WO2009045403A2 - MENIN REGULATION OF β-ISLET CELL PROLIFERATION - Google Patents

Abstract

The present invention is directed to the regulation of β-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. In certain aspects, the subject methods diagnose, monitor, detect, and/or prevent diabetes, pre-diabetes, or gestational diabetes. Also provided herein are methods for screening for agents which modulate β-islet cell proliferation and methods of treating a subject for diabetes, pre-diabetes, or gestational diabetes.

Description

MENIN REGULATION OF B-ISLET CELL PROLIFERATION

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims benefit of the filing date of U.S. Provisional Patent Application Ser. No. 60/960,457 filed October 1, 2007, which is incorporated herein by reference in its entirety.

TECHNICAL FIELD OF THE INVENTION

[0002] The field of this invention generally relates to the regulation of β-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 β-islet proliferation.

BACKGROUND OF THE INVENTION

[0003] 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 β-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.

[0004] The incidence of 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 β-islet cells under various conditions. Maternal pancreatic islet expansion in rodents and humans suggests that adaptive islet cell growth is a mechanism for ensuring metabolic balance in pregnancy, a physiological state marked by increased insulin demand. (F. A. Van Assche, L. Aerts, F. De Prins Br J Obstet Gynaecol 85, 818 (1978); J.A. Parsons, T.C. Brelie, R.L. Sorenson. Endocrinology 130, 1459 (1992); R. L. Sorenson, T.C. Brelje, Hormone Metabolism Research 29, 301 (1997)). Descriptive studies with rats (Parsons et al. Endocrinology 130, 1459 (1992); R. L. Sorenson, et al. Hormone Metabolism Research 29, 301 (1997) support the hypothesis that proliferation of insulin-secreting islet β-cells is the principal mechanism of β-cell expansion in pregnancy, but the molecular basis of facultative maternal β-cell proliferation is unknown. Moreover, it is unclear if impaired maternal β-cell proliferation leads to reduced insulin levels and gestational diabetes (J. Boloker, SJ. Gertz, R.A. Simmons, Diabetes 51, 1499 (2002)).

[0006] Hyperplasia of the maternal pituitary and islets in pregnancy is reminiscent of endocrine proliferation in Multiple Endocrine Neoplasia Type 1 (MENl), a human cancer syndrome characterized by synchronous tumors of the pituitary, endocrine pancreas and parathyroid. (E. Horvath, K. Kovacs, B. W. Scheithauer, Pituitary 1, 169 (1999)) The majority of MENl cases result from mutation of Menl, whose protein product is menin (C. Larsson, B. Skogseid, K. Oberg, Y. Nakamura, M. Nordenskjold, Nature 332, 85 (1988); S.K. Agarwal et al. Ann N Y AcadSci. 1014, 189 (2004)). In mice and humans, mutation and . pathological Menl loss promotes neuroendocrine tumors, including islet β-cell tumors (S.K. Agarwal et al. Ann N Y AcadSci. 1014, 189 (2004); JJ. Heit, S.K. Karnik, S.K. Kim, Annu Rev Cell Dev Biol 22, 311 (2006)). It may be postulated that physiological changes in Menl expression might regulate facultative maternal β-cell growth in pregnancy.

[0007] 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).). This complex promotes tri-methylation of histone H3 on lysine 4 (H3K4), an epigenetic mark associated with transcriptionally-active chromatin. Menin-dependent histone methylation maintains expression of p27^Kψ] and /?/S^INK4C (h_ereaft_er) /λ27 and pl8), which encode cyclin-dependent kinase (CDK) inhibitors that prevent islet proliferation (S. K. Karnik, et al., Proc Natl AcadSci USA 102, 14659 (2005); T.A. Milne et al, Proc Natl Acad Sci USA 102, 749 (2005); R. W. Schnepp et al., Cancer Res. 66, 5707 (2006); D.S. Franklin, V.L. Godfrey, D.A. O'Brien, C. Deng, Y. Xiong, MoI Cell Biol. 20, 6147 (2000)). Consistent with prior findings in islet tumors, reduced islet menin levels in pregnancy after 8 days post-coitum (dpc) were accompanied by reduced p27 andpl8 mRNA and protein levels. (Figure 1, Panel C; Karnik et αl. Science 318: 806-809 (2007)).

[0008] Recognizing the impossibility of islet isolation from humans, recent studies have focused on searching for more accessible cells which might serve as surrogates to measure the robustness of the Bcl6-menin response to β-cell mitogens. Like islet β-cells, subsets of blood leukocytes (like B lymphocytes and plasma cells) have a physiology partially dedicated to protein secretion. Moreover, advances in myeloma genetics have revealed that plasma cell growth and fate is regulated by CCNDl, CCND2, pl 8INK4C, Bcl6, MafA, MafB, Ezh2 and FGFR3 (Tonon G. 2007. Molecular pathogenesis of multiple myeloma. Hematol Oncol Clin North Am. 21 :985-1006), known regulators of islet β-cell growth and function (Heit JJ, Karnik SK, Kim SK. 2006a. Intrinsic regulators of pancreatic beta-cell proliferation. Annu Rev Cell Dev Biol. 22:31 1-38; Karnik SK, Chen H, McLean GW, Heit JJ, Gu X, Zhang AY, Fontaine M, Yen MH, Kim SK. 2007. Menin controls growth of pancreatic beta-cells in pregnant mice and promotes gestational diabetes mellitus. Science 318:806-9; Edlund H. 2002. Pancreatic organogenesis—developmental mechanisms and implications for therapy. Nat Rev Genet 3:524-32; Murtaugh LC. 2007. Pancreas and beta-cell development: from the actual to the possible. Development 134:427-38).

[0009] Likewise, work by the Kim group and others has revealed common regulators or drugs that impact lymphocyte and islet cell development and proliferation, including the phosphatase calcineurin, NFATcI , Pbxl, Pbx2, MLLl and the drugs FK506 and cyclosporine A (Heit JJ, Apelqvist AA, Gu X, Winslow MM, Neilson JR, Crabtree GR, Kim SK. 2006b. Calcineurin/NFAT signaling regulates pancreatic beta-cell growth and function. Nature 443:345-349; Kim SK, L. Selleri, J.S. Lee, A. Zhang, X. Gu, Y. Jacobs, and M.L. Cleary. 2002. Pbxl inactivation disrupts pancreas development and in Ipfl -deficient mice promotes diabetes mellitus. Nature Genetics 30: 430-435; Karnik SK, Hughes CM, Gu X, Rozenblatt- Rosen O, McLean GW, Xiong Y, Meyerson M, Kim SK. 2005. Menin regulates pancreatic islet growth by promoting histone methylation and expression of genes encoding p27Kipl and pl8INK4c. Proc Natl Acad Sci USA. 102:14659-64; Milne, T. A. et al. 2005. Menin and MLL cooperatively regulate expression of cyclin dependent kinase inhibitors. Proc Natl Acad Sci USA 102, 749-5; Yokoyama, A., Somervaille, T.C., Smith, K.S., Rozenblatt-Rosen, O., Meyerson, M. & Cleary, M.L. 2005. The menin tumor suppressor protein is an essential oncogenic cofactor for MLL-associated leukemogenesis. Cell 123, 207-18; H. Chen, L. Selleri and S.K., unpubl. results). Prior work also revealed that menin regulates cell growth and differentiation in human and murine lymphocytic leukemias (Yokoyama et al 2005). Thus, there are remarkable similarities in the physiological and genetic programs that control lymphocytes and islet β-cells.

SUMMARY OF THE INVENTION

[0010] The present invention relates to the regulation of β-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 β-islet cell proliferation by measuring in surrogate cells the expression of genes related to β-islet cell proliferation, their corresponding gene products and antibodies specific for the gene products. In one aspect, the genes related to β-islet cell proliferation comprise menin and/or menin-related factors and the abnormalities in β-islet cell proliferation relate to a diabetes, pre-diabetes or a condition related to diabetes. In certain aspects, 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 β-islet cell proliferation and methods of treating a subject for diabetes, pre-diabetes, or gestational diabetes.

[0011] 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. [0012] In certain aspects of the invention, 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.

[0013] In certain embodiments of the invention, the one or more menin-related factors are selected from BCl-6, Menl, and HoxA9.

[0014] In certain aspects of the invention, the measured expression level of BCl-6 is greater than the measured expression level of the control. In another aspect, the measured expression level of Menl is lower than the measured expression level of the control. In still another aspect, the measured expression level of HoxA9 is lower than the measured expression level of the control.

[0015] In some embodiments of the invention, the measurement of gene expression level comprises a quantitative measurement.

[0016] In some embodiments of the invention, the collection of blood cells is leukocytes. In certain aspects, the leukocytes comprise at least one of lymphocytes, B- lymphocytes, T- lymphocytes, neutrophils, eosinophils, basophils, and monocytes.

[0017] In certain aspects of the invention, 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.

[0018] In certain embodiments of the invention, 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.

[0019] In certain aspects of the invention, the measurement of gene expression level comprises a polymerase chain reaction (PCR).

[0020] In certain embodiments of the invention, 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.

[0021] In certain aspects of the invention, the measurement of gene expression level comprises a quantitative measurement of protein level. [0022] In certain aspects of the invention, the protein level is measured by an immunoassay selected from RIA, ELISA, EMIT, CEDIA, Western blot analysis, or FIA.

[0023] In certain aspects of the invention, the protein level is measured by mass spectrometry.

[0024] 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.

[0025] In certain aspects of the invention, the subject at risk of developing diabetes, prediabetes, or gestational diabetes is obese.

[0026] In certain aspects of the invention, the subject at risk has a genetic propensity for developing diabetes, pre-diabetes, or gestational diabetes.

[0027] In certain aspects of the invention, the subject at risk of developing diabetes, prediabetes, or gestational diabetes is pregnant.

[0028] Aspects of the invention further include 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, the kits 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.

[0029] In certain aspects of the invention, the kits optionally include instructions for carrying out a method of aiding in the diagnosis of diabetes, pre-diabetes, or gestational diabetes.

[0030] In certain aspects of the invention, the reagent specific for the menin-related factor is an antibody, or fragment thereof, that is specific for the menin-related factor.

[0031] In certain aspects of the invention, the kits further comprise an amount of at least one menin-related factor suitable for normalizing data. [0032] Aspects of the invention further include methods of screening for an agent effective in regulation of mammalian β-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 β-islet cell growth.

[0033] In certain aspects of the invention, 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.

[0034] In certain aspects of the invention, the candidate agent is effective for treating or preventing a risk of developing diabetes, pre-diabetes, or gestational diabetes in a subject.

[0035] Aspects of the invention include assays to measure human biomarkers for diabetes diagnosis and prognosis. In certain aspects, the invention provides methods of using more accessible lymphocyte cells as surrogates to measure the robustness of the Bcl6-menin response to β-cell mitogens. In one embodiment, the β-cell mitogen is leptin while in another embodiment, the β-cell mitogen is PRL.

[0036] In aspects of the invention, menin is shown to be a factor in the proliferation of β- islet cells, such that increasing amounts of menin results in inhibition of proliferation of the β-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.

[0037] Aspects of the invention include markers from accessible tissues like blood useful for (1) predicting diabetes risk, (2) monitoring ongoing β-cell proliferation and β-cell mass changes, (3) predicting individual 'potential' for β-cell expansion or regeneration and (4) detecting 'pre-clinical' diabetes.

[0038] Aspects of the invention will become apparent to the skilled artisan by the following description of the invention. BRIEF DESCRIPTION OF THE FIGURES

[0039] Figure 1 provides the results of maternal β cell mass, body mass, and real time RT- PCR analysis on islets during pregnancy. Panel A depicts maternal β cell mass data and 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.

[0040] Figure 2 provides the results of blood glucose during ad libitum feeding, fasting, and glucose tolerance testing in nonpregnant βMenl mice. Panel A depicts RT-PCR analysis and Panel B depicts Western blot analysis of Menl transgene expression in islets from βMenl mice and controls (wild type mice, 'WT¹: TRE-Menl mice, TRE'; RJP-rtTA mice, 'RIP'). Panel C depicts the results of a glucose tolerance test in non-pregnant βMenl and control mice. Panel D provides blood glucose levels in non-pregnant βMenl mice and controls fed ad libitum. Panel E provides blood glucose levels in fasted non-pregnant βMenl mice and controls. Panels F and G depict the results of real-time RT-PCR quantification of mRNA levels of indicated genes in islets isolated from Dox-administered pregnant βMenl mice, pregnant controls, both at 17 dpc, and nonpregnant controls. n=3-6 mice per group.

[0041] Figure 3 depicts blood glucose levels during ad libitum feeding and fasting in pregnant βMenl 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 βMenl mice fed ad libitum during pregnancy. Panel C: Blood glucose levels in fasted pregnant control and pregnant βMenl mice at 16 dpc.

[0042] Figure 4 shows maternal mass, litter size and mass, and insulin secretion in βMenl mice. Panel A: maternal mass; Panels B: litter size; and Panel C: birth weight of pups at 18 dpc on post-natal day 1 (Pl) from βMenl mothers and controls. Panel D depicts insulin content, Panel E depicts insulin secretion following glucose stimulation and Panel F depicts insulin secretion following arginine stimulation in 16 dpc islets purified from pregnant βMenl mice, and pregnant controls, both on Dox. Panel G depicts the quantification of activated caspase3⁺ expression in islets from pregnant βMenl mice and pregnant controls, both on Dox.

[0043] 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.

[0044] 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. In Panel N, c- myc is a gene whose mRNA levels are unchanged in MIN6 cells exposed to prolactin or other treatments.

[0045] 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.

[0046] 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. Other Primer Pairs (PP2 and PP3) flank segments of Bcl6 lacking 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.

[0047] 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.

[0048] Figure 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.

[0049] Figure 11 shows that Bcl6-Menl regulation in blood leukocytes mirrors pancreatic islets during obesity in humans.

[0050] Figure 12 depicts mRNA levels in PRL-exposed leukocytes in indicated patient groups. * P<0.05, ** PO.01.

[0051] Figure 13 depicts the BcL6 linkage to GDM history.

[0052] Figure 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

[0053] Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, 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, since the scope of the present invention will be limited only by the appended claims.

[0054] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0055] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

[0056] It must be noted that as used herein and in the appended claims, the singular forms "a," "and," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a menin-related factor" includes a plurality of such menin- related factors and reference to "an agent" includes reference to one or more agents and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

[0057] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0058] The present invention relates to the regulation of β-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. In certain aspects, the subject methods diagnose, monitor, detect, and/or prevent diabetes, prediabetes, or gestational diabetes. Also provided herein are methods for screening for agents which modulate β-islet cell proliferation and methods of treating a subject for diabetes, prediabetes, or gestational diabetes.

[0059] In aspects of the invention, menin is shown to be a factor in the proliferation of β- islet cells, such that increasing amounts of menin results in inhibition of proliferation of the β-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. In certain aspects of the invention, the one or more menin-related factors are selected from BCl-6, Menl, and HoxA9.

[0060] 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). A "Menl polynucleotide" refers to a polynucleotide encoding a Menl polypeptide (also referred to in the literature as "menin"). For example, 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. Exemplary 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.

[0061] 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. For example, 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. Exemplary 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.

[0062] 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. For example, 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. Exemplary 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.

[0063] The term "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.

[0064] The terms "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. As used herein, known means that the value represents an understood parameter, e.g., a level of expression of a marker gene.

[0065] As used herein, 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.

[0066] In certain aspects of the invention, the measured expression level of BCl-6 is greater than the measured expression level of the control. In another aspect, the measured expression level of Menl is lower than the measured expression level of the control. In still another aspect, the measured expression level of HoxA9 is lower than the measured expression level of the control.

[0067] A "diabetes-related condition" according to the present invention 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. In some embodiments, the diabetes is a Type 2 diabetes-related condition. In some embodiments, the diabetes-related condition is related to abnormal growth or condition of β-islet cells. In some embodiments, the β-islet cells are insulin-producing cells.

[0068] In some embodiments, the monitoring, detection or diagnosis of a diabetes-related condition comprises one or more of: (1) predicting diabetes risk, (2) monitoring ongoing β- cell proliferation and β-cell mass changes, (3) predicting individual 'potential' for β-cell expansion or regeneration and (4) detecting 'pre-clinical' diabetes.

[0069] Markers suitable for the detection, diagnosis or monitoring of a diabetes-related condition according to the invention are collectively described as "menin-related factors." Examples of menin-related factors include, but are not limited to, Menl, BCl-6, HoxA9, CCNDl, CCND2, pl 8^INK4C, MafA, Mafβ, phosphatase calcineurin, NFATcI, Pbx2, and MLLl . In some embodiments the menin-related factors comprise Menl, BCl-6, and HoxA9. [0070] Desirably, the target of a menin-related factor would be one that is regulated by menin or other protein of interest in the menin pathway. For example, in lymphocytes HoxA9 should follow the same directional changes in cell level as confirming the menin result. Alternatively, one may use a housekeeping protein or other normal protein unaffected by the menin pathway as a standard.

[0071] As compared to a normal level, where the state of the host is considered to determine the normal level, e.g. pregnant or non-pregnant, generally 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. In the pregnant state as compared to the non-pregnant state, menin levels are at least 2 fold higher.

[0072] While 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.

[0073] In determining the menin or other relevant protein level, namely a protein in the pathway controlling β-islet cell population, or a standard or control protein, one can use blood cells, particularly leucocytes. One can isolate the leucocytes by conventional ways, centrifuging the blood and isolating the buffy coat. If one desires a particular leukocyte population, e.g. lymphocytes, including B- and T-cells, neutrophils, eosinophils, basophils, monocytes, etc., one can add 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.

[0074] In one embodiment, methods for detection or diagnosis of diabetes-related conditions in an individual are provided. In particular embodiments, 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.

[0075] In one embodiment, the diagnostic/detection agent is an antibody, preferably a monoclonal antibody, preferably linked to a detectable agent.

[0076] In some embodiments 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)).

[0077] In one embodiment, 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)).

Frequently, it is desirable to amplify the nucleic acid sample prior to hybridization. One of skill in the art will appreciate that whatever amplification method is used, if a quantitative result is desired, care must be taken to use a method that maintains or controls for the relative frequencies of the amplified nucleic acids.

[0078] Methods of "quantitative" amplification are well known to those of skill in the art. For example, 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.

[0079] 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).

[0080] Other suitable amplification methods include, but are not limited to polymerase chain reaction (PCR) (Innis, et al., PCR Protocols, A guide to Methods and Application. Academic Press, Inc. San Diego, (1990)), ligase chain reaction (LCR) (see Wu and Wallace, Genomics, 4:560 (1989), Landegren, et al., Science, 241 :1077 (1988) and Barringer, et al., Gene, 89:117 (1990), transcription amplification (Kwoh, et al., Proc. Natl. Acad. Sci. USA, 86:1173 (1989)), and self-sustained sequence replication (Guatelli, et al., Proc. Nat. Acad. Sci. USA, 87:1874 (1990)).

[0081] In an embodiment for use with chips or other surface supported nucleic acid array, 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. After synthesis of double-stranded cDNA, 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. Methods of in vitro polymerization are well known to those of skill in the art (see, e.g., Sambrook, supra.) and this particular method is described in detail by Van Gelder, et al., Proc. Natl. Acad. Sci. USA, 87: 1663-1667 (1990) who demonstrate that in vitro amplification according to this method preserves the relative frequencies of the various RNA transcripts. Moreover, Eberwine et al. {Proc. Natl. Acad. Sci. USA, 89: 3010-3014) provide a protocol that uses two rounds of amplification via in vitro transcription to achieve greater than 10⁶ fold amplification of the original starting material thereby permitting expression monitoring even where biological samples are limited.

[0082] When using a nucleic acid chip array, the direct transcription method described above provides an antisense (aRNA) pool. Where antisense RNA is used as the target nucleic acid, the oligonucleotide probes provided in the array are chosen to be complementary to subsequences of the antisense nucleic acids. Conversely, where 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. Finally, where the nucleic acid pool is double stranded, the probes may be of either sense as the target nucleic acids include both sense and antisense strands.

[0083] 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. For example, 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. Other suitable cloning systems include phage lambda vectors designed for Cre-loxP plasmid subcloning (see e.g., Palazzolo et al., Gene, 88: 25-36 (1990)).

[0084] Desirably, a high activity RNA polymerase (e.g. about 2500 units/μL for T7, available from Epicentre Technologies) is used.

[0085] 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. Thus, for example, polymerase chain reaction (PCR) with labeled primers or labeled nucleotides will provide a labeled amplification product. In one embodiment, 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.

[0086] Alternatively, 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). [0087] 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., Dynabeads™), 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; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241.

Means of detecting such labels are well known. Thus, for example, 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.

[0088] The label may be added to the target (sample) nucleic acid(s) prior to, or after the hybridization. So called "direct labels" are detectable labels that are directly attached to or incorporated into the target (sample) nucleic acid prior to hybridization. In contrast, so called "indirect labels" are joined to the hybrid duplex after hybridization. Often, the indirect label is attached to a binding moiety that has been attached to the target nucleic acid prior to the hybridization. Thus, for example, 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. For a detailed review of methods of labeling nucleic acids and detecting labeled hybridized nucleic acids see Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 24: Hybridization With Nucleic Acid Probes, P. Tijssen, ed. Elsevier, N.Y., (1993)).

[0089] Fluorescent labels are preferred and easily added during an in vitro transcription reaction. In a preferred embodiment, fluorescein labeled UTP and CTP are incorporated into the RNA produced in an in vitro transcription reaction as described above. [0090] 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.

[0091] Instead of the mRNA, one may determine the proteins 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. There are numerous commercially available protocols that may be adapted for the subject invention where the 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. 4,067,774; 4,517,288; 4,708,929; 4,778,767; 5,200,084; 6,436,296 and 7,045,296. Also available are methods using mass spectrometry to identify and quantitate proteins in a biological sample, where the sample may have undergone preliminary treatment before being introduced into the mass spectrometer.

[0092] In the immunoassays, 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. Alternatively, one may have an antibody for one idiotype of the protein bound to a surface and a second labeled antibody to a different idiotype, where the protein serves as a bridge to bind the second antibody to the surface. The signal at the surface may then be determined.

[0093] The status of the β-islet cells is established by investigating the proteins in the menin pathway, such as menin which diminishes in conjunction with expansion of β-islet cells are appropriate and Bcl6 which increases in conjunction with expansion of β-islet cells. The determination may be prospective in determining how a patient will respond to a condition that normally results in increased β-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. Depending upon whether proliferation or inhibition of proliferation is desired, the human patient may then be counseled or treated to correct the dysfunction. In those individuals that have a dysfunction in that the menin pathway inhibits β-islet cell proliferation, changes in life style can be prescribed. For those who are obese, a diet change would be indicated to permit the reduction in weight. For those who do not have characteristics related to enhancing the probability of diabetes except for the menin pathway dysfunction, they can be directed to an appropriate diet, encouraged to reduce stress, etc. From a clinical standpoint, it is very advantageous in counseling a patient to be able to refer to a specific cause of the condition and be able to monitor such cause as the patient responds to the medical advice.

[0094] Aspects of the invention include methods of counseling humans having a dysregulation potential of menin modulation of β-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 β-islet population. Menin levels or related factor, e.g. Bcl6, associated with the pathway including menin associated with β-islet proliferation or contraction is determined from a blood sample. The determination can be correlated with the ability of β-islet cell expansion or contraction. One can monitor whether the β-islet cells are properly responding to the condition of the individual that warrants an expansion or contraction of the number of β-islet cells. One or more of the proteins or their mRNAs may be determined. In addition, one may use an internal control, such as a target in the cells being assayed.

[0095] The subject invention demonstrates that menin and/or menin related factors play a role in regulating β-islet cell proliferation. These factors, particularly menin, can be determined by measuring mRNA or protein levels. It is shown that the menin pathway regulates the β-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 β- islet cell menin in that the level of menin in the white blood cells tracks with the level in the β-islet cells.

[0096] The opportunity to determine whether a patient responds to the need for expansion or contraction of the number of β-islet cells allows health practitioners to advise patients of their need to change their life style, be aware of glucose consumption, monitor their insulin and glucose and appropriate intervals and live a healthier life style. With the evidence in hand of dysregulation of β-islet cell proliferation, a health practitioner is in a much stronger position to counsel a patient and to monitor the patient as to their ability to regulate the expansion of β-islet cells. During pregnancy, one can determine whether the woman has properly responded by expanding the β-islet cell population during her pregnancy and whether post-partum, the population has diminished by monitoring the level of menin in lymphocytes.

[0097] In one embodiment, a method of modulating β-islet cell proliferation is provided. In another embodiment, methods of treating cells or individuals with a diabetes-related condition are provided.

[0098] The method comprises administration of an agent . In particular embodiments, the cancer inhibitor is an antisense molecule, a pharmaceutical composition, a therapeutic agent or small molecule, or a monoclonal, polyclonal, chimeric or humanized antibody. In particular embodiments, a therapeutic agent is coupled with a an antibody, preferable a monoclonal antibody.

[0099] (a) Antisense Molecules

[0100] In one embodiment, 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. The ability to derive an antisense or a sense oligonucleotide, based upon a cDNA sequence encoding a given protein is described in, for example, Stein and Cohen, Diabetes related conditions Res. 48:2659, (1988) and van der Krol et al., BioTechniques 6:958, (1988).

[0101] 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.

[0102] 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. Preferably, 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. Alternatively, 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.

[0103] (b) RNA Interference

[0104] 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. This mechanism appears to be different from the interferon response that results from dsRNA mediated activation of protein kinase PKR and 2',5'-oligoadenylate synthetase resulting in non-specific cleavage of mRNA by ribonuclease L. (reviewed in Sharp, P. A., RNA interference-2001, Genes & Development 15:485-490 (2001)).

[0105] 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). Elbashir, S. M. et al. Nature 411 :494-498 (2001); Caplen, N. J. et al. Proc. Natl. Acad. Sci. USA 98:9742-9747 (2001); Harborth, J. et al. J. Cell Sci. 1 14:4557- 4565 (2001). The term "short interfering RNA" or "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). As used herein, 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).

[0106] 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. When introduced into a cell, they assemble with yet-to-be-identified proteins of an endonuclease complex (RNA-induced silencing complex), which then guides target mRNA cleavage. As a consequence of degradation of the targeted mRNA, cells with a specific phenotype characteristic of suppression of the corresponding protein product are obtained. The small size of 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.

[0107] Intracellular transcription of small RNA molecules is achieved by cloning the siRNA templates into RNA polymerase II (Pol III) transcription units, which normally encode the small nuclear RNA (snRNA) U6 or the human RNase P RNA Hl . 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. 20, 497-500 (2002).); in the second, 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)).

[0108] Co-expression of sense and antisense siRNAs mediate silencing of target genes, whereas expression of sense or antisense siRNA alone do not greatly affect target gene expression. Transfection of plasmid DNA, rather than synthetic siRNAs, may appear advantageous, considering the danger of RNase contamination and the costs of chemically synthesized siRNAs or siRNA transcription kits. Stable expression of siRNAs allows new gene therapy applications, such as treatment of persistent viral infections. Considering the high specificity of siRNAs, the approach also allows the targeting of disease-derived transcripts with point mutations, such as RAS or TP53 oncogene transcripts, without alteration of the remaining wild-type allele. Finally, by high-throughput sequence analysis of the various genomes, 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)).

[0109] 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)).

[0110] 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. In an embodiment, 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. In another method a short double strand 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. In various embodiments, the nucleic acid molecule is at least a 7 mer, at least a 10 mer, or at least a 20 mer. In a further embodiment, the sequence is unique.

[0111] (c) Pharmaceutical Compositions

[0112] Pharmaceutical 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. For purposes of this invention, 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.

[0113] The compositions can be used to treat or prevent diabetes or a diabetes-related condition. In addition, the pharmaceutical compositions can be used in conjunction with conventional methods of diabetes treatment. The terms "treatment", "treating", "treat" and the like are 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" as used herein 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.

[0114] Where 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.

[0115] 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.

[0116] The term "therapeutically effective amount" as used herein 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. For purposes of the present invention, 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.

[0117] A pharmaceutical composition can also contain a pharmaceutically acceptable carrier. The term "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. Auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, can also be present in such vehicles. Typically, 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. A thorough discussion of pharmaceutically acceptable excipients is available in Remington: The Science and Practice of Pharmacy (1995) Alfonso Gennaro, Lippincott, Williams, & Wilkins.

[0118] The pharmaceutical 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.

[0119] The pharmaceutical compositions of the present invention comprise a menin-related factor protein in a form suitable for administration to a patient. In the preferred embodiment, 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. "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. [0120] The pharmaceutical 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. Additives are well known in the art, and are used in a variety of formulations.

[0121] 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. Depending upon the manner of introduction, 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. Once formulated, the 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.

[0122] 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. Generally, delivery of 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.

[0123] Once differential expression of a gene corresponding to a menin-related factor polynucleotide described herein has been found to correlate with a diabetes-related condition, 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.). In other embodiments, 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 β-islet cells relative to normal cells or as an agonist for gene products that are decreased in expression in diabetic β-islet cells (e.g., to promote the activity of gene products that act as suppressors).

[0124] 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. For example, administration of polynucleotide therapeutic compositions agents includes local or systemic administration, including injection, oral administration, particle gun or catheterized administration, and topical administration. Preferably, 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. For example, a small metastatic lesion is located and the therapeutic composition injected several times in several different locations within the body of tumor. Alternatively, 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.

[0125] 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. Sci. (USA) (1990) 87:3655; Wu et al., J. Biol. Chem. (1991) 266:338. Therapeutic 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 μg to about 2 mg, about 5 μg to about 500 μg, and about 20 μg to about 100 μg 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.

[0126] 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.

[0127] 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. 2,200,651; EP 0 345 242; and WO 91/02805), 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)), and adeno- associated virus (AAV) vectors (see, e.g., WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655). Administration of DNA linked to killed adenovirus as described in Curiel, Hum. Gene Ther. (1992) 3:147 can also be employed.

[0128] 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.

[0129] Further, 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. Moreover, 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).

[0130] The administration of the 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. In some instances, for example, in the treatment of wounds and inflammation, the menin-related factor proteins and modulators may be directly applied as a solution or spray.

[0131] In a preferred embodiment, menin-related factor proteins and modulators are administered as therapeutic agents, and can be formulated as outlined above. Similarly, 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. [0132] Thus, in one embodiment, methods of modulating menin-related factor gene activity in cells or organisms are provided. In one embodiment, 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. Alternatively, 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. In a preferred embodiment, for example when the menin-related factor sequence is down-regulated in diabetes-related conditions, 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. In a preferred embodiment, 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. Alternatively, for example when the menin-related factor sequence is up-regulated in diabetes related conditions, the activity of the endogenous menin-related factor gene is decreased, for example by the administration of a menin-related factor antisense nucleic acid.

[0133] (d) Vaccines

[0134] In a preferred embodiment, 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).

[0135] In one embodiment, 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. In a preferred embodiment a patient is immunized with a DNA vaccine comprising a plurality of nucleotide sequences derived from a menin-related factor gene. Similarly, it is possible to immunize a patient with a plurality of menin-related factor genes or portions thereof. Without being bound by theory, expression of the polypeptide encoded by the DNA vaccine, cytotoxic T-cells, helper T-cells and antibodies are induced that recognize and destroy or eliminate cells expressing menin-related factor proteins.

[0136] In a preferred embodiment, the DNA vaccines include a gene encoding an adjuvant molecule with the DNA vaccine. Such 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.

[0137] (e) Antibodies

[0138] In one embodiment, a menin-related factor modulator is an antibody as discussed above. In one embodiment, 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. Similarly, 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. In a preferred embodiment, 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. For example, 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.

[0139] 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. [0140] 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. In general, the specific antibody will be detectably labeled, either directly or indirectly. Direct labels include radioisotopes; enzymes whose products are detectable (e.g., luciferase, β-galactosidase, and the like); fluorescent labels (e.g., fluorescein isothiocyanate, rhodamine, phycoerythrin, and the like); fluorescence emitting metals, e.g., ¹⁵²Eu, 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.

[0141] In some embodiments, the methods are adapted for use in vivo, e.g., to locate or identify sites where diabetes related conditions cells are present. In these embodiments, 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.

[0142] (f) Screening Methods

[0143] 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.

[0144] In some embodiments the activity of the menin-related factors is increased or decreases by at least 10%, 20%, 25%, 30%, 40%, 50%, 70%, 90% or 99%.

[0145] 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.

[0146] Screening may be directed to known pharmacologically active compounds and chemical analogs thereof, or to new agents with unknown properties such as those created through rational drug design. In certain embodiments, 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. In certain aspects, the candidate agent is effective for treating or preventing a risk of developing diabetes, pre-diabetes, or gestational diabetes in a subject.

[0147] 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. In multi-step screening processes of the subject invention, 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. Following the initial screening in the non-mammalian animal models of the subject invention, the positive compounds are then screened in non-human mammalian animal models, including non- human mammalian animal models. In addition, 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.

[0148] (g) Kits

[0149] Also provided by the subject invention are kits for use in aiding detection or diagnosis of a subject's risk of developing diabetes, pre-diabetes, or gestational diabetes. The kits 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. In certain aspects, 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. In certain aspects, the kit may further include an amount of at least one menin-related factor suitable for normalizing data.

[0150] Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following examples are illustrative only, and not limiting of the remainder of the disclosure in any way whatsoever.

EXAMPLES Example 1: β-cell mass in pregnant C57B16 mice

[0151] To investigate mechanisms controlling maternal islet expansion, β-cell mass in pregnant C57B16 mice were examined. The results demonstrated that maternal β-cell mass increased by 2-fold (Figure 1, Panel A), accommodating increases in maternal body mass (Figure 1, Panel B). Following parturition, maternal β-cell mass and body mass returned to pre-paitum levels (Figure 1, Panels A-B). To assess maternal islet cell proliferation, labeling studies were performed with bromodeoxyuridine (BrdU). β-cell proliferation increased in pregnant mice until 15 days post coitum (dpc) and then declined to pre-partum levels. Thus, maternal islet β-cell expansion and mass is dynamic in mice.

Example 2: Generation of mice that permit conditional Menl expression in β-cells

[0152] To test if adaptive maternal β-cell proliferation might require reduced Menl expression, mice were generated that permitted conditional Menl expression in β-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 β- cells directed by the rat insulin promoter ("RIP;" J. J. Heit et al, Nature 443, 345 (2006)). In bi-transgenic RlP-rtTA, TRE-Menl mice (abbreviated βMenl), administration of doxycycline (Dox) allows rtTA binding to the TRE element and stimulates β-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 βMenl 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 β-cells stimulated expression of known menin-target genes. However, TRE-Menl expression in male or virgin female βMenl mice did not alter serum insulin or glucose control (Figure 2, Panels C-E), indicating that TRE-Me/?/ expression alone did not disrupt β-cell function.

Example 3: Consequences of menin mis-expression during pregnancy

[0153] To assess the consequences of menin mis-expression during pregnancy, 10 week-old βMenl females were continuously exposed to Dox for 8 weeks, a period encompassing mating, gestation and delivery. Western blot and RT-PCR analysis revealed that Menl mRNA and menin levels were increased in islets from Dox-exposed pregnant βMenl females compared to controls, and immunohistology confirmed that menin was increased in islet β- cells from βMenl mothers (Figure 2, Panel Y). p27 and p!8 mRNA and protein in βMenl islets also increased to levels indistinguishable from those in pre-partum islets (Figure 2, Panel F). By contrast, mRNAs encoding Insulinl (Insl), Insulin2 {Ins2), Glut2, and Pdxl, factors that govern β-cell function, were unchanged in βMenl islets (Figure 2, Panel G). Glucose regulation, islet gene expression and β-cell growth were measured in female βMenl mice starting at 10 weeks of age. Unlike age-matched pregnant controls or virgin βMenl females exposed to Dox, pregnant βMenl females exposed to Dox and fed ad libitum developed moderate hyperglycemia by 9 dpc, which worsened until delivery (Figure 3, Panels A-B). Compared to controls, mean glucose levels in fasted βMenl mothers on Dox were also significantly higher (Figure 3, Panel C). Intraperitoneal glucose challenge similarly revealed impaired glucose tolerance in pregnant βMenl females on Dox . Consistent with these findings, serum insulin levels were reduced in pregnant βMenl mice. Other phenotypes, including body mass, litter size, and average birth weight of pups were indistinguishable for βMenl mothers and controls (Figure 4, Panels A-C). In women with gestational diabetes, serum glucose concentration often returns to normal after delivery. Likewise in βMenl mothers on Dox, blood glucose levels decreased after birth of the pups, and were indistinguishable from those of control mothers. Thus, βMenl mice recapitulated features of human gestational diabetes.

Example 4: Whether impaired insulin production or secretion might underlie hypoinsulinemia in βMenl mice

[0154] To investigate whether impaired insulin production or secretion might underlie hypoinsulinemia in βMenl mice, insulin content and secretion were measured in isolated islets. Insulin content per islet cell was similar in pregnant βMenl and control mice (Figure 4, Panel D). Likewise, insulin secretion by βMenl islets following stimulation with glucose or arginine (Figure 4, Panels E-F) was indistinguishable from that of controls. The pancreata of pregnant βMenl and control mice we assessed to determine if gestational hypoinsulinemia and hyperglycemia in βMenl mice reflected impaired β-cell expansion. In Dox-treated pregnant βMenl mice at the end of gestation, β-cell mass was significantly reduced compared to pregnant controls and appeared indistinguishable from β-cell mass in pre-partum βMenl mice. In pregnant βMenl mice, BrdU studies revealed reduced β-cell proliferation at 17 dpc. Thus, gestational reduction of menin, p27 and pi 8 levels was prevented in βMenl islets, leading to impaired β-cell expansion. Moreover, no differences were detected in islet cell apoptosis in βMenl and control littermate mice (Figure 4, Panel G), suggesting that reduced β-cell mass in βMenl mice did not result from cell death. Collectively, these findings indicate that attenuation of maternal islet menin levels permits adaptive β-cell expansion in pregnancy.

[0155] Prolactin and placental lactogens are hormonal regulators of pregnancy that stimulate β-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)) We investigated if prolactin signaling regulates Menl in β-cells. Lactogenic hormones stimulate phosphorylation and nuclear accumulation of Signal transducer and transactivator 5 (STAT5), which induces expression of targets like Bcl6 (F. A. Scheeren et al., Nat Immunol. 6, 303 (2005)). In islets from pregnant mice, nuclear STAT5 accumulated in β-cells, and levels of phospho-STAT5 and STAT5 occupancy at consensus STAT5 binding sequences (C. C. Chang, B. H. Ye, R. S. Chaganti, R. Dalla-Favera, Proc Natl Acad Sci USA. 93, 6947 (1996)) in Bcl6 increased (Figure 5, Panel A). Bcl6 mRNA and protein increased (Figure 5, Panel B) and ChIP revealed direct association of Bcl6, a transcriptional repressor (C. C. Chang, et al., Proc Natl Acad Sci USA. 93, 6947 (1996); V. L. Seyfert, D. Allman, Y. He, L. M. Staudt, Oncogene 12, 2331 (1996)), with Menl during pregnancy. Mis-expression of Bcl6 in MIN6 cells, a murine insulinoma-derived β-cell line responsive to normal growth cues (S. K. Karnik, et al, Proc Natl Acad Sci USA 102, 14659 (2005); J. Miyazaki et al, Endocrinology 127, 126 (1990)), was sufficient to reduce expression of endogenous Menl, pi 8 and p27 mRNA (Figure 5, Panel C). Moreover, Bcl6 expression reduced transcription of MewZ-luciferase reporters harboring consensus Bcl6 binding sequences (Figure 5, Panels D- E). Thus, Bcl6 directly associated with and repressed Menl transcription in β-cells. Prior studies have shown that steroids like progesterone and dexamethasone can inhibit the mitogenic effects of prolactin on β-cells, but the underlying mechanism is unknown. Simultaneous exposure of isolated mouse islets to prolactin and progesterone attenuated changes in Menl,pl8, p27, and Bcl6 expression provoked by prolactin alone. (A. J. Weinhaus, N. V. Bhagroo, T. C. Brelje, R. L. Sorenson, Endocrinology 141, 1384 (2000)). Thus, multiple hormonal inputs likely regulate β-cell Menl expression. Bcl6-dependent changes in Menl, pi 8 and p27 expression provoked by prolactin in MIN6 cells or in cultured human islets (Figures 6-8) corroborated these findings, and showed that Menl regulation by lactogen signaling is evolutionarily conserved.

Example 5: Lactogen signaling sufficient to reduce Menl expression and increase β-cell proliferation in vivo

[0156] To test if lactogen signaling was sufficient to reduce Menl expression and increase β- 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 β-cell proliferation in vivo. Compared with islets from vehicle-infused controls, 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 β-cells. Thus, short-term prolactin infusion was sufficient to reduce Menl expression in vivo and to stimulate proliferation of adult islet β-cells. Lactogenic hormone regulation of Menl may govern other features that affect β-cell expansion, such as β-cell size and survival (L. Scaglia, F.E. Smith, S. Bonner-Weir, Endocrinology 136:5451 (1995)).

Example 6: Menin regulation of adaptive β-cell expansion in obesity

[0157] To determine if menin might regulate adaptive β-cell expansion in obesity, another common physiological state that stimulates adaptive islet expansion, 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 β-cell proliferation.

[0158] The subject work shows that menin functions as a physiological regulator of adaptive β-cell expansion in pregnancy and probably other common states linked to Type 2 diabetes, such as obesity. The data indicate that menin may integrate β-cell growth signals in physiological islet expansion, controlling dynamic histone modifications that govern β-cell fate and proliferation. These findings that Menl expression is regulated by prolactin and progesterone offers the consideration that defects in signaling pathways regulated by lactogenic or steroid hormones underlie specific forms of Type 2 diabetes, including gestational diabetes, and endocrine neoplasias linked to Menl inactivation, including carcinoid and insulinoma (S.K. Agarwal et αl. Ann N YAcαdSci. 1014, 189 (2004)). These results also indicate that manipulation of regulators, co-factors and targets of menin could serve as a therapeutic strategy for expanding functional pancreatic islets in diabetes mellitus.

Example 7: Isolation of RNA from lymphocytes

[0159] The following protocol can be used to harvest lymphocytes and isolate RNA from mice and humans and used to determine the level of mRNA related to the proteins of interest discussed above. In one study, mouse lymphocytes were isolated and analyzed from pregnant and control matched mice.

[0160] Harvesting and treatment of lymphocytes were carried out as follows:

[0161] 1. On the day of blood draw, lymphocytes were purified from erthyrocytes by loading buffy coat onto a Ficoll-plaque column (Sigma) and spinning at 800xg in a centrifuge.

[0162] 2. Lymphocyte number and viability were assessed on a hemocytometer and by Trypan blue staining.

[0163] 3. Typically the total yield was IxIO⁷ cells. 4 2.5xlO⁶ aliquots were plated per well in a 12 well tissue culture plate (Corning).

[0164] 4. Samples were then treated with Vehicle (PBS), 5ng/ml human prolactin (PRL, R&D Systems), 50ng/ml PRL, or 500ng/ml PRL.

[0165] Isolation of RNA from mouse and human lymphocytes is carried out according to the following protocol:

[0166] Cells are grown in suspension and lymphocytes pipetted off from tissue culture plates.

[0167] 1. Transfer to 1.5 ml Eppendorf tube. [0168] 2. Centrifuge at 1000 rpm for 5 min.

[0169] 3. Discard supernate.

[0170] 4. Resuspend the cellular pellet in 500ml of Trizol (Invitrogen).

[0171] 5. Vortex the pellet for 15 sees.

[0172] 6. Incubate on the benchtop for 3 minutes.

[0173] 7. Add 100ml of chloroform.

[0174] 8. Invert tubes for 15-30 sees.

[0175] 9. Incubate on benchtop for 5 minutes.

[0176] 10. Centrifuge at 14K for 15 minutes at 4°C.

[0177] 1 1. Transfer the aqueous layer to a fresh Eppendorf tube.

[0178] 12. Add 250ml of isopropanol to the aqueous layer.

[0179] 13. Invert tube 4-6 times.

[0180] 14. Incubate on benchtop for 15 minutes.

[0181] 15. Centrifuge at 14K for 15 minutes at 4°C.

[0182] 16. Aspirate supernate.

[0183] 17. Wash pellet in 70% RNase free EtOH.

[0184] 18. Resuspend pellet in 10- 15ml RNase-free H₂O.

[0185] 19. RNA is quantified on a spectrophotometer using A260 reading. Total yield is usually around 100-300ng of RNA.

Example 8: Determination of mRNA levels

[0186] Reverse-Transcriptase reactions were carried out according to the following protocol:

[0187] 1. ~100-300ng of RNA was used per reverse transciptase reaction

[0188] 2. 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 80⁰C 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.

[0189] 3. Once complete, the final reverse transciptase reaction product was diluted 1 :1 in RNase-free H₂O.

[0190] Real time PCR analysis was carried out according to the following protocol:

[0191] The resulting cDNA from the reverse-transciptase reactions were used as template for all real time PCR reactions.

[0192] 1. Each real time reaction was replicated in triplicate/reaction. [0193] 2. Each real time reaction had a final loading volume of 25ml.

[0194] 3. Each real time reaction consisted of the following: 2x Master Mix (comprised of buffer and polymerase), cDNA, MiIIiQ H₂O, and 2Ox probe set

[0195] 4. All samples were normalized to a β-actin endogenous control.

[0196] The results for the mRNA determinations are shown in Figure 9 and 1OB for mouse, and Figure 1OC for human.

Example 9: Isolation of protein from lymphocytes

[0197] 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

[0202] 5. Resuspend the cellular pellet in detergent lysis buffer, and the soluble fraction is isolated for further fractionation by polyacrylamide gel electrophoresis before Western blotting.

[0203] Similar methods can be used for other tissues. Example 10: Expression of Bcl6, Menl and HoxΛ9 (a menin target) in blood 'buffy coat¹ leukocytes from pregnant or obese subjects

[0204] Expression ofBclό, Menl and HoxA9 (a menin target) in blood 'buffy coat' leukocytes were measured from pregnant (Figure 10) or obese subjects (Figure 1 1). In both humans and mice, Bcl6 mRNA levels increase, and Menl mRNA level decrease in maternal leukocytes during gestation (Figure 10, Panels A-C). In humans, this is correlated with reduced expression of HoxA9, a known target of menin (Figure 10, Panel C). Reduced levels of HoxA9 mRNA indicate that menin protein or activity is reduced in blood lymphocytes in pregnancy.

[0205] Likewise, in obese humans (BMI=31.9 vs. 20.9), who are known to have increased β- cell mass compared to lean controls (Butler et al 2003), 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 β-cell proliferation or changes in β-cell mass.

Example 11: Expression of Bcl6, Menl and HoxA9 is response to mitogens like PRL or leptin

[0206] These results suggested that leukocytes could be used to measure responses to mitogens like PRL or leptin. To test this possibility, human leukocytes were exposed to PRL, which resulted in increased Bcl6 expression, while Menl and the menin target HoxA9 decreased (Figure 12, Panel A). Moreover, the magnitude of the changes in Bcl6 and Menl mRNA provoked by PRL in leukocytes was indistinguishable from that in PRL-treated islets (Figure 12, Panel B). To investigate if this blood-based assay could be used to measure diabetes risk, studies were performed of patients with a prior history of GDM (n=7) and matching control women (n=l 1): at the time of, neither group had evidence of diabetes or other ongoing disease. It was reasoned that the Bcl6-menin response in subjects with GDM history might be impaired. Leukocytes from the GDM group failed to alter expression of Bcl6, Menl or HoxA9 when exposed to PRL (Figure 12, Panel B). This suggests that the cellular machinery for responding to mitogens like PRL is dysfunctional in patients with a history of GDM. These results indicate that the islet β-cells in these patients may also harbor this defective machinery: if so, then diabetes in may be a manifestation of defects in the 'proliferative potential' of β-cells. Example 12: Expression of Bcl6 in patients with a history of GDM

[0207] To explore the possibility that regulation of Bcl6 may be impaired in patients with a history of GDM, the 'baseline' levels of Bcl6 mRNA were compared in blood leukocytes that were untreated. As shown in Figure 13, a significant elevation of BcW mRNA was measured in these patients compared to controls. As a group, the patients with GDM history were indistinguishable from controls for other characteristics, including age and body mass index (Figure 14)

[0208] These findings in combination with the results showing that Bcl6 in patients with prior GDM is relatively uninducible following mitogen challenge, support the possibility that cis-regulatory sites regulating Bcl6 expression may be impaired. If so, genomic DNA-based studies like SNP mapping or DNA sequencing of the Bcl6 locus, one of the most highly polymorphic loci in the human genome (Migliazza A, Martinotti S, Chen W, Fusco C, Ye BH, Knowles DM, Offit K, Chaganti RS, Dalla Favera R. 1995. Frequent somatic hypermutation of the 5' noncoding region of the BCL6 gene in B-cell lymphoma. Proc Natl AcadSci USA 92:12520; Peng HZ, Du MQ, Koulis A, Aiello A, Dogan A, Pan LX, Isaacson PG. 1999. Nonimmunoglobulin gene hypermutation in germinal center B cells. Blood 93:2167-72), may ultimately identify linked molecular changes corresponding to the altered Bcl6 expression we observe. Moreover, these findings demonstrate a significant change in this biomarker in frozen blood leukocytes raises the possibility of using 'archived' human tissues sources to measure Bcl6 or other candidate biomarkers of β-cell proliferation or mass. Use of archived tissue could greatly accelerate studies to validate such a marker. In summary, the results of these examples demonstrate that markers from accessible tissues like blood may be useful for (1) predicting diabetes risk, (2) monitoring ongoing β-cell proliferation and β- cell mass changes, (3) predicting individual 'potential' for β-cell expansion or regeneration and (4) detecting 'pre-clinical' diabetes.

[0209] All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0210] The invention now being fully described, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. 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 comprising: 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.

2. The method according to claim 1, wherein the menin-related factors comprise one or more of selected from BCl-6, Menl, HoxA9, CCNDl, CCND2, pl8^INK4C, MafA, MafB, phosphatase calcineurin, NFATcI, Pbx2, and MLLl .

3. The method of claim 1 or 2, wherein the one or more menin-related factors is selected from BCl-6, Menl, and HoxA9.

4. The method of claim 3, wherein the measured expression level of BCl-6 is greater than the measured expression level of the control.

5. The method of claim 3, wherein the measured expression level of Menl is lower than the measured expression level of the control.

6. The method of claim 3, wherein the measured expression level of HoxA9 is lower than the measured expression level of the control.

7. The method according to any of claims 1-6, wherein the measurement of gene expression level comprises a quantitative measurement.

8. The method according to any of claims 1-7, wherein the blood cells are leukocytes.

9. The method of claim 8, wherein the leukocytes comprises at least one of lymphocytes, B- lymphocytes, T-lymphocytes, neutrophils, eosinophils, basophils, and monocytes.

10. The method according to any of claims 1-9, wherein the level of gene expression is measured by the level of mRNA of one or more menin-related factors in the cells.

1 1. The method according to any of claims 1-9, wherein the level of gene expression is measured by the level of protein of one or more menin-related factors in the cells.

12. The method of claim 10, further comprising isolating mRNA from the leukocytes prior to measuring the RNA level expressed by the one or more menin-related factor.

13. The method of claim 12, wherein the measurement of gene expression level comprises a polymerase chain reaction (PCR).

14. The method of claim 10, further comprising isolating a menin-related factor protein from the leukocytes prior to measuring the protein level expressed by the one or more menin- related factor.

15. The method of claim 14, wherein the measurement of gene expression level comprises a quantitative measurement of protein level.

16. The method of claim 11, wherein protein level is measured by an immunoassay selected from RIA, ELISA, EMIT, CEDIA, Western blot analysis, or FIA.

17. The method of claims 11, wherein protein level is measured by mass spectrometry.

18. A method 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.

19. The method of claim 18, wherein the blood cells are leukocytes.

20. The method of claim 18, wherein the level of gene expression of one or more menin- related factors is measured as mRNA expression or protein expression.

21. The method according to any of claims 1-3 and 18-20, wherein the one or more menin related factor is selected from BCl-6, Menl, HoxA9, CCNDl, CCND2, plβ¹™⁴⁰, MafA, Maffi, phosphatase calcineurin, NFATcI, Pbx2, and MLLl.

22. The method according to any of claims 1-3 and 18-20, wherein the subject is at risk of developing diabetes, pre-diabetes, or gestational diabetes is obese.

23. The method according to any of claims 1-3 and 18-20, wherein the subject has a genetic propensity for developing diabetes, pre-diabetes, or gestational diabetes.

24. The method according to any of claims 1-3 and 18-20, wherein the subject at risk of developing diabetes, pre-diabetes, or gestational diabetes is pregnant.

25. A kit 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, the kit comprising: at least one reagent specific for detecting a menin-related factor selected from: BCl-6, Menl, HoxA9, CCNDl, CCND2, pl8^1NK4C, MafA, MafB, phosphatase calcineurin, NFATcI, Pbx2, and MLLl; and optionally, instructions for carrying out a method of aiding in the diagnosis of diabetes, pre-diabetes, or gestational diabetes.

26. The kit of claim 25 wherein the reagent specific for the menin-related factor is an antibody, or fragment thereof, that is specific for the menin-related factor.

27. The kit of claim 25 further comprising an amount of at least one menin-related factor suitable for normalizing data.

28. A method of screening for an agent effective in regulation of mammalian β-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 β-islet cell growth.

29. The method of claim 29, wherein the one or more menin-related factor is selected from BCl-6, Menl, HoxA9, CCNDl, CCND2, pl8^INK4C, MafA, Maffi, phosphatase calcineurin, NFATcI, Pbx2, and MLLl

30. The method of claim 29, wherein the blood cell is a leukocyte.

31. The method of claim 29, wherein the level of gene expression of one or more menin- related factors is measured as mRNA expression or protein expression.

32. The method of claim 29 wherein the candidate agent is 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.

33. The method according to any of claims 29-32 wherein the candidate agent is effective for treating or preventing a risk of developing diabetes, pre-diabetes, or gestational diabetes in a subject.