WO2016040987A1

WO2016040987A1 - Treating and/or preventing psoriasis

Info

Publication number: WO2016040987A1
Application number: PCT/AU2015/000569
Authority: WO
Inventors: Allison June Cowin; Zlatko KOPECKI; Heng Teck CHONG
Original assignee: Abregen Pty Ltd
Priority date: 2014-09-15
Filing date: 2015-09-15
Publication date: 2016-03-24

Abstract

The present invention relates to psoriasis and to the treatment and prevention thereof. Specifically, a biomarker of psoriasis has been identified, namely Flightless I. Accordingly, the present invention provides methods for the treatment and/or prevention of psoriasis by decreasing the expression and/or activity of Flightless I. Methods of diagnosing psoriasis are also provided together with methods of determining susceptibility to psoriasis. Pharmaceutical compositions including agents that decrease the expression and/or activity of Flightless I are also provided for the treatment and/or prevention of psoriasis. Finally, kits for diagnosing psoriasis or for determining susceptibility to psoriasis are encompassed by the present invention.

Description

TREATING AND/OR PREVENTING PSORIASIS

PRIORITY CLAIM

[0001] This application claims priority from Australian provisional patent application number 2014903677 filed on 15 September 2014, the contents of which are to be taken as incorporated herein by this reference.

FIELD OF THE INVENTION

[0002] The present invention relates generally to methods and compositions for treating and/or preventing psoriasis. More particularly, the present invention relates the methods and compositions for treating and/or preventing psoriasis by modulating the activity and/or expression of Flightless I.

BACKGROUND OF THE INVENTION

[0003] Psoriasis is a chronic cutaneous skin condition affecting around 0.5-1 % of children and 2-4% of adults. When psoriasis develops, patches of skin thicken, redden, and become covered with silvery scales. These patches are generally referred to as plaques which are usually itchy and can burn. Psoriasis most often occurs on the elbows, knees, scalp, lower back, face, palms and soles of the feet. The scaling occurs when the cells in the outer layer of the skin reproduce faster than normal and accumulate on the surface of the skin.

[0004] Histopathological features of psoriatic plaques include growth and dilation of the superficial blood vessels (which causes redness) and hyperproliferation (hyperplasia) of the epithelium. The psoriatic epidermis is characterised by elongated rete pegs, marked thickening (acanthosis), and abnormal differentiation of the keratinocytes. Terminal differentiation of the keratinocytes is incomplete, such that squamous keratinocytes retain nuclei (parakeratosis), and the cells of the stratum corneum are poorly adherent resulting in the characteristic scale and flake of psoriatic plaques. These histological changes are accompanied by disruption of the epidermal permeability barrier.

[0005] Psoriasis occurs in all age groups and affects men and women equally. People affected by psoriasis suffer from discomfort, restricted joint motion and emotional distress. About 10% of people suffering from psoriasis have joint inflammation that produces symptoms similar to arthritis. Both psoriasis of the skin and psoriatic arthritis are frequently accompanied by impairment of the quality of life. The clinical severity of psoriasis is measured using standard methods. These methods include the Psoriasis Area and Severity Index (PASI), which evaluates skin lesions of affected subjects by their characteristics of erythema, induration and scaling as well as by the surface area involved. Body surface area (BSA) which determines the area involved in relation to the whole body surface and physician's global assessment (PGA) which is an overall evaluation of lesion severity are also clinical parameters of psoriasis severity.

[0006] The pathogenesis of psoriasis has yet to be characterised; however, it is known that it is based on a complicated interplay between genetic and environmental factors. For example, in one study it was shown that 4.5-91 % of pediatric cases had a positive family history of the condition, and in other studies 71 % of children suffering from psoriasis had a first-degree relative with the condition. Intrinsic and extrinsic environmental influences appear to play a pivotal role in the initiation and exacerbation of psoriasis. For example, upper respiratory infections, emotional stress, and drugs are factors that have been implicated with the manifestation or aggravation of the condition.

[0007] A variety of treatments and methods have been used over the years to combat psoriasis. However, it is recognised that psoriasis is a life-long condition and so the purpose of treatments are primarily to manage psoriatic episodes and to alleviate the symptoms associated with those episodes.

[0008] Traditional oral systemic therapies have included methotrexate and cyclosporine. Methotrexate is a folic acid analog that reversibly inhibits dihydrofolate reductase, therefore interfering with DNA synthesis and repair and repair. Cyclosporine is an immunosuppressant agent which induces general immunosuppression by preventing T cell activation and cytokine expression. Although both compunds have been effective in some patients, their long-term use is complicated by several toxicities such as nausea, lost appetite, diarrhea, bone marrow toxicity, pulmonary toxicity, hepatotoxicity, nephrotoxicity and arterial hypertension.

[0009] Topical corticosteroids are considered the first-line treatment agents for psoriasis, and their role in the management of the condition is based on their anti-inflammatory and antiproliferative properties. However, corticosteroid use is not without its complications. For example, potent corticosteroids must be avoided in such anataomical sites as the face, genitals, and flexural areas where the skin is thinner. Prolonged use of topical corticosteroids also leads to adverse events such as thinning of the skin, bruising, striae, telangiectasias, acneiform eruption, tachyphylaxis and risks associted with systemic absorption. [0010] Analogues of vitamin D3 metabolites have also been used to treat psoriasis. The most well-known analogues are calcipotriol, which is sold under the brand name Dovonex®, and tacalcitol, which is sold under the brand name Curatoderm®. Vitamin D3 analogs contribute to the treatment of psoriatic episodes by their capacity to stimulate keratinocyte differentiation and to inhibit their proliferation. Although these agents are generally well tolerated, there are a number of side effects observed, with the most common being irritation of the skin at the site of application. Studies have indicated that up to 20% of patients experience this side effect. The risk and severity of the side effects is increased dramatically when these agents are occluded, or covered, and therefore agents such as Dovonex® and Curatoderm® are not recommended for "skin fold" areas, where skin can occlude other skin. These drugs are also not recommended for the face, where the skin is particularly sensitive.

[0011] Other traditional treatments have included phototherapy (such as UV irradiation) which come with their own negative side-effects. Retinoids such as acitretin are also in use; however, as with all retinoids, use of these agents bears a high teratogenic risk. Coal-tar compounds which have antiproliferative and antipruritic properties have also been used; however, due to their strong odour and staining capacity their use is limited.

[0012] Biologic agents are increasingly being used for psoriasis which are agents that target specific portions of the immune system. The first biologies approved for psoriasis (such as efalizumab and alefacept) were primarily directed against receptors involved in T cell transmigration or activation, but their use has been discontinued. More recently, biologies have been generated to neutralise cytokines such as TNF. A number of TNF antagonists are available for psoriasis treatment including anti-TNF antibodies such as adalimumab, infliximab, golimumab and certolizumab, and the TNF receptor fusion protyein etanercept. However, TNF antagonists are linked to certain safety concerns such as serious infection, increased risk of non-melanoma skin cancers, congestive heart failure, and infusion reactions. Other monoclonal antibodies are also available such as ustekinumab which targets the p40 subunit shared by IL-12 and IL-23; however, serious infections, malignancies and adverse cardiovascular events have been observed under therapy with neutralising p40 antibodies.

[0013] In light of the complex etiology of psoriasis, there is a significant need for the identification of new molecular targets responsible for the development and progression of psoriasis. Such targets will serve as a basis for the therapeutic intervention and diagnosis of psoriasis. [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 its encoded protein in psoriasis. These studies have shown that an increased level of Flightless I is associated with the development and progression of psoriasis in vivo.

[0016] Accordingly, in a first aspect, the present invention provides a method of treating and/or preventing psoriasis in a subject, the method including the step of decreasing the expression and/or activity of Flightless I in the subject.

[0017] In some embodiments, 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. In one embodiment, the agent is selected from one or more of the group consisting of a drug, a small molecule, a nucleic acid, an oligonucleotide, an oligopeptide, a polypeptide, a protein, an enzyme, a polysaccharide, a glycoprotein, a hormone, a receptor, a ligand for a receptor, a co-factor, an antisense oligonucleotide, a ribozyme, a small interfering RNA, a microRNA, short hairpin RNA, a lipid, an aptamer, a virus, and an antibody or an antigen binding part thereof.

[0018] In some embodiments, the agent is an antibody to Flightless I, or an antigen binding part thereof.

[0019] In some embodiments, epidermal thickening of an affected area of skin of the subject is reduced. In some embodiments, cellular proliferation and differentiation of an affected area of skin of the subject is decreased.

[0020] In some embodiments, the agent is administered topically to the skin of the subject.

[0021] In a second aspect, the present invention provides a method of diagnosing psoriasis in a subject, the method including the steps of:

measuring the level of expression and/or activity of Flightless I in 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 psoriasis in the subject on the basis of the comparison.

[0022] In some embodiments of the second aspect of the invention, 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 psoriasis in the subject.

[0023] In some embodiments of the second aspect of the invention, the level of expression and/or activity of Flightless I is measured in a sample obtained from the subject. In some embodiments, measuring the level of expression of Flightless I includes measuring the level of Flightless I RNA or protein. For example, the level of Flightless I mRNA may be measured.

[0024] In a third aspect, the present invention provides a method of determining if a subject is susceptible to developing psoriasis, the method including the steps of:

measuring the level of expression and/or activity of Flightless I in 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

determining if the subject is susceptible to developing psoriasis on the basis of the comparison.

[0025] In some embodiments of the third aspect of the invention, 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 indicates that the subject is susceptible to developing psoriasis.

[0026] In some embodiments of the third aspect of the invention, the level of expression and/or activity of Flightless I is measured in a sample obtained from the subject. In some embodiments, measuring the level of expression of Flightless I includes measuring the level of Flightless I RNA or protein. For example, the level of Flightless I mRNA may be measured.

[0027] In a fourth aspect, the present invention provides a pharmaceutical composition when used for treating and/or preventing psoriasis in a subject, the composition including an effective amount of an agent that decreases expression and/or activity of Flightless I.

[0028] In some embodiments of the fourth aspect of the invention, the agent is selected from one or more of the group consisting of a drug, a small molecule, a nucleic acid, an oligonucleotide, an oligopeptide, a polypeptide, a protein, an enzyme, a polysaccharide, a glycoprotein, a hormone, a receptor, a ligand for a receptor, a co-factor, an antisense oligonucleotide, a ribozyme, a small interfering RNA, a microRNA, short hairpin RNA, a lipid, an aptamer, a virus, and an antibody or an antigen binding part thereof.

[0029] In some embodiments of the fourth aspect of the invention, the agent is an antibody to Flightless I, or an antigen binding part thereof.

[0030] In some embodiments of the fourth aspect of the invention, the pharmaceutical composition is a topical composition. In some embodiments, the pharmaceutical composition is a systemic composition.

[0031] In a fifth aspect, the present invention provides a kit when used for diagnosing psoriasis in a subject, or determining if a subject is susceptible to developing psoriasis, the kit including means for measuring the level of expression and/or activity of Flightless I in the subject.

[0032] In some embodiments of the fifth aspect of the invention, a level of expression and/or activity of Flightless I in the subject that is higher than a reference level of expression and/or activity for Flightless I diagnoses psoriasis in the subject, or is indicative that the subject is susceptible to developing psoriasis.

[0033] In some embodiments of the fifth aspect of the invention, the level of expression and/or activity of Flightless I is measured in a sample obtained from the subject.

[0034] In some embodiments of the fifth aspect of the invention, measuring the level of expression of Flightless I includes measuring the level of Flightless I RNA or protein. For example, the level of Flightless I mRNA may be measured.

BRIEF DESCRIPTION OF THE FIGURES

[0035] 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 drawings.

[0036] FIGURE 1 - shows that the expression of Flii is increased in human psoriatic skin compared to normal human skin (A). Representative images are shown (B). [0037] FIGURE 2 - shows that skin erythema is reduced in Flii-deficient mice. In a mouse model of psoriasis, daily Imiquimod cream was used to induce psoriasis features on the shaved dorsa. The degree of erythema was measured and the results show that genetically Flii-deficient mice (FIN+/-) (n=6) exhibit significantly less erythema compared to wild-type (WT) and Flii-overexpressing (FliiTg/Tg) mice (n=6 each) (A). Representative images of the dorsa are shown (B).

[0038] FIGURE 3 - shows that epidermal thickness and rete pegs length (epidermal extensions into the dermis) are reduced in Flii-deficient mice with psoriasis. Compared to wild-type (WT) and Flii-overexpressing (FliiTg/Tg) mice, genetically Flii-deficient mice (FIN+/-) with psoriasis exhibit reduced epidermal thickness and rete pegs length. Representative images are shown in (A) and presented graphically in (B). Both features are characteristics of psoriasis.

[0039] FIGURE 4 - shows that markers of inflammation are reduced in Flii-deficient mice. Genetically Flii-deficient mice (FIN+/-) have reduced specific markers of inflammation including neutrophils infiltrate, NF-kB and IL-17 (A) compared to wild-type (WT) and Flii-overexpressing (FliiTg/Tg) mice (n=6 each). Representative images are shown (B).

[0040] FIGURE 5 - shows that cellular proliferation and cytokine markers are reduced in Flii- deficient mice compared to Flii-overexpressing mice. Flii-deficient mice (FIN+/-) have reduced PCNA positive cells in the epidermis (A), reduced IL-6 mRNA (C) and reduced IL-22 mRNA (D). Representative images are shown (B).

[0041] FIGURE 6 - shows that administration of anti-Flii antibodies reduce erythema and epidermal thickness. Treatment of wild-type mice with anti-Flii antibodies in a topical cream leads to reduced erythema (A and C) and epidermal thickness (B and D) in a mouse model of psoriasis compared to IgG control (n=6 each).

DETAILED DESCRIPTION OF THE INVENTION

[0042] 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

[0042] As set out above, the present invention is predicated, in part, on the identification of an association between the expression of Flightless I and psoriasis. For example, the inventors have determined that the level of Flightless I protein is increased in human psoriatic skin cells and that decreasing expression and/or activity of Flightless I leads to a decrease in measures of psoriasis.

[0043] Accordingly, the present invention provides methods, compositions and kits that have one or more combinations of advantages. For example, some of the advantages of the embodiments disclosed herein include one or more of the following: a marker of psoriasis; a target for psoriasis treatment and/or prevention; a method for the treatment and/or prevention of psoriasis; a method of diagnosing proriasis; a method of determining susceptibility to psoriasis; a composition for treating and/or preventing psoriasis; a kit for diagnosing psoriasis; and to address one or more problems and/or to provide one or more advantages, or to provide a commercial alternative. Other advantages of certain embodiments of the present disclosure are also disclosed herein.

[0044] In a first aspect, the present invention provides a method of treating and/or preventing psoriasis in a subject, the method including the step of decreasing the expression and/or activity of Flightless I in the subject.

[0045] The present invention also provides use of an agent that decreases the expression and/or activity of Flightless I in the manufacture of a medicament for treating and/or preventing psoriasis in a subject. Furthermore, the present invention provides use of an agent that decreases the expression and/or activity of Flightless I for treating and/or preventing psoriasis in a subject.

[0046] 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 Flighless I protein is highly conserved suggesting that it carries out important, conserved functions.

[0047] 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.

[0048] 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 "FIN" and "FIN"

[0049] 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.

[0050] 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.

[0051] The method of the first aspect of the invention requires the step of decreasing the 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 Leuicine Rich Repeat domain allowing Flightless I to act as a multifunctional protein with major roles in wound healing. Flightless I negatively regulates wound healing through regulating cellular migration and proliferation, cellular adhesion and spreading. Recent findings have confirmed its role in actin polymerisation and capping of actin monomers. [0052] 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.

[0053] 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.

[0054] 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 al., 2004 {NEJM, 351 (27): 2817-2826), or Anderson et al., 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.

[0055] 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 skin biopsy sample (and corresponding normal or unaffected skin 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.

[0056] 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.

[0057] 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 Flighltess 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.

[0058] 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. [0059] 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.

[0060] Also contemplated are traditional immunoassays including, for example, sandwich immunoassays including ELISA or fluorescence-based immunoassays, as well as other enzyme immunoassays. 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).

[0061] 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 1/1 13085.

[0062] 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. [0063] 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.

[0064] 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 al., 1988, Rapid Commun. Mass Spectrom., 2: 151 -153; and Norris JL and Caprioli RM, 2013, Chem Rev., 1 13: 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 al., 2004, Mass Spec. Reviews, 23: 34-44; and U.S. Patents Nos. 5,719,060 and 6,225,047).

[0065] 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). [0066] Other methods which may be employed to determine if the level of Flighltess 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 Flighltess I protein has a leucine-rich repeat which is known to bind proteins such as FLAP-1 (Wilson SA et al., 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 al., 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.

[0067] 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 Flighltess I may be assayed according to a concomitant change in gene transcription as mediated by the Flightless I protein. Flightless I also has a major role in wound healing. Accordingly, an assay based on an assessment of wound healing may be used to measure changes in Flightless I activity. Given that Flightless I negatively regulates wound healing through regulating 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.

[0068] 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 psoriasis.

[0069] For example, the terms "treat", "treating" or "treatment," as used herein are to be understood to include within their scope one or more of the following outcomes: (i) alleviation of, reduction of, and/or providing relief from, itching, (ii) alleviation of, reduction of, and/or providing relief from, red scaling skin (iii) alleviation of, reduction of, and/or providing relief from, lichenification; (iv) alleviation of, reduction of, and/or providing relief from, papules or small raised bumps, on the skin; (v) alleviation of, reduction of, and/or providing relief from, ichthyosis (dry, rectangular scales on the skin); (vi) alleviation of, reduction of, and/or providing relief from, keratosis pilaris (small, rough bumps, generally on the face, upper arms, and thighs), (vii) alleviation of, reduction of, and/or providing relief from, hyperlinear palms (increased number of skin creases on the palms), (viii) alleviation of, reduction of, and/or providing relief from urticaria (red, raised bumps, often after exposure to an allergen, at the beginning of flares or after exercise or a hot bath); (ix) alleviation of, reduction of, and/or providing relief from, cheilitis (inflammation of the skin on and around the lips); (x) alleviation of, reduction of, and/or providing relief from, atopic pleat (an extra fold of skin that develops under the eye), (xi) alleviation of, reduction of, and/or providing relief from, hyperpigmented eyelids (eyelids that have become darker in colour from inflammation or hay fever); (xii) relieving a condition in a subject that is caused by psoriasis; and (xiii) stopping symptoms in a subject that are associated with psoriasis.

[0070] The terms "treat", "treating" or "treatment," as used herein are to be understood to also include within their scope one or more of the following outcomes: (i) alleviation of, reduction of, and/or providing relief from, plaques; (ii) alleviation of, reduction of, and/or providing relief from, scaling. For example, decreased redness, decreased thickness of the plaques, decreased scaling, decreased area of involvement, clearing of plaques, and/or decrease in PASI score is included.

[0071] The terms "prevent" or "preventing" as used herein are to be understood to include within their scope inhibiting the onset of the symptoms referred to above under definition of the terms "treat", "treating" or "treatment".

[0072] In one embodiment of the first aspect of the invention, 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.

[0073] 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 a neutralizing antibody (or an antigen binding part thereof), an antisense nucleic acid that binds to Flightless I imRNA 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, a microRNA, a short hairpin RNA, a ribozyme that can cleave Flightless I mRNA, an aptamer, and an agent that interacts with or binds to the Flightless I protein (or a regulator of Flightless I) and inhibits its activity, such as a drug, small molecule, protein, polypeptide or oligopeptide.

[0074] 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.

[0075] 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 a/., 1991 (Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, U.S. Government Printing Office). [0076] 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).

[0077] 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 V_H-C_Hi 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 a/., 1990, Nature 348:552-554).

[0078] 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.

[0079] 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 al., 1984, Proc. Natl. Acad. Sci. USA, 81 : 6851 -6855; Morrison and Oi, 1988, Adv. Immunol., 44: 65-92; Verhoeyen et al., 1988, Science, 239: 1534-1536; Padlan, 1991 , Molec. Immun., 28: 489-498; and Padlan, 1994, Molec. Immun., 31 : 169-217.

[0080] 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.

[0081] 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 al., 1995, Nature 374: 168-173; Nuttall et al., 2001 , Mol. Immunol., 38: 313-326; Diaz et al., 2002, Immunogenetics 54: 501-512; and Nuttall et al., 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 ): 1 126-1 136). 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).

[0082] 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. [0083] In some embodiments, the antibody to Flightless I is a neutralising antibody. 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.

[0084] 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.

[0085] 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.

[0086] 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 al., 1975, Nature 256: 495-497; Kozbor et al., 1985, J. Immunol. Methods 81 :31-42; Cote et al., 1983, Proc. Natl. Acad. Sci. USA 80: 2026-2030; and Cole et al., 1984, Mol. Cell Biochem. 62: 109-120).

[0087] 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 al., 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 al., 1995, J. Immunol. Methods 182: 41 -50; Ames et al., 1995, J. Immunol. Methods 184: 177-186; Kettleborough et al., 1994, Eur. J. Immunol. 24: 952-958; Persic et al., 1997, Gene 187: 9-18; Burton et al., 1994, Advances in Immunology 57: 191 -280; PCT application number PCT/GB91/01 134; PCT publications numbers WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/1 1236; 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.

[0088] 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 al., 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.

[0089] 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 ).

[0090] 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., 201 1 , 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.

[0091] 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.

[0092] 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.

[0093] 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.

[0094] 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 al., 1998, Nature 391 : 806-81 1 ). 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, ef a/., 2005, Current Opinion in Structural Biology, 15: 331-3341 .

[0095] 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).

[0096] 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.

[0097] 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 et 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.

[0098] 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.

[0099] 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 imRNAs, 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.

[0100] 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.

[0101] 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'-0-propyl and T- methoxyethoxy ribose modifications stabilize oligonucleotides to nucleases in cell culture and in vivo.

[0102] 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.

[0103] As indicated above, decreasing the expression and/or activity of Flightless I may be achieved by various gene-targeted silencing strategies. Specific examples include mutageneis and gene knock-down using zinc-finger nucleases (see Carroll D, 201 1 , Genetics 188: 773-782; Sander JD et a/., 201 1 , Nat. Methods 8: 67-69; and Miller JC et ai, 2007, Nat. Biotechnol., 25: 778-785), mutageneis and gene knock-down using transcription activator-like effector nuclease (TALEN) systems (see Bogdanove AJ and Voytas DF, 201 1 , Science 333: 1843-1846; Streubel J et a/., 2012, Nat. Biotechnol., 30: 593-595; Cermak T et a/., 201 1 , 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 al., 2014, Nat. Biotechnol., 32: 947-951 ), and mutageneis and gene knock-down using the 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 ). These gene knock-down technologies are merely representative and are not limiting to other mecahisms that may be employed.

[0104] 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.

[0105] 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.

[0106] In psoriasis, skin at the affected area is often cracked and covered by a thick opaque scale. This scale represents a thickened layer of surface keratin (hyperkeratosis), which overlies an epidermis markedly thickened by an increase in the number of cells in the various layers (particularly stratum spinosum and the granular layer). Such thickening, termed acanthosis, is a common feature of chronic psoriasis. Accordingly, in some embodiments, decreasing expression and/or activity of Flightless I reduces epidermal thickening of an affected area of skin of the subject.

[0107] By "reducing" epidermal thickening, is meant that the thickness of the epidermis is reduced to an extent that resembles, or near resembles, the thickness of an unaffected area of the skin of the subject.

[0108] As indicated above, the inventors have determined that the level of Flightless I protein is increased in affected skin cells during psoriasis. The inventors have also established that decreasing expression of Flightless I leads to a decrease in the severity of symptoms of psoriasis. This differential expression of Flightless I indicates that it is a suitable biomarker which can form the basis of diagnostic and prognostic testing for psoriasis.

[0109] 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) as compared with another phenotypic status (e.g. not having the disease). 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.

[0110] Accordingly, in a second aspect, the present invention provides a method of diagnosing psoriasis in a subject, the method including the steps of:

diagnosing psoriasis in the subject on the basis of the comparison.

[0111] Furthermore, in a third aspect, the present invention provides a method of determining if a subject is susceptible to developing psoriasis, the method including the steps of:

[0112] The identification of differential expression of Flightless I in psoriasis also enables methods for assessing the therapeutic efficacy in a subject of a treatment for psoriasis.

[0113] 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.

[0114] 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.

[0115] The sample which is obtained from the subject will typically be a skin sample taken from the affected area, including a corresponding normal skin sample. However, 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.

[0116] 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 psoriasis or is known to be found in non-affected skin 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 psoriasis. 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 psoriasis. 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.

[0117] In an embodiment of the second and third aspects of the invention, 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 psoriasis in the subject or indicates that the subject is susceptible to developing psoriasis. [0118] 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.

[0119] In a fourth aspect, the present invention provides a pharmaceutical composition when used for treating and/or preventing psoriasis in a subject, the composition including 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 with respect to the first aspect of the invention.

[0120] 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.

[0121] 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.

[0122] 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.

[0123] 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., and these preparations can be administered topically, as intramuscular or subcutaneous injections, as an embedded preparation, or as a transmucosal preparation through nasal cavity, rectum, uterus, vagina, lung, etc. The composition may also be administered in the form of oral preparations (for example solid preparations such as tablets, capsules, granules or powders; liquid preparations such as syrup, emulsions or suspensions).

[0124] 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.

[0125] 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.

[0126] For these purposes, the composition may be administered topically, orally, 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, intraperitoneal, intrathecal, intraventricular, intrasternal, and intracranial injection or infusion techniques.

[0127] 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.

[0128] 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.

[0129] 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.

[0130] 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.

[0131] 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).

[0132] 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.

[0133] 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.

[0134] 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-d iff user vehicle. [0135] 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.

[0136] 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.

[0137] 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.

[0138] 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.

[0139] 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.

[0140] 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.

[0141] 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 psoriatic episodes at their site of occurrence in the body.

[0142] 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.

[0143] In a fifth aspect, the present invention provides a kit for diagnosing psoriasis in a subject, or for determining if a subject is susceptible to developing psoriasis, the kit including means for measuring the level of expression and/or activity of Flightless I in the subject. In some embodiments, a level of expression and/or activity of Flightless I in the subject that is higher than a reference level of expression and/or activity for Flightless I diagnoses psoriasis in the subject, or is indicative that the subject is susceptible to developing psoriasis.

[0144] Means and methods for measuring the level of expression and/or activity of Flightless I in the subject according to the fifth aspect of the invention are described in detail above.

[0145] 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 biospecfi^'c capture reagents, the kit can include a solid support with a reactive surface, and a container including the biospecific capture reagent.

[0146] 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.

[0147] 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.

[0148] 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.

[0149] 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 values in between these limits.

[0150] Furthermore, 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.

[0151] 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 and Sambrook, 2012 (supra).

[0152] 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.

[0153] 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 Psoriasis

Methodology

Human Psoriatic Skin and Flightless Expression

[0154] Normal human skin and 7 psoriatic skin samples were obtained from the South Australian Pathology archive after ethics approval was given from the Royal Adelaide Hospital Research Ethics Committee. The diagnosis was confirmed with the pathologist. Sections of these paraffin-embedded tissues were stained for expression of Flii using the immunohistochemistry protocol detailed below.

Murine model of chronic psoriasis

[0155] Mice skin was induced with inflammation using an established model of psoriasis (van der Fits L et ai, 2009, J. Immunol., 182(9): 5836-45) on six-eight week old female wild-type mice (n=6), Flii heterozygous (FIN+/-) mice (n=6) and Flii transgenic (FliiTg/Tg) mice (n=6). Anaesthesia was induced using 5% isoflurane at 2 L oxygen per minute and maintained using 2% isoflurane at 500 ml oxygen per minute throughout the procedure. Each mouse was shaved on the dorsum and hair further removed using Veet hair removal cream prior to treatment application. Each mouse received a daily topical dose of 62.5 mg of commercially available IMQ cream (5%) (Aldara; 3M Pharmaceuticals) for 6 consecutive days, translating in a daily dose of 3.125 mg of the active compound. Mice were euthanized using C0₂ and cervical dislocation at day 7 post induction. The negative control group (n = 3) received sterile saline (100 μΙ) applied using the same procedure. Mice dorsa were photographed. Severity of the skin inflammation was examined by measuring the level of skin erythema using a DermaLab Unit (Cortex Technology).

Histology and Immunohistochemistry

[0156] Skin tissue harvested from mice dorsa were fixed in 10% formalin overnight, followed by processing in a Leica TP1020 tissue processor which dehydrated the tissues in a graded alcohol series (70% for 120 mins, 80% for 60 mins, 90% for 105 mins and 100% for 240 mins). They were then cleared in transitional solvent xylene for 180 mins followed by 240 mins of tissue infiltration with paraffin wax. Tissue sections (4 μιη) were cut from paraffin- embedded fixed tissue using a Leica RM2235 microtome. Prior to staining, skin sections were dewaxed by a series of xylene (30 mins) and graduated ethanol washes (bringing sections to water) (100% for 1 min, 70% for 1 min and 30% for 1 min) before further processing. Skin sections were either stained with Haematoxylin and Eosin (H&E) or subjected to antigen retrieval and immunohistochemistry. Staining the sections in H&E involved bringing sections to water as mentioned above, followed by staining in Lillie's-Mayer's Haematoxylin for 6 min, "blueing" sections in bicarbonate water for 15 sec, differentiating Haematoxylin in 0.25% Acid Alcohol for 6 sec, staining in alcohol based Eosin stain for 2 min, dehydrating in graded alcohol series (30% for 30 sec, 70% for 30 sec, 100% for 1 min) and clearing in transitional solvent xylene for 2 min before mounting in DePeX mounting medium. H&E stained tissue was examined histologically for epidermal, dermal and overall skin thickness measurements in the skin tissues.

[0157] Immunohistochemistry experiments were undertaken on all samples following a standard antigen retrieval procedure using the primary and secondary antibodies mentioned below. All primary antibodies were at a concentration of 200 μg/0.1 ml and were used at a 1 :200 dilution, while all secondary antibodies were at a concentration of 2 mg/ml and were used at a 1 :400 dilution. Primary antibodies - Flightless I rabbit polyclonal IgG (H-300) (sc- 30046), Neutrophil Marker (NIMP-R14) rat polyclonal lgG2b (sc-59338), Proliferating Cell Nucelar Antigen (PCNA) (PC10) mouse polyclonal lgG2a (sc-56), lnterleukin-17 IL-17 (E-19) goat polyclonal IgG (sc-6077), and N FKB (H-1 19) rabbit polyclonal IgG (sc-7178) were all obtained from Santa Cruz Biotechnology Inc. (Texas, USA). Species specific secondary antibodies - Alexa Fluor 488 goat anti-rabbit polyclonal IgG (A1 1006), Alexa Fluor 488 goat anti-mouse polyclonal IgG (A1 1001 ), Alexa Fluor 488 donkey anti-goat polyclonal IgG (A1 1055) and Alexa Fluor 568 goat anti-rabbit polyclonal IgG (A1 101 1 ) were all purchased from Lifetechnologies Australia Pty Ltd (Victoria, Australia).

[0158] Sections were dewaxed by a series of xylene changes (30 min) and gradual ethanol washes (100% for 1 min, 70% for 1 min and 30% for 1 min), before being rinsed in 1x Phosphate Buffered Saline (PBS) and pre-treated with 250 ml Target Retrieval Solution (TRS) solution (2.8g Citric Acid, 3.76g Glycine, 0.372g EDTA, pH 5.9 in 1 L 1 xPBS). The sections were then microwaved for 2 min on "high" after which a "ballast" pot of water was added to help absorb some heat and pre-treatment continued for 2 x 5 min with regular "airing" to let the steam out and ensure that temperature reached 94°C but not 100°C. Sections were then cooled to 50°C on ice before they were washed in fresh 1 x PBS and enzyme digested with 0.0625g of Trypsin (Sigma-Aldrich, Sydney, Australia) dissolved in 1xPBS and pre-warmed to 37°C. Following the 3 min enzyme digestion at 37°C, sections were washed in 1 xPBS and then incubated for 30 min in NHS blocking solution (3%NHS in 1xPBS). Slides were then washed in 1 xPBS for 2 min and then incubated in primary antibody in a humid air tight box overnight at 4°C. Sections were then washed 3 x 2 min in 1xPBS and then incubated in Alexa Flour fluorescent species specific secondary antibody for 1 hr in a dark humid box. Slides were then washed 3 x 2 min in 1xPBS to remove any unspecific binding and mounted in Dako Fluorescent Mounting Medium (DAKO Corporation, Botany, Australia). Slides were stored in the dark at -20°C. Integrated fluorescence intensity was determined using AnalySIS software package (Soft-Imaging System GmbH, Munster, Germany). Negative controls were included to demonstrate antibody staining specificity. Control samples undergo the exact same staining procedure outlined except omitting the primary or secondary antibody. All control sections had negligible immunofluorescence.

Flightless Antibody treatment

[0159] Mice received a daily topical application of FnAb (250 μΙ of 5% FnAb in Sorbolene Cream) or a dose matched IgG control (n=6 in each group) from 3 days prior to the induction of psoriasis for a total of 9 days. Flightless antibody G12 was made in-house by immunising mice with the Flightless I peptide sequence H-CKLEHLSVSHN-OH. Methods for generating antibodies in this manner are described in detail above. Control IgG from mouse serum (I8765), technical grade, >80% (SDS-PAGE), and buffered aqueous solution was purchased from Sigma Aldrich (Castle Hill, Australia). Induction of psoriasis followed the protocol set out above. Severity of skin inflammation was examined using clinical photographs and degree of erythema as detailed above. On day 7, the mice were euthanized using C0₂ overdose followed by cervical dislocation prior to collection of psoriatic skin lesions which were used for histology and immunohistochemical analysis.

Real Time PCR

[0160] Tissue harvested was snap-frozen in liquid nitrogen and stored at -80°C for up to two months before isolating RNA. For RNA extraction, half of the tissue was cut into small pieces, up to 5 mm x 5 mm, and added to a microtube (1.5 ml Eppendorf Safe-Lock). The remaining half of the skin was immediately returned to storage at -80°C. Total RNA was isolated from the tissue using Ultraclean Tissue and Cell RNA Isolation Kit (MoBio Laboratories, Carlsbad, CA). Briefly, tissues (-20 mg) were homogenized in lysis binding solution (provided in the kit), with 2-mercaptoethanol (100 mM), and using the Bullet Blender and stainless steel beads (Next Advance, Averill Park, NY), and RNA was extracted according to the manufacturer's protocol. RNA quality and concentration were assessed using a Nanodrop UV spectrophotometer (Thermo Fisher Scientific, VIC, Australia). Only samples that had a 260/280 ratio of >1 .8 were used. Total cDNA was synthesized using iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA) in S1000 Thermal Cycler (Bio-Rad Laboratories, Hercules, CA) according to the manufacturer's protocol. Quantitative PCR was performed using iQ SYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA). Reactions were performed in triplicates. The plate was then placed in a CFX Connect Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA). Reactions underwent 30s at 95°C, then 40 cycles of 5s at 95°C and 20s at 60°C, and 10s at 95°C before determination of melt curve between 65°C and 95°C. GAPDH was used as a reference gene. For relative comparison, the cycle threshold value (Ct) using was analysed using the AACt method and reported the data as Ct normalized to GAPDH. Sequences for the PCR primers (Geneworks, Adelaide, Australia) were: IL-6, forward, 5'-CCTCTCTGCAAGAGACTTCCAT-3' (SEQ ID NO: 8, reverse, 5'-AGTCTCCTCTCCGGACTTGT-3' (SEQ ID NO: 9); IL-22, forward, 5 - CAGCTCCTGTCACATCAGCGGT-3' (SEQ ID NO: 10), reverse, 5'- AGGTCCAGTTCCCCAATCGCCT-3' (SEQ ID NO: 1 1 ); and GAPDH, forward, 5 - GGGCTCTCTGCTCCTCCCTGT-3' (SEQ ID NO: 12), reverse, 5 - CGGCCAAATCCGTTCACACCG-3' (SEQ ID NO: 13).

Results

Flii is upregulated in human psoriatic skin

[0161] Samples collected from patients with psoriasis (n=7) were assessed for Flii protein expression using immunohistochemistry and compared to normal human skin. As can be seen in Figure 1 , expression of Flii is increased in human psoriatic skin compared to normal human skin.

Decreasing Flii gene expression reduces the severity of psoriasis

[0162] Using an established model of psoriasis, the effect of reducing Flii gene expression on the development of psoriasis was examined. In this model, decreasing the level of Flii by 50%, as occurs in Flii-deficient (FIN+/-) mice, reduced the amount of erythema in the skin as determined by spectrophotometric analysis compared to wild-type (WT) and Flii- overexpressing (FliiTg/Tg) mice (see Figure 2).

[0163] Figure 2 shows skin erythema is reduced in Flii-deficient mice. In the mouse model of psoriasis, daily Imiquimod cream was used to induce psoriasis features on the shaved dorsa. The degree of erythema was measured and the results show that genetically Flii-deficient mice (FIN+/-) (n=6) exhibited significantly less erythema compared to wild-type (WT) and Flii- overexpressing (FliiTg/Tg) mice (n=6 each). This data demonstrates the severity of psoriasis is significantly reduced in Flii-deficient mice. Reduced Flightless leads to reduced epidermal thickening

[0164] Psoriasis induced Flii-deficient mice showed significantly reduced epidermal thickening and reduced length of rete pegs compared to wild-type (WT) and Flii- overexpressing (FliiTg/Tg) mice indicative of reduced disease severity (see Figure 3).

[0165] Figure 3 shows epidermal thickness and rete pegs length (epidermal extensions into the dermis) are reduced in Flii-deficient mice with psoriasis. Compared to wild-type and Flii- overexpressing mice, genetically Flii-deficient mice with psoriasis exhibit reduced epidermal thickness and rete pegs length. As can be seen in Figure 3B, Flii-deficient mice with psoriasis also demonstrate reduced cell differentiation. This data demonstrates significantly reduced epidermal thickening, reduced length of rete pegs, and reduced cell differentiation, in Flii deficient mice supporting knock-down of Flii for the treatment of psoriasis.

Inflammatory cell infiltrate and cytokine production is decreased in Flii reduced mice

[0166] The effect of Flii on inflammation was determined by measuring the amount of neutrophil infiltration, NF-κΒ activation and subsequent IL-17A cytokine secretion. In agreement with histological analysis of psoriatic induced skin, Flii-deficient mice were found to have reduced neutrophil infiltration, NF-κΒ activation and subsequent 11-17A cytokine (see Figure 4).

[0167] Figure 4 shows markers of inflammation are reduced in Flii deficient mice. Genetically Flii-deficient mice have reduced specific markers of inflammation including neutrophils infiltrate, NF-kB and IL-17 (A) compared to wild-type and Flii-overexpressing mice (n=6 each). Representative images are shown (B). This data demonstrates inflammatory cell infiltrate and cytokine production is decreased in Flii reduced mice.

Cellular proliferation is decreased in psoriasis when Flii levels are decreased

[0168] Flii-deficient mice psoriasitic skin showed decreased numbers of proliferative PCNA positive cells in the epidermis as well as decreased expression of pro-inflammatory cytokines IL-6 and IL-22, compared to Flii overexpressing counterparts (see Figure 5).

[0169] Figure 5 shows that cellular proliferation and pro-inflammatory markers are reduced in Flii-deficient mice. Flii-deficient mice have reduced CNA positive cells in the epidermis, reduced IL-6 imRNA and reduced IL-22 mRNA (n=6 each). Representative images are shown (B). This data demonstrates cellular proliferation and differentiation is decreased in psoriasis when Flii levels are decreased. Flii antibodies (FnAbs) reduce psoriasis when applied to the skin

[0170] Topical FnAbs were applied to the skin of psoriasis induced mice in a cream vehicle and compared with IgG control. FnAb decreased erythema as measured by spectrophotometer (see Figure 6). Decreased epidermal thickening was observed histologically (Figure 6B), consistent with reduced disease severity.

[0171] Figure 6 shows reduced erythema and epidermal thickness following administration of anti-Flii antibodies. Treatment of wild-type mice with a topical cream leads to reduced erythema (A and C) and epidermal thickness (B and D) in the mouse model of psoriasis compared to the IgG control (n=6 each). This data also demonstrates Flii antibodies reduce psoriasis when applied to the skin.

[0172] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to, or indicated in this specification, individually or collectively, and any and all combinations of any two or more of the steps or features.

Claims

1. A method of treating and/or preventing psoriasis in a subject, the method including the step of decreasing the expression and/or activity of Flightless I in the subject.

2. The method of claim 1 , wherein 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.

3. The method of claim 2, wherein the agent is selected from one or more of the group consisting of a drug, a small molecule, a nucleic acid, an oligonucleotide, an oligopeptide, a polypeptide, a protein, an enzyme, a polysaccharide, a glycoprotein, a hormone, a receptor, a ligand for a receptor, a co-factor, an antisense oligonucleotide, a ribozyme, a small interfering RNA, a microRNA, short hairpin RNA, a lipid, an aptamer, a virus, and an antibody or an antigen binding part thereof.

4. The method of claim 2 or claim 3, wherein the agent is an antibody to Flightless I, or an antigen binding part thereof.

5. The method of any one of claims 1 to 4, wherein epidermal thickening of an affected area of skin of the subject is reduced.

6. The method of any one of claims 1 to 5, wherein cellular proliferation and differentiation of an affected area of skin of the subject is decreased.

7. The method of any one of claims 2 to 6, wherein the agent is administered topically to the skin of the subject.

8. A method of diagnosing psoriasis in a subject, the method including the steps of: measuring the level of expression and/or activity of Flightless I in the subject;

diagnosing psoriasis in the subject on the basis of the comparison.

9. The method of claim 8, wherein 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 psoriasis in the subject.

10. The method of claim 8 or claim 9, wherein the level of expression and/or activity of Flightless I is measured in a sample obtained from the subject.

1 1 . The method of any one of claims 8 to 10, wherein measuring the level of expression of Flightless I includes measuring the level of Flightless I RNA or protein.

12. The method of claim 1 1 , wherein the Flightless I RNA is Flightless I mRNA.

13. A method of determining if a subject is susceptible to developing psoriasis, the method including the steps of:

14. The method of claim 13, wherein 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 indicates that the subject is susceptible to developing psoriasis.

15. The method of claim 13 or claim 14, wherein the level of expression and/or activity of Flightless I is measured in a sample obtained from the subject.

16. The method of any one of claims 13 to 15, wherein measuring the level of expression of Flightless I includes measuring the level of Flightless I RNA or protein.

17. The method of claim 16, wherein the Flightless I RNA is Flightless I mRNA.

18. A pharmaceutical composition when used for treating and/or preventing psoriasis in a subject, the composition including an effective amount of an agent that decreases expression and/or activity of Flightless I.

19. The pharmaceutical composition of claim 18, wherein the agent is selected from one or more of the group consisting of a drug, a small molecule, a nucleic acid, an oligonucleotide, an oligopeptide, a polypeptide, a protein, an enzyme, a polysaccharide, a glycoprotein, a hormone, a receptor, a ligand for a receptor, a co-factor, an antisense oligonucleotide, a ribozyme, a small interfering RNA, a microRNA, short hairpin RNA, a lipid, an aptamer, a virus, and an antibody or an antigen binding part thereof.

20. The pharmaceutical composition of claim 18 or claim 19, wherein the agent is an antibody to Flightless I, or an antigen binding part thereof.

21 . The pharmaceutical composition of any one of claims 18 to 20, wherein the pharmaceutical composition is a topical composition.

22. The pharmaceutical composition of any one of claims 18 to 21 , wherein the pharmaceutical composition is a systemic composition.

23. A kit when used for diagnosing psoriasis in a subject, or determining if a subject is susceptible to developing psoriasis, the kit including means for measuring the level of expression and/or activity of Flightless I in the subject.

24. The kit of claim 23, wherein a level of expression and/or activity of Flightless I in the subject that is higher than a reference level of expression and/or activity for Flightless I diagnoses psoriasis in the subject, or is indicative that the subject is susceptible to developing psoriasis.

25. The kit of claim 23 or claim 24, wherein the level of expression and/or activity of Flightless I is measured in a sample obtained from the subject.

26. The kit of any one of claims 23 to 25, wherein measuring the level of expression of Flightless I includes measuring the level of Flightless I RNA or protein.

27. The kit of claim 26, wherein the Flightless I RNA is Flightless I mRNA.