R = -0.43, p-value < 0.001). ii) Gene set enrichment analysis performed on Module 17, showing a significant loss of DNAm in CD organoids compared to healthy controls and UC in TL Names of MHC-I pathway genes are highlighted in bold. (C) Schematic summary of the MHC-I pathway. (D) DNAm (beta value) of four representative MHC-I related DMPs showing CD associated loss of DNAm in Tl and SC but not DUO organoids (DUO = 54, Tl = 127 and SC = 131). (E) Average DNAm (beta value) of all CpGs located in MHC-I related genes for lEOs obtained from three gut segments comparing CD, UC and control patients. (F) Average MHC-I (i) and NLRC5 (ii) DNAm as well as NLRC5 gene expression (iii) in Tl lEOs stimulated with pro-inflammatory cytokines I FNy and TN Fa (n=5). (FDR* < 0.05, FDR **< 0.01, FDR***< 0.001, FDR**** < 0.0001, ns = not significant).

[0052] Figure 2. Loss of MHCI DNAm correlates with increased gene expression in primary intestinal epithelium of CD patients. (A) Overview of patient cohort, sample preparation and data generation. (B and C) DNAm and gene expression in the purified Tl (B) and SC (C) epithelium, i) Average DNAm (beta value) of all CpGs located in MHC-I pathway related genes, and selected CpGs showing significant, CD associated loss of DNAm. ii) Correlation between the beta values and corresponding gene expression (R=Spearman's Rank Correlation). (D) NLRC5 promoter DNAm in the intestinal epithelium of healthy, UC and CD patients at the point of diagnosis and during reassessment. (E) Correlation of NLRC5 promoter DNAm in primary lEOs obtained from the same patient at diagnosis and reassessment (Spearman's Rank Correlation). (F) NLRC5 promoter DNAm in Tl lEOs derived from CD, UC and control patients (n=3 lines per condition, two-way ANOVA with Turkey's test for multiple comparisons. ****p < 0.0001, ns = not significant). (G) NLRC5 mRNA expression in Tl lEOs derived from controls, UC and CD patients at baseline and upon IFNy treatment (lOng / ml for 6h). Data are normalised to the mean of control lines and shown as mean ± SEM (two-way ANOVA with Turkey's test for multiple comparisons. **p < 0.01, *p <0.05, ns = not significant). N = 3 IEO lines in each group for three independent experiments.

[0053] Figure 3. IEC MHC-I DNAm stratifies patients into distinct subgroups that correlate with phenotype and disease severity. (A) i) Unsupervised hierarchical clustering of average MHC-I DNAm in Tl (n = 127) lEOs (Cohort 1). Average MHC-I DNAm score is shown for each sample (middle annotation bar) and grouped by cluster (bottom annotation bar). Distribution of lEOs based on the MHC-I DNAm score cluster is shown on the right, ii) Unsupervised hierarchical clustering of average MHC-I DNAm in primary purified Tl (n = 70) lECs (Cohort 2). Distribution of primary purified lECs based on the MHC-I DNAm score cluster split by diagnosis is shown on the right. (B) Average MHC-I DNAm score in Tl lEOs derived from CD patients comparing disease outcome and phenotype: i) patients with and without requirement for treatment with azathioprine (AZA); ii) patients with and without treatment escalation to biologies; iii) patients with and without the presence of perianal disease; iv) patients with overall severe versus mild / moderate disease outcome. (C) i) Unsupervised hierarchical clustering of average MHC-I DNAm in Tl CD (n=55) lEOs (Cohort 1). Average MHC-I DNAm score is shown for each sample (middle annotation plot) and grouped by cluster (bottom annotation bar). Distribution of lEOs based on the MHC-I DNAm score cluster split by disease severity is shown on the right, ii) Box plot of average MHC-I DNAm and selected 28 prognostic CpGs in Tl CD (n=55) lEOs (Cohort 1), for CD samples with severe and mild / moderate disease outcomes respectively. P values were calculated by two-way Welch's t-test (** p < 0.01). iii) ROC curves and AUC scores of logistic regression classifiers for CD severity prognosis using Tl CD (n=55) lEOs (Cohort 1), based on selected 28 prognostic CpGs, 628 MHC- I CpGs and median of random selections of 628 CpGs respectively. ROC and AUC are both estimated means through repeated cross-validations, iv) Fagan's nomogram of CD severity risk stratification by prognostic risk scores (Figure 4C) based on selected 28 CpGs in Tl CD (n=55) lEOs (Cohort 1). Prior probability of severe CD is 43.6% (95% Cl: 31%-58%). Likelihood ratio for risk score > 0.65 is 3.44 (blue line, 95% Cl: 1.0-11.6), the posterior probability for severe CD is 72.7% (95% Cl: 44%-90%). Likelihood ratio for risk score < 0.33 is 0.29 (orange line, 95% Cl: 0.11-0.74), the posterior probability for severe CD is 18.2% (95% Cl: 8%-36%).

[0054] Figure 4. IEC MHC-I DNAm stratifies patients into distinct subgroups that correlate with phenotype and disease severity. (A) i) Unsupervised hierarchical clustering of average MHC-I DNAm in SC (n = 131) lEOs (Cohort 1). Average MHC-I DNAm score is shown for each sample (middle annotation bar) and grouped by cluster (bottom annotation bar). Distribution of lEOs based on the MHC-I DNAm score cluster is shown on the right, ii) Unsupervised hierarchical clustering of average MHC-I DNAm in primary purified SC (n = 72) lECs (Cohort 2). Average MHC-I DNAm is shown for each sample (middle annotation bar) and grouped by cluster (bottom annotation bar). Distribution of primary purified lECs based on the MHC-I DNAm score cluster split by diagnosis is shown on the right. (B) NLRC5 and selected MHC-I CpG DNAm in Tl and SC lEOs comparing CD patients with severe versus mild / moderate disease outcomes, i) NLRC5 promoter CpGs (cg07839457 and cg07862320); ii) HLA-E (cg20105257); and iii) PSMB9 (cg00045690). Comparisons for panels C and D were calculated using Wilcox tests (* p < 0.05, ** p < 0.01). (C) Box plot of average MHC-I DNAm and selected 53 diagnostic CpGs in Tl (n = 127) lEOs (Cohort 1), for CD and non-CD (UC patient or healthy control) samples respectively. P values were calculated by two-way Welch's t-test (**** p < 0.0001). ii) ROC curves and AUC scores of logistic regression classifiers for CD diagnosis using Tl (n = 127) lEOs (Cohort 1), based on selected 53 diagnostic CpGs, 628 MHC-I CpGs and median of random selections of 628 CpGs respectively. ROC and AUC are both estimated means through repeated cross validations, iii) Box plot of CD severity prognostic risk score based on methylation of selected 28 CpGs processed by logistic regression classifiers, for CD samples with severe and mild / moderate disease outcomes respectively. P values were calculated by two-way Welch's t-test (** p < 0.01).

[0055] Figure 5. MHC-I gene transcription of primary IEC and mucosal biopsies separates patients into distinct subgroups across multiple patient and sample cohorts. (A) Unsupervised hierarchical clustering of selected MHC-I pathway gene expression in lECs from Cohort 2 in Tl (n = 70). Samples are labelled by diagnosis (top annotation bar). Average MHC-I pathway gene expression score is shown for each sample (middle annotation bar) and grouped by cluster (bottom annotation bar). Distribution of Tl lECs based on average MHC-I pathway gene expression split by diagnosis is shown on the right.

[0056] (B) Clustering of selected MHC-I pathway gene expression in lECs from Cohort 2 in SC (n = 72). Samples are labelled by diagnosis (top annotation bar). Average MHC-I pathway gene expression score is shown for each sample (middle annotation bar) and grouped by cluster (bottom annotation bar). Distribution of SC lECs based on average MHC-I pathway gene expression split by diagnosis is shown on the right.

[0057] (C) Unsupervised hierarchical clustering of whole biopsy gene expression of selected MHC-I pathway genes from Cohort 6 (n = 322). Samples are labelled by diagnosis (top annotation bar), tissue inflammation (second row annotation bar) and presence or absence of deep ulcers (third row annotation bar). Average MHC-I pathway gene expression is shown for each sample (fourth row annotation bar) and grouped by cluster (bottom annotation bar). (D) Unsupervised hierarchical clustering of whole biopsy gene expression of selected MHC-I pathway genes from Cohort 7 Tl samples (n = 78). Samples are labelled by diagnosis (top annotation bar), tissue inflammation (second row annotation bar) and disease activity (third row annotation bar). Average MHC-I pathway gene expression is shown for each sample (forth row annotation bar) and grouped by cluster (bottom annotation bar). (E) Unsupervised hierarchical clustering of whole biopsy gene expression of selected MHC-I pathway genes from Cohort 7 colon samples (n = 116). Samples are labelled by diagnosis (top annotation bar), tissue inflammation (second row annotation bar) and disease activity (third row annotation bar). Average MHC-I expression is shown for each sample (fourth row annotation bar) and grouped by cluster (bottom annotation bar).

[0058] DETAILED DESCRIPTION OF THE INVENTION

[0059] The diagnosis and clinical management of IBD presents significant challenges, largely because the molecular mechanisms underlying IBD are ill defined. The Inventors have overcome these challenges by identifying DNA methylation and gene expression signatures that are associated with both the presence and severity of IBD.

[0060] In more detail, the Inventors generated 312 mucosal stem cell derived IEO lines obtained from 168 patients diagnosed with Crohn's Disease (CD), non-IBD / healthy controls and Ulcerative Colitis (UC). Genome-wide epigenetic and transcriptomic profiling of lEOs and primary purified epithelium revealed highly stable, IBD associated loss of DNA methylation in MHC-I (major histocompatibility complex class I) related genes which correlated with increased gene expression of said genes. The Inventors confirmed that decreased DNA methylation of MHC-I related genes results in increased expression of MHC-I. As demonstrated herein, intestinal epithelial cells present antigens via MHC-I and so increased expression of MHC-I results in increased activation of mucosal lymphocytes. Without wishing to be bound by theory, the Inventors believe that increased expression of MHC-I by intestinal epithelial cells drives chronic intestinal inflammation in IBD through increased interaction with and activation of mucosal lymphocytes. Identifying epigenetically regulated intestinal epithelial MHC-I as a novel mechanism in IBD pathogenesis supports a paradigm shift towards the role of the intestinal epithelium as a non-classical antigen-presenting cell type directly driving chronic intestinal inflammation through increased interaction with mucosal lymphocytes.

[0061] Using Crohn's Disease as a model system, the Inventors made the surprising discovery that the degree of DNA methylation and expression of MHC-I related genes provides a reliable biomarker for the severity of IBD. Importantly, the DNA methylation and expression signature of the invention allows patients to be stratified based on the severity of IBD. This ability to stratify IBD patients advantageously allows therapeutic interventions to be optimised based on the severity of disease. For example, patients identified as having more severe IBD can be prioritised for rapid and often more aggressive treatment strategies. Patients identified as having less severe (e.g. mild) IBD may be subjected to less aggressive treatment strategies than those with more severe IBD, thereby limiting exposure to side effects associated with aggressive treatment strategies. Likewise, treatment for patients identified as having moderate IBD can be tailored accordingly. Importantly, IBD patients can be monitored over time to track the progress of disease and to identify patients who may be relapsing or exhibiting increased disease severity. The methods of the invention can also be used to determine the efficacy of therapeutic agents by comparing the level of DNA methylation of MHC-I related genes and / or the level of expression of MHC-I related genes before and after treatment.

[0062] Unexpectedly, the Inventors found that DNA methylation changes were retained in patient derived lEOs and remained stable even over prolonged periods of in vitro culture indicating that epigenetic changes affect intestinal stem-cells and are being inherited during mitosis even in the absence of an inflammatory milieu. The stability of DNA methylation changes, and the associated changes in gene expression, not only supports the reliability of DNA methylation as a biomarker for IBD, but also validates the utility of patient derived lEOs as translational research tools. These lEOs provide powerful in vitro systems for screening agents for therapeutic activity against IBD, and for predicting the likelihood that a patient will respond to therapy.

[0063] As described above, the Inventors advantageously discovered that the level of DNA methylation and / or the level or expression of MHC-I related genes can be used to assess the severity of IBD (e.g. the severity of Crohn's Disease and / or the severity of Ulcerative Colitis) in a patient. Thus, the invention provides a method of assessing the severity of IBD in a patient, the method comprising: (a) comparing the level of DNA methylation of one or more MHC-I related gene(s) and / or the level of expression of one or more MHC-I related gene(s) in a biological sample obtained from the patient to the level of DNA methylation of said one or more MHC-I related gene(s) and / or the level of expression of said one or more MHC-I related gene(s) in a control, wherein the control is associated with a known severity of IBD; and (b) determining the severity of IBD based on the comparison performed in step (a). The invention also provides use of the level of DNA methylation of one or more MHC-I related gene(s) and / or the level of expression of one or more M HC-I related gene(s) for assessing the severity of IBD.

[0064] A lower level of DNA methylation of said one or more MHC-I related gene(s) and / or a higher level of expression of said one or more MHC-I related gene(s) in the biological sample as compared to the level of DNA methylation of said one or more MHC-I related gene(s) and / or the level of expression of said one or more MHC-I related gene(s) in the control is typically indicative that the patient has a higher severity of IBD as compared to the severity level associated with the control. The same level of DNA methylation of said one or more MHC-I related gene(s) and / or the same level of expression of said one or more MHC-I related gene(s) in the biological sample as compared to the level of DNA methylation of said one or more MHC-I related gene(s) and / or the level of expression of said one or more MHC-I related gene(s) in the control is typically indicative that the patient has the same severity of IBD as the severity level associated with the control. A higher level of DNA methylation of said one or more MHC-I related gene(s) and / or a lower level of expression of said one or more MHC-I related gene(s) in the biological sample as compared to the level of DNA methylation of said one or more MHC-I related gene(s) and / or the level of expression of said one or more MHC-I related gene(s) in the control is typically indicative that the patient has a lower severity of IBD as compared to the severity level associated with the control.

[0065] In some embodiments, the control is a level of DNA methylation of said one or more MHC-I related gene(s) and / or a level of expression of said one or more MHC-I related gene(s) that is associated with mild IBD (e.g. mild Crohn's Disease or mild Ulcerative Colitis).

[0066] In some embodiments, the control is a level of DNA methylation of said one or more MHC-I related gene(s) and / or a level of expression of said one or more MHC-I related gene(s) that is associated with moderate IBD (e.g. moderate Crohn's Disease or moderate Ulcerative Colitis).

[0067] In some embodiments, the control is a level of DNA methylation of said one or more MHC-I related gene(s) and / or a level of expression of said one or more MHC-I related gene(s) that is associated with severe IBD (e.g. severe Crohn's Disease or severe Ulcerative Colitis).

[0068] In some embodiments, the method comprises comparing the level of DNA methylation of said one or more MHC-I related gene(s) and / or the level of expression of one or more MHC-I related gene(s) in the biological sample to the level of DNA methylation of said one or more MHC-I related gene(s) and / or the level of expression of said one or more MHC-I related gene(s) in more than one control, wherein said more than one control comprise controls associated with different severities of IBD (e.g. a control associated with mild IBD and a control associated with moderate IBD; a control associated with mild IBD and a control associated with severe IBD; a control associated with moderate IBD and a control associated with severe IBD; or a control associated with mild IBD, a control associated with moderate IBD and a control associated with severe IBD).

[0069] In some embodiments, the method comprises determining whether the IBD is mild, moderate, or severe based on the comparison to one or more control(s).

[0070] The severity of IBD is typically determined based on disease severity over time using methods known in the art. For example, the severity of IBD may be determined based on various parameters including, but not limited to, patient reported symptoms, imaging, the response to induction treatment, the number of bowel movements per day, presence of blood in stool, temperature, heart rate, presence of anaemia, level of abdominal pain, level of dehydration, presence of abdominal mass, the level of inflammation markers (e.g. C-reactive protein), frequency of disease relapses, need for surgery, requirement for more potent treatment, need for rapid treatment escalation, occurrence of complications and impact on quality of life. IBD severity may be defined as described in Howell, K. J. et al. Gastroenterology 154, 585-598 (2018). In some embodiments, a patient may be classified as having mild IBD if they exhibit full response to single induction treatment (e.g. the patient enters remission following induction treatment), maintenance treatment (e.g. with 5-ASA or azathioprine) is effective in maintaining remission, and the IBD does not exhibit progression. In some embodiments, a patient may be classified as having severe IBD if they require second line treatment (e.g. with biologies) and / or IBD related surgery to control symptoms and / or they have had more than two additional induction treatments post diagnosis. It will be understood that an induction treatment typically refers to a therapy administered to the patient following diagnosis or during a flare up of disease which aims to reduce the severity of disease and / or (re-)establish remission. It will also be understood that a maintenance treatment aims to maintain remission and / or prevent progression of the IBD. Severe IBD is typically treatment resistant and involves frequent relapse.

[0071] In some embodiments, the severity of IBD is defined as mild, moderate, or severe.

[0072] In some embodiments, wherein the IBD is Crohn's Disease, disease severity is defined using the Crohn's Disease Activity Index (CDAI). The Crohn's Disease Activity Index (CDAI) is well known in the art and provides a scoring system of between 0 and 600 for defining disease severity. In some embodiments, a CDAI of <220 indicates mild disease, a CDAI of 220-450 indicates moderate disease, and a CDAI of >450 indicates severe disease.

[0073] In some embodiments, wherein the IBD is Ulcerative Colitis, disease severity is defined using the Truelove-Witts classification of disease severity. The Truelove-Witts classification of disease severity is well known in the art and defines disease severity as mild, moderate or severe based on: (i) the number of bowel movements per day; (ii) the amount or presence of blood in stool; (iii) temperate; (iv) pulse rate; (v) haemoglobin levels (presence of anaemia); and (vi) erythrocyte sedimentation rate. For example, a patient having fewer than 4 bowel movements per day, no or small amounts of blood in stool, a temperature of <37.8°C, a pulse <90 bpm, no anaemia and an erythrocyte sedimentation rate of <30 is typically diagnosed with mild disease. A patient having between 4 and 5 bowel movements per day, mild or severe amounts of blood in stool, a temperature of <37.8°C, a pulse <90 bpm, no anaemia and an erythrocyte sedimentation rate of <30 is typically diagnosed with moderate disease. A patient having 6 or more bowel movements per day, visible blood in stool, a temperature of >37.8°C, a pulse >90 bpm, anaemia and an erythrocyte sedimentation rate of >30 is typically diagnosed with severe disease.

[0074] In some embodiments, the method comprises assessing the severity of IBD in samples obtained from the patient at intervals, e.g. samples obtained every month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 8 months, every 10 months, or every 12 months.

[0075] In some embodiments, wherein the patient is identified as having IBD, the method further comprises treating said patient for IBD. In some embodiments, such as wherein the patient is identified as having mild or moderate IBD, said treating comprises administering to the patient one or more of a 5-ASA, a corticosteroid, and / or an immunosuppressant. In some embodiments, such as wherein the patient is identified as having severe IBD, said treating comprises administering to the patient one or more of cyclosporine, a biologic, a JAK inhibitor, and / or ozanimod. In some embodiments, such as wherein the patient is identified as having severe IBD, said treating comprises surgery, optionally resection or colectomy.

[0076] In some embodiments, wherein the patient is identified as having IBD, the method further comprises administering to the patient a therapeutic agent which reduces or inhibits the activity of one or more proteins encoded by one or more MHC-I related gene(s).

[0077] The invention also provides a method for monitoring the progression of IBD in a patient, the method comprising: (a) comparing: (i) the level of DNA methylation of one or more MHC-I related gene(s) and / or the level of expression of one or more MHC-I related gene(s) in a biological sample obtained from the patient at a first time point with (ii) the level of DNA methylation of said one or more MHC-I related gene(s) and / or the level of expression of said one or more MHC-I related gene(s) in a biological sample obtained from the patient at a second subsequent time point; and (b) determining whether the IBD has progressed between the first and second time point based on the comparison performed in step (a). The invention also provides use of the level of DNA methylation of one or more MHC-I related gene(s) and / or the level of expression of one or more MHC-I related gene(s) for monitoring the progression of IBD.

[0078] It will be understood that IBD that has "progressed" typically exhibits increased severity at the second time point as compared to the first time point. IBD that has not "progressed" typically exhibits the same severity or reduced severity at the second time point as compared to the first time point.

[0079] In some embodiments, the second subsequent time point is at least one week after the first time point. In some embodiments, the second subsequent time point is at least 2 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 8 months, at least 10 months, or at least 12 months after the first time point.

[0080] In some embodiments, the method comprises comparing the level of DNA methylation of one or more MHC-I related gene(s) and / or the level of expression of one or more MHC-I related gene(s) in a biological sample obtained from the patient at a first time point with the level of DNA methylation of said one or more MHC-I related gene(s) and / or the level of expression of said one or more MHC-I related gene(s) in several biological samples each obtained from the patient at different, subsequent time points.

[0081] In some embodiments, a lower level of DNA methylation of said one or more MHC-I related gene(s) and / or a higher level of expression of said one or more MHC-I related gene(s) in the biological sample at the first time point as compared to the level of DNA methylation of said one or more MHC-I related gene(s) and / or the level of expression of said one or more MHC-I related gene(s) in the biological sample at the second time point indicates that the IBD has not progressed between the first and second time points and / or the IBD has reduced in severity at the second time point as compared to the first time point.

[0082] In some embodiments, the same level of DNA methylation of said one or more MHC-I related gene(s) and / or the same level of expression of said one or more MHC-I related gene(s) in the biological sample at the first time point as compared to the level of DNA methylation of said one or more MHC-I related gene(s) and / or the level of expression of said one or more MHC-I related gene(s) in the biological sample at the second time point indicates that the IBD has not progressed between the first and second time points and / or the IBD has the same severity at the first and second time points.

[0083] In some embodiments, a higher level of DNA methylation of said one or more MHC-I related gene(s) and / or a lower level of expression of said one or more MHC-I related gene(s) in the biological sample at the first time point as compared to the level of DNA methylation of said one or more MHC-I related gene(s) and / or the level of expression of said one or more MHC-I related gene(s) in the biological sample at the second time point is indicative that the IBD has progressed between the first and second time points.

[0084] In some embodiments, wherein the IBD has progressed between the first and second time points, the method comprises treating said patient if the patient was not already being treated for IBD, or increasing the level of treatment (e.g. to a more aggressive treatment strategy) if the patient was already being treated for IBD. For example, if the IBD has progressed, the patient may be administered an induction treatment as opposed to a maintenance treatment. If the IBD has not progressed, the patient may be maintained on their current treatment or may be administered a less aggressive treatment to avoid unnecessary treatment.

[0085] The invention also provides a method for determining the efficacy of a therapeutic agent in a patient having IBD, the method comprising: (a) comparing: (i) the level of DNA methylation of one or more MHC-I related gene(s) and / or the level of expression of one or more MHC-I related gene(s) in a biological sample obtained from the patient prior to administration of a therapeutic agent with (ii) the level of DNA methylation of said one or more MHC-I related gene(s) and / or the level of expression of said one or more MHC-I related gene(s) in a biological sample obtained from the patient after administration of the therapeutic agent; and (b) determining the efficacy of the therapeutic agent based on the comparison performed in step (a). The invention also provides use of the level of DNA methylation of one or more MHC-I related gene(s) and / or the level of expression of one or more MHC- I related gene(s) for determining the efficacy of a therapeutic agent in a patient having IBD.

[0086] It will be understood that the efficacy of the therapeutic agent will depend on the type of therapy that the therapeutic agent is being used for. For example, maintenance therapy typically aims to prevent progression of IBD and / or to ensure the patient remains in remission. Thus, a therapeutic agent for use in maintenance therapy would be considered efficacious if the patient does not exhibit progression / increased severity of IBD following treatment and / or exhibits a decreased severity of IBD following treatment. However, a therapeutic agent for use in induction therapy (which aims to reduce the severity of disease and / or establish remission) would be considered efficacious only if the patient exhibits reduced severity of IBD following treatment.

[0087] In some embodiments, the therapeutic agent comprises one or more of a 5-ASA, a corticosteroid, an immunosuppressant, cyclosporine, a biologic, a JAK inhibitor, and ozanimod. In some embodiments, the therapeutic agent reduces or inhibits the activity of one or more proteins encoded by one or more MHC-I related gene(s).

[0088] In some embodiments, a lower level of DNA methylation of said one or more MHC-I related gene(s) and / or a higher level of expression of said one or more MHC-I related gene(s) in the biological sample obtained prior to administration of the therapeutic agent as compared to the level of DNA methylation of said one or more MHC-I related gene(s) and / or the level of expression of said one or more MHC-I related gene(s) in the biological sample obtained after administration of the therapeutic agent is indicative that the therapeutic agent is efficacious.

[0089] In some embodiments, a higher level of DNA methylation of said one or more MHC-I related gene(s) and / or a lower level of expression of said one or more MHC-I related gene(s) in the biological sample obtained prior to administration of the therapeutic agent as compared to the level of DNA methylation of said one or more MHC-I related gene(s) and / or the level of expression of said one or more MHC-I related gene(s) in the biological sample obtained after administration of the therapeutic agent is indicative that the therapeutic agent is not efficacious.

[0090] In some embodiments, the same level of DNA methylation of said one or more MHC-I related gene(s) and / or the same level of expression of said one or more MHC-I related gene(s) in the biological sample obtained prior to administration of the therapeutic agent as compared to the level of DNA methylation of said one or more MHC-I related gene(s) and / or the level of expression of said one or more MHC-I related gene(s) in the biological sample obtained after administration of the therapeutic agent is indicative that the therapeutic agent may be efficacious. For example, no change in the level of DNA methylation of said one or more MHC-I related gene(s) and / or the level of expression of said one or more MHC-I related gene(s) may indicate that the therapeutic agent has prevented progression of IBD. If the patient is in remission, no change in the level of DNA methylation of said one or more MHC- I related gene(s) and / or the level of expression of said one or more MHC-I related gene(s) may indicate that the therapeutic agent is efficacious in maintaining a state of remission.

[0091] In some embodiments, the biological sample obtained from the patient after administration of the therapeutic agent is obtained at least one week after the administration of therapeutic agent. In some embodiments, the biological sample obtained from the patient after administration of the therapeutic agent is obtained at least 2 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 8 months, at least 10 months, or at least 12 months after administration of the therapeutic agent.

[0092] In some embodiments, the method comprises monitoring the efficacy of the therapeutic agent in samples obtained from the patient at multiple time points during administration of the therapeutic agent. In some embodiments, the method comprises monitoring the efficacy of the therapeutic agent every month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 8 months, every 10 months, or every 12 months.

[0093] In some embodiments, the method further comprises assessing the severity of IBD prior to and after administration of a therapeutic agent e.g. by a method described herein.

[0094] In some embodiments, the method further comprises monitoring the progression of IBD in the patient after completion of a therapeutic intervention e.g. by a method described herein.

[0095] Advantageously, the invention achieves differential diagnosis of Ulcerative Colitis and Crohn's Disease in patients having or suspected of having IBD. In some embodiments, the patient is suspected of having IBD. For example, the patient may exhibit one or more symptoms associated with IBD including, but not limited to, persistent diarrhoea that lasts longer than four weeks, abdominal pain, blood or mucus in their stool, rectal bleeding, exhaustion, unexplained weight loss, and / or anaemia. In some embodiments, the patient has previously been diagnosed with IBD.

[0096] The invention provides a method of differentially diagnosing Ulcerative Colitis or Crohn's Disease in a patient having or suspected of having IBD, the method comprising: (a) comparing the level of DNA methylation of one or more MHC-I related gene(s) and / or the level of expression of one or more MHC- I related gene(s) in a biological sample obtained from the patient to the level of DNA methylation of said one or more MHC-I related gene(s) and / or the level of expression of said one or more MHC-I related gene(s) in a control; and (b) determining if the patient has Crohn's Disease or Ulcerative Colitis based on the comparison performed in step (a). The invention also provides use of the level of DNA methylation of one or more MHC-I related gene(s) and / or the level of expression of one or more MHC- I related gene(s) for the differential diagnosis of Crohn's Disease or Ulcerative Colitis.

[0097] The control is typically a level of DNA methylation of said one or more MHC-I related gene(s) and / or a level of expression of said one or more MHC-I related gene(s) that can be used to differentiate between samples derived from patients that have Crohn's Disease and patients that have Ulcerative Colitis.

[0098] In some embodiments, the control is a level of DNA methylation of one or more MHC-I related gene(s) and / or a level of expression of said one or more MHC-I related gene(s) that is associated with the presence of Crohn's Disease. In some embodiments a difference between the level of DNA methylation of said one or more MHC-I related gene(s) and / or the level of expression of said one or more MHC-I related gene(s) in the biological sample as compared to the level of DNA methylation of said one or more MHC-I related gene(s) and / or the level of expression of said one or more MHC-I related gene(s) in the control is indicative of the absence of Crohn's Disease. No difference between the level of DNA methylation of said one or more MHC-I related gene(s) and / or the level of expression of said one or more MHC-I related gene(s) in the biological sample as compared to the level of DNA methylation of said one or more MHC-I related gene(s) and / or the level of expression of said one or more MHC-I related gene(s) in the control is typically indicative of the presence of Crohn's Disease. In some embodiments, a difference between the level of DNA methylation of said one or more MHC-I related gene(s) and / or the level of expression of said one or more MHC-I related gene(s) in the biological sample as compared to the level of DNA methylation of said one or more MHC-I related gene(s) and / or the level of expression of said one or more MHC-I related gene(s) in the control is indicative of the presence of Ulcerative Colitis. In some embodiments, the control is a level of DNA methylation of said one or more MHC-I related gene(s) and / or a level of expression of said one or more MHC-I related gene(s) that is associated with the presence of Ulcerative Colitis. In some embodiments a difference between the level of DNA methylation of said one or more MHC-I related gene(s) and / or the level of expression of said one or more MHC-I related gene(s) in the biological sample as compared to the level of DNA methylation of said one or more MHC-I related gene(s) and / or the level of expression of said one or more MHC-I related gene(s) in the control is indicative of the absence of Ulcerative Colitis. No difference between the level of DNA methylation of said one or more MHC-I related gene(s) and / or the level of expression of said one or more MHC-I related gene(s) in the biological sample as compared to the level of DNA methylation of said one or more MHC-I related gene(s) and / or the level of expression of said one or more MHC-I related gene(s) in the control is typically indicative of the presence of Ulcerative Colitis. In some embodiments, a difference between the level of DNA methylation of said one or more MHC- I related gene(s) and / or the level of expression of said one or more MHC-I related gene(s) in the biological sample as compared to the level of DNA methylation of said one or more MHC-I related gene(s) and / or the level of expression of said one or more MHC-I related gene(s) in the control is indicative of the presence of Crohn's Disease.

[0099] In some embodiments, prior to differentially diagnosing Ulcerative Colitis or Crohn's Disease, the method further comprises: (i) comparing the level of DNA methylation of one or more MHC-I related gene(s) and / or the level of expression of one or more MHC-I related gene(s) in a biological sample obtained from the patient to the level of DNA methylation of said one or more MHC-I related gene(s) and / or the level of expression of said one or more MHC-I related gene(s) in a control, wherein the control is a level of DNA methylation of one or more MHC-I related gene(s) and / or a level of expression of said one or more MHC-I related gene(s) that can be used to differentiate between samples derived from patients having IBD and patients who do not have IBD; and (ii) determining if the patient has IBD based on the comparison performed in step (a).

[0100] In some embodiments, the control is a level of DNA methylation of said one or more MHC-I related gene(s) and / or a level of expression of said one or more MHC-I related gene(s) that is associated with the presence of IBD. In this embodiment, (i) a higher level of DNA methylation of said one or more MHC-I related gene(s) and / or a lower level of expression of said one or more MHC-I related gene(s) in the biological sample as compared to the level of DNA methylation of said one or more MHC-I related gene(s) and / or the level of expression of said one or more MHC-I related gene(s) in the control is indicative of the absence of IBD; and (ii) the same or a lower level of DNA methylation of said one or more MHC-I related gene(s) and / or the same or a higher level of expression of said one or more MHC- I related gene(s) in the biological sample as compared to the level of DNA methylation of said one or more MHC-I related gene(s) and / or the level of expression of said one or more MHC-I related gene(s) in the control is indicative of the presence of IBD.

[0101] In some embodiments, the control is a level of DNA methylation of said one or more MHC-I related gene(s) and / or a level of expression of said one or more MHC-I related gene(s) that is associated with the absence of IBD. In this embodiment, (i) a lower level of DNA methylation of said one or more MHC- I related gene(s) and / or a higher level of expression of said one or more MHC-I related gene(s) in the biological sample as compared to the level of DNA methylation of said one or more MHC-I related gene(s) and / or the level of expression of said one or more MHC-I related gene(s) in the control is indicative of the presence of IBD; and (ii) the same or a higher level of DNA methylation of said one or more MHC-I related gene(s) and / or the same or a lower level of expression of said one or more MHC- I related gene(s) in the biological sample as compared to the level of DNA methylation of said one or more MHC-I related gene(s) and / or the level of expression of said one or more MHC-I related gene(s) in the control is indicative of the absence of IBD.

[0102] In some embodiments, if the patient is identified as having Crohn's Disease, the method further comprises treating said patient for Crohn's Disease. In some embodiments, if the patient is identified as having Ulcerative Colitis, the method further comprises treating said patient for Ulcerative Colitis.

[0103] In some embodiments, treating a patient for Crohn's Disease or Ulcerative Colitis comprises administering to the patient one or more agents selected from an aminosalicylate (5-ASA, e.g. mesalazine, sulfasalazine, olsalazine and balsalazide), a corticosteroid (e.g. prednisolone or budesonide), an immunosuppressant (e.g. azathioprine, mercaptopurine or methotrexate), cyclosporine, a biologic (e.g. adalimumab, infliximab, vedolizumab or ustekinumab), a JAK inhibitor (e.g. tofacitinib or filgotinib), and ozanimod. In some embodiments, treating a patient for Crohn's Disease or Ulcerative Colitis comprises surgery, optionally resection or colectomy.

[0104] In some embodiments, treating a patient for Crohn's Disease or Ulcerative Colitis comprises administering to the patient a therapeutic agent which reduces or inhibits the activity of one or more proteins encoded by one or more MHC-I related gene(s).

[0105] The methods of the invention can be combined in any way to improve clinical management of IBD.

[0106] In some embodiments, the use of the level of DNA methylation of one or more MHC-I related gene(s) and / or the level of expression of one or more MHC-I related gene(s) is an in vitro use.

[0107] References herein to IBD embrace Crohn's Disease and Ulcerative Colitis. In some embodiments, the IBD is Crohn's Disease. In some embodiments, the IBD is Ulcerative Colitis. The level of DNA methylation is typically the level of DNA methylation at a CpG site within a gene or within the promoter region of that gene. In some embodiments, the level of DNA methylation of said one or more MHC-I related gene(s) comprises the level of DNA methylation in a promoter region of said one or more MHC-I related gene(s).

[0108] As used herein, the term "MHC-I related genes" embraces genes which exhibit expression levels that correlate with MHC-I expression. As used herein, this term includes, but is not limited to, NLRC5, B2M, TAPI, TAP2, HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, HLA-DPA1, PSMB8, PSMB9, IFIT1, and IRF1.

[0109] In some embodiments, the level of DNA methylation is the level of DNA methylation at one or more CpG sites in one or more MHC-I related gene(s) wherein the CpG site(s) are differentially methylated in patients who have IBD and those who do not. In some embodiments, the level of DNA methylation is the level of DNA methylation at one or more CpG sites in one or more MHC-I related gene(s) wherein the CpG site(s) are differentially methylated in patients who have Crohn's Disease and those who do not. In some embodiments, the level of DNA methylation is the level of DNA methylation at one or more CpG sites in one or more MHC-I related gene(s) wherein the CpG site(s) are differentially methylated in patients who have Ulcerative Colitis and those who do not. In some embodiments, the level of DNA methylation is the level of DNA methylation at one or more CpG sites in one or more MHC-I related gene(s) wherein the CpG site(s) are differentially methylated in patients who have Crohn's Disease and patients who have Ulcerative Colitis.

[0110] In some embodiments, the level of DNA methylation is the level of DNA methylation at one or more CpG sites in one or more MHC-I related gene(s) wherein the CpG site(s) are differentially methylated in patients who have severe IBD and those who do not have severe IBD (e.g. those who have mild or moderate IBD). In some embodiments, the level of DNA methylation is the level of DNA methylation at one or more CpG sites in one or more MHC-I related gene(s) wherein the CpG site(s) are differentially methylated in patients who have mild IBD and those who do not have mild IBD (e.g. those who have moderate or severe IBD). In some embodiments, the level of DNA methylation is the level of DNA methylation at one or more CpG sites in one or more MHC-I related gene(s) wherein the CpG site(s) are differentially methylated in patients who have moderate IBD and those who do not have moderate IBD (e.g. those who have mild or severe IBD).

[0111] The Inventors have identified a number of CpG sites in MHC-I related gene which are differentially methylated in patients who have IBD. Thus, in some embodiments, the level of DNA methylation is the level of DNA methylation at one or more (e.g. 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 or more, 50 or more, 55 or more, 60 or more, 65 or more, 70 or more, 75 or more, 80 or more, 85 or more, 90 or more, 95 or more, 100 or more, 200 or more, 300 or more, 400 or more, 500 or more, or 600 or more) CpG sites in one or more MHC-I related gene(s), wherein the one or more CpG sites are selected from cg27587780, cgl5375424, cgl7394978, cgll666365, cgll086820, cg20287640, cgl3907973, cg25410123, cg05941961, cgl8890864, cg21428887, cg05915281, cg06942904, cg08977266, cg00483888, cgll971752, cgl5421363, cg00578828, cg26382286, cg21138405, cg00255919, cgl5018934, cg08698936, cgll201654, cg22298860, cgll768167, cg04186657, cgl4738290, cg03278703, cgll576659, cg02688650, cgl4402472, cg24703717, cg05237001, cg03851143, cgllll6776, cg23912231, cg09805507, cgl6728223, cg05603292, cgl7625506, cg20891813, cgl3924755, cgl9348622, cgl8676033, cgl0262357, cg26069562, cgll423684, cg07016276, cg20927242, cgl2588917, cg23892836, cg24351901, cgl5331332, cg00504902, cgll617938, cg01405582, cgll587584, cg05358170, cgl4140375, cgl0601943, cg22310628, cgl3040666, cg02945359, cg06036836, cg04892672, cg25197880, cg01393604, cg24952408, cg26987604, cg25252977, cgl6650906, cg23148731, cgl2550837, cgl6891968, cgl7264941, cg03176423, cgl8371410, cg21632181, cgl4317321, cg24177217, cgl5180617, cg08179037, cg08755130, cgl6289618, cgl5628633, cgl4574237, cg23611220, cg21727129, cg08708750, cg09430946, cg03432955, cg09865095, cg00126638, cg03521696, cg21529533, cgl2287358, cg03997176, cgl9141489, cg22257941, cgll363230, cgl5174396, cgl4546076, cgl3130352, cg26511972, cg23110482, cg03298800, cgl0807461, cg01818016, cgll682706, cg21855392, cgl5595495, cg05523662, cgl5319255, cgl7678719, cg23489273, cgll808100, cg05839762, cgl4772439, cgl4018363, cg08039587, cgl6742075, cg20408505, cg25637655, cgl7608381, cgll946459, cgl6367518, cgl6507955, cg21297314, cg08416870, cgl0794086, cg09275023, cgll861940, cg04807188, cgl0124767, cg02942965, cg20644330, cgl3223754, cg04305858, cgl7475918, cg26175526, cg01462744, cg22952566, cg20534075, cg20662397, cg25293221, cg08899786, cgl4849431, cgl5118824, cg02751503, cg01365762, cg21292025, cgl0457005, cg08180063, cg25664704, cg05384135, cgl5816267, cg09609649, cgl8183435, cg09620840, cg21176130, cg09326440, cgl2700271, cgl3007871, cg00340855, cgll594821, cg21758773, cg05201185, cg02188225, cg20652371, cg21366673, cg23235965, cg27486585, cgl7615629, cg20105257, cg02678305, cgl9109457, cg09569347, cgl3036546, cgl6535080, cgll019014, cg24710520, cgll247343, cgl3872627, cg01064373, cgl8511546, cg26392102, cgl7096289, cgl2965927, cgl8020334, cgl8747378, cg24877963, cgl7974398, cg05619024, cgl3155295, cg01006124, cg25355501, cg06659144, cg02827714, cgl8785300, cg27358585, cg08309069, cg05030953, cg23533285, cg06422189, cg23923934, cg27529346, cg05139028, cglll87245, cg01517384, cgl3902357, cg05932159, cg09588770, cgl2730088, cgl7731992, cg06646969, cg01035015, cg26706521, cgl0542672, cg04981410, cg22016094, cgll239749, cg09889987, cg21454965, cg02010152, cg03477130, cgl0294956, cgll469594, cg08905753, cg00409309, cg27246453, cg04576021, cg01530082, cgl7369196, cg01493678, cgl7103109, cg01200893, cgl9811863, cg00411901, cgl9121661, cgl2333338, cg06707784, cg21021279, cg25798341, cg22619784, cg05592483, cg02633767, cg25823314, cgl8243910, cg08985301, cgl3991568, cg27533873, cg26187287, cg00167916, cg08327277, cgl8050892, cg06121167, cg09408973, cgl4571125, cg00340328, cg26070383, cg07023208, cgl9887824, cg25744682, cgl0924050, cg27176262, cg09871008, cgl8511153, cgl3347198, cg02151752, cg01448551, cg02670545, cg09351471, cgll709793, cg07353306, cg01283574, cgll752893, cgl2121080, cgl3600652, cg00720839, cgl6026549, cgl2761728, cg07015256, cg24813704, cg21359558, cg09187413, cg24469596, cgl8045172, cg08513422, cgl4997446, cg20397692, cg25139471, cgl5785389, cg08358188, cgl2671948, cg08354908, cg00386460, cgl0451089, cg20224850, cgl8266257, cgl0894807, cgl0627913, cgl7390106, cg27618777, cg04255770, cgl5672065, cgl2854186, cg22940798, cg08998192, cgl3563634, cg23560159, cgl0088320, cg22230640, cgll668794, cg20724405, cg05729731, cg26260794, cg03026929, cg20898522, cgl9221044, cgll439192, cg02779707, cg07546106, cgl7253307, cg21780812, cgl2180249, cg00857968, cgl8607757, cgl9655544, cg03544736, cgl7670640, cg25728415, cg24364491, cgl3477582, cgl4867383, cg08557029, cg20558646, cgl6855458, cg05420994, cg23059310, cgl6951654, cg22798247, cgl4493899, cgl3871037, cg23750151, cg25693349, cgl2048225, cgl2094903, cgl8505752, cg08331398, cg21621645, cgll954557, cgl9646975, cgl2718404, cg22510079, cg08992729, cg26988373, cg22158175, cgl6009764, cg20060630, cg00971309, cg03078854, cg24031377, cg21593554, cgll453837, cg24898914, cg00533183, cg08675092, cg08099136, cg01309328, cgl4022881, cgll990058, cgl8696632, cg00777968, cg27232057, cg24893844, cg24625115, cg05115162, cg24767041, cg22007922, cg09006592, cg05545172, cg05872513, cg24326130, cg26829379, cg20848216, cg00889031, cg05242261, cg21322855, cg05135714, cg21568368, cgl0128166, cg07456831, cgl3853192, cg05629721, cgl8963307, cg01295600, cgl7392730, cg08772265, cg03995852, cgl5451100, cgl3603062, cg21535207, cg01702338, cgl0087598, cg20630683, cg06531943, cg03166550, cg06870868, cgl0192196, cglll41874, cg05697726, cgl6483840, cg02657012, cgl6890093, cgll706729, cg07218288, cgl9136673, cg05693489, cgl2253437, cgl3165140, cg09354037, cg25384897, cg02756056, cg09154880, cg02561720, cgll313335, cg05412855, cg05480623, cgl2499157, cg07072394, cg20880499, cg02351231, cgll969117, cg08351785, cg03807983, cg00240875, cg01255458, cg20357228, cg23132351, cg01979171, cg08151204, cgl7973694, cgl4293027, cg05262533, cg26033526, cg01673307, cg24111025, cg25042789, cg02181920, cg06473288, cg26234900, cgl0666909, cg08818207, cg04773990, cg24154161, cg23880953, cg26700949, cgl5926590, cgl8436324, cg05412930, cg06634056, cgl3403689, cg25110530, cg05826626, cg08880054, cg25524784, cgl4286550, cgl7378691, cg09968749, cg01170201, cg08466770, cgl4925994, cgl6501436, cgl9776032, cg01897517, cg01547742, cg09374375, cg27652200, cgll012835, cg23922920, cg27572016, cg05246277, cg25110511, cgl7199468, cgl8903583, cg20194454, cg23806084, cg06852255, cg03299066, cg00704844, cgl6494397, cg07504780, cg02439889, cgl4272068, cgl2641838, cgl2862002, cgl3274644, cg01692674, cg24718356, cg20729846, cg02551145, cg08766503, cg20813491, cg07250080, cg03735531, cgl2762680, cg02567488, cg07156249, cg03465320, cgl6853860, cg06791592, cg21826978, cgl0817441, cg04908668, cg21764921, cgl6229954, cgl9412007, cgl9760441, cg26265820, cg26811065, cg08209711, cg02878284, cgl9994157, cg00045690, cg04913118, cg06043617, cg09322555, cgl3869180, cg24461669, cgl8696027, cgl8555073, cg21445676, cgl0081723, cg07192821, cg08459087, cg00079638, cg08350173, cg24134304, cgl3964393, cg03355298, cg00837838, cgl9404757, cgl2468675, cgl9721944, cg08188779, cg27467953, cg03425812, cgl2828896, cg27537252, cg05475649, cgl6039157, cg02988397, cg21979287, cg22138564, cg07839457, cgl6411857, cg06862692, cg06754565, cg03056682, cg06181531, cg00881185, cg26963976, cg02493440, cg02169333, cg08558449, cg04097610, cg07862320, cg05723552, cgl2256630, cg24341550, cg03089589, cg07223648, cg21333585, cg02644232, cgl6677488, cg05757530, cg04601693, cgl0561843, cg20740356, cgl8440314, cg26661738, cgl6007266, cgl6362955, cgl7047468,

[0112] Cg24661931, Cgl4196251, cgll345288, cg04799664, cgl7625032, cg09561419, cg02705619,

[0113] Cgll987321, cg02610906, cg06265737, cg09174467, cg05154222, cgl0854209, cg03754428, cg27462748, cgl7340138, cgl8398267, cg09624807, cg23314414, cg00125947, cg01834111, cg06600851, cgl0553082, cg23820818, cg07990412, cgl9840565, cgl6213529, cg07054754, cgl5875239, cg04344414, cg01517277, cgl8258710, cg00589550, cgl2439595, cgl2928479, cglll01747, cg23848181, cgl8060687, cg24399801, cg09714724, cg23079860, cg04972244, and cgl4946319.

[0114] Importantly, the Inventors have found that several of the CpG sites described herein are differentially methylated depending on the severity of IBD. Said CpG sites include, but are not limited to, cglll87245, cg00340855, cg05475649, cg24341550, cgl2256630, cgl0601943, cgl2761728, cgl0817441, cgl5375424, cg23923934, cgl4140375, cg01405582, cg02181920, cgll617938, cgl8511546, cgl8696027, cg26033526, cg05697726, cg25384897, cg22310628, cg05358170, cgl7615629, cg07862320, cgll594821, cg07218288, cg24111025, cg07839457, and cgl3007871. The Inventors have surprisingly found that prognostic signatures comprising one or more of these 28 CpG sites provide an accurate prediction of disease severity in IBD. For example, a prognostic marker comprising each of these 28 CpG sites was found to predict a severe disease outcome in Crohn's Disease patients with an area under the curve (AUC) of 0.72.

[0115] Thus, in some embodiments, the level of DNA methylation is the level of DNA methylation at one or more (e.g. 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, 25 or more, 26 or more, 27 or more, or 28) CpG sites in one or more MHC-I related gene(s), wherein the one or more

[0116] CpG sites are selected from cglll87245, cg00340855, cg05475649, cg24341550, cgl2256630, cgl0601943, cgl2761728, cgl0817441, cgl5375424, cg23923934, cgl4140375, cg01405582, cg02181920, cgll617938, cgl8511546, cgl8696027, cg26033526, cg05697726, cg25384897, cg22310628, cg05358170, cgl7615629, cg07862320, cgll594821, cg07218288, cg24111025, cg07839457, and cgl3007871. In some embodiments, the level of DNA methylation is the level of DNA methylation at CpG sites comprising cglll87245, cg00340855, cg05475649, cg24341550. cgl2256630, cgl0601943, cgl2761728, cgl0817441, cgl5375424, cg23923934, cgl4140375. cg01405582, cg02181920, cgll617938, cgl8511546, cgl8696027, cg26033526, cg05697726. cg25384897, cg22310628, cg05358170, cgl7615629, cg07862320, cgll594821, cg07218288. cg24111025, cg07839457, and cgl3007871.

[0117] The Inventors also have discovered that several of the CpG sites described herein are differentially methylated depending on the type of IBD, e.g. Crohn's Disease or Ulcerative Colitis. Said CpG sites include, but are not limited to, cg27529346, cglll87245, cgl6890093, cg05201185, cgll706729, cg09354037, cg00340855, cg06473288, cg05475649, cg24341550, cg02756056, cgl2256630. cgl0601943, cgl0817441, cgl5375424, cg05693489, cg23923934, cg04576021, cg27176262. cg01673307, cgl4140375, cg27537252, cg26234900, cg01405582, cg02181920, cgl9136673. cg02988397, cg25042789, cgl8511546, cg06791592, cg26033526, cg05697726, cg25384897. cg05412855, cg27486585, cgl8243910, cg06422189, cg24898914, cgll453837, cgl2253437. cg23235965, cg09187413, cg00533183, cgl7615629, cg22310628, cg05358170, cg07862320. cgll594821, cg07218288, cg24111025, cgll587584, cg07839457, and cgl3007871. The Inventors have surprisingly found that diagnostic signatures comprising one or more of these CpG sites can be used to differentially diagnose Crohn's Disease and Ulcerative Colitis. For example, a diagnostic marker comprising all 53 of these CpG sites can be used to diagnose Crohn's Disease with an AUC of 0.82.

[0118] Thus, in some embodiments, the level of DNA methylation is the level of DNA methylation at one or more (e.g. 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, 25 or more, 26 or more, 27 or more, 28 or more, 29 or more, 30 or more, 31 or more, 32 or more, 33 or more, 34 or more, 35 or more, 36 or more, 37 or more, 38 or more, 39 or more, 40 or more, 41 or more, 42 or more, 43 or more, 44 or more, 45 or more, 46 or more, 47 or more, 48 or more, 49 or more, 50 or more, 51 or more, 52 or more, or 53) CpG sites in one or more MHC-I related gene(s), wherein the one or more CpG sites are selected from cg27529346, cglll87245, cgl6890093, cg05201185. cgll706729, cg09354037, cg00340855, cg06473288, cg05475649, cg24341550, cg02756056. cgl2256630, cgl0601943, cgl0817441, cgl5375424, cg05693489, cg23923934, cg04576021. cg27176262, cg01673307, cgl4140375, cg27537252, cg26234900, cg01405582, cg02181920. cgl9136673, cg02988397, cg25042789, cgl8511546, cg06791592, cg26033526, cg05697726. cg25384897, cg05412855, cg27486585, cgl8243910, cg06422189, cg24898914, cgll453837. cgl2253437, cg23235965, cg09187413, cg00533183, cgl7615629, cg22310628, cg05358170. cg07862320, cgll594821, cg07218288, cg24111025, cgll587584, cg07839457, and cgl3007871. In some embodiments, the level of DNA methylation is the level of DNA methylation at one or more CpG sites in one or more MHC-I related gene(s), wherein the one or more CpG sites are selected from cg27529346, cglll87245, cgl6890093, cg05201185, cgll706729, cg09354037, cg00340855, cg06473288, cg05475649, cg24341550, cg02756056, cgl2256630, cgl0601943, cgl0817441, cgl5375424, cg05693489, cg23923934, cg04576021, cg27176262, cg01673307, cgl4140375, cg27537252, cg26234900, cg01405582, cg02181920, cgl9136673, cg02988397, cg25042789, cgl8511546, cg06791592, cg26033526, cg05697726, cg25384897, cg05412855, cg27486585, cgl8243910, cg06422189, cg24898914, cgll453837, cgl2253437, cg23235965, cg09187413, cg00533183, cgl7615629, cg22310628, cg05358170, cg07862320, cgll594821, cg07218288, cg24111025, cgll587584, cg07839457, and cgl3007871.

[0119] In some embodiments, the one or more MHC-I related gene(s) are selected from NLRC5, B2M, TAPI, TAP2, HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, HLA-DPA1, PSMB8, PSMB9, IFIT1, and IRF1. In some embodiments, the one or more MHC-I related gene(s) comprise 2 or more (e.g. 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, or all 15) of NLRC5, B2M, TAPI, TAP2, HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, HLA-DPA1, PSMB8, PSMB9, IFIT1, and IRF1.

[0120] NLRC5 encodes nucleotide-binding domain, leucine-rich repeat CARD Domain Containing 5 (NLRC5). As demonstrated herein, NLRC5 acts as transcriptional transactivator of MHC-I in the intestinal epithelium. In some embodiments, the one or more MHC-I related gene(s) comprise NLRC5. In some embodiments, the level of DNA methylation comprises the level of DNA methylation at one or more CpG site(s) located within NLRC5, optionally wherein the one or more CpG sites are selected from cg07839457, cgl6411857, cg06862692, cg06754565, cg03056682, cg06181531, cg00881185, cg26963976, cg02493440, cg02169333, cg08558449, cg04097610, cg07862320, cg05723552, cgl2256630, cg24341550, cg03089589, cg07223648, cg21333585, cg02644232, cgl6677488, cg05757530, cg04601693, cgl0561843, cg20740356, eg 18440314, cg26661738, cgl6007266, cgl6362955, cgl7047468, cg24661931, cgl4196251, cgll345288, cg04799664, cgl7625032, cg09561419, cg02705619, cgll987321, cg02610906, cg06265737, cg09174467, cg05154222, cgl0854209, cg03754428, cg27462748, cgl7340138, cgl8398267, cg09624807, cg23314414, cg00125947, cg01834111, cg06600851, cgl0553082, cg23820818, cg07990412, cgl9840565, cgl6213529, cg07054754, cgl5875239, cg04344414, cg01517277, cgl8258710, cg00589550, cgl2439595, cgl2928479, cglll01747, cg23848181, cgl8060687, cg24399801, cg09714724, cg23079860, cg04972244, and cgl4946319. In some embodiments, the level of DNA methylation comprises the level of DNA methylation at CpG site(s) located within the promoter region of NLRC5, wherein the CpG sites comprise cg07839457 and / or cg07862320.

[0121] B2M encodes beta-2-microglobulin which is a component of the class I major histocompatibility complex (MHC). In some embodiments, the one or more MHC-I related gene(s) comprise B2M.

[0122] In some embodiments, the level of DNA methylation comprises the level of DNA methylation at one or more CpG site(s) located within B2M, optionally wherein the one or more CpG sites are selected from cgl8696027, cgl8555073, cg21445676, cgl0081723, cg07192821, cg08459087, cg00079638, cg08350173, cg24134304, cgl3964393, cg03355298, cg00837838, cgl9404757, cgl2468675, cgl9721944, cg08188779, cg27467953, cg03425812, cgl2828896, cg27537252, cg05475649, cgl6039157, cg02988397, cg21979287, and cg22138564.

[0123] TAPI encodes antigen peptide transporter 1 which is an ABC transporter associated with antigen processing. In some embodiments, the one or more MHC-I related gene(s) comprise TAPI.

[0124] In some embodiments, the level of DNA methylation comprises the level of DNA methylation at one or more CpG site(s) located within TAPI, optionally wherein the one or more CpG sites are selected from cgl0192196, cglll41874, cg05697726, cgl6483840, cg02657012, cgl6890093, cgll706729, cg07218288, cgl9136673, cg05693489, cgl2253437, cgl3165140, cg09354037, cg25384897, cg02756056, cg09154880, cg02561720, cgll313335, cg05412855, cg05480623, cgl2499157, cg07072394, cg20880499, cg02351231, cgll969117, cg08351785, cg03807983, cg00240875, cg01255458, cg20357228, cg23132351, cg01979171, cg08151204, cgl7973694, cgl4293027, cg05262533, cg26033526, cg01673307, cg24111025, cg25042789, cg02181920, cg06473288, cg26234900, cgl0666909, cg08818207, cg04773990, cg24154161, cg23880953, cg26700949. cgl5926590, cgl8436324, cg05412930, cg06634056, cgl3403689, cg25110530, cg05826626. cg08880054, cg25524784, cgl4286550, cgl7378691, cg09968749, cg01170201, cg08466770. cgl4925994, cgl6501436, cgl9776032, cg01897517, cg01547742, cg09374375, cg27652200. cgll012835, cg23922920, cg27572016, cg05246277, cg25110511, cgl7199468, cgl8903583. cg20194454, cg23806084, cg06852255, cg03299066, cg00704844, cgl6494397, cg07504780. cg02439889, cgl4272068, cgl2641838, cgl2862002, cgl3274644, cg01692674, cg24718356. cg20729846, cg02551145, cg08766503, cg20813491, cg07250080, cg03735531, cgl2762680. cg02567488, cg07156249, cg03465320, and cgl6853860.

[0125] TAP2 encodes antigen peptide transporter 2 which is an ABC transporter associated with antigen processing. In some embodiments, the one or more MHC-I related gene(s) comprise TAP2.

[0126] In some embodiments, the level of DNA methylation comprises the level of DNA methylation at one or more CpG site(s) located within TAP2, optionally wherein the one or more CpG sites are selected from cg22016094, cgll239749, cg09889987, cg21454965, cg02010152, cg03477130, cgl0294956, cgll469594, cg08905753, cg00409309, cg27246453, cg04576021, cg01530082, cgl7369196, cg01493678, cgl7103109, cg01200893, cgl9811863, cg00411901, Cgl9121661, cgl2333338, cg06707784, cg21021279, cg25798341, cg22619784, cg05592483, cg02633767, cg25823314, cgl8243910, cg08985301, cgl3991568, cg27533873, cg26187287, cg00167916, cg08327277, cgl8050892, cg06121167, cg09408973, cgl4571125, cg00340328, cg26070383, cg07023208, cgl9887824, cg25744682, cgl0924050, cg27176262, cg09871008, cgl8511153, cgl3347198, cg02151752, cg01448551, cg02670545, cg09351471, cgll709793, cg07353306, cg01283574, cgll752893, cgl2121080, cgl3600652, cg00720839, cgl6026549, cgl2761728, cg07015256, cg24813704, cg21359558, cg09187413, cg24469596, cgl8045172, cg08513422, cgl4997446, cg20397692, cg25139471, cgl5785389, cg08358188, cgl2671948, cg08354908, cg00386460, cgl0451089, cg20224850, cgl8266257, cgl0894807, cgl0627913, cgl7390106, cg27618777, cg04255770, cgl5672065, cgl2854186, cg22940798, cg08998192, cgl3563634, cg23560159, cgl0088320, cg22230640, cgll668794, cg20724405, cg05729731, cg26260794, cg03026929, cg20898522, cgl9221044, cgll439192, cg02779707, cg07546106, cgl7253307, cg21780812, cgl2180249, cg00857968, cgl8607757, cgl9655544, cg03544736, cgl7670640, cg25728415,

[0127] Cg24364491, cgl3477582, cgl4867383, cg08557029, cg20558646, cgl6855458, cg05420994, cg23059310, cgl6951654, cg22798247, cgl4493899, and cgl3871037. HLA-A encodes HLA class I histocompatibility antigen, A alpha chain which is an antigen-presenting major histocompatibility complex class I (MHCI) molecule. In some embodiments, the one or more MHC-I related gene(s) comprise HLA-A.

[0128] In some embodiments, the level of DNA methylation comprises the level of DNA methylation at one or more CpG site(s) located within HLA-A, optionally wherein the one or more CpG sites are selected from cg05523662, cgl5319255, cgl7678719, cg23489273, cgll808100, cg05839762, cgl4772439, cgl4018363, cg08039587, cgl6742075, cg20408505, cg25637655, cgl7608381, and cgll946459.

[0129] HLA-B encodes HLA class I histocompatibility antigen, B alpha chain which is an antigen-presenting major histocompatibility complex class I (MHCI) molecule. In some embodiments, the one or more MHC-I related gene(s) comprise HLA-B.

[0130] In some embodiments, the level of DNA methylation comprises the level of DNA methylation at one or more CpG site(s) located within HLA-B, optionally wherein the one or more CpG sites are selected from cg23533285, cg06422189, cg23923934, cg27529346, cg05139028, cglll87245, cg01517384, cgl3902357, cg05932159, cg09588770, cgl2730088, cgl7731992, cg06646969, cg01035015, cg26706521, cgl0542672, and cg04981410.

[0131] HLA-C encodes HLA class I histocompatibility antigen, C alpha chain which is an antigen-presenting major histocompatibility complex class I (MHCI) molecule. In some embodiments, the one or more MHC-I related gene(s) comprise HLA-C.

[0132] In some embodiments, the level of DNA methylation comprises the level of DNA methylation at one or more CpG site(s) located within HLA-C, optionally wherein the one or more CpG sites are selected from cg24710520, cgll247343, cgl3872627, cg01064373, cgl8511546, cg26392102, cgl7096289, cgl2965927, cgl8020334, cgl8747378, cg24877963, cgl7974398, cg05619024, cgl3155295, cg01006124, cg25355501, cg06659144, cg02827714, cgl8785300, cg27358585, cg08309069, and cg05030953.

[0133] HLA-E encodes HLA class I histocompatibility antigen, alpha chain E which is a non-classical MHClb molecule. In some embodiments, the one or more MHC-I related gene(s) comprise HLA-E.

[0134] In some embodiments, the level of DNA methylation comprises the level of DNA methylation at one or more CpG site(s) located within HLA-E, optionally wherein the one or more CpG sites are selected from cgl6367518, cgl6507955, cg21297314, cg08416870, cgl0794086, cg09275023, cgll861940, cg04807188, cgl0124767, cg02942965, cg20644330, cgl3223754, cg04305858, cgl7475918, cg26175526, cg01462744, cg22952566, cg20534075, cg20662397, cg25293221, cg08899786, cgl4849431, cgl5118824, cg02751503, cg01365762, cg21292025, cgl0457005, cg08180063, cg25664704, cg05384135, cgl5816267, cg09609649, cgl8183435, cg09620840, cg21176130, cg09326440, cgl2700271, cgl3007871, cg00340855, cgll594821, cg21758773, cg05201185, cg02188225, cg20652371, cg21366673, cg23235965, cg27486585, cgl7615629, cg20105257, cg02678305, cgl9109457, cg09569347, cgl3036546, cgl6535080, and cgll019014.

[0135] HLA-F encodes HLA class I histocompatibility antigen, alpha chain F which is a non-classical MHClb molecule. In some embodiments, the one or more MHC-I related gene(s) comprise HLA-F.

[0136] In some embodiments, the level of DNA methylation comprises the level of DNA methylation at one or more CpG site(s) located within HLA-F, optionally wherein the one or more CpG sites are selected from cgl5018934, cg08698936, cgll201654, cg22298860, cgll768167, cg04186657, cgl4738290, cg03278703, cgll576659, cg02688650, cgl4402472, cg24703717, cg05237001, cg03851143. cgllll6776, cg23912231, cg09805507, cgl6728223, cg05603292, cgl7625506, cg20891813. cgl3924755, cgl9348622, cgl8676033, cgl0262357, cg26069562, cgll423684, cg07016276. cg20927242, cgl2588917, cg23892836, cg24351901, cgl5331332, cg00504902, cgll617938. cg01405582, cgll587584, cg05358170, cgl4140375, cgl0601943, cg22310628, cgl3040666. cg02945359, cg06036836, cg04892672, cg25197880, cg01393604, cg24952408, cg26987604. cg25252977, cgl6650906, cg23148731, cgl2550837, cgl6891968, cgl7264941, cg03176423. cgl8371410, cg21632181, cgl4317321, cg24177217, cgl5180617, cg08179037, cg08755130. cgl6289618, and cgl5628633.

[0137] HLA-G encodes HLA class I histocompatibility antigen, alpha chain G which is a non-classical MHClb molecule. In some embodiments, the one or more MHC-I related gene(s) comprise HLA-G.

[0138] In some embodiments, the level of DNA methylation comprises the level of DNA methylation at one or more CpG site(s) located within HLA-G, optionally wherein the one or more CpG sites are selected from cgl4574237, cg23611220, cg21727129, cg08708750, cg09430946, cg03432955, cg09865095, cg00126638, cg03521696, cg21529533, cgl2287358, cg03997176, cgl9141489, cg22257941, cgll363230, cgl5174396, cgl4546076, cgl3130352, cg26511972, cg23110482, cg03298800, cgl0807461, cg01818016, cgll682706, cg21855392, and cgl5595495.

[0139] PSMB8 encodes proteasome subunit beta type-8 which is involved in antigen processing to generate class I binding peptides. In some embodiments, the one or more MHC-I related gene(s) comprise PSMB8.

[0140] In some embodiments, the level of DNA methylation comprises the level of DNA methylation at one or more CpG site(s) located within PSMB8, optionally wherein the one or more CpG sites are selected from cg23750151, cg25693349, cgl2048225, cgl2094903, cgl8505752, cg08331398, cg21621645, cgll954557, cgl9646975, cgl2718404, cg22510079, cg08992729, cg26988373, cg22158175, cgl6009764, cg20060630, cg00971309, cg03078854, cg24031377, cg21593554, cgll453837, cg24898914, cg00533183, cg08675092, cg08099136, cg01309328, cgl4022881, cgll990058, cgl8696632, cg00777968, cg27232057, cg24893844, cg24625115, cg05115162, cg24767041, cg22007922, cg09006592, cg05545172, cg05872513, cg24326130, cg26829379, cg20848216, cg00889031, cg05242261, cg21322855, cg05135714, cg21568368, cgl0128166, cg07456831, cgl3853192, cg05629721, cgl8963307, cg01295600, cgl7392730, cg08772265, cg03995852, cgl5451100, cgl3603062, cg21535207, cg01702338, cgl0087598, cg20630683, cg06531943, cg03166550, cg06870868, cgl0192196, cglll41874, cg05697726, cgl6483840, cg02657012, cgl6890093, cgll706729, cg07218288, cgl9136673, cg05693489, cgl2253437, cgl3165140, cg09354037, cg25384897, cg02756056, and cg09154880.

[0141] PSMB9 encodes proteasome subunit beta type-9 which is involved in antigen processing to generate class I binding peptides. In some embodiments, the one or more MHC-I related gene(s) comprise PSMB9.

[0142] In some embodiments, the level of DNA methylation comprises the level of DNA methylation at one or more CpG site(s) located within PSMB9, optionally wherein the one or more CpG sites are selected from cg06791592, cg21826978, cgl0817441, cg04908668, cg21764921, cgl6229954, cgl9412007, cgl9760441, cg26265820, cg26811065, cg08209711, cg02878284, cgl9994157, cg00045690, cg04913118, cg06043617, cg09322555, cgl3869180, cg24461669, cgll453837, cg24898914, cg00533183, cg08675092, cg08099136, cg01309328, cgl4022881, cgll990058, cgl8696632, cg00777968, cg27232057, cg24893844, cg24625115, cg05115162, cg24767041, cg22007922, cg09006592, cg05545172, cg05872513, cg24326130, cg26829379, cg20848216, cg00889031, cg05242261, cg21322855, cg05135714, cg21568368, cgl0128166, cg07456831, cgl3853192, cg05629721, cgl8963307, cg01295600, cgl7392730, cg08772265, cg03995852, cgl5451100, cgl3603062, cg21535207, cg01702338, cgl0087598, cg20630683, cg06531943, cg03166550, cg06870868, cgl0192196, cglll41874, cg05697726, cgl6483840, cg02657012, cgl6890093, cgll706729, cg07218288, cgl9136673, cg05693489, cgl2253437, cgl3165140, cg09354037, cg25384897, cg02756056, cg09154880, cg02561720, cgll313335, cg05412855, cg05480623, cgl2499157, cg07072394, cg20880499, cg02351231, cgll969117, cg08351785, cg03807983, cg00240875, cg01255458, cg20357228, cg23132351, cg01979171, cg08151204, cgl7973694, cgl4293027, cg05262533, cg26033526, cg01673307, cg24111025, cg25042789, cg02181920, cg06473288, cg26234900, cgl0666909, cg08818207, cg04773990, cg24154161, cg23880953, cg26700949, cgl5926590, cgl8436324, cg05412930, cg06634056, cgl3403689, cg25110530, cg05826626, cg08880054, cg25524784, cgl4286550, cgl7378691, cg09968749, cg01170201, cg08466770, cgl4925994, cgl6501436, cgl9776032, cg01897517, cg01547742, cg09374375. cg27652200, cgll012835, cg23922920, cg27572016, cg05246277, cg25110511, cgl7199468. cgl8903583, cg20194454, cg23806084, cg06852255, cg03299066, cg00704844, cgl6494397. cg07504780, cg02439889, cgl4272068, cgl2641838, cgl2862002, cgl3274644, cg01692674. cg24718356, cg20729846, cg02551145, cg08766503, cg20813491, cg07250080, cg03735531. cgl2762680, cg02567488, cg07156249, cg03465320, and cgl6853860.

[0143] IRF1 encodes interferon regulatory factor 1 which is a transcriptional regulator. In some embodiments, the one or more MHC-I related gene(s) comprise IRF1.

[0144] In some embodiments, the level of DNA methylation comprises the level of DNA methylation at one or more CpG site(s) located within IRF1, optionally wherein the one or more CpG sites are selected from cg27587780, cgl5375424, cgl7394978, cgll666365, cgll086820, cg20287640, cgl3907973, cg25410123, cg05941961, cgl8890864, cg21428887, cg05915281, cg06942904, cg08977266, cg00483888, cgll971752, cgl5421363, cg00578828, cg26382286, cg21138405, and cg00255919.

[0145] HLA-DPA1 encodes HLA class II histocompatibility antigen, DP alpha 1 chain which binds peptides derived from antigens that access the endocytic route of antigen presenting cells and presents them on the cell surface for recognition by the CD4 T-cells. In some embodiments, the one or more MHC-I related gene(s) comprise HLA-DPA1.

[0146] IFIT1 encodes Interferon-induced protein with tetratricopeptide repeats 1. In some embodiments, the one or more MHC-I related gene(s) comprise IFIT1.

[0147] It will be readily understood that comparing the level of DNA methylation of one or more MHC-I related gene(s) and / or the level of expression of one or more MHC-I related gene(s) in a biological sample obtained from the patient to the level of DNA methylation of said one or more MHC-I related gene(s) and / or the level of expression of said one or more MHC-I related gene(s) in a control may comprise calculating a risk score and comparing the risk score to a threshold value. The threshold level is typically a level of risk score which can be used to distinguish between outcomes, e.g. between different disease severity levels or between different diagnoses. In some embodiments, the risk score is compared to multiple thresholds which each distinguish between different groups. For example, a first threshold may distinguish patients having severe or moderate IBD from patients having mild IBD, and a second threshold may distinguish between patients having severe IBD from patients having moderate or mild IBD. An exemplary method for calculating the risk score is provided in the Examples, see "Risk Score System and Patient Stratification". A patient identified as having IBD by a method of the invention may be treated with any known IBD therapeutic. The present invention helps ensure that the treatment is appropriate for the severity of IBD in a patient. Suitable IBD therapeutics include, but are not limited to, aminosalicylates (5-ASA, e.g. mesalazine, sulfasalazine, olsalazine and balsalazide), corticosteroids (e.g. prednisolone or budesonide), immunomodulators or immunosuppressants (e.g. thiopurine, azathioprine, mercaptopurine or methotrexate), cyclosporine, biologies (e.g. adalimumab, infliximab, vedolizumab or ustekinumab), JAK inhibitors (e.g. tofacitinib or filgotinib), and ozanimod.

[0148] Based on the type and severity of IBD and the dosage regimen employed, an IBD therapeutic may provide an induction treatment and / or a maintenance treatment. For example, in some embodiments, a patient is treated with an induction treatment selected from 5-ASA, corticosteroids, biologies, JAK inhibitors, and ozanimod. In some embodiments, a patient is treated with a maintenance treatment selected from 5-ASA, immunosuppressants, biologies, JAK inhibitors, and ozanimod.

[0149] Patients who exhibit more severe IBD will typically be treated using more aggressive therapeutic agents (e.g. a 5-ASA, a corticosteroid, or an immunosuppressant) or surgery. Patients who exhibit milder IBD are typically treated with less aggressive therapeutic interventions (e.g. cyclosporine, a biologic, a JAK inhibitor, and / or ozanimod).

[0150] The invention also provides a method of treating a patient identified as having IBD by a method of the invention, wherein the treating comprises: (i) administering to the patient one or more of a 5-ASA, a corticosteroid, an immunosuppressant, cyclosporine, a biologic, a JAK inhibitor, and ozanimod; and / or (ii) surgery, optionally by resection or colectomy.

[0151] The invention also provides a method of treating a patient identified as having IBD by a method of the invention, wherein the treating comprises: (i) administering to the patient a therapeutic agent which reduces or inhibits the activity of one or more proteins encoded by one or more MHC-I related gene(s); and / or (ii) surgery, optionally by resection or colectomy.

[0152] In some embodiments, a method of the invention further comprises determining the level of DNA methylation of one or more MHC-I related gene(s) and / or the level of expression of one or more MHC- I related gene(s) in the biological sample obtained from the patient.

[0153] In some embodiments, the level of DNA methylation is determined by sodium bisulphite conversion assay, next generation sequencing (NGS), Oxford Nanopore sequencing, differential enzymatic cleavage of DNA, a methylated DNA immunoprecipitation assay, methylation-specific PCR, methylation-sensitive melting assay, and / or a bisulphite-free detection method. In some embodiments, the level of expression is determined by RNAseq, microarray, and / or quantitative PCR (qPCR).

[0154] In some embodiments, a method of the invention further comprises obtaining the biological sample from the patient. In some embodiments, the biological sample is obtained from an intestinal biopsy and / or a faecal sample. In some embodiments, the biological sample is derived from the Terminal Ileum (Tl) and / or the Sigmoid Colon (SC).

[0155] In some embodiments, the biological sample comprises intestinal epithelial cells. In some embodiments, the biological sample comprises intestinal epithelial organoids (lEOs). lEOs can be generated from mucosal stem cells and retain epigenetic signatures of host-derived tissues. Importantly, lEOs provide a novel opportunity to investigate the contribution of IEC intrinsic molecular mechanisms in vitro. lEOs closely mimic many aspects of intestinal epithelial physiology as they self-organise into 3-dimensional crypt-villus structures that contain all epithelial cell subsets and follow the same cell renewal and differentiation dynamics. A major advantage of lEOs in the context of IBD is the ability to study lECs without exposure to their local inflammatory environment, thereby eliminating potentially confounding factors influencing gene expression and cellular function. Most importantly, as lEOs are derived from constantly dividing intestinal mucosal stem cells, disease associated alterations that persist in culture reflect intestinal epithelial cell-intrinsic and heritable pathologies.

[0156] Methods for generating lEOs are known in the art. For example, lEOs may be generated by isolating intestinal crypts from human mucosal biopsies and culturing them in a growth medium as described in Kraiczy, J. et al. Gut 68, 49-61 (2019) and Edgar, R. D. et al. Cell Mol Gastroenter 14, 1295-1310 (2022).

[0157] The invention provides a method for predicting the likelihood that a patient will respond to therapy with a therapeutic agent, the method comprising: (a) exposing intestinal epithelial organoids (lEOs) derived from the patientto the therapeutic agent; and (b) quantifying expression of MHC-I in the lEOs; wherein reduced expression of MHC-I as compared to the expression of MHC-I in the absence of the therapeutic agent indicates an increased likelihood that the patient will respond to therapy.

[0158] In some embodiments, the lEOs are derived from mucosal stem cells obtained from an intestinal biopsy.

[0159] In some embodiments, the method comprises exposing lEOs having the same level of DNA methylation of one or more MHC-I related gene(s) and / or same level of expression of one or more MHC-I related gene(s) as the patient to the therapeutic agent. In some embodiments, the method comprises exposing the therapeutic agent to lEOs derived from another patient or a group of patients having the same severity of IBD as the patient.

[0160] The invention also provides a method for screening an agent for therapeutic activity against IBD, the method comprising: (a) exposing intestinal epithelial organoids (lEOs) derived from at least one patient having IBD to the agent; and (b) quantifying expression of MHC-I and / or one or more MHC-I related gene(s) in the lEOs; wherein reduced expression of MHC-I and / or said one or more MHC-I related gene(s) as compared to expression of MHC-I and / or said one or more MHC-I related gene(s) in the absence of the agent is indicative of therapeutic activity against IBD.

[0161] In some embodiments, the lEOs are derived from mucosal stem cells obtained from an intestinal biopsy.

[0162] In some embodiments, the method further comprises exposing the lEOs to IFN-y. Advantageously, the Inventors have discovered that exposing lEOs to IFN-y stimulates MHC-I expression thereby providing a larger effect size.

[0163] In some embodiments, the therapeutic agent is selected from a biologic, an immunomodulator, a JAK inhibitor, a 5-ASA, a corticosteroid, and an immunosuppressant. In some embodiments, the therapeutic agent is selected from a small molecule, a peptide, an aptamer, and a peptidomimetic. In some embodiments, the therapeutic agent reduces or inhibits the activity of one or more proteins encoded by one or more MHC-I related gene(s).

[0164] Expression of MHC-I and / or one of more MHC-I related gene(s) may be measured using any suitable method known in the art. For example, quantifying expression of MHC-I may involve RNAseq, microarray, and / or quantitative PCR (qPCR).

[0165] The invention also provides a kit comprising (a) reagents for determining the level of DNA methylation of one or more MHC-I related gene(s) and / or the level of expression of one or more MHC-I related gene(s); and (b) instructions for use in: (i) screening for the presence of IBD; (ii) assessing the severity of IBD; (iii) monitoring the progression of IBD; and / or (iv) determining the efficacy of a therapeutic agent in a patient having IBD.

[0166] In some embodiments, the reagents comprise primer(s) and / or probe(s) for detecting the level of methylation of one or more MHC-I related gene(s). In some embodiments, the kit comprises one or more primer(s) specific to a methylated sequence. In some embodiments, the kit comprises one or more primer(s) specific to an unmethylated sequence. In some embodiments, the kit comprises a methylation-specific restriction enzyme. EXAMPLES

[0167] The invention will be further clarified by the following examples, which are intended to be purely exemplary of the invention and are in no way limiting.

[0168] Example 1

[0169] Crohn's Disease (CD) is one of the main forms of Inflammatory Bowel Diseases (IBD) causing chronic, relapsing inflammation of the intestinal mucosa. Altered function of the intestinal epithelium is considered to play a key role in CD pathogenesis, however exact mechanisms contributing towards lifelong relapsing mucosal inflammation remain ill defined. DNA methylation (DNAm) is a key epigenetic mechanism known to determine cellular identity by regulating gene transcription, with alterations increasingly being implicated in IBD pathogenesis.

[0170] The Inventors generated 312 mucosal stem cell derived intestinal epithelial organoid (IEO) lines obtained from mucosal biopsies of small (duodenum (DUO) and terminal ileal (Tl)) and large bowel (sigmoid colon (SC)) of 168 patients diagnosed with CD (n=72), UC (Ulcerative Colitis (UC, n=23) and healthy controls (n=73). The Inventors performed genome wide molecular profiling including DNAm, bulk and single cell RNA sequencing. Organoids were subjected to gene editing and the functional consequences of DNAm changes were evaluated using an organoid-lymphocyte co-culture.

[0171] The Inventors identified highly stable, CD associated loss of DNAm at MHC-I loci including nucleotide- binding domain, leucine-rich repeat CARD Domain Containing 5 (NLRC5) and cognate gene upregulation. Single cell RNA sequencing of primary mucosal tissue and lEOs confirmed the role of NLRC5 as transcriptional transactivator in the intestinal epithelium. MHC-I DNAm in lEOs showed a significant correlation with CD disease phenotype and outcomes. Application of machine learning approaches enabled the development of a disease prognostic epigenetic molecular signature.

[0172] In summary, the Inventors have identified epigenetically regulated intestinal epithelial MHC-I as a novel mechanism in CD pathogenesis.

[0173] Molecular profiling of patient derived lEOs reveals stable loss of MHC-I and NLRC5 DNAm in the intestinal epithelium of CD patients.

[0174] Generation of lEOs from patient samples offers unique opportunities to investigate IEC intrinsic mechanisms by eliminating disease associated confounding factors such as mucosal inflammation. lEOs from 168 patients diagnosed with CD (n=72), UC (n=23) and non-IBD, healthy controls (n=73) were generated. Following in vitro culture of organoids for a minimum of 2 passages (approximately 2-3 weeks), genome-wide DNAm profiling was performed, and frozen organoid stocks stored in a living organoid biobank (Figure 1A). Organoid growth, viability and structure were monitored over time with no major disease associated differences observed on a microscopic level (Figure lAii). Next, in order to identify disease associated DNAm changes that are retained in lEOs, the Inventors applied a weighted gene co-expression network analyses (WGCNA) to Tl organoid methylation. WGCNA enables unsupervised identification of highly correlated groups of CpGs (termed modules) as well as their correlation with a specific phenotype (i.e. CD, UC, Controls). As shown in Figure IB, analyses revealed several modules that significantly correlated with a diagnosis of CD with the most strongly correlated Module (Module 17, p<0.001) demonstrating a loss of DNAm in CD patient derived lEOs (Figure IBi). Gene ontology (GO) analyses preformed on genes containing CpGs forming Module 17 showed significant enrichment scores for pathways related to 'antigen processing and presentation via MHC- / '. Genes encoding key components of MHC-I signalling include PSMB8 / 9, HLA-B, -C and -F, TAP1 / 2 and B2M (Figure IBii and 1C). Similar results were obtained when applying WGCNA analyses to lEOs derived from the Sigmoid Colon (SC) with the most strongly CD correlated Module (i.e. Module 9) containing MHC-I enriched hypomethylated CpGs, half of which overlapping with Module 17. These results were further confirmed by performing differential DNAm analyses, identifying a total of 234 hypomethylated CpGs in both Tl and SC organoids derived from CD patients that were found to be enriched for MHC-I related pathways. Importantly, amongst the most significant loci showing loss of DNAm in CD patient derived lEOs, were two CpGs located within the promoter region of Nucleotide- binding Oligomerization Domain, Leucine-rich repeat and caspase recruitment domain (CARD) containing 5 (NLRC5, cg07839457 and cg07862320) a known transcriptional regulator / transactivator of MHC-1 (Figure ICi and ii).

[0175] Average DNAm loss of NLRC5 and MHC-I related genes in CD compared with control organoids were found to reach 20% in both Tl and SC, whilst no differences were observed in the DUO (Figure ID). To investigate intestinal epithelial (IE) MHC-I DNAm more broadly, a summary methylation score was computed by calculating average DNAm levels of all CpGs associated with genes known to be involved in the MHC-I pathway (Figure ID and methods). As shown (Figure ID and IE), global loss of MHC-I DNAm was observed in CD patient derived lEOs compared with non-IBD / healthy controls in both the Tl and SC, but not in the DUO. Loss of MHC-I DNAm was also observed in the SC of UC patients compared with controls. The fact that disease associated loss of MHC-I DNAm is still present in lEOs following multiple passages / cell divisions indicates a high degree of stability, as well as mitotic heritability, of these epigenetic signatures. Indeed, such CD associated DNAm changes remained stable even over prolonged periods (i.e. over several months) of in vitro culture and in the absence of inflammatory stimuli. Furthermore, stimulation of lEOs with IBD relevant inflammatory cytokines I FNy and TNFa, whilst inducing strong transcriptional changes, did not alter MHC-I DNAm (Figure IF), further highlighting the stability of these epigenetic marks and their independence of the inflammatory milieu.

[0176] Taken together, these findings suggested stable loss of DNAm in MHC-I pathway genes, including the promoter region of NLRC5 in the intestinal epithelium of CD patients.

[0177] CD associated loss of intestinal epithelial DNAm in MHC-I is associated with increased gene transcription in vivo and in vitro.

[0178] Next, genome-wide DNAm and transcriptional profiles of primary intestinal epithelium obtained from patients newly diagnosed with CD, UC and matched healthy controls were analysed (Cohort 2, Figure 2Ai). Results confirmed CD associated global loss of MHC-I DNAm and at individual CpGs including NLRC5 in both Tl and SC epithelium (Figure 2Bi & Ci). A significant loss of MHC-I DNAm was also observed in the SC epithelium of UC patients, although to a lesser extent than CD (Figure 2Ci). As with DNAm, to survey broad intestinal epithelial MHC-I transcription, a global MHC-I expression score was calculated as the average of the main genes known to be involved in the MHC-I pathway (Methods). A highly significant inverse correlation was found between MHC-I DNAm and gene expression in both Tl and SC epithelium (Figure 2Bii and 2Cii). Next, the stability of intestinal epithelial MHC-I DNAm in vivo was tested by analysing primary IEC DNAm obtained from IBD patients at the time of diagnosis and several months after the initiation of treatment. Consistent with the findings in patient derived lEOsorganoids, CD associated epigenetic changes including loss of NLRC5 promoter DNAm were also found to be highly stable marks remaining present in patients even after several months of treatment (Figure 2D). Consistent with these findings, the Inventors observed a highly significant correlation between genome-wide IEC DNAm at diagnosis and repeat assessment (Figure 2E). Lastly, to confirm the impact of disease associated loss of MHC-I DNAm on gene transcription in vitro, NLRC5 expression levels in CD patient derived lEOs were compared with UC and healthy controls. As shown in Figure 2F and 2G, CD patient derived lEOs harbouring lower NLRC5 promoter DNAm had significantly higher NLRC5 expression levels compared with UC and non-IBD controls leading to a significant inverse correlation. Importantly, lower NLRC5 promoter DNAm levels in CD patients was also found to be associated with a higher level of gene expression in response to I FNy compared with organoids derived from control patients (Figure 2G).

[0179] These findings validated In summary, the above results validate a highly stable loss of MHC-I and NLRC5 DNAm in primary epithelium of CD patients as well as showing cognate gene upregulation both in primary patient samples and derived lEOs in vitro and demonstrate an associated increase in gene expression in vivo and in vitro. Intestinal epithelial MHC-I DNAm signatures stratify CD patients and correlate with disease behaviour.

[0180] The findings described herein demonstrate a stable loss of MHC-I and NLRC5 DNAm in CD patients, leading to increased gene transcription in their small and large bowel intestinal epithelium. Combined with the high degree of stability in vitro and in vivo, the Inventors speculated that these highly stable epigenetic alterations already present at diagnosis may contribute towards persistence and / or chronic relapsing inflammation in CD and hence impact on long-term disease course, phenotype and severity. Consistent with this hypothesis, major variations in the degree of molecular changes amongst patients with CD were observed, raising the potential of distinct molecular subgroups. To test this hypothesis, the Inventors performed unsupervised hierarchical clustering based on MHC-I DNAm profiles of patient derived Tl and SC lEOs and primary purified IEC (Figure 3A and 4A). Differences in intestinal epithelial MHC-I DNAm profiles resulted in the separation of patients into three distinct subgroups. Specifically, clusters of MHC-I DNA hypomethylation (MHC-I DNAm low) were enriched for patients with CD in both small (Tl) and large bowel (SC) epithelium, whilst non-IBD / healthy controls represent the majority of samples within the MHC-I DNAm 'high' cluster (Figure 3A and 4A, right panels). However, a subset of CD patient samples displayed higher MHC-I DNAm profiles similar to non- IBD / healthy controls, highlighting molecular differences amongst the wide range of patients currently classified as having CD. A clear molecular stratification of CD patients was also achieved based on unsupervised clustering of primary purified IEC or whole biopsy MHC-I gene expression profiles across multiple patient cohorts (Figure 5). Importantly, as shown in Figure 3B and 4B, average MHC-I DNAm in Tl organoids was found to differ significantly between patients with different clinically relevant disease phenotypes and outcome parameters. These included the presence of perianal disease, the need to intensify medical treatment (e.g. use of a thiopurine immunomodulator or biologies) as well as an overall more severe disease (based on a compound summary disease outcome / severity score, see Methods). As these data highlight the potential for organoid derived MHC-I DNAm signatures as clinical biomarkers, the Inventors next aimed to develop a refined prognostic signature using combined WGCNA and machine learning approaches (see Materials and Methods). As shown in Figure 3C, results of analyses identified a refined signature containing 28 out of 628 MHC-I related CpGs (Tables 2 and 3) which demonstrates a clear stratification of CD patients into distinct molecular subgroups (Figure 3Ci) and improved separation of CD patients with severe CD from those with mild / moderate disease (Figure 3Cii). Development of a risk score based on these 28 CpGs (see methods) allowed prediction of a severe disease outcome in CD patients with Receiver Operator Analyses returning 72% Area Under the Curve (AUC 0.721, Figure 3Ciii). Additionally, as shown in Figure 3Civ, using a cut-off risk score of >0.65 increases the prediction of a severe disease outcome from 43.6% to 72.7%, whilst in patients with a risk score <0.33, the posterior probability of a severe disease course drops to 18.2% (Figure 3Civ). Lastly, the Inventors also tested the potential diagnostic value of organoid derived MHC-I DNAm. As shown in Figure 4C, using the same computational approaches 53 CpGs were identified that provided a high diagnostic accuracy for CD with an AUC of 0.82.

[0181] In summary, substantial variation in IEC MHC-I DNAm allows stratification of CD patients into distinct molecular subgroups that correlate with disease phenotype and long-term outcome / disease severity highlighting their potential value as clinical biomarkers enabling a proactive, personalised treatment of disease.

[0182] Discussion

[0183] Altered function of the intestinal epithelium has long been considered a key factor in IBD pathogenesis. However, whilst major attention has been placed on potential mechanisms leading to impaired barrier function, the potential role of the intestinal epithelium as an antigen presenting and processing cell type has largely been neglected. Here, the Inventors have identified highly stable DNAm changes as the novel underlying mechanism leading to increased MHC-I expression in the intestinal epithelium of IBD patients. Generating adult stem cell derived lEOs allowed the Inventors to eliminate the potentially confounding impact of mucosal inflammation on DNAm as well as demonstrate the stability of DNAm in vitro. Specifically, the fact that IBD DNAm changes were retained in patient derived lEOs and remained stable even over prolonged periods of in vitro culture indicates that epigenetic changes affect intestinal stem cells and are being inherited / passed on during mitosis in the absence of an inflammatory milieu. Furthermore, IBD associated DNAm changes observed in lEOs were validated in primary purified lECs and found to correlate with gene transcription (Figure 2). Indeed, highly significant increased MHC-I gene expression was also confirmed in mucosal biopsies from two large independent cohorts of adult and paediatric patients with CD.

[0184] Several studies have reported on the role of NLRC5 as a transcriptional regulator of MHC-I in other cell types, including dendritic cells, lymphocytes and pancreatic beta cells. Indeed, increased NLRC5 expression and associated autoantigen presentation via MHC-I in human pancreatic beta cells have recently been linked to disease pathogenesis of type 1 diabetes (T1D). Conversely, hypermethylation of the NLRC5 promoter associated with reduced MHC-I expression has been proposed as a key mechanism leading to immune evasion in several tumours including colon cancer. Interestingly, the level of NLRC5 promoter DNAm in tumour tissue was associated with disease outcome, indicating that variation in these cell type specific epigenetic changes can directly influence disease progression. In keeping with these findings, the Inventors observed major variation in the degree of DNAm changes in the intestinal epithelium of patients with CD, to an extent that allowed stratification of patients into distinct molecular subgroups (Figure 3). Importantly, the degree of both DNAm loss within the NLRC5 promoter and MHC-I genes correlated with clinical disease phenotypes and outcome.

[0185] The findings described herein provide further evidence to support the emerging concept of inflammatory memory and trained innate immunity. Specifically, exposure to environmental triggers, occurring at critical periods during development, can lead to enduring epigenetic changes that determine cellular function. Together with the continued exposure to an ever-wider range of new pathogens, pollutants and allergens, this mechanism has already been demonstrated in epithelial cells of the skin, lung and intestinal epithelium. The well-known increase in incidence of IBD in countries such as India, China and the Middle East has been linked to the adoption of a more 'urban' lifestyle, highlighting the impact of recent changes in social environment on pathogenesis. Compelling evidence for this concept was generated in a study by Christ and colleagues who found that exposure of mice to a 'western' diet caused lasting epigenetic changes in monocytes, leading to augmented immune responses to infection long after mice were returned to a 'normal' diet.

[0186] Epidemiological studies on populations migrating from rural to industrialised areas also support this hypothesis. As the Inventors observed epigenetic changes in the intestinal stem cell compartment of CD epithelium, these findings suggest an important role of immune-tissue stem cell interactions as a fundamental mechanism leading to the persistence of gut inflammation in IBD. Importantly, this study demonstrates that lEOs faithfully retain patient specific, CD associated DNAm changes in culture, further validating them as powerful translational research tools. Specifically, observed correlation between IEO derived MHC-I DNAm signatures and disease severity highlights the major potential for these molecular signatures as clinical biomarkers. Indeed, the analyses described herein using machine learning approaches led to the development of a DNAm based, molecular risk score which enables the prediction of a severe disease outcome in CD patients with up to 70% accuracy. Importantly, the generation of a large living organoid biobank, containing over 300 well characterised lines, now provides unique opportunities for the development of novel clinical biomarkers, as well treatments specifically targeting MHC-I.

[0187] The Inventors have identified epigenetically regulated intestinal epithelial MHC-I as a novel mechanism in IBD pathogenesis. These findings support a paradigm shift towards the role of the intestinal epithelium as a non-classical antigen presenting cell type directly driving chronic intestinal inflammation through increased interaction with mucosal lymphocytes.

[0188] Advantageously, the DNA methylation and gene expression signatures of the invention can be used to diagnose IBD and to assess the severity of IBD in patients. The ability to stratify IBD patients based on disease severity advantageously allows therapeutic interventions to be optimised for a particular patient, thereby increasing the likelihood that a patient will receive appropriate treatment and reducing overtreatment of patients with mild disease.

[0189] Materials and Methods

[0190] Ethical approval

[0191] Ethical approval was obtained from the local research committee (REC 12 / EE / 0482 and REC 17 / EE / 0265) and patients were prospectively recruited following informed patient and / or carer consent. All investigations were carried out according to the Declaration of Helsinki and Good Clinical Practice Guidelines.

[0192] Patient recruitment, sample collection and clinical data recording

[0193] Patients were prospectively recruited at Cambridge University Hospitals - NHS Foundation Trust, and biopsy samples obtained during routine clinical endoscopy at point of diagnosis. Diagnosis of Crohn's Disease (CD) and Ulcerative Colitis (UC) was made according to international guidelines (revised Porto criteria) (Levine A, Koletzko S, Turner D, et al. ESPGHAN Revised Porto Criteria for the Diagnosis of Inflammatory Bowel Disease in Children and Adolescents. Journal of Pediatric Gastroenterology and Nutrition 2014;58:795-806). Patients undergoing endoscopic examination as part of their routine clinical care with normal macroscopic and histological appearance of their intestinal mucosa and complete resolution of any gastrointestinal symptoms were classified as non-IBD, healthy controls. All patients were followed for a minimum of 18 months post-diagnosis in the Cambridge paediatric gastroenterology unit, and detailed clinical data was prospectively recorded (details provided in Supplementary Materials and Methods).

[0194] Mucosal biopsies were obtained from proximal and distal small bowel (i.e. Duodenum = DUO, Terminal Ileum = Tl) and distal large bowel (i.e. Sigmoid Colon = SC). Inflammation status of a mucosal sample (inflamed vs. non-inflamed) was based on histological assessment of paired samples taken within 2 cm.

[0195] Human intestinal epithelial organoid culture (lEOs) generation and biobank

[0196] Intestinal crypts were isolated from human mucosal biopsies and cultured in a growth medium, as previously described, to generate human intestinal epithelial organoids (lEOs) (Kraiczy J, Nayak KM, Howell KJ, et al. DNA methylation defines regional identity of human intestinal epithelial organoids and undergoes dynamic changes during development. Gut 2019;68:49-61; and Edgar RD, Perrone F, Foster AR, et al. Culture-Associated DNA Methylation Changes Impact on Cellular Function of Human Intestinal Organoids. Cell Mol Gastroenterol Hepatol 2022;14:1295-1310).

[0197] Following the expansion of lEOs over a minimum of two weeks in culture, frozen stocks were generated and stored in a living biobank and / or further expanded for experiments including cytokine stimulation (Supplementary Materials and Methods).

[0198] RNA / DNA extraction, Reverse transcription and quantitative PCR

[0199] DNA and / or RNA from human or mouse organoids were extracted using the AHPrep DNA / RNA mini kit (Qiagen, Hilden, Germany). RNA was reverse-transcribed and used to perform a quantitative PCR (qPCR) as previously reported (Kraiczy J, Nayak KM, Howell KJ, et al. DNA methylation defines regional identity of human intestinal epithelial organoids and undergoes dynamic changes during development. Gut 2019;68:49-61). Relative expression was calculated using the AACt method. Statistical analysis for qPCR data was performed using GraphPad Prism V.9.00 (GraphPad). Genomewide DNA methylation profiling was performed on bisulfite-converted DNA using the EZ DNA methylation Gold kit (Zymo Research, Irvine, CA).

[0200] DNA Methylation and Bulk RNA-seq Data and analysis

[0201] Genome-wide DNA methylation was profiled using either the Illumina Infinium Human Methylation 450 Beadchip, or the Illumina EPIC platform (Illumina, Cambridge, UK) (Pidsley R, Zotenko E, Peters TJ, et al. Critical evaluation of the Illumina MethylationEPIC Beadchip microarray for whole-genome DNA methylation profiling. Genome Biology 2016;17:208; and Price ME, Cotton AM, Lam LL, et al. Additional annotation enhances potential for biologically-relevant analysis of the Illumina Infinium HumanMethylation450 Beadchip array. Epigenetics Chromatin 2013;6:4). DNAm data was processed as described previously with minor modifications. Further details on WGCNA, differential DNAm, pathway enrichment analyses and machine learning approaches to generate a prognostic and diagnostic DNAm signatures as well as risk scores are provided in the Supplementary Materials and Methods. Bulk RNA-seq data was processed using standard computational approaches as described previously (Howell KJ, Kraiczy J, Nayak KM, et al. DNA Methylation and Transcription Patterns in Intestinal Epithelial Cells From Pediatric Patients With Inflammatory Bowel Diseases Differentiate Disease Subtypes and Associate With Outcome. Gastroenterology 2018;154:585-598). Further details are provided in the supplement. Average MHC-I Expression Score

[0202] An average MHC-I gene expression score comprising the genes NLRC5, TAP1 / 2, PSMB8 / 9, HLA-A / -B / - C / -E / -F / -G, IRF1 and B2M was calculated, subtracted by the aggregated expression of 100 control gene sets. All analysed genes were binned based on average expression, and control genes randomly selected from each bin.

[0203] Statistical analysis

[0204] Statistical analysis was performed using GraphPad Prism software or R. Sample sizes (n) and statistical tests used are indicated in figure legends. Plots with confidence intervals show mean ± standard error of mean (SEM) unless otherwise indicated. For in vivo experiments, sample sizes were determined based on previous experiments (Castro-Dopico T, Dennison TW, Ferdinand JR, et al. Anti-commensal IgG Drives Intestinal Inflammation and Type 17 Immunity in Ulcerative Colitis. Immunity 2019;50:1099-1114.el0). For T-tests, assumptions of normality and equal variance were confirmed using the Shapiro-Wilk test and by confirming higher-to-lower standard deviation ratios were below 2.

[0205] Supplementary Materials and Methods

[0206] Patient recruitment and clinical data recording

[0207] All patients recruited to this study were followed for a minimum of 18 months post-diagnosis in the Cambridge pediatric gastroenterology unit, and detailed clinical data was prospectively recorded. This included phenotypic parameters at diagnosis (e.g. presence of perianal disease) and information on disease course and outcomes. The latter covered number of treatment escalations, treatment history for surgical intervention, treatment with biologies, and use of immunomodulator (i.e. Azathioprine). To account for the fact that disease outcome in patients suffering from IBD is not restricted to a single measure, the Inventors also calculated a compound severity score. The score, which has been previously published (with minor modifications), considered number of treatment escalations, escalation to treatment with biologies, presence of peri-anal disease, and the requirement of IBD related surgery1.

[0208] Briefly, the score was calculated at 12 months from diagnosis and patients were categorised into mild (full response to single induction treatment, maintenance treatment with 5AZA for UC and azathioprine for CD, no further escalation), severe (requirement for escalation to second line treatment with biologies, requirement for IBD related surgery to control symptoms, more than 2 additional induction treatments post diagnosis), moderate (remaining cases). All patients were treated according to a standard protocol and treatment decisions were made by a multi-disciplinary team resulting in highly comparable treatment courses and outcome measures.

[0209] Sample Collection

[0210] At diagnostic endoscopy, mucosal biopsies were obtained from proximal and distal small bowel (i.e. Duodenum = DUO, Terminal Ileum = Tl) and distal large bowel (i.e. Sigmoid Colon = SC). Inflammation status of a mucosal sample (inflamed vs. non-inflamed) was based on histological assessment of paired samples taken within 2 cm. Histological assessment was performed by an experienced histopathologist. Biopsies were processed immediately for the generation of intestinal epithelial organoids or molecular profiling.

[0211] Human intestinal epithelial organoid culture (lEOs) generation and biobank lEOs were frozen after centrifugation at 800g for 5 minutes, resuspension of the pellet in 1ml of Recovery Cell Culture Freezing Medium (Gibco), placement in a cryo-freezing container ("Mr. Frosty," Thermo Fisher Scientific) for at least 24h, and then transfer to -150°C for long-term storage.

[0212] Each organoid line is accompanied by detailed clinical information, quality control data including bright field images (taken using an EVOS FL system (Life Technologies)) to illustrate organoid growth, cell numbers as well as culture conditions used. cDATA ANALYSES

[0213] DNA Methylation Data and analysis

[0214] Genome-wide DNA methylation was profiled using either the Illumina Infinium Human Methylation 450 Beadchip, or the Illumina EPIC platform (Illumina, Cambridge, UK)3-4. DNAm data was processed using the minfi package and normalised based on control probes on each array using functional normalisation5. Samples were removed if they had high average detection p-values (>0.05) across all probes. Batch effect was removed by using ComBat6and probes were filtered for quality leaving 799,922 and 799,049 CpGs in the IEO and IEC EPIC array samples respectively, and 414,293 CpGs in the IEC 450K array samples3-4. As the IEC samples were assayed on both Illumina EPIC and 450K arrays, only the 384,394 CpGs measured on both platforms were used for differential DNAm analysis in this cohort (Cohort 2). Of a total of 142 at diagnosis IEC samples, 82 were present on the 450K array. All IEO samples were assayed on the Illumina EPIC array (Cohort 1). Accession Numbers: E-MTAB-12841, E-MTAB-5463. Previously published genome-wide DNAm profiles of primary purified IEC samples2were subjected to in-silico correction for potential cellular contamination. Briefly, cell composition was estimated using existing / public datasets from the Gene Expression Omnibus (GEO)7and ArrayExpress8then IEC DNAm values were adjusted for intraepithelial lymphocyte proportion9.

[0215] Differential DNA Co-Methylation

[0216] Epigenome-wide weighted gene co-expression network analysis (WGCNA)10was performed on both Tl and SC IEO samples (Cohort 1). The top 10% variant probes, identified across all samples (Tl: n=127, SC: n=131), were retained after filtering for low variance beta values, resulting in 79,757 CpGs. Comethylation network analysis was carried out using the WGCNA R package (v.1.72.). An appropriate soft-thresholding power (P = 8) was selected to fit a scale-free network. Signed topological overlap matrix (TOM) was calculated to measure the pairwise Pearson's correlation between all CpGs. The resulting hierarchical clustering dendrogram was generated, followed by module detection with a minimum module size 100 and a minimum cut height of 0.995, using the Dynamic Tree Cut algorithm. Subsequently, the first principal components of each module and their correlation with disease traits were calculated to identify the set of co-methylated CpGs strongly associated with CD.

[0217] Pathway Enrichment

[0218] To explore pathways related to differential methylation with IBD, the enrichment of gene ontology (GO)11groups in the genes adjacent to hypomethylated CpGs was performed using fgsea (vl.24.0)12and GO biological processes as pathways. Only GO groups between 15 and 400 genes were used.

[0219] DNAm and Expression Correlation

[0220] Correlation between DNAm and expression was explored by calculating Spearman's correlations and compared to random sets on comparable CpG gene pairs. Ensembl gene IDs with the highest mean count in the IEC data were used, which left 29,411 unique genes. Correlations between DNAm at CpGs adjacent to a gene and that gene's expression (26,963 correlations; 2-1019 CpGs / gene, mean 28 CpG per gene; 1-21 gene / CpG, mean 1.1) were calculated for all CpG-gene pairs.

[0221] Expression Array Data

[0222] Publicly available Tl (n=78) and colon (n=116) biopsy expression array data from adult patients profiled on the Affymetrix Human Gene 1.0 ST expression array were downloaded from the GEO (Cohort 7, n=75 CD, 97 UC and 22 non-IBD controls)13. Accession Number: GSE75214. Raw signal intensity data were normalised using the Robust Multi-array Average (RMA) method using affy vl.70.0 and quality control was performed using a rrayQ.ua lity Metrics v3.48.014 16. The normalised dataset was annotated using the pd.hugene.l.O.st.vl v3.14.1 annotation package17.

[0223] Bulk RNA-seq Data

[0224] Paediatric purified epithelium from terminal ileum (Tl) and sigmoid colon (SC), Cohort 2 (n = 20 CD, 22 UC and 22 non-IBD controls), were transcriptionally profiled using RNA-seq by University of Kiel, Germany using an established pipeline as described previously. Accession Number: E-MTAB-5464.

[0225] Unmodified human, PB NLRC5-mCherry and Nlrc5+ / +and Nlrc5^ mouse organoid lines treated with inflammatory cytokines as described above were transcriptionally profiled at Cambridge Genomic Services (Dept. Pathology, Tennis Court Road, University of Cambridge, Cambridge). Accession Numbers: E-MTAB-11548; NLRC5-mCherry and mouse data available on request.

[0226] In addition, publicly available Tl biopsy RNA-seq data from 322 paediatric patients were downloaded from the GEO using SRA Explorer (Cohort 6, n = 218 CD, 62 UC and 42 non-IBD controls)18. Accession Number: GSE57945.

[0227] Raw FastQ. files for all bulk RNA-seq samples were first pre-processed using BBMap v38.2619to remove adapters and low-quality sequence. FastQCvO.il.9 was used to ascertain data quality before and after trimming and filtering20. Reads were next aligned to the human or mouse genome (Ensembl GRCh38 and GRCm38, release 99) using STAR v2.5.0 and indexed using SamTools vl.ll. Finally, raw counts were extracted using featurecounts, (subread package, v2.0.3) and assessed for quality using the NOISeq v2.36.0 and DESeq2 vl.32.0, R packages21-25.

[0228] Differential DNA Methylation

[0229] Epigenome-wide association tests were performed in both the IEO and IEC DNAm cohorts (Cohorts 1 and 2). In the IEC samples, covariates for histological based inflammation measure (binary: inflamed or not), sex and age were incorporated with linear models using Umma. The difference in group mean betas (delta beta, db) was also calculated as a measure of effect size. As the lEOs are a pure population of epithelial cells with no immune cell component, inflammation status was irrelevant and was not included as a covariate in the model. Age and sex were included as covariates (Tables 1-3).

[0230] Statistical Univariate Analysis of DNA Methylation and Biomarker Selection

[0231] For CD severity prognostic methylation biomarkers, Tl CD (n=55) lEOs (Cohort 1) samples were classified into two groups, severe CD and mild / moderate CD. For each individual CpG, two groups of samples were used to generate two empirical distributions, and their difference was assessed by 2- sample Anderson-Darling test26. The CpGs with smaller p-values have more distinct distributions between the two groups, so they were selected as informative features for the prediction of whether a sample is from one group or the other. Using the method described above, 78 CpGs candidates for diagnosis and 72 CpGs candidates for prognosis were selected. Taking the intersection with significant CpGs from WGCNA DNA Co-Methylation network analysis, 53 CpGs were selected as CD diagnostic biomarker (Table 2, p-value for intersection < le-5), while 28 CpGs were selected as CD severity prognostic biomarker (Table 3, p-value for intersection < le-5).

[0232] Machine Learning Predictors for CD Prognosis and Diagnosis

[0233] To develop CD severity prognostic prediction model, preliminary model selection using default hyperparameters were carried out to test different machine learning classification algorithms. With 628 MHC-I CpGs (Table 1) or 28 prognostic CpGs (Table 3) of Tl (n=127) lEOs (Cohort 1), logistic regression (AUC 0.71 / 0.72) performs consistently better than support vector machine (AUC 0.71 / 0.62)27, random forest (AUC 0.67 / 0.66)28, XGBoost (AUC 0.64 / 0.62)29and perceptron (AUC O.71 / O.62)30. Therefore, logistic regression classifier was selected for the subsequent analysis. Healthy and disease control (UC) samples were labelled as non-severe CD and used in training data to provide more context for classifiers, while only CD samples were used in testing. Repeated cross-validation was chosen as the resampling method to estimate model performance. Cross-validations were stratified such that each split has the same number of control, mild CD, moderate CD and severe CD samples. Cross-validation split number was chosen to be 5. Other split numbers were tested and produced similar results. Evaluation metrics for predictors were chosen to be ROC curve and corresponding AUC scores, and their estimated population means obtained from repeated cross- validation were used for comparison. To illustrate the prognostic value of 628 MHC-I CpGs (Table 1), permutation analysis was done by randomly selecting 628 CpGs from all measured CpGs and running the aforementioned prediction procedure. By AUC scores, predictors with 628 MHC-I CpGs as input perform better than 95.8% (95% Cl size < 2%) of randomly selected CpG sets. The median performer among randomly selected CpG sets was included in the ROC plot. Machine learning classification algorithms, cross-validation and evaluation metrics were from scikit-learn library31. The same procedure was also implemented to develop CD diagnostic prediction model.

[0234] Risk Score System and Patient Stratification

[0235] The prognostic risk score system was developed by further processing the output of machine learning prediction model. Machine learning binary classifier operates by generating probability predictions for each sample and comparing it to a fixed classification threshold between 0 and 1 to make a decision. Each logistic regression classifier from repeated cross validation generated a probability prediction of the binary outcome (severe / non-severe) for each sample in its testing set. Since each sample appears in the testing set of multiple classifiers, the cross-sectional mean of probability predictions for each sample was taken to generate a stable estimate for mean risk of developing severe CD. To ensure the consistency of cross-sectional operation between different classifiers, a coherent methodology to choose classification threshold and adjust probability predictions accordingly for each classifier was needed. After a comparison of methods to select the optimal threshold, the method of always choosing the top left corner of ROC (minimising the top left index, (1 - TPR)A2+FPRA2) was selected32. To generate reliable risk scores, cross-validation was repeated until risk score of every sample converged (size of 95% Cl of mean < 0.01). The same procedure was also implemented to develop CD diagnostic risk score system. To demonstrate the risk score system's potential for prognostic stratification, two cut-offs (risk>0.65 and risk<0.33) were chosen to detect high and low risk patients respectively and were represented on Fagan's Nomogram. 60% (95% Cl: 46%-73%) of CD patients are expected to be stratified by one of the two cut-offs.

[0236] References - supplementary materials and methods

[0237] 1. Howell KJ, Kraiczy J, Nayak KM, et al. DNA Methylation and Transcription Patterns in Intestinal Epithelial Cells From Pediatric Patients With Inflammatory Bowel Diseases Differentiate Disease Subtypes and Associate With Outcome. Gastroenterology 2018;154:585-598.

[0238] 2. Edgar RD, Perrone F, Foster AR, et al. Culture-Associated DNA Methylation Changes Impact on Cellular Function of Human Intestinal Organoids. Cell Mol Gastroenterol Hepatol 2022;14:1295-1310.

[0239] 3. Pidsley R, Zotenko E, Peters TJ, et al. Critical evaluation of the Illumina MethylationEPIC Beadchip microarray for whole-genome DNA methylation profiling. Genome Biology 2016;17:208.

[0240] 4. Price ME, Cotton AM, Lam LL, et al. Additional annotation enhances potential for biologically- relevant analysis of the Illumina Infinium HumanMethylation450 Beadchip array. Epigenetics Chromatin 2013;6:4.

[0241] 5. Aryee MJ, Jaffe AE, Corrada-Bravo H, et al. Minfi: a flexible and comprehensive Bioconductor package for the analysis of Infinium DNA methylation microarrays. Bioinformatics 2014;30:1363-9.

[0242] 6. Leek JT, Johnson WE, Parker HS, et al. The sva package for removing batch effects and other unwanted variation in high-throughput experiments. Bioinformatics 2012;28:882-3.

[0243] 7. Edgar R, Domrachev M, Lash AE. Gene Expression Omnibus: NCBI gene expression and hybridization array data repository. Nucleic Acids Res 2002;30:207-10.

[0244] 8. Parkinson H, Kapushesky M, Shojatalab M, et al. ArrayExpress— a public database of microarray experiments and gene expression profiles. Nucleic Acids Research 2006;35:D747- D750.

[0245] 9. Houseman EA, Accomando WP, Koestler DC, et al. DNA methylation arrays as surrogate measures of cell mixture distribution. BMC Bioinformatics 2012;13:86.

[0246] 10. Langfelder P, Horvath S. WGCNA: an R package for weighted correlation network analysis. BMC Bioinformatics 2008;9:559.

[0247] 11. The Gene Ontology resource: enriching a GOId mine. Nucleic Acids Res 2021;49:D325-d334.

[0248] 12. Korotkevich G, Sukhov V, Budin N, et al. Fast gene set enrichment analysis. bioRxiv 2021:060012. 13. Vancamelbeke M, Vanuytsel T, Farre R, et al. Genetic and Transcriptomic Bases of Intestinal Epithelial Barrier Dysfunction in Inflammatory Bowel Disease. Inflammatory Bowel Diseases 2017;23:1718-1729.

[0249] 14. Gautier L, Cope L, Bolstad BM, et al. affy-analysis of Affymetrix GeneChip data at the probe level. Bioinformatics 2004;20:307-15.

[0250] 15. Irizarry RA, Hobbs B, Collin F, et al. Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics 2003;4:249-264.

[0251] 16. Kauffmann A, Gentleman R, Huber W. arrayQualityMetrics— a bioconductor package for quality assessment of microarray data. Bioinformatics 2008;25:415-416.

[0252] 17. Carvalho B. pd. hugene. 1.0. st. vl: Platform Design Info for Affymetrix HuGene-l_0-st-vl. R package 2015.

[0253] 18. Haberman Y, Tickle TL, Dexheimer PJ, et al. Pediatric Crohn disease patients exhibit specific ileal transcriptome and microbiome signature. J Clin Invest 2014;124:3617-33.

[0254] 19. B. B-B.

[0255] 20. de Sena Brandine G, Smith AD. Falco: high-speed FastQC emulation for quality control of sequencing data. FlOOORes 2019;8:1874.

[0256] 21. Dobin A, Davis CA, Schlesinger F, et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 2013;29:15-21.

[0257] 22. Liao Y, Smyth GK, Shi W. featurecounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 2014;30:923-30.

[0258] 23. Tarazona S, Furid-Tari P, Turra D, et al. Data quality aware analysis of differential expression in RNA-seq with NOISeq R / Bioc package. Nucleic Acids Research 2015;43:el40-el40.

[0259] 24. Love Ml, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 2014;15:550.

[0260] 25. Li H, Handsaker B, Wysoker A, et al. The Sequence Alignment / Map format and SAMtools. Bioinformatics 2009;25:2078-9.

[0261] 26. Scholz FW, Stephens MA. K-Sample Anderson-Darling Tests. Journal of the American Statistical Association 1987;82:918-924.

[0262] 27. Cortes C, Vapnik V. Support-vector networks. Machine Learning 1995;20:273-297.

[0263] 28. Breiman L. Random Forests. Machine Learning 2001;45:5-32.

[0264] 29. Chen T, Guestrin C. XGBoost: A Scalable Tree Boosting System. Proceedings of the 22nd ACM SIGKDD International Conference on Knowledge Discovery and Data Mining. San Francisco, California, USA: Association for Computing Machinery, 2016:785-794.

[0265] 30. Rosenblatt F. The perceptron: A probabilistic model for information storage and organization in the brain. Psychological Review 1958;65:386-408.

[0266] 31. Pedregosa F, Varoquaux G, Gramfort A, et al. Scikit-learn: Machine Learning in Python. J. Mach. Learn. Res. 2011;12:2825-2830.

[0267] 32. De Borre M, Che H, Yu Q, et al. Cell-free DNA methylome analysis for early preeclampsia prediction. Nat Med 2023;29:2206-2215.

[0268] TABLES

[0269] Table 1. List of 628 MHC-I differential methylated CpGs with gene names.

[0270] Table 2. List of 28 prognostic CpGs with gene names.

[0271] Table 3. List of 53 diagnostic CpGs with gene names.

Claims

CLAIMS1. A method of assessing the severity of IBD in a patient, the method comprising:(a) comparing the level of DNA methylation of one or more MHC-I related gene(s) and / or the level of expression of one or more MHC-I related gene(s) in a biological sample obtained from the patient to the level of DNA methylation of said one or more M HC-I related gene(s) and / or the level of expression of said one or more MHC-I related gene(s) in a control, wherein the control is associated with a known severity of IBD; and(b) determining the severity of IBD based on the comparison performed in step (a).

2. A method for monitoring the progression of IBD in a patient, the method comprising:(a) comparing: (i) the level of DNA methylation of one or more MHC-I related gene(s) and / or the level of expression of one or more MHC-I related gene(s) in a biological sample obtained from the patient at a first time point with (ii) the level of DNA methylation of said one or more MHC-I related gene(s) and / or the level of expression of said one or more MHC-I related gene(s) in a biological sample obtained from the patient at a second subsequent time point; and(b) determining whether the IBD has progressed between the first and second time point based on the comparison performed in step (a).

3. A method for determining the efficacy of a therapeutic agent in a patient having IBD, the method comprising:(a) comparing: (i) the level of DNA methylation of one or more MHC-I related gene(s) and / or the level of expression of one or more MHC-I related gene(s) in a biological sample obtained from the patient prior to administration of the therapeutic agent with (ii) the level of DNA methylation of said one or more MHC-I related gene(s) and / or the level of expression of said one or more MHC-I related gene(s) in a biological sample obtained from the patient after administration of the therapeutic agent; and(b) determining the efficacy of the therapeutic agent based on the comparison performed in step (a).

4. A method of differentially diagnosing Crohn's Disease or Ulcerative Colitis in a patient having or suspected of having IBD, the method comprising:(a) comparing the level of DNA methylation of one or more MHC-I related gene(s) and / or the level of expression of one or more MHC-I related gene(s) in a biological sample obtained from the patient to the level of DNA methylation of said one or more MHC-I related gene(s) and / or the level of expression of said one or more MHC-I related gene(s) in a control; and(b) determining if the patient has Crohn's Disease or Ulcerative Colitis based on the comparison performed in step (a).

5. The method of any preceding claim, wherein the biological sample comprises intestinal epithelial cells or intestinal epithelial organoids (lEOs).

6. The method of any preceding claim, wherein the IBD is selected from Crohn's Disease and Ulcerative Colitis.

7. The method of any preceding claim, wherein the one or more MHC-I related gene(s) are selected from NLRC5, B2M, TAPI, TAP2, HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, HLA-DPA1, PSMB8, PSMB9, IFIT1, and IRF1.

8. The method of claim 7, wherein the one or more MHC-I related gene(s) comprise NLRC5.

9. The method of any preceding claim, wherein the method further comprises determining the level of DNA methylation of one or more MHC-I related gene(s) and / or the level of expression of one or more MHC-I related gene(s) in the biological sample obtained from the patient.

10. The method of any preceding claim, wherein the method comprises determining the level of DNA methylation at one or more CpG site(s) of one or more MHC-I related gene(s); optionally wherein the one or more CpG site(s) are selected from cg07839457, cg07862320, cg27587780, cgl5375424, cgl7394978, cgll666365, cgll086820, cg20287640, cgl3907973, cg25410123, cg05941961, cgl8890864, cg21428887, cg05915281, cg06942904, cg08977266, cg00483888, cgll971752, cgl5421363, cg00578828, cg26382286, cg21138405, cg00255919, cgl5018934, cg08698936, cgll201654, cg22298860, cgll768167, cg04186657, cgl4738290, cg03278703, cgll576659, cg02688650, cgl4402472, cg24703717, cg05237001, cg03851143, cgllll6776, cg23912231, cg09805507, cgl6728223, cg05603292, cgl7625506, cg20891813, cgl3924755, cgl9348622,cgl8676033, cgl0262357, cg26069562, cgll423684, cg07016276, cg20927242, cgl2588917, cg23892836, cg24351901, cgl5331332, cg00504902, cgll617938, cg01405582, cgll587584, cg05358170, cgl4140375, cgl0601943, cg22310628, cgl3040666, cg02945359, cg06036836, cg04892672, cg25197880, cg01393604, cg24952408, cg26987604, cg25252977, cgl6650906, cg23148731, cgl2550837, cgl6891968, cgl7264941, cg03176423, cgl8371410, cg21632181, cgl4317321, cg24177217, cgl5180617, cg08179037, cg08755130, cgl6289618, cgl5628633, cgl4574237, cg23611220, cg21727129, cg08708750, cg09430946, cg03432955, cg09865095, cg00126638, cg03521696, cg21529533, cgl2287358, cg03997176, cgl9141489, cg22257941, cgll363230, cgl5174396, cgl4546076, cgl3130352, cg26511972, cg23110482, cg03298800, cgl0807461, cg01818016, cgll682706, cg21855392, cgl5595495, cg05523662, cgl5319255, cgl7678719, cg23489273, cgll808100, cg05839762, cgl4772439, cgl4018363, cg08039587, cgl6742075, cg20408505, cg25637655, cgl7608381, cgll946459, cgl6367518, cgl6507955, cg21297314, cg08416870, cgl0794086, cg09275023, cgll861940, cg04807188, cgl0124767, cg02942965, cg20644330, cgl3223754, cg04305858, cgl7475918, cg26175526, cg01462744, cg22952566, cg20534075, cg20662397, cg25293221, cg08899786, cgl4849431, cgl5118824, cg02751503, cg01365762, cg21292025, cgl0457005, cg08180063, cg25664704, cg05384135, cgl5816267, cg09609649, cgl8183435, cg09620840, cg21176130, cg09326440, cgl2700271, cgl3007871, cg00340855, cgll594821, cg21758773, cg05201185, cg02188225, cg20652371, cg21366673, cg23235965, cg27486585, cgl7615629, cg20105257, cg02678305, cgl9109457, cg09569347, cgl3036546, cgl6535080, cgll019014, cg24710520, cgll247343, cgl3872627, cg01064373, cgl8511546, cg26392102, cgl7096289, cgl2965927, cgl8020334, cgl8747378, cg24877963, cgl7974398, cg05619024, cgl3155295, cg01006124, cg25355501, cg06659144, cg02827714, cgl8785300, cg27358585, cg08309069, cg05030953, cg23533285, cg06422189, cg23923934, cg27529346, cg05139028, cglll87245, cg01517384, cgl3902357, cg05932159, cg09588770, cgl2730088, cgl7731992, cg06646969, cg01035015, cg26706521, cgl0542672, cg04981410, cg22016094, cgll239749, cg09889987, cg21454965, cg02010152, cg03477130, cgl0294956, cgll469594, cg08905753, cg00409309, cg27246453, cg04576021, cg01530082, cgl7369196, cg01493678, cgl7103109, cg01200893, cgl9811863, cg00411901, cgl9121661, cgl2333338, cg06707784, cg21021279, cg25798341, cg22619784, cg05592483, cg02633767, cg25823314, cgl8243910, cg08985301, cgl3991568, cg27533873, cg26187287, cg00167916, cg08327277, cgl8050892, cg06121167, cg09408973, cgl4571125, cg00340328, cg26070383, cg07023208, cgl9887824, cg25744682, cgl0924050, cg27176262, cg09871008, cgl8511153, cgl3347198, cg02151752, cg01448551, cg02670545, cg09351471, cgll709793, cg07353306, cg01283574, cgll752893, cgl2121080, cgl3600652, cg00720839, cgl6026549, cgl2761728,cg07015256, cg24813704, cg21359558, cg09187413, cg24469596, cgl8045172, cg08513422, cgl4997446, cg20397692, cg25139471, cgl5785389, cg08358188, cgl2671948, cg08354908, cg00386460, cgl0451089, cg20224850, cgl8266257, cgl0894807, cgl0627913, cgl7390106, cg27618777, cg04255770, cgl5672065, cgl2854186, cg22940798, cg08998192, cgl3563634, cg23560159, cgl0088320, cg22230640, cgll668794, cg20724405, cg05729731, cg26260794, cg03026929, cg20898522, cgl9221044, cgll439192, cg02779707, cg07546106, cgl7253307, cg21780812, cgl2180249, cg00857968, cgl8607757, cgl9655544, cg03544736, cgl7670640, cg25728415, cg24364491, cgl3477582, cgl4867383, cg08557029, cg20558646, cgl6855458, cg05420994, cg23059310, cgl6951654, cg22798247, cgl4493899, cgl3871037, cg23750151, cg25693349, cgl2048225, cgl2094903, cgl8505752, cg08331398, cg21621645, cgll954557, cgl9646975, cgl2718404, cg22510079, cg08992729, cg26988373, cg22158175, cgl6009764, cg20060630, cg00971309, cg03078854, cg24031377, cg21593554, cgll453837, cg24898914, cg00533183, cg08675092, cg08099136, cg01309328, cgl4022881, cgll990058, cgl8696632, cg00777968, cg27232057, cg24893844, cg24625115, cg05115162, cg24767041, cg22007922, cg09006592, cg05545172, cg05872513, cg24326130, cg26829379, cg20848216, cg00889031, cg05242261, cg21322855, cg05135714, cg21568368, cgl0128166, cg07456831, cgl3853192, cg05629721, cgl8963307, cg01295600, cgl7392730, cg08772265, cg03995852, cgl5451100, cgl3603062, cg21535207, cg01702338, cgl0087598, cg20630683, cg06531943, cg03166550, cg06870868, cgl0192196, cglll41874, cg05697726, cgl6483840, cg02657012, cgl6890093, cgll706729, cg07218288, cgl9136673, cg05693489, cgl2253437, cgl3165140, cg09354037, cg25384897, cg02756056, cg09154880, cg02561720, cgll313335, cg05412855, cg05480623, cgl2499157, cg07072394, cg20880499, cg02351231, cgll969117, cg08351785, cg03807983, cg00240875, cg01255458, cg20357228, cg23132351, cg01979171, cg08151204, cgl7973694, cgl4293027, cg05262533, cg26033526, cg01673307, cg24111025, cg25042789, cg02181920, cg06473288, cg26234900, cgl0666909, cg08818207, cg04773990, cg24154161, cg23880953, cg26700949, cgl5926590, cgl8436324, cg05412930, cg06634056, cgl3403689, cg25110530, cg05826626, cg08880054, cg25524784, cgl4286550, cgl7378691, cg09968749, cg01170201, cg08466770, cgl4925994, cgl6501436, cgl9776032, cg01897517, cg01547742, cg09374375, cg27652200, cgll012835, cg23922920, cg27572016, cg05246277, cg25110511, cgl7199468, cgl8903583, cg20194454, cg23806084, cg06852255, cg03299066, cg00704844, cgl6494397, cg07504780, cg02439889, cgl4272068, cgl2641838, cgl2862002, cgl3274644, cg01692674, cg24718356, cg20729846, cg02551145, cg08766503, cg20813491, cg07250080, cg03735531, cgl2762680, cg02567488, cg07156249, cg03465320, cgl6853860, cg06791592, cg21826978, cgl0817441, cg04908668, cg21764921, cgl6229954, cgl9412007, cgl9760441, cg26265820,cg26811065, cg08209711, cg02878284, cgl9994157, cg00045690, cg04913118, cg06043617, cg09322555, cgl3869180, cg24461669, cgl8696027, cgl8555073, cg21445676, cgl0081723, cg07192821, cg08459087, cg00079638, cg08350173, cg24134304, cgl3964393, cg03355298, cg00837838, cgl9404757, cgl2468675, cgl9721944, cg08188779, cg27467953, cg03425812, cgl2828896, cg27537252, cg05475649, cgl6039157, cg02988397, cg21979287, cg22138564, cgl6411857, cg06862692, cg06754565, cg03056682, cg06181531, cg00881185, cg26963976, cg02493440, cg02169333, cg08558449, cg04097610, cg05723552, cgl2256630, cg24341550, cg03089589, cg07223648, cg21333585, cg02644232, cgl6677488, cg05757530, cg04601693, cgl0561843, cg20740356, eg 18440314, cg26661738, cgl6007266, cgl6362955, cgl7047468, cg24661931, cgl4196251, cgll345288, cg04799664, cgl7625032, cg09561419, cg02705619, cgll987321, cg02610906, cg06265737, cg09174467, cg05154222, cgl0854209, cg03754428, cg27462748, cgl7340138, cgl8398267, cg09624807, cg23314414, cg00125947, cg01834111, cg06600851, cgl0553082, cg23820818, cg07990412, cgl9840565, cgl6213529, cg07054754, cgl5875239, cg04344414, cg01517277, cgl8258710, cg00589550, cgl2439595, cgl2928479, cglll01747, cg23848181, cgl8060687, cg24399801, cg09714724, cg23079860, cg04972244, and cgl4946319.

11. The method of claim 10, wherein the one or more CpG site(s) are selected from cg07839457, cg07862320, cglll87245, cg00340855, cg05475649, cg24341550, cgl2256630, cgl0601943, cgl2761728, cgl0817441, cgl5375424, cg23923934, cgl4140375, cg01405582, cg02181920, cgll617938, cgl8511546, cgl8696027, cg26033526, cg05697726, cg25384897, cg22310628, cg05358170, cgl7615629, cgll594821, cg07218288, cg24111025, and cgl3007871.

12. The method of claim 10, wherein the one or more CpG site(s) are selected from cg07839457, cg07862320, cg27529346, cglll87245, cgl6890093, cg05201185, cgll706729, cg09354037, cg00340855, cg06473288, cg05475649, cg24341550, cg02756056, cgl2256630, cgl0601943, cgl0817441, cgl5375424, cg05693489, cg23923934, cg04576021, cg27176262, cg01673307, cgl4140375, cg27537252, cg26234900, cg01405582, cg02181920, cgl9136673, cg02988397, cg25042789, cgl8511546, cg06791592, cg26033526, cg05697726, cg25384897, cg05412855, cg27486585, cgl8243910, cg06422189, cg24898914, cgll453837, cgl2253437, cg23235965, cg09187413, cg00533183, cgl7615629, cg22310628, cg05358170, cgll594821, cg07218288, cg24111025, cgll587584, and cgl3007871.

13. The method of any preceding claim, wherein the method further comprises obtaining the biological sample from the patient.

14. The method of any preceding claim, wherein the biological sample is obtained from an intestinal biopsy and / or a faecal sample.

15. The method of any preceding claim, wherein the level of DNA methylation is determined by sodium bisulphite conversion assay, next generation sequencing (NGS), differential enzymatic cleavage of DNA, a methylated DNA immunoprecipitation assay, methylation-specific PCR, methylation-sensitive melting assay, and / or a bisulphite-free detection method.

16. The method of any preceding claim, wherein the level of expression is determined by RNAseq, microarray, and / or quantitative PCR (qPCR).

17. The method of any one of claims 4-16, wherein:(a) if the patient is identified as having Crohn's Disease, the method further comprises treating said patient for Crohn's Disease; or(b) if the patient is identified as having Ulcerative Colitis, the method further comprises treating said patient for Ulcerative Colitis.

18. The method of any one of claims 1 or 5-16, wherein if the patient is identified as having moderate or severe IBD, the method further comprises treating said patient for IBD.

19. The method of any one of claims 1 or 5-16, wherein if the patient is identified as having severe IBD, the method further comprises treating said patient for IBD.

20. The method of any one of claims 17-19, wherein said treating comprises administering to the patient one or more of an aminosalicylate (5-ASA, e.g. mesalazine, sulfasalazine, olsalazine and balsalazide), a corticosteroid (e.g. prednisolone or budesonide), an immunosuppressant (e.g. azathioprine, mercaptopurine or methotrexate), cyclosporine, a biologic (e.g. adalimumab, infliximab, vedolizumab or ustekinumab), a JAK inhibitor (e.g. tofacitinib or filgotinib), and ozanimod.

21. The method of any one of claims 17-20, wherein said treating comprises surgery, optionally resection or colectomy.

22. The method of any one of claims 3 or 5-16, wherein the therapeutic agent comprises one or more of a 5-ASA (e.g. mesalazine, sulfasalazine, olsalazine and balsalazide), a corticosteroid (e.g. prednisolone or budesonide), an immunosuppressant (e.g. azathioprine, mercaptopurine or methotrexate), cyclosporine, a biologic (e.g. adalimumab, infliximab, vedolizumab or ustekinumab), a JAK inhibitor (e.g. tofacitinib or filgotinib), and ozanimod.

23. A method of treating a patient identified as having Crohn's Disease or Ulcerative Colitis by a method of any one of claims 4-16, wherein the treating comprises: (i) administering to the patient one or more of a 5-ASA (e.g. mesalazine, sulfasalazine, olsalazine and balsalazide), a corticosteroid (e.g. prednisolone or budesonide), an immunosuppressant (e.g. azathioprine, mercaptopurine or methotrexate), cyclosporine, a biologic (e.g. adalimumab, infliximab, vedolizumab or ustekinumab), a JAK inhibitor (e.g. tofacitinib or filgotinib), and ozanimod; and / or (ii) surgery, optionally resection or colectomy.

24. A method of treating a patient identified as having moderate or severe IBD by a method of any one of claims 1 or 5-16, wherein the treating comprises: (i) administering to the patient one or more of a 5-ASA (e.g. mesalazine, sulfasalazine, olsalazine or balsalazide), a corticosteroid (e.g. prednisolone or budesonide), an immunosuppressant (e.g. azathioprine, mercaptopurine or methotrexate), cyclosporine, a biologic (e.g. adalimumab, infliximab, vedolizumab or ustekinumab), a JAK inhibitor (e.g. tofacitinib or filgotinib), and ozanimod; and / or (ii) surgery, optionally resection or colectomy.

25. A method of treating a patient identified as having severe IBD by a method of any one of claims 1 or 5-16, wherein the treating comprises: (i) administering to the patient one or more of cyclosporine, a biologic, a JAK inhibitor, and / or ozanimod; and / or (ii) surgery, optionally resection or colectomy.

26. A method of treating IBD in a patient, wherein the method comprises administering to the patient a therapeutic agent which reduces or inhibits the activity of one or more proteins encoded by one or more MHC-I related gene(s).

27. Use of the level of DNA methylation of one or more MHC-I related gene(s) and / or the level of expression of one or more MHC-I related gene(s) for assessing the severity of IBD.

28. Use of the level of DNA methylation of one or more MHC-I related gene(s) and / or the level of expression of one or more MHC-I related gene(s) for monitoring the progression of IBD.

29. Use of the level of DNA methylation of one or more MHC-I related gene(s) and / or the level of expression of one or more MHC-I related gene(s) for determining the efficacy of a therapeutic agent in a patient having IBD.

30. Use of the level of DNA methylation of one or more MHC-I related gene(s) and / or the level of expression of one or more MHC-I related gene(s) for the differential diagnosis of Crohn's Disease or Ulcerative Colitis.

31. A kit comprising (a) reagents for determining the level of DNA methylation of one or more MHC-I related gene(s) and / or the level of expression of one or more MHC-I related gene(s); and (b) instructions for use in: (i) assessing the severity of IBD; (ii) monitoring the progression of IBD; (iii) determining the efficacy of a therapeutic agent in a patient having IBD; and / or (iv) differential diagnosis of Crohn's Disease or Ulcerative Colitis.

32. The kit of claim 31, wherein the reagents comprise primer(s) and / or probe(s) for detecting the level of methylation of one or more MHC-I related gene(s).

33. The kit of claim 31 or claim 32, wherein the kit comprises: (i) one or more primer(s) specific to a methylated sequence; and / or (ii) one or more primer(s) specific to an unmethylated sequence.

34. The kit of any of claims 31-33, wherein the one or more MHC-I related gene(s) are selected from NLRC5, B2M, TAPI, TAP2, HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, HLA-DPA1, PSMB8, PSMB9, IFIT1, and IRF1.

35. A method for predicting the likelihood that a patient will respond to therapy with a therapeutic agent, the method comprising:(a) exposing intestinal epithelial organoids (lEOs) derived from the patient to the therapeutic agent; and(b) quantifying the level of expression of MHC-I and / or the level of expression of one or more MHC-I related gene(s) in the lEOs;wherein reduced expression of MHC-I and / or reduced expression of said one or more MHC-I related gene(s) as compared to the level of expression of MHC-I and / or the level of expression of said one or more MHC-I related gene(s) in the absence of the therapeutic agent indicates an increased likelihood that the patient will respond to therapy.

36. A method for screening an agent for therapeutic activity against IBD, the method comprising:(a) exposing intestinal epithelial organoids (lEOs) derived from at least one patient having IBD to the agent; and(b) quantifying the level of expression of MHC-I and / or the level of expression of one or more MHC-I related gene(s) in the lEOs; wherein reduced expression of MHC-I and / or reduced expression of said one or more MHC-I related gene(s) as compared to the level of expression of MHC-I and / or the level of expression of said one or more MHC-I related gene(s) in the absence of the agent is indicative of therapeutic activity against IBD.

37. The method of claim 35 or claim 36, wherein the method further comprises exposing the lEOs to IFN-y.

38. The method of any one of claims 35-37, wherein the lEOs are derived from mucosal stem cells obtained from an intestinal biopsy.

39. The method of any of claims 35-38, wherein the one or more MHC-I related gene(s) are selected from NLRC5, B2M, TAPI, TAP2, HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, HLA-DPA1, PSMB8, PSMB9, IFIT1, and IRF1.

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