WO2005067708A2

WO2005067708A2 - Zebrafish model for autoimmune diseases

Info

Publication number: WO2005067708A2
Application number: PCT/GB2005/000082
Authority: WO
Inventors: Ruth Barrett; Paul Goldsmith; Angeleen Fleming
Original assignee: Daniolabs Limited
Priority date: 2004-01-14
Filing date: 2005-01-13
Publication date: 2005-07-28
Also published as: WO2005067708A3

Abstract

Generation and use of animal models, including fish such as zebrafish, for instance in assays to identify and investigate genes and substances involved in physiological processes and in disease and disease treatment, identification and use of drug targets. Fluid from a second animal species is administered to a non-human test animal to modify one or more aspects of a physiological process or to induce one or more symptoms or signs of disease in the test animal.

Description

ANIMAL MODELS AND USES THEREOF

The present invention relates to generation and use of animal models, for instance in assays to identify and investigate genes and substances involved in physiological processes and in disease and disease treatment, identification and use of drug targets. In particular, the present invention relates to generation and use of model animals for physiological processes and diseases. The invention further relates to use of fish, e.g. zebrafish, as models for physiological processes and for diseases.

Our understanding of the physiological processes of the animal body, in particular the components and mechanisms underlying them, is still far from complete. This is true for certain physiological processes more than others. For example, various phenomena may be due to secreted factors which are hard to identify. As an example, during the induction of sleep, an unknown hormone may be released into the CSF. By administering samples of CSF to zebrafish, and then iterating through particular fractions of that CSF, each time assessing the resulting zebrafish phenotype, it is possible to identify the active constituent of the CSF.

The same is true for diseases of the animal body, some of which have been well characterized, while the mechanisms underlying others are far from clear. For example, in a paraneoplastic condition, effects remote from the cancer are seen. It is possible to identify the circulating factor responsible for these effects by administering fractions of plasma from patients to zebrafish, gradually narrowing down the fraction to the active constituent.

For example, autoimmune conditions are major causes of morbidity and mortality. Normally, immunological tolerance to one's own tissue develops such that the body doesn't produce antibodies directed against itself. Complex control mechanisms exist to keep the potential development of ^λforbidden clones' of cells that could produce antibodies directing against self in check. If this breaks down, autoimmunity develops - the loss of tolerance to self antigens .

Autoantibodies have been detected against a whole range of antigens involving both specific organs (e.g. intrinsic factor antibodies in pernicious anaemia) and multiple tissues and organs (e.g. anti-nuclear factor antibodies in systemic lupus erythematosis) (Chiorazzi 2003).

Disease may ensue through several different mechanisms (Isenberg and Morrow 1995), including: 1. Immune complex formation, in which antibody-antigen^' complexes deposit in various tissues, such as the renal glomeruli . 2. Sensitised T cells directly injure cells, or release lymphokines to indirectly damage the cells . 3. Circulating autoantibodies recognise and bind to antigens resulting in release of inflammatory mediators, complement activation, cytotoxic cell activation, blockade of normal receptor function. As well as classical autoimmune conditions such as rheumatoid arthritis or pernicious anaemia, many other diseases are recognised to have an autoimmune component which may initiate, exacerbate or propagate the disease process (e.g. ulcerative colitis and multiple sclerosis) .

In order to fully understand the components and mechanisms underlying physiological processes, and assess factors that interact with and affect those components and mechanisms, it is essential to have models of physiological processes which recapitulate key aspects of the processes in humans.

Similarly, in order to fully understand the pathogenesis, develop new treatments or assess existing treatments for diseases in general, it is essential to have disease models which recapitulate key aspects of the disease process in humans. For example, it is possible to try and identify which causative agents are responsible for a disease effect through the administration of isolated human sera or isolated human antibodies, to cultures of cells in vitro . However, this comes with all of the limitations of in vitro biology, with cells not being present in their natural state.

To continue the example, for many autoimmune diseases, animal models have been created. The standard method of modelling autoimmune disease in vivo is to introduce a component of the tissue of interest from a foreign species to induce the test animal to develop antibodies against this tissue. These antibodies may then cross-react with the self-tissue. A typical example is in attempts to model multiple sclerosis through the injection of foreign myelin basic protein, a component of the myelin sheath, since demyelination is an important feature of the disease process in humans. For example, myelin basic protein from cows may be injected into rodents to induce autoimmune disease in rodents.

The advantage of using mammalian models is their greater similarity to humans; however a major problem with such mechanisms of disease modelling is that the induced antibodies are not necessarily the same as the actual pathogenic antibodies in humans. Indeed it can be very difficult to identify what exactly is the pathogenic antibody (s), as:

1. Measured antibodies may be epiphenomenon and not directly responsible for the disease effect (O'Connor, Chitnis et al. 2003) . 2. It may be a particular combination or set of antibodies which are responsible for the disease effect.

It is therefore difficult to accurately model autoimmune disease in particular, and diseases in general. For example, experimental allergic encephalomyelitis is currently the standard animal model for multiple sclerosis, yet differs substantially from the human disease state (T Hart and Amor 2003) .

While the identification of genes involved in a physiological process is useful in helping to elucidate the components involved in the process, it does not necessarily lead to a complete understanding of the underlying events and/or mechanism (s) . It would be very useful to have a model system in which screens can be carried out to identify a gene or substance that affects or modifies a physiological process. Such a system would be particularly relevant to human physiological processes if performed in a vertebrate and in vivo.

Similarly, while the identification of disease causing genes is useful in helping elucidate the mechanisms of disease, it does not necessarily lead to strategies for treatment. It would be very useful to have a model system in which screens can be carried out to identify a gene or substance to cure a previously existing disease. Such a system would be ' particularly relevant to human disease if performed in a vertebrate and in vivo .

It would therefore be desirable to have an in vivo system to accurately model physiological processes and diseases, for example, an in vivo system to model an autoimmune disease. Provided by the present invention are animal (e.g. fish) models for physiological processes and diseases which are not only representative of the underlying process or disease, but are also particularly amenable for use in subsequent screening. This allows in turn the identification of genes or substances involved in physiological processes and in disease and disease treatment, and the identification of drug targets and human therapeutics.

The invention is generally applicable to any of a variety of physiological processes, diseases and disorders, and a range of examples is specifically set out herein. For example, phenomena which may be mediated by circulating or secreted factors, as yet unknown, could be analysed this way, such as sleep induction, sleep maintenance, hunger, satiation and level of sexuality.

Thus, in general, the present invention lies in providing an animal model for a physiological process or disease state and uses thereof. In general, the present invention is concerned with generating an animal model by administering a fluid from an animal species to a test animal.

The present invention provides means, specifically a non- human test animal of a first species for modelling a physiological process wherein fluid from a second animal species is administered to the non-human test animal to modify one or more aspects of the physiological process in the test animal.

The present invention further provides means, specifically a non-human test animal of a first species for modelling a disease state wherein fluid from a second animal species having the disease is administered to the non-human test animal to induce one or more symptoms or signs of the disease state in the test animal.

The present invention yet further provides means, specifically a non-human test animal of a first species for modelling an autoimmune disease state wherein fluid from a second animal species having the disease is administered to the non-human test animal to induce one or more symptoms or signs of the disease state in the test animal. The non-human test animal may have one or more modified aspects of a physiological process or one or more symptoms or signs of disease prior to the modification of the physiological process o or the induction of the disease state, respectively, to be subsequently modelled. The symptoms may be a mild form of the disease state, such that administering fluid from a second animal species having the disease results in the non-human test animal developing the full disease state.

In one embodiment the second animal species may be a human.

Alternatively the fluid may be administered with or prior to the administration of a substance which also modifies one or more aspects of a physiological process or induces one or more symptoms or signs of the disease. Preferably the substance is administered prior to the administration of the fluid. The substance may be a chemical. For example, in the case of Inflammatory Bowel Disease the substance may be trinitrobenzene sulfonic acid (TNBS) . Alternatively the substance may be dextran sulphate sodium (DSS).

The non-human test animal may have a genetic predisposition for the modified physiological process or disease state. The genetic predisposition may result in one or more modified aspects, or one or more symptoms of the disease, which result in a mild form of the disease state. Administration of fluid from a second animal species having the disease may result in the non-human test animal developing the full disease state. In animals having a genetic predisposition to the modified process or disease, the genetic condition may be introduced by means of a GAL4/UAS system.

The non-human test animal of a first species may include, but is not limited to a monkey, cow, goat, sheep, dog, cat, rabbit or rodent, for example rat or mouse.

Preferably, the non-human test animal is a non-mammalian test animal.

More preferably, the non-human test animal is a fish. Most preferably, the non-human test animal is a zebrafish. The zebrafish is an organism which combines many of the advantages of mammalian and invertebrate model systems. It is a vertebrate and thus more relevant in models of human disease than Drosophila or other invertebrates, but unlike other vertebrate models it can be used to perform genetic screens .

A number of peer reviewed papers highlight and validate the use of zebrafish as a species in which to model human disorders. [Dooley K and Zon LI (2000) Zebrafish: a model system for the study of human disease. Current Opinion in Genetics and Development 10 : 252-6 -Barut BA and Zon LI

(2000) Realising the potential of Zebrafish as a model for human disease Physiological Genomics 13: 49-51 - Fishman MC

(2001) Zebrafish: The Canonical Vertebrate. Science 294: 1290-1] . The use of vertebrates offers the opportunity to perform sophisticated analyses to identify genes and substances involved in physiological processes and to identify genes and processes involved in disease.

The inventors have appreciated that zebrafish offer the unique combination of invertebrate scalability and vertebrate modelling capabilities. They develop rapidly, with the basic body plan already having been laid out within 24 hours of fertilization. Moreover, their ex-utero development within a transparent capsule allows the easy in vivo visualisation of internal organs through a dissecting microscope. Many physiological processes and disease states can be modelled within the first week of life, at which time the embryos are only a few millimetres long and capable of living in very small volumes of fluid, for example, 100 μl of fluid. This permits analysis of individual embryos in multi-channel format, such as 96 well plate format. This is particularly useful for drug screening, with many chemicals being arranged in 96 well plate format.

Alternatively, a population of fish in a petri dish or a tank may be employed. A population of fish may be treated together, and may be tested together, e.g. via addition of one or more or a combination of test substances to the water. The zebrafish has a short maturation period of two to three months and is highly fecund, with a single pair of adults capable of producing 100 to 200 offspring per week. Both embryos and adults are small, embryos being a few mm and adults 2-3 cm long. They are cheap and easy to maintain. The ability to generate large numbers of offspring in a small place offers the potential of large scalability.

In addition to Zebrafish, other fish such as fugu, goldfish, medaka and giant rerio are amenable to manipulation, mutation and study, and use in aspects and embodiments of the present invention as disclosed herein.

Thus, fish, in particular zebrafish, have the advantages of providing for the testing of large numbers of experimental animals as well as the use or testing of small volumes of fluids or test substances. This latter feature allows not only for the testing of fluids or test substances only available in small amounts, but also allows for the testing of sub-components of fluids or test substances, such that it is possible to proceed through iterations to narrow down an active fraction or constituent of a fluid or test substance.

In preferred embodiments, the fluid to be administered to the test animal is obtained from a human. For example, in the case of models for physiological processes, the fluid may be obtained from a normal human. In the case of disease models, the fluid may be obtained from a human patient having the disease, such as an autoimmune disease. The fluid may be serum, CSF, joint aspirate, peritoneal fluid or bronchiolar lavage fluid.

In certain embodiments, the fluid is a fluid obtainable only in small amounts, for example one to a few μl, for example, CSF and joint aspirate. The fluid may also be a sub- component or fraction of a fluid, for example, a subcomponent or fraction of CSF or joint aspirate.

In preferred embodiments, the fluid comprises an antibody, a hormone, . a growth factor and/or a cytokine. Thus, the causative agent in generating the model may be, but is not limited to, an antibody, a hormone, a growth factor or a cytokine, for example, IL-6, IL-8 and/or IL-10.

In other embodiments, the fluid may be a purified or isolated sub-component or fraction of a fluid, such that the subcomponent or fraction has been separated from at least one other component or fraction of the original fluid. For example, a purified or isolated sub-component or fraction may be an antibody, a hormone, a growth factor or a cytokine, such that the active agent of the fluid may consist only of an antibody, a hormone, a growth factor or a cytokine, for example. The person skilled in the art is aware of the types of molecule that may be components of various fluids obtainable from an animal species, for example, that antibodies may be a sub-component of serum.

In one embodiment, a small volume, for example a μl, of CSF from a normal human is administered to the test animal and the effect on one or more aspects of one or more physiological processes observed. The CSF may then be fractionated and re-tested to determine the causative agent responsible for the modified aspect (s) of the physiological process. The fraction containing the causative agent may be subject to further procedures as known in the art, to purify or isolate the causative agent. In a further embodiment, a disease such as an autoimmune condition is induced in vast numbers of the test animal at once, for example in 10 000 zebrafish, as a prelude to a high-throughput drug screen.

The non-human test animals may be treated with fluid in a number of ways. The test animal may be contacted with the fluid, or the fluid may be touched or rubbed on their surface or injected into them. Where the non-human test animal is a fish, the fluid may be added to water in which the fish are. Different fluids may be added to each well of a multi-well plate, such as a 96 well plate.

In some preferred embodiments, the fluid obtained from a human patient having the disease is administered to the non- human test animal. Administration may be locally, via injection into the pericardium, joint space, peritoneum or CNS or via a nasogastric or rectal tube.

Alternatively, the non-human test animal may be bathed in medium comprising the fluid.

In one embodiment, the fluid may comprise an antibody and the antibody may be purified or concentrated from the fluid prior to administration to the test animal. In yet other embodiments, the fluid may comprise a hormone and/or a cytokine and the hormone and/or cytokine may be purified or concentrated from the fluid prior to administration to the test animal. 13

For example, serum from patients with multiple sclerosis, or CSF (containing oligocloncal bands - a diagnostic criteria for multiple sclerosis) may be administered to zebrafish, either alone or in combination with an administration of a conventional disease inducing agent such as myelin basic protein. This may be by either immersion, injection into the pericardium, peritoneum, or intracerebrally .

In another embodiment, the present invention provides an antibody molecule comprising one or more specific binding members. The antibody molecule is able to bind an antigen of the non-human test animal to which antibodies from a human with an autoimmune disease bind when administered to a non- human test animal and which result in one or more symptoms of an autoimmune disease. The specific binding members may be naturally derived or wholly or partially synthetically produced. The method includes bringing into contact a library of specific binding members according to the antigen, and selecting one or more specific binding members of the library able to bind the antigen.

The term "antibody molecule" can be construed as covering any specific binding member or substance having an antibody antigen-binding domain with the required specificity. Thus, this term covers antibody fragments and derivatives, including any polypeptide comprising an immunoglobulin binding domain, whether natural or wholly or partially synthetic. Chimeric molecules comprising an immunoglobulin binding domain, or equivalent, fused to another polypeptide are therefore included. The library may be displayed on the surface of bacteriophage particles, each particle containing nucleic acid encoding the antibody VH variable domain displayed on its surface, and optionally also a displayed VL domain if present.

Following selection of specific binding members able to bind the antigen and displayed on bacteriophage particles, nucleic acid may be taken from a bacteriophage particle displaying a said selected specific binding member. Such nucleic acid may be used in subsequent production of a specific binding member or an antibody VH variable domain (optionally an antibody VL variable domain) by expression from nucleic acid with the sequence of nucleic acid taken from a bacteriophage particle displaying a said selected specific binding member.

Physiological processes which can be modelled in the test animal can be selected from, but are not limited to, the following: sleep induction, sleep maintenance, awakening, hunger, food preference and emotional states.

Diseases which can be modelled in the test animal can be selected from, but are not limited to, the following: hypersomnolence, hyperactivity, joint disease, osteoporosis and obesity.

An autoimmune disease which can be modelled in the test animal can be selected from the following: Graves disease, Myasthenia Gravis, Lambert Eaton myasthenic syndrome, SLE, Pernicious anaemia, Autoimmune haemolytic anaemia, Vitiligo, Alopecia, Anklosing Spondylitis, Mixed Connective Tissue

Disease, Autoimmune Addison's Disease, Multiple Sclerosis, Autoimmune Hepatitis, Pemphigus Vulgaris, Behcet's Disease, Pernicious Anemia, Bullous Pemphigoid, Polyarteritis Nodosa, Polychondritis, Celiac Disease, Polymyalgia Rheumatica, Polymyositis and Dermatomyositis, Chronic Inflammatory Demyelinating Polyneuropathy, Primary Agammaglobulinemia, Churg-Strauss Syndrome, Primary Biliary Cirrhosis, Cicatricial Pemphigoid, Psoriasis, CREST Syndrome, Cold Agglutinin Disease, Reiter's Syndrome, Crohn ' s Disease, Rheumatic Fever, Discoid Lupus, Rheumatoid Arthritis, Scleroderma, Grave's Disease, Sjogren's Syndrome, Guillain- Barre syndrome, Stiff-Man Syndrome, Hashimoto's Thyroiditis, Takayasu Arteritis, Idiopathic Pulmonary Fibrosis, Temporal Arteritis/Giant Cell Arteritis, Idiopathic Thrombocytopenia Purpura (ITP), Ulcerative Colitis, IgA Nephropathy, Insulin Dependent Diabetes (Type I), or Lichen Planus.

In one embodiment the autoimmune disease is inflammatory bowel disease (IBD) .

A particularly important aspect in selecting drug targets or compounds for possible development is assessing the likely degree of effect on the physiological process or disease state, rather than just an assessment of whether it has a detectable effect or not. It is valuable to be able to quantitate the potential therapeutic effect at the earliest opportunity and rank this against other candidate compounds and targets . Only those compounds or targets with a large enough effect to translate into a clinically useful effect can then be selected for further development. Equally, only those compounds or targets with a large enough effect to be detected in the clinical trial with the degree of power which will subsequently be run can then be selected for further development .

In another aspect of the present invention, the antibody may be from an animal other than human, for example murine. These antibodies have similar binding characteristics as human antibodies and are therefore capable of eliciting similar immune responses in the non-human test animal as human antibodies. The fluid or antibody is administered to a species of animal different to the animal species from which they are taken.

In a further embodiment, the physiological process or disease model in fish is generated by means of expression of a transgene that induces an effect on an aspect of behaviour and/or physiology of the fish, a measurable and preferably gradable phenotype .

The expression construct may encode antisense RNA. Thus a series of lines of zebrafish expressing various antisense RNAs may be generated and crossed to, or expressed in, the physiological process or disease situation. This may also be combined with the Gal4/UAS system with bidirectional promoters in the cases of processes or diseases modelled using the Gal4/UAS system.

One form of administration of an antibody or a protein component of a fluid comprises a transgenic non-human test animal wherein the transgene is under the control of a promoter. The promoter used to control expression, which may be tissue-specific expression may be inducible, which may facilitate establishment and/or screening of a fish line. For example, expression of antibodies may be under the control of an inducible promoter such that antibody expression can be switched on by inducing the promoter. The antibodies when expressed result in the development of a disease phenotype. These antibodies may have the same binding characteristics as human antibodies.

Tissue-specific and/or inducible expression can be used to overcome difficulties with lethality, and allows for provision of a gradable phenotype to screen (e.g. skin pigmentation, auditory response, swimming) .

In particular embodiments the physiological process or disease gene when expressed results in a physiological process or disease phenotype in a dominant fashion. Some embodiments of the invention involves placing the gene under the control of a promoter, rather than its own natural promoter, to avoid lethality, the promoter for example being inducible and/or tissue-specific. The modified process or disease is then only manifest under conditions in which the promoter is induced and/or in tissues in which the promoter is active. This can be used to allow fish to reach an age of viability and fecundity. For example, using an inducible promoter allows for the disease process to be switched off before the fish die and not switched on until any pleiotropic actions have terminated.

A preferred inducible promoter for use in embodiments of the present invention is a heat-shock promoter, such as HSP70

[Yeh, 2000] . Use of such a promoter allows for modification of a physiological process or induction of a disease state in a controlled fashion by means of alteration of temperature. Fish are amenable to prolonged expression from a heat-shock promoter, which may be necessary before a phenotype is detectable, as temperature of water within which fish reside can be easily adjusted and maintained. The inventors have observed that zebrafish can survive at a wide variety of temperatures. Only minor changes in temperature are necessary to activate heat shock promoters [D'Avino, 1999].

A gene to be employed in an embodiment of the present invention may be any gene in wild-type or mutant form which, when expressed in a non-human animal such as fish, results in the modification of one or more aspects of one or more physiological processes.

A disease gene to be employed in an embodiment of the present invention may be any gene in wild-type or mutant form which, when expressed in a non-human animal such as fish results in abnormal development, dysfunction or degeneration of tissue or cell function.

In further preferred embodiments of the present invention, a Gal4/UAS system is used. GAL4 encodes a yeast { Saccharomyces cerevisiae) protein of 881 amino acids, that regulates genes induced by galactose. It does so by directly binding to four related 17bp sites, together defining an Upstream Activating Sequences (UAS) element, analogous to a multicellular eukaryotes enhancer element. GAL4 can function in a wide variety of organisms to activate transcription from the UAS element. This permits targeted gene expression in a temporal and spatial fashion in vivo . To achieve this, transcription of the responder gene is controlled by presence of the UAS element. To activate their transcription, responder lines are mated to lines expressing GAL4 in a particular tissue of interest, termed the driver. The resulting progeny then express the responder in the desired tissue of interest.

This can be used to overcome difficulties with lethality, allows the same driver lines to be used for several processes or diseases and facilitates creation of mutated lines for subsequent genetic rescue. The Gal4/UAS system was originally developed as a means of tissue specific gene expression in Drosophila [Brand, 1993; Brand, 1994) . Such a system is now also being used to assess developmental genes in zebrafish [Scheer, 2002] .

The invention provides for manipulation of nucleic acid in order to modify cells of non-human test animals such as fish, as disclosed. Nucleic acid of a disease gene or physiological process-modifying gene to be expressed in fish in accordance with the invention is to be integrated into the chromosome of cells. Integration may be promoted by inclusion of sequences which promote recombination with the genome, in accordance with techniques available in the art. The gene may be heterologous to the fish, e.g. may be heterologous to zebrafish (e.g. mammalian, such as human), and may be in wild-type form or in any allelic or mutant form. The gene may be a zebrafish or other fish gene, in wild-type or mutated form, e.g. to provide an extra copy of a zebrafish or other fish gene, such as in a mutated form. Nucleic acid sequences encoding the peptides or polypeptides of the present invention may be readily prepared by the skilled person using the information and references contained herein and techniques known in the art (for example, see Sambrook and Russell "Molecular Cloning, A Laboratory

Manual", Third Edition, Cold Spring Harbor Laboratory Press, 2001, and Ausubel et al, Current Protocols in Molecular Biology, John Wiley and Sons, 1992, or later edition thereof). See Detrich et al. (1998) The Zebrafish: Biology. Methods in Cell Biology. Volume 59, and Detrich et al. (1998) The Zebrafish: Genetics and Genomics. Methods in Cell Biology. Volume 60 for techniques of zebrafish maintenance, utagenesis, transgenesis and mapping.

The desired coding sequence may be incorporated in a construct having one or more control sequences operably linked to the nucleic acid to control its expression. Appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate may be included.

Regions responsible for promoter and enhancer activity of a gene known to be expressed in a desirable pattern such as only under certain conditions or in certain tissue, may be isolated by ligating stretches of sequence from upstream of the translation start codon in the gene to a reporter gene. Constructs with deletions in putative promoter and/or enhancer regions are generated and the constructs tested for tissue specific gene expression in transgenic animal, e.g. transgenic fish such as zebrafish, fugu, goldfish, medaka and giant rerio.

A selectable marker, for example gene encoding a fluorescent protein such as Green Fluorescent Protein (GFP) may be included to facilitate selection of clones in which the gene construct has inserted into the genome. Where a fluorescent marker is used, embryos may be screened under a fluorescent dissecting microscope. Embryos, or fish into which they grow, may be screened for the presence of a defect resulting from the transgene. In another approach, embryos may be pooled prior to extraction of genomic DNA and analysis of the genomic DNA by PCR and/or restriction enzyme digest. Positive clones may be expanded and developed into breeding fish. These fish may then be bred to produce fish which carry one copy of the gene construct in the germ line. These heterozygous fish may then be bred to produce fish carrying the gene homozygously .

A further aspect provides a method which includes introducing a nucleic acid construct wherein a coding sequence of a desired physiological process-modifying gene or disease gene is placed under control of a promoter into an embryo cell of a fish, e.g. zebrafish. DNA may be injected directly in vivo into cells of an early embryo. With the establishment of embryonic stem cell culture, other methods generally referred to without limitation as "transformation", may be employed, for instance selected from any method available in the art, such as using calcium phosphate transfection, DEAE-Dextran, electroporation, liposome-mediated transfection and transduction using retrovirus or other virus. Marker genes such as antibiotic resistance or sensitivity genes may be used in identifying clones containing nucleic acid of interest, as is well known in the art.

In a further aspect, the present invention provides a method of making a non-human test animal, such as a fish, useful in or for use in a screen as disclosed herein and discussed further below. Such a method may comprise providing a gene construct wherein a coding sequence of a physiological process-modifying gene or disease gene is operably linked to a promoter that has the desired inducibility and/or tissue specificity, in the fish, introducing the gene construct into a fish embryo, causing or allowing the gene construct to integrate into the fish embryo genome, and growing the fish embryo into a viable fish.

A viable and reproductive non-human animal, e.g. fish, may mate with one or more other fish, establishing a line of fish, e.g. zebrafish, transgenic for the gene construct comprising the physiological process-modifying gene or disease gene operably linked to, and under regulatory control of, the promoter. A line of such fish, e.g. zebrafish, is useful in screens as disclosed.

In order to introduce a gene into a fish embryo, e.g. a zebrafish embryo, a gene construct is made, using techniques available to those skilled in the art. The construct may be released from a vector by restriction digest, and gel purified, for example by elution in IxTE (pH8.0) and dilution to a working concentration of 50-100 ug/ml KC1 containing a marker dye such as tetramethyl-rhodamine dextran (0.125%). Typically, 1 to 3 nl of this solution may be injected into single celled zebrafish embryos. Several thousand embryos may be injected.

Injected embryos are grown up and then mated with each other or to a non-transgenic wild-type fish. Transmission of the transgene to the subsequent generation is usually mosaic, ranging from 2 to 90%. At least 100 offspring are typically analysed to establish whether the founder fish carriers the transgene.

Fish demonstrating a desired phenotype and/or genotype may be grown up and may be mated with wild-type fish. The parents and offspring may be matched and the offspring similarly assessed for phenotype and/or genotype. Those offspring with a particular phenotype, and hence likely germline transmission of an integrated physiological process-modifying gene or disease gene construct, can be selectively bred. Some of the offspring may be sacrificed for more detailed analysis, e.g. to confirm the nature of the modification to the process or the disease. This analysis may include in si tu hybridisation studies using sense and anti-sense probes to the introduced gene to check for expression of the construct in cells of the fish, anatomical assessment such as with plastic sections to check for an effect on tissue or cells, and terminal deoxyuridine nucleotide end labelling (TUNEL) to check for apoptotic cell death in cells.

Families from which fish with the appropriate characteristics came may be maintained through subsequent generations. This maintenance then allows this new mutant strain to be entered into a secondary screen in accordance with further aspects of the invention.

Another aspect of the present invention provides cells of a transgenic non-human animal e.g. transgenic fish, such as zebrafish, fugu, goldfish, medaka and giant rerio as disclosed, whether isolated cells or cell lines derived from the fish, optionally immortalised using standard techniques.

A gene such as a physiological process-modifying gene or a disease gene sequence (e.g. heterologous to non-human test animal, such as heterologous to fish e.g. zebrafish) to be employed in aspects and embodiments of the present invention may employ a wild-type gene or a mutant, variant or derivative sequence may be employed. The sequence may differ from wild-type by a change which is one or more of addition, insertion, deletion and substitution of one or more nucleotides of the sequence shown. Changes to a nucleotide sequence may result in an amino acid change at the protein level, or not, as determined by the genetic code.

As noted, non-human test animals having a modified physiological process or one or more symptoms of a disease, such as an autoimmune disease, may be generated by administering fluid or antibodies, in which case the invention also provides for screening for a gene that has an effect on the aspect of behaviour of physiology that is affected by the fluid or antibody administration. Thus, the aspects of the invention involve genetic rescue of an induced phenotype. Fish such as Zebrafish are particularly amenable to genetic rescue experiments.

Mutagens such as ethylnitrosourea (ENU) may be used to generate mutated lines for rescue screening, in either the Fl-3 (for dominant) or F3 (for recessive) generations. (It is only by the third generation that recessive mutations can be bred to homozygosity. ) ENU introduces point mutations with high efficiency, so any phenotype is most likely to be recessive. Retroviral vectors may be used for mutagenesis, and although they are an order of magnitude less effective than ENU they offer the advantage of rapid cloning of a mutated gene (see e.g. Golling et al.(2002) Nat Genet 31, 135-40. Mariner/To family transposable elements have been successfully mobilised in the zebrafish genome and may be used as mutagenic agents (Raz et al. (1998) Curr Biol 8, 82- 8. ENU remains the most efficient and easy method available at the moment, and so is preferred for now.

Rescue strains are then created and the underlying genes mapped. However, in the case of a dominant transgenic, gal4 driven disease or a HSP line, additional steps are preferably performed in accordance with aspects and embodiments of the present invention, as follows.

If the model is a dominant transgenic, then only 75% of the offspring from a cross of 2 adult transgenics will harbour the mutant gene. Thus in 25% of cases, one will not know whether rescue has occurred or whether no mutant gene has been inherited. Selective breeding strategies are therefore needed to generate a homozygous transgenic line. Heterozygous carriers are grown up and incrossed. Some of these progeny will be homozygous carriers . They can be identified as such through crossing to responder lines. They will be capable of driving expression of the responder gene in all cases in which the response element is inherited. The next step is then to mutate this line at random, such as with ENU, and to screen the offspring for deviation from the expected phenotype. All of the offspring of an outcross should be carriers. Any possible rescued fish are grown up then retested. Alternatively, an adult heterozygote is mutated at random and the offspring screened. Normal offspring, or those in which one suspects the phenotype may have been partially rescued, are genotyped from a sample of cells or tissue taken from the non-human animal, e.g. via fin clipping or equivalent method. If the non-human animal is a carrier, then it is grown up and the new line generated. The underlying rescue gene is then mapped.

If the model is under the UAS promoter, a related problem to that described above occurs. The solution in this case is to generate homozygous GAL4 driver lines, mutate these at random, cross these with the UAS lines, then screen for dominant rescue, as described in detail earlier.

The mapping of mutant genes is comparatively easy. The density of markers on the fish genetic map, for example, is already considerably greater than that of the mouse map, despite the relatively recent popularity of zebrafish. Consult the harvard website on zebrafish, findable using any available web browser using terms "zebrafish" AND "harvard", currently (28 November 2002) found at

(http : //zebrafish .mgh . harvard . edu/mapping/ssr_map_index . tml ) , The Sanger Centre has begun to sequence the zebrafish genome with sequence currently (28 November 2002) published at www.ensembl.org/Danio_rerio/. The site can be found using any web browser using terms "danio rerio" and "Sanger" or "ENSEMBL". Around 70,000 ESTs have been identified and are being mapped on a radiation-hybrid map.

Another strategy for introducing effects, which may be random, on an aspect of behaviour or physiology in accordance with the present invention, is to down-regulate the function or activity of a gene, for instance employing a gene silencing or antisense technique, such as RNA interference or morpholinos. These can be either targeted against candidate genes, or generated against an array of genes as part of a systematic screen. It is relatively easy to inject RNA, DNA, chemicals, morpholinos or fluorescent markers into fish embryos, including zebrafish embryos, given their ex utero development.

A morpholino is a modified oligonucleotide containing A, C, G or T linked to a morpholine ring which protects against degradation and enhances stability. Antisense morpholinos bind to and inactivate RNAs and seem to work particularly well in zebrafish. Some disadvantages with this approach include the a priori need to know the gene sequence, the need to inject the chemical into the early embryo, potential toxic side effects and the relatively short duration of action. Additionally, they knock down the function of a gene, and thus do not offer the same repertoire of allele alterations as point mutations.

A further strategy for altering the function of a gene or protein as part of an in vivo screen, coupled to any of the various other components of the screening strategy disclosed herein, is to generate transgenic lines expressing protein aptamers, crossing these with the disease lines, or inducing disease by other means, then assaying for an altered disease state. Protein aptamers provide another route for drug discovery [Colas, 1996] but the ability to assay their effectiveness in vivo in accordance with the present invention markedly increasing their usefulness beyond in vitro screening methods.

A mutant non-human animal such as a mutant zebrafish transgenic for a physiological process-modifying gene or disease gene under control of a particular promoter and containing a mutation within a suppressor gene that lessens activity or effect of the physiological process-modifying gene or disease gene on an aspect of behaviour or physiology of the animal is itself useful in a further assay for a test substance able to modulate or affect, preferably potentiate or increase the suppression effect of the suppressor gene. Clearly, the same applies where a mutation in a gene is identified that enhances or increases activity of a second gene .

Of course, the person skilled in the art will design any appropriate control experiments with which to compare results obtained in test assays. In various further aspects, the present invention thus provides a pharmaceutical composition, medicament, drug or other composition comprising a suppressor gene or other gene or gene product or substance found to affect the physiological process-modifying gene or disease gene of interest or suppression of the physiological process- modifying gene or disease gene of interest, the use of such a material in a method of medical treatment, a method comprising administration of such a material to a patient, e.g. for treatment (which may include preventative treatment) of a medical condition, use of such a material in the manufacture of a composition, medicament or drug for administration for such a purpose, e.g. for treatment of a proliferative disorder, and a method of making a pharmaceutical composition comprising admixing such a material with a pharmaceutically acceptable excipient, vehicle or carrier, and optionally other ingredients.

One or more small molecules may be preferred therapeutics identified or obtained by means of the present invention. However, the invention may be used to identify appropriate targets for antibody mediated therapy, therapy mediated through gene targeting or protein targeting, or any of a variety of gene silencing techniques, including RNAi, antisense and morpholinos.

Alternatively, instead of or as well as an attempt to rescue the phenotype through the induction of genetic mutations, rescue may be achieved through application of a test substance, e.g. one or more chemicals. In this situation, all of the above methods will not require the mutation step for rescue (but will still require the mutation step if this is part of the procedure for modification of the physiological process or induction of the disease state) .

In a further aspect of the present invention, a non-human test animal in which one or more aspects of a physiological process has been modified or one or more symptoms of a disease, for example, an autoimmune condition, has been induced, may be treated with a test substance to screen for a substance capable of affecting the physiological process or the development of the disease. The effect of the test substance may be assessed by comparing an aspect of behaviour or physiology of treated non-human test animals with that aspect of behaviour or physiology of untreated animals to identify any treated non-human test animal with altered behaviour or physiology compared with an untreated non-human test animal, thereby to identify a test substance that affects a physiological process or development of the disease state.

The present invention provides means, specifically non-human test animals such as fish for use in methods of screening for a test substance which when administered to the non-human test animal affects a physiological process or ameliorates symptoms of a disease state, such as an autoimmune disease state .

The test substance may be any substance desired to be tested for affecting a physiological process or a disease state. Thus, in certain embodiments, the test substance is a candidate drug compound. The test substance may an individual compound or may be a library of candidate drug compounds. In yet other embodiments, the test substance is a known drug.

In certain embodiments, the test substance is fluid obtainable from an animal species. In preferred embodiments, the fluid is obtainable from a human, preferably a normal human. The fluid may be serum, CSF, joint aspirate, peritoneal fluid or bronchiolar lavage fluid.

In other preferred embodiments, the test substance is fluid that may be obtainable only in small amounts, for example one to a few μl, for example, CSF and joint aspirate. The fluid may also be a sub-component or fraction of a fluid, for example, a sub-component or fraction of CSF or joint aspirate.

The test substance may be fluid that comprises an antibody, a hormone, a growth factor and/or a cytokine. Thus, the active component in the fluid may be, but is not limited to, an antibody, a hormone, a growth factor and/or a cytokine, for example, IL-6, IL-8 and/or IL-10.

In other embodiments, the test substance may be fluid that may be a purified or isolated sub-component or fraction of fluid, such that the sub-component or fraction has been separated form at least one other component or fraction of the original fluid. For example, a purified or isolated subcomponent or fraction may be an antibody , a hormone, a growth factor or a cytokine, such that the active component of the fluid may consist only of an antibody, a hormone, a growth factor or a cytokine. The person skilled in the art is aware of the types of molecule that may be components of various fluids obtainable from an animal species, for example, that antibodies may be a sub-component of serum. The non-human test animal may be treated with the test substance in a number of ways. For example, fish may be contacted with the test substance, it may be touched or rubbed on their surface or injected into them.

A further advantage of fish, especially zebrafish is the fact they live in water. This makes administration of test substances easy as they may be added to water in which the fish are. Zebrafish and other fish also readily absorb chemicals. The effective concentration of chemicals in the water often equates to the effective plasma concentration in mammals .

Different test substances may be added to each well of a multi-well plate, such as a 96 well plate, to identify that test substance exhibiting a beneficial or deleterious effect. There may be one or multiple fish in each well exposed to the test substance.

Moreover, the inventors have discovered that zebrafish are- also DMSO (dimethyl sulphoxide) tolerant. This is important as DMSO is used as a solvent to dissolve many drugs. The inventors have established that zebrafish can tolerate 1% DMSO. Thus, a candidate drug or other test substance may be dissolved in DMSO and administered to zebrafish by adding to the fish water to give a final concentration of DMSO of at least up to 1%. This is employed in various preferred aspects and embodiments of the present invention.

The test substance may be added prior to, or concurrent with, the modification of a physiological process or the onset of the disease phenotype. Preferably the test substance may be added subsequent to the modification of a physiological process or the onset of the disease phenotype. The modification of a physiological process or the onset of the disease phenotype may be caused by the administration of fluid or antibody to the non-human test animal. Preferably the modification of a physiological process or onset of the disease phenotype may be caused by the administration of human antibodies or the administration of human hormones, growth factors and/or cytokines .

The same test substance may be added to different wells at a different concentration. For example, test substance 1 may be added to well Al at a concentration of lmM, to well A2 at a concentration of lOOuM, to well A3 at a concentration of lOuM, to well A4 at a concentration of luM and to well A5 at a concentration of O.luM. Then test substance 2 to well BI etc. The panel of test substances may be known drugs or new chemical entities .

Additionally, the test substances may be added in combination. For example, well A2 may contain test substance

1 and 2, well A3 test substance 1 and 3, well B2 test substance 2 and 3. Alternatively, every well may contain test substance x, with individual wells containing a panel of additional test substances.

In other options, a population of fish in a petri dish or a tank may be employed and treated together, e.g. via addition of one or more or a combination of test substances in the water.

Thus, zebrafish enable the entire biological pathway of a vertebrate to be screened in a high-throughput fashion.

The present invention in certain aspects and embodiments provides for screening for and preferably identifying or obtaining a substance that provides a synergistic combination with another substance, or for screening for and preferably identifying or obtaining two or more substances that together provide an additive or synergistic combination. Clinical benefit is often derived from synergistic combinations of drugs. Use of an in vivo system in accordance with the present invention allows for identification of such synergistic combinations.

Thus, in certain embodiments the invention comprises treating the non-human test animal, as discussed, with two or more substances, at least one of which is a test substance, and comparing the effect of the two or more substances in combination to determine the optimum effect (whether simultaneously or sequentially applied) on an aspect of behaviour or physiology with the effect of either or both of the two or more substances when applied individually or alone. Either all (or both) of the substances applied may each be a test substance, or one of the substances may be a drug known to have a beneficial effect in the disease that is the subject of the model, for example, or at least an effect in the treated non-human test animal.

The invention thus provides for screening for and preferably identifying or obtaining a substance that provides an additive effect to a known drug or compound or a synergistic effect with the known drug or compound. It also provides for screening for and preferably identifying or obtaining a combination of two or more substances that provide a synergistic effect, compared with the effect of the two substances when employed individually or alone.

Add-on therapies are useful because it is difficult to conduct clinical trials in which an existing drug is withdrawn from a patient and replaced with a new drug. The patient is deprived of a drug which has at least got some proven efficacy and some confidence in its side-effect profile. Additionally, the patient will be vulnerable to their disease during the phases of withdrawal of the existing drug and build up of the test drug.

In addition to a test substance, the non-human test animal may be a mutated animal rather than a wild-type animal. It is then possible to assay for interacting effects, either beneficial synergistic effects, or deleterious effects, of the mutation plus the test substances. Alternatively, the analysis may be of the known therapeutic agent and the genetic mutation to discover either a new drug target of benefit in combination with the known drug, or a genetic marker of use in predicting which patients are most likely to benefit (or not benefit) from prescription of the known drug.

In another embodiment, a combination of potential agents is administered to a non-human test animal having one or more symptoms of a disease, for example, immunosuppressive agents where the animal has symptoms of an autoimmune disease, which may be generated through addition of pathogenic antibodies to the non-human test animal or where the animal is a fish, to the fish water or through expression or knock out of a gene, to assess whether the combination is more effective than either of the individual agents.

For example, there are a variety of immunosuppressive agents, either in clinical trials or currently prescribed. For the sake of this example, assume there are 11 drugs to be tested. It is possible that the various drugs act at different pinch points in biological pathways and that by judicious co- prescribing, an optimal combination may be found that is better than any drug alone, whilst with no worse a side effect profile. It would be very difficult to do clinical trials, or indeed mammalian studies to determine the optimum combination. The present invention allows this.

The present invention also provides for screening for and preferably identifying or obtaining a substance that ameliorates one or more side effects of an active substance, e.g. a therapeutically active substance. There are many drugs which have been discontinued in clinical trials, or are marketed but infrequently prescribed, not because they are not therapeutically effective, but because their side-effect profile is limiting. The side-effects may be relatively benign, but significant to the patient, such as renal damage (e.g. cyclosporin) . It is desirable to allow the administration of such drugs, with proven beneficial effects, through the co-administration of an additional agent to improve the side-effect profile.

In accordance with the present invention, such agents are screened for in animals in which administration of the active substance induces a side-effect or other phenotype reflective or indicative of a side-effect. Thus in embodiments of the invention, an active agent is administered to non-human test animals having one or more symptoms of an autoimmune disease and the side-effect of other phenotype is assessed for such animals when subjected to one or more test substances. This does not require a priori knowledge of action of the co- administered agent. In other embodiments, agents that achieve the desired therapeutic effect with a reduction of side-effects can be screened for and preferably identified or obtained by means of assessment of disease phenotype and side-effect phenotype. As with other aspects and embodiments of the present invention, this may involve co-administration of a primary compound together with either a battery of candidate substances, or together with randomly induced genetic mutation. With the latter approach, i.e. mutation, subsequent steps are needed to identify the appropriate co- therapeutic following identification of fish with a mutation that provides an ameliorative effect. A diverse library of drug-like compounds, such as the LOPAC library (Sigma) may be used, or the Chembridge PHARMACOphore diverse combinatorial library. Other targeted libraries against particular targets classes may be used, such as ion channel libraries or G protein libraries.

Still further provided by the present invention is a method of identifying mutations, genotypes, allelic variations, haplotypes and genetic profiles associated with responsiveness to a therapeutic. There is an increasing move towards targeted prescribing, whereby the choice of therapeutic is influenced by genotyping the patient. Particular polymorphisms have been found to predict both the therapeutic effectiveness of a compound, and also the likelihood of suffering certain side effects. Such rationalised prescribing is cost-effective. It also makes clinical trials easier to run, as likely responders can be targeted, thus necessitating a smaller sample size to achieve statistical significance. However, for the moment, most drugs, both already prescribed or in development, do not have an appropriate test.

The present invention provides for assessing the effectiveness of various medications in combination with random genetic mutations to identify those mutations which either enhance or decrease the therapeutic effectiveness and/or alter the side effect profile. This allows for identification of genes, polymorphisms, mutations, alleles and haplotypes associated with a particular response to a drug or other treatment, enabling development of appropriate genetic assays in humans to permit rationalised prescribing. It is possible to introduce random mutations into the zebrafish genome, for example with the use of chemical mutagenesis (Solnica-Krezel et al . , Genetics 1994, 136(4): ^'1401-20) . It is also possible to make transgenic fish carrying exogenous genes.

In a further embodiment, rather than target the prescribing of a beneficial agent, or improve the efficacy of an already beneficial agent, the invention may be used to reduce the side effects of an agent which otherwise might not be prescribed because of its negative side effect profile. In this situation the deleterious side effect is assayed, with an improvement of this deleterious side effect being examined for through the result of an additional chemical or interactor gene.

For example, mitoxantrone has useful clinical effects in the treatment of multiple sclerosis [van de Wyngaert, 2001] , but its use is severely curtailed on account of the cardiac side effect profile [Ghalie, 2002] . Fish with random mutations or bathed in a test substance are exposed to mitoxantrone and examined for a change in cardiac function compared to baseline. An improvement in function from baseline treatment with mitoxantrone alone, leads to the identification of the interactor gene, or chemical and subsequently therapeutic, to be co-prescribed with mitoxantrone in multiple sclerosis patients .

It is well known that pharmaceutical research leading to the identification of a new drug may involve the screening of very large numbers of candidate substances, both before and even after a lead compound has been found. This is one factor which makes pharmaceutical research very expensive and time-consuming. Means for assisting in the screening process can have considerable commercial importance and utility.

Such means for screening for substances potentially useful in affecting a physiological process, or treating or preventing a disorder or disease is provided by non-human test animal such as fish according to the present invention. Modifier genes, such as enhancer or suppressor genes identified using the invention, and substances that affect activity of such suppressor genes represent an advance in the fight against disease since they provide basis for design and investigation of therapeutics for in vivo use, as do test substances able to affect activity or effect of a treatment, and substances that affect activity or effect of expression of a disease gene in a fish.

In various further aspects the present invention relates to screening and assay methods and means, and substances identified thereby.

Whatever the material used in a method of medical treatment of the present invention, administration is preferably in a "prophylactically effective amount" or a "therapeutically effective amount" (as the case may be, although prophylaxis may be considered therapy) , this being sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated.

Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors.

Pharmaceutical compositions according to the present invention, and for use in accordance with the present invention, may include, in addition to active ingredient, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration, which may be oral, or by injection, e.g. cutaneous, subcutaneous or intravenous.

Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may include a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.

For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.

Examples of techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed), 1980.

Vectors such as viral vectors have been used in the prior art to introduce nucleic acid into a wide variety of different target cells. Typically the vectors are exposed to the target cells so that transfection can take place in a sufficient proportion of the cells to provide a useful therapeutic or prophylactic effect from the expression of the desired peptide. The transfected nucleic acid may be permanently incorporated into the genome of each of the targeted cells, providing long lasting effect, or alternatively the treatment may have to be repeated periodically.

A variety of vectors, both viral vectors and plasmid vectors, are known in the art, see US Patent No. 5,252,479 and WO 93/07282. In particular, a number of viruses have been used as gene transfer vectors, including papovaviruses, such as SV40, vaccinia virus, herpesviruses, including HSV and EBV, and retroviruses . Many gene therapy protocols in the prior art have used disabled murine retroviruses.

As an alternative to the use of viral vectors in gene therapy other known methods of introducing nucleic acid into cells includes mechanical techniques such as microinjection, transfer mediated by liposomes and receptor-mediated DNA transfer, also administration of naked DNA or RNA, by simple administration, e.g. injection, of nucleic acid such as a plasmid, for instance to muscle.

The application of a test substance may then be as follows, in accordance with embodiments of the present invention:

1. A test substance is added to the non-human test animal (e.g. a fish) either prior to the modification of the physiological process or appearance of the disease state, at the time of the modification of the physiological process or induction of the disease state, or after the modification of the physiological process or the induction of the disease state. The first two situations are more likely to identify a prophylactic chemical, the latter a drug which reverts the process or disease state back to normal. The test substance may be a chemical and may be a random chemical administered in a high-throughput fashion to fish in 96 well plate format, or a selected chemical administered to a clutch of fish in a Petri dish.

2. The animal is then screened for deviation from the initial process or disease state.

The following additional steps are highly desirable in screening, and their use is provided by the present invention in preferred embodiments:

Rather than add a single chemical, a combination of chemicals is added. For instance, a known therapeutic agent may be administered to all animals at a dose at which a further beneficial effect could still be detected. A random chemical library is then added to animals such as fish and an incremental effect screened for.

1. Induce an autoimmune disease state by the administration of antibodies to fish.

2. Administer drug 1 to row 1, drug 2 to row 2, etc.

3. Administer drug 1 to column 1, drug 2 to column 2 etc.

4. Compare the immunosuppressive effect of all wells with that of the drugs when given alone .

A further embodiment of the above allows for detection of augmentation of a particular drug through a particular mutation, as follows:

1. Induce genetic mutation through any of the above.

2. Induce disease state through any of the above.

3. Administer test chemical.

4. Assess whether the combination of the mutation plus chemical is greater than either alone.

5. The mutated gene is then used as a beneficial target, as described above. A further embodiment of the invention allows identification of genetic factors which help determine the appropriateness of a particular therapeutic agent for a given patient. If the mutation augments the effect of the drug, that mutation is searched for in human homologues. Patients with this mutation should be preferentially prescribed the drug. If the mutation leads to a deleterious effect or lack of effect, then patients should avoid this drug.

A further embodiment of the invention allows identification of genetic or chemical factors which help prevent the side effects of an otherwise toxic drug. The following is an illustrative embodiment with reference to multiple sclerosis, but may be applied in other contexts for other diseases:

1. Mitoxantrone has a beneficial effect on multiple sclerosis (MS) patients, but causes cardiotoxicity.

2. An MS model of zebrafish is created which responds to treatment with mitoxantrone, but with the added complication of cardiac pathology.

3. The treated fish are co-treated with a panel of chemicals, (or alternatively are mutagenised as a route to a drug target) .

4. Those fish which no longer show the cardiac effects, but still show the beneficial effects are selected. The chemical is then used as a co-agent in patients to allow the safer administration of the mitoxantrone, (or alternatively the mutagenised gene is mapped and used to develop the co-agent) . A further embodiment of the present invention involves attempting to modify the initial phenotype through a protein aptamer, rather than through a genetic mutation of chemical means. For example, a method may be performed in accordance with the following:

1. A construct coding for the desired aptamer (or random constructs for random aptamers) is injected into embryos to generate lines expressing the aptamer.

2. These lines are then crossed to the disease-expressing lines, or alternatively the disease state is induced in these lines .

3. The lines are then tested for deviation from the expected or initial phenotype.

4. If deviation occurs, the aptamer has in vivo proof of action and is used to derive a therapeutic agent.

Having identified fish with a mutation that confers rescue on a disease phenotype, the following steps may be performed:

1. The human homologue of the zebrafish rescue gene is cloned.

2. The same type of mutation is introduced into the human homologue 3. The wild-type and mutated constructs are injected into the embryos.

4. The disease state is induced and assessed.

5. If the wild-type gene prevents the rescue, but the mutant gene retains it, this provides further evidence that the mutation is beneficial. However, a negative result does not necessarily rule out benefit.

6. The protein encoded by the human homologue is used for direct drug screens in vitro or directed in vivo screening.

Zebrafish are DMSO tolerant

The inventors have discovered that zebrafish are DMSO tolerant. The inventors have established that zebrafish can tolerate 1% DMSO. Thus, a candidate drug or other test substance may be dissolved in DMSO and administered to zebrafish by adding to the fish water to give a final concentration of DMSO of at least upto 1%.

Zebrafish of tup background were tested in batches of 10. DMSO was added to fish water on day 3 and fish examined daily up to day 9. At 1% DMSO (lOul/ml) , no abnormality was detected, as assessed by detailed examination under a dissecting microscope to 5x magnification. Motoric behaviour was also normal. Examples

Part 1

A method for the visualisation of the gut and induction of a disease state resembling inflammatory bowel disease through the administration of trinitrobenzene sulfonic acid (TNBS) has been identified. The severity of the disease phenotype observed correlates with the dose of TNBS to which the fish are exposed. 75 μg/ml TNBS is used to induce a full disease phenotype. This model has many of the recognised features in human inflammatory bowel disease, including: 1. Biological a. TNF alpha upregulation b. Region specific changes c. Changes in gut motility 2. Pathological a. Transdifferentiation b. Metaplasia c. Mast cell proliferation 3. Clinical a. Resolves with steroids/5-ASA

However, for the reasons described above, this model has limitations with regards to the known autoimmune component to inflammatory bowel disease.

Autoimmunity has been emphasized in the pathogenesis of ulcerative colitis (UC) . A putative autoantigen, tropomyosin 5, has been isolated from colon epithelial cells that can induce a significant T cell response in UC patients (Taniguchi et al . 2001, WO99/48508, WO01/58927) . It is likely that this normally intracellular protein is chaperoned to the cell surface by Colon Epithelial Protein (CEP) . Evidence for this comes from the finding that tropomyosin 5 forms a complex with CEP, as determined by co-immunoprecipitation (Kesari et al. 1989, WO01/58927) . Autoantibodies that recognize tropomyosin 5 are spontaneously produced by lamina propria mononuclear cells that infiltrate the inflamed UC tissue (Biancone et al . 1995); 95% of UC patients' blood contains such anti-tropomyosin 5 autoantibodies (Taniguchi et al . 2001) . Furthermore, CEP and tropomyosin 5 can induce T- lymphocyte-mediated oral tolerance when fed to rats, supporting the role of autoimmunity in UC. CEP is known to be upregulated in UC patients (Ref 5) . The model for pathogenesis in UC is therefore that upregulation of CEP in the disease state causes externalization of tropomyosin 5, which is then recognized by autoantibodies (WO01/58927) . CEP may also be acting to externalize tropomyosin 5 in the other tissues affected in UC, for example the skin (WO01/58927) .

Part 2

CEP is present in zebrafish distal gut and upregulated in IBD. The antibody used to detect zebrafish CEP is in fact made against human CEP, but the two species are similar enough to allow cross-reactivity, important in creation of the autoimmune model. CEP is also expressed in chrondrocytes and a subset of skin cells in zebrafish, a similar distribution to that reported in man (Kesari et al 1985 and Ref 6) . These tissues are often sites of extra-colonic inflammation in UC patients (Ref 4) .

Part 3

Using anti-sera from pooled from patients with severe UC or from non-UC patients (control), immunoreactivity in control and TNBS-induced IBD zebrafish samples has been examined and the degree of staining and determined a difference in the degree of immunoreactivity between control and UC anti-sera assessed. This indicates that disease sera, containing a high antibody titre, can interact with and recognize antigens presented on the zebrafish gut lining that are upregulated in the disease state. While there is some staining using control anti-sera, staining is stronger in samples stained with UC anti-sera.

Part 4

Control and TNBS-induced IBD zebrafish were exposed to pooled anti-sera from a number of UC patients to determine whether UC sera had properties pathogenic to zebrafish. TNBS was used at 10 μg/ml, a dose that does not induce the full IBD phenotype. We hypothesised that the combined low dose of TNBS and UC patient sera would result in the full IBD phenotype. When either healthy or TNBS-treated fish were exposed to UC sera an upregulation of CEP expression around the anus and in the skin was observed, as well as exacerbation of the IBD phenotype in the distal gut. Specifically, this involved disruption of the rectal epithelium. Histological analysis suggests that UC sera causes cytotoxicity in rectal epithelium, possibly by inducing apoptosis.

All documents mentioned anywhere in this specification are incorporated by reference.

References

1. Taniguchi M, Geng X, Glazier KD, Dasgupta A, Lin JJ, Das KM. Cellular immune response against tropomyosin isoform 5 in ulcerative colitis.

Clin Immunol. 2001 Dec; 101 (3) : 289-95.

2. Kesari KV, Yoshizaki N, Geng X, Lin JJ, Das KM. Externalization of tropomyosin isoform 5 in colon epithelial cells .

Clin Exp Immunol. 1999 Nov; 118 (2) : 219-27.

3. Biancone L, Mandal A, Yang H, Dasgupta T, Paoluzi AO, Marcheggiano A, Paoluzi P, Pallone F, Das KM.

Production of immunoglobulin G and Gl antibodies to cytoskeletal protein by lamina propria cells in ulcerative colitis. Gastroenterology. 1995 Jul; 109 (1) : 3-12.

4. Das KM.

Relationship of extraintestinal involvements in inflammatory bowel disease: new insights into autoimmune pathogenesis. Dig Dis Sci. 1999 Jan; 44 (1) : 1-13. Review.

Ref 5 http : //www2. gastroj ournal . org/scripts/om. dll/serve?action=searchD

B&searchDBfor=art&artType=abs&id=pml688539&nav=abs

Ref 6 http: //www2. gastrojournal. org/scripts/om. dll/serve?action=searchD

B&searchDBfor=art&artType=abs&id=pm8020652&nav=abs Patents

WO99/48508 - Treatment of Ulcerative Colitis with Tropomyosin

Isoforms and Monoclonal Antibodies to Tropomyosin Isoforms.

WO01/58927 - Therapeutic and Diagnostic Methods for Ulcerative Colitis and Associated Disorders.

Chiorazzi, N. (2003). Immune mechanisms and disease. New York, New York Academy of Sciences.

Isenberg, D. and J. Morrow (1995) . Friendly fire : explaining autoimmune disease. Oxford, Oxford University Press.

O'Connor, K. C, T. Chitnis, et al. (2003). "Myelin basic protein-reactive autoantibodies in the serum and cerebrospinal fluid of multiple sclerosis patients are characterized by low- affinity interactions." J Neuroimmunol 136(1-2): 140-8.

T Hart, B. A. and S. Amor (2003) . "The use of animal models to investigate the pathogenesis of neuroinflammatory disorders of the central nervous system." Curr Opin Neurol 16(3): 375-83.

Claims

1. A non-human test animal of a first species for modelling a physiological process, wherein fluid from a second animal species is administered to the non-human test animal to modify one or more aspects of the physiological process in the test animal.

2. A non-human test animal of a first species for modelling a disease state, wherein fluid from a second animal species having the disease is administered to the non-human test animal to induce one or more symptoms or signs of the disease in the test animal .

3. The non-human test animal according to claim 1, wherein the disease state modelled has an autoimmune component.

4. The non-human test animal according to claim 2 or 3, wherein the disease state modelled is an autoimmune disease state.

5. A test animal according to any one of the preceding claims , wherein the second animal species having the disease is a human patient.

6. The non-human test animal according to any one of the preceding claims, wherein the non-human test animal is a non- mammalian test-animal.

7. A test animal according to any one of the preceding claims, wherein the fluid is serum, CSF, joint aspirate, peritoneal fluid or bronchial lavage fluid.

8. The test animal according to any one of the preceding claims, wherein the fluid comprises an antibody, a hormone, a growth factor and/or a cytokine.

9. A test animal according to any one of the preceding claims, wherein the animal is bathed in medium comprising the fluid.

10. A test animal according to any one of claims 1 to 8, wherein the fluid is administered locally, via injection into the pericardium, joint space, peritoneum or central nervous system or via a nasogastric or rectal tube.

11. A test animal according to claimδ, wherein the antibody, hormone, growth factor and/or cytokine is purified, isolated or concentrated from the fluid for administration to the test animal .

12. A test animal according to claim 11 for modelling a disease state , wherein the antibody is obtained from fluid from a human patient having the disease.

13. A test animal according to any one of claims 3 to 12, wherein the disease is an autoimmune disease.

14. A non-human test animal for modelling an autoimmune disease state, wherein an antibody molecule is administered to the non- human test animal to induce one or more symptoms of the autoimmune disease in the animal, wherein the antibody molecule is able to bind an antigen of the non-human test animal to which antibodies from a human with the autoimmune disease bind and induce one or more symptoms of the autoimmune disease when administered to the non-human test animal.

15. A test animal according to any one of claims 1 to 14, wherein the non-human test animal has one or more modified aspects of a physiological process or one or more inflammatory symptoms or signs of disease prior to the administration of the fluid.

16. A test animal according to any one of claims 1 to 14, wherein a substance which modifies one or more aspects of a physiological process or induces one or more inflammatory symptoms or signs of disease is administered prior to administration of the fluid.

17. A test animal according to claim 16, wherein the symptoms or signs of disease are induced by administration of a substance such as TNBS or DSS to the test animal.

18. A test animal according to any one of claims 1 to 14, wherein the non-human test animal has a genetic predisposition for the modified physiological process or disease state.

19. A test animal according to any one of claims 1 to 18, wherein the non-human test animal is a fish.

20. A test animal according to claim 19, wherein the fish is a zebrafish.

21. A test animal according to any one of claims 4 to 20, wherein the autoimmune disease is selected from the group consisting of: Graves disease, Myasthenia Gravis, Lambert Eaton myasthenic syndrome, SLE, Pernicious anaemia, Autoimmune haemolytic anaemia, Vitiligo, Alopecia, Anklosing Spondylitis, Mixed Connective Tissue Disease, Autoimmune Addison's Disease, Multiple Sclerosis, Autoimmune Hepatitis, Pemphigus Vulgaris, Behcet ' s Disease, Pernicious Anemia, Bullous Pemphigoid, Polyarteritis Nodosa, Polychondritis, Celiac Disease, Polymyalgia Rheumatica, Polymyositis and Dermatomyositis, Chronic Inflammatory Demyelinating Polyneuropathy, Primary Agammaglobulinemia, Churg-Strauss Syndrome, Primary Biliary Cirrhosis, Cicatricial Pemphigoid, Psoriasis, CREST Syndrome, Cold Agglutinin Disease, Reiter's Syndrome, Crohn ' s Disease, Rheumatic Fever, Discoid Lupus, Rheumatoid Arthritis, Scleroderma, Grave's Disease, Sjogren's Syndrome, Guillain-Barre syndrome, Stiff-Man Syndrome, Hashimoto's Thyroiditis, Takayasu Arteritis, Idiopathic Pulmonary Fibrosis, Temporal Arteritis/Giant Cell Arteritis, Idiopathic Thrombocytopenia Purpura (ITP), UlcerativeColitis, IgA Nephropathy, Insulin Dependent Diabetes (Type I), or Lichen Planus .

22. A test animal according to any one of claims 4 to 20, wherein the autoimmune disease is inflammatory bowel disease.

23. A method of screening for a test substance that affects a physiological process comprising: treating a test animal according to any one of claims 1 to 11 and 15 to 20, with the test substance; comparing an aspect of behaviour or physiology of the treated non-human test animal with that aspect of behaviour or physiology of an untreated non-human test animal in order to identify any treated test animal with altered behaviour or physiology compared with an untreated animal; thereby to identify a test substance that affects the physiological process.

24. A method of screening for a test substance that affects the development of a disease state comprising: treating a test animal according to any one of claims 2 to 20, with the test substance; comparing an aspect of behaviour or physiology of the treated non-human test animal with that aspect of behaviour or physiology of an untreated non-human test animal in order to identify any treated test animal with altered behaviour or physiology compared with an untreated animal; thereby to identify a test substance that affects development of the disease state.

25. A method of screening for a test substance that affects the development of an autoimmune disease state comprising: treating a test animal according to any one of claims 1 to 22, with the test substance; comparing an aspect of behaviour or physiology of the treated non-human test animal with that aspect of behaviour or physiology of an untreated non-human test animal in order to identify any treated test animal with altered behaviour or physiology compared with an untreated animal; thereby to identify a test substance that affects development of the autoimmune disease state.

26. A method according to any one of claims 23 to 25, wherein the test substance is dissolved in a solvent, wherein the solvent is DMSO.

27. A method according to claim 26, wherein the method comprises administering the test substance dissolved in DMSO to water inhabited by the animal.

28. A method according to claims 26 or 27, wherein the substance dissolved in DMSO is added to water inhabited by the fish to give a final concentration of DMSO of 1% or less.

29. A method according to any one of claims 23 to 28, wherein the test substance is administered at an equal concentration in multiple wells.

30. A method according to any one of claims 23 to 28, wherein the test substance is administered at various concentrations in each well .

31. A method of screening for an optimum combination of two test substances, drugs, genes or drug targets that affect a physiological process or the development of a disease state comprising: treating a test animal according to claims 1 to 22 with the test substances, drugs, genes or drug targets; comparing an aspect of behaviour or physiology of the treated non-human test animal with that aspect of behaviour or physiology of an untreated non-human test animal in order to identify any treated test animal with altered behaviour or physiology compared with an untreated animal; thereby to identify a combination of two substances that affect the physiological process or development of the disease state .

32. A method according to claim 31, wherein a first test substance is administered at an equal concentration in multiple wells, and to each individual well a further test substance is added, and the effect of each test substance in various combinations is compared to determine the optimum combination.

33. A method according to claim 31, wherein the effect of each further test substance in various combinations is compared to determine deleterious combinations.

34. A method according to claim 31, comprising determining the optimum combination of two potential drug targets, by comparing the effect of mutations or genetic alterations in various combinations .

35. A method according to any one of claims 31 to 33, comprising testing combinations of 3, 4, 5 or any higher number of test substances.

36. A method of determining whether two or more test substances, drugs, genes or drug targets, that affect a physiological process or the development of a disease state, have an additive or synergistic effect when present simultaneously, the method comprising: treating a test animal according to claims 1 to 22 with the test substances, drugs, genes or drug targets; comparing an aspect of behaviour or physiology of the treated non-human test animal with that aspect of behaviour or physiology of an untreated non-human test animal in order to identify any treated test animal with altered behaviour or physiology compared with an untreated animal; thereby to identify a combination of two substances that have an additive or synergistic effect when present simultaneously to affect the physiological process or development of the disease state.

37. A method according to claim 36 comprising administering two test substances separately and in combination.

38. A method according to claim 37 wherein the effects of two test substances both separately and in combination are assayed at a range of possible combinations from their respective dose- response curves when given in isolation.

39. A method according to claim 36 or claim 37 wherein the effect when given in combination is compared to see whether it is greater than the summation of the effect of the two test substances at the same concentration given separately.

40. A method according to claim 36 comprising determining the additive or synergistic effect of two or more potential drug targets, by comparing the effect of mutations or genetic alterations in various combinations.

41. A method according to any one of claims 36 to 39 comprising testing combinations of 3, 4, 5 or any higher number of test substances or drug targets.

42. The method according to any one of claims 31 to 41, wherein the disease state has an autoimmune component.

43. The method according to any one of claims 31 to 42, wherein the disease state is an autoimmune disease state.

44. A method of identifying a test substance, drug, gene or drug target that lessens an otherwise deleterious effect or side effect of a second test substance, drug, gene or drug target, the method comprising: treating a test animal according to claims 1 to 22 with the test substance, drug, gene or drug target; comparing an aspect of behaviour or physiology of the treated non-human test animal with that aspect of behaviour or physiology of an untreated non-human test animal in order to identify any treated test animal with altered behaviour or physiology compared with an untreated animal; thereby to determine whether a substance, drug, gene or drug target affects activity or effect of the second test substance, drug, gene or drug target, whereby a substance that lessens said otherwise deleterious effect or side effect is identified.

45. A method according to claim 44, wherein said second test substance, drug, gene or drug target has a beneficial effect, but also a negative effect in isolation, whereas in combination with test substance, the negative effect is lessened.

46. A method according to claim 44 or claim 45, wherein the effect is assayed in multiwell format with a range of possible ameliorating compounds or genetic mutations.

47. A method according to any one of the preceding claims wherein a gene or mutation is identified for a patient population which is more likely to respond to a particular substance or drug, or which is less likely to respond to a particular substance or drug, or which may demonstrate a negative side effect when administered a particular substance or drug.

48. A method according to claim 47 wherein the effect of a particular mutation or polymorphism on the efficacy or side effect profile of a test substance or genetic alteration is compared with wild type response.

49. A method according to claim 47 or claim 48 wherein the effect is assayed in a medium or high throughput fashion in zebrafish to identify such possible polymorphisms or genetic factors resulting in variations in drug responsiveness.

50. A method according to any one of claims 48 to 49 wherein one of more genetic factors identified in zebrafish are used to identify corresponding human genetic factors.