Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
  • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
  • A01K67/027—New or modified breeds of vertebrates
  • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
  • A01K67/0276—Knock-out vertebrates
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K2217/00—Genetically modified animals
  • A01K2217/07—Animals genetically altered by homologous recombination
  • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K2227/00—Animals characterised by species
  • A01K2227/10—Mammal
  • A01K2227/105—Murine
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K2267/00—Animals characterised by purpose
  • A01K2267/03—Animal model, e.g. for test or diseases
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K2267/00—Animals characterised by purpose
  • A01K2267/03—Animal model, e.g. for test or diseases
  • A01K2267/035—Animal model for multifactorial diseases
  • A01K2267/0368—Animal model for inflammation

Definitions

• the present invention relates to an animal model for inflammatory bowel disease.
• the model is based on a mammal not expressing a functional mdrla gene product or wherein the mdrla gene product is inhibited, which develops inflammatory bowel disease when subjected to elevated chlorine concentrations. More specifically, the mammal is a transgenic mouse lacking mdrla.
• This model facilitates the characterisation of the pathogenic mechanisms of inflammatory bowel disease and the development of diagnostics, therapies and therapeutic compounds.
• IBD Inflammatory bowel disease
• UC ulcerative colitis
• CD Crohn's disease
• Intestinal flora may also be an important co-factor in the pathogenesis of intestinal inflammation.
• spontaneous colitis can be prevented if animals are maintained in a germ-free environment (Dianda L et al (1997) Am J Pathol 150, 91-97, Taurog JD et al (1994) J Exp Med 180, 2359-2364).
• Other studies have shown that intestinal inflammation can be successfully treated with oral antibiotics (Panwala CM et al. (1998) J Immunol 161, 5733-5744).
• patients with IBD have adverse and enhanced reactivity to their autologous resident flora (MacDonald TT. (1995) Clin Exp Immunol 102, 445-447).
• intestinal flora may be important in the pathogenesis of intestinal inflammation.
• trefoil factor Manton et al (1996) Science 274, 262-265) or N-cadherin (Hermiston ML & Gordon JI. (1995) Science 270, 1203- 1207).
• These mice also have evidence of inflammation in the gastrointestinal tract.
• intestinal flora it is plausible to hypothesize that food constituents may contribute to the inflammatory response observed in rodents with defective epithelial cell barriers, since they would potentially have access to areas of the gut lumen which they would not have in animals with an intact epithelial barrier.
• Mdr genes encode P-glycoproteins, which are drug efflux pumps. These pumps are localised in the plasma membranes of many tissues and have a natural role to play in protecting the host from potentially harmful compounds.
• mice Three mdrl genes have been identified in rodents (mdrla, mdrlb and mdr2) and two in humans (mdrl and mdr2).
• mdrla P-glycoprotein is also expressed in the blood- brain and blood-testis barrier
• mdrlb P-glycoprotein is expressed in adrenal glands, pregnant uterus and ovaries (Bommhardt U et al. (1994) Eur J Immunol 24, 2974-2981). Both mdrla and mdrlb P-glycoprotein are expressed in the liver, kidney, spleen and heart.
• mice with a targeted deletion of the mdrla gene were generated. These mice had an increased sensitivity to certain drugs, but did not appear to have any constitutive abnormalities (Schinkel AH et al (1994) Cell 77, 491-502). Mdrla " " mice are routinely used in studies to evaluate the involvement of P-glycoprotein in the absorption of compounds, brain penetration of compounds and the hepatobiliary excretion of compounds that are potentially substrates for P-glycoprotein. Interestingly, a recent study reported that approximately 25% of mdrla " " mice spontaneously developed colonic inflammation that is histologically similar to human IBD (Panwala CM et al (1998) J Immunol 161, 5733-5744).
• mice being used for drug metabolism studies, and preliminary data has been reported (Banner KH et al. (2001) Gastroenterology 120, A693).
• clinical symptoms included acute body weight loss and soft "sticky" faeces and histopathological analysis revealed a marked diffuse colitis.
• this is not a reliable model that can be used on a routine basis for this disease.
• the invention is based on the surprising observation that a very high incidence of colitis (with symptoms and histology closely resembling human IBD) is observed in mdrla " " mice when they are supplied with chlorinated drinking water. This observation has led to the development of a robust animal model for IBD, which is useful for testing compounds for their efficacy in treating, curing or preventing IBD, as well as identifying candidate genes which may be involved in the pathogenesis of IBD.
• One aspect of the invention is therefore a method of inducing IBD-like symptoms in a mammal, wherein the mammal (i) is a non-human mammal not expressing a functional mdrla gene product, and (ii) is subjected to elevated chlorine concentrations.
• Another aspect of the invention is a method of inducing IBD-like symptoms in a mammal, wherein the mammal is treated with an inhibitor of the mdrla gene product or with a compound that inhibits the expression of the mdrla gene product in the gut, and is subjected to elevated chlorine concentrations.
• the elevated chlorine concentrations are given by supply of chlorinated drinking water to the mammal.
• the chlorinated drinking water has a chlorine concentration of above lppm, preferably above 3ppm, most preferably 5ppm or above.
• Other ways of subjecting the mammals to elevated chlorine concentrations may include adding chlorine to the food supply, giving the animals chlorine tablets, or using chlorine-bleached bedding for the mammal, exposing the animals to bleach, etc.
• the mammal is a transgenic rodent, preferably a transgenic mouse, not expressing a functional mdrla gene.
• a preferred aspect of the invention is a method as described above wherein more than 50%, preferably more than 70%, more preferably more than 80%, even more preferably more than 90%, and most preferably 100% of the animals show IBD-like symptoms.
• the preferred mammal used in the method is a transgenic mdrla "7" knockout mouse, even more preferred transgenic mouse strain FNB.129P2-Rgy5"* ; ⁇ 7.
• any mammal not expressing the mdrla gene product may be used. This may include a mammal with a naturally occurring mutation in both alleles of the mdrla gene or its promoter. This may also include a wildtype mammal treated with a compound that inhibits the expression of the mdrla gene in the mammal, e.g.
• a cAMP dependant protein kinase inhibitor a sub- therapeutic dose of mitomycin C or an antisense oligonucleotide, ribozyme, siRNA (short interfering RNA) or other molecules interfering with the transcription or translation of the mdrla gene or mRNA, specific for the mdrla orthologue in the mammal of choice.
• the mammal of choice expressing a functional mdrla gene product may be used in the model provided the mdrla gene product is effectively inhibited, e.g. by treatment with a compound that effectively inhibits the mdrla gene product in the mammal, e.g.
• MDR inhibitors by treatment with a compound or compounds that have been identified as MDR inhibitors or by blocking antibodies specific for mdrla.
• Various compounds and mechanisms to inhibit MDR have been reviewed recently (C. Avendano & J.C. Menendez (2002) Curr. Med. Chem. 9, 159-193).
• Another aspect of the invention is a method of screening a candidate compound for its efficacy in ameliorating the symptoms of IBD, the method comprising the following steps:
• a further aspect of the invention is a method of screening a candidate compound for its efficacy
• Yet another aspect of the invention is a method of screening for genes that may be involved in the pathogenesis of IBD and therefore may be novel targets for the development of drugs for the treatment of IBD, comprising the following steps:
• RNA samples wherein a RNA which shows a difference in these samples indicates a gene that may be implicated in the pathogenesis of IBD.
• a further aspect of the invention is a method of screening for genes that may be involved in the pathogenesis of IBD comprising the following steps:
• RNA samples wherein a RNA which shows a difference between the samples of (a) and (c), but not a similar difference between the samples of (b) and (d), is a gene that may be implicated in the pathogenesis of IBD.
• RNA samples mentioned above can be carried out by expression profiling, preferably by differential display PCR or subtractive hybridisation methods, even more preferably by microarray analysis.
• the preferred mammal used in the methods described above is a transgenic mdrla " ' " knockout mouse, even more preferred transgenic mouse strain FVB ⁇ 29 ' P2-Pgy3" nl N7.
• Another aspect of the invention is the identification of the human homologue of any candidate gene identified as described above. This can be carried out by using the mammalian candidate gene as a probe to screen a human cDNA library from suitable human gut tissue, followed by sequence analysis of the positive clones; it can also be done by searching databases such as Genbank (Nucl Acids Res. 30, 17-20 (2002)), available for example on the NCBI web site
• a further aspect of the invention is a method of preparing a composition, which comprises (a) identifying a compound that is capable of ameliorating the symptoms of IBD by the method comprising the following steps:
• Yet a further aspect of the invention is a method of preparing a composition which comprises (a) identifying a compound that is capable of preventing or delaying the development of IBD by the method comprising the following steps:
• Figure 1 Effect of Dexamethasone on colon to body weight ratio in mdrla "7" mice (mean ⁇ sem).
• Figure 2 Effect of Dexamethasone on caecum to body weight in mdrla "7" mice (mean ⁇ sem).
• Figure 3 Height of the colon/rectum mucosa throughout the whole colon rectum.
• mice Forty male mdrla "7" mice (strain FNB.129P2-Pgy3"* 7 ⁇ 7) and six control male mice (mdrla +/+ ) from the same genetic background (FNB; strain FNB/ ⁇ Tac) (6-8 weeks old) were obtained from Taconic Farms (U.S.A). Mice were individually housed in solid bottom cages containing autoclaved Bee Kay bedding from Bantin & Kingman (U.K.) with the addition of Envirodri, a nesting material made from virgin paper, free from deodorising chemicals, to provide enrichment.
• Envirodri a nesting material made from virgin paper, free from deodorising chemicals, to provide enrichment.
• mice Prior to commencing the study, all mice (40 mdrla "7" and 6 mdrla +7+ ) acclimatised for 14 days. From a pilot study (Banner, K.H. et al (2001) Gastroenterology 120, A693) body weight and soft faeces score were found to be most predictive of GI inflammation and thus in the present study these clinical signs were monitored. A scoring system was devised with different weightings for different clinical signs (Table 1). The intention was that the score should reflect the severity of the colitis.
• Scoring system (pilot study) Scoring system (present studjy)
• Body weight (choose one)
• Body weight (choose one)
• the 6 mdrla + + mice received vehicle (water).
• mice were euthanised (one from the vehicle-treated group and one from the group treated with 0.05mg/kg dexamethasone).
• All mice were euthanased.
• the colon and caecum from each mouse was removed and weighed. Each colon was then divided longitudinally so that one section could be processed for histological analysis and the other for cytokine measurement (see below).
• the animals were necropsied.
• the colon/rectum and caecum were weighed and colon/rectum, caecum, ileum, jejunum, duodenum, stomach, mesenteric nodes, thymus, kidney, spleen were sampled and fixed in 10% buffered formalin solution.
• the colon/rectum and ileum were prepared in a "Swiss roll” technique (Moolenbeek, C. &
• Colitis appeared to begin within the proximal region of the colon as this was the main region affected in scores 1 and 2. When the severity of changes increased (scores 3 and 4), lesions were more prominent in the middle and distal regions of the colon and in the rectum.
• the colitis was characterized histologically by variable infiltration of inflammatory cells within the lamina intestinal, mainly macrophages, lymphocytes and polynuclear neutrophils and mucosal thickening. Increase in crypt lengths, increased basophilia and number of mitoses of lining enterocytes, goblet cell loss and interstitial oedema were observed. While only a few erosions were seen in score 2, obliteration of normal architecture, surperficial to transmural ulcerations and crypt abscesses were seen from score 3.
• pathogens such as Helicobacter hepaticus, Helicobacter bilis, Helicobacter rodentium have been associated with the development of gastrointestinal inflammation in rodents.
• Three separate techniques were therefore used to confirm the presence or absence of all known murine pathogens in faecal and colon samples. Faecal samples were analysed using PCR, by two independent collaborators. Colon samples were analysed by electron microscopy and histology using Warthin Starry and Fite Faraco stains. Serological and bacteriological studies were also carried out according to FELASA recommendations (Report of FELASA working group (1996) Laboratory Animals 30, 193-208 & (1994) Laboratory Animals 28; 1-12).
• Colitis severity score is an ordered categorical measure graded on a scale of 0-4. This type of data is generally analysed using a proportional odds model, however due to the small sample size this was not appropriate and ANOVA was used. This makes the assumption of having normally distributed data, which although we had categorical data, seemed to hold approximately true. All analysis was carried out using Genstat 5 Release 4.2 for Windows.
• Table 3 Growth Rate and body weight over study period (allowing for culling group). *: Two sample t test: *P ⁇ 0.05 vs mdrla +7+
• Dexamethasone treatment however, had no effect on levels of either cytokine at any of the doses used (Table 4).
• Table 4 Levels of IL-8 and IFN- ⁇ in homogenised colon samples from mdrla +/+ and mdr 1 a " " mice (allowing for culling group)
• Table 5 Effect of dexamethasone (0-2mg/kg ) on colitis severity scores graded histologically.
• mice were free of viruses, bacteria and parasites known to be pathogenic as specified in FELASA recommendations (Report of FELASA working group (1996) Laboratory Animals 30, 193-208 & (1994) Laboratory Animals 28; 1-12).
• Example 2 Controlled study
• mice and 16 mdrla ++ mice of otherwise identical genetic background are acclimatised with unchlorinated drinking water. The animals are then divided into groups as shown in the Table below (Table 6):
• mice are then monitored for soft faeces and absence of body weight gain for up to 5 weeks.
• mice 40 mdrla-/- mice are acclimatised, and then given 5ppm chlorinated drinking water for 2 days. They are then divided into 4 groups:
• Symptoms of IBD are monitored for 3-4 weeks, using one of the methods described in
• Example 1 A test compound is considered promising for the treatment of IBD if a significant, dose-related improvement is observed in the animals treated with the test compound as compared with the animals which are given vehicle only.
• Example 4 Prevention of IBD
• mice 40 mdrla-/- mice are acclimatised for 2 days, during which they are given unchlorinated drinking water. They are then divided into 4 groups: 10 animals are given vehicle only
• test compound 10 animals are given 0.05 mg kg of test compound 10 animals are given 0.5 mg/kg of test compound 10 animals are given 5 mg/kg of test compound
• the animals are supplied with chlorinated (5ppm) drinking water. The animals are then monitored for the onset of IBD-like symptoms.
• test compound is considered promising for the prevention of IBD if a significant, dose- related decrease or delay in the onset of symptoms is observed in the animals treated with the test compound as compared with the animals which are given vehicle only.
• Example 5 Comparison of changes in gene expression in colons from mdrla “7” vs wild type (FNB) mice, and the effect of dexamethasone on these changes
• the aims of the study were to compare changes in gene expression in colons from FVB wildtype vs. mdrla " " mice and to examine the effect of dexamethasone on these changes.
• mice Male FVB wildtype and mdrla "7" mice (9-10 weeks old) were dosed with dexamethasone (2mg/kg i.p.) or vehicle (water) daily for 7 days. Mice were culled and colons removed for histological assessment.
• R ⁇ A was purified from a single donor from each group using a modified Trizol and R ⁇ easy protocol, yielding greater than 50 ug R ⁇ A/sample, with a A260/A280 purity greater than 1.9.
• RNA quality and hybridisation signal were good in all samples.
• Mouse U74A data was normalised to 300. Total number of probe sets (-genes) 12,639.
• mdrla "7" mice showed clear evidence of increased inflammation as indicated by, for example, upregulation of inflammatory markers such as complement components, MHC molecules, leukocyte markers (indicating infiltration of leukocytes into the tissue), cytokines, chemokines, integrins, cyclooxygenase-2 and TNF family molecules and ligand, as well as showing evidence of tissue destruction as indicated by, for example, upregulation of metalloproteinase expression.
• known drug targets for IBD in the mdrla "7" mice as compared to the control animals, such as phospholipase A2 (about 12x upregulated), or inducible nitric oxide synthase (about 5x upregulated). This clearly indicates the suitability of the method for the identification of new drug targets that will aid in the identification of effective treatments for IBD.
• Candidate drug targets are, for example, genes that are upregulated in mdrla "7" mice and that are brought down to near normal levels by the dexamethasone treatment.
• Candidate biomarkers useful in diagnosis and assessment of disease status, progression, and success of therapy, can also be identified by such an experiment; for example, we found known markers for IBD such as interleukin-l ⁇ (61 fold upregulated), matrix metalloproteinase 3 (MMP-3; 29 fold upregulated) or granzyme B (6 fold upregulated), upregulated in the mdrla "7' mice as compared to the control animals. Other genes showing the same pattern of regulation in this experiment are therefore also candidate biomarkers or diagnostic tools for IBD.
• Example 6 Time course of changes in gene expression in mdrla "7" mice after switch to chlorinated drinking water as compared to wildtype mice.
• mice Male mice (9-10 weeks old) (same number of wildtype and mdrla "7" ) are acclimatised, receiving unchlorinated drinking water, for 2 days. The water is then changed to chlorinated drinking water (5ppm), and mice from each group in parallel are culled and colons removed for histological assessment and RNA isolation just before and at various time points after they have started drinking the chlorinated water. Suitable time points would be 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 48 hours, 72 hours.
• Genes that are candidates for being involved in the pathogenesis of IBD are those that change in expression in mdrla " " mice but not (or not to the same extent) in wildtype mice. Especially interesting are genes that change early after switching the water supply, as these may be genes involved in the onset of the disease, and inhibiting those may prevent the onset of the disease.
• genes that are candidates for being useful biomarkers for IBD can also be identified by this experiment.
• such genes may encode proteins that are found in serum, and useful biomarkers may be upregulated or downregulated in the mdrla "7" mice as compared to the wildtype mice.

Landscapes

• Life Sciences & Earth Sciences (AREA)
• Health & Medical Sciences (AREA)
• Genetics & Genomics (AREA)
• Engineering & Computer Science (AREA)
• Biotechnology (AREA)
• Zoology (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Organic Chemistry (AREA)
• Biomedical Technology (AREA)
• General Engineering & Computer Science (AREA)
• Environmental Sciences (AREA)
• General Health & Medical Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Veterinary Medicine (AREA)
• Wood Science & Technology (AREA)
• Microbiology (AREA)
• Physics & Mathematics (AREA)
• Biochemistry (AREA)
• Plant Pathology (AREA)
• Biophysics (AREA)
• Animal Behavior & Ethology (AREA)
• Animal Husbandry (AREA)
• Biodiversity & Conservation Biology (AREA)
• Molecular Biology (AREA)
• Investigating Or Analysing Biological Materials (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Applications Claiming Priority (3)

Publications (1)

Family

ID=9952805

Family Applications (1)

Country Status (4)

Families Citing this family (4)

• 2003
  • 2003-02-11 GB GB0303085A patent/GB0303085D0/en not_active Ceased
• 2004
  • 2004-02-03 WO PCT/IB2004/000312 patent/WO2004071186A1/en not_active Ceased
  • 2004-02-03 EP EP04707600A patent/EP1594358A1/de not_active Withdrawn
  • 2004-02-03 JP JP2006502410A patent/JP2006520192A/ja active Pending

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events