WO2009022119A1 - Endométriose de murin modélisée par activation du k-ras d'un endomètre menstruel - Google Patents

Endométriose de murin modélisée par activation du k-ras d'un endomètre menstruel Download PDF

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WO2009022119A1
WO2009022119A1 PCT/GB2008/002713 GB2008002713W WO2009022119A1 WO 2009022119 A1 WO2009022119 A1 WO 2009022119A1 GB 2008002713 W GB2008002713 W GB 2008002713W WO 2009022119 A1 WO2009022119 A1 WO 2009022119A1
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rodent
endometriosis
nucleic acid
donor
endometrium
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PCT/GB2008/002713
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David Stephen Charnock-Jones
Cristin Print
Ching-Wen Cheng
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Cambridge Enterprise Limited
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    • 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
    • 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
    • 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
    • A01K2217/05—Animals comprising random inserted nucleic acids (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
    • 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
    • A01K2267/0331—Animal model for proliferative diseases

Definitions

  • This invention relates to models of endometriosis, in particular rodent endometriosis models which may be useful, for example, in the development of treatments for this condition.
  • Endometriosis is one of the most common causes of pelvic pain and infertility in women, which afflicts approximately 5-10% of women of reproductive age [1-3] .
  • the pathological diagnosis of endometriosis is characterized by the presence of benign endometrial glands and stroma outside the uterus [1, 4] .
  • Endometriosis occurs naturally in human and some non-human primates, and non-human primates have been used widely for endometriosis research [6-8] .
  • ethical considerations and the cost of keeping non-human primates limit the use of these animal models.
  • small laboratory animals such as rodents do not naturally menstruate nor develop endometriosis
  • a model of endometriosis using small laboratory animals is a more ethically and economically attractive option.
  • the autotransplantation model of endometriosis in which pieces of uterine horn are surgically transposed onto connective tissue supplied by the internal mesenteric artery, has been performed in rat and mouse [9-12] ; and has also been applied to rabbit [13] .
  • This model is the most commonly used one among the three existing murine models, however the inclusion of myometrium in the ectopic uterine fragments is different from pathogenesis of human endometriosis and is the main weakness of this model.
  • Histologically nude mouse implants are characterised by the presence of endometrial glandular acini and stromal cells although lesions can contain necrosis, haemosiderin and an infiltrate of macrophages and other leucocytes.
  • estrogen supplementation is associated with larger lesions and a proliferative histological pattern whereas danazol resulted in atrophic tissue changes [19] .
  • An unfavourable aspect of the nude mouse model is that the proportion of mice that develop lesions has been documented to vary from 33-100% [18-20] .
  • the SCID mouse model has been reported to show higher success rate in lesion development [21, 22], and this might be due to their defective natural killer cell function [21] .
  • NOD-SCID non-obese diabetic SCID
  • little difference in lesion development frequency was observed [23] .
  • the xenograft model is the first established endometriosis model using small laboratory animals, it is not as widely used as the homologous model, probably due to the difficulty of collecting human endometrial tissue.
  • the compromised immune function in host mice is a significant key limitation of this model .
  • the three current murine models of endometriosis all have their limitations. During menstruation, the superficial layer of endometrium breaks down and is sloughed from the basal layer. These tissues are stressed, undergoing degeneration involving many apoptotic cells and the overall tissue is rather fragile.
  • One common difference between the existing models and human endometriosis is that the donor tissues from all these three models are intact rather than stressed and fragile as is the case in menstruating endometrium.
  • the endometrium of small laboratory animals does not decidualize spontaneously without embryo implantation. Furthermore these species do not menstruate.
  • a murine model of decidualization and menstruation based on Finn and Pope's report has been developed [24, 25] .
  • sloughed decidualized endometrium was isolated from a progesterone-withdrawn donor.
  • the menstruating endometrium barely survived when transplanted into immunocompromised nude mice but not at all when transferred to immunocompetent recipients of the same strain.
  • a murine endometriosis model based on the activation of an oncogenic K-ras allele using transgenic mice was reported in 2005 [26] . This model requires the injection of a cre-encoding retrovirus into the ovarian bursa, which is a surgical procedure.
  • the present inventors have discovered that the implantation of menstruating endometrium from K-ras activated mice into wild type recipients produces viable endometriosis-like lesions. This allows the production of a rodent model of endometriosis which may be useful in the development of therapeutics .
  • a first aspect of the invention provides a method of producing a rodent endometriosis model comprising providing a female donor rodent having an activatable oncogenic nucleic acid sequence, activating the oncogenic nucleic acid sequence, inducing menstruation of endometrium comprising activated oncogenic nucleic acid sequence from the donor rodent, and implanting the menstruating endometrium into a female recipient rodent.
  • endometrial lesions develop in the recipient rodent. Endometrial lesions are glands of tissue containing both stromal and glandular epithelial cells as well as blood vessels and fibrous collagen structures.
  • the recipient rodent thus represents a model of endometriosis.
  • the donor rodent may be any suitable laboratory species, including mouse, hamster, rat or guinea pig. In preferred embodiments, the rodent may be a mouse. A donor mouse may be of any suitable murine strain.
  • the donor rodent is ovariectomised.
  • the recipient rodent has intact ovaries, i.e. is not ovariectomised, and/or is not treated with exogenous oestrogen, e.g estradiol.
  • a suitable oncogenic nucleic acid may confer a growth advantage or increased resistance to apoptosis on a host cell expressing the nucleic acid, without causing tumourigenesis
  • An activatable oncogenic nucleic acid sequence is a nucleic acid which is inactive and is not expressed to produce an active product within the donor rodent until a suitable stimulus is applied. Upon exposure to the stimulus, the oncogenic nucleic acid is activated and active product is produced within those cells of the donor rodent which are subjected to the stimulus.
  • Suitable stimuli for activating the oncogenic nucleic acid are described below.
  • the activatable oncogenic nucleic acid sequence may be located extrachromosomally or more preferably within the genome of the donor rodent .
  • the oncogenic nucleic acid is an oncogenic K-ras nucleic acid.
  • K-ras protein is a 21-kD GTPase which is a member of the ras signalling pathway and modulates cellular proliferation and differentiation.
  • Wild-type K-ras may be converted into an oncogenic K-ras allele by mutation of one or more amino acids within the wild-type K-ras sequence, typically within the GTPase domain.
  • Oncogenic K-ras alleles cause constitutive activation of ras-signalling pathway, leading to unregulated cellular proliferation and impaired differentiation.
  • An oncogenic K-ras nucleic acid sequence encodes an oncogenic K- ras allele.
  • a suitable sequence may encode the sequence of wild- type K-ras with an oncogenic mutation.
  • Suitable oncogenic mutations include the substitution of GIy at position 12 or 13 in the K-ras sequence.
  • GIy 12 may be substituted for Ser, VaI or Asp.
  • the GIy 12 may be substituted for VaI (i.e. K-ras V12) .
  • an oncogenic K-ras nucleic acid may have the murine K-ras sequence with one or more oncogenic mutations .
  • Murine K-ras has an amino acid sequence having the database accession number NP_067259.3 GI: 84370270 and is encoded by a nucleic acid having the database accession number NM_021284.4 GI: 142374722.
  • the oncogenic K-ras nucleic acid sequence may be a heterologous sequence, for example from a non-murine or non-rodent mammalian species, with one or more oncogene- activating mutations.
  • Suitable heterologous oncogenic K-ras nucleic acids include the human K-ras isoform a which has an amino acid sequence having the database accession number NP_004976.2 GI: 15718761 which is encoded by a nucleic acid having the database accession number NM_004985.3 GI: 34485723 or the human K-ras isoform b which has an amino acid sequence having the database accession number NP_203524.1 GI: 15718763 which is encoded by a nucleic acid having the database accession number NM_033360.2 GI: 34485724
  • a heterologous nucleic acid is a nucleic acid that is outside its natural environment i.e. it is not naturally occurring within the donor rodent.
  • a heterologous nucleic acid may be recombinant.
  • the heterologous nucleic acid may, for example, have been introduced into the donor rodent or an ancestor thereof by conventional recombinant techniques.
  • the donor rodent may be homozygous or heterozygous for the activatable oncogenic nucleic acid sequence.
  • the donor rodent may be a K-ras +/ ⁇ mouse, such as a K-ras vl2+/ ⁇ mouse.
  • the activated oncogenic nucleic acid may be operably linked to appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate.
  • the regulatory sequences drive the expression of the oncogenic nucleic acid in rodent cells following activation.
  • tissue-specific regulatory sequences may drive expression of the activated oncogenic nucleic acid sequence in specific tissues, such as the endometrium.
  • the regulatory sequences may be operably linked to the oncogenic nucleic acid sequence before the oncogenic nucleic acid sequence is activated or may only become operably linked to the oncogenic nucleic acid sequence during activation.
  • Activation of the oncogenic nucleic acid may be systemic throughout the rodent or may only occur in specific tissues such as the endometrium. Tissue specific activation may be achieved, for example, by the application of the stimulus to specific tissues .
  • any suitable stimulus may be employed to activate the oncogenic nucleic acid.
  • the stimulus may be expression of a site specific recombinase.
  • the oncogenic nucleic acid may be activatable by a site specific recombinase.
  • Suitable site specific recombinases include Cre and FLP.
  • Cre recombinase is a Type I topoisomerase from bacteriophage Pl that catalyzes the site-specific recombination of DNA between loxP sites (Abremski, K. and Hoess, R. (1984) J. Biol. Chem., 259, 1509-1514) .
  • loxP sites which are recognised by Cre recombinase are 34 base pair (bp) sequences comprised of two 13 bp inverted repeats flanking an 8 bp spacer region (Metzger, D. and Feil,
  • Cre recombinase may, for example, have the amino acid sequence having the database accession number YP__006472.1 GI: 46401628. Nucleic acid encoding Cre recombinase may be provided using conventional techniques. The nucleic acid sequence of Cre recombinase is found between nucleotides 436 to 1467 of the Pl phage genome of NC_005856.1 GI: 46401624.
  • FLP recombinase is a site specific integrase from the 2 micron plasmid of S. cerevisiae that catalyzes the site-specific recombination of DNA between FLT sites.
  • the FLT sites which are recognised by FLP recombinase are comprised of three 13 bp repeats and an 8 bp spacer region.
  • the use of FLP recombinase to mediate recombination between FLT sites is well known in the art (see for example Pan et al MoI Cell Biol. (1993) 13(6) 3167- 3175) .
  • FLP recombinase may, for example, have the amino acid sequence having the database accession number NP_040488.1 GI11466068.
  • Nucleic acid encoding FLP recombinase may be provided using conventional techniques.
  • the nucleic acid sequence of FLP recombinase is found between nucleotides 5570 to 6318 and 1 to 523 of the 2 micron plasmid sequence of NC 001398.1 GI11466067.
  • the oncogenic nucleic acid sequence may be associated in the genome of the donor rodent with target sites for the site specific recombinase (e.g.
  • the oncogenic nucleic acid sequence and target sites may be in any suitable arrangement which causes activation of the oncogenic nucleic acid sequence following recombinase mediated recombination between the target sites.
  • a blocking sequence flanked by target sites may be positioned within the oncogenic nucleic acid.
  • the blocking sequence prevents transcription of active oncogenic nucleic acid.
  • the blocking sequence is excised by recombinase-mediated recombination between the target sites, allowing transcription of the active oncogenic nucleic acid.
  • a blocking sequence may be positioned between the oncogenic nucleic acid and a promoter. Excision of the blocking sequence operably links the promoter and the oncogenic nucleic acid, allowing transcription of the oncogenic nucleic acid.
  • the oncogenic nucleic acid may be flanked by target sites and positioned in an inverted orientation with respect to its promoter, such that it is not expressed in an active form.
  • the oncogenic nucleic acid is activated by expression of site specific recombinase, which mediates inversion of the oncogenic nucleic acid between the inverted target sites, allowing transcription of the active oncogenic nucleic acid.
  • the oncogenic nucleic acid may comprise a STOP codon flanked by target sites.
  • the STOP codon prevents the translation of active oncogenic protein from the nucleic acid.
  • Site specific recombinase mediates excision of the STOP codon from the nucleic acid, allowing active oncogenic protein to be translated from the oncogenic nucleic acid.
  • the donor rodent further comprises a nucleic acid sequence encoding a site specific recombinase such as Cre or FLP. Expression of the nucleic acid produces the site specific recombinase which activates the oncogenic nucleic acid sequence.
  • a site specific recombinase such as Cre or FLP.
  • the nucleic acid encoding site specific recombinase may be operably linked to appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate.
  • the regulatory sequences drive the expression of site-specific recombinase in rodent cells.
  • expression of the site- specific recombinase is tissue specific.
  • site-specific recombinase not expressed in all cells of the donor rodent.
  • Site-specific recombinase may, for example, be specifically expressed in epithelial cells, in particular cells of the endometrium.
  • the nucleic acid encoding the site-specific recombinase gene may be operably linked to tissue specific, in particular epithelial cell specific, regulatory sequences, such as promoters.
  • tissue specific, in particular epithelial cell specific, regulatory sequences such as promoters.
  • epithelial promoters are known in the art and include the Ah promoter .
  • the nucleic acid encoding the site-specific recombinase gene may be operably linked to inducible regulatory sequences, such as promoters .
  • Inducible regulatory sequences may provide expression of the site-specific recombinase only in cells or tissues which are exposed to an inducing agent.
  • Site-specific recombinase is expressed following exposure of cells or tissues to the inducing agent and the expressed site-specific recombinase activates the oncogenic nucleic acid sequence in the cells or tissues of the donor rodent which are exposed to the inducing agent.
  • the tissue specificity of site-specific recombinase expression may be increased by applying the inducing agent selectively to the uterus and/or endometrium of the donor rodent, such that expression of the site-specific recombinase is only induced in the endometrium and adjacent tissues and not systemically in all tissues of the donor rodent.
  • inducible promoters include Ah, Tet-on, ecdysone or tamoxifen controlled promoters.
  • the site-specific recombinase gene may be operably linked to the Ah promoter, which is induced by administration of ⁇ -napthoflavone ( ⁇ -NF) .
  • the Ah promoter is especially preferred because it is both inducible and epithelial cell specific (Kemp et al Nucl Acid Res 2004 32 e92; Ireland et al (2004) Gastroenterology 126 1236-1246).
  • the inducing agent which induces expression of the site-specific recombinase may be administered systemically, for example by intraperitoneal injection, or may be administered specifically to the uterus and/or endometrium of the donor rodent, for example by injection into one or both horns of the uterus, such that site-specific recombinase expression is induced in the endometrium and adjacent tissues and not systemically in all tissues of the donor rodent.
  • the donor rodent may be heterozygous or, more preferably, homozygous for the nucleic acid encoding site specific recombinase,
  • the donor rodent may be a AhCre +/+ mouse.
  • the donor rodent may further comprise a nucleic acid encoding an activatable first reporter gene.
  • a reporter gene is a nucleic acid which encodes a detectable gene product.
  • a reporter gene may encode an enzyme which mediates a luminescent, fluorescent or chromogenic reaction which produces a detectable signal.
  • Suitable reporter genes are well known in the art and include green fluorescent protein (GFP) , luciferase, and enhanced yellow fluorescent protein (eYFP) and variants thereof, alkaline phosphatase, ⁇ - galactosidase (lacZ) , and ⁇ -glucuronidase.
  • the reporter gene may be inactive (i.e. not expressed in an active form within the donor rodent) until the stimulus which activates the oncogenic nucleic acid is applied. Upon exposure to this stimulus, the reporter gene is activated and expressed in an active form within tissues of the donor rodent which are subjected to the stimulus, in addition to the oncogenic nucleic acid. Expression of the reporter gene in a tissue or cell is therefore indicative of expression of the oncogenic nucleic acid in the tissue or cell.
  • the stimulus is preferably expression of site-specific recombinase i.e. expression of the reporter gene may be activatable by site-specific recombinase.
  • the activatable reporter gene may be associated in the genome of the donor rodent with target sites which allow the activation of the reporter gene by site-specific recombinase.
  • the reporter gene and target sites may be in any suitable arrangement which allows activation of the reporter gene by recombination between the target sites. Suitable arrangements are described in more detail above.
  • a Cre recombinase activatable reporter gene such as lacZ, may be present at the ROSA26 locus of the donor rodent .
  • the donor rodent may further comprise a nucleic acid encoding a second reporter gene which is expressed consitutively .
  • a second reporter gene which is expressed consitutively .
  • the implanted menstruating endometrium may constitutive express luciferase, and may be detected in the recipient rodent using real-time in vivo imaging techniques (Xenogen Corp, MA USA) .
  • the donor rodent may be heterozygous or, more preferably, homozygous for the activatable reporter gene.
  • the donor rodent may be a ROSA26R-LacZ +/+ mouse.
  • a suitable donor rodent may comprise an inducible site specific recombinase nucleic acid sequence, a site specific recombinase activatable oncogenic nucleic acid sequence and a site specific recombinase activatable reporter gene.
  • the donor rodent is heterozygous for the oncogenic nucleic acid sequence and homozygous for the site specific recombinase and reporter gene.
  • the donor rodent may be a Jf- ras vl2+/' /AhCre +/+ /ROSA26R-LacZ +/+ mouse.
  • a donor rodent may comprise one or more additional genetic modifications to those set out above. Suitable modifications include deletions, insertions or substitutions of one or more nucleotides. A genetic modification may inactivate or modify the activity of a target sequence. Suitable target sequences include genes which encode chemokines, cytokines, growth factors and their receptors, proteases, protease inhibitors, immune regulators or adhesion molecules.
  • Nucleic acid sequences as described above may be comprised within a vector in the genome of the donor rodent.
  • Vectors may be plasmids, viral e.g. 'phage, or phagemid, as appropriate.
  • plasmids viral e.g. 'phage, or phagemid, as appropriate.
  • Molecular Cloning a Laboratory Manual: 3rd edition, Russell et al., 2001, Cold Spring Harbor Laboratory Press, and Protocols in Molecular Biology, Second Edition, Ausubel et al . eds . John Wiley & Sons, 1992.
  • a viral vector suitable for expression in rodent cells may be employed.
  • Suitable viral vectors include adenovirus, adeno-associated virus (AAV) , for example AAV serotype 2 virus, retrovirus, lentivirus, recombinant adenovirus, ⁇ gutless' adenovirus, herpes simplex virus, and poliovirus vectors.
  • a viral vector may be packaged into a viral particle comprising one or more capsid proteins prior to transfection of host cells.
  • suitable techniques may include DEAE-dextran, polyethyleneimine, electroporation, liposome-mediated transfection and transduction using retrovirus or other virus, e.g. adenovirus, AAV, lentivirus or vaccinia.
  • Transgenic donor rodents as described herein may be produced in accordance with standard techniques.
  • a heterologous nucleic acid may be introduced into a rodent germ line cell, and a transgenic rodent generated from said rodent germ line cell.
  • a suitable rodent germ-line cell may include an egg, oocyte or embryonic stem (ES) cell.
  • the nucleic acid may be introduced into a germ line cell that is comprised in an early stage embryo, such as a blastocyst.
  • the heterologous nucleic acid or vector may be introduced into the germ line cell using any method known in the art, including, for example, pronuclear microinjection; retrovirus mediated gene transfer into germ lines; gene targeting in embryonic stem cells; electroporation of embryos; sperm-mediated gene transfer; calcium phosphate/DNA co-precipitates, microinjection of DNA into the nucleus, bacterial protoplast fusion with intact cells, transfection, and polycations, e.g., polybrene, polyornithine, etc., (See, for example Van der Putten, et al . , 1985, Proc. Natl. Acad. Sci., USA 82:6148-6152; Thompson, et al .
  • the cells in which the heterologous nucleic acid has successfully incorporated into the rodent germ- line cell genome may be identified.
  • Cells comprising the heterologous nucleic acid may be identified, for example, by detecting the expression of a marker gene.
  • transformed cells may be treated with a selective agent that selects either cells expressing or cells not expressing the selectable marker.
  • selectable markers and agents are known in the art. For example, cells expressing the introduced neomycin resistance gene are resistant to the compound G418, while cells that do not express the neo gene marker are killed by G418.
  • Successful insertion of the heterologous nucleic acid into the genome may be confirmed by analyzing the DNA of the selected cells using routine techniques, such as PCR and/or Southern analysis .
  • a rodent may be generated from a cell comprising the heterologous nucleic acid or vector using standard techniques (see for example Piedrahita et al (1992) PNAS USA 89 4471-4475, Roller et al (1989) PNAS USA 86 8927-8931; Transgenic Animal Technology: A Laboratory Handbook, Pinkert CA (2002) Academic Press) .
  • the cell may be introduced into a blastocyst.
  • the injection of transformed cells injected into a rodent blastocyst may lead to the formation of chimeras (see e.g., Bradley, A. in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E. J. Robertson, ed., IRL, Oxford, pp.
  • germ-line cells identified as comprising the heterologous nucleic acid may be allowed to aggregate with dissociated rodent embryo cells to form the aggregation chimera.
  • a chimeric embryo can then be implanted into a suitable pseudopregnant female foster rodent and the embryo brought to term.
  • Chimeric progeny harbouring the heterologous nucleic acid in their germ cells can be used to breed mice in which all cells of the rodent comprise the heterologous nucleic acid.
  • Rodents such as mice, which are produced as described may be crossed with mice of the same or other genotypes to produce descendents .
  • the genotype of the rodent or descendent may be determined.
  • a method may include determining that the rodent or its descendent specifically expresses the heterologous nucleic acid(s). Methods of determining the genotypes of rodents are well-known in the art.
  • Menstruation of the endometrium of the donor rodent may be induced following or simultaneous with activation of the oncogenic nucleic acid.
  • menstruation is induced by lowering progesterone levels in the endometrium of the donor rodent. Suitable methods are described in Finn, C. A. & Pope, M. (1984) J Endocrinol. 200, 295-300 and Cheng, C. W., et al (2007) Biol Reprod. 76 871- 883.
  • menstruation may be induced by treating the donor rodent with progesterone and oestrogen, for example 17 ⁇ - oestradial (E2), inducing decidualisation of the donor rodent endothelium and lowering progesterone levels, for example by withdrawing or stopping further progesterone treatment.
  • progesterone and oestrogen for example 17 ⁇ - oestradial (E2)
  • decidualisation of the donor rodent endothelium for example by withdrawing or stopping further progesterone treatment.
  • the rodent may initially be treated with progesterone and oestrogen for a number of days, for example up to 6 days, up to 8 days or up to 10 days. Suitable regimens are described in Finn, C. A. et al (1984) J Endocrinol. 100, 295-300 and Cheng, C. W. et al (2007) Biol Reprod. 76 871-883.
  • a course of treatment may comprise administration of oestrogen on day 1 and day 2, and administration of progesterone and oestrogen on day 6> day 7, and day 8, as shown in Table 1.
  • Progesterone and oestrogen may be administered by subcutaneous injection.
  • Decidualisation may be induced in the endometrium of the donor rodent by any convenient technique including physical means such as crushing or drawing a .thread through the lumen.
  • decidualisation is induced by administration of oil, such as maize or peanut oil, to the uterus of the donor rodent, for example by injection.
  • Decidualisation may be induced after or simultaneously with activation of the oncogenic nucleic acid.
  • Progesterone levels may be lowered concomitantly with decidualisation or after decidualisation. Progesterone levels may be lowered by withdrawing or stopping progesterone treatment and/or by administering an antiprogesterone.
  • Antiprogesterones include progesterone receptor antagonists such as mifepristone (RU486) and RU38486 and anti-progesterone antibodies. Withdrawing progesterone treatment or administering antiprogesterone has the effect of lowering progesterone levels in the endometrium of the donor rodent, causing degeneration of the donor rodent endometrium and leading to the production of menstruating endometrial tissue. Menstruating endometrial tissue may be collected from the donor rodent 48 to 84 hours, preferably 50 to 70 hours after the final administration of progesterone, more preferably around 60 hours.
  • Menstruating endometrial tissue may be resuspended in a suitable medium, such as a collagen or fibrin gel or matrigelTM (BD Biosciences, Oxford UK), after collection from the donor rodent.
  • a suitable medium such as a collagen or fibrin gel or matrigelTM (BD Biosciences, Oxford UK)
  • the menstruating endometrial tissue may be treated with drugs, antibodies and/or subjected to genetic modification, for example by transfection with plasmid or viral vectors or other nucleic acids such as antisense oligonucleotides or RNAi.
  • the collected menstruating endometrial tissue may be labelled with fluorescent dyes to allow tracking of the tissue after implantation.
  • the menstruating endometrial tissue may be implanted into the recipient rodent by any suitable technique.
  • the tissue may be sub-cutaneously injected into the abdominal side of the recipient rodent.
  • the menstruating endometrial tissue is implanted within 30 mins, preferably less than 10 mins of collection from the donor rodent .
  • the recipient rodent may be any suitable laboratory species, including mouse, hamster, rat or guinea pig.
  • the recipient rodent may be a mouse.
  • a recipient mouse may be of any suitable murine strain.
  • the recipient rodent is the same species and strain as the donor rodent.
  • the recipient rodent may be an immunocompetent rodent.
  • Suitable strains include C57BL/6 mice
  • the recipient rodent may be treated with an oestrogen, such as 17 ⁇ -estradiol, before implantation of the menstruating endometrial tissue. In other embodiments, the recipient rodent is not treated with an oestrogen, such as 17 ⁇ - estradiol, before implantation of the menstruating endometrial tissue .
  • the recipient rodent may comprise one or more additional genetic modifications to those set out above. Suitable modifications include deletions, insertions or substitutions of one or more nucleotides and may inactivate or modify the activity of a target sequence. Suitable target sequences include genes which encode chemokines, cytokines, growth factors and their receptors, proteases, protease inhibitors, immune regulators or adhesion molecules.
  • endometrial lesions form in the recipient rodent.
  • the lesions may form within 28 days following implantation, typically in 7 to 28 days.
  • the endometrial lesions may be stable in the recipient rodent for 7 days or more, 14 days or more or 21 days or more
  • Endometrial lesions may be detected in the recipient rodent by conventional techniques.
  • endometrial lesions may be detected in samples obtained from the recipient rodent by standard immunohistochemical techniques.
  • endometrial lesions may be detected in situ using real time in vivo imaging techniques. Suitable imaging techniques are known in the art (see for example Masuda et al
  • a method of screening for an agent for use in the treatment of endometriosis may comprise: producing a rodent endometriosis model by a method described above, administering a test compound to the rodent endometriosis model, and determining the effect of said compound on endometriosis in said model.
  • the effect of the compound on the implanted menstruating endometrium may be determined.
  • the size or amount of endometriotic lesions formed by the implanted menstruating endometrium may be determined.
  • the cellular composition of the lesions can be determined
  • This may be carried out using standard techniques, such as immunohistochemistry or in vivo imaging.
  • test compound is a putative agent which may be useful in the treatment of endometriosis.
  • Test compounds which may be screened using the methods described herein may be natural or synthetic chemical compounds used in drug screening programmes. Extracts of plants, microbes or other organisms which contain several characterised or uncharacterised components may also be used.
  • Combinatorial library technology provides an efficient way of testing a potentially vast number of different compounds for ability to modulate an interaction.
  • Such libraries and their use are known in the art, for all manner of natural products, small molecules and peptides, among others.
  • the use of peptide libraries may be preferred in certain circumstances.
  • test compound or compound which may be added to a method of the invention will normally be determined by trial and error depending upon the type of compound used.
  • amount of test compound or compound which may be added to a method of the invention will normally be determined by trial and error depending upon the type of compound used.
  • from about 0.00InM to ImM or more of putative inhibitor compound may be used, for example from 0.0InM to lOO ⁇ M, e.g. 0.1 to 50 ⁇ M, such as about lO ⁇ M.
  • test compounds for screening include small organic molecules, peptides, polypeptides, cells, nucleic acids, and vectors .
  • Suitable polypeptides for screening may include antibodies, antibody derivatives or other specific binding proteins, chemokines, cytokines, growth factors, proteases, protease inhibitors, immune regulators, adhesion molecules.
  • Nucleic acids may include aptamers or sense or anti-sense suppression constructs which reduce or abolish expression of a target gene.
  • Vectors may include plasmid or viral vectors.
  • a vector may comprise a heterologous nucleic acid for expression in the rodent model.
  • a heterologous nucleic acid may encode a polypeptide of interest or mediate the suppression of expression of a gene of interest in the model.
  • a method described herein may comprise identifying a test compound as useful in the treatment of endometriosis. Following identification of a compound using a method described above, the compound may be isolated and/or synthesised.
  • the identified compound may be synthesised using conventional chemical synthesis methodologies. Methods for the development and optimisation of synthetic routes are well known to persons skilled in this field.
  • a compound identified using a method described herein may be assessed or investigated further using one or more secondary screens.
  • the toxicology and/or biological effect of the compound may be determined in wild-type non-human animals.
  • the donor and or recipient mice may be sacrificed or euthanized.
  • a compound identified using a method described herein may be modified to optimise its pharmaceutical properties.
  • the modified compound may be tested using the methods described herein to see whether it has the target property, or to what extent it is exhibited.
  • Modified compounds include mimetics of the lead compound. Further optimisation or modification can then be carried out to arrive at one or more final compounds for in vivo or clinical testing.
  • test compound may be used in preparation, i.e. manufacture or formulation, of a composition such as a medicament, pharmaceutical composition or drug. These may be administered to individuals, e.g. for the treatment of a condition described herein.
  • a method may comprise formulating the test compound or the modified test compound in a pharmaceutical composition with a pharmaceutically acceptable excipient, vehicle or carrier. Suitable acceptable excipients, vehicles and carriers are well- known in the art .
  • pharmaceutically acceptable refers to compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgement, suitable for use in contact with the tissues of a subject (e.g., human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • a subject e.g., human
  • Each carrier, excipient, etc. must also be “acceptable” in the sense of being compatible with the other ingredients of the formulation.
  • Suitable carriers, excipients, etc. can be found in standard pharmaceutical texts, for example, Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Company, Easton, Pa., 1990.
  • a rodent endometriosis model may comprise: a recipient rodent implanted with menstruating endometrium from a donor rodent, wherein the menstruating endometrium of the donor rodent has an activated oncogenic nucleic acid sequence.
  • a suitable rodent endometriosis model may be obtainable by a method described above.
  • a rodent endometriosis models is produced by the methods described above.
  • Menstruating endometrium with an activated oncogenic nucleic acid sequence may be produced from a donor rodent by inducing menstruation in the donor rodent, for example by lowering progesterone levels in the endometrium, and collecting the menstruating endometrium from the donor rodent.
  • the induction of menstruation, collection and implantation of menstruating endometrium from a donor rodent is described in more detail above .
  • the rodent endometriosis model may be useful, for example, in investigating the pathogenesis of endometriosis, identifying target genes or other factors associated with endometriosis and screening for agents useful in the treatment of endometriosis.
  • a method of identifying or screening for a gene involved in endometriosis may comprise; altering the expression of a candidate gene in a rodent endometriosis model as described above; and, determining the effect of said activation on endometriosis in said model.
  • the expression of a candidate gene may be altered in the donor rodent and/or the recipient rodent. In some embodiments, the expression of the candidate gene may be altered in the endometrium of the donor rodent.
  • a gene identified as involved in endometriosis may be a useful therapeutic target for drugs for the treatment of endometriosis.
  • expression of the candidate gene may be increased, for example by gene activation or overexpression from a recombinant expression vector.
  • expression of the candidate gene may be decreased or reduced, for example by standard ⁇ knock out' techniques, or antisense or sense suppression techniques, such as RNAi.
  • a gene may be inactivated in the donor rodent endometrium by site-specific recombinase-mediated ablation
  • candidate gene suspected of involvement in endometriosis such as a growth factor, cytokine, protease or other factor, may be inactivated in the donor and/or the recipient rodent and the effect of the inactivation on the development of endometriosis determined relative to controls without the inactivation.
  • the effect on endometriosis may be determined by determining the effect of the alteration in gene expression on the size or amount or cellular composition of endometriotic lesions formed by the implanted menstruating endometrium in the rodent model. This may be carried out, for example, by in vivo imaging or standard immunohistochemical techniques.
  • a change in the size, number or cellular composition of endometrial lesions in mice with altered candidate gene expression relative to control mice with unaltered candidate gene expression is indicative that the gene is involved in endometriosis .
  • a decrease or reduction in the size or number of endometrial lesions in mice with increased candidate gene expression or an increase in the size or number of endometrial lesions in mice with reduced candidate gene expression relative to control mice with unaltered candidate gene expression is indicative that agonists of the candidate gene product may be useful in the treatment of endometriosis.
  • a decrease or reduction in the size or number of endometrial lesions in mice with decreased candidate gene expression or an increase in the size or number of endometrial lesions in mice with increased candidate gene expression, relative to control mice with unaltered candidate gene expression is indicative that antagonists of the product of the candidate gene may be useful in the treatment of endometriosis.
  • a method described herein may comprise identifying a candidate gene as involved in endometriosis.
  • the candidate gene and its product may be characterised further and employed in further research or drug discovery, for example to identify agonists or antagonists of the identified gene product which are useful in the treatment of endometriosis
  • Figure 1 shows haematoxylin/eosin staining of endometriosis lesions collected from murine models. Arrows indicate glandular structure; when using wild type donors, the gland-like structure is disintegrating, however when using K-ras expressed donors, glands are more organized. Vessels are also visible in the K-ras induced lesions, indicated as * signs.
  • Figure 2 shows drug induced Cre-recombinase expression resulted in ⁇ -galatosidase gene recombination and expression in murine endometrium. The ⁇ -galatosidase positive cells are stained dark. There is tense staining in glandular epithelial cells; however some cells within the stroma area are also stained positively.
  • Figure 3 shows immunohistochemistry results which confirm the presence of glandular epithelial cells and stromal cells.
  • the left panels show positive staining of glandular epithelial cells at different time-points after tissue implantation.
  • the right panels show the staining of stromal cells.
  • Figure 4 shows the staining of blood vessels using an antibody against endothelial cell marker CD31.
  • FIG. 5 shows the presence of leukocytes within the lesions using an antibody against leukocyte common antigen CD45
  • Figure 6 shows collagen and fibrous structure within the lesions using Van Gieson's staining.
  • Figure 7 shows an example of a lesion on the peritoneal surface of a recipient mouse after dissection.
  • the divisions on the ruler are lmm. Lesions were generated in the absence of matrigel, and are sensitive to estadiol ablation through administration of the estrogen receptor antagonist ICI 182,780 (Fulvestrant) .
  • Figure 8 (A) and (B) show endometriotic cysts in induced murine endometriosis. The cells stained dark around the perimeter of the cysts are LacZ positive, indicating ere mediated recombination. Lesions were generated in the absence of matrigel, and are sensitive to estadiol ablation through administration of the estrogen receptor antagonist ICI 182,780 (Fulvestrant) .
  • Figure 9 (A) and (B) show high power images (x400) of glandular structures in murine lesions (H&E stain)_in induced murine endometriosis. Again, lesions were generated in the absence of matrigel, and are sensitive to estadiol ablation through administration of the estrogen receptor antagonist ICI 182,780 (Fulvestrant) .
  • Figure 10 A shows collagen and fibrous structure within lesions identified using Van Gieson' s staining in human endometriosis.
  • B shows collagen and fibrous structure within lesions identified using Van Gieson' s stain in induced murine endometriosis. Lesions were generated in the absence of matrigel, and are sensitive to estadiol ablation through administration of the estrogen receptor antagonist ICI 182,780 (Fulvestrant) . In both A and B collagen is visible as elongate fibres, nuclei as darker stained dots and blood cells are stained light gray.
  • Figure 11 shows CD45+ve cells (darker stained) in a murine endometriotic lesion in induced murine endometriosis. Glandular and epithelial cells lining the cyst are also visible. Lesions were generated in the absence of matrigel, and are sensitive to estadiol ablation through administration of the estrogen receptor antagonist ICI 182,780 (Fulvestrant) .
  • Figure 12 shows cytokeratin staining (black) of epithelial cells in the cyst wall of a murine lesion in induced murine endometriosis. Lesions were generated in the absence of matrigel, and are sensitive to estadiol ablation through administration of the estrogen receptor antagonist ICI 182,780 (Fulvestrant) .
  • Figure 13 shows a negative control for immunostaining of a murine endometriotic lesion in induced murine endometriosis using an irrelevant primary antibody. Darker stained regions indicate LacZ +ve cells. This demonstrates that the staining seen in Figure 11 and 12 is specific. Lesions were generated in the absence of, and are sensitive to estadiol ablation through administration of the estrogen receptor antagonist ICI 182,780 (Fulvestrant) .
  • mice All procedures and care of the animals were performed following United Kingdom Home Office regulations.
  • Adult female C57BL/6 mice were purchased (Harlan UK, Oxon, United Kingdom) and K- ras vl2+/ ⁇ /AhCre +/+ /ROSA26R-LacZ +/+ transgenic mice were bred on site. All of the mice were maintained in standard housing.
  • the K-ras vl2+/' /AhCre +/+ /ROSA26R-LacZ +/+ transgenic mice allow transient endometrial, (predominantly epithelial), expression of Cre protein, that can mediate recombination of the K-rasV12 transgene and ROSA26R-LacZ reporter gene.
  • K-ras vl2 /AhCre transgenic mice were crossed with Rosa26R mice to generated K- ras vl2+/ -/AhCre +/ ⁇ /ROSA26R-LacZ +/ ⁇ transgenic mice.
  • Temgesic (GenusXpress, 0.03mg per animal) was also administered intra-muscularly in the end of the surgery.
  • Murine Endometriosis Model Female K-ras vl2+/' /AhCre +/+ /ROSA26R-LacZ +/+ transgenic mice were used as donor animals. They were ovariectomised and allowed to recover for at least seven days and then treated sequentially with steroid hormones as previously described [24, 25] . On days 1 and 2, animals were injected subcutaneously with lOOng of 17 ⁇ - oestradial (E2) dissolved in 0.1ml arachis oil (Sigma, Poole, Dorset, United Kingdom) . There was no hormone treatment on days 3, 4 and 5.
  • E2 17 ⁇ - oestradial
  • the collected menstruating endometrial tissue was re-suspended in matrigel (BD biosciences, Oxford, United Kingdom) .
  • Adult female C57BL/6 mice were used as recipients. These mice were given a long-lasting estradiol pellet (Innovative Research of America, FL, USA) subcutaneously the day before tissue implantation.
  • estradiol pellet Innovative Research of America, FL, USA
  • lOO ⁇ l of the menstruating tissue suspension was carefully injected subcutaneously to the abdominal side of the recipient and the incision was sealed with superglue and followed by spray plaster (Boots, London, UK) .
  • anti-cytokeratin epidermal cells, wide spectrum, DAKO, Ely, United Kingdom
  • anti-human-smooth muscle actin clone 1A4, DAKO
  • anti-mouse-CD31 endothelial cells, clone MBC 13.3, BD Biosciences
  • anti-mouse-CD45 leukocytes, clone 30-F11, BD Biosciences
  • a mouse on mouse (M.0. M.) kit (Vector Laboratories, Peterborough, United Kingdom) was used for staining using the monoclonal anti-smooth actin antibody.
  • All the other anti-mouse primary antibodies were detected by a biotin conjugated goat polyclonal antibody against rat IgG (Zymed, San Francisco, CA) . Streptavidin (Vector Laboratories) and DAB (Sigma) were used for secondary antibody detection and final visualization, and the cell nuclei were counter-stained with haematoxylin.
  • K-ras promotes the formation of endometriosis lesions
  • the recipient animals were sacrificed at Day 7, 14, 21 and 28 after tissue implantation, and lesions developed around implantation site were collected.
  • FIG. 1 shows an example of uterus section after Cre protein expression is induced. All the endometrial glands are positively stained (shown as dark), and some of the stromal cells are also positively stained.
  • an endometriotic lesion contains both the stromal and some glandular cells [1, 4] . Therefore endometriosis lesions collected at different time-points were stained for glandular epithelial cells and stromal cells. As shown in Figure 2, both glandular epithelial cells and stromal cells were detectable in all lesions (positive staining shown as dark) . Many of the positive stained glandular epithelial cells were also positively stained with LacZ staining and were surrounded by stroma. The presence of both gland epithelial cells and stroma cells coheres with the definition of endometriosis.
  • endothelial cells were visible in the endometriotic lesions. We observed macroscopically that a few small vessels were visible around the lesion and seemed to be growing into it, and the presence of endothelial cells within the lesions was in concord with our observation.
  • Fibrous material was found around the endometriotic lesion as is observed in human lesions.
  • the lesions were stained with Van Gieson's stain to identify the fibrous structure and compared with human endometriosis and lesions collected from Nude mice model.
  • Ras-induced murine endometriosis shows similar collagen deposition when compared with human endometriosis and nude mouse model.
  • lesions can also form in mouse recipients which have intact ovaries and which are not treated with exogenous estradiol. This is advantageous as human endometriosis patients typically have intact ovaries and are not treated with estradiol.
  • one of the modalities of treatment is to ablate estradiol production and thus reduce lesion growth in humans .

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Abstract

Cette invention porte sur la production de modèles rongeurs d'endométriose par implantation chez des rongeurs receveurs de type sauvage d'un endomètre menstruel provenant de rongeurs donneurs dans lesquels un oncogène, tel que K-ras, a été activé. L'invention porte ainsi sur des modèles rongeurs d'endométriose ainsi que sur des procédés de production et d'utilisation de tels modèles dans le criblage d'agents thérapeutiques.
PCT/GB2008/002713 2007-08-10 2008-08-11 Endométriose de murin modélisée par activation du k-ras d'un endomètre menstruel WO2009022119A1 (fr)

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WO2012112883A1 (fr) * 2011-02-18 2012-08-23 Yale University Variant de kras et endométriose

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US20020066116A1 (en) * 1997-03-26 2002-05-30 Jeffrey Boyd Endometriosis mouse model

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US20020066116A1 (en) * 1997-03-26 2002-05-30 Jeffrey Boyd Endometriosis mouse model

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CHENG CHING-WEN; BIELBY HOLLI; LICENCE DI; SMITH STEPHEN K; PRINT CRISTIN G; CHARNOCK-JONES D STEPHEN: "Quantitative cellular and molecular analysis of the effect of progesterone withdrawal in a murine model of decidualization", BIOLOGY OF REPRODUCTION, vol. 76, no. 5, May 2007 (2007-05-01), pages 871 - 883, XP002506013 *
DINULESCU DM, INCE TA, QUADE BJ, SHAFER SA, CROWLEY D, JACKS T.: "Role of K-ras and Pten in the development of mouse models of endometriosis and endometrioid ovarian cancer.", NAT MED, vol. 11, no. 1, 26 December 2004 (2004-12-26) - January 2005 (2005-01-01), pages 63 - 70, XP002506011 *
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