(12) Patent Application Publication (10) Pub. No.: US 2015/0038552 A1 Rothenberg Et Al

US 20150.038552A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2015/0038552 A1 Rothenberg et al. (43) Pub. Date: Feb. 5, 2015

(54) ESOPHAGEAL MICRORNA EXPRESSION (52) U.S. Cl. PROFILES INEOSINOPHILIC ESOPHAGITIS CPC ...... CI2O I/6883 (2013.01); C12O 2600/178 (2013.01) (71) Applicant: CHILDRENS HOSPITAL USPC ...... 514/44A: 514/44 R: 514/169; 506/9: MEDICAL CENTER, Cincinnati, OH 506/16: 435/6.12 (US) (72) Inventors: Marc E. Rothenberg, Cincinnati, OH (57) ABSTRACT (US); Thomas Xuefeng Lu, Cincinnati, OH (US) Methods and compositions disclosed herein generally relate to methods of treating eosinophilic esophagitis (EE) and eosi (21) Appl. No.: 14/380,672 nophilic disorders by providing or enhancing a diagnosis of EE and eosinophilic disorders. In particular, the invention (22) PCT Filed: Feb. 22, 2013 relates to obtaining a sample from a patient, then quantifying from the sample an amount of one or more microRNAs (miR (86). PCT No.: PCT/US2013/027503 NAs) associated with EE, wherein an altered level of the S371 (c)(1) miRNA correlates with a positive diagnosis of EE. An EE (2) Date: s Aug. 22, 2014 diagnosis can then be provided or enhanced, based upon the 9 quantifying step, and an appropriate treatment can be admin Related U.S. Application Data istered to the patient. The invention further relates to diag (60) Provisional application No. 61/602,897, filed on Feb. nostic kits, tests, and/or arrays that can be used to quantify the 24, 2012. one or more miRNAs associated with EE, as well as treat s ments developed to up-regulate or down-regulate one or more Publication Classification miRNAs and/or their downstream pathways relevant to EE or asthma. The invention further relates to the use of IGF1 and (51) Int. Cl. IGF1R inhibitors for the treatment of EE and eosinophilic CI2O I/68 (2006.01) disorders. Patent Application Publication Feb. 5, 2015 Sheet 1 of 44 US 2015/0038552 A1

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ESOPHAGEAL MICRORNA EXPRESSION then commonly followed by biopsy analysis. EE provides an PROFILES INEOSINOPHILIC ESOPHAGITIS opportunity to closely study human inflammatory diseases, as obtaining tissue specimens via endoscopy is routine standard CROSS REFERENCE TO RELATED of-care, and the biopsy material is amenable to molecular APPLICATION analysis (see, e.g., Liacouras, C. et al. J. Allergy Clin. Immu 0001. The present application claims the benefit of priority mol. 128:3-20 (2011); Abonia, J. et al. J. Allergy Clin. Immu under 35 U.S.C. S 119(e) to U.S. Provisional Application No. mol. 126:140-9 (2010)). 61/602,897, ESOPHAGEAL MICRORNA EXPRESSION PROFILES IN EOSINOPHILIC ESOPHAGITIS, filed on SUMMARY Feb. 24, 2012, which is currently co-pending herewith and 0008 Embodiments of the invention encompass methods which is incorporated by reference in its entirety. of treating a patient with eosinophilic esophagitis (EE), including obtaining a sample from a patient, analyzing the STATEMENT REGARDING sample to determine a level of one or more miRNAs associ FEDERALLY SPONSORED RESEARCH ated with EE, determining whether the level of the one or 0002. This invention was made with U.S. Government more miRNAS is up-regulated or down-regulated relative to a support. This work was supported in part by the NHLBI Ruth level of the one or more miRNAs measured in a normal L. Kirschstein National Research Service Award for indi individual, wherein the presence of an elevated or reduced vidual predoctoral MD/PhD fellows F30HL104892 and NIH level of one or more miRNAs associated with EE results in the grants R01 DK076893, R01AI083450, T32GM063483, patient being diagnosed with EE, and treating the patient with T32HD046387, U19AIO70235, P3ODK078392, and an appropriate therapeutic strategy based upon the diagnosis. AI070235. The U.S. Government has certain rights in the 0009. In some embodiments of the methods, the one or subject matter hereof. more miRNAs associated with EE can include, for example, miR-886-5p, miR-886-3p, miR-222*, miR-7, miR-29b, miR FIELD OF THE INVENTION 642, miR-339-5p, miR-21, miR-21*, miR-142-5p, miR 146a, miR-146b, miR-142-3p, miR-132, miR-212, miR-592, 0003. The invention disclosed herein generally relates to miR-92a-1*, miR-223*, miR-223, miR-801, miR-106b, diagnosis, treatment, and/or management of eosinophilic miR-375, miR-211, miR-210, miR-365, miR-203, miR esophagitis, asthma, and/or allergic diseases, disorders, and/ 193a-5p, miR-193b, miR-193a-3p, let-7c, miR-144*, or or conditions arising therefrom and/or related thereto. miR-30a-3p. In some embodiments, the one or more miRNAs associated with EE can include, for example, miR-21, miR BACKGROUND 223, miR-375, miR-142-3p, miR-146a, or miR-146b. In 0004 All publications mentioned herein are incorporated some embodiments, the one or more miRNAs associated with by reference to the same extent as if each individual publica EE can include, for example, miR-21, miR-223, or miR-375. tion or patent application was specifically and individually 0010. In some embodiments, the determination of whether indicated to be incorporated by reference. The following the level(s) of the one or more miRNAs associated with EE description includes information that can be useful in under are elevated or reduced relative to a level of the one or more standing the present Subject matter. It is not an admission that miRNAs measured in a normal individual can be combined any of the information provided herein is prior art or relevant with a determination of a level(s) of one or more additional to the presently claimed Subject matter, or that any publica biomarkers associated with EE. In some embodiments, the tion specifically or implicitly referenced is prior art. one or more additional biomarkers associated with EE can 0005 Eosinophilic esophagitis (EE, also abbreviated EoE include, for example, an mRNA biomarker. In some embodi in some publications) is an emerging worldwide disease char ments, the one or more additional biomarkers associated with acterized by marked eosinophil infiltration of the esophageal EE can include, for example, eotaxin-3. mucosal epithelium (>15 eosinophils/high power field (hpf)) 0011. In some embodiments, the sample can be, for that is refractory to acid Suppressive therapy and is associated example, an esophageal tissue sample. In some embodiments, with chronic symptoms from childhood into adulthood (see, the sample can be, for example, a plasma or serum sample. In e.g., Furuta, G. et al. Gastroenterology 133: 1342-63 (2007): Some embodiments, the sample can be, for example, a buccal Assa'ad, A. et al. JAllergy Clin. Immunol. 119:731-8 (2007): sample, an oral Swish, or saliva. Straumann, A. and Simon, H. J. Allergy Clin. Immunol. 115: 0012. In some embodiments, the appropriate therapeutic 418-9 (2005); Liacouras, C. et al. J. Allergy Clin. Immunol. strategy for a patient diagnosed with EE can include, for 128:3-20 (2011)). First described in the late 1970s, the inci example, allergen removal, Steroid treatment, dietary man dence and prevalence of EE has been increasing, and EE is agement, proton pump inhibitor (PPI) therapy, administration now a global disease reported in every continent except Africa of one or more topical glucocorticoids, humanized antibodies (see, e.g., Liacouras, C. et al. J. Allergy Clin. Immunol. 128: against one or more relevant cytokines and/or mediators, one 3-20 (2011)). or more small molecule inhibitors of an eosinophil and/or 0006 EE symptoms mimic gastroesophageal reflux dis allergic disease activation pathway, one or more Small mol ease (GERD) and can vary with age. Patients with EE can ecule inhibitors capable of modulating miRNA levels and/or have gastrointestinal complains that typically include, but are as severing as stem-loop processing inhibitors, and/or any not limited to, failure to thrive, vomiting, abdominal pain, combination thereof. In some embodiments, the topical glu dysphagia, and food impactions (see, e.g., Furuta, G. et al. cocorticoid can include, for example, fluticasone, budes Gastroenterology 133:1342-63 (2007); Liacouras, C. et al. J onide, and/or ciclesonide. In some embodiments, the human Pediatr. Gastroenterol. Nutr. 45:370-91 (2007)). ized antibody against a relevant cytokine or mediator can 0007 EE diagnosis involves endoscopy, which is an inva include, for example, an antibody against eotaxin-1, eotaxin sive and inconvenient procedure. The endoscopy procedure is 3, IL-13, IL-5, IL-5RC, CD49D, SIGLEC-8, IgE, CD300A, US 2015/0038552 A1 Feb. 5, 2015

TSLP, and/or IL-33. In some embodiments, the small mol level of miR-675 following treatment indicates that the ecule inhibitor can include, for example, a notch-signaling patient is responsive to steroid treatment. inhibitor or an inhibitor or antagonist of CCR3. CCL11, 0019. In some embodiments, a patient diagnosed with EE VLA4, CRTH2, prostaglandin D2, histamine H4 receptor, can be determined to be likely to be responsive or non-re IL-13, IL-4, and/or the common B chain. sponsive to anti-IL-13 treatment, wherein an elevated level of 0013. In some embodiments, the appropriate therapeutic one or more miRNAs associated with periostin levels indi strategy includes using any of the one or more miRNA(s) cates that the patient is likely to be responsive to anti-IL-13 associated with EE found to be elevated relative to the level(s) treatment. In some embodiments, the one or more miRNAs of the one or more miRNAs measured in a normal individual associated with periostin levels can include, for example, or using one or more corresponding modified miRNA(s) as a miR-223 and/or miR-375. therapeutic target or agent. In some embodiments, the appro 0020 Embodiments of the invention are also directed to priate therapeutic strategy includes administering to the methods of diagnosingapatient with eosinophilic esophagitis patient one or more agents such as, for example, an anti (EE), the methods including obtaining a sample from a miRNA oligonucleotide (antagomir), an antisense oligo patient, analyzing the sample to determine a level of one or nucleotide, a locked nucleic acid, an RNA competitive inhibi more miRNAs associated with EE in adult patients, and deter tor or decoy, and/or a viral vector expressing one or more mining whether the level of the one or more miRNAs are miRNA genes, and the like. In some embodiments, the up-regulated or down-regulated relative to a level of the one antagomir can be directed against a miRNA found to be or more miRNAs measured in a normal individual, wherein elevated relative to the level(s) of the one or more miRNAs the presence of an elevated or reduced level of one or more measured in a normal individual. In some embodiments, the miRNAs associated with EE results in the patient being diag antagomir against the up-regulated miRNA can be, for nosed with EE. example, a miR-21, miR-223, miR-146a, and/or miR-146b 0021 Embodiments of the invention are also directed to antagomir, and the like. In some embodiments, the competi methods of treating a patient with an eosinophilic disorder, tive inhibitor against the elevated miRNA can be, for the methods including, obtaining a sample from a patient, example, an IGF1 or IGF1R inhibitor, and the like. In some analyzing the sample to determine a level of one or more embodiments, the IGF1R inhibitor can be, for example, NVP miRNAs associated with an eosinophilic disorder, determin AEW541 and/or pricopodophyllin, and the like. In some ing whether the level of the one or more miRNAs is up embodiments, the viral vector expressing one or more regulated or down-regulated relative to a level of the one or miRNA genes can be, for example, a lentiviral vector, an more miRNAs measured in a normal individual, wherein the adenoviral vector, and/or an adeno-associated virus, and the presence of an elevated or reduced level of one or more like. miRNAs associated with an eosinophilic disorder results in 0014. Some embodiments of the methods further include a the patient being diagnosed with an eosinophilic disorder, and determination of eosinophilic esophagitis or chronic esoph treating the patient with an appropriate therapeutic strategy agitis, wherein the presence of a non-elevated or non-reduced based upon the diagnosis. level of one or more miRNAs associated with eosinophilic 0022. In some embodiments, the eosinophilic disorder can esophagitis results in the patient being diagnosed with be, for example, an eosinophilic gastrointestinal disorder chronic esophagitis. In some embodiments, the appropriate (EGID). In some embodiments, the eosinophilic disorder can therapeutic strategy can include, for example, antacid admin be, for example, asthma. In some embodiments, the one or istration, H2 agonist administration, and/or PPI therapy. more miRNAS associated with asthma can be, for example, 0.015. Some embodiments of the methods further include a miR-375. In some embodiments, the sample can include, for determination of active eosinophilic esophagitis or eosino example, lung and/or lung epithelial cells. philic esophagitis in remission, wherein the one or more 0023 Embodiments of the invention are also directed to a miRNAs can include, for example, miR-886-5p, miR-886 diagnostic kit, test, or array, including materials for quantifi 3p, miR-222*, miR-7, miR-29b, miR-642, miR-339-5p, cation of at least two analytes, wherein the at least two ana miR-21, miR-21*, miR-142-5p, miR-146a, miR-146b, miR lytes are miRNAS associated with eosinophilic esophagitis 142-3p, miR-132, miR-212, miR-592, miR-92a-1*, miR (EE). 223*, miR-223, miR-801, miR-106b, miR-375, miR-211, 0024. In some embodiments, the at least two analytes can miR-210, miR-365, miR-203, miR-193a-5p, miR-193b, include, for example, miR-21, miR-223, and miR-375. In miR-193a-3p, let-7c, miR-144*, and miR-30a-3p. Some embodiments, the at least two analytes can include, for example, miR-886-5p, miR-886-3p, miR-222*, miR-7, miR 0016 Some embodiments of the methods further include a 29b, miR-642, miR-339-5p, miR-21, miR-21*, miR-142-5p, determination of EE disease severity, wherein a highly up miR-146a, miR-146b, miR-142-3p, miR-132, miR-212, regulated or highly down-regulated level of the one or more miR-592, miR-92a-1*, miR-223*, miR-223, miR-801, miR miRNAs relative to the level(s) of the one or more miRNAs 106b, miR-375, miR-211, miR-210, miR-365, miR-203, measured in a normal individual is indicative of a severe case miR-193a-5p, miR-193b, miR-193a-3p, let-7c, miR-144*, of EE. and miR-30a-3p. In some embodiments, the at least two ana 0017. In some embodiments, a patient diagnosed with EE lytes can include, for example, miR-21, miR-223, miR-375, can be determined to be compliant with and/or exposed to miR-142-3p, miR-146a, and miR-146b. In some embodi steroid treatment, wherein an elevated level of miR-675 fol ments, the at least two analytes can include, for example, lowing treatment indicates that the patient is compliant with miR-21, miR-223, miR-375, miR-142-3p, miR-146a, and and/or exposed to steroid treatment. miR-146b. 0018. In some embodiments, a patient diagnosed with EE 0025. In some embodiments, the at least two analytes can and treated with a steroid can be determined to be responsive include all of miR-21, miR-223, and miR-375. In some or non-responsive to steroid treatment, wherein an elevated embodiments, the at least two analytes can include all of US 2015/0038552 A1 Feb. 5, 2015

miR-21, miR-223, miR-375, miR-142-3p, miR-146a, and 5A illustrates the identification of the pri-miR-21 region in miR-146b. In some embodiments, the at least two analytes the RNA-Seq analysis. The normalized coverage tracks for can include all of miR-886-5p, miR-886-3p, miR-222*, miR the mRNA-Seq are displayed, along with the spliced reads 7, miR-29b, miR-642, miR-339-5p, miR-21, miR-21*, miR that are present in exons of VMP1. The lack of spliced reads 142-5p, miR-146a, miR-146b, miR-142-3p, miR-132, miR present between the regions outside of the exons of VMP1 212, miR-592, miR-92a-1*, miR-223*, miR-223, miR-801, indicates that it is a portion of pri-miR-21. The regions anno miR-106b, miR-375, miR-211, miR-210, miR-365, miR tated as “exon' or “intron’ are not necessarily true exons and 203, miR-193a-5p, miR-193b, miR-193a-3p, let-7c, miR introns of a gene but regions that showed significant expres 144*, and miR-30a-3p. sion patterns similar to that of a gene's exons and introns. 0026. In some embodiments, the diagnostic kit, test, or They were annotated as such to facilitate the identification of array can include a gene chip. In some embodiments, the gene the region to use for the correlation analysis. FIG. 5B illus chip includes a low density array. trates the identification of the pri-miR-223 region in the 0027. In some embodiments, the diagnostic kit, test, or RNA-Seq analysis. The normalized coverage tracks for the array can include a surface with a DNA array. mRNA-seq are displayed, but there are no reads spanning BRIEF DESCRIPTION OF THE DRAWINGS junctions because there are no splicing events present in this region. The regions annotated as “exon' are not necessarily 0028. Those of skill in the art will understand that the drawings, described below, are for illustrative purposes only. true exons of a gene but regions that showed significant The drawings are not intended to limit the scope of the present expression patterns similar to that of a gene’s exons. They teachings in any way. were annotated as such to facilitate the identification of the 0029 FIG. 1 depicts microRNA (miRNA) expression pro region to use for the correlation analysis. files in normal patients and EE patients. The figure presents a 0034 FIGS. 6A-C depict gene enrichment analyses of heatmap of 21 up-regulated and 11 down-regulated miRNAs miR-21 and miR-223 co-regulated genes in EE patients, with in EE patients compared to normal controls. extensive enrichment of genes with functional features asso 0030 FIGS. 2A-F depict quantitative real time poly ciated with T cell polarization, IFNY signaling, and regulation merase chain reaction (qRT-PCR) verification of a selected of eosinophilia among genes whose expression is correlated set of differentially expressed miRNAs in normal patients and with miR-21 and miR-223 expression in the esophageal biop EE patients. The graphs in FIGS. 2A-E depict the expression sies. The networks are shown as Cytoscape (open source of 5 miRNAs, namely (2A) miR-21, (2B) miR-223, (2C) software, see http 0.05. responder patients, and fluticasone propionate non-responder 0037 FIG. 9 depicts miR-142-3p expression levels in patients. Relative expression levels were determined by qRT patient plasma samples. Expression of miR-142-3p was PCR normalized to U6. N=7-11 patients per group; data are determined in plasma samples from active EE patients and EE represented as meant SEM. remission patients compared to normal controls. The relative 0033 FIGS. 5A-B depict regions identified as primary expression levels were normalized to miR-16. N=13-14 transcripts for miR-21 and miR-223 (pri-miR-21 and pri plasma samples per group; data are represented as miR-223) during RNA sequencing (RNA-Seq) analysis. FIG. meaniSEM; NS: not significant, with P>0.05. US 2015/0038552 A1 Feb. 5, 2015

0038 FIG. 10 depicts esophageal eosinophil counts for days; eosinophils are identified as CCR3. Siglec-Fi cells. wild type (WT) mice and miR-21 gene knockout (KO) mice FIG. 17C illustrates miR-223 expression levels during the under Saline control (Sal) or upon exposure to the allergen eosinophil differentiation culture. N=3 per group; data are Aspergillus fumigatus (Asp). represented as meant SEM. 0039 FIGS. 11A-B depict miR-21 induction during eosi 0046 FIGS. 18A-C depict the growth of eosinophil pro nophil differentiation. FIG. 11A illustrates the purity of cul genitor cells and morphology of mature eosinophils from tured eosinophils at day 14, eosinophils are identified as miR-223" and miR-223 cultures during the ex vivo eosi CCR3"Siglec-F" cells. FIG. 11B illustrates miR-21 expres nophil differentiation culture. FIG. 18A illustrates the total sion levels during the eosinophil differentiation culture, as cell number of eosinophils in cultures derived from miR determined by quantitative polymerase chain reaction 223" and miR-223 mice, as determined by cell counting (qPCR) normalized to U6. N=3 per group; data are repre using a hemacytometer. N=6 per group; data are represented sented as meantSEM. as mean-SEM. FIG. 18B illustrates the morphology of miR 0040 FIGS. 12A-C depict the growth of eosinophil pro 223" and miR-223 eosinophil progenitor culture at day 8, genitor cells from miR-21 mice and miR-21" controls 10, and 12, as determined by modified Giemsa (Diff-Quik) during the ex vivo eosinophil culture. FIG. 12A illustrates the staining FIG. 18C illustrates the morphology of miR-223" total cell number of eosinophil cultures: FIG. 12B illustrates and miR-223 culturedeosinophils at day 14, as determined the total number of neutrophil cultures derived from miR by Diff-Quik (Fisher Scientific) staining. 21" and miR-21 mice. N=6 per group; data are repre 0047 FIG. 19 depicts levels of IGF1R during eosinophil sented as meant-SEM. FIG. 12C displays the morphology of differentiation culture from the miR-223' and miR-223 miR-21" and miR-21 cultured eosinophils at day 12, as mice. The figure presents a western blot showing levels of determined by Diff-Quik (Fisher Scientific, Pittsburgh, Pa.) pre-IGF1R and IGF1R in eosinophil differentiation cultures staining. derived from miR-223" and miR-223 mice from day 4 to 0041 FIG. 13 depicts levels of apoptosis in the eosinophil day 14. The housekeeping gene glyceraldehyde 3-phosphate progenitor culture from the miR-21" and miR-21 mice. dehydrogenase (GAPDH) was used as a loading control. Levels of annexin V and 7AAD staining during eosinophil 0048 FIGS. 20 A-C depict the finding that the increased differentiation culture were determined by fluorescence-ac growth observed in cultures derived from miR-223 mice tivated cell sorting (FACS). The viable cells are annexin can be reversed by an IGF1R inhibitor. FIG. 20A displays V/7AAD. The early apoptotic cells areannexin V/7AAD, data for bone marrow-derived eosinophils from miR-223" and the late apoptotic cells are annexin V/7AAD". and miR-223 mice; eosinophils were treated with 2 um 0042 FIGS. 14A-C depict blood eosinophil percentage picropodophyllin (PPP, an IGF1R inhibitor) or an equivalent and bone marrow eosinophil colony forming unit capacity in volume of dimethyl sulfoxide (DMSO). Growth was mea the miR-21" and miR-21 mice. FIG. 14A illustrates sured by cell counting using a hemacytometer. FIG. 20B blood eosinophil percentage from miR-21" and miR-21 presents a western blot showing levels of IGF1R expression mice, as determined by FACS staining for CCR3. Siglec-F" in miR-223" and miR-223 cells after 2-day treatment cells; n=9-10 mice per group. FIG. 14B illustrates bone mar with PPP. GAPDH was used as a loading control. FIG. 20O row eosinophil colony forming unit (CFU-Eos), and FIG. presents a dose response study of different concentrations of 14C illustrates neutrophil colony forming unit (CFU-G) PPP on the proliferative response of eosinophil cultures capacity from miR-21" and miR-21 mice. N=4 per derived from miR-223' and miR-223 mice. N=3 per group; data are represented as meantSEM. group; data are represented as meantSEM. 0043 FIGS. 15A-D present heatmaps of differentially 0049 FIGS. 21A-B depict the increased growth observed regulated genes between miR-21" and miR-21 eosino in eosinophil progenitor cultures derived from miR-223 phil progenitor cultures at day 8 and day 12. FIG. 15A pre mice, coupled with a delay in eosinophil progenitor differen sents a heatmap of differentially regulated genes at day 8 of tiation. FIG. 21A displays CCR3 expression at day 8, day 10, the eosinophil differentiation culture. FIG. 15B presents a and day 12 of the eosinophil progenitor culture in miR-223 heatmap of differentially regulated genes at day 12 of the cultures compared to miR-223" cultures, as measured by eosinophil differentiation culture. FIG. 15C depicts the quan qPCR normalized to HPRT1. N=3 per group; data are repre titative RT-PCR verification of a selected set of differentially sented as mean+SEM. FIG. 21B displays levels of CCR3 expressed genes between miR-21" and miR-21 eosino expression during eosinophil differentiation culture, as deter phil progenitor cultures. FIG. 15D illustrates a functional mined by FACS staining of surface CCR3 and Siglec Flevels: enrichment analysis of differentially regulated genes in the mature eosinophils are identified as CCR3"Siglec-F" cells. eosinophil progenitor cultures at day 12. The networks are 0050 FIGS. 22A-B depict the mature eosinophil levels in shown as Cytoscape (open source Software) graph networks the blood and eosinophil progenitor levels in the bone marrow generated from ToppCluster (Cincinnati Children’s Hospital of miR-223' and miR-223 mice. FIG. 24A depicts the Medical Center) network analysis. level of mature eosinophils in the blood of miR-223" and 0044 FIG. 16 depicts a biological function enrichment miR-223 mice. Blood eosinophil levels were determined analysis of differentially regulated genes in eosinophil pro by CCR3 and SiglecF staining. The double positive cells are genitor cultures at day 8. The figure illustrates an analysis of eosinophils. N=7-8 mice per group. FIG. 24B depicts the the most significant biological functions represented by the eosinophil progenitor levels in the bone marrow of miR differentially regulated genes between miR-21" and miR 223" and miR-223 mice. Eosinophil progenitor levels 21 eosinophil progenitor cultures. were determined by CD34 and IL5RC. staining. The double 004.5 FIGS. 17A-C depict miR-223 induction during positive cells are eosinophil progenitors. N=3-4 mice per eosinophil differentiation. FIG. 17A presents a schematic of group. Data are represented as meantS.E.M. the ex vivo bone marrow-derived eosinophil culture. FIG. 0051 FIGS. 23A-D present heatmaps of differentially 17B illustrates the purity of cultured eosinophils after 14 regulated genes between miR-223" and miR-223 eosino US 2015/0038552 A1 Feb. 5, 2015 phil progenitor cultures at day 8 and day 12, along with their 0057 FIG. 29 depicts miR-375 expression levels in differ most strongly associated biological functions. FIG.22A pre ent cell types. The relative expression level of miR-375 in sents a heatmap of differentially regulated genes at day 8 of different cell types was determined by qPCR normalized to the eosinophil differentiation culture. FIG. 22B illustrates a U6. N=3 per group; data are represented as meant SEM. functional enrichment analysis of differentially regulated 0058 FIGS. 30A-B depict genes differentially regulated genes in the eosinophil progenitor cultures at day 8. The by miR-375 in esophageal epithelial cells before and after networks are shown as Cytoscape (open source Software) IL-13 stimulation. FIG. 30A presents a heatmap showing graph networks generated from ToppCluster (Cincinnati genes differentially expressed in esophageal epithelial cell Children’s Hospital Medical Center) network analysis. FIG. line transduced with either a control vector or a pre-miR-375 22C presents a heatmap of differentially regulated genes at expression vector, before and after IL-13 stimulation. FIG. day 12 of the eosinophil differentiation culture. FIG. 22D 30B displays a functional enrichment analysis of pathways illustrates an analysis of the most significant biological func affected by miR-375 under IL-13-stimulated conditions. The tions represented by the differentially regulated genes networks are shown as Cytoscape (open source Software) between miR-223" and miR-223 eosinophil progenitor graph networks generated from ToppCluster (Cincinnati cultures at day 12. Children’s Hospital Medical Center) network analysis. 0052 FIG. 24 depicts a biological function enrichment 0059 FIG. 31 depicts a biological function enrichment analysis of differentially regulated genes in eosinophil pro analysis of all miR-375-regulated genes. The figure illustrates genitor cultures at day 8. The figure illustrates an analysis of an analysis of the most significant diseases and disorders the most significant biological functions represented by the represented by all genes differentially regulated by miR-375. differentially regulated genes between miR-223" and miR 0060 FIG.32 depicts the expression levels of thymic stro 223 eosinophil progenitor cultures. mal lymphopoietin (TSLP) in polyinosinic:polycytidylic 0053 FIGS. 25A-B depict the miRNA expression profile acid (poly(I:C)) stimulated pre-miR-375-transduced TE-7 in human esophageal epithelial cells and human bronchial esophageal epithelial cells compared to controls. Control epithelial cells after 24 hours of IL-13 stimulation. FIG. 25A transduced and pre-miR-375-transduced TE-7 cells were presents a heatmap of 4 down-regulated and 2 up-regulated stimulated with 25 g/mL poly(I:C) for 0, 2, 4, and 8 hours. miRNAs in IL-13-stimulated human esophageal epithelial Expression levels were determined by qPCR normalized to cells compared to controls. FIG.25B presents a heatmap of 4 HPRT1. N=4 per group; data are represented as mean-SEM. down-regulated and 2 up-regulated miRNAs in IL-13-stimu lated human bronchial epithelial cells compared to controls. DETAILED DESCRIPTION OF THE INVENTION 0054 FIGS. 26A-D depict qRT-PCR verification of miR 0061 All references cited herein are incorporated by ref 375 expression in IL-13-stimulated human esophageal epi erence in their entirety. Also incorporated herein by reference thelial cells and human bronchial epithelial cells. Expression in their entirety include: U.S. Patent Application No. 60/633, of miR-375 was determined in (FIG. 26A) IL-13-stimulated 909, EOTAXIN-3 IN EOSINOPHILIC ESOPHAGITIS, human esophageal epithelial cells compared to controls and filed on Dec. 27, 2004; U.S. Pat. No. 8,030.003, DIAGNOSIS (FIG. 26B) IL-13-stimulated human bronchial epithelial cells OF EOSINOPHILIC ESOPHAGITIS BASED ON PRES compared to controls. FIG. 26C displays a kinetic analysis of ENCE OF ANELEVATED LEVEL OF EOTAXIN-3, issued miR-375 expression in IL-13-stimulated esophageal epithe Oct. 4, 2011 and filed as U.S. patent application Ser. No. lial cells. FIG. 26D displays a kinetic analysis of miR-375 11/721,127 on Jun. 7, 2007; U.S. patent application Ser. No. expression in IL-13-stimulated normal human bronchial epi 12/492,456, EVALUATION OF EOSINOPHILIC ESOPH thelial cells. The relative expression levels were normalized AGITIS, filed on Jun. 26, 2009; U.S. patent application Ser. to U6. N=4 per group; data are represented as mean-SEM; No. 12/628,992, IL-13 INDUCED GENE SIGNATURE NS: not significant. FOR EOSINOPHILIC ESOPHAGITIS, filed on Dec. 1, 2009; U.S. Provisional Application No. 61/430,453, A 0055 FIG. 27 depicts expression of miR-375 in a doxy STRIKING LOCAL ESOPHAGEAL CYTOKINE cycline-induced IL-13 lung transgenic experimental asthma EXPRESSION PROFILE INEOSINOPHILICESOPHAGI model. The relative expression levels of miR-375 were deter TIS, filed on Jan. 6, 2011; U.S. patent application Ser. No. mined by qPCR normalized to U6. N-6 mice per group; data 13/051,873, METHODS AND COMPOSITIONS FOR are represented as meant-SEM. MITIGATING EOSINOPHILIC ESOPHAGITIS BY 0056 FIGS. 28A-C depict expression of miR-375 in MODULATINGLEVELS AND ACTIVITY OF EOTAXIN esophageal biopsies from EE patients and the correlation with 3, filed on Mar. 18, 2011; U.S. patent application Ser. No. esophageal eosinophil counts and EE signature genes. FIG. 13/132,884, DETERMINATION OF EOSINOPHILIC 28A displays the miR-375 expression levels in normal con ESOPHAGITIS, filed on Jun. 3, 2011; U.S. Provisional trols, EE patients, chronic esophagitis patients, EE patients Application No. 61/497,796, NEGATIVE REGULATION responsive to glucocorticoid therapy (fluticasone proprion OF EOSINOPHIL PRODUCTION BY TOLL-LIKE ate), EE patients unresponsive to glucocorticoid therapy, and RECEPTORS, filed on Jun. 16, 2011; U.S. Patent Application EE patients responsive to diet modification. Expression levels No. 61/571,115, DIAGNOSTIC METHODS OF EOSINO were determined by qPCR normalized to U6. N=8-15 PHILIC ESOPHAGITIS, filed on Jun. 21, 2011; U.S. Provi patients per group; data are represented as meantSEM. FIG. sional Application No. 61/500,508, MOLECULAR DIAG 28B displays the correlation between miR-375 expression NOSTIC PANEL OF EOSINOPHILIC and esophageal eosinophil counts. FIG. 28C displays the GASTROINTESTINAL DISORDERS, filed on Jun. 23, correlation between miR-375 expression and EE signature 2011; U.S. patent application Ser. No. 13/132,295, METH genes. The significance of the correlation was plotted as the ODS OF DETERMINING EFFICACY OF GLUCOCORTI negative log of p value for each gene. The dashed line repre COID TREATMENT OF EOSINOPHILIC ESOPHAGITIS, sents significance level after false discovery rate correction. filed on Aug. 22, 2011; PCT Patent Application No. US2012/ US 2015/0038552 A1 Feb. 5, 2015

020556, ESOPHAGEALCYTOKINE EXPRESSION PRO has not yet been diagnosed as having it; (b) inhibiting the FILES IN EOSINOPHILIC ESOPHAGITIS, filed on Jan. 6, disease, i.e., arresting its development; and (c) relieving the 2012; U.S. Provisional Application No. 61/602,897, ESOPH disease, i.e., causing regression of the disease and/or relieving AGEAL MICRORNA EXPRESSION PROFILES INEOSI one or more disease symptoms. "Treatment can also encom NOPHILIC ESOPHAGITIS, filed on Feb. 24, 2012: PCT pass delivery of an agent or administration of a therapy in Patent Application No. US2012/42985, BLOCKADE OF order to provide for a pharmacologic effect, even in the EOSINOPHIL PRODUCTION BY TOLL-LIKE RECEP absence of a disease or condition. The term “treatment” is TORS, filed on Jun. 18, 2012: PCT Patent Application No. used in Some embodiments to refer to administration of a US2012/043640, DIAGNOSTIC METHODS FOR EOSI compound of the present invention to mitigate a disease or a NOPHILIC ESOPHAGITIS, filed on Jun. 21, 2012; and PCT disorder in a host, preferably in a mammalian Subject, more Patent Application No. US2012/044061, MOLECULAR preferably in humans. Thus, the term “treatment can include DIAGNOSTIC PANEL OF EOSINOPHILIC GAS includes: preventing a disorder from occurring in a host, TROINTESTINAL DISORDERS, filed on Jun. 25, 2012. particularly when the host is predisposed to acquiring the 0062 Unless otherwise noted, terms are to be understood disease, but has not yet been diagnosed with the disease; according to conventional usage by those of ordinary skill in inhibiting the disorder, and/or alleviating or reversing the the relevant art. disorder. Insofar as the methods of the present invention are 0063 As used herein, the term “subject” refers to any directed to preventing disorders, it is understood that the term member of the animal kingdom. In some embodiments, a “prevent does not require that the disease state be completely Subject is a human patient. thwarted (see Webster's Ninth Collegiate Dictionary). 0064. As used herein, the term “sample encompasses a Rather, as used herein, the term preventing refers to the ability sample obtained from a subject or patient. The sample can be of the skilled artisan to identify a population that is suscep of any biological tissue or fluid. Such samples include, but are tible to disorders, such that administration of the compounds not limited to, sputum, saliva, buccal sample, oral sample, of the present invention can occur prior to onset of a disease. blood, serum, mucus, plasma, urine, blood cells (e.g., white The term does not mean that the disease state must be com cells), circulating cells (e.g. stem cells or endothelial cells in pletely avoided. the blood), tissue, core or fine needle biopsy samples, cell 0069. As used herein, the terms “modulated' or “modula containing body fluids, free floating nucleic acids, urine, tion.” or “regulated' or “regulation' and “differentially regu stool, peritoneal fluid, and pleural fluid, liquor cerebrospina lated can refer to both up regulation (i.e., activation or stimu lis, tear fluid, or cells therefrom. Samples can also include lation, e.g., by agonizing or potentiating) and downregulation sections of tissues such as frozen or fixed sections taken for (i.e., inhibition or Suppression, e.g., by antagonizing, decreas histological purposes or microdissected cells or extracellular ing or inhibiting), unless otherwise specified or clear from the parts thereof. A sample to be analyzed can be tissue material context of a specific usage. from a tissue biopsy obtained by aspiration or punch, excision 0070. As used herein, the term “marker' or “biomarker or by any other Surgical method leading to biopsy or resected refers to a biological molecule, such as, for example, a nucleic cellular material. Such a sample can comprise cells obtained acid, peptide, protein, hormone, and the like, whose presence from a subjector patient. In some embodiments, the sample is or concentration can be detected and correlated with a known a body fluid that include, for example, blood fluids, serum, condition, Such as a disease state. It can also be used to refer mucus, plasma, lymph, asciitic fluids, gynecological fluids, or to a differentially expressed gene whose expression pattern urine but not limited to these fluids. In some embodiments, can be utilized as part of a predictive, prognostic or diagnostic the sample can be a saline Swish, a buccal scrape, a buccal process in healthy conditions or a disease state, or which, Swab, and the like. alternatively, can be used in methods for identifying a useful 0065. As used herein, “blood can include, for example, treatment or prevention therapy. plasma, serum, whole blood, blood lysates, and the like. 0071. As used herein, the term “expression levels' refers, 0066. As used herein, the term “assessing includes any for example, to a determined level of biomarker expression. form of measurement, and includes determining if an element The term “pattern of expression levels' refers to a determined is present or not. The terms “determining.” “measuring.” level of biomarker expression compared either to a reference "evaluating.” “assessing and “assaying can be used inter (e.g. a housekeeping gene or inversely regulated genes, or changeably and can include quantitative and/or qualitative other reference biomarker) or to a computed average expres determinations. sion value (e.g. in DNA-chip analyses). A pattern is not lim 0067. As used herein, the term “diagnosing or monitor ited to the comparison of two biomarkers but is more related ing with reference to a disease state or condition refers to a to multiple comparisons of biomarkers to reference biomar method or process of determining if a Subject has or does not kers or samples. A certain pattern of expression levels can have a particular disease state or condition or determining the also result and be determined by comparison and measure severity or degree of the particular disease state or condition. ment of several biomarkers as disclosed herein and display 0068. As used herein, the terms “treatment.” “treating.” the relative abundance of these transcripts to each other. “treat, and the like, refer to obtaining a desired pharmaco 0072. As used herein, a “reference pattern of expression logic and/or physiologic effect. The effect can be prophylac levels' refers to any pattern of expression levels that can be tic in terms of completely or partially preventing a disease or used for the comparison to another pattern of expression symptom thereof and/or can be therapeutic in terms of a levels. In some embodiments of the invention, a reference partial or complete cure for a disease and/or adverse effect pattern of expression levels is, for example, an average pat attributable to the disease. "Treatment, as used herein, cov tern of expression levels observed in a group of healthy or ers any treatment of a disease in a Subject, particularly in a diseased individuals, serving as a reference group. human, and includes: (a) preventing the disease from occur 0073 Eosinophilic esophagitis (EE, also referred to as ring in a Subject which may be predisposed to the disease but EoE in some publications) is a condition characterized by US 2015/0038552 A1 Feb. 5, 2015 elevated esophageal levels of eosinophils. EE is considered to Allergy Clin. Immunol. 128:23-32 (2011)). Although several be a T2-associated disease (see, e.g., Blanchard, C. et al. J. phenotypic subsets of EE patients have emerged, EE esoph Allergy Clin. Immunol. 120:1292-300 (2007); Blanchard, C. ageal transcriptome analysis has revealed a highly conserved et al. J. Allergy Clin. Immunol. 127:208-17 (2011); Strau expression profile irrespective of patient phenotype (as mann, A. etal.J. Allergy Clin. Immunol. 108:954-61 (2001)). defined by sex, atopic status, and familial clustering). How 0074 EE diagnosis requires endoscopy with biopsy analy ever, the sensitivity of the EE transcriptome has not been fully sis. Reliable, non-invasive techniques for the diagnosis ofEE, established (see, e.g., Blanchard, C. et al. J. Allergy Clin. such as biomarker detection methods, would be preferable to Immunol. 118: 1054-9 (2006); Blanchard, C. and Rothenberg, endoscopic techniques. While blood levels of potential EE M. Gastrointest. Endosc. Clin. N. Am. 18:133-43 (2008)). In biomarkers, such as eosinophils, eotaxin-3, eosinophil-de addition to acquired gene expression changes in the esopha rived neurotoxin, and IL-5 proteins, are known to be elevated gus, EE is also an inherited disease that involves a complex in EE, such non-invasive techniques have heretofore not been combination of genetic and environmental factors (see, e.g., widely used because their sensitivity and specificity are gen Sherrill, J. and Rothenberg, M. J. Allergy Clin. Immunol. erally too low to be clinically helpful (see, e.g., Konikoff M. 128:23-32 (2011)). et al. Gastroenterology 131:1381-91 (2006)). (0079 EE studies have uncovered the key interplay of the 0075 Eosinophils are multifunctional effector cells pro adaptive and innate immune system, including the key role of duced in the bone marrow from eosinophil lineage-commit IL-13-driven epithelial cell gene responses, including ted progenitor cells. Eosinophils are implicated in the patho eotaxin-3. Characterization of gene expression differences genesis of a variety of diseases, including asthma, between patients with EE and non-EE subjects via esoph hypereosinophilic syndrome, eosinophil gastrointestinal dis ageal microarray expression analysis has established orders, and parasitic infections, including helminth infection eotaxin-3 as the most overexpressed gene in patients with EE: (see, e.g., Broide, D. et al. J. Allergy Clin. Immunol. 127:689 this finding has been replicated in independent studies (see, 95 (2011); Venge, P. Clin. Respir:J. 4 Suppl. 1:15-19 (2010): e.g., Blanchard, C. et al. Int. J. Biochem. Cell Biol. 37:2559 Anthony, R. et al. Nat. Rev. Immunol. 7:975-87 (2007): 73 (2005); Bhattacharya, B. et al. Hum. Pathol. 38:1744-53 Hogan, S. et al. Clin. Exp. Allergy 38:709-50 (2008)). (2007); Lucendo, A. et al. Am. J. Gastroenterol. 103:2184-93 0076 Eosinophils differentiate from hematopoietic stem (2008)). cells via a common myeloid progenitor cell in mice through 0080 IL-13-induced epithelial gene and protein expres an intermediate granulocyte/macrophage progenitor, then via sion changes are central to the pathogenesis of multiple aller an eosinophil lineage committed progenitor marked by gic diseases, including EE and asthma. IL-13 is an adaptive CD34" and CD125 (see, e.g., Iwasaki, H. et al. J. Exp. Med. immune cytokine that is involved in mediating the effector 201:1891-7 (2005)). The cytokine IL-5 is particularly impor functions of T2 responses. The central role of IL-13 in tant in eosinophil lineage development, as it promotes the allergic disorders has been demonstrated by the attenuation of selective differentiation of eosinophils and also stimulates the experimental allergic diseases in animals with blockade and/ release of mature eosinophils from the bone marrow (see, or gene deletion of IL-13 and/or its receptor signaling com e.g., Hogan, S. et al. Clin. Exp. Allergy 38:709-50 (2008)). ponents (see, e.g., Leigh, R. et al. Am. J. Respir: Crit. Care IL-5 has also been shown to promote eosinophil survival by Med 169:860-7 (2004): Yang, M. et al. J. Immunol. 177: activating MAP kinase, Lyn tyrosine kinase, and PI3 kinase 5595-603 (2006); Grunig, G. et al. Science 282:2261-3 signaling (see, e.g., Kouro, T. et al. Int. Immunol. 21:1303-9 (1998); Junttila, I. et al. J. Exp. Med. 205:2595-608 (2008); (2009); Rosas, M. et al. J. Leukoc. Biol. 80:186-95 (2006)). Lee, P. et al. J. Clin. Invest. 116:163–73 (2006)). 0077. A lineage-committed eosinophil progenitor popula I0081. One of the critical functions of IL-13 is to modify tion that gives rise exclusively to eosinophils was identified in epithelial gene expression at sites of inflammation. IL-13 both murine and human bone marrow (Iwasaki, H. et al. J. induced gene expression changes in epithelial cells in vitro Exp. Med. 201:1891-7 (2005); Mori, Y. et al. J. Exp. Med. have been shown to significantly overlap with gene expres 206:183-93 (2009)). This eosinophil lineage committed pro sion changes seen in patients in Vivo (see, e.g., Wills-Karp, M. genitor population is IL-5RC. positive, while the non-eosino Immunol. Rev. 202: 175-90 (2004); Liacouras, C. et al. J. phil lineage committed progenitors are IL-5RC. negative, and Allergy Clin. Immunol. 128:3-20 (128); Rothenberg, M. Gas IL-5 was shown to induce the growth and maturation of troenterology 137:1238-49 (2009); Blanchard, C. et al. J. eosinophils (see, e.g., Iwasaki, H. et al. J. Exp. Med. 201: Allergy Clin. Immunol. 120:1292-1300 (2007); Lee, J. et al. 1891-7 (2005); Dyer, K. et al. J. Immunol. 181:4004-9 Am. J. Respir: Cell. Mol. Biol. 25:474-85 (2001); Laprise, C. (2008)). Although key transcription factors have been identi et al. BMC Genomics 5:21 (2004); Woodruff, P. et al. Proc. fied to regulate eosinophil lineage commitment (e.g. C/EBP. Natl. Acad. Sci. U.S.A. 104:15858-63 (2007); Zhen, G. et al. GATA1 and PU. 1), the mechanisms controlling the growth Am. J. Respir: Cell. Mol. Biol. 36:244-54 (2007)). The epi and proliferation of eosinophil progenitor cells in response to thelial cell has been shown to be a key target cell type for IL-5 have heretofore not been well understood. IL-13 mediated responses, making it an attractive model for 0078 Multiple studies have demonstrated that EE is asso investigation. For example, epithelial cells are required for ciated with marked changes in gene expression, particularly IL-13-induced airway hyper-reactivity and mucus production in the esophagus, where ~1% of the human genome has an (Kuperman, D. et al. Nat. Med. 8:885-9 (2002)), and IL-13 altered, tissue-specific expression pattern, collectively induced epithelial cell gene expression changes have a critical referred to as the EE transcriptome, that is largely but not fully role in the pathogenesis of EE (Blanchard, C. et al. J. Allergy reversible following disease remission with glucocorticoid Clin. Immunol. 120:1292-1300 (2007)). therapy (see, e.g., Blanchard, C. etal.J. Clin. Invest. 116:536 I0082 Recent early clinical studies with IL-13-neutraliz 47 (2006); Blanchard, C. et al. J. Allergy Clin. Immunol. ing agents provide evidence that anti-IL-13 holds promise for 120:1292-300 (2007); Abonia, J. et al. J. Allergy Clin. Immu the treatment of allergic disorders, especially in patient Sub mol. 126:140-9 (2010); Sherrill, J. and Rothenberg, M. J. groups, based on the expression profiles of various genes US 2015/0038552 A1 Feb. 5, 2015

(especially periostin) (Corren, J. et al. N. Engl. J. Med. 365: I0087. Different hematopoietic lineages have significant 1088-98 (2011)). Therefore, study of IL-13-mediated differences in their miRNA expression (see, e.g., Navarro, F. responses and the pathways that regulate IL-13-induced gene and Lieberman, J.J. Immunol. 184:5939-47 (2010); Petriv, O. expression will provide insight into therapeutic strategies, et al. Natl. Acad. Sci. U.S.A. 107:15443-8 (2010)). While especially for allergic disorders characterized by IL-13 over various miRNAs have been shown to regulate the differentia production, Such as EE and asthma. tion and lineage commitment of hematopoietic progenitor 0083. Most studies concerning the regulation of the EE cells (see, e.g., Navarro, F. and Lieberman, J. J. Immunol. transcriptome have focused on the induction and regulation of 184:5939-47 (2010); Georgantas, R. et al. Proc. Nat. Acad. in situ gene expression by cytokines (e.g. IL-13), transcrip Sci. U.S.A. 104:2750-5 (2007)), the mechanism for miRNA tion factors, and co-activators (e.g. STAT6 and CBP) (see, regulation over the development of hematopoietic cells after e.g., Blanchard, C. et al. J. Allergy Clin. Immunol. 120:1292 lineage commitment, including regulation of eosinophil pro 300 (2007); Lim, E. et al. J. Biol. Chem. 28.6:13193-204 genitor cell growth by miRNAs, has heretofore not been (2011): Blanchard, C. etal.J. Immunol. 184:4033-41 (2010)). well-described. However, other regulatory processes. Such as those involving I0088 Although multiple cytokines (including IL-3, IL-5, microRNAs (miRNAs), have not been explored. and GM-CSF) and transcription factors (including Gata1 and EE-Associated miRNAs PU1.1) have been shown to regulate the growth of eosinophil 0084 As disclosed herein, certain miRNAs are associated progenitors (see, e.g., Rothenberg, M. and Hogan, S. Annu. with EE. Additionally, miRNA expression profiles can be Rev. Immunol. 24: 147-174 (2006)), other regulatory mol studied in EE, which has a highly conserved, disease-specific ecules, such as miRNAS, can have a role in regulating or transcript profile. A patient’s miRNA plasma or serum levels fine-tuning this process. One recent report has shown that can be measured to provide or contribute to an EE diagnosis: miR-21* can regulate the pro-survival effect of GM-CSF on this information can be used to determine an appropriate eosinophils (Wong, C. et al. Immunobiology, 218:255-62 treatment for the patient. (2013)). 0085 MiRNAs are single-stranded, non-coding RNA I0089 Although the miRNA let-7 has been shown to target molecules of 19-25 nucleotides in length that regulate gene IL-13 directly, and miR-155 has been shown to target expression post-transcriptionally to silence target genes by IL-13RC.1 (Martinez-Nunez, R. et al. J. Biol. Chem. 286: either inhibiting protein translation or facilitating the degra 1786-94 (2011); Kumar, M. et al. J. Allergy Clin. Immunol. dation of target mRNAs (see, e.g., Sayed, D. and Abdellatif, 128:1077–85, e1-10 (2011); Polikepahad, S. et al. J. Biol. M. Physiol. Rev. 91:827-87 (2011); Winter, J. et al. Nat. Cell. Chem. 285:30139-49 (2010)), there has heretofore been little Biol. 11:228-34 (2009)). In animals, miRNAs base pair with evidence to demonstrate that IL-13-induced miRNAs can the complementary regions in the 3' untranslated regions of regulate or fine tune IL-13 mediated-responses. mRNA and induce translational repression and/or mRNA degradation depending on the degree of complementarity of the base pairing (Carthew, R. and Sontheimer, E. Cell 136: MiRNA Expression Profiles 642-55 (2009)). MiRNAs represent a key class of regulators 0090. As described herein, a comprehensive analysis of of messenger RNA (mRNA) expression and translation and global miRNA expression profiles was conducted on the have diverse roles in fundamental biological processes, such esophageal tissues of patients with active EE, active chronic as cell proliferation, differentiation, apoptosis, stress esophagitis, EE patients in remission after glucocorticoid response, and immune response, among many others (see, treatment who have a mainly normalized EE transcriptome e.g., Sayed, D. and Abdellatif, M. Physiol. Rev. 91:827-87 (see, e.g., Blanchard, C. et al. J. Allergy Clin. Immunol. 120: (2011)). To date, few studies have examined the role of miR 1292-300 (2007); Caldwell, J. et al. J. Allergy Clin. Immunol. NAS in human allergic and/or esophageal diseases, except for 125:879-888 (2010)), and normal control subjects; this study the setting of asthma and esophageal cancers (see, e.g., Smith, established a miRNA signature for EE patients. This EE C. et al. World J. Gastroenterol. 16:531-7 (2010); Mattes, J. et miRNA signature is distinct from that of chronic esophagitis al. Proc. Natl. Acad. Sci. U.S.A. 106:18704-9 (2009); Kumar, patients and is largely reversible upon disease remission. M. et al. J. Allergy Clin. Immunol. 128:1077-85 (2011); Col lison, A. et al. J. Allergy Clin. Immunol. 128:160-7 (2011): 0091 Specifically, 21 upregulated and 11 downregulated Jiang, X. Mol. and Cell. Biochem. 353:35-40 (2011): Lu, T. et miRNAs were identified in patients with active EE, including al. J. Immunol. 182:4994-5002 (2009); Lu, T. et al. J. Immu miR-21 and miR-223 as the most upregulated miRNAs and mol. 187:3362-73 (2011)). miR-375 as the most downregulated miRNA in patients with I0086 MiRNAs represent a particularly attractive class of EE. These miRNAs can therefore serve as biomarkers for EE molecules in the regulation of the EE transcriptome, as a alone or in combination with other biomarkers. Three of the single miRNA can target hundreds of genes and can mediate differentially regulated miRNAs in the esophageal biopsies, the epigenetic mechanisms underlying gene-environment namely miR-146a, miR-146b, and miR-223, were also dif interactions, which can have a key but heretofore unexplored ferentially regulated in EE patient plasma samples; these role in EE (see, e.g., Sato, F. et al. Febs J. 278: 1598-609 miRNAs can therefore be used as non-invasive biomarkers (2011)). In addition, miRNA involvement in EE is interesting for EE alone or in combination with other biomarkers. due to the recent identification of a key role of a specific T 0092. This EE-associated miRNA signature correlated helper type 2 (T2)-associated miRNA, namely miR-21, in with the degree of tissue eosinophilia and was distinct from critically regulating T helper cell polarization, as EE involves patients with chronic (non-eosinophilic) esophagitis. The dif a local polarized T2 response (see, e.g., Blanchard, C. et al. ferential miRNA expression was largely reversible in patients J. Allergy Clin. Immunol. 127:208-17 (2011): Lu, T. et al. J. that responded to glucocorticoid therapy. These results there Immunol. 182:4994-5002 (2009); Straumann, A. et al. J. fore demonstrate the dynamic expression of miRNAs in a Allergy Clin. Immunol. 108:954-61 (2001)). human allergic disease and the role for tissue and blood US 2015/0038552 A1 Feb. 5, 2015

miRNAS as biomarkers to provide insight into disease diag 1:289-96 (2008)), and which has recently shown to be a key nosis, response to therapy, and the degree of allergic inflam biomarker for anti-IL-13 responsiveness in human asthma mation. (Corren, J. etal. N. Engl.J.Med. 365:1088-98 (2011)). These 0093. As described herein, the role of miRNA in regulat data provide the first human evidence to substantiate the ing T2-associated diseases, including EE, was established. recent findings that miR-21 critically regulates the polariza Studies on the roles and regulations of the miRNAs differen tion of adaptive immunity in mice (Lu, T. et al. J. Immunol. tially regulated in EE can lead to improved patient diagnosis 187:3362-73 (2011)), supporting the previous finding that and facilitate the development of miRNA mimics and inhibi miR-21 regulates T1 VS. T.2 balances by targeting tors as therapies for EE patients. IL-12p35 expression (Lu, T. etal.J. Immunol. 182:4994-5002 0094. MiR-21, which has been shown to regulate IL-12 (2009)). expression and the balance of Til Vs. T2 responses in mice 0099. As described herein, let-7c was found to be down (see, e.g., Lu, T. etal.J. Immunol. 182:4994-5002 (2009); Lu, regulated, meaning let-7c can be used to regulate IL-13 levels T. et al. J. Immunol. 187:3362-73 (2011)), was found to be (Polikepahad, S. et al. J. Biol. Chem. 285:30139-49 (2010)). one of the most up-regulated miRNAs in EE patients. Due to Up-regulation of miR-146a was found in EE patients. As the high level of species conservation of the miR-21 binding miR-146a has recently been demonstrated to selectively regu site in the 3' untranslated region of IL12p35, miR-21 can have late Treg-mediated suppression of T1 cells (Lu, L. et al. Cell a similar role in human allergic inflammation (see, e.g., Lu, T. 142:914-29 (2010)), up-regulation of miR-146a can suppress et al. J. Immunol. 182:4994-5002 (2009)). The results T1 responses and promote T2 responses. described herein provide the first set of human data that 0100. These findings support a model whereby multiple Substantiate that miR-21 can have a similar role in human miRNAS coordinate polarized T. responses in the pathogen allergic inflammation. esis of EE. Recent human studies on two other T2 associated 0095. Both miR-223 and miR-21 were recently found to diseases, namely atopic dermatitis and ulcerative colitis, have be up-regulated in eosinophilic esophagitis patients (Lu, T. et identified a role for miRNA in regulating T cell proliferation al. J. Allergy Clin. Immunol., 129:1064-1075 e1069 (2012)). and epithelial-derived chemokine production, as well as up They are the top two miRNAs correlated with eosinophil regulation of miR-21 in ulcerative colitis and down-regula levels in patient esophageal biopsies. Using systems biology tion of let-7 in atopic dermatitis (Wu, F. et al. Gastroenterol analysis, miR-223 and miR-21 were found to co-regulate a set ogy 135:1624-1635 (2008); Sonkoly, E. et al. J. Allergy Clin. of interacting target genes involved in eosinophil prolifera Immunol. 126:581-9 (2010)). tion and differentiation (Lu, T. etal. J. Allergy Clin. Immunol., 01.01 One of the defining histological features of EE is 129:1064-1075 e1069 (2012)). Because miR-21 promotes intense eosinophil infiltration in the esophagus. As described cell proliferation, the up-regulation of miR-223 can provide a herein, a majority of the dysregulated miRNAs demonstrate check and balance in the system given the ability of miR-223 significant correlation between miRNA expression level and to promote eosinophil maturation. esophageal eosinophil count, reflecting disease severity. 0096. Furthermore, because miR-21*, a complementary Functional enrichment analyses were performed for the two miRNA of miR-21, was up-regulated after GM-CSF treat miRNAs that most strongly correlated with eosinophil levels, ment and can inhibit the apoptosis of eosinophils (Wong, C. et namely miR-21 and miR-223; these analyses empirically pre al. Immunobiology, 218:255-62 (2013)), the minor miRNAs dicted that both miRNAs regulate levels of tissue eosino can also have a role in regulating the proliferation of eosino philia, demonstrating the interplay between these two miR phil progenitors, adding another level of complexity. Thera NAs in allergic inflammation. Both miRNAs correlated pies targeting miRNAS, including miR-21, miR-223, and significantly with IL-5, a key eosinophil growth factor shown their minor miR* forms, can allow fine-tuning of the eosino to be contributory in murine models of EE and human EE phil level in various diseases. (see, e.g., Mishra, A. et al. Gastroenterology 134:204-14 0097 Upregulation of miR-21 in patients with EE can (2008); Assaad, A. et al. Gastroenterology 141: 1593-604 partially explain the increased T2 cytokine levels and T2 (2011)). responses seen in EE patients. As described herein, esoph 0102) Another significant histological finding in EE ageal miR-21 levels were found to be inversely correlated patients is epithelial basal layer hyperplasia. In particular, with esophageal IL-12p35 levels. Up-regulation of miR-21 in miR-203 is known to repress epithelial cell proliferation and EE patients can therefore partially explain the increased T2 promote epithelial cell differentiation (see, e.g., Yi, R. et al. cytokines and T2 responses seen in EE patients. Studies Nature 452:225-9 (2006)). As such, repression of miR-203 using human data can further elucidate the role of miR-21 in can in part explain the observed epithelial hyperplasia. human allergic inflammation. (0103. Several of the EE-associated miRNAs have been 0098 Co-regulated miR-21 target genes in EE patients linked with esophageal squamous carcinoma or with Bar were found to be significantly enriched in the regulation of T rett's esophagus, including let-7 (Liu, Q. etal. Mol. Biol. Rep. cell polarization and IFNY production. Direct analysis of a 39:1239-46 (2012)), miR-142-3p (Lin, R. et al. J. Surg. myriad of esophageal transcripts for correlation with miR-21 Oncol. 105:175-82 (2011)), miR-203 (Yuan, Y. et al. BMC demonstrated strong correlations with key elements of the EE Cancer 11:57 (2011)), miR-210 (Tsuchiya, S. et al. J. Biol. transcriptome, including cell-specific markers for key inflam Chem. 286:420-8 (2011)), miR-223 (Li, S. et al. J. Biomed. matory cells (such as eosinophils and mast cells), as well as Sci. 18:24 (2011)), miR-375 (Li, X. et al. Dig. Dis. Sci. CCL26 (also known as eotaxin-3), which is functionally 56:2849-56 (2011)), and miR-21 (Matsushima, K. et al. involved in eosinophil recruitment (see, e.g., Shinkai, A. etal. Digestion 82:138-44 (2010)). Some miRNAs, such as miR J. Immunol. 163:1602-10 (1999)) and POSTN (periostin), 21, have been shown to be oncomirs and/or tumor Suppressors which is involved in tissue remodeling (see, e.g., Stansfield, (see, e.g., Medina, P. et al. Nature 467:86-90 (2010); Hatley, W. et al. Ann. Thorac. Surg. 88:1916-21 (2009)) and eosino M. et al. Cancer Call 18:282-93 (2010)). While EE is not philia (see, e.g., Blanchard, C. et al. Mucosal Immunol. considered to be a pre-malignant condition, EE involves US 2015/0038552 A1 Feb. 5, 2015

marked epithelial cell hyperplasia, and these miRNAs can e.g., Assa'ad, A. et al. J. Allergy Clin. Immunol. 119:731-8 have a role in this feature of EE. (2007)). Therefore, an elevated level of miR-146b can pre 0104. As described herein, miR-675 was found to be the dispose EE patients in remission to a relapse. only disease remission-induced miRNA. MiR-675 is derived Targeted Ablation of miR-21 Decreases Murine Eosinophil from the H19 gene, which is a paternally imprinted gene (see, Progenitor Cell Growth e.g., Cai, X. and Cullen, B. RNA 13:313-6 (2007)). The over 0109 MiR-21 has been reported to be up-regulated in a expression of H19 is commonly associated with various can variety of disorders associated with eosinophilia, including cers (see, e.g., Tsang, W. Carcinogenesis 31:350-8 (2010)). asthma (see, e.g., Lu, T. et al. J. Immunol. 182:4994-5002 H19 was previously found to be induced in glucocorticoid (2009)), ulcerative colitis (see, e.g., Wu, F. et al. Gastroen responder patients compared to EE patients or normal con terology 135:1624-35 e1624 (2008)), and eosinophilic esoph trols; this induction was not seen in patients that did not agitis (Lu, T. et al. J. Allergy Clin. Immunol., 129:1064-75 respond to glucocorticoid therapy (Caldwell, J. et al. J. (2012)). MiR-21 has been reported to be pro-proliferative and Allergy Clin. Immunol. 125:879-888 (2010)). anti-apoptotic by targeting multiple tumor Suppressor genes 0105. As described herein, the miR-675 expression pat (see, e.g., Krichevsky, A. and Gabriely, G. J. Cell. Mol. Med. tern closely resembles that of H 19. Since the exact roles of 13:39-53 (2009); Papagiannakopoulos, T. et al. Cancer Res. H19 and its miRNA product, namely miR-675, in the disease 68:8164-72 (2008); Hatley, M. et al. Cancer Cell 18:282-93 remission process have heretofore been unknown, elucidat (2010)). Several of the miR-21 target genes, including Apafl ing their functions can provide information regarding the and PTEN, have been implicated in inhibiting eosinophil disease remission process in EE. Based on the role H19 and proliferation or promoting its apoptosis (see, e.g., Jakiela, B. miR-675 can have in DNA methylation responses and the etal. Rheumatology (Oxford) 48:1202-7 (2009); Adachi, T. et unique overexpression of this miRNA specifically within al. J. Immunol. 17.9:8105-11 (2007)). Although the transcrip patients in remission, miR-675 can be involved in epigenetic tion factor Gfil has been found to repress miR-21 expression programming in the esophageal cells of EE remission during the transition from common myeloid progenitor to the patients. granulocyte/macrophage progenitor stage, thereby promot 0106. As described herein, the expression levels of a ing neutrophil/monocyte differentiating conditions (see, e.g., selected set of EE-associated miRNAs in EE patient plasma Velu, C. et al. Blood 113:4720-8 (2009)), little information samples were measured. MiR-146a, miR-146b, and miR-223 has heretofore been available regarding the function of miR were found to be up-regulated in the EE plasma samples 21 during eosinophil differentiation. compared to controls (allergic individuals without EE). These 0110. As described herein, miR-21 was found to be among miRNAs can therefore serve as non-invasive biomarkers for the most up-regulated miRNAs in patients with EE and has EE alone or in combination with other non-invasive biomar the highest correlation with esophageal eosinophil levels. kers. MiR-21 has been identified as a regulator of eosinophil pro 0107 Plasma miRNAs have been reported to exist both genitor growth and is the first miRNA proven to have a role in within exosomes and in protein-bound vesicle-free form (see, directly regulating eosinophil development. MiR-21 was e.g., Arroyo, J. etal. Proc. Natl. Acad. Sci. U.S.A. 108:5003-8 found to be up-regulated during eosinophil differentiation (2011); Rabinowits, G. et al. Clin. Lung Cancer 10:42-6 from eosinophil progenitors, and targeted ablation of miR-21 (2009)). The circulating miRNAs can be taken up by cells was found to decrease eosinophil progenitor growth. through exosome uptake or pinocytosis (see, e.g., Valadi, H. et al. Nat. Cell Biol. 9:654-9 (2007); Tian, T. et al. J. Cell. 0111. MiR-21 is shown to be progressively up-regulated Biochem. 111:488-96 (2010)). Mast cells have been found to during IL-5-driven eosinophil differentiation from progenitor release exosomes containing miRNA (see, e.g., Valadi, H. et cells in vivo. Eosinophil progenitor cultures derived from al. Nat. Cell Biol. 9:654-9 (2007)). Mast cells also express miR-21 mice were found to have increased levels of apo high levels of both miR-146a and miR-146b (see, e.g., ptosis as indicated by increased levels of annexin V positivity Sonkoly, E. et al. PLoS One 2:e610 (2007); Mayoral, R. et al. compared to those of miR-21" mice. MiR-21 mice were J. Immunol. 182:433-45 (2009)). EE patients have concomi found to have decreased eosinophil colony forming unit tant esophageal mastocytosis (see, e.g., Abonia, J. et al. J. capacity in the bone marrow and reduced blood eosinophil levels in vivo. Therefore, targeted ablation of miR-21 in the Allergy Clin. Immunol. 126:140-9 (2010); Aceves, S. et al. J. eosinophil progenitor cultures leads to reduced eosinophil Allergy Clin. Immunol. 126:1198-204 (2010): Dellon, E. etal. progenitor growth capacity. Am. J. Gastroenterol. 106:264-71 (2011)), which could account for the elevated miR-146a and miR-146b levels seen 0112 Whole genome microarray analysis of miR-2 in the serum of EE patients. Since miR-146a has been found and miR-21 eosinophil progenitor cultures identified dif to selectively promote Treg-mediated Suppression of Til ferentially regulated genes between miR-21" and miR response, an increased circulating level of plasma miR-146a 21 eosinophil progenitor cultures. These included genes can further propagate or help maintain the T2 responses seen involved in cell proliferation (e.g. Ms4a3, Grb7, and Pik3ró), in EE patients. cell cycle, and immune response; therefore, these pathways 0108. As described herein, while plasma miR-146a and were identified as those pathways in eosinophils most signifi miR-223 returned to baseline levels during EE remission, cantly affected by miR-21. miR-146b remained elevated in EE remission patients. While 0113. These results demonstrate that miR-21 can directly the specific role of miR-146b in regulating adaptive immune regulate the development of eosinophils by influencing the responses has not been investigated, miR-146a and miR-146b growth capacity of eosinophil progenitors. Since mature eosi have an identical seed sequence that is critical for miRNA nophils lose their proliferative capacity and do not divide, the mediated target gene expression. Therefore, miR-146b can up-regulation of miR-21 can prevent premature loss of the also suppress T1 responses and promote T2 responses. EE proliferative potential of eosinophil progenitors. Further elu patients in remission often relapse as time progresses (see, cidation of the roles of miR-21 in regulating eosinophil levels US 2015/0038552 A1 Feb. 5, 2015 and immunoinflammatory responses can lead to therapeutic (Krichevsky, A. and Gabriely, G.J. Cell. Mol. Med. 13:39-53 options for eosinophilic disorders. (2009); Hatley, M. et al. Cancer Cell 18:282-93 (2010)). As 0114. No differentially regulated genes were identified at described herein, increased levels of apoptosis were found in day 4 of the eosinophil progenitor culture, Supporting previ miR-21 eosinophil cultures compared to miR-21" cul ous findings that progenitor cell growth under the influence of tures. Potential eosinophil hematopoiesis defects were inves stem cell factor (SCF) and Flt-3L is not regulated by miR-21. tigated in the miR-21 mice in vivo. The miR-21 mice There were 38 differentially regulated genes that were iden were found to have both decreased eosinophils in the blood tified between days 8 and 14, with only one (Psrc 1) being a and decreased eosinophil colony forming unit capacity in the predicted target of miR-21. Computational analysis identified bone marrow, consistent with the observed phenotype in the an overall functional effect exactly in the pathways (e.g. ex vivo eosinophil cultures. regulation of cell proliferation and cell cycle) associated with 0119. MiR-21 has been found to be over-expressed in the observed phenotype and the known role of miR-21 in allergic diseases with significant eosinophilia, including other systems (Krichevsky, A. and Gabriely, G.J. Cell. Mol. experimental asthma in mice and human EE (Lu, T. et al. J. Med. 13:39-53 (2009); Hatley, M. et al. Cancer Cell 18:282 Immunol. 182:4994-5001 (2009); Wu, F. et al. Gastroenter 93 (2010)). As such, miR-21 can exert some effects on direct ology 135:1624-1635 e1624 (2008); Lu, T. et al. J. Immunol. targets that synergistically interact to ultimately regulate eosi 187:3362-73 (2011): Lu, S. et al. PLoS One, 7:e40676 nophilopoeisis. Moreover, miR-21 can regulate additional (2012)). MiR-21 was previously found to be capable of regu genes at the protein level that were not identified by the lating immunoinflammatory responses by targeting the IL12/ genomic screen in the current study. The observed decreased IFNY pathway (Lu, T. et al. J. Immunol. 182:4994-5001 growth capacity of the miR-21 eosinophil progenitors is (2009): Lu, T. et al. J. Immunol. 187:3362-73 (2011)). Regu likely due to modest regulation of a combination of miR-21 lation of inflammatory response was also found to be one of targets. the significantly enriched pathways represented by the differ 0115 More than a dozen anti-proliferative genes have entially expressed genes in the miR-21 deficient eosinophil been reported as miR-21 targets, including Pdcd4, Pten, progenitor cultures. These results indicate that miR-21 can Tpm1, Apafl. Btg2, Map2k3, RhoB, and Ski, among many have additional roles in regulating the immunoinflammatory others (Krichevsky, A. and Gabriely, G. J. Cell. Mol. Med. responses beyond regulation of the IL12/IFNY pathway. 13:39-53 (2009); Hatley, M. et al. Cancer Cell 18:282-93 Because miR-21 can also affect eosinophil progenitor (2010)). Among these, over-expression of Pten has been growth, therapeutic interventions targeting miR-21 can shown to attenuate eosinophil Survival (Adachi, T. et al. J. reduce the levels of eosinophilia in Some circumstances. Immunol. 179:8105-11 (2007)), and Apaf-1 is part of the I0120 In summary, miR-21 has been identified as a regu intrinsic apoptosis pathway that is Suppressed by IL-5 signal lator of eosinophil progenitor growth. This represents the first ing (Dewson, G. et al. Blood 98.2239-47 (2001)). The miRNA demonstrated to have a direct role in regulating eosi observed decreased growth capacity of the miR-21 eosi nophil development. Further elucidating and understanding nophil progenitors can be due to modest regulation of a com the roles of miR-21 in regulating the levels of eosinophils and bination of miR-21 targets. in immunoinflammatory responses can lead to additional 0116 Psrc 1, one of the up-regulated genes, is a predicted therapeutic options for eosinophilic disorders. target of miR-21 based on sequence conservation and binding site potential (Lu, T. et al. J. Immunol. 182:4994-5002 MiR-223 Deficiency Increases Eosinophil Progenitor Cell (2009)). Over-expression of Psrc1 has been shown to sup Growth press colony formation in lung carcinoma cells (Lo, P. et al. I0121. MiR-223 has been found to be over-expressed in Oncogene 18:7765-74 (1999)). The up-regulation of Psrc1 asthma, EE, and atopic dermatitis, where eosinophils are could potentially contribute to the decreased growth of miR implicated in the disease pathogenesis to varying degrees 21 eosinophil progenitors. (Lu, T. etal.J. Immunol. 182:4994-5002 (2009); Garbacki, N. 0117. As described herein, the gene Pik3ró, a regulatory et al. PLoS One 6:e16509 (2011); Sonkoly, E. et al. J. Allergy Subunit for phosphoinositide 3-kinase (PI3 kinase) gamma, Clin. Immunol. 126:581-9 (2010); Mattes, J. et al. Proc. Natl. was over-expressed in both day 8 and day 12 in the miR-21 Acad. Sci. U.S.A. 106:18704-0 (2009)). MiR-223 has been eosinophil progenitor cultures. PI3 kinase signaling has been shown to target the IGF1 receptor (IGF1R) (see, e.g., Johnni shown to be essential for IL-5 mediated eosinophil Survival dis, J. et al. Nature 451:1125-9 (2008)), which is the major (Rosas, M. et al. J. Leukoc. Biol. 80:186-95 (2006)). Pik3ró physiologic receptor for IGF1 (see, e.g., Smith, T. Pharma has been shown to be expressed primarily in the hematopoi col. Rev. 62: 199-236 (2010)). IGF1 is a major anabolic hor etic compartment and can potentially compete with Pik3r5 mone that stimulates cell growth and is a potent inhibitor of for binding with p110y (Suire, S. et al. Curr. Biol. 15:566-70 programmed cell death; therefore IGF1R can be differentially (2005)). The Pik3ró/p110y heterodimer is four-fold less sen regulated by miR-223. Although IGF1 has not been previ sitive than the Pik3r5/p110y heterodimer (Suire, S. etal. Curr: ously examined for its impact on eosinophil progenitors, Biol. 15:566-70 (2005)). As described herein, Pik3ró and IGF1 and IGF1R inhibitors can be clinically useful for eosi Pik3r5 are expressed at similar levels in the wild type eosi nophilic disorders. The expression of miR-223 has been nophil progenitors. Up-regulation of Pik3ró can lead to an shown to be mediated by myeloid transcription factors PU. 1 increased level of Pik3ró/p110y heterodimer and a decreased and C/EBP, factors that are important in eosinophilopoiesis level of Pik3r5/p110y heterodimer, thereby attenuating PI3 (see, e.g., Fukao, T. et al. Cell 129:617-31 (2007)). kinase signaling. This can in part account for the decreased I0122. As described herein, miR-223 was found to regulate growth seen in miR-21 eosinophil progenitors. the proliferation and differentiation of eosinophil progeni 0118. MiR-21 has been known to promote cell growth in tors; miR-223 was also found to be up-regulated during eosi various cell types, most notably in tumor cells, by targeting a nophil differentiation in an ex vivo bone marrow-derived variety of pro-apoptotic genes both directly and indirectly eosinophil culture model. MiR-223-deficient eosinophil pro US 2015/0038552 A1 Feb. 5, 2015 genitor cells were found to have a hyperproliferative capacity. of miR223, although the former is regulated by miR223. Mechanistic analysis identified a contributory role for the Therefore, IGF1 is a new pathway involved in eosinophil IGF1 receptor (IGF1R) in mediating eosinophil progenitor development for which pharmacological blockade (indepen cell proliferation. dent of miR223) demonstrates a positive effect. This is the 0123 Gene expression analysis followed by systems bio first report of the relationship between IGF1 and IGF1R in logical analysis identified the role of miR-223 in hematopoi eosinophilia. etic development and cellular growth and function. Consis I0129. While the up-regulation of IGF1R can have a con tent with this prediction, miR-223 mice had a delay in tributory role in the increased growth seen in the miR-223 eosinophil differentiation as assessed by CCR3 expression. eosinophil progenitor cultures, additional pathways can be These data demonstrate that miRNAs can directly regulate involved as well. As described herein, whole genome the development of eosinophils by influencing the prolifera microarray analysis identified multiple additional growth tion and differentiation of eosinophil progenitor cells. and proliferation-related genes differentially regulated 0.124. MiR-223 was found to regulate the growth and dif between miR-223" and miR-223 eosinophil progenitor ferentiation of eosinophil progenitors. MiR-223 was found to cultures at day 8 of the culture, before the onset of the be up-regulated in an ex vivo bone marrow-derived eosino increased growth, and at day 12. There was a specific enrich phil differentiation culture. Targeted ablation of miR-223 ment in genes that regulate hematologic cell development leads to an increase in eosinophil progenitor growth, as miR known to be involved in eosinophilopoiesis. These include 223 cells had a markedly increased growth in response to down-regulation of NAD(P)H:cquinone oxidoreductase 1 the eosinophil growth factor IL-5. In addition, miR-223 defi (NOO1), a cytosolic protein protecting cells against oxidative ciency led to a defect in eosinophil maturation, as indicated stress, and inhibitor of DNA binding 2 (ID2), an inhibitor of by a delayed up-regulation of surface CCR3 expression. the basic helix-loop-helix family of transcription factors. 0.125 Up-regulation was found for the miR-223 target NQO1-deficient mice have been shown to have a significant gene IGF1R in eosinophil cultures derived from miR-223 increase in blood granulocytes, including eosinophils (Long, mice compared to miR-223" littermate controls (Johnnidis, D. et al. Cancer Res.62:3030-6 (2002)). Silencing of ID2 has J. et al. Nature 451:1125-9 (2008)). The up-regulation of been shown to cause increased eosinophil progenitor growth IGF1R coincided with the onset of the increased growth seen and delayed eosinophil progenitor differentiation (Buiten in the miR-223 eosinophil culture. MiR-223 has therefore huis, M. et al. Blood 105:4272-81 (2005)). been identified as a regulator of eosinophil IGF1R levels. 0.130. Analysis of eosinophil maturation of the bone mar 0.126 The increased growth observed in the eosinophil row-derived eosinophils indicated that the increased growth cultures derived from miR-223 mice was found to be of the miR-223 eosinophil progenitor was associated with reversible upon treatment with an IGF1R inhibitor. The a delay in differentiation. The finding that miR-223 can regu growth of miR-223" eosinophil progenitors can also be late the development of eosinophils by influencing eosinophil inhibited by an IGF1R inhibitor. These data demonstrate that progenitor growth and differentiation can therefore be used to the increased proliferation seen in miR-223 eosinophil design potential therapeutic interventions targeting or affect cultures has not bypassed the IGF1R pathway and provide the ing levels of miR-223. first demonstration that IGF1R is involved in eosinophil I0131. In summary, miR-223 has been identified as a regu development. lator of eosinophil progenitor proliferation. IGF1R is up 0127. Several IGF1R inhibitors are currently under devel regulated during in eosinophil development, and miR-223 is opment for the treatment of various types of cancer (Yee, D. a regulator of IGF1R levels. The roles and regulations of Journal of the National Cancer Institute, 104:975-981 miRNAs during eosinophil development can be utilized to (2012)). These data indicate that the IGF1R inhibitors can lead to novel therapeutic targets for eosinophilic disorders. potentially also be used to treat patients with eosinophilia, MiR-375 Regulates an IL-13-Induced Epithelial Such as the hypereosinophilic syndrome (Arefi. M. et al. Transcriptome International Journal of Hematology, 96:320-326 (2012)). Furthermore, miR-223 has been identified as a regulator of 0.132. As described herein, a lentiviral strategy and whole eosinophil IGF1R levels. While the up-regulation of IGF1R transcriptome analysis were used in epithelial cells to dem likely has a contributory role in the increased proliferation onstrate that miR-375 over-expression was sufficient to mark seen in the miR-223 eosinophil progenitor cultures, this edly modify IL-13-associated immunoinflammatory does not preclude the involvement of additional pathways. In pathways in epithelial cells in vitro, further Substantiating particular, this microarray analysis identified multiple addi interactions between miR-375 and IL-13. These results dem tional growth and proliferation-related genes differentially onstrate that miRNAS have a key role in regulating and fine regulated between miR-223" and miR-223 cultures. tuning IL-13 mediated responses, and miR-375 is a key These include down-regulation of NAD(P)H:cquinone oxi downstream mediator of IL-13-induced responses. doreductase 1 (NQO1), where NQO1 deficient mice have 0.133 MiRNA array analysis was used to determine the been found to have a significant increase in blood granulo differentially expressed miRNAs after IL-13 stimulation in cytes including eosinophils (Long, D. et al. Cancer Res., two distinct human epithelial cell types, namely esophageal 62:3030-3036 (2002)). Down-regulation of inhibitor of DNA squamous cells and bronchial columnar cells. Among the binding 2 (ID2), whose knockdown has been shown to cause IL-13-regulated miRNAs, miR-375 showed a conserved pat increased eosinophil progenitor growth and delayed eosino tern of down-regulation between these two epithelial cell phil progenitor differentiation, was also observed (Buiten types. MiR-375 levels were analyzed in an IL-13-induced huis, M. et al. Blood, 105:4272-4281 (2005)). murine asthma model, and down-regulation was observed in 0128 IGF1R is expressed by eosinophil progenitors, and the murine asthmatic lungs. an IGF1R inhibitor (pricopodphyllin) inhibits eosinophil pro 0.134 Direct examination of human allergic tissue, from genitor cell growth. Both of these findings occur independent esophageal biopsies from patients with EE, indicated that US 2015/0038552 A1 Feb. 5, 2015

miR-375 was inversely related to the degree of allergic Immunol. 126:581-9 (2010)) and ulcerative colitis (Wu, F. et inflammation, including esophageal eosinophil levels and al. Gastroenterology 135:1624-35 (2008)), and hyperprolif gene expression levels of T2 cytokines and mast cell specific erative diseases, such as esophageal squamous carcinoma proteases. MiR-375 over-expression was sufficient to mark (Kong, K. et al. Gut 61:33-42 (2011)), up-regulation of miR edly modify IL-13-associated immunoinflammatory path 375 in a T2-associated disease in humans has not been ways in epithelial cells in vitro. These results support a key reported. Therefore, IL-13 can have the long-term effect of role of miRNAs, particularly miR-375, in regulating and fine down-regulating miR-375 expression. tuning IL-13 mediated responses. 0.141. MiR-375 has been previously shown to enhance 0135 MiRNAs were identified that were differentially goblet cell differentiation by repressing KLF5 expression regulated after IL-13 stimulation in human bronchial colum (Biton, M. et al. Nat. Immunol. 12:239-46 (2011)) and has nar and esophageal squamous epithelial cells. Among the also been shown to attenuate cell proliferation by targeting IL-13-regulated miRNAs, miR-375 was found to be the only IGF1R, PDK1, andYWHAQ (Kong, K. et al. Gut 70:2239-49 miRNA that was down-regulated in both epithelial cell types (2011); Tsukamoto, Y. et al. Cancer Res. 70:2339-49 (2010)). after IL-13 stimulation in EE patient samples compared to As described herein, neither of these pathways was affected in control patients. EE patients or upon analysis of miR-375-regulated genes in 0136. As described herein, analysis of different human esophageal epithelial cells (Blanchard, C. etal.J. Clin. Invest. cell types involved in allergic inflammation identified the 116:536-47 (2006)), indicating that the activity of miR-375 highest expression of miR-375 in epithelial cells. MiR-375 may be dependent on the cellular context, consistent with was found to be inversely correlated with the level of esoph previous reports (Tsukamoto, Y. et al. Cancer Res. 70:2339 ageal eosinophils and expression of the mast cell specific 49 (2010); de Souza Rocha Simonini, P. et al. Cancer Res. genes CPA3 and TPSAB1. The down-regulation of miR-375 70:9175-84 (2010)). was specific to EE patients; the chronic esophagitis patients 0142. MiR-375 has been previously reported to regulate had miR-375 expression levels comparable to normal con TSLP expression in an HT-29 human colonic adenocarci trols. Disease remission with either fluticasonetherapy or diet noma cell line (Biton, M. et al. Nat. Immunol. 12:239-46 modification was associated with normalization of miR-375 (2011)). In this study, TSLP and miR-375 were concomi levels; this can be due to the result of reduced IL-13; patients tantly induced by IL-13 in HT-29 cells, and knockdown of that did not respond to fluticaSone therapy continued to have miR-375 inhibited TSLP production. In addition, over-ex repressed miR-375 levels. pression of miR-375 induced TSLP expression in HT-29 cells 0.137 Modulation of miR-375 levels was found to be suf (Biton, M. etal. Nat. Immunol. 12:239-46 (2011)). TSLP has ficient to regulate IL-13 mediated gene expression, particu been shown to have an important role in EE pathogenesis larly with pathways involved in immunoinflammatory pro (see, e.g., Rothenberg, M. et al. Nat. Genet. 42:289-91 cesses. Levels of miR-375 markedly inversely correlated with (2010); Sherrill, J. et al. J. Allergy Clin. Immunol. 126:160-5 a large set of immunoinflammatory genes (including IL-13) (2010)). As described herein, the ability of miR-375 to regu in the esophagus of patients with EE. late TSLP expression in esophageal epithelial cells was ana 0.138. As described herein, a genome-wide transcriptome lyzed. Additionally, miR-375 was found to have no effect on based approach was used to demonstrate that miR-375 can TSLP production, and there was no correlation between miR potentiate and repress IL-13-mediated effects, indicating the 375 and TSLP in the esophageal samples. The disparity complex interaction between cytokine and miRNA-mediated between these results and previous studies could be due to the gene regulation; miR-375 was therefore sufficient to regulate use of different cell types and/or different mechanisms in an IL-13-induced epithelial transcriptome. The inflammatory TSLP induction in these cells, since previous studies report diseases and immunological diseases are the two most sig that IL-13 induces TSLP expression in HT-29 cells but not in nificantly over-represented disease states regulated by miR esophageal epithelial cells (Blanchard, C. et al. J. Allergy 375. These include allergy-associated genes, such as MMP12 Clin. Immunol. 120:1292-1300 (2007); Biton, M. et al. Nat. and MUC4 (Mukhopadhyay, S. et al. J. Allergy Clin. Immu Immunol. 12:239-46 (2011)). mol. 126:70-6 (2010); Lavigne, M. et al. Biochem. Biophys. 0.143 IL-13 has been found to down-regulate miR-375 Res. Comm. 324:534-54 (2004); Fahy, J. Am. J. Respir: Crit. and modulate IL-13 regulated gene expression in bronchial Care Med. 164:S46-51 (2001); Pouladi, M. et al. Am. J. epithelial cells; this is relevant to asthma and other IL-13 Respir: Cell. Mol. Biol. 30:84-90 (2004)). mediated diseases. However, it is unclear whether over-ex 0.139. As further described herein, miR-375 expression pression of miR-375 can correct the allergic phenotype in was found to be unchanged after 2 hours of IL-13 stimulation, asthma and EE. This can be resolved in future studies utilizing despite the finding of up-regulation of miR-375 after 2 hours miR-375 lung and/or esophageal epithelial specific trans of IL-13 stimulation reported in a previous study (Biton, M. et genic mice. al. Nat. Immunol. 12:239-46 (2011)). This inconsistency 0144 Previous reports have indicated that the miRNAs could be due to the use of the HT-29 human colon adenocar miR-203 and miR-223 are differentially regulated in T2 cinoma cell line, as opposed to human esophageal squamous associated diseases (Wu, F. etal. Gastroenterology 135:1624 cells and bronchial columnar cells. MiR-375 was found to be 35 (2008); Sonkoly, E. et al. J. Allergy Clin. Immunol. 126: down-regulated after 24 and 48 hours of IL-13 stimulation, 581-9 (2010)). As described herein, 10 other miRNAs in which is consistent with the previous finding that miR-375 addition to miR-375 were identified that were differentially levels were at or below baseline after 16 hours of IL-13 regulated in either human esophageal epithelial cells or stimulation (Biton, M. et al. Nat. Immunol. 12:239-46 human bronchial epithelial cells, reflecting cell type-specific (2011)). effects of IL-13 stimulation. 0140. While miR-375 down-regulation has been reported (0145 MiR-375 expression levels were found to reflect in patient samples from multiple T2-associated diseases, disease activity, normalize with remission, and inversely cor Such as atopic dermatitis (Sonkoly, E. et al. J. Allergy Clin. relate with the degree of allergic inflammation. MiR-375 was US 2015/0038552 A1 Feb. 5, 2015

strongly associated with parameters germane to allergic NAS are selected from FIG.1. In some embodiments, at least responses, including eosinophil levels, gene expression levels 4 miRNAs are selected from FIG.1. In some embodiments, at of the T2 cytokines IL-5 and IL-13, the mast cell-specific least 5 miRNAs are selected from FIG. 1. In some embodi enzymes CPA3 and TPSAB1, and POSTN (the gene that ments, at least 10 miRNAs are selected from FIG.1. In some encodes periostin). Periostin has been demonstrated to have a embodiments, at least 15 miRNAs are selected from FIG. 1. key role in IL-13 associated remodeling responses (Blan In some embodiments, at least 20 miRNAs are selected from chard, C. et al. Mucosal. Immunol. 1:289-96 (2008)), and its FIG. 1. In some embodiments, at least 25 miRNAs are level predicts responsiveness to anti-IL-13 therapy in humans selected from FIG.1. In some embodiments, at least 30 miR (Corren, J. etal. N. Engl. J. Med. 365:1088-98 (2011)); there NAS are selected from FIG. 1. In some embodiments, all of fore, the finding that miR-375 strongly correlates with human the miRNAS are selected from FIG. 1. POSTN levels in vivo, as described herein, can therefore be used to design potential therapeutic interventions that modu 0152. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, or 9 late levels of miR-375. miRNAs are selected from FIG.1. In some embodiments, 10, Treatment Based on miRNA Levels 11, 12, 13, 14, 15, 16, 17, 18, or 19 miRNAs are selected from 0146 Embodiments of the invention are directed to meth FIG.1. In some embodiments, 20, 21, 22, 23, 24, 25, 26, 27, ods of treating EE in a patient, wherein the methods comprise 28, or 29 miRNAs are selected from FIG.1. In some embodi analyzing the sample from a patient to determine a level of ments, 30, 31, or 32 miRNAs are selected from FIG. 1. one or more miRNAs associated with EE, determining whether the level of the one or more miRNAs is up-regulated 0153. In some embodiments, anywhere between 1 to 10 or down-regulated relative to a level of the one or more miRNAs are selected from FIG. 1. In some embodiments, miRNAs measured in a normal individual, wherein the pres anywhere between 1-20 miRNAs are selected from FIG.1. In ence of an elevated or reduced level of one or more miRNAs some embodiments, anywhere between 1-30 miRNAs are associated with EE results in the patient being diagnosed with Selected from FIG. 1. EE, and treating the patient with an appropriate therapeutic 0154. In some embodiments, the miRNAs associated with strategy based upon the diagnosis. EE are measured using one or more methods and/or tools, 0147 Embodiments of the invention are also directed to including for example, but not limited to, Taqman (Life Tech methods of distinguishing EE from other disorders in a sub nologies, Carlsbad, Calif.), Light-Cycler (Roche Applied ject, wherein the methods comprise analyzing the sample Science, Penzberg, Germany), ABI fluidic card (Life Tech from a patient to determine a level of one or more miRNAs nologies), NanoString R (NanoString Technologies, Seattle, associated with EE, determining whether the level of the one Wash.), NANODROPR) technology (Thermo Fisher Scien or more miRNAs is up-regulated or down-regulated relative tific (Wilmington, Del.), fluidic card, and the like. The person to a level of the one or more miRNAs measured in a normal of skill in the art will recognize such other formats and tools, individual, wherein the presence of an elevated or reduced which can be commercially available or which can be devel level of one or more miRNAs associated with EE results in the oped specifically for Such analysis. patient being diagnosed with EE or with another disorder, and treating the patient with an appropriate therapeutic strategy (O155 Determination of the miRNA level(s) as described based upon the diagnosis. In some embodiments, the other herein can be combined with determination of the levels of disorder is chronic esophagitis. one or more non-miRNA biomarkers associated with EE. For 0148 Embodiments of the invention are also directed to example, determination of the miRNA level(s) as described methods of determining whether a subject with EE has active herein can be combined with determination of the levels of EE or remission EE, wherein the methods comprise analyzing one or more genes of the EE transcriptome. Such a determi the sample from a patient to determine a level of one or more nation can include measurement of the gene DNA or RNA, or miRNAs associated with EE, determining whether the level the gene product. Such genes can include, for example, of the one or more miRNAS is up-regulated or down-regu eotaxin-3, and the like. lated relative to a level of the one or more miRNAs measured 0156 Embodiments of the invention are also directed to in a normal individual, wherein the presence of an elevated or methods of treating an eosinophilic disorder (other than EE) reduced level of one or more miRNAs associated with EE in a patient, wherein the methods comprise analyzing the results in the patient being diagnosed with active EE or remis sample from a patient to determine a level of one or more sion EE, and treating the patient with an appropriate thera miRNAs associated with an eosinophilic disorder, determin peutic strategy based upon the diagnosis. ing whether the level of the one or more miRNAs is up 0149. In embodiments of the invention, the one or more regulated or down-regulated relative to a level of the one or EE-associated miRNAs are selected from the list of genes more miRNAs measured in a normal individual, wherein the dysregulated in EE provided in FIG. 1. presence of an elevated or reduced level of one or more 0150. In some embodiments, at least one miRNA is miRNAs associated with an eosinophilic disorder results in selected from FIG.1. In some embodiments the at least one the patient being diagnosed with an eosinophilic disorder, and miRNA includes miR-21. In some embodiments, at least one treating the patient with an appropriate therapeutic strategy miRNA is selected from FIG.1. In some embodiments the at based upon the diagnosis. Eosinophilic disorders other than least one miRNA includes miR-21. In some embodiments the EE include, for example, eosinophilic gastrointestinal disor at least one miRNA includes miR-223. In some embodiments der (EGID) outside of the esophagus, asthma, and the like. In the at least one miRNA includes miR-375. In some embodi embodiments of the invention, miRNAs associated with ments the at least one miRNA includes miR-146a. In some asthma include miR375. The method of Claim 29, wherein embodiments the at least one miRNA includes miR-146b. the one or more miRNAS associated with asthma comprises 0151. In some embodiments, at least 2 miRNAs are miR375. In some embodiments, the sample comprises lung selected from FIG.1. In some embodiments, at least 3 miR and/or lung epithelial cells. US 2015/0038552 A1 Feb. 5, 2015

EE Therapies cific antibodies for IL-33 blockade, notch signaling inhibitors O157 Certain embodiments of the invention involve for notch inhibition, and the like. Specific examples of such administering EE therapies, including allergen removal, Ste compounds include, for example, IL-33-specific antibodies, roid treatment, dietary management, and the combination of notch signaling inhibitors. Such as Semagacestat, and the like steroid treatment and dietary management. EE therapies also 0.161. In some embodiments, EE can be treated through include the use of proton pump inhibitors (PPIs), topical the blockade of eosinophil production, such as through IL-5R glucocorticoids, such as fluticasone, budesonide, or blockade, and the like. Compounds that can be used for these ciclesonide, humanized antibodies against relevant cytokines purposes include, for example, IL-5RC-specific antibodies and/or mediators, such as eotaxin-1, eotaxin-3, IL-13, IL-5, for IL-5R blockade, and the like. Specific examples of such IL-5RC, CD49D, SIGLEC-8, IgE, CD300A, TSLP, and/or compounds include, for example, IL-5RC.-specific antibod IL-33, small molecule inhibitors of an eosinophiland/or aller ies, such as benralizumab, and the like. gic disease activation pathway, such as a notch-signaling 0162 Certain embodiments of the invention involve using inhibitor or an inhibitor or antagonist of CCR3. CCL11, miRNAs or modified miRNAs as therapeutic targets or VLA4. CRT2, prostaglandin D2, histamine H4 receptor, agents. For example, any miRNA(s) associated with EE IL-13, IL-4, and/or the common 0 chain, and Small molecule found to be elevated relative to the level(s) of the one or more inhibitors capable of modulating miRNA levels and/or as miRNAS measured in a normal individual or using one or severing as stem-loop processing inhibitors. more corresponding modified miRNA(s) can be used as a 0158. In some embodiments, EE can be treated through therapeutic target or agent. the blockade of eosinophil recruitment, such as through 0163. In some embodiments, EE can be treated by modu CCR3 and/or CCL11 inhibition, adhesion molecule inhibi lating one or more miRNAs via one or more of a number of tion, CRTH2 and prostaglandin D2 inhibition, histamine H4 approaches, including the use of anti-miRNA oligonucle receptor inhibition, IL-13 and/or IL-4 blockade, and the like. otides (antagomirs, or AMOS), antisense oligonucleotides Compounds that can be used for these purposes include, for (ASOs), locked nucleic acids (LNAs) which modify miRNAs example, Small molecule CCR3 antagonists and/or eotaxin or serve as modified antisense oligonucleotides, RNA com 1-specific antibodies for CCR3 and/or CCL11 inhibition, petitive inhibitors or decoys (miRNA sponges), small mol CD49D-specific antibodies and/or small molecule VLA4 ecule inhibitors of miRNA stem-loop processing, and viral antagonists for adhesion molecule inhibition, CRT2 antago vectors expressing one or more miRNA genes, including nists for CRT2 and prostaglandin D2 inhibition, Small mol lentiviral vectors (LVs), adenoviral vectors (AVs), and adeno ecule histamine H4 receptor antagonists for histamine H4 associated virus (AAV), and the like. For example, EE receptor inhibition, and IL-13-specific antibodies, IL-4RC. therapy can involve the administration of an antagomir antagonists, IL-4 variants for IL-13 and/or IL-4 blockade, and directed against a miRNA found to be elevated relative to the the like. Specific examples of Such compounds include, for level(s) of the one or more miRNAs measured in a normal example, Small molecule CCR3 antagonists, such as individual, a miR-21, miR-223, miR-146a, and/or miR-146b LH31407, eotaxin-1-specific antibodies, such as bertili antagomir, an IGF1 or IGF1R inhibitor, such as NVP mumab, CD49D-specific antibodies, such as natalizubam, AEW541 and/or pricopodophyllin, and the like. Small molecule VLA4 antagonists, such as compound 1. 0164. The example targeting strategies and compounds CRT2 antagonists, such as OC000459, small molecule his presently provided are intended to be representative. One of tamine H4 receptor antagonists, such as INCB38579, IL-13 skill in the art will recognize that different compounds from specific antibodies, such as lebrikizumab, IL-4RC. antago those listed above can be used to achieve a comparable out nists. Such as AMG 317, IL-4 variants, such as pitrakinra, and come and how to identify Such compounds. the like. 0.165 Competitive antagonists of a given miRNA can be 0159. In some embodiments, EE can be treated through generated using previously described techniques (Krutzfeldt the inhibition of eosinophil survival, such as through IL-5 J. et al. Nature. 438:685-9 (2005); Burnett, J. and Rossi, J. and/or IL-5RC. blockade, SIGLEC-8 agonism, IgE blockade, Chem. Biol. 19:60-71 (2012); van Rooij, E. et al. Circ. Res. activation of inhibitory receptors, TSLP inhibition, and the 110:496-507 (2012)); these include modification of the phos like. Compounds that can be used for these purposes include, phorothioate (PS) backbone, LNA modification, chemical for example, IL-5-specific antibodies, IL-5RC.-specific anti Substitution at the 2'-position of the Sugar ring, cholesterol bodies, and/orantisense oligonucleotides directed against the conjugation at the 3' or 5' end through a hydroxyprolinol common 0 chain for IL-5 and/or IL-5RC. blockade, SIGLEC linkage, and use of a 'guide Strand designed to mimic the 8-specific antibodies for SIGLEC-8 agonism, IgE-specific miRNA of interest, and the like. The chemistry involved in the antibodies for IgE blockade, CD300A-specific antibodies for targeting of miRNAs is the same as that involved in other activation of inhibitory receptors, TSLP-specific antibodies RNA-directed therapies, such as siRNA, shRNA, and the like, for TSLP inhibition, and the like. Specific examples of such and the relevant issues relating to drug delivery are the same. compounds include, for example, IL-5-specific antibodies, 0166 Heretofore unknown anti-miRNA therapeutics can Such as mepolizumab and reslizumab, IL-5RC.-specific anti be developed by the screening of various compounds. Com bodies. Such as benralizumab, antisense oligonucleotides pounds that can be screened to determine their utility as directed against the common B chain, such as TPI ASM8. anti-miRNA therapeutics include for example, but are not SIGLEC-8-specific antibodies, IgE-specific antibodies, such limited to, libraries of known compounds, including natural as omalizumab, CD300A-specific antibodies, TSLP-specific products, such as plant or animal extracts, synthetic chemi antibodies, such as AMG 157, and the like cals, biologically active materials including proteins, pep 0160. In some embodiments, EE can be treated through tides such as soluble peptides, including but not limited to the inhibition of eosinophil activation, such as through IL-33 members of random peptide libraries and combinatorial blockade, notch inhibition, and the like. Compounds that can chemistry derived molecular libraries made of D- or L-con be used for these purposes include, for example, IL-33-spe figuration amino acids, or both, phosphopeptides (including, US 2015/0038552 A1 Feb. 5, 2015 but not limited to, members of random or partially degener involve administering chronic esophagitis therapies, includ ate, directed phosphopeptide libraries), antibodies (includ ing antacid administration, H2 agonist administration, and/or ing, but not limited to, polyclonal, monoclonal, chimeric, PPI therapy. human, anti-idiotypic or single chain antibodies, and Fab, F(ab') and Fab expression library fragments, and epitope Administration binding fragments thereof), organic and inorganic molecules, (0171 The miRNAs, modified miRNAs, or anti-miRNAs and the like. used as therapeutic targets or agents can be administered via 0167. In addition to the more traditional sources of test oral or parenteral delivery routes (Subcutaneous or intrave compounds, computer modeling and searching technologies nous), as has been described previously (van Rooij. E. et al. permit the rational selection of test compounds by utilizing Circ. Res. 110:496-507 (2012)). Such therapeutics can be structural information from the ligand binding sites relevant administered by any pharmaceutically acceptable carrier, proteins. Such rational selection of test compounds can including, for example, any and all solvents, dispersion decrease the number of test compounds that must be screened media, coatings, antibacterial and antifungal agents, isotonic in order to identify a therapeutic compound. Knowledge of and absorption delaying agents, and the like, compatible with the sequences of relevant proteins allows for the generation of pharmaceutical administration. The use of Such media and models of their binding sites that can be used to screen for agents for pharmaceutically active Substances is known in the potential ligands. This process can be accomplished in several art. Except insofar as any conventional medium or agent is manners known in the art. A preferred approach involves incompatible with the active compound, Such media can be generating a sequence alignment of the protein sequence to a used in the compositions of the invention. Supplementary template (derived from the crystal structures or NMR-based active compounds can also be incorporated into the compo model of a similar protein(s), conversion of the amino acid sitions. A pharmaceutical composition of the invention is structures and refining the model by molecular mechanics formulated to be compatible with its intended route of admin and visual examination. If a strong sequence alignment can istration. Routes of administration include for example, but not be obtained then a model can also be generated by build are not limited to, intravenous, intramuscular, and oral, and ing models of the hydrophobic helices. Mutational data that the like. Additional routes of administration include, for point towards residue-residue contacts can also be used to example, Sublingual, buccal, parenteral (including, for position the helices relative to each other so that these con example, Subcutaneous, intramuscular, intraarterial, intrader tacts are achieved. During this process, docking of the known mal, intraperitoneal, intracisternal, intravesical, intrathecal, ligands into the binding site cavity within the helices can also or intravenous), transdermal, oral, transmucosal, and rectal be used to help position the helices by developing interactions administration, and the like. that would stabilize the binding of the ligand. The model can 0172 Solutions or suspensions used for appropriate routes be completed by refinement using molecular mechanics and of administration, including, for example, but not limited to loop building using standard homology modeling techniques. parenteral, intradermal, or Subcutaneous application, and the (General information regarding modeling can be found in like, can include, for example, the following components: a Schoneberg, T. et. al. Molecular and Cellular Endocrinology sterile diluent such as water for injection, saline solution, 151:181-93 (1999); Flower, D. Biochimica et Biophysica fixed oils, polyethylene glycols, glycerine, propylene glycol Acta 1422:207-34 (1999); and Sexton, P. Current Opinion in or other synthetic solvents; antibacterial agents such as ben Drug Discovery and Development 2:440-8 (1999).) Zyl alcohol or methyl parabens; antioxidants such as ascorbic 0168 Once the model is completed, it can be used in acid or sodium bisulfate; chelating agents such as ethylene conjunction with one of several existing computer programs diaminetetraacetic acid; buffers such as acetates, citrates, or to narrow the number of compounds to be screened by the phosphates and agents for the adjustment of tonicity Such as screening methods of the present invention, like the DOCK sodium chloride or dextrose, and the like. The pH can be program (UCSF Molecular Design Institute, San Francisco, adjusted with acids or bases, such as, for example, hydrochlo Calif.). In several of its variants it can screen databases of ric acid or sodium hydroxide, and the like. The parenteral commercial and/or proprietary compounds for Steric fit and preparation can be enclosed in, for example, ampules, dispos rough electrostatic complementarity to the binding site. able Syringes, or multiple dose vials made of glass or plastic, Another program that can be used is FLEXX (Tripos Inc., St. and the like. Louis, Mo.). 0173 Pharmaceutical compositions suitable for injectable use include, for example, sterile aqueous solutions (where Chronic Esophagitis Therapies water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or 0169. Any anti-gastroesophgeal reflux disease (GERD) dispersion, and the like. For intravenous administration, Suit therapy can be used to treat chronic esophagitis. There are able carriers include, for example, physiological saline, bac various definitions of GERD; some include esophagitis (his teriostatic water, Cremophor ELTM (BASF, Parsippany, N.J.) tological finding of epithelial hyperplasia with acute non orphosphate buffered saline (PBS), and the like. In all cases, eosinophilic inflammation), others are purely clinical (heart the composition should be fluid to the extent that easy Syring burn), and others are based on measurement of esophageal ability exists. The carrier can be a solvent or dispersion acid levels (e.g. pH probes). Accordingly, GERD and chronic medium containing, for example, water, ethanol, polyol (for (non-eosinophilic) esophagitis can be considered to be example, glycerol, propylene glycol, and liquid polyethylene equivalent as a first approximation, and anti-GERD therapies glycol, and the like), and Suitable mixtures thereof, and the can be used to treat chronic esophagitis. like. The proper fluidity can be maintained, for example, by 0170 Anti-GERD therapies include, for example, antacid the use of a coating Such as lecithin, by the maintenance of the administration, H2 agonist administration, and/or PPI required particle size in the case of dispersion and by the use therapy, and the like. Certain embodiments of the invention of Surfactants. Prevention of the action of microorganisms US 2015/0038552 A1 Feb. 5, 2015

can be achieved by various antibacterial and antifungal 0.178 The compounds can also be prepared in the form of agents, such as, for example, parabens, chlorobutanol, phe Suppositories (e.g., with conventional Suppository bases Such nol, ascorbic acid, thimerosal, and the like. In many cases, it as cocoa butter and other glycerides) or retention enemas for can be preferable to include isotonic agents, such as, for rectal delivery. example, Sugars, polyalcohols such as mannitol, Sorbitol, and 0179. In one embodiment, the active compounds are pre Sodium chloride, and the like, in the composition. Prolonged pared with carriers that will protect the compound against absorption of the injectable compositions can be brought rapid elimination from the body, such as a controlled release about by including in the composition an agent which delays formulation, including implants and microencapsulated absorption Such as, for example, aluminum monoStearate and delivery systems, and the like. Biodegradable, biocompatible gelatin, and the like. polymers can be used, such as, for example, ethylene vinyl 0.174 Sterile injectable solutions can be prepared by acetate, polyanhydrides, polyglycolic acid, collagen, poly incorporating the active compound in the required amount in orthoesters, and polylactic acid, and the like. Methods for an appropriate solvent with one or a combination of ingredi preparation of Such formulations will be apparent to those ents enumerated above, as required, followed by filtered ster skilled in the art. The materials can also be obtained commer ilization. Generally, dispersions are prepared by incorporat cially from Alza Corporation and Nova Pharmaceuticals, Inc. ing the active compound into a sterile vehicle that contains a Liposomal Suspensions (including liposomes targeted to basic dispersion medium and the required other ingredients infected cells with monoclonal antibodies to viral antigens) from those enumerated above. In the case of sterile powders can also be used as pharmaceutically acceptable carriers. for the preparation of sterile injectable solutions, the pre These can be prepared according to methods known to those ferred methods of preparation are vacuum drying and freeze skilled in the art, for example, as described in U.S. Pat. No. drying which yields a powder of the active ingredient plus any 4.522,811, which is incorporated herein by reference in its additional desired ingredient from a previously sterile-fil entirety. tered solution thereof. 0180. It is especially advantageous to formulate oral or 0175 Oral compositions generally include an inert diluent parenteral compositions in dosage unit form for ease of or an edible carrier. They can be enclosed in gelatin capsules administration and uniformity of dosage. “Dosage unit form' or compressed into tablets, for example. For oral administra as used herein refers to physically discrete units Suited as tion, the agent can be contained in enteric forms to Survive the unitary dosages for the Subject to be treated; each unit con stomach or further coated or mixed to be released in a par taining a predetermined quantity of active compound calcu ticular region of the gastrointestinal (GI) tract by known lated to produce the desired therapeutic effect in association methods. For the purpose of oral therapeutic administration, with the required pharmaceutical carrier. The details for the the active compound can be incorporated with excipients and dosage unit forms of the invention are dictated by and directly used in the form of tablets, troches, or capsules, or the like. dependent on the unique characteristics of the active com Oral compositions can also be prepared using a fluid carrier pound and the particular therapeutic effect to be achieved, and for use as a mouthwash, wherein the compound in the fluid the limitations inherent in the art of compounding Such an carrier is applied orally and Swished and expectorated or active compound for the treatment of individuals. Such Swallowed. Pharmaceutically compatible binding agents, details are known to those of skill in the art. and/or adjuvant materials can be included as part of the com 0181 Certain embodiments of the invention include using position. The tablets, pills, capsules, troches, and the like can quantification data from a gene-expression analysis and/or contain any of the following exemplary ingredients, or com from a miRNA analysis, either from an esophageal biopsy pounds of a similar nature: a binder Such as microcrystalline sample, or from a sample of esophageal mucosa, or from a cellulose, gum tragacanth or gelatin; an excipient Such as blood sample. Embodiments of the invention include not only starch or lactose, a disintegrating agent Such as alginic acid, methods of conducting and interpreting such tests but also Primogel(R), or corn starch; a lubricant such as magnesium include reagents, kits, assays, and the like, for conducting the Stearate; a glidant Such as colloidal silicon dioxide; a Sweet ening agent Such as Sucrose or saccharin; or a flavoring agent testS. Such as peppermint, methyl salicylate, or orange flavoring, or 0182 Diagnostic-testing procedure performance is com the like. monly described by evaluating control groups to obtain four critical test characteristics, namely positive predictive value 0176 For administration by inhalation, the compounds (PPV), negative predictive value (NPV), sensitivity, and can be delivered in the form of an aerosol spray from pres specificity, which provide information regarding the effec Sured container or dispenser, which contains a Suitable pro tiveness of the test. The PPV of a particular diagnostic test pellant, e.g., a gas such as carbon dioxide, or a nebulizer, or represents the proportion of subjects with a positive test result the like. who are correctly diagnosed; for tests with a high PPV, a 0177 Systemic administration can also be by transmu positive test indicates the presence of the condition in ques cosal or transdermal means. For transmucosal or transdermal tion. The NPV of a particular diagnostic test represents the administration, penetrants appropriate to the barrier to be proportion of Subjects with a negative test result who are permeated are used in the formulation. Such penetrants are correctly diagnosed; for tests with a high NPV, a negative test generally known in the art, and include, for example, for indicates the absence of the condition. Sensitivity represents transmucosal administration, detergents, bile salts, and the proportion of correctly identified subjects who are actual fusidic acid derivatives, and the like. Transmucosal adminis positives; for tests with high sensitivity, a positive test indi tration can be accomplished through the use of nasal sprays or cates the presence of the condition in question. Specificity Suppositories. For transdermal administration, the active represents the proportion of correctly identified subjects who compounds are formulated into ointments, Salves, gels, or are actual negatives; for tests with high specificity, a negative creams as generally known in the art. test indicates the absence of the condition. US 2015/0038552 A1 Feb. 5, 2015

0183. The correlations disclosed herein, between EE and EXAMPLES miRNA levels and/or mRNA levels and/or gene expression levels, provide a basis for conducting a diagnosis of EE, or for 0187. The following non-limiting examples are provided enhancing the reliability of a diagnosis of EE by combining to further illustrate embodiments of the invention disclosed the results of a quantification of miRNA with results from herein. It should be appreciated by those of skill in the art that other tests or indicia of EE. For example, the results of a the techniques disclosed in the examples that follow represent quantification of miRNA could be combined with the results approaches that have been found to function well in the prac of a quantification of one or more cytokines or mRNAS. Thus, tice of the invention, and thus can be considered to constitute even in situations in which a given miRNA, cytokine, or examples of modes for its practice. However, those of skill in mRNA correlates only moderately or weakly with EE, pro the art should, in light of the present disclosure, appreciate viding only a relatively small PPV. NPV, specificity, and/or that many changes can be made in the specific embodiments sensitivity, the correlation can be one indicium, combinable that are disclosed and still obtain a like or similar result with one or more others that, in combination, provide an without departing from the spirit and scope of the invention. enhanced clarity and certainty of diagnosis. Accordingly, the Example 1 methods and materials of the invention are expressly contem plated to be used both alone and in combination with other MiRNA Expression Profiling in EE Patients tests and indicia, whether quantitative or qualitative in nature. 0184 The disclosure, figures, and tables herein make 0188 A study was undertaken to identify miRNAs that are mention of statistical significance and “p values.” While p differentially expressed in EE patients. Esophageal biopsy values below 0.05 are considered to be statistically signifi samples from EE patients and normal controls were analyzed cant, it is within the scope of embodiments of the present for their miRNA expression profiles, as described below. invention to make use of correlations having a reported p value above 0.05 as well as below 0.05. For example, in a Human Esophageal Tissues study having a small sample size but a genuine correlation, a 0189 Patients were selected without regard to age, race, or p value can be above 0.05, such as, for example, 0.06, 0.07, sex. Normal patients had symptoms consistent with gastroe 0.08, 0.09, 0.10, 0.15, or more. Since p value is affected by sophageal reflux disease or EE, but endoscopic and histologic sample size, two studies can have the same proportion of appearances were normal (Abonia, J. et al. J. Allergy Clin. outcomes, and a study with a smaller sample size can have a Immunol. 126:140-9 (2010)). The inclusion criteria for active p value above 0.05, while the study with the larger sample size EE patients included a clinical diagnosis ofEE and eosinophil can have a p value below 0.05, even though the correlation is counts of >24 per 400x high power field (hpf) in the esoph proportionally the same. Thus, whileap value below 0.05, for ageal biopsies; most patients had lack of response to proton any sample size, is a strong indication of a statistically sig pump inhibitor (PPI) therapy (Table 1). The inclusion criteria nificant correlation, a genuine correlation can exist, that is for active chronic esophagitis patients include a clinical diag tested with a small sample size, and the p value of Such a test nosis of esophagitis and eosinophil counts of 1-15 per 400x can be above 0.05. hpf in the esophageal biopsies. Patients with systemic or 0185. The illustrative embodiments described in the swallowed topical glucocorticoid use were excluded from the detailed description, drawings, and claims are not meant to be selection of active EE or active chronic esophagitis patients. limiting. Having described the invention in detail, it will be The inclusion criteria for EE patients responding to glucocor apparent that modifications, variations, and equivalent ticoid treatment included a history of EE, treatment with embodiments are possible without departing from the spirit or Swallowed topical glucocorticoid, response as indicated by Scope of the Subject matter presented herein. eosinophil count <2 per 400xhpf, and normalization of his 0186. Having described the invention in detail, it will be tological features of the disease. The EE patients not respond apparent that modifications, variations, and equivalent ing to glucocorticoid treatment had a history ofEE, treatment embodiments are possible without departing the scope of the with Swallowed topical glucocorticoid, and lack of response invention defined in the appended claims. Furthermore, it as indicated by esophageal eosinophil count >24 per 400xhpf should be appreciated that all examples in the present disclo in biopsies obtained at follow-up visits after therapy. These Sure are provided as non-limiting examples. patient characteristics are listed in Table 1. TABLE 1.

Patient clinical characteristics. Chronic EE Responding EE not Responding Normal EE Esophagitis to Steroids to Steroids

# of Patients 23 31 9 2O 8 Male, n (%) 11 (48) 23 (74) 7 (78) 16 (80) 6 (75) Age (yr)

Mean 9.26 9.29 9.06 8.32 10.96 Range 1.43-1901 189-32.26 4.5-16.12 3.75-1943 4.3-16.41 Medications 6 (26) 23 (74) 2 (22) 16 (80) 7 (88) Steroids, n (%) O O O 20 (100) 8 (100) *The percentages listed reflect total number of patients studied who had taken at least 8 weeks of PPI therapy prior to a diagnostic endoscopy, US 2015/0038552 A1 Feb. 5, 2015 19

0190. Eight normal controls, 10 active EE patients, 5 normal controls and EE patients identified 21 up-regulated chronic esophagitis patients, and 6 EE patients responding to and 11 down-regulated miRNAs (FIG. 1). The most up-regu glucocorticoid treatment were included in the microarray lated miRNAs included miR-21 and miR-223, and the most analysis. The remaining patients were included in the quan down-regulated miRNA was miR-375. titative PCR (qPCR) studies only. (0196. The differentially expressed miRNAs were evalu (0191 Patients were selected for miRNA microarray ated by performing quantitative real time polymerase chain analysis and qPCR studies based on systematic enrollment. reaction (qRT-PCR) on a selected set of differentially Patients enrolled in this study between November 2007 and expressed miRNAs, including miR-21, miR-223, miR-375, July 2009 were included in the microarray analysis. Addi let-7c, and miR-203 (FIG. 2A-E). There was a strong corre tional patients enrolled between July 2009 and June 2011 lation between the qRT-PCR and microarray data, with a who met the inclusion criteria and had RNA samples that Pearson correlation coefficient of 0.99 and p<0.01 (FIG.2F). passed the RNA quality analysis were included in the PCR analysis. TABLE 2 RNA Extraction and miRNA Microarray Analysis 0.192 Total RNA, including miRNA, from patient esoph Logo fold change of all miRNAs expressed above background levels. ageal biopsy samples was isolated using the miRNeasy Mini Chronic Fluticasone Kit (Qiagen, Valencia, Calif.), according to the manufactur Gene Symbol Normal Esophagitis EE Responders er's instructions. RNA quality was assessed using the 2100 Bioanalyzer (Agilent Technologies, Santa Clara, Calif.), and U6 O.OOS O.O10 O.O70 -O.065 RNU24 O.OS2 -0.2O3 O.040 O.170 only samples with RNA integrity number-8 were included in RNU43 -3.572 -3.124 -0.249 O.O2O the analyses. MiRNA expression was profiled using the Taq RNU44 -OOOS -0.010 -O.O70 O.06S Man Human MicroRNA Array V2.0 (Applied Biosystems, RNU48 -O.O23 -0.031 O.249 O.093 RNU6B 0.055 -O.319 -O.OSO -O.O39 Carlsbad, Calif.), which includes probes for 667 human miR hsa-let-7a O.046 O.O26 -0.019 O.007 NAS, as annotated in release 10.0 of the miRBase microRNA hsa-let-7b O.254 O140 -0.057 -O.230 Registry (Griffiths-Jones, S. et al. Nucleic Acids Res. hsa-let-7c O. 114 -O.O92 -O.973 -O.O99 34:D140-4 (2006)), according to the manufacturer's proto hsa-let-7d O.06S O.O33 O.OO1 O.152 cols (Applied Biosystems). Data analyses were carried out hsa-let-7e -0.074 -0.141 -O.390 -0.103 hsa-let-7f-2* -0.033 -O.259 -O153 -0.109 using GeneSpring software (Agilent Technologies). The hsa-let-7f -0.138 O.146 -O.250 O.151 microarray data have been deposited into the Array Express hsa-let-7g -OOOS O.261 O.O89 0.553 database, found at http

US 2015/0038552 A1 Feb. 5, 2015

TABLE 2-continued 0198 The chronic esophagitis patients had a miRNA expression profile that was similar to normal healthy controls Logo fold change of all miRNAs expressed above background levels. and distinct from EE patients (FIG. 3A). No miRNAs that Chronic Fluticasone were differentially regulated between normal controls and Gene Symbol Normal Esophagitis EE Responders chronic esophagitis patients were identified. hsa-miR-502 -O.OSO 0.414 O.395 O.874 Example 3 hsa-miR-505* O.O90 -0.424 O469 -O.065 hsa-miR-532-3p -0.012 -O.O71 -0.142 O.239 hsa-miR-532 O.036 -O.263 -0.024 -0.019 Reversibility of Differentially Expressed miRNA in hsa-miR-545 O.O24 1.117 O.S60 1.007 EE Patients hSn-miR-565 O.O39 -0.143 O.907 O.143 hsa-miR-574-3p O.OS1 -0.452 -0.657 -0.234 0199 A subsequent study was undertaken to determine hsa-miR-576-3p -0.150 -O-269 -OOO8 O.064 whether the EE miRNA expression signature was fixed or hsa-miR-579 -O.O15 O.O73 -O.291 O.445 hsa-miR-590-5p O.O09 -0.127 O.OS6 O.O16 reversible in patients who responded to glucocorticoid treat hsa-miR-592 -0.135 O.816 1663 O.OS4 ment and had normalization of esophageal histology, includ hsa-miR-597 O.244 O.S82 O668 0.774 ing eosinophil counts. Active EE patients were compared to hsa-miR-598 O.OO3 O.O37 -O3O2 O.285 EE patients that responded to fluticasone propionate therapy. hsa-miR-625* O.129 -0.100 O664 O.168 hsa-miR-625 O.O34 O.236 O.839 O.S16 (0200. Of the 32 differentially expressed miRNA, 27 were hsa-miR-628-5p O.O41 O.O79 O.863 -0.103 normalized. The reversible miRNAs included all of the most hsa-miR-629 -O.O13 -0.114 -O.200 O.226 highly up-regulated and down-regulated miRNAs (FIG. 3A). hsa-miR-629 -O.O68 -0.584 -O.378 O.187 There were 5 up-regulated miRNAs that were still dysregu hsa-miR-642 -0.041 O.403 1.16S 1110 hsa-miR-650 O.166 -3.439 9.6O2 1.714 lated in glucocorticoid responder patients, including miR-7. hsa-miR-652 O.2O6 O.S.04 O.366 O622 miR-29b, miR-642, miR-339-5p, and miR-92a-1*. hsa-miR-660 -0.046 O.OO2 O.O38 O433 hsa-miR-661 O.329 -0.214 O.82O -O.477 Example 4 hsa-miR-671-3p O.O12 O.O69 O.S22 O.062 hsa-miR-675 O.1SO -0.208 -4.643 7.873 hsa-miR-7-1* -O.259 -0.202 0.257 O.229 Correlation of miRNA Expression Levels with hsa-miR-7 O.158 O.399 1.221 O640 Esophageal Eosinophil Counts hsa-miR-708 O.O79 -018O -0.563 O.407 hsa-miR-744* -OOOS -0.212 -0.678 O.09S 0201 A subsequent study was undertaken to determine hsa-miR-744 0.055 O.O34 -0.335 O.107 whether the magnitude of miRNA expression changes corre hsa-miR-760 O.194 -0.633 -0.054 -O.896 hsa-miR-766 -2.682 -0.455 1.014 O.S8O lated to the eosinophil counts in the biopsies of EE patients. hsa-miR-768-3p -OOOS -O.O81 0.770 O.167 Out of the 32 miRNAs that were differentially regulated in EE hsa-miR-769-5p -0.064 -O.285 O.263 O.196 patients, 24 significantly correlated with esophageal eosino hsa-miR-801 O.O84 -0.460 3.908 -0.568 phil counts (Table 3). The most up-regulated miRNAs, hsa-miR-877 O.233 -0.078 0.571 -0.423 hsa-miR-885-5p -0.184 O.OO3 -O.O27 O.781 namely miR-21 and miR-223, also had the strongest correla hsa-miR-886-3p -O.O32 -O.O99 1.583 -O.316 tion of their expression level to the eosinophil counts. hsa-miR-886-5p O.O94 0.177 1528 -O.277 hsa-miR-9 -0.123 O.O18 -0.222 O.145 TABLE 3 hsa-miR-923 O.853 O.445 -O.866 -O.802 hsa-miR-92a-I* -0.333 O.151 1.129 O.872 Correlation between miRNA expression level and peak hsa-miR-92a -0.052 O.107 O.198 O491 eosinophil counts in EE patient biopsies. hsa-miR-93* -0.051 O.OO6 O.242 O.308 hsa-miR-93 -0.008 O.OO)4 O.385 -O.O81 P value Significance P<05 RSquare hsa-miR-942 -0.347 O.OO8 O.164 O.S30 hsa-miR-944 O.007 -0.182 -O.O81 O.130 hsa-miR-21 O.OOO2 ------Yes O663 hsa-miR-95 -0.051 O.229 O.301 0.565 hsa-miR-223 O.OOO2 ------Yes 0.657 hsa-miR-96 -0.063 -0.129 -O.S25 O.195 hsa-miR-132 O.OOO3 ------Yes O642 hsa-miR-99a O.158 -0.326 -0.600 O.285 hsa-miR-223* O.OOOS ------Yes O.617 hsa-miR-99a -0.052 -0.113 -0.619 O.274 hsa-miR-146b O.OOOS ------Yes O.615 hsa-miR-99b -O.O88 O.298 O.204 1290 hsa-miR-801 O.OOOS ------Yes O.617 hsa-miR-99b O.043 -O.OSS O.131 -0.018 hsa-miR-29b O.OOOS ------Yes O.614 hsa-miR-21 O.OOO6 ------Yes O606 The asterisk(*) after the name of the miRNA indicates the minor form of the miRNA derived hsa-miR-375 O.OO15 ---- Yes 0.552 from the passenger strand. hsa-miR-212 O.OO15 ---- Yes 0.551 hsa-miR-203 O.OO19 ---- Yes O.S38 hsa-miR-142-5p O.OOS2 ---- Yes O-463 Example 2 hsa-miR-211 O.OO60 ---- Yes O4S2 hsa-let-7c O.OO6S ---- Yes O.447 hsa-miR-642 O.OO85 ---- Yes O.425 Specificity of Differentially Expressed miRNA in EE hsa-miR-210 O.O12 -- Yes O.396 Patients hsa-miR-222* O.O12 -- Yes O.393 hsa-miR-365 O.O13 -- Yes O.390 hsa-miR-142-3p O.O21 -- Yes O.348 0.197 A subsequent study was undertaken to determine hsa-miR-886-3p O.O21 -- Yes O346 whether the miRNA expression signature identified in hsa-miR-193b O.O22 -- Yes O.343 Example 1 was specific to EE. The miRNA expression profile hsa-miR-92a-1* O.O23 -- Yes O.339 hsa-miR-339-5p O.O34 -- Yes O.302 of EE patients was compared to that of healthy controls, as hsa-miR-886-5p O.O3S -- Yes O.298 well as patients who presented with symptoms of EE but were hsa-miR-146a O.OS1 S No O.261 ultimately diagnosed with chronic (non-eosinophilic) esoph hsa-miR-592 O.O60 S No O.246 agitis. US 2015/0038552 A1 Feb. 5, 2015 22

TABLE 3-continued Example 5 Correlation between miRNA expression level and peak Correlation of miR-21 and miR-223 Expression eosinophil counts in EE patient biopsies. Levels with Major EE Signature Genes P value Significance P<05 R square 0202) A subsequent study was undertaken to determine hsa-miR-106b O.066 S No O.236 whether the expression of miR-21 and miR-223 could be hsa-miR-193a-5p O.O68 S No O-234 correlated to previously identified EE signature genes. hsa-miR-193a-3p O.1SO S No O.152 qRT-PCR hsa-miR-7 O.240 S No O.104 0203 Esophageal mRNA from EE patients was reverse hsa-miR-30a-3p O.810 S No O.OOS transcribed to cDNA by using the High Capacity cDNA hsa-miR-144* O.999 S No O.OOO Reverse Transcription Kit (Applied Biosystems) following +++: P<0.001; the manufacturer's protocol, using the TaqManreagents for ++: P<0.01; amplification of EE signature genes (Applied Biosystems) +: P<0.05; (Abonia, J. et al. J. Allergy Clin. Immunol. 126:140-9 (2010); ins; not significant, Ps 0.05. The asterisk(*) after the name of the miRNA indicates the minor form of the miRNA derived Blanchard, C. et al. J. Clin. Invest. 116:536-47 (2006); Blan from the passenger strand. chard, C. et al. J. Allergy Clin. Immunol. 120: 1292-300 (2007)). The EE signature genes are listed in Table 4. TABLE 4 List of EE signature genes used for correlation analysis. Entrez Gene ID Gene Name Description 55 ACPP Acid phosphatase, prostate 72 ACTG2 Actin, gamma 2, Smooth muscle, enteric 246 ALOX15 Arachidonate 15-lipoxygenase 383 ARG1 Arginase, liver 10344 CCL26 Chemokine (C-C motif) ligand 26 1232 CCR3 Chemokine (C-C motif) receptor 3 131450 CD2OOR1 CD200 receptor 1 978 CDA Cytidine deaminase 60437 CDH26 Cadherin-like 26 3426 CFI Complement factor I 10370 CITED2 Cbp/p300-interacting transactivator, with Gluf Asp-rich carboxy-terminal domain, 2 11.78 CLC Charcot-Leyden crystal protein 9071 CLDN10 Claudin 10 1215 CMA1 Chymase 1, mast cell 1296 COL8A2 Collagen, type VIII, alpha 2 1359 CPA3 Carboxypeptidase A3 (mast cell) 10321 CRISP3 Cysteine-rich secretory protein 3 8727 CTNNAL1 Catenin (cadherin-associated protein), alpha-like 1 1828 DSG1 Desmoglein 1 2009 EML.1 Echinoderm microtubule associated protein like 1 2312 FLG Filaggrin 9245 GCNT3 Glucosaminyl (N-acetyl) transferase 3, mucin type 134266 GRPEL2 GrpE-like 2, mitochondrial (E. coli) 27306 HPGDS Hematopoietic prostaglandin D Synthase 3269 HRH1 Histamine receptor H1 3512 G mmunoglobulin J polypeptide, linker protein for immunoglobulin alpha and mu polypeptides 3596 L13 interleukin 13 3565 L4 interleukin 4 3567 LS interleukin 5 (colony-stimulating factor, eosinophil) 3568 LSRA interleukin 5 receptor, alpha 3576 L8 interleukin 8 3759 KCNV2 Potassium inwardly-rectifying channel, Subfamily J, member 2 25984 KRT23 Keratin 23 (histone deacetylase inducible) 4321 MMP12 Matrix metallopeptidase 12 (macrophage elastase) 4585 MUC4 Mucin 4, cell Surface associated 4747 NEFL Neurofilament, light polypeptide 68 kDa 5367 PMCH Pro-melanin-concentrating hormone 10631 POSTN Periostin, osteoblast specific factor 64092 SAMSN1 SAM domain, SH3 domain and nuclear localization signals 1 912O SLC16A6 Solute carrier family 16, member 6 (monocarboxylic acid transporter 7) 5172 SLC26A4 Solute carrier family 26, member 4 846S1 SPINK7 Serine peptidase inhibitor, Kazal type 7 (putative) 7130 TNFAIP6 Tumor necrosis factor, alpha-induced protein 6 7177 TPSAB1 Tryptase alpha/beta 1 8548O TSLP Thymic stromal lymphopoietin 23554 TSPAN12 Tetraspanin 12 11045 UPK1A Uroplakin 1A 7348 UPK1B Uroplakin 1B US 2015/0038552 A1 Feb. 5, 2015

0204 Real-time PCR amplification was performed on a as previously published mRNA gene expression microarray Taqman 7900HT Real-Time PCR System (Applied Biosys experiments (see, e.g., Blanchard, C. et al. J. Clin. Invest. tems). The amplification protocol consisted of a hot start of 116:536-47 (2006); Abonia, J. etal.J. Allergy Clin. Immunol. 95°C. for 10 min, followed by 40 cycles of 15 seconds at 95° 126:140-9 (2010)). C. and 1 minute at 60°C. The expression correlation study Analysis of miR-21 and miR-223 Co-Regulated Genes by between miR-21, miR-223, and 48 EE genes was performed RNA-Seq and Gene Expression Microarray on GraphPad Prism (GraphPad Software, La Jolla, Calif.) 0210 RNA-Seq data were obtained from esophageal RNA software. samples from 11 EE patients by the Genomic Sequencing 0205 Negative log of p values from Pearson correlation Core Laboratory at Cincinnati Children’s Hospital Medical analysis were plotted to demonstrate correlation significance Center. The RNA-Seq was aligned to the GrCh37 build of the with EE genes. To control for the increased risk of false human genome using the Ensembl annotations as a guide for positives due to the number of statistical tests performed, a TopFlat (Johns Hopkins University, Baltimore, Md., Univer Bonferroni correction was applied based on the number of sity of California, Berkeley, Calif.; Harvard University, Cam gene expression profiles compared. Because the average pair bridge, Mass.) (Trapnell, C. et al. Bioinformatics 25:1105-11 wise correlation between gene expression profiles was 0.54, a (2009)). The resulting files were then analyzed with Cufflinks principal components analysis was applied to determine the ((Johns Hopkins University, Baltimore, Md., University of effective number of independent comparisons (Cheverud, J. California, Berkeley, Calif.; California Institute of Technol Heredity 87:52-8 (2001)). Using this approach, a p-value of ogy) to test for differential expression and differential regu 0.002 was required to achieve a family-wise error rate of 0.05. lation (Trapnell, C. et al. Nat. Biotechnol. 28:511-1 (2010)). 0211. The 3' region of miR-21 was identified as the best Results representation of pri-miR-21 (labeled as exon 3 in FIG. 5A). The region 3' of miR-223 was identified as the best represen 0206. MiR-21 was found to be significantly correlated tation of pri-miR-223 (labeled as exon 2 in FIG.5B) (Saini, H. with the esophageal expression of genes involved in inflam et al. BMC Genomics 9:564 (2008)). mation, including CCL26 (eotaxin-3), remodeling including 0212. The pri-miR-21 region was found to be significantly POSTN (periostin), eosinophilia including IL-5, and cell spe enriched in the EE patients compared to the control patients. cific markers for eosinophils (CLC) and mast cells (CPA3 and The expression pattern of the pri-miR-21 was then correlated TPSAB1) (FIG.3B). In addition, miR-21 significantly corre with the other gene expression patterns present from the lated with the gene CTNNAL1, which has been implicated in RNA-Seq expression profiles. MiR-223 was analyzed in a cell growth, proliferation, and wound repair (FIG. 3B) similar fashion. Gene expression microarray experiments (Xiang, Y. et al. J. Cell. Biochem. 103:920-30 (2008)). MiR from EE patients were also analyzed (Abonia, J. et al. J. 223 had the highest correlation with POSTN, IL-5, and CLC Allergy Clin. Immunol. 126:140-9 (2010); Blanchard, C. etal. (FIG. 3C). J. Clin. Invest. 116:536-47 (2006)). Example 6 0213. MiR-21 and miR-223 were correlated using the Human U133 probes (Affymetrix, Santa Clara, Calif.) that MiR-675 is Found to be a Disease best corresponded to the miRNA. A correlated gene is one Remission-Induced miRNA in ER whose expression follows similar or inverse trends with the miRNA in question. These probes were then correlated with 0207. A subsequent study was undertaken to determine microarray expression data, and 470 probes correlated well whether any miRNAs were differentially regulated in with at least one of the miRNA probes. The genes that showed response to treatment. One miRNA, miR-675, was up-regu the highest correlations and the most highly differentially lated in glucocorticoid responder patients compared to nor regulated genes in the previously published EE transcriptome mal. EE, or chronic esophagitis patients (FIG. 4A). were used as a training set alongside genes related to T1/T2 0208. To determine whether miR-675 is a glucocorticoid differentiation and eosinophilia that are target genes of miR induced or EE remission-induced miRNA, miR-675 expres 21 and miR-223 (Chen, J. et al. Nucleic Acids Res. 37:W305 sion levels were measured in normal controls, EE patients, EE 11 (2009)). The genes that showed patterns related to miR-21 patients that responded to glucocorticoid treatment, and EE or miR-223 were then analyzed to determine significant path patients that did not respond to glucocorticoid treatment, as ways and annotations related to T1/T2 differentiation and described in Example 1. The study found that miR-675 was eosinophil development and function. The genes were then induced in patients that responded to glucocorticoid treat clustered based on the strength of the annotations associated ment. MiR-675 was not induced in patients that did not with them. The results were rendered as a Cytoscape (open respond to glucocorticoid treatment (FIG. 4B). source software) map (FIGS. 6A-B). Example 7 Results MiR-21 and miR-223 Co-Regulated Genes are 0214. MiR-21 and miR-223 were found to interactively Significantly Enriched in Pathways Involved in regulate many similar pathways, including leukocyte prolif Adaptive Immunity and Regulation of Eosinophils eration, leukocyte activation, cytokine production, and immune response (FIG. 6A). The co-regulated target genes of 0209 Because miR-21 and miR-223 were found to be the miR-21 and miR-223 were involved in adaptive immune sys top two upregulated miRNAs in patients with EE, a subse tem polarization, IFNY signaling, and regulation of eosino quent study was undertaken to identify mRNA expression philia (FIG. 6B, FIG. 7). The significantly enriched pathways patterns that significantly correlated with the expression of included regulation of interleukin secretion, interferon pro miR-21 and miR-223. The study was conducted on esoph duction and signaling, and T-cell differentiation and activa ageal RNA samples subjected to RNA-Seq analysis, as well tion (FIG. 6B). Esophageal IL-12p35 levels showed a strong US 2015/0038552 A1 Feb. 5, 2015 24 inverse correlation with esophageal miR-21 levels (FIG. 6C). reaction using the TaqMan miRNA Assays (Applied Biosys This is consistent with the previous finding that miR-21 regu tems), following the manufacturer's protocols. lates T1 versus T2 balance by targeting IL-12p35 expres sion (Lu, T. et al.J. Immunol., 182:494-5002 (2009): Lu, T. et Results al. J. Immunol., 187:3362-73 (2011)). 0220. Using plasma samples from EE patients and normal controls, the expression levels of the 10 most differentially Example 8 regulated miRNAs associated with EE were determined. This analysis included 6 up-regulated miRNAS, namely miR-21. Identification of Differentially Expressed miRNAs miR-132, miR-142-3p, miR-146a, miR-146b, and miR-223, from EE Patient Plasma Samples and four down-regulated miRNAs, namely miR-203, miR 0215. MiRNAs have recently been reported to be present 210, miR-365, and miR-375. in plasma samples in a stable form protected from endog 0221 None of the down-regulated miRNAs was detected enous RNAse activities (Mitchell, P. et al. Proc. Natl. Acad. in the EE or normal control plasma samples after 40 cycles of Sci. U.S.A., 105:10513-8 (2008)). Plasma miRNAs canthere PCR. Of the up-regulated miRNAs, the expression of miR fore be used as noninvasive biomarkers ((Mitchell, P. et al. 142-3p, miR-146a, miR-146b, and miR-223 was detected in Proc. Natl. Acad. Sci. U.S.A., 105:10513-8 (2008); Zahm, A. all of the plasma samples. The expression of miR-21 and et al. J. Pediatr. Gastroenterol. Nutr., 53:26-33 (2011); Shen, miR-132 were only detected in some of the samples at low J. et al. Lab. Invest., 91:579-87 (2011)). Accordingly, a study levels and were not analyzed further. was undertaken to evaluate a subset of the miRNAs found to 0222 Because miR-16 has been reported to have a con be differentially regulated in the esophageal biopsies of EE stant expression level in plasma samples (Shen, J. et al. Lab. patients to determine whether these miRNAs could also be Invest. 91:579-87 (2011)), miR-16 was used as an endog differentially regulated in patient plasma samples. enous control to determine the differential expression of the Patient Selection for Plasma miRNA Analysis miRNAs between EE patient plasma samples and normal 0216 For the plasma miRNA level analysis, normal atopic controls. There were no significant differences in the average controls (defined as patients having asthma, allergic rhinitis, C. value of miR-16 between normal and EE patients (25. and/or eczema) were selected to control for the atopy com 5+0.4 vs. 26.0+1.1, respectively). monly seen in EE patients. The active EE patients were 0223 Comparing EE patients to normal controls, miR selected based on a clinical diagnosis of EE and eosinophil 146a, miR-146b, and miR-223 were found to be up-regulated counts of 224 per 400xhpf in the esophageal biopsies. in EE patient plasma samples (FIGS. 8A-C). While miR-146a Patients with systemic or swallowed topical glucocorticoid and miR-223 returned to baseline levels in EE remission use were excluded from the selection of normal atopic control patients, miR-146b remained elevated (FIGS. 8A-C). Using a or active EE patients. The EE remission patients were 1.5-fold change cut-off, the positive predictive value and selected based on a history ofEE and treated with Swallowed negative predicative value of normal controls vs. active EE topical glucocorticoid, with response as indicated by eosino patients and active EE patients vs. EE patients in remission phil counts 1 per 400xhpf and normalization of histological were calculated (Table 5). The levels of miR-142-3p were not features of the disease. changed between any of the groups (FIG. 9). 0217 Patients were selected retrospectively from cell-free plasma samples collected between November 2007 and June TABLE 5 2011 based on the selection criteria outlined and the avail Positive predictive value and negative predictive value of ability of sufficient quantity of plasma samples for RNA differentially regulated Serum miRNAS. isolation. A total of 13 normal, 13 EE, and 14 EE remission patients were included in the plasma miRNA analysis. Three Normal vs. Active EE vs. normal, 4 EE, and 3 EE remission patients included in the Active EE EE Remission plasma miRNA analysis overlapped with patients selected for Positive Negative Positive Negative esophageal miRNA microarray analysis. Other patients Predictive Predictive Predictive Predictive included in the esophageal miRNA microarray analysis did Value Value Value Value not have Sufficient quantities of plasma samples available for miR-146a O.82 O73 O.6O O.66 RNA isolation. miR-146b O.91 O.30 OSO O.SO RNA. Isolation from Human Plasma Samples miR-223 O.77 O.64 O.63 O.62 0218 Cell-free human plasma samples (750 ul) were mixed with Trizol LS reagent (Life Technologies, Carlsbad, Calif.) at a 1:3 ratio, with 1 lug of RNA from the bacteriophage Example 9 MS2 added as carrier RNA. The bacteriophage MS2 RNA was selected as a carrier RNA because it does not contain Elucidation of the Functional Role of miR-21 in EE miRNAs. The RNA was then extracted using the miRNEasy 0224. A study was undertaken to determine the functional Mini Kit (Qiagen) according to the manufacturer's protocols. role for miR-21, as this was the most induced miRNA in the Detection of Plasma miRNAs esophagus of EE patients. Wild type (WT) and miR-21 gene 0219 Plasma (prepared from Na-EDTA tubes) miRNAs knockout (KO) mice (Lu, T. et al. J. Immunol. 187:3362-73 were reverse transcribed using MegaPlex RT primers (Ap (2011)) were subjected to an experimental model of EE, plied Biosystems), Human pool set V2.1 with pre-amplifica elicited by repeated exposure to the allergen Aspergillus fumi tion (Applied Biosystems), according to the manufacturers gatus, as reported (Mishra, A. et al. J. Clin. Invest. 107:83-90 protocols. The pre-amplified samples were diluted 1:40, and (2001)). Saline challenge was used on WT and KO mice as a 1.5 ul of the diluted product was then used in a 15 ul PCR control. Following allergen exposure or saline challenge, the US 2015/0038552 A1 Feb. 5, 2015 number of eosinophils in the esophagus was quantitated by nuclear RNA. Relative expression was calculated using the anti-major basic protein immunohistochemistry, followed by comparative C method, as previously described (Livak, K. et morphometric analysis. al. Method. 25:402-8 (2001)). 0225. As shown in FIG. 10, miR-21 KO mice were mark edly protected from the development of experimental EE. Statistical Analysis These findings demonstrate a functional role for miR-21 in the elicitation of experimental EE and further support the 0231 Statistical analyses were performed with the stu utility of anti-miR-21 therapeutic agents, such as miR-21 dent's t-test or one-way ANOVA with Tukey post-hoc test antagomirs. where appropriate. Statistical significance and the p values are indicated on the figures where appropriate. P values less Example 10 than 0.05 were considered statistically significant.

Expression of miR-21 in an Ex Vivo Culture of Bone Results Marrow-Derived Eosinophils 0226. A study was conducted to determine the role of 0232 High purity ex vivo bone marrow-derived eosino miR-21 in eosinophil development and function. A murine ex phils were obtained, as determined by FACS staining for vivo bone marrow-derived eosinophil culture model that gen CCR3 and Siglec-F double positive cells, on day 14 (FIG. erates >90% eosinophils after 14 days of culture was utilized 11A). Up-regulation of miR-21 was observed during the eosi (Dyer, K. et al. J. Immunol. 181:4004-9 (2008)). nophil differentiation from day 4 to day 14 (FIG. 11B). Mice Example 11 0227. MiR-21 gene targeted mice were backcrossed for 5 generations into the C57BL/6 background, described by a Arrested Eosinophil Progenitor Growth in previous protocol (Lu, T. et al. J. Immunol. 187:3362-73 Eosinophil Cultures Derived from miR-21. Mice (2011)). Littermate controls were used for all experiments. 0233. In a subsequent study, the effect of miR-21 on eosi All animals used were housed under specific pathogen-free nophil development was determined. Progenitor cells were conditions in accordance with institutional guidelines. cultured into eosinophils, and a profound phenotype in cel lular growth was noted. The miR-21 mice had a marked Bone Marrow-Derived Cell Cultures arrest in eosinophil progenitor growth compared to controls, 0228 Bone marrow cells were collected from femurs and with the most prominent effect seen between days 8 and 12 tibia of the mice, and the stem/progenitor cell-enriched low (FIG. 12A). By comparison, neutrophil progenitor growth density fraction was isolated by gradient centrifugation using was not significantly changed (FIG. 12B). The miR-21" and the Histopaque 1083 (Sigma, St. Louis, Mo.), according to miR-21 bone marrow-derived eosinophils were morpho the manufacturer's protocol. The low density fraction of bone logically indistinguishable from each other at day 12 (FIG. marrow cells were cultured in Iscove’s Modified Dulbecco's 12C). Media (IMDM) with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 ug/ml streptomycin Supplemented Example 12 with 100 ng/mL stem cell factor and 100 ng/mL FLT-3 ligand (Peprotech, Rocky Hill, N.J.) from day 0 to day 4 at a con Increased Apoptosis in Eosinophil Cultures Derived centration of 1x10/mL in 6-well plates. from miR-21. Mice 0229. For eosinophil differentiation, the stem cell factor and FLT-3 ligand were replaced with 10 ng/mL, IL-5 on day 4, 0234. In the same study, the levels of apoptosis in the and the cells were cultured for an additional 10 days in the miR-21 and miR-21" eosinophil progenitor cultures were presence of IL-5 (Dyer, K. et al. J. Immunol. 181:4004-9 measured by Annexin V and 7AAD staining. The viable cells (2008)). One half of the culture media was replaced with fresh are Annexin V and 7AAD double negative. The early apop media every other day. The cells were counted and concen totic cells are Annexin V positive and 7AAD negative. The tration was adjusted to 1x10°/mL during each media change. late apoptotic cells are Annexin V and 7AAD double positive. Eosinophil maturity was assessed by fluorescence-activated Compared to miR-21" cultures, the miR-21 eosinophil cell sorting (FACS) staining for CCR3 and Siglec-F and/or progenitor cultures have increased levels of both the Annexin Diff-Quik (Fisher Scientific) staining of cytospin prepara V 7AAD population and the AnnexinV 7AAD" popula tions. For neutrophil differentiation, the stem cell factor and tion, indicative of increased levels of apoptosis in the miR FLT-3 ligand were replaced with 20 ng/mL granulocyte 21 cultures (FIG. 13). colony-stimulating factor (G-CSF) on day 4, and the cells were cultured for an additional 6 days in the presence of Example 13 G-CSF. Eosinophil and neutrophil progenitor growth was assessed by counting the cells every 2 days using a hemacy Reduced Eosinophilia in the Blood of miR-21 tOmeter. Mice Quantitative Assessment of miRNA Levels 0230 Levels of miRNA expression were measured quan 0235 A subsequent study was undertaken to determine titatively by using the TaqMan MicroRNA Assay (Applied the consequences of miR-21 on eosinophil hematopoiesis in Biosystems), following the manufacturer's protocol, and vivo. This was achieved by determining the blood eosinophil assayed on the 7900HT Real-Time PCR System (Applied level in the miR-21 mice, using FACS to measure blood Biosystems). Normalization was performed using U6 small eosinophil percentage. US 2015/0038552 A1 Feb. 5, 2015 26

Determination of Blood Eosinophil Percentage by FACS probes was generated by requiring a minimum raw expres 0236 Red blood cells were lysed from mouse blood by sion level of the 20" percentile out of all probes on the using red blood cell (RBC) lysis buffer (Sigma) twice for 5 microarray. The resulting probe sets were then baseline trans minutes each time. The eosinophil percentage was deter formed and filtered on at least a 1.5-fold difference between mined by FACS staining of blood cells with fluorescein miR-21" and miR-21 eosinophil progenitor cultures. (FITC)-conjugated anti-CCR3 (R&D Systems, Minneapolis, 0243 Statistical significance was determined at p-0.05 Minn.) and PE-conjugated anti-Siglec-F (BD Biosciences, with Benjamini Hochberg false discovery rate correction. The San Diego, Calif.). The eosinophils analyzed were the CCR3 resulting list of genes was clustered using hierarchical clus and Siglec-F double positive cells, as described by a previous tering, and a heatmap was generated (FIGS. 15A-B). Biologi protocol (Fulkerson, P. et al. Proc. Natl. Acad. Sci. U.S.A. cal functional enrichment analysis was carried out using Top 103: 16418-16423 (2006)). pGene/ToppCluster (Cincinnati Children’s Hospital Medical Center) (Chen, J. et al. Nucleic Acids Res. 37:W305-11 Results (2009): Kaimal, V. et al. Nucleic Acids Res. 38:W96-102 (2010)). The microarray data have been deposited into the 0237. The miR-21 mice were found to have a decreased Array Express database, found at http

TABLE 6 List of differentially regulated genes between miR-21' and miR-21 eosinophil progenitor cultures at day 8. Transcript ID Gene Symbol Gene Description Fold Change Regulation 10362442 Trdn Triadin 1.53 Up-regulated 10363224 Fabp7 Fatty acid binding protein 7, brain 1.73 Up-regulated 10377286 Pik3ró Phosphoinositide-3-kinase, regulatory subunit 6 1.79 Up-regulated 10380226 Cuedc1 CUE domain containing 1 2.00 Up-regulated 10536294 Peg10 Paternally expressed 10 1.52 Up-regulated 10544610 Igf2bp3 Insulin-like growth factor 2 mRNA binding protein 3 1.74 Up-regulated 10583.056 Mmp12 Matrix metallopeptidase 12 1.67 Up-regulated

TABLE 7 List of differentially regulated genes between miR-21" and miR-21 eosinophil progenitor cultures at day 12. Transcript ID Gene Symbol Gene Description Fold Change Regulation O354732 Hspci1 Heat shock protein 1 (chaperonin) 54 Up-regulate O364093 Derl3 Derl-like domain family, member 3 53 Up-regulate O377286 Pik3ró Phosphoinositide-3-kinase, regulatory subunit 6 .67 Up-regulate O384398 Grb10 Growth factor receptor bound protein 10 .76 Up-regulate 0425287 Koelr3 KDEL (Lys-Asp-Glu-Leu) endoplasmic reticulum 53 Up-regulate protein retention receptor 3 O466224 MS4a3 Membrane-spanning 4-domains, Subfamily A, 88 Up-regulate member 3 0489343 Ptpla Protein tyrosine phosphatase-like (proline instead of 53 Up-regulate catalytic arginine), member a O495316 Psrc1 Proline?serine-rich coiled-coil 1 S1 Up-regulate OS27323 Ba15 Basic helix-loop-helix family, member a15 57 Up-regulate OS42857 Fair2 Fatty acyl CoA reductase 2 52 Up-regulate O548105 Cond2 Cyclin D2 51 Up-regulate OSS9818 NA NA SO Up-regulate OS6S570 463243411Rik RIKEN cDNA 4632434I11 gene 66 Up-regulate 0574572 2210023G05Rik RIKEN cDNA 2210023GO5 gene S8 Up-regulate O589413 Nme6 Non-metastatic cells 6, protein expressed in 52 Up-regulate LOC100046163| (nucleoside-diphosphate kinase) similar to Nme6 LOC100046157 protein hypothetical protein LOC100046157 O345752 II1r2 interleukin 1 receptor, type II -2.16 Down-regulate O368343 Arg1 Arginase, liver -4.84 Down-regulate O374248 Abca.13 ATP-binding cassette, sub-family A (ABC1), member -1.90 Down-regulate 3 O377782 Cec10a C-type lectin domain family 10, member A -2.69 Down-regulate O378.816 Slcóa4 Solute carrier family 6 (neurotransmitter transporter, -3.98 Down-regulate Serotonin), member 4 O37963O Sf2 Schlafen 2 -2.06 Down-regulate O3797.10 E230016K23Rik RIKEN cDNA E230016K23 gene -1.64 Down-regulate O380566 Phosphol| Phosphatase, orphan 1|ABI gene family, member 3 -1.59 Down-regulate Abi3 O380927 Grbf Growth factor receptor bound protein 7 -1.56 Down-regulate 0436095 Retmla Resistin like alpha -2.99 Down-regulate O467979 Sc1 Stearoyl-Coenzyme A desaturase 1 -2.00 Down-regulate O483228 Scm3a Sodium channel, voltage-gated, type III, alpha -155 Down-regulate 0491313 Cldn11 Claudin 11 -1.62 Down-regulate 0570291 F10 Coagulation factor X -2.01 Down-regulate OS85068 FanSSd Family with sequence similarity 55, member D -1.51 Down-regulate 0588691 Hyal 1| Hyaluronoglucosaminidase 1 N-acetyltransferase 6 -150 Down-regulate Nat6 O598O10 CCr11 Chemokine (C-C motif) receptor 1-like 1 -155 Down-regulate

Example 16 Mice Expression of miR-223 in an Ex Vivo Culture of 0249 MiR-223 gene targeted mice were backcrossed for 5 Bone Marrow Derived Eosinophils generations into the C57BL/6 background, as described in a previous protocol (Lu, T. et al. J. Immunol. 187:3362-73 (2011)). Littermate controls were used for all experiments. 0248. A study was conducted to determine the role of All animals used were housed under specific pathogen-free miR-223 in eosinophil development and function. A murine conditions in accordance with institutional guidelines. The ex vivo bone marrow-derived eosinophil culture model that Institutional Animal Care and Use Committee of the Cincin generates >90% eosinophils after 14 days of culture was nati Children’s Hospital Medical Center approved the use of utilized (Dyer, K. et al. J. Immunol. 181:4004-9 (2008)). animals in these experiments. US 2015/0038552 A1 Feb. 5, 2015 28

Bone Marrow-Derived Eosinophil Cultures had a markedly increased proliferation, with the most promi nent effect seen between day 10 and day 14 (FIG. 18A). 0250 Bone marrow cells were collected from femurs and Morphology was monitored at days 8, 10, and 12 (FIG. 18B). tibia of the mice, and the stem/progenitor cell-enriched low The miR-223" and miR-223 bone marrow-derived eosi density fraction was isolated by gradient centrifugation using nophils were morphologically indistinguishable from each the Histopaque 1083 (Sigma), according to the manufactur other at day 14 (FIG. 18C). er's protocol. The low density fraction of bone marrow cells were cultured in IMDM with 10% FBS, 100 U/ml penicillin, Example 18 and 100 g/ml streptomycin supplemented with 100 ng/mL stem cell factor and 100 ng/mL FLT-3 ligand (Peprotech) Up-Regulation of IGF1 Receptor in Eosinophil from day 0 to day 4. Cultures Derived from miR-223 Mice 0251. The stem cell factor and FLT-3 ligand were replaced (0256 In the same study, IGF1 receptor (IGF1R) levels with 10 ng/mL, IL-5 on day 4, and the cells were cultured for were evaluated to determine whether they were differentially an additional 10-12 days in the presence of IL-5 (Dyer, K. et regulated between eosinophil cultures derived from miR al. J. Immunol. 181:4004-9 (2008)). The culture media was 223 mice and miR-223** littermate controls. IGF1 has changed every other day, and cells were counted and concen heretofore not been examined for its impact on eosinophils or tration was adjusted to 1x10°/mL during each media change. their progenitors. Eosinophil maturity was assessed by FACS staining for CCR3 and Siglec-F and/or Diff-Quik (Fisher Scientific) Preparation of Total Cell Lysates and Western Blot staining of cytospin preparations. Eosinophil progenitor 0257 Cells were rapidly washed in phosphate buffered growth was assessed by counting the cells every 2 days using saline and lysed in M-PER mammalian protein extraction a hemacytometer. The schematic for the culture of bone mar reagent (Pierce, Rockford, Ill.), according to the manufactur row-derived eosinophils is shown in FIG. 17A. er's protocol. Protease inhibitor cocktails (Pierce) and phos Quantitative Assessment of miRNA Levels phatase inhibitor cocktails (Pierce) were added to the M-PER 0252 Total RNA was isolated using the miRNeasy Mini protein extraction reagent immediately before lysis. Western Kit (Qiagen), according to the manufacturer's protocols. Lev blot analysis was performed, as described in a previous pro els of miRNA expression were measured quantitatively by tocol (Lim, E. et al. J. Biol. Chem. 286:13193-204 (2011)). using the TaqMan MicroRNA Assay (Applied Biosystems), The assay was conducted using an anti-IGF1 Rantibody (Cell following the manufacturer's protocol, and assayed on the Signaling Technology, Danvers, Mass.) and an anti-GAPDH 7900HT Real-Time PCR System (Applied Biosystems). Nor antibody (Abcam, Cambridge, UK). malization was performed using U6 small nuclear RNA. Relative expression was calculated using the comparative C, Results method, as described in a previous protocol (Livak, K. et al. 0258 IGF1R was not found to be expressed at day 4, Method. 25:402-8 (2001)). indicating that proliferation of progenitor cells under the influence of stem cell factor and FLT-3 ligand is not depen Statistical Analysis dent on IGF levels. Significant levels of IGF expression were found from day 10 to day 14 of the culture, coinciding with 0253 Statistical analyses were performed with students the increased proliferation seen in both the miR-223" and t-test or one-way ANOVA with Tukey post-hoc test where miR-223 eosinophil cultures (FIG. 18A, FIG. 19). How appropriate. Statistical significance and the p values were ever, in the miR-223" cultures, the IGF1R level progres indicated on the figures where appropriate. P values less than sively decreased from day 10 to day 14, reflecting that eosi 0.05 were considered statistically significant. nophil progenitors gradually lose their proliferation capacity during the differentiation process (FIG. 18A, FIG. 19). Com Results pared to the miR-223" cultures, the miR-223 cultures have significantly increased levels of IGF1R at both day 12 0254 High purity ex vivo bone marrow-derived eosino and day 14 (FIG. 19). phils were obtained, as determined by FACS staining for CCR3 and Siglec-F double positive cells, on day 14 (FIG. Example 19 17B). Up-regulation of miR-223 was observed during the eosinophil differentiation culture from day 4 to day 14 (FIG. Reversibility of Increased Proliferation in 17C), with the most prominent difference seen between days miR-223 Eosinophil Cultures by Treatment with 8 and 14. an IGF1R Inhibitor 0259 A subsequent study was undertaken to determine Example 17 whether the up-regulation of IGF1R was responsible for the increased proliferation seen in eosinophil cultures derived Enhanced Eosinophil Progenitor Proliferation in from the miR-223 mice. Eosinophil Cultures Derived from miR-223 Mice Analysis of Cell Proliferation after Picropodophyllin Treat ment 0255. In the same study, the effect of miR-223 on the 0260 Bone marrow-derived eosinophils were re-sus proliferation of eosinophil progenitor cells was determined pended at a concentration of 1x10° cells/ml and treated with by culturing bone marrow-derived eosinophils from miR DMSO or 2 uM of picropodophyllin at day 8 (Yin, S. et al. 223 mice. Compared to cultures from the wild type litter Neuro. Oncol. 12:19-27 (2010)). Cell growth was determined mate controls, the miR-223 eosinophil progenitor cultures by cell counting using a hemacytometeronday 10 and day 12. US 2015/0038552 A1 Feb. 5, 2015 29

Cell lysates were collected on day 10, and levels of IGF1R PE (BD Bioscience), which are markers for mature eosino expression were determined by western blot. phils. Staining was performed on ice for 30 minutes in stain 0261 Bone marrow-derived eosinophils were resus ing buffer (0.5% BSA, 0.01% NaNs in 1xEHBSS), according pended at a concentration of 1x10° cells/mL and plated in a 96 to the manufacturer's protocol (R&D Systems and BD Bio well plate at 100 uL per well on day 9 to determine a dose sciences). Data was acquired on a BD FACS Canto I flow response curve of picropodophyllin. The cells were treated cytometer (BD Biosciences) and analyzed using FlowJo with increasing concentrations of picropodophyllin, and the (TreeStar, Ashland, Oreg.). level of cell growth was determined using Cell-Titer Glo luminescent cell viability assay (Promega, Madison, Wis.), Results according to the manufacturer's protocol. (0268. MiR-223 eosinophil progenitors were found to have delayed up-regulation of CCR3 compared with miR Results 223" eosinophil progenitors at all three time points (FIG. 0262 Eosinophil cultures were treated on day 8 with 2 uM 21A), indicative of delayed maturation of the miR-223/ of picropodophyllin, an IGF1R inhibitor, or an equivalent eosinophil progenitor cells. The mature eosinophils were volume of dimethyl sulfoxide (DMSO) as a control. DMSO CCR3 and Siglec-F". There were less than 2% CCR3"Siglec treatment had no effect on eosinophil proliferation. F" cells during the eosinophil culture on day 8, indicating that 10263. The miR-223 cultures treated with DMSO had nearly all cells were in the progenitor stage in both the miR significant increases in growth compared to DMSO-treated 223* and miR-223 cultures (FIG. 21B). While CCR3" miR-223" cultures (FIG. 20A), confirming the results SiglecF cells begin to appear on day 10, there were substan shown in FIG. 18C. In contrast, treatment with 2 uM of tially fewer CCR3"SiglecF" cells in the miR-223 cultures picropodophyllin inhibited the growth of both miR-223" than in the miR-223" cultures. This difference is most pro and miR-223 cultures to a similar extent (FIG.20A), com nounced during day 10 and day 12 of the eosinophil progeni pletely reversing the increased proliferation seen in miR tor culture (FIG. 21B), concomitant with the onset of the 223 cultures. increased proliferation seen in the miR-223 cultures (FIG. 0264. The levels of IGF1R expression were analyzed on 18A). day 10 with or without picropodophyllin treatment. 0269. The blood eosinophil levels were measured in vivo, Picropodophyllin was found to induce a nearly complete and no difference was found between the miR-223" and down-regulation of IGF1 receptor in both the miR-223" and miR-223 mice (FIG. 22A). This is likely due to in vivo miR-223 cultures (FIG. 20B). Dose-response demon compensation at the stage where multipotent progenitors are strated that picropodophyllin inhibited miR-223" and differentiated into eosinophillineage-committed progenitors. miR223 eosinophil progenitor growth with similar ICso When the level of IL5Ra"CCR3' eosinophil lineage-commit (FIG.20C). ted progenitors was measured in Vivo, a decreased level of eosinophil lineage-committed progenitors was found in the Example 20 miR-223 mice (FIG.22B). These results demonstrate that the increased proliferation of the miR-223 eosinophil pro Association Between Increased Proliferation genitors is associated with a delay in eosinophil differentia Observed in miR-223 Eosinophil Progenitor tion. Cultures and Delay in Differentiation 0265. In the same study, a delayed up-regulation of CCR3 Example 21 and delayed down-regulation of CD34 in miR-223 eosino phil progenitor cultures was observed compared to miR Genes Found to be Differentially Regulated Between 223" eosinophil progenitor cultures, indicative of delayed miR-223 and miR-223 Eosinophil Progenitor maturation of the miR-223 eosinophil progenitor cells. Cultures qPCR analysis of the CCR3 level was performed on day 8, 0270 Having identified a decreased differentiation of day 10, and day 12 eosinophil progenitor cultures. To deter miR223 eosinophils using a targeted approach (focusing mine the surface expression of CCR3 during the eosinophil on CCR3 and Siglec-F), a subsequent study was conducted to progenitor culture, FACS analysis of CCR3 and Siglec-F extend this finding at a genome-wide level to identify genes expression was performed from day 8 to day 16 of eosinophil differentially expressed during the eosinophil progenitor cul culture. ture. A gene expression microarray analysis was performed qRT-PCR for mRNA on the eosinophil progenitor cultures on days 4, 8, and 12. 0266 Total RNA was reverse transcribed using the High Mouse Genome-Wide mRNA Microarray Capacity cDNA Reverse Transcription kit (Applied Biosys (0271 The Mouse Gene 1.0ST array (Affymetrix) was tems). All primer/probe sets were obtained from Applied used to compare gene expression profiles between miR Biosytems (Applied Biosystems). Samples were analyzed by 223" and miR-223 eosinophil progenitor cultures at day the TaqMan qRT-PCR (Applied Biosystems) for CCR3. (As 4, day 8, and day 12. Microarray data were analyzed using say ID: Mm01216172 ml) and normalized to HPRT1 (Assay GeneSpring software (Agilent Technologies). Global scaling ID: Mm00446968 m1). Relative expression was calculated was performed to compare genes from chip to chip, and a base using the comparative C method. set of probes was generated by requiring a minimum raw Flow Cytometry Analysis of Eosinophil Surface CCR3 expression level of 20" percentile out of all probes on the Expression microarray. The resulting probe sets were then baseline trans formed and filtered on at least a 1.5-fold difference between 0267 One million cultured eosinophil progenitor cells miR-223" and miR-223 eosinophil progenitor cultures. were stained with CCR3-FITC (R&D Systems) and SiglecF Statistical significance was determined at p-0.05 with Ben US 2015/0038552 A1 Feb. 5, 2015 30 jamini Hochberg false discovery rate correction. The result Flt-3L is not dependent on miR-223. At day 8 of the culture, ing list of genes was clustered using hierarchical clustering, 17 genes were found to be down-regulated, and 16 genes were and a heatmap was generated. Biological functional enrich found to be up-regulated (FIG. 23A). The full gene list ment analysis was carried out using Ingenuity Pathway appears in Table 8. Functional enrichment analysis identified Analysis (Ingenuity Systems, Redwood City, Calif.) and Top the top affected biological functions to be hematological sys pGene/ToppCluster (Cincinnati Children’s Hospital Medical tem development and function, cell growth, and regulation of Center) (Chen, J. et al. Nucleic Acids Res. 37:W305-11 immune response (FIG. 23B, FIG. 24), consistent with the (2009): Kaimal, V. et al. Nucleic Acids Res. 38:W96-102 earlier finding that miR-223 affects proliferative responses in (2010)). The microarray data have been deposited into the eosinophils. Analysis of genes differentially regulated at day Array Express database, found at http

O3529OS C34 CD34 antigen Up-regulate O36O338 Fcer1a Fc receptor, IgE, high affinity I, alpha polypeptide Up-regulate O364529 Pirtl3 Proteinase 3 Up-regulate O364535 Elane Elastase, neutrophil expressed Up-regulate O3841SO Purb Purine rich element binding protein B Up-regulate 0398.039 Serpina3f Serine (or cysteine) peptidase inhibitor, clade A, member 3Flserine (or cysteine) Up-regulate Serpina3g peptidase inhibitor, clade A, member 3G 0403821 Tcrg-V3| T-cell receptor gamma, variable 3 IT-cell receptor gamma, variable 2 Up-regulate Tcrg-V2 0407940 Naip3| NLR family, apoptosis inhibitory protein 3T-cell receptor gamma, variable 21 Up-regulate Tcrg-V2| T-cell receptor gamma, variable 1 Tcrg-V1 O411215 Crhbp Corticotropin releasing hormone binding protein Up-regulate O419578 Ndrg2 N-myc downstream regulated gene 2 Up-regulate 0420261 Ctsg Cathepsin G Up-regulate O427436 C7 Complement component 7 Up-regulate O435288. Muc13 Mucin 13, epithelial transmembrane Up-regulate 0451 123 Slc29a1 Solute carrier family 29 (nucleoside transporters), member 1 Up-regulate OSS48OO Rab38 RAB38, member of RAS oncogene family Up-regulate OS74O23 Mt. Metallothionein 2 Up-regulate 0349157 Serpinb2 Serine (or cysteine) peptidase inhibitor, clade B, member 2 Down-regulate O38O381 Tob1 Transducer of ErbB-2.1 Down-regulate O383717 Inpp5i nositol polyphosphate 5-phosphatase J Down-regulate O389022 Myold Myosin ID Down-regulate O389025 Myold Myosin ID Down-regulate O399.691 Id2 inhibitor of DNA binding 2 Down-regulate 0424683 Ly6g Lymphocyte antigen 6 complex, locus G Down-regulate O439296 Stfa2 Stefin A2 Down-regulate O439299 Stfa3 Stefin A3 Down-regulate O475517 AA467197 Expressed sequence AA467197 microRNA 147 Down-regulate Mr147 O493.820 S100a.6 S100 calcium binding protein A6 (calcyclin) Down-regulate OS 17791 Padi4 Peptidyl arginine deiminase, type IV Down-regulate 0539739 Asprv1 Aspartic peptidase, retroviral-like 1 Down-regulate 0551696 Rasgrp4 RAS guanyl releasing protein 4 Down-regulate 0556302 Ampd3 Adenosine monophosphate deaminase 3 Down-regulate 0581538 Ngo1 NAD(P)H dehydrogenase, quinone 1 Down-regulate O590628 CCr3 Chemokine (C-C motif) receptor 3 Down-regulate US 2015/0038552 A1 Feb. 5, 2015 31

TABLE 9 List of differentially regulated genes between miR-223' and miR-223 eosinophil progenitor cultures at day 12. Transcript ID Gene Symbol Gene Description Regulation

O356470 Glutamine repeat protein 1 Regu 8. O379866 Carbonic anhydrase 4 Regu 8. O398O39 Serine (or cysteine) peptidase inhibitor, clade A, member 3Flserine Regu 8. (or cysteine) peptidase inhibitor, clade A, member 3G O403821 T-cell receptor gamma, variable 3|T-cell receptor gamma, variable 2 Regu NLR family, apoptosis inhibitory protein 3T-cell receptor gamma, Regu variable 2 T-cell receptor gamma, variable 1

O427436 Complement component 7 Regu O432675 RIKEN cDNA I730030J21 gene Regu O435288 Mucin 13, epithelial transmembrane Regu O442762 Protease, serine, 34 Regu O444284 Histocompatibility 2, Oregion beta locus Regu OSO6274 DnaJ (Hsp40) homolog, subfamily C, member 6 Regu OS42120 C-type lectin domain family 2, member i Regu O566254 Hemoglobin, beta adult major chain hemoglobin, beta adult minor Regu chain O566258 Hemoglobin, beta adult major chain hemoglobin, beta adult minor Regu 8. e chain O588S92 Calcium channel, voltage-dependent, alpha 2 delta Subunit 2 Regu 8. O344966 Lymphocyte antigen 96 Down-regu O3S3.192 Eyes absent 1 homolog (Drosophila) Down-regu O3S4374 Solute carrier family 40 (iron-regulated transporter), member 1 Down-regu O3S4649 Post-GPI attachment to proteins 1 Down-regu O355456 Melanoregulin Down-regu O35916.1 Sterol O-acyltransferase 1 Down-regu O360377 Down-regu O362S11 Glutathione S-transferase, mu3 Down-regu O363735 Early growth response 2 Down-regu O365559 Insulin-like growth factor 1 Down-regu O369413 Sphingosine phosphate lyase 1 Down-regu O372988 Solute carrier family 16 (monocarboxylic acid transporters), Down-regu member 7 O374248 Abca.13 ATP-binding cassette, sub-family A (ABC1), member 13 Down-regu O378.253 Camkk1 Calcium calmodulin-dependent protein kinase kinase 1, alpha Down-regu O397645 Gpró5 G-protein coupled receptor 65 Down-regu O4O1968 Galc Galactosylceramidase Down-regu O405587 Tgfbi Transforming growth factor, beta induced Down-regu O4O6928 Cd18O CD180 antigen Down-regu O416251 Egr3 Early growth response 3 Down-regu O439895 Alcam Activated leukocyte cell adhesion molecule Down-regu O448278 Mmp25 Matrix metallopeptidase 25 Down-regu O458S47 Gm8615 Glucosamine-6-phosphate deaminase 1 pseudogene Down-regu Gnpda1 O467124 Acta2 Actin, alpha 2, Smooth muscle, aorta Down-regu O467258 Myof MyOferlin Down-regu O469816 II1rn Interleukin 1 receptor antagonist Down-regu O47O614 11900O2A17Rik RIKEN cDNA 11900O2A17 gene Down-regu O487588 II1a Interleukin 1 alpha Down-regu O488.382 C93 CD93 antigen Down-regu O492021 Postn Periostin, osteoblast specific factor|RIKEN cDNA A630052E07 Down-regu A63OOS2EO7Rik gene O492682 Fam198b. Family with sequence similarity 198, member B Down-regu O498827 Fnip2 Folliculin interacting protein 2 Down-regu O499861 S100a S100 calcium binding protein A9 (calgranulin B) Down-regu OSO1164 CSfl. Colony stimulating factor 1 (macrophage) Down-regu OSO 1229 Gstm1 Glutathione S-transferase, mu 1 Down-regu 0505517 Tra. Toll-like receptor 4 Down-regu OS19983 Fgl2 Fibrinogen-like protein 2 Down-regu OS21667 BSt1 Bone marrow stromal cell antigen 1 Down-regu OS26853 Fam20c Family with sequence similarity 20, member C Down-regu O5282O7 C36 CD36 antigen Down-regu O535282 Card 11 Caspase recruitment domain family, member 11 Down-regu OS41599 Cec4b2 C-type lectin domain family 4, member b2 Down-regu 0542355 Emp1 Epithelial membrane protein 1 Down-regu OS4S101 Hpgds Hematopoietic prostaglandin D Synthase Down-regu OS47621 Apobec1 Apollipoprotein B mRNA editing enzyme, catalytic polypeptide 1 Down-regu OS47664 Clec4e C-type lectin domain family 4, member e Down-regu O560242 C5ar1 Complement component 5a receptor 1 Down-regu O562709 C33 CD33 antigen Down-regu OS64539 Mctp2 Multiple C2 domains, transmembrane 2 Down-regu OS6902O Ifitmé Interferon induced transmembrane protein 6 Down-regu US 2015/0038552 A1 Feb. 5, 2015 32

TABLE 9-continued List of differentially regulated genes between miR-223' and miR-223 eosinophil progenitor cultures at day 12. Transcript ID Gene Symbol Gene Description Regulation 10572130 Lp1 Lipoprotein lipase Down-regulated 10583.044 Mmp13 Matrix metallopeptidase 13 Down-regulated 10583100 Mmp8 Matrix metallopeptidase 8 Down-regulated 10583112 Mmp27 Matrix metallopeptidase 27 Down-regulated 106O1385 Tr13 Toll-like receptor 13 Down-regulated 10607870 Trf Toll-like receptor 7 Down-regulated

TABLE 10 List of overlapping differentially regulated genes between miR-223' and miR-223' eosinophil progenitor cultures at day 8 and day 12. Transcript ID Gene Symbol Gene Description Regulation 10427436 C7 Complement component 7 Up-regulated 10435288. Muc13 Mucin 13, epithelial transmembrane Up-regulated 10407940 Naip3| NLR family, apoptosis inhibitory protein 3T-cell receptor gamma, Up-regulated Tcrg-V2 variable 2IT-cell receptor gamma, variable 1 Tcrg-V1 10403821 Tcrg-V3. T-cell receptor gamma, variable 3|T-cell receptor gamma, variable 2 Up-regulated Tcrg-V2 10398.039 Serpina3f Serine (or cysteine) peptidase inhibitor, clade A, member 3Flserine (or Up-regulated Serpina3g cysteine) peptidase inhibitor, clade A, member 3G

Example 22 vious protocol (Blanchard, C. et al. J. Allergy Clin. Immunol. 120:1292-300 (2007)). The human bronchial epithelial cell Expression Profiling of miRNA in IL-13-Stimulated line HBEC was cultured as previously described (Ramirez, R. Epithelial Cells et al. Cancer Res. 64:9027-34 (2004)). The normal human 0274. A study was undertaken to identify miRNAs differ bronchial epithelial cells (Lonza, Basel, Switzerland) were entially expressed in epithelial cells in response to IL-13 cultured, as described in a previous protocol (Kariyawasam, stimulation. MiRNA expression in IL-13-stimulated human H. et al. J. Allergy Clin. Immunol. 124:454-462 (2009)). The bronchial and esophageal epithelial cells was determined U937 monocytes and Jurkat T cells were cultured in RPMI using miRNA microarrays. 1640 medium (Fisher Scientific) supplemented with 10% FBS, 100 U/mL penicillin, and 100 ug/ml streptomycin. The Human Esophageal Tissues CCD-16Lu fibroblasts were cultured in Eagle's Minimum 0275 Patients were selected without regard to age, race, or Essential Medium (ATCC, Manassas, Va.) supplemented sex. Normal patients presented to the clinic with symptoms with 10% FBS, 100 U/mL penicillin, and 100 g/ml strepto consistent with gastroesophageal reflux disease or EE, but the mycin. endoscopic and histologic findings for these patients were RNA Extraction and miRNA Microarray Analysis normal. The active EE patients had a clinical diagnosis of EE 0277 Human esophageal epithelial cells and bronchial and eosinophil counts of 224 per 400xhpf in the esophageal epithelial cells were stimulated with media or 100 ng/mL biopsies. The active chronic esophagitis patients had eosino IL-13 for 24 hours. Total RNA, including miRNA, was iso phil counts of 1-15 per 400xhpf in the esophageal biopsies. lated using the miRNeasy Mini Kit (Qiagen), according to the Patients with systemic or swallowed topical glucocorticoid manufacturers instructions. RNA quality was assessed using use were excluded from the selection of active EE or active the 2100 bioanalyzer (Agilent Technologies), and only chronic esophagitis patients. The EE remission patients samples with RNA integrity number >8 were used. MiRNA responding to steroid treatment had a clinical history of EE, expression from human bronchial epithelial cells was profiled treatment with Swallowed topical glucocorticoids, and using the TaqMan Human MicroRNA Array v1.0 (Applied responsiveness as indicated by an eosinophil count of s1 per Biosystems), which includes probes for 365 human miRNAs, 400xhpf and normalization of histological features of the according to the manufacturer's protocols. Data analysis was disease. The EE remission patients responding to diet treat carried out using GeneSpring Software (Agilent Technolo ment had a clinical history of EE, treatment with diet modi gies). fication, and responsiveness, as described above. The EE (0278. The miRNAs differentially regulated between patients not responding to glucocorticoid treatment had a unstimulated and IL-13 stimulated samples were identified clinical history of EE, treatment with swallowed topical glu by normalizing the expression data to the average of two cocorticoid, and non-responsiveness as indicated by an eosi endogenous control probes, namely RNU44 and RNU48, nophil count of -24 per 400xhpf. then filtered on cycle threshold values <35 and at least a 2-fold change between unstimulated and IL-13-stimulated samples. Cell Culture Statistical significance was determined at p-0.05 with Ben 0276 Human esophageal epithelial cells derived from jamini Hochberg false discovery rate correction. The list of human patient biopsies were cultured, as described in a pre differentially expressed miRNAs was clustered using hierar US 2015/0038552 A1 Feb. 5, 2015 chical clustering, and a heatmap was generated. A similar Experimental Asthma Induction in IL-13 Bitransgenic Mice analysis was carried out comparing unstimulated and IL-13 stimulated human esophageal epithelial cells, except the Taq 0284 Bitransgenic mice bearing CCSP-rtTA and (tetO) Man Human MicroRNA Array V2.0 (Applied Biosystems) ,CMV-IL-13 transgenes have been previously described (Lu, was used, which includes probes for 667 human miRNAs. T. et al. J. Immunol. 182:4994-5002 (2009)). Experimental The microarray data have been deposited into the Array asthma was induced in IL-13 bitransgenic mice by feeding Express database, found at http

(Ackerman, S. et al. J. Biol. Chem. 277:14859-68 (2002)), generated. Biological functional enrichment analysis was mast cells (CPA3 and TPSAB1) (Xing, W. et al. Proc. Natl. carried out using Ingenuity Pathway Analysis (Ingenuity Sys Acad. Sci. U.S.A. 108: 14210-5 (2011)), and epithelial cells tems) and Toppgene/Toppcluster (Cincinnati Children’s Hos (FLG) (Blanchard, C. et al. J. Immunol. 184:4033-41 (2010)) pital Medical Center) (Chen, J. et al. Nucleic Acids Res. (FIGS. 28B-C). 37:W305-11 (2009): Kaimal, V. et al. Nucleic Acids Res. Example 24 38:W96-102 (2010)). The microarray data have been depos ited into the Array Express database, found at http 99% GFP+ after selection. Human Genome-Wide mRNA Microarray 0299 MiR-375 was able to repress a large set of genes at 0295) The human Gene 1.0ST array (Affymetrix) was baseline, consistent with the function of miRNAs as repres used to compare the gene expression profiles of control sors of gene expression (FIG. 29A). A smaller set of genes transduced TE-7 cells and pre-miR-375-transduced TE-7 were induced at baseline; this can be though miR-375 medi cells before and after IL-13 treatment. Microarray data were ated repression of transcriptional repressors (FIG. 30A). analyzed using GeneSpring Software (Agilent Technologies, MiR-375 was able to both potentiate and antagonize a subset San Diego, Calif.), as described in a previous protocol (Lu, T. of IL-13 mediated gene signatures, indicating a complex et al. J. Immunol. 187:3362-73 (2011)). interaction between miR-375 and effects of IL-13 (FIG. 0296 Global scaling was performed to compare genes 30A). from chip to chip, and a base set of probes was generated by 0300 Functional analysis indicated that the pathways requiring a minimum raw expression level of the 20th per affected by miR-375 under IL-13-stimulated conditions were centile out of all probes on the microarray. The resulting enriched for processes involved in extracellular matrix orga probe sets were then baseline transformed and filtered on at nization, cellular junctions, and inflammation (FIG. 30B). least a 1.2-fold difference between control-transduced and The differentially regulated genes are listed in Table 11. pre-miR-375-transduced cells with or without IL-13 treat 0301 Analysis of all miR-375-regulated genes indicated ment to identify miR-375 regulated genes. Statistical signifi that inflammatory diseases and immunological diseases are cance was determined at p-0.05 with Benjamini Hochberg the two most significantly over-represented disease states false discovery rate correction. The resulting list of genes was (FIG. 31). The miR-375-regulated genes in each of these clustered using hierarchical clustering, and a heatmap was disease processes are listed in Table 12. US 2015/0038552 A1 Feb. 5, 2015 35

TABLE 11 List of genes differentially regulated by miR-375 in esophageal epithelial cells before and after IL-13 stimulation. Transcript ID Gene Symbol Gene Description Regulation 7900129 C1orf113 Chromosome 1 open reading frame 113 family with sequence Repressed by miR-375 a FAM176B similarity 176, member B baseline 7905339 GABPB2 GA binding protein transcription factor, beta Subunit 2 Repressed by miR-375 a baseline 790.7531 GPR52 G protein-coupled receptor 52 Repressed by miR-375 a baseline 7910379 DUSPSP Dual specificity phosphatase 5 pseudogene Repressed by miR-375 a 3.Selle 7914322 SNORD103A Small nucleolar RNA, CD box 103 Asmall nucleolar RNA, CD Repressed by miR-375 a SNORD103B box 103B baseline 7914324 SNORD103A Small nucleolar RNA, CD box 103 Asmall nucleolar RNA, CD Repressed by miR-375 a SNORD103B box 103B baseline 7920664 THEBS3 Thrombospondin 3 Repressed by miR-375 a baseline 7920852 KIAAO907 KIAAO907 Repressed by miR-375 a baseline 7934278 P4HA1 Prolyl 4-hydroxylase, alpha polypeptide I Repressed by miR-375 a baseline 7942332 FOLR1 Folate receptor 1 (adult) Repressed by miR-375 a baseline 7944867 SIAE Sialic acid acetylesterase Repressed by miR-375 a baseline 7951297 MMP12 Matrix metallopeptidase 12 (macrophage elastase) Repressed by miR-375 a baseline 79542O8 PIK3C2G Phosphoinositide-3-kinase, class 2, gamma polypeptide Repressed by miR-375 a 3.Selle 7956038 MMP19 Matrix metallopeptidase 19hypothetical LOC4401.04 Repressed by miR-375 a LOC44O104 baseline 795.6876 LLPH LLP homolog, long-term synaptic facilitation (Aplysia) Repressed by miR-375 a baseline 7963261 LOC4941SO Prohibitin pseudogene Repressed by miR-375 a baseline 7964119 STAT2 Signal transducer and activator of transcription 2, 113 kDa Repressed by miR-375 a baseline 796.7210 LOC338799 LOC338799 Repressed by miR-375 a baseline 7971461 LCP Lymphocyte cytosolic protein 1 (L-plastin) Repressed by miR-375 a baseline 7978718 SEC23A Sec23 homotog A (S. cerevisiae) Repressed by miR-375 a baseline 7994,582 SULT1A3 Sulfotransferase family, cytosolic, 1A, phenol-preferring, member 3 Repressed by miR-375 a GIYD1 GIY-YIG domain containing 1|GIY-YIG domain containing 21 baseline GIYD2 Sulfotransferase family, cytosolic, 1A, phenol-preferring, member 2 SULT1A2 Sulfotransferase family, cytosolic, 1A, phenol-preferring, member 4 SULT1A4 7994781 SULT1A3 Sulfotransferase family, cytosolic, 1A, phenol-preferring, member 3 Repressed by miR-375 at G1YD1 G1Y-YIG domain containing 1|GIY-YIG domain containing 2 baseline GIYD2 Sulfotransferase family, cytosolic, 1A, phenol-preferring, member 2 SULT1A2 Sulfotransferase family, cytosolic, 1A, phenol-preferring, member 4 SULT1A4 7996430 HSF4IFBXL8 Heat shock transcription factor 4 I F-box and leucine rich repeat Repressed by miR-375 a protein 8 baseline 7998664 SNORA10 Small nucleolar RNA, H/ACA box 10 Repressed by miR-375 a baseline 8000757 DOC2A Double C2-like domains, alphaINO80 complex subunit E Repressed by miR-375 a INO8OE baseline 8000799 GDPD3 Glycerophosphodiester phosphodiesterase domain containing 3 Repressed by miR-375 a baseline 8O13364 SLC47A2 Solute carrier family 47, member 2 Repressed by miR-375 a baseline 8O13622 SGK494 Uncharacterized serine/threonine-protein kinase Sgk294 Repressed by miR-375 a FL2SOO6 baseline 8O14903 GSDMB Gasclermin B Repressed by miR-375 a baseline 8O16259 LRRC37A2 Leucine rich repeat containing 37, member A2 leucine rich repeat Repressed by miR-375 a LRRC37A3 containing 37Aleucine rich repeat containing 37, member A3 baseline LOC10O2943.35 similar to c114 SLIT-like testicular protein 8O31176 LENG8 Leukocyte receptor cluster (LRC) member 8 Repressed by miR-375 a baseline 8O32249 ADAMTSLS ADAMTS-like 5 Repressed by miR-375 a baseline US 2015/0038552 A1 Feb. 5, 2015 36

TABLE 1 1-continued List of genes differentially regulated by miR-375 in esophageal epithelial cells before and after IL-13 stimulation. Transcript ID Gene Symbol Gene Description Regulation 8033257 C3 Complement component 3 Repressed by miR-375 a baseline 8041204 SNORA10 Small nucleolar RNA, H/ACA box 10 Repressed by miR-375 a baseline 80446OS LOC654433 LOC654433 Repressed by miR-375 a baseline 8045,539 KYNU Kynureninase (L-kynurenine hydrolase) Repressed by miR-375 a baseline 8046906 GULP1 GULP, engulfment adaptor PTB domain containing 1 Repressed by miR-375 a 3.Selle 8048381 STK36 Serine/threonine kinase 36, fused homotog (Drosophila) Repressed by miR-375 a baseline 8053200 DQX1 DEAQ box RNA-dependent ATPase 1 Repressed by miR-375 a baseline 8056151 PLA2R1 Phospholipase A2 receptor 1, 180 kDa Repressed by miR-375 a baseline 8066786 ZMYND8 Zinc finger, MYND-type containing 8 Repressed by miR-375 a baseline 8066822 SULF2 Sulfatase 2 Repressed by miR-375 a baseline 8074577 PI4KAP2 Phosphatidylinositol 4-kinase, catalytic, alpha pseudogene 2 Repressed by miR-375 a P4KAP1 phosphatidylinositol 4-kinase, catalytic, alpha pseudogene 1 baseline 8085.716 SATB1 SATB homeobox 1 Repressed by miR-375 a baseline 8O86627 ALS2CL ALS2C-terminal like Repressed by miR-375 a baseline 8O87433 NICN1 Nicolin 1|aminomethyltransterase Repressed by miR-375 a AMT baseline 8092978 MUC4 Mucin 4, cell Surface associated Repressed by miR-375 a baseline 80984.41 ODZ3 Odz, Odd Oz ten-m homolog 3 (Drosophila) Repressed by miR-375 a baseline 8102792 PCDH18 Protocadherin 18 Repressed by miR-375 a baseline 81 03853 MGC4S800 Hypothetical LOC90768 Repressed by miR-375 a baseline 81OSO77 CARD6 Caspase recruitment domain family, member 6 Repressed by miR-375 a baseline 8109086 ADRB2 Adrenergic, beta-2-, receptor, Surface Repressed by miR-375 a baseline 8116921 EDN Endothelin 1 Repressed by miR-375 a baseline 8117714 OR2J3 Olfactory receptor, family 2, Subfamily J, member 3 olfactory Repressed by miR-375 a OR21 receptor, family 2, subfamily J, member 1 baseline 81246.54 GABBR1 Gamma-aminobutyric acid (GABA) B receptor, 1 Repressed by miR-375 a baseline 8124691 HCG8 HLA complex group 8 Repressed by miR-375 a baseline 812S687 RGL2 Ral guanine nucleotide dissociation stimulator-like 2 Repressed by miR-375 a baseline 8126O18 STK38 Serine/threonine kinase 38 Repressed by miR-375 a baseline 8126428 TRERF1 Transcriptional regulating factor 1 Repressed by miR-375 a baseline 8133633 NSUNSP2 NOP2/Sun domain family, member 5 pseudogene 2INOP2 Sun Repressed by miR-375 a NSUNSP1 domain family, member 5 pseudogene 1|NOL1/NOP2 Sun domain baseline NSUNSB family, member 5BINOL1/NOP2/Sun domain family, member 5| NSUNSC NOL1/NOP2/Sun domain family, member 5Cltripartite motif TRIM73 containing 73 8140386 STYXL1 Serine/threonine?tyrosine interacting-like 1 Repressed by miR-375 at baseline 81.48149 ZHX2 Zinc fingers and homeoboxes 2 Repressed by miR-375 at baseline 8153550 NRBP2 Nuclear receptor binding protein 2 Repressed by miR-375 at baseline 8156060 TLE4 Transducin-like enhancer of split 4 (E(sp1) homolog, Drosophila) Repressed by miR-375 at baseline 8157800 MIR181A2 MicroRNA 181 a-2 Repressed by miR-375 at baseline 8158662 SNORD62A Small nucleolar RNA, CD box 62A small nucleolar RNA, CD box Repressed by miR-375 at SNORD62B 62B baseline 8158864 SNORD62A Small nucleolar RNA, CD box 62A small nucleolar RNA, CD box Repressed by miR-375 at SNORD62B 62B baseline US 2015/0038552 A1 Feb. 5, 2015 37

TABLE 1 1-continued List of genes differentially regulated by miR-375 in esophageal epithelial cells before and after IL-13 stimulation. Transcript ID Gene Symbol Gene Description Regulation 8161.373 LOC100289528 LOC100289528 Repressed by miR-375 a baseline 8161554 LOC100289528 LOC100289528 Repressed by miR-375 a baseline 8170921 PLXNA3 Plexin A3 Repressed by miR-375 a baseline 8175666 GABRE Gamma-aminobutyric acid (GABA) A receptor, epsilon Repressed by miR-375 a baseline 8177694 OR2J3 Olfactory receptor, family 2, Subfamily J, member 3 olfactory Repressed by miR-375 a OR21 receptor, family 2, subfamily J, member 1 baseline 8178298 GABBR1 Gamma-aminobutyric acid (GABA) B receptor, 1 Repressed by miR-375 a baseline 81789SS RGL2 Ral guanine nucleotide dissociation stimulator-like 2 Repressed by miR-375 a baseline 8179003 OR2J3 Olfactory receptor, family 2, Subfamily J, member 3 olfactory Repressed by miR-375 a OR21 receptor, family 2, subfamily J, member 1 baseline 8179595 GABBR1 Gamma-aminobutyric acid (GABA) B receptor, 1 Repressed by miR-375 a baseline 818O144 RCL2 Ral guanine nucleotide dissociation stimulator-like 2 Repressed by miR-375 a baseline 7904364 WDR3 WD repeat domain 3 induced by miR-375 at baseline 7916219 C1orf163 Chromosome 1 open reading frame 163 induced by miR-375 at baseline 7927267 FAM35B Family with sequence similarity 35, member Bfamily with induced by miR-375 at baseline FAM35B2 sequence similarity 35, member B2|family with sequence similarity FAM3SA 35, member A 7927288 FAM35B2 Family with sequence similarity 35, member B2|family with induced by miR-375 at baseline FAM3SA sequence similarity 35, member Bfamily with sequence similarity 35, member A 792.9072 IFITS Interferon-induced protein with tetratricopeptide repeats 5 induced by miR-375 at baseline 7932765 MPP7 Membrane protein, palmitoylated 7 (MAGUK p55 subfamily induced by miR-375 at baseline member 7) 7935707 CHUK Conserved helix-loop-helix ubiquitous kinase induced by miR-375 at baseline 7944656 SCSDL Sterol-C5-desaturase (ERG3 delta-5-desaturase homolog, induced by miR-375 at baseline S. cerevisiae)-like 794-5169 TMEM4SB Transmembrane protein 45B induced by miR-375 at baseline 7948908 SNORD26 Small nucleolar RNA, C/D box 26 small nucleolar RNA host gene induced by miR-375 at baseline SNEHG1 1 (non-protein coding) 8059026 MIR375 MicroRNA375 induced by miR-375 at baseline 7951O36 SNORDS Small nucleolar RNA, C/D box 5 ITATA box binding protein induced by miR-375 at baseline TAF1D (TBP)-associated factor, RNA polymerase I, D, 41 kDa 7.981964 SNORD116-8 Small nucleolar RNA, CD box 116-8 small nucleolar RNA, CD induced by miR-375 at baseline SNORD11 6-3 box 116-3|small nucleolar RNA, CD box 116-9 SNORD11 6-9 7981976 SNORD116-14 Small nucleolar RNA, CD box 116-14 induced by miR-375 at baseline 7981978 SNORD116-15 Small nucleolar RNA, CD box 116-15 induced by miR-375 at baseline 7981982 SNORD116-17 Small nucleolar RNA, CD box 116-17small nucleolar RNA, CD induced by miR-375 at baseline SNORD116-19 box 116-19small nucleolar RNA, CD box 116-15small nucleolar SNORD11 6-15 RNA, CD box 116-16small nucleolar RNA, CD box 116-18 SNORD11 6-16 Small nucleolar RNA, CD box 116-21 small nucleolar RNA, CD SNORD116-18 box 116-22small nucleolar RNA, CD box 116-14|small nucleolar SNORD11 6-21 RNA, CD box 116-20 small nucleolar RNA, CD box 116 cluster SNORD11 6-22 Small nuclear ribonucleoprotein polypeptide N SNORD116-14 SNORD11 6-2O SNORD116(a) SNRPN 7981986 SNORD116-17 Small nucleolar RNA, CD box 116-17small nucleolar RNA, CD Induced by miR-375 at baseline SNORD116-19 box 116-19small nucleolar RNA, CD box 116-15small nucleolar SNORD11 6-15 RNA, CD box 116-16small nucleolar RNA, CD box 116-18 SNORD11 6-16 Small nucleolar RNA, CD box 116-21 small nucleolar RNA, CD SNORD116-18 box 116-22small nucleolar RNA, CD box 116-14|small nucleolar SNORD11 6-21 RNA, CD box 116-20 small nucleolar RNA, CD box 116 cluster SNORD11 6-22 Small nuclear ribonucleoprotein polypeptide N SNORD116-14 SNORD11 6-2O SNORD116(a) SNRPN 7981988 SNORD116-20 Small nucleolar RNA, CD box 116-20 small nucleolar RNA, CD Induced by miR-375 at baseline SNORD116(a) box 116 cluster 798.1996 SNORD11 6-24 Small nucleolar RNA, CD box 116-24 Induced by miR-375 at baseline 8OO6634 PIGW Phosphatidylinositol glycan anchor biosynthesis, class W Induced by miR-375 at baseline 8OO9380 SNORA38B Small nucleolar RNA, H/ACA box 38B (retrotransposed) Induced by miR-375 at baseline US 2015/0038552 A1 Feb. 5, 2015 38

TABLE 1 1-continued List of genes differentially regulated by miR-375 in esophageal epithelial cells before and after IL-13 stimulation. Transcript ID Gene Symbol Gene Description Regulation 8106193 UTP15 U3 small nucleolar RNA-associated protein 15 homolog induced by miR-375 at baseline 8122440 LTV1 Yeast LTV1 homotog induced by miR-375 at baseline 8132897 LANCL2 Lanc lantibiotic synthetase component C-like 2 (bacterial) induced by miR-375 at baseline 8132922 MRPS17 Mitochondrial ribosomal protein S17 glioblastoma amplified induced by miR-375 at baseline GBAS sequence Zinc finger protein 713 ZNF713 8146685 RRS1 Homolog of yeast ribosome biogenesis regulatory protein RRS1 induced by miR-375 at baseline 8146921 RDH10 Retinol dehydrogenase 10 (all-trans) induced by miR-375 at baseline 8150537 SLC20A2 Solute carrier family 20 (phosphate transporter), member 2 induced by miR-375 at baseline MPAD1 nositol monophosphatase domain containing 1 induced by miR-375 at baseline 7904106 MAGI3 Membrane associated guanylate kinase, WW and PDZ domain 3-me induction containing 3 agonize by miR-375 7908204 HMCN1 Hemicentin 1 3-me iate induction agonize by miR-375 7917912 DPYD Dihydropyrimidine dehydrogenase 3-me iate induction agonize by miR-375 7920687 GBAP1 Glucosidase, beta, acid pseudogene 1 3-me iate induction agonize by miR-375 7968872 DNAJC15 DnaJ (Hsp40) homolog, subfamily C, member 15 3-me iate induction agonize by miR-375 798.5752 NCRNAOOOS2 Non-protein coding RNA52 3-me iate induction agonize by miR-375 798836O DUOX2 Dual oxidase 2 3-me iate induction agonize by miR-375 804O249 PDA6 Protein disulfide isomerase family A, member 6 ATPase, H+ 3-me iate induction ATP6V1C2 transporting, lysosomal 42 kDa, V1 subunit C2 agonize by miR-375 8043036 LOC1720 Dihydrofolate reductase pseudogene 3-me iate induction agonize by miR-375 8076113 LOC646851 Similar to OTTHUMPOOOOOO2872O 3-me iate induction RP1-199H16.1 agonize by miR-375 8130129 NUP43 Nucleoporin 43 kDa 3-me iate induction agonize by miR-375 SEMA3D Sema domain, immunoglobulin domain (Ig), short basic domain, 3-me iate induction secreted, (semaphorin) 3D agonize by miR-375 SAMD9L. Sterile alpha motif domain containing 9-like 3-me iate induction agonize by miR-375 7905154 C1 orf51 Chromosome 1 open reading frame 51 3-me iate repression agonize by miR-375 7975062 SIPA1L2 Signal-induced proliferation-associated 1 like 2 3-me iate repression agonize by miR-375 79.38485 MICAL2 Microtubule associated monoxygenase, calponin and LIM domain 3-me iate repression containing 2 agonize by miR-375 8069795 CLDN8 Claudin 8 3-me iate repression agonize by miR-375 8020740 DSG4 Desmoglein 4 3-me iate repression agonize by miR-375 8042283 HSPC159 Galectin-related protein 3-me iate repression agonize by miR-375 ACVR1 Activin A receptor, type 1 3-me iate repression agonize by miR-375 8102800 SLC7A11 Solute carrier family 7, (cationic amino acid transporter, y+ system) 3-me iate repression member 11 agonize by miR-375 811S623 ATP1 OB ATPase, class V, type 10B 3-me iate repression agonize miR-375 8164215 SNORA6S Small nucleolar RNA, H/ACA box 65 3-me ia e repression agonize miR-375 792.7936 DDX21 DEAD (Asp-Giu-Ala-Asp) box polypeptide 21 3-medi18 induction entiate R-375 792.9047 IFIT2 Interferon-induced protein with tetratricopeptide repeats 2 3-me induction entiate l R-375 7938702 DKFZp686O24166 DKFZp686O24166 3-me induction entiate l R-375 79.54382 PYROXD1 Pyridine nucleotide-disulphide oxidoreductase domain 1|RecQ 3-me induction RECQL protein-like (DNA helicase Q1-like) entiate l R-375 8042830 MTHFD2 Methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 2, 3-me induction methenyltetrahydrofolate cyclohydrolase entiate l R-375 8047788 ADAM 23 ADAM metallopeptidase domain 23 3-me induction o entiate l R-375 8062211 RPF2 Ribosome production factor 2 homolog (S. cerevisiae) brix domain 3-me induction BXDC1 containing 1 entiate l R-375 8069532 HSPA13 Heat shock protein 70 kDa family, member 13 3-me uction O entiate h R -3 7 5 US 2015/0038552 A1 Feb. 5, 2015 39

TABLE 1 1-continued List of genes differentially regulated by miR-375 in esophageal epithelial cells before and after IL-13 stimulation. Transcript ID Gene Symbol Gene Description Regulation 8O84O64 MTHFD2 Methyenetetrahydrofolate dehydrogenase (NADP+ dependent) 2, L-13-mediated induction methenyltetrahydrofolate cyclohydrolase potentiated by miR-375 8135774 PTPRZ1 Protein tyrosine phosphatase, receptor-type, Z polypeptide 1 L-13-mediated induction potentiated by miR-375 81.42S4O FAM3C Family with sequence similarity 3, member C L-13-mediated induction potentiated by miR-375 8166442 FAM3C Family with sequence similarity 3, member C L-13-mediated induction potentiated by miR-375 8171248 KAL1 Kallmann syndrome 1 sequence L-13-mediated induction potentiated by miR-375 792.445O DUSP10 Dual specificity phosphatase 10 L-13-mediated repression potentiated by miR-375 796237S PRICKLE1 Prickle homolog 1 (Drosophila) L-13-mediated repression potentiated by miR-375 81.27563 COL12A1 Collagen, type XII, alpha 1 L-13-mediated repression potentiated by miR-375 8133688 SNORA14A Small nucleolar RNA, H/ACA box 14A L-13-mediated repression potentiated by miR-375 81756.83 MIR224 MicroRNA 224 gamma-aminobutyric acid (GABA) A receptor, L-13-mediated repression GABRE epsilon potentiated by miR-375 796S4O3 LUM Lumican L-13-mediated repression potentiated by miR-375 8O26490 UCA1 Urothelial cancer associated 1 (non-protein coding) L-13-mediated repression potentiated by miR-375 8033674 MUC16 Mucin 16, cell Surface associated L-13-mediated repression potentiated by miR-375 8049123 ALPP Alkaline phosphatase, placental (Regan isozyme) L-13-mediated repression potentiated by miR-375 80491.28 ALPPL2 Alkaline phosphatase, placental-like 2 L-13-mediated repression potentiated by miR-375 8058765 FN1 Fibronectin 1 L-13-mediated repression potentiated by miR-375 8104693 PDZD2 PDZ domain containing 2 L-13-mediated repression potentiated by miR-375 812682O GPR110 G protein-coupled receptor 110 L-13-mediated repression potentiated by miR-375

TABLE 12 List of miR-375-regulated genes that are involved in immunoinflammatory responses. Gene ID Gene Symbol Gene Description Disease Process 90 ACVR1 Activin A receptor, type 1 inflammatory diseases 154 ADRB2 Adrenergic, beta-2-, receptor, Surface inflammatory diseases 2SO ALPP Alkaline phosphatase, placental inflammatory diseases 275 AMT Aminomethyltransferase inflammatory diseases 718 C3 Complement component 3 inflammatory diseases 1147 CHUK Conserved helix-loop-helix ubiquitous kinase inflammatory diseases 8448 DOC2A Double C2-like domains, alpha inflammatory diseases 1806 DPYD Dihydropyrimidine dehydrogenase inflammatory diseases SOSO6 DUOX2 Dual oxidase 2 inflammatory diseases 11221 DUSP10 Dual specificity phosphatase 10 inflammatory diseases 2335 FN1 Fibronectin 1 inflammatory diseases 2SSO GABBR1 Gamma-aminobutyric acid (GABA) B receptor, 1 inflammatory diseases 2564 GABRE Gamma-aminobutyric acid (GABA) A receptor, epsilon inflammatory diseases SS876 GSDMB Gasclermin B inflammatory diseases 8942 KYNU Kynureninase (L-kynurenine hydrolase) inflammatory diseases 3936 LCP1 Lymphocyte cytosolic protein 1 (L-plastin) inflammatory diseases 6468.51 LOC646851 Hypothetical LOC646851 inflammatory diseases 4060 LUM Lumican inflammatory diseases 260425 MAG13 Membrane-associated guanylate kinase, WW and PDZ domain containing 3 inflammatory diseases 964.5 MICAL2 Microtubule-associated monoxygenase, calponin and LIM domain containing 2 inflammatory diseases 4321. MMP12 Matrix metallopeptidase 12 (macrophage elastase) inflammatory diseases 143098 MPP7 Membrane protein, palmitoylated 7 (MAGUK p55 subfamily member 7) inflammatory diseases 84276 NICN1 Nicolin 1 inflammatory diseases SS714 ODZ3 Odz, Odd Oziten-m homolog 3 (Drosophila) inflammatory diseases 23O37 PDZD2 PDZ domain containing 2 inflammatory diseases S288 PIK3C2G Phosphoinositide-3-kinase, class 2, gamma polypeptide inflammatory diseases US 2015/0038552 A1 Feb. 5, 2015 40

TABLE 12-continued List of miR-375-regulated genes that are involved in immunoinflammatory responses. Gene ID Gene Symbol Gene Description Disease Process 2292S PLA2R1 Phospholipase A2 receptor 1, 180 kDa inflammatory diseases 14416S PRICKLE1 Prickle homolog 1 (Drosophila) inflammatory diseases 58O3 PTPRZ1 Protein tyrosine phosphatase, receptor-type, Z polypeptide 1 inflammatory diseases S863 RGL2 Ral guanine nucleotide dissociation stimulator-like 2 inflammatory diseases 21928S SAMD9L. Sterile alpha motif domain containing 9-like inflammatory diseases 223.117 SEMA3D Sema domain, immunoglobulin domain (Ig), short basic domain, Secreted, inflammatory diseases (semaphorin) 3D 23657 SLC7A11 Solute earner family 7+ (cationic amino acid transporter, y+ system) member 11 inflammatory diseases 66.38 SNRPN Small nuclear ribonucleoprotein polypeptide N inflammatory diseases 2.7148 STK36 Serine/threonine kinase 36, fused homolog (Drosophila) inflammatory diseases 55959 SULF2 Sulfatase 2 inflammatory diseases 120224 TMEM4SB Transmembrane protein 45B inflammatory diseases 84135 UTP15 UTP15, U3 Small nucleolar ribonucleoprotein, homolog (S. cerevisiae) inflammatory diseases 22882 ZHX2 Zinc fingers and homeoboxes 2 inflammatory diseases 23613 ZMYND8 Zinc finger, MYND-type containing 8 inflammatory diseases 8745 ADAM23 ADAM metallopeptidase domain 23 mmunological diseases 54 ADRB2 Adrenergic, beta-2-, receptor, Surface mmunological diseases 718 C3 Complement component 3 mmunological diseases 1147 CHUK Conserved helix-loop-helix ubiquitous kinase mmunological diseases 8448 DOC2A Double C2-like domains, alpha mmunological diseases 1806 DPYD Dihydropyrimiddline dehydrogenase mmunological diseases 165545 DQX1 DEAQ box polypeptide 1 (RNA-dependent ATPase) mmunological diseases 11221 DUSP10 Dual specificity phosphatase 10 mmunological diseases 1906 EDN1 Endothelin 1 mmunological diseases 2335 FN1 Fibronectin 1 mmunological diseases 2SSO GABBR1 Gamma-aminobutyric acid (GABA) B receptor, 1 mmunological diseases 2S53 GABPB2 GA binding protein transcription factor, beta Subunit 2 mmunological diseases 2564 GABRE Gamma-aminobutyric acid (GABA) A receptor, epsilon mmunological diseases 3433 IFIT2 Lnterferon-induced protein with tetratricopeptide repeats 2 mmunological diseases 3936 LCP1 Lymphocyte cytosolic protein 1 (L-plastin) mmunological diseases 6468.51 LOC646851 Hypothetical LOC646851 mmunological diseases 260425 MAGI3 Membrane-associated guanylate kinase, WW and PDZ domain containing 3 mmunological diseases 964.5 MICAL2 Microtubule-associated monoxyrenase, calponin and LIM domain containing 2 mmunological diseases 143098 MPP7 Membrane protein palmitoylated 7 mmunological diseases SS714 ODZ3 Odz, Odd Oziten-m homolog 3 (Drosophila) mmunological diseases 23O37 PDZD2 PDZ domain containing 2 mmunological diseases S288 PIK3C2G Phosphoinositide-3-kinase, class 2, gamma polypeptide mmunological diseases 2292S PLA2R1 Phospholipase A2 receptor 1, 180 kDa mmunological diseases 14416S PRICKLE1 Prickle homolog 1 (Drosophila) mmunological diseases S863 RGL2 Ral guanine nucleotide dissociation stimulator-like 2 mmunological diseases 21928S SAMD9L. Sterile alpha motif domain containing 9-like mmunological diseases S7568 SIPA1L2 Signal-induced proliferation-associated 1 like 2 mmunological diseases 23657 SLC7A11 Solute carrier family 7, (cationic amino acid transporter, y+ system) member 11 mmunological diseases 66.38 SNRPN Small nuclear ribonucleoprotein polypeptide N mmunological diseases 11329 STK38 Serine/threonine kinase 38 mmunological diseases 84135 UTP15 UTP15, U3 Small nucleolar ribonucleoprolein, homolog (S. cerevisiae) mmunological diseases 22882 ZHX2 Zinc fingers and homeoboxes 2 mmunological diseases

0302 MiR-375 was found to have no effect on TSLP 0304. The treatment method is carried out on a patient to production (FIG. 31), and there was no correlation between determine the patients level(s) of miRNAs associated with miR-375 and TSLP in the esophageal samples. The control EE and whether the level of the one or more miRNAs is transduced cells and pre-miR-375-transduced cells expressed up-regulated or down-regulated relative to a level of the one TSLP at similar levels without stimulation and have similar or more miRNAs measured in a normal individual, wherein levels of induction after polyinosinic:polycytidylic acid the presence of an elevated or reduced level of one or more miRNAs associated with EE results in the patient being diag (polyI:C) stimulation (FIG. 32). nosed with EE. A patient sample is analyzed for expression of at least one of the miRNAs, or a subset of the miRNAs or all Example 26 of the miRNAs, as listed in Tables 2 and 3. The data is analyzed to determine expression levels of the miRNAs as Treatment of EE Based on Determination of miRNA disclosed herein to establish an EE diagnosis, which is then Levels used to determine an appropriate therapeutic strategy depend ing on the diagnosis. 0303 Determination of level(s) of miRNAs associated Example 27 with EE, as described herein, can be used to treat EE. For example, determination of level(s) of miRNAs associated Optimizing EE Treatment Based on Specific miRNA with EE can be used to establish an EE diagnosis, which can Levels then be used to determine an appropriate therapeutic strategy 0305 Determination of level(s) of miRNAs associated depending on the diagnosis. with EE, as described herein, can be used to treat EE to US 2015/0038552 A1 Feb. 5, 2015

determine if a particular drug is or could potentially be effec EE and whether the level of the one or more miRNAs is tive. For example, the determination of level(s) of miRNAs up-regulated or down-regulated relative to a level of the one associated with EE can be used to determine if a therapy or more miRNAs measured in a normal individual, wherein specific for a molecule involved in EE disease pathogenesis the presence of an elevated or reduced level of one or more up- or down-regulates certain EE-associated miRNAs. miRNAs associated with EE results in the patient being diag 0306 As described in Example 6, miR-675 was found to nosed with EE. A patient sample is analyzed for expression of be the only disease remission-induced miRNA, as miR-675 is at least one of the miRNAs, or a subset of the miRNAs or all up-regulated in glucocorticoid responder patients compared of the miRNAs, as listed in Tables 2 and 3. The data is to normal. EE, or chronic esophagitis patients. Accordingly, analyzed to determine expression levels of the miRNAs as miR-675 can be used to identify, and thereby determine an disclosed herein to establish an EE diagnosis. effective treatment strategy for, glucocorticoid responder patients, as well as EE patients who do not respond to gluco Example 29 corticoid treatment. 0307 Periostin has been demonstrated to have a key role Diagnosis of Eosinophilic Disorders Based on in IL-13 associated remodeling responses. Accordingly, Determination of miRNA Levels determination of level(s) of miRNAs associated with perios 0314 Determination of miRNAs level(s), as described tin can be used to identify, and thereby determine an effective herein, can be used to treat eosinophilic disorders other than treatment strategy for, anti-IL-13 responder patients, as well EE. For example, determination of level(s) of miRNAs asso as EE patients who do not respond to anti-IL-13 treatment. ciated with an eosinophilic disorder can be used to establish 0308 The treatment method is carried out on a patient to an eosinophilic disorder diagnosis, which can then be used to determine the patients level(s) of miRNAs associated with determine an appropriate therapeutic strategy depending on EE and whether the level of the one or more miRNAs is the diagnosis. up-regulated or down-regulated relative to a level of the one 0315. The miRNAs associated with EE can also be asso or more miRNAs measured in a normal individual, wherein ciated with eosinophilic disorders other than EE, such as the presence of an elevated or reduced level of one or more EGID and asthma, given that they relate to eosinophil prolif miRNAs associated with EE results in the patient being diag eration. For example, as described in Example 23, miR-375 nosed with EE. A patient sample is analyzed for expression of has been shown to be involved in asthma. Accordingly, miR at least one of the miRNAs, or a subset of the miRNAs or all 375 can be used as a biomarker alone or in combination with of the miRNAs, as listed in Tables 2 and 3. The data is other miRNAs shown to be involved with asthma, such as analyzed to determine expression levels of the miRNAs as miR-21 and miR-223, and the like. disclosed herein to establish an EE diagnosis, which is then 0316 The treatment method is carried out on a patient to used to determine an appropriate therapeutic strategy depend determine the patients level(s) of miRNAs associated with ing on the diagnosis. an eosinophilic disorder and whether the level of the one or 0309 The patient diagnosed with EE is evaluated to deter more miRNAS is up-regulated or down-regulated relative to a mine whether the patient is compliant with and/or exposed to level of the one or more miRNAs measured in a normal steroid treatment by determining the patient’s miR-675 level, individual, wherein the presence of an elevated or reduced wherein an elevated level of miR-675 indicates that the level of one or more miRNAs associated with an eosinophilic patient is compliant with and/or exposed to steroid treatment. disorder results in the patient being diagnosed with an eosi 0310. A patient diagnosed with EE for whom steroid nophilic disorder. A patient sample is analyzed for expression therapy has been determined to be the appropriate therapeutic of at least one of the miRNAs, or a subset of the miRNAs or strategy is evaluated following treatment to determine all of the miRNAs, as listed in Tables 2 and 3. The data is whether the patient is responsive or non-responsive to steroid analyzed to determine expression levels of the miRNAs as treatment, wherein an elevated level of miR-675 following disclosed herein to establish an eosinophilic disorder diagno treatment indicates that the patient is responsive to steroid sis, which is then used to determine an appropriate therapeu treatment. tic strategy depending on the diagnosis. 0311. The patient diagnosed with EE is also evaluated to 0317. The various methods and techniques described determine whether the patient is likely to be responsive or above provide a number of ways to carry out the application. non-responsive to anti-IL-13 treatment, wherein an elevated Of course, it is to be understood that not necessarily all level of one or more miRNAs associated with periostin levels, objectives or advantages described can be achieved in accor such as miR-223 and/or miR-375, indicates that the patient is dance with any particular embodiment described herein. likely to be responsive to anti-IL-13 treatment. Thus, for example, those skilled in the art will recognize that the methods can be performed in a manner that achieves or Example 28 optimizes one advantage or group of advantages as taught Diagnosis of EE Based on Determination of miRNA herein without necessarily achieving other objectives or Levels advantages as taught or Suggested herein. A variety of alter natives are mentioned herein. It is to be understood that some 0312 Determination of level(s) of miRNAs associated preferred embodiments specifically include one, another, or with EE, as described herein, can be used to diagnose EE. For several features, while others specifically exclude one, example, determination of level(s) of miRNAs associated another, or several features, while still others mitigate a par with EE can be used to establish an EE diagnosis, which can ticular feature by inclusion of one, another, or several advan then be used to determine an appropriate therapeutic strategy tageous features. depending on the diagnosis. 0318. Furthermore, the skilled artisan will recognize the 0313 The diagnostic method is carried out on a patient to applicability of various features from different embodiments. determine the patients level(s) of miRNAs associated with Similarly, the various elements, features and steps discussed US 2015/0038552 A1 Feb. 5, 2015 42 above, as well as other known equivalents for each Such 0323 All patents, patent applications, publications of element, feature or step, can be employed in various combi patent applications, and other material. Such as articles, nations by one of ordinary skill in this art to perform methods books, specifications, publications, documents, things, and/ in accordance with the principles described herein. Among or the like, referenced herein are hereby incorporated herein the various elements, features, and steps some will be specifi by this reference in their entirety for all purposes, excepting cally included and others specifically excluded in diverse any prosecution file history associated with same, any of embodiments. same that is inconsistent with or in conflict with the present 0319. Although the application has been disclosed in the document, or any of same that may have a limiting affect as to context of certain embodiments and examples, it will be the broadest scope of the claims now or later associated with understood by those skilled in the art that the embodiments of the present document. By way of example, should there be the application extend beyond the specifically disclosed any inconsistency or conflict between the description, defini embodiments to other alternative embodiments and/or uses tion, and/or the use of a term associated with any of the and modifications and equivalents thereof. incorporated material and that associated with the present 0320 In some embodiments, the numbers expressing document, the description, definition, and/or the use of the quantities of ingredients, properties such as molecular term in the present document shall prevail. weight, reaction conditions, and so forth, used to describe and 0324. In closing, it is to be understood that the embodi claim certain embodiments of the application are to be under ments of the application disclosed herein are illustrative of the stood as being modified in Some instances by the term principles of the embodiments of the application. Other modi "about.” Accordingly, in Some embodiments, the numerical fications that can be employed can be within the scope of the parameters set forth in the written description and attached application. Thus, by way of example, but not of limitation, claims are approximations that can vary depending upon the alternative configurations of the embodiments of the applica desired properties sought to be obtained by a particular tion can be utilized in accordance with the teachings herein. embodiment. In some embodiments, the numerical param Accordingly, embodiments of the present application are not eters should be construed in light of the number of reported limited to that precisely as shown and described. significant digits and by applying ordinary rounding tech 1. A method of treating a patient with eosinophilic esoph niques. Notwithstanding that the numerical ranges and agitis (EE), the method comprising: parameters setting forth the broad scope of Some embodi obtaining a sample from a patient; ments of the application are approximations, the numerical analyzing the sample to determine a level of one or more values set forth in the specific examples are reported as pre miRNAs associated with EE: cisely as practicable. determining whether the level of the one or more miRNAs 0321. In some embodiments, the terms “a” and “an and is up-regulated or down-regulated relative to a level of “the and similar references used in the context of describing the one or more miRNAs measured in a normal indi a particular embodiment of the application (especially in the vidual, wherein the presence of an elevated or reduced context of certain of the following claims) can be construed to level of one or more miRNAs associated with EE results cover both the singular and the plural. The recitation of ranges in the patient being diagnosed with EE; and of values herein is merely intended to serve as a shorthand treating the patient with an appropriate therapeutic strategy methodofreferring individually to each separate value falling based upon the diagnosis. within the range. Unless otherwise indicated herein, each 2. The method of claim 1, wherein the one or more miR individual value is incorporated into the specification as if it NAS associated with EE are selected from the group consist were individually recited herein. All methods described ing of miR-886-5p, miR-886-3p, miR-222*, miR-7, miR herein can be performed in any suitable order unless other 29b, miR-642, miR-339-5p, miR-21, miR-21*, miR-142-5p, wise indicated herein or otherwise clearly contradicted by miR-146a, miR-146b, miR-142-3p, miR-132, miR-212, context. The use of any and all examples, or exemplary lan miR-592, miR-92a-1*, miR-223*, miR-223, miR-801, miR guage (for example, "Such as”) provided with respect to cer 106b, miR-375, miR-211, miR-210, miR-365, miR-203, tain embodiments herein is intended merely to better illumi miR-193a-5p, miR-193b, miR-193a-3p, let-7c, miR-144*, nate the application and does not pose a limitation on the and miR-30a-3p. Scope of the application otherwise claimed. No language in 3. (canceled) the specification should be construed as indicating any non 4. (canceled) claimed element essential to the practice of the application. 5. The method of claim 1, wherein the determination of 0322 Preferred embodiments of this application are whether the level(s) of the one or more miRNAs associated described herein, including the best mode knownto the inven with EE are elevated or reduced relative to a level of the one tors for carrying out the application. Variations on those pre or more miRNAS measured in a normal individual is com ferred embodiments will become apparent to those of ordi bined with a determination of a level(s) of one or more addi nary skill in the art upon reading the foregoing description. It tional biomarkers associated with EE. is contemplated that skilled artisans can employ such varia 6. (canceled) tions as appropriate, and the application can be practiced 7. The method of claim 5, wherein the one or more addi otherwise than specifically described herein. Accordingly, tional biomarkers associated with EE comprises eotaxin-3. many embodiments of this application include all modifica 8. (canceled) tions and equivalents of the Subject matter recited in the 9. The method of claim 1, wherein the sample comprises a claims appended hereto as permitted by applicable law. plasma or serum sample. Moreover, any combination of the above-described elements 10. (canceled) in all possible variations thereof is encompassed by the appli 11. The method of claim 1, wherein the appropriate thera cation unless otherwise indicated herein or otherwise clearly peutic strategy for a patient diagnosed with EE comprises contradicted by context. allergen removal, steroid treatment, dietary management, US 2015/0038552 A1 Feb. 5, 2015

proton pump inhibitor (PPI) therapy, administration of one or elevated level of miR-675 following treatment indicates that more topical glucocorticoids, humanized antibodies against the patient is responsive to steroid treatment. one or more relevant cytokines and/or mediators, one or more 28. The method of claim 1, wherein a patient diagnosed Small molecule inhibitors of an eosinophil and/or allergic with EE is determined to be likely to be responsive or non disease activation pathway, one or more Small molecule responsive to anti-IL-13 treatment, wherein an elevated level inhibitors capable of modulating miRNA levels and/or as of one or more miRNAs associated with periostin levels indi serving as stem-loop processing inhibitors, and/or any com cates that the patient is likely to be responsive to anti-IL-13 bination thereof. treatment. 29. (canceled) 12. (canceled) 30. A method of diagnosing a patient with eosinophilic 13. (canceled) esophagitis (EE), the method comprising: 14. (canceled) obtaining a sample from a patient; 15. The method of claim 1, wherein the appropriate thera analyzing the sample to determine a level of one or more peutic strategy comprises using any of the one or more miR miRNAs associated with EE in adult patients; and NA(s) associated with EE found to be elevated relative to the determining whether the level of the one or more miRNAs level(s) of the one or more miRNAs measured in a normal are up-regulated or down-regulated relative to a level of individual or using one or more corresponding modified miR the one or more miRNAs measured in a normal indi NA(s) as a therapeutic target or agent. vidual, wherein the presence of an elevated or reduced 16. The method of claim 15, wherein the appropriate thera level of one or more miRNAs associated with EE results peutic strategy comprises administering to the patient one or in the patient being diagnosed with EE. more agents comprising an anti-miRNA oligonucleotide (an 31. (canceled) tagomir), an antisense oligonucleotide, a locked nucleic acid, 32. (canceled) an RNA competitive inhibitor or decoy, and/or a viral vector 33. (canceled) expressing one or more miRNA genes. 34. (canceled) 17. (canceled) 35. (canceled) 18. (canceled) 36. A diagnostic kit, test, or array, comprising materials for 19. (canceled) quantification of at least two analytes, wherein the at least two 20. (canceled) analytes are miRNAS associated with eosinophilic esophagi 21. (canceled) tis (EE). 22. The method of claim 1, further comprising a determi 37. (canceled) nation of eosinophilic esophagitis or chronic esophagitis, 38. The diagnostic kit, test, or array of claim 36, wherein wherein the presence of a non-elevated or non-reduced level the at least two analytes are selected from the group consist of one or more miRNAs associated with eosinophilic esoph ing of miR-886-5p, miR-886-3p, miR-222*, miR-7, miR agitis results in the patient being diagnosed with chronic 29b, miR-642, miR-339-5p, miR-21, miR-21*, miR-142-5p, esophagitis and wherein the appropriate therapeutic strategy miR-146a, miR-146b, miR-142-3p, miR-132, miR-212, comprises antacid administration, H2 agonist administration, miR-592, miR-92a-1*, miR-223*, miR-223, miR-801, miR and/or PPI therapy. 106b, miR-375, miR-211, miR-210, miR-365, miR-203, 23. (canceled) miR-193a-5p, miR-193b, miR-193a-3p, let-7c, miR-144*, 24. (canceled) and miR-30a-3p. 25. (canceled) 39. (canceled) 26. The method of claim 1, wherein a patient diagnosed 40. (canceled) with EE is determined to be compliant with and/or exposed to 41. (canceled) steroid treatment, wherein an elevated level of miR-675 fol 42. (canceled) lowing treatment indicates that the patient is compliant with 43. (canceled) and/or exposed to steroid treatment. 44. (canceled) 27. The method of claim 11, wherein a patient diagnosed 45. (canceled) with EE and treated with a steroid is determined to be respon 46. (canceled) sive or non-responsive to steroid treatment, wherein an