WO2020077397A1

WO2020077397A1 - Methods and compositions for the treatment of mucosal lesions

Info

Publication number: WO2020077397A1
Application number: PCT/AU2019/051117
Authority: WO
Inventors: Allison June Cowin; Zlatko KOPECKI
Original assignee: Abregen Pty Ltd
Priority date: 2018-10-15
Filing date: 2019-10-15
Publication date: 2020-04-23

Abstract

The present invention relates to the treatment of mucosal lesions in the gastrointestinal tract. Specifically, methods and pharmaceutical compositions are provided for the treatment of a mucosal lesion associated with a disorder of the gastrointestinal tract. The methods comprise administering to a subject an effective amount of an agent which decreases expression and/or activity of Flightless I in the subject. Treatment of disorders such as inflammatory bowel disease (including ulcerative colitis and Crohn's disease), coeliac disease, peptic ulcer disease, and regional ileitis are contemplated. Methods and associated kits for diagnosing a disorder of the gastrointestinal tract or for assessing the progression of a disorder of the gastrointestinal tract in a subject are also provided.

Description

METHODS AND COMPOSITIONS FOR THE TREATMENT OF MUCOSAL LESIONS

PRIORITY CLAIM

[0001] This application claims priority from Australian provisional patent application number 2018903891 filed on 15 October 2018, the content of which is to be taken as incorporated herein by this reference.

FIELD OF THE INVENTION

[0002] The present invention relates generally to the treatment of mucosal lesions in the gastrointestinal tract. Specifically, methods and compositions are provided for the treatment of blistered or ulcerated mucosal tissue associated with a disorder of the gastrointestinal tract.

BACKGROUND OF THE INVENTION

[0003] The gastrointestinal tract comprises a series of organs which have sophisticated, and autonomous functions coordinated over a range of length and time scales. The primary functions of the gastrointestinal tract are digestion, absorption, excretion, and protection. The stomach and small intestine are principally responsible for digestion and absorption, a process incorporating both physical (e.g. retropulsion in the stomach) and chemical (e.g. bile and enzymes in the small intestine) mechanisms. The large intestine is primarily concerned with desiccation and compaction of waste, with storage in the sigmoid colon and rectum prior to elimination. The whole system provides a protective function from pathogens.

[0004] A gastrointestinal wall surrounds the lumen of the gastrointestinal tract and is made up of four distinct layers of specialised tissue. These include the mucosa, submucosa, muscular layer, and serosa. The mucosa is the innermost layer of the gastrointestinal tract and is the layer exposed to the bolus as it passes through the tract. The mucosa itself is made up of three layers - the epithelium (the innermost layer), the lamina propria, and the muscularis mucosae. Accordingly, the mucosal epithelia are the layer of cells which constitute an efficient physical barrier that allow exchanges between the external and internal environments, while at the same time protecting the host from environmental pathogens and harmful substances produced locally by gut microflora.

[0005] Clearly, compromise of the epithelial layer of the mucosa in the gastrointestinal tract of subjects can lead to complications, sometimes severe, in both digestion and physical barrier functions. Indeed, many digestive diseases and disorders are common, and the pathogenesis of several can be traced, at least in part, to epithelial dysfunction or disruption. For example, if the integrity of the epithelium is compromised, even commensal bacteria, and/or their products, can gain access to subepithelial compartments and trigger immune and inflammatory responses that may further derange mucosal homeostasis.

[0006] As an example, animal models have provided a wealth of information about mucosal immunology as it relates to the maintenance of intestinal homeostasis or the disruption of such homeostasis and the intestinal inflammation associated with inflammatory bowel disease (IBD). IBD is a chronic idiopathic multifactorial disease that manifests in the gastrointestinal tract of immunocompetent individuals. IBD encompasses two disorders, ulcerative colitis and Crohn’s disease, each characterised by reoccurring flare-up periods resulting in lifelong relapses. Characteristic symptoms arise from aggressive, cytokine driven, non-infectious chronic inflammation that overcomes the mucosa by disrupting normal intestinal structure and function. Prevalence of the disease is increasing, with a current incidence rate of approximately 0.3% in Australia, and 0.4% to 0.6% in North America. Additionally, annual healthcare costs attributed to the disease in North America alone exceed US$1.7 billion. Currently, there is no effective preventative or curative treatment for IBD.

[0007] Ulcerative colitis (UC) is charactrised by cytokine driven inflammation that disrupts the mucosa and impedes intestinal structure and function. UC is typified by ulceration of intestinal epithelia, primarily in the rectum. However, it has been identified throughout the entirety of the colon though never extending to the small intestine. The primary symptoms of ulcerative colitis are rectal bleeding, bloody diarrhea, abdominal pain and inflammation of the rectal mucosa (proctitis). UC has an incidence of 7.6 to 13.9 cases per 100,000 people in Westernised industrialised nations. UC may present at any age with men and women equally affected; however, incidence peaks in young adults and to lesser extent in the elderly. In adults at presentation, about 55% have proctitis, 30% left sided colitis, and 15% extensive colitis or pan colitis. In children, only 25% present with proctitis alone, 30% have left sided colitis, and in 45% the disease extends to the transverse colon or beyond. UC is a chronic lifelong condition that, untreated, has a relapsing and remitting course.

[0008] T reatments for UC include corticosteroids, aminosalicylates, immunomodulators and biologies such as anti-tumour necrosis factor-a (TNF-a) antibody, and surgical resection. Randomised controlled trials have demonstrated that infliximab and adalimumab, TNF-a antibody therapies, are effective for patients with moderate to severe colitis significantly improving mucosal healing and rates of disease remission hence decreasing the need for colectomy. However, access and cost of this therapy is still a limiting factor for many UC patients, highlighting the need for novel targeted therapies. Additionally, only about two- thirds of subjects with UC respond well to treatment and in severe disease with pancolitis there is a cumulative risk of colon cancer that increases with time due to chronic inflammation.

[0009] Crohn’s disease differs from ulcerative colitis in that inflammatory processes begin in the mucosa and spread outwards, affecting all layers and regions of the gastrointestinal tract and leading to submucosal inflammation and oedema. In terms of distribution of the disease, 25% of the patients have colitis only, 25% have ileitis only, and 50% have ileocolitis. The primary symptoms of Crohn’s disease include abdominal pain, diarrhea, fever and weight loss.

[0010] Another disorder associated with compromised gastrointestinal mucosa includes peptic ulcer disease. The disease encompasses breaks in the lining of the stomach (manifesting as gastric ulcers), first part of the small intestine (manifesting as a duodenal ulcers), or occasionally the lower esophagus. The most common symptom of a duodenal ulcer is waking at night with upper abdominal pain or upper abdominal pain that improves with eating. With a gastric ulcer, the pain may worsen with eating, and is often described as a burning or dull ache. Other symptoms include belching, vomiting, weight loss, or poor appetite. About a third of older people have no symptoms. Complications may include bleeding, perforation and blockage of the stomach, with bleeding occurring in as many as 15% of people.

[0011] Peptic ulcer disease is commonly caused by the bacteria Helicobacter pylori, and by non-steroidal anti-inflammatory drugs. Other less common causes include tobacco smoking, stress due to serious illness, Behcet disease, Zollinger-Ellison syndrome, Crohn’s disease and liver cirrhosis. The declining prevalence of Helicobacter pylori infection and widespread use of potent anti-secretory drugs has meant that peptic ulcer disease has become substantially less prevalent than it was two decades ago. Management has, however, become more challenging than ever because of the threat of increasing antimicrobial resistance worldwide and widespread use of complex anti-thrombotic therapy in the ageing population. Peptic ulcers not associated with Helicobacter pylori infection or the use of non-steroidal anti-inflammatory drugs, are now also imposing substantial diagnostic and therapeutic challenges.

[0012] The epithelial layer of the mucosa is also affected in Coeliac disease which primarily affects the small intestine. Specifically, biopsy of tissue from the small intestine of patients shows blunting of villi, crypt hypertrophy, and lymphocyte infiltration of crypts. The disease occurs in about 1% of people in most populations; however, diagnosis rates are increasing, and this seems to be due to a true rise in incidence rather than increased awareness and detection. Coeliac disease develops in genetically susceptible individuals who, in response to unknown environmental factors, develop an immune response that is subsequently triggered by the ingestion of gluten. Classic symptoms include gastrointestinal problems such as chronic diarrhea, abdominal distention, malabsorption, loss of appetite and among children failure to grow normally. The only known effective treatment of Coeliac disease is a strict lifelong gluten-free diet, which leads to recovery of the intestinal mucosa, improves symptoms and reduces risk of developing complications in most people. If untreated, the disease may result in cancers such as intestinal lymphoma and a slightly increased risk of early death.

[0013] Accordingly, there is a clear need for new effective therapies for the treatment of mucosal lesions in the gastrointestinal tract, including those associated with the disorders and conditions described above.

[0014] The discussion of documents, acts, materials, devices, articles and the like is included in this specification solely for the purpose of providing a context for the present invention. It is not suggested or represented that any or all of these matters formed part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

SUMMARY OF THE INVENTION

[0015] The present invention arises out of studies into the role of the Flightless I gene and encoded protein in gastrointestinal disorders. These studies have shown that an increased level of Flightless I in cells and tissues of the gastrointestinal tract are found in human subjects with UC, are associated with disease severity in a mouse model of IBD, and are associated with a delay in remediation of blistered mucosal tissue. [0016] Accordingly, in a first aspect, the present invention provides a method of treating a mucosal lesion in the gastrointestinal tract of a subject, the method comprising administering to the subject an effective amount of an agent which decreases expression and/or activity of Flightless I in the subject.

[0017] In some embodiments, the expression and/or activity of Flightless I is decreased in the mucosa of the gastrointestinal tract of the subject. In some embodiments, the mucosal lesion is an ulcer or blister. In some embodiments, the mucosal lesion is associated with a disorder of the gastrointestinal tract.

[0018] In a second aspect, the present invention provides a method of decreasing mucosal damage in the gastrointestinal tract of a subject, the method comprising administering to the subject an effective amount of an agent which decreases the expression or activity of Flightless I in the subject.

[0019] In some embodiments of the second aspect of the invention, the expression and/or activity of Flightless I is decreased in the mucosa of the gastrointestinal tract of the subject. In some embodiments, the agent decreases mucosal ulceration or blistering. In some embodiments, the subject is suffering from a disorder of the gastrointestinal tract.

[0020] In a third aspect, the present invention provides a method of treating an ulcer or blister in the mucosa of the gastrointestinal tract of a subject, the method comprising administering to the subject an effective amount of an agent which decreases expression and/or activity of Flightless I in the mucosa of the subject. In some embodiments, the ulcer or blister is associated with a disorder of the gastrointestinal tract.

[0021] In some embodiments of the first to third aspects of the invention, the disorder is selected from the group consisting of inflammatory bowel disease, coeliac disease, peptic ulcer disease, and regional ileitis. In some embodiments, the inflammatory bowel disease includes ulcerative colitis and Crohn’s disease. In some embodiments, the peptic ulcer is a gastric ulcer or a duodenal ulcer.

[0022] In a fourth aspect, the present invention provides a method of treating inflammatory bowel disease in a subject, the method comprising administering to the subject an effective amount of an agent which decreases expression and/or activity of Flightless I in the subject. ln some embodiments, the expression and/or activity of Flightless I is decreased in the mucosa of the gastrointestinal tract of the subject.

[0023] In some embodiments of the fourth aspect of the invention, the agent treats an ulcer or blister of the mucosa associated with the inflammatory bowel disease. In some embodiments, the inflammatory bowel disease includes ulcerative colitis and Crohn’s disease.

[0024] In some embodiments of the first to fourth aspects of the invention, the expression and/or activity of Flightless I protein is decreased by the agent. In some embodiments, the expression of Flightless I mRNA is decreased by the agent.

[0025] In some embodiments of the first to fourth aspects of the invention, the agent is selected from one or more of the group consisting of an antibody to Flightless I or an antigen binding part thereof, an antisense nucleic acid that binds to Flightless I mRNA and which interferes with translation, a small interfering RNA specific for Flightless I mRNA, a microRNA specific for Flightless I mRNA, a short hairpin RNA specific for Flightless I mRNA, a ribozyme that can cleave Flightless I mRNA, an aptamer specific for Flightless I mRNA, or a drug, small molecule, protein, polypeptide or oligopeptide, that interacts with or binds to the Flightless I protein (or a regulator of Flightless I) and inhibits its expression and/or activity. In some embodiments, the agent is a neutralising antibody to Flightless I, or an antigen binding part of a neutralising antibody to Flightless I.

[0026] In a fifth aspect, the present invention provides a method of diagnosing a disorder of the gastrointestinal tract in a subject, the method including the steps of:

measuring the level of expression and/or activity of Flightless I in the mucosa of the gastrointestinal tract of the subject;

comparing the level of expression and/or activity of Flightless I in the subject to a reference level of expression and/or activity of Flightless I; and

diagnosing a disorder of the gastrointestinal tract in the subject on the basis of the comparison.

[0027] In some embodiments of the fifth aspect of the invention, a level of expression and/or activity of Flightless I in the mucosa of the subject that is higher than the reference level of expression and/or activity for Flightless I is indicative of a disorder of the gastrointestinal tract in the subject. [0028] In a sixth aspect, the present invention provides a method of assessing the progression of a disorder of the gastrointestinal tract in a subject, the method including the steps of:

assessing the progression of a disorder of the gastrointestinal tract in the subject on the basis of the comparison.

[0029] In some embodiments of the sixth aspect of the invention, the subject is undergoing treatment for the disorder.

[0030] In some embodiments of the sixth aspect of the invention, a level of expression and/or activity of Flightless I in the mucosa of the subject that is higher than the reference level of expression and/or activity for Flightless I is indicative of progression of a disorder of the gastrointestinal tract in the subject.

[0031] In some embodiments of the fifth and sixth aspects of the invention, the level of expression and/or activity of Flightless I is measured in a mucosal sample obtained from the subject. In some embodiments, measuring the level of expression of Flightless I includes measuring the level of Flightless I protein or Flightless I RNA. In some embodiments, the level of Flightless I protein is measured using an antibody to Flightless I or an antigen binding part thereof. In some embodiments, the Flightless I RNA is Flightless I mRNA.

[0032] In some embodiments of the fifth and sixth aspects of the invention, the disorder is selected from the group consisting of inflammatory bowel disease, coeliac disease, peptic ulcer disease, and regional ileitis. In some embodiments, the inflammatory bowel disease includes ulcerative colitis and Crohn’s disease. In some embodiments, the peptic ulcer is a gastric ulcer or a duodenal ulcer.

[0033] In a seventh aspect, the present invention provides a pharmaceutical composition when used for, or for use in, treating a mucosal lesion in the gastrointestinal tract of a subject, the composition including an effective amount of an agent that decreases expression and/or activity of Flightless I. [0034] In some embodiments of the seventh aspect of the invention, the agent decreases the expression and/or activity of Flightless I in the mucosa of the gastrointestinal tract of the subject. In some embodiments, the mucosal lesion is an ulcer or blister. In some embodiments, the mucosal lesion is associated with a disorder of the gastrointestinal tract.

[0035] In an eighth aspect, the present invention provides a pharmaceutical composition when used for, or for use in, decreasing mucosal damage in the gastrointestinal tract of a subject, the composition including an effective amount of an agent that decreases expression and/or activity of Flightless I.

[0036] In some embodiments of the eighth aspect of the invention, the expression and/or activity of Flightless I is decreased in the mucosa of the gastrointestinal tract of the subject. In some embodiments, the agent decreases mucosal ulceration or blistering. In some embodiments, the subject is suffering from a disorder of the gastrointestinal tract.

[0037] In a ninth aspect, the present invention provides a pharmaceutical composition when used for, or for use in, treating an ulcer or blister in the mucosa of the gastrointestinal tract of a subject, the composition including an effective amount of an agent which decreases expression and/or activity of Flightless I in the mucosa of the subject. In some embodiments, the ulcer or blister is associated with a disorder of the gastrointestinal tract.

[0038] In some embodiments of the seventh to ninth aspects of the invention, the disorder is selected from the group consisting of inflammatory bowel disease, coeliac disease, peptic ulcer disease, and regional ileitis. In some embodiments, the inflammatory bowel disease includes ulcerative colitis and Crohn’s disease. In some embodiments, the peptic ulcer is a gastric ulcer or a duodenal ulcer.

[0039] In a tenth aspect, the present invention provides a pharmaceutical composition when used for, or for use in, treating inflammatory bowel disease in a subject, the method comprising administering to the subject an effective amount of an agent which decreases expression and/or activity of Flightless I in the subject.

[0040] In some embodiments of the tenth aspect of the invention, the agent decreases the expression and/or activity of Flightless I in the mucosa of the gastrointestinal tract of the subject. In some embodiments, the agent treats an ulcer or blister of the mucosa associated with the inflammatory bowel disease. In some embodiments, the inflammatory bowel disease includes ulcerative colitis and Crohn’s disease.

[0041] In some embodiments of the seventh to tenth aspects of the invention, the agent decreases the expression and/or activity of Flightless I protein. In some embodiments, the agent decreases the expression of Flightless I mRNA.

[0042] In some embodiments of the seventh to tenth aspects of the invention, the pharmaceutical composition of any one of claims 59 to 67, wherein the agent is selected from one or more of the group consisting of an antibody to Flightless I or an antigen binding part thereof, an antisense nucleic acid that binds to Flightless I mRNA and which interferes with translation, a small interfering RNA specific for Flightless I mRNA, a microRNA specific for Flightless I mRNA, a short hairpin RNA specific for Flightless I mRNA, a ribozyme that can cleave Flightless I mRNA, an aptamer specific for Flightless I mRNA, or a drug, small molecule, protein, polypeptide or oligopeptide, that interacts with or binds to the Flightless I protein (or a regulator of Flightless I) and inhibits its expression and/or activity. In some embodiments, the agent is a neutralising antibody to Flightless I, or an antigen binding part of a neutralising antibody to Flightless I.

[0043] In an eleventh aspect, the present invention provides kit when used for, or for use in, diagnosing a disorder of the gastrointestinal tract in a subject, or for assessing the progression of a disorder of the gastrointestinal tract in a subject, the kit including means for measuring the level of expression and/or activity of Flightless I in the subject.

[0044] In some embodiments of the eleventh aspect of the invention, the level of expression and/or activity of Flightless I is measured in the mucosa of the gastrointestinal tract of the subject.

[0045] In some embodiments of the eleventh aspect of the invention, a level of expression and/or activity of Flightless I in the mucosa of the subject that is higher than the reference level of expression and/or activity for Flightless I is indicative of a disorder of the gastrointestinal tract in the subject. In some embodiments, the level of expression and/or activity of Flightless I is measured in a mucosal sample obtained from the subject.

[0046] In some embodiments of the eleventh aspect of the invention, measuring the level of expression of Flightless I includes measuring the level of Flightless I protein or Flightless I RNA. In some embodiments, the level of Flightless I protein is measured using an antibody to Flightless I or an antigen binding part thereof. In some embodiments, the Flightless I RNA is Flightless I mRNA.

[0047] In some embodiments of the eleventh aspect of the invention, the disorder is selected from the group consisting of inflammatory bowel disease, coeliac disease, peptic ulcer disease, and regional ileitis. In some embodiments, the inflammatory bowel disease includes ulcerative colitis and Crohn’s disease. In some embodiments, the peptic ulcer is a gastric ulcer or a duodenal ulcer.

[0048] In a twelfth aspect, the present invention provides use of an agent which decreases the expression or activity of Flightless I in a subject in the manufacture of a medicament for treating a mucosal lesion in the gastrointestinal tract of the subject.

[0049] In a thirteenth aspect, the present invention provides use of an agent which decreases the expression or activity of Flightless I in a subject in the manufacture of a medicament for decreasing mucosal damage in the gastrointestinal tract of a subject.

[0050] In a fourteenth aspect, the present invention provides use of an agent which decreases the expression or activity of Flightless I in a subject in the manufacture of a medicament for treating an ulcer or blister in the mucosa of the gastrointestinal tract of a subject.

[0051] In a fifteenth aspect, the present invention provides use of an agent which decreases the expression or activity of Flightless I in a subject in the manufacture of a medicament for treating inflammatory bowel disease in a subject.

BRIEF DESCRIPTION OF THE FIGURES

[0052] For a further understanding of the aspects and advantages of the present invention, reference should be made to the following detailed description, taken in conjunction with the accompanying figures which illustrate certain embodiments of the present invention.

[0053] FIGURE 1 - outlines the process for targeted disruption of the Flii gene and generation of Flii transgenic mice with ubiquitous overexpression of Flii gene. A: Schematic representation of the domain structure of the targeting vector, relevant portion of the Flii gene and the targeted allele after homologous recombination. Restriction enzymes sites, BspEi is denoted by B, EcoRV by E and Ncol by N. Flii exons are represented by the numbered open boxes. The tk-neo and pgk-thymidine kinase casettes are indicated. B: Three primer PCR indicating wild-type (210 bp) and mutant allele (538 bp) products. Animals with one wild-type copy of the Flii gene and one mutant allele expressed no more than 50% of the normal wild-type Flii expression levels. C: Domain structure of the cosmid containing the Flii gene which was used to generate Flii transgenic mice. The cosmid contains the Flii gene, the SMCR7 gene, and parts of the TOP3A and LLGL1 genes. Restriction sites on the cosmid are also illustrated. D: Characterization of the Flii^T9/T9 mouse strain using HRM analysis of species-specific products. Green - Melt curve of human FLII amplicon obtained by PCR of genomic DNA; Red - Melt curve of the mouse Flii amplicon following PCR of cDNA from six tissues (brain, heart, lung, muscle, skin and spleen) of BALB/c animal; Blue - analysis of amplicons obtained by PCR of cDNA from six tissues of Fiii^T9/T9 _animals. The melt curve is intermediate between the mouse (red) and human (green) amplicons indicating that the cDNA contains a mixture of two species. E: Western analysis of total Flii protein in heart, skin and muscle of BALB/c wild-type mice and age and sex matched Flii^T9/T9 counterparts illustrating clear Flii overexpression in transgenic mice. Figures adopted from Campbell HD et ai, 2002, Mol. Cell. Biol., 22: 3518-3526; and Thomsen N et ai, 201 1 , Genesis, 49: 681-688.

[0054] FIGURE 2 - shows the results of Flightless I protein expression analysis in distal colonic tissue of healthy controls and subjects with ulcerative colitis (UC). A significantly increased number of Flii positive cells were observed in colonic tissue sourced from UC patients. A: Representative H&E stained sections of colonic tissue from UC patients (upper left panel) and healthy controls (lower left panel), and Flightless I (Flii) stained sections of UC patients (upper right panel) and healthy controls (lower right panel). UC patients have a clear increase in inflammatory cell infiltrate and increased numbers of Flii positive inflammatory cells (red) in lamina propria compared to healthy control tissue. DAPI nuclear marker is stained blue. Magnification x 20. Scale bar = 50pms. B: A graph representing the tissue section experiments in A showing the number of Flii positive cells in lamina propria of UC patients compared to healthy control patients. Mean +/- SEM. *p<0.05.

[0055] FIGURE 3 - are graphs showing the decreased clinical disease severity in Flightless I heterozygous mice (Flii^+/_) compared to wild type (WT) and Flightless I over-expressing mice (Flii ^9/T9) following DSS consumption in the DSS model of inflammatory bowel disease. Increased clinical disease severity is observed in Flii over-expressing mice following DSS consumption. A: All Flii overexpressing mice showed evidence of rectal bleeding and a significantly increased disease activity index on day 7 of the experiment compared to wild- type mice. Flii deficient mice show no evidence of rectal bleeding and significantly decreased disease activity index from day 3 of the experiment. B: Colon lengths of colitis- induced Flii^+/ wild-type and Flii^T9/ra mce were compared to colon lengths in water control counterpart mice, and percentage change in colon shortening analysed. Colitis-induced Flii^+/ mice have significantly reduced percentage of colon shortening while Flii^T9/T9 have significantly increased percentage of colon shortening compared to wild-type counterparts, suggestive of increased UC damage in mice with high Flii levels. n=8/genotype. Mean +/- SEM. *p<0.05.

[0056] FIGURE 4 - shows the results of histological analysis of distal colonic tissue in Flightless I heterozygous mice (Flii^+/ ) compared to wild type (WT) and Flightless I over expressing mice (Flii ^9/T9) in the DSS model of inflammatory bowel disease. Increased disease histological severity is observed in Flii over-expressing mice. A: H&E stained sections of Flii^+A (upper panels), WT (middle panels) and Flii ^9/T9 (lower panels) mice. The results are represented graphically with respect to the histological disease severity score across all mice groups. Flii overexpressing mice showed significantly higher histological disease severity compared with Flii deficient mice, with significantly damaged mucosal tissue and high level of pro-inflammatory cell infiltrate. B: H&E stained sections of Flii^+A (upper panels), WT (middle panels) and Flii ^9/T9 (lower panels) mice. Flii overexpression resulted in significantly delayed healing of blistered mucosal tissue with significantly reduced distal colon crypt depth compared to both wild-type controls and Flii deficient mice counterparts. The results are represented graphically with respect to the distal colon crypt depth in all mice groups. Magnification x4 (left panels) and x20 (right panels). Scale Bar = 200pm. n=8/genotype. Mean +/- SEM. *p<0.05.

[0057] FIGURE 5 - shows the results of histological analysis of distal colonic tissue in Flightless I heterozygous mice (Flii^+/_) compared to wild type (WT) and Flightless I over expressing mice (Flii ^9/T9) in the DSS model of inflammatory bowel disease. High Flii levels promote mucosal damage in DSS model of UC. H&E stained sections of Flii^+A (upper panels), WT (middle panels) and Flii ^9/T9 (lower panels) mice are shown. The results are represented graphically with respect to crypt area index in all mice groups. Flii overexpressing mice show significantly decreased crypt area index indicative of increased disease severity compared to wild-type controls, while Flii deficient mice have significantly increased crypt area index indicative of decreased mucosal blistering and gut damage in the DSS model of UC. n=8/genotype. Magnification x4 (left panels) and x20 (right panels). Scale Bar = 200pm. Mean +/- SEM. *p<0.05.

[0058] FIGURE 6 - shows the effect of Flightless I expression levels on cell proliferation and inflammation in the DSS model of inflammatory bowel disease. A: Representative images (centre panels) of staining and graphical representation (left) of the numbers of proliferating cell nuclear antigen (PCNA) positive cells per cypt. Altering Flii expression has no effect on enterocyte proliferation in the DSS model of UC. B: A graph showing the effect of Flightless I on cell inflammation assessed by measuring the level of MPO activity in the distal colon of DSS induced IBD in Flii^+A, wild-type and Flii ^9/T9 mice. The level of myeloperoxidase (units per gram of tissue) in Flii^+A, WT and Flii ^9/T9 mice is shown. Decreasing Flii levels significantly reduces the myeloperoxidase levels in mucosal tissue indicative of significantly reduced pro-inflammatory cell infiltrate in colitis-induced Flii deficient mice compared to wild-type controls. n=8/genotype. Magnification x10. Scale Bar = 100pm. Mean +/- SEM. *p<0.05.

[0059] FIGURE 7 - shows that Flii regulates immune responses in a DSS-induced model of UC. Representative images (A) and graphical analysis (B) of TNF-a levels in distal colon of colitis-induced Flii^+/ wild-type and Flii^T9/T9m ce. TNF-a levels were predominantly present in apical enterocytes and significantly lower levels were observed in Flii^+/ colitis-induced mice. Magnification x10. Scale Bar = 100pm. C: A graph showing that overexpression of Flii increases distal colon inflammation with significant upregulation of both Thi and Th₂ pro- inflammatory cytokine profiles, while Flii reduction decreases inflammatory responses in a DSS-induced mouse model of UC. n = 6. Mean +/- SEM. *p<0.05.

[0060] FIGURE 8 - shows that Flii over-expression inhibits Wnt^-catenin signalling in DSS- induced colitis. Representative images (A and C) and graphical analysis (B and D) of b- catenin and Axin-2 levels in distal colon of colitis-induced Flii^+/ wild-type and Flii^T9/Ta mce. b-catenin staining was most prominent at the base of colonic crypts while punctate Axin-2 staining was detected throughout the colonic epithelium. Flii overexpression resulted in decreased b-catenin levels while Flii deficiency resulted in decreased Axin-2 levels. Magnification x20. Scale Bar = 50pm. n = 6. Mean +/- SEM. *p<0.05. E: Graphs showing the effects of Flii gene expression on Axin-2 (left graph) and Lgr6 (right graph) mRNA levels by RT-PCR analysis n = 6. Mean +/- SEM. *p<0.05. F: Representative images of b-catenin and Flii colocalization in DSS-induced wild-type colitis mice (left panel) but not in Flii^T9/T9 counterparts (right panel). Magnification x40. Scale Bar = 50pm. n = 6. Mean +/- SEM. *p<0.05. G-H: Effects of Flii gene expression on Axin-2 and b-catenin levels were also confirmed using Western Blotting. Pooled samples, representative blots of repeated experiment n = 6. Mean +/- SEM. *p<0.05. I: Schematic illustration of Flii regulation of activated Wnt^-catenin signalling pathway.

[0061] FIGURE 9 - are graphs showing the effects of systemic administration of a neutralizing antibody to Flightless I in the DSS model of inflammatory bowel disease. A: Disease activity index score over a 7 day period in DSS mice following treatment with control IgG or neutralizing antibody to Flightless I (FnAb). B: Colonic length in DSS mice following treatment with control IgG or FnAb. n=8/genotype. Mean +/- SEM. *p<0.05.

[0062] FIGURE 10 - shows the results of histological analysis of distal colonic tissue following systemic administration of a neutralizing antibody to Flightless I in the DSS model of inflammatory bowel disease. A: H&E stained sections of control IgG-treated (upper panels) and FnAb-treated (lower panels) mice. The results are represented graphically with respect to the histological disease severity score across both mice groups. B: H&E stained sections of control IgG-treated (upper panels) and FnAb-treated (lower panels) mice. The results are represented graphically with respect to the distal colon crypt depth in both mice groups. Magnification x4 (left panels) and x20 (right panels). Scale Bar = 200pm. n=8/genotype. Mean +/- SEM. *p<0.05.

[0063] FIGURE 11 - shows the results of histological analysis of distal colonic tissue following systemic administration of a neutralizing antibody to Flightless I (FnAb) in the DSS model of inflammatory bowel disease. Control IgG-treated (upper panels) and FnAb-treated (lower panels) mice are shown. The results are represented graphically with respect to crypt area index in both mice groups. n=8/genotype. Magnification x4 (left panels) and x20 (right panels). Scale Bar = 200pm. Mean +/- SEM. *p<0.05.

[0064] FIGURE 12 - is a graph of Transepithelial Resistance (TER) measurement in cultured IEC-6 (normal small intestine epithelial rat cell line) enterocytes over a period of 14 days following treatment with a neutralizing antibody to Flightless I (FnAb) or IgG control. Data are representative of two independent experiments. n=6. Mean +/- SEM; *p<0.05. DETAILED DESCRIPTION OF THE INVENTION

[0065] Nucleotide sequences are referred to herein by a sequence identifier number (SEQ ID NO:). A summary of the sequence identifiers is provided in Table 1. A sequence listing has also been provided at the time of filing this application.

TABLE 1

Summary of Sequence Identifiers

[0066] As set out above, the present invention is predicated, in part, on the identification of an association between the expression of Flightless I and gastrointestinal disorders. For example, it has been found that increased levels of Flightless I in cells of the mucosal layer of the gastrointestinal tract are an indicator of mucosal lesions and that decreasing expression and/or activity of Flightless I leads to a decrease in measures of mucosal damage. Flightless I therefore represents a therapeutic target for the treatment of disorders of the gastrointestinal tract.

[0067] Accordingly, certain disclosed embodiments provide methods, compositions, products, and kits, that have one or more advantages. For example, some of the advantages of some embodiments disclosed herein include one or more of the following: a target for the treatment of disorders of the gastrointestinal tract; methods and pharmaceutical compositions for treating a mucosal lesion in the gastrointestinal tract; methods and pharmaceutical compositions for decreasing mucosal damage in the gastrointestinal tract; methods and pharmaceutical compositions for treating an ulcer or blister in the mucosa of the gastrointestinal tract; methods and pharmaceutical compositions for treating inflammatory bowel disease; methods for diagnosing a disorder of the gastrointestinal tract; methods for assessing the progression of a disorder of the gastrointestinal tract; kits for diagnosing a disorder of the gastrointestinal tract, or for assessing the progression of a disorder of the gastrointestinal tract; or the provision of a commercial alternative to existing methods and compositions. Other advantages of some embodiments of the present disclosure are provided herein.

[0068] The present invention provides, amongst other things, methods for treating a mucosal lesion in the gastrointestinal tract of a subject, or for decreasing mucosal damage in the gastrointestinal tract of a subject. The methods comprise administering to the subject an effective amount of an agent which decreases expression and/or activity of Flightless I in the subject.

[0069] As would be understood by a person skilled in the art, the gastrointestinal tract comprises a large muscular tube that extends from the mouth to the anus, where the movement of muscles, along with the release of hormones and enzymes, enables the digestion of food and subsequent elimination of waste products of the digestive process. The gastrointestinal tract is also referred to the Gl tract, alimentary canal or digestive tract. The gastrointestinal tract comprises a series of interconnected organs, which include the esophagus, stomach, small intestine, and large intestine. [0070] The lumen of the gastrointestinal tract is surrounded by the gastrointestinal wall which is made up of four layers - the mucosa, submucosa, muscular layer, and serosa. The mucosa is the innermost layer and is the layer exposed to the bolus of food as it passes through the tract. The mucosa itself is made up of three layers - the epithelium (the innermost layer), the lamina propria, and the muscularis mucosae.

[0071] As used herein a “mucosal lesion” refers to damaged mucosal tissue of the gastrointestinal tract. The damage may be as a cause and/or consequence of a gastrointestinal disorder. Alternatively, the damage may not be related to a gastrointestinal disorder but may be due to other factors such as pathological infection, excessive alcohol consumption, side effects of drug use (e.g. nonsteroidal anti-inflammatory drug use and salicylate use), lysolecithin, bile acids, acidosis, sepsis, stress, malnutrition, enteritis, uremia, and hypoxemia. Any condition or factor that leads to mucosal lesions and mucosal damage is encompassed by the present invention.

[0072] In some embodiments, the mucosal lesion or mucosal damage may manifest as an ulcer or blister of the mucosal layer, including of the epithelial mucosa. Mucosal damage may also be evidenced as erosion of the mucosal layer, a decrease in colon length and reduced distal colon crypt depth (indicative of more severe mucosal damage), and rectal bleeding.

[0073] Accordingly, the present invention also provides methods for treating an ulcer or blister in the mucosa of the gastrointestinal tract of a subject, the method comprising administering to the subject an effective amount of an agent which decreases expression and/or activity of Flightless I in the mucosa of the subject.

[0074] A disorder of the gastrointestinal tract with reference to the present invention is any disorder which gives rise to mucosal lesions and mucosal damage. Specific examples include inflammatory bowel disease, coeliac disease, peptic ulcer disease, and regional ileitis. This list of disorders is non-limiting and so others are contemplated. Inflammatory bowel disease includes ulcerative colitis and Crohn’s disease, and peptic ulcer disease encompasses lesions to the stomach (manifesting as gastric ulcers), first part of the small intestine (manifesting as a duodenal ulcers), or occasionally the lower esophagus. [0075] Accordingly, the present invention also provides methods for treating inflammatory bowel disease in a subject, the method comprising administering to the subject an effective amount of an agent which decreases expression and/or activity of Flightless I in the subject.

[0076] As used herein,“Flightless I” is to be understood to refer to a gene that encodes a protein with a gelsolin-like actin binding domain and an N-terminal leucine-rich repeat- protein protein interaction domain. Flightless I was originally identified in Drosophila where mutations in the gene caused defects in the flight muscles which, consequently, were unable to support flight. The Flightless I gene has since been found to be present in a number of species, including human, chimpanzee, baboon, monkey, mouse, zebrafish, frog, dog and yeast. Indeed, between the higher order species, the Flightless I protein is highly conserved suggesting that it carries out important, conserved functions.

[0077] The human Flightless I gene encodes a 140 kD protein which is a member of the gelsolin family of proteins. The human gene encodes three isoforms variants, the mRNA and amino acid sequences of which are set out in SEQ ID NOs: 1 to 6, and represented by GenBank Accession Numbers NM_002018.3 and NP_002009.1 (variant 1), NM_001256264.1 and NP_001243193.1 (variant 2), and NM_001256265.1 and NP_001243194.1 (variant 3). Further details of the Flightless I gene in human and other species may be accessed from the GenBank database at the National Centre for Biotechnology Information (NCBI) (www.ncbi.nlm.nih.gov). For example, the Gene ID number for human Flightless I is 2314, for chimpanzee is 454486, for baboon is 10101901 1 , for monkey is 700471 , for mouse is 14248, for zebrafish is 560281 , for frog is 444748, for dog is 479521 , and for yeast is 176215.

[0078] Further details regarding the Flightless I gene in other species can be found at the UniGene portal of the NCBI (i.e. UniGene Hs. 513984 http://www.ncbi.nlm.nih.gov/UniGene/clust.cgi?ORG=Hs&CID=513984&ALLPROT=1). Alternatively, details of the nucleotide and amino acid sequence for Flightless I can be accessed from the UniProt database (www.uniprot.org) wherein the UniProt ID for human Flightless I is Q13045 (variant 1 and 2), and F5H407 (variant 3). The contents of the GenBank and UniProt records are incorporated herein by reference. Human Flightless I will also be referred to herein as“Flii” and“FIN”.

[0079] It is to be made clear that reference herein to Flightless I, includes a reference to its naturally-occurring variants. In this regard, a“variant” of Flightless I may exhibit a nucleic acid or an amino acid sequence that is at least 80% identical, at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or at least 99.9% identical to native Flightless I. In some embodiments, a variant of Flightless I is expected to retain native biological activity or a substantial equivalent thereof.

[0080] As would be understood by a person skilled in the art, the term“gene” refers to a region of genomic nucleotide sequence (nuclear or mitochondrial) associated with a coding region which is transcribed and translated into a functional biomolecule (protein) composed primarily of amino acids. Accordingly, the term "gene" with respect to Flightless I may include regulatory regions (e.g. promoter regions), transcribed regions, protein coding exons, introns, untranslated regions and other functional and/or non-functional sequence regions associated with Flightless I.

[0081] The methods of the present invention, as referred to above, require the step of administering an effective amount of an agent that decreases expression and/or activity of Flightless I. As would be understood by a person skilled in the art, the term“expression” includes: (1) transcription of the Flightless I gene into a messenger RNA (mRNA) molecule; and/or (2) translation of the mRNA into the Flightless I protein. In effect, the expression of the Flightless I gene can be decreased at the RNA and/or protein stages of expression. With respect to the term“activity”, this should be taken to mean the normal function of the translated Flightless I protein. For example, Flightless I belongs to the Gelsolin family of actin severing proteins which function in the cytoplasm of cells where they control actin organisation. This is achieved by severing pre-existing actin filaments, capping the fast growing filament ends and nucleating or bundling actin filaments to enable filament reassembly into new cytoskeletal structures. Several members of this Gelsolin family, including Flightless I, also have roles in regulating gene transcription and act as nuclear receptor co-activators. Flightless I is a multifunctional protein with a unique structure containing both a gelsolin domain and a Leucine Rich Repeat (LRR) domain allowing Flightless I to act as a multifunctional protein with major roles in regulating cellular migration and proliferation, cellular adhesion and spreading. Recent findings have confirmed its role in actin polymerisation and capping of actin monomers.

[0082] Reference herein to“decrease” with respect to the expression of Flightless I, whether at the transcriptional (mRNA) or translational (protein) stage is intended to mean, for example, at least a 1%, at least a 5%, at least a 10%, at least a 20%, at least a 30%, at least a 40%, at least a 50%, at least a 60%, at least a 70%, at least a 80%, at least a 90%, at least a 100% or greater reduction in the level of Flightless I mRNA or protein in the affected subject. In one embodiment, the expression of Flightless I will be decreased to a level to that observed in a healthy non-affected subject or to that observed in a non-affected tissue (e.g. normal healthy tissues) of the subject.

[0083] Reference herein to“decrease” with respect to the activity of Flightless I is intended to mean a reduction in the function of Flightless I in the affected subject. In effect, the activity of Flightless I in the affected subject is to be reduced to a level commensurate with that observed in a healthy non-affected subject and/or in normal healthy tissues of the subject. In some embodiments, the activity of Flightless I may be reduced by at least 1 %, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100% or greater in the affected subject.

[0084] Methods which can be used to measure the level of expression (and decrease thereof) of Flightless I in the subject would be known in the art. With respect to measuring a decrease in the transcription of the Flightless I gene into mRNA, levels of mRNA may be measured by techniques which include, but are not limited to, Northern blotting, RNA in situ hybridisation, reverse-transcriptase PCR (RT-PCR), real-time (quantitative) RT-PCR, microarrays, or “tag based” technologies such as SAGE (serial analysis of gene expression). Microarrays and SAGE may be used to simultaneously quantitate the expression of more than one gene. Primers or probes may be designed based on nucleotide sequence of the Flightless I gene or transcripts thereof. Methodology similar to that disclosed in Paik et ai, 2004 ( NEJM , 351 (27): 2817-2826), or Anderson et ai, 2010 ( Journal of Molecular Diagnostics, 12(5): 566-575) may be used to measure the expression of the Flightless I gene. Many methods are also disclosed in standard molecular biology text books such as Green MR and Sambrook J, Molecular Cloning: A Laboratory Manual (4th edition), Cold Spring Harbor Laboratory Press, 2012.

[0085] With respect to RT-PCR, the first step is typically the isolation of total RNA from a sample obtained from the subject under investigation. A typical sample in this instance would be a biopsy sample of mucosal tissue from a relevant section of the gastrointestinal tract (and corresponding normal or unaffected tissue of the subject), although other sample sources are contemplated as described below. Messenger RNA (mRNA) may be subsequently purified from the total RNA sample. The total RNA sample (or purified mRNA) is then reverse transcribed into cDNA using a suitable reverse transcriptase. The cDNA derived from the reverse transcription reaction then serves as a template for a typical PCR reaction. In this regard, two oligonucleotide PCR primers specific for the Flightless I gene are used to generate a PCR product. A third oligonucleotide, or probe, designed to detect a nucleotide sequence located between the other two PCR primers may also used in the PCR reaction. In this regard, the probe is non-extendible by the Taq DNA polymerase enzyme used in the PCR reaction, and is labelled with a reporter fluorescent dye and a quencher fluorescent dye. Any laser-induced emission from the reporter dye is quenched by the quenching dye when the two dyes are located close together, as they are on the probe. During the PCR amplification reaction, the Taq DNA polymerase enzyme cleaves the probe in a template-dependent manner. The resultant probe fragments disassociate in solution, and signal from the released reporter dye is freed from the quenching effect of the second fluorophore. One molecule of reporter dye is liberated for each new molecule synthesized, and detection of the unquenched reporter dye provides the basis for quantitative interpretation of the data.

[0086] In real-time RT-PCR the amount of product formed, and the timing at which the product is formed, in the PCR reaction correlates with the amount of starting template. RT- PCR product will accumulate quicker in a sample having an increased level of mRNA compared to a standard or“normal” sample. Real-time RT-PCR measures either the fluorescence of DNA intercalating dyes such as Sybr Green into the synthesized PCR product, or can measure PCR product accumulation through a dual-labelled fluorigenic probe (i.e. , TaqMan probe). The progression of the RT-PCR reaction can be monitored using PCR machines such as the Applied Biosystems' Prism 7000 or the Roche LightCycler which measure product accumulation in real-time. Real-time RT-PCR is compatible both with quantitative competitive PCR and with quantitative comparative PCR. The former uses an internal competitor for the target sequence for normalization, while the latter uses a normalization gene contained within the sample, or a housekeeping gene for RT-PCR.

[0087] The production and application of microarrays for measuring the level of expression of the Flightless I gene at the transcriptional level are well known in the art. In general, in a microarray, a nucleotide sequence (for example an oligonucleotide, a cDNA, or genomic DNA) representing a portion or all of the Flightless I gene occupies a known location on a substrate. A nucleic acid target sample (for example total RNA or mRNA) obtained from a subject of interest is then hybridized to the microarray and the amount of target nucleic acid hybridized to each probe on the array is quantified and compared to the hybridisation which occurs to a standard or“normal” sample. One exemplary quantifying method is to use confocal microscope and fluorescent labels. The Affymetrix GeneChip™ Array system (Affymetrix, Santa Clara, Calif.) and the Atlas™ Human cDNA Expression Array system are particularly suitable for quantifying the hybridization; however, it will be apparent to those of skill in the art that any similar systems or other effectively equivalent detection methods can also be used. Fluorescently labelled cDNA probes may also represent the Flightless I nucleic acid target sample. Such probes can be generated through incorporation of fluorescent nucleotides during reverse transcription of total RNA or mRNA extracted from a sample of the subject to be tested. Labelled cDNA probes applied to the microarray will hybridize with specificity to the equivalent spot of DNA on the array. Quantitation of hybridization of each arrayed element allows for assessment of corresponding mRNA abundance in the sample compared to the abundance observed in a standard or“normal” sample. With dual colour fluorescence, separately labelled cDNA probes generated from two sources of RNA are hybridized pairwise to the array. The relative abundance of the transcripts from the two sources corresponding to each specified gene is thus determined simultaneously. The miniaturized scale of the hybridization using microarray analysis affords a convenient and rapid evaluation of the expression pattern for large numbers of genes. Such methods have been shown to have the sensitivity required to detect rare transcripts, which are expressed at a few copies per cell, and to reproducibly detect at least approximately two-fold differences in the expression levels.

[0088] Methods which can be used to measure a decrease in the level of expression of Flightless I at the translational level (protein level) would be known in the art. For example, the level of Flightless I protein may be measured by techniques which include, but are not limited to, antibody-based testing (including Western blotting, immunoblotting, enzyme- linked immunosorbant assay (ELISA), radioimmunoassay (RIA), immunoprecipitation and dissociation-enhanced lanthanide fluoro immuno assay (DELFIA)), proteomics techniques, surface plasmon resonance (SPR), versatile fibre-based SPR, chemiluminescence, fluorescent polarization, phosphorescence, immunohistochemistry, immunofluorescence, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), as described in WO 2009/004576 (including surface enhanced laser desorption/ionization mass spectrometry (SELDI-MS), especially surface-enhanced affinity capture (SEAC), protein microarrays, surface-enhanced need desorption (SEND) or surface-enhanced photo label attachment and release (SEPAR)), matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, microcytometry, microarray, microscopy, fluorescence activated cell sorting (FACS), and flow cytometry. [0089] With respect to antibody-based testing methods such as immunohistochemistry and immunoblotting, antibodies or antisera, preferably polyclonal antisera, and most preferably monoclonal antibodies specific for the Flightless I protein are used to detect protein abundance in the subject. The antibodies can be detected by direct labelling of the antibodies themselves, for example with radioactive labels, fluorescent labels, hapten labels such as, biotin, or an enzyme such as horseradish peroxidase or alkaline phosphatase. Alternatively, unlabelled primary antibody may be used in conjunction with a labelled secondary antibody, comprising antisera, polyclonal antisera or a monoclonal antibody specific for the primary antibody. Immunohistochemistry protocols and kits are well known in the art and are commercially available. Antibodies can be produced by methods well known in the art, for example, by immunizing animals with the protein under investigation. Further detailed description is provided below.

[0090] Also contemplated are traditional immunoassays including, for example, sandwich immunoassays including ELISA or fluorescence-based immunoassays, as well as other enzyme immunoassays. Commercially available ELISA kits for Flightless I detection are available. A list of available suppliers is provided at https://www.biocompare.eom/pfu/1 10627/soids/26285/ELISA_Kit/Flightless_l , and includes for example MyBioSource.com, LifeSpan Biosciences, Biomatik, Abbexa Ltd and DLdevelop. Another commercial source is Generon Ltd (Fairview, Clontarf, Dublin, Ireland). Nephelometry is an assay performed in liquid phase, in which antibodies are in solution. Binding of the Flightless I protein to the antibody results in changes in absorbance, which are measured. In the SELDI-based immunoassay, a biospecific capture reagent for the Flightless I protein is attached to the surface of an MS probe, such as a pre-activated ProteinChip array (see below). The protein is then specifically captured on the biochip through this reagent, and the captured protein is detected by mass spectrometry (see below).

[0091] A further technique for assessing protein levels using an antibody-based platform involves the versatile fibre-based surface plasmon resonance (VeSPR) biosensor, as described in PCT International Publication No. WO 201 1/113085.

[0092] Proteomics can also be used to analyse the expression level of Flightless I protein present in a sample at a certain point of time. In particular, proteomic techniques can be used to assess the global changes of protein expression in a sample (also referred to as expression proteomics). Proteomic analysis typically includes: (i) separation of individual proteins in a sample by 2-D gel electrophoresis (2-D PAGE); (ii) identification of the individual polypeptides recovered from the gel, for example by mass spectrometry or N- terminal sequencing; and (iii) analysis of the data using bioinformatics.

[0093] Protein microarrays (also termed biochips) may also be used to determine the level of Flightless I protein in a sample. Many protein biochips are described in the art, including for example protein biochips produced by Ciphergen Biosystems, Inc. (Fremont, CA), Zyomyx (Hayward, CA), Invitrogen (Carlsbad, CA), Biacore (Uppsala, Sweden) and Procognia (Berkshire, UK). Examples of such protein biochips are described in the following patents or published patent applications: U.S. Patent Nos. 6,225,047, 6,537,749, 6,329,209, and 5,242,828, and PCT International Publication Nos. WO 00/56934 and WO 03/048768.

[0094] The level of Flightless I protein can also be measured by mass spectrometry, a method that employs a mass spectrometer to detect gas phase ions. Examples of mass spectrometers are time-of-flight, magnetic sector, quadrupole filter, ion trap, ion cyclotron resonance, electrostatic sector analyzer and hybrids of these. The mass spectrometer may be a laser desorption/ionization (LDI) mass spectrometer. In laser desorption/ionization mass spectrometry, the Flightless I protein to be detected is placed on the surface of a mass spectrometry probe, a device adapted to engage a probe interface of the mass spectrometer and to present the protein to ionizing energy for ionization and introduction into a mass spectrometer. A laser desorption mass spectrometer employs laser energy, typically from an ultraviolet laser, but also from an infrared laser, to desorb analytes from a surface, to volatilize and ionize them and make them available to the ion optics of the mass spectrometer. The analysis of Flightless I protein by LDI can take the form of matrix-assisted laser desorption/ionization (MALDI - as described for example in Karas M and Hillenkamp F, 1988, Anal. Chem., 60: 2299-2301 ; Tanaka K et ai, 1988, Rapid Commun. Mass Spectrom., 2: 151-153; and Norris JL and Caprioli RM, 2013, Chem Rev., 113: 2309-2342) or of surface-enhanced laser desorption/ionization (SELDI - as described for example in Hutchens T and Yip T, 1993, Rapid Commun. Mass Spectrom., 7: 576-580; Tang N et ai., 2004, Mass Spec. Reviews, 23: 34-44; and U.S. Patents Nos. 5,719,060 and 6,225,047).

[0095] Other laser desorption mass spectrometry methods which may be employed include surface-enhanced neat desorption (SEND - as described for example in U.S. Patent No. 6, 124, 137 and PCT International Publication No. WO 03/64594), SEAC/SEND (a version of laser desorption mass spectrometry in which both a capture reagent and an energy absorbing molecule are attached to the sample presenting surface), and surface-enhanced photolabile attachment and release (SEPAR - which involves the use of probes having moieties attached to the surface that can covalently bind Flightless I protein, and then release the protein through breaking a photolabile bond in the moiety after exposure to light, e.g. to laser light).

[0096] Other methods which may be employed to determine if the level of Flightless I protein has decreased in a subject include assays which rely on known protein/protein interactions. These assays may also be used as an indicator of a decrease in activity of Flightless I in a subject. For example, Flightless I protein has an actin-binding domain, and so assays which measure the amount or level of binding between the Flightless I protein and actin will be a reflection of the level and/or activity of Flightless I protein in a particular sample. This level can be compared to the level of binding in a normal control sample. Furthermore, the Flightless I protein has a leucine-rich repeat which is known to bind proteins such as FLAP- 1 (Wilson SA et ai, 1998, Nucleic Acids Res., 26: 3460-3467), and Flightless I has been shown to bind directly to the diaphanous-related formins Daaml and mDial (Higashi T et ai, 2010, J. Biol. Chem., 285: 16231-16238). Therefore, assays which measure the amount or level of binding between the Flightless I protein and one or more of these other proteins will be a reflection of the level and/or activity of Flightless I protein in a particular sample.

[0097] Further assays which may be used to measure the level of decrease in activity of the Flightless I protein will be dictated by the function of the protein. As indicated above, Flightless I regulates gene transcription and acts as a nuclear receptor co-activator. Therefore, a decrease in the activity of Flightless I may be assayed according to a concomitant change in gene transcription as mediated by the Flightless I protein. Given that Flightless I regulates cellular migration and proliferation, cellular adhesion and spreading, assays which measure for changes in cell migration or proliferation, for example, may also be used to measure the activity of the Flightless I protein.

[0098] The terms "treat", "treating" or "treatment," as used herein are to be understood to include within their scope one or more of the alleviation of, reduction of, and/or providing relief from, a symptom of a mucosal lesion or mucosal damage in the gastrointestinal tract, including an ulcer or blister (for example those associated with inflammatory bowel disease), rectal bleeding, diarrhea, abdominal cramps and pain, anemia, and fatigue.

[0099] In the methods of the present invention, as referred to above, decreasing the expression and/or activity of Flightless I in the subject includes administration to the subject of an effective amount of an agent that decreases the expression and/or activity of Flightless I. The term“effective amount” as used herein is the quantity which, when administered to a subject, improves the prognosis and/or health state of the subject. The amount to be administered to a subject will depend on the particular characteristics of one or more of the level or amount of resistance to the agent in the subject, and characteristics such as the general health, other diseases, age, sex, genotype, and body weight of the subject. A person skilled in the art will be able to determine appropriate dosages depending on these and other factors. The effective amount of the agent to be used in the various embodiments of the invention is not particularly limited.

[0100] The agent may be any agent that is capable of decreasing the expression and/or activity of Flightless I. For example, the agent may be selected from one or more of the group consisting of an antibody to Flightless I or an antigen binding part thereof (including a neutralizing antibody to Flightless I or an antigen binding part of a neutralising antibody to Flightless I), an antisense nucleic acid (oligonucleotide) that binds to Flightless I mRNA and which interferes with translation, a molecule that can specifically repress transcription of endogenous Flightless I mRNA such as a specific DNA or RNA binding protein, a nucleic acid capable of forming a triple helix structure, a small interfering RNA specific for Flightless I mRNA, a microRNA specific for Flightless I mRNA, a short hairpin RNA specific for Flightless I mRNA, a ribozyme that can cleave Flightless I mRNA, an aptamer specific for Flightless I mRNA, and an agent that interacts with or binds to the Flightless I protein (or a regulator of Flightless I) and inhibits its expression and/or activity, such as a drug, small molecule, protein, polypeptide or oligopeptide. However, the above list is not exhaustive and so other agents are contemplated.

[0101] In one embodiment, the agent which decreases the expression and/or activity of Flightless I is an antibody, or an antigen binding part thereof, to the Flightless I protein. As would be understood by a person skilled in the art, an "antibody" refers to a polypeptide comprising a framework region from an immunoglobulin gene or fragments thereof that specifically binds and recognizes an antigen, in this case the Flightless I protein. The recognised immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the multitude of immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. [0102] Naturally occurring immunoglobulins have a common core structure in which two identical light chains (about 24 kD) and two identical heavy chains (about 55 or 70 kD) form a tetramer. The amino-terminal portion of each chain is known as the variable (V) region and can be distinguished from the more conserved constant (C) regions of the remainder of each chain. Within the variable region of the light chain is a C-terminal portion known as the J region. Within the variable region of the heavy chain, there is a D region in addition to the J region. Most of the amino acid sequence variation in immunoglobulins is confined to three separate locations in the V regions known as hypervariable regions or complementarity determining regions (CDRs) which are directly involved in antigen binding. Proceeding from the amino-terminus, these regions are designated CDRI, CDR2 and CDR3, respectively. The CDRs are held in place by more conserved framework regions (FRs). Proceeding from the amino-terminus, these regions are designated FRI, FR2, FR3, and FR4, respectively. The locations of CDR and FR regions and a numbering system have been defined for example by Kabat et ai, 1991 (Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, U.S. Government Printing Office).

[0103] The term "antigen binding part" is to be understood to mean the antigen-binding portion of an antibody molecule, including a Fab, Fab’, F(ab’)2, Fv, a single-chain antibody (scFv), a chimeric antibody, a diabody or any polypeptide that contains at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding, such as a molecule including one or more CDRs (see further detail below).

[0104] Antibodies exist as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases. Therefore, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)'2, a dimer of Fab which itself is a light chain joined to VH-CHI by a disulfide bond. The F(ab)'2 may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)'2 dimer into an Fab' monomer. The Fab' monomer is essentially Fab with part of the hinge region. While various antibody fragments are defined in terms of the digestion of an intact antibody, a person skilled in the art would appreciate that such fragments may be synthesized de novo either chemically or by using recombinant DNA methodology. Therefore, the term antibody, as used herein, also includes antibody fragments either produced by the modification of whole antibodies, or those synthesized de novo using recombinant DNA methodologies (e.g. single chain Fv) or those identified using phage display libraries (see for example McCafferty et ai, 1990, Nature 348:552-554). [0105] A "chimeric antibody" is an antibody molecule in which (a) the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and/or species, or an entirely different molecule which confers new properties to the chimeric antibody, e.g. an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigen specificity. The chimeric antibodies may be monovalent, divalent, or polyvalent immunoglobulins. For example, a monovalent chimeric antibody is a dimer (HL) formed by a chimeric H chain associated through disulfide bridges with a chimeric L chain, as noted above. A divalent chimeric antibody is a tetramer (H2 L₂) formed by two HL dimers associated through at least one disulfide bridge. A polyvalent chimeric antibody is based on an aggregation of chains.

[0106] In some embodiments, the antibody may be a humanised antibody. A "humanised" antibody is an antibody that retains the reactivity of a non-human antibody while being less immunogenic in humans. This can be achieved, for example, by retaining the non-human CDR regions and replacing the remaining parts of the antibody with their human counterparts. See for example Morrison et ai, 1984, Proc. Natl. Acad. Sci. USA, 81 : 6851- 6855; Morrison and Oi, 1988, Adv. Immunol., 44: 65-92; Verhoeyen et ai, 1988, Science, 239: 1534-1536; Padlan, 1991 , Molec. Immun., 28: 489-498; and Padlan, 1994, Molec. Immun., 31 : 169-217.

[0107] In some embodiments, the antibody may be a fully human antibody. As would be understood by a person skilled in the art, a fully human antibody is an antibody in which both the variable and constant regions are of human origin. Methods for producing or identifying such antibodies are described below.

[0108] Additional antibody types are also contemplated by the present invention. These include antibodies sourced from a non-mammalian animal such as a cartilaginous fish (e.g. sharks) or modified human protein scaffolds that provide functionality similar to shark antibodies, such as i-bodies. Shark antibodies are also called Ig new antigen receptors (IgNARs). They are disulphide-bonded homodimers consisting of five constant domains (CNAR), one variable domain (VNAR), and no light chains (Greenberg et ai, 1995, Nature 374: 168-173; Nuttall et ai, 2001 , Mol. Immunol., 38: 313-326; Diaz et ai, 2002, Immunogenetics 54: 501-512; and Nuttall et ai, 2003, Eur. J. Biochem., 270: 3543-3554). Antibodies sourced from camels (camelid antibodies), dromedaries and llamas are also contemplated by the present invention. Such antibodies consist of only two heavy chains and are devoid of light chains. Due to the heavy chain dimer structure of camelid and shark antibodies, they are sometimes termed“heavy-chain mini-antibodies” (mnHCAbs) or“mini antibodies” (mnAbs) (Holliger and Hudson, 2005, Nat. Biotechnol., 23(1): 1 126-1136). Without the light chain, these antibodies bind to their antigens by a single domain - the variable antigen binding domain of the heavy chain immunoglobulin, referred to as Vab (camelid antibodies) or V-NAR (chark antibodies).

[0109] Affibodies are also contemplated by the present invention. Affibody molecules are a class of affinity proteins based on a 58-amino acid residue protein domain, derived from one of the IgG-binding domains of staphylococcal protein A. This three helix bundle domain has been used as a scaffold for the construction of combinatorial phagemid libraries, from which Affibody variants that target the desired molecules can be selected using phage display technology (Nord K et al., 1997, Nat. Biotechnol., 15: 772-777; Ronmark J et al., 2002, Eur. J. Biochem., 269: 2647-2655). Further details about Affibodies and methods of production thereof are also disclosed in US Patent No 5831012.

[0110] In some embodiments, the antibody to Flightless I is a neutralising antibody to Flightless I, or an antigen binding part of a neutralising antibody to Flightless I. In some embodiments, the antibody binds specifically to the leucine rich repeat domain of the Flightless I protein. As would be understood by a person skilled in the art, a neutralising antibody is and antibody that can reduce or neutralise the expression and/or activity of Flightless I.

[0111] For the production of antibodies, various hosts including rabbits, rats, goats, mice, humans, and others may be immunised by injection with a Flightless I polypeptide or with any fragment, peptide or oligopeptide thereof which has immunogenic properties. Various adjuvants may be used to increase immunological response and include, but are not limited to, Freund's, mineral gels such as aluminum hydroxide, and surface-active substances such as lysolecithin. Adjuvants used in humans include BCG (bacilli Calmette-Guerin) and Corynebacterium parvum.

[0112] It is preferred that the Flightless I oligopeptides, peptides, or fragments used to induce antibodies have an amino acid sequence consisting of at least 5 amino acids, and, more preferably, of at least 10 amino acids of Flightless I. It is also preferable that these oligopeptides, peptides, or fragments are identical to a portion of the amino acid sequence of the natural protein and contain the entire amino acid sequence of a small, naturally occurring molecule. In one non-limiting example, the peptide H-CKLEHLSVSHN-OH (SEQ ID NO: 7) may be used to immunise mice for the production of an antibody to Flightless I. Short stretches of amino acids from Flightless I, including the stretch of amino acids listed above, may be fused with those of another protein, such as keyhole limpet haemocyanin (KLH), and antibodies to the chimeric molecule may be produced.

[0113] Monoclonal antibodies to Flightless I may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique (for example, see Kohler et ai, 1975, Nature 256: 495-497; Kozbor et ai, 1985, J. Immunol. Methods 81 :31-42; Cote et ai, 1983, Proc. Natl. Acad. Sci USA 80: 2026-2030; and Cole et ai, 1984, Mol. Cell Biochem. 62: 109- 120).

[0114] Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature (for example, see Orlandi et ai, 1989, Proc. Natl. Acad. Sci. USA 86: 3833-3837; and Winter and Milstein, 1991 , Nature 349: 293-299). Antibodies may also be generated using phage display. For example, functional antibody domains are displayed on the surface of phage particles that carry the polynucleotide sequences encoding them. Such phage can be utilized to display antigen-binding domains expressed from a repertoire or combinatorial antibody library (e.g. human or murine). Phage expressing an antigen binding domain that binds Flightless I can be selected or identified with Flightless I, e.g. using labeled Flightless I or a portion thereof. Phage used in these methods are typically filamentous phage including fd and Ml 3 binding domains expressed from phage with Fab, Fv or disulfide stabilised Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein. Examples of phage display methods that can be used to make the antibodies may include those disclosed in Brinkman et ai, 1995, J. Immunol. Methods 182: 41-50; Ames et ai, 1995, J. Immunol. Methods 184: 177-186; Kettleborough et ai, 1994, Eur. J. Immunol. 24: 952-958; Persic et ai, 1997, Gene 187: 9- 18; Burton et ai, 1994, Advances in Immunology 57: 191-280; PCT application number PCT/GB91/01134; PCT publications numbers WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401 ; and US Patent Numbers

5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821 ,047; 5,571 ,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969, 108; each of which is incorporated herein by reference in its entirety.

[0115] Techniques which can be used to produce single-chain Fvs and antibodies include those described in US Patent Numbers 4,946,778 and 5,258,498; Huston et ai, 1991 , Methods in Enzymology 203: 46-88; Shu et al., 1993, Proc. Natl. Acad. Sci. USA 90: 7995- 7999; and Skerra et al., 1988, Science 240: 1038-1040.

[0116] Antibody fragments which contain specific binding sites for Flightless I may be generated using standard techniques known in the art. For example, F(ab')2 fragments may be produced by pepsin digestion of a Flightless I antibody molecule and Fab fragments generated by reducing the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (for example, see Huse et al., 1989, Science 246: 1275-1281).

[0117] Fully human antibodies may be produced using a number of techniques. These include using display technologies as mentioned above in which human antibodies or antibody fragments are displayed on the surface of a phage for example. In another method (Lonberg N, 2008, Handb. Exp. Pharmacol., 69-97), first generation human antibodies to Flightless I may be produced by utilising transgenic animals that produce antibodies from human genes. When challenged with an antigen (i.e. Flightless I or an oligopeptide, peptide, or fragment thereof), these animals produce human antibodies avoiding the humanisation steps. Human antibodies can also be produced from B cells isolated from humans using a technisue described in Crowe JE Jr, 2009, Vaccine 27: 47-51. Other techniques for human antibody production are described in PCT international publication number WO 2013/168150 and Duvall M et al., 2011 , mAbs 3(2): 203-208, amongst others. For example, Duvall et al utilises technology which produces human IgG antibody libraries from naive B cells isolated from human tonsil tissue. The antibodies are produced from human genes and are therefore 100% human antibodies.

[0118] Monoclonal antibodies to Flightless I may also be purchased from commercial sources, including AbCam (Cambridge, United Kingdom), BosterBio (California, USA), Novus Biologicals (Colorado, USA), Sigma Aldrich (Missouri, USA), Santa Cruz Biotechnology (California, USA), and Life Technologies (California, USA). Additional sources of such antibodies can be found at www.biocompare.com/Search- Antibodies/?search=Flightless&said=0.

[0119] Various immunoassays may be used for screening to identify antibodies having the desired specificity. Numerous protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies with established specificities are well known in the art. Such immunoassays typically involve the measurement of complex formation between a protein and its specific antibody. A two-site, monoclonal-based immunoassay utilising antibodies reactive to two non-interfering epitopes is preferred, but a competitive binding assay may also be employed.

[0120] In one embodiment, decreasing the expression and/or activity of Flightless I may be achieved by antisense or gene-targeted silencing strategies. Accordingly, such strategies utilise agents including antisense oligonucleotides, antisense RNA, antisense RNA expression vectors, small interfering RNAs (siRNA), microRNAs (miRNAs) and short hairpin RNAs (shRNAs). Still further, catalytic nucleic acid molecules such as nucleases, aptamers, DNAzymes and ribozymes may be used for gene silencing. These molecules function by cleaving their target mRNA molecule rather than merely binding to it as in traditional antisense approaches.

[0121] An "antisense oligonucleotide” encompassed by the present invention corresponds to an RNA sequence as well as a DNA sequence coding therefor, which is sufficiently complementary to the Flightless I mRNA molecule, for which the antisense RNA is specific, to cause molecular hybridisation between the antisense RNA and the Flightless I mRNA such that translation of the mRNA is inhibited. Such hybridisation can occur under in vitro and in vivo conditions. The antisense molecule must have sufficient complementarity to Flightless I gene so that the antisense RNA can hybridize to the Flightless I gene (or mRNA) and inhibit its expression regardless of whether the action is at the level of splicing, transcription, or translation. In some embodiments, the complementary antisense sequence is about 15 to 30 nucleotides in length, for example, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides, or longer or shorter, as desired. Antisense oligonucleotides can include sequences hybridisable to any of several portions of the Flightless I gene, including the coding sequence, 3 ' or 5' untranslated regions, or intronic sequences. [0122] The terms "small interfering RNA" and "siRNA" interchangeably refer to short double- stranded RNA oligonucleotides that mediate RNA interference (also referred to as "RNA- mediated interference," or RNAi). RNAi is a highly conserved gene silencing event functioning through targeted destruction of individual mRNA by a homologous double- stranded small interfering RNA (siRNA) (Fire, A et ai., 1998, Nature 391 : 806-811). Mechanisms for RNAi are reviewed, for example, in Bayne and Allshire, 2005, Trends in Genetics, 21 : 370-73; Morris, 2005, Cell Mol. Life Sci., 62: 3057-3066; and Filipowicz, et ai, 2005, Current Opinion in Structural Biology, 15: 331-3341.

[0123] For the purposes of the present invention, RNAi can be effected by introduction or expression in the subject of siRNAs specific for Flightless I. The double stranded oligonucleotides used to effect inhibition of expression, at either the transcriptional or translational level, can be of any convenient length. siRNA molecules are typically from about 15 to about 30 nucleic acids in length, for example, about 19-25 nucleic acids in length, for example, about 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30 nucleic acids in length. Optionally the dsRNA oligonucleotides can include 3' overhang ends. Exemplary 2-nucleotide 3' overhangs can be composed of ribonucleotide residues of any type and can be composed of 2'-deoxythymidine resides, which lowers the cost of RNA synthesis and can enhance nuclease resistance of siRNAs in the cell culture medium and within transfected cells (see Elbashir et ai., 2001 , Nature 41 1 : 494-498).

[0124] Longer dsRNAs of 50, 75, 100 or even 500 base pairs or more can also be utilised. Exemplary concentrations of dsRNAs for effecting Flightless I inhibition are about 0.05 nM, 0.1 nM, 0.5 nM, 1.0 nM, 1.5 nM, 25 nM or 100 nM, although other concentrations can be utilised depending upon the nature of the cells treated and other factors readily discernable to the skilled artisan.

[0125] Exemplary dsRNAs can be synthesized chemically or produced in vitro or in vivo using appropriate expression vectors. Exemplary synthetic RNAs include 21 nucleotide RNAs chemically synthesised using methods known in the art. Synthetic oligonucleotides are preferably deprotected and gel-purified using methods known in the art (see for example Elbashir ef a/., 2001 , Genes Dev. 15: 188-200). Alternatively the dsRNAs can be transcribed from a mammalian expression vector. A single RNA target, placed in both possible orientations downstream of an appropriate promoter for use in mammalian cells, will transcribe both strands of the target to create a dsRNA oligonucleotide of the desired target sequence. Any of the above RNA species should be designed to include a portion of nucleic acid sequence represented in a target nucleic acid.

[0126] The specific sequence utilised in design of the siRNA oligonucleotides can be any contiguous sequence of nucleotides contained within the expressed gene message of the Flightless I target. Programs and algorithms, known in the art, may be used to select appropriate target sequences within the Flightless I gene (for example see the Ambion website at ambion.com). In addition, optimal sequences can be selected utilising programs designed to predict the secondary structure of a specified single stranded nucleic acid sequence and allow selection of those sequences likely to occur in exposed single stranded regions of a folded mRNA. Methods and compositions for designing appropriate siRNA oligonucleotides may be found, for example, in US patent number 6,251 ,588, the contents of which are incorporated herein by reference.

[0127] As would be understood by a person skilled in the art, ribozymes are enzymatic RNA molecules capable of catalyzing specific cleavage of RNA. The composition of a ribozyme molecule of the present invention should include one or more sequences complementary to Flightless I mRNA, and the well-known catalytic sequence responsible for mRNA cleavage or a functionally equivalent sequence (see for example US patent number 5,093,246, which is incorporated herein by reference in its entirety). Ribozyme molecules designed to catalytically cleave Flightless I mRNA transcripts can also be used to prevent translation of Flightless I mRNA. While ribozymes that cleave mRNA at site-specific recognition sequences can be used to destroy target mRNAs, the use of hammerhead ribozymes is preferred. Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA. Preferably, the target mRNA has the following sequence of two bases: 5'-UG-3'. The construction and production of hammerhead ribozymes is well known in the art.

[0128] Flightless I targeting ribozymes of the present invention necessarily contain a hybridising region complementary to two regions, each of at least 5 and preferably each 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 contiguous nucleotides in length, of the target Flightless I mRNA. In addition, the ribozymes should possess highly specific endoribonuclease activity, which autocatalytically cleaves the Flightless I sense mRNA.

[0129] With regard to antisense, siRNA or ribozyme oligonucleotides, phosphorothioate oligonucleotides can be used. Modifications of the phosphodiester linkage as well as of the heterocycle or the sugar may provide an increase in efficiency. Phophorothioate is used to modify the phosphodiester linkage. An N3'-P5' phosphoramidate linkage has been described as stabilising oligonucleotides to nucleases and increasing the binding to RNA. Peptide nucleic acid (PNA) linkage is a complete replacement of the ribose and phosphodiester backbone and is stable to nucleases, increases the binding affinity to RNA, and does not allow cleavage by RNAse H. Its basic structure is also amenable to modifications that may allow its optimisation as an antisense component. With respect to modifications of the heterocycle, certain heterocycle modifications have proven to augment antisense effects without interfering with RNAse H activity. An example of such modification is C-5 thiazole modification. Finally, modification of the sugar may also be considered. 2'- O-propyl and T-methoxyethoxy ribose modifications stabilize oligonucleotides to nucleases in cell culture and in vivo.

[0130] Inhibitory oligonucleotides can be delivered to a subject or the cell of a subject by direct transfection or transfection and expression via an expression vector. Appropriate expression vectors include mammalian expression vectors and viral vectors, into which has been cloned an inhibitory oligonucleotide with the appropriate regulatory sequences including a promoter to result in expression of the antisense RNA in a host cell. Suitable promoters can be constitutive or development-specific promoters. Transfection delivery can be achieved by liposomal transfection reagents, known in the art (e.g. Xtreme transfection reagent, Roche, Alameda, CA; Lipofectamine formulations, Invitrogen, Carlsbad, CA). Delivery mediated by cationic liposomes, by retroviral vectors and direct delivery are efficient. Another possible delivery mode is targeting using antibody to cell surface markers for the target cells.

[0131] As indicated above, decreasing the expression and/or activity of Flightless I may be achieved by various gene-targeted silencing strategies. Specific examples include mutagenesis and gene knock-down using zinc-finger nucleases (ZNFs) (see Carroll D, 201 1 , Genetics 188: 773-782; Sander JD et ai., 2011 , Nat. Methods 8: 67-69; and Miller JC et ai, 2007, Nat. Biotechnol., 25: 778-785), mutagenesis and gene knock-down using transcription activator-like effector nuclease (TALEN) systems (see Bogdanove AJ and Voytas DF, 2011 , Science 333: 1843-1846; Streubel J et ai., 2012, Nat. Biotechnol., 30: 593-595; Cermak T et ai., 2011 , Nucl. Acids Res., 39: e82; Chen K and Gao C, 2013, J. Genet. Genomics 40: 271-279; Voytas DF, 2013, Ann. Rev. Plant Biol., 64: 327-350; and Wang Y et ai., 2014, Nat. Biotechnol., 32: 947-951), and mutagenesis and gene knock down using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system or equivalently adapted systems (see Belhaj K et al., 2015, Current Opinion in Biotechnology, 32: 76-84; Shan Q et al., 2014, Nature Protocols, 9: 2395-2410; and Wang Y et al., 2014, Nat. Biotechnol., 32: 947-951).

[0132] Recent developments in DNA manipulation allows for effective introduction of exogenous DNA into the chromosome of a host cell and allows insertion of a mutation at a particular locus on a chromosome. For example, a point mutation can be introduced into the Flightless I gene by knock-in, wherein the mutation may introduce a premature stop codon thereby disrupting production of wild-type protein. A mutation may also be introduced into a regulatory region of the gene (for example in the promoter) to reduce or eliminate expression of the gene. In many circumstances, gene knock-in involves homologous recombination mechanisms. Under natural circumstances, the probability of homologous recombination between an exogenous targeting vector and the genome of a cell is very low (about 1/10⁵ to 1/10⁶). Spontaneous gene targeting typically occurs at a very low frequency in mammalian cells with an efficiency of 1 in a million cells. The presence of a double-strand break is often recombinogenic and increases the homologous recombination frequency by several thousand-fold (see Jasin M, 1996, Trends Genet., 12(6): 224-228).

[0133] Recently developed techniques, including ZFNs, TALENs, CRISPR, and other site- directed nuclease technologies, enable the creation of double-strand DNA breaks at desired locus sites. These controlled double-strand breaks can promote homologous recombination at such specific locus sites. This process relies on targeting specific sequences of nucleic acid molecules, such as chromosomes, with endonucleases that recognize and bind to such sequences and induce a double-strand break in the nucleic acid molecule.

[0134] A nuclease-mediated double-stranded DNA break in the genome can be repaired by two main mechanisms: Non-Homologous End Joining (NHEJ), which frequently results in the introduction of non-specific insertions and deletions (indels), or homology directed repair (HDR), which incorporates a homologous strand as a repair template. When a sequence-specific nuclease is delivered along with a homologous donor DNA construct containing a desired mutation, gene targeting efficiencies are increased by 1000-fold compared to just the donor construct alone.

[0135] Alternative methods have been developed to accelerate the process of genome modification by directly injecting DNA or mRNA of site-specific nucleases into a cell to generate a DNA double strand break (DSB) at a specified locus. DSBs induced by these site-specific nucleases can then be repaired by either error-prone non-homologous end joining (NHEJ) resulting, for example, in mutants carrying deletions or insertions at the cut site. If a donor plasmid with homology to the ends flanking the DSB is co-injected, high- fidelity homologous recombination can produce plants with targeted integrations.

[0136] The CRISPR type II system has been used to edit the genomes of a broad spectrum of species (see for example Friedland AE et al., 2013, Nat. Methods, 10(8): 741-743; Mali P et al. , 2013, Science, 339(6121): 823-826; Hwang WY etal., 2013, Nat. Biotechnol., 31 (3): 227-229; Jiang W ef al., 2013, Nat. Biotechnol., 31 (3): 233-239; Jinek M et al., 2013, eLife, 2: e00471 ; Cong L et al., 2013, supra). CRISPR is particularly customizable because the active form consists of an invariant Cas9 protein and an easily programmable single guide RNA (sgRNA). Of the various CRISPR orthologs, the Streptococcus pyogenes (Sp) CRISPR is the most well-characterized and widely used. The Cas9-gRNA complex first probes DNA for the protospacer-adjacent motif (PAM) sequence (-NGG for Sp Cas9), after which Watson-Crick base-pairing between the sgRNA and target DNA proceeds in a ratchet mechanism to form an R-loop. Following formation of a ternary complex of Cas9, sgRNA, and target DNA, the Cas9 protein generates two nicks in the target DNA, creating a blunt double-strand break (DSB) that is repaired by the non-homologous end joining (NHEJ) pathway or template-directed homologous recombination (HR). CRISPR methods are disclosed, for example, in US 8,697,359.

[0137] The aforementioned knockout, knockdown or downregulation technologies are merely representative and are not limiting to other mechanisms that may be employed.

[0138] The agent in the various embodiments of the present invention may also cause an alteration in the intracellular and/or extracellular localisation of Flightless I. For example, the agent may cause re-localisation of Flightless I from the cytoplasm of the cell to the nucleus of the cell, or re-localisation of Flightless I from the nucleus to the cytoplasm.

[0139] As indicated above, the Flightless I gene is evolutionary conserved across a number of species. Accordingly, the term“subject” as used in the present invention should be taken to encompass any subject which expresses the Flightless I gene. In some embodiments, the subject is a human or animal subject. The animal subject may be a mammal, a primate, a livestock animal (e.g. a horse, a cow, a sheep, a pig, or a goat), a companion animal (e.g. a dog, a cat), a laboratory test animal (e.g. a mouse, a rat, a guinea pig, a bird), an animal of veterinary significance, or an animal of economic significance. [0140] The inventors have determined that the level of Flightless I protein is increased in cells of the mucosal layer of the gastrointestinal tract acting as an indicator of mucosal lesions. The inventors have also established that decreasing expression of Flightless I leads to a decrease in measures of mucosal damage. This differential expression of Flightless I indicates that it is a suitable biomarker which can form the basis of diagnostic and prognostic testing for mucosal lesions and mucosal damage in subjects, including lesions and damage associated with gastrointestinal disorders such as irritable bowel syndrome.

[0141] A biomarker is effectively an organic biomolecule which is differentially present in a sample taken from a subject of one phenotypic status (e.g. having a disease or disorder) as compared with another phenotypic status (e.g. not having the disease or disorder). A biomarker is differentially present between different phenotypic status groups if the mean or median expression level of the biomarker is calculated to be different (i.e. higher or lower) between the groups. Therefore, biomarkers, alone or in combination, provide an indication that a subject belongs to one phenotypic status or another.

[0142] Accordingly, the present invention provides a method of diagnosing a disorder of the gastrointestinal tract in a subject, the method including the steps of:

[0143] The identification of differential expression of Flightless I also enables methods for assessing the therapeutic efficacy in a subject of a treatment for a disorder of the gastrointestinal tract.

[0144] Accordingly, the present invention also provides a method of assessing the progression of a disorder of the gastrointestinal tract in a subject, the method including the steps of:

comparing the level of expression and/or activity of Flightless I in the subject to a reference level of expression and/or activity of Flightless I; and assessing the progression of a disorder of the gastrointestinal tract in the subject on the basis of the comparison.

[0145] Methods and assays which may be used to measure expression and/or activity of Flightless I (and the level thereof) have been described in detail above. The aforementioned methods and assays may measure the level of expression of Flightless I at the transcriptional (mRNA) or translational (protein) stage of expression.

[0146] In the subject, the level of expression and/or activity of Flightless I may be measured directly, or in an alternative embodiment, the level of expression and/or activity of Flightless I may be measured in a sample obtained from a subject. It is to be made clear that the sample obtained from the subject that is analysed by the methods of the present invention may have previously been obtained from the subject, and, for example, stored in an appropriate repository. In this instance, the sample would have been obtained from the subject in isolation of, and therefore separate to, the methods of the present invention.

[0147] The sample which is obtained from the subject will typically be a biopsy sample of mucosal tissue taken from a relevant section of the gastrointestinal tract, including a sample corresponding to normal or unaffected tissue of the subject. However, given the systemic expression of Flightless I, a sample form any tissue in which Flightless I is expressed may be obtained. In this regard, the sample may also include a blood sample, or a sample derived from blood (for example a serum sample or a plasma sample or a fraction of a blood, serum or plasma sample, blood cells), saliva, buccal swab, stool sample, bladder washing, semen, and urine. In certain circumstances, the sample may be manipulated in any way after procurement, such as by treatment with reagents, solubilization, or enrichment for certain components, such as the relevant protein or polynucleotide under investigation.

[0148] Once the level of expression and/or activity of Flightless I been measured in the subject, or in a sample obtained from the subject, the level of expression and/or activity is compared to a reference level of expression and/or activity for Flightless I. The reference level of expression and/or activity for Flightless I is a level of expression and/or activity that is associated with a known status, i.e. a level of expression and/or activity which is known to be found in a subject not suffering from a disorder of the gastrointestinal tract or not suffering from a mucosal lesion or mucosal damage, or is known to be found in non-affected gastrointestinal tissue of the subject (a“normal subject” or“normal sample” in the context of the present invention). A reference level of expression and/or activity of Flightless I may be derived from at least one normal subject and is preferably derived from an average of normal subjects (e.g. n=2 to 100 or more), wherein the subject or subjects have no prior history of mucosal lesions or damage. A reference level of expression and/or activity of Flightless I can also be obtained from one or more“normal samples” from a subject suspected to have a gastrointestinal disorder and/or a mucosal lesion or mucosal damage. For example, a reference level of expression and/or activity of Flightless I may be obtained from at least one normal sample and is preferably obtained from an average of normal samples (e.g. n=2 to 100 or more) from the subject.

[0149] In some embodiments, a level of expression and/or activity of Flightless I in the subject that is higher than the reference level of expression and/or activity for Flightless I is indicative of a disorder of the gastrointestinal tract in the subject or is indicative of progression of a disorder of the gastrointestinal tract in the subject.

[0150] In some embodiments of the present invention, a level of expression and/or activity of Flightless I is measured at more than one time point. Such "serial" sampling is well suited, for example, to monitoring the progression of psoriasis. Serial sampling can be performed for any desired timeline, such as monthly, quarterly (i.e. every three months), semi-annually, annually, biennially, or less frequently. The comparison between the measured expression level in the subject and the reference expression level may be carried out each time a new sample is measured, or the data relating to levels may be held for less frequent analysis.

[0151] In some embodiments, the subject may be undergoing an existing treatment for a gastrointestinal disorder or existing treatment for a mucosal lesion or mucosal damage. Examples of existing treatments include, but are not limited to, anti-inflammatory drugs such as mesalamine (also known as Asacol HD, Delzicol, etc), balsalazide (Colazal) and olsalazine (Dipentum) depending on which part of colon is affected; immunosuppressant drugs such as azathioprine (also known as Azasan or Imuran), mercaptopurine (Purinethol, Purixan), cyclosporine (Gengraf, Neoral, Sandimmune) and methotrexate (Trexall); antibiotics such as ciprofloxacin (Cipro) and metronidazole (Flagyl); anti-diarrheal medication such as psyllium powder (Metamucil) or methylcellulose (Citrucel); general pain relievers; iron and vitamin supplements; or even life-style and diet choices.

[0152] Therefore, the aforementioned assessment method may be used to monitor the progress and efficacy of an existing treatment in the subject. As an extension to this, the assessment method may also be used to perform clinical trials of a new drug, or monitor the progress of a subject on the new drug. Therapy or clinical trials involve administering the drug being tested in a particular regimen. The regimen may involve a single dose of the drug or multiple doses of the drug over time. The doctor or clinical researcher monitors the effect of the drug on the subject over the course of administration. If the drug has the desired pharmacological impact, the level of expression and/or activity of Flightless I will approximate or be identical to the reference level of expression and/or activity of Flightless I. Therefore, the trending of the expression and/or activity levels of Flightless I can be monitored in the subject during the course of treatment. The level of expression and/or activity of Flightless I can be determined using the methods described in detail above. One embodiment of this method involves determining the level of expression and/or activity of Flightless I for at least two different time points during a course of drug therapy, e.g. a first time and a second time, and comparing the change in expression and/or activity level over that time, if any. For example, the level of expression and/or activity of Flightless I can be measured before and after drug administration or at two different time points during drug administration. The effect of therapy is determined based on these comparisons. If a treatment is effective, the level of expression and/or activity of Flightless I will approximate or be identical to the reference level of expression and/or activity of Flightless I, while if treatment is ineffective, the level of expression and/or activity of Flightless I will remain higher than the reference level.

[0153] The present invention also provides a pharmaceutical composition when used for, or for use in, treating a mucosal lesion in the gastrointestinal tract of a subject, decreasing mucosal damage in the gastrointestinal tract of a subject, treating an ulcer or blister in the mucosa of the gastrointestinal tract of a subject, or treating inflammatory bowel disease in a subject. In this regard, the composition includes an effective amount of an agent that decreases expression and/or activity of Flightless I. Examples of suitable agents have been described in detail above. The meaning of “decreasing the expression and/or activity of Flightless I” has also been described in detail above.

[0154] The delivery or administration of the agent in the various embodiments of the present invention may be delivery or administration of the agent alone, or delivery or administration of the agent formulated into a suitable pharmaceutical composition, as referred to above.

[0155] In this regard, the pharmaceutical composition may also include the use of one or more pharmaceutically acceptable additives, including pharmaceutically acceptable salts, amino acids, polypeptides, polymers, solvents, buffers, excipients and bulking agents, taking into consideration the particular physical and chemical characteristics of the agent to be administered.

[0156] The preparation of such pharmaceutical compositions is known in the art, for example as described in Remington's Pharmaceutical Sciences, 18th ed., 1990, Mack Publishing Co., Easton, Pa. and U.S. Pharmacopeia: National Formulary, 1984, Mack Publishing Company, Easton, Pa.

[0157] For example, the agent can be prepared into a variety of pharmaceutical compositions in the form of, for example, an aqueous solution, an oily preparation, a fatty emulsion, an emulsion, a gel, a cream, etc., depending on the location of the gastrointestinal tract which requires treatment. These preparations can be administered as intramuscular, intravenous, intraperitoneal or subcutaneous injections, as an embedded preparation, as a transmucosal preparation, or in the form of an oral preparation (for example solid preparations such as tablets, capsules, granules or powders; liquid preparations such as syrup, emulsions or suspensions).

[0158] Compositions containing the agent may also contain a preservative, stabiliser, dispersing agent, pH controller or isotonic agent. Examples of suitable preservatives are glycerin, propylene glycol, phenol or benzyl alcohol. Examples of suitable stabilisers are dextran, gelatin, a-tocopherol acetate or alpha-thioglycerin. Examples of suitable dispersing agents include polyoxyethylene (20), sorbitan mono-oleate (Tween 80), sorbitan sesquioleate (Span 30), polyoxyethylene (160) polyoxypropylene (30) glycol (Pluronic F68) or polyoxyethylene hydrogenated castor oil 60. Examples of suitable pH controllers include hydrochloric acid, sodium hydroxide and the like. Examples of suitable isotonic agents are glucose, D-sorbitol or D-mannitol.

[0159] The administration of the agent in the various embodiments of the present invention may also be in the form of a composition containing a pharmaceutically acceptable carrier, diluent, excipient, suspending agent, lubricating agent, adjuvant, vehicle, delivery system, emulsifier, disintegrant, absorbent, preservative, surfactant, colorant, flavorant or sweetener, taking into account the physical and chemical properties of the agent being administered. [0160] For these purposes, the composition may be administered orally, topically, parenterally, by inhalation spray, adsorption, absorption, rectally, nasally, bucally, vaginally, intraventricularly, via an implanted reservoir in dosage formulations containing conventional non-toxic pharmaceutically-acceptable carriers, or by any other convenient dosage form. The term parenteral as used herein includes subcutaneous, intravenous, intramuscular, intradermal, intraperitoneal, intrathecal, intraventricular, intrasternal, and intracranial injection or infusion techniques.

[0161] When administered parenterally, the composition will normally be in a unit dosage, sterile injectable form (solution, suspension or emulsion) which is preferably isotonic with the blood of the recipient with a pharmaceutically acceptable carrier. Examples of such sterile injectable forms are sterile injectable aqueous or oleaginous suspensions. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable forms may also be sterile injectable solutions or suspensions in non-toxic parenterally-acceptable diluents or solvents, for example, as solutions in 1 ,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, saline, Ringer's solution, dextrose solution, isotonic sodium chloride solution, and Hanks' solution. In addition, sterile, fixed oils are conventionally employed as solvents or suspending mediums. For this purpose, any bland fixed oil may be employed including synthetic mono- or di-glycerides, corn, cottonseed, peanut, and sesame oil. Fatty acids such as ethyl oleate, isopropyl myristate, and oleic acid and its glyceride derivatives, including olive oil and castor oil, especially in their polyoxyethylated versions, are useful in the preparation of injectables. These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants.

[0162] The carrier may contain minor amounts of additives, such as substances that enhance solubility, isotonicity, and chemical stability, for example anti-oxidants, buffers and preservatives.

[0163] When administered orally, the agent will usually be formulated into unit dosage forms such as tablets, cachets, powder, granules, beads, chewable lozenges, capsules, liquids, aqueous suspensions or solutions, or similar dosage forms, using conventional equipment and techniques known in the art. Such formulations typically include a solid, semisolid, or liquid carrier. Exemplary carriers include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, mineral oil, cocoa butter, oil of theobroma, alginates, tragacanth, gelatin, syrup, methyl cellulose, polyoxyethylene sorbitan monolaurate, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and the like.

[0164] A tablet may be made by compressing or moulding the agent optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing, in a suitable machine, the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, surface active, or dispersing agent. Moulded tablets may be made by moulding in a suitable machine, a mixture of the powdered active ingredient and a suitable carrier moistened with an inert liquid diluent.

[0165] The administration of the agent in the various embodiments of the present invention may also utilise controlled release technology. The agent may also be administered as a sustained-release pharmaceutical. To further increase the sustained release effect, the agent may be formulated with additional components such as vegetable oil (for example soybean oil, sesame oil, camellia oil, castor oil, peanut oil, rape seed oil); middle fatty acid triglycerides; fatty acid esters such as ethyl oleate; polysiloxane derivatives; alternatively, water-soluble high molecular weight compounds such as hyaluronic acid or salts thereof (weight average molecular weight: ca. 80,000 to 2,000,000), carboxymethylcellulose sodium (weight average molecular weight: ca. 20,000 to 400,000), hydroxypropylcellulose (viscosity in 2% aqueous solution: 3 to 4,000 cps), atherocollagen (weight average molecular weight: ca. 300,000), polyethylene glycol (weight average molecular weight: ca. 400 to 20,000), polyethylene oxide (weight average molecular weight: ca. 100,000 to 9,000,000), hydroxypropylmethylcellulose (viscosity in 1 % aqueous solution: 4 to 100,000 cSt), methylcellulose (viscosity in 2% aqueous solution: 15 to 8,000 cSt), polyvinyl alcohol (viscosity: 2 to 100 cSt), polyvinylpyrrolidone (weight average molecular weight: 25,000 to 1 ,200,000).

[0166] Alternatively, the agent may be incorporated into a hydrophobic polymer matrix for controlled release over a period of days. The agent may then be moulded into a solid implant, or externally applied patch, suitable for providing efficacious concentrations of the agent over a prolonged period of time without the need for frequent re-dosing. Such controlled release films are well known to the art. Other examples of polymers commonly employed for this purpose that may be used include nondegradable ethylene-vinyl acetate copolymer a degradable lactic acid-glycolic acid copolymers which may be used externally or internally. Certain hydrogels such as poly(hydroxyethylmethacrylate) or poly(vinylalcohol) also may be useful, but for shorter release cycles than the other polymer release systems, such as those mentioned above.

[0167] The carrier may also be a solid biodegradable polymer or mixture of biodegradable polymers with appropriate time release characteristics and release kinetics. The agent may then be moulded into a solid implant suitable for providing efficacious concentrations of the agent over a prolonged period of time without the need for frequent re-dosing. The agent can be incorporated into the biodegradable polymer or polymer mixture in any suitable manner known to one of ordinary skill in the art and may form a homogeneous matrix with the biodegradable polymer, or may be encapsulated in some way within the polymer, or may be moulded into a solid implant.

[0168] In some embodiments, the pharmaceutical composition is a topical composition. For topical administration, the composition of the present invention may be in the form of a solution, spray, lotion, cream (for example a non-ionic cream), gel, paste or ointment. Alternatively, the composition may be delivered via a liposome, nanosome, or nutri-diffuser vehicle.

[0169] A cream is a formulation that contains water and oil and is stabilized with an emulsifier. Lipophilic creams are called water-in-oil emulsions, and hydrophilic creams oil- in-water emulsions. The cream base for water-in-oil emulsions are normally absorption bases such as vaseline, ceresin or lanolin. The bases for oil-in-water emulsions are mono- , di-, and tri-glycerides of fatty acids or fatty alcohols with soaps, alkyl sulphates or alkyl polyglycol ethers as emulsifiers.

[0170] A lotion is an opaque, thin, non-greasy emulsion liquid dosage form for external application to the skin, which generally contains a water-based vehicle with greater than 50% of volatiles and sufficiently low viscosity that it may be delivered by pouring. Lotions are usually hydrophilic, and contain greater than 50% of volatiles as measured by LOD (loss on drying). A lotion tends to evaporate rapidly with a cooling sensation when rubbed onto the skin.

[0171] A paste is an opaque or translucent, viscous, greasy emulsion or suspension semisolid dosage form for external application to the skin, which generally contains greater than 50% of hydrocarbon-based or a polyethylene glycol-based vehicle and less than 20% of volatiles. A paste contains a large proportion (20-50%) of dispersed solids in a fatty or aqueous vehicle. An ointment tends not to evaporate or be absorbed when rubbed onto the skin.

[0172] An ointment is an opaque or translucent, viscous, greasy emulsion or suspension semisolid dosage form for external application to the skin, which generally contains greater than 50% of hydrocarbon-based or a polyethylene glycol-based vehicle and less than 20% of volatiles. An ointment is usually lipophilic, and contains >50% of hydrocarbons or polyethylene glycols as the vehicle and <20% of volatiles as measured by LOD. An ointment tends not to evaporate or be absorbed when rubbed onto the skin.

[0173] A gel is usually a translucent, non-greasy emulsion or suspension semisolid dosage form for external application to the skin, which contains a gelling agent in quantities sufficient to impart a three-dimensional, cross-linked matrix. A gel is usually hydrophilic, and contains sufficient quantities of a gelling agent such as starch, cellulose derivatives, carbomers, magnesium-aluminum silicates, xanthan gum, colloidal silica, aluminium or zinc soaps.

[0174] The composition for topical administration may further include drying agents, anti foaming agents; buffers, neutralizing agents, agents to adjust pH; colouring agents and decolouring agents; emollients; emulsifying agents, emulsion stabilizers and viscosity builders; humectants; odorants; preservatives, antioxidants, and chemical stabilizers; solvents; and thickening, stiffening, and suspending agents, and a balance of water or solvent.

[0175] Each of the formulated pharmaceutical compositions referred to above may be systemic in nature once administered to a subject. That is, once the pharmaceutical composition has been delivered, it is capable of spreading throughout the body of the subject thereby treating the mucosal lesions and mucosal damage at their site of occurrence in the body.

[0176] It should also be appreciated that other methods of delivery of an agent to modulate the expression and/or activity of Flightless I are contemplated. For example, the agent may be delivered by way of a nucleic acid or vector that allows for expression of the agent in the appropriate target cells. For example, the agent may be delivered by way of a viral vector that causes expression of the agent in target cells.

[0177] The present invention also provides a kit when used for, or for use in, diagnosing a disorder of the gastrointestinal tract in a subject, or for assessing the progression of a disorder of the gastrointestinal tract in a subject, the kit including means for measuring the level of expression and/or activity of Flightless I in the subject. In some embodiments, the level of expression and/or activity of Flightless I is measured in the mucosa of the gastrointestinal tract of the subject. In some embodiments, a level of expression and/or activity of Flightless I in the mucosa of the subject that is higher than the reference level of expression and/or activity for Flightless I is indicative of a disorder of the gastrointestinal tract in the subject.

[0178] Means and methods for measuring the level of expression and/or activity of Flightless I in the subject are described in detail above.

[0179] In one embodiment, the kit includes a solid support, such as a chip, sensor, a microtiter plate or a bead or resin having a capture reagent attached thereon, wherein the capture reagent binds Flightless I. Therefore, for example, a kit of the present invention can comprise mass spectrometry probes for SELDI, such as ProteinChip^® arrays, or a versatile fibre-based SPR sensing device. In the case of biospecfic capture reagents, the kit can include a solid support with a reactive surface, and a container including the biospecific capture reagent.

[0180] In one embodiment, the kit can also include a washing solution or instructions for making a washing solution, in which the combination of the capture reagent and the washing solution allows capture of Flightless I on the solid support for subsequent detection by, for example, mass spectrometry. The kit may include more than one type of adsorbent, each present on a different solid support.

[0181] In some embodiments, such a kit can include instructions for suitable operational parameters in the form of a label or separate insert. For example, the instructions may inform a consumer about how to collect the sample, how to wash the probe or the Flightless I to be detected.

[0182] In some embodiments, the kit can include one or more containers with samples that represent a reference expression level for Flightless I, and are therefore to be used as a standard for calibration. [0183] It is to be noted that where a range of values is expressed, it will be clearly understood that this range encompasses the upper and lower limits of the range, and all numerical values or sub-ranges in between these limits as if each numerical value and sub range is explicitly recited. The statement "about X% to Y%" has the same meaning as "about X% to about Y%," unless indicated otherwise.

[0184] The term“about” as used in the specification means approximately or nearly and in the context of a numerical value or range set forth herein is meant to encompass variations of +/- 10% or less, +/- 5% or less, +/- 1% or less, or +/- 0.1 % or less of and from the numerical value or range recited or claimed.

[0185] As used herein, the singular forms“a,”“an,” and“the” may refer to plural articles unless specifically stated otherwise.

[0186] Throughout this specification, unless the context requires otherwise, the word “comprise”, or variations such as“comprises” or“comprising”, will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

[0187] All methods described herein can be performed in any suitable order unless indicated otherwise herein or clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the example embodiments and does not pose a limitation on the scope of the claimed invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential.

[0188] It will be apparent to the person skilled in the art that while the invention has been described in some detail for the purposes of clarity and understanding, various modifications and alterations to the embodiments and methods described herein may be made without departing from the scope of the inventive concept disclosed in this specification.

[0189] Furthermore, the description provided herein is in relation to several embodiments which may share common characteristics and features. It is to be understood that one or more features of one embodiment may be combinable with one or more features of the other embodiments. In addition, a single feature or combination of features of the embodiments may constitute additional embodiments. [0190] The subject headings used herein are included only for the ease of reference of the reader and should not be used to limit the subject matter found throughout the disclosure or the claims. The subject headings should not be used in construing the scope of the claims or the claim limitations.

[0191] Finally, reference is made to standard textbooks of molecular biology that contain methods for carrying out basic techniques encompassed by the present invention. See, for example, Green MR and Sambrook J, 2012, supra.

[0192] The invention is further illustrated in the following example. The example is for the purpose of describing particular embodiments only and is not intended to be limiting with respect to the above description.

EXAMPLE 1

Flightless I Expression and Mucosal Lesions of the Gl Tract

[0193] Mucosal lesions of the gastrointestinal tract, such as those which manifest as a result of inflammatory bowel disease, are a significant health concern for affected subjects. The purpose of the present study was to examine the role that Flightless I (Flii) plays in said mucosal lesions with a view to the development of a potential therapeutic intervention.

Materials and Methods

Human studies

[0194] Human biopsy samples were obtained via colonoscopies performed at The Queen Elizabeth Hospital (TQEH, Adelaide). Samples were taken from ten adult subjects with ulcerative colitis and from ten normal adult subjects with non-inflammatory conditions, such as irritable bowel syndrome or who attended for colon cancer screening were included in the study. All experimental protocols were approved by the Human Ethics Committee of the TQEH in accordance with relevant guidelines and regulations. Approval was given to perform additional biopsy for research and to archive biopsies for future studies and written informed consent was obtained from all participants. Colonic biopsies in histologic paraffin blocks were retrieved and were sectioned transversely at 4 pm. Histological sections were stained with haematoxylin and eosin (H&E) and standard immunohistochemistry staining protocols (see below) for Flii (2 mg/ml; anti-Flightless I sc-30046 rabbit IgG; Santa Cruz Biotechnology, CA, USA) and 4’6-diamidino-2-phenylindole (DAPI; 0.1 mg/ml; D1306; Live Technologies Australia, VIC, AUS) as previously described (Chong HT et ai, 2017, Br. J. Dermatol., 176: 705-712). Animal studies

[0195] Female Balb/c mice were maintained according to the Australian Standards for Animal Care under the protocols approved by the Child, Youth and Women’s Health Service Animal Ethics Committee, The University of Adelaide Animal Ethics Committee and University of South Australia Animal Ethics Committee (AEC 962/12/16 and AEC 137a/13). All strains were BALB/c-congenic and were maintained as homozygous colonies or by continuous backcrossing to BALB/c animals. Wild-type controls were obtained from BALB/c inbred litters. Mice (aged 8 weeks, weighing 18-22g) were acclimatised to single cages under controlled room temperature (25°C) and photoperiod (12: 12-hr light-dark cycle) conditions. Animals were provided with ad libitum access to tap water and a standard 18% casein-based diet (National Research Council. 1995. Nutrient Requirements of Laboratory Animals, Fourth Revised Edition, 1995. Washington, DC: The National Academies Press. https://doi.Org/10.17226/4758).

[0196] The murine alleles of Flii used in this study included: a heterozygous carrier of the murine Flightless I gene (Flii): Flii tmI Hdc (MGL2179825) written as Flii^+/", and Tg(FLII)2Hdc (MGL4939366), a transgenic strain expressing exogenous human flightless I (FLII) (Cowin AJ et ai., 2007, J. Pathol., 21 1 : 572-581 ; Campbell HD et ai., 2002, Mol. Cell. Biol., 22: 3518-3526). Flii^+I were generated by a loss of function mutation in the Flii gene via homologous recombination in embryonic stem cells and passage of these cells through the germ line following chimera production (Thomsen N et ai, 201 1 , Genesis, 49: 681-688). The generation of F//7^+/ mice and the resulting mutation are described in detail in Campbell HD et ai, 2002, supra, and a diagram of the targeting strategy is illustrated in Figure 1A. The heterozygous mice were identified using three PCR primer sets that amplified products specific to the wild-type or targeted allele as illustrated in Figure 1 B. The PCR was performed on DNA extracted from ear biopsies of potential heterozygotes. The animals with one wild-type copy of the Flii gene and one mutant copy of the Flii gene express no more than 50% of the normal Flii gene expression.

[0197] Mice homozygous for the transgene used in this study (Flii^T9/T9 mice) had two copies of the human FLII transgene and had significantly elevated levels of Flii protein compared to wild-type mice (Thomsen N et ai., 2011 , supra). Mice carrying additional copies of the Flii gene were generated by introduction of a cosmid construct into the mouse genome using transgenesis. At the time of strain production, the cosmid contained the human Flii gene and the surrounding sequences with the extent of the construct being defined via restriction mapping (Thomsen N et ai., 2011 , supra). The availability of the mouse genome allowed for the estimation of the extent of the cosmid. Currently, it is known that the cosmid contains all the neighbouring SMCR7 gene and parts of the Topo and LLGL1 genes Figure 1C. The transgenic strain was backcrossed to BALB/c animals for 10 generations before being intercrossed and homozygous animals were classified via progeny testing following established protocols. The mouse colony was subsequently maintained by intercross of animals homozygous for the transgene. The expression of the Human FLII gene was examined using species specific RT-PCR showing FLII expression in all tissues examined (adult brain, heart, lung, muscle, spleen and skin) (Figure 1 D). An upregulation in the level of Flii protein was confirmed using semi-quantitative Western analysis that showed total (mouse + human) protein levels increased up to 1.52 times compared to that of wild-type mice (Figure 1 E).

[0198] Colonic inflammation was induced in mice using Dextran Sulphate Sodium (DSS; colitis grade; MW 36,000 - 50,000; #02160110; MP Biomedicals, Jomar Life Research, SA, AUS) (Yazbeck R et aL, 2011 , J. Cell Physiol., 226(12): 3219-3224). The DSS induced model of UC has the phenotypic features of human disease including clinical symptoms of diarrhoea, rectal bleeding and weight loss and histological features of ulceration, oedema, crypt and epithelial cell damage, and increased lymphocyte, monocyte and granulocyte infiltration (Yazbeck R et aL, 201 1 , J. Cell Physiol., 226(12): 3219-3224). Briefly, 2% DSS was introduced in drinking water of mice heterozygous for Flightless I (i.e. mice expressing a single copy of Flightless I - Flii^+A), wild-type mice (i.e. mice expressing both copies of Flightless I), and transgenic mice overexpressing Flightless I (i.e. mice expressing extra copies of Flightless I - Flii ^9/T9) over a period of seven days (n=8/genotype). Body weights were recorded daily and a disease activity index (DAI) was calculated daily from a range of criteria to provide an overall score of mucosal damage severity over seven days. Criteria included dual coat, poor posture, pale/sunken eyes, behavioural change, dehydration, excess squealing, reluctance to move, stool consistency, rectal bleeding, and weight loss. On day 7 of the experiment, mice were euthanized by CO2 asphyxiation followed by cervical dislocation and distal colon sections were collected for RTq-PCR, immunohistochemistry, myeloperoxidase (MPO) analysis and histological processing. MPO is an enzyme located in the intracellular granules of neutrophils and is used as a measure of neutrophil content indicative of acute tissue inflammation. Distal colon samples were homogenized in a phosphate buffer and kinetic absorbance measured at 450nm as described in Yazbeck R et ai, 2011 , supra; and Kim JJ et ai, 2012, J. Vis. Exp., 60: e3678. Histology and Immunohistochemistry

[0199] Paraffin embedded, fixed tissue samples were stained with H&E or subjected to antigen retrieval and immunohistochemistry following manufacturer’s protocols (DAKO Corporation, DK). H&E stained sections were used for standardised measurements of colon length, crypt depth, crypt area index and histological disease severity following well established protocols (Cheah KY et ai, 2013, Dig. Dis. Sci., 58: 970-977; Yazbeck R et ai, 2013, supra). Briefly, crypt depth and area index were determined in a blinded study using Image-Pro Plus software (Media Cybernetics, MD, USA) and 40 well orientated crypts per tissue per mouse were analysed and a mean value obtained (Cheah KY etai., 2013, supra). A semi-quantitative assessment of histological mucosal damage was used to obtain a disease severity score by assessment of 7 histological parameters including: enterocyte, crypt, and crypt cell disruption, reduction in goblet cells, lymphocytic and polymorhonuclear cell infiltration, and thickening/oedema of the submucosa and muscularis externa as detailed in Yazbeck R et ai., 2011 , supra.

[0200] For immunohistochemistry, following blocking in 3% normal goat serum, primary antibodies were applied at 2 mg/ml (4°C) overnight in a humidified chamber. Primary antibodies included: anti-Flightless I sc-30046 rabbit IgG, anti-PCNA sc- 56 mouse lgG2a, and anti-TNF-a sc-52746 mouse IgG, anti-Flightless I sc-21716 mouse monoclonal IgG, anti^-tubulin sc-51670 mouse monoclonal IgG, and anti^-catenin sc-7963 rabbit polyclonal IgG, all purchased from Santa Cruz Biotechnology (California, USA). Isotype control mouse lgG2a antibody (ab170191) and anti-Axin-2 (ab32197) rabbit polyclonal IgG were purchased from Abeam (Victoria, Australia). Species specific secondary antibodies used included Alexa Fluor goat anti-rabbit 488 (A11006), goat anti-mouse 633 (A21050), goat anti-mouse 488 (A11001) and goat anti-rabbit 633 (A31577), which were purchased from Life Technologies Australia (Victoria, Australia).

[0201] The nuclei were counterstained with 4’6-diamidino-2-phenylindole (DAPI; 0.1 mg/ml; D1306; Life Technologies Australia, Victoria, Australia) for 3 minutes at room temperature prior to mounting sections in Fluorescence Mounting Medium (DAKO Corporation, DK). Images were captured on an Olympus microscope and CellSense Live Science Imaging Software program (Olympus, Germany) was used to determine the integrated fluorescence intensity. Negative controls and isotype control antibody were included to demonstrate antibody staining specificity. Control samples underwent the same staining procedure outlined except the primary or secondary antibody was omitted. All control sections had negligible immunofluorescence. [0202] A similar study was repeated using 4% DSS in wild-type mice treated daily with 100 pi IP injections of a neutralising antibody to Flightless I (FnAb - 100pg/ml) over a seven day period. The Flightless I neutralising antibody was made in-house and was an affinity-purified mouse monoclonal anti-Flightless (FnAb) IgGi antibody raised against the Flightless I protein. Disease severity was analysed macroscopically using the DAI score and distal colon sections were collected and analysed histologically using established protocols as described above.

Myeloperoxidase assay

[0203] Myeloperoxidase assay (MPO) was performed to detect neutrophil infiltration using protocols previously published (Chartier LC et ai, 2018, Dig. Dis. Sci., 63: 135-145). Briefly, distal colon tissue was homogenized and centrifuged at 13,000g for 12 min, supernatant was discarded, and 0.5% hexadecyltrimethyl ammonium bromide buffer (Sigma Aldrich, NSW, Australia) was used to re-suspend the tissue homogenate. After vortexing and centrifuging for 2 min, water control and test samples were aliquoted (50 mI) into duplicate wells of the 96-well plate. A reaction solution of 4.2 mg of O-dianisidine dihydrochloride reagent, 12.5 mI H₂C>_2, 2.5 ml_ potassium phosphate buffer (pH 6.0), 22.5 ml_ distilled water was added to each well (200 mI) and absorbance was measured at 450 nm at 1 min intervals for 15 min using a spectrophotometer (Victor X4 Multilabel Reader, Perkin Elmer, SGP).

Q-PCR

[0204] Harvested tissue was snap-frozen in liquid nitrogen and total RNA was isolated from 1 cm of distal colon per sample (n=6 / genotype) using the Ultraclean Tissue and Cell RNA Isolation Kit (MoBio Laboratories, CA, USA) according to the manufacture’s protocol. Total cDNA was reverse-transcribed from equal amounts of RNA (200 ng) per sample using the iScript cDNA synthesis kit (Bio-Rad Laboratories, CA, USA) according to the manufacturer’s protocol. The PCR reaction mix consisted of 2 mI RT reaction mix, 5 mI 5xPCR buffer, RNA and water making a total volume of 20 mI. The reaction was initiated by incubation at 25°C for 5 min, followed by annealing at 42°C for 30 min and final incubation at 85°C for 5 min followed by 10 min at 4°C. Quantitative PCR was performed using iQ SYBR Green Supermix (Bio-Rad Laboratories, CA, USA) in triplicate reactions in the CFX connect real time PCR system and analysed by CFX Maestro software (Bio-Rad Laboratories, CA, USA). Each Q-PCR reaction mix consisted of 10 mI supermix, 1 mI of cDNA, primers and water making a total volume of 20 mI. A three-step PCR was carried out with initial denaturation for 30 sec at 95°C, followed by 39 cycles of denaturation for 5 sec at 95°C and annealing for 20 sec at 60°C with a final extension of denaturation for 10 sec at 95°C and annealing for 5 sec at 60°C. CyPA and GAPDH were used as reference genes and the inter-reaction calculator method was applied for all plates. For relative comparison, the cycle threshold value (Ct) was analysed using the AACt method and data reported as Ct normalized to reference genes. Gene expression was expressed as fold change of the WT value. Sequences for PCR primers are listed in Table 2.

TABLE 2

Primer Sequences for Q-PCR

Western Blotting

[0205] Protein was extracted from distal colon tissue sections of colitis-induced Flii^+/, wild- type, and Flii^T9/T9 mice by homogenising tissue in a lysis buffer (50 mM Tris pH 7.5, 1 mM EDTA, 50 mM NaCI, 0.5% Triton X-100) containing protease inhibitor tablet (1 per 10 ml; Complete, Mini (Roche, Australia). Samples were centrifuged, and supernatants collected. The BCA kit was used to quantify protein levels and 50 pg of protein was run on 10% SDS- PAGE gels at 100V for 1 hour and transferred to nitrocellulose membrane using standard Towbins Buffer with 20% Methanol at 100V for 1 hour. Following blocking in 12% milk blocking buffer for 15 minutes. Primary antibodies including anti^-catenin sc-7963 rabbit polyclonal IgG (1 :400), anti-Axin-2 (ab32197) rabbit polyclonal IgG (1 :400) and anti-b- tubulin sc-51670 mouse monoclonal IgG (1 :3000) were diluted in buffer and applied to the membrane at 4°C overnight. Species-specific secondary horseradish peroxidase- conjugated antibodies were diluted in 5% milk-blocking buffer and applied to the membrane at room temperature for 1 hour. Protein bands were detected using Super Signal West Femto (Pierce Biotechnology, Rockford, I L) and visualized with GeneSys analysis software (Syngene, MD).

PCNA Analysis

[0206] Distal colonic tissue was analyzed for the presence of proliferating enterocytes in the colon crypts of DSS induced IBD in Flii^+/ wild-type, and Flii^T9/T9 m ce, by staining the tissue for proliferating cell nuclear antigen (PCNA). Details of the methodology used can be found in Chong HT et ai, 2017, supra.

TER Measurements

[0207] Transepithelial Resistance (TER) measurement in cultured I EC-6 (normal small intestine epithelial rat cell line) enterocytes was conducted over a period of 14 days following treatment with a neutralizing antibody to Flightless I (FnAb) or IgG control. Details of the methodology used can be found in Kopecki Z et ai, 2014, J. Pathol., 232(5): 541-552.

Statistical analysis

[0208] Parametric data were expressed as mean ± standard error of the mean (SEM). Histological crypt depth and MPO activity were analysed using a one-way ANOVA with Tukey’s post hoc tests. Disease activity index were analysed using repeated measures ANOVA with least significance difference to compare the differences both between and within groups. Non-parametric data included histological damage severity scores and were analysed using a Kruskal Wallis test with Mann Whitney U tests, expressed as median range. Data were tested for normality (Kolmogorov-Smirnov and Shapiro-Wilk test) and homogeneity (Levenes test) using IBM SPSS Statistics (Version 24.0, IBM Statistical Package for Social Science Statistics for Windows, SPSS Inc., Chicago, IL, USA). In some experiments, statistical differences were calculated using a paired student t-test on Microsoft Excel software. For all statistical analysis, p<0.05 was considered statistically significant. Results

Flightless I is significantly increased in human UC colonic tissue

[0209] Histological analysis of human samples revealed classic morphological characteristics associated with UC including increased inflammatory infiltrate in lamina propria, crypt distortion and shortening when compared to healthy control (Figure 2A). No Flii staining was observed in colonocytes lining the lumen or colonic crypts. However strong Flii staining was observed in the inflammatory infiltrate present in the lamina propria (Figure 2A). Cell counts of Flii positive cells in the human colonic tissue revealed significantly elevated levels of Flii in UC patients compared to healthy controls (Figure 2B). Isotype control staining revealed negligible fluorescence (data not shown).

Overexpression of Flightless I increases clinical disease severity following DSS consumption

[0210] While only 37.5% of wild-type mice exhibited evidence of rectal bleeding, 100% of Flii overexpressing mice ( Flii^{T9/T ®}) showed signs of rectal bleeding and all had significantly higher average disease activity index on day 7 compared to either Flii^+/ mice or wild-type counterparts (Figure 3A). In contrast, mice with low Flii ( Flii^+/) exhibited no rectal bleeding and significantly decreased average disease activity index from day 3 of the experiment (Figure 3A). Examining the visceral and gastrointestinal organ weights showed no significant differences between the three genotypes (data not shown). However, when comparing the degree of colon shortening in colitis-induced mice by analysing the change in colon lengths in colitis-induced vs water control mice results showed that colitis-induced Flii^+/ mice have reduced percentage of colon shortening compared to wild-type counterparts. Flii^T9/T9 mice had a significant increase in percentage of colon shortening compared to both F//7^+/ and wild-type counterparts suggestive of greater disease severity in response to higher levels of Flii (Figure 3B).

Increased histological disease severity is observed in colitis-induced Flii^T9/T9 mice

[0211] The results of histological analysis of tissue sections of the distal colon obtained from the DSS model experiments are shown in Figure 4. Distal colonic tissue from Flii^+/ , wild-type and Flii^T9/T9 colitis-induced animals was examined and a clear increase in colitis severity was observed in mice with elevated levels of Flii including elevated polymorphonuclear infiltration (Figure 4A). Overall there was a statistically significant increase in histological disease severity between Flii^T9/T9 colitis-induced animals compared to Flii^+/ counterparts (Figure 4A). Additionally, colitis-induced Flii^T9/T9 mice showed significantly delayed healing of damaged mucosal tissue as demonstrated by significantly reduced distal colon crypt depth compared to both F//7^+/ and wild-type mice counterparts (Figure 4B).

[0212] Given that the expression of Flightless I protein was increased in human subjects with UC, and given that mice overexpressing Flightless I protein had increased disease severity using the DSS model of IBD, Flightless I represents a target for disease intervention. This is further evidenced in Figure 5 which shows that decreasing Flightless I protein expression levels significantly decreased mucosal damage in the DSS model of IBD. Specifically, increased disease histological severity in colitis-induced Flii^T9/T9 mice was observed following analysis of crypt area index revealing a significantly decreased crypt area index in Flii^T9/T9 mice compared to F///^+/ and wild-type mice counterparts (Figure 5). Conversely, mice with low levels of Flii showed a significant increase in crypt area index compared to both normal and Flii^T9/Ta mce suggestive of decreased colitis severity.

Decreased Flii levels lead to a reduced inflammation in DSS-induced colitis

[0213] To ascertain the effect of differential Flii on mucosal healing of colitis-induced mice, enterocyte proliferation and total tissue inflammation were assessed. No effect of Flii altering levels were observed on enterocyte proliferation as demonstrated by analysis of the numbers of PCNA positive cells in the crypts of colitis-induced F//7^+/ , wild-type and Flii^T9/T9 mice (Figure 6A). However, assessment of total tissue inflammation by MPO analysis revealed significantly decreased levels of tissue inflammation in colitis-induced Flii^+/ mice compared to both wild-type and Flii^T9/T9 counterparts (Figure 6B). Additionally, distal colons of colitis-induced F//7^+/ mice showed significantly lower levels of TNF-a compared to wild- type and Flii^T9/T9 counterparts with staining observed in only apical enterocytes (Figure 7A- B). Increased levels of Flii also resulted in exacerbation of both Thi and Th₂ immune responses in colitis-induced mice with significantly greater levels of TNF-a, IFN-g, IL-5 and IL-13 (Figure 7C) being observed. Similarly, mice with low Flii exhibited a reduced inflammatory response in their distal colons with significantly decreased levels of TNF-a, IL- 17A and IL-5 (Figure 7C).

Flii over-expression inhibits Wnt^-catenin signalling and impairs regeneration of colonic crypts in DSS-induced colitis

[0214] To determine the effect of differential Flii gene expression on regeneration of distal colonic crypts in colitis-induced mice, Wnt^-catenin signalling was assessed. Flii overexpression was found to inhibit Wnt^-catenin signalling, with significantly decreased levels of Lgr6 receptor and intracellular b-catenin levels while Flii deficiency resulted in a significantly decreased number of Axin-2 positive cells (Figures 8A-E). These findings were further confirmed using PCR and Western Blotting (Figure 8E and Figures 8G-H). Co localisation of Flii with b-catenin was observed in wild-type colitis-induced mice however this was absent in Flii over-expressing counterparts (Figure 8F). These findings suggest that Flii effects on Wnt^-catenin signalling pathway may underpin the impaired regeneration of colonic crypts observed in Flii over-expressing mice (Figure 81).

[0215] Mice treated with daily systemic administration of a neutralizing antibody to Flightless I (FnAb) showed reduced incidence of rectal bleeding and significantly decreased disease activity index score from day 4 of the experiment in the mouse model of IBD (Figure 9A). Furthermore, treatment of mice with systemic FnAb resulted in a trend towards longer colon length in the DSS model of IBD suggestive of decreased mucosal damage (Figure 9B).

[0216] FnAb treatment reduced histological disease severity in mice with DSS-induced mucosal damage. As shown in Figure 10A, FnAb treated animals had significantly decreased inflammatory infiltrate and a reduced histological disease severity score compared to IgG treated control mice. However, no significant difference was observed in the distal colon crypt depth between the two mice groups (Figure 10B).

[0217] As shown in Figure 1 1 , decreasing Flii expression levels using FnAb treatment significantly decreased mucosal damage in the DSS model of IBD. Specifically, FnAb treated mice had a significantly increased crypt area index compared to IgG controls indicative of decreased disease severity.

[0218] Figure 12 shows that IEC-6 enterocytes treated with FnAb had an increased tight junction barrier function. Transepithelial resistance (TER) of cultured IEC-6 enterocytes was determined over a period of 14 days showing significantly improved enterocyte cell tight junction function at day 10 of the experiment compared to IgG control treated cells. TER is the measurement of electrical resistance across a cellular monolayer and is a very sensitive and reliable method to confirm the integrity and permeability of the monolayer providing an indication of cell ability to heal blistered mucosal areas and re-establish the barrier between the lumen and the colonic tissue by forming the tight junctions. As cells become confluent TER is expected to increase, reaching maximum resistance by day 10 of the assay, and then decreasing as cells start to die off due to contact inhibition. In this experiment, addition of FnAbs resulted in significantly higher TER compared to control indicating that IEC-6 enterocytes treated with FnAb have improved tight junction barrier function. This points to FnAb treatment providing better recovery of mucosa following blistering.

Discussion

[0219] This study has shown that human UC lesions have significantly elevated levels of Flightless I (Flii), a cytoskeletal protein previously shown to impair healing responses and to be upregulated in response to tissue inflammation in a number of different inflammatory skin disease conditions including human psoriasis, dermatitis and inflammation mediated epidermolysis bullosa acquisita. In the current study, Flii was prominent in the inflammatory infiltrate of human lamina propria surrounding the distal colon crypts suggesting an involvement in the inflammatory pathway of human colitis.

[0220] This study examined the extent of Flii involvement in UC and mucosal healing using an acute model of DSS-induced colitis which closely resembles clinical and histopathological features of human UC (Nagaoka M and Radi ZA, 2012, Front Biosci (Schol. Ed.), 4: 1295-1314. While the mechanism of DSS-induced damage in the colon remains unclear, damage is most prominent in the distal colon and is believed to be caused by alterations in colonic microflora, direct cytotoxic effects on the epithelium and increased macrophage and neutrophil activity resulting in free radical production (Yazbeck R et ai, 201 1 , supra. Clear differences in disease severity were observed in response to altered Flii levels including higher degree of colon shortening, decreased crypt depth and increased inflammation in animals with high Flii levels. In contrast, reducing Flii expression resulted in significantly reduced levels of colon shortening, no evidence of rectal bleeding, significantly decreased disease severity and significantly higher crypt area index compared to mice with normal levels of Flii. Together, these results indicate that high levels of Flii in the gut of patients with UC, may exert a negative influence on clinical disease progression and recurrence.

[0221] Acute DSS-induced colitis is characterized by an increase in pro-inflammatory cytokines TNF-a and IFN-g which are the major proinflammatory cytokines that synergistically drive epithelial barrier dysfunction and apoptosis, particularly during colitis. Chronic DSS-induced colitis comprises focal Wnt^-catenin mediated epithelial regeneration and both Thi and Th₂ cytokine profiles (Dieleman LA et ai, 1998, Clinical and Experimental Immunology, 114: 385-391 ; Perse M and Cerar A, 2012, J. Biomedicine and Biotechnol. , 718617, doi: 10.1 155/2012/718617 (2012); Egger B et ai., 2000, Digestion, 62: 240-248). In patients, IFN-g has been causatively involved in UC epithelial homeostasis and intestinal inflammation (Ito H et al., 2006, Nihon Naika Gakkai, 95: 2246-2250) while IL-17A is associated with increased UC disease activity and ability to trigger and amplify multiple inflammatory pathways regulating gut inflammation (Iboshi Y et al., 2-17, J. Gastroenterol., 52: 315-326). Flii has been demonstrated to regulate inflammation through its effects on TLR4 signalling pathway both intracellularly and extracellularly (Dai P et a!., 2009, J. Immunol., 182: 3450-3460; Lei N et al., 2012, J. Cell Sci., 125: 4288-4296). Its intracellular effect on TLR4 signalling and subsequent NF-kB secretion is mediated via interactions with Myd88 and has been shown to affect inflammation signalling in inflammatory mediated psoriasiform dermatitis (Chong HT et al., 2017, supra). Flii is secreted through a non- classical late endosome/lysosome mediated pathway by both fibroblasts and macrophages, and is present in both acute and chronic human wound fluids (Cowin AJ et al., 2007, supra ; Cowin AJ et al., 2012, Commun. Integr. Biol., 5(6): 546-549; Lei N et al., 2012, supra). Like its family member gelsolin, plasma Flii functions to scavenge extracellular actin following injury and mediate inflammatory responses (Lei N et al., 2012, supra ; Hu Y et al., 2013, Lupus, 22: 1455-1461). Plasma Flii binding to lipopolysaccharide alters macrophage activation and subsequent macrophage secretion of TNF-a (Lei N et al., 2012, supra). Additionally, a recent study has shown that Flii alters inflammatory responses in inflammation mediated atopic dermatitis, where high Flii correlates with increased inflammatory responses resulting in a skewed Th₂ response (Kopecki Z et al., 2018, Frontiers in Immunol., 9, doi: 10.3389/fimmu.2018.01833).

[0222] In this study, a significantly increased inflammatory cell infiltrate was observed in the distal colon of colitis-induced Flii overexpressing animals compared to controls while colitis- induced mice with low levels of Flii showed significantly decreased MPO activity in the distal colon suggesting a role for Flii in augmentation of UC mediated inflammation and mucosal healing. Furthermore, examining the effect of Flii on cytokines known to drive UC mediated tissue inflammation revealed that reducing Flii expression results in a decrease in tissue inflammation and significantly lower levels of pro-inflammatory cytokines including TNF-a, IL-17A and IL-5; all of which favour decreased UC disease severity. In contrast, but in agreement with increased UC disease severity observed in Flii^T9/T9 mice, distal colons of these colitis-induced mice showed an exacerbated immune response with significantly increased expression of Thi and Th₂ cytokines including TNF-a, IFN-g, IL-5 and IL-13. Indeed, this atypical Th₂ response with increased IL-5 and IL-13 levels has been observed in chronic UC patients (Rosen MJ et al., 2017, Gastroenterology, 152: 1345-1357). The observed effects of Flii on Thi/Th₂ immune responses are also in agreement with previous reports showing high levels of Flii alter immune responses in inflammation mediated conditions including psoriasiform dermatitis and atopic dermatitis (Chong HT et ai, 2017, supra ; Kopecki Z et ai, 2018, supra). Together, these findings indicate that Flii plays an important role in UC, and that its effect on inflammation promotes a Th₂ mediated response in UC which would favour a more chronic disease state.

[0223] Studies investigating skin homeostasis and wound healing indicate that Flii negatively regulates epidermal stem cell activation via its effects on Wnt signaling. How the Wnt^-catenin signalling pathway contributes to mucosal healing during colitis has yet to be formally established. Here we demonstrated that Flii overexpression led to inhibition of Wnt signalling with decreased expression of b-catenin and leucine-rich repeat-containing G protein-coupled receptor 6 (Lgr6) receptor required for R-spondin amplification of canonical Wnt signalling. This agrees with previous studies showing that Flii can inhibit Wnt signalling by binding to negative regulators of the Wnt signalling pathway through Dishevelled (Dvl) protein interactions (Liu J et ai, 2005, Proc. Natl. Acad. Sci. USA, 102: 1927-1932). We further showed that Flii colocalization with cytoplasmic b-catenin may lead to decreased b- catenin stabilisation and increased ubiquitin mediated and proteasomal b-catenin degradation. Regulation of Wnt^-catenin signalling by Flii was also evident in Flii deficient mice which showed decreased Axin-2 expression, supporting studies which suggested that Flii may impact b-catenin via Axin-2 regulation at a transcriptional level. Further studies are required to identify the specific molecular mechanisms governing Flii involvement in Wnt signalling pathway and its subsequent effects on activation and proliferation of intestinal stem cells.

[0224] In conclusion, we have demonstrated that Flii is upregulated in the distal colon of human UC patients. High levels of Flii correlate with greater inflammation and exacerbated Thi/ Th₂ immune responses resulting in increased disease severity in mouse models of DSS-induced colitis, while reducing Flii levels promotes decreased gut inflammation and improved mucosal healing. Although the exact mechanism of Flii function in UC is yet to be elucidated, our results indicate that Flii negatively regulates Wnt^-catenin signalling required for regeneration of colonic crypts. Together these results show that manipulation of Flii expression and/or activity represents a novel therapeutic intervention by which UC disease severity, tissue inflammation and mucosal healing can be improved.

[0225] Further experiments can be conducted using additional models of IBD including but not limited to assessing the effect of reducing Flightless I in a model of chronic colitis using a T cell-mediated murine model (CD4⁺CD45RB^hi9h transfer to RAG2-/-) (Ostanin et ai, 2009, Am. J. Physiol. Gastrointest. Liver Physiol. 296(2):G135-146). A review of additional models of IBD can be found in Kiesler P et ai, 2015, Cell. Mol. Gastroenterol. Hepatol., 1 : 154-170; Jiminez JA et ai, 2015, Gut Pathogens, 7: 29 DOI 10.1186/s 13099-015-0076-y; and Rezende KS et ai, March 21 , 2018,“Use of Animal Models in the Study of Colitis, Experimental Animal Models of Human Diseases - An Effective Therapeutic Strategy”, Ibeh Bartholomew, IntechOpen, DOI: 10.5772/intechopen.75608. Available from: https://www.intechopen.com/books/experimental-animal-models-of-human-diseases- an-effective-therapeutic-strategy/use-of-animal-models-in-the-study-of-colitis.

[0226] Experimental models to confirm efficacy of Flightless I modulation for the treatment of coeliac disease can be found in Stoven S et ai, 2013, Expert Opin. Drug Discov., 8(4): 445-457. Experimental models to confirm efficacy of Flightless I modulation for the treatment of peptic ulcer disease can be found in Weiner H, 1996, Psychosom. Med., 58(6): 524-545; and Adinortey MB et ai, 2013, Ulcers, Article ID 796405, http://dx.doi.org/10.1155/2013/796405. Experimental models to confirm efficacy of Flightless I modulation for the treatment of Crohn’s disease can be found in Pizarro T et ai, 2003, Trends Mol. Med., 9(5): 218-222. Experimental models to confirm efficacy of Flightless I modulation for the treatment of conditions such as ileitis can be found in Cominelli F et ai, 2017, Cell. Mol. Gastroenterol. Hepatol., 4(1): 19-32.

Claims

1. A method of treating a mucosal lesion in the gastrointestinal tract of a subject, the method comprising administering to the subject an effective amount of an agent which decreases expression and/or activity of Flightless I in the subject.

2. The method of claim 1 , wherein the expression and/or activity of Flightless I is decreased in the mucosa of the gastrointestinal tract of the subject.

3. The method of claim 1 or claim 2, wherein the mucosal lesion is an ulcer or blister.

4. The method of any one of claims 1 to 3, wherein the mucosal lesion is associated with a disorder of the gastrointestinal tract.

5. The method of claim 4, wherein the disorder is selected from the group consisting of inflammatory bowel disease, coeliac disease, peptic ulcer disease, and regional ileitis.

6. The method of claim 5, wherein the inflammatory bowel disease includes ulcerative colitis and Crohn’s disease.

7. The method of claim 5, wherein the peptic ulcer is a gastric ulcer or a duodenal ulcer.

8. The method of any one of claims 1 to 7, wherein the expression and/or activity of Flightless I protein is decreased by the agent.

9. The method of any one of claims 1 to 7, wherein the expression of Flightless I mRNA is decreased by the agent.

10. The method of any one of claims 1 to 9, wherein the agent is selected from one or more of the group consisting of an antibody to Flightless I or an antigen binding part thereof, an antisense nucleic acid that binds to Flightless I mRNA and which interferes with translation, a small interfering RNA specific for Flightless I mRNA, a microRNA specific for Flightless I mRNA, a short hairpin RNA specific for Flightless I mRNA, a ribozyme that can cleave Flightless I mRNA, an aptamer specific for Flightless I mRNA, or a drug, small molecule, protein, polypeptide or oligopeptide, that interacts with or binds to the Flightless I protein (or a regulator of Flightless I) and inhibits its expression and/or activity.

11. The method of any one of claims 1 to 10, wherein the agent is a neutralising antibody to Flightless I, or an antigen binding part of a neutralising antibody to Flightless I.

12. A method of decreasing mucosal damage in the gastrointestinal tract of a subject, the method comprising administering to the subject an effective amount of an agent which decreases the expression or activity of Flightless I in the subject.

13. The method of claim 12, wherein the expression and/or activity of Flightless I is decreased in the mucosa of the gastrointestinal tract of the subject.

14. The method of claim 12 or claim 13, wherein the agent decreases mucosal ulceration or blistering.

15. The method of any one of claims 12 to 14, wherein the subject is suffering from a disorder of the gastrointestinal tract.

16. The method of claim 15, wherein the disorder is selected from the group consisting of inflammatory bowel disease, coeliac disease, peptic ulcer disease, and regional ileitis.

17. The method of claim 16, wherein the inflammatory bowel disease includes ulcerative colitis and Crohn’s disease.

18. The method of claim 16, wherein the peptic ulcer is a gastric ulcer or a duodenal ulcer.

19. The method of any one of claims 12 to 18, wherein the expression and/or activity of Flightless I protein is decreased by the agent.

20. The method of any one of claims 12 to 18, wherein the expression of Flightless I mRNA is decreased by the agent.

21. The method of any one of claims 12 to 20, wherein the agent is selected from one or more of the group consisting of an antibody to Flightless I or an antigen binding part thereof, an antisense nucleic acid that binds to Flightless I mRNA and which interferes with translation, a small interfering RNA specific for Flightless I mRNA, a microRNA specific for Flightless I mRNA, a short hairpin RNA specific for Flightless I mRNA, a ribozyme that can cleave Flightless I mRNA, an aptamer specific for Flightless I mRNA, or a drug, small molecule, protein, polypeptide or oligopeptide, that interacts with or binds to the Flightless I protein (or a regulator of Flightless I) and inhibits its expression and/or activity.

22. The method of any one of claims 12 to 21 , wherein the agent is a neutralising antibody to Flightless I, or an antigen binding part of a neutralising antibody to Flightless I.

23. A method of treating an ulcer or blister in the mucosa of the gastrointestinal tract of a subject, the method comprising administering to the subject an effective amount of an agent which decreases expression and/or activity of Flightless I in the mucosa of the subject.

24. The method of claim 23, wherein the ulcer or blister is associated with a disorder of the gastrointestinal tract.

25. The method of claim 24, wherein the disorder is selected from the group consisting of inflammatory bowel disease, coeliac disease, peptic ulcer disease, and regional ileitis.

26. The method of claim 25, wherein the inflammatory bowel disease includes ulcerative colitis and Crohn’s disease.

27. The method of claim 25, wherein the peptic ulcer is a gastric ulcer or a duodenal ulcer.

28. The method of any one of claims 23 to 27, wherein the expression and/or activity of Flightless I protein is decreased by the agent.

29. The method of any one of claims 23 to 27, wherein the expression of Flightless I mRNA is decreased by the agent.

30. The method of any one of claims 23 to 29, wherein the agent is selected from one or more of the group consisting of an antibody to Flightless I or an antigen binding part thereof, an antisense nucleic acid that binds to Flightless I mRNA and which interferes with translation, a small interfering RNA specific for Flightless I mRNA, a microRNA specific for Flightless I mRNA, a short hairpin RNA specific for Flightless I mRNA, a ribozyme that can cleave Flightless I mRNA, an aptamer specific for Flightless I mRNA, or a drug, small molecule, protein, polypeptide or oligopeptide, that interacts with or binds to the Flightless I protein (or a regulator of Flightless I) and inhibits its expression and/or activity.

31. The method of any one of claims 23 to 30, wherein the agent is a neutralising antibody to Flightless I, or an antigen binding part of a neutralising antibody to Flightless I.

32. A method of treating inflammatory bowel disease in a subject, the method comprising administering to the subject an effective amount of an agent which decreases expression and/or activity of Flightless I in the subject.

33. The method of claim 32, wherein the expression and/or activity of Flightless I is decreased in the mucosa of the gastrointestinal tract of the subject.

34. The method of claim 32 or claim 33, wherein the agent treats an ulcer or blister of the mucosa associated with the inflammatory bowel disease.

35. The method of any one of claims 32 to 34, wherein the inflammatory bowel disease includes ulcerative colitis and Crohn’s disease.

36. The method of any one of claims 32 to 35, wherein the expression and/or activity of Flightless I protein is decreased by the agent.

37. The method of any one of claims 32 to 35, wherein the expression of Flightless I mRNA is decreased by the agent.

38. The method of any one of claims 32 to 37, wherein the agent is selected from one or more of the group consisting of an antibody to Flightless I or an antigen binding part thereof, an antisense nucleic acid that binds to Flightless I mRNA and which interferes with translation, a small interfering RNA specific for Flightless I mRNA, a microRNA specific for Flightless I mRNA, a short hairpin RNA specific for Flightless I mRNA, a ribozyme that can cleave Flightless I mRNA, an aptamer specific for Flightless I mRNA, or a drug, small molecule, protein, polypeptide or oligopeptide, that interacts with or binds to the Flightless I protein (or a regulator of Flightless I) and inhibits its expression and/or activity.

39. The method of any one of claims 32 to 38, wherein the agent is a neutralising antibody to Flightless I, or an antigen binding part of a neutralising antibody to Flightless I.

40. A method of diagnosing a disorder of the gastrointestinal tract in a subject, the method including the steps of:

41. The method of claim 40, wherein a level of expression and/or activity of Flightless I in the mucosa of the subject that is higher than the reference level of expression and/or activity for Flightless I is indicative of a disorder of the gastrointestinal tract in the subject.

42. The method of claim 40 or claim 41 , wherein the level of expression and/or activity of Flightless I is measured in a mucosal sample obtained from the subject.

43. The method of any one of claims 40 to 42, wherein measuring the level of expression of Flightless I includes measuring the level of Flightless I protein or Flightless I RNA.

44. The method of claim 43, wherein the level of Flightless I protein is measured using an antibody to Flightless I or an antigen binding part thereof.

45. The method of claim 43, wherein the Flightless I RNA is Flightless I mRNA.

46. The method of any one of claims 40 to 45, wherein the disorder is selected from the group consisting of inflammatory bowel disease, coeliac disease, peptic ulcer disease, and regional ileitis.

47. The method of claim 46, wherein the inflammatory bowel disease includes ulcerative colitis and Crohn’s disease.

48. The method of claim 46, wherein the peptic ulcer is a gastric ulcer or a duodenal ulcer.

49. A method of assessing the progression of a disorder of the gastrointestinal tract in a subject, the method including the steps of:

50. The method of claim 49, wherein the subject is undergoing treatment for the disorder.

51. The method of claim 49 or claim 50, wherein a level of expression and/or activity of Flightless I in the mucosa of the subject that is higher than the reference level of expression and/or activity for Flightless I is indicative of progression of a disorder of the gastrointestinal tract in the subject.

52. The method of any one of claims 49 to 51 , wherein the level of expression and/or activity of Flightless I is measured in a mucosal sample obtained from the subject.

53. The method of any one of claims 49 to 52, wherein measuring the level of expression of Flightless I includes measuring the level of Flightless I protein or Flightless I RNA.

54. The method of claim 53, wherein the level of Flightless I protein is measured using an antibody to Flightless I or an antigen binding part thereof.

55. The method of claim 53, wherein the Flightless I RNA is Flightless I mRNA.

56. The method of any one of claims 49 to 55, wherein the disorder is selected from the group consisting of inflammatory bowel disease, coeliac disease, peptic ulcer disease, and regional ileitis.

57. The method of claim 56, wherein the inflammatory bowel disease includes ulcerative colitis and Crohn’s disease.

58. The method of claim 56, wherein the peptic ulcer is a gastric ulcer or a duodenal ulcer.

59. A pharmaceutical composition when used for, or for use in, treating a mucosal lesion in the gastrointestinal tract of a subject, the composition including an effective amount of an agent that decreases expression and/or activity of Flightless I.

60. The pharmaceutical composition of claim 59, wherein the agent decreases the expression and/or activity of Flightless I in the mucosa of the gastrointestinal tract of the subject.

61. The pharmaceutical composition of claim 59 or claim 60, wherein the mucosal lesion is an ulcer or blister.

62. The pharmaceutical composition of any one of claims 59 to 61 , wherein the mucosal lesion is associated with a disorder of the gastrointestinal tract.

63. The pharmaceutical composition of claim 62, wherein the disorder is selected from the group consisting of inflammatory bowel disease, coeliac disease, peptic ulcer disease, and regional ileitis.

64. The pharmaceutical composition of claim 63, wherein the inflammatory bowel disease includes ulcerative colitis and Crohn’s disease.

65. The pharmaceutical composition of claim 63, wherein the peptic ulcer is a gastric ulcer or a duodenal ulcer.

66. The pharmaceutical composition of any one of claims 59 to 65, wherein the agent decreases the expression and/or activity of Flightless I protein.

67. The pharmaceutical composition of any one of claims 59 to 65, wherein the agent decreases the expression of Flightless I mRNA.

68. The pharmaceutical composition of any one of claims 59 to 67, wherein the agent is selected from one or more of the group consisting of an antibody to Flightless I or an antigen binding part thereof, an antisense nucleic acid that binds to Flightless I mRNA and which interferes with translation, a small interfering RNA specific for Flightless I mRNA, a microRNA specific for Flightless I mRNA, a short hairpin RNA specific for Flightless I mRNA, a ribozyme that can cleave Flightless I mRNA, an aptamer specific for Flightless I mRNA, or a drug, small molecule, protein, polypeptide or oligopeptide, that interacts with or binds to the Flightless I protein (or a regulator of Flightless I) and inhibits its expression and/or activity.

69. The pharmaceutical composition of any one of claims 59 to 68, wherein the agent is a neutralising antibody to Flightless I, or an antigen binding part of a neutralising antibody to Flightless I.

70. A pharmaceutical composition when used for, or for use in, decreasing mucosal damage in the gastrointestinal tract of a subject, the composition including an effective amount of an agent that decreases expression and/or activity of Flightless I.

71. The pharmaceutical composition of claim 70, wherein the expression and/or activity of Flightless I is decreased in the mucosa of the gastrointestinal tract of the subject.

72. The pharmaceutical composition of claim 70 or claim 71 , wherein the agent decreases mucosal ulceration or blistering.

73. The pharmaceutical composition of any one of claims 70 to 72, wherein the subject is suffering from a disorder of the gastrointestinal tract.

74. The pharmaceutical composition of claim 73, wherein the disorder is selected from the group consisting of inflammatory bowel disease, coeliac disease, peptic ulcer disease, and regional ileitis.

75. The pharmaceutical composition of claim 74, wherein the inflammatory bowel disease includes ulcerative colitis and Crohn’s disease.

76. The pharmaceutical composition of claim 74, wherein the peptic ulcer is a gastric ulcer or a duodenal ulcer.

77. The pharmaceutical composition of any one of claims 70 to 76, wherein the agent decreases the expression and/or activity of Flightless I protein.

78. The pharmaceutical composition of any one of claims 70 to 77, wherein the agent decreases expression of Flightless I mRNA.

79. The pharmaceutical composition of any one of claims 70 to 78, wherein the agent is selected from one or more of the group consisting of an antibody to Flightless I or an antigen binding part thereof, an antisense nucleic acid that binds to Flightless I mRNA and which interferes with translation, a small interfering RNA specific for Flightless I mRNA, a microRNA specific for Flightless I mRNA, a short hairpin RNA specific for Flightless I mRNA, a ribozyme that can cleave Flightless I mRNA, an aptamer specific for Flightless I mRNA, or a drug, small molecule, protein, polypeptide or oligopeptide, that interacts with or binds to the Flightless I protein (or a regulator of Flightless I) and inhibits its expression and/or activity.

80. The pharmaceutical composition of any one of claims 70 to 79, wherein the agent is a neutralising antibody to Flightless I, or an antigen binding part of a neutralising antibody to Flightless I.

81. A pharmaceutical composition when used for, or for use in, treating an ulcer or blister in the mucosa of the gastrointestinal tract of a subject, the composition including an effective amount of an agent which decreases expression and/or activity of Flightless I in the mucosa of the subject.

82. The pharmaceutical composition of claim 81 , wherein the ulcer or blister is associated with a disorder of the gastrointestinal tract.

83. The pharmaceutical composition of claim 82, wherein the disorder is selected from the group consisting of inflammatory bowel disease, coeliac disease, peptic ulcer disease, and regional ileitis.

84. The pharmaceutical composition of claim 83, wherein the inflammatory bowel disease includes ulcerative colitis and Crohn’s disease.

85. The pharmaceutical composition of claim 83, wherein the peptic ulcer is a gastric ulcer or a duodenal ulcer.

86. The pharmaceutical composition of any one of claims 81 to 85, wherein the agent decreases to expression and/or activity of Flightless I protein.

87. The pharmaceutical composition of any one of claims 81 to 85, wherein the agent decreases expression of Flightless I mRNA.

88. The pharmaceutical composition of any one of claims 81 to 87, wherein the agent is selected from one or more of the group consisting of an antibody to Flightless I or an antigen binding part thereof, an antisense nucleic acid that binds to Flightless I mRNA and which interferes with translation, a small interfering RNA specific for Flightless I mRNA, a microRNA specific for Flightless I mRNA, a short hairpin RNA specific for Flightless I mRNA, a ribozyme that can cleave Flightless I mRNA, an aptamer specific for Flightless I mRNA, or a drug, small molecule, protein, polypeptide or oligopeptide, that interacts with or binds to the Flightless I protein (or a regulator of Flightless I) and inhibits its expression and/or activity.

89. The pharmaceutical composition of any one of claims 81 to 88, wherein the agent is a neutralising antibody to Flightless I, or an antigen binding part of a neutralising antibody to Flightless I.

90. A pharmaceutical composition when used for, or for use in, treating inflammatory bowel disease in a subject, the method comprising administering to the subject an effective amount of an agent which decreases expression and/or activity of Flightless I in the subject.

91. The pharmaceutical composition of claim 90, wherein the agent decreases the expression and/or activity of Flightless I in the mucosa of the gastrointestinal tract of the subject.

92. The pharmaceutical composition of claim 90 or claim 91 , wherein the agent treats an ulcer or blister of the mucosa associated with the inflammatory bowel disease.

93. The pharmaceutical composition of any one of claims 90 to 92, wherein the inflammatory bowel disease includes ulcerative colitis and Crohn’s disease.

94. The pharmaceutical composition of any one of claims 90 to 93, wherein the agent decreases the expression and/or activity of Flightless I protein.

95. The pharmaceutical composition of any one of claims 90 to 93, wherein the agent decreases the expression of Flightless I mRNA.

96. The pharmaceutical composition of any one of claims 90 to 95, wherein the agent is selected from one or more of the group consisting of an antibody to Flightless I or an antigen binding part thereof, an antisense nucleic acid that binds to Flightless I mRNA and which interferes with translation, a small interfering RNA specific for Flightless I mRNA, a microRNA specific for Flightless I mRNA, a short hairpin RNA specific for Flightless I mRNA, a ribozyme that can cleave Flightless I mRNA, an aptamer specific for Flightless I mRNA, or a drug, small molecule, protein, polypeptide or oligopeptide, that interacts with or binds to the Flightless I protein (or a regulator of Flightless I) and inhibits its expression and/or activity.

97. The pharmaceutical composition of any one of claims 90 to 96, wherein the agent is a neutralising antibody to Flightless I, or an antigen binding part of a neutralising antibody to Flightless I.

98. A kit when used for, or for use in, diagnosing a disorder of the gastrointestinal tract in a subject, or for assessing the progression of a disorder of the gastrointestinal tract in a subject, the kit including means for measuring the level of expression and/or activity of Flightless I in the subject.

99. The kit of claim 98, wherein the level of expression and/or activity of Flightless I is measured in the mucosa of the gastrointestinal tract of the subject.

100. The kit of claim 99, wherein a level of expression and/or activity of Flightless I in the mucosa of the subject that is higher than the reference level of expression and/or activity for Flightless I is indicative of a disorder of the gastrointestinal tract in the subject.

101. The kit of any one of claims 98 to 100, wherein the level of expression and/or activity of Flightless I is measured in a mucosal sample obtained from the subject.

102. The kit of any one of claims 98 to 101 , wherein measuring the level of expression of Flightless I includes measuring the level of Flightless I protein or Flightless I RNA.

103. The kit of claim 102, wherein the level of Flightless I protein is measured using an antibody to Flightless I or an antigen binding part thereof.

104. The kit of claim 102, wherein the Flightless I RNA is Flightless I mRNA.

105. The kit of any one of claims 98 to 104, wherein the disorder is selected from the group consisting of inflammatory bowel disease, coeliac disease, peptic ulcer disease, and regional ileitis.

106. The kit of claim 105, wherein the inflammatory bowel disease includes ulcerative colitis and Crohn’s disease.

107. The kit of claim 105, wherein the peptic ulcer is a gastric ulcer or a duodenal ulcer.

108. Use of an agent which decreases the expression or activity of Flightless I in a subject in the manufacture of a medicament for treating a mucosal lesion in the gastrointestinal tract of the subject.

109. Use of an agent which decreases the expression or activity of Flightless I in a subject in the manufacture of a medicament for decreasing mucosal damage in the gastrointestinal tract of a subject.

1 10. Use of an agent which decreases the expression or activity of Flightless I in a subject in the manufacture of a medicament for treating an ulcer or blister in the mucosa of the gastrointestinal tract of a subject.

1 11. Use of an agent which decreases the expression or activity of Flightless I in a subject in the manufacture of a medicament for treating inflammatory bowel disease in a subject.