WO2009045403A2 - Régulation par la ménine de la prolifération des cellules d'îlots bêta - Google Patents

Régulation par la ménine de la prolifération des cellules d'îlots bêta Download PDF

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WO2009045403A2
WO2009045403A2 PCT/US2008/011318 US2008011318W WO2009045403A2 WO 2009045403 A2 WO2009045403 A2 WO 2009045403A2 US 2008011318 W US2008011318 W US 2008011318W WO 2009045403 A2 WO2009045403 A2 WO 2009045403A2
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menin
diabetes
level
expression
cells
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PCT/US2008/011318
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WO2009045403A3 (fr
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Seung Kim
Satyajit Karnik
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The Board Of Trustees Of The Leland Stanford Junior University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the field of this invention generally relates to the regulation of ⁇ -islet cell proliferation by menin and/or menin-related factors.
  • the present invention provides methods of measuring the expression of menin and/or menin-related factors, their corresponding gene products and antibodies specific for the gene products in the detection, diagnosis, monitoring, prevention and/or treatment of diabetes, pre-diabetes, or gestational diabetes as well as methods of screening for identifying agents effective in regulating ⁇ -islet proliferation.
  • Endocrine disorders of the pancreas affect over 20 million people in the United States. These human conditions are linked to a deficiency in insulin producing ⁇ -cells, of which, the most common type of undergrowth disorder is diabetes mellitus. Diabetes mellitus is classified into several types: 1) Type 1 diabetes mellitus; 2) Type 2 diabetes mellitus; 3) inherited forms of diabetes mellitus; 4) gestational diabetes mellitus; 5) medically induced diabetes mellitus; and 6) diabetes mellitus induced from malnutrition, infection and illness.
  • Type 2 diabetes mellitus has greatly increased as the society as a whole has become more obese. This and other factors have made Type 2 diabetes mellitus a major medical problem of almost epidemic proportions. At the present time there is no convenient manner to identify the early stages of Type 2 diabetes mellitus. While there is no present cure for the loss of the controlled production of insulin, there are actions that can be taken that would substantially prolong the period before onset of the symptoms of the disease. [0005] Because of the great interest in understanding and preventing Type 2 diabetes mellitus, investigators have been studying the role of ⁇ -islet cells under various conditions.
  • MENl Multiple Endocrine Neoplasia Type 1
  • Menl whose protein product is menin
  • menin C. Larsson, B. Skogseid, K. Oberg, Y. Nakamura, M. Nordenskjold, Nature 332, 85 (1988); S.K. Agarwal et al.
  • Menin functions in a histone methyltransferase protein complex containing Mixed Lineage Leukemia-1 (MLL) (C. M. Hughes et al. Molecular Cell 13, 587 (2004); A. Yokoyama, et al., MoI Cell Biol. 24, 5639 (2004).).
  • MLL Mixed Lineage Leukemia-1
  • H3K4 histone H3 on lysine 4
  • Menin-dependent histone methylation maintains expression of p27 K ⁇ ] and /?/S INK4C (h erea ft er) / ⁇ 27 and pl8), which encode cyclin-dependent kinase (CDK) inhibitors that prevent islet proliferation (S.
  • CDK cyclin-dependent kinase
  • the present invention relates to the regulation of ⁇ -islet cell proliferation by menin and/or menin-related factors.
  • the present invention provides methods of diagnosing or monitoring a disease or condition associated with abnormalities in ⁇ -islet cell proliferation by measuring in surrogate cells the expression of genes related to ⁇ -islet cell proliferation, their corresponding gene products and antibodies specific for the gene products.
  • the genes related to ⁇ -islet cell proliferation comprise menin and/or menin-related factors and the abnormalities in ⁇ -islet cell proliferation relate to a diabetes, pre-diabetes or a condition related to diabetes.
  • the subject methods diagnose, monitor, detect, treat and/or prevent diabetes, pre-diabetes, or gestational diabetes. Also provided herein are methods for screening for agents which modulate ⁇ -islet cell proliferation and methods of treating a subject for diabetes, pre-diabetes, or gestational diabetes.
  • aspects of the invention include a method of diagnosing a disease or condition selected from a risk of developing diabetes, pre-diabetes, or gestational diabetes in a subject.
  • the method includes a) providing a collection of blood cells from the subject; b) measuring a level of gene expression of one or more menin-related factors in the cells; c) comparing the measured level of gene expression of the one or more menin-related factors to a control sample from a normal individual; and d) detecting an alteration in expression of the one or more menin-related factors relative to the control sample, wherein an altered level of expression indicates a diagnosis of risk of developing diabetes, pre-diabetes or gestational diabetes.
  • the menin-related factors comprise one or more of selected from BCl-6, Menl, HoxA9, CCNDl, CCND2, pl8INK4C, MafA, MafB, phosphatase calcineurin, NFATcI, Pbx2, and MLLl.
  • the one or more menin-related factors are selected from BCl-6, Menl, and HoxA9.
  • the measured expression level of BCl-6 is greater than the measured expression level of the control.
  • the measured expression level of Menl is lower than the measured expression level of the control.
  • the measured expression level of HoxA9 is lower than the measured expression level of the control.
  • the measurement of gene expression level comprises a quantitative measurement.
  • the collection of blood cells is leukocytes.
  • the leukocytes comprise at least one of lymphocytes, B- lymphocytes, T- lymphocytes, neutrophils, eosinophils, basophils, and monocytes.
  • the level of gene expression is measured by the level of mRNA of one or more menin-related factors in the cells. In other aspects, the level of gene expression is measured by the level of protein of one or more menin-related factors in the cells.
  • the methods of diagnosing a disease or condition further comprise isolating mRNA from the leukocytes prior to measuring the RNA level expressed by the one or more menin-related factor.
  • the measurement of gene expression level comprises a polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • the methods of diagnosing a disease or condition further comprise isolating a menin-related factor protein from the leukocytes prior to measuring the protein level expressed by the one or more menin-related factor.
  • the measurement of gene expression level comprises a quantitative measurement of protein level.
  • the protein level is measured by an immunoassay selected from RIA, ELISA, EMIT, CEDIA, Western blot analysis, or FIA.
  • the protein level is measured by mass spectrometry.
  • aspects of the invention further include methods of monitoring progression of a disease or condition selected from a risk of developing diabetes, pre-diabetes, or gestational diabetes in a subject, comprising: measuring a level of gene expression of one or more menin-related factors in a blood cell sample from the subject at one or more or time points, wherein monitoring progression of the risk of developing diabetes, pre-diabetes, or gestational diabetes, comprises detecting a change in measured levels of gene expression of the one or more menin-related factors over time.
  • the subject at risk of developing diabetes, prediabetes, or gestational diabetes is obese.
  • the subject at risk has a genetic propensity for developing diabetes, pre-diabetes, or gestational diabetes.
  • the subject at risk of developing diabetes, prediabetes, or gestational diabetes is pregnant.
  • kits for use in aiding detection or diagnosis of a disease or condition selected from a risk of developing diabetes, pre-diabetes, or gestational diabetes in a subject comprising: at least one reagent specific for detecting a menin-related factor selected from: BCl-6, Menl, HoxA9, CCNDl, CCND2, pl8INK4C, MafA, Maffi, phosphatase calcineurin, NFATcI, Pbx2, and MLLl.
  • kits optionally include instructions for carrying out a method of aiding in the diagnosis of diabetes, pre-diabetes, or gestational diabetes.
  • the reagent specific for the menin-related factor is an antibody, or fragment thereof, that is specific for the menin-related factor.
  • kits further comprise an amount of at least one menin-related factor suitable for normalizing data.
  • aspects of the invention further include methods of screening for an agent effective in regulation of mammalian ⁇ -islet cell growth, the method comprising: a) providing a collection of blood cells; and b) determining a difference in level of gene expression of one or more menin-related factors in the blood cells in the presence or absence of a candidate agent, wherein an altered level of expression in the presence of the candidate agent indicates that the candidate agent is effective in regulation of mammalian ⁇ -islet cell growth.
  • the candidate agent is a small molecule, a peptide, a polyclonal antibody, a monoclonal antibody, an antisense RNA, a small interfering RNA (siRNA), a ribozyme, a short hairpin RNA (shRNA), a polypeptide, polysaccharide, lipid, nucleic acid, or combination thereof.
  • siRNA small interfering RNA
  • shRNA short hairpin RNA
  • the candidate agent is effective for treating or preventing a risk of developing diabetes, pre-diabetes, or gestational diabetes in a subject.
  • aspects of the invention include assays to measure human biomarkers for diabetes diagnosis and prognosis.
  • the invention provides methods of using more accessible lymphocyte cells as surrogates to measure the robustness of the Bcl6-menin response to ⁇ -cell mitogens.
  • the ⁇ -cell mitogen is leptin while in another embodiment, the ⁇ -cell mitogen is PRL.
  • menin is shown to be a factor in the proliferation of ⁇ - islet cells, such that increasing amounts of menin results in inhibition of proliferation of the ⁇ -islet cells.
  • the level of menin or other member of the pathway controlling the production of menin or in the downstream pathway controlled by menin is determined using a portion of the leukocyte blood population and assaying for the mRNA or protein.
  • the menin or related factor level is employed in counseling individuals in relation to glucose regulation, particularly as to diabetes.
  • aspects of the invention include markers from accessible tissues like blood useful for (1) predicting diabetes risk, (2) monitoring ongoing ⁇ -cell proliferation and ⁇ -cell mass changes, (3) predicting individual 'potential' for ⁇ -cell expansion or regeneration and (4) detecting 'pre-clinical' diabetes.
  • Figure 1 provides the results of maternal ⁇ cell mass, body mass, and real time RT- PCR analysis on islets during pregnancy.
  • Panel A depicts maternal ⁇ cell mass data
  • Panel B depicts maternal body mass data in C57B16 mice during pregnancy. Note the X axis is not drawn to linear scale.
  • Panel C depicts real time RT-PCR analysis of pi 8 and p27 mRNA levels in maternal islets during pregnancy. Data here and in other figures are presented as means ⁇ s.d. Here and in other figures one asterisk, PO.05; two asterisks, P ⁇ 0.01 ; three asterisks, PO.001.
  • Figure 2 provides the results of blood glucose during ad libitum feeding, fasting, and glucose tolerance testing in nonpregnant ⁇ Menl mice.
  • Panel A depicts RT-PCR analysis and
  • Panel B depicts Western blot analysis of Menl transgene expression in islets from ⁇ Menl mice and controls (wild type mice, 'WT 1 : TRE-Menl mice, TRE'; RJP-rtTA mice, 'RIP').
  • Panel C depicts the results of a glucose tolerance test in non-pregnant ⁇ Menl and control mice.
  • Panel D provides blood glucose levels in non-pregnant ⁇ Menl mice and controls fed ad libitum.
  • Panel E provides blood glucose levels in fasted non-pregnant ⁇ Menl mice and controls.
  • Figure 3 depicts blood glucose levels during ad libitum feeding and fasting in pregnant ⁇ Menl mice and controls.
  • Panels A-C show scatter plots of blood glucose levels in mice at indicated times. The mean for data points is indicated by a horizontal line.
  • Panel A Blood glucose levels in control wild type mice fed ad libitum during pregnancy.
  • Panel B Blood glucose levels in ⁇ Menl mice fed ad libitum during pregnancy.
  • Panel C Blood glucose levels in fasted pregnant control and pregnant ⁇ Menl mice at 16 dpc.
  • Figure 4 shows maternal mass, litter size and mass, and insulin secretion in ⁇ Menl mice.
  • Panel A maternal mass
  • Panels B litter size
  • Panel C birth weight of pups at 18 dpc on post-natal day 1 (Pl) from ⁇ Menl mothers and controls.
  • Panel D depicts insulin content
  • Panel E depicts insulin secretion following glucose stimulation
  • Panel F depicts insulin secretion following arginine stimulation in 16 dpc islets purified from pregnant ⁇ Menl mice, and pregnant controls, both on Dox.
  • Panel G depicts the quantification of activated caspase3 + expression in islets from pregnant ⁇ Menl mice and pregnant controls, both on Dox.
  • Figure 5 depicts Bcl6 regulation of menin in maternal islets and MIN6 cells.
  • Panel A ChIP studies of STAT5 associated with Bcl6 promoter from nonpregnant, pregnant (13 dpc), postpartum (P4), and postpartum (P21) maternal islets.
  • Panel B Real time RT-PCR for Bcl6 expression in maternal islets during pregnancy.
  • Panel C Real time RT-PCR for Bcl6 expression in MIN6 cells transfected with either a Bcl6 expression plasmid (black bars) or a control plasmid (grey bars).
  • Panel D Luciferase assay strategy and Panel E: relative luciferase activity in MIN6 cells transfected with Menl reporter constructs. Red crosses denote mutation of Bcl6 binding sites within the Luc24 reporter.
  • Figure 6 depicts the prolactin signaling effects on MIN6 cells.
  • Panels A-B BrdU (green)/DAPI (blue) detection in MIN6 cells exposed to vehicle.
  • Panels C-D BrdU (green)/DAPI (blue) detection in MIN6 cells exposed to prolactin.
  • Panel E Quantification of BrdU incorporation and
  • Panel F MIN6 cell number.
  • Panels G-H Stat5 (green)/DAPI (blue) detection in MIN6 cells exposed to vehicle.
  • Panel I-J Stat5 (green)/DAPI (blue) detection in MIN6 cells exposed to prolactin.
  • Panel K Quantification of pStat5 nuclear localization and Panel L: pStat5 + BrdU* immunostained MIN6 cells following prolactin exposure.
  • Panel M Real-time RT-PCR analysis of Menl, pi 8 and p27 or
  • Panel N BcW and c-myc mRNA levels in MIN6 cells exposed to vehicle, prolactin or prolactin with dexamethasone.
  • c- myc is a gene whose mRNA levels are unchanged in MIN6 cells exposed to prolactin or other treatments.
  • Figure 7 depicts prolactin-dependent repression of Menl in MIN6 cells.
  • Panel A PCR primer pairs (PP) flanked by indicated genomic segments assessed by ChIP detection of Bcl6 protein association with intended Menl cw-regulatory regions. Arrows mark putative Bcl6 binding sites.
  • Panel B ChIP detection of Bcl6 association with the Menl locus in MIN6 cells exposed to vehicle, prolactin, or prolactin with dexamethasone.
  • Panel C ChEP detection of Bcl6 association with the Menl, pi 8 and p27 loci in MIN6 cells exposed to vehicle, prolactin, or prolactin with dexamethasone.
  • Panel D Me»7-luciferase reporter constructs containing specified regions of the Menl promoter region. Arrows indicate putative Bcl6 binding sites. Red crosses indicate mutation of Bcl6 binding sites in the Luc24 reporter.
  • Panel E Luciferase reporter assay in transfected MIN6 cells treated with vehicle, prolactin or prolactin with dexamethasone.
  • Figure 8 depicts the prolactin-dependent regulation of Bcl6, Menl, and menin targets in human islets.
  • Panel A depicts the Real-time RT-PCR analysis of BcW and
  • Panel B depicts RT-PCR analysis of Menl, pi 8 and p27 in human islets exposed to vehicle (Veh), prolactin (PRL), or prolactin with dexamethasone (PRL+Dex).
  • Panels C-F ChIP studies of human islets exposed to Veh, PRL or PRL+Dex.
  • Panel C Association of Stat5 protein with the islet Bcl6 locus.
  • Bcl6 Primer Pair 1 (PPl) amplifies a region containing STAT5 binding sites.
  • Panel D Association of Bcl6 protein with the islet Menl locus. Menl PP2 amplifies a region containing Bcl6 binding sites. Other Primer Pairs (PPl and PP3) flank segments of Menl lacking Bcl6 binding sites.
  • Panel E Reduced association of menin protein with the pi 8 locus in prolactin-exposed islets.
  • Panel F Reduced association of menin protein with the p27 locus in prolactin-exposed islets.
  • Figure 9 depicts altered Menl and Bcl6 expression in maternal blood. mRNA levels of indicated factors in lymphocytes are determined from maternal blood as compared to nonpregnant hosts for Panel A: Menl ; Panel B: Bcl6; Panel C: p21 ; Panel D: pl 8; and Panel E: p27.
  • FIG 10 depicts the results of expression levels of BcW, Menl, and HoxA9 in pregnant mice and humans.
  • Panel A shows that Bcl6-Menl regulation in blood leukocytes mirrors pancreatic islets during pregnancy in Panel B: mice and Panel C: humans.
  • Figure 11 shows that Bcl6-Menl regulation in blood leukocytes mirrors pancreatic islets during obesity in humans.
  • Figure 12 depicts mRNA levels in PRL-exposed leukocytes in indicated patient groups. * P ⁇ 0.05, ** PO.01.
  • Figure 13 depicts the BcL6 linkage to GDM history.
  • FIG 14 shows that age and body mass index (BMI) are indistinguishable in control subjects compare to subjects with a GDM history DETAILED DESCRIPTION OF THE EMBODIMENTS
  • the present invention relates to the regulation of ⁇ -islet cell proliferation by menin and/or menin-related factors.
  • the present invention provides methods of diagnosing or monitoring a disease or condition by measuring the expression of menin and/or menin-related factors, their corresponding gene products and antibodies specific for the gene products.
  • the subject methods diagnose, monitor, detect, and/or prevent diabetes, prediabetes, or gestational diabetes.
  • methods for screening for agents which modulate ⁇ -islet cell proliferation and methods of treating a subject for diabetes, prediabetes, or gestational diabetes are also provided herein.
  • menin is shown to be a factor in the proliferation of ⁇ - islet cells, such that increasing amounts of menin results in inhibition of proliferation of the ⁇ -islet cells.
  • the level of menin or other member of the pathway controlling the production of menin or in the downstream pathway controlled by menin is determined using a portion of the leukocyte blood population and assaying for the mRNA or protein.
  • the one or more menin-related factors are selected from BCl-6, Menl, and HoxA9.
  • Menin is a 610 amino acid nuclear protein produced from the Menl tumor suppressor gene, which is mutated in familial multiple endocrine neoplasia Type 1. Mutation in the menin gene promotes pathogenesis of Type 1 multiple endocrine neoplasia (MENl) syndrome and sporadic neuroendocrine tumors of the parathyroid, endocrine pancreas and anterior pituitary in humans. Chandrashekharappa SC, et al. Science 276:404-407 (1997) Menin is expressed in a variety of human cells. Wautot V et al. Int. J. Cancer 85:877-881 (1997).
  • MENl multiple endocrine neoplasia
  • a “Menl polynucleotide” refers to a polynucleotide encoding a Menl polypeptide (also referred to in the literature as "menin”).
  • a MENl polynucleotide can be an mRNA that encodes a Menl polypeptide or a chromosomal DNA sequence that comprises the gene sequences encoding the above-referenced mRNA.
  • Menl polynucleotide or polypeptide sequences are publicly available.
  • Menl polynucleotide or polypeptide sequences include, but are not limited to, the sequences set forth in GenBank (accessible at: www.ncbi.nlm.nih.gov/Genbank/) accession numbers: U93236, U93237, AJ297487, AJ297488 and AJ297489, herein incorporated by reference.
  • BC16 (B-cell CLL/lymphoma 6) is a gene that encodes a zinc finger transcription factor and contains an N-terminal POZ domain.
  • a "BC16 polynucleotide” refers to a polynucleotide encoding a BC16 polypeptide.
  • a BC16 polynucleotide can be an mRNA that encodes a BC16 polypeptide or a chromosomal DNA sequence that comprises the gene sequences encoding the above-referenced mRNA.
  • BC16 polynucleotide or polypeptide sequences are publicly available.
  • BC16 polynucleotide or polypeptide sequences include, but are not limited to, the sequences set forth in GenBank accession numbers: AW470156, BC146796, BC150184, CN401512, AW337942, EU883531, Z21943, AI624861, DB025893 and UOOl 15, herein incorporated by reference.
  • HoxA9 (homeobox A9) is a gene that encodes a DNA-binding transcription factor which may regulate gene expression, morphogenesis, and differentiation.
  • a " HoxA9 polynucleotide” refers to a polynucleotide encoding a HoxA9 polypeptide.
  • a HoxA9 polynucleotide can be an mRNA that encodes a HoxA9 polypeptide or a chromosomal DNA sequence that comprises the gene sequences encoding the above- referenced mRNA.
  • HoxA9 polynucleotide or polypeptide sequences are publicly available.
  • BC16 polynucleotide or polypeptide sequences include, but are not limited to, the sequences set forth in Genbank accession numbers: AW612618, BC006537, BG258601, and CA442923, herein incorporated by reference.
  • assessing includes any form of measurement, and includes determining if an element is present or not.
  • the terms “determining”, “measuring”, “evaluating”, “assessing” and “assaying” are used interchangeably and include quantitative and qualitative determinations. Assessing may be relative or absolute. “Assessing the presence of includes determining the amount of something present, and/or determining whether it is present or absent. As used herein, the terms “determining,” “measuring,” and “assessing,” and “assaying” are used interchangeably and include both quantitative and qualitative determinations.
  • reference and “control” are used interchangeably to refer to a known value or set of known values against which an observed value may be compared.
  • known means that the value represents an understood parameter, e.g., a level of expression of a marker gene.
  • the phrase "increased or decreased expression” is used to mean an increase or decrease in the transcription of one or more genes of interest, resulting in an increase or decrease in the levels of mRNA for each gene, respectively.
  • the phrase is also used to mean an increase or decrease in the levels of the corresponding gene product (e.g., protein) in the cell, independent of transcription levels or rates. For example, an increase in degradation rate of an mRNA encoding the protein in question, without a change in the transcription rate, may result in a decrease in the levels of protein in the cell.
  • the measured expression level of BCl-6 is greater than the measured expression level of the control.
  • the measured expression level of Menl is lower than the measured expression level of the control.
  • the measured expression level of HoxA9 is lower than the measured expression level of the control.
  • a "diabetes-related condition" comprises one or more of the following: 1) Type 1 diabetes mellitus; 2) Type 2 diabetes mellitus; 3) inherited forms of diabetes mellitus; 4) gestational diabetes mellitus; 5) medically induced diabetes mellitus; and 6) diabetes mellitus induced from malnutrition, infection and illness.
  • the diabetes is a Type 2 diabetes-related condition.
  • the diabetes-related condition is related to abnormal growth or condition of ⁇ -islet cells.
  • the ⁇ -islet cells are insulin-producing cells.
  • the monitoring, detection or diagnosis of a diabetes-related condition comprises one or more of: (1) predicting diabetes risk, (2) monitoring ongoing ⁇ - cell proliferation and ⁇ -cell mass changes, (3) predicting individual 'potential' for ⁇ -cell expansion or regeneration and (4) detecting 'pre-clinical' diabetes.
  • menin-related factors Markers suitable for the detection, diagnosis or monitoring of a diabetes-related condition according to the invention are collectively described as "menin-related factors.”
  • menin-related factors include, but are not limited to, Menl, BCl-6, HoxA9, CCNDl, CCND2, pl 8 INK4C , MafA, Maf ⁇ , phosphatase calcineurin, NFATcI, Pbx2, and MLLl .
  • the menin-related factors comprise Menl, BCl-6, and HoxA9.
  • the target of a menin-related factor would be one that is regulated by menin or other protein of interest in the menin pathway.
  • HoxA9 should follow the same directional changes in cell level as confirming the menin result.
  • the state of the host is considered to determine the normal level, e.g. pregnant or non-pregnant
  • the normal level e.g. pregnant or non-pregnant
  • there will be at least about a 1.5 fold difference between normal and dysfunctional usually at least about a 2 fold difference and may be as high as 4 fold or more.
  • menin levels are at least 2 fold higher.
  • the subject method is primarily directed to humans, it may also serve for other mammals, such as murine, mouse and rat, ovine, equine, feline, canine, bovine, etc.
  • the menin or other relevant protein level namely a protein in the pathway controlling ⁇ -islet cell population, or a standard or control protein
  • blood cells particularly leucocytes.
  • a particular leukocyte population e.g. lymphocytes, including B- and T-cells, neutrophils, eosinophils, basophils, monocytes, etc.
  • a labeled antibody e.g. fluorescent labeled monoclonal antibody, specific for the desired population, and cell sort in a fluorescence activated cell sorter.
  • a number of cells are selected that provides a response assay response.
  • the cells may then be lysed in a conventional lysing buffer, numerous lysing buffers are commercially available.
  • the lysate is then assayed for the menin concentration, usually providing a control, such as cells from a normal population, a cell line modified to provide a known level of menin, menin in an appropriate medium, menin mRNA, or a plot obtained with known amounts of menin or mRNA from a reproducible source.
  • the diagnostic/detection agent is a small molecule that preferentially binds to a menin-related factor according to the invention.
  • "Small molecule” as used herein, is meant to refer to a composition, which has a molecular weight of less than about 5 kD and preferably less than about 2.5 kD.
  • Small molecules can be nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids or other organic (carbon containing) or inorganic molecules.
  • Many pharmaceutical companies have extensive libraries of chemical and/or biological mixtures comprising arrays of small molecules, often fungal, bacterial, or algal extracts, which can be screened with any of the assays of the invention.
  • the diagnostic/detection agent is an antibody, preferably a monoclonal antibody, preferably linked to a detectable agent.
  • the expression of menin-related factors is monitored at the mRNA and/or protein level.
  • Methods of isolating total mRNA are well known to those of skill in the art. For example, methods of isolation and purification of nucleic acids are described in detail in Chapter 3 of Laboratory Techniques in Biochemistry and Molecular Biology: Hybridization With Nucleic Acid Probes, Part I. Theory and Nucleic Acid Preparation, P. Tijssen, ed. Elsevier, N. Y. (1993) and Chapter 3 of Laboratory Techniques in Biochemistry and Molecular Biology: Hybridization With Nucleic Acid Probes, Part I. Theory and Nucleic Acid Preparation, P. Tijssen, ed. Elsevier, N.Y. (1993)).
  • the total nucleic acid is isolated from a given sample using, for example, an acid guanidinium-phenol-chloroform extraction method and polyA + mRNA is isolated by oligo-dT column chromatography or by using (dT)n magnetic beads (see, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual (2nd ed.), VoIs. 1-3, Cold Spring Harbor Laboratory, (1989), or Current Protocols in Molecular Biology, F. Ausubel et al., ed. Greene Publishing and Wiley-Interscience, New York (1987)).
  • Methods of "quantitative" amplification are well known to those of skill in the art.
  • quantitative PCR involves simultaneously co-amplifying a known quantity of a control sequence using the same primers. This provides an internal standard that may be used to calibrate the PCR reaction. The assay method may then include probes specific to the internal standard for quantification of the amplified nucleic acid.
  • One internal standard is a synthetic A W 106 cRNA.
  • the AW 106 cRNA is combined with RNA isolated from the sample according to standard techniques known to those of skill in the art.
  • the RNA is then reverse transcribed using a reverse transcriptase to provide copy DNA.
  • the cDNA sequences are then amplified (e.g., by PCR) using labeled primers.
  • the amplification products are separated, typically by electrophoresis, and the amount of radioactivity (proportional to the amount of amplified product) is determined.
  • the amount of mRNA in the sample is then calculated by comparison with the signal produced by the known AW 106 RNA standard.
  • Detailed protocols for quantitative PCR are provided in PCR Protocols, A Guide to Methods and Applications, Innis et al., Academic Press, Inc. N.Y., (1990).
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • the sample mRNA is reverse transcribed with a reverse transcriptase and a primer consisting of oligo dT and a sequence encoding the phage T7 promoter to provide single stranded DNA template.
  • the second DNA strand is polymerized using a DNA polymerase.
  • T7 RNA polymerase is added and RNA is transcribed from the cDNA template. Successive rounds of transcription from each single cDNA template results in amplified RNA.
  • the direct transcription method described above provides an antisense (aRNA) pool.
  • aRNA antisense
  • the oligonucleotide probes provided in the array are chosen to be complementary to subsequences of the antisense nucleic acids.
  • the target nucleic acid pool is a pool of sense nucleic acids
  • the oligonucleotide probes are selected to be complementary to subsequences of the sense nucleic acids.
  • the probes may be of either sense as the target nucleic acids include both sense and antisense strands.
  • the protocols cited above include methods of generating pools of either sense or antisense nucleic acids. Indeed, one approach can be used to generate either sense or antisense nucleic acids as desired.
  • the cDNA can be directionally cloned into a vector (e.g., Stratagene's pBluescript II KS (+) phagemid) such that it is flanked by the T3 and T7 promoters. In vitro transcription with the T3 polymerase will produce RNA of one sense (the sense depending on the orientation of the insert), while in vitro transcription with the T7 polymerase will produce RNA having the opposite sense.
  • a vector e.g., Stratagene's pBluescript II KS (+) phagemid
  • In vitro transcription with the T3 polymerase will produce RNA of one sense (the sense depending on the orientation of the insert), while in vitro transcription with the T7 polymerase will produce RNA having the opposite sense.
  • Other suitable cloning systems include phage lambda
  • RNA polymerase e.g. about 2500 units/ ⁇ L for T7, available from Epicentre Technologies.
  • the hybridized nucleic acids are detected by detecting one or more labels attached to the sample nucleic acids.
  • the labels may be incorporated by any of a number of means. However, desirably, the label is simultaneously incorporated during the amplification step in the preparation of the sample nucleic acids.
  • PCR polymerase chain reaction
  • labeled primers or labeled nucleotides will provide a labeled amplification product.
  • transcription amplification as described above, using a labeled nucleotide (e.g. fluorescein-labeled UTP and/or CTP) incorporates a label into the transcribed nucleic acids.
  • a label may be added directly to the original nucleic acid sample (e.g., mRNA, polyA mRNA, cDNA, etc.) or to the amplification product after the amplification is completed.
  • Means of attaching labels to nucleic acids are well known and include, for example nick translation or end-labeling (e.g. with a labeled RNA) by kinasing of the nucleic acid and subsequent attachment (ligation) of a nucleic acid linker joining the sample nucleic acid to a label (e.g., a fluorophore).
  • Detectable labels suitable for use in the present invention include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means.
  • Useful labels in the present invention include biotin for staining with labeled streptavidin conjugate, magnetic beads (e.g., DynabeadsTM), fluorescent dyes (e.g., fluorescein, Texas Red ® , rhodamine, green fluorescent protein, and the like), radiolabels (e.g., 3 H, 125 1, 35 S, 14 C, or 32 P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and colorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads.
  • Patents teaching the use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,9
  • radiolabels may be detected using photographic film or scintillation counters, while fluorescent markers may be detected using a photodetector to detect emitted light.
  • Enzymatic labels are typically detected by providing the enzyme with a substrate and detecting the reaction product produced by the action of the enzyme on the substrate, and colorimetric labels are detected by simply visualizing the colored label.
  • the label may be added to the target (sample) nucleic acid(s) prior to, or after the hybridization.
  • direct labels are detectable labels that are directly attached to or incorporated into the target (sample) nucleic acid prior to hybridization.
  • indirect labels are joined to the hybrid duplex after hybridization.
  • the indirect label is attached to a binding moiety that has been attached to the target nucleic acid prior to the hybridization.
  • the target nucleic acid may be biotinylated before the hybridization. After hybridization, an avidin-conjugated fluorophore will bind the biotin bearing hybrid duplexes providing a label that is easily detected.
  • Fluorescent labels are preferred and easily added during an in vitro transcription reaction.
  • fluorescein labeled UTP and CTP are incorporated into the RNA produced in an in vitro transcription reaction as described above.
  • Numerous references describe methods of quantitating mRNA from a cellular sample. See, for example, U.S. Patent nos. 5,219,727; 6,063,568; 6,300,058; and 7,070,925, and U.S. Patent application nos. 20050137388; 20060257889; 20070059690; 20070072229; and 20070190560, and references contained therein.
  • the proteins may be determined directly.
  • the amount of protein is most conveniently determined using an immunoassay employing labeled antibodies or labeled protein competitors, particularly monoclonal antibodies, specific for each of the proteins of interest.
  • labeled monoclonal antibodies are not commercially available.
  • the preparation of monoclonal antibodies is well established and amply described in the literature.
  • Commercially available assays include RIA, ELISA, EMIT®, CED IA®, Western blot, FIA, etc. Descriptions of these and other assays may be found in U.S. Patent nos.
  • the isolated cells may be lysed, particulate material removed by centrifugation, and the supernatant analyzed by mixing an aliquot of the supernatant with a labeled competitor for the protein and obtaining a signal.
  • a labeled competitor for the protein for the protein and obtaining a signal.
  • the status of the ⁇ -islet cells is established by investigating the proteins in the menin pathway, such as menin which diminishes in conjunction with expansion of ⁇ -islet cells are appropriate and Bcl6 which increases in conjunction with expansion of ⁇ -islet cells.
  • the determination may be prospective in determining how a patient will respond to a condition that normally results in increased ⁇ -islet cells, e.g. pregnancy, obesity, or other condition requiring enhanced insulin production, at the time of the condition or retrospective in analyzing the cause of a particular condition associated with glucose metabolism, e.g. diabetes mellitus.
  • the human patient may then be counseled or treated to correct the dysfunction.
  • aspects of the invention include methods of counseling humans having a dysregulation potential of menin modulation of ⁇ -islet cell proliferation.
  • the condition of such dysregulation may involve a propensity for Type 2 diabetes mellitus, may be involved with pregnancy or other condition requiring the expansion or contraction of the ⁇ -islet population.
  • Menin levels or related factor, e.g. Bcl6, associated with the pathway including menin associated with ⁇ -islet proliferation or contraction is determined from a blood sample. The determination can be correlated with the ability of ⁇ -islet cell expansion or contraction.
  • One can monitor whether the ⁇ -islet cells are properly responding to the condition of the individual that warrants an expansion or contraction of the number of ⁇ -islet cells.
  • One or more of the proteins or their mRNAs may be determined.
  • an internal control such as a target in the cells being assayed.
  • menin and/or menin related factors play a role in regulating ⁇ -islet cell proliferation.
  • menin can be determined by measuring mRNA or protein levels. It is shown that the menin pathway regulates the ⁇ -islet cell proliferation where higher concentrations of cellular menin results in inhibition of proliferation, while lower concentrations allow for cellular expansion. It is also shown that white blood cells, particularly lymphocytes, can be used as a surrogate for the ⁇ - islet cell menin in that the level of menin in the white blood cells tracks with the level in the ⁇ -islet cells.
  • a method of modulating ⁇ -islet cell proliferation is provided.
  • methods of treating cells or individuals with a diabetes-related condition are provided.
  • the method comprises administration of an agent .
  • the cancer inhibitor is an antisense molecule, a pharmaceutical composition, a therapeutic agent or small molecule, or a monoclonal, polyclonal, chimeric or humanized antibody.
  • a therapeutic agent is coupled with a an antibody, preferable a monoclonal antibody.
  • the menin-related factor modulator is an antisense molecule.
  • Antisense molecules as used herein include antisense or sense oligonucleotides comprising a single-stranded nucleic acid sequence (either RNA or DNA) capable of binding to target mRNA (sense) or DNA (antisense) sequences for menin-related factor molecules.
  • Antisense or sense oligonucleotides, according to the present invention comprise a fragment generally at least about 14 nucleotides, preferably from about 14 to 30 nucleotides.
  • Antisense molecules can be modified or unmodified RNA, DNA, or mixed polymer oligonucleotides. These molecules function by specifically binding to matching sequences resulting in inhibition of peptide synthesis (Wu-Pong, November 1994, BioPharm, 20-33) either by steric blocking or by activating an RNase H enzyme. Antisense molecules can also alter protein synthesis by interfering with RNA processing or transport from the nucleus into the cytoplasm (Mukhopadhyay & Roth, 1996, Crit. Rev. in Oncogenesis 7, 151-190). In addition, binding of single stranded DNA to RNA can result in nuclease-mediated degradation of the heteroduplex (Wu-Pong, supra).
  • Backbone modified DNA chemistry which has thus far been shown to act as substrates for RNase H are phosphorothioates, phosphorodithioates, borontrifluoridates, and 2'-arabino and 2'-fluoro arabino-containing oligonucleotides.
  • Antisense molecules may be introduced into a cell containing the target nucleotide sequence by formation of a conjugate with a ligand binding molecule, as described in WO 91/04753.
  • Suitable ligand binding molecules include, but are not limited to, cell surface receptors, growth factors, other cytokines, or other ligands that bind to cell surface receptors.
  • conjugation of the ligand binding molecule does not substantially interfere with the ability of the ligand binding molecule to bind to its corresponding molecule or receptor, or block entry of the sense or antisense oligonucleotide or its conjugated version into the cell.
  • a sense or an antisense oligonucleotide may be introduced into a cell containing the target nucleic acid sequence by formation of an oligonucleotide-lipid complex, as described in WO 90/10448. It is understood that the use of antisense molecules or knock out and knock in models may also be used in screening assays as discussed above, in addition to methods of treatment.
  • RNA interference refers to the process of sequence-specific post transcriptional gene silencing in animals mediated by short interfering RNAs (siRNA) (Fire et al., Nature, 391, 806 (1998)). The corresponding process in plants is referred to as post transcriptional gene silencing or RNA silencing and is also referred to as quelling in fungi. The presence of dsRNA in cells triggers the RNAi response though a mechanism that has yet to be fully characterized.
  • siRNA short interfering RNAs
  • RNA interference Small interfering RNAs
  • siRNAs are powerful sequence-specific reagents designed to suppress the expression of genes in cultured mammalian cells through a process known as RNA interference (RNAi).
  • RNAi RNA interference
  • siRNA refers to a double stranded nucleic acid molecule capable of RNA interference "RNAi", (see Kreutzer et al., WO 00/44895; Zernicka-Goetz et al. WO 01/36646; Fire, WO 99/32619; Mello and Fire, WO 01/29058).
  • RNAi RNA interference
  • siRNA molecules are limited to RNA molecules but further encompass chemically modified nucleotides and non-nucleotides. siRNA gene-targeting experiments have been carried out by transient siRNA transfer into cells (achieved by such classic methods as liposome-mediated transfection, electroporation, or microinjection).
  • Molecules of siRNA are 21- to 23-nucleotide RNAs, with characteristic 2- to 3- nucleotide 3'-overhanging ends resembling the RNase III processing products of long double- stranded RNAs (dsRNAs) that normally initiate RNAi.
  • dsRNAs long double- stranded RNAs
  • RNA-induced silencing complex an endonuclease complex
  • siRNAs compared with traditional antisense molecules, prevents activation of the dsRNA-inducible interferon system present in mammalian cells. This avoids the nonspecific phenotypes normally produced by dsRNA larger than 30 base pairs in somatic cells.
  • RNA polymerase II RNA polymerase II
  • snRNA small nuclear RNA
  • RNA polymerase II RNA polymerase II
  • Two approaches have been developed for expressing siRNAs: in the first, sense and antisense strands constituting the siRNA duplex are transcribed by individual promoters (Lee, N. S. et al. Nat. Biotechnol. 20, 500-505 (2002). Miyagishi, M. & Taira, K. Nat. Biotechnol.
  • siRNAs are expressed as fold-back stem-loop structures that give rise to siRNAs after intracellular processing (Paul, C. P. et al. Nat. Biotechnol. 20:505-508 (2002)).
  • the endogenous expression of siRNAs from introduced DNA templates is thought to overcome some limitations of exogenous siRNA delivery, in particular the transient loss of phenotype.
  • U6 and Hl RNA promoters are members of the type III class of Pol III promoters. (Paule, M. R. & White, R. J. Nucleic Acids Res. 28, 1283-1298 (2000)).
  • the DNA-based methodology may also be a cost-effective alternative for automated genome-wide loss-of-function phenotypic analysis, especially when combined with miniaturized array-based phenotypic screens. (Ziauddin, J. & Sabatini, D. M. Nature 411:107-110 (2001)).
  • dsRNAs The presence of long dsRNAs in cells stimulates the activity of a ribonuclease III enzyme referred to as dicer.
  • Dicer is involved in the processing of the dsRNA into short pieces of dsRNA known as short interfering RNAs (siRNA) (Berstein et al., 2001, Nature, 409:363 (2001)).
  • Short interfering RNAs derived from dicer activity are typically about 21- 23 nucleotides in length and comprise about 19 base pair duplexes.
  • Dicer has also been implicated in the excision of 21 and 22 nucleotide small temporal RNAs (stRNA) from precursor RNA of conserved structure that are implicated in translational control (Hutvagner et al., Science, 293, 834 (2001)).
  • the RNAi response also features an endonuclease complex containing a siRNA, commonly referred to as an RNA-induced silencing complex (RISC), which mediates cleavage of single stranded RNA having sequence homologous to the siRNA. Cleavage of the target RNA takes place in the middle of the region complementary to the guide sequence of the siRNA duplex Elbashir et al., Genes Dev., 15, 188 (2001)).
  • RISC RNA-induced silencing complex
  • This invention provides an expression system comprising an isolated nucleic acid molecule comprising a sequence capable of specifically hybridizing to the menin-related factor sequences.
  • the nucleic acid molecule is capable of inhibiting the expression of the menin-related factor protein.
  • a method of inhibiting expression of menin- related factor inside a cell by a vector-directed expression of a short RNA which short RNA can fold in itself and create a double strand RNA having menin-related factor mRNA sequence identity and able to trigger posttranscriptional gene silencing, or RNA interference (RNAi), of the menin-related factor gene inside the cell.
  • RNAi RNA interference
  • RNA having menin-related factor mRNA sequence identity is delivered inside the cell to trigger posttranscriptional gene silencing, or RNAi, of the menin-related factor gene.
  • the nucleic acid molecule is at least a 7 mer, at least a 10 mer, or at least a 20 mer.
  • the sequence is unique.
  • compositions encompassed by the present invention include as active agent, the polypeptides, polynucleotides, antisense oligonucleotides, or antibodies of the invention disclosed herein in a therapeutically effective amount.
  • An "effective amount" is an amount sufficient to effect beneficial or desired results, including clinical results.
  • An effective amount can be administered in one or more administrations.
  • an effective amount of an adenoviral vector is an amount that is sufficient to palliate, ameliorate, stabilize, reverse, slow or delay the progression of the disease state.
  • compositions can be used to treat or prevent diabetes or a diabetes-related condition.
  • pharmaceutical compositions can be used in conjunction with conventional methods of diabetes treatment.
  • treatment used herein to generally refer to obtaining a desired pharmacologic and/or physiologic effect.
  • the effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete stabilization or cure for a disease and/or adverse effect attributable to the disease.
  • Treatment covers any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease or symptom from occurring in a subject which may be predisposed to the disease or symptom but has not yet been diagnosed as having it; (b) inhibiting the disease symptom, i.e., arresting its development; or (c) relieving the disease symptom, i.e., causing regression of the disease or symptom.
  • the pharmaceutical composition comprises an antibody that specifically binds to a gene product encoded by a differentially expressed polynucleotide
  • the antibody can be coupled to a drug for delivery to a treatment site or coupled to a detectable label to facilitate imaging of a site comprising lymphocytes or leukocytes.
  • Methods for coupling antibodies to drugs and detectable labels are well known in the art, as are methods for imaging using detectable labels.
  • a "patient” for the purposes of the present invention includes both humans and other animals, particularly mammals, and organisms. Thus the methods are applicable to both human therapy and veterinary applications. In the preferred embodiment the patient is a mammal, and in the most preferred embodiment the patient is human.
  • therapeutically effective amount refers to an amount of a therapeutic agent to treat, ameliorate, or prevent a desired disease or condition, or to exhibit a detectable therapeutic or preventative effect.
  • the effect can be detected by, for example, chemical markers or antigen levels.
  • Therapeutic effects also include reduction in physical symptoms, such as decreased body temperature.
  • the precise effective amount for a subject will depend upon the subject's size and health, the nature and extent of the condition, and the therapeutics or combination of therapeutics selected for administration. The effective amount for a given situation is determined by routine experimentation and is within the judgment of the clinician.
  • an effective dose will generally be from about 0.01 mg/kg to about 5 mg/kg, or about 0.01 mg/kg to about 50 mg/kg or about 0.05 mg/kg to about 10 mg/kg of the compositions of the present invention in the individual to which it is administered.
  • a pharmaceutical composition can also contain a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier refers to a carrier for administration of a therapeutic agent, such as antibodies or a polypeptide, genes, and other therapeutic agents. The term refers to any pharmaceutical carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which can be administered without undue toxicity.
  • Suitable carriers can be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles. Such carriers are well known to those of ordinary skill in the art.
  • Pharmaceutically acceptable carriers in therapeutic compositions can include liquids such as water, saline, glycerol and ethanol.
  • the therapeutic compositions are prepared as injectables, either as liquid solutions or suspensions, solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared.
  • Liposomes are included within the definition of a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable salts can also be present in the pharmaceutical composition, e.g., mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like.
  • compositions can be prepared in various forms, such as granules, tablets, pills, suppositories, capsules, suspensions, salves, lotions and the like.
  • Pharmaceutical grade organic or inorganic carriers and/or diluents suitable for oral and topical use can be used to make up compositions containing the therapeutically-active compounds.
  • Diluents known to the art include aqueous media, vegetable and animal oils and fats. Stabilizing agents, wetting and emulsifying agents, salts for varying the osmotic pressure or buffers for securing an adequate pH value, and skin penetration enhancers can be used as auxiliary agents.
  • compositions of the present invention comprise a menin-related factor protein in a form suitable for administration to a patient.
  • the pharmaceutical compositions are in a water soluble form, such as being present as pharmaceutically acceptable salts, which is meant to include both acid and base addition salts.
  • “Pharmaceutically acceptable acid addition salt” refers to those salts that retain the biological effectiveness of the free bases and that are not biologically or otherwise undesirable, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like
  • organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid,
  • “Pharmaceutically acceptable base addition salts” include those derived from inorganic bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Particularly preferred are the ammonium, potassium, sodium, calcium, and magnesium salts.
  • Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine.
  • compositions may also include one or more of the following: carrier proteins such as serum albumin; buffers; fillers such as microcrystalline cellulose, lactose, corn and other starches; binding agents; sweeteners and other flavoring agents; coloring agents; and polyethylene glycol.
  • carrier proteins such as serum albumin
  • buffers such as buffers
  • fillers such as microcrystalline cellulose, lactose, corn and other starches
  • binding agents such as microcrystalline cellulose, lactose, corn
  • the compounds having the desired pharmacological activity may be administered in a physiologically acceptable carrier to a host, as previously described.
  • the agents may be administered in a variety of ways, orally, parenterally e.g., subcutaneously, intraperitoneally, intravascularly, etc.
  • the compounds may be formulated in a variety of ways.
  • the concentration of therapeutically active compound in the formulation may vary from about 0.1-100% wt/vol.
  • compositions contemplated by the invention can be (1) administered directly to the subject (e.g., as polynucleotide, polypeptides, small molecule agonists or antagonists, and the like); or (2) delivered ex vivo, to cells derived from the subject (e.g., as in ex vivo gene therapy).
  • Direct delivery of the compositions will generally be accomplished by parenteral injection, e.g., subcutaneously, intraperitoneally, intravenously, intramuscularly, or to the interstitial space of a tissue.
  • Other modes of administration include oral and pulmonary administration, suppositories, and transdermal applications, needles, and gene guns or hyposprays.
  • Dosage treatment can be a single dose schedule or a multiple dose schedule.
  • Methods for the ex vivo delivery and reimplantation of transformed cells into a subject are known in the art and described in e.g., International Publication No. WO 93/14778.
  • Examples of cells useful in ex vivo applications include, for example, stem cells, particularly hematopoietic, lymph cells, macrophages, dendritic cells, or tumor cells.
  • nucleic acids for both ex vivo and in vitro applications can be accomplished by, for example, dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei, all well known in the art.
  • the condition can be amenable to treatment by administration of a therapeutic agent based on the provided polynucleotide, corresponding polypeptide or other corresponding molecule (e.g., antisense, ribozyme, etc.).
  • a therapeutic agent based on the provided polynucleotide, corresponding polypeptide or other corresponding molecule (e.g., antisense, ribozyme, etc.).
  • the disorder can be amenable to treatment by administration of a small molecule drug that, for example, serves as an inhibitor (antagonist) of the function of the encoded gene product of a gene having increased expression in diabetic ⁇ -islet cells relative to normal cells or as an agonist for gene products that are decreased in expression in diabetic ⁇ -islet cells (e.g., to promote the activity of gene products that act as suppressors).
  • a small molecule drug that, for example, serves as an inhibitor (antagonist) of the function of the encoded gene product of a gene having increased expression in diabetic ⁇ -islet cells relative to normal cells or as an agonist for gene products that are decreased in expression in diabetic ⁇ -islet cells (e.g., to promote the activity of gene products that act as suppressors).
  • the dose and the means of administration of the inventive pharmaceutical compositions are determined based on the specific qualities of the therapeutic composition, the condition, age, and weight of the patient, the progression of the disease, and other relevant factors.
  • administration of polynucleotide therapeutic compositions agents includes local or systemic administration, including injection, oral administration, particle gun or catheterized administration, and topical administration.
  • the therapeutic polynucleotide composition contains an expression construct comprising a promoter operably linked to a polynucleotide of at least 12, 22, 25, 30, or 35 contiguous nucleotides of the menin-related factor polynucleotides disclosed herein.
  • Various methods can be used to administer the therapeutic composition directly to a specific site in the body.
  • a small metastatic lesion is located and the therapeutic composition injected several times in several different locations within the body of tumor.
  • arteries that serve a tumor are identified, and the therapeutic composition injected into such an artery, in order to deliver the composition directly into the tumor.
  • a tumor that has a necrotic center is aspirated and the composition injected directly into the now empty center of the tumor.
  • An antisense composition is directly administered to the surface of the tumor, for example, by topical application of the composition.
  • X-ray imaging is used to assist in certain of the above delivery methods.
  • Targeted delivery of therapeutic compositions containing an antisense polynucleotide, subgenomic polynucleotides, or antibodies to specific tissues can also be used.
  • Receptor- mediated DNA delivery techniques are described in, for example, Findeis et al., Trends Biotechnol. (1993) 1 1 :202; Chiou et al., Gene Therapeutics: Methods And Applications Of Direct Gene Transfer (J. A. Wolff, ed.) (1994); Wu et al., J. Biol Chem. (1988) 263:621; Wu et al., J. Biol. Chem. (1994) 269:542; Zenke et al., Proc. Natl. Acad.
  • compositions containing a polynucleotide are administered in a range of about 100 ng to about 200 mg of DNA for local administration in a gene therapy protocol. Concentration ranges of about 500 ng to about 50 mg, about 1 ⁇ g to about 2 mg, about 5 ⁇ g to about 500 ⁇ g, and about 20 ⁇ g to about 100 ⁇ g of DNA can also be used during a gene therapy protocol.
  • Factors such as method of action (e.g., for enhancing or inhibiting levels of the encoded gene product) and efficacy of transformation and expression are considerations that will affect the dosage required for ultimate efficacy of the antisense subgenomic polynucleotides. Where greater expression is desired over a larger area of tissue, larger amounts of antisense subgenomic polynucleotides or the same amounts re-administered in a successive protocol of administrations. In all cases, routine experimentation in clinical trials will determine specific ranges for optimal therapeutic effect.
  • the therapeutic polynucleotides and polypeptides of the present invention can be delivered using gene delivery vehicles.
  • the gene delivery vehicle can be of viral or non-viral origin (see generally, Jolly, Diabetes related conditions Gene Therapy (1994) 1 :51 ; Kimura, Human Gene Therapy (1994) 5:845; Connelly, Human Gene Therapy (1995) 1 :185; and Kaplitt, Nature Genetics (1994) 6:148).
  • Expression of such coding sequences can be induced using endogenous mammalian or heterologous promoters. Expression of the coding sequence can be either constitutive or regulated.
  • Viral-based vectors for delivery of a desired polynucleotide and expression in a desired cell are well known in the art.
  • Exemplary viral-based vehicles include, but are not limited to, recombinant retroviruses (see, e.g., WO 90/07936; WO 94/03622; WO 93/25698; WO 93/25234; U.S. Pat. No. 5,219,740; WO 93/11230; WO 93/10218; U.S. Pat. No. 4,777,127; GB Patent No.
  • alphavirus-based vectors e.g., Sindbis virus vectors, Semliki forest virus (ATCC VR-67; ATCC VR-1247), Ross River virus (ATCC VR-373; ATCC VR-1246) and Venezuelan equine encephalitis virus (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532)
  • AAV adeno- associated virus
  • Non-viral delivery vehicles and methods can also be employed, including, but not limited to, polycationic condensed DNA linked or unlinked to killed adenovirus alone (see, e.g., Curiel, Hum. Gene Ther. (1992) 3:147); ligand-linked DNA (see, e.g., Wu, J. Biol. Chem. (1989) 264:16985); eukaryotic cell delivery vehicles cells (see, e.g., U.S. Pat. No. 5,814,482; WO 95/07994; WO 96/17072; WO 95/30763; and WO 97/42338) and nucleic charge neutralization or fusion with cell membranes. Naked DNA can also be employed.
  • Exemplary naked DNA introduction methods are described in WO 90/11092 and U.S. Pat. No. 5,580,859.
  • Liposomes that can act as gene delivery vehicles are described in U.S. Pat. No. 5,422,120; WO 95/13796; WO 94/23697; WO 91/14445; and EP 0524968. Additional approaches are described in Philip, MoI. Cell Biol. (1994) 14:2411, and in Woffendin, Proc. Natl. Acad. Sci. (1994) 91 : 1581.
  • non-viral delivery suitable for use includes mechanical delivery systems such as the approach described in Woffendin et al., Proc. Natl. Acad. Sci. USA (1994) 91(24): 1 1581.
  • the coding sequence and the product of expression of such can be delivered through deposition of photopolymerized hydrogel materials or use of ionizing radiation (see, e.g., U.S. Pat. No. 5,206,152 and WO 92/11033).
  • Other conventional methods for gene delivery that can be used for delivery of the coding sequence include, for example, use of hand-held gene transfer particle gun (see, e.g., U.S. Pat. No. 5,149,655); use of ionizing radiation for activating transferred gene (see, e.g., U.S. Pat. No. 5,206,152 and WO 92/11033).
  • menin-related factor proteins and modulators of the present invention can be done in a variety of ways as discussed above, including, but not limited to, orally, subcutaneously, intravenously, intranasally, transdermally, intraperitoneal Iy, intramuscularly, intrapulmonary, vaginally, rectally, or intraocularly.
  • the menin-related factor proteins and modulators may be directly applied as a solution or spray.
  • menin-related factor proteins and modulators are administered as therapeutic agents, and can be formulated as outlined above.
  • menin-related factor genes (including the full-length sequence, partial sequences, or regulatory sequences of the menin-related factor coding regions) can be administered in gene therapy applications, as is known in the art.
  • These menin-related factor genes can include antisense applications, either as gene therapy (i.e. for incorporation into the genome) or as antisense compositions, as will be appreciated by those in the art.
  • methods of modulating menin-related factor gene activity in cells or organisms are provided.
  • the methods comprise administering to a cell an anti-menin-related factor antibody that reduces or eliminates the biological activity of an endogenous menin-related factor protein.
  • the methods comprise administering to a cell or organism a recombinant nucleic acid encoding a menin-related factor protein. As will be appreciated by those in the art, this may be accomplished in any number of ways.
  • the activity of the menin-related factor gene product is increased by increasing the amount of menin-related factor expression in the cell, for example by overexpressing the endogenous menin-related factor gene or by administering a gene encoding the menin-related factor sequence, using known gene-therapy techniques.
  • the gene therapy techniques include the incorporation of the exogenous gene using enhanced homologous recombination (EHR), for example as described in PCT/US93/03868, hereby incorporated by reference in its entirety.
  • EHR enhanced homologous recombination
  • the activity of the endogenous menin-related factor gene is decreased, for example by the administration of a menin-related factor antisense nucleic acid.
  • menin-related factor genes are administered as DNA vaccines, either single genes or combinations of menin-related factor genes. Naked DNA vaccines are generally known in the art. Brower, Nature Biotechnology, 16: 1304-1305 (1998).
  • menin-related factor genes of the present invention are used as DNA vaccines.
  • Methods for the use of genes as DNA vaccines are well known to one of ordinary skill in the art, and include placing a menin-related factor gene or portion of a menin-related factor gene under the control of a promoter for expression in a patient with diabetes related conditions.
  • the menin-related factor gene used for DNA vaccines can encode full-length menin-related factor proteins, but more preferably encodes portions of the menin-related factor proteins including peptides derived from the menin-related factor protein.
  • a patient is immunized with a DNA vaccine comprising a plurality of nucleotide sequences derived from a menin-related factor gene.
  • the DNA vaccines include a gene encoding an adjuvant molecule with the DNA vaccine.
  • adjuvant molecules include cytokines that increase the immunogenic response to the menin-related factor polypeptide encoded by the DNA vaccine. Additional or alternative adjuvants are known to those of ordinary skill in the art and find use in the invention.
  • a menin-related factor modulator is an antibody as discussed above.
  • the menin-related factor proteins of the present invention may be used to generate polyclonal and monoclonal antibodies to menin-related factor proteins, which are useful as described herein.
  • the menin-related factor proteins can be coupled, using standard technology, to affinity chromatography columns. These columns may then be used to purify menin-related factor antibodies.
  • the antibodies are generated to epitopes unique to a menin-related factor protein; that is, the antibodies show little or no cross-reactivity to other proteins. These antibodies find use in a number of applications.
  • the menin-related factor antibodies may be coupled to standard affinity chromatography columns and used to purify menin-related factor proteins.
  • the antibodies may also be used therapeutically as blocking polypeptides, as outlined above, since they will specifically bind to the menin-related factor protein.
  • the present invention further provides methods for detecting the presence of and/or measuring a level of a polypeptide in a biological sample, which menin-related factor polypeptide is encoded by a menin-related factor polynucleotide that is differentially expressed in a diabetes related conditions cell, using an antibody specific for the encoded polypeptide.
  • the methods generally comprise: a) contacting the sample with an antibody specific for a polypeptide encoded by a menin-related factor polynucleotide that is differentially expressed in a prostate diabetes related conditions cell; and b) detecting binding between the antibody and molecules of the sample.
  • Detection of specific binding of the antibody specific for the encoded diabetes related conditions-associated polypeptide, when compared to a suitable control is an indication that encoded polypeptide is present in the sample.
  • Suitable controls include a sample known not to contain the encoded menin-related factor polypeptide or known not to contain elevated levels of the polypeptide; such as normal tissue, and a sample contacted with an antibody not specific for the encoded polypeptide, e.g., an anti-idiotype antibody.
  • a variety of methods to detect specific antibody-antigen interactions are known in the art and can be used in the method, including, but not limited to, standard immunohistological methods, immunoprecipitation, an enzyme immunoassay, and a radioimmunoassay.
  • the specific antibody will be detectably labeled, either directly or indirectly.
  • Direct labels include radioisotopes; enzymes whose products are detectable (e.g., luciferase, ⁇ -galactosidase, and the like); fluorescent labels (e.g., fluorescein isothiocyanate, rhodamine, phycoerythrin, and the like); fluorescence emitting metals, e.g., 152 Eu, or others of the lanthanide series, attached to the antibody through metal chelating groups such as EDTA; chemiluminescent compounds, e.g., luminol, isoluminol, acridinium salts, and the like; bioluminescent compounds, e.g., luciferin, aequorin (green fluorescent protein), and the like.
  • the antibody may be attached (coupled) to an insoluble support, such as a polystyrene plate or a bead.
  • Indirect labels include second antibodies specific for antibodies specific for the encoded polypeptide ("first specific antibody"), wherein the second antibody is labeled as described above; and members of specific binding pairs, e.g., biotin-avidin, and the like.
  • the biological sample may be brought into contact with and immobilized on a solid support or carrier, such as nitrocellulose, that is capable of immobilizing cells, cell particles, or soluble proteins.
  • the support may then be washed with suitable buffers, followed by contacting with a detectably- labeled first specific antibody. Detection methods are known in the art and will be chosen as appropriate to the signal emitted by the detectable label. Detection is generally accomplished in comparison to suitable controls, and to appropriate standards.
  • the methods are adapted for use in vivo, e.g., to locate or identify sites where diabetes related conditions cells are present.
  • a detectably-labeled moiety e.g., an antibody, which is specific for a diabetes related conditions-associated polypeptide is administered to an individual (e.g., by injection), and labeled cells are located using standard imaging techniques, including, but not limited to, magnetic resonance imaging, computed tomography scanning, and the like. In this manner, diabetes related conditions cells are differentially labeled.
  • the invention also relates to a method for identifying a candidate agent which regulates the activity of one or more of the menin-related factors, comprising: (a) providing a cell comprising said one or more menin-related factors (b) providing a candidate agent library; (c) selecting a candidate agent selected from the library; (d) contacting the cell with the candidate agent; and (e) measuring the activity of the one or more menin-related factors, wherein an increase or decrease of the activity of the one or more menin-related factors by at least 10% relative to the activity of the menin-related factors in the cell, wherein the cell is not contacted with the candidate agent, identifies the candidate agent as an agent which regulates the activity of said one or more polypeptides.
  • the activity of the menin-related factors is increased or decreases by at least 10%, 20%, 25%, 30%, 40%, 50%, 70%, 90% or 99%.
  • Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs. New potential therapeutic agents may also be created using methods such as rational drug design or computer modeling.
  • the candidate agent may be an agent selected from a small molecule, a small molecule, a peptide, a polyclonal antibody, a monoclonal antibody, an antisense RNA, a small interfering RNA (siRNA), a ribozyme, a short hairpin RNA (shRNA), a polypeptide, polysaccharide, lipid, nucleic acid, or combination thereof.
  • the candidate agent is effective for treating or preventing a risk of developing diabetes, pre-diabetes, or gestational diabetes in a subject.
  • the above screening methods may be part of a multi-step screening process of evaluating candidate therapeutic agents for their efficacy (and safety) in regulating the activity of one or more of the menin-related factors for treating or preventing a risk of developing diabetes, pre-diabetes, or gestational diabetes in mammalian hosts, e.g. humans.
  • a candidate compound or library of compounds is subjected to screening in a second in vivo model, e.g. a mouse model, following screening in the subject non-mammalian animal model.
  • the positive compounds are then screened in non-human mammalian animal models, including non- human mammalian animal models.
  • a pre in vivo screening step may be employed, in which the compound is first subjected to an in vitro screening assay for its potential as a therapeutic agent in regulating the activity of one or more of the menin-related factors. Any convenient in vitro screening assay may be employed, where a variety of suitable in vitro screening assays is known to those of skill in the art.
  • kits for use in aiding detection or diagnosis of a subject's risk of developing diabetes, pre-diabetes, or gestational diabetes may include at least one reagent specific for detecting a menin-related factor selected from: BCl-6, Menl, HoxA9, CCNDl, CCND2, pl8 INK4C , MafA, MafB, phosphatase calcineurin, NFATcI, Pbx2, and MLLl.
  • the kits may optionally include instructions for carrying out a method of aiding in the diagnosis of diabetes, pre-diabetes, or gestational diabetes.
  • the reagent specific for the menin-related factor may be an antibody, or fragment thereof, that is specific for the menin-related factor.
  • the kit may further include an amount of at least one menin-related factor suitable for normalizing data.
  • Example 2 Generation of mice that permit conditional Menl expression in ⁇ -cells
  • mice were generated that permitted conditional Menl expression in ⁇ -cells.
  • Transgenic mice producing hemagglutinin-tagged menin under control of the tetracycline response element (JRE-Menl; Karnik, SA, et al. Science 318: 806-809 (2007)) were generated and mated with mice expressing the reverse tetracycline trans-activator (rtTA) in ⁇ - cells directed by the rat insulin promoter ("RIP;" J. J. Heit et al, Nature 443, 345 (2006)).
  • JRE-Menl hemagglutinin-tagged menin under control of the tetracycline response element
  • rtTA reverse tetracycline trans-activator
  • TRE-Menl mice In bi-transgenic RlP-rtTA, TRE-Menl mice (abbreviated ⁇ Menl), administration of doxycycline (Dox) allows rtTA binding to the TRE element and stimulates ⁇ -cell expression of Menl mRNA and menin protein ( Figure 2, Panels A-B). Exposure ofRIP-rtTA or TRE-Menl single transgenic mice to Dox did not induce changes in menin levels ( Figure 2, Panels A-B). In islets isolated from male or virgin female ⁇ Menl on dox mice continuously exposed to Dox after 10 weeks of age, we detected increased levels of Menl, p27 and pl8 mRNA. Thus, conditional induction of menin in ⁇ -cells stimulated expression of known menin-target genes. However, TRE-Menl expression in male or virgin female ⁇ Menl mice did not alter serum insulin or glucose control ( Figure 2, Panels C-E), indicating that TRE-Me/?/ expression alone did not disrupt ⁇
  • Prolactin and placental lactogens are hormonal regulators of pregnancy that stimulate ⁇ -cell proliferation in rodent and human islets, but the molecular basis for their mitogenic effect is unknown.
  • T. C. Brelje et al. Endocrinology 132, 879 (1993); J.H. Nielsen Endocrinology 110, 600 (1982); L. Labriola et al, MoI Cell Endocrinol. 264, 16 (2007)
  • STAT5 Signal transducer and transactivator 5
  • Example 5 Lactogen signaling sufficient to reduce Menl expression and increase ⁇ -cell proliferation in vivo
  • mice were transplanted with osmotic micropumps to deliver prolactin for 6 days (T. Shingo et al, Science 299, 117 (2003)) to test if lactogen signaling was sufficient to reduce Menl expression and increase ⁇ -cell proliferation in vivo.
  • islets from prolactin-infused mice had a 4-fold increase in Bcl6 expression, a 50% reduction of Menl, p!8, and p27 mRNA, and a 2.5 fold increase of BrdU incorporation by ⁇ -cells.
  • prolactin infusion was sufficient to reduce Menl expression in vivo and to stimulate proliferation of adult islet ⁇ -cells.
  • Lactogenic hormone regulation of Menl may govern other features that affect ⁇ -cell expansion, such as ⁇ -cell size and survival (L. Scaglia, F.E. Smith, S. Bonner-Weir, Endocrinology 136:5451 (1995)).
  • Example 6 Menin regulation of adaptive ⁇ -cell expansion in obesity
  • islet menin levels were measured in A y mice, a well-characterized model of hyperphagic obesity (reviewed in T.T. Yen et al, FASEB J. 8, 479 (1994)). At three months, when A y mice are obese but normoglycemic, A y islet levels of Menl mRNA, menin, and p27 and pi 8 mRNA and protein were reduced compared to islets from wild type controls. These results indicate that in obesity, menin attenuation regulates adaptive ⁇ -cell proliferation.
  • menin functions as a physiological regulator of adaptive ⁇ -cell expansion in pregnancy and probably other common states linked to Type 2 diabetes, such as obesity.
  • the data indicate that menin may integrate ⁇ -cell growth signals in physiological islet expansion, controlling dynamic histone modifications that govern ⁇ -cell fate and proliferation.
  • Example 7 Isolation of RNA from lymphocytes
  • mice and humans were used to determine the level of mRNA related to the proteins of interest discussed above.
  • mouse lymphocytes were isolated and analyzed from pregnant and control matched mice.
  • lymphocytes were purified from erthyrocytes by loading buffy coat onto a Ficoll-plaque column (Sigma) and spinning at 800xg in a centrifuge.
  • RNA from mouse and human lymphocytes is carried out according to the following protocol:
  • RNA is quantified on a spectrophotometer using A260 reading. Total yield is usually around 100-300ng of RNA.
  • Example 8 Determination of mRNA levels
  • RT reactions were performed using the Retroscript Kit (Ambion) according to the manufacturer's protocol. Briefly: 10ml of RNA (100-300ng), 4ml of 25mM dNTP's, 2ml of random decamers were combined in a PCR reaction tube and incubated at 80 0 C for 3 min. The tube was then incubated on ice for 3 min. at which time the following was added in: ImI of RNase inhibitor, 2ml of 1Ox reverse transcriptase buffer, and reverse transcriptase enzyme. The reaction was incubated at 42°C for 1 hour.
  • Each real time reaction consisted of the following: 2x Master Mix (comprised of buffer and polymerase), cDNA, MiIIiQ H 2 O, and 2Ox probe set
  • Protein can be obtained from the lymphocytes according to the following protocol: [0198] 1. Pipette off lymphocytes from tissue culture plates (cells are grown in suspension) [0199] 2. Transfer to 1.5 ml Eppendorf tube [0200] 3. Centrifuge at 1000 rpm for 5 min. [0201] 4. Discard supernate
  • Example 10 Expression of Bcl6, Menl and Hox ⁇ 9 (a menin target) in blood 'buffy coat 1 leukocytes from pregnant or obese subjects
  • Bcl6 mRNA was elevated and Menl mRNA was reduced ( Figure 11). This suggests that Bcl6 mRNA and Menl mRNA levels in peripheral blood cells may be useful surrogate biomarkers that reflect ⁇ -cell proliferation or changes in ⁇ -cell mass.
  • Example 11 Expression of Bcl6, Menl and HoxA9 is response to mitogens like PRL or leptin
  • results of these examples demonstrate that markers from accessible tissues like blood may be useful for (1) predicting diabetes risk, (2) monitoring ongoing ⁇ -cell proliferation and ⁇ - cell mass changes, (3) predicting individual 'potential' for ⁇ -cell expansion or regeneration and (4) detecting 'pre-clinical' diabetes.

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Abstract

L'invention concerne la régulation par la ménine et/ou les facteurs liés à la ménine de la prolifération des cellules d'îlots bêta. Elle concerne aussi des procédés de diagnostic ou de contrôle de maladie ou d'affection consistant à mesurer l'expression de la ménine et/ou des facteurs liés à la ménine, de leurs produits géniques correspondants et anticorps spécifiques à ces produits. Selon certains aspects, les procédés décrits visent à diagnostiquer, contrôler, détecter et/ou prévenir le diabète, le prédiabète ou le diabète de gestation. L'invention concerne également des procédés de détection d'agents qui modulent la prolifération considérée et des procédés de traitement d'un patient souffrant de diabète, de prédiabète ou de diabète de gestation.
PCT/US2008/011318 2007-10-01 2008-09-30 Régulation par la ménine de la prolifération des cellules d'îlots bêta WO2009045403A2 (fr)

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EP2521785A2 (fr) * 2010-01-06 2012-11-14 CuRNA, Inc. Traitement de maladies associées à un gène de développement pancréatique par inhibition du produit de la transcription anti-sens naturel en un gène de développement pancréatique
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EP2521785A4 (fr) * 2010-01-06 2013-09-25 Curna Inc Traitement de maladies associées à un gène de développement pancréatique par inhibition du produit de la transcription anti-sens naturel en un gène de développement pancréatique
WO2013154709A1 (fr) * 2012-04-13 2013-10-17 Diabetomics, Llc Biomarqueurs maternels pour le diabète gestationnel
US8852873B2 (en) 2012-04-13 2014-10-07 Diabetomics, Llc Maternal biomarkers for gestational diabetes
US8975034B2 (en) 2012-04-13 2015-03-10 Diabetomics, Llc Maternal biomarkers for gestational diabetes
US9383370B2 (en) 2012-04-13 2016-07-05 Diabetomics, Inc. Maternal biomarkers for gestational diabetes
WO2018160534A1 (fr) * 2017-02-28 2018-09-07 Joslin Diabetes Center Protection de cellules bêta contre une attaque immunitaire
US11708561B2 (en) 2017-02-28 2023-07-25 Joslin Diabetes Center Protection of beta cells from immune attack

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