JP2007509923A - Inhibition of phosphodiesterase 9 as a treatment for obesity-related conditions - Google Patents

Abstract

The invention relates to animals, for example, in the treatment of overweight or obese patients (eg, human or companion animals) or as a means for producing low-fat meat in food stock animals (eg, cows, chickens, pigs). For reducing body weight and / or body fat and methods for treating eating disorders by administering a PDE9 inhibitor to a patient in need of treatment for eating disorders (eg, bulimia or bulimia) About. The invention also features biological tools for further studying PDE9 function, ie, genetically modified mice and animal cells having a PDE9 gene disruption.
[Selection] Figure 7

Description

  The present invention relates to methods for reducing body weight and / or body fat in the treatment of, for example, overweight and / or obese patients, and eating disorders (eg bulimia and polyphagia) by administering a phosphodiesterase 9 (PDE9) inhibitor. A method of treating a symptom). The present invention also features genetically modified mammalian cells and genetically modified mice with PDE9 gene disruption.

  Individuals who are diagnosed as obese or overweight are, for example, coronary heart disease, stroke, hypertension, type 2 diabetes, dyslipidemia, sleep apnea, osteoarthritis, gallbladder disease, depression, And suffering great risks of developing other health conditions, such as certain cancers (eg, endometrial cancer, breast cancer, prostate cancer and colon cancer). The adverse health consequences of obesity make obesity the second leading cause of preventable death in the United States and have a significant economic and psychosocial impact on society (eg, McGinnis and Foege, JAMA 270: 2207-2212, 1993).

  Obesity has become a major public health problem due to its increasing morbidity and is now recognized as a chronic disease that requires treatment to reduce its associated health risks. While weight loss itself is an important treatment outcome, one of the primary goals of obesity management is to improve cardiovascular and metabolic values to reduce morbidity and mortality associated with obesity. It has been found that weight loss of 5-10% of body weight can substantially improve metabolic values such as blood glucose level, blood pressure and lipid concentration. For this reason, it is conceivable that intentional weight loss of 5-10% of body weight can reduce morbidity and mortality.

  Cyclic nucleotide phosphodiesterases (PDEs) are cyclic nucleotides such as, for example, the second messenger cAMP (cyclic adenosine 3 ′, 5′-monophosphate) and cGMP (cyclic guanine 3 ′, 5′-monophosphate). Catalyze hydrolysis. Thus, PDE plays a crucial regulatory role in a very diverse signaling pathway (Beavo, Physiol. Rev. 75: 725-748, 1995). For example, PDE is visual ((McLaughlin et al., Nat. Genet. 4: 130-134, 1993), olfaction (Yan et al., Proc. Natl. Acad. Sci. USA 92: 9677-81, 1995). Platelet aggregation (Dickinson et al. Biochem. J. 323: 371-377, 1997), aldosterone synthesis (MacFarland et al., J. Biol. Chem. 266: 136-142, 1991), insulin secretion (Zhao et al. , J. Clin. Invest. 102: 869-873, 1998), T cell activation (Li et al., Science 283: 848-51, 1999), smooth muscle relaxation (Boolell et al., Int. J. Impot. Res. 8: 47-52, 1996; Ballard et al., J. Urol. 159: 2164-171, 1998).

  PDE forms a superfamily of enzymes that are subdivided into 11 major gene families (Beavo, Physiol. Rev. 75: 725-748, 1995; Beavo et al., Mol. Pharmacol. 46: 399- 405, 1994; Soderling et al., Proc. Natl. Acad. Sci. USA 95: 8991-8996, 1998; Fisher et al., Biochem. Biophys. Res. Commun. 246: 570-577, 1998; Hayashi et al , Biochem. Biophys. Res. Commun. 250: 751-756, 1998; Soderling et al., J. Biol. Chem. 273: 15553-58, 1998; Fisher et al., J. Biol. Chem. 273: 15559-15564, 1998; Soderling et al., Proc. Natl. Acad. Sci. USA 96: 7071-7076, 1999; and Fawcett et al., Proc. Natl. Acad. Sci. USA 97: 3702-3707, 2000 ).

  Each PDE gene family encodes phosphodiesterases that are functionally distinguished by unique enzymatic characteristics and pharmacological profiles. In addition, each family exhibits distinct tissue, cell, and subcellular expression patterns (Beavo et al., Mol. Pharmacol. 46: 399-405, 1994; Soderling et al., Proc. Natl Acad. Sci. USA 95: 8991-8996, 1998; Fisher et al., Biochem. Biophys. Res. Commun. 246: 570-577, 1998; Hayashi et al., Biochem. Biophys. Res. Commun. 250: 751-756, 1998; Soderling et al., J. Biol. Chem. 273: 15553-15558, 1998; Fisher et al., J. Biol. Chem. 273: 15559-15564, 1998; Soderling et al., Proc Natl. Acad. Sci. USA 96: 7071-7076, 1999; Fawcett et al., Proc. Natl. Acad. Sci. USA 97: 3702-3707, 2000; Boolell et al., Int. J. Impot. Res 8: 47-52, 1996; Ballard et al., J. Urol. 159: 2164-71, 1998; Houslay, Semin. Cell Dev. Biol. 9: 161-67, 1998; and Torphy et al., Pulm. Pharmacol. Ther. 12: 131-135, 1999). Therefore, by administering a compound that selectively modulates the activity of one family or subfamily of PDE enzymes, the cAMP and / or cGMP signaling pathway can be modulated in a cell- or tissue-specific manner. Is possible.

  Fisher et al. (J. Biol. Chem. 273: 15559-15564, 1998) has identified the PDE9 enzyme as a new member of the PDE enzyme family that selectively hydrolyzes cGMP over cAMP. PDE9 is present in a variety of human tissues, including testis, brain, small intestine, skeletal muscle, heart, lung, thymus and spleen. PDE9 inhibitors have been reported as useful for the treatment of cardiovascular disorders (WO 03/037899) and insulin resistance syndrome, hypertension and / or type 2 diabetes (WO 03/037432).

SUMMARY OF THE INVENTION In a first aspect, the present invention provides a method of treating an animal for reducing body fat, comprising administering to the animal in need thereof a therapeutically effective amount of a PDE9 inhibitor. Features. Preferably, the animal is a human or companion animal (eg, dog, cat, horse) and is overweight, more preferably the animal is obese. In other preferred embodiments, the animal is a food stock animal (eg, chicken, cow, pig) and such treatment is done to produce lean meat. In another preferred embodiment, the PDE9 inhibitor is a PDE9 selective inhibitor, or the PDE9 inhibitor is administered orally.

  In a second aspect, the invention features a method of treating an animal for eating disorders, comprising administering to the animal in need thereof a therapeutically effective amount of a PDE9 inhibitor. . Preferably, the eating disorder is bringe eating disorder or bulimia and the PDE9 inhibitor is a PDE9 selective inhibitor or the PDE9 inhibitor is administered orally .

  In a third aspect, the invention features a method of treating an animal for metabolic syndrome, comprising administering to an animal in need thereof a therapeutically effective amount of a PDE9 inhibitor. Preferably, the PDE9 inhibitor is a PDE9 selective inhibitor or the PDE9 inhibitor is administered orally.

  The present invention further relates to genetically-modified mice that are homozygous for the disruption of the PDE9 gene, after 6 weeks of high fat diet, compared to wild type mice after 6 weeks of high fat diet. Characterized by genetically modified mice that exhibit weight loss or fat mass reduction during adipose depot. In a preferred embodiment, the mouse expresses an exogenous reporter gene under the control of regulatory sequences of the PDE9 gene, or the mouse exhibits undetectable PDE9 activity. In a related aspect, the present invention provides a cultured genetically modified murine cell derived from the mouse. In another related aspect, the present invention provides the above-described method for producing a mouse, wherein (a) a DNA sequence comprising a PDE9 gene targeting construct that disrupts the PDE9 gene upon recombination with the PDE9 gene is inserted into a mouse ES cell. A step of introducing; (b) a step of selecting a mouse ES cell whose genome contains a disruption of the PDE9 gene; (c) a step of introducing the ES cell selected in step (b) into a mouse blastocyst or morula (D) transferring the blastocysts or morulas of step (c) into pseudopregnant mice; (e) developing transferred blastocysts or morulas to a period of time producing chimeric mice; And (f) mating sexually mature chimeric mice with mice heterozygous for PDE9 disruption to obtain homozygous mice for PDE9 gene disruption. Step; wherein, the mice after a high fat diet for 6 weeks, as compared to wild type mice after a high fat diet for 6 weeks, a method of indicating the lipid loss during weight loss or fat accumulation.

  The present invention further relates to genetically modified cultured mammalian cells, wherein the cells are homozygous for the disruption of the PDE9 gene and the cells or progeny cells derived from the cells exhibit undetectable PDE9 polypeptide activity. Wherein the cells or progeny cells are characterized by genetically modified cultured mammalian cells that exhibit PDE9 polypeptide activity without the homozygous disruption. In a preferred embodiment, the cell is an embryonic stem (ES) cell, more preferably the cell is a murine ES cell or a human ES cell.

  In another embodiment, the invention provides an isolated nucleic acid molecule comprising a PDE11 gene targeting construct, wherein the construct disrupts the PDE9 gene upon recombination with the PDE9 gene. Provide molecules.

  Those skilled in the art will fully understand the terms used in this application to describe the present invention in the specification and claims. Nonetheless, the following terms are immediately described below unless otherwise specified herein.

“PDE9 inhibitor” means an agent that reduces or reduces the biological activity of a PDE9 polypeptide. Such agents can be proteins (eg, anti-PDE9 antibodies), nucleic acids (eg, PDE9 antisense or RNA interference ((RNAi) nucleic acids), amino acids, peptides, carbohydrates, small molecules (organic or inorganic), or in cells. Includes any other compound or composition that reduces the activity of the PDE9 polypeptide, either by effectively reducing the amount of PDE9 present, or by reducing the enzymatic activity of the PDE9 polypeptide. The compounds that are include all solvates, hydrates, pharmaceutically acceptable salts, tautomers, stereoisomers and prodrugs of the compounds, preferably small molecules for use in the present invention. PDE9 inhibitor is less than 10 [mu] M, more preferably less than 1 [mu] M, even more preferably an _{IC 50} of less than 0.1μM Any PDE9 inhibitor used in the present invention may further include some or all other PDEs, preferably PDE1A, PDE1B, PDE1C, PDE2, PDE3A, PDE3B, PDE4A, PDE4B, PDE4C, PDE4D, PDE5, PDE6, PDE7A , PDE7B, PDE8A, PDE8B, PDE10 and / or PDE11 are also preferably selective.

By “selective” PDE9 inhibitor is meant an agent that inhibits PDE9 activity with an IC ₅₀ that is at least 10-fold, preferably at least 100-fold lower than the IC ₅₀ for inhibition of one or more other PDEs. Preferably, such agents are combined with a pharmaceutically acceptable delivery vehicle or carrier. Antisense oligonucleotides directed to the PDE9 gene or mRNA to inhibit the expression of the PDE9 gene or mRNA are made by standard methods (eg, Agrawal et al. Methods in Molecular Biology: Protocols for Oligonucleotides and Analogs, Vol. 20, see 1993). Similarly, RNA molecules that function to reduce the production of PDE9 enzyme in cells can be made by standard methods known to those skilled in the art (eg, Hannon, Nature 418: 244-251, 2002; Shi, Trends in Genetics 19: 9-12, 2003; Shuey et al., Drug Discovery Today 7: 1040-1046, 2002). Examples of PDE9 inhibitors are described herein and in WO 03/037899, WO 03/037432 and US Provisional Patent Application No. 60 / 466,639 (filed April 30, 2003), incorporated herein by reference. Has been.

  “Reduced PDE9 activity” refers to the result of administration of a pharmacological agent that decreases the level of functional PDE9 polypeptide in a cell, as a result of gene disruption or manipulation of PDE9 gene function, or inhibits PDE9 activity. As an artificial reduction in the polypeptide activity of the PDE9 enzyme.

  The phrase “pharmaceutically acceptable” means that the specified carrier, vehicle, diluent, excipient (s) and / or salt is generally chemically and / or other ingredients contained in the formulation. Or it means being physically compatible and physiologically compatible with the recipient.

  The term “prodrug” refers to a compound that is a drug precursor that releases a drug in vivo, after administration, by a chemical or physiological process (eg, when at physiological pH or by enzymatic activity). . Considerations on the synthesis and use of prodrugs include Higuchi and Stella's Prodrugs as Novel Delivery Systems, vol. 14 of the ACS Symposium Series, and Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press , 1987.

The terms “salt” and “pharmaceutically acceptable salt” refer to organic and inorganic salts of compounds, stereoisomers of compounds or prodrugs of compounds.
“Overweight” and a more severe “obesity” condition are over the ideal body weight (more specifically, over the ideal body fat) in adults over 18 years of age. Generally defined by body mass index (BMI), BMI correlates with total fat and also with the relative risk of suffering premature death or disability due to disease as a result of being overweight or obese. The health risk increases with an increase in excessive body fat. The BMI is divided by dividing the weight in kg by the square of the height in m (kg / m ² ) or by multiplying the weight in pounds by 703 and dividing by the square of the height in inches (lbsx703 / in ² ). “Overweight” typically results in a BMI of 25.0-29.9. “Obesity” is typically defined as 30 or more BMI (eg, National Heart, Lung, and Blood Institute, Clinical Guidelines on the Identification, Evaluation, and Treatment of Overweight and Obesity in Adults, The Evidence Report, Washington DC: US Department of Health and Human Services, NIH publication no. 98-4083, 1998). In muscular individuals, the correlation between BMI, body fat and disease risk is weaker than in other individuals. Therefore, an assessment of whether such a muscular individual is actually overweight or obese is more likely by other means, such as a direct measurement of total fat or a waist-hip ratio assessment. May be done accurately.

  By “high fat diet” as administered to genetically modified or wild type mice is meant a diet consisting of at least 45% kcal fat (fat), preferably at least 58% fat. Specific diets include the Surwit diet (Surwit et al., Metabolism 47: 1354-1359; Surwit et al., Metabolism 47: 1089-1096, 1998; Surwit et al., J. Biol. Chem. 271: 9437- 9440, 1996; and Surwit et al., Metabolism 44: 645-651, 1995), D12451 Rodent diet (45% kcal fat, Research Diets, Inc., New Brunswick, NJ), and D12331 Rodent diet (58% kcal fat , Research Diets, Inc.).

As defined herein, and Adult Treatment Panel III (ATP III; National Institutes of Health: Third Report of the National Cholesterol Education Program Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III), Executive Summary; Bethesda, MD, National Institutes of Health, National Heart, Lung and Blood Institute, 2001 (NIH pub.no.01-3670). more than:
1. Abdominal obesity: waist circumference> 102 cm for men, waist circumference> 88 cm for women;
2. Hypertriglyceridemia: ≧ 150 mg (1.695 mmol / l);
3. Low HDL cholesterol: <40 mg / dl (1.036 mmol / l) for men, <50 mg / dl (1.295 mmol / l) for women;
4). Hypertension: ≧ 130/85 mmHg;
5). High fasting blood glucose: ≧ 110 mg / dl (≧ 6.1 mmol / l)
Occurs when having; or
According to the World Health Organization criteria (Alberti and Zimmet, Diabet. Med. 15: 539-53, 1998), in addition to diabetes, impaired glucose tolerance, fasting blood glucose, or insulin resistance, more than:
1. Hypertension: ≧ 160/90 mmHg;
2. Hyperlipidemia: triglyceride concentration ≧ 150 mg / dl (1.695 mmol / l) and / or HDL cholesterol <35 mg / dl (0.9 mmol / l) in men, <39 mg / dl (1.0 mmol / l) in women );
3. Central obesity: waist-to-hip ratio> 0.90 for men,> 0.85 for women, and / or BMI> 30 kg / m ² ;
4). Microproteinuria: urinary albumin excretion rate ≧ 20 μg / min or albumin / creatinine ratio ≧ 20 mg / kg
It occurs when you have

“Therapeutically effective” means causing a reduction in body fat.
A “PDE9 gene disrupted” is a cell that normally expresses a wild-type version of the PDE9 gene, wherein the cellular activity of the PDE9 polypeptide encoded by the disrupted gene is reduced or preferably eliminated. As such, it means a PDE9 gene that has been genetically modified. The genetic modification effectively eliminates all of the wild type copy of the PDE9 gene in the cell (eg, the genetically modified non-human mammal or animal cell is homozygous for the PDE9 gene disruption or originally If only the wild-type copy of the existing PDE9 gene is now destroyed), the genetic modification reduces the PDE9 polypeptide activity compared to control cells expressing the wild-type PDE9 gene. This decrease in PDE9 polypeptide activity results from a decrease in PDE9 gene expression (ie, PDE9 mRNA levels are effectively reduced resulting in a decrease in PDE9 polypeptide levels) and / or a decrease in PDE9 polypeptide activity is: A disrupted PDE9 gene results because it encodes a mutant polypeptide with altered (eg, reduced) function compared to the wild-type PDE9 polypeptide. Preferably, the PDE9 polypeptide activity in the genetically modified non-human mammal or animal cell is 50% or less of the wild type level, more preferably 25% or less, even more preferably 10% of the wild type level. Below, it drops. Most preferably, homozygous PDE9 gene disruption results in undetectable PDE9 activity in a cell type that demonstrates wild-type PDE9 activity.

  A “genetically modified non-human mammal” containing a disrupted PDE9 gene refers to a non-human mammal produced by genetic manipulation so as to contain a disrupted PDE9 gene, and the disrupted PDE9 gene. It means the offspring of such a non-human mammal, as inherited by inheritance. The genetically modified non-human mammal, for example, produces an undifferentiated germ cell or embryo having a desired genetic modification, and then transplants the undifferentiated germ cell or embryo into a foster mother for intrauterine development. Can be generated. In the case of a genetically modified undifferentiated germ cell or embryo, in the case of a mouse, transplanting the genetically modified embryonic stem (ES) cell into a mouse undifferentiated germ cell or aggregating ES cells into a tetraploid embryo Can be produced. Alternatively, various species of genetically modified embryos can be obtained by nuclear transfer. In the case of nuclear transfer, the donor cells are somatic or pluripotent stem cells that are engineered to contain the desired genetic modification that disrupts the PDE9 gene. The nucleus of this cell is then transferred into an enucleated fertilized oocyte or parthenogenetic oocyte; the resulting embryo is reconstituted and developed into undifferentiated germ cells. The genetically modified undifferentiated germ cells obtained by either of the above methods are then transplanted into foster mothers by standard methods well known to those skilled in the art. “Gene-modified non-human mammal” encompasses all progeny of the non-human mammal produced by the above method as long as the progeny inherit at least one copy of the genetic modification that disrupts the PDE9 gene. It is preferred that all somatic cells and germ cells of the genetically modified non-human mammal contain the modification. Preferred non-human mammals that are genetically modified to contain a disrupted PDE9 gene include rodents (eg, mice and rats), cats, dogs, rabbits, guinea pigs, hamsters, sheep, pigs and ferrets. To do.

  A “genetically modified animal cell” containing a disrupted PDE9 gene is an animal cell (preferably a mammalian cell), including a human cell, produced by genetic manipulation to contain a disrupted PDE9 gene, and It means cells differentiated from daughter cells and genetically modified parental ES cells or stem cells that inherit the disrupted PDE9 gene. These cells can be genetically modified in culture by any standard method known in the art. As an alternative to genetically modifying cells during culture, non-human mammalian cells can also be isolated from genetically modified non-human mammals containing a PDE9 gene disruption. The animal cells of the present invention can be obtained from tumorigenic or transformed cell lines adapted to primary cells or tissue preparations and cultures. These cells and cell lines include, for example, endothelial cells, epithelial cells, islet cells, neurons and other neural tissue-derived cells, mesothelial cells, bone cells, lymphocytes, chondrocytes, hematopoietic cells, immune cells, major glands or Cells of organs (eg, testis, liver, lung, heart, stomach, pancreas, kidney, and skin), muscle cells (including skeletal muscle, smooth muscle and heart muscle), exocrine or endocrine cells, fibroblasts, and embryos and Derived from other totipotent or pluripotent stem cells (eg, ES cells, ES-like cells, embryonic germ cells and other stem cells such as progenitor cells and tissue-derived stem cells). Preferred genetically modified cells are ES cells, more preferably mouse or rat ES cells, most preferably human ES cells and cells differentiated from genetically modified ES cells.

  A “genetically modified” non-human mammal or animal cell is heterologous or homozygous for a modification that is genetically manipulated into a non-human mammal or animal cell or into a precursor non-human mammal or animal cell. It is sex. Standard methods of genetic manipulation that can be used to transfer modifications include homologous recombination, viral vector gene trapping, irradiation, chemical mutation, and nucleotides that encode antisense RNA alone or in combination with catalytic ribozymes. Includes transgenic expression of the sequence. A preferred method of genetic modification to disrupt a gene is to modify the endogenous gene by inserting a “heterologous nucleic acid sequence” into the locus (eg, by homologous recombination or viral vector gene trapping). A “heterologous nucleic acid sequence” is an exogenous sequence that occurs non-naturally in a gene. This insertion of heterologous DNA can occur in any region of the PDE9 gene (eg, in an enhancer, promoter, regulatory region, non-coding region, coding region, intron or exon). The most preferred method of genetic manipulation for gene disruption is homologous recombination, in which heterologous nucleic acid sequences are inserted in a targeted manner, either alone or with deletion of a portion of an endogenous gene sequence. Is done.

  “Homozygosity” means a non-human mammalian or animal cell having a disruption of all alleles of the PDE9 gene when it relates to the PDE9 gene disruption in the non-human mammal or animal cell. However, the PDE9 gene sequence of each of these disrupted alleles need not be identical. For example, in non-human mammals, one allele of PDE9 is destroyed as a result of deletion of one region of the gene sequence, and the other allele is destroyed as a result of deletion of another region of the gene sequence. In such cases, it can be homozygous for PDE9 failure.

“ES cells” or “ES-like cells” are pluripotent stem cells derived from embryos, primitive germ cells, or teratocarcinomas that can differentiate into cell types that represent infinite self-renewal as well as all three germ layers, Means pluripotent stem cells.
“Microarray” means an arrangement of identifiable polynucleotides or polypeptides on a substrate, as described in more detail herein.

  “Wild type”, when referring to a non-human mammal or animal cell, means a non-human mammal or animal cell that optionally does not contain a disrupted PDE9 gene. For example, when comparing a particular characteristic of a non-human mammal of the present invention to that characteristic of a wild-type mammal, the term wild-type refers to a non-human mammal that does not contain a disrupted PDE9 gene (ie, its Mammals in which the PDE9 gene is a wild type). Preferably, the wild type non-human mammal is substantially similar to the non-human mammal of the present invention, more preferably substantially the same, except for non-destruction or disruption of the PDE9 gene. . Similarly, for example, when comparing a particular characteristic of an animal cell of the present invention to that characteristic of a wild type animal cell, the term wild type refers to an animal cell that does not contain a disrupted PDE9 gene (ie, its PDE9 Cell whose gene is wild type). Preferably, the wild-type animal cell is substantially similar to the animal cell of the present invention, more preferably substantially the same, except for non-destruction or disruption of the PDE9 gene.

  Other features and advantages of the invention will be more fully apparent from the following detailed description, and from the claims. While the invention will be described in connection with specific embodiments, it will be understood that other variations and modifications that can be implemented are part of the invention and are within the scope of the claims. This application confirms that any equivalents, variations, uses or adaptations of the invention described below are generally within the scope of known or customary practice within the art and without undue experimentation. The principles of the present invention, including deviations from the present disclosure, are intended to be covered. Further guidance on the production and use of nucleic acids and polypeptides can be found in standard textbooks of molecular biology, protein chemistry and immunology (eg, Davis et al., Basic Methods in Molecular Biology, Elsevir Sciences Publishing , Inc., New York, NY, 1986; Hames et al., Nucleic Acid Hybridization, IL Press, 1985; Molecular Cloning, Sambrook et al., Current Protocols in Molecular Biology, Eds. Ausubel et al., John Wiley and Sons , 2001; Current Protocols in Human Genetics, Eds.Dracopoli et al., John Wiley and Sons, 1994; Current Protocols in Protein Science, Eds. John E. Coligan et al., John Wiley and Sons, 2002; and Current Protocols in Immunology, Eds. John E. Coligan et al., John Wiley and Sons, 1994). All publications mentioned herein, including published patent applications and issued patents, are hereby incorporated by reference in their entirety.

Detailed Description of the Invention The present invention is for example in the treatment of overweight or obese patients (e.g. human or companion animals) or to produce lean meat in foodstock animals (e.g. cows, chickens, pigs). A method for reducing body weight and / or body fat in an animal and a method for treating eating disorders (eg bulimia and bulimia) as a means of inhibiting PDE9 inhibition in patients in need thereof The present invention relates to a method by administering an agent. The invention also features biological tools for further studying PDE9 function, ie genetically modified mouse and animal cells with PDE9 gene disruption. As disclosed in the Examples herein, administration of a PDE9 inhibitor reduces weight gain in an ob / ob mouse model of obesity, and PDE9 knockout mice gain weight after being fed a high fat diet. And fat accumulation enhancement is relatively unlikely. In both examples, reducing PDE9 activity is an effective way to reduce body weight and / or body fat, eg, for treating animal patients who are overweight, obese or suffering from eating disorders. Demonstrates that it can be used and can be used in foodstock animal species to produce lean meat.

Exemplary PDE9 Inhibitors Any PDE9 inhibitor can be used in the present invention. PDE9 inhibitors are known to those skilled in the art and can be determined by standard assays known to those skilled in the art, such as, for example, in WO 03/037899 and WO 03/037432. PDE9 inhibitors used in the methods of the present invention are disclosed in WO 03/037899 and WO 03/037432, and US Provisional Patent Application No. 60 / 466,639 (filed April 30, 2003), which is incorporated herein by reference. PDE9 inhibitors that are present. The compounds disclosed as PDE9 inhibitors in the above US provisional patent applications include the following compounds:
3-Isopropyl-5- [2- (2-morpholin-4-yl-ethoxy) -benzyl] -1,6-dihydro-pyrazolo [4,3-d] pyrimidin-7-one (hereinafter “Compound A”) Called);
1-{[2- (3-Isopropyl-7-oxo-6,7-dihydro-1H-pyrazolo [4,3-d] pyrimidin-5-ylmethyl) -phenoxy] -acetyl} -pyrrolidine-2-carboxylic acid ;
3-Isopropyl-5- [2- (2-oxo-2-piperazin-1-yl-ethoxy) -benzyl] -1,6-dihydro-pyrazolo [4,3-d] pyrimidin-7-one trifluoro Acetate salt;
3-isopropyl-5- [2- (2-morpholin-4-yl-2-oxo-ethoxy) -benzyl] -1,6-dihydro-pyrazolo [4,3-d] pyrimidin-7-one;
3-isopropyl-5- [2- (2-oxo-2-pyrrolidin-1-yl-ethoxy) -benzyl] -1,6-dihydro-pyrazolo [4,3-d] pyrimidin-7-one;
N, N-diethyl-2- [2- (3-isopropyl-7-oxo-6,7-dihydro-1H-pyrazolo [4,3-d] pyrimidin-5-ylmethyl) -phenoxy] -acetamide;
1-{[2- (3-Isopropyl-7-oxo-6,7-dihydro-1H-pyrazolo [4,3-d] pyrimidin-5-ylmethyl) -phenoxy] -acetyl} -pyrrolidine-2-carboxylic acid Methyl ester;
4-{[2- (3-Isopropyl-7-oxo-6,7-dihydro-1H-pyrazolo [4,3-d] pyrimidin-5-ylmethyl) -phenoxy] -acetyl} -piperazine-1-carboxylic acid tert-butyl ester;
N- (2-dimethylamino-ethyl) -2- [2- (3-isopropyl-7-oxo-6,7-dihydro-1H-pyrazolo [4,3-d] pyrimidin-5-ylmethyl) -phenoxy] -Acetamide;
[2- (3-Isopropyl-7-oxo-6,7-dihydro-1H-pyrazolo [4,3-d] pyrimidin-5-ylmethyl) -phenoxy] -acetic acid;
3-isopropyl-5- [2- (5-chloro-2-morpholin-4-yl-ethoxy) -benzyl] -1,6-dihydro-pyrazolo [4,3-d] pyrimidin-7-one;
3-isopropyl-5- [2- (2-pyrrolidin-1-yl-ethoxy) -benzyl] -1,6-dihydro-pyrazolo [4,3-d] pyrimidin-7-one;
3-isopropyl-5- [2- (2-morpholin-4-yl-ethoxy) -cyclohexylmethyl] -1,6-dihydro-pyrazolo [4,3-d] pyrimidin-7-one;
5- [5-fluoro-2- (2-morpholin-4-yl-ethoxy) -benzyl] -3isopropyl-1,6-dihydro-pyrazolo [4,3-d] pyrimidin-7-one;
3-cyclopentyl-5- [5-fluoro-2- (2-morpholin-4-yl-ethoxy) -benzyl] -1,6-dihydro-pyrazolo [4,3-d] pyrimidin-7-one;
9- (1,2-dimethyl-propyl) -2- [2- (2-morpholin-4-yl-ethoxy) -benzyl] -1,9-dihydro-purin-6-one;
2- [2- (2-morpholin-4-yl-ethoxy) -benzyl] -9- (tetrahydro-furan-3-yl) -1,9-dihydro-purin-6-one;
5- [2- (2-diethylamino-ethoxy) -benzyl] -3-isopropyl-1,6-dihydro-pyrazolo [4,3-d] pyrimidin-7-one;
3-cyclopentyl-5- [2- (2-morpholin-4-yl-ethoxy) -benzyl] -1,6-dihydro-pyrazolo [4,3-d] pyrimidin-7-one;
9- (1 (R), 2-dimethyl-propyl)-[2- (2-morpholin-4-yl-ethoxy) -benzyl] -1,9-dihydro-purin-6-one;
9- (2-methyl-butyl) -2- [2- (2-morpholin-4-yl-ethoxy) -benzyl] -1,9-dihydro-purin-6-one;
9-cyclopentyl-2- [2- (2-morpholin-4-yl-ethoxy) -benzyl] -1,9-dihydro-purin-6-one;
5- [2- (2-morpholin-4-yl-ethoxy) -benzyl] -3-pyridin-3-yl-1,6-dihydro-pyrazolo [4,3-d] pyrimidin-7-one;
9- (1,2-dimethyl-propyl) -2- [2- (2-morpholin-4-yl-ethoxy) -benzyl] -1,9-dihydro-purin-6-one;
9-isopropyl-2- [2- (2-morpholin-4-yl-ethoxy) -benzyl] -1,9-dihydro-purin-6-one;
2- [2- (2-morpholin-4-yl-ethoxy) -benzyl] -9- (tetrahydro-furan-2-ylmethyl) -1,9-dihydro-purin-6-one;
9- (1-Isopropyl-2-methyl-propyl) -2- [2- (2-morpholin-4-yl-ethoxy) -benzyl] -1,9-dihydro-purin-6-one;
9- (1-ethyl-propyl) -2- [2- (2-morpholin-4-yl-ethoxy) -benzyl] -1,9-dihydro-purin-6-one; and N- [2- (3 -Isopropyl-7-oxo-6,7-dihydro-1H-pyrazolo [4,3-d] pyrimidin-5-ylmethyl) -cyclohexyl] -2-pyrrolidin-1-yl-acetamide.

  It will be understood by those skilled in the art that all stereoisomers, tautomers, solvates, hydrates, prodrugs and pharmaceutically acceptable salts of the compounds listed above are also encompassed. I will.

Methods of Treatment Agents identified as PDE9 inhibitors are administered at a dosage sufficient to reduce body weight or body fat (eg, by reducing the mass of one or more fat stores). Such a therapeutically effective amount depends, for example, on the condition of the patient or animal, the route of administration, the formulation, the judgment of the practioner, and other factors that will be apparent to those skilled in the art in view of this disclosure, It will be determined using routine optimization techniques.

  PDE9 inhibitors suitable for use in the present invention can be administered alone, but in the case of human therapy, generally suitable pharmaceutical doses selected for the intended route of administration and standard pharmaceutical practice. It will be administered as a mixture with a form, diluent or carrier.

  For example, a suitable PDE9 inhibitor or salt or solvate thereof for use in the present invention can be administered orally, buccal or sublingually, immediate-, delayed-, modulated-, sustained-, two-step -In the form of tablets, capsules (including soft gel capsules), multiparticulates, gels, films, ovules, elixirs, solutions or suspensions for controlled-release or pulse delivery applications And can contain flavoring agents or coloring agents. Such compounds can also be administered by rapidly dispersing or rapidly dissolving dosage forms or in the form of high energy dispersions or as coated particles. Suitable pharmaceutical formulations may be in coated or uncoated form as required.

  Such solid pharmaceutical compositions, such as tablets, are applied for example as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate, glycine and starch (preferably corn, potato or tapioca starch). Forming agents such as disintegrants such as sodium starch glycolate, croscarmellose sodium and certain complex silicates, and eg polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC), succinic acetate Granulating binders such as hydroxypropyl methylcellulose (HPMCAS), sucrose, gelatin and gum arabic can be included. In addition, lubricants such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.

  A similar type of solid composition can also be used as a filler in gelatin capsules or HPMC capsules. Preferred excipients in this regard include lactose, starch, cellulose, lactose or high molecular weight polyethylene glycols. For aqueous suspensions and / or elixirs, PDE9 inhibitor compounds and various sweetening or flavoring agents, colorants or dyes, emulsifiers and / or suspending agents, eg water, ethanol, propylene Diluents such as glycol and glycerin, and combinations thereof can be combined.

  Controlled release and pulsed release dosage forms can be prepared using, for example, excipients as detailed for immediate release dosage forms, release rate modifiers (which are applied onto the body of the device and / or in the body of the device). Can be included with additional excipients that act as Release rate modifiers include HPMC, HPMCAS, methylcellulose, sodium carboxymethylcellulose, ethylcellulose, cellulose acetate, polyethylene oxide, xanthan gum, carbomer, ammonio methacrylate copolymer, hydrogenated castor oil, carnauba wax, paraffin wax, cellulose acetate phthalate, It includes, but is not limited to, hydroxypropylmethylcellulose phthalate, methacrylic acid copolymers and mixtures thereof. Modified release and pulsed release dosage forms may contain one or a combination of release rate modifying excipients. Release rate modifying excipients can be present both within the dosage form (ie, within the matrix) and / or on the dosage form (ie, on the surface or coating).

  Rapidly dispersible or dissolvable dosage form (FDDF) is composed of the following components: aspartame, acesulfame potassium, citric acid, croscarmellose sodium, crospovidone, diascorbic acid, ethyl acrylate, ethyl cellulose, gelatin, hydroxypropyl methylcellulose, magnesium stearate , Mannitol, methyl methacrylate, mint flavor, polyethylene glycol, fumed silica, silicon dioxide, sodium starch glycolate, sodium stearyl fumarate, sorbitol, xylitol. As used herein to describe FDDF, the term “dispersibility or solubility” depends on the solubility of the drug substance used. That is, when the drug substance is insoluble, a rapidly dispersible dosage form can be produced, and when the drug substance is soluble, a rapidly soluble dosage form can be produced.

  Suitable PDE9 inhibitors for use according to the invention are parenterally, for example, intracavernous, intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, intramuscular or subcutaneous. It is also possible to administer. Alternatively, the PDE9 inhibitor can be administered by infusion or needle-free techniques. For such parenteral administration, the PDE9 inhibitor is in the form of a sterile aqueous solution that may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood. Is best used. The aqueous solution is appropriately buffered (preferably at a pH of about 3-9) as needed. The manufacture of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical methods well known to those skilled in the art.

  For oral or parenteral administration to human patients, the daily dosage level of the PDE9 inhibitor used in the present invention is usually 1 to 500 mg (in single or divided doses). A preferred dosage range is from about 1 mg to about 100 mg. The dosage can be a single dose, a divided daily dose, or a multiple daily dose. Alternatively, for example, continuous administration such as by controlled release dosage forms (in which case such continuous dosage forms can be administered on a daily scale, or such continuous administration can be administered over 2 days at a time. By a slow release formulation).

  Thus, for example, a PDE9 inhibitor tablet or capsule suitable for use in accordance with the present invention may suitably contain 1 mg to 250 mg of active compound for administration of one or more at a time. . Preferred tablets or capsules may suitably contain from about 1 mg to about 50 mg of the active compound for administration of one or more at a time. In any case, the physician will determine the actual dosage that will be most appropriate for any individual patient and will vary with the age, weight and response of the particular patient. There can, of course, be individual instances where higher or lower dosage ranges are merited, and such dosage ranges are within the scope of this invention.

  PDE9 inhibitors suitable for use according to the present invention can also be administered intranasally or by inhalation, in the form of a dry powder inhaler or aerosol spray presentation, from a pressurized container, pump, spray or nebulizer. Propellants [eg, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, hydrofluoroalkanes (eg, 1,1,1,2-tetrafluoroethane (HFA134A ™) or 1,1,1,2, 3,3,3-heptafluoropropane (HFA227EA ™), carbon dioxide or other suitable gas]. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. Pressurized containers, pumps, sprays or nebulizers use a mixture of ethanol and propellant as a solvent, which may contain a solution or suspension of the active compound (eg, additionally a lubricant (eg, sorbitan trioleate)). Used). Capsules or cartridges (eg, made of gelatin) for use in inhalers or insufflators should be manufactured to contain a powder mix of a compound of the invention and a suitable powder base (eg, lactose or starch). Can do.

  Aerosol or dry powder formulations are preferably prepared so that each metered amount or “puff” contains 1-50 mg of a PDE9 inhibitor for delivery to the animal to be treated. The total daily dose with an aerosol will be within the range of 1-50 mg, which may be administered in a single dose or, more usually, in divided doses throughout the day.

  A PDE9 inhibitor suitable for use according to the present invention can also be formulated for delivery by an atomizer. Formulations for atomizer devices include, as solubilizers, emulsifiers or suspending agents, the following ingredients: water, ethanol, glycerol, propylene glycol, low molecular weight polyethylene glycol, sodium chloride, fluorocarbon, polyethylene glycol ether, sorbitan trioleate, Oleic acid.

  Alternatively, a PDE9 inhibitor suitable for use according to the present invention can be administered in the form of a suppository or pessary, or the PDE9 inhibitor can be in the form of a gel, hydrogel, lotion, solution, cream, ointment or dusting powder. Can be applied topically. PDE9 inhibitors suitable for use according to the present invention can also be administered dermally or transdermally, for example by use of a skin patch. The inhibitor can also be administered by the pulmonary or rectal route.

  The PDE9 inhibitor can also be administered by the ocular route. For ophthalmic use, the compounds are isotonic as a fine suspension in isotonic pH-adjusted sterile saline, or preferably in combination with a preservative such as, for example, benzylalkonium chloride. Can be formulated as a solution in sterile physiological saline with adjusted pH. Alternatively, the PDE9 inhibitor can be formulated in an ointment such as petrolatum.

  For topical application to the skin, PDE9 inhibitors suitable for use according to the present invention include, for example: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and water. It can be formulated as a suitable ointment containing the active ingredient or agent suspended or dissolved in a mixture with one or more. Alternatively, the PDE9 inhibitor is, for example, one of the following: mineral oil, sorbitan monostearate, polyethylene glycol, liquid paraffin, polysorbate 60, cetyl ester wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. It can be formulated as a suitable lotion or cream suspended or dissolved in the above mixture.

  PDE9 inhibitors suitable for use according to the present invention can also be used in combination with cyclodextrins. Cyclodextrins are known to form inclusion and non-inclusion complexes with drug molecules. Formation of the drug-cyclodextrin complex can modulate the solubility, dissolution rate, bioavailability and / or stability properties of the drug molecule. Drug-cyclodextrin complexes are generally useful for most dosage forms and administration routes. As an alternative to direct complexation with the drug, cyclodextrin can be used as an auxiliary additive, for example as a carrier, diluent or solubilizer. α-, β- and γ-cyclodextrins are some of the most commonly used examples, suitable examples being described in WO 91/11172, WO 94/02518 and WO 98/55148.

  In general, oral administration is the preferred route and most convenient for humans. In situations where the recipient suffers from dysphagia or impaired drug absorption after oral administration, the drug can be administered parenterally, sublingually or buccally.

  For veterinary use, the PDE9 inhibitor is administered as a well-accepted formulation by routine veterinary practice, and the veterinarian determines the dosing schedule and route that is most appropriate for the particular animal. Will. Such animals include companion animals that are overweight, obese, or overweight or at risk for obesity. Other animals that can be treated according to the present invention are food stock animals for obtaining less fat meat than can be obtained without treatment according to the present invention.

The therapeutic efficacy of such PDE9 inhibitors is measured in view of this disclosure, for example, with respect to the determination of ED ₅₀ (a dose that is therapeutically effective in 50% of the population) by cell culture or standard therapeutic methods in experimental animals. be able to.

Data obtained from cell culture assays and animal studies can be used to determine dosage ranges for use in humans. The dosage may vary depending on, for example, the formulation and route of administration. For any PDE9 inhibitor used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. Dosages can be determined in animal models to obtain a circulating plasma concentration range that includes the IC ₅₀ determined in cell culture. Such information can be used to more accurately determine effective doses for humans. Plasma levels can be measured, for example, by high performance liquid chromatography.

The PDE9 inhibitors used according to the present invention can also be used with other agents for the treatment of the diseases, conditions and / or disorders described herein. Therefore, a method of treatment comprising administering a PDE9 inhibitor in combination with other agents is also provided. Suitable agents that can be used in combination with the compounds of the present invention include obesity inhibitors, such as β3-adrenergic receptor agonists, apolipoprotein-B secretion / microsomal triglyceride transfer protein (apo-B / MTP) inhibitors, Peptide YY _3-36 and analogs thereof, MCR-4 agonists, cholecystokinin-A (CCK-A) agonists, monoamine reuptake inhibitors (eg, sibutramine), sympathomimetic drugs, cannabinoid receptor antagonists (eg, rimonabant) (SR-141,716A)), dopamine agonist (eg bromocriptine), melanocyte stimulating hormone receptor analog, 5HT2c agonist, melanin-concentrating hormone antagonist, leptin (OB protein), leptin analog, leptin receptor agonis , Galanin antagonists, lipase inhibitors (eg tetrahydrolipstatin or orlistat), appetite suppressants (eg bombesin agonists), neuropeptide-Y antagonists, thyroid-like agents, dehydroepiandrosterone or analogs thereof, glucocorticoid receptors Agonists or antagonists, orexin receptor antagonists, glucagon-like peptide-1 receptor agonists, ciliary neurotrophic factor (eg, Axokine (available from Regeneron Pharmaceuticals, Inc. Tarrytown, NY and Procter & Gamble Company, Cincinnati, OH) TM)), human agouti-related protein (AGRP), ghrelin receptor antagonist, histamine 3 receptor antagonist or inverse agonist, neuromedin U receptor agonist, IIβ-hydroxy Steroid dehydrogenase-1 inhibitor and the like. Other anti-obesity agents, including the preferred agents listed below, are well known to those skilled in the art or will be readily apparent to those skilled in the art in view of the present disclosure.

Particularly preferred are obesity-suppressing drugs selected from the group consisting of orlistat, sibutramine, bromocriptine, ephedrine, leptin, pseudoephedrine and peptide _YY3-36 (including analogs thereof). It is preferred to administer the compounds and combination therapies of the invention in conjunction with a dietary regimen suitable for exercise and purpose.

Representative anti-obesity agents for use in the combinations, pharmaceutical compositions and methods of the present invention can be prepared using methods known to those skilled in the art, for example, sibutramine is described, for example, in US Pat. No. 4,929,629. As described; bromocriptine is described, for example, in U.S. Patent Nos. 3,752,814 and 3,752,888; orlistat is described in, for example, U.S. Patent Nos. 5,274,143; No. 5,420,305; No. 5,540,917; and No. 5,643,874. As well as PYY _3-36 (including analogs) can be prepared, for example, as described in US Patent Application Publication No. 2002/0141985 and WO 03/027637.

  Skilled technicians have certain factors that include, but are not limited to, the severity of a mammalian disease or disorder, treatment history, general health and / or age, and other existing diseases. It will be appreciated that it will affect the dosage and timing required to effectively treat the animal. Further, treatment of a mammal with a therapeutically effective amount of a PDE9 inhibitor can include a single treatment or, preferably, can include a series of treatments.

Genetically modified non-human mammals and cells Genetically modified animal cells, including genetically modified non-human mammals and human cells of the invention, are atypical or homozygous for modifications that disrupt the PDE9 gene. The animal cells can be derived by genetic manipulation of the cells in culture, or in the case of non-human mammalian cells, the cells can be isolated from the genetically modified non-human mammal.

Disruption of the PDE9 gene To produce the genetically modified non-human mammals and mammalian cells of the present invention, chemical mutagenesis (Rinchik, Trends in Genetics 7: 15-21, 1991, Russell, Environmental & Molecular Mutagenesis 23 ( Suppl. 24): 23-29, 1994), irradiation (Russell, supra), transgenic expression of PDE9 gene antisense RNA, alone or in combination with catalytic RNA ribozyme sequences (Luyckx et al., Proc. Natl. Acad Sci. 96: 12174-79, 1999; Sokol et al., Transgenic Research 5: 363-71, 1996; Efrat et al., Proc. Natl. Acad. Sci. USA 91: 2051-55, 1994; Larsson et al., Nucleic Acids Research 22: 2242-48, 1994) and known in the art, including disruption of the PDE9 gene by insertion of a heterologous nucleic acid sequence into the PDE9 locus, as discussed further below. Genetic modification techniques can be used to disrupt the PDE9 locus. Preferably, the heterologous sequence is inserted by homologous recombination or by insertion of a viral vector. Most preferably, the method of PDE9 gene disruption for making genetically modified non-human mammals and mammalian cells of the present invention is homologous recombination and includes a deletion of a portion of the endogenous PDE9 gene sequence.

  Heterologous sequence integration may involve the following mechanisms: interfering with the PDE9 gene transcription or translation process (eg, by interfering with promoter recognition, or by inserting a transcription stop site or translation stop codon into the PDE9 gene); or Distort so that it no longer encodes a normally functioning PDE9 polypeptide (eg, by introducing a heterologous coding sequence into the PDE9 gene coding sequence, or by introducing a frameshift mutation or amino acid (s) substitution) Or, in the case of a double crossover event, the PDE9 gene is disrupted by one or more of (by deletion of part of the PDE9 gene coding sequence that is required for the expression of functional PDE9 protein).

  In order to insert the heterologous sequence into the PDE9 locus in the genome of the cell to produce the genetically modified non-human mammals and animal cells of the invention based on the present specification, the heterologous DNA sequence is, for example, electroporated. Transfection, calcium phosphate precipitation, retroviral infection, microinjection, biolistics, liposome transfection, DEAE-dextran transfection, or transfer infection, and are introduced into cells by standard methods known in the art ( For example, Neumann et al., EMBO J. 1: 841-845, 1982; Potter et al., Proc. Natl. Acad. Sci USA 81: 7161-65, 1984; Chu et al., Nucleic Acids Res. 15: 1311-26, 1987; Thomas and Capecchi, Cell 51: 503-12, 1987; Baum et al., Biotechniques 17: 1058-62, 1994; Biewenga et al., J. Neuroscience Methods 71: 67-75, 1997; Zhang e t al., Biotechniques 15: 868-72, 1993; Ray and Gage, Biotechniques 13: 598-603, 1992; Lo, Mol. Cell. Biol. 3: 1803-14, 1983; Nickoloff et al., Mol. Biotech 10: 93-101, 1998; Linney et al., Dev. Biol. (Orlando) 213: 207-16, 1999; Zimmer and Gruss, Nature 338: 150-153, 1989; and Robertson et al., Nature 323 : 445-48, 1986). A preferred method for introducing heterologous DNA into cells is electroporation.

A homologous recombination method using the homologous recombination PDE9 gene as a target for destruction is carried out by introducing a PDE9 gene targeting vector into a cell containing the PDE9 gene. The ability of the vector to target the PDE9 gene for disruption results from using a nucleotide sequence in the vector that is homologous to the PDE9 gene, ie, associated with the PDE9 gene. The homologous region facilitates hybridization between the vector and the endogenous sequence of the PDE9 gene. When hybridization occurs, the probability that a crossover event will occur between the targeting vector and the genomic sequence is greatly increased. As a result of the crossover event, integration of the vector sequence into the PDE9 locus and functional disruption of the PDE9 gene occurs.

  General principles for the construction of vectors used for targeting are reviewed in Bradley et al. (Biotechnol. 10: 534, 1992). DNA can be inserted by homologous recombination using two different types of vectors: insertion vectors or replacement vectors. The insertion vector is a circular DNA containing a region of PDE9 gene homology with double-strand breaks. After hybridization of the homology region with the endogenous PDE9 gene, a single crossover event at double-strand break results in the insertion of the entire vector sequence into the endogenous gene at the site of crossover.

  For producing the genetically modified non-human mammals and animal cells of the invention by homologous recombination, a more preferred vector is a replacement vector that is collinear rather than circular. Replacement vector integration into the PDE9 gene requires a double crossover event, ie, a crossover at two sites of hybridization between the targeting vector and the PDE9 gene. As a result of the double crossover event, the integration of the vector sequence sandwiched between the two site crossovers into the PDE9 gene and the corresponding endogenous PDE9 gene that essentially bridges between the two site crossovers Deletion of the sequence occurs (eg Thomas and Capecchi et al., Cell 51: 503-12, 1987; Mansour et al., Nature 336: 348-52, 1988; Mansour et al., Proc. Natl. Acad Sci. USA 87: 7688-7692, 1990; and Mansour, GATA 7: 219-227, 1990).

  The region of homology in the targeting vector used to produce the genetically modified non-human mammals and animal cells of the present invention is generally at least 100 nucleotides in length. Most preferably, the homology region is at least 1-5 kilobases (kb) long. Although the minimum correlation length or minimum correlation required for homologous regions has not been demonstrated, targeting efficiency for homologous recombination generally depends on the correlation length and correlation between the targeting vector and the PDE9 locus. Correspond. If a replacement vector is used and a portion of the endogenous PDE9 gene is deleted upon homologous recombination, a further problem is the size of the deleted portion of the endogenous PDE9 gene. If the length of this part of the endogenous PDE9 gene is greater than 1 kb, a targeting cassette having a homology region longer than 1 kb is desirable to enhance recombination efficiency. Based on this specification, further guidance on selecting and using sequences effective for homologous recombination can be found in the literature (eg, Deng and Capecchi, Mol. Cell. Biol. 12: 3365-3371, 1992; Bollag et al. al., Annu. Rev. Genet. 23: 199-225, 1989; and Waldman and Liskay, Mol. Cell. Biol. 8: 5350-5357, 1988).

  As will be appreciated by those skilled in the art based on the present invention, pBluescript-related plasmids (eg Bluescript KS + 11), pQE70, pQE60, pQE-9, as a vector backbone for the construction of the PDE9 gene targeting vector of the present invention. pBS, pD10, phage script, phiX174, pBK Phagemid, pNH8A, pNH16a, pNH18Z, pNH46A, ptrc99a, pKK223-3, pKK233-3, pDR540, and pRIT5 PWLNEO, pSV2CAT, pXT1, pSG (Stratagene), pSVK3, PBPV, PMSG And a wide variety of cloning, including vectors based on pSVL, pBR322 and pBR322, pMB9, pBR325, pKH47, pBR328, pHC79, phage Charon 28, pKB11, pKSV-10, pK19-related plasmids, pUC plasmids and pGEM series plasmids Vectors can be used. These vectors are available from a variety of commercial sources (eg, Boehringer Mannheim Biochemicals, Indianapolis, IN; Qiagen, Valencia, CA; Stratagene, La Jolla, CA; Promega, Madison, WI; and New England Biolabs, Beverly, MA). Is available from However, any other vector, such as a plasmid, virus or portion thereof, can be used as long as they are replicable and viable in the desired host. The vector may also contain sequences that allow its replication in the host whose genome is to be modified. The use of such vectors can extend the interaction period during which recombination can occur and can increase targeting efficiency (see Molecular Biology, ed. Ausubel et al, Unit 9.16, Fig. 9.16.1).

  The particular host used to construct the targeting vector of the present invention is not critical. Examples include E. coli K12 RR1 (Bolivar et al., Gene 2: 95, 1977), E. coli K12 HB101 (ATCC No. 33694), E. coli MM21 (ATCC No. 336780), E. coli DH1 (ATCC No. 33849), E. coli strain DH5α, and E. coli STBL2. Alternatively, hosts such as C. cerevisiae or B. subtilis can be used. Such hosts are commercially available (eg, Stratagene, La Jolla, CA; and Life Technologies, Rockville, MD).

  To create the targeting vector, the PDE9 gene targeting construct is added to the vector backbone. The PDE9 gene targeting construct of the present invention has at least one PDE9 gene homology region. In order to produce the PDE9 gene homology region, the PDE9 genome or cDNA sequence is used as the basis for PCR primer production. These primers are used to amplify the desired region of the PDE9 sequence by high fidelity PCR amplification (Mattila et al., Nucleic Acids Res. 19: 4967, 1991; Eckert and Kunkel 1: 17,1991; and US Patent No. .4,683,202). The genomic sequence is obtained from a genomic clone library or from the preparation of genomic cDNA (preferably from the animal species to be targeted for PDE9 gene disruption), and the PDE9 cDNA sequence is used to create a PDE9 targeting vector. (Eg, GenBank® NM008804 (murine) or GenBank® NM002606 (human)).

  Preferably, the targeting construct of the present invention further comprises an exogenous nucleotide sequence encoding a positive marker protein. Stable expression of the positive marker after vector integration provides the cell with identifiable characteristics, ideally without compromising cell viability. Therefore, in the case of a replacement vector, the marker gene is located between two flanking homology regions and after a double crossover event (in a format in which the marker gene is arranged to be expressed after integration). It is integrated into the PDE9 gene.

  The positive marker protein is a selectable protein; the stable expression of such a protein in the cell is a selectable phenotypic characteristic (ie the survival of the cell under lethal conditions without the characteristic) It is preferable to provide a characteristic that enhances Thus, by imposing selectable conditions, cells that stably express a positive selectable marker coding vector sequence can be isolated on the basis of viability from other cells that have not successfully incorporated the vector sequence. it can. Examples of positive selectable marker proteins (and their selective agents) are neo (G418 or canomycin), hyg (hygromycin), hisD (histidinol), gpt (xanthine), ble (bleomycin) and hprt (hypoxanthine). (See, eg, Capecchi and Thomas, US Pat. No. 5,464,764, and Capecchi, Science 244: 1288-92, 1989). Other positive markers that can also be used as an alternative to selectable markers include reporter proteins such as, for example, β-galactosidase, firefly luciferase, or green fluorescent protein (e.g., Current Protocols in Cytometry, Unit 9.5, And Current Protocols in Molecular Biology, Unit 9.6, John Wiley & Sons, New York, NY, 2000).

  The positive selection step does not distinguish cells that have integrated the vector by targeted homologous recombination at the PDE9 locus from cells that randomly and non-homologously integrate the vector sequence at any chromosomal location. Therefore, when producing a genetically modified non-human mammal and animal cell of the present invention using a replacement vector for homologous recombination, it is also preferable to include a nucleotide sequence encoding a negative selectable marker protein. . Negative selectable marker expression causes cells expressing the marker to lose viability when exposed to certain agents (ie, under certain selection conditions, the marker protein Become lethal to the cells). Examples of negative selectable markers (and their lethal agents) are herpes simplex virus thymidine kinase (ganciclovir or 1,2-deoxy-2-fluoro-α-d-arabinofurancil-5-iodouracil) , Hprt (6-thioguanine or 6-thioxanthine), and diphtheria toxin, ricin toxin, and cytosine deaminase (6-fluorocytosine).

  The nucleotide sequence encoding the negative selectable marker is placed outside the two homology regions of the replacement vector. Given this arrangement, if random non-homologous recombination results in integration; a sequence where homologous recombination between the PDE9 gene and two homology regions in the targeting construct encodes the negative selectable marker When excluding from integration, the cell incorporates a negative selectable marker and is stably expressed. Thus, by applying negative conditions, cells that have incorporated the targeting vector by random non-homologous recombination lose viability.

  The above combinations of positive and negative selectable markers are preferred in targeting constructs used for the production of genetically modified non-human mammals and animal cells of the present invention. Because a series of positive and negative selection steps are designed to more effectively select only those cells that have undergone vector integration by homologous recombination (hence, cells that probably have a disrupted PDE9 gene). Because it can. Further examples of positive-negative selection schemes, selectable markers and targeting constructs are described, for example, in US Pat. No. 5,464,764, WO 94/06908, US Pat. No. 5,859,312 and Valancius and Smithies, Mol. Cell. Biol. 11: 1402, 1991.

  In order for the marker protein to be stably expressed upon vector integration, the targeting vector can be designed such that the marker coding sequence is operably linked to the endogenous PDE9 gene promoter upon vector integration. The marker expression is then driven by a PDE9 gene promoter in the cell that normally expresses the PDE9 gene. Alternatively, each marker in the targeting construct of the vector contains its own promoter that drives expression independent of the PDE9 gene promoter. This latter scheme has the advantage of allowing expression of the marker in cells that typically do not express the PDE9 gene (Smith and Berg, Cold Spring Harbor Symp. Quant. Biol. 49: 171, 1984; Sedivy and Sharp, Proc. Natl. Acad. Sci. (USA) 86: 227, 1989; Thomas and Capecchi, Cell 51: 503, 1987).

  Exogenous promoters that can be used to drive marker gene expression include cell-specific or stage-specific promoters, constitutive promoters, and inducible or regulated promoters. Non-limiting examples of these promoters include herpes simplex thymidine kinase promoter, cytomegalovirus (CMV) promoter / enhancer, SV40 promoter, PGK promoter, PMC1-neo, metallothionein promoter, adenovirus rate promoter, vaccine virus 7.5K promoter , Avian β globin promoter, histone promoter (eg, mouse histone H3-614), β actin promoter, neuron specific enolase, muscle actin promoter and cauliflower mosaic virus 35S promoter (generally Sambrook et al., Molecular Cloning, Vols. I-III, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989 and Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, 2000; St ratagene, La Jolla, CA).

  During production of the genetically modified non-human mammals and animal cells of the invention, primers that are specific for the desired vector integration event in order to ascertain whether the cells have integrated the vector sequence into the target PDE9 locus or Genomic probes can be used in combination with polymerase chain reaction (PCR) or Southern blot analysis to identify the presence of desired vector integration into the PDE9 locus (Erlich et al., Science 252: 1643-51, 1991; Zimmer and Gruss, Nature 338: 150, 1989; Mouellic et al., Proc. Natl. Acad. Sci. (USA) 87: 4712, 1990; and Shesely et al., Proc. Natl. Acad. Sci. USA) 88: 4294, 1991).

Gene Trapping Based on the present specification, another available method that is effective for inserting heterologous nucleic acid sequences into the PDE9 locus to disrupt the PDE9 gene is gene trapping. This method
In order to randomly insert a gene trap vector coding sequence into a gene, a splice is used for exon mRNA, which is a cellular machinery present in all mammalian cells. Once inserted, the gene trap vector produces a mutation that can disrupt the trapped PDE9 gene. In contrast to homologous recombination, this mutagenesis system produces large-scale random mutations. Therefore, to obtain genetically modified cells containing the disrupted PDE9 gene, cells containing this particular mutation must be identified and selected from a pool of cells containing random mutations in the diverse genes. I must.

  Gene trapping systems and vectors have been published for use in genetic modification of murine cells and other cell types (eg, Allen et al., Nature 333: 852-55, 1988; Bellen et al., Genes Dev. 3 : 1288-1300, 1989; Bier et al., Genes Dev. 3: 1273-1287, 1989; Bonnerot et al., J. Virol. 66: 4982-91, 1992; Brenner et al., Proc. Nat. Acad Sci. USA 86: 5517-21, 1989; Chang et al., Virology 193: 737-47, 1993; Friedrich and Soriano, Methods Enzymol. 225: 681-701, 1993; Friedrich and Soriano, Genes Dev. 5: 1513-23, 1991; Goff, Methods Enzymol. 152: 469-81, 1987; Gossler et al., Science 244: 463-65, 1989; Hope, Develop. 113: 399-408, 1991; Kerr et al., Cold Spring Harb. Symp. Quant. Biol. 2: 767-776, 1989; Reddy et al., J. Virol. 65: 1507-1515, 1991; Reddy et al., Proc. Natl. Acad. Sci. USA 89 : 6721-25, 1992; Skarnes et al., Genes Dev. 6: 903-918, 1992; von Melchner and Ruley, J. Virol. 63: 3227-3233, 1989; and Yoshida et al., Transgen Res. 4: 277-87, 1995).

  Individual mutant cell lines containing the disrupted PDE9 gene are identified in the mutant cell population using, for example, reverse transcription (RT) and PCR to identify mutations in the PDE9 gene sequence. The This method can be simplified by pooling clones. For example, to find individual clones containing a disrupted PDE9 gene, RT-PCR is performed using one primer fixed in the gene trap vector and the other primer in the PDE9 gene sequence. . Positive RT-PCR results demonstrate that the vector sequence is encoded in the PDE9 gene transcript and that the PDE9 gene has already been disrupted by a gene trap integration event (eg, Sands et al ., WO 98/14614, U.S. Pat. No. 6,080,576).

Temporal, Spatial and Induced PDE9 Gene Disruption In certain embodiments of the present invention, functional disruption of the endogenous PDE9 gene is at a specific developmental or cell cycle stage (temporal disruption) or in a specific cell type. (Spatial destruction) occurs. In other embodiments, PDE9 gene disruption is induced when certain conditions are present. Recombinant excision systems such as the Cre-Lox system can be used to activate or inactivate the PDE9 gene at specific developmental stages, in specific tissues or cell types, or under specific environmental conditions . In general, methods using Cre-Lox technology are performed as described by Torres and Kuhn, Laboratory Protocols for Conditional Gene Targeting, Oxford University Press, 1997. The FLP-FRT system can also be used using a methodology similar to that described for the Cre-Lox system. Further guidance regarding the use of recombinase excision systems to conditionally disrupt genes by homologous recombination or viral insertion can be found, for example, in US Patent No. 5,626,159; US Patent No. 5,527,695; US Patent No. 5,434,066; WO 98/29533. U.S. Patent No. 6,228,639; Orban et al., Proc. Nat. Acad. Sci. USA 89: 6861-65, 1992; O'Gorman et al., Science 251: 1351-55, 1991; Sauer et al., Nucleic Acids Research 17: 147-61, 1989; Barinaga, Science 265: 26-28, 1994; and Akagi et al., Nucleic Acids Res. 25: 1766-73, 1997. Two or more recombinase systems can be used to genetically modify the non-human mammal or animal cell of the present invention.

  When using homologous recombination to disrupt the PDE9 gene in a temporal, spatial and inducible manner using a recombinase system such as the Cre-Lox system, a portion of the PDE9 gene coding region is loxP The site is replaced by a targeting construct containing the flanking PDE9 gene coding region. Non-human mammals and animal cells with this genetic modification contain a functional loxP-flanked PDE9 gene. The temporal, spatial or inducible aspects of PDE9 gene modification are additional transgenes expressed in non-human mammals or animal cells under the desired spatial, temporal or inducible promoter control, respectively. Induced by the expression pattern of the Cre recombinase transgene. Cre recombinase targets the loxP site for recombination. Therefore, when Cre expression is activated, the loxP undergoes recombination and excises the flanked PDE9 gene coding sequence, resulting in functional disruption of the PDE9 gene ((Rajewski et al. , J. Clin. Invest. 98: 600-03, 1996; St.-Onge et al., Nucleic Acids Res. 24: 3875-77, 1996; Agah et al., J. Clin. Invest. 100: 169- 79, 1997; Brocard et al., Proc. Natl. Acad. Sci. USA 94: 14559-63, 1997; Feil et al., Proc. Natl. Acad. Sci. USA 93: 10887-90, 1996; and Kuhn et al., Science 269: 1427-29, 1995).

  Cells containing both the Cre recombinase transgene and the loxP flanking PDE9 gene can be obtained by standard transgenic techniques, or, in the case of genetically modified non-human mammals, one parental loxP gene under the control of the desired promoter. It can be made by cross-breeding of a genetically modified non-human mammal containing the ranking PDE9 gene and the other parent containing the Cre recombinase transgene. Further guidance on the use of the recombinase system and specific promoters to disrupt the PDE9 gene temporally, spatially or conditionally can be found in, for example, Sauer, Meth. Enz. 225: 890-900, 1993; Gu et al ., Science 265: 103-06, 1994; Araki et al., J. Biochem. 122: 977-82, 1997; Dymecki, Proc. Natl. Acad. Sci. 93: 6191-96, 1996; and Meyers et al. ., Nature Genetics 18: 136-41, 1998.

  Inducible disruption of the PDE9 gene can also be achieved using a tetracycline reaction binary system (Gossen and Bujard, Proc. Natl. Acad. Sci. USA 89: 5547-51, 1992). This system involves genetic modification of cells by introducing a Tet promoter into an endogenous PDE9 gene regulatory element and a tetracycline-regulated repressor (TetR) expression transgene. In such cells, administration of tetracycline activates TetR, which in turn inhibits PDE9 gene expression, thus destroying the PDE9 gene (St.-Onge et al., Nucleic Acids Res. 24: 3875 -77, 1996; US Patent No. 5,922,927).

  The above systems for temporal, spatial and inducible disruption of the PDE9 gene are described, for example, in WO98 / 29533 and US Pat. No. 6,288,639, for producing genetically modified non-human mammals and animal cells of the present invention. As described above, when gene trapping is used as a gene modification method, it can also be employed.

Production of genetically modified non-human mammals and animal cells In order to produce the genetically modified animal cells of the present invention, the above gene modification method is used for PDE9 gene disruption in any kind of somatic cells or stem cells derived from animals. be able to. Genetically modified animal cells of the present invention include, but are not limited to, mammalian cells including human cells and avian cells. These cells can be derived from genetic manipulation of any animal cell line, such as, for example, culture adaptive cell lines, tumorigenic or transformed cell lines, differentiated genetically engineered ES cells, or these These cells can be isolated from a genetically modified non-human mammal having the desired PDE9 genetic modification.

  These cells can be heterozygous or homozygous for the disrupted PDE9 gene. To obtain cells that are homozygous for the PDE9 gene disruption (-/-), direct sequential targeting of both alleles can be performed. These methods can be facilitated by recycling positive selectable markers. According to this scheme, the nucleotide sequence encoding a positive selectable marker is removed after disruption of one allele using the Cre-LoxP system. Thus, the same vector can be used in subsequent targeting rounds to disrupt the allele of the second PDE9 gene (Abuin and Bradley, Mol. Cell. Biol. 16: 1851-56, 1996; Sedivy et al., TIG 15: 88-90, 1999; Cruz et al., Proc. Natl. Acad. Sci. (USA) 88: 7170-74, 1991; Mortensen et al., Proc. Natl. Acad. Sci. (USA) 88 : 7036-40, 1991; te Riele et al., Nature (London) 348: 649-651, 1990).

  An alternative scheme to obtain ES cells that are PDE9 − / − is homogenotization of cells from a population that is heterozygous for PDE9 gene disruption (PDE9 +/−). This method uses a scheme that selects PDE9 +/− target clones expressing selectable drug resistance markers against very high drug concentrations; this selection expresses two copies of the sequence encoding the drug resistance marker. Therefore, preference is given to cells that are homozygous for the PDE9 gene disruption (Mortensen et al., Mol. Cell. Biol. 12: 2391-95, 1992). In addition, as described in detail below, genetically modified animal cells can be obtained from genetically modified PDE9 − / − non-human mammals produced by mating non-human mammals that are PDE9 +/− in germ cells. .

  After genetic modification of the desired cell or cell line, the PDE9 locus can be identified as a modified site by PCR analysis by standard PCR or Southern blotting known to those skilled in the art (eg, US Pat. No. 4,683,202; And Erlich et al., Science 252: 1643, 1991). If PDE9 gene messenger RNA (mRNA) levels and / or PDE9 polypeptide levels are reduced in cells that normally express the PDE9 gene, functional disruption of the PDE9 gene will be further demonstrated. RT-PCR, Northern blot analysis or in situ hybridization can be used to measure PDE9 gene mRNA levels. Quantification of PDE9 polypeptide levels produced by the cells can be performed, for example, by standard immunoassay methods known in the art. Such immunoassays include, but are not limited to, for example, RIA (radioimmunoassay), ELISA (enzyme-linked immunosorbent assay), “sandwich” immunoassay, immunoradiometric assay, gel diffusion precipitation reaction, immunodiffusion assay, in situ immunoassay ( Competitive and non-using techniques such as Western blot (using colloidal gold, enzyme or radioisotope labels), two-dimensional gel analysis, precipitation reactions, immunofluorescence assays, protein A assays and immunoelectrophoresis assays Competitive assay systems are included.

  Preferred genetically modified animal cells of the present invention are embryonic stem (ES) cells and ES-like cells. These cells are described, for example, in mice (Evans et al., Nature 129: 154-156, 1981; Martin, Proc. Natl. Acad. Sci., USA, 78: 7634-7638, 1981), pigs and sheep (Notanianni). et al., J. Reprod. Fert. Suppl., 43: 255-260, 1991; Campbell et al., Nature 380: 64-68, 1996) and primates including humans (Thomson et al., US patent) No. 5,843,780, Thomson et al., Science 282: 1145-1147, 1995; and Thomson et al., Proc. Natl. Acad. Sci. USA 92: 7844-7848, 1995). Derived from pre-floor embryos and undifferentiated germ cells. Successful homologous recombination-mediated gene disruption in human ES cells has been reported (Zwaka and Thomson, Nature Biotech. 21: 319-21, 2003).

  These types of cells are pluripotent, that is, under appropriate conditions, differentiate into a very diverse cell type derived from all three germ layers: endoderm, mesoderm and ectoderm. Depending on the culture conditions, ES cell samples can be differentiated indefinitely as stem cells and can be allowed to differentiate into a very diverse variety of different cell types in a single sample. Or, for example, macrophage-like cells, neuronal cells, cardiomyocytes, chondrocytes, adipocytes, smooth muscle cells, endothelial cells, skeletal muscle cells, keratinocytes, and hematopoietic cells, such as eosinophils, mast cells, erythroid progenitor cells, or megakaryocytes It can be guided to differentiate into specific cell types such as spheres. For example, Keller et al., Curr. Opin. Cell Biol. 7: 862-69, 1995; Li et al., Curr. Biol. 8: 971, 1998; Klug et al., J. Clin. Invest. 98: 216-24, 1996; Lieschke et al., Exp. Hematol. 23: 328-34, 1995; Yamane et al., Blood 90: 3516-23, 1997; and Hirashima et al., Blood 93: 1253-63, As detailed in 1999, directed differentiation is achieved by including specific growth factors or matrix components in the culture conditions.

  The particular embryonic stem cell line used for genetic modification is not critical; exemplary murine ES cell lines are AB-1 (McMahon and Bradley, Cell 62: 1073-85, 1990), E14 (Hooper et al., Nature 326: 292-95, 1987), D3 (Doetschman et al., J. Embryol. Exp. Morph. 87: 27-45, 1985), CCE (Robertson et al, Nature 323: 445-48, 1986), RW4 (Genome Systems, St. Louis, MO), and DBA / 1lacJ (Roach et al., Exp. Cell Res. 221: 520-25, 1995); an exemplary human ES cell line is H1. 1 cell (Zwaka and Thomson, Nature Biotech. 21: 319-321, 2003). Using genetically modified murine ES cells, genetically modified mice can be produced by published methods (Robertson, 1987, Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, Ed. EJ Robertson, Oxford: IRL Press, pp. 71-112, 1987; Zjilstra et al., Nature 342: 435-438, 1989; and Schwartzberg et al., Science 246: 799-803, 1989).

  After confirming that the ES cells contain the desired functional disruption of the PDE9 gene, these ES cells are transformed into a suitable undifferentiated germ cell host for production of chimeric mice according to methods known in the art. (Capecchi, Trends Genet. 5: 70, 1989). The particular mouse undifferentiated germ cells used in the present invention are not critical. Examples of such undifferentiated germ cells include undifferentiated germ cells derived from C57BL6 mice, C57BL6 albino mice, Swiss outbred mice, CFLP mice, and MFI mice. Alternatively, ES cells can be sandwiched between tetraploid embryos in an aggregation well (Nagy et al., Proc. Natl. Acad. Sci. USA 90: 8424-8428, 1993).

  Next, embryonic undifferentiated germ cells containing genetically modified ES cells are transplanted into pseudopregnant female mice and developed in utero (Hogan et al., Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory press, Cold Spring Harbor, NY 1988; and Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, EJ Robertson, ed., IRL Press, Washington, DC, 1987). Children born to foster mothers can be screened to identify children that are chimeric with respect to PDE9 gene disruption. In general, such children contain some cells derived from genetically modified donor ES cells as well as other cells derived from the original undifferentiated germ cells. In this situation, if using a coat color scheme, the child should first be screened for mosaic coat color to distinguish cells derived from donor ES cells from other cells of undifferentiated germ cells. Can do. Alternatively, DNA from genetically modified cells can be identified using DNA from a child's tail tissue.

  Crossbreeding of chimeric mice containing the PDE9 gene disruption in germline cells gives rise to progeny that have the PDE9 gene disruption in all germline and somatic cells. Next, mice that are heterozygous for the PDE9 gene disruption can be bred to produce homozygotes (see, eg, US Patent No. 5,557,032; and US Patent No. 5,532,158).

  An alternative to the above ES cell technology for transferring genetic modifications from cells to whole animals is to use nuclear transfer. This method involves other genetically modified non-human mammals other than mice, such as sheep (McCreath et al., Nature 29: 1066-69, 2000; Campbell et al., Nature 389: 64-66, 1996; and Schnieke et al., Science 278: 2130-33, 1997) and cattle (Cibelli et al., Science 280: 1256-58, 1998). Briefly, somatic cells (eg, fibroblasts) or pluripotent stem cells (eg, ES-like cells) are selected as nuclear donors and genetically modified to contain a functional disruption of the PDE9 gene. When a DNA vector is inserted into a somatic cell to mutate the PDE9 gene, a promoter-less marker is added to the vector so that incorporation of the vector into the PDE9 gene results in expression of the marker under the control of the PDE9 gene promoter. Preferably used (Sedivy and Dutriaux, TIG 15: 88-90, 1999; McCreath et al., Nature 29: 1066-69, 2000). Next, nuclei from donor cells with the appropriate PDE9 gene disruption are transferred to enucleated fertilized oocytes or parthenogenetic oocytes (Campbell et al., Nature 380: 64, 1996; Wilmut et al. al., Nature 385: 810, 1997). Embryos are reconstructed, cultured, developed to the morula / undifferentiated germ cell stage, and transferred to foster mothers for whole-intrauterine development.

  The invention also encompasses genetically modified non-human mammals and progeny of genetically modified animal cells. Even if the offspring is heterozygous or homozygous for a genetic modification that disrupts the PDE9 gene, the progeny can also be a genetic modification of the original gene disruption of the PDE9 gene that can occur in subsequent generations, Due to mutations or environmental effects, it may not be genetically the same as the parent non-human mammal and animal cell.

  Cells from the genetically modified non-human animal can be isolated from the tissue or organ using techniques known to those skilled in the art. In one embodiment, the genetically modified cells of the present invention are immortalized. According to this embodiment, the cell can be immortalized by genetically engineering into the cell a telomerase gene, an oncogene, such as mos or v-src, or an apoptosis inhibitor gene, such as bcl-2. Alternatively, the cells can be immortalized by fusion with a hybridization partner using techniques known to those skilled in the art.

A genetically modified non-human mammal and animal cell (non-human) of the present invention containing a “humanized” non-human mammal and an endogenous PDE9 gene that has been disrupted by animal cells, so as to express a human PDE9 sequence. Can be modified (referred to herein as “humanization”). A preferred method for humanization of cells involves replacing endogenous PDE9 sequences with nucleic acid sequences encoding human PDE9 sequences by homologous recombination (Jakobsson et al., Proc. Natl. Acad. Sci. USA 96 : 7220-25, 1999). The vector is similar to the vectors traditionally used as targeting vectors with respect to the 5 ′ and 3 ′ homology arms and positive / negative selection schemes. However, the vector further has, after recombination, a human PDE9 coding sequence in place of the endogenous sequence or a base pair change, exon substitution or codon substitution that modifies the endogenous sequence to encode human PDE9. Also includes sequences to be performed. Once homologous recombination has been identified, sequences based on any selection (eg, neo) can be deleted using Cre or Flp-mediated site-directed recombination (Dymecki, Proc. Natl. Acad Sci. 93: 6191-96, 1996).

        When human PDE9 sequences are used instead of the endogenous sequences, it is preferable to introduce these changes directly downstream of the endogenous translation start site. This positioning protects the endogenous temporal and spatial expression pattern of the PDE9 gene. The human sequence can be a full-length human cDNA sequence or a whole genome sequence with a polyA tail attached to the 3 ′ end for proper processing (Shiao et al., Transgenic Res. 8: 295-302, 1999). ). Further guidance on these methods of genetically modifying cells and non-human mammals to replace the expression of an endogenous gene with its human counterpart can be found, for example, in Sullivan et al., J. Biol. Chem. 272: 17972-17980, 1997, Reaume et al., J. Biol. Chem. 271: 23380-23388, 1996 and Scott et al., US Pat. No. 5,777,194.

  Other methods for creating such “humanized” organisms include disruption of endogenous genes and subsequent introduction of transgenes encoding human sequences by pronuclear microinjection into knockout embryos. It is a two-step method.

Use of genetically modified non-human mammals and animal cells PDE9 function and therapeutic relevance, for example, as described in the examples below, PDE9-/-phenotypes of non-human mammals and animal cells of the present invention Further studies can further elucidate. For example, using genetically modified PDE9 − / − non-human mammals and animal cells, certain disease models such as obesity, eating disorders, cardiovascular disorders, insulin resistance syndrome, hypertension, and / or type 2 It can be determined whether PDE9 plays a role in the development or prevention of symptoms or phenotypes in diabetes. If the symptom or phenotype is different in a PDE9 − / − non-human mammal or animal cell compared to a wild type (PDE9 + / +) or PDE9 +/− non-human mammal or animal cell, The peptide will play a role in the regulatory function associated with the symptom or phenotype. Examples of tests that can be used to assess PDE9 function include body weight, body fat, blood pressure, glucose / insulin metabolism (eg glucose uptake in isolated tissues, glycogen metabolizing enzymes) in PDE9 − / − mice and wild type mice. Changes in activity, changes in glycogen levels in the liver or muscle, and / or changes in body composition), and comparisons regarding changes in activity or phosphorylation status of components of the insulin signaling pathway.

  In addition, the agent has been identified as a PDE9 agonist or antagonist (eg, when the agent is administered to a PDE9 + / + or PDE9 +/− non-human mammal or animal cell, the agent has a PDE9 polypeptide activity). In situations where one or more of these are significantly modified), the genetically modified PDE9 − / − non-human mammals and animal cells of the present invention have effects other than those known to occur as a result of antagonism (agonism) of PDE9. Are useful for characterizing any other effect elicited by the agent (ie, the non-human mammals and animal cells can be used as negative controls). For example, if administration of the agent elicits an effect in a PDE9 + / + non-human mammal or animal cell that is not known to be associated with PDE9 polypeptide activity, the agent alone exhibits this effect. Or the modulation of PDE9 by administration of the agent to the corresponding PDE9 − / − non-human mammal or animal cell. If this effect is not present in PDE9 − / − non-human mammals or animal cells or is significantly less, then at least part of this effect will be mediated by PDE9. However, if the PDE9 − / − non-human mammal or animal cell shows an effect comparable to the PDE9 + / + or PDE9 +/− non-human mammal or animal cell, the effect is Mediated by a route that does not involve transmission.

  Furthermore, PDE9 − / − non-human mammals or animal cells are useful as a negative control to test this hypothesis if the agent is suspected to have an effect primarily through the PDE9 pathway. If the agent actually acts through PDE9, the PDE9 − / − non-human mammal or animal cell is found in the PDE9 + / + non-human mammal or animal cell upon administration of the agent. Does not show the same effect.

  Using the genetically modified non-human mammals or animal cells of the present invention, the expression of PDE9 + / + or PDE9 +/- non-human mammals or animal cells is discriminatory compared to their respective wild type controls. It is also possible to identify genes that are regulated. Such genes can be identified based on this specification using techniques known to those skilled in the art. For example, DNA arrays can be used to identify genes whose expression is differentially regulated in PDE9 expression in PDE9 +/− or PDE9 − / − mice. DNA arrays are known to those skilled in the art (e.g., Aigner et al., Arthritis and Rheumatism 44: 2777-89, 2001; U.S. Patent No. 5,965,352; Schena et al., Science 270: 467-470, 1995; Schena et al., Proc. Natl. Acad. Sci. USA 93: 10614-10619, 1996; DeRisi et al., Nature Genetics 14: 457-460, 1996; Shalon et al., Genome Res. 6: 639-645, 1996; and Schena et al., Proc. Natl. Acad. Sci. (USA) 93: 10539-11286, 1995; U.S. Patent No. 5,474,796; US Pat.No. 5,605,662; WO 95/25116; WO 95/35505; Heller et al., Proc. Natl. Acad. Sci. 94: 2150-2155, 1997).

  Array elements can be synthesized on the substrate surface using chemical coupling methods and inkjet devices. An array similar to a dot or slot blot can be used to place and connect the elements to the surface of the substrate using thermal, UV, chemical or mechanical coupling methods. A typical array can be made by hand or using available methods and equipment and can contain any suitable number of elements. After hybridization, the non-hybridized probe is removed and the level and pattern of fluorescence are measured using a scanner. The degree of complementarity and relative abundance of each probe that hybridizes to elements on the microarray can be assessed by analysis of the scan image.

  Full-length cDNAs, expressed sequence tags (ESTs) or fragments thereof can include microarray elements. Fragments suitable for hybridization can be selected using software well known in the art, such as LASERGENE software (DNASTAR). A full-length cDNA, EST or fragment thereof corresponding to one of the nucleotide sequences of the present invention or randomly selected from a cDNA library related to the present invention is placed on a suitable substrate (eg glass slide) Deploy. The cDNA is fixed to the slide using, for example, UV crosslinking followed by thermal and chemical treatment followed by drying. A fluorescent probe is prepared and used for hybridization to elements on the substrate. The substrate is analyzed by methods well known in the art, for example, by scanning and analyzing a microarray image.

  Furthermore, using the genetically modified non-human mammals and animal cells of the present invention, expression profiles or post-translational modifications in PDE9 +/− or PDE9 − / − non-human mammals are compared to their respective wild type controls. Any protein that has changed can be identified. Based on this specification, techniques known to those skilled in the art can be used to identify such proteins. For example, proteomic assays can be used to identify proteins whose expression profile or post-translational modification in PDE9 +/− or PDE9 − / − mice has been altered to compensate for deficiencies in PDE9 expression. Proteome assays are known to those skilled in the art (eg, Conrads et al., Biochem. Biophys. Res. Commun. 290: 896-890, 2002; Dongre et al., Biopolymers 60: 206-211, 2001; Van Eyk , Curr Opin Mol Ther 3: 546-553, 2001; Cole et al., Electrophoresis 21: 1772-1781, 2000; Araki et al., Electrophoresis 21: 180-1889, 2000).

1. Preparation of PDE9 targeting vector A targeting vector construct was designed according to the scheme shown in FIG. This construct contained two arms homologous to the murine PDE9 genomic sequence: a 0.9 kb 5 ′ homology arm and a 4.3 kb 3 ′ homology arm. These arms sandwiched the LacZ-Neo construct. DNA containing the targeting construct was inserted into ES R1 cells by electroporation (Deng et al., Dev. Biol. 185: 42-54, 1997; Udy et al., Exp. Cell Res. 231: 296 -301, 1997). Upon homologous recombination, base pairs 142-175 of the PDE9 cDNA coding sequence shown in FIG. 2 (base pairs 142-175 are underlined) were deleted from the endogenous gene and replaced by the LacZ-Neo cassette. . ES cells that are neomycin resistant were analyzed by Southern blot to confirm the disruption of the PDE9 gene. These target ES cells were then used to produce chimeric mice by injecting these target ES cells into undifferentiated germ cells and transplanting the undifferentiated embryo cells into pseudopregnant female mice (Capecchi et al. al., Trends Genet. 5: 70, 1989, Hogan et al., Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; and Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, EJ Robertson, ed., IRL Press, Washington, DC, 1987). Next, the chimeric mouse was bred with a C57BL / 6 (Jackson Laboratories, Bar Harbor, ME) mouse to produce an F1 PDE9 +/− heterozygote, and the zygote was mated to produce an F2 PDE9. -/-Homozygous mice were produced (Charles River Laboratories, Wilmington, MA). Functional disruption of the PDE9 gene in the heterozygote and homozygote was confirmed by PCR and Southern blot analysis.

2. Method of effect of PDE9 gene disruption on body weight, body composition and metabolites Male and female PDE9 KO (− / −) mice and wild type (+ / +) littermate controls, as previously described, were at least prior to the start of the study. Acclimatized to the environment for one week and free access to water and D11 mouse chow (Purina, Brentwood, MO).

  Male and female mice (17-19 weeks old) were divided into 4 experimental groups with 2-5 mice per cage. One group of control mice for each gender remains a D11 mouse chow and the remaining group for each gender is a diet composed of 58 kcal% fat (D12331 Rodent Diet, Research Diets, Inc., New Brunswick, NJ). The test period was changed over a week. Body weight was measured on day 0 and monitored weekly. The mass of fat accumulation was analyzed on day 0 and at the end of the study as described further below.

  On day 1, plasma glucose was measured by a retroorbital blood sample. 25 μl of blood was added to 100 μl of 0.025% heparinized saline in a microtube (Denville Scientific, Inc., Metuchen, NJ). These tubes were spun for 2 minutes at the best setting on a Beckman Microfuge 12. Plasma was collected for plasma glucose and triglyceride measurements as described further below. During the course of this study, body weight and food intake were assessed and, as described further below, for plasma glucose and triglyceride measurements, approximately 8a. m. A blood sample was taken.

  On the morning of the last day of the study, a blood sample was taken through the retroorbital sinus for plasma glucose and triglyceride measurements. The mice were then killed and approximately 1 ml of blood was collected in a Microtainer® plasma separator tube (Becton-Dickinson, Inc., Franklin Lakes, NJ) containing lithium heparin. These tubes were spun for 5 minutes at the highest setting on a Beckman Microfuge 12. Plasma was collected in 1.5 ml Eppendorf tubes, snap frozen in liquid nitrogen and stored at −80 ° C. until analyzed for insulin, fructosamine or cGMP levels.

  Plasma glucose, triglycerides and fructosamine were measured using a Roche / Hitachi 912 Clinical Chemistry Analyzer (Roche Diagnostics Corp., Indianapolis, IN). Plasma cGMP was measured using a BioTrak ™ enzyme-immunoassay system (Amersham, Piscataway, NJ). Plasma insulin was evaluated by a similar method using the Mercodia ELISA Insulin kit supplied by ALPCO (Uppsala, Sweden). All assays were performed according to each manufacturer's instructions.

  Fat accumulation mass was quantified 5 days before the end of the test. To assess fat accumulation mass, a commercially available micro-computed tomography (CT) system (MicroCAT®, ImTek Inc., Oak Ridge, TN) was used with a high resolution CCD / phosphorus screen detector. A 360 ° radioscopic image was obtained. The scanner consisted of a 36 mm / 36 mm cylinder diameter / long field view with a spatial resolution of less than 50 μM. The X-ray source was biased to 40 KeV with an anode current set at 0.4 mA. Anesthetized mice were placed on a radiolucent mouse bed in an anatomically correct supine position with the tail end in close contact with the micro CT and the tip of the mouth in contact with the anesthesia delivery tube in place. Initial radiographic images were acquired at 90 ° to the surface of the mouse bed so that the scan plane was centered at the level of the iliac crest of each mouse to allow accurate positioning of the mouse. Each mouse was scanned when accurate aiming was ensured. Each scan consisted of 196 individual projections with an exposure time of 250 μs / projection; the total image capture time was about 12 minutes at 145 μM resolution.

  Manipulating the 196 projection obtained with a mouse micro CT scan to produce a two-dimensional cross-sectional image of the mouse, image reconstruction was performed using MicroCAT® Reconstruction, Visualization and Analysis Software (ImTek Inc., Oak Ridge, TN). (Paulus et al., Neoplasia 2: 62-70, 2000). Two sets of reconstructed images were created per scan for each mouse to know the individual lipid accumulation mass. The first set of 6 reconstructed images formed a montage for analysis of inguinal and accessory testicular adipose tissue accumulation mass. The second reconstruction set consists of nine slices, determined by both intervertebral and midvertebral landmarks, and this set consists of retroperitoneal and mesenteric fat accumulation tissue accumulation Used to determine mass.

  The reconstructed bitmap image was converted to a TIFF image for image analysis. Subsequently, the TIFF images were analyzed and the lipid accumulation mass was determined using Scion Image for Windows (registered trademark) (Scion Corporation, Frederick MD). A paint brush tool (pixel size # 3) was used to draw boundaries separating individual lipid accumulations and the total pixel count of each fat region was determined by Scion Image software. In this test, with the upper and lower pixel intensity thresholds selected, a lookup table (LUT) of 115-187 was found to be optimal for measuring fat accumulation.

The average number of pixels between each slice was calculated ((slice _n + slice _{n + 1} ) / 2). The total number of pixels representing individual lipid accumulation was calculated by multiplying the average number of pixels between each slice by the average number of pixels in each slice. Finally, the pixel count was converted to the accumulated tissue mass according to the following formula: accumulated mass (mg) = 0.000915 g / ul × 0.000757 μl / voxel × 1000 mg / g × voxel count. The first factor corrects for the specific gravity of glyceryl trioleate, which is representative of the density of the major storage form (ie, triglycerides) of lipids in adipose tissue. The second factor is the amount per pixel, and the third factor converts the resulting mass to mg.

Results PDE9 disruption resulted in decreased weight gain and weight loss when both male and female KO mice were fed a high fat diet compared to their wild type counterparts (FIG. 3). Female mice also demonstrated a 6% reduction in height. Male and female KO mice also demonstrated a decrease in lipid content during various fat accumulations along with weight loss (FIG. 4). In male KO mice there was a significant decrease in post-peritoneal and mesenteric fat accumulation; in female KO mice there was a significant decrease in inguinal, gonad and post-peritoneal accumulation. In comparison, no difference in body weight was observed between KO mice and wild-type mice in females fed a standard chow diet (Fig. 5), and the fat lipid content decreased only in gonad fat accumulation. (FIG. 6).

  Regarding plasma metabolites after high fat diet, female KO mice demonstrated elevated cGMP, decreased glucose and decreased insulin (Table 1); male KO mice demonstrated increased cGMP and decreased glucose (Table 1). .

3. How to influence pharmacological PDE9 inhibition on body weight, body composition and food intake in ob / ob mice
Female ob / ob mice 6-10 weeks old obtained from Jackson Laboratories (Bar Harbor, ME) were used. Mice were housed at 5 per cage and had free access to water and initially D11 mouse chow. After an acclimatization period of 1 week, switch to a powdered diet (Mouse Breeder / Auto-JL K20 mouse chow, PMI Feeds, Inc., St. Louis, MO) for 3 days, and to the diet before the start of the PDE9 inhibitor administration period Adapted.

  The PDE9 inhibitor compound (Compound A) was administered as a compound / chow mixture in a custom ground powdered mouse chow (Research Diets, Inc., New Brunswick, NJ); Mixed with the feed, the designated dose in the range of 1-200 mg / kg / day was ingested. In addition to the control group without compound, a group taking darglitazone (1 mg / kg / day) was also included as a positive control.

  Mice were randomly assigned to 10 groups of 5 per cage. Body weight was measured on day 0 and weekly thereafter. On day 1, retroorbital blood samples were taken and plasma glucose was measured as previously described. On the last day of the test, blood samples were taken for glucose, triglyceride, insulin and cGMP measurements as previously described.

Results The following results represent results from several separate tests using the same protocol as above. FIG. 7 shows the decrease in weight gain in ob / ob mice given 100 mg / kg / day of PDE9 inhibitor Compound A compared to mice given either no compound control diet or darglitazone treated diet. Compound A induced a dose-dependent effect on both a decrease in normal body weight gain (FIG. 8A) and a further decrease in food intake (FIG. 8B) after 2 and 4 days of treatment. In the late stage of the study (Figure 9), the PDE9 effect on food intake may be transient if there is no effect on food intake at an intermediate dose of 100 mg / kg / day sell.

  An intervening dose of 100 mg / kg / day of Compound A also resulted in a decrease in glucose, triglycerides and fructosamine. Typical results are shown in FIGS. 10, 11 and 12, respectively.

  In both examples, reducing PDE9 activity is an effective way to reduce body weight and / or body fat, for example treating overweight, obese or suffering from eating disorders Demonstrates that it can be used in animal food stock species to produce lean meat.

FIG. 1 is a schematic diagram of a targeting construct used to disrupt the PDE9 gene. The 5 'and 3'-homology arms complementary to the PDE9 genomic sequence, 0.9 kb and 4.3 kb, respectively, sandwich the LacZ-Neo cassette. A portion of the genomic sequence of each homology arm is shown as SEQ ID NO: 1 and SEQ ID NO: 2. FIG. 2 shows the cDNA sequence of murine PDE9 (SEQ ID NO: 3). Upon homologous recombination with the targeting construct, the underlined sequence, base pairs 142-175, is deleted and replaced with LacZ-Neo. FIG. 3 is a line graph detailing changes in body weight in wild-type (WT) and genetically modified mice homozygous for PDE9 gene disruption (PDE9 knockout (KO) mice) during a 6-week high-fat diet course. It is. 4A (male) and FIG. 4B (female) are bar graphs showing the mass of some fat accumulation in WT and PDE9 KO mice after a 6-week high-fat diet. SC—subcutaneous; TBW—total body weight. FIG. 5 is a bar graph comparing the body weights of female WT mice and PDE9 KO mice after a 6-week control chow diet. FIG. 6A (baseline) and FIG. 6B (after 6 weeks chow diet) are bar graphs showing the mass of fat accumulation in female WT and PDE9 KO mice (Ing-groin subcutaneous; Gon-gonad; RP-post-peritoneal) ; Mes-mesentery). FIG. 7 is a line graph detailing the time course of weight gain in female ob / ob mice in the control group, the compound A treated (100 mg / kg / day) group and the darglitazone treated group. FIG. 8A is a bar graph showing Compound A dose effect on body weight on days 2 and 4 in female ob / ob mice. FIG. 8B is a bar graph showing the dose effect of Compound A on food consumption on days 2 and 4 in female ob / ob mice. FIG. 9 is a bar graph comparing the time course of food consumption in control, Compound A treated (100 mg / kg / day) and darglitazone treated female ob / ob mice. FIG. 10 is a line graph comparing plasma glucose in control, Compound A treated (100 mg / kg / day) and darglitazone treated female ob / ob mice. FIG. 11 is a line graph showing plasma triglycerides on days 1, 2 and 4 in control and Compound A treated (50 and 100 mg / kg / day) female ob / ob mice. FIG. 12 is a bar graph comparing plasma fluxamine on day 16 in control, Compound A (100 mg / kg / day) and darglitazone treated female ob / ob mice.

Claims (15)

  A method of treating an animal for reducing body fat, comprising administering to an animal in need thereof a therapeutically effective amount of a phosphodiesterase 9 (PDE9) inhibitor.   The method of claim 1, wherein the animal is overweight.   The method of claim 1, wherein the animal is obese.   2. The method of claim 1, wherein the PDE9 inhibitor is a PDE9 selective inhibitor.   A method of treating an animal for eating disorders, comprising administering to the animal in need thereof a therapeutically effective amount of a PDE9 inhibitor.   6. The method of claim 5, wherein the PDE9 inhibitor is a PDE9 selective inhibitor.   Genetically modified mice that are homozygous for the disruption of the PDE9 gene, after 6 weeks of high-fat diet, decreased body weight loss or lipid mass during fat accumulation compared to wild-type mice after 6 weeks of high-fat diet A genetically modified mouse showing   8. The mouse of claim 7, which expresses an exogenous reporter gene under the control of the regulatory sequence of the PDE9 gene.   8. The mouse of claim 7, exhibiting undetectable PDE9 activity.   A genetically modified cultured mammalian cell that is homozygous for the disruption of the PDE9 gene, wherein the cell or a progeny cell derived from the cell exhibits undetectable PDE9 polypeptide activity, wherein the cell or progeny cell is Genetically engineered cultured mammalian cells that exhibit PDE9 polypeptide activity without conjugation disruption.   11. The genetically modified mammalian cell of claim 10, wherein the cell is an embryonic stem (ES) cell.   The genetically modified cell of claim 11, wherein the cell is a murine ES cell.   The genetically modified cell of claim 11, wherein the cell is a human ES cell. The following steps:
(A) introducing a DNA sequence containing a PDE9 gene targeting construct that disrupts the PDE9 gene upon recombination with the PDE9 gene into mouse ES cells;
(B) selecting a mouse ES cell whose genome contains a PDE9 gene disruption;
(C) introducing the ES cells selected in step (b) into a mouse blastocyst or morulae;
(D) a step of transplanting the blastocyst or morula in step (c) into a foster mother mouse;
(E) by developing a migrated blastocyst or morula to a period in which a chimeric mouse is generated; and (f) by mating the chimeric mouse of step (e) with a mouse heterozygous for PDE9 disruption. Obtaining mice homozygous for the PDE11 gene disruption;
A method for producing a mouse according to claim 10, comprising:
A method wherein said mice that are homozygous for PGE9 gene disruption show a decrease in body weight or lipid content during fat accumulation after 6 weeks of high fat diet compared to wild type mice after 6 weeks of high fat diet.   An isolated nucleic acid molecule comprising a PDE9 gene targeting construct, wherein the construct destroys the PDE9 gene upon recombination with the PDE9 gene.

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