WO2002046421A2 - Methodes et compositions d'analyse de recepteurs muscariniques m3 de l'acetylcholine - Google Patents

Methodes et compositions d'analyse de recepteurs muscariniques m3 de l'acetylcholine Download PDF

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WO2002046421A2
WO2002046421A2 PCT/US2001/051110 US0151110W WO0246421A2 WO 2002046421 A2 WO2002046421 A2 WO 2002046421A2 US 0151110 W US0151110 W US 0151110W WO 0246421 A2 WO0246421 A2 WO 0246421A2
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human animal
receptor
mice
expression
animal
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WO2002046421A3 (fr
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Jurgen Wess
Masahisa Yamada
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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
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0276—Knock-out vertebrates
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70571—Receptors; Cell surface antigens; Cell surface determinants for neuromediators, e.g. serotonin receptor, dopamine receptor
    • 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
    • A01K2267/0362—Animal model for lipid/glucose metabolism, e.g. obesity, type-2 diabetes

Definitions

  • the present invention provides methods and compositions for screening drugs and other compositions for their effect on appetite and weight control, including compositions suitable for the treatment of obesity in humans and other animals.
  • the present invention provides animals that lack expression of the muscarinic acetylcholine receptor M3.
  • Muscarinic receptors mediate most of the inhibitory and excitatory effects of the neurotransmitter acetylcholine in the heart, smooth muscle, glands, and neurons (both presynaptic and postsynaptic) in the autonomic and central nervous systems (Eglen and Watson, Pharmacol. Toxicol., 78:59-68 [1996]). Indeed, muscarinic cholinergic neurotransmission plays a pivotal role in the regulation of a great number of important physiological processes, including cognitive, sensory, vegetative, and extrapyramidal motor functions (Brown and Taylor, in Hardman et al.
  • the muscarinic receptors are predicted to conform to a generic protein fold consisting of seven hydrophobic transmembrane helices joined by alternative intracellular and extracellular loops, an extracellular amino-terminal domain, and a cytoplasmic carboxyl-terminal domain.
  • the mammalian muscarinic receptors display a high degree of sequence identity, particularly in the transmembrane domains, sharing approximately 145 invariant amino acids (Wess, TIPS 14:308-313 [1993]).
  • muscarinic receptors All of the mammalian muscarinic receptors contain a very large third cytoplasmic loop which, except for the membrane-proximal portions, displays virtually no sequence identity among the different family members (Bonner, Trends Neurosci., 12:148-151 [1989]). Ligand binding to the receptor is believed to trigger conformational changes within the helical bundle, which are then transmitted to the cytoplasmic domain, where the interaction with specific G proteins occurs.
  • the present invention provides methods and compositions for screening drugs and other compositions for their effect on appetite and weight control, including compositions suitable for the treatment of obesity in humans and other animals.
  • the present invention provides animals that lack expression of the muscarinic acetylcholine receptor M3.
  • the present invention provides non-human animals with abnormal expression of the muscarinic acid M3 receptor.
  • the non-human animal is selected from the order Rodentia.
  • the animal is a mouse.
  • the genome of the mouse contains a non-naturally occurring mutation within the M3 receptor locus.
  • the non-human animal does not express the wild-type M3 receptor.
  • the non-human animal displays hypophagia.
  • the non-human animal shows increased hypothalamic levels of agouti- related peptide mRNA and reduced expression of proopiomelanocortin mRNA.
  • the non-human animal shows significantly reduced levels of melanin- concentrating hormone hypothalamic mRNA.
  • the present invention provides non-human animals in which the M3 receptor is selectively inactivated.
  • the invention provides a non-human animal, wherein the animal expresses a reduced level of the M3 receptor relative to a corresponding wild-type animal.
  • the present invention provides animals that lack functional expression of the M3 receptor. It is not intended that the mutation in the genome of the animals of the present invention be limited to a particular mutation, as the mutations may involve deletions in one or more exons of the M3 receptor gene, as well as frameshift mutations within the M3 receptor gene.
  • a neo cassette that replaces a 1.6-kilobase (kb) Xbal- SseS337I genomic fragment that includes the translation start site and the region coding for the first 389 amino acids of the M3 receptor protein is used to produce the M3 mutant animals of the present invention.
  • the genome of the animal further comprises a heterologous selectable marker gene.
  • the non-human animal of the invention is further characterized by being capable of expressing wild-type levels of other muscarinic receptors (i.e., Ml, M2, M4, and M5).
  • the present invention further relates to the use of these animals for screening candidate therapeutic compounds.
  • the non-human animal of the present invention is a member of the order Rodentia.
  • the non-human animal of the present invention is a mouse.
  • the present invention also provides methods for screening compounds for weight control activity, comprising: providing a non-human animal lacking expression of the M3 receptor, and a composition comprising a test compound in a form suitable for administration; administering the test compound to the non-human animal; and determining the weight of the non-human animal.
  • the methods further comprise the step of measuring a change in the weight of the non-human animal, wherein the test compound provides for weight control in the non-human animal.
  • the test compound is selected from the group consisting of M3 agonists and antagonists.
  • the non-human animal is selected from the order Rodentia.
  • the non- human animal is a mouse.
  • the genome of the mouse contains a non-naturally occurring mutation within the M3 receptor locus.
  • the non-human animal does not express the wild-type M3 receptor.
  • the non-human animal displays hypophagia.
  • the non-human animal shows increased hypothalamic levels of agouti-related peptide mRNA and reduced expression of proopiomelanocortin mRNA.
  • the non-human animal shows significantly reduced levels of melanin- concentrating hormone hypothalamic mRNA.
  • the genome of the non-human animal further comprises a heterologous selectable marker gene.
  • the present invention also provides methods for screening a test compound for weight control activity, wherein the methods comprise: providing (a) first and second non-human animals, wherein the first non-human animal expresses a reduced level of M3 relative to a corresponding wild-type animal, and the second non-human animal is a wild-type animal, and (b) a composition comprising the test compound; and administering the test compound to the first non-human animal to produce a treated test animal and administering the test compound to the second non-human animal to produce a control animal.
  • the invention further comprises comparing the weight of the animals after administration of the test compound.
  • the invention further comprises comparing the food intake of the animals over a given time period.
  • the invention comprises comparing the serum leptin and/or insulin levels of the animals.
  • the peripheral fat deposits of the animals are compared.
  • the treated test animal displays a reduction in food take, decreased body weight, reduced peripheral fat deposits, and/or very low serum and/or insulin levels, as compared to the control animal, while in other embodiments, the control animal displays a reduction in food take, decreased body weight, reduced peripheral fat deposits, and/or very low serum and/or insulin levels, as compared to the treated test animal.
  • the present invention also provides methods for screening compounds for weight control activity, comprising: providing a non-human animal lacking expression of the M3 receptor, a non-human animal expressing the M3 receptor, and a composition comprising a test compound in a form suitable for administration; administering the test compound to the non-human animal lacking expression of the M3 receptor and the non-human animal expressing the M3 receptor; and determining the weight control activity of the test compound.
  • the methods comprise measuring changes in the weights of the non-human animal lacking expression of the M3 receptor and the non- human animal expressing the M3 receptor, thereby identifying a compound as therapeutic.
  • the test compound is selected from the group consisting of M3 agonists and antagonists.
  • the non-human animal lacking expression of the M3 receptor and the non-human animal expressing the M3 receptor are selected from the order Rodentia.
  • the non-human animal lacking expression of the M3 receptor and the non-human animal expressing the M3 receptor are both mice.
  • the genome of the mouse contains a non-naturally occurring mutation within the M3 receptor locus.
  • the non-human animal lacking expression of the M3 receptor displays hypophagia.
  • the non-human animal shows increased hypothalamic levels of agouti-related peptide mRNA and reduced expression of proopiomelanocortin mRNA.
  • the non-human animal shows reduced levels of melanin-concentrating hormone hypothalamic mRNA.
  • the genome of the non-human animal lacking expression of the M3 receptor further comprises a heterologous selectable marker gene.
  • the test compound of the methods of the invention is an anti-obesity compound.
  • the test compound is an appetite suppressant.
  • the test compound affects the M3 receptor-dependent cholinergic pathway that regulates appetite at a site downstream of the immediate hypothalamic leptin targets.
  • the test compound effects the M3 receptor-dependent cholinergic pathway that regulates appetite at a site downstream of the immediate hypothalamic leptin targets and upstream of the MCH system.
  • Figure 1 provides schematics of the targeted disruption of the mouse M3 muscarinic receptor gene.
  • Panel A shows the structure of the wild-type allele, gene- targeting vector, and mutated allele.
  • Panel B provides results from Southern blot analysis of EcoRN-digested genomic D ⁇ A from a properly targeted ⁇ S cell clone and of mouse tail D ⁇ A prepared from F 2 pups generated by intermating of F, heterozygotes.
  • Panel C provides results from a Western blot analysis of mouse cerebral cortex membranes.
  • Panel D provides immunoprecipitation results for muscarinic receptor subtypes from mouse cerebral cortex (CTX) and the hypothalamus/thalamus (T/H) region.
  • CTX mouse cerebral cortex
  • T/H hypothalamus/thalamus
  • Figure 2 provides results from analyses of body weight, peripheral fat pad, food intake, metabolic rate and salivation responses of wild-type and 5R " ⁇ mice.
  • Panel A shows growth curves for wild-type (open circles) and M3R ' (filled circles) male mice fed standard food pellets.
  • Panel B shows the growth curves for wild-type (open circles) and ?R " " (filled circles) female mice fed standard food pellets.
  • Panel I provides results for muscarinic receptor-mediated salivation experiments conducted using 24-week old male mice (n - 5 or 6 per group).
  • Figure 3 provides results from glucose and insulin tolerance tests of wild-type and M3R ' mice.
  • Panel A provides the results of glucose tolerance tests, while Panel B provides the results of insulin tolerance tests.
  • Figure 4 provides results of semi -quantitative RT-PCR analysis to observe the expression of hypothalamic neuropeptide mRNA in wild-type and M3R 'A mice.
  • Panel A provides data showing the expression of hypothalamic neuropeptide mRNAs in wild-type and JR ⁇ mice.
  • Panel B provides data showing the feeding response to intra- cerebro ventricular administration of orexigenic neuropeptides.
  • Panel C shows the co- expression of MCH and M3 receptors in neurons of the lateral hypofhalamus.
  • the present invention provides methods and compositions for screening drugs and other compositions for their effect on appetite and weight control, including compositions suitable for the treatment of obesity in humans and other animals.
  • the present invention provides animals that lack expression of the muscarinic acetylcholine receptor M3.
  • M3 muscarinic acetylcholine receptors are believed to play a role in parasympathetic stimulation of smooth muscle contraction and glandular secretion. Indeed, the M3 receptor is present on most glandular and smooth muscle tissues (Brown and Taylor supra; Wess et al, supra; and Caulfield et al, supra), and is widely expressed, though at relatively low abundance throughout the central nervous system, including the diencephalon (Levey et al, Neurosci., 63:207-221 [1994]). To date, only limited information is available regarding the functions of the M3 receptor.
  • mice with a targeted disruption of the M3 receptor gene were created (i.e., M3 receptor knockout mice; M3R 'A ). These mice lacked functional M3 receptors. Strikingly, the M3 receptor-deficient mice showed significant reductions in their food intake, decreases in body weight, reduced peripheral fat deposits, and very low serum leptin and insulin levels.
  • mice showed increased hypothalamic levels .of agouti- related peptide (AGRP) mRNA and reduced expression of proopiomelanocortin (POMC) mRNA.
  • AGRP agouti- related peptide
  • POMC proopiomelanocortin
  • M3R 'A mice hypothalamic mRNA levels of melanin-concentrating hormone (MCH), which are normally upregulated in fasted animals leading to an increase in food intake (Qu, Nature 380:243-247 [1996]; and Shimada et al, Nature 396:670-674 [1998]), were significantly reduced in M3R 'A mice.
  • Intra-cerebroventricular injection experiments showed that an agouti-related peptide analogue lacked orexigenic (appetite-stimulating) activity in M3R 'A mice. However, M3R 'A mice remained responsive to the orexigenic effects of melanin-concentrating hormone (MCH).
  • hypothalamic leptin/melanocortin AGP/POMC
  • the M3R ' mice of the present invention were produced using homologous recombination in embryonic stem cells as known in the art (See e.g., Gomeza et al, Proc. Natl. Acad. Sci. USA 96:1692-1697 [1999]; and Gomeza et al, Proc. Nail. Acad. Sci. USA 96:10483-10488 [1999]).
  • Homozygous M3R A mice were obtained with the expected Mendelian frequency and did not differ from their wild-type littermates in overall health, fertility, and longevity. No abnormalities were observed in histological examinations of brain, heart, lung, liver, spleen and pancreas tissues from these animals.
  • M3R M3 receptor
  • M3 receptor M3 receptor
  • Western blot analysis and immunoprecipitation studies using M3 receptor selective antibodies See, Figure 1, Panel C and D. Immunoprecipitation studies also indicated that the M3 receptor is expressed at relatively low densities (20-50 fmol/mg) in all principal brain areas, including the cerebral cortex and diencephalon (i.e., thalamus and hypofhalamus), as shown in Figure 1, Panel D.
  • lack of M3 receptors did not lead to compensatory changes in the expression levels of the remaining four muscarinic receptor subtypes Ml, M2, M4, and M5.
  • M3R 'A mice During the first post-natal week, the body weight of M3R 'A mice did not significantly differ from that of their wild-type littermates (See, Figure 2, Panels A and B). However, starting at the end of post-natal week two (females) or three (males), M3R ⁇ A mice displayed a significant decrease in body weight and reduced weight gain (See, Figure 2, Panel A). For example, at post-natal week 12, male and female M3R 'A mice weighed about 22% less (PO.0001) than their wild-type littermates (See, Figure 2, Panels A and B). A similar percent weight difference persisted throughout the entire observation period (i.e., more than one year). No significant reduction in body weight was observed with heterozygous M3R +A mutant mice.
  • M3R 'A mice (10- and 22-week old males) exhibited a 50-60% reduction in the weight of gonadal (epididymal) fat pads. Since the weight of mouse gonadal fat pads closely correlates with total body fat mass (Rogers and Webb, Brit. J. Nutr., 43:83-86 [1980]), reduced body fat content is predicted to represent a major factor contributing to the decreased body weight displayed by the M3R ' mice.
  • mice that carried the M3 receptor mutation in a pure genetic background (129SvEv) were also studied.
  • isogenic mice that carried the M3 receptor mutation in a pure genetic background (129SvEv) were also studied.
  • M3 muscarinic receptors are believed to be involved in mediating cholinergic stimulation of salivary gland secretion (Brown and Taylor, supra; Wess et al, supra; and Caulfield et al, supra).
  • the reduced body weight of M3 -receptor-deficient mice has been proposed to be caused by the absence of muscarinic receptor-mediated salivary flow (Matsui et al, Proc. Natl. Acad. Sci., 97:9579-9584 [2000]).
  • M3R ' was wet, suggesting that basal salivary secretion was sufficient to permit normal feeding.
  • mice were offered a wet mash diet rather than dry food pellets. These experiments showed that consumption of wet mash food instead of dry standard mouse chow had little effect on the reduction in body weight and food intake observed with the M3R 'A mice (See, Figure 2, Panels C, D and F). Together, these results argue against the idea that impaired salivation is responsible for the hypophagic phenotype displayed by the 3R " mice.
  • M3 and M2 receptors are co-expressed on gastrointestinal smooth muscle where they are thought to promote gastric emptying and gut contractility (Eglen et al, Pharmacol. Rev., 48:531-565 [1996]).
  • IGF-I insulin-like growth factor I
  • T3 and T4 serum levels of thyroid hormones
  • corticosterone did not differ significantly among M3R 'A mice and their wild-type littermates, as also shown in Table 1. Strikingly, however, M3R ⁇ A mice exhibited drastically reduced (9-fold) serum insulin levels, as shown in Table 1.
  • M3R ' mice Despite low circulating insulin levels, M3R ' mice showed normal or slightly reduced blood (serum) glucose levels (as also shown in Table 1), and did not become hyperglycemic throughout the entire observation period (i.e., more than one year; data not shown). Moreover, a glucose tolerance test (2 g/kg administered intraperitoneally) demonstrated that M3R ' were able to clear glucose from the blood at least as efficiently as wild-type mice, as shown in Figure 3, Panel A. Similar results were obtained when the glucose load (2 g/kg) was administered orally (data not shown), indicating that the M3R ' mice maintain normal glucose tolerance despite marked hypoinsulinemia.
  • M3R ⁇ mice showed a much more pronounced and prolonged hypoglycemia than their wild-type littermates, indicative of an increase in insulin sensitivity (Figure 3, Panel B).
  • the low serum insulin levels found in M3R 'A mice are believed to be probably due to the reduction in total body fat, since insulin, like leptin, circulates at levels proportional to body fat content (Schwartz et al, supra).
  • pancreatic M3 receptors which may be involved in insulin release (Boschero et al, Am. J. Physiol., 268:E336-E342 [1995]) makes an additional contribution to the observed reduction in serum insulin levels.
  • the rectal temperatures of wild-type and M3R 'A mice were also determined.
  • the low body weight in M3R 'A is primarily attributable to a reduction in food intake.
  • Energy homeostasis and food intake are known to be under the strict control of the regulatory centers in the hypothalamus (Flier and Maratos-Flier, supra; Elmquist et al, supra; and Schwartz et al, supra).
  • hypothalamic neuropeptides proopiomelanocortin [POMC], agouti-related peptide [AGRP], prepro-orexin, and melanin-concentrating hormone [MCH]
  • POMC proopiomelanocortin
  • AGRP agouti-related peptide
  • MCH melanin-concentrating hormone
  • MCH expression is increased by reduced food intake and leptin deficiency in wild-type animals (Flier and Maratos-Flier, supra; Inui, supra; Elmquist, supra; Schwartz et al, supra; Qu, supra, and Shimada et al, supra).
  • MCH-deficient mice are hypophagic and lean (Shimada et al, supra), indicating that intact MCH signaling is essential for proper regulation of food intake.
  • impaired MCH signalling may contribute to the pathogenesis of certain forms of anorexia (Mystkowski et al, J. Neurosci., 20:8637-8642 [2000]).
  • MCH is almost exclusively expressed in neurons of the lateral hypothalamus (Nahon, supra), a region that receives abundant ascending cholinergic projections from the pedunculopontine and laterodorsal tegmental nuclei (Bayer et al, Neurosci., 91:1087-1101 [1999]).
  • Immunocytochemical studies suggest that the distribution of MCH-containing neurons overlaps with that of the M3 receptor protein in the lateral hypothalamus. It is therefore likely that hypothalamic MCH expression is under stimulatory cholinergic control mediated by M3 receptor activation.
  • hypothalamic slice preparations have shown that activation of hypothalamic muscarinic receptors leads to a robust increase in MCH mRNA expression (Bayer et al, supra).
  • an understanding of the mechanism(s) is not necessary in order to use the present invention.
  • Hypophagia associated with reduced MCH expression is therefore likely to represent the major cause of the lean phenotype displayed by the M3R ' mice.
  • MCH mRNA-containing neurons >90%> located in the lateral hypothalamus co-express M3 muscarinic receptors.
  • M3 receptor protein was localized to both MCH cell bodies and dendritic processes.
  • Hypothalamic MCH neurons receive abundant ascending cholinergic projections from the pedunculopontine and laterodorsal tegmental nuclei and activation of hypothalamic muscarinic receptors leads to a robust increase in MCH mRNA expression (Bayer et al, Neurosci., 91 : 1087-1101 [1998]).
  • the data obtained during the development of the present invention are consistent with the concept that hypothalamic MCH expression is under stimulatory cholinergic control mediated by M3 muscarinic receptors.
  • the observations made during the development of the present invention indicate that there is an M3 receptor-dependent cholinergic pathway that regulates appetite at a site downstream of the immediate hypothalamic leptin targets and probably upstream of the MCH system.
  • an understanding of the mechanism(s) involved is not necessary in order to use the present invention. Nonetheless, the present invention provides compositions and methods suitable for the development of antagonists of the central M3 muscarinic receptors for use in weight control and management (e.g., treatment of obesity).
  • muscarinic acetylcholine receptor refers to any of the subtypes of the muscarinic acetylcholine receptor family. In particularly preferred embodiments, the present invention pertains to the "M3 receptor.”
  • knock-out animal refers to an animal that has a mutation that shuts off or alters gene expression. "Knock-out” mutations shut off or alter gene expression and are currently used to produce a phenotype in the whole animal which reflects the function of the knocked-out gene.
  • the knock-out animals of the present invention lack expression of functional M3 muscarinic acetylcholine receptors.
  • non-human animals comprise any non-human animal whose genome contains an oligonucleotide sequence (e.g. , a gene) encoding a modified form of the M3 receptor.
  • the modification renders the animal incapable of expressing the M3 receptor (i.e., a knock-out animal), as detected, for example, by Western blot analysis and enzyme-linked immunosorbent assay (ELISA).
  • Such non-human animals include vertebrates such as rodents, non-human primates, ovines, bovines, ruminants, lagomorphs, porcines, caprines, equines, canines, felines, aves, etc.
  • Non-human animals are selected from the order Rodentia which includes murines (e.g. , rats and mice), most preferably mice.
  • the "non-human animals having a genetically engineered genotype” of the invention are preferably produced by experimental manipulation of the genome of the germline of the non-human animal. These genetically engineered non-human animals may be produced by several methods including the use of ES stem cells to produce knock out animals as described herein.
  • the term also encompasses animals containing a "transgene” comprising nucleic acid (usually DNA) into an embryonal target cell or integration into a chromosome of the somatic and/or germ line cells of a non-human animal by way of human intervention, such as by the methods known in the art and/or described herein.
  • Non-human animals which contain a transgene are referred to as "transgenic non-human animals.”
  • a transgenic animal is an animal whose genome has been altered by the introduction of a transgene.
  • the term "gene” means the deoxyribonucleotide sequences comprising the coding region of a structural gene and including sequences located adjacent to the coding region on both the 5' and 3' ends for a distance of several kb on either end such that the gene corresponds to the length of the full-length mRNA.
  • the sequences which are located 5' of the coding region and which are present on the mRNA are referred to as 5' non-translated sequences.
  • the sequences which are located 3' or downstream of the coding region and which are present on the mRNA are referred to as 3' non-translated sequences.
  • genomic form or clone of a gene contains coding sequences, termed exons, alternating with non-coding sequences termed “introns” or “intervening regions” or “intervening sequences.”
  • Introns are segments of a gene which are transcribed into heterogenous nuclear RNA (hnRNA); introns may contain regulatory elements such as enhancers. Introns are removed or “spliced out” from the nuclear or primary transcript; introns therefore are absent in the messenger RNA (mRNA) transcript.
  • genomic forms of a gene may also include sequences located on both the 5 ' and 3 ' end of the sequences which are present on the RNA transcript.
  • flanking sequences or regions are located 5' or 3' to the non-translated sequences present on the mRNA transcript.
  • the 5' flanking region may contain regulatory sequences such as promoters and enhancers which control or influence the transcription of the gene.
  • the 3 ' flanking region may contain sequences which direct the termination of transcription, posttranscriptional cleavage and polyadenylation.
  • coding region when used in reference to a structural gene refers to the nucleotide sequences which encode the amino acids found in the nascent polypeptide as a result of translation of an mRNA molecule.
  • the coding region is bounded, in eukaryotes, on the 5' side by the nucleotide triplet "ATG" which encodes the initiator methionine and on the 3' side by one of the three triplets which specify stop codons (i.e., TAA, TAG, TGA).
  • structural gene refers to a DNA sequence coding for RNA or a protein.
  • regulatory genes are structural genes which encode products (e.g., transcription factors) which control the expression of other genes.
  • regulatory element refers to a genetic element which controls some aspect of the expression of nucleic acid sequences.
  • a promoter is a regulatory element which facilitates the initiation of transcription of an operably linked coding region.
  • Other regulatory elements are splicing signals, polyadenylation signals, termination signals, enhancer elements, etc. Promoters and enhancers consist of short arrays of DNA sequences that interact specifically with cellular proteins involved in transcription (Maniatis et al, Science 236:1237 [1987]).
  • Promoter and enhancer elements have been isolated from a variety of eukaryotic sources including genes in yeast, insect and mammalian cells and viruses (analogous control elements [i.e., promoters], are also found in prokaryotes). The selection of a particular promoter and enhancer depends on what cell type is to be used to express the protein of interest. Some eukaryotic promoters and enhancers have a broad host range while others are functional in a limited subset of cell types (for review see Voss, et al, Trends Biochem. Sci., 11:287 [1986]; and Maniatis, et al, Science 236:1237 [1987]).
  • the SV40 early gene enhancer is very active in a wide variety of cell types from many mammalian species and has been widely used for the expression of proteins in mammalian cells (Dijkema, et al, EMBO J. 4:761 [1985]).
  • Other examples of promoter/enhancer elements active in a broad range of mammalian cell types are those from the human elongation factor 1 gene (Uetsuki et al, J. Biol. Chem., 264:5791 [1989]; Kim et al, Gene 91:217 [1990]; and Mizushima and Nagata, Nucl. Acids.
  • promoter element refers to a DNA sequence that is located at the 5' end of (i.e., precedes) a gene in a DNA polymer and provides a site for initiation of the transcription of the gene into mRNA.
  • gene of interest and “nucleotide sequence of interest” refer to any gene or nucleotide sequence, respectively, the manipulation of which may be deemed desirable for any reason by one of ordinary skill in the art.
  • expression vector refers to a recombinant DNA molecule containing a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a particular host organism.
  • Nucleic acid sequences necessary for expression in prokaryotes include a promoter, optionally an operator sequence, a ribosome binding site and possibly other sequences.
  • Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals.
  • a "modification" as used herein in reference to a nucleic acid sequence refers to any change in the structure of the nucleic acid sequence. Changes in the structure of a nucleic acid sequence include changes in the covalent and non-covalent bonds in the nucleic acid sequence. Illustrative of these changes are mutations, mismatches, strand breaks, as well as covalent and non-covalent interactions between a nucleic acid sequence (which contains unmodified and/or modified nucleic acids) and other molecules.
  • Illustrative of a covalent interaction between a nucleic acid sequence and another molecule are changes to a nucleotide base (e.g., formation of thymine glycol) and covalent cross-links between double-stranded DNA sequences which are introduced by, for example, ultraviolet radiation or by cis-platinum.
  • a covalent interaction between a nucleic acid sequence and another molecule includes covalent binding of two nucleic acid sequences to psoralen following ultraviolet irradiation.
  • Non- covalent interactions between a nucleic acid sequence and another molecule include non- covalent interactions of a nucleic acid sequence with a molecule other than a nucleic acid sequence and other than a polypeptide sequence.
  • Non-covalent interactions between a nucleic acid sequence with a molecule other than a nucleic acid sequence and other than a polypeptide sequence are illustrated by non-covalent intercalation of ethidium bromide or of psoralen between the two strands of a double- stranded deoxyribonucleic acid sequence.
  • the present invention contemplates modifications which cause changes in a functional property (or properties), such changes manifesting themselves at a variety of time points.
  • allelic series when made in reference to a gene refers to wild-type sequences of the gene.
  • An "allelic series of modifications" as used herein in reference to a gene refers to two or more nucleic acid sequences of the gene, where each of the two or more nucleic acid sequences of the gene contains at least one modification when compared to the wild-type sequences of the gene.
  • mutation refers to a deletion, insertion, or substitution.
  • a “deletion” is defined as a change in a nucleic acid sequence in which one or more nucleotides is absent.
  • An “insertion” or “addition” is that change in a nucleic acid sequence which has resulted in the addition of one or more nucleotides.
  • substitution results from the replacement of one or more nucleotides by a molecule which is a different molecule from the replaced one or more nucleotides.
  • a nucleic acid may be replaced by a different nucleic acid as exemplified by replacement of a thymine by a cytosine, adenine, guanine, or uridine.
  • a nucleic acid may be replaced by a modified nucleic acid as exemplified by replacement of a thymine by thymine glycol.
  • mismatch refers to a non-covalent interaction between two nucleic acids, each nucleic acid residing on a different polynucleic acid sequence, which does not follow the base-pairing rules. For example, for the partially complementary sequences 5'-AGT-3' and 5'-AAT-3', a G-A mismatch is present.
  • nucleic acid and unmodified nucleic acid refer to any one of the known four deoxyribonucleic acid bases (i.e., guanine, adenine, cytosine, and thymine).
  • modified nucleic acid refers to a nucleic acid whose structure is altered relative to the structure of the unmodified nucleic acid. Illustrative of such modifications would be replacement covalent modifications of the bases, such as alkylation of amino and ring nitrogens as well as saturation of double bonds.
  • modified cell refers to a cell which contains at least one modification in the cell's genomic sequence.
  • nucleic acid sequence-modifying agent refers to an agent which is capable of introducing at least one modification into a nucleic acid sequence.
  • Nucleic acid sequence-modifying agents include, but are not limited to, chemical compounds (e.g., N-ethyl-N-nitrosurea (E ⁇ U), methylnitrosourea (M ⁇ U), procarbazine hydrochloride (PRC), triethylene melamine (TEM), acrylamide monomer (AA), chlorambucil (CHL), melphalan (MLP), cyclophosphamide (CPP), diethyl sulfate (DES), ethyl methane sulfonate (EMS), methyl methane sulfonate (MMS), 6-mercaptopurine (6MP), mitomycin-C (MMC), procarbazine (PRC), N-methyl-N'-nitro-N-nitrosoguanidine (M ⁇ G), 3 H 2 O, and urethane (UR)),
  • wild-type when made in reference to a gene refers to a gene which has the characteristics of that gene when isolated from a naturally occurring source.
  • a wild-type gene is that which is most frequently observed in a population and is thus arbitrarily designated the “normal” or “wild-type” form of the gene.
  • modified or mutant refers to a gene or gene product which displays modifications in sequence and/or functional properties (i.e., altered characteristics) when compared to the wild-type gene or gene product. It is noted that naturally-occurring mutants can be isolated; these are identified by the fact that they have altered characteristics when compared to the wild-type gene or gene product.
  • mutant e.g., knock-out
  • a mutant animal refers to an animal which expresses a reduced level of M3 receptors in comparison with normal wild-type animals.
  • a "corresponding wild-type animal” refers to an animal which is isogenic (i.e., has the same genetic background) and contains a wild-type M3 receptor gene.
  • a "variant of the M3 receptor” is defined as an amino acid sequence which differs by one or more amino acids from the wild-type M3 receptor sequence.
  • the variant may have conservative changes, wherein a substituted amino acid has similar structural or chemical properties (e.g., replacement of leucine with isoleucine). More rarely, a variant may have nonconservative changes (e.g., replacement of a glycine with a tryptophan). Similar minor variations may also include amino acid deletions or insertions (i.e., additions), or both.
  • Guidance in determining which and how many amino acid residues may be substituted, inserted or deleted without abolishing biological or immunological activity may be found using computer programs well known in the art, for example, D ⁇ AStar software.
  • targeting vector and “targeting construct” are used interchangeably to refer to oligonucleotide sequences comprising a M3 receptor gene. It is preferred that the targeting vector also comprise a selectable marker gene.
  • the targeting vector contains M3 receptor gene sequences sufficient to permit the homologous recombination of the targeting vector into at least one allele of the M3 receptor gene resident in the chromosomes of the target or recipient cell (e.g., ES) cells.
  • the targeting vector contains 2 kb to 10 kb of DNA homologous to the M3 receptor gene. This 2 kb to 10 kb of DNA may be located downstream or upstream of the selectable marker gene, or may be divided on each side of the selectable marker gene.
  • the selectable marker gene is located upstream of the M3 receptor gene.
  • the targeting vector may contain more than one selectable maker gene.
  • the targeting vector preferably contains a positive selectable marker (e.g. , the neo gene) and a negative selectable marker (e.g., the diphtheria toxin (dt gene) or Herpes simplex virus tk (HSV- tk) gene).
  • dt gene diphtheria toxin
  • HSV- tk Herpes simplex virus tk
  • the presence of the negative selectable marker permits the identification of recipient cells containing the targeting vector at the targeted site (i.e., which has integrated by virtue of homologous recombination into the target site); cells which survive when grown in medium which selects against the expression of the negative selectable marker do not contain a copy of the negative selectable marker.
  • the targeting vectors of the present invention are of the "replacement-type;" integration of a replacement-type vector results in the insertion of a selectable marker into the target gene.
  • Replacement-type targeting vectors may be employed to disrupt a gene resulting in the generation of a null allele (i.e., an allele incapable of expressing a functional protein; null alleles may be generated by deleting a portion of the coding region, deleting the entire gene, introducing an insertion and/or a frameshift mutation, etc.) or may be used to introduce a modification (e.g., one or more point mutations) into a gene.
  • selectable marker or “selectable gene product” as used herein refer to the use of a gene which encodes an enzymatic activity that confers resistance to an antibiotic or drug upon the cell in which the selectable marker is expressed.
  • Selectable markers may be "positive”; positive selectable markers typically are dominant selectable markers (i.e., genes which encode an enzymatic activity which can be detected in any mammalian cell or cell line [including ES cells]).
  • dominant selectable markers include, but are not limited to, (1) the bacterial aminoglycoside 3' phosphotransferase gene (also referred to as the neo gene) which confers resistance to the drug G418 in mammalian cells, (2) the bacterial hygromycin G phosphotransferase (hyg) gene which confers resistance to the antibiotic hygromycin and (3) the bacterial xanthine- guanine phosphoribosyl transferase gene (also referred to as the gpt gene) which confers the ability to grow in the presence of mycophenolic acid.
  • the bacterial aminoglycoside 3' phosphotransferase gene also referred to as the neo gene
  • hyg bacterial hygromycin G phosphotransferase
  • gpt bacterial xanthine- guanine phosphoribosyl transferase gene
  • Selectable markers may be "negative"; negative selectable markers encode an enzymatic activity whose expression is cytotoxic to the cell when grown in an appropriate selective medium.
  • the HSN-t/V gene and the dt gene are commonly used as a negative selectable marker. Expression of the HSV-tk gene in cells grown in the presence of gancyclovir or acyclovir is cytotoxic; thus, growth of cells in selective medium containing gancyclovir or acyclovir selects against cells capable of expressing a functional HSN TK enzyme. Similarly, the expression of the dt gene selects against cells capable of expressing the diphtheria toxin;
  • An animal whose genome "comprises a heterologous selectable marker gene” is an animal whose genome contains a selectable marker gene not naturally found in the animal's genome which is introduced by means of molecular biological methods.
  • a heterologous selectable marker is distinguished from an endogenous gene naturally found in the animal's genome in that expression or activity of the heterologous selectable marker can be selected for or against.
  • operable combination refers to the linkage of nucleic acid sequences in such a manner that a nucleic acid molecule capable of directing the transcription of a given gene and/or the synthesis of a desired protein molecule is produced.
  • the term also refers to the linkage of amino acid sequences in such a manner so that a functional protein is produced.
  • an oligonucleotide having a nucleotide sequence encoding a gene means a nucleic acid sequence comprising the coding region of a gene (i.e. the nucleic acid sequence which encodes a gene product).
  • the coding region may be present in either a cD ⁇ A, genomic D ⁇ A or R ⁇ A form.
  • the oligonucleotide may be single-stranded (t.e., the sense strand) or double- stranded.
  • Suitable control elements may be placed in close proximity to the coding region of the gene, if needed to permit proper initiation of transcription and/or correct processing of the primary RNA transcript.
  • the coding region utilized in the expression vectors of the present invention may contain endogenous enhancers, splice junctions, intervening sequences, polyadenylation signals, or other sequences, or a combination of both endogenous and exogenous control elements.
  • an oligonucleotide sequence comprising at least a portion of the M3 receptor gene refers to a polynucleotide sequence (i.e., a nucleic acid sequence) containing a nucleotide sequence derived from the M3 receptor gene. This sequence may encode a portion of the M3 receptor (i.e., not the entire sequence); alternatively, this sequence may encode the entire sequence or may simply contain non-coding regions derived from the M3 receptor gene or a combination of coding and non-coding regions.
  • the oligonucleotide may be RNA or DNA and may be of genomic or synthetic origin.
  • portion when in reference to a gene refers to fragments of that gene.
  • the fragments may range in size from 10 nucleotides to the entire gene sequence minus one nucleotide.
  • an oligonucleotide comprising at least a portion of a gene may comprise small fragments of the gene or nearly the entire gene.
  • substantially purified refers to molecules, either nucleic or amino acid sequences, that are removed from their natural environment, isolated or separated, and are at least 60%> free, preferably 75%> free, and most preferably 90%> free from other components with which they are naturally associated.
  • An "isolated polynucleotide” is therefore a substantially purified polynucleotide.
  • PCR polymerase chain reaction
  • the mixture is denatured and the primers then annealed to their complementary sequences within the target molecule.
  • the primers are extended with a polymerase so as to form a new pair of complementary strands.
  • the steps of denaturation, primer annealing and polymerase extension can be repeated many times (i.e., denaturation, annealing and extension constitute one "cycle”; there can be numerous "cycles") to obtain a high concentration of an amplified segment of the desired target sequence.
  • the length of the amplified segment of the desired target sequence is determined by the relative positions of the primers with respect to each other, and therefore, this length is a controllable parameter.
  • PCR polymerase chain reaction
  • PCR it is possible to amplify a single copy of a specific target sequence in genomic DNA to a level detectable by several different methodologies (e.g., hybridization with a labeled probe; incorporation of biotinylated primers followed by avidin-enzyme conjugate detection; incorporation of 32 P-labeled deoxynucleotide triphosphates, such as dCTP or dATP, into the amplified segment).
  • any oligonucleotide sequence can be amplified with the appropriate set of primer molecules.
  • the amplified segments created by the PCR process itself are, themselves, efficient templates for subsequent PCR amplifications.
  • Amplified target sequences may be used to obtain segments of DNA (e.g., genes) for the construction of targeting vectors, transgenes, etc..
  • PCR product and “amplification product” refer to the resultant mixture of compounds after two or more cycles of the PCR steps of denaturation, annealing and extension are complete. These terms encompass the case where there has been amplification of one or more segments of one or more target sequences.
  • RNA is reverse transcribed using a single primer (e.g., an oligo-dT primer) prior to PCR amplification of the desired segment of the transcribed DNA using two primers.
  • a single primer e.g., an oligo-dT primer
  • the term "primer” refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is induced, (i.e., in the presence of nucleotides and of an inducing agent such as DNA polymerase and at a suitable temperature and pH).
  • the primer is preferably single stranded for maximum efficiency in amplification, but may alternatively be double stranded. If double stranded, the primer is first treated to separate its strands before being used to prepare extension products.
  • the primer is an oligodeoxyribonucleotide.
  • the primer must be sufficiently long to prime the synthesis of extension products in the presence of the inducing agent. The exact lengths of the primers will depend on many factors, including temperature, source of primer and the use of the method.
  • probe refers to an oligonucleotide (i.e., a sequence of nucleotides), whether occurring naturally as in a purified restriction digest or produced synthetically, recombinantly or by PCR amplification, which is capable of hybridizing to another oligonucleotide of interest.
  • a probe may be single- stranded or double-stranded. Probes are useful in the detection, identification and isolation of particular gene sequences.
  • any probe used in the present invention will be labeled with any "reporter molecule,” so that it is detectable in any detection system, including, but not limited to enzyme (e.g., ELISA, as well as enzyme-based histochemical assays), fluorescent, radioactive, and luminescent systems. It is not intended that the present invention be limited to any particular detection system or label.
  • restriction endonucleases and “restriction enzymes” refer to bacterial enzymes, each of which cut double- or single- stranded nucleic acid at or near a specific nucleotide sequence.
  • the term "compound” refers to any chemical entity, pharmaceutical, drug, and the like that can be used to treat or prevent a disease, illness, sickness, or disorder of bodily function. Compounds comprise both known and potential therapeutic compounds.
  • a compound can be determined to be therapeutic by screening, e.g., using the screening methods of the present invention.
  • a "known therapeutic compound” refers to a therapeutic compound that has been shown (e.g., through animal trials or prior experience with administration to humans) to be effective in such treatment or prevention.
  • a compound is said to be "in a form suitable for administration” when the compound may be administered to an animal by any desired route (e.g. , oral, intravenous, subcutaneous, intramuscular, etc.) and the compound or its active metabolites appear in the desired cells, tissue or organ of the animal in an active form.
  • terapéutica amount refers to that amount of a compound required to neutralize undesirable pathologic effects in a subject.
  • C degrees Centigrade
  • rpm repetitions per minute
  • BSA bovine serum albumin
  • CFA complete Freund's adjuvant
  • IFA incomplete Freund's adjuvant
  • IgG immunoglobulin G
  • IM intramuscular
  • IP intraperitoneal
  • IN intravenous or intravascular
  • SC subcutaneous
  • H 2 O water
  • HCl hydrochloric acid
  • aa amino acid
  • bp base pair
  • kb kilobase pair
  • kD kilodaltons
  • gm grams
  • g micrograms
  • mg milligrams
  • ng nanograms
  • 1 microliters
  • ml milliliters
  • mm millimeters
  • nm nanom
  • M micromolar
  • M micromolar
  • U units
  • a mouse M3 muscarinic receptor genomic clone was isolated from a 129Sv/J mouse genomic library (Genome Systems) by using a PCR fragment corresponding in sequence to the central portion of the third intracellular loop of the receptor as a probe.
  • the primers used in the PCR were: sense: 5'-GGT TCA CCA CCA AGA GCT GG-3' (SEQ ID NO:l) anti-sense: 5'-GGT CTT GCC TGT GTC CAC GG-3' (SEQ ID NO:2)
  • sense 5'-GGT TCA CCA CCA AGA GCT GG-3'
  • anti-sense 5'-GGT CTT GCC TGT GTC CAC GG-3' (SEQ ID NO:2)
  • PCR 94°C for 1 min; 60°C for 2 min; and 72°C for 3 min
  • a 50 ⁇ l reaction volume containing 200 ng mouse genomic DNA
  • Tris-HCl (20 mM, pH 8.4 KCl (50 mM), MgCl 2 (1.5 niM), dNTPs (0.2 mM each )
  • primers (1 ⁇ g each) and Taq DNA polymerase (2.5 U).
  • the PCR product was 380 bp in size.
  • the pPN2T vector (Paszty et al, Nat. Genet., 11:33-39 [1995]) was used as the backbone for the generation of the M3 receptor targeting construct.
  • the final targeting vector contained two copies of the herpes simplex virus thymidine kinase gene (TK) and a PGK-PGK-neomycin resistance cassette (neo) which replaced a 1.6 kb -Yb I-Ss'e8337I genomic fragment that included the translation start site and the region coding for the first 389 amino acids of the M3 receptor protein (See, Figure 1, Panel A).
  • the targeting vector was linearized and introduced into TCI (129 SvEv) embryonic stem (ES) cells by electroporation (Deng et al, Cell 84:911-921 [1996]). Clones resistant to G418 and gancyclovir were isolated and the occurrence of homologous recombination was then confirmed by Southern hybridization as known in the art.
  • mice Properly targeted ES cell clones were microinjected into C57BL/6J blastocysts to generate male chimeric offspring, which in turn, were mated with female C57BL/6J mice (Jackson) to generate F, offspring. F, animals heterozygous for the M3 receptor mutation were then intermated to produce homozygous M3 receptor mutant mice (F 2 ). All of the mice used in the development of the present invention were littermates of the F 2 generation (C57BL/6J x 129SvEv hybrids).
  • the neo cassette used in these experiments replaced a 1.6-kilobase (kb) -Yb ⁇ I-Ss-e8337I genomic fragment that included the translation start site and the region coding for the first 389 amino acids of the M3 receptor protein.
  • Panel A provides a schematic of the targeted disruption of the mouse M3 muscarinic receptor gene.
  • Panel A shows the structure of the wild-type allele, gene-targeting vector, and mutated allele.
  • the M3 receptor coding region is represented by a shaded box.
  • Panel B provides the results from Southern blot analysis of EcoRV-digested genomic DNA from a properly targeted ⁇ S cell clone and of mouse tail DNA prepared from F 2 pups generated by intermating of F, heterozygotes.
  • the probes used for this Southern analysis are indicated ( ⁇ , EcoRV; K, Kpnl; N, Notl; Sa, Sail; Sm, Sm l; Ss, Sse8337I; Xb, Xbal; and Xh, Xhol).
  • the 13 and 9.3 kb bands represent the wild-type and mutant M3 receptor alleles respectively.
  • M1-M5 muscarinic receptor-specific rabbit polyclonal antisera were raised against non-conserved regions of the third cytoplasmic loops of the mouse M1-M5 receptor proteins, as known in the art (See e.g., Levey et al, J. Neurosci., 11 :3218-3226 [1991]).
  • Membranes prepared from different mouse brain regions obtained from 20-week old male mice were incubated with 2 nM [ 3 H]quinuclidinyl benzilate (QNB) to label M1-M5 muscarinic receptors (the M3 genotypes are indicated in Figure 1, Panel D).
  • [H]QNB-labeled receptors were solubilized with 1%> digitonin and subjected to immunoprecipitation by M1-M5 receptor- specific antisera as known in the art (See e.g., Gomeza et al, Proc. Natl. Acad. Sci. USA 96:1692-1697 [1999]; and Gomeza et al, Proc. Natl. Acad. Sci. USA 96:10483-10488 [1999]).
  • M3R M3 receptor
  • M3 receptor M3 receptor
  • Western blot analysis and immunoprecipitation studies using M3 receptor selective antibodies See, Figure 1, Panel C and D.
  • Immunoprecipitation studies also indicated that the M3 receptor is expressed at relatively low densities (20-50 fmol/mg) in all major brain areas, including cerebral cortex and diencephalon (i.e., thalamus and hypothalamus), as shown in Figure 1, Panel D.
  • lack of M3 receptors did not lead to compensatory changes in the expression levels of the remaining four muscarinic receptor subtypes Ml, M2, M4, and M5.
  • mice For food intake measurements, wild-type and M3R 'A mutant mice were individually housed for 4 days prior to the start of the experiments. Consumption of standard dry rodent food pellets (i.e., chow; Zeigler), or wet mash food (prepared by mixing powder made from CA-1 (CLEA) pellets (3.45 kcal/g), with twice the weight of sterilized water) and body weight were then measured for 5 consecutive days. Cages were carefully monitored for food spillage. The rectal temperatures of the animals were measured at 10:00 AM with a rectal probe (Thermalert).
  • standard dry rodent food pellets i.e., chow; Zeigler
  • wet mash food prepared by mixing powder made from CA-1 (CLEA) pellets (3.45 kcal/g)
  • the left portion of the Panel shows the average daily food intake per mouse, while the right portion of the Panel shows the average daily food consumption per gram of body weight.
  • the data are shown as means ⁇ s.e.m.
  • the asterisk (*) indicates RO.05
  • the dagger (j) indicates RO.001.
  • the statistical significance was determined using an unpaired two-tailed Student's t-test.
  • M3R ' mice During the first post-natal week, the body weight of M3R ' mice did not significantly differ from that of their wild-type littermates (See, Figure 2, Panels A and B). However, starting at the end of post-natal week two (females) and three (males), M3R 'A mice displayed a significant decrease in body weight and reduced weight gain (See, Figure 2, Panel A). For example, at post-natal week 12, male and female M3R ⁇ A mice weighed about 22%> less (RO.0001) than their wild-type littermates (See, Figure 2, Panels A and B). A similar percent weight difference persisted throughout the entire observation period (i.e., more than one year). No significant reduction in body weight was observed with heterozygous M3R +A mutant mice.
  • M3R 'A mice (10- and 22-week old males) exhibited a 50-60%> reduction in the weight of gonadal (epididymal) fat pads. Since the weight of mouse gonadal fat pads closely correlates with total body fat mass (Rogers and Webb, Brit. J. Nutr., 43:83-86 [1980]), reduced body fat content is predicted to represent a major factor contributing to the decreased body weight displayed by the M3R 'A mice.
  • mice that carried the M3 receptor mutation in a pure genetic background (129SvEv) were also observed.
  • isogenic mice that carried the M3 receptor mutation in a pure genetic background (129SvEv) were also observed.
  • mice were offered a wet mash diet rather than dry food pellets. These experiments showed that consumption of wet mash food instead of dry standard mouse chow had little effect on the reduction in body weight and food intake observed with the M3R 'A mice (See, Figure 2, Panels C, D and F). Together, these results argue against the idea that impaired salivation is responsible for the hypophagic phenotype displayed by the 5R " mice. Serum Chemistry and Hormone Measurements
  • mice 18 week old male mice were fasted for at least 8 hours, and then given glucose (2 g/kg) via intraperitoneal injection or oral gavage.
  • glucose 2 g/kg
  • human insulin (0.75 U/kg) (Lilly) was administered intraperitoneal ly to freely fed male mice (28 weeks old). Blood samples were taken via retroorbital sinus puncture immediately before and 15, 30, 60, and 120 minutes after the administration of glucose or insulin. Blood glucose levels were measured by glucose strips (ExacTech RSG glucose meter; Medisense).
  • Results from glucose and insulin tolerance tests for wild-type and M3R ' mice are shown in Figure 3.
  • Panel A shows the results for glucose tolerance tests
  • Panel B shows the results for insulin tolerance tests.
  • the asterisk (*) indicates P ⁇ 0.05
  • ) indicates P ⁇ 0.001.
  • the statistical significance was determined using an unpaired two-tailed Student's /-test.
  • M3R 'A mice exhibited drastically reduced (9-fold) serum insulin levels, as shown in Table 1.
  • M3R ' mice showed normal or slightly reduced blood (serum) glucose levels (as also shown in Table 1), and did not become hyperglycemic throughout the entire observation period (i.e., up to one year).
  • the glucose tolerance test demonstrated that M3R 'A were able to clear glucose from the blood at least as efficiently as wild-type mice, as shown in Figure 3, Panel A. Similar results were obtained when the glucose load (2 g/kg) was administered orally, indicating that the M3R 'A mice remain fully insulin sensitive.
  • M3R 'A mice showed a much more pronounced and prolonged hypoglycemia than their wild-type littermates, indicative of an increase in insulin sensitivity ( Figure 3, Panel B).
  • the low serum insulin levels found in M3R 'A mice are believed to be probably due to the reduction in total body fat, since insulin, like leptin, circulates at levels proportional to body fat content (Schwartz et al, supra).
  • the levels of many other serum parameters e.g., total protein, total albumin, sodium, potassium, chloride, bicarbonate, calcium, alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, alkaline phosphatase, creatine phosphokinase, urea nitrogen, creatinine, cholesterol, and bilirubin, were not significantly affected by the lack of functional M3 receptors (See, Table 1).
  • Oxygen consumption and carbon dioxide production were measured by using a four chamber Oxymax system (Columbus) with one mouse per chamber, as known in the art (See e.g., Gong et al, J. Biol. Chem., 275:16251-16257 [2000]).
  • mice were subjected to a charcoal transit test. Prior to the experiments, mice were fasted for 24 hours, but had free access to water. Each mouse received 0.2 ml suspension of 2%> activated charcoal in 1.2%) gum arabic intragastrically (via oral gavage). The mice were sacrificed by cervical dislocation 20 minutes after charcoal administration. The stomach and small intestine were carefully removed and the omentum separated, avoiding stretching. The total length of the small intestine from the pyloric sphincter to the ileocecal junction and the distance traveled by the charcoal were measured. Gastrointestinal transit was expressed as the distance traveled by the charcoal per total length of the small intestine.
  • mice were anesthetized with pentobarbital (50 ⁇ g/kg, IP). Salivation responses to pilocarpine (1, 5 and 15 mg per kilogram, SC) were quantitated in 5 minute intervals over a 30 minute observation period by filter paper method as known in the art (See e.g., Parkes and Parks, Br. J. Pharmacol, 46:315-323 [1972]).
  • filter paper method See e.g., Parkes and Parks, Br. J. Pharmacol, 46:315-323 [1972]).
  • Figure 2 Panel I.
  • data are given as mean ⁇ s.e.m., "*" indicates RO.05, and "j" indicates PO.001, using Student's t-test.
  • M3R light 24,254 ⁇ 2,934
  • M3R + + light, 29,439 ⁇ 4,140
  • M3R dark, 59,011 ⁇ 8,011
  • RT-PCR methods used in the development of the present invention are described.
  • Freely fed male mice (20-weeks old) were implanted with a guide cannula into the left lateral ventricle (0.3 mm posterior and 3.0 mm ventral to bregma) as known in the art (See e.g., Sakurai et al, Cell 92:573-585 [1998]).
  • peptides were dissolved in 2 ⁇ l of 0.9%> saline and administered through the guide cannula at 2 hours into the light cycle. Food intake was measured during the 2 hours after injections. Mice had at least 48 hours between treatments to regain normal feeding patterns.
  • AGRP 83.132 (human) and MCH were obtained from Phoenix, while orexin-A (human) was obtained from the Peptide Institute.
  • PCR amplifications were performed with the following primer pairs: AGRP (GenBank # ⁇ M007427): 5'-TGACTGCAATGTTGCTGAGTTGTG-3' (forward primer; SEQ ID NO:3), and 5'-CTAGGTGCGACTACAGAGGTTCGAG-3' (reverse primer; SEQ ID NO:4) (product size: 392 bp); POMC (GenBank #NM008895): 5'-TTTCCTGGCAACGGAGATGA-3' (forward primer; SEQ ID NO:5), and 5'- CCACCGTAACGCTTGTCCTT-3' (reverse primer; SEQ ID NO:6) (product size: 449 bp); MCH: 5'-CAGCTTCCAAGTCCATAAGG-3' (forward primer; SEQ ID NO:7), and 5'-ACTCTTCCCAGCATACACCT-3' (reverse primer; SEQ ID NO:8) (product size: 498; See also, Shimada et al, supra); prepro-orexin (GenBank
  • Panel A provides data showing the expression of hypothalamic neuropeptide mRNAs in wild-type and M3R mice.
  • Total RNA was isolated from hypothalami of freely fed, 20-week old, male M3R A mice (-) and their wild-type littermates (+), and subjected to semi-quantitative RT-PCR analysis using the above primers (i.e., primers specific for AGRP, POMC, MCH, prepro-orexin, and -actin [internal standard]). For each sample, the ratio was formed between the integrated optical density of the neuropeptide band and that of the internal standard.
  • the asterisk (*) indicates R ⁇ 0.05
  • ) indicates a RO.001.
  • Statistical significance was determined with an unpaired Student's t-test.
  • hypothalamic MCH expression was significantly reduced in M3R A mice, as shown in Figure 4, Panel A. This finding was also confirmed by Northern blotting analysis.
  • Panel B provides data from experiments conducted to demonstrate the feeding response to intracerebroventricular administration of orexigenic neuropeptides.
  • Sections were subsequently processed for M3 receptor immunohistochemistry using an M3-receptor-specific rabbit polyclonal antibody (diluted 1:50) directed against the C-terminus of the M3 receptor, as known in the art (See e.g., Zeng et al, J. Biol. Chem., 274:16629-16640 [1999]). Bound antibody was visualized by using an avidin/biotin/peroxidase method (brown reaction product), as known in the art (See, Elias et al, supra).
  • M3 receptor immunoreactivity was observed in several regions of the hypothalamus, including the arcuate nucleus and the lateral hypothalamus. In addition, the presence of M3 receptor protein was observed in neurons of the lateral hypothalamus expressing MCH mRNA. As indicated in Figure 4, Panel C, there is co-expression of MCH and M3 muscarinic acid receptors in neurons of the lateral hypothalamus (LH), a secondary feeding center. MCH neurons receive synaptic input from both the AGRP/POMC system of the arcuate nucleus (ARC)(Elias et al, J. Comp. Neurol, 402:442-459 [1998]; Broberger et al, J. Comp.
  • ARC arcuate nucleus

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Abstract

Méthodes et compositions permettant de cribler des médicaments et d'autres compositions à la recherche de leur effet sur l'appétit et le contrôle du poids, y compris compositions pour traiter l'obésité chez les humains et les animaux. En particulier, la présente invention concerne des animaux chez qui est absente l'expression du récepteur muscarinique M3 de l'acétylcholine.
PCT/US2001/051110 2000-10-30 2001-10-26 Methodes et compositions d'analyse de recepteurs muscariniques m3 de l'acetylcholine WO2002046421A2 (fr)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001029176A2 (fr) * 1999-10-15 2001-04-26 Genaissance Pharmaceuticals, Inc. Isogenes cibles de medicaments: polymorphismes dans le gene 3 muscarinique, de recepteur cholinergique

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001029176A2 (fr) * 1999-10-15 2001-04-26 Genaissance Pharmaceuticals, Inc. Isogenes cibles de medicaments: polymorphismes dans le gene 3 muscarinique, de recepteur cholinergique

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ELMQUIST JOEL K ET AL: "From lesions to leptin: Hypothalamic control of food intake and body weight." NEURON, vol. 22, no. 2, February 1999 (1999-02), pages 221-232, XP002237440 ISSN: 0896-6273 *
LEVEY A I ET AL: "LOCALIZATION OF MUSCARINIC M3 RECEPTOR PROTEIN AND M3 RECEPTOR BINDING IN RAT BRAIN" NEUROSCIENCE, NEW YORK, NY, US, vol. 63, no. 1, 1994, pages 207-221, XP000612457 ISSN: 0306-4522 *
MATSUI MINORU ET AL: "Multiple functional defects in peripheral autonomic organs in mice lacking muscarinic acetylcholine receptor gene for the M3 subtype." PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES, vol. 97, no. 17, 15 August 2000 (2000-08-15), pages 9579-9584, XP002237439 August 15, 2000 ISSN: 0027-8424 *
YAMADA MASAHISA ET AL: "Mice lacking the M3 muscarinic acetylcholine receptor are hypophagic and lean." NATURE (LONDON), vol. 410, no. 6825, 2001, pages 207-212, XP002237441 ISSN: 0028-0836 *

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