WO2013088110A1

WO2013088110A1 - Annexin-a1 transgenic animals

Info

Publication number: WO2013088110A1
Application number: PCT/GB2012/000905
Authority: WO
Inventors: Fulvio D'acquisto; Mauro Perretti; Giuseppa PIRAS
Original assignee: Queen Mary & Westfield College, University Of London
Priority date: 2011-12-14
Filing date: 2012-12-14
Publication date: 2013-06-20
Also published as: GB201121561D0

Abstract

The present invention provides a transgenic non-human animal model for obsessive compulsive disorder (OCD) or a disease related to OCD wherein the cells of said animal contain an exogenous nucleic acid encoding Annexin-Al and wherein said exogenous nucleic acid encoding Annexin-Al is expressed in the T cells of said animal, and the use of such a non-human transgenic animal model for the study of OCD or a disease related to OCD. The invention also provides a method of producing a transgenic non-human animal embryo and a method of producing a transgenic non-human animal model for obsessive compulsive disorder (OCD) or a disease related to OCD, a cell line derived from the transgenic non- human animal model of the invention, and methods of screening candidate molecules to determine whether they are effective for the treatment of OCD or a disease related to OCD or for the treatment of a T cell mediated disease.

Description

ANNEXIN-A1 TRANSGENIC ANIMALS

Field of the Invention

The present invention relates to a transgenic non-human animal model for obsessive compulsive disorder (OCD) or a disease related to OCD and the use of such an animal model to screen for candidate molecules as treatments for OCD and related diseases.

Background to the Invention

Obsessive compulsive disorder (OCD) is a chronic, relapsing psychiatric affliction with a lifetime prevalence of 1-3%. According to the Diagnostic and Statistical Manual of Mental Disorders (4th ed; DSM IV), the essential features of this disease are recurrent obsessions and/or compulsions (e.g., doubting, checking, washing) that are time consuming (i.e., they take more than 1 hour a day) or cause marked distress or significant impairment. The most effective treatments for mental disorders like OCD are antipsychotic and behavioural treatments. Yet, around 30% of the patients are refractory to pharmaco- and behavioural therapy. In addition, side effects such as agranulocytosis (loss of the white blood cells that help a person fight infection) and changes in a person's metabolism (leading to diabetes) are serious problems that limit the use of these drugs. There is therefore a need in the art for animal models to screen candidate compounds to identify drugs that are effective to treat such diseases without causing these unwanted side effects.

Mouse models of genetically-induced OCD and related diseases have recently attracted the attention of several scientists for their potential in the development of novel therapeutic approaches for these pathologies. Each of the models has strengths and limitations, which dictate the aim(s) it can serve (Wang L, Simpson HB, Dulawa SC. Behav Pharmacol. 2009 Mar; 20(2): 119-33). However, one common feature of all these models is that the genetic modifications are mainly targeting cells or tissues of the nervous system. Summary of the Invention

The present inventors have previously shown that a protein called Annexin-Al (Anx-Al) plays an immunomodulatory role in T cells. In particular they have found that high levels of Anx-Al cause T cell hyperactivation and favour the development of autoimmune diseases. The present inventors have now surprisingly found that overexpression of Anx-Al in T cells in transgenic mice exacerbates the development of autoimmune diseases and, at the same time, induces the manifestation of signs of OCD and related diseases.

Accordingly, in a first aspect the present invention provides a transgenic non-human animal model for obsessive compulsive disorder (OCD) or a disease related to OCD wherein the cells of said animal contain an exogenous nucleic acid encoding Annexin-Al and wherein said exogenous nucleic acid encoding Annexin-Al is expressed in the T cells of said animal.

Detailed Description of the Invention

The present invention provides a transgenic non-human animal model for obsessive compulsive disorder (OCD) or a disease related to OCD wherein the cells of said animal contain an exogenous nucleic acid encoding Annexin-Al and wherein said exogenous nucleic acid encoding Annexin-Al is expressed in the T cells of said animal.

The term "transgenic" as used herein with respect to transgenic non-human animals is meant to define a non-human animal that contains an exogenous nucleic acid as defined herein. The term "transgenic" as used herein thus encompasses animals that contain a gene or nucleic acid that is heterologous to the animal (i.e. from a different species of animal) or, more typically, a homologous gene or nucleic acid (i.e. from the same species of animal) that is overexpressed.

Typically, the non-human animal is a non-human mammal. The non-human mammal is typically an experimentally useful animal and is therefore typically a rodent, for example a mouse, rat or guinea pig, and is typically a mouse or a rat, most typically a mouse. However, the non-human mammal may be from another mammalian species, such as rabbit, a canine (dog) or feline (cat), or an ungulate species such as an ovine (sheep), porcine (pig), equine (horse), caprine (goat) or bovine (cow). The transgenic non-human animal model of the invention is an animal model for obsessive compulsive disorder (OCD) or a disease related to OCD. Diseases related to OCD include trichotillomania, dermatillomania, Tourette's Syndrome (TS), Asperger's syndrome, anorexia, hnlimia, depression, panic disorder, panic attacks, bipolar disorder, hypochondriasis, post- traumatic stress disorder (PTSD), social anxiety disorder, schizophrenia, attention deficit hyperactivity disorder (ADHD) and body dysmorphic disorder (BDD).

The cells of the transgenic non-human animal of the invention contain an exogenous nucleic acid encoding Annexin-Al . The term "exogenous" means from outside the animal of interest. Thus an "exogenous" nucleic acid that is present in the cells of an animal means a nucleic acid sequence from outside that particular animal. An exogenous nucleic acid can be an additional copy of a nucleic acid that is homologous to a nucleic acid present in the animal or a nucleic acid that is not normally present in the animal, for example a nucleic acid having a different sequence from the nucleic acid present in the animal or a nucleic acid from a different species of animal (i.e. a nucleic acid that is heterologous to the animal). Typically, the exogenous nucleic acid is an additional copy of a nucleic acid that is homologous to a nucleic acid present in the animal. The term "endogenous" is the converse of "exogenous" and therefore means from within the animal of interest.

In some embodiments, the transgenic non-human animal of the invention contains an endogenous gene or nucleic acid that is overexpressed. The term "overexpressed" as used herein means that a nucleic acid is expressed at higher than usual levels, for example due to the addition of one or more copies of an exogenous gene or nucleic acid sequence that corresponds to the endogenous gene or nucleic acid sequence, or by activation of an endogenous gene. The term "nucleic acid" as used herein takes its usual meaning in the art. A nucleic acid comprises a plurality of nucleotides, which are sometimes referred to as bases (in single stranded nucleic acid molecules) or as base pairs (bp, in double stranded nucleic acid molecules). The term "nucleic acid" is used interchangeably herein with the term "polynucleotide". A "nucleic acid" or "polynucleotide" as defined herein includes a plurality of oligonucleotides as defined herein.

Nucleic acids for use in the present invention are typically the naturally-occurring nucleic acids DNA or RNA, but can also be artificial nucleic acids such as PNA (peptide nucleic acid), LNA (locked nucleic acid), UNA (unlocked nucleic acid), GNA (glycol nucleic acid) and TNA (threose nucleic acid). Nucleic acids such as DNA for use in the invention can be synthetic or natural. Nucleic acids for use in the present invention typically consist of the nucleotides adenine (A), cytosine (C), guanine (G), thymine (T) and uracil (U), but modified nucleotides can also be used in the present invention.

The length of a nucleic acid or polynucleotide sequence can be measured in terms of the number of nucleotides it contains. The term "kilobase" (kb) means 1000 nucleotides. As used herein, the term "oligonucleotide" means a polymer of nucleotides (i.e. at least 2 nucleotides) that is shorter in length than a "nucleic acid" of "polynucleotide" as defined herein. Typically, an oligonucleotide consists of up to 40 nucleotides or bases, more typically up to 60 nucleotides or bases. Typically, an oligonucleotide is sufficiently short that it has no secondary or tertiary structure. An oligonucleotide is therefore a fragment of a nucleic acid or polynucleotide.

The term "gene" takes its usual meaning in the art, and includes a DNA or RNA sequence encoding a polypeptide, together with regions preceding and following the coding region, as well as intervening sequences (introns) between individual coding segments (exons).

In all definitions, the singular and plural are used interchangeably.

The cells of the transgenic non-human animal of the present invention contain an exogenous nucleic acid encoding Annexin-Al. As defined herein, an exogenous nucleic acid is a nucleic acid sequence from outside the transgenic non-human animal of the invention.

Annexins are a group of calcium- and phospholipid-binding cellular proteins and are also known as lipocortins. The annexin family has 13 members in humans, including Annexin Ai, Annexin A2 and Annexin A5, and also 13 members in mice. Annexin-Al is also known as Annexin-1 and is referred to herein as "Αηχ-ΑΓ'. Human annexin-1 (Anx-Al) is a 37-kDa protein and was originally described as a mediator of the actions of glucocorticoids. Over the last few years evidence has shown than Anx-Al plays a homeostatic role in the adaptive immune system, in particular T cells, by modulating the strength of T cell receptor (TCR) signalling. In humans, Anx-AI acts as an endogenous down-regulator of inflammation in cells of the innate immune system in vivo. Figure 1 is a ribbon diagram showing the three- dimensional structure of human Anx-AI . A number of studies have shown that an N-terminal peptide of human Anx-AI named Ac.2- 26 acts as a bioactive surrogate of the whole protein (see e.g. Lim et al, Proc Natl Acad Sci USA 95, 14535-9, 1998).

Anx-AI and its N-terminal derived bioactive peptides mediate their biological effects through members of the formyl peptide receptor (FPR) family. The full-length protein Anx-AI exerts its counterregulatory actions on neutrophil extravasation and innate immunity by direct binding and activation of one member of this family, formyl peptide receptor like-1 (FPRL-1), also known as formyl peptide receptor 2 (FPR-2/ALX). The present inventors have previously found that stimulation of T cells in the presence of hr Anx-AI increases T cell activation via stimulation of FPRL-1 FPR-2 ALX (D'Acquisto et al, Blood 109: 1095-1102, 2007).

The peptide Ac.2-26 binds to a different member of the FPR family, the N-formylpeptide receptor 1 (FPR-1). A recent study (Gao et al. Behav Genet. 41(5): 724-733, 2011) has shown that mice deficient for FPR-1 exhibited increased exploratory activity, reduced anxiety- like behavior, and impaired fear memory. However, FPR-1 is the receptor for a number of ligands and there is no evidence in this study to show that the effects demonstrated in the mice were as a result of blocking annexin binding. Annexins have homologues in a wide variety of animal species, including mice, rats, cats, dogs and chimpanzees.

In the mouse {Mus musculus), there are eight transcripts of the gene that encodes Anx-AI . Of these, only two are translated and thus there are two isoforms of Anx-AI, designated Arrxal- 001 and Anxal-201. The nucleotide and protein sequences are available from the Ensembl website (www.ensembl.org). The sequences are designated ENSMUST00000025561 (Anxal-001 transcript sequence), ENSMUSP000000255 1 (Anxal-001 protein sequence), ENSMUST00000099584 (Anxal-201 transcript sequence) and ENSMUSP00000097180 (Anxal-201 protein sequence). The nucleotide and amino acid sequences of one isoform of mouse Annexin-1 (Anx-Al), Anxal-001, are shown in Figure 2A (SEQ ID NOs: 1 and 2 respectively). The nucleotide and amino acid and sequences of the other isoform of mouse Anx-Al, Anxal-201, are shown in Figure 2B (SEQ ID NOs: 3 and 4 respectively).

In the rat (Rattus norvegic s), there are two transcripts of the gene that encodes Anx-Al . Both are translated and thus there are two isoforms of Anx-Al, designated Anxal and D3ZVZ4_RAT. The nucleotide and protein sequences are available from the Ensembl website (www.ensembl.org). The sequences are designated ENSRNOT00000023664 (Anxal transcript sequence), ENSRNOP00000023664 (Anxal protein sequence), ENSRNOT00000054790 (D3ZVZ4_RAT transcript sequence) and ENSRNOP00000051674 (D3ZVZ4_RAT protein sequence). The nucleotide and amino acid sequences of one isoform of rat Annexin-1 (Anx-Al), Anxal, are shown in Figure 2C (SEQ ID NOs: 5 and 6 respectively). The nucleotide and amino acid and sequences of the other isoform of rat Anx- Al , D3ZVZ4_RAT, are shown in Figure 2D (SEQ ID NOs: 7 and 8 respectively).

There are eight human nucleotide sequences which encode Anx-Al. Of these, only four are translated and thus there are four isoforms of Anx-Al, designated AN Al-002, ANXA 1-003, ANXAl-004 and ANXA1-006. These sequences are available from the Ensembl website (www.ensembl.org) and are designated ENST00000257497 (ANXAl-002 transcript sequence), ENSP00000257497 (ANXAl-002 protein sequence), ENST00000376911 (ANXA1-003 transcript sequence), ENSP00000366109 (ANXA1-003 protein sequence), ENST00000456643 (ANXAl-004 transcript sequence), ENSP00000412489 (ANXAl-004 protein sequence), ENST00000415424 (ANXA 1-006 transcript sequence) and ENSP00000414013 (A XA1-006 protein sequence). The nucleotide and amino acid sequences of one isoform of human Annexin-1 (Anx-Al), ANXA1-003, are shown in Figure 2E (SEQ ID NOs: 9 and 10 respectively). The nucleotide and amino acid sequences of another isoform, ANXAl-002, are shown in Figure 2F (SEQ ID NOs: 1 1 and 12 respectively). The nucleotide and amino acid sequences of another isoform, ANXAl-004, are shown in Figure 2G (SEQ ID NOs: 13 and 14 respectively). The nucleotide and amino acid sequences of another isoform, ANXA1-006, are shown in Figure 2H (SEQ ID NOs: 15 and 16 respectively). As can be seen from Figure 2, isoforms ANXAl-002, ANXAl-004 and ANXA1-006 are either short splice variants of ANXA1-003 or variants of ANXA1-003 with a small number of amino acid changes.

The nucleotide sequence of the mouse Anx-Al isoform Anxal-001 shown in Figure 2A (SEQ ID NO: 1) is as follows:

1 ATGGCAATGGTAT

14 CAGAATTCCTCAAGCAGGCCCGTTTTCTTGAAAATCAAGAACAGGAATATGTTCAAGCTG

74 TAAAATCATACAAAGGTGGTCCTGGGTCAGCAGTGAGCCCCTACCCTTCCTTCAATGT T

134 CCTCGGATGTTGCTGCCTTGCACAAAGCTATCATGGTTAAAGGTGTGGATGAAGCAACCA

1 94 TCATTGACATTCTTACCAAGAGGACCAATGCTCAGCGCCAGCAGATCAAGGCCGCGTACT

25 TACAGGAGAATGGAAAGCCCTTGGATGAAGTCTTGAGAAAAGCCCTTACAGGCCACCTGG

3 1 AGGAGGTTGTTTTGGCTATGCTAAAAACTCCAGCTCAGTTTGATGCAGATGAACTCCGTG

37 GTGCCATGAAGGGACTTGGAACAGATGAAGACACTCTCATTGAGATTTTGACAACAAGAT

434 CTAACGAACAAATCAGAGAGATTAATAGAGTCTACAGAGAAGAGCTGAAAAGAGATCTGG

4 94 CCAAAGACATCACTTCAGATACATCTGGAGACTTTCGGAAAGCCTTGCTTGCTCTTGCCA

554 AGGGTGACCGTTGTCAGGACTTGAGTGTGAATCAAGATTTGGCTGATACAGATGCCAGGG

614 CTTTGTATGAAGCTGGAGAAAGGAGAAAGGGGACAGACGTGAACGTCTTCACCACAATTC

674 TGACCAGCAGGAGCTTTCCTCATCTTCGCAGAGTGTTTCAGAATTACGGAAAGTACAGTC

734 AACATGACATGAACAAAGCTCTGGATCTGGAACTGAAGGGTGACATTGAGAAGTGCCTCA

7 94 CAACCATCGTGAAGTGTGCCACCAGCACTCCAGCTTTCTTTGCCGAGAAGCTGTACGAAG

854 CCATGAAGGGTGCCGGAACTCGCCATAAGGCATTGATCAGGATTATGGTCTCCCGTTCGG

91 AAATTGACATGAATGAAATCAAAGTATTTTACCAGAAGAAGTATGGAATCTCTCTTTGCC

974 AAGCCATCCTGGATGAAACCAAAGGAGACTATGAAAAAATCCTGGTGGCTCTGTGTGGTG

1034 GAAACTAG

In one embodiment, the exogenous nucleic acid encoding Annexin-Al comprises (a) nucleotides 4 to 1038 of the nucleotide sequence shown in Figure 2A (SEQ ID NO: 1) or (b) a nucleotide sequence having at least 70% identity to nucleotides 4 to 1038 of the nucleotide sequence shown in Figure 2 A (SEQ ID NO: 1).

In another embodiment, the exogenous nucleic acid encoding Annexin-Al comprises (a) the nucleotide sequence shown in Figure 2A (SEQ ID NO: 1) or (b) a nucleotide sequence having at least 70% identity to the nucleotide sequence shown in Figure 2A (SEQ ID NO: 1).

In one embodiment, the exogenous nucleic acid encoding Annexin-Al comprises (a) a nucleotide sequence encoding the amino acid sequence shown in Figure 2A (SEQ ID NO: 2) or (b) a nucleotide sequence having at least 70% identity to a nucleotide sequence encoding the amino acid sequence shown in Figure 2A (SEQ ID NO: 2).

In another embodiment, the exogenous nucleic acid encoding Annexin-Al comprises (a) nucleotides 4 to 1065 of the nucleotide sequence shown in Figure 2B (SEQ ID NO: 3) or (b) a nucleotide sequence having at least 70% identity to nucleotides 4 to 1065 of the nucleotide sequence shown in Figure 2B (SEQ ID NO: 3).

In another embodiment, the exogenous nucleic acid encoding Annexin-Al comprises (a) the nucleotide sequence shown in Figure 2B (SEQ ID NO: 3) or (b) a nucleotide sequence having at least 70% identity to the nucleotide sequence shown in Figure 2B (SEQ ID NO: 3).

In one embodiment, the exogenous nucleic acid encoding Annexin-Al comprises (a) a nucleotide sequence encoding the amino acid sequence shown in Figure 2B (SEQ ID NO: 4) or (b) a nucleotide sequence having at least 70% identity to a nucleotide sequence encoding the amino acid sequence shown in Figure 2B (SEQ ID NO: 4).

Typically, the exogenous nucleic acid encoding Annexin-Al is from the same species as the transgenic non-human animal of the invention. Accordingly, in these embodiments of the invention, the transgenic non-human animal is typically a mouse.

In another embodiment, the exogenous nucleic acid encoding Annexin-Al comprises (a) nucleotides 4 to 1038 of the nucleotide sequence shown in Figure 2C (SEQ ID NO: 5) or (b) a nucleotide sequence having at least 70% identity to nucleotides 4 to 1038 of the nucleotide sequence shown in Figure 2C (SEQ ID NO: 5).

In another embodiment, the exogenous nucleic acid encoding Annexin-Al comprises (a) the nucleotide sequence shown in Figure 2C (SEQ ID NO: 5) or (b) a nucleotide sequence having at least 70% identity to the nucleotide sequence shown in Figure 2C (SEQ ID NO: 5).

In one embodiment, the exogenous nucleic acid encoding Annexin-Al comprises (a) a nucleotide sequence encoding the amino acid sequence shown in Figure 2C (SEQ ID NO: 6) or (b) a nucleotide sequence having at least 70% identity to a nucleotide sequence encoding the amino acid sequence shown in Figure 2C (SEQ ID NO: 6).

In another embodiment, the exogenous nucleic acid encoding Annexin-Al comprises (a) nucleotides 4 to 1155 of the nucleotide sequence shown in Figure 2D (SEQ ID NO: 7) or (b) a nucleotide sequence having at least 70% identity to nucleotides 4 to 1155 of the nucleotide sequence shown in Figure 2D (SEQ ID NO: 7).

In another embodiment, the Annexin-Al is encoded by a nucleotide sequence comprising (a) the nucleotide sequence shown in Figure 2D (SEQ ID NO: 7) or (b) a nucleotide sequence having at least 70% identity to the nucleotide sequence shown in Figure 2D (SEQ ID NO: 7).

In one embodiment, the exogenous nucleic acid encoding Annexin-Al comprises (a) a nucleotide sequence encoding the amino acid sequence shown in Figure 2D (SEQ ID NO: 8) or (b) a nucleotide sequence having at least 70% identity to a nucleotide sequence encoding the amino acid sequence shown in Figure 2D (SEQ ID NO: 8).

Typically, the exogenous nucleic acid encoding Annexin-Al is from the same species as the transgenic non-human animal of the invention. Accordingly, in these embodiments of the invention, the transgenic non-human animal is typically a rat.

In one embodiment, the exogenous nucleic acid encoding Annexin-Al comprises (a) nucleotides 4 to 1038 of the nucleotide sequence shown in Figure 2E (SEQ ID NO: 9) or (b) a nucleotide sequence having at least 70% identity to nucleotides 4 to 1038 of the nucleotide sequence shown in Figure 2E (SEQ ID NO: 9).

In another embodiment, the exogenous nucleic acid encoding Annexin-Al comprises (a) the nucleotide sequence shown in Figure 2E (SEQ ID NO: 9) or (b) a nucleotide sequence having at least 70% identity to the nucleotide sequence shown in Figure 2E (SEQ ID NO: 9).

In one embodiment, the exogenous nucleic acid encoding Annexin-Al comprises (a) a nucleotide sequence encoding the amino acid sequence shown in Figure 2E (SEQ ID NO: 10) or (b) a nucleotide sequence having at least 70% identity to a nucleotide sequence encoding the amino acid sequence shown in Figure 2E (SEQ ID NO: 10).

In one embodiment, the exogenous nucleic acid encoding Annexin-Al comprises (a) nucleotides 4 to 1038 of the nucleotide sequence shown in Figure 2F (SEQ ID NO: 11) or (b) a nucleotide sequence having at least 70% identity to nucleotides 4 to 1038 of the nucleotide sequence shown in Figure 2F (SEQ ID NO: 11).

In another embodiment, the exogenous nucleic acid encoding Annexin-Al comprises (a) the nucleotide sequence shown in Figure 2F (SEQ ID NO: 11) or (b) a nucleotide sequence having at least 70% identity to the nucleotide sequence shown in Figure 2F (SEQ ID NO: 11).

In one embodiment, the exogenous nucleic acid encoding Annexin-Al comprises (a) a nucleotide sequence encoding the amino acid sequence shown in Figure 2F (SEQ ID NO: 12) or (b) a nucleotide sequence having at least 70% identity to a nucleotide sequence encoding the amino acid sequence shown in Figure 2F (SEQ ID NO: 12).

In one embodiment, the exogenous nucleic acid encoding Annexin-Al comprises (a) nucleotides 4 to 612 of the nucleotide sequence shown in Figure 2G (SEQ ID NO: 13) or (b) a nucleotide sequence having at least 70% identity to nucleotides 4 to 612 of the nucleotide sequence shown in Figure 2G (SEQ ID NO: 13).

In another embodiment, the exogenous nucleic acid encoding Annexin-Al comprises (a) the nucleotide sequence shown in Figure 2G (SEQ ID NO: 13) or (b) a nucleotide sequence having at least 70% identity to the nucleotide sequence shown in Figure 2G (SEQ ID NO: 13).

In one embodiment, the exogenous nucleic acid encoding Annexin-Al comprises (a) a nucleotide sequence encoding the amino acid sequence shown in Figure 2G (SEQ ID NO: 14) or (b) a nucleotide sequence having at least 70% identity to a nucleotide sequence encoding the amino acid sequence shown in Figure 2G (SEQ ID NO: 1 ).

In one embodiment, the exogenous nucleic acid encoding Annexin-Al comprises (a) nucleotides 4 to 345 of the nucleotide sequence shown in Figure 2H (SEQ ID NO: 15) or (b) a nucleotide sequence having at least 70% identity to nucleotides 4 to 345 of the nucleotide sequence shown in Figure 2H (SEQ ID NO: 15). In another embodiment, the exogenous nucleic acid encoding Annexin-Al comprises (a) the nucleotide sequence shown in Figure 2H (SEQ ID NO: 15) or (b) a nucleotide sequence having at least 70% identity to the nucleotide sequence shown in Figure 2H (SEQ ID NO: 15). In one embodiment, the exogenous nucleic acid encoding Annexin-Al comprises (a) a nucleotide sequence encoding the amino acid sequence shown in Figure 2H (SEQ ID NO: 16) or (b) a nucleotide sequence having at least 70% identity to a nucleotide sequence encoding the amino acid sequence shown in Figure 2H (SEQ ID NO: 16). In some embodiments, the exogenous nucleic acid encoding Annexin-Al has some sequence variations compared to the sequence of an endogenous nucleic acid encoding Annexin-Al. This will be the case, for example, when the endogenous nucleic acid encoding Annexin-AI in a particular animal encodes one isoform of Annexin-Al from that species of animal, whilst the exogenous nucleic acid encoding Annexin-Al encodes a different isoform from the same species of animal.

In some embodiments, the exogenous nucleic acid encoding Annexin-Al comprises a nucleotide sequence having at least 75%, 80%, 82%, 83%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to any one of the nucleotide sequences described above.

It will be understood by a person of skill in the art that a nucleotide sequence requires a start codon (ATG) and a stop codon (TAG, TAA or TGA) in order to be translated into a peptide. The sequences shown in Figure 2 generally include start and stop codons. Accordingly, the invention encompasses the use of fragments of the sequences shown in Figure 2, most typically those fragments that do not include the start and stop codons. Such fragments are described herein, in particular the fragments consisting of nucleotides 4 to 1038 of the nucleotide sequence shown in Figure 2 A (SEQ ID NO: 1) nucleotides 4 to 1065 of the nucleotide sequence shown in Figure 2B (SEQ ID NO: 3), nucleotides 4 to 1038 of the nucleotide sequence shown in Figure 2C (SEQ ID NO: 5), nucleotides 4 to 1155 of the nucleotide sequence shown in Figure 2D (SEQ ID NO: 7), nucleotides 4 to 1038 of the nucleotide sequence shown in Figure 2E (SEQ ID NO: 9), nucleotides 4 to 1038 of the nucleotide sequence shown in Figure 2F (SEQ ID NO: 11), nucleotides 4 to 612 of the nucleotide sequence shown in Figure 2G (SEQ ID NO: 13) or nucleotides 4 to 345 of the nucleotide sequence shown in Figure 2H (SEQ ID NO: 15).

In some embodiments of the invention, the exogenous nucleic acid encoding Annexin-Al further includes nucleotide sequences that represent genetic elements useful for transcription and/or nucleotide sequences encoding peptide markers or tags, as described in detail below. In these embodiments, further nucleotide sequences will be included after the start codon and/or before the stop codon. In various embodiments of the invention, therefore, the nucleic acid sequence comprises nucleotides 4 to 1038 of the nucleotide sequence shown in Figure 2 A (SEQ ID NO: 1), nucleotides 4 to 1065 of the nucleotide sequence shown in Figure 2B (SEQ ID NO: 3), nucleotides 4 to 1038 of the nucleotide sequence shown in Figure 2C (SEQ ID NO: 5), nucleotides 4 to 1155 of the nucleotide sequence shown in Figure 2D (SEQ ID NO: 7), nucleotides 4 to 1038 of the nucleotide sequence shown in Figure 2E (SEQ ID NO: 9), nucleotides 4 to 1038 of the nucleotide sequence shown in Figure 2F (SEQ ID NO: 11), nucleotides 4 to 612 of the nucleotide sequence shown in Figure 2G (SEQ ID NO: 13) or nucleotides 4 to 345 of the nucleotide sequence shown in Figure 2H (SEQ ID NO: 15) together with a start codon preceding the sequence and a stop codon following the sequence. In these embodiments, the nucleic acid optionally includes one or more further nucleotide sequences which are typically located immediately after the start codon and or immediately prior to the stop codon.

As described above, in some embodiments of the invention, the exogenous nucleic acid encoding Annexin-Al further encodes a tag, such as an epitope tag, or a marker, such as a fluorescent marker. The addition of one or more such markers or tags allows the overexpressed Annexin-Al (encoded by the exogenous nucleic acid encoding Annexin-Al) to be differentiated from endogenous Annexin-Al. The different Annexin-Al proteins (overexpressed and endogenous) can then be differentiated using biological or physical assay techniques. The marker or tag may be at the amino terminus or carboxy termmus of the overexpressed Annexin-Al protein or may be inserted internally with respect to the amino acid sequence of the overexpressed Annexin-Al protein.

The tag or marker is typically a peptide, typically a peptide consisting of 5 to 250 amino acids. Typically, the peptide consists of 5 to 50, 10 to 60, 20 to 70, 30 to 80, 40 to 90, 50 to 100, 60 to 150, 70 to 200 or 80 to 250 amino acids. In one embodiment, the tag is an epitope tag. An epitope is a short peptide that is a defined amino acid sequence from a protein with a cognate antibody. The skilled person can select such epitopes based on sequences identified as possessing antigenic properties. These tags are particularly useful for western blotting, immunofluorescence and immunoprecipitation experiments, although they also find use in antibody purification. In certain embodiments of the invention the epitope tag has one of the following amino acid sequences: FLAG tag (KDDDDKYD, SEQ ID NO: 17), c-myc (EQKLISEEDL, SEQ ID NO: 18), simian virus V5 (G PIPNPLLGLDST, SEQ ID NO. 19), Haemagglutinin (HA) (YPYDVPDYA, SEQ ID NO: 20), ClonelOO (NVRFSTIVRRRA, SEQ ID NO: 21), rabl la (KQMSDRRENDMSPS, SEQ ID NO: 22), DOB (SGNEVSRAVLLPQSC, SEQ ID NO: 23), SG11 (SSLS YTNPAVAATSANL, SEQ ID NO: 24), erbB4 (RSTLQHPDYLQEYST, SEQ ID NO: 25), ARF (VSTLLRWERFPGHRQA, SEQ ID NO: 26), RY (KFQQLVQCLTEFHAALGAYV, SEQ ID NO: 27), WILPEP1 (QEQCQEVWRKRVISAFLKSP, SEQ ID NO: 28) or HAFIO (RLSDKTGPVAQEKS, SEQ ID NO: 29).

Preferably the epitope tag is recognised by its cognate antibody irrespective of whether it is located at the amino terminus, carboxy terminus or in an internal domain of Annexin-Al .

In one embodiment, the nucleic acid encodes a FLAG tag. In this embodiment, the nucleic acid includes the sequence GATTACAAGGATGACGACGATAAG (SEQ ID NO: 30).

In another embodiment, the tag possesses enzymatic activity that converts a substrate to a form that is readily detectable by an assay, for example a kinase activity specifying phosphorylation of another protein or peptide substrate that could be added to a secreted or excreted analyte along with a phosphate group donor. Detection could be achieved using an immunological assay based on detection by an antibody specifically recognising the phosphorylated version of the tagged reporter protein or alternatively by the use of phosphate radiolabeled with an isotope of phosphorous such as P or P. Other enzymic modifications include for example acetylation, sulphation and glycosylation. Another possibility is a peptide tag that is a catalytic sequence of an enzyme such as Glutathione-S-transferase (GST) where enzyme activity can be detected by means of an activity assay or by antibody reactivity. In one embodiment, the marker is a fluorescent marker. Such markers are used to give visual readout on a protein. Fluorescent markers include green fluorescent protein (GFP) and red fluorescent protein (RFP).

Typically, the nucleotide encoding the marker or tag is contiguous with the coding sequence for the Annexin-Al, such as the coding sequence shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7, and as described above is typically included after the coding sequence but prior to the stop codon (for example TAG, TGA or TAA). In another embodiment, a linker nucleic acid sequence may be inserted between the two sequences that encodes a short sequence of amino acids.

In some embodiments of the invention, the exogenous nucleic acid encoding Annexin-Al further includes one or more nucleotide sequences encoding other genetic elements. For example, a Kozak sequence can be included to enhance transcription. A suitable Kozak sequence is GCCACC (SEQ ID NO: 31). Other genetic elements that could usefully be included will be known to a person skilled in the art.

The sequence of Anx-Al FLAG inserted into the mice generated in the Examples is shown in Figure 3B (SEQ ID NO: 32). As can be seen from Figure 3B, in addition to the coding sequence for murine Anx-Al, the sequence includes Smal (and isoschizomer Xmal) recognition sites (CCCGGG, SEQ ID NO: 33), Kozak sequence (GCCACC, SEQ ID NO: 31), Anx-Al FLAG start and stop codons (ATG and TAG respectively) as well the coding sequence for the incorporated FLAG epitope (GATTACAAGGATGACGACGATAAG, SEQ ID NO: 30). The forward and reverse primers are also shown. The resulting expressed protein has the incorporated FLAG epitope (KDDDDKYD, SEQ ID NO: 17) at the C-terminus of the protein.

"Identity", as will be known to a person of skill in the art, is the relationship between two or more polynucleotide sequences or two or more polypeptide sequences, as determined by comparing the sequences, typically along their whole length. In the art, identity also means the degree of sequence relatedness between polynucleotide or polypeptide sequences, as the case may be, as determined by the match between strings of such sequences. While there exist a number of methods to measure identity between two polynucleotide or two polypeptide sequences, methods commonly employed to determine identity are codified in computer programs.

Computational approaches to sequence alignment generally fall into two categories: global alignments and local alignments. A global alignment attempts to align every residue in every sequence and thus forces the alignment to span the entire length of all query sequences. Global alignments are most useful when the sequences in the query set are similar and of approximately equal size. A general global alignment technique is the Needleman-Wunsch algorithm, which is based on dynamic programming. In contrast, local alignments identify regions of similarity within long sequences that can be widely divergent overall. Local alignments are often preferable, but can be more difficult to calculate because of the additional challenge of identifying the regions of similarity. Local alignments are more useful for dissimilar sequences that are suspected to contain regions of similarity or similar sequence motifs within a larger sequence. The Smith- Waterman algorithm is a general local alignment method and is also based on dynamic programming. With sufficiently similar sequences, there is no difference between local and global alignments. Hybrid methods, known as semiglobal or "glocal" (short for global-local) methods, attempt to find the best possible alignment that includes the start and end of one or the other sequence. This can be especially useful when the downstream part of one sequence overlaps with the upstream part of the other sequence. Preferred computer programs to determine identity between two sequences include, but are not limited to, BLAST (Altschul et al., J. Mol. Biol. 215, 403 (1990), available at http://blast.ncbi.nIm.nih.gov BIast.cgi), including BLASTp (for proteins), BLASTn and BLASTx (for nucleotides), gapped BLAST and PSI-BLAST (for proteins, Altschul et al., Nucleic Acids Research 25 (17): 3389-402, 1997), FASTA (available at htt : // www.ebi .ac .uk/Tools/sss ) . CiustalW/CIustalX (Thompson et al., Nucleic Acids Research 22 (22): 4673-4680 (1994), latest version is 2.1) and the GCG program package (Devereux et al., Nucleic Acids Research, 12, 387 (1984)). The Clustal program can be used to compare both nucleotide and amino acid sequences. This program compares sequences and finds the optimal alignment by inserting spaces in either sequence as appropriate. It is possible to calculate amino acid identity or similarity (identity plus conservation of amino acid type) for an optimal alignment. A program like BLASTx will align the longest stretch of similar sequences and assign a value to the fit. It is thus possible to obtain a comparison where several regions of similarity are found, each having a different score. Both types of identity analysis are contemplated in the present invention.

The percent identity of two amino acid sequences or of two nucleic acid sequences can be determined by aligning the sequences for optimal comparison purposes (e.g., gaps can be introduced in the one sequence for best alignment with the other sequence) and comparing the amino acid residues or nucleotides at corresponding positions. The "best alignment" is an alignment of two sequences which results in the highest percent identity. The percent identity is determined by the number of identical amino acid residues or nucleotides in the sequences being compared (i.e., % identity = number of identical positions/total number of positions x 100).

As described above, determination of percent identity between two sequences can be accomplished using a mathematical algorithm known to those of skill in the art. An example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990), modified as in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993). The BLASTn and BLASTx programs of Altschul et al., J. Mol. Biol. 2Ϊ5, 403 (1990) have incorporated such an algorithm,, BLAST nucleotide searches can be performed with the BLASTn program, score = 100, wordlength ~ 12 to obtain nucleotide sequences homologous to nucleic acid molecules of the invention. BLAST protein searches can be performed with the BLASTp program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilised as described in Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402. Alternatively, PSI-Blast can be used to perform an iterated search which detects distant relationships between molecules (Id.). When utilising BLAST, Gapped BLAST, and PSI- Blast programs, the default parameters of the respective programs (e.g., BLASTx and BLASTn) can be used. See http://www.ncbi.nlm.nih.gov. Another example of a mathematical algorithm utilised for the comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). The ALIGN program (version 2.0) which is part of the CGC sequence alignment software package has incorporated such an algorithm. Other algorithms for sequence analysis known in the art include ADVANCE and ADAM as described in Torellis and Robotti (1994) Comput. Appl. Biosci., 10 :3-5; and FASTA described in Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85:2444-8. Within FASTA, ktup is a control option that sets the sensitivity and speed of the search.

Typically, the nucleotide sequences used in the invention have at least 70% identity, using the default parameters of the BLAST computer program (Atschul et al., J. Mol. Biol. 215, 403- 410 (1990)) provided by HGMP (Human Genome Mapping Project), at the nucleotide level, to the nucleotide sequences described herein. More typically, the nucleotide sequence has at least 75%, 80%, 82%, 83%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity, at the nucleotide level, to the nucleotide sequences described herein. In some embodiments, the nucleotide sequence has 83.6%, 93.8% or 97.3% identity to the nucleotide sequence shown in Figure 2A (SEQ ID NO: 1).

The exogenous nucleic acid encoding Annexin-Al is expressed in the T cells of the transgenic non-human animal of the present invention. This expression pattern is typically achieved by the nucleic acid encoding Annexin-Al being under the control of a suitable promoter, i.e. a T cell-specific promoter. A suitable T cell-specific promoter for use in the invention is the CD2 promoter. This promoter drives expression in both immature and mature T cells. The human or mouse CD2 promoter can be used. Alternatively, one of the promoters of the human T- cell- or lymphocyte-specific gene, lck, can be used. This gene encodes a tyrosine kinase and is a member of the src family . The promoters preferentially drive the expression of transgenic genes at the level of immature T cells (thymocytes).

A promoter for use in the invention can be a constitutive promoter or an inducible promoter. The promoter is typically a constitutive promoter, but an inducible promoter can alternatively be used such that the T cell specific overexpression of Annexin-Al occurs in response to a particular stimulus. A nucleic acid according to the invention is suitably inserted into a vector for expression in the transgenic non-human animal of the invention. The vector is typically an expression vector that contains nucleic acid sequences as defined above. The term "vector" or "expression vector" generally refers to any nucleic acid vector which may be RNA, DNA or cDNA. The term "expression vector" may include, among others, chromosomal, episomal, and virus-derived vectors, for example, vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids. Generally, any vector suitable to maintain, propagate or express nucleic acid to express a polypeptide in a host can be used for expression in this regard. Expression vectors will include various genetic elements, including but not limited to an origin of replication, a suitable promoter as defined above (typically a T cell-specific promoter) and/or enhancer and/or a locus control region (LCR), and also any necessary ribosome binding sites, polyadenylation regions, splice donor and acceptor sites, transcriptional termination sequences, and 5'-flanking non-transcribed sequences that are necessary for expression. Optionally, an expression vector for use in the invention can include a selectable marker to permit isolation of vector containing cells after exposure to the vector. The expression vectors can also include selectable markers, such as antibiotic resistance, which enable the vectors to be propagated. In one embodiment, the expression vector for use in the invention includes an origin of replication, a promoter and an LCR. As described above, the promoter is typically a T cell- specific promoter, for example a CD2 promoter or an LCK promoter.

The vector used in the Examples herein is the VACD2 vector having the structure shown in Figure 4. The vector includes components of the prokaryotic pBS SK- vector, the human CD2 promoter and LCR, as well as the exogenous nucleic acid encoding Anx-Al . However, any suitable vector can be used. The nucleic acid encoding Annexin-Al is typically stably integrated into the genome of the transgenic non-human animal of the invention. In this embodiment, the cells of the offspring of the transgenic non-human animal of the present invention also contain the same exogenous nucleic acid encoding Annexin-Al as cells of the transgenic non-human animal of the present invention.

The transgenic non-human animal of the present invention can be prepared by any suitable method known in the art. Suitable methods include but are not limited to pronuclear injection, methods involving transformation of embryonic stem cells and somatic cell nuclear transfer.

In one embodiment, the transgenic non-human animal of the present invention is prepared by the technique of pronuclear microinjection. This technique is well known in the art and was first described by Gordon et al (Proc Natl Acad Sci U S A. 1980 December; 77(12): 7380- 7384). The technique involves directly injecting DNA into the pronucleus of a fertilized egg and then implanting the egg into an animal such as a mouse. In the present invention, the DNA encodes Annexin-Al and is under the control of a T cell-specific promoter. As described herein, the DNA is typically in the form of a vector.

In an alternative embodiment, the transgenic non-human animal of the present invention is prepared by transforming embryonic stem cells with a DNA sequence of interest, injecting the embryonic stem cells into the inner cell mass of a blastocyst and then implanting the blastocyst into an animal such as a mouse. In the present invention, the DNA encodes Annexin-Al and is under the control of a T cell-specific promoter. As described herein, the DNA is typically in the form of a vector.

In a further alternative embodiment, the transgenic non-human animal of the present invention is prepared using a nuclear transfer cloning procedure, typically somatic cell nuclear transfer (SCNT), This technique was first described in relation to the cloning of "Dolly" the sheep by Campbell et al. (Nature 380 (6569): 64-6 (1996)). The technique involves removing the nucleus from a donor cell and inserting it into an enucleated oocyte (the recipient). Alternatively, the entire donor cell may be fused with the enucleated oocyte. This allows the donor nucleus to be reprogrammed by the cytoplast of the enucleated oocyte. The oocyte is then allowed to develop and eventually goes on to form a blastocyst. In preparing a transgenic non-human animal of the invention, the non-human animal may be subjected to further transgenesis, in which the transgenesis is the introduction of an additional gene or genes or a protein-encoding nucleic acid sequence or sequences that encode proteins other than Annexin-Al. The transgenesis may be carried out by any of the methods described herein for producing the transgenic non-human animal of the invention, but the additional exogenous nucleic acid encodes a different protein from Annexin-Al.

In one embodiment, the present invention provides a transgenic non-human animal model for obsessive compulsive disorder (OCD) or a disease related to OCD wherein an exogenous nucleic acid encoding Annexin-Al is expressed in the T cells of said animal. In one embodiment, Annexin-Al is overexpressed in human T cells using adenoviral, retroviral or electroporation methods. The human T cells are then transferred into an immunodeficient non-human animal such as an immunodeficient mouse, also known as a severe combined immunodeficiency (SCID) mouse. SCID mice, which lack both T and B lymphocytes and accept xenogenic cells, have been used for human cell transfer for evaluating the pathogenesis of a variety of human diseases. In this case, SCID mice receiving mock- transfected/transduced human T cells can be compared for their behaviour to SCID mice receiving human T cells transfected/transduced with AnxAl cDNA. In this embodiment, the exogenous nucleic acid encoding Annexin-Al typically encodes a human or mouse Annexin- Al as described in detail herein and may have any one of the sequences set out in SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 or SEQ ID NO: 15 or SEQ ID NO: 1 or SEQ ID NO: 3, or a fragment thereof such as a coding sequence, as described herein. In a second aspect, the present invention provides a method of producing a transgenic non- human animal embryo, comprising inserting an exogenous nucleic acid encoding Annexin-Al under control of a T cell-specific promoter into a fertilized egg, blastocyst or enucleated oocyte of a non-human animal. As described above, the method can be carried out using pronuclear microinjection, by transforming embryonic stem cells with a DNA sequence of interest and injecting the embryonic stem cells into the inner cell mass of a blastocyst or by using somatic cell nuclear transfer. In this embodiment of the invention, an embryo is to be construed broadly as being from the blastocyst stage up to birth. In some embodiments, the blastocyst may be stored, for example by freezing, subsequent to being produced by this method. In a third aspect, the present invention provides a method of producing a transgenic non- human animal model for obsessive compulsive disorder (OCD) or a disease related to OCD, comprising:

(a) carrying out the method of the second aspect of the invention to produce a non-human animal embryo;

(b) implanting said non-human animal embryo into a female non-human animal; and (c) causing a non-human animal to develop to term from the embryo.

The non-human animal embryo can be implanted into a female non-human animal by any suitable method, which will be known to a person of skill in the art. In one embodiment, the method of the third aspect of the invention further comprises breeding from the non-human animal. The result of this method is offspring of the non-human animal of the first aspect of the invention.

It may also be of interest to study cells derived from the transgenic non-human animal of the invention.

In a fourth aspect, the present invention therefore provides a cell line derived from the transgenic non-human animal model of the first aspect of the invention. The cell line will typically be a blood cell line, for example a cell line of any cell derived from the haematopoietic lineage. In one embodiment, the cell line is a haematopoietic stem cell line. The cell line is typically a lymphocyte cell line and is more typically a T cell line. As described herein, an exogenous nucleic acid encoding Annexin-Al is expressed in the T cells of the transgenic non-human animal of the first aspect of the invention. Accordingly, T cells derived from the transgenic non-human animal of the first aspect of the invention are useful for scientific study, for example to investigate relationships between T cells and OCD. Such a T cell line can be prepared, for example, by extracting T cells from a transgenic non-human animal model of the first aspect of the invention and then culturing these cells in vitro or by transferring them into "recipient" mice (adoptive cell transfer) to see if signs of OCD can be transferred by these cells. In both cases, T cells can be collected from spleen and lymph nodes and optionally further purified by commercially available kits. The inventors have found that overexpressing Annexin-A in the T cells of transgenic mice exacerbates the development of autoimmune diseases and, at the same time, induces the manifestation of signs of OCD and related diseases. The transgenic non-human animal model of the present invention is therefore a useful tool for studying the symptoms and progression of OCD and related diseases. The invention therefore also extends to the use of tire transgenic non-human animal of the first aspect of the invention for the study of OCD or a disease related to OCD. The invention also extends to the use of the transgenic non-human animal of the first aspect of the invention as an animal model for OCD or a disease related to OCD.

Furthermore, the transgenic non-human animal model of the present invention is a useful tool to investigate the link between T cells and the development of OCD and related diseases. The invention therefore also extends to the use of the transgenic non-human animal of the first aspect of the invention to investigate the link between T cells and the development of OCD or a disease related to OCD. For instance, investigating the gene expression profile of AnxAl¹⁸ T cells (transgenic T cells overexpressing Annexin-Al) should provide information on the specific phenotype of these cells and its impact on known neuronal pathways involved in the development of OCD. The availability of this information might also help to understand the behavioural changes observed in immunocompromised or immunosuppressed patients.

The transgenic non-buman animal model of the present invention is also a valuable tool to test for drugs for use in the treatment of OCD and related diseases.

Accordingly, in a fifth aspect, the present invention provides a method of screening a candidate molecule to determine whether said candidate molecule is effective for the treatment of OCD or a disease related to OCD comprising:

(a) administering said candidate molecule to the non-human transgenic animal model of the first aspect of the invention; and (b) detennining whether said non-human transgenic animal model exhibits reduced symptoms of OCD or a disease related to OCD after administration of said candidate molecule. If the non-human transgenic animal exhibits reduced symptoms of OCD or a disease related to OCD after administration of the candidate molecule compared to the symptoms of OCD or a disease related to OCD prior to administration of the candidate molecule, the candidate molecule is effective for the treatment of OCD or a disease related to OCD. In this embodiment, the candidate molecule can be any molecule that is suitable for administering to a human or animal for treatment of a disease. For example, the candidate molecule can be a chemical entity or a biological molecule such as an antibody. The candidate molecule can, for example, be a T cell modulating drug or an anti-psychotic or anxiolytic drug.

Step (a) of the fifth aspect of the invention comprises administering the candidate molecule to the non-human transgenic animal model of the first aspect of the invention. The candidate molecule can be administered to the animal in any suitable manner, but will typically by administered orally or by injection, for example by intravenous, intraperitoneal or subcutaneous injection. The candidate molecule can be formulated with one or more pharmacologically acceptable excipients or diluents for testing in a method according to this aspect of the invention.

Step (b) of the fifth aspect of the invention comprises determining whether said non-human transgenic animal model exhibits reduced symptoms of OCD or a disease related to OCD after administration of the candidate molecule. This step will be carried out after the candidate molecule is administered to the non-human transgenic animal model. Step (b) of this aspect of the invention is typically carried out by monitoring the behaviour of the non- human transgenic animal model after administration of the candidate molecule. For example, in a transgenic mouse model, the reduction of marble burying behaviour or digging behaviour (both proposed to reflect compulsive behaviour) after administration of the candidate molecule compared to the level of such behaviour prior to administration of the candidate molecule indicates that the candidate molecule is effective at treating OCD or a disease related to OCD.

The method of the fifth aspect of the invention is a straightforward and cost-effective method for drug screening, since the spontaneous mouse model of OCD and related disorders requires no behavioural training and no pharmacological manipulation.

As set out above, the inventors have found that overexpressing Annexin-Al in the T cells of transgenic mice exacerbates the development of autoimmune diseases. The transgenic non- human animal model of the present invention can also be used to test for drugs for use in the treatment of T cell mediated diseases.

Accordingly, in a sixth aspect the present invention provides a method of screening a candidate molecule to determine whether said candidate molecule is effective for the treatment of a T cell mediated disease comprising:

(a) administering said candidate molecule to the non-human transgenic animal model of the first aspect of the invention; and

(b) determining whether said non-human transgenic animal model exhibits reduced symptoms of the T cell mediated disease after administration of said candidate molecule.

If the non-human transgenic animal exhibits reduced symptoms of the T cell mediated disease after administration of the candidate molecule compared to the symptoms of the T cell mediated disease prior to administration of the candidate molecule, the candidate molecule is effective for the treatment of the T cell mediated disease.

The T cell mediated disease can be, for example, graft-versus-host disease, graft rejection, atherosclerosis, HIV, AIDS, psoriasis, miscarriage or an autoimmune disease. Autoimmune diseases include rheumatoid arthritis (RA), multiple sclerosis (MS)_; systemic lupus erythematosus (SLE), Addison's disease, Grave's disease, scleroderma, polymyositis, diabetes, autoimmune uveoretinitis, ulcerative colitis, pemphigus vulgaris, inflammatory bowel disease, autoimmune thyroiditis, uveitis, Behcet's disease and Sjogren's syndrome. In this embodiment, the candidate molecule can be any molecule that is suitable for administering to a human or animal for treatment of a disease. For example, the candidate molecule can be a chemical entity or a biological molecule such as an antibody. Step (a) of the sixth aspect of the invention is the same as step (a) of the fifth aspect of the invention and is thus as described herein in relation to step (a) of the fifth aspect of the invention.

Step (b) of the sixth aspect of the invention comprises determining whether said non-human transgenic animal model exhibits reduced symptoms of the T cell mediated disease after administration of the candidate molecule. This step will be carried out after the candidate molecule is administered to the non-human transgenic animal model. The symptoms could for example be symptoms of MOG 3-5s-i duced experimental autoimmune encephalomyelitis (EAE) or collagen-induced arthritis (CIA), which are mouse models of multiple sclerosis (MS) and rheumatoid arthritis (RA) respectively.

Preferred features for the second and subsequent aspects of the invention are as for the first aspect mutatis mutandis. The Anx-Al^tg mice produced by the present inventors show signs of spontaneous anxiety/OCD behaviour as seen in other genetically modified mice. However, the genetic modification introduced in these mice (i.e. overexpression of AnxAl) is specifically restricted to T cells. Therefore, these mice provide a unique opportunity to investigate how alteration in T cell functions can in turn influence the development of neuropsychiatric disorders. In these mice, Annexin-1 over-expression is strong, T cell specific, copy-number dependent and genome integration position independent.

In one embodiment, the present invention provides a transgenic mouse model for obsessive compulsive disorder (OCD) or a disease related to OCD wherein the cells of said mouse contain an exogenous nucleic acid encoding Annexin-Al and comprising nucleotides 4 to 1038 of the nucleotide sequence shown in Figure 2A (SEQ ID NO: 1) and wherein said exogenous nucleic acid encoding Annexin-Al is expressed in the T cells of said animal. The transgenic mouse overexpresses Annexin-Al in its T cells. In one embodiment, the exogenous nucleic acid encoding Annexin-Al is under control of the human CD2 promoter. In one embodiment, the exogenous nucleic acid further comprises a nucleotide sequence encoding a FLAG tag, for example the nucleotide sequence GATTACAAGGATGACGACGATAAG (SEQ ID NO: 30). In one embodiment, the cells of said mouse contain an exogenous nucleic acid having the sequence shown in Figure 3B (SEQ ID NO: 32).

In one embodiment, the transgenic mouse is generated by inserting C-terminus FLAG-tagged Anx-Al into the mouse genome using the technique of pronuclear microinjection and using a VACD2 cassette vector. In one embodiment, the C-terminus FLAG-tagged Anx-Al is inserted into the mouse genome as part of a vector, typically the VACD2 vector which is 15425 bp in length and contains the appropriate elements for in vivo expression of the inserted gene in mouse. The vector typically includes the human CD2 Locus Control Region (hCD2LCR), the human CD2 promoter and the prokaryotic pBS SK- vector. In one embodiment, restriction enzymes such as Notl and Sail are used to remove the prokaryotic sequences from the pBS SK- vector and a restriction enzyme such as Smal used to insert the Anx-Al FLAG gene. After removal of prokaryotic sequences, the construct is typically inserted into the mouse genome by pronuclear microinjection. Briefly, the (optionally purified) construct is microinjected into the pronucleus of embryos, typically embryonic day 0.5 (E0.5) embryos. Microinjected embryos that survive injection are transferred to the oviduct of a foster mother, typically an E0.5 pseudopregnant foster mother. Offspring are then born approximately 20 days later.

The present invention will now be further described by way of reference to the following Examples which are present for the purposes of illustration only. In the Examples, reference is made to a number of Figures in which:

FIGURE 1 is a ribbon diagram of annexin-1 structure showing the four annexin repeats and the N-terminal domain.

FIGURE 2 A shows (i) the nucleotide sequence (SEQ ID NO: 1) and (ii) the amino acid sequence (SEQ ID NO: 2) of mouse Annexin-1 (Anx-Al), isoform Anxal-001. Figure 2B shows (i) the nucleotide sequence (SEQ ID NO: 3) and (ii) the amino acid sequence (SEQ ID NO: 4) of mouse Armexin-1 (Anx-Al), isoform Anxal-201. Figure 2C shows (i) the nucleotide sequence (SEQ ID NO: 5) and (ii) the amino acid sequence (SEQ ID NO: 6) of rat Annexin-1 (Anx-Al), isoform Anxal . Figure 2D shows (i) the nucleotide sequence (SEQ ID NO: 7) and (ii) the amino acid sequence (SEQ ID NO: 8) of rat Annexin-1 (Anx-Al), isoform D3ZVZ4_RAT. Figure 2E shows (i) the nucleotide sequence (SEQ ID NO: 9) and (ii) the amino acid sequence (SEQ ID NO: 10) of human Annexin-1 (Anx-Al), isoform ANXA1-003. Figure 2F shows (i) the nucleotide sequence (SEQ ID NO: 11) and (ii) the amino acid sequence (SEQ ID NO: 12) of human Annexin-1 (Anx-Al), isoform ANXA1-002. Figure 2G shows (i) the nucleotide sequence (SEQ ID NO: 13) and (ii) the amino acid sequence (SEQ ID NO: 14) of human Annexin-1 (Anx-Al), isoform ANXA1-004. Figure 2H shows (i) the nucleotide sequence (SEQ ID NO: 15) and (ii) the amino acid sequence (SEQ ID NO: 16) of human Annexin-1 (Anx-Al), isoform ANXA1-006.

FIGURE 3 shows the generation of the Anx-Al FLAG. (A) Agarose gel. analysis of Anx-Al FLAG PCR product (1041 bp) generated from RAW264.7 cell cDNA. (B) Sequence of Anx-Al FLAG (SEQ ID NO: 32) showing the Anx-Al coding sequence (between start codon and FLAG epitope), the Smal (and isoschizomer Xmal) recognition sites (CCCGGG, SEQ ID NO: 33), Kozak sequence (GCCACC, SEQ ID NO: 31), Anx-Al FLAG start and stop codons (ATG and TAG respectively) as well the coding sequence for the incorporated FLAG epitope (GATTACAAGGATGACGACGATAAG, SEQ ID NO: 30). The forward and reverse primers are also shown. The resulting expressed protein has the incorporated FLAG epitope (KDDDDKYD, SEQ ID NO: 17) at the C-terminus of the protein.

FIGURE 4 (A) shows the VACD2/FLAG Anx-Al construct used for the generation of Anx-Al transgenic mice. The VACD2 vector is 15425 bp in length and contains the appropriate elements for in vivo expression of the inserted gene in mouse. Shown here, the human CD2 Locus Control Region (hCD2LCR), the human CD2 promoter and the prokaryotic pBS SK- vector. Enzymes that were used to manipulate the vector prior to microinjection to the mouse genome are shown. Notl and Sail were used to remove the prokaryotic sequences and Smal used to insert the Anx-Al FLAG gene. (B) is a schematic representation of the VACD2 Anx-Al Flag construct showing the CD2 promoter region, the inserted Anx-Al Flag cDNA and the locus control region (LCR). The figure also shows a schematic representation of the Anx-Al Flag with the 4 annexin repeats (I-II-III-IV) and the amino acid sequence of the Flag epitope used to tag the protein.

FIGURE 5 shows that Anx-Al^tg mice exhibit an increase in marble burying behavior. (A) C57/BL6 and AnxAl^tg male mice (6-8 weeks old) were tested for the marble- burying behavior as detailed in Material and Methods. Data are means ± SEMs. N =8-10 mice per value. ** P<0.01 compared with C57/BL6 wild-type controls. (B) Representative pictures of the experimental setting used for the marble burying test: cage with marble before the experiment (Start), cage after the test with C57 BL6 (C57/BL6) or AnxAl'^s (AnxAl^tg) mice.

FIGURE 6 shows that Anx-Al ^tg mice exhibit an increased digging behavior. (A) C57/BL6 and AnxAl*⁸ male mice (6-8 weeks old) were tested for the digging behavior as detailed in Material and Methods. Data are means ± SEMs. N =8-10 mice per value. ** P<0.01 compared with C57 BL6 wild-type controls. (B) Representative pictures showing the typical behavior C57/BL6 (C57 BL6) or AnxAl'⁸ (AnxAl^tg) mice in their home cage.

FIGURE 7 shows T cell repertoire and response of Anx-Al^t T cells. (A) Spleen (top panels) and lymph node lymphocytes from wt C57 BL6 and Anx-Al^t8 mice were analysed for CD4 and CD8 expression. Numbers in dot plots indicate the percentage of cells falling in the quadrant for the respective marker (CD4 and CD8). Results are representative of 4 experiments. (B) Naive CD4+ T cells were stimulated with indicated concentrations of plate-bound anti-CD3/CD28 for 12-16 hrs and then analyzed for their profile of CD25 and CD69 activation markers. Numbers in dot plots indicate the percentage of CD25/CD69 double positive. Results are representative of 3 experiments.

FIGURE 8 shows increased development of MOG35-55-induced EAE in Anx-Al^¾ mice. C57BL/6 or AnxAl¹⁸ mice were immunized with MOG35-55 in CFA as described in Materials and Methods and monitored daily for signs of EAE (A) and for weight gain/loss (B) for 22 days. Results are means ± SEM (n = 6/group). *** p < 0.001 vs C57/BL6 controls. Example 1 - Generation of Anx-Al^¾ mice

Transgenic mice overexpressing Anx-Al (Anx-Al ^tg) in T cells were generated by inserting C- terminus FLAG-tagged Anx-Al into the mouse genome using a VACD2 cassette vector and the technique of pronuclear microinjection. Briefly, the murine Anx-Al gene was amplified and tagged with the FLAG epitope and then cloned into the pcDNA3.1 vector. The Anx-Al FLAG was recovered from the pcDNA3.1 vector and then ligated into linearised VACD2 vector. The VACD2 Anx-Al FLAG construct was then modified and purified, then inserted into the mouse genome by pronuclear injection. The methods will now be described in detail. Generation of FLAG tagged version of Anx-Al - Polymerase chain reaction (PCR)

In order to differentiate the overexpressed Anx-Al from the endogenous one, a C-terminal FLAG-tagged version of Anx-Al was cloned using the primers (Thermo Electron, UK) shown in Table 1. For each reaction the following reagents were combined (final volume 30 μΐ): 1 μΐ cDNA template (containing ~1 g cDNA), 17.2 μΐ water, 3 μΐ ΙΟχ PCR buffer, 2.4 μΐ MgCl₂, 2.4 μΐ dNTP mix, 1 μΐ sense primer (100 pmol/μΐ), 1 μΐ anti-sense primer (100 pmol/μΐ), 1 μί Taq DNA polymerase (all sourced from Promega, UK). The primer pairs used and the reaction conditions are shown in Table 1. PCR was performed with sense and antisense oligonucleotides containing the sequence CCCGGG (SEQ ID NO: 33), a recognition site for restriction endonuclease Smal to be incorporated just before the start codon (ATG) and after the stop codon (TAG) of the Anx-Al gene. In between the start codon and the Smal site a Kozak sequence was inserted to enhance transcription. Finally and most importantly the sequence for the FLAG epitope was incorporated in frame with the coding sequence. Figure 3 A shows agarose gel analysis of the Anx-Al FLAG PCR product. Figure 3B shows the sequence of Anx-Al FLAG (SEQ ID NO: 32) showing the Anx-Al coding sequence, the Smal (and isoschizomer Xmal) recognition sites, Kozak sequence, Anx-Al FLAG start and stop codons as well the coding sequence for the incorporated FLAG epitope. The forward and reverse primers are also shown. Table 1. Primers used for the amplification of Anx-Al FLAG and reaction conditions.

(Top) The CCCGGG sequence (SEQ ID NO: 33) is the recognition site for Smal (and its isoschizomer Xmal), the KOZAK sequence GCCACC (SEQ ID NO: 31) is contiguous with the ATG start codon. The sequence CTACTTATCGTCGTCATCCTTGTAATC (SEQ ID NO: 34) includes the coding sequence for the FLAG epitope DYKDDDD (SEQ ID NO: 35) and a stop codon and was incorporated in frame with the Anx-Al coding region. (Bottom) PCR conditions used for the reaction.

Table 1

Oligonucleotides

(5'-3')

TCCCCCGGGGGAGCCACCATGGCAATGGTATCAGAATTCCTC (SEQ [D NO: 36] Sense

TCCCCCGGGGGACTACTTATCGTCGTCATCCTTGTAATCGTTTCCACCACACAGAGCC (SEQ ID NO: 37) Anti- sense

PCR reaction conditions

Temperature No. of cycles Step

94°C, 4min xl Denaturation

94° C, lmin x35 Denaturation/extension

55°C, lmin x35 Primer annealing

72°C, lmin x35 Product elongation

72°C, 7min xl Elongation extended

Cloning of FLAG tagged Anx-Al into pcDNA3.1 vector

Annexin-1 FLAG cDNA was cloned into the Smal site of pcDNA3.1 (+) vector (Invitrogen). The PCR product and the host vector pcDNA3.1 were digested with Smal for 2 hours. Both the insert and the linearized vector digest products were analysed and purified by agarose gel electrophoresis (1 % agarose gels, TAE).

The two linearized fragments to be ligated were treated with 10 units of bovine alkaline phosphatase (Calf Intestine Phosphatase (CIP) Roche) in 1 Ox enzyme buffer for 1 hour at 37 °C. This dephosphorylates the ends of the fragments (removal of 5' phosphates), prevents re- ligation and improves ligation efficiency. CIP-treated DNA were quantified and ligated using high concentrated T4 Ligase enzyme (NEB, UK) and molar ratio of vector to insert 1:3, 1 :1 and 3:1. The reaction contained 10-50 ng of vector, 5 x ligase buffer, 5 units of T4 DNA ligase and water up to 20 μΐ. The reactions were incubated for 16 hours at RT. Mock ligation controls were always included using only the vector or only the insert. Subcloning into the VACD2 cassette

The VACD2 vector (kindly provided by D.Kioussis, National Institute for Medical Research, London) contains the human CD2 promoter and the locus control region of the human CD2 gene conferring tissue specific expression of the adjacent gene after integration into the mouse genome.

The Anx-Al FLAG was recovered from pcDNA3.1 vector following Smal digestion that produces 'blunt-ended' fragments as well as by Xmal (isoschizomer) digestion that produces 'sticky-ends'. The VACD2 vector was linearised with Smal and Xmal digestion. Both digestions were performed as follows; 1 μg of DNA (in 5 μί) was mixed with 1 μΐ of Smal (or Xmal) enzyme (10 units, NEB, UK), 2 μΐ ΙΟχ enzyme buffer and 12 μΐ of water. The samples were incubated for 1 h at 25 °C and separated on 0.8 % agarose gels. The 15.4 kb linearised fragment of the VACD2 vector and the 1 kb Anx-Al FLAG insert were gel purified.

The two fragments were quantified and treated with 10 units of CIP. Ligation of blunt ended DNA fragments was performed at molar ratio of vector to insert 1:3, 1:1 and 3:1. The reaction contained 50 ng of vector, 5 x ligase buffer, 5 units of T4 DNA ligase and water. Ligation reactions were incubated at RT for 16 hours. Mock ligation controls were included using only vector and only insert.

Pronuclear microinjection and production of transgenic mice

Microinjection in CBAxC57BL/6 mice was performed in the Transgenic core facility of Queen Mary University of London (http://www.icms.qmul.ac.uk/corefacilities/transgenic/index.html). The VACD2 Anx-Al FLAG construct was modified by removal of prokaryotic sequences and purified for pronuclear microinjection into the mouse genome. The purified construct was microinjected into the pronucleus of embryonic day 0.5 (E0.5) embryos. Microinjected embryos that survived injection were transferred to the oviduct of an E0.5 pseudopregnant foster mother. Offspring were then born approximately 20 days later.

Example 2 - Testing for disease in Anx-Al mice Materials and Methods

Marble-burying behaviour

Fifteen glass marbles were placed, evenly spaced in 5 rows of 3, on a 5 cm layer of sawdust bedding, lightly pressed down to make a flat even surface, in a plastic cage approximately 20 x 30 cm. A mouse is placed in each cage and left for 30 min after which the number of marbles buried (to 2/3 their depth) with sawdust is counted.

Digging Test

Mice (male or female; 6-7 weeks of age) were observed for their burying/digging performance in cages filled with 5 cm layer of sawdust bedding, lightly pressed down to make a flat even surface.. Measurements included the latency to start digging and the number of digging bouts. Test duration was 15 min. MOG^.^-induced experimental autoimmune encephalomyelitis (EAE)

Male C57/BL6 mice were immunized with MOG3_3-5₅/CFA as previously described (Paschalidis et al., J Neuroinilarnmation. 2009; 6:33). Briefly, mice were immunized subcutaneously on day 0 with 300μ1 of emulsion consisting of 300 g of MOG35-55 in PBS combined with an equal volume of CFA containing 300pg heat-killed M. tuberculosis H37Ra. The emulsion was injected in both flanks and followed by an intraperitoneal injection of B. pertussis toxin (500ng 100 μΐ) in 100 μΐ of saline on days 0 and 2. Mice were observed daily for signs of EAE and weight loss. Disease severity was scored on a 6-point scale: 0 = no disease; 1 = partial flaccid tail; 2 =complete flaccid tail; 3 = impaired righting reflex; 4 = partial hind limb paralysis; 5 = complete hind limb paralysis; 6 =moribund or dead animal.

Results

The results of these experiments are as follows:

1. Using the marble-burying test (MBT), an animal model proposed to reflect compulsive behaviour, it was observed that Anx-Al*⁸ mice show a significant increase in the number of buried marbles compared to wild-type C57/BL6 mice (Figure 5). 2. Measuring the digging behaviour in their home cage, it was found that Anx-Al ^g mice a reduced latency and increased duration of digging compared to wild-type C57/BL6 mice (Figure 6). 3. T cells from Anx- A 1 ^,g mice have a reduced threshold of activation compared to T cells from wild-type C57/BL6 mice (Figure 7).

4. Using a mouse model of multiple sclerosis, the experimental autoimmune encephalomyelitis (EAE), it was found that Ahx-Al^tg mice show a significant increase development of sign of disease compared to wild-type C57 BL6 mice (Figure 8).

Together these results suggest that overexpression of Anx-Al in T cells exacerbates the development of autoimmune diseases and, at the same time, induces the manifestation of signs of OCD and related diseases.

Claims

1. A transgenic non-human animal model for obsessive compulsive disorder (OCD) or a disease related to OCD wherein the cells of said animal contain an exogenous nucleic acid encoding Annexin-Al and wherein said exogenous nucleic acid encoding Annexin-Al is expressed in the T cells of said animal.

2. The transgenic non-human animal model o claim 1, wherein the non-human animal is a mammal.

3. The transgenic non-human animal model of claim 2, wherein the mammal is a rodent.

4. The transgenic non-human animal model of claim 3, wherein the rodent is a mouse or rat.

5. The transgenic non-human animal model of claim 4, wherein the rodent is a mouse.

6. The transgenic non-human animal model of any one of the preceding claims, wherein the exogenous nucleic acid sequence encoding Annexin-Al comprises (a) nucleotides 4 to 1038 of the nucleotide sequence shown in Figure 2 A (SEQ ID NO: 1); (b) a nucleotide sequence having at least 70% identity to nucleotides 4 to 1038 of the nucleotide sequence shown in Figure 2 A (SEQ ID NO: 1); (c) a nucleotide sequence encoding the amino acid sequence shown in Figure 2A (SEQ ID NO: 2) or (d) a nucleotide sequence having at least 70% identity to a nucleotide sequence encoding the amino acid sequence shown in Figure 2A (SEQ ID NO: 2).

7. The transgenic non-human animal model of any one of the preceding claims, wherein said exogenous nucleic acid encoding Annexin-Al iurther encodes a tag or marker.

8. The transgenic non-human animal model of any one of the preceding claims, wherein said exogenous nucleic acid encoding Annexin-Al is under control of a T cell specific promoter.

9. The transgenic non-human animal model of claim 8, wherein the T cell specific promoter is the CD2 promoter.

10. The transgenic non-human animal model of any one of the preceding claims, wherein the disease related to OCD is selected from the group consisting of trichotillomania, dermatillomania, Tourette's Syndrome (TS), Asperger's syndrome, anorexia, bulimia, depression, panic disorder, panic attacks, bipolar disorder, hypochondriasis, post-traumatic stress disorder (PTSD), social anxiety disorder, schizophrenia, attention deficit hyperactivity disorder (ADHD) and body dysmorphic disorder (BDD).

11. A method of producing a transgenic non-human animal embryo, comprising inserting an exogenous nucleic acid encoding Annexin-Al under control of a T cell-specific promoter into a fertilized egg, blastocyst or enucleated oocyte of a non-human animal.

12. The method of claim 11, wherein the method is carried out using pronuclear microinjection.

13. The method of claim 11 , wherein the method is carried out by transforming embryonic stem cells with a DNA sequence of interest and injecting the embryonic stem cells into the inner cell mass of a blastocyst.

14. The method of claim 11, wherein the method is carried out using somatic cell nuclear transfer.

15. A method of producing a transgenic non-human animal model for obsessive compulsive disorder (OCD) or a disease related to OCD, comprising:

(a) carrying out the method of any one of claims 11 to 14 to produce a non-human animal embryo;

16. The method of claim 15, further comprising breeding from the non-human animal.

17. A cell line derived from the transgenic non-human animal model of any one of claims 1 to 10.

18. The cell line of claim 17, which is a T cell line.

19. A method of screening a candidate molecule to determine whether said candidate molecule is effective for the treatment of OCD or a disease related to OCD comprising:

(a) administering said candidate molecule to the non-human transgenic animal model of any one of claims 1 to 10; and

(b) determining whether said non-human transgenic animal model exhibits reduced symptoms of OCD or a disease related to OCD after administration of said candidate molecule.

20. The method of claim 19, wherein the disease related to OCD is selected from the group consisting of trichotillomania, dermatillomania, Tourette's Syndrome (TS), Asperger's syndrome, anorexia, bulimia, depression, panic disorder, panic attacks, bipolar disorder, hypochondriasis, post-traumatic stress disorder (PTSD), social anxiety disorder, schizophrenia, attention deficit hyperactivity disorder (ADHD) and body dysmorphic disorder (BDD).

21. A method of screening a candidate molecule to determine whether said candidate molecule is effective for the treatment of a T cell mediated disease comprising:

(b) determining whether said non-human transgenic animal model exhibits reduced symptoms of a T cell mediated disease after administration of said candidate molecule.

22. The method of claim 21, wherein the T cell mediated disease is selected from the group consisting of graft-versus-host disease, graft rejection, atherosclerosis, HIV, AIDS, psoriasis, miscarriage and autoimmune diseases.

23. The method of claim 22, wherein the autoimmune disease is selected from the group consisting of rheumatoid arthritis (RA), multiple sclerosis (MS), systemic lupus erythematosus (SLE), Addison's disease, Grave's disease, scleroderma, polymyositis, diabetes, autoimmune uveoretinitis, ulcerative colitis, pemphigus vulgaris, inflammatory bowel disease, autoimmune thyroiditis, uveitis, Behcet's disease and Sjogren's syndrome.

24. Use of the non-human transgenic animal model of any one of claims 1 to 10 for the study of OCD or a disease related to OCD.