EP1855523A2 - Resistance a l'insuline et la leptine avec de l'hyperletptinemie dans des souris deficientes en recepteur d'androgene - Google Patents

Resistance a l'insuline et la leptine avec de l'hyperletptinemie dans des souris deficientes en recepteur d'androgene

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Publication number
EP1855523A2
EP1855523A2 EP06717647A EP06717647A EP1855523A2 EP 1855523 A2 EP1855523 A2 EP 1855523A2 EP 06717647 A EP06717647 A EP 06717647A EP 06717647 A EP06717647 A EP 06717647A EP 1855523 A2 EP1855523 A2 EP 1855523A2
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Prior art keywords
diabetes
subject
activity
type
mice
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German (de)
English (en)
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EP1855523A4 (fr
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Chawnshang Chang
Hung-Yun Lin
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University of Rochester
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University of Rochester
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0276—Knock-out vertebrates
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • C07K14/721—Steroid/thyroid hormone superfamily, e.g. GR, EcR, androgen receptor, oestrogen receptor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • G01N33/5088—Supracellular entities, e.g. tissue, organisms of vertebrates
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/035—Animal model for multifactorial diseases
    • A01K2267/0362—Animal model for lipid/glucose metabolism, e.g. obesity, type-2 diabetes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/30—Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
    • G01N2333/723—Steroid/thyroid hormone superfamily, e.g. GR, EcR, androgen receptor, oestrogen receptor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders
    • G01N2800/042—Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders

Definitions

  • Androgen receptor is a member of the steroid hormone superfamily of nuclear receptors. Androgen receptor has been implicated in many cancers in an androgen dependent way. Disclosed herein androgen receptor is also involved in the development of Type ⁇ Diabetes and coronary heart disease (CHD). Furthermore, while antiandrogens, such as hydroxyflutamide have been used to treat AR dependent cancers for many years, these same treatments can increase the factors that contribute to Type ⁇ Diabetes and CHD.
  • this invention in one aspect, relates to Type II Diabetes and Coronary Heart Disease (CHD) and related diseases and conditions.
  • CHD Type II Diabetes and Coronary Heart Disease
  • Figure 1 shows that AR "/y mice developed excess adiposity.
  • Figure IA shows that the growth curves of mice were determined at indicated time points between the ages of 5 and 40 weeks. Values are mean ⁇ SE from 9-10 mice per group. *P ⁇ 0.05, **P ⁇ 0.01, AR "/y versus male WT.
  • Figure IB shows the epididymal and perirenal fat pads isolated from 35-week-old mice (upper panel). Sections of WAT (white adipose tissue) are shown at 4Ox magnification (lower panel) of 35-week-old mice, scale bar, 200 ⁇ m.
  • C Distribution of cell size of epididymal WATs from 35-week-old mice.
  • Figure 2 shows that AR " y mice are insulin resistant, and glucose intolerant.
  • Figure 2A shows the GTT (glucose tolerance test) (oral bolus 2 mg/g body weight) of 35- week-old mice after 14-h fast.
  • Figure 2B shows the AUC analysis of GTT.
  • Figure 2C shows the ITT (insulin tolerance test ) (intraperitoneal 1 U/kg body weight) of 25-week-old mice.
  • Figure 2D shows the ITT (intraperitoneal 1 U/kg body weight) of 35-week-old mice. Values are mean ⁇ SE from 5-6 mice per group. *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001, AR " /y versus male WT.
  • Figure 2E shows that in vivo PI3K activity was measured in phosphotyrosine immunoprecipitates prepared from skeletal muscles, adipose tissues, and livers from 14-h fast of 35-week-old mice. Tissues were collected 3 min after intraperitoneal injection of insulin or isotonic saline. The results were quantified by
  • PhosphoLnaging Values are representative of 3 mice of each group. **P ⁇ 0.01, AR "/y with insulin versus WT with insulin.
  • Figure 2F shows the skeletal muscles and livers that were removed from 35-week-old, ad libitum-fed mice. Values of each group were converted into milligrams of TG (triglyceride) per gram of tissue (wet weight) after comparison with a glycerol standard. Values are mean ⁇ SE from 5 mice per group. ***P ⁇ 0.001, AR "/y versus WT.
  • Figure 3 shows the Effects of AR deficiency on adipocyte-derived adipokine.
  • Figure 3A shows the fed leptin concentrations in 8-, 25-, or 35-week-old mice.
  • Figure 3B shows the relationship between fed leptin concentrations and body weight of 35-week-old mice.
  • Figure 3 C shows the adiponectin concentrations in 35-week-old mice.
  • Figure 4 shows that Aging AR " y mice developed leptin resistance but not younger mice.
  • Figure 4A shows the food intake per 24-h of 35-week-old mice.
  • Figure 4B shows the weight changes per 24-h of 35 -week-old mice.
  • Figure 4C shows the food intake per 24-h of 20-week-old mice.
  • Figure 4D shows the weight changes per 24-h of 20-week-old mice.
  • Values are mean -t SE from 3-4 mice per group.
  • Figure 5 shows the recombination of AR-loxP allele in the liver of AR (L"/y) mice, growth curve, cumulative weight gain, and serum glucose in WT and AR (L"/y) mice on high fat diet (HFD). Mice were given HFD from 8 weeks as described in Methods.
  • Figure 5(a) shows the detection of intact (wt) versus recombined (ko) allele and SIp by RT- PCR from liver.
  • Figure 5(b) shows the body weight and (c) cumulative weight gain on
  • HFD of the animals measured weekly. Also shown are fasting serum glucose levels (d) at 24 weeks, (e) at 36 weeks, and (f) at 52 weeks on HFD of the animals.
  • Figure 6 shws the fat accumulation in the liver of treated WT and AR (Wy) mice on HFD.
  • Figures 6(a-b) show images of gross appearance and H&E staining of liver sections of WT mice treated with the normal chow (a), HFD (b), AR (Wy -* mice with normal chow (c), and HFD (d) at 16 weeks for 8 weeks. The clear vacuoles in the liver section are identified by arrows in b and d.
  • Figure 7 shows that fed HFD in AR (L"/y) mice results in defects in glucose homeostasis.
  • Figure 7(a) shows that glucose tolerance tests were performed. Blood samples were collected and glucose measured at the times indicated. Results represent blood glucose concentrations are expressed as mean ⁇ SEM.
  • Figure 7(b) shows the serum insulin concentration at the times indicated.
  • Figure 7(c) shows PI3K activity in PY- immunoprecipitates, and a representative ⁇ hosphatidylinositol-(3,4,5) trisphosphate (PIP3) level. Bars represent mean ⁇ SEM.
  • Figure 7(d) show the serum insulin concentration at 24 weeks.
  • Figure 8 shows decreased adiponectin, increased FFA, and leptin levels in AR (L" /y) mice.
  • Figure 8(a) shows serum free fatty acid levels in random-fed mice.
  • Figure 8(b) shows serum cholesterol levels in random-fed mice.
  • Figure 8(c) shows serum adiponectin levels of random-fed mice.
  • Figure 8(d) shows serum leptin levels of random-fed mice. Bars represent mean ⁇ SEM.
  • compositions and methods are not limited to specific synthetic methods, specific recombinant biotechnology methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. 17. It is estimated that by the year 2030 there will be approximately 366 million people affected by type 2 diabetes in worldwide (WiId 5 S, et al. (2004) Diabetes Care 27:1047-1053) with many of those affected in the elderly age group ⁇ Diabetes Care 21 :296- 309, 1998). Although the primary factors causing this disease are elusive, insulin resistance and leptin insensitivity may play a major role in its development (Saad, MF 5 et al. (1989) Lancet 1:1356-1359).
  • Testosterone and its metabolite, dihydrotestosterone (DHT) 5 can activate androgen receptor (AR) to exert their androgenic actions.
  • AR androgen receptor
  • the proper or maximal androgen (A) action typically requires interaction with selective coregulators in selective tissues (Chang,CS, et al. (1988) Science 240:324-326; Heinlein,CA, and Chang,C. (2002) Endocr.Rev. 23:175-200).
  • Leptin the adipocyte-derived adipokine product of the ob gene, has been shown to induce a negative energy balance by reducing appetite and increasing energy expenditure (Friedman, JM 5 and HalaasJL. (1998) Nature 395:763-770). Leptin circulates in serum at levels that parallel the mass of body fat. However, obese individuals have been found to be resistant to the negative regulatory function of circulating leptin (Hamilton, BS 5 et al. (1995) Nat Med 1 :953-956).
  • mice that lack leptin or are leptin resistant, respectively, are profoundly hyperphagic and hypometabolic, leading to an obese phenotype, and manifest numerous abnormalities, such as type 2 daibetes with severe insulin resistance, hypothermia and cold intolerance, infertility, and decrease in lean mass (Burks, DJ, et al. (2000) Nature 407:377-382; Chen, H, et al. (1996) Cell 84:491-495; Chua, SCJ, et al. (1996) Science 271 :994-996; Hausberg,M, et al. (2002) Diabetes 51:2434-2440; Lee,GH, et al. (1996) Nature 379:632-635).
  • Ranges can be expressed herein as from “about” one particular value, and/or to "about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10" is also disclosed.
  • Primers are a subset of probes which are capable of supporting some type of enzymatic manipulation and which can hybridize with a target nucleic acid such that the enzymatic manipulation can occur.
  • a primer can be made from any combination of nucleotides or nucleotide derivatives or analogs available in the art which do not interfere with the enzymatic manipulation.
  • Probes are molecules capable of interacting with a target nucleic acid, typically in a sequence specific manner, for example through hybridization. The hybridization of nucleic acids is well understood in the art and discussed herein. Typically a probe can be made from any combination of nucleotides or nucleotide derivatives or analogs available in the art.
  • Decrease refers to lower or less than and typically in reference to a control or other embodiment.
  • a level of a subject activity can be decreased either in reference to a control level of activity, the activity of a control performed in parallel with the subject activity, or the activity of a second subject activity.
  • a level of a subject activity can be increased either in reference to a control level of activity, the activity of a control performed in parallel with the subject activity, or the activity of a second subject activity.
  • AR activity is shown herein to have a profound effect on leptin sensitivity (resistance), insulin resistance, glucose resistance, triglyceride levels, high-density lipoprotein levels (HDL), and low-density lipoprotein (LDL) levels. Specifically, it is disclosed herein, that a decrease in AR activity results in increased insulin, glucose, and leptin resistance, increased HDL levels, increased uric acid, and decreased LDL levels. Thus, decreased AR activity can play a prime role in the development of Type II diabetes as well as coronary heart disease. 31.
  • animals which lack functional AR such as an AR knockout animal have leptin sensitivity (resistance), insulin resistance, glucose resistance, triglyceride levels, increased HDL, and decreased LDL levels. These animals are shown herein as a model or part of a model for Type II diabetes.
  • models for type II diabetes that comprise an animal which is an AR knock out as well as models comprising an AR knock out animal and a particular assay, condition, or state, which is related to diabetes or heart disease, such as Type II diabetes, as well as animals that have a particular level of blood glucose as controlled, by for example, having been fed or fasted for a particular period of time.
  • compositions as models for type II diabetes comprising an androgen receptor knockout animal and an assay for type II diabetes. a) Androgen receptor knockouts
  • an AR knock out animal is any animal with a disrupted AR loci or gene.
  • the AR loci can be disrupted by, for example, disrupting one of the exons, such that a stop codon terminates translation of the AR peptide early or where the exon is completely taken out.
  • the AR loci would include any exon or intron associated with the AR gene on the X chromosome.
  • the AR gene is considered any sequence associated with the AR locus. Thus, it would at least include the chromosomal nucleic acid contained within any organism that expresses an AR, such as, the introns, exons, 5' upstream sequence involved with the AR coding and non-coding sequence, and 3' downstream sequence involved with the AR coding and non coding sequence.
  • a disrupted AR loci can be any AR loci that does not produce a native AR protein.
  • a disrupted AR loci would also include any AR loci wherein the nucleic acid of the natural AR gene, including exons and introns has been altered.
  • the altering of the AR gene will cause a disruption in AR function, by for example, preventing DNA binding in the AR gene product or ligand binding in the AR gene product or transactivating activity in the AR gene product.
  • the disrupted AR loci can be made using any known technique, including homologous recombination techniques.
  • the disrupted loci can be an alteration of any exon to produce a non-functional AR protein.
  • constructs and methods to mutate any exon in the AR through homologous recombination via the surrounding introns are constructs and methods to mutate any exon in the AR through homologous recombination via the surrounding introns.
  • the AR knock outs can be produced so that they are conditional, by for example, being dependent on recombinase activity, such as Cre.
  • Cre recombinase activity
  • the Cre-lox system has been successfully used herein to generate AR knockout mice (ARKO).
  • This principle has been successfully applied for tissue-specific transgene expression (Orban PC, 1992), for site specific gene targeting (Gu, 1994) and for exchange of gene sequence by the "knock-in" method (Hank M, 1995).
  • the ARKO animal can have a liver-specific knock out by using the albumin promoter. It is understood and herein contemplated that other ARKO animals can be used utilizing other tissue specific promoters known in the art.
  • exon 1 can be floxed through addition of a lox site in sequence that will homologously recombine with Ihtron 1 and inron 2.
  • lox sites could be inserted into sequence which would homologously recombine with intron 2 and intron 3 for exon 2, intron 3 and intron 4 for exon 3, intron 4 and intron 5 for exon 4, intron 5 and intron 6 for exon 5, and so forth for each exon which are considered disclosed herein.
  • the disrupted AR loci can be in any cell that contains an AR loci, such as an embryonic stem cell, an embryonic germ cell, a breast cell, a breast cancer cell, an ovary cell, an ovary cancer cell, and any cell line of cells that contain AR genes which are expressed, such as prostate cells, testis, bone, brain, neural, and muscle.
  • an AR loci such as an embryonic stem cell, an embryonic germ cell, a breast cell, a breast cancer cell, an ovary cell, an ovary cancer cell, and any cell line of cells that contain AR genes which are expressed, such as prostate cells, testis, bone, brain, neural, and muscle.
  • the ARKOs can be in any cell or in any animal, such as a rat, mouse, non- human primate (including but not limited to monkey and chimpanzee), rabbit, porcine, ovine, or bovine.
  • a rat, mouse, non- human primate including but not limited to monkey and chimpanzee
  • rabbit, porcine, ovine, or bovine can be in any mammal. They can be used via any technique and the animals produced as well as their progeny are disclosed.
  • Disclosed are methods of making a model for type II diabetes comprising a) generating a mouse with an AR gene or necessary portion thereof for AR actvity, between two loxP sites, b) generating a mouse with Cre recombinase under the control of a ⁇ -actin promoter (ACTB Cre+) and heterozygous for the AR gene, and c) mating a female ACTB Cre+ mouse with and ARloxP mouse creating an AR Cre-lox mouse.
  • ACTB Cre+ ⁇ -actin promoter
  • Cre-loxP system is described herein to create the model, it is understood and herein contemplated that any inducible promoter system such as the FIp recombinase and the tetracycline inducible promoter system, may be used in place of the Cre-loxP inducible promoter system. Also disclosed are methods of generating a model for type II diabetes comprising generating an androgen receptor knock out animal and assaying the animal for characteristics of type II diabetes.
  • a model for Coronary Heart Disease comprising a) generating a mouse with an AR gene or necessary portion thereof for activity, between two loxP sites, b) generating a mouse with Cre recombinase under the control of a ⁇ -actin promoter (ACTB Cre+) and heterozygous for the AR gene, and c) mating a female ACTB Cre+ mouse with and ARloxP mouse creating an AR Cre-lox mouse.
  • methods of generating a model for Coronary Heart Disease (CHD) comprising generating an androgen receptor knock out animal and assaying the animal for characteristics of coronary heart disease. b) Assays, conditions, or states related to diabetes
  • the models include an assay or a condition or a state that is related to diabetes which is either monitored or tested, for example.
  • a model could include the ARKO animal disclosed herein, along with a state the animal is put into, such as a fed state or a fasting state for a period of time. It is understood and herein contemplated that periods of fasting can occur for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours prior to assay. It is also understood that feeding can occur within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours of the assay period. This can, for example, produce conditions which mimic a Type II diabetic situation.
  • models for Type ⁇ diabetes comprising an androgen receptor knockout animal and an assay for type ⁇ diabetes, wherein the assay is performed on an animal that fasted for at least 6, 8, 10, 12, 14, 16, 18, 20, 22, 24 hours or any number of hours in between. It is also understood that feeding can occur after a period of fasting.
  • the animal can be put into a fed state 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours prior to the assay period following 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours of fasting.
  • the ARKO animal can have its blood glucose level tested or an insulin tolerance test (ITT) or glucose tolerance test (GTT) can be performed.
  • ITT insulin tolerance test
  • GTT glucose tolerance test
  • models for Type ⁇ diabetes comprising an androgen receptor knockout animal and an assay for type II diabetes, wherein the assay for Type II diabetes comprises an insulin tolerance test (ITT).
  • models for Type II diabetes comprising an androgen receptor knockout animal and an assay for type II diabetes, wherein the assay for Type II diabetes comprises a glucose tolerance test (GTT).
  • the ARKO animal can have its leptin levels monitored or tested, or its triglyceride or HDL or LDL levels monitored or tested. Fatty acids, triglycerides, and cholesterol can also be measured.
  • C- reactive peptide CBP
  • Adiponectin and Resistin markers for Diabetes, also can be assayed by Elisa method. It is understood that these and other types of tests can also be performed on a subject to determine if they have a particular disases condition, even if they are not an ARKO mouse, such as a human.
  • the disclosed models can be used in a variety of ways.
  • the models can be used to test the effect a particular composition, such as a potential drug for Type II diabetes, has on the knockout animal.
  • the effect a composition has on the model can be determined using one or more of the assays disclosed herein alone or in combination.
  • the models can also be used to screen libraries of compounds to identify molecules having a particular effect on the model animal. Molecules having the desired effect can be identified and further synthesized or tested for example.
  • the models can also be used to test existing pharmaceuticals to see what their effect is the disclosed models.
  • the information gained from perfoming tests on existing pharmaceuticals can be used to make treatment decisions regarding that particular composition or one could make the decision to start other therapies in conjunction with that composition, if for example, the composition had a negative effect in the Type II diabetes model, one might look for a combination therapy for Type II diabetes or one might further choose to monitor diabetic indicators, such as glucose levels or insulin resistance, when taking that particular pharmaceutical.
  • methods for screening drugs for an effect on Type II Diabetes comprising administering the drug to an androgen deficient mouse and assaying for AR activity, wherein a increase in AR activity indicates a drug that can treat Type II Diabetes.
  • Diabetes comprising administering the drug to an androgen deficient mouse and assaying for AR activity, wherein a increase in AR activity indicates a drug that can treat Type II Diabetes, further comprising performing an assay for a characteristic of type II diabetes.
  • a control may be used as a basis for assessing the effect of a composition being screened.
  • methods of testing a composition for its effect on Type II diabetes comprising administering the composition to an androgen receptor knockout animal, and performing an assay related to Type II diabetes, wherein a change in the assay relative to a control indicates the compound has an effect on Type II diabetes.
  • methods for screening drags for an effect on CHD comprising administering the drug to an Androgen deficient mouse and assaying for AR activity, wherein a increase in AR activity indicates a drug that can be used to treat CHD.
  • Methods of diagnosis and prognosis 43 The information that lack of androgen receptor leads to characteristics and conditions related to diabetes, such as Type II diabetes, and CHD indicate that a variety of screening and testing methods are disclosed related to subjects and the presence of or likelihood to have or get diabetes, such as Type II diabetes, or CHD. 44.
  • subjects who have diabetes or Type II diabetes or CHD can have their AR activity tested.
  • To test a human subject's AR activity one could indirectly assay the serum levels of Testosterone or 5-alpha-dihydrotestosterone. Besides, one could assay the number of CAG repeats of AR in subject, which is inversely correlated with AR activity.
  • AR activity can also be performed using methods as described herein, for example, for assaying AR activity in tissue or cell samples from mice disclosed herein. An AR activity lower than that of a control would indicate that the diabetes or CHD of the subject could be due to the loss of AR activity. Steps could then be taken, as discussed herein, to elevate the activity of the AR activity to help alleviate or reduce the diabetes or CHD. In another sitruation, a subject could have their AR activity tested.
  • the techniques and methods disclosed herein can be used to assess the likelihood a subject will develop a condition due to decreased AR activity.
  • methods for diagnosing the likelihood of a subject to develop Type II Diabetes comprising taking a tissue sample from the subject and assaying for AR activity, wherein a decrease in AR activity indicates Type II Diabetes. It is understood that subjects with decreased AR activity can have increased leptin resistance and increased insulin resistance. Therefore, it is understood and herein contemplated that a subject with decreased AR activity will likely develop Type II diabetes.
  • One measure of AR activity would be to assay the expression of AR dependent genes in the sample.
  • Another measure of AR acivity comprises assaying the amount of AR present in the sample.
  • the disclosed methods can be used to diagnose the likelihood a subject will develop coronary heart disease CHD.
  • methods for diagnosing the likelihood of a subject to develop CHD comprising taking a tissue sample from the subject and assaying for AR activity, wherein a decrease in AR activity indicates CHD. It is understood and herein contemplated that subjects with decreased AR activity can have increased uric acid levels, increased HDL, and decreased LDL, all of which contribute to CHD. It is also understood that a subject can be a cell, mammal, mouse, or human. 46.
  • the disclosed methods can also be used to diagnose a condition.
  • a method of diagnosing a subject with Type II diabetes comprising a) obtaining a tissue sample, and b) assaying for AR activity, wherein a lack of AR indicates Type ⁇ diabetes.
  • a method of diagnosing a subject with CHD comprising a) obtaining a tissue sample, and b) assaying for AR activity, wherein a lack of AR indicates CHD.
  • various tissue samples can be used with the disclosed methods. Specifically disclosed are methods, wherein the tissue sample is blood, white adipose tissue (WAT), liver tissue, or skeletal muscle.
  • Androgen ablation thereapy is a commonly used method of treating cancer and in particular prostate cancer.
  • the diminishment of AR activity can have dramatic consequences in the patient.
  • the patient can develop leptin resistance and insulin resistance which are two of the main components of Type ⁇ Diabetes.
  • decreased AR activity can lead to increased high density lipoproteins, increased uric acid output, and decreased low-density lipoproteins associated with CHD.
  • a patient being treated for cancer with an androgen ablatement mitin can develope secondary problems associated with the treatment that left unchecked can put the patients life at risk.
  • a patient that developes Type II diabetes or CHD while under treatment with androgen ablatement therapy can be assessed to determine if AR is the reason for the developing Type II diabetes or CHD. IfAR activity is decreased in the patient and tests indicate that diabetes or CHD is a problem, a new treatment can be prescribed to avoid complications associated with Type II diabetes and CHD.
  • Also disclosed are methods for evaluating whether a treatment with a compound should be performed due to the effect the treatment has on CHD, wherein the compound modulates the androgen activity the method comprising a) exposing a subject Q to the compound, and b) evaluating androgen activity in the presence of the compound, wherein a change in the androgen activity of the subject, relative to the androgen activity of a subject that has not been exposed to the compound, indicates that the compound modulates androgen activity, and wherein a decrease in androgen activity indicates a negative effect on CHD, providing an indication that treatment with the compound may not 5 be indicated.
  • AR activators Due to the effects of AR activity on prinicipal activities (e.g., leptin resistance, insulin resistance, increased HDL) associated with Type II dabetes and CHD, agents that can increase AR activity can be used to treat these conditions, herein called AR activators.
  • Q AR activators can be any molecule discussed herein as increasing AR activity including coactivators and androgen receptor ligands and anlogs as discussed herein and understood.
  • a reduced level can be determined by either comparing the amount of the testosterone or one of its precursors or metabolites or molecules in the testosterone synthesis pathway in a subject and comparing it to the amount that had been present in the subject at a prior determination of the same.
  • a reduced level can also include a comparison of the level of testosterone or one of its precursors or metabolites or molecules in the testosterone synthesis pathway in a subject to a known standard or average amount for the same.
  • average levels of testosterone for males can be 300-l,000ng/dl or 300-1, 100ng/dl.
  • Average testosterone levels in women can be about 10 times lower, such as between 15-70 ng/DL for women aged 20-39 and between 4-70 ng/dL for women aged 40-59.
  • Disclosed are methods of treating a subject with Type ⁇ Diabetes comprising administering to the subject an agent that modulates AR activity, wherein an increase in
  • AR activity reduces Type II Diabetes.
  • methods of treating a subject with CHD comprising administering to the subject an agent that modulates AR activity, wherein an increase in AR activity reduces CHD.
  • agent for treating CHD or Type II diabetes can include hormones such as testosterone and DHT.
  • methods of treating a subject with Type II diabetes or CHD wherein the agent comprises testosterone or CHD.
  • compositions which act as AR coactivators/coregulators including ARA24, ARA54, ARA55, ARA70, ARA267, gelsolin, and supervillian. A discussion of these co-regulators can be found in United States Patent Application Nos.
  • the N-terminus is quite polymorphic in terms of sequence and length between NRs.
  • the N-terminus is more likely to provide unique surfaces to recruit distinct factors that contribute to the specific action of a certain NR.
  • the AR has a large N-termius (ARN) and there are two distinct regions important for its transactivation function residing within the ARN: residues 141-338, which are required for full ligand-inducible transactivation, and residues 360-494, where the ligand-independent activation function-1 (AF-I) region is located (Heinlein, C.A., et al. 2002. Endocr. Rev. 23 : 175-200).
  • Coactivators and corepressors have been identified to interact with ARN (Hsiao, P., et al. 1999. J Biol. Chem. 274:22373-22379, Hsiao, P., et al. 1999. J. Biol. Chem. 274:20229-20234, Knudsen, K.E., et al. 1999. Cancer Res. 59:2297- 2301, Lee, D.K., et al. 2000. J. Biol. Chem. 275:9308-9313, Markus, S.M., et al. 2002. MoI. Biol. Cell 13:670-682, Petre, C.E., et al. 2002. J. Biol.
  • ARN extends to more than one half of the full length protein, its associated proteins are relatively fewer compared to those associated with AR DBD and AR LBD, presumably due to the existence of the AF-I region which limits the application of conventional yeast-two hybrid system by using ARN as bait. It's likely there are still more ARN associated proteins remaining to be identified.
  • AR is classified with glucocorticoid receptor (GR), mineralocorticoid receptor and progesterone receptor (PR) as one group within the nuclear receptor (NR) superfamily, since they share high homology in the DBD and recognize very similar hormone response elements (Forman, B.M. et al. 1990. MoI. Endocrinol. 4:1293-1301, Laudet, V., et al. 1992. EMBO J. 11 : 1003-1013).
  • GR glucocorticoid receptor
  • PR progesterone receptor
  • Androgen receptor signalling 54 Androgen exerts its effects via the intracellular AR, a member of the superfamily of nuclear receptors (Chang, C. S., et al. (1988) Science 240 (4850), 324-6, Mangelsdorf, D. J., et al. (1995) Cell 83 (6), 835-9).
  • AR Upon androgen binding, AR dissociates from the heat-shock proteins and binds to androgen response elements, resulting in upregulation or downregulation of the transcription of AR target genes.
  • the AR In addition to responding to ligands, the AR is affected by kinase signaling pathways which directly or indirectly alter the biological response to androgens. This phenomenon is mediated by the AR, as antiandrogens have been shown to block kinase-induced transcriptional activation (Sadar, M. D. (1999) JBiol Chem 21 A (12), 7777-83). Growth factors, cytokines, and neuropeptides have been implicated in various in vitro and in vivo models of human malignancies, including prostate cancers (Burfeind, P., et al. (1996) Proc Nat! Acad Sd US A 93 (14), 7263-8).
  • IGF-I insulin-like growth factor- 1
  • KGF keratinocyte growth factor
  • EGF epidermal growth factor
  • MAPK and Akt kinase cascades have been shown to be involved in growth factor-mediated AR activation (Yeh, S., et al. (1999) Proc Natl Acad Sci USA 96 (10), 5458-63, Wen, Y., et al. (2000) Cancer Res 60 (24), 6841-5, Lin, H. K., et al. (2001) Proc Natl A cad Sci USA 98 (13), 7200-5).
  • AR can be activated and have increased activity, such as transcription activity, as described herein, when AR is bound to its ligand or an activating derivative of said ligand, a precursor to said ligand, a metabolite of said ligand, or a composition that increases a protein or small molecule in the pathway that produces testosterone or an androgen receptor ligand.
  • AR is activated by androgens such as testosterone and 5- ⁇ - dihydrotestosterone.
  • Principal mammalian androgens are testosterone and 5- ⁇ -dihydrotestosterone.
  • androgens There are variety of classes of androgens including Delta-3,4-3-keto androgens, which included Delta-4-androstenedione (Adione), Testosterone, and 11-Beta-hydroxy- androstenedione (11-Adione); Delta-5,6-3-keto androgens, which include, Dehydroepiandrosterone, also known as dehydroisoandrosterone (DHEA), DHEA sulfate (DHEAS); and Delta-5-androstenedion; 5 -Alpha-reduced androgens, which include, 5- Alpha-dihydrotestosterone (dihydrotestosterone, DHT) and 5-Alpha-androstanediol; 17- Keto-steroids, which include DHEA, DHEAS, Delta-4-androstenedione (Adione), Delta
  • testosterone contains a 17- ⁇ -OH (hydroxyl) and a 3-keto group which are necessary for activity. Oxidation of the 17-/3-OH to a 17-keto group, or conversion to a 17 ⁇ -OH, results in steroids with decreased androgen activity. Likewise, the 3 -OH instead of 3-keto group is much less active partly due to increased metabolism. (http://www.iieuiOSci.pharm.utoledo.edu/MBC3320/andiOgens.htm, Dr. William S. Messer, Jr, This page was last updated on Wedneday, April 19, 2000 at 5:23 p.m.)
  • testosterone analogs increase oral activity, prolonged action, and/or selectivity for either anabolic or androgenic activity, but not both.
  • Various testosterone analogs and testosterone and other androgen activators can be administered as a topical cream.
  • Testosterone analogs show increased activity if Reducing the double bond between carbons 4 and 5 ( ⁇ 4) increases testosterone analog activity. This reduction can be catalyzed by the action of 5- ⁇ reductase.
  • Introduction of a fluorine at C9, as for glucocorticoids, increases activity.
  • addition of an alkyl group to position 17 increases stability by preventing oxidation to an inactive 17-keto form. Oxidation to the 17- keto form typically occurs in the liver and can be an obstacle to oral administration.
  • the 17- ⁇ methyl testosterone has twice the activity of native testosterone and can enter through the buccal mucosa (mouth membranes). Loss of the 19-CH 3 enhances selectivity for anabolic activity by decreasing seletively the androgenic effect.
  • Danazol (Danocrine®) is a testosterone derivative having lower androgen agonist without estrogenic activity. Danazol has an acetylene group at Cl 7 and couples a heterocyclic (oxazole) ring to the steroid A ring. Danazol suppresses FSH and LH release from the anterior pituitary and to increase levels of C4 component of complement. (http://www.neurosci.pharm.utoledo.edu/MBC3320/androgens.htm, Dr. William S. Messer, Jr, This page was last updated on Wedneday, April 19, 2000 at 5:23 p.m.).
  • Fluoxymesterone (Halotestin®) possesses a combination of features including a 17- ⁇ methyl group, a 9- ⁇ fluorine, and a 11-OH group. Andronergic activity is increased 10 times and anabolic activity increased 20 times as compared to that of 17- ⁇ methyl testosterone.
  • Testosterone (Andro®, Andryl®, Delatest®, Depotest®, Duratest®, Everone®, Histerone®, Tesanone®, Testex®, Testrin®P.A.) and 17- ⁇ methyl testosterone (Android®, Metandren®, Oreton® Methyl, Virilon®) can be used, as well as the other AR activators, as described herein.
  • Nandrolone derivatives (Androlone®, Duraboline®, HybolinTM Improved, Neo-Durabolic) are examples of this type of analog.
  • the prodrug form contains a des-C19 methyl group.
  • Oxandrolone having an oxygen instead of C2 and a 17- ⁇ methyl group exhibits approximately three times more anabolic activity with only slight andronergic activity compared to testosterone. flittp://www.neurosci.pharm.utoledo.edu/MBC3320/androgens.htm. Dr. William S. Messer, Jr, This page was last updated on Wedneday, April 19, 2000 at 5:23 p.m.).
  • antibodies that are disclosed herein in particular antibodies that bind AR in any of its various forms, sujch as logand bound, as well as binding of coactivators-or co-regulators of AR. Also disclosed are antibodies that bind proteins whose production is regulated by AR. The disclosed antibodies can be used in assays for determining the presence of AR either directly or indirectly and thus, can be used to assay for AR activity either directly or indirectly. Also disclosed are antibodies which are capable of increasing the AR activity, or binding other ligands, such that the diabetic related effects disclosed herein, are reduced. a) Antibodies Generally, antibodies that bind AR in any of its various forms, sujch as logand bound, as well as binding of coactivators-or co-regulators of AR. Also disclosed are antibodies that bind proteins whose production is regulated by AR. The disclosed antibodies can be used in assays for determining the presence of AR either directly or indirectly and thus, can be used to assay for AR activity either directly or indirectly. Also disclosed are antibodies which are capable of increasing the AR activity, or binding other
  • antibodies is used herein in a broad sense and includes both polyclonal and monoclonal antibodies, hi addition to intact immunoglobulin molecules, also included in the term “antibodies” are fragments or polymers of those immunoglobulin molecules, and human or humanized versions of immunoglobulin molecules or fragments thereof, as described herein.
  • the antibodies are tested for their desired activity using the in vitro assays described herein, or by analogous methods, after which their in vivo therapeutic and/or prophylactic activities are tested according to known clinical testing methods.
  • the term "monoclonal antibody” as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies within the population are identical except for possible naturally occurring mutations that may be present in a small subset of the antibody molecules.
  • the monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they exhibit the desired antagonistic activity (See, U.S. Pat. No. 4,816,567 and Morrison et al., Proc. Natl. Acad. ScL USA, 81:6851-6855 (1984)). 70.
  • Monoclonal antibodies of the invention can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975).
  • a hybridoma method a mouse or other appropriate host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent.
  • the lymphocytes may be immunized in vitro, e.g., using the HTV Env-CD4-co-receptor complexes described herein.
  • the monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567 (Cabilly et al.).
  • DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).
  • Libraries of antibodies or active antibody fragments can also be generated and screened using phage display techniques, e.g., as described in U.S. Patent No. 5,804,440 to Burton et al. and U.S. Patent No. 6,096,441 to Barbas et al. 72. In vitro methods are also suitable for preparing monovalent antibodies.
  • Digestion of antibodies to produce fragments thereof, particularly, Fab fragments can be accomplished using routine techniques known in the art. For instance, digestion can be performed using papain. Examples of papain digestion are described in WO 94/29348 published Dec. 22, 1994 and U.S. Pat. No. 4,342,566. Papain digestion of antibodies typically produces two identical antigen binding fragments, called Fab fragments, each with a single antigen binding site, and a residual Fc fragment. Pepsin treatment yields a fragment that has two antigen combining sites and is still capable of cross-linking antigen.
  • the fragments can also include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acids residues, provided the activity of the antibody or antibody fragment is not significantly altered or impaired compared to the non-modified antibody or antibody fragment. These modifications can provide for some additional property, such as to remove/add amino acids capable of disulfide bonding, to increase its bio-longevity, to alter its secretory characteristics, etc.
  • the antibody or antibody fragment must possess a bioactive property, such as specific binding to its cognate antigen.
  • Functional or active regions of the antibody or antibody fragment may be identified by mutagenesis of a specific region of the protein, followed by expression and testing of the expressed polypeptide.
  • antibody can also refer to a human antibody and/or a humanized antibody.
  • Many non-human antibodies e.g., those derived from mice, rats, or rabbits
  • are naturally antigenic in humans and thus can give rise to undesirable immune responses when administered to humans. Therefore, the use of human or humanized antibodies in the methods of the invention serves to lessen the chance that an antibody administered to a human will evoke an undesirable immune response.
  • Human antibodies e.g., those derived from mice, rats, or rabbits
  • the human antibodies of the invention can be prepared using any technique. Examples of techniques for human monoclonal antibody production include those described by Cole et al. (Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77, 1985) and byBoerner et al. (J. Immunol, 147 (l):86-95, 1991). Human antibodies of the invention (and fragments thereof) can also be produced using phage display libraries (Hoogenboom et al., J. MoI. Biol, 227:381, 1991; Marks et al., J. MoI Biol, 222:581, 1991). 76. The human antibodies of the invention can also be obtained from transgenic animals.
  • transgenic, mutant mice that are capable of producing a full repertoire of human antibodies, in response to immunization, have been described (see, e.g., Jakobovits et al., Proc. Natl. Acad. ScL USA, 90:2551-255 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann et al., Year in Immunol., 7:33 (1993)).
  • the antibodies are generated in other species and "humanized” for administration in humans.
  • Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as scFv, sFv, FV, Fab, Fab', F (ab')2, or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin.
  • Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, ret or rabbit having the desired specificity, affinity and capacity, hi some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • CDR complementary determining region
  • donor antibody such as mouse, ret or rabbit having the desired specificity, affinity and capacity, hi some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • Humanized antibodies may also comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences, hi general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., Nature, 321 :522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); and Presta, Curr. Op. Struct.
  • Fc immunoglobulin constant region
  • Humanization can be essentially performed following the method of Winter and co- workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies (U.S. Pat. No.
  • humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
  • variable domains both light and heavy
  • the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable domain sequences.
  • the human sequence which is closest to that of the rodent is then accepted as the human framework (FR) for the humanized antibody (Sims et al., J. Immunol., 151:2296 (1993) and Chothia et al., J. MoI. Biol., 196:901 (1987)).
  • Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework may be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci.
  • humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three dimensional models of the parental and humanized sequences. Three dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences.
  • the term "monoclonal antibody” as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies within the population are identical except for possible naturally occurring mutations that may be present in a small subset of the antibody molecules.
  • the monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain (s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they exhibit the desired antagonistic activity (See, U.S. Pat. No. 4,816,567 and Morrison et al., Proc. Natl. Acad. ScL U.S.A., 81:6851-6855 (1984)).
  • Monoclonal antibodies of the invention can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975).
  • a hybridoma method a mouse or other appropriate host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent.
  • the lymphocytes may be immunized in vitro, e.g., using the complexes described herein.
  • Transgenic animals e.g., mice
  • J (H) antibody heavy chain joining region
  • transfer of the human germ-line immunoglobulin gene array in such germ- line mutant mice will result in the production of human antibodies upon antigen challenge (see, e.g., Jakobovits et al., Proc. Natl. Acad. Sci.
  • Human antibodies can also be produced in phage display libraries (Hoogenboom et al., J. MoI. Biol, 227:381 (1991); Marks et al., J. MoL Biol, 222:581 (1991)).
  • the techniques of Cote et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol., 147 (l):86-95 (1991)).
  • peripheral blood lymphocytes are used in methods of producing monoclonal antibodies if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired.
  • the lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, "Monoclonal Antibodies: Principles and Practice” Academic Press, (1986) pp. 59-103).
  • Immortalized cell lines are usually transformed mammalian cells, including myeloma cells of rodent, bovine, equine, and human origin.
  • rat or mouse myeloma cell lines are employed.
  • the hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.
  • a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.
  • the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (“HAT medium”), which substances prevent the growth of HGPRT-deficient cells.
  • HAT medium hypoxanthine, aminopterin, and thymidine
  • Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium.
  • More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the SaIk Institute Cell Distribution Center, San Diego, Calif, and the American Type Culture Collection, Rockville, Md. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., "Monoclonal Antibody Production Techniques and Applications” Marcel Dekker, Inc., New York, (1987) pp. 51-63).
  • the culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against ARA67, AR, GSK2B, or hRad9, for example.
  • the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunoabsorbent assay
  • the clones maybe subcloned by limiting dilution or FACS sorting procedures and grown by standard methods. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells may be grown in vivo as ascites in a mammal.
  • the monoclonal antibodies secreted by the subclones may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, protein G, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
  • the monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567 (Cabilly et al.).
  • DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).
  • Libraries of antibodies or active antibody fragments can also be generated and screened using phage display techniques, e.g., as described in U.S. Patent No. 5,804,440 to Burton et al. and U.S. Patent No. 6,096,441 to Barbas et al.
  • In vitro methods are also suitable for preparing monovalent antibodies.
  • Digestion of antibodies to produce fragments thereof, particularly, Fab fragments can be accomplished using routine techniques known in the art. For instance, digestion can be performed using papain. Examples of papain digestion are described in WO 94/29348 published Dec. 22, 1994 and U.S. Pat. No. 4,342,566.
  • Papain digestion of antibodies typically produces two identical antigen binding fragments, called Fab fragments, each with a single antigen binding site, and a residual Fc fragment. Pepsin treatment yields a fragment that has two antigen combining sites and is still capable of cross-linking antigen.
  • Antibody fragments 89. Also disclosed are fragments of antibodies which have bioactivity.
  • the polypeptide fragments of the present invention can be recombinant proteins obtained by cloning nucleic acids encoding the polypeptide in an expression system capable of producing the polypeptide fragments thereof, such as an adenovirus or baculovirus expression system.
  • an expression system capable of producing the polypeptide fragments thereof, such as an adenovirus or baculovirus expression system.
  • amino acids found to not contribute to either the activity or the binding specificity or affinity of the antibody can be deleted without a loss in the respective activity.
  • amino or carboxy-terminal amino acids are sequentially removed from either the native or the modified non-immunoglobulin molecule or the immunoglobulin molecule and the respective activity assayed in one of many available assays.
  • a fragment of an antibody comprises a modified antibody wherein at least one amino acid has been substituted for the naturally occurring amino acid at a specific position, and a portion of either amino terminal or carboxy terminal amino acids, or even an internal region of the antibody, has been replaced with a polypeptide fragment or other moiety, such as biotin, which can facilitate in the purification of the modified antibody.
  • a modified antibody can be fused to a maltose binding protein, through either peptide chemistry or cloning the respective nucleic acids encoding the two polypeptide fragments into an expression vector such that the expression of the coding region results in a hybrid polypeptide.
  • the hybrid polypeptide can be affinity purified by passing it over an amylose affinity column, and the modified antibody receptor can then be separated from the maltose binding region by cleaving the hybrid polypeptide with the specific protease factor Xa. (See, for example, New England Biolabs Product Catalog, 1996, pg. 164.). Similar purification procedures are available for isolating hybrid proteins from eukaryotic cells as well.
  • the fragments include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acids residues, provided the activity of the fragment is not significantly altered or impaired compared to the nonmodified antibody or antibody fragment. These modifications can provide for some additional property, such as to remove or add amino acids capable of disulfide bonding, to increase its bio-longevity, to alter its secretory characteristics, etc. In any case, the fragment must possess a bioactive property, such as binding activity, regulation of binding at the binding domain, etc. Functional or active regions of the antibody may be identified by mutagenesis of a specific region of the protein, followed by expression and testing of the expressed polypeptide.
  • a variety of immunoassay formats may be used to select antibodies that selectively bind with a particular protein, variant, or fragment.
  • solid-phase ELISA immunoassays are routinely used to select antibodies selectively immunoreactive with a protein, protein variant, or fragment thereof. See Harlow and Lane. Antibodies, A Laboratory Manual. Cold Spring Harbor Publications, New York, (1988), for a description of immunoassay formats and conditions that could be used to determine selective binding.
  • the binding affinity of a monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980). f) Administration of antibodies
  • Antibodies of the invention are preferably administered to a subject in a pharmaceutically acceptable carrier.
  • Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. A.R. Gennaro, Mack Publishing Company, Easton, PA 1995.
  • an appropriate amount of a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic.
  • the pharmaceutically-acceptable carrier include, but are not limited to, saline, Ringer's solution and dextrose solution.
  • the pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5.
  • Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of antibody being administered.
  • the antibodies can be administered to the subject, patient, or cell by injection (e.g., intravenous, intraperitoneal, subcutaneous, intramuscular), or by other methods such as infusion that ensure its delivery to the bloodstream in an effective form. Local or intravenous injection is preferred.
  • Effective dosages and schedules for administering the antibodies may be determined empirically, and making such determinations is within the skill in the art. Those skilled in the art will understand that the dosage of antibodies that must be administered will vary depending on, for example, the subject that will receive the antibody, the route of administration, the particular type of antibody used and other drugs being administered.
  • the AR, Rb, ARA54, ARA55, ARA24, ARA70, ARA267, gelsolin, supervillin, and SRC-I antibodies and antibody fragments can also be administered' to patients or subjects as a nucleic acid preparation (e.g., DNA or RNA) that encodes the antibody or antibody fragment, such that the patient's or subject's own cells take up the nucleic acid and produce and secrete the encoded antibody or antibody fragment.
  • a nucleic acid preparation e.g., DNA or RNA
  • compositions identified by screening with disclosed compositions / combinatorial chemistry a) Combinatorial chemistry
  • compositions can be used as targets for any combinatorial technique to identify molecules or macromolecular molecules that interact with the disclosed compositions in a desired way.
  • Particular disclosed are assays for identifying molecules that modulate AR or another ligand, such that the diabetic related effects of a loss of AR as disclosed herein are reduced or decreased.
  • compositions such as macromolecular molecules
  • molecules such as macromolecular molecules
  • the molecules identified and isolated when using the disclosed compositions such as, AR, RB, ARA54, ARA55, ARA24, ARA70, ARA267, gelsolin, supervillin, and SRC-I, are also disclosed.
  • the products produced using the combinatorial or screening approaches that involve the disclosed compositions such as, AR, RB, ARA54, ARA55, ARA24, ARA70, ARA267, gelsolin, supervillin, and SRC-I, are also considered herein disclosed.
  • Combinatorial chemistry includes but is not limited to all methods for isolating small molecules or macromolecules that are capable of binding either a small molecule or another macromolecule, typically in an iterative process.
  • Proteins, oligonucleotides, and sugars are examples of macromolecules.
  • oligonucleotide molecules with a given function, catalytic or ligand-binding can be isolated from a complex mixture of random oligonucleotides in what has been referred to as "in vitro genetics" (Szostak, TIBS 19:89, 1992).
  • Combinatorial techniques are particularly suited for defining binding interactions between molecules and for isolating molecules that have a specific binding activity, often called aptamers when the macromolecules are nucleic acids.
  • RNA molecule is generated in which a puromycin molecule is covalently attached to the 3 '-end of the RNA molecule.
  • An in vitro translation of this modified RNA molecule causes the correct protein, encoded by the RNA to be translated.
  • the puromycin a peptdyl acceptor which cannot be extended, the growing peptide chain is attached to the puromycin which is attached to the RNA.
  • the protein molecule is attached to the genetic material that encodes it. Normal in vitro selection procedures can now be done to isolate functional peptides.
  • nucleic acid manipulation procedures are performed to amplify the nucleic acid that codes for the selected functional peptides.
  • new RNA is transcribed with puromycin at the 3 '-end, new peptide is translated and another functional round of selection is performed.
  • protein selection can be performed in an iterative manner just like nucleic acid selection techniques.
  • the peptide which is translated is controlled by the sequence of the RNA attached to the puromycin. This sequence can be anything from a random sequence engineered for optimum translation (i.e. no stop codons etc.) or it can be a degenerate sequence of a known RNA molecule to look for improved or altered function of a known peptide.
  • Cohen et al. modified this technology so that novel interactions between synthetic or engineered peptide sequences could be identified which bind a molecule of choice.
  • the benefit of this type of technology is that the selection is done in an intracellular environment.
  • the method utilizes a library of peptide molecules that attached to an acidic activation domain.
  • a peptide of choice for example an extracellular portion of AR, Rb, ARA54, ARA55, ARA24, ARA70, ARA267, gelsolin, supervillin, and SRC-I is attached to a DNA binding domain of a transcriptional activation protein, such as Gal 4.
  • Combinatorial libraries can be made from a wide array of molecules using a number of different synthetic techniques. For example, libraries containing fused 2,4- pyrimidinediones (United States patent 6,025,371) dihydrobenzopyrans (United States Patent 6,017,768and 5,821,130), amide alcohols (United States Patent 5,976,894), hydroxy-amino acid amides (United States Patent 5,972,719) carbohydrates (United States patent 5,965,719), l,4-benzodiazepin-2,5-diones (United States patent 5,962,337), cyclics (United States patent 5,958,792), biaryl amino acid amides (United States patent 5,948,696), thiophenes (United States patent 5,942,387), tricyclic Tetrahydroquinolines (United States patent 5,925,527), benzofurans (United States patent 5,919,955), isoquino
  • the disclosed compositions can be used as targets for any molecular modeling technique to identify either the structure of the disclosed compositions or to identify potential or actual molecules, such as small molecules, which interact in a desired way with the disclosed compositions.
  • the nucleic acids, peptides, and related molecules disclosed herein can be used as targets in any molecular modeling program or approach.
  • CHARMm performs the energy minimization and molecular dynamics functions.
  • QUANTA performs the construction, graphic modeling, and analysis of molecular structure.
  • QUANTA allows interactive construction, modification, visualization, and analysis of the behavior of molecules with each other.
  • a number of articles review computer modeling of drugs interactive with specific proteins, such as Rotivinen, et al., 1988 Acta Pharmaceutica Fennica 97, 159-166; Ripka, New Scientist 54-57 (June 16, 1988); McKinaly and Rossmann, 1989 Annu. Rev. Pharmacol. Toxiciol.
  • SEQ ID NO: 2 sets forth a particular sequence of an AR
  • SEQ E) NO: 1 sets forth a particular sequence of the protein encoded by SEQ ID NO: 2, an AR protein.
  • variants of these and other genes and proteins herein disclosed which have at least, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 percent homology to the stated sequence.
  • the homology can be calculated after aligning the two sequences so that the homology is at its highest level.
  • hybridization typically means a sequence driven interaction between at least two nucleic acid molecules, such as a primer or a probe and a gene.
  • Sequence driven interaction means an interaction that occurs between two nucleotides or nucleotide analogs or nucleotide derivatives in a nucleotide specific manner. For example, G interacting with C or A interacting with T are sequence driven interactions. Typically sequence driven interactions occur on the Watson-Crick face or Hoogsteen face of the nucleotide.
  • the hybridization of two nucleic acids is affected by a number of conditions and parameters known to those of skill in the art. For example, the salt concentrations, pH, and temperature of the reaction all affect whether two nucleic acid molecules will hybridize.
  • selective hybridization conditions can be defined as stringent hybridization conditions.
  • stringency of hybridization is controlled by both temperature and salt concentration of either or both of the hybridization and washing steps.
  • the conditions of hybridization to achieve selective hybridization may involve hybridization in high ionic strength solution (6X SSC or 6X SSPE) at a temperature that is about 12-25 0 C below the Tm (the melting temperature at which half of the molecules dissociate from their hybridization partners) followed by washing at a combination of temperature and salt concentration chosen so that the washing temperature is about 5°C to 20°C below the Tm.
  • the temperature and salt conditions are readily determined empirically in preliminary experiments in which samples of reference DNA immobilized on filters are hybridized to a labeled nucleic acid of interest and then washed under conditions of different stringencies. Hybridization temperatures are typically higher for DNA-RNA and RNA-RNA hybridizations. The conditions can be used as described above to achieve stringency, or as is known in the art. (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989; Kunkel et al. Methods Enzymol. 1987:154:367, 1987 which is herein incorporated by reference for material at least related to hybridization of nucleic acids).
  • a preferable stringent hybridization condition for a DNA:DNA hybridization can be at about 68 0 C (in aqueous solution) in 6X SSC or 6X SSPE followed by washing at 68°C.
  • Stringency of hybridization and washing if desired, can be reduced accordingly as the degree of complementarity desired is decreased, and further, depending upon the G-C or A-T richness of any area wherein variability is searched for.
  • stringency of hybridization and washing if desired, can be increased accordingly as homology desired is increased, and further, depending upon the G-C or A-T richness of any area wherein high homology is desired, all as known in the art.
  • selective hybridization conditions are by looking at the amount (percentage) of one of the nucleic acids bound to the other nucleic acid. For example, in some embodiments selective hybridization conditions would be when at least about, 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 percent of the limiting nucleic acid is bound to the non-limiting nucleic acid.
  • the non-limiting primer is in for example, 10 or 100 or 1000 fold excess.
  • This type of assay can be performed at under conditions where both the limiting and non-limiting primer are for example, 10 fold or 100 fold or 1000 fold below their kd, or where only one of the nucleic acid molecules is 10 fold or 100 fold or 1000 fold or where one or both nucleic acid molecules are above their k d .
  • Another way to define selective hybridization is by looking at the percentage of primer that gets enzymatically manipulated under conditions where hybridization is required to promote the desired enzymatic manipulation.
  • selective hybridization conditions would be when at least about, 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 91, 98, 99, 100 percent of the primer is enzymatically manipulated under conditions which promote the enzymatic manipulation, for example if the enzymatic manipulation is DNA extension, then selective hybridization conditions would be when at least about 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 percent of the primer molecules are extended.
  • Preferred conditions also include those suggested
  • compositions and methods are used herein, as an example, to exemplify the disclosed compositions and methods. It is understood that the description related to this sequence is applicable to any sequence disclosed herein unless specifically indicated otherwise. Those of skill in the art understand how to resolve sequence discrepancies and differences and to adjust the compositions and methods relating to a particular sequence to other related sequences (i.e. sequences of AR). Primers and/or probes can be designed for any AR sequence given the information disclosed herein and known in the art. 3. Delivery of the compositions to cells
  • compositions and methods which can be used to deliver nucleic acids to cells, either in vitro or in vivo. These methods and compositions can largely be broken down into two classes: viral based delivery systems and non- viral based delivery systems.
  • the nucleic acids can be delivered through a number of direct delivery systems such as, electroporation, lipofection, calcium phosphate precipitation, plasmids, viral vectors, viral nucleic acids, phage nucleic acids, phages, cosmids, or via transfer of genetic material in cells or carriers such as cationic liposomes.
  • Transfer vectors can be any nucleotide construction used to deliver genes into cells (e.g., a plasmid), or as part of a general strategy to deliver genes, e.g., as part of recombinant retrovirus or adenovirus (Ram et al. Cancer Res. 53:83-88, (1993)).
  • plasmid or viral vectors are agents that transport the disclosed nucleic acids, such as Ab, RB, ARA54, ARA55, ARA24, ARA70, ARA267, gelsolin, supervillin, and SRC-I into the cell without degradation and include a promoter yielding expression of the gene in the cells into which it is delivered.
  • Viral vectors are, for example, Adenovirus, Adeno-associated virus, Herpes virus, Vaccinia virus, Polio virus, ADDS virus, neuronal trophic virus, Sindbis and other RNA viruses, including these viruses with the HIV backbone. Also preferred are any viral families which share the properties of these viruses which make them suitable for use as vectors. Retroviruses include Murine Maloney Leukemia virus (MMLV) and retroviruses that express the desirable properties of MMLV as a vector. Retroviral vectors are able to carry a larger genetic payload, i.e., a transgene or marker gene, than other viral vectors, and for this reason are a commonly used vector. However, they are not as useful in non-proliferating cells.
  • MMLV Murine Maloney Leukemia virus
  • Retroviral vectors are able to carry a larger genetic payload, i.e., a transgene or marker gene, than other viral vectors, and for this reason are a commonly used vector
  • Adenovirus vectors are relatively stable and easy to work with, have high titers, and can be delivered in aerosol formulation, and can transfect non-dividing cells.
  • Pox viral vectors are large and have several sites for inserting genes, they are thermostable and can be stored at room temperature.
  • a preferred embodiment is a viral vector which has been engineered so as to suppress the immune response of the host organism, elicited by the viral antigens.
  • Preferred vectors of this type will carry coding regions for Interleukin 8 or 10.
  • Viral vectors can have higher transaction (ability to introduce genes) abilities than chemical or physical methods to introduce genes into cells.
  • viral vectors contain nonstructural early genes, structural late genes, an RNA polymerase HI transcript, inverted terminal repeats necessary for replication and encapsidation, and promoters to control the transcription and replication of the viral genome.
  • viruses When engineered as vectors, viruses typically have one or more of the early genes removed and a gene or gene/promotor cassette is inserted into the viral genome in place of the removed viral DNA. Constructs of this type can carry up to about 8 kb of foreign genetic material.
  • the necessary functions of the removed early genes are typically supplied by cell lines which have been engineered to express the gene products of the early genes in trans.
  • a retrovirus is an animal virus belonging to the virus family of Retro viridae, including any types, subfamilies, genus, or tropisms.
  • Retroviral vectors in general, are described by Verma, I.M., Retroviral vectors for gene transfer. In Microbiology-1985, American Society for Microbiology, pp. 229-232, Washington, (1985), which is incorporated by reference herein. Examples of methods for using retroviral vectors for gene therapy are described in U.S. Patent Nos. 4,868,116 and 4,980,286; PCT applications WO 90/02806 and WO 89/07136; and Mulligan, (Science 260:926-932 (1993)); the teachings of which are incorporated herein by reference.
  • a retrovirus is essentially a package which has packed into it nucleic acid cargo.
  • the nucleic acid cargo carries with it a packaging signal, which ensures that the replicated daughter molecules will be efficiently packaged within the package coat.
  • a packaging signal In addition to the package signal, there are a number of molecules which are needed in cis, for the replication, and packaging of the replicated virus.
  • a retroviral genome contains the gag, pol, and env genes which are involved in the making of the protein coat. It is the gag, pol, and env genes which are typically replaced by the foreign DNA that it is to be transferred to the target cell.
  • Retrovirus vectors typically contain a packaging signal for incorporation into the package coat, a sequence which signals the start of the gag transcription unit, elements necessary for reverse transcription, including a primer binding site to bind the tRNA primer of reverse transcription, terminal repeat sequences that guide the switch of RNA strands during DNA synthesis, a purine rich sequence 5' to the 3' LTR that serves as the priming site for the synthesis of the second strand of DNA synthesis, and specific sequences near the ends of the LTRs that enable the insertion of the DNA state of the retrovirus to insert into the host genome.
  • a packaging signal for incorporation into the package coat a sequence which signals the start of the gag transcription unit, elements necessary for reverse transcription, including a primer binding site to bind the tRNA primer of reverse transcription, terminal repeat sequences that guide the switch of RNA strands during DNA synthesis, a purine rich sequence 5' to the 3' LTR that serves as the priming site for the synthesis of the second strand of DNA synthesis, and specific sequences near the ends of the
  • gag, pol, and env genes allow for about 8 kb of foreign sequence to be inserted into the viral genome, become reverse transcribed, and upon replication be packaged into a new retroviral particle. This amount of nucleic acid is sufficient for the delivery of one to many genes depending on the size of each transcript. It is preferable to include either positive or negative selectable markers along with other genes in the insert. 130. Since the replication machinery and packaging proteins in most retroviral vectors have been removed (gag, pol, and env), the vectors are typically generated by placing them into a packaging cell line.
  • a packaging cell line is a cell line which has been transfected or transformed with a retrovirus that contains the replication and packaging machinery, but lacks any packaging signal.
  • the vector carrying the DNA of choice When the vector carrying the DNA of choice is transfected into these cell lines, the vector containing the gene of interest is replicated and packaged into new retroviral particles, by the machinery provided in cis by the helper cell. The genomes for the machinery are not packaged because they lack the necessary signals.
  • viruses have been shown to achieve high efficiency gene transfer after direct, in vivo delivery to airway epithelium, hepatocytes, vascular endothelium, CNS parenchyma and a number of other tissue sites (Morsy, J. Clin. Invest. 92:1580-1586 (1993); Kirshenbaum, J. Clin. Invest. 92:381-387 (1993); Roessler, J. Clin. Invest.
  • Recombinant adenoviruses achieve gene transduction by binding to specific cell surface receptors, after which the virus is internalized by receptor-mediated endocytosis, in the same manner as wild type or replication-defective adenovirus (Chardonnet and Dales, Virology 40:462-477 (1970); Brown and Burlingham, J. Virology 12:386-396 (1973); Svensson and Persson, J. Virology 55:442-449 (1985); Seth, et al., J. Virol. 51:650-655 (1984); Seth, et al., MoL Cell. Biol. 4:1528-1533 (1984); Varga et al., J. Virology 65:6061-6070 (1991); Wickham et al., Cell 73:309-319 (1993)).
  • a viral vector can be one based on an adenovirus which has had the El gene removed and these virons are generated in a cell line such as the human 293 cell line. In another preferred embodiment both the El and E3 genes are removed from the adenovirus genome.
  • AAV adeno-associated virus
  • This defective parvovirus is a preferred vector because it can infect many cell types and is nonpathogenic to humans.
  • AAV type vectors can transport about 4 to 5 kb and wild type AAV is known to stably insert into chromosome 19. Vectors which contain this site specific integration property are preferred.
  • An especially preferred embodiment of this type of vector is the P4.1 C vector produced by Avigen, San Francisco, CA, which can contain the herpes simplex virus thymidine kinase gene, HSV-tk, and/or a marker gene, such as the gene encoding the green fluorescent protein, GFP.
  • the AAV contains a pair of inverted terminal repeats (ITRs) which flank at least one cassette containing a promoter which directs cell- specific expression operably linked to a heterologous gene.
  • ITRs inverted terminal repeats
  • Heterologous in this context refers to any nucleotide sequence or gene which is not native to the AAV or Bl 9 parvovirus.
  • the vectors of the present invention thus provide DNA molecules which are capable of integration into a mammalian chromosome without substantial toxicity.
  • the inserted genes in viral and retroviral usually contain promoters, and/or enhancers to help control the expression of the desired gene product.
  • a promoter is generally a sequence or sequences of DNA that function when in a relatively fixed location in regard to the transcription start site.
  • a promoter contains core elements required for basic interaction of RNA polymerase and transcription factors, and may contain upstream elements and response elements. (4) Large payload viral vectors
  • herpes simplex virus (HSV) and Epstein-Barr virus (EBV) have the potential to deliver fragments of human heterologous DNA > 150 kb to specific cells. EBV recombinants can maintain large pieces of DNA in the infected B-cells as episomal DNA.
  • compositions can be delivered to the target cells in a variety of ways.
  • the compositions can be delivered through electroporation, or through lipofection, or through calcium phosphate precipitation. The delivery mechanism chosen will depend in part on the type of cell targeted and whether the delivery is occurring for example in vivo or in vitro.
  • the compositions can comprise, in addition to the disclosed AR, Rb,
  • lipids such as liposomes, such as cationic liposomes (e.g., DOTMA, DOPE, DC-cholesterol) or anionic liposomes.
  • Liposomes can further comprise proteins to facilitate targeting a particular cell, if desired.
  • Administration of a composition comprising a compound and a cationic liposome can be administered to the blood afferent to a target organ or inhaled into the respiratory tract to target cells of the respiratory tract.
  • liposomes see, e.g., Brigham et al. Am. J. Resp. Cell. MoI.
  • the compound can be administered as a component of a microcapsule that can be targeted to specific cell types, such as macrophages, or where the diffusion of the compound or delivery of the compound from the microcapsule is designed for a specific rate or dosage. 142.
  • delivery of the compositions to cells can be via a variety of mechanisms. As one example, delivery can be via a liposome, using commercially available liposome preparations such as LIPOFECTIN, LBPOFECTAMINE (GIBCO-BRL, Inc., Gaithersburg, MD),
  • nucleic acid or vector of this invention can be delivered in vivo by electroporation, the technology for which is available from Genetronics, Inc. (San Diego, CA) as well as by means of a SONOPORATION machine (hnaRx Pharmaceutical Corp., Arlington, AZ).
  • the materials may be in solution, suspension (for example, incorporated into microparticles, liposomes, or cells). These may be targeted to a particular cell type via antibodies, receptors, or receptor ligands.
  • the following references are examples of the use of this technology to target specific proteins to tumor tissue (Senter, et al., Bioconiugate
  • Vehicles such as "stealth” and other antibody conjugated liposomes (including lipid mediated drug targeting to colonic carcinoma), receptor mediated targeting of DNA through cell specific ligands, lymphocyte directed tumor targeting, and highly specific therapeutic retroviral targeting of murine glioma cells in vivo.
  • the following references are examples of the use of this technology to target specific proteins to tumor tissue (Hughes et al., Cancer Research, 49:6214-6220, (1989); and Litzinger and Huang, Biochimica et Biophysica Acta, 1104:179-187, (1992)).
  • receptors are involved in pathways of endocytosis, either constitutive or ligand induced.
  • receptors cluster in clathrin-coated pits, enter the cell via clathrin-coated vesicles, pass through an acidified endosome in which the receptors are sorted, and then either recycle to the cell surface, become stored intracellularly, or are degraded in lysosomes.
  • the internalization pathways serve a variety of functions, such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, dissociation and degradation of ligand, and receptor-level regulation. Many receptors follow more than one intracellular pathway, depending on the cell type, receptor concentration, type of ligand, ligand valency, and ligand concentration. Molecular and cellular mechanisms of receptor-mediated endocytosis has been reviewed (Brown and Greene, DNA and Cell Biology 10(6) 399-409 (1991)).
  • Nucleic acids that are delivered to cells which are to be integrated into the host cell genome typically contain integration sequences. These sequences are often viral related sequences, particularly when viral based systems are used. These viral integration systems can also be incorporated into nucleic acids which are to be delivered using a non- nucleic acid based system of delivery, such as a liposome, so that the nucleic acid contained in the delivery system can be come integrated into the host genome.
  • Other general techniques for integration into the host genome include, for example, systems designed to promote homologous recombination with the host genome. These systems typically rely on a sequence flanking the nucleic acid to be expressed that has enough homology with a target sequence within the host cell genome that recombination between the vector nucleic acid and the target nucleic acid takes place, causing the delivered nucleic acid to be integrated into the host genome. These systems and the methods necessary to promote homologous recombination are known to those of skill in the art. c) In vivo/ex vivo
  • compositions can be administered in a pharmaceutically acceptable carrier and can be delivered to the subject' s cells in vivo and/or ex vivo by a variety of mechanisms well known in the art (e.g., uptake of naked DNA, liposome fusion, intramuscular injection of DNA via a gene gun, endocytosis and the like).
  • cells or tissues can be removed and maintained outside the body according to standard protocols well known in the art.
  • the compositions can be introduced into the cells via any gene transfer mechanism, such as, for example, calcium phosphate mediated gene delivery, electroporation, microinjection or proteoliposomes.
  • the transduced cells can then be infused (e.g., in a pharmaceutically acceptable carrier) or homotopically transplanted back into the subject per standard methods for the cell or tissue type. Standard methods are known for transplantation or infusion of various cells into a subject. 4.
  • the nucleic acids that are delivered to cells typically contain expression controlling systems.
  • the inserted genes in viral and retroviral systems usually contain promoters, and/or enhancers to help control the expression of the desired gene product.
  • a promoter is generally a sequence or sequences of DNA that function when in a relatively fixed location in regard to the transcription start site.
  • a promoter contains core elements required for basic interaction of RNA polymerase and transcription factors, and may contain upstream elements and response elements.
  • promoters controlling transcription from vectors in mammalian host cells may be obtained from various sources, for example, the genomes of viruses such as: polyoma, Simian Virus 40 (SV40), adenovirus, retroviruses, hepatitis-B virus and most preferably cytomegalovirus, or from heterologous mammalian promoters, e.g. beta actin promoter.
  • the early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment which also contains the SV40 viral origin of replication (Fiers et al., Nature, 273: 113 (1978)).
  • the immediate early promoter of the human cytomegalovirus is conveniently obtained as a Hindi ⁇ E restriction fragment (Greenway, PJ. et al., Gene 18: 355-360 (1982)).
  • promoters from the host cell or related species also are useful herein.
  • Enhancer generally refers to a sequence of DNA that functions at no fixed distance from the transcription start site and can be either 5' (Laimins, L. et al., Proc. Natl. Acad. Sci. 78: 993 (1981)) or 3' (Lusky, M.L., et al., MoI. Cell Bio. 3: 1108 (1983)) to the transcription unit. Furthermore, enhancers can be within an intron (Banerji, J.L. et al., Cell 33: 729 (1983)) as well as within the coding sequence itself (Osborne, T.F., et al., MoI. Cell Bio. 4: 1293 (1984)).
  • Enhancers function to increase transcription from nearby promoters. Enhancers also often contain response elements that mediate the regulation of transcription. Promoters can also contain response elements that mediate the regulation of transcription. Enhancers often determine the regulation of expression of a gene. While many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, fetoprotein and insulin), typically one will use an enhancer from a eukaryotic cell virus for general expression.
  • Preferred examples are the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
  • the promotor and/or enhancer may be specifically activated either by light or specific chemical events which trigger their function.
  • Systems can be regulated by reagents such as tetracycline and dexamethasone.
  • the promoter and/or enhancer region can act as a constitutive promoter and/or enhancer to maximize expression of the region of the transcription unit to be transcribed, m certain constructs the promoter and/or enhancer region may be active in all eukaryotic cell types, even if it is only expressed in a particular type of cell at a particular time.
  • a preferred promoter of this type is the CMV promoter (650 bases).
  • Other preferred promoters are SV40 promoters, cytomegalovirus (full length promoter), and retroviral vector LTF.
  • GFAP glial fibrillary acetic protein
  • Expression vectors used in eukaryotic host cells may also contain sequences necessary for the termination of transcription which may affect mRNA expression. These regions are transcribed as polyadenylated segments in the untranslated portion of the mRNA encoding tissue factor protein. The 3' untranslated regions also include transcription termination sites. It is preferred that the transcription unit also contain a polyadenylation region. One benefit of this region is that it increases the likelihood that the transcribed unit will be processed and transported like mRNA.
  • the identification and use of polyadenylation signals in expression constructs is well established. It is preferred that homologous polyadenylation signals be used in the transgene constructs.
  • the polyadenylation region is derived from the SV40 early polyadenylation signal and consists of about 400 bases. It is also preferred that the transcribed units contain other standard sequences alone or in combination with the above sequences improve expression from, or stability of, the construct. b) Markers
  • the viral vectors can include nucleic acid sequence encoding a marker product. This marker product is used to determine if the gene has been delivered to the cell and once delivered is being expressed.
  • Preferred marker genes are the E. CoIi lacZ gene, which encodes ⁇ -galactosidase, and green fluorescent protein.
  • the marker may be a selectable marker.
  • suitable selectable markers for mammalian cells are dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hydromycin, and puromycin.
  • DHFR dihydrofolate reductase
  • thymidine kinase thymidine kinase
  • neomycin neomycin analog G418, hydromycin
  • puromycin puromycin.
  • selectable markers When such selectable markers are successfully transferred into a mammalian host cell, the transformed mammalian host cell can survive if placed under selective pressure.
  • These cells lack the ability to grow without the addition of such nutrients as thymidine or hypoxanthine. Because these cells lack certain genes necessary for a complete nucleotide synthesis pathway, they cannot survive unless the missing nucleotides are provided in a supplemented media.
  • An alternative to supplementing the media is to introduce an intact DHFR or TK gene into cells lacking the respective genes, thus altering their growth requirements, individual cells which were not transformed with the DHFR or TK gene will not be capable of survival in non-supplemented media.
  • the second category is dominant selection which refers to a selection scheme used in any cell type and does not require the use of a mutant cell line. These schemes typically use a drug to arrest growth of a host cell. Those cells which have a novel gene would express a protein conveying drug resistance and would survive the selection. Examples of such dominant selection use the drugs neomycin, (Southern P. and Berg, P., JL Molec. Appl. Genet. 1: 327 (1982)), mycophenolic acid, (Mulligan, R.C. and Berg, P. Science 209: 1422 (1980)) or hygromycin, (Sugden, B. et al, MoI. Cell. Biol. 5: 410-413 (1985)).
  • AR protein and gelsolin protein there are numerous variants of the AR protein and gelsolin protein that are known and herein contemplated.
  • derivatives of the AR proteins which also function in the disclosed methods and compositions. Protein variants and derivatives are well understood to those of skill in the art and can involve amino acid sequence modifications. For example, amino acid sequence modifications typically fall into one or more of three classes: substitutional, insertional, or deletional variants. Insertions include amino and/or carboxyl terminal fusions as well as intrasequence insertions of single or multiple amino acid residues. Insertions ordinarily will be smaller insertions than those of amino or carboxyl terminal fusions, for example, on the order of one to four residues.
  • Immunogenic fusion protein derivatives are made by fusing a polypeptide sufficiently large to confer immunogenicity to the target sequence by cross- linking in vitro or by recombinant cell culture transformed with DNA encoding the fusion.
  • Deletions are characterized by the removal of one or more amino acid residues from the protein sequence. Typically, no more than about from 2 to 6 residues are deleted at any one site within the protein molecule.
  • These variants ordinarily are prepared by site specific mutagenesis of nucleotides in the DNA encoding the protein, thereby producing DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture.
  • substitution mutations at predetermined sites in DNA having a known sequence are well known, for example Ml 3 primer mutagenesis and PCR mutagenesis.
  • Amino acid substitutions are typically of single residues, but can occur at a number of different locations at once; insertions usually will be on the order of about from 1 to 10 amino acid residues; and deletions will range about from 1 to 30 residues.
  • Deletions or insertions preferably are made in adjacent pairs, i.e. a deletion of 2 residues or insertion of 2 residues. Substitutions, deletions, insertions or any combination thereof may be combined to arrive at a final construct. The mutations must not place the sequence out of reading frame and preferably will not create complementary regions that could produce secondary mRNA structure. Substitutional variants are those in which at least one residue has been removed and a different residue inserted in its place. Such substitutions generally are made in accordance with the following Tables 1 and 2 and are referred to as conservative substitutions.
  • Substantial changes in function or immunological identity are made by selecting substitutions that are less conservative than those in Table 2, i.e., selecting residues that differ more significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.
  • substitutions which in general are expected to produce the greatest changes in the protein properties will be those in which (a) a hydrophilic residue, e.g. seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g.
  • an electropositive side chain e.g., lysyl, arginyl, or histidyl
  • an electronegative residue e.g., glutamyl or aspartyl
  • substitutions include combinations such as, for example, GIy, Ala; VaI, He, Leu; Asp, GIu; Asn, GIn; Ser, Thr; Lys, Arg; and Phe, Tyr.
  • conservatively substituted variations of each explicitly disclosed sequence are included within the mosaic polypeptides provided herein.
  • Substitutional or deletional mutagenesis can be employed to insert sites for N-glycosylation (Asn-X-Thr/Ser) or O-glycosylation (Ser or Thr).
  • Deletions of cysteine or other labile residues also maybe desirable.
  • Deletions or substitutions of potential proteolysis sites, e.g. Arg is accomplished for example by deleting one of the basic residues or substituting one by glutaminyl or histidyl residues.
  • Certain post-translational derealizations are the result of the action of recombinant host cells on the expressed polypeptide. Glutaminyl and asparaginyl residues are frequently post-translationally deamidated to the corresponding glutamyl and asparyl residues. Alternatively, these residues are deamidated under mildly acidic conditions. Other post-translational modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the o- amino groups of lysine, arginine, and histidine side chains (T.E. Creighton, Proteins: Structure and Molecular Properties, W. H. Freeman & Co., San Francisco pp 79-86 [1983]), acetylation of the N-terminal amine and, in some instances, amidation of the C- terminal carboxyl.
  • variants and derivatives of the disclosed proteins herein are through defining the variants and derivatives in terms of homology/identity to specific known sequences.
  • SEQ JD NO: 2 sets forth a particular sequence of AR
  • SEQ ID NO: 1 sets forth a particular sequence of an AR protein.
  • variants of these and other proteins herein disclosed which have at least, 70% or 75% or 80% or 85% or 90% or 95% homology to the stated sequence.
  • the homology can be calculated after aligning the two sequences so that the homology is at its highest level.
  • each particular nucleic acid sequence may not be written out herein, it is understood that each and every sequence is in fact disclosed and described herein through the disclosed protein sequence.
  • one of the many nucleic acid sequences that can encode the protein sequence set forth in SEQ ID NO: 1 is set forth in SEQ ID NO:2. It is understood that for this mutation all of the nucleic acid sequences that encode this particular derivative of the AR are also disclosed. It is also understood that while no amino acid sequence indicates what particular DNA sequence encodes that protein within an organism, where particular variants of a disclosed protein are disclosed herein, the known nucleic acid sequences that encodes that protein are herein disclosed and described.
  • compositions can also be administered in vivo in a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject, along with the nucleic acid or vector, without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.
  • the carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art. 170.
  • compositions may be administered orally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, transdermally, extracorporeally, topically or the like, although topical intranasal administration or administration by inhalant is typically preferred.
  • topical intranasal administration means delivery of the compositions into the nose and nasal passages through one or both of the nares and can comprise delivery by a spraying mechanism or droplet mechanism, or through aerosolization of the nucleic acid or vector. The latter may be effective when a large number of animals is to be treated simultaneously.
  • Administration of the compositions by inhalant can be through the nose or mouth via delivery by a spraying or droplet mechanism.
  • compositions can also be directly to any area of the respiratory system (e.g., lungs) via intubation.
  • the exact amount of the compositions required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the allergic disorder being treated, the particular nucleic acid or vector used, its mode of administration and the like. Thus, it is not possible to specify an exact amount for every composition. However, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein. 171.
  • Parenteral administration of the composition, if used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions. A more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained. See, e.g., U.S. Patent No. 3,610,795, which is incorporated by reference herein.
  • the materials may be in solution, suspension (for example, incorporated into microparticles, liposomes, or cells). These may be targeted to a particular cell type via antibodies, receptors, or receptor ligands.
  • the following references are examples of the use of this technology to target specific proteins to tumor tissue (Senter, et al.,
  • Vehicles such as "stealth” and other antibody conjugated liposomes (including lipid mediated drug targeting to colonic carcinoma), receptor mediated targeting of DNA through cell specific ligands, lymphocyte directed tumor targeting, and highly specific therapeutic retroviral targeting of murine glioma cells in vivo.
  • the following references are examples of the use of this technology to target specific proteins to tumor tissue (Hughes et al., Cancer Research. 49:6214-6220, (1989); and Litzinger and Huang, Biochimica et Biophvsica Acta. 1104:179-187, (1992)).
  • receptors are involved in pathways of endocytosis, either constitutive or ligand induced.
  • receptors cluster in clathrin-coated pits, enter the cell via clathrin-coated vesicles, pass through an acidified endosome in which the receptors are sorted, and then either recycle to the cell surface, become stored intracellularly, or are degraded in lysosomes.
  • the internalization pathways serve a variety of functions, such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, dissociation and degradation of ligand, and receptor-level regulation. Many receptors follow more than one intracellular pathway, depending on the cell type, receptor concentration, type of ligand, ligand valency, and ligand concentration. Molecular and cellular mechanisms of receptor-mediated endocytosis has been reviewed (Brown and Greene. DNA and Cell Biology 10:6, 399-409 (1991)). a) Pharmaceutically Acceptable Carriers
  • compositions including antibodies, can be used therapeutically in combination with a pharmaceutically acceptable carrier.
  • compositions can be administered intramuscularly or subcutaneously. Other compounds will be administered according to standard procedures used by those skilled in the art.
  • compositions may include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice.
  • Pharmaceutical compositions may also include one or more active ingredients such as antimicrobial agents, antiinflammatory agents, anesthetics, and the like.
  • the pharmaceutical composition may be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. Administration may be topically (including ophthalmically, vaginally, rectally, intranasally), orally, by inhalation, or parenterally, for example by intravenous drip, subcutaneous, intraperitoneal or intramuscular injection.
  • the disclosed antibodies can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally.
  • Preparations for parenteral administration include sterile aqueous or nonaqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable
  • organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
  • Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.
  • Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
  • compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders may be desirable. 180.
  • compositions may potentially be administered as a pharmaceutically acceptable acid- or base- addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkyl and aryl amines and substituted ethanolamines.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid
  • organic acids such as formic acid, acetic acid, propionic acid, glyco
  • the dosage ranges for the administration of the compositions are those large enough to produce the desired effect in which the symptoms disorder are effected.
  • the dosage should not be so large as to cause adverse side effects, such as unwanted cross- reactions, anaphylactic reactions, and the like.
  • the dosage will vary with the age, condition, sex and extent of the disease in the patient and can be determined by one of skill in the art.
  • the dosage can be adjusted by the individual physician in the event of any counterindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days.
  • compositions 182 Methods of making the compositions 182.
  • the compositions disclosed herein and the compositions necessary to perform the disclosed methods can be made using any method known to those of skill in the art for that particular reagent or compound unless otherwise specifically noted.
  • animals produced by the process of transfecting a cell within the animal with any of the nucleic acid molecules disclosed herein Disclosed are animals produced by the process of transfecting a cell within the animal any of the nucleic acid molecules disclosed herein, wherein the animal is a mammal. Also disclosed are animals produced by the process of transfecting a cell within the animal any of the nucleic acid molecules disclosed herein, wherein the mammal is mouse, rat, rabbit, cow, sheep, pig, or primate.
  • ⁇ -Actin is a housekeeping gene and is universally expressed in every tissue; therefore, the ⁇ -Actin promoter driven Cre (ACTB-Cre; Jackson Laboratories, Bar Harbor, ME) will express and delete floxed AR fragments in all the tissues.
  • the AR-/y mice were genotyped by PCR, as described previously (Yeh,S, et al. (2002) Proc Natl Acad Sd USA 99:13498-13503). Animals were housed in pathogen free facilities, maintained on a 12-hour light/dark schedule (light on at 0600) and had free access to standard laboratory chow (no. 5010; PMI Lab Diet, St. Louis, MO) and water.
  • Serum tumor necrosis factor-alpha (TNF- ⁇ ) levels were determined in duplicate in 20 ⁇ l serum samples using a mouse TNF- ⁇ ELISA kit (eBioscience, San Diego, CA) according to the manufacturer's protocol.
  • GTT glucose tolerance test
  • mice were given an oral bolus of D-glucose (2 g/kg body weight) and the blood glucose concentration was measured in samples taken at 0, 30, 60, 90, and 120 min after the glucose bolus.
  • An Insulin tolerance test (ITT) was performed on 6-h fasting mice by intraperitoneal injection of 1 U/kg body weight human insulin (Sigma-Aldrich, St. Louis, MO). Blood glucose concentration was determined at 0, 30, 60, 90, and 120 min after insulin administration.
  • Triglyceride (TG) levels in serum from fasting animals were determined using the GPO-Trinder Assay (Sigma-Aldrich).
  • Serum free fatty acid (FFA) levels in fasting animals were measured using NEFA-Kit-U (Wako Pure Chemical USA Inc., Richmond, VA).
  • FFA serum free fatty acid
  • tissue TG content 50-100 mg tissue pieces were homogenized on ice in pH 7.3 extraction buffer (20 mM Tris, 1 niM ⁇ -mercaptoethanol, 1 mM EDTA). Following centrifugation, the glycerol content of the supernatants was determined using the GPO-Trinder assay (Sigma- Aldrich) according to the manufacturer's instructions.
  • Phosphoinositide-3-OH kinase (PI3K) activity (4) Phosphoinositide-3-OH kinase (PI3K) activity.
  • mice were subjected to 14-h fast, injected intraperitonelly with saline or insulin (10 U per kg (body weight)) and sacrificed 3 min after injection. Tissues were collected and frozen. PI3K activity was measured in phosphotyrosine immunoprecipitates (p-Tyr, Ab-4; EMD Biosciences, Inc., San Diego, CA) from white adipose tissue (WAT), skeletal muscle, and liver lysates as previously described (Goodyear,LJ, et al. (1995) J Clin Invest 95:2195-2204).
  • the medium was changed again 24 h after transfection, and the cells were treated with 1 nM of DHT or 1 ⁇ M of HF for 24 h. The cells were then harvested and whole cell extracts were used for CAT or Luc assay. The CAT activity was quantitated with a Phosphorlmager (Molecular Dynamics). The Luc assay was determined using a Dual-Luciferase Reporter Assay System (Promega) and luminometer. (6) Glutathione S-transferase (GST) pull-down assay
  • the purified GST proteins were then resuspended in 100 ⁇ l of interaction buffer (20 mM HEPES/pH 7.9, 150 mM KCl, 5 mM MgCl 2 , 0.5 mM EDTA, 0.5 mM Dithiothreitol, 0.1% (v/v) NP-40, 0.1% (w/v) BSA and 1 mM PMSF) and mixed with 5 ⁇ l of [ 35 S]-labeled TNT AR protein in the presence or absence of 1 ⁇ M ligand at 4oC for 3 hours. After several washes with NETN buffer, the bound proteins were separated by SDS/8% PAGE and visualized using autoradiography. (7) Western Blot
  • DUl 45 cells were transiently cotransfected with a GAL4-hybrid expression plasmid, a VP16-hybrid expression plasmid, the reporter plasmid pG5-CAT, and the pCMV- ⁇ -gal internal control plasmid. Transfections and CAT assays were performed as described above.
  • mice were divided into 2 groups and were treated once daily with equal volumes of intraperitoneal injections of saline or mouse leptin (R&D Systems Inc., Minneapolis, MN) dissolved in saline at 5 /xg/g (body weight) doses for 6 days. Food intake and changes in body weight were measured to estimate the effects of exogenous leptin administration.
  • MEF cell lines were self-immortalized following the 3T9 protocol. Briefly, primary WT and ARVy MEFs were isolated from E 12.5 littermate embryos and cultured in
  • DMEM plus 10% (v/v) FBS DMEM plus 10% (v/v) FBS.
  • MEFs were then plated at a density of 2.5 x 106 cells per 25 ml flask. Every 3 days, cells were gently trypsinized and re-plated at the same density. Cells were immortalized after 5 months of continuous culture.
  • Mouse 3T3-L1 preadipocytes (CLl 73; ATCC, Manassas, VA), WT, and AR-/y MEF cell lines were maintained in DMEM containing 10% (v/v) FBS.
  • the medium was changed to DMEM supplemented with 10% (v/v) FBS, 10 ⁇ g/ml insulin, 0.5 mM 3-isobutyl-l-methylxanthine, and 1 ⁇ M dexamethasone at 2 days after reaching confluence and then incubated with 10 nM DHT or 100 nM DHT and with or without hydroxyflutamide (HF).
  • the media containing either of the drugs were renewed every other day.
  • Each PCR reaction contained 1 ⁇ cDNA, 50 ⁇ M primers and 12.5 ⁇ iQTM SYBR green supermix reagent (Bio-Rad Laboratories) and was triplicated. 18s was used as an internal control.
  • a list of the primer sequences for real-time PCR is available in Table 3.
  • ACTB-Cre mice mice lacking AR in mixed C57BL/6/129Sv/FVB background were generated (Yeh,S, et al. (2002) Proc Natl Acad Sd U.S.A. 99:13498-13503).
  • Male WT and knockout AR-/y mice were used for the present studies. Since the AR-/y mice were phenotypically female in appearance, female WT mice were also used as an additional comparison group for phenotypic analysis.
  • the growth curves of male WT, female WT, and AR-/y mice showed that AR-/y mice exhibited a significant decrease in the body weight gain throughout the 20th week as compared to male WT mice (Fig. IA).
  • Serum NEFA 0.25 ⁇ 0.02 0.25 ⁇ 0.02 0.71 ⁇ 0.04 $
  • Serum cholesterol (mmol/1) 2.28 ⁇ 0.05 2.28 ⁇ 0.06 2.44 ⁇ 0.05
  • mRNA from these tissues were further analyzed.
  • the mRNA levels of four lipid metabolism genes, peroxisome proliferators-activated receptor gamma (PP AR ⁇ ), CCAAT/enhancer-binding protein alpha (C/EBP ⁇ ), adipocyte fatty acid binding protein/adipocyte P2 (aP2), and sterol regulatory element-binding protein Ic (SREBPIc) were higher in WAT of AR '/y as compared to WT mice (Table 5), indicating loss of AR can contribute to the increase of adipogenesis and lipogenesis via stimulation of several genes, such as lipid metabolism genes.
  • estradiol is converted not only from estrone but also T, therefore it was also impossible to exclude another possibility that the abnormalities in AR "/y mice simply reflected less estrogen converted from T, but it was found that serum estradiol levels, as well as levels of the prohormone androstenedione remained unchanged in AR "/y mice compared to WT mice s O I
  • TG total TG
  • NEFA nonesteri ⁇ ed fatty acids for mice.
  • the IR index is the product versus sham male WT.
  • the marked hyperinsulinemia and hyperglycemia in AR "/y mice clearly demonstrate that loss of AR can reduce insulin sensitivity.
  • a relatively small increase in body weight (-15%) is associated with a remarkable reduction in insulin sensitivity ( ⁇ 65%) in AR "/y mice, and insulin resistance that occurs as early as 20 weeks of age in non-obese AR "/y mice, indicating that loss of AR can directly reduce insulin sensitivity in target tissues without first increasing body weight significantly.
  • the excess fat mass in AR '/y mice can be due to an impaired ability of skeletal muscle to use lipid as fuel substrate and of liver to catabolize lipid, leading to a shunting of lipid to adipose tissue.
  • the unchanged food intake in AR "/y mice indicates that the excess weight gain and adiposity can be due to normal energy input coupled with reduced lipid oxidation and increased lipid storage.
  • impairment in hepatic lipid oxidation resulted in elevated circulating FFA, and hepatic steotosis in PPAR ⁇ -null mice (Kersten,S, et al. (1999) J.Clin.Invest 103:1489-1498).
  • PP ARa Activation of PP ARa by fibrates reduces adiposity in fa/fa Zucker diabetic fatty (ZDF) rats by the activation of several peroxisomal and mitochondrial fatty acid oxidation genes (Guerre- MiIIo 5 M, et al. (2003) J.Biol.Chem. 275:16638-16642). Given that skeletal muscle and liver are the major sites of lipid oxidation and deposition, respectively, it is possible that loss of PP ARo; could produce such an effect.
  • skeletal muscle and hepatic insulin resistance in AR "/y mice can be secondary to altered release of adipokine.
  • Leptin increased insulin sensitivity and concomitantly reduced TG content by promoting lipid oxidation in animal models of insulin resistance and in humans with lipodystrophic diabetes (Petersen,KF, et al. (2002) J.Clin.Invest 109:1345-1350).
  • the AR-Iy mice with elevated leptin however, have significant leptin resistance when exogenous leptin is administered.
  • PP ARa is necessary for the lipopenic action of hyperleptinemia on white adipose and liver tissues, hi PPAR ⁇ -null mice infused with adenovirus-leptin, up- regulation of carnitine palmitoyl transferase- 1 rnENA in fat, down-regulation of acetyl CoA carboxylase in liver, and up-regulation of PPAR ⁇ coactivator-1 alpha rnRNA in both tissues are abolished, as is the reduction in their TG content, indicating that leptin action may be mediated through PP ARa (Lee, Y, et al.
  • AR-/y mice progressively develop leptin resistance and insulin resistance, resulting in obesity. This differs from the ob/ob and db/db mice that display early onset genetic obesity and have leptin inactivity from a very early stage (Tschop,M, and Heiman,ML. (200I) E*/?. Clin. Endocrinol. Diabetes. 109:307-319). ARrIy mice have milder obesity due to the late onset of leptin and insulin resistance. However, leptin resistance in AR-/y mice that is similar with db/db mice, whereas dysfunctional leptin receptor is likely to be a major factor.
  • AR "/y mice not only have an AR deficiency but also have decreased serum levels of androgens.
  • treatment with nonaromatizable DHT restored the physiological serum DHT levels, whereas estrogen levels remained unchanged as compared to WT mice.
  • ERa estrogen receptor alpha
  • AR mice provide an in vivo model showing that loss of AR increases serum leptin concentration and skeletal muscle/hepatic TG content, which can result in the development of obesity, leptin resistance, and insulin resistance.
  • obesity and progressive insulin resistance can lead to type 2 diabetes and an increased risk of cardiovascular diseases (Fernandez-Real,JM, and Gört,W (2003) Endocr Rev 24:278- 301), a better understanding of the molecular mechanisms involved, and dissection of the roles of A-AR in insulin and leptin resistance can help in the development of better therapeutic approaches to type 2 diabetes, obesity, and other cardiovascular diseases.
  • hepatic androgen receptor (AR) hepatic androgen receptor
  • AlC y ' mice hepatic AR knockout mice were generated by crossbreeding floxAR mice with albumin-Cre mice.
  • the AR ⁇ " y ' mice showed similar growth curve to wild-type mice ( Figure IA-B.
  • HFD high-fat diet
  • AR (L"/y) mice exhibit greater cumulative weight gain and increased serum glucose levels while being fed on a HFD for 24, 36, and 52 weeks (Figure 5C-F). AR (L"/y) mice were also marked by increase fat vacuoles in the liver ( Figure 6). Additionally, compared with WT mice, AR ⁇ mice on a HFD displayed more hepatic steatosis, and a type 2 diabetes phenotype, characterized by hypertriglyceridemia, hyperinsulinemia, hyperglycemia, and insulin resistance possibly due to a decreased insulin-mediated PI3K activation (Figure 7, Figure 8, and Table 8).
  • GTT Glucose tolerance test
  • IR insulin resistance
  • the mRNA levels of the gluconeogenic enzymes, phosphoenolpyruvate carboxykinase, and hepatic nuclear factor-4 alpha were upregulated in livers of ABS Uy ⁇ mice as compared with wt mice fed with high-fat diet.
  • Loss of hepatic AR increased sterol regulatory element-binding protein- Ic, a key regulator of lipogenic gene transcription, and its downstream target mRNAs, as well as increased hepatic lipid accumulation. Loss of hepatic AR was also associated with a decrease in peroxisomal proliferator-activated receptor alpha mRNA expression, and a trend toward decreased fatty acid /3-oxidation.
  • Haluzik,M, Gavrilova,O, LeRoith,D Peroxisome proliferator-activated receptor-alpha deficiency does not alter insulin sensitivity in mice maintained on regular or high-fat diet: hyperinsulinemic-euglycemic clamp studies. Endocrinology 145:1662-1667, 2004
  • Heine,PA, Taylor, JA, Iwamoto,GA, Lubahn,DB, Cooke,PS Increased adipose tissue in male and female estrogen receptor-alpha knockout mice.
  • Heinlein,CA, Chang,C Induction and repression of peroxisome proliferator-activated receptor alpha transcription by coregulator ARA70. Endocrine. 21 : 139-146, 2003
  • Hotamisligil,GS, Johnson,RS, Distel,RJ, Ellis,R, Papaioannou,VE, Spiegelman,BM Uncoupling of obesity from insulin resistance through a targeted mutation in aP2, the adipocyte fatty acid binding protein. Science 274:1377-1379, 1996
  • Hotamisligil The role of TNFalpha and TNF receptors in obesity and insulin resistance. J Intern Med 245:621-625, 1999
  • Aromatase-deficient mice have a phenotype of increased adiposity. Proc. Natl. Acad. Sd. U.S.A 97:12735-12740, 2000

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Abstract

La présente invention a trait à des compositions et des procédés pour la maladie liée au diabète et à la maladie coronarienne.
EP06717647A 2005-01-07 2006-01-06 Resistance a l'insuline et la leptine avec de l'hyperletptinemie dans des souris deficientes en recepteur d'androgene Withdrawn EP1855523A4 (fr)

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WO2003012394A2 (fr) * 2001-07-27 2003-02-13 University Of Rochester Animaux transgeniques a inactivation du recepteur androgene
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FAN WUQIANG ET AL: "Androgen receptor null male mice develop late-onset obesity caused by decreased energy expenditure and lipolytic activity but show normal insulin sensitivity with high adiponectin secretion" DIABETES, vol. 54, no. 4, April 2005 (2005-04), pages 1000-1008, XP002581155 ISSN: 0012-1797 *
MAYES J S ET AL: "Direct effects of sex steroid hormones on adipose tissues and obesity." OBESITY REVIEWS : AN OFFICIAL JOURNAL OF THE INTERNATIONAL ASSOCIATION FOR THE STUDY OF OBESITY NOV 2004 LNKD- PUBMED:15458395, vol. 5, no. 4, November 2004 (2004-11), pages 197-216, XP002581154 ISSN: 1467-7881 *
SATO TAKASHI ET AL: "Late onset of obesity in male androgen receptor-deficient (AR KO) mice." BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 3 JAN 2003 LNKD- PUBMED:12480537, vol. 300, no. 1, 3 January 2003 (2003-01-03), pages 167-171, XP002581152 ISSN: 0006-291X *
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