WO2001034833A2 - Compositions et methodes pour reguler l'inhibiteur endogene de l'atp-synthase, et pour le traitement du diabete - Google Patents

Compositions et methodes pour reguler l'inhibiteur endogene de l'atp-synthase, et pour le traitement du diabete Download PDF

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WO2001034833A2
WO2001034833A2 PCT/US2000/030862 US0030862W WO0134833A2 WO 2001034833 A2 WO2001034833 A2 WO 2001034833A2 US 0030862 W US0030862 W US 0030862W WO 0134833 A2 WO0134833 A2 WO 0134833A2
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organelle
atp
cells
sequence
ifl
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WO2001034833A3 (fr
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Christen M. Anderson
William Clevenger
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Mitokor
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Priority to JP2001536758A priority patent/JP2003527835A/ja
Priority to AU17599/01A priority patent/AU1759901A/en
Publication of WO2001034833A2 publication Critical patent/WO2001034833A2/fr
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/04—Fusion polypeptide containing a localisation/targetting motif containing an ER retention signal such as a C-terminal HDEL motif
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/07—Fusion polypeptide containing a localisation/targetting motif containing a mitochondrial localisation signal
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • the present invention relates generally to diabetes mellitus, and in particular to compositions and methods for the diagnosis, prognosis and treatment of type 2 diabetes. More specifically, the invention relates to compositions and methods for the direct delivery of extracellular polypeptides to organelles, to the use of increased mitochondrial ATP production to treat diabetes, to methods of using IFl (the inhibitory factor of mitochondrial ATPase) and derivatives thereof as therapeutic agents for diabetes mellitus, and to methods of using IFl and derivatives thereof as reagents for assays designed to identify agents that either (i) cause or contribute to, or (ii) ameliorate or treat, diabetes mellitus.
  • IFl the inhibitory factor of mitochondrial ATPase
  • Type 2 diabetes mellitus or '"late onset” diabetes, is a common, degenerative disease affecting 5 to 10 percent of the population in developed countries.
  • the propensity for developing type 2 diabetes mellitus (“tyP e 2 DM”) is reportedly maternally inherited, suggesting a mitochondrial genetic involvement. (Alcolado, J.C. and Alcolado, R., Br. Med. J. 302:1178-1180 (1991); Reny, S.L., International J.
  • Diabetes is a heterogeneous disorder with a strong genetic component; monozygotic twins are highly concordant and there is a high incidence of the disease among first degree relatives of affected individuals.
  • oral agents that are designed to lower blood glucose levels.
  • oral agents include (i) the sulfonylureas, which act by enhancing the sensitivity of the pancreatic beta cell to glucose, thereby increasing insulin secretion in response to a given glucose load; (ii) the biguanides, which improve glucose disposal rates and inhibit hepatic glucose output; (iii) the thiazolidinediones, which improve peripheral insulin sensitivity through interaction with nuclear peroxisome proliferator- activated receptors (PPAR, see, e.g., Spiegelman, 1998 Diabetes 47:507-514; Schoonjans et al., 1997 Curr. Opin. Lipidol.
  • PPAR nuclear peroxisome proliferator- activated receptors
  • the degenerative phenotype that may be characteristic of late onset diabetes mellitus includes indicators of altered mitochondrial respiratory function, for example impaired insulin secretion, decreased ATP synthesis and increased levels of reactive oxygen species.
  • type 2 DM may be preceded by or associated with certain related disorders. For example, it is estimated that forty million individuals in the U.S. suffer from impaired glucose tolerance (IGT). Following a glucose load, ciruculating glucose concentrations in IGT patients rise to higher levels, and return to baseline levels more slowly, than in unaffected individuals. A small percentage of IGT individuals (5-10%) progress to non-insulin dependent diabetes (NIDDM) each year. This form of diabetes mellitus.
  • IGT impaired glucose tolerance
  • type 2 DM is associated with decreased release of insulin by pancreatic beta cells and a decreased end-organ response to insulin.
  • Other symptoms of diabetes mellitus and conditions that precede or are associated with diabetes mellitus include obesity, vascular pathologies, peripheral and sensory neuropathies and blindness.
  • none of the current pharmacological therapies corrects the underlying biochemical defect in type 2 DM. Neither do any of these currently available treatments improve all of the physiological abnormalities in type 2 DM such as impaired insulin secretion, insulin resistance and/or excessive hepatic glucose output.
  • treatment failures are common with these agents, such that multi-drug therapy is frequently necessary.
  • Mitochondria are the main energy source in cells of higher organisms, and provide direct and indirect biochemical regulation of a wide array of cellular respiratory, oxidative and metabolic processes. Such processes include electron transport chain (ETC) activity, which drives oxidative phosphorylation to produce metabolic energy in the form of adenosine triphosphate (ATP), and which controls mitochondrial regulation of intracellular and intramitochondrial calcium homeostasis.
  • ETC electron transport chain
  • ATP adenosine triphosphate
  • Mitochondrial ultrastructural characterization reveals the presence of an outer mitochondrial membrane that serves as an interface between the organelle and the cytosol, a highly folded inner mitochondrial membrane that appears to form attachments to the outer membrane at multiple sites, and an intermembrane space between the two mitochondrial membranes.
  • the subcompartment within the inner mitochondrial membrane is commonly referred to as the mitochondrial matrix.
  • the cristae originally postulated to occur as infoldings of the inner mitochondrial membrane, have recently been characterized using three-dimensional electron tomography as also including tube-like conduits that may form networks, and that can be connected to the inner membrane and/or the intermembrane space by open, circular (30 nm diameter) junctions (Perkins et al., 1997, Journal of Structural Biology 119:260-272).
  • the inner mitochondrial membrane While the outer membrane is freely permeable to ionic and non-ionic solutes having molecular weights less than about ten kilodaltons, the inner mitochondrial membrane exhibits selective and regulated permeability for many small molecules, including certain cations, and is impermeable to large (> -10 kDa) molecules.
  • Z stands for -2.303 RT/F.
  • the value of Z is -59 at 25°C when ⁇ p and ⁇ m are expressed in mV and ⁇ pH is expressed in pH units (see, e.g., Ernster et al., J. Cell Biol 91:227s, 1981 and references cited therein).
  • ⁇ m provides the energy for phosphorylation of adenosine diphosphate (ADP) to yield ATP by ETC Complex V, a process that is coupled stoichiometrically with transport of a proton into the matrix.
  • ADP adenosine diphosphate
  • ⁇ m is also the driving force for the influx of cytosolic Ca 2+ into the mitochondrion.
  • Even fundamental biological processes, such as translation of mRNA molecules to produce polypeptides, may be dependent on ⁇ m (Cote et al., J. Biol. Chem. 265:7532-7538, 1990).
  • ETC Complex V the primary means whereby protons can return to the matrix.
  • MPT mitochondrial permeability transition
  • protons are able to bypass the conduit of Complex V without generating ATP, thereby uncoupling respiration from ATP production.
  • MPT mitochondrial permeability transition
  • ⁇ m collapses and mitochondrial membranes lose the ability to selectively regulate permeability to solutes both small (e.g., ionic Ca 2" , Na + , K + and H + ) and large (e.g., proteins).
  • Loss of mitochondrial potential also appears to be a critical event in the progression of diseases associated with altered mitochondrial function such as diabetes mellitus, and also including degenerative diseases such as Alzheimer's Disease; Parkinson's Disease; Huntington's disease; dystonia; Leber's hereditary optic neuropathy; schizophrenia; mitochondrial encephalopathy, lactic acidosis, and stroke (MELAS); cancer; psoriasis; hyperproliferative disorders; mitochondrial diabetes and deafness (MIDD) and myoclonic epilepsy ragged red fiber syndrome.
  • biochemical energy is produced by mitochondrial oxidative phosphorylation, whereby electrons are transported along the ETC from donor NADH and ultimately transferred to acceptor oxygen in a process coupled to ATP synthesis.
  • ETC Complex V ATP synthase, also referred to as F 0 F, ATPase or ATPase herein
  • F 0 F ATPase
  • ATP synthase occurs as a multi-component complex of at least 16 differenct polypeptides (Walker et al., 1994 PEBS Lett. 346:39), including the transmembrane F 0 portion in which resides proton pump activity, and the F, extramembrane portion having catalytic (e.g., ATP synthesis or ATP hydrolysis) activity.
  • the globular catalytic F, ATP synthase portion comprises six polypeptides (subunits ⁇ , ⁇ , ⁇ , ⁇ , ⁇ and the ATP synthase inhibitor protein IF,), which are encoded by nuclear genes and are imported into the mitochondria during mitochdrial biogenesis. Enzyme complexes similar to mammalian ATP synthase are found in all cell types and in chloroplast and bacterial membranes.
  • IFl also notated IF, which inhibits catalytic activity of the ATP synthase F, portion
  • Mature IFl protein is approximately 84 amino acids in length (9.6 kDa) and is synthesized as an approximately 105 amino acid precursor protein from which the N-terminal signal sequence is cleaved after importation into mitochondria.
  • IFl features pH-sensitive, primarily alpha-helical structure that is highly conserved in eukaryotes such as yeast and mammals (Lebowitz et al. 1993 Arch. Biochem. Biophys. 301 :64).
  • IFl In the alpha helix conformation IFl is inactive as an ATP synthase inhibitor, but at pH ⁇ 6.7 IFl loses its helical structure and is activated to bind to the catalytic portion ⁇ (and possibly ⁇ ) subunit and inhibit ATP synthase (Jackson et al., 1988 FEBS Lett. 229:224; Mimura et al., 1993 J. Biochem. 113:350). IFl inhibition of ATPase activity may also be influenced by mitochondrial membrane potential and/or by IFl interactions with phospholipids (see, e.g., Solaini et al., 1997 Biochem J. 327:443 and references cited therein). IFl and related proteins are described, for example, in WO98/33909 and references cited therein.
  • Mitochondrial energy production is related to glucose homeostasis primarily through the regulation of glucose stimulated insulin secretion (GSIS).
  • GSIS glucose stimulated insulin secretion
  • the initial step is uptake of glucose into the ⁇ -cells via glut 2 glucose transporters ( Figure 2). Uptake significantly exceeds glucose utilization and is therefore not rate-limiting for the sequence of events that triggers insulin release (Matschinsky, Diabetes, 45:223- 241 (1996); Newgard and McGarry, Annu Rev Biochem, 64:689-719 (1995)). Rather, it is the subsequent phosphorylation of glucose to glucose-6-phosphate (G6P) that appears to define the setpoint at which secretion is initiated.
  • GSIS glucose stimulated insulin secretion
  • the Km of glucokinase is 6-11 mM, while that of hexokinase I is 10-100 ⁇ M.
  • hexokinase I is inhibited by its product, G6P, while glucokinase is not.
  • the majority of glucose phosphorylating activity in ⁇ -cells is accounted for by the high Km glucokinase.
  • the low Km hexokinase I is believed to be inactive in the islets due to inhibition by G6P.
  • insulin secretion normally occurs when the blood glucose begins to rise above the physiological level of ⁇ 5.5 mM.
  • Glucokinase is bound to the outer surface of the mitochondria in beta cells through its interaction with the mitochondrial membrane protein porin (also called VDAC or voltage-dependent anion channel) (Malaisse-Lagae and Malaisse, Biochem Med Metab Biol, 39:80-89, (1988); Sener et al., Arch Biochem Biophys, 251 :61-67 (1986); Muller et al., Arch Biochem Biophys, 308:8-23 (1994)).
  • VDAC voltage-dependent anion channel
  • porin is associated with the mitochondrial adenine nucleotide translocator (ANT)
  • binding of GK to the pore may facilitate delivery of oxidatively produced ATP to the enzyme, which preferentially uses ATP produced by the mitochondria (Rasschaert and Malaisse, Biochim Biophys Acta, 1015:353-360 (1990)).
  • Delivery of ADP back to the mitochondria for resynthesis of ATP by complex V may also be a function of this association (Laterveer et al., In Gnaiger E, Gellerich FN, and Wyss M (eds.): Modern Trends in Biothermokinetics 3, New York: Plenum, pp.186-190 (1994)).
  • glucokinase as the glucose sensor is illustrated by the development of diabetes in individuals who have mutations of the glucokinase gene (Froguel et al., TV Engl J Med, 328:697-702 (1993); Bell et al., Annu Rev Physiol, 58:171-186 (1996)).
  • Maturity Onset Diabetes of the Young (MODY) is a form of diabetes mellitus that resembles NIDDM clinically, but has its onset before the age of 25, is generally milder, and has an autosomal dominant mode of transmission. At least three distinct mutations have been identified in MODY families (Bell et al., Annu Rev Physiol, 58:171-186 (1996)).
  • MODY 2 is characterized by mutations of the glucokinase gene, resulting in a predicted 50-100% decrease in glucokinase activity and impaired insulin secretion (Froguel et al., N Engl J Med, 328:697-702 (1993); Hattersley et al., Lancet, 339:1307-1310 (1992)).
  • the occurrence of diabetes in heterozygous individuals who have some residual glucokinase activity underscores the role of glucokinase as the rate limiting glucose sensor of the beta cell.
  • glycolytic pathway distal to G6P appears particularly important for insulin secretion with regards to the production of NADH (Dukes et al., J Biol Chem, 269:10979-10982 (1994); MacDonald and Fahien, Arch Biochem Biophys, 279:104-108 (1990)), which is efficiently shuttled from the cytosol to the mitochondria. There, it enters the electron transport chain at complex 1 and fuels oxidative production of ATP.
  • glucose-stimulated insulin secretion can be abrogated at the cellular level by a variety of metabolic inhibitors, including oligomycin, azide, antimycin A, rotenone, cyanide, and the uncoupler FCCP (MacDonald and Fahien, Arch Biochem Biophys, 279:104-108 (1990); Detimary et al., Biochem J, 297:455-461 (1994); Dukes et al., J Biol Chem, 269:10979-10982 (1994); Kiranadi et al., FEBS Lett, 283:93-96 (1991)).
  • metabolic inhibitors including oligomycin, azide, antimycin A, rotenone, cyanide, and the uncoupler FCCP (MacDonald and Fahien, Arch Biochem Biophys, 279:104-108 (1990); Detimary et al., Biochem J, 297:455-461 (1994); Dukes et al., J Biol
  • p° INS-1 cells produced using ddC retained normal basal insulin secretion, but failed to increase insulin secretion in response to a glucose challenge.
  • a normal insulin secretory response to KC1 was observed in these cells, suggesting that the insulin secretory machinery distal to the mitochondria was intact.
  • intracellular ATP levels did not change in response to glucose in these p° INS-1 cells (Anderson, Drug Development Research, 46:57-67 (1999)).
  • the shift from oxidative to glycolytic ATP production in this p° cell line was also demonstrated by an increase in lactate production by the p° cells as compared to the parental INS-1 cells.
  • hexokinase associates with the mitochondria in skeletal muscle, resulting in activation of the enzyme (De Vos et al., Biochem Int, 24:117-121 (1991); Adams, Biochim Biophys Acta, 932:195-205 (1988); Weiler, Biochem Med, 33:223-235 (1985)), and facilitating delivery of ATP to the enzyme.
  • the specific effects of mitochondrial mutations and mitochondrial dysfunction on the activity of hexokinase remain to be determined, but may contribute to impaired insulin-mediated glucose utilization.
  • apoptosis e.g., cancer, autoimmune diseases and possibly certain forms of diabetes in the first instance, and stroke damage and neurodegeneration in Alzheimer's disease in the latter case.
  • apoptosis e.g., cancer, autoimmune diseases and possibly certain forms of diabetes in the first instance, and stroke damage and neurodegeneration in Alzheimer's disease in the latter case.
  • mitochondria e.g., apoptosis, and the role of mitochondria therein, see, e.g., Green and Reed (Science 2#7: 1309-1312, 1998), Green (Cell 94:695-698, 1998) and Kroemer (N ⁇ twre Medicine 3:614-620, 1997).
  • the present invention provides compositions and methods related to exploiting the regulation of glucose-stimulated insulin secretion by mitochondrial energy production to at least partially overcome the inadequate GSIS in type 2 DM, and offers other related advantages.
  • the endogenous inhibitor of ATP synthase is an IFl
  • the IFl is a mammalian IFl
  • the mammalian IFl is a mouse IFl, a rat IFl, a rabbit IFl, a bovine IFl, a canine IFl , a non-human primate IFl or a human IFl .
  • the invention provides a method for identifying an agent that alters mitochondrial ATP production, comprising contacting, in the absence and presence of a candidate agent, an isolated IFl polypeptide and an isolated mitochondrial ATP synthase, wherein the ATP synthase is capable of ATP synthesis, under conditions and for a time sufficient for ATP production to occur; and comparing a level of ATP production by the ATP synthase in the presence of the candidate agent to a level of ATP production in the absence of the candidate agent, and therefrom identifying an agent that alters mitochondrial ATP production.
  • the invention provides a method of treating diabetes comprising administering to a patient in need thereof an effective amount of a compound that (a) increases the synthesis of mitochondrial ATP in cells, (b) decreases the hydrolosis of mitochondrial ATP in cells, or (c) does both (a) and (b).
  • the compound is a composition that inhibits one or more activities of IFl or a composition that mimics IFl .
  • the invention provides a method of identifying agents useful for treating diabetes, comprising contacting a sample comprising mitochondria with a candidate agent and determining an effect of the compound on the amount of ATP in the sample, wherein a compound that results in increased ATP in the sample is identified as an agent useful for treating diabetes.
  • a method of identifying an agent useful for treating diabetes comprising contacting a sample comprising mitochondria with a candidate agent and determining an effect of the compound on the amount of ATP in the mitochondria, wherein a compound that results in increased ATP in the mitochondria is identified as an agent useful for treating diabetes.
  • an organelle-targeted fusion protein comprising: (a) a first polypeptide portion comprising an organelle targeting sequence, wherein the organelle targeting sequence is capable of promoting the localization of a protein to a selected organelle; (b) a second polypeptide portion comprising a tat sequence; and (c) a third polypeptide portion having an amino acid sequence distinct from the first or the second polypeptide portion, wherein the organelle-targeted fusion protein is taken up by cells upon contact and is preferentially localized to the selected organelle.
  • an organelle-targeted compound comprising: (a) a first polypeptide portion comprising an organelle targeting sequence, wherein the organelle targeting sequence is capable of promoting the localization of a protein to a selected organelle; (b) a second polypeptide portion comprising a "tat sequence"; and (c) a nucleic acid portion, wherein the organelle-targeted compound is taken up by cells upon contact and is preferentially localized to the selected organelle.
  • the nucleic acid portion is a DNA, an RNA, an oligonucleotide, a ribozyme, an expression cassette, an expression construct, or a peptide nucleic acid.
  • the organelle-targeted compound further comprises a detectable label.
  • the selected organelle is a mitochondrion, and the organelle targeted sequence is a mitochondrial targeting sequence, which in certain further embodiments is SEQ ID NO: 10, SEQ ID NOT 1 or SEQ ID NO: 14.
  • the selected organelle is a Golgi apparatus, and the organelle targeted sequence is a Golgi targeting sequence.
  • the selected organelle is a nucleus, and the organelle targeted sequence is a nuclear targeting sequence.
  • the selected organelle is a chloroplast, and the organelle targeted sequence is a chloroplast targeting sequence.
  • the selected organelle is the endoplasmic reticulum
  • the organelle targeted sequence is an ER targeting sequence.
  • the present invention provides a method of delivering an organelle-targeted fusion protein to an organelle in a cell comprising contacting the cell with the organelle- targeted fusion protein as just described.
  • the invention provides an expression construct encoding an organelle-targeted fusion protein, wherein the organelle-targeted fusion protein comprises: (a) a first polypeptide portion comprising an organelle targeted sequence wherein the organelle targeted sequence is capable of promoting the localization of a protein to a selected organelle; (b) a second polypeptide portion comprising a tat sequence; and (c) a third polypeptide portion having an amino acid sequence distinct from the first or the second polypeptide portion, wherein the organelle-targeted fusion protein is taken up by cells upon contact and is preferentially localized to the selected organelle.
  • the invention provides a host cell comprising the expression construct as just described.
  • the invention provides a method of producing an organelle-targeted fusion protein comprising culturing the host cell just described, and recovering the organelle- targeted fusion protein therefrom.
  • Figure 1 shows the loss over time of mitochondrial DNA (mtDNA) from INS-1 cells treated with ddC (panel 1 A) and the secretion of insulin by these cells and the parent INS-1 cells in response to glucose treatment (panel IB).
  • Figure 2 shows the results of experiments in which INS-1 cells and mtDNA-depleted INS-1 cells are treated with glucose and measured for their ability to produce ATP (panel 2A) or lactate (panel 2B).
  • Figure 3 shows the inhibition of purified FI -ATPase by Aurovertin-B.
  • Figure 4 shows the inhibition of purified FI -ATPase by partially purified bovine cardiac IFl.
  • Figure 5 shows the amino acid sequence of rat IFl (SEQ ID NO: 13) and results from experiments in which two different synthetic polypeptides derived from rat IFl were tested for their ability to inhibit purified FI -ATPase or F0-F1 -ATPase.
  • Figure 6 shows the results of an experiment in which a synthetic polypeptide corresponding to amino acids 42-58 of rat IFl was tested for its ability to inhibit F0-F1 -ATPase in rat alkaline submitochondrial particles.
  • Figure 7 shows gel electrophoretic (Coomassie stain) and western blot characterization of recombinant IFl fusion proteins.
  • Figure 8 shows inhibition of ATP hydrolase activity in rat liver submitochondrial particles by a recombinant IF 1 fusion protein.
  • Figure 9 shows enhancement of glucose-stimulated insulin secretion (GSIS) by a recombinant IFl fusion protein.
  • the present invention relates to compositions and methods for treating type 2 DM, including methods for identifying an agent that alters mitochondrial ATP production.
  • the invention is therefore directed in pertinent part to the unexpected observation that regulation of glucose-stimulated insulin secretion (GSIS) by mitochondrial energy production can be manipulated in a manner that permits restoration of some or all of the inadequate GSIS present in type 2 DM.
  • GSIS glucose-stimulated insulin secretion
  • mitochondrial function may be altered (e.g., increased or decreased in a statistically significant manner relative to an appropriate control, and in certain highly preferred embodiments, increased) by alteration of interactions between IFl and ATP synthase as described herein.
  • IFl interactions with ATP synthase may be altered, for example, by altering the binding of IFl to ATP synthase, and in certain embodiments the ability of IFl to alter or regulate ATP synthase catalytic activity, which may include ATP synthase activity and/or ATP hydrolysis activity, may be altered.
  • the invention therefore contemplates compositions and methods for altering the association of IFl with at least one ATP synthase subunit, or for altering the expression level of IFl, or for altering the activity of IFl .
  • the invention contemplates agents, and screening assays to identify them, that interfere with IFl binding to ATP synthase subunits in a manner that prevents IFl inhibition of ATP synthase catalytic synthesis of ATP; the invention also contemplates agents, and screening assays to identify them, that interfere with IF1- encoding gene expression; the invention also contemplates mutant IFl that is altered in its ability to interact with ATP synthase.
  • IFl may bind to sites on ATP synthase FI ⁇ and/or ⁇ subunits, such that the invention also contemplates mutant ATP synthase subunits which, by virtue of their mutation(s), are altered in their ability to functionally interact with IFl .
  • the present invention is also directed in part to organellar-targeted fusion proteins, and in particular embodiments, to IFl fusion proteins comprising organelle-selective or organelle-specific targeting sequences (OTS). Examples of organelles for which polypeptide targeting domains are known in the art are briefly described here.
  • Mitochondria As described above, mitochondria are the main energy source in cells of higher organisms, and provide direct and indirect biochemical regulation of a wide array of cellular respiratory, oxidative and metabolic processes, including oxidative phosphorylation to produce ATP, intracellular calcium homeostasis and apoptosis.
  • agents including mitochondrially targeted fusion proteins comprising mitochondrial targeting sequences, which fusion proteins further comprise polypeptide domains able to interact with and/or influence mitochondrial components might have a variety of remedial, therapeutic, palliative, rehabilitative, preventative, prophylactic or disease-impeditive uses.
  • green fluorescent protein (GFP) fusion protein derivatives have been targeted to the mitochondrial matrix using cytochrome c oxidase subunit IV protein sequences sequences (Llopis et al., Proc. Natl. Acad. Sci. U.S.A. 95:6803-6808, 1993), to the mitochondrial intermembrane space using cytochrome c protein sequences (Mahajan et al., Nature Biotech. 76:547-552, 1998), and to the outer membrane of mitochondria using hexokinase (Sui et al., Arch. Biochem. Biophys.
  • Aequorin fusion protein derivatives have been targeted to mitochondria using cytochrome c oxidase protein sequences (Pinton et al., Biofactors 5:243-253, 1998; Rizzuto et al., Nature 358:325-327, 1992).
  • Other fusion proteins have been described that target mitochondrial sites using protein sequences from mitochondrial (or bacterial) thiolases (Arakawa et al., J. Biochem., Tokyo, 707: 160-164, 1990), F 0 - ATPase subunit 9 (J. Biol. Chem. 277:25208-25212, 1996), manganese superoxide dismutase (Balzan et al., Proc. Natl Acad. Sci. U.S.A. 92:4219-4223, 1995), and P-450(SCC) (Kumamoto et al., Biochem., Tokyo, 105:72-78, 1989).
  • Chloroplasts The chloroplast is an organelle found in plant cells wherein photosynthesis takes place. Photosynthesis, in addition to being an integral part of a plant cell's metabolism, is an important process that impacts many other living organisms as well. The reason for this is twofold: photosynthesis "fixes" atmospheric CO 2 into biologically usable carbohydrate (CHO) n molecules and also produces O 2 which is required by all aerobic organisms. Like mitochondria, chloroplasts have a double (outer and inner) membrane, contain their own DNA and have translation factors (ribosomes, tRNAs, etc.) that are distinct from those found in the cytoplasm.
  • Electron microscopy demonstrates that, like mitochondria, chloroplasts have a highly organized internal ultrastructure which includes flattened membranous bodies known as lamellae or thykaloid discs. Chloroplasts are, however, typically much larger than mitochondria; in higher plants they are generally cylindrical in shape and range from about 5 to 10 ⁇ in length and from 0.5 to 2 ⁇ in diameter. Like mitochondria, which are present in greater numbers in certain tissues (e.g., liver) than others, chloroplasts have greater copy numbers in some tissues than others. For example, mature leaves contain many chloroplasts and the total amount of chloroplast DNA in such leaves is about twice that of nuclear DNA (Jope et al., J. Cell Biol 79:631-636, 1978).
  • fusion proteins have been targeted to the chloroplast outer membrane by use of the SCE70 heat shock protein targeting sequence (Wu et al., J. Biol Chem. 265:19384-19391, 1993).
  • Other targeting sequences such as those from the Rieske iron-sulfiur protein (Madueno et al., J. Biol. Chem. 269:17458-17463, 1994), direct fusion proteins across the chloroplast thylakoid membrane.
  • fusion proteins comprising dual targeting polypeptide sequences may be employed as described (Creissen et al, Plant J. 5: 167-175, 1995; Huang et al., Plant Cell 2:1249-1260, 1990).
  • care must be taken to ensure that a dual targeting sequence is not employed.
  • the nucleus is the organelle that comprises most (from the standpoint of information, if not mass) of a cell's DNA in the form of several chromosomes (Mitochondria and chloroplasts have their own DNA molecules that are typically much smaller than the nuclear genomes, and thus encode fewer functions; however, as a cell contains only one nucleus and may contain many mitochondria and/or chloroplasts, the total mass of the DNA molecules in these organelles may approach that of the nuclear DNA.)
  • the nucleus is bounded by two membranes collectively called the nuclear envelope (the membranes are known as the inner and outer nuclear membranes).
  • RNA molecules are conveyed to or from the cytosol through openings in the nuclear envelope called nuclear pores.
  • aequorin fusion protein derivatives have been targeted to the nucleus using nucleoplasmin protein sequences (Badminton et al., J. Biol Chem. 277:31210-31214, 1997).
  • Endoplasmic Reticulum The endoplasmic reticulum (ER) is composed of a series of flattened sheets, tubes and sacs that enclose a large intracellular space.
  • the membrane of the ER is in structural continuity with the outer nuclear membrane and extends throughout the cytoplasm. Some functions of the ER include the synthesis and transport of membrane proteins and lipids.
  • two types of ERs may exist in a cell. Smooth ER are generally tubular in shape and are typically devoid of attached ribosomes; one major function of smooth ER is lipid metabolism.
  • Rough ER typically occurs as flattened sheets, the cytosolic side of which is usually associated with many active (protein-synthesizing) ribosomes.
  • aequorin fusion protein derivatives have been targeted to the endoplasmic reticulum using calreticulin protein sequences (Kendall et al., Biochem. Biophys. Res. Commun. 759:1008-1016, 1992).
  • the Golgi Apparatus is a system of stacked, flattened and membrane-enclosed sacs and is generally thought to be involved in the modification, sorting and packaging of macromolecules for secretion or for delivery to other subcellular compartments. Numerous small (> ⁇ 50 nM) membrane-enclosed vesicles which are thought to comprise macromolecules in order to carry out the transport thereof between the Golgi apparatus and other subcellular compartments.
  • Aequorin fusion protein derivatives for example, have been targeted to the Golgi membrane using galactosyltransferase, SNAP-25, connexin and 5-HT 1 ⁇ - receptor protein sequences (Burton et al., Mol Cell. Biol 7:419-434, 1996; Marsault et al., EMBO J. 76:1575-1581, 1997; Daguzan et al., Int. J. Dev. Biol. 39:653-657, 1995).
  • GFP fusion proteins have been targeted to the Golgi apparatus using galactosyltransferase protein sequences (Llopis et al., Proc. Natl Acad. Sci. U.S.A. 95:6803-6808, 1993).
  • organelle-targeted molecules of the invention have the following structures:
  • OET indicates an optional epitope tag, for example, a His tag (SEQ ID NO:l), a FLAG® epitope (SEQ ID NO:2), an AU1 epitope (SEQ ID NO:3), an AU5 epitope (SEQ ID NO:4), a c-mvc epitope (SEQ ID NO:5), a Glu-Glu epitope (SEQ ID NO:6), an HA.l l epitope (SEQ ID NO:7), an IRS eptiope (SEQ ID NO:8), or a KT3 epitope (SEQ ID NO:9);
  • CTS indicates a cellular transport sequence, a preferred sequence being that described as “TAT CTS” (SEQ ID NOS: 10, 11 and 27) herein;
  • OTS indicates an organellar targeting sequence;
  • MOI indicates a molecule of interest that one desires to target to a specific organelle, for example, a polypeptide or a nucleic acid.
  • the MOI is an IFl polypeptide and in certain other preferred embodiments the MOI is a nucleic acid sequence encoding an IFl polypeptide or a fragment, derivative, mutant or variant thereof as provided herein.
  • the MOI may be an antisense IFl nucleic acid, for example, the reverse complement of a portion of an IFl encoding nucleic acid sequence (e.g., containing the reverse complement of the ATG sequence encoding initiating methionine).
  • the MOI may be a mutated IFl polypeptide (or a nucleic acid encoding such a mutated IFl) selected for its failure to bind to ATP synthase. In other embodiments, the MOI may be a mutated IF 1 polypeptide (or a nucleic acid encoding such a mutated IFl) selected for its ability to activate ATP synthase catalytic activity.
  • these elements are arranged from the amino (N-) terminal end on the left to the carboxy (C-) terminal end on the right. It will be appreciated by those skilled in the art that the order of these elements can be altered, and additional elements can be added to the organelle-targeted molecules so long as the functionality of the various elements is retained and delivery to the desired organelles is not impaired.
  • the CTS (cellular transport sequence) is a polypeptide capable of delivering a covalently attached molecule into a target cell.
  • a preferred CTS is a HIV-1 tat protein or a HIV-1 tat-derived polypeptide, such as are described herein or in U.S. Patents 5,670,617; 5,674,980; 5,747,641 ; or 5,804,604.
  • Tat proteins from other viruses such as HIV-2 (Guyader et al., Nature 326:662-669, 1987), equine infectious anemia virus (Carroll et al., J.
  • TAT polypeptides derived from those tat proteins fall within the scope of the present invention.
  • TAT polypeptides comprising the region that mediates entry and uptake into cells can be defined using known techniques (see, e.g., Frankel et al., Proc. Natl Acad Sci. U.S.A. 56:7397-7401, 1989) and the present disclosure as a guide.
  • the present invention is directed in part to compositions and methods for the modulation of mitochondrial energy production and GSIS for the treatment of type 2 diabetes mellitus (type 2 DM).
  • Certain useful embodiments of the invention include:
  • a method for treating diabetes that includes increasing mitochondrial ATP synthesis or decreasing hydrolysis of mitochondrial ATP (or both) in cells of an individual in need thereof;
  • a method of screening for or identifying an agent that alters (e.g., increases or decreases) the binding interaction of at least one IF 1 protein and at least one ATP synthase subunit that comprises comparing the level of IFl binding to an ATP synthase subunit after contacting at least one IFl protein and at least one ATP synthase subunit in the presence or absence of one or more candidate agents or test compounds under conditions and for a time sufficient to permit IFl binding to the ATP synthase subunit(s);
  • a method of screening for or identifying an agent that alters the effect of an IFl protein on an ATP synthase catalytic activity comprises comparing the level of ATP synthase catalytic activity (e.g., synthase activity and/or hydrolase activity) in the presence of a candidate agent to the level of activity in the absence of the agent by contacting an IFl protein and a catalytically competent ATP synthase under conditions and for a time sufficient to detect ATP synthase catalytic activity in the presence or absence of one or more candidate agents or test compounds, and determining the activity of the ATP synthase;
  • the level of ATP synthase catalytic activity e.g., synthase activity and/or hydrolase activity
  • a method of screening for or identifying an agent that alters the expression of a nucleic acid that encodes an IFl protein comprises contacting at least one cell comprising a nucleic acid that encodes an IFl protein with one or more candidate agents or test compounds and measuring expression of an IF 1 protein.
  • Membrane permeant derivative refers to a chemical derivative of a compound that increases membrane permeability of the compound. These derivatives are made better able to cross cell membranes because hydrophilic groups are masked to provide more hydrophobic derivatives. Also, the making groups can be designed to be cleaved from the compound within a cell to make the compound more hydrophilic once within the cell. Because the substrate is more hydrophilic than the membrane permeant derivative, it preferentially localizes within the cell (U.S. Patent No. 5,741,657 to Tsien et al., issued April 21, 1998).
  • isolated polynucleotide refers to a polynucleotide of genomic, cDNA, PCR or synthetic origin, or some combination thereof, which by virtue of its origin, the isolated polynucleotide (1) is not associated with the cell in which the isolated polynucleotide is found in nature, or (2) is operably linked to a polynucleotide that it is not linked to in nature.
  • the isolated polynucleotide can optionally be linked to promoters, enhancers, or other regulatory sequences.
  • isolated protein refers to a protein of cDNA, recombinant RNA, or synthetic origin, or some combination thereof, which by virtue of its origin the isolated protein (1) is not associated with proteins normally found within nature, or (2) is isolated from the cell in which it normally occurs, or (3) is isolated free of other proteins from the same cellular source, for example, free of cellular proteins), or (4) is expressed by a cell from a different species, or (5) does not occur in nature.
  • Polypeptide is used herein as a generic term to refer to native protein, fragments, or analogs of a polypeptide sequence.
  • Active fragment refers to a fragment of a parent molecule, such as an organic molecule, nucleic acid molecule, or protein or polypeptide, or combinations thereof, that retains at least one activity of the parent molecule.
  • Naturally occurring refers to the fact that an object can be found in nature.
  • a polypeptide or polynucleotide sequence that is present in an organism, including viruses, that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally occurring.
  • Operaably linked refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner.
  • a control sequence operably linked to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.
  • Control sequences refer to polynucleotide sequences that effect the expression of coding and non-coding sequences to which they are ligated. The nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal biding site, and transcription termination sequences; in eukaryotes, generally, such control sequences include promoters and transcription termination sequences.
  • the term control sequences is intended to include components whose presence can influence expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.
  • Polynucleotide refers to a polymeric form of nucleotides of a least ten bases in length, either ribonucleotides or deoxyribonucleotides or a modified from of either type of nucleotide.
  • the term includes single and double stranded forms of DNA or RNA.
  • Genomic polynucleotide refers to a portion of the nuclear genome.
  • Mitochondrial genomic polynucleotide refers to a portion of the mitochondria genome.
  • Active genomic polynucleotide or active portion of a genome refer to regions of a genome (nuclear or mitrochondrial) that can be up regulated, down regulated or both, either directly or indirectly, by a biological process.
  • Ribozyme means enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA.
  • the mechanism of ribozyme action involves sequence- specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage.
  • Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target RNA target for ribozyme cleavage sites which include the sequences GUA, GUU and GUC. Once identified, short RNA sequences between 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for secondary structural features which may render the oligonucleotide inoperable.
  • the suitability of candidate targets can also be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays.
  • Directly in the context of a biological process or processes, refers to direct causation of a process that does not require intermediate steps, usually caused by one molecule contacting or binding to another molecule (the same type or different type of molecule). For example, molecule A contacts molecule B, which causes molecule B to exert effect X that is part of a biological process.
  • “Indirectly” in the context of a biological process or precesses refers to indirect causation that requires intermediate steps, usually caused by two or more direct steps. For example, molecule A contacts molecule B to exert effect X which in turn causes effect Y.
  • Sequence identity refers to the proportion of base matches between two nucleic acid sequences or the proportion of amino acid matches between two amino acid sequences. When sequence identity is expressed as a percentage, for example 50%, the percentage denotes the proportion of matches of the length of sequences from a desired sequence that is compared to some other sequence. Gaps (in either of the two sequences) are permitted to maximize matching; gap lengths of 15 bases or less are usually used, 6 bases or less are preferred with 2 bases or less more preferred.
  • the sequence identity between the target nucleic acid and the oligonucleotide sequence is preferably not less than 10 target base matches out of 20 (50% identity) and more preferably not less than about 60% identity, 70% identity, 80% identity or 90%> identity), and most preferably not less than 95% identity.
  • “Selectively hybridize” refers to detectably and specifically bind.
  • Polynucleotides, oligonucleotides and fragments thereof selectively hybridize to target nucleic acid strands, under hybridization and wash conditions that minimize appreciable amounts of detectable binding to nonspecific nucleic acids.
  • High stringency conditions can be used to achieve selective hybridization conditions as known in the art.
  • the nucleic acid sequence identity between the polynucleotides, oligonucleotides, and fragments thereof and a nucleic acid sequence of interest will be at least 30%, and more typically and preferably of at least 40%, 50%, 60%, 70%, 80% or 90%.
  • Hybridization and washing conditions are typically performed at high stringency according to conventional hybridization procedures. Positive clones are isolated and sequenced. For example, a full length polynucleotide sequence can be labeled and used as a hybridization probe to isolate genomic clones from an appropriate target library as they are known in the art. Typical hybridization conditions and methods for screening plaque lifts and other pu ⁇ oses are known in the art (Benton and Davis, Science 196:180 (1978); Sambrook et al., supra, (1989)).
  • Two amino acid sequences have share identity if there is a partial or complete identity between their sequences. For example, 85% identity means that 85% of the amino acids are identical when the two sequences are aligned for maximum matching. Gaps (in either of the two sequences being matched) are allowed in maximizing matching; gap lengths of 5 or less are preferred with 2 or less being more preferred.
  • two protein sequences (or polypeptide sequences derived from them of at least 30 amino acids in length) share identity, as this term is used herein, if they have an alignment score of at least 5 (in standard deviation units) using the program ALIGN with the mutation data matrix and a gap penalty of 6 or greater (Dayhoff, in Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, volume 5, pp. 101-110 (1972) and Supplement 2, pp. 1-10).
  • “Corresponds to” refers to a polynucleotide sequence that shares identity (for example is identical) to all or a portion of a reference polynucleotide sequence, or that a polypeptide sequence is identical to all or a portion of a reference polypeptide sequence.
  • the term “complementary to” is used herein to mean that the complementary sequence is homologous to all or a portion of a reference polynucleotide sequence.
  • the nucleotide sequence TATAC corresponds to a reference sequence TATAC and is complementary to a reference sequence GTATA.
  • a reference sequence is a defined sequence used as a basis for a sequence comparison; a reference sequence can be a subset of a larger sequence, for example, as a segment of a full length cDNA or gene sequence given in a sequence listing, or may comprise a complete cDNA or gene sequence. Generally, a reference sequence is at least 20 nucleotides in length, frequently at least 25 nucleotides in length, and often at least 50 nucleotides in length.
  • two polynucleotides can each (1) comprise a sequence (for example a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) may further comprise a sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity.
  • a comparison window refers to a conceptual segment of at least 20 contiguous nucleotide positions wherein a polynucleotide sequence may be compared to a reference sequence of at least 20 contiguous nucleotides and wherein the portion of the polynucleotide sequence in the comparison window can comprise additions and deletions (for example, gaps) of 20 percent or less as compared to the reference sequence (which would not comprise additions or deletions) for optimal alignment of the two sequences.
  • Optimal alignment of sequences for aligning a comparison window can be conducted by the local identity algorithm (Smith and Waterman, Adv. Appl Math. 2:482 (1981)), by the identity alignment algorithm (Needleman and Wunsch, J.
  • Percentage of sequence identity is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (for example, the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
  • Substantial identity denotes a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 30 percent sequence identity, preferably at least 50 to 60 percent sequence, more usually at least 60 percent sequence identity as compared to a reference sequence over a comparison window of at least 20 nucleotide positions, frequently over a window of at least 25 to 50 nucleotides, wherein the percentage of sequence identity is calculated by comparing the reference sequence to the polynucleotide sequence that may include deletions or addition which total 20 percent or less of the reference sequence over the window of comparison.
  • Substantial identity as applied to polypeptides herein means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 30 percent sequence identity, preferably at least 40 percent sequence identity, and more preferably at least 50 percent sequence identity, and most preferably at lest 60 percent sequence identity. Preferably, residue positions, which are not identical, differ by conservative amino acid substitutions.
  • Constant amino acid substitutions refer to the interchangeability of residues having similar side chains.
  • a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine
  • a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine
  • a group of amino acids having amide-containing side chains is asparagine and glutamine
  • a group of amino acids having aromatic side chains is phenylalanine, tyrosine and tryptophan
  • a group of amino acids having basic side chains is lysine, arginine and histidine
  • a group of amino acids having sulfur-containing side chan is cystein and methionine.
  • Preferred conservative amino acid substitution groups are: valine-leucine-isoleucine; phenylalanine-tyrosine; lysine-arginine; alanine-valine; glutamic-aspartic; and asparagine-glutamine.
  • “Modulation” refers to the capacity to either enhance or inhibit a functional property of a biological activity or process, for example, enzyme activity or receptor binding. Such enhancement or inhibition may be contingent on the occurrence of a specific event, such as activation of a signal transduction pathway and/or may be manifest only in particular cell types.
  • Module refers to a chemical (naturally occurring or non-naturally occurring), such as a biological macromolecule (for example, nucleic acid, protein, non- peptide or organic molecule) or an extract made from biological materials, such as prokaryotes, bacteria, eukaryotes, plants, fungi, multicellular organisms or animals, invertebrates, vertebrates, mammals and humans, including, where appropriate, extracts of: whole organisms or portions of organisms, cells, organs, tissues, fluids, whole cultures or portions of cultures, or environmental samples or portions thereof.
  • a biological macromolecule for example, nucleic acid, protein, non- peptide or organic molecule
  • an extract made from biological materials such as prokaryotes, bacteria, eukaryotes, plants, fungi, multicellular organisms or animals, invertebrates, vertebrates, mammals and humans, including, where appropriate, extracts of: whole organisms or portions of organisms, cells, organs, tissues, fluids, whole cultures or portions of cultures, or
  • Modulators are typically evaluated for potential activity as inhibitors or activators (directly or indirectly) of a biological process or processes (for example, agonist, partial antagonist, partial agonist, antagonist, antineoplastic, cytotoxic, inhibitors of neoplastic transformation or cell proliferation, cell proliferation promoting agents, antiviral agents, antimicrobial agents, antibacterial agents, antibiotics, and the like) by inclusion in assays described herein.
  • a biological process or processes for example, agonist, partial antagonist, partial agonist, antagonist, antineoplastic, cytotoxic, inhibitors of neoplastic transformation or cell proliferation, cell proliferation promoting agents, antiviral agents, antimicrobial agents, antibacterial agents, antibiotics, and the like.
  • the activity of a modulator may be known, unknown or partially known.
  • Test chemical refers to a chemical or extract, including an agent or compound such as a “test compound”, to be tested by at least one method of the present invention to be a putative modulator.
  • a test chemical is usually not known to bind to the target of interest.
  • Control test chemical refers to a chemical known to bind to the target (for example, a known agonist, antagonist, partial agonist or inverse agonist).
  • Test chemical does not typically include a chemical added to a mixture as a control condition that alters the function of the target to determine signal specificity in an assay.
  • control chemicals or conditions include chemicals that (1) non-specifically or substantially disrupt protein structure (for example denaturing agents such as urea or guandium, sulfhydryl reagents such as dithiotritol and beta-mercaptoethanol), (2) generally inhibit cell metabolism (for example mitochondrial uncouples) and (3) non- specifically disrupt electrostatic or hydrophobic interactions of a protein (for example, high salt concentrations or detergents at concentrations sufficient to non-specifically disrupt hydrophobic or electrostatic interactions).
  • test chemical also does not typically include chemicals known to be unsuitable for a therapeutic use for a particular indication due to toxicity of the subject. Usually, various predetermined concentrations of test chemicals are used for determining their activity.
  • the concentration of test chemical used can be expressed on a weight to volume basis.
  • the following ranges of concentrations can be used: between about 0.001 micrograms/ml and about 100 milligram/ml, preferably between about 0.01 micrograms/ml and about 10 milligrams/ml, and more preferably between about 0.1 micrograms/ml and about 1 milligrams/ml or between about 1 microgram/ml and about 100 micrograms/ml .
  • “Target” refers to a biochemical entity involved in a biological process. Targets are typically proteins that play a useful role in the physiology or biology of an organism. A therapeutic chemical typically binds to a target to alter or modulate its function.
  • targets can include, but not be limited to, cell surface receptors, G-proteins, G-protein coupled receptors, kinases, phosphatases, ion channels, lipases, phosholipases, nuclear receptors, intracellular structures, tubules, tubulin, and the like.
  • Label refers to inco ⁇ oration of a detectable marker, for example by inco ⁇ oration of a radiolabled compound or attachment to a polypeptide of moieties such as biotin that can be detected by the binding of a section moiety, such as marked avidin.
  • a detectable marker for example by inco ⁇ oration of a radiolabled compound or attachment to a polypeptide of moieties such as biotin that can be detected by the binding of a section moiety, such as marked avidin.
  • Various methods of labeling polypeptide, nucleic acids, carbohydrates, and other biological or organic molecules are known in the art.
  • Such labels can have a variety of readouts, such as radioactivity, fluorescence, color, chemiluminescence or other readouts known in the art or later developed.
  • the readouts can be based on enzymatic activity, such as beta-galactosidase, beta-lactamase, horseradish peroxidase, alkaline phosphatase, luciferase; radioisotopes such as 3 H, 14 C, 35 S, l25 I or l3l I); fluorescent proteins, such as green fluorescent proteins; or other fluorescent labels, such as FITC, rhodamine, and lanthanides. Where appropriate, these labels can be the product of the expression of reporter genes, as that term is understood in the art. Examples of reporter genes are beta-lactamase (U.S. Patent No.
  • substantially pure refers to an object species or activity that is the predominant species or activity present (for example on a molar basis it is more abundant than any other individual species or activities in the composition) and preferably a substantially purified fraction is a composition wherein the object species or activity comprises at least about 50 percent (on a molar, weight or activity basis) of all macromolecules or activities present.
  • a substantially pure composition will comprise more than about 80 percent of all macromolecular species or activities present in a composition, more preferably more than about 85%, 90%, 95% and 99%.
  • the object species or activity is purified to essential homogeneity, wherein contaminant species or activities cannot be detected by conventional detection methods) wherein the composition consists essentially of a single macromolecular species or activity.
  • an activity may be caused, directly or indirectly, by a single species or a plurality of species within a composition, particularly with extracts.
  • “Pharmaceutical agent or drug” refers to a chemical, composition or activity capable of inducing a desired therapeutic effect when property administered by an appropriate dose, regime, route of administration, time and delivery modality.
  • “Pharmaceutical agent or drug” refers to a chemical, composition or activity capable of inducing a desired therapeutic effect when property administered by an appropriate dose, regime, route of administration, time and delivery modality.
  • a “bioactive compound” refers to a compound that exhibits at least one bioactivity.
  • a “bioactivity” refers to a composition that exhibits at least one activity that modulates a biological process, cellular process or disease state.
  • Preferred bioactivities include, but are not limited to activities that modulate at least one mitochondrial activity or mitochondrial function as provided herein (such as the production of ATP) or mitochondrial mass, such as by an increase (mitochondrial biogenesis) or decrease in the number of mitochondria or the amount of mitochondrial DNA.
  • Another preferred bioactivity includes an activity that modulates a cellular process, such as the production or secretion of insulin.
  • a further preferred bioactivity includes an activity that modulates a disease states such as diabetes type I (type 1 DM) or diabetes type II (type 2 DM).
  • a "mitochondrial biogenesis activity” is an activity that modulates the production of active, inactive or defective mitochondria, preferably active mitochondria.
  • a “mitoclastic activity” is an activity that modulates the destruction of mitochondria.
  • An “anti -diabetic activity” is an activity that modulates the disease state of diabetes, including diabetes type I and diabetes type II.
  • an anti-diabetic activity is also, directly or indirectly, a mitochondrial biogenesis activity.
  • bioactive derivative refers to a modification of a bioactive compound or bioactivity that retains at least one characteristic activity of the parent compound.
  • a “bioactive precursor” refers to a precursor of a bioactive compound or bioactivity that exhibits at least one characteristic activity of the resulting bioactive compound or bioactivity.
  • a “patient” or “subject” refers a whole organism in need of treatment, such as a farm animal, companion animal or human. An animal refers to any non- human animal.
  • an “indicator of mitochondrial function” is any parameter that is indicative of mitochondrial function that can be measured by one skilled in the art.
  • the indicator of mitochondrial function is a mitochondrial electron transport chain enzyme, a Krebs cycle enzyme, a mitochondrial matrix component, a mitochondrial membrane component or an ATP biosynthesis factor.
  • the indicator of mitochondrial function is mitochondrial number per cell or mitochondrial mass per cell.
  • the indicator of mitochondrial function is an ATP biosynthesis factor.
  • the indicator of mitochondrial function is the amount of ATP per mitochondrion, the amount of ATP per unit mitochondrial mass, the amount of ATP per unit protein or the amount of ATP per unit mitochondrial protein.
  • the indicator of mitochondrial function comprises free radical production. In other embodiments, the indicator of mitochondrial function comprises a cellular response to elevated intracellular calcium. In other embodiments, the indicator of mitochondrial function is the activity of a mitochondrial enzyme such as, by way of non-limiting example, citrate synthase, hexokinase II, cytochrome c oxidase, phosphofructokinase, glyceraldehyde phosphate dehydrogenase, glycogen phosphorylase, creatine kinase, NADH dehydrogenase, glycerol 3 -phosphate dehydrogenase, triose phosphate dehydrogenase or malate dehydrogenase. In other embodiments, the indicator of mitochondrial function is the realtive or absolute amount of mitochondrial DNA per cell in the patient.
  • a mitochondrial enzyme such as, by way of non-limiting example, citrate synthase, hexokinase II, cytochrome
  • “Improving mitochondrial function” may refer to (a) substantially restoring to a normal level at least one indicator of glucose responsiveness in cells having reduced glucose responsiveness and reduced mitochondrial mass and/or impaired mitochondrial function; or (b) substantially restoring to a normal level, or increasing to a level above and beyond normal levels, at least one indicator of mitochondrial function in cells having impaired mitochondrial function or in cells having normal mitochondrial function, respectively.
  • Improved mitochondrial function may result from changes in extramitochondrial structures or events, as well as from mitochondrial structures or events, in direct interactions between mitochondrial and extramitochondrial genes and/or their gene products, or in structural or functional changes that occur as the result of interactions between intermediates that may be formed as the result of such interactions, including metabolites, catabolites, substrates, precursors, cofactors and the like.
  • Impaired mitochondrial function may include impairments in the level and/or rate of any respiratory, metabolic or other biochemical or biophysical activity in some or all cells of a biological source.
  • markedly impaired ETC activity may be related to impaired mitochondrial function, as may be generation of increased ROS or defective oxidative phosphorylation.
  • altered mitochondrial membrane potential, induction of apoptotic pathways and formation of atypical chemical and biochemical crosslinked species within a cell, whether by enzymatic or non-enzymatic mechanisms, may all be regarded as indicative of mitochondrial function.
  • mitochondrial function is determined as the amount of ATP per cell, per unit protein or per mitochondrion in a sample, and in certain embodiments the rate of ATP synthesis in the sample is determined. In certain embodiments an ATP biosynthesis factor as provided herein is determined.
  • ATP biosynthesis factor refers to any naturally occurring cellular component that contributes to the efficiency of ATP production in mitochondria.
  • a cellular component may be a protein, polypeptide, peptide, amino acid, or derivative thereof; a lipid, fatty acid or the like, or derivative thereof; a carbohydrate, saccharide or the like or derivative thereof, a nucleic acid, nucleotide, nucleoside, purine, pyrimidine or related molecule, or derivative thereof, or the like.
  • An ATP biosynthesis factor includes at least the components of the ETC and of the Krebs cycle (see, e.g., Lehninger, Biochemistry, 1975 Worth Publishers, New York; Voet and Voet, Biochemistry, 1990 John Wiley & Sons, New York; Mathews and van Holde, Biochemistry, 1990 Benjamin Cummings, Menlo Park, California) and any protein, enzyme or other cellular component that participates in ATP synthesis, regardless of whether such ATP biosynthesis factor is the product of a nuclear gene or of an extranuclear gene (e.g., a mitochondrial gene).
  • Participation in ATP synthesis may include, but need not be limited to, catalysis of any reaction related to ATP synthesis, transmembrane import and/or export of ATP or of an enzyme cofactor, transcription of a gene encoding a mitochondrial enzyme and/or translation of such a gene transcript.
  • Compositions and methods for determining whether a cellular component is an ATP biosynthesis factor are well known in the art, and include methods for determining ATP production (including determination of the rate of ATP production in a sample) and methods for quantifying ATP itself.
  • the contribution of an ATP biosynthesis factor to ATP production can be determined, for example, using an isolated ATP biosynthesis factor that is added to cells or to a cell-free system.
  • the ATP biosynthesis factor may directly or indirectly mediate a step or steps in a biosynthetic pathway that influences ATP production.
  • an ATP biosynthesis factor may be an enzyme that catalyzes a particular chemical reaction leading to ATP production.
  • an ATP biosynthesis factor may be a cofactor that enhances the efficiency of such an enzyme.
  • an ATP biosynthesis factor may be an exogenous genetic element introduced into a cell or a cell-free system that directly or indirectly affects an ATP biosynthetic pathway. Those having ordinary skill in the art are readily able to compare ATP production by an ATP biosynthetic pathway in the presence and absence of a candidate ATP biosynthesis factor.
  • Routine determination of ATP production may be accomplished using any known method for quantitative ATP detection, for example by way of illustration and not limitation, by differential extraction from a sample optionally including chromatographic isolation; by spectrophotometry; by quantification of labeled ATP recovered from a sample contacted with a suitable form of a detectably labeled ATP precursor molecule such as, for example, 32 P; by quantification of an enzyme activity associated with ATP synthesis or degradation; or by other techniques that are known in the art.
  • the amount of ATP in a biological sample or the production of ATP (including the rate of ATP production) in a biological sample may be an indicator of mitochondrial function.
  • ATP may be quantified by measuring luminescence of luciferase catalyzed oxidation of D- luciferin, an ATP dependent process.
  • Enzyme catalytic activity refers to any function performed by a particular enzyme or category of enzymes that is directed to one or more particular cellular function(s).
  • ATP biosynthesis factor catalytic activity refers to any function performed by an ATP biosynthesis factor as provided herein that contributes to the production of ATP.
  • enzyme catalytic activity is manifested as facilitation of a chemical reaction by a particular enzyme, for instance an enzyme that is an ATP biosynthesis factor, wherein at least one enzyme substrate or reactant is covalently modified to form a product.
  • enzyme catalytic activity may result in a substrate or reactant being modified by formation or cleavage of a covalent chemical bond, but the invention need not be so limited.
  • Various methods of measuring enzyme catalytic activity are known to those having ordinary skill in the art and depend on the particular activity to be determined.
  • enzymes including mitochondrial enzymes or enzymes that are ATP biosynthesis factors as provided herein
  • quantitative criteria for enzyme catalytic activity are well established. These criteria include, for example, activity that may be defined by international units (IU), by enzyme turnover number, by catalytic rate constant (K cat ), by Michaelis-Menten constant (K m ), by specific activity or by any other enzymological method known in the art for measuring a level of at least one enzyme catalytic activity.
  • Specific activity of a mitochondrial enzyme such as an ATP biosynthesis factor, may be expressed as units of substrate detectably converted to product per unit time and, optionally, further per unit sample mass (e.g., per unit protein or per unit mitochondrial mass).
  • enzyme catalytic activity may be expressed as units of substrate detectably converted by an enzyme to a product per unit time per unit total protein in a sample. In certain particularly preferred embodiments, enzyme catalytic activity may be expressed ' as units of substrate detectably converted by an enzyme to product per unit time per unit mitochondrial mass in a sample. In certain highly preferred embodiments, enzyme catalytic activity may be expressed as units of substrate detectably converted by an enzyme to product per unit time per unit mitochondrial protein mass in a sample. Products of enzyme catalytic activity may be detected by suitable methods that will depend on the quantity and physicochemical properties of the particular product.
  • detection may be, for example by way of illustration and not limitation, by radiometric, colorimetric, spectrophotometric, fluorimetric, immunometric or mass spectrometric procedures, or by other suitable means that will be readily apparent to a person having ordinary skill in the art.
  • detection of a product of enzyme catalytic activity may be accomplished directly, and in certain other embodiments detection of a product may be accomplished by introduction of a detectable reporter moiety or label into a substrate or reactant such as a marker enzyme, dye, radionuclide, luminescent group, fluorescent group or biotin, or the like.
  • a detectable reporter moiety or label such as a marker enzyme, dye, radionuclide, luminescent group, fluorescent group or biotin, or the like.
  • the amount of such a label that is present as unreacted substrate and/or as reaction product, following a reaction to assay enzyme catalytic activity is then determined using a method appropriate for the specific detectable reporter moiety or label. For radioactive groups, radionuclide decay monitoring, scintillation counting, scintillation proximity assays (SPA) or autoradiographic methods are generally appropriate.
  • SPA scintillation proximity assays
  • suitably labeled antibodies may be prepared including, for example, those labeled with radionuclides, with fluorophores, with affinity tags, with biotin or biotin mimetic sequences or those prepared as antibody-enzyme conjugates (see, e.g., Weir, D.M., Handbook of Experimental Immunology, 1986, Blackwell Scientific, Boston; Scouten, W.H., Methods in Enzymology 735:30-65, 1987; Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; Haugland, 1996 Handbook of Fluorescent Probes and Research Chemicals- Sixth Ed., Molecular Probes, Eugene, Oregon; Scopes, R.K., Protein Purification: Principles and Practice, 1987, Springer- Verlag, New York; Hermanson, G.T.
  • Spectroscopic methods may be used to detect dyes (including, for example, colorimetric products of enzyme reactions), luminescent groups and fluorescent groups. Biotin may be detected using avidin or streptavidin, coupled to a different reporter group (commonly a radioactive or fluorescent group or an enzyme). Enzyme reporter groups may generally be detected by the addition of substrate (generally for a specific period of time), followed by spectroscopic, spectrophotometric or other analysis of the reaction products. Standards and standard additions may be used to determine the level of enzyme catalytic activity in a sample, using well known techniques.
  • enzyme catalytic activity of an ATP biosynthesis factor may further include other functional activities that lead to ATP production, beyond those involving covalent alteration of a substrate or reactant.
  • an ATP biosynthesis factor that is an enzyme may refer to a transmembrane transporter molecule that, through its enzyme catalytic activity, facilitates the movement of metabolites between cellular compartments.
  • Such metabolites may be ATP or other cellular components involved in ATP synthesis, such as gene products and their downstream intermediates, including metabolites, catabolites, substrates, precursors, cofactors and the like.
  • an ATP biosynthesis factor that is an enzyme may, through its enzyme catalytic activity, transiently bind to a cellular component involved in ATP synthesis in a manner that promotes ATP synthesis.
  • a binding event may, for instance, deliver the cellular component to another enzyme involved in ATP synthesis and/or may alter the conformation of the cellular component in a manner that promotes ATP synthesis.
  • conformational alteration may be part of a signal transduction pathway, an allosteric activation pathway, a transcriptional activation pathway or the like, where an interaction between cellular components leads to ATP production.
  • an ATP biosynthesis factor may include, for example, a mitochondrial membrane protein.
  • Suitable mitochondrial membrane proteins include such mitochondrial components as the adenine nucleotide transporter (ANT; e.g., Fiore et al., 1998 Biochimie 50:137; Klingenberg 1985 Ann. N. Y.Acad. Sci. 456:279), the voltage dependent anion channel (VDAC, also referred to as porin; e.g., Manella, 1997 J. Bioenergetics Biomembr. 29:525), the malate-aspartate shuttle, the mitochondrial calcium uniporter (e.g., Litsky et al., 1997 Biochem.
  • ANT adenine nucleotide transporter
  • VDAC voltage dependent anion channel
  • porin e.g., Manella, 1997 J. Bioenergetics Biomembr. 29:525
  • the malate-aspartate shuttle e.g., Litsky et al., 1997 Biochem.
  • uncoupling proteins UCP-1 , -2, -3; see e.g., Jezek et al., 1998 7 «t. J. Biochem. Cell Biol. 30:1163
  • a hexokinase a peripheral benzodiazepine receptor
  • a mitochondrial intermembrane creatine kinase cyclophilin D
  • a Bcl-2 gene family encoded polypeptide the tricarboxylate carrier (e.g., locobazzi et al., 1996 Biochim. Biophys. Acta 1284:9; Bisaccia et al., 1990 Biochim. Biophys.
  • Affinity techniques are particularly useful in the context of isolating an enzyme or an ATP biosynthesis factor protein or polypeptide for use according to the methods of the present invention, and may include any method that exploits a specific binding interaction involving an enzyme or an ATP biosynthesis factor to effect a separation.
  • an enzyme or an ATP biosynthesis factor protein or polypeptide may contain covalently attached oligosaccharide moieties
  • an affinity technique such as binding of the enzyme (or ATP biosynthesis factor) to a suitable immobilized lectin under conditions that permit carbohydrate binding by the lectin may be a particularly useful affinity technique.
  • affinity techniques include immunological and other biochemical affinity techniques for isolating and/or detecting a specific protein or polypeptide antigen (e.g., an enzyme or ATP biosynthesis factor) and/or a specific binding interaction between biomolecules such as proteins, which techniques rely on specific binding interaction between antibody combining sites for antigen and antigenic determinants present on the factor, or between protein-protein binding sites, ligand- receptor binding sites, receptor-counterreceptor binding sites or the like.
  • a specific protein or polypeptide antigen e.g., an enzyme or ATP biosynthesis factor
  • Binding of an antibody or other affinity reagent to an antigen or other cognate ligand, receptor or counterreceptor is "specific" where the binding interaction involves a Ka of greater than or equal to about 10 ⁇ M ⁇ l, preferably of greater than or equal to about 10 ⁇ M ⁇ l, more preferably of greater than or equal to about 10 ⁇ M ⁇ l and still more preferably of greater than or equal to about 10 ⁇ M ⁇ l .
  • Affinities of binding partners or antibodies can be readily determined using conventional techniques, for example those described by Scatchard et al., Ann. N Y. Acad. Sci. 57:660 (1949).
  • Immunological techniques include, but need not be limited to, immunoaffinity chromatography, immunoprecipitation, solid phase immunoadso ⁇ tion or other immunoaffinity methods.
  • affinity techniques see, for example, Scopes, R.K., Protein Purification: Principles and Practice, 1987, Springer- Verlag, New York; Weir, D.M., Handbook of Experimental Immunology, 1986, Blackwell Scientific, Boston; and Hermanson, G.T. et al., Immobilized Affinity Ligand Techniques, 1992, Academic Press, Inc., California; which are hereby inco ⁇ orated by reference in their entireties, for details regarding techniques for isolating and characterizing complexes, including affinity techniques.
  • Samples of cells for the present invention can be provided as cells in culture or from a subject, such as a tissue, fluid or organ or a portion of any of the foregoing.
  • cells can preferably be from tissues that are involved in glucose metabolism, such as pancreatic cells, islates of Langerhans, pancreatic beta cells, muscle cells, liver cells or other appropriate cells.
  • cells are provided in culture and can be a primary cell line or a continuous cell line and can be provided as a clonal population of cells or a mixed population of cells.
  • the cells are insulin producing (and more preferably insulin secreting) cells in that they naturally produce and optionally secrete insulin or have been engineered to produce and optionally secrete insulin under appropriate stimuli, such as in the presence of glucose.
  • Preferred cells include, but are not limited to, a glucose-responsive, insulin-producing cell line such as the rat-derived INS-1 cell line; cells (particularly beta cells) derived from Zucker diabetic fatty rat (ZDF) or cells (particularly beta cells) from Zucker lean control rates (ZLC) ) (Shafrir et al., J. Basic Clin. Physiol. Pharmacol. 9:347-385, 1988).
  • rho-zero derivatives of the above cell lines that have been depleted of their mitochondrial DNA (mtDNA); such cells are commonly referred to as "p " ("rho-zero").
  • cybrid cells i.e., derivatives of the above cell lines in which the endogenous mtDNA has been replaced by mtDNA from an individual suffering from diabetes or another mitochondrial disease of interest.
  • General methods for preparing, using and assaying the mitochondrial functions of rho-zero and cybrid cells are described in U.S. Patent No. 5,888,438, published PCT applications WO 95/26973 and WO 98/17826, King and Attardi (Science 246:500-503, 1989), Chomyn et al.
  • Cybrid cells comprising mitochondria dervied from diabetic individuals are described in published PCT applications WO 95/26973 and WO 98/17826. Cybrid cells can be made using mitochondria from healthy subjects or from subjects that may have mitochondrial defects. Briefly, a host cell line is treated with ethidium bromide, or an antiviral agent (as described in copending U.S. patent applications 09/069,489 and 09/237,999) such as ddC, to substantially deplete cells of mitochondrial DNA (mtDNA). Platelets, or other sources of mitochondria, are fused with the mitochondria depleted cells to form a hybrid cell that includes the nuclear genome of the host cell and the mitochondria (and thus mitochondrial genome) of the subject.
  • ethidium bromide or an antiviral agent (as described in copending U.S. patent applications 09/069,489 and 09/237,999) such as ddC
  • mtDNA mitochondrial DNA
  • Platelets, or other sources of mitochondria are fused with the mitochondria deplete
  • Ceramide particularly C2 ceramide, but not C2 dihydroceramide
  • nitric oxide are stimulated by FAA (oleate:palmitate).
  • C6 ceramide can induce caspase 3 activation in INS-1 cells.
  • sodium nitroprusside (SNP) can induce INS-1 cell death.
  • Biological samples may comprise any tissue or cell preparation in which at least one mitochondrial function can be detected (and which in certain preferred embodiments pertains to mitochondrial ATP production and/or an ATP biosynthesis factor as provided herein), and may vary in nature accordingly, depending on the particular indicator(s) to be compared.
  • Biological samples may be provided by obtaining a blood sample, biopsy specimen, tissue explant, organ culture or any other tissue or cell preparation from a subject or a biological source.
  • the subject or biological source may be a human or non-human animal, a primary cell culture or culture adapted cell line including but not limited to genetically engineered cell lines that may contain chromosomally integrated or episomal recombinant nucleic acid sequences, immortalized or immortalizable cell lines, somatic cell hybrid or cytoplasmic hybrid "cybrid" cell lines, differentiated or differentiatable cell lines, transformed cell lines and the like.
  • the subject or biological source may be suspected of having or being at risk for having type 2 diabetes mellitus, and in certain preferred embodiments of the invention the subject or biological source may be known to be free of a risk or presence of such as disease.
  • signs and symptoms of type 2 diabetes may be used to so designate a subject or biological source, for example clinical signs referred to in Gavin et al. (Diabetes Care 22(suppl. 1):S5-S19, 1999, American Diabetes Association Expert Committee on the Diagnosis and Classification of Diabetes Mellitus) and references cited therein, or other means known in the art for diagnosing type 2 diabetes.
  • biological samples may be obtained from the subject or biological source before and after contacting the subject or biological source with a candidate agent, for example to identify a candidate agent capable of effecting a change in the level of a mitochondrial function, relative to the level before exposure of the subject or biological source to the agent.
  • Candidate agents for use in screening assay methods provided by the present invention such as methods for identifying an agent that alters mitochondrial ATP production or methods for identifying an agent for treating diabetes, may be provided as "libraries” or collections of compounds, compositions or molecules. Such molecules typically include compounds known in the art as “small molecules” and having molecular weights less than 10 5 daltons, preferably less than 10 4 daltons and still more preferably less than 10 3 daltons. For example, members of a library of test compounds can be administered to a plurality of samples, and then assayed for their ability to increase or decrease the level of at least one indicator of mitochondrial function.
  • Candidate agents further may be provided as members of a combinatorial library, which preferably includes synthetic agents prepared according to a plurality of predetermined chemical reactions performed in a plurality of reaction vessels.
  • various starting compounds may be prepared employing one or more of solid- phase synthesis, recorded random mix methodologies and recorded reaction split techniques that permit a given constituent to traceably undergo a plurality of permutations and/or combinations of reaction conditions.
  • the resulting products comprise a library that can be screened followed by iterative selection and synthesis procedures, such as a synthetic combinatorial library of peptides (see e.g., PCT/US91/08694, PCT/US91/04666, which are hereby inco ⁇ orated by reference in their entireties) or other compositions that may include small molecules as provided herein (see e.g., PCT/US94/08542, EP 0774464, U.S. 5,798,035, U.S. 5,789,172, U.S. 5,751,629, which are hereby inco ⁇ orated by reference in their entireties).
  • Those having ordinary skill in the art will appreciate that a diverse assortment of such libraries may be prepared according to established procedures, and tested for their influence on an indicator of mitochondrial function, according to the present disclosure.
  • the invention provides a method of treating a patient having type 2 DM by administering to the patient an agent that substantially increases mitochondrial ATP synthesis in cells, and/or that substantially decreases mitochondrial ATP hydrolysis in cells, and/or that restores at least one mitochondrial function to a level found in control or normal subjects.
  • the restored or increased mitochondrial function is the amount of ATP produced.
  • an agent that substantially restores or alters (e.g., increases or decreases in a statistically significant fashion) mitochondrial ATP biosynthetic and/or mitochondrial ATP hydrolytic function to a normal level effects the return of the level of ATP to a level found in control subjects.
  • the agent that substantially restores such mitochondrial function confers a clinically beneficial effect on the subject.
  • the agent that substantially restores such mitochondrial function promotes a statistically significant change the mitochondrial function.
  • an agent that substantially restores at least one mitochondrial function to a normal level may include an agent capable of fully or partially restoring such level.
  • a preferred agent comprises a composition that inhibits (i.e., impairs, hinders or otherwise down-regulates) one or more activities of IFl .
  • a preferred agent may comprise a composition that mimics IFl, which relates to a composition that structurally and/or functionally resembles, imitates, supplants, supplements, augments, enhances, substitutes or otherwise replaces all or a portion of a native IFl molecule, for example by possessing a three-dimensional structure capable of binding interactions with binding partners to which IFl binds, or as another example, by exhibiting greater stability and/or specificity under physiological conditions than might be expected of IFl .
  • the nucleotide sequence of interest such as a nucleotide sequence encoding an IFl , or functional equivalents thereof, is inserted into an appropriate "expression vector," i.e., a genetic element, often capable of autonomous replication, which contains the necessary elements for the transcription and, in instances where the gene product is a protein, translation of the inserted nucleotide sequence.
  • an expression vector i.e., a genetic element, often capable of autonomous replication, which contains the necessary elements for the transcription and, in instances where the gene product is a protein, translation of the inserted nucleotide sequence.
  • a genetic element that comprises an expression vector and a nucleic acid of interest in an arrangement appropriate for expression of a gene product of interest is referred to herein as an "expression construct.”
  • a variety of expression vector/host systems may be utilized to contain and express a nucleotide sequence of interest. These include but are not limited to microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., baculovirus); plant cell systems transfected with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with bacterial expression vectors (e.g., Ti or pBR322-based plasmids); or animal cell systems.
  • microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., baculovirus); plant cell systems transfected with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco
  • control elements or "regulatory sequences” of these systems, which may vary in their strength and specificities, are those nontranslated regions of the vector, enhancers, promoters, and 5' and 3' untranslated regions, which interact with host cellular proteins to carry out transcription and, where the gene product of interest is a protein, translation.
  • control elements or “regulatory sequences” of these systems, which may vary in their strength and specificities, are those nontranslated regions of the vector, enhancers, promoters, and 5' and 3' untranslated regions, which interact with host cellular proteins to carry out transcription and, where the gene product of interest is a protein, translation.
  • any number of suitable transcription and translation elements including constitutive and inducible promoters, may be used.
  • inducible promoters including hybrid promoters, such as lacZ promoter of the BluescriptTM phagemid (Stratagene, La Jolla, CA.) or pSportl (Life Technologies, Inc., Rockville, MD) and ptrp-lac hybrids and the like may be used.
  • hybrid promoters such as lacZ promoter of the BluescriptTM phagemid (Stratagene, La Jolla, CA.) or pSportl (Life Technologies, Inc., Rockville, MD) and ptrp-lac hybrids and the like
  • the baculovirus polyhedrin promoter may be used.
  • Promoters and/or enhancers derived from the genomes of plant cells e.g., heat shock, RUBISCO; and storage protein gene promoters
  • plant viruses or pathogens e.g., viral or Agrobacterium-based promoters or leader sequences
  • promoters from mammalian genes or from mammalian viruses are appropriate. If it is necessary to generate a cell line that contains multiple copies of the nucleotide sequence of interest, vectors based on SV40 or EBV may be used with an appropriate selectable marker.
  • a number of expression vectors may be selected depending upon the use intended for expressed gene product of interest. For example, when large quantities of a protein of interest are needed for the induction of antibodies, vectors which direct high level expression of the protein of interest, or fusion proteins derived therefrom that are more readily assayed and/or purified, may be desirable.
  • Such vectors include, but are not limited to, Escherica coli cloning and expression vectors such as pET (Stratagene, La Jolla, California), pRSET (Invitrogen, Carlsbad, California) or pGEMEXTM (Promega, Madison, WI) vectors, in which the sequence encoding a protein of interest is ligated downstream from a bacteriophage T7 promoter and ribosome binding site so that, when the expression construct is transformed into E.
  • Escherica coli cloning and expression vectors such as pET (Stratagene, La Jolla, California), pRSET (Invitrogen, Carlsbad, California) or pGEMEXTM (Promega, Madison, WI) vectors, in which the sequence encoding a protein of interest is ligated downstream from a bacteriophage T7 promoter and ribosome binding site so that, when the expression construct is transformed into E.
  • coli expressing the T7 RNA polymerase large levels of the polypeptide of interest are produced; pGEMTM vectors (Promega), in which inserts into sequences encoding the lacZ ⁇ -peptide may be detected using colorimetric screening; and the like.
  • pGEMTM vectors Promega
  • Plasmids such as pGEX vectors (Amersham Pharmacia Biotech, Piscataway, NJ) may be used to express polypeptides of interest as fusion proteins.
  • Such vectors comprise a promoter operably linked to a glutathione S-transferase (GST) gene from Schistosoma japonicum (Smith et al., 1988, Gene 67:31-40), the coding sequence of which has been modified to comprise a thrombin cleavage site-encoding nucleotide sequence immediately 5' from a multiple cloning site.
  • GST glutathione S-transferase
  • GST fusion proteins can be detected by Western blots with anti-GST or by using a colorimetric assay; the latter assay utilizes glutathione and 1 -chloro-2-4-dinitrobenzene (CDNB) as substrates for GST and yields a yellow product detectable at 340 nm (Habig et al., 1974, J. Biol. Chem. 249:7130-7139).
  • GST fusion proteins produced from expression constructs derived from this expression vector can be purified by, e.g. , adso ⁇ tion to glutathione- agarose beads followed by elution in the presence of free glutathione.
  • Another series of expression vectors of this type are the pBAD/His vectors (Guzman et al., J. Bact. 777:4121-4130, 1997; Invitrogen, Carlsbad, CA), which contains the following elements operably linked in a 5' to 3' orientation: the inducible, but tightly regulatable, araBAD promoter; optimized E.
  • Fusion proteins made from pBAD/His expression constructs can be purified using substrates or antibodies that specifically bind to the His-tag, and assayed by Western analysis using the Anti- XpressTM antibody.
  • Proteins made in such systems are designed to include heparin, thrombin, enterokinase, factor XA or other protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety by treatment with the appropriate protease.
  • Expression vectors derived from bacteriophage including cosmids and phagemids, may also be used to express nucleic acids of interest in bacterial cells.
  • Such vectors include, but are not limited to, ZAP ExpressTM, Lambda ZAPTM, and Lambda gtl l bacteriophage vectors, pBluescriptTM phagemids, (all available from Stratagene) and the pSLl 180 Superlinker Phagemid (Amersham Pharmacia Biotech).
  • yeast such as Saccharomyces cerevisiae or Pichia pastoris, a number of vectors containing constitutive or inducible promoters such as those for mating factor alpha, GALl, TEF1, AOX1 or GAP may be used.
  • Appropriate expression vectors include various pYES, pYD and pTEF derivatives (Invitrogen) (see, for example, Grant et al., Methods in Enzymology 753:516-544, 1987; Lundblad et al., Units 13.4 to 13.7 of Chapter 13 in: Short Protocols in Molecular Biology, 2nd Ed., Ausubel et al., eds., John Wiley & Sons, New York, New York, 1992, pages 13-19 to 13-33).
  • the expression of a nucleotide sequence of interest may be driven by any of a number of promoters.
  • viral promoters such as the 35S and 19S promoters of CaMV (Brisson et al., Nature 370:51 1-514, 1984) may be used alone or in combination with the omega leader sequence from TMV (Takamatsu et al., EMBO J. 3:17-311, 1987).
  • plant promoters such as the promoter of the gene encoding the small subunit of RUBISCO (Coruzzi et al.., EMBO J.
  • Another expression system which may be used to express a gene product of interest is an insect system.
  • Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae.
  • the nucleotide sequence of interest may be cloned into a nonessential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter. Successful insertion of the sequence of interest will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein.
  • the recombinant viruses are then used to infect S frugiperda cells or Trichoplusia larvae in which the gene product of interest is expressed (see "Piwnica- Worms, Expression of Proteins in Insect Cells Using Baculovirus Vectors," Section II of Chapter 16 in: Short Protocols in Molecular Biology, 2nd Ed., Ausubel et al., eds., John Wiley & Sons, New York, New York, 1992, pages 16-32 to 16-48; L ⁇ pez-Ferber et al., Chapter 2 in: Baculovirus Expression Protocols, Methods in Molecular Biology, Vol. 39, CR. Richardson, Ed., Humana Press, Totawa, New Jersey, 1995, pages 25-63).
  • S. frugiperda cells Sf9, Sf21 or High FiveTM cells
  • appropriate baculovirus transfer vectors are commercially available from, e.g., Invitrogen.
  • Expression systems utilizing Drosophila S2 cells may also be utilized.
  • Expression constructs for expressing nucleic acids of interest in mammalian cells are prepared in a step-wise process.
  • expression cassettes that comprise a promoter (and associated regulatory sequences) operably linked to a nucleic acid of interest are constructed in bacterial plasmid-based systems; these expression cassette-comprising constructs are evaluated and optimized for their ability to produce the gene product of interest in mammalian cells that are transiently transfected therewith.
  • these expression cassettes are transferred to viral systems that produce recombinant proteins during lytic growth of the virus (e.g., SV40, BPV, EBV, adenovirus; see below) or from a virus that can stably integrate into and transduce a mammalian cellular genome (e.g., a retroviral expression construct).
  • virus e.g., SV40, BPV, EBV, adenovirus; see below
  • a virus that can stably integrate into and transduce a mammalian cellular genome e.g., a retroviral expression construct.
  • SV40 late promoter expression vectors e.g., pSVL, Amersham Pharmacia Biotech
  • glucocorticoid- inducible promoter expression vectors e.g., pMSG, Amersham Pharmacia Biotech
  • Rous sarcoma enhancer-promoter expression vectors e.g., pRc/RSV, Invitrogen
  • CMV immediate early promoter expression vectors including derivatives thereof having selectable markers to agents such as Neomycin, Hygromycin or Z ⁇ OCINTM (e.g., pRc/CMV2, pCDM8, pcDNAl.l, pcDNAl.l/Amp, pcDNA3.1, pcDNA3.1, p
  • a control plasmid such as pCHHO (Pharmacia) may be cotransfected with the expression construct being examined so that levels of the gene product of interest can be normalized to a gene product expressed from the control plasmid.
  • Preferred expression cassettes consisting essentially of a promoter and associated regulatory sequences operably linked to a nucleic acid of interest, are identified by the ability of cells transiently transformed with a vector comprising a given expression cassette to express high levels of the gene product of interest, or a fusion protein derived therefrom, when induced to do so. Expression may be monitored by Northern or Western analysis or, in the case of fusion proteins, by a reporter moiety such as an enzyme or epitope. Effective expression cassettes are then inco ⁇ orated into viral expression vectors. Nucleic acids, preferably DNA, comprising preferred expression cassettes are isolated from the transient expression constructs in which they were prepared, characterized and optimized.
  • a preferred method of isolating such expression cassettes is by amplification by PCR, although other methods (e.g., digestion with appropriate restriction enzymes) can be used.
  • Preferred expression cassettes are introduced into viral expression vectors, preferably retroviral expression vectors, in the following manner.
  • a DNA molecule comprising a preferred expression cassette is introduced into a retroviral transfer vector by ligation.
  • retroviral transfer vectors Two types are known in the art: replication-incompetent and replication-competent.
  • Replication-incompetent vectors lack viral genes necessary to produce infectious particles but retain cis-acting viral sequences necessary for viral transmission. Such exacting sequences include the ⁇ packaging sequence, signals for reverse transcription and integration, and viral promoter, enhancer, polyadenylation and other regulatory sequences.
  • Replication-competent vectors retain all these elements as well as genes encoding virion structural proteins (typically, those encoded by genes designated gag, ol and env) and can thus infectious particles.
  • a packaging cell line i.e., a cell line that produces mRNAs encoding gag, pol and env genes but lacking the ⁇ packaging sequence. See, generally, Cepko, Unit 9.10 of Chapter 9 in: Short Protocols in Molecular Biology, 2nd Ed., Ausubel et al., eds., John Wiley & Sons, New York, New York, 1992, pages 9-30 to 9-35.
  • a retroviral construct comprising an expression cassette comprising a nucleic acid of interest produces RNA molecules comprising the cassette sequences and the ⁇ packaging sequence.
  • These RNA molecules correspond to viral genomes that are encapsidated by viral structural proteins in an appropriate cell line (by "appropriate” it is meant that, for example, a packaging cell line must be used for constructs based on replication-incompetent retroviral vectors).
  • Infectious viral particles are then produced, and released into the culture supernatant, by budding from the cellular membrane.
  • the infectious particles which comprise a viral RNA genome that includes the expression cassette for the gene product of interest, are prepared and concentrated according to known methods.
  • helper virus i.e., viral particles which do not comprise the expression cassette for the gene product of interest. See, generally, Cepko, Units 9.11, 9.12 and 9.13 of Chapter 9 in: Short Protocols in Molecular Biology, 2nd Ed., Ausubel et al., eds., John Wiley & Sons, New York, New York, 1992, pages 9-36 to 9-45.
  • Viral particles comprising an expression cassette for the gene product of interest are used to infect in vitro (e.g., cultured cells) or in vivo (e.g., cells of a rodent, or of an avian species, which are part of a whole animal). Tissue explants or cultured embryos may also be infected according to methods known in the art. See, generally, Cepko, Unit 9.14 of Chapter 9 in: Short Protocols in Molecular Biology, 2nd Ed., Ausubel et al., eds., John Wiley & Sons, New York, New York, 1992, pages 9-45 to 9- 48. Regardless of the type of cell used, production of the gene product of interest is directed by the recombinant viral genome.
  • host cells may be chosen for its ability to modulate the expression of the inserted sequences or, when the gene product of interest is a protein, to process the protein of interest in the desired fashion.
  • modifications of proteins include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation.
  • Post-translational processing which cleaves a "prepro" form of the protein of interest may also be important for its correct intracellular localization, folding and/or function.
  • Different host cells such as CHO, HeLa, MDCK, HEK293, WI38, etc. have specific cellular machinery and characteristic mechanisms for such post-translational activities and may be chosen to ensure the correct modification and processing of a protein of interest.
  • Expression systems of the invention also include the few systems in which a nucleic acid of interest is expressed from an organellar genome.
  • Means for the genetic manipulation of the mitochondrial genome of Saccharomyces cerevisiae (Steele et al., Proc. Natl. Acad. Sci. U.S.A. 93:5253-5257, 1996) and systems for the genetic manipulation of plant chlo ⁇ lasts (U.S. Patent No. 5,693,507; Daniell et al., Nature Biotechnology 76:345-348, 1998) have been described.
  • nucleic acids that encode polypeptide sequences may have to be altered in organellar expression systems in order to reflect the differences in the genetic codes of organelles (see, e.g., Table 1).
  • RNA of interest is used to generate a cDNA molecule that can be used to detect nucleic acids having the sequence of interest, or to produce a polypeptide encoded by the sequence of the RNA of interest.
  • cDNA reverse complementary DNA
  • RNA portion of the resultant (RNA:DNA) hybrid may then be displaced or enzymatically degraded, after which the single-stranded DNA (ssDNA) is used as a template for one or more rounds of DNA polymerization, the product of which is a double-stranded DNA (dsDNA) molecule.
  • the dsDNA molecule includes the sequence of the RNA of interest (except that uridine residues in the RNA are replaced by thymidine residues in the DNA).
  • the nucleotide sequence of the dsDNA is then determined and analyzed; additionally or alternatively, the dsDNA is cloned, i.e., inco ⁇ orated into a vector DNA that is capable of replication in an appropriate host cell. If the dsDNA molecule includes a sequence that encodes a polypeptide, a preferred vector is an expression vector.
  • a DNA molecule prepared according to the methods of the invention can be a full-length cDNA, i.e., one comprising a nucleotide sequence that encodes an entire protein.
  • a full-length cDNA will encompass a "start” (translation initiating) codon, a “stop” (translation terminating) codon, and all the polypeptide- encoding sequences in-between.
  • a DNA molecule prepared according to the methods of the invention can be an Expressed Sequence Tag (EST), i.e., one which does not comprise a complete full length cDNA but which does comprise a nucleotide sequence that is a portion of an full length cDNA or of a mRNA comprising a full length cDNA.
  • An EST is useful in and of itself as, e.g., a probe in methods for detecting a mRNA of interest. Because a full-length cDNA is required for, e.g., recombinant DNA expression of a protein encoded by a mRNA interest, it may also be desirable to use an EST as a tool to isolate a full-length cDNA according to a variety of methods.
  • a nucleic acid comprising an EST sequence of interest can be labeled and used to probe preparations of cellular DNA, cDNA or RNA for hybridizing sequences, and such hybridizing sequences can be isolated, amplified and cloned according to known methods.
  • the sequence of an EST can be used to prepare primers for inverse PCR, a process by which sequences flanking an EST of interest can be determined (see, e.g., Benkel and Fong, Genet.
  • a nucleic acid of interest can be used to examine tissue- or temporal-specific patterns of expression of a nucleic acid of interest in a variety of methods known in the art.
  • the nucleic acid of interest can be detectably labeled and used to probe (i) an immobilized collection of mRNA molecules (e.g., RNA Master BlotsTM or Multiple Tissue Northern, MTNTM, Blots from Clontech) or (ii) a cDNA library (prepared according to methods known in the art or available from, e.g., Clontech or from depositories such as the American Type Culture Collection, ATCC, Manassas, VA).
  • mRNA molecules e.g., RNA Master BlotsTM or Multiple Tissue Northern, MTNTM, Blots from Clontech
  • a cDNA library prepared according to methods known in the art or available from, e.g., Clontech or from depositories such as the American Type Culture Collection, ATCC, Manassas, VA).
  • a sequence of interest can be used to design specific PCR primers that can be used in amplification reactions in 96-well plates wherein each well comprises first strand cDNAs from a particular tissue (such as, e.g., the Rapid-ScanTM gene expression panel from OriGene Technologies, Inc., Rockville, MD).
  • a particular tissue such as, e.g., the Rapid-ScanTM gene expression panel from OriGene Technologies, Inc., Rockville, MD.
  • automated, semi-automated or robotic means may be used to carry out such assays.
  • Mammalian tissues that may be examined include but are not limited to brain (including, by way of example but not limitation, whole brain and subsections thereof, e.g., amygdala, caudate nucleus, cerebellum, cerebral cortex, frontal lobe, hippocampus, medulla oblongata, occipital lobe, putamen, substantia nigra, temporal lobe, thalamus, acumens, subthalamic nucleus, inferio temporal cortex, medial frontal cortex, occipital pole), heart, kidney, spleen, liver, colon, lung, small intestine, stomach, skeletal muscle, smooth muscle, testis, uterus, bladder, lymph nodes, spinal cord, dorsal root ganglia, trachea, bone marrow, placenta, salivary glands, thyroid glands, thymus, adrenal glands, pancreas, ovary, uterus, prostate, skin,
  • tissue or cells from which a cDNA corresponding to an EST of interest can optimally be prepared In order to identify tissues or cells from which a cDNA corresponding to an EST of interest can optimally be prepared, mRNA or cDNA libraries or arrays derived from the organism from which the EST of interest was isolated are probed. Tissues or cells having a high level of expression of the nucleic acid of interest are preferably used as sources for full-length nucleic acids, i.e., nucleic acids containing all the genetic information required to express a complete gene product of interest.
  • the full-length nucleic acids are used, e.g., to express the gene product (i.e., RNA or protein) of interest or to prepare manipulated cells or transgenic animals in which the level of expression or activity, or tissue- or temporal-specific patterns of expression, of the gene product of interest is altered relative to the wildtype condition.
  • the gene product i.e., RNA or protein
  • manipulated cells or transgenic animals in which the level of expression or activity, or tissue- or temporal-specific patterns of expression, of the gene product of interest is altered relative to the wildtype condition.
  • ESTs and full-length cDNAs are to search in silico for corresponding protein sequences, in order to identify proteins of interest encoded thereby and to prepare antibodies thereto.
  • the nucleotide sequence of an EST or cDNA of interest is translated in silico in all six potential reading frames (three reading frames on each strand of a dsDNA), and the resulting amino acid sequences are used as probes to search protein databases for a match to a portion of a protein having a known amino acid sequence.
  • mitochondrial proteins it is desirable to perform such in silico translations using both the "universal" genetic code and the somewhat different genetic code utilized in mitochondria (TABLE 1), as different amino acid sequences will result in each case.
  • Nucleic acids having or comprising a sequence of interest can be prepared by a variety of methods known in the art. For example, such nucleic acids can be made using molecular biology or synthetic techniques (Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press (1989)). Many equivalent bases, both naturally occurring and synthetic, in nucleotide sequences are known in the art. For example, thymine (T) residues in DNA are transcribed into uracil (U) residues in RNA molecules but, because both T and U specifically pair with adenine (A) residues, these changes do not impact hybridization specificity. Nucleic acids comprising such equivalent substitutions are within the scope of the disclosure.
  • nucleic acids of the invention may have one or more non-nucleotide moieties.
  • non-nucleotides and their use in ribozymes are described in U.S. Patent No. 5,891,683 and includes polyethers, polyamines, polyamides, polyhydrocarbons and abasic nucleotides.
  • nucleic acids can be oligonucleotides, including oligodeoxyribonucleotides and oligoribonucleotides synthesized in vitro by, for example, the phosphotriester, phosphoramidite or H-phosphanate methodologies (see, respectively, Christodoulou, "Oligonucleotide Synthesis: Phosphotriester Approach," Chapter 2 In: Protocols for Oligonucleotides and Analogs: Synthesis and Properties, Agrawal, ed., Methods in Molecular Biology Vol.
  • the length of a nucleic acid according to the present invention can be chosen by one skilled in the art depending on the particular pu ⁇ ose for which the nucleic acid is intended.
  • the length of the nucleic acid is preferably from about 10 to about 100 base nucleotides (nt), more preferably from about 12 nt to about 60 nt, and most preferably from about 15 nt to about 30 nt.
  • the length of the nucleic acid is preferably from about 20 nt to about 200 nt, more preferably from about 30 nt to about 100 nt, and most preferably from about 40 nt to about 80 nt.
  • the length of the nucleic acid is preferably from about 10 nt to about 5,000 nt, more preferably from about 15 to about 1,000 nt, and most preferably from about 20 nt to about 500 nt.
  • nucleic acids of the invention are also readily chosen by one skilled in the art. Such modifications may include, for example, means by which the nucleic acid is detectably labeled for use as a probe. Typical detectable labels include radioactive moieties and reporter groups such as, e.g., enzymes and fluorescent or luminescent moieties. Other chemical modifications appropriate for particular uses, such as antisense applications, as explained herein.
  • nucleic acids of the invention can be provided in kit form, e.g., in a single or separate container, along with other reagents, buffers, enzymes or materials to be used in practicing at least one method of the invention.
  • the kit can be provided in a container that can optionally include instructions or software for performing a method of the invention. Such instructions or software can be provided in any language or human- or machine- readable format.
  • a variety of methods for detecting nucleic acids may be used in the methods of the invention. Such methods include, without limitation, the following methodologies. It should be noted that, regardless of which method is used to identify candidate differentially expressed genes, a second independent method should be used to verify the results obtained from the first method.
  • cells that do not express an IFl are used as a first cell and cells that express the IFl are used as the second cell such that differential display of the first cell and the second cell is determined.
  • the first cell and the second cell can be the same cell, however, the second cell has been induced to express a particular IFl by an appropriate inducer, such as tetracycline, in a construct such as that described in FIG. 1.
  • Subtractive Hybridization In a typical procedure for applying the technique of subtraction hybridization (Hedrick et al., Nature 305:149-153, 1984) to investigate differences in the expression of genes of a certain sample of test or target cells, e.g. from tumor tissues or tissues in a disease state, such as tissues affected by diabetes, as compared with the expression of genes of a sample of reference cells, e.g. cells from corresponding normal tissue, total cell mRNA is extracted (using any preferred method) from both samples of cells.
  • the mRNA in the extract from the test or target cells is then used in a conventional manner to synthesize corresponding single stranded cDNA using an appropriate primer and a reverse transcriptase in the presence of the necessary deoxynucleoside triphosphates, and the template mRNA is subsequently degraded by alkaline hydrolysis or RNase H to leave only the single stranded cDNA.
  • the single stranded cDNA thus derived from the mRNA expressed by the test or target cells is then mixed under hybridizing conditions with an excess quantity of the mRNA extract from the reference (normal) cells; this mRNA is generally termed the subtraction hybridization "driver" since it is this mRNA or other single stranded nucleic acid present in excess which "drives" the subtraction process.
  • driver subtraction hybridization
  • cDNA strands having common complementary sequences anneal with the mRNA strands to form mRNA/cDNA duplexes and are thus subtracted from the single stranded species present.
  • the only single stranded DNA remaining is then the unique cDNA that is derived specifically from the mRNA produced by genes which are expressed solely by the test or target cells.
  • the reference cells may be used as a source of single-stranded DNA
  • the test or target cells may be used as a source of driver RNA.
  • the remaining single-stranded DNA is derived from mRNA produced by genes expressed in the reference cells but not in the target cells.
  • HAP hydroxyapatite
  • strept streptavidin-biotin
  • One or more repeat rounds of the subtraction hybridization may be carried out to improve the degree of removal of commonly expressed sequences, although other means may be employed (see, e.g., U.S. Patent No. 5,589,339).
  • the single-stranded cDNA may be converted to double-stranded DNA by methods or means know in the art.
  • multiple copies of a single nucleotide for example deoxycytidine may be added, onto the 3' end of the single-stranded DNA molecules using an enzyme such as terminal transferase, and then an oligonucleotide of complementary sequence, e.g. poly G to prime synthesis of the complementary strand using any of a number of commercially available DNA polymerases can be used.
  • an enzyme such as terminal transferase
  • an oligonucleotide of complementary sequence e.g. poly G to prime synthesis of the complementary strand using any of a number of commercially available DNA polymerases
  • the cDNA sequences obtained from subtractive hybridization may be used to produce labeled probes that may perhaps then be used for detecting or identifying corresponding cloned copies in a cDNA clone colony or cDNA library (labeling of such probes is frequently introduced by using labeled deoxynucleoside triphosphates in synthesis of the cDNA),
  • High Density Arrays Multiple sample nucleic acid hybridization analysis can be carried out on micro-formatted multiplex or matrix devices (e.g., DNA or RNA chips, filters and microarrays) (see, e.g., Bains, Bio/Technology 70:757-758, 1992). These hybridization formats are micro-scale versions of the conventional "dot blot" and “sandwich” hybridization systems. In these methods, specific DNA sequences are typically attached to, or synthesized on, very small specific areas of a solid support, allowing large numbers of different DNA sequences to be placed in a small area.
  • the high density arrays comprise target elements, i.e., target nucleic acid molecules bound to a solid support.
  • the nucleic acids for both the target elements and the probes may be, for example, RNA, DNA, or cDNA.
  • target elements comprising nucleic acid elements that are short synthetic oligonucleotides derived from mRNA, cDNA or EST sequences are used to carry out serial analysis of gene expression (SAGE; U.S. Patent No. 5,866,330).
  • nucleic acid molecules in the test and control collections (which may be, e.g., mRNA preparations from a diseased and undiseased human) are detectably labeled.
  • the first and second labeled probes thus formed are each contacted to an identical high density array comprising a plurality of target elements under conditions such that nucleic acid hybridization to the target elements can occur.
  • a binding ratio >1 indicates that nucleic acids hybridizing to the particular target element are "up-regulated” in the nucleic acid collection prepared from the diseased patient relative to the nucleic acid prepared from the control individual, whereas a binding ratio ⁇ 1 indicates that nucleic acids hybridizing to the particular target element are "down-regulated" in the diseased patient.
  • High density cDNA arrays that may be used in the invention include but are not limited to GeneChipTM arrays comprising synthetic oligonucleotides
  • One type of high density array uses electronic hybridization, i.e., a method that directs sample DNA molecules to, and concentrates them at, test sites on a microchip that can be electronically activated by a positive charge. Because DNA molecules in solution have strong negative charges, they are attracted to activated sites.
  • the electronic hybridization of sample DNA molecules at each test site promotes rapid hybridization of the sample DNAs with the nucleic acids of the target elements.
  • Materials for electronic hybridization are available from Nanogen (San Diego, CA) and the method is described in U.S. Patent No. 5,849,486.
  • RNA may be reverse transcribed and amplified with specific primer sets, and the resulting amplification products from the two samples compared (Hipfel R, et al. (1998) J. Biochem Biophys. Methods 37: 131-135; Bosch TC and Lohmann JU (1998) Mthods Mol Biol 86: 153-160). Total cell RNA is extracted (using any preferred method) from both samples of cells.
  • RNA from both samples is reverse transcribed using a set of twelve primers containing a sequences of poly (T) terminating in one of either AA, AC, AG, AT, CA, CC, CG, CT, GA,GC, GG, or GT.
  • the single stranded cDNAs of the resulting cDNA/mRNA hybrids are then amplified in separate reactions, with each reaction using one of the set of twelve "3' " primers used in the reverse transcription reaction and one of a set of "5' "primers.
  • a set of about twenty 5' primers is used, each with a different arbitrary sequence.
  • the resulting amplification products are labeled, preferably by using primers which have inco ⁇ orated a fluorescent dye, but other labeling methods and other labels may be used, and electrophoresed such as on gels.
  • the products resulting from reverse transcription and amplification of RNA from two different samples with the same primer sets are compared. Bands which are overexpressed or underexpressed in one sample when compared with another sample may be excised from the gel, reamplified, cloned, and sequenced to identify genes with different levels of expression in the two samples.
  • Various antisense-based methodologies may be used to modulate (reduce or eliminate) the expression of a nucleic acid of interest, and the corresponding gene product, in organelles, cells, tissues, organs and organisms. Such antisense modulation may be used to validate the role of a gene of interest in a disease or disorder or, when the causes or symptoms of a disease or disorder result from the over-expression of a nucleic acid of interest, as therapeutic agents.
  • the expression of IFl can be increased by interfering with the transcription or translation of inhibitors of IFl transcription or translation.
  • the expression of IFl can be decreased by interfering with the transcription or translation of activators of IFl transcription or translation or by interfering with the transcription or translation of IFl itself.
  • antisense refers to nucleic acids that comprise one or more sequences that are the reverse complement of the "sense" strand of a gene, i.e., the strand that is transcribed and, in the case of protein-encoding sequences, translated. Because antisense nucleic acids bind with high specificity to their targeted nucleic acids, selectivity is high and toxic side effects resulting from misdirection of the compounds can be minimal.
  • antisense compositions are of two types: (i) synthetic antisense oligonucleotides, including enzymatic ones such as, e.g., ribozymes; and (ii) antisense expression constructs.
  • synthetic antisense oligonucleotides including enzymatic ones such as, e.g., ribozymes
  • antisense expression constructs One skilled in the art will be able to utilize either modality as is appropriate to the given situation.
  • Synthetic antisense oligonucleotides are prepared from the reverse complement of a nucleic acid of interest.
  • An antisense oligonucleotide consists of nucleic acid sequences corresponding to the reverse complement of a differentially expressed RNA.
  • the antisense oligonucleotides specifically bind to the RNA molecules and interfere with their function by preventing secondary structures from forming or blocking the binding of regulatory or RNA-stabilizing factors.
  • oligonucleotides can inhibit RNA splicing, polyadenylation or protein translation, thus limiting or preventing the amount of protein made from such mRNAs.
  • such oligoncuelotides can bind to double-stranded DNA molecules and form triplexes therewith, and thus interfere with the transcription of such sequences.
  • PNAs peptide nucleic acids
  • the sugar-phosphate backbone of biological nucleic acids has been replaced with a polypeptide-like chain.
  • Targeting sequences that direct proteins to organelles can be conjugated to the backbone of antisense PNAs, with the result being that such conjugates are preferentially delivered to the targeted organelle (see, for example, published PCT applications WO 97/41150 and WO 99/05302.
  • Antisense oligonucleotides may be inherently enzymatic in nature, that is, capable of degrading the RNA molecule towards which they are targeted; such molecules are generally referred to as "ribozymes.”
  • ribozymes A variety of increasingly short synthetic ribozyme frameworks that can be modified to comprise a nucleic acid sequence of interest have been described (Couture and Stinchcomb, Trends Genet. 12:510-515, 1996), including but not limited to hai ⁇ in ribozymes (Hampel, Prog. Nucleic Acid Res. Mol Biol. 55:1-39, 1998), hammerhead ribozymes (Birikh et al., Eur. J. Biochem. 245:1-16, 1997) and minizymes (Kuwabara et al., Nature Biotechnology 76:961-965, 1998).
  • antisense modulation of gene expression in a cell can also be achieved by expression constructs that direct the transcription of the reverse complement of a nucleotide sequence of interest in vivo.
  • expression constructs that direct the transcription of the reverse complement of a nucleotide sequence of interest in vivo.
  • all that may be required is the "flipping" (i.e., reversing the orientation) of a nucleic acid of interest that has been cloned into a mammalian or plant expression vector, respectively.
  • an antisense expression construct of this type is a promoter operably linked to the reverse complement of a nucleic acid of interest. It is also possible to design expression constructs that express ribozymes in cells. Antisense and ribozyme expression constructs are also used to produce transgenic animals in which the level of expression of a gene of interest can be modulated in a temporal- or tissue-specific manner (see Sokol and Murray, Transgenic Res. 5:363-371, 1996, for a review).
  • Nucleic acid sequences derived according to the present invention may also be used to design "RNA decoys," i.e., short RNA molecules corresponding to exacting regulatory sequences that bind tr ⁇ «s-acting regulatory factors. When overexpressed in a cell or administered in excess thereto, such RNA decoys competitively inhibit the binding and thus action of the tr ⁇ «.y-acting regulatory factors, and thus limit or prevent the ability of such factors to carry out processes that stabilize (or destabilize) the RNA of interest, or enhance (or decrease) the polyadenylation, splicing nuclear transport, or translation of the RNA (Sullenger et al., J. Virol. 65:6811- 6816, 1991). Expression of the RNA of interest may thus be either enhanced or decreased for therapeutic pu ⁇ oses.
  • the nucleic acids of interest identified according to the methods of the invention may encode amino acid sequences. Such amino acid sequences may correspond to a full-length protein or to a polypeptide portion thereof.
  • the present invention also includes polypeptides that are derivatives of IFl, or polypeptides that have at least one activity of IFl .
  • certain polypeptides according to the present invention may comprise IF 1 mutant, variant, derivative, analog, fusion or fragment polypeptides or the like, which are unable to bind to an ATP synthase subunit or which, upon binding to ATP synthase, activate rather than inhibit ATP synthase activity. Identification, construction, expression, detection and functional assays of such polypeptides are readily performed by the person having ordinary skill in the art based upon the present disclosure.
  • the protein may be a known protein that is commercially available or one to which antibodies are known and can be used to isolate the protein from appropriate biological samples. If a full-length protein of the invention has not previously been described, it may be produced via recombinant DNA methodologies for example, using the expression systems described previously, or prepared from biological samples using known biochemical techniques. Short (i.e., having less than about 30 amino acids) polypeptides that are encoded by short (i.e., having less than about 100 nucleotides) nucleic acids of the invention or derived from the amino acid sequences encoded by longer nucleic acids or from full-length proteins can be synthesized in vitro by methods known in the art. Fusion proteins comprising amino acid sequences of interest may also be prepared and are included within the scope of the polypeptides and proteins of the invention.
  • polypeptides and proteins of the invention have a variety of applications. They may be used to generate antibodies or to screen for ligands that may serve as therapeutic agents, or may themselves be used as therapeutic agents. Full-length proteins of the invention may have the activity of the wildtype protein and may thus be used to treat conditions resulting from a loss of such activity. Polypeptides of the invention may also have such activities, or may competitively inhibit a protein of interest in vivo by binding a ligand of the protein. If the ligand is an activator of the protein, such polypeptides may be used to treat conditions resulting from the over-expression or over-activation of the protein in vivo.
  • the ligand is a toxin or activator of cell death (apoptosis or necrosis)
  • administration of a protein or polypeptide that binds such a ligand to a patient in need thereof will have the beneficial effect of competitively inhibiting the action of the toxin or cell death activator.
  • Antibodies to a protein or polypeptide of interest are prepared according to a variety of methods known in the art.
  • antibodies that bind with IFl or a label sequence, such as FLAG can be used to detect IFl or a label sequence, particularly in a cell, using labeled antibodies that bind with such polypeptides.
  • such antibodies may be polyclonal, monoclonal or monospecific antibodies.
  • Primary antibodies of the invention bind specifically to a particular protein or polypeptide of interest and are thus used in assays to detect and quantitate such proteins and polypeptides.
  • the invention also includes active fragments or active portions that exhibit the binding specificity or the substantial binding specificity of the intact antibody they were derived from.
  • a primary antibody of the invention is detectably labeled or is specifically recognized and monitored by a detectably labeled secondary antibody or a combination of a secondary antibody and a tertiary molecule (which may also be an antibody) that is detectably labeled.
  • the primary antibody of the invention provides a means by which a protein or polypeptide of interest is specifically bound and subsequently detected.
  • One preferred assay format is the Enzyme-Linked Immunosorbent Assay (ELISA) format.
  • a nucleic acid of interest may encode a known protein or a portion thereof, or a polypeptide sequence that is homologous to a known protein.
  • antisera to the known protein, or the known protein itself may be commercially available.
  • the known or recombinantly-produced protein can be used to immunize a mammal of choice (e.g., a rabbit, mouse or rat) in order to produce antisera from which polyclonal antibodies can be prepared (see, e.g., Cooper and Paterson, Units 1 1.12 and 1 1.13 in Chapter 11 in: Short Protocols in Molecular Biology, 2nd Ed., Ausubel et al., eds., John Wiley & Sons, New York, New York, 1992, pages 11-37 to 11-41).
  • nucleic acid sequence of interest encodes a polypeptide sequence for which no complete protein (or homolog thereof) is known, is too short to encode more than about 30 amino acids (i.e., the nucleic acid of interest is less than about 100 nucleotides in length), or encodes more than one polypeptide sequence of potential interest, such candidate amino acid sequences can be used to synthesize one or more polypeptide molecules, each of which has a defined amino acid sequence.
  • Such synthetic polypeptides can then be used to immunize animals (e.g., rabbits) according to methods known in the art (Collawn and Paterson, Units 11.14 and 11.15 in Chapter 11 in: Short Protocols in Molecular Biology, 2nd Ed., Ausubel et al., eds., John Wiley & Sons, New York, New York, 1992, pages 11-42 to 11-46; Cooper and Paterson, Units 11.12 and 11.13 in Chapter 11 in: Short Protocols in Molecular Biology, 2nd Ed., Ausubel et al., eds., John Wiley & Sons, New York, New York, 1992, pages 11-37 to 11-41).
  • the resulting antisera may then be used to probe cells from which the nucleic acid of interest was isolated.
  • a positive response to a given antiserum indicates that the candidate reading frame from which the synthetic polypeptide used to raise the antiserum was derived is a reading frame used to encode at least one protein in the cell(s) so examined.
  • such an antiserum can be used to identify proteins of interest in the cells from which the nucleic acid of interest was isolated.
  • monoclonal antibodies are often the preferred type of antibody for a variety of applications.
  • Methods for producing and preparing monoclonal antibodies are known in the art (see, e.g., Fuller et al., Units 11.4 to 1 1.11 in Chapter 1 1 in: Short Protocols in Molecular Biology, 2nd Ed., Ausubel et al., eds., John Wiley & Sons, New York, New York, 1992, pages 11-22 to 11-36).
  • Murine monoclonal antibodies may be "humanized” to reduce their antigenicity in humans and used as therapeutic agents (see, e.g., G ⁇ ssow and Seemann, Methods in Enzymology 203:99-121, 1991; Vaughan et al., Nature Biotechnology 16:535-539, 1998).
  • Antibodies to proteins and polypeptides of interest are used to detect such proteins and polypeptides in a variety of assay formats. Such immunoassays may useful in diagnostic, prognostic or pharmacogenetic methods of the invention, or in methods in which various cell types, tissues or organs are probed for the presence of a protein of interest. Monoclonal antibodies are generally preferred for such methods due to their high degree of specificity and homogeneity.
  • Assays for or utilizing one or more of the antibodies, polypeptides and proteins, ligands therefor and nucleic acids of the invention are used in diagnostic, prognostic and pharmacogenetic methods of the invention.
  • diagnostic refers to assays that provide results which can be used by one skilled in the art, typically in combination with results from other assays, to determine if an individual is suffering from a disease or disorder of interest such as diabetes, including type I and type II
  • prognostic refers to the use of such assays to evaluate the response of an individual having such a disease or disorder to therapeutic or prophylactic treatment.
  • the term "pharmacogenetic” refers to the use of assays to predict which individual patients in a group will best respond to a particular therapeutic or prophylactic composition or treatment.
  • the terms "disease” and “disorder” refers to diabetes, either type I or type II.
  • samples from individuals are assayed with regard to the relative or absolute amounts of a "marker,” i.e., a nucleic acid or protein of interest, or an endogenous ligand of or antibody to a nucleic acid or protein of interest.
  • a marker i.e., a nucleic acid or protein of interest, or an endogenous ligand of or antibody to a nucleic acid or protein of interest.
  • An increased or decreased level of a marker relative to control levels indicates that the individual from which the sample was taken has, has had, or is likely to develop the disease or disorder of interest.
  • control level refers to the level of marker present in samples taken from one or more individuals known to not have the disease or disorder of interest, or to the level of marker present in a sample taken from the individual in question before or after the diagnostic sample.
  • nucleic acids of the invention may be used to screen for single nucleotide polymo ⁇ hisms (SNPs) and other mutations such as gene deletions or insertions, by hybridization methods (Sapolsky RJ et al. Genet. Anal. (1999) 14: 187-192), or other methods as they are known or later developed in the art.
  • SNPs single nucleotide polymo ⁇ hisms
  • patients suffering from a disease or disorder of interest are stratified with regards to desirable or undesirable responses to a potential treatment using one or more assays of the invention.
  • a therapeutic composition and/or treatment known to be more effective, or which produces fewer side-effects, in some patients as compared to others is administered a group of patients suffering from a disease or disorder of interest.
  • a method of identifying which patients having the disease are more likely to respond to a therapeutic composition and/or treatment comprises providing samples from a group of patients having said disease; measuring the amount or molecular attribute of a protein or polypeptide of interest, or of a nucleic acid of interest, or a ligand therefor or antibody thereto, or any combination thereof present in said samples; providing the therapeutic composition and/or treatment to the patients; measuring the degree, frequency, rate or extent of responses of the patients to the therapeutic composition and/or treatment; and determining if a correlation exists between the amount or molecular attributes of a nucleic acid of interest, or the amount or molecular attributes of a protein or polypeptide of interest, or a ligand therefor or antibody thereto present in said samples and the degree, frequency, rate or extent of such responses.
  • the resulting correlations are used to stratify patients in the following manner. If such a correlation is a positive correlation, the presence of such correlation indicates that patients yielding samples having an increased or decreased amount, relative to the established normal range, of the protein or polypeptide of interest, or the ligand or antibodies therefor, or nucleic acid molecules, or an increase or decreased amount, relative to the established normal range, of the nucleic acid of interest, are more likely to respond to said treatment. In contrast, if the correlation is a negative correlation, the presence of said correlation indicates that patients yielding samples having an increased amount of the protein or polypeptide of interest, or the ligand therefor, or of the nucleic acid of interest are less likely to respond to said treatment.
  • molecular attributes of nucleic acids and/or polypeptides of the invention may correlate positively or negatively with patients' responses to therapeutic compositions and treatments, and methods to screen for the relevant molecular attributes to stratify patients to determine optimal therapeutic courses are also part of the invention.
  • the response(s) that are measured in these methods can be desirable response(s), in which case it is preferred to provide the therapeutic composition and/or treatment to patients having a relatively high level of the protein or polypeptide of interest, or the ligand therefor, or of the nucleic acid of interest present.
  • the response(s) that are measured in these methods can be undesirable response(s), in which case it is preferred to avoid providing the therapeutic composition and/or treatment to patients having a relatively high level of the protein or polypeptide of interest, or the ligand therefor, or of the nucleic acid of interest.
  • kits for performing one or more assays of the invention are preferred.
  • Antibodies, polypeptides and proteins, ligands therefor and nucleic acid probes and primers of the invention can be provided in kit form, e.g., in a single or separate container, along with other reagents, buffers, enzymes or materials to be used in practicing at least one method of the invention.
  • kits can be provided in a container that can optionally include instructions or software for performing a method of the invention.
  • Such instructions or software can be provided in any language or human- or machine-readable format.
  • the nucleic acids, proteins, polypeptides, antibodies and transgenic animals of the invention may be used to validate the role of a gene product of interest in a particular disease, disorder or undesirable response, and to screen for conditions or compounds that can be used to treat such diseases, disorders and undesirable responses, preferably using high-throughput screening methods such as they are known in the art or later developed.
  • Such treatment can be remedial, therapeutic, palliative, rehabilitative, preventative, impeditive or prophylactic in nature.
  • Diseases and disorders to which the invention may be applied include diabetes, including type I and type II.
  • undesirable response refers to a biological or biochemical response by one or more cells of an organism to one or more physical conditions, chemical agents, or combinations thereof that leads to an undesirable consequence.
  • An undesirable response can occur at the organellar level (e.g., loss of ⁇ in mitochondria), the cellular level (e.g., cell death such as apoptosis or necrosis), in tissues (e.g., ischemia), in organs (e.g., ischemic heart disease) or to the organism as a whole (e.g., death; loss of reproductive capacity or cognitive processes).
  • Physical conditions that may produce an undesirable response include, without limitation, hypothermia, hyperthermia, dehydration, exposure to ultraviolet and other types of radiation, micro-gravity, physical trauma, tensile stress, and exposure to electrical or magnetic fields.
  • Chemical agents that may produce an undesirable response include without limitation reactive oxygen species (ROS), apoptogens, and the like.
  • Nucleic acids of the invention are used to screen for conditions or compounds that can be used to treat disease states and undesirable responses in the following manner. Treatment of cells with antisense molecules, including ribozymes, or introduction thereinto of antisense constructs specific for a given gene product of interest, should result in such cells demonstrating at least one of the biochemical or biological defects associated with the disease or disorder for which the gene product is being validated.
  • transgenic animals comprising constructs directing the over-expression of a gene of interest, or an antisense or ribozyme expression construct, or animals to which antisense, ribozyme or molecular decoy oligonucleotides are administered, will demonstrate at least one of the biochemical or biological defects associated with the disease or disorder of interest if the nucleic acid encodes a gene product that is a valid target for the disease or disorder.
  • SNPs or mutant forms of the gene identified by the invention and correlated with diseases or disorders may be introduced into cells or animals by homologous recombination. Such cells or animals or cells derived from such animals, may be used to assess responses to conditions or compounds that can be used to treat disease states by any of a variety of assays or physiological assessments/measurements.
  • polypeptides of interest that may be targets for therapeutic intervention
  • cells may be contacted with one or more antibodies specific for the polypeptide, and the presentation of responses associated with the disease or disorder will be seen with valid targets.
  • Polypeptides and proteins of the invention are also used to screen for conditions or compounds that can be used to treat disease states and undesirable responses.
  • the protein of interest, or a polypeptide derived therefrom having at least one activity of the protein of interest is produced by recombinant DNA methods or in vitro synthetic techniques.
  • the protein or polypepeptide which may be attached to a solid support, is contacted with a detectably labeled ligand (including, for example, an antibody).
  • a compound is then introduced to the reaction vessel, and active compounds are identified as those that cause the release of the detectably labeled ligand.
  • Assays involving nucleic acids, polypeptides, or antibodies of the invention may be automated for rapid screening of multiple compounds.
  • the invention includes high throughput screens that may be developed as having particular applicability to the nucleic acids, polypeptides, antibodies, and genetically manipulated cells of the invention, and also high throughput screens as they are currently known in the art (for example, Stockwell, BR et al. (1999) Chem. Biol 6: 71-83; McDonald, OB et al. (1999) Anal. Biochem. 268: 318-329; Sapolsky, RJ et al. Genet. Anal. (1999) 14: 187-192; Swartzmann, EE et al. (1999) Anal Biochem. 271 : 143-151; Gonzalez, JE and Neglescu PA (1998) Curr. Opin. Biotech. 624-631), and as may be adapted for the pu ⁇ oses of the invention.
  • the present invention exploits the binding interaction between IFl and ATP synthase as described herein to provide a method of identifying an agent which alters the interactions between IFl and ATP synthase.
  • the binding interaction between an IFl and at least one ATP synthase subunit and or an ATP synthase multi-subunit complex may result in the formation of a complex, which refers to a specific intermolecular association that results from an affinity interaction between an IFl and ATP synthase. as provided herein.
  • An IF 1 and ATP synthase complex may be identified by any of a variety of techniques known in the art for demonstrating an intermolecular interaction between two polypeptides, for example, co-purification, co-precipitation, co- immunoprecipitation, radiometric or fluorimetric assays, western immunoblot analyses, affinity capture including affinity techniques such as solid-phase ligand-counterligand sorbent techniques, affinity chromatography and surface affinity plasmon resonance, and the like. Determination of the presence of a complex may employ antibodies, including monoclonal, polyclonal, chimeric and single-chain antibodies, and the like, that specifically bind to the IFl as provided herein and/or to the ATP synthase. Labeled IFl polypeptides as provided herein and/or one or more labeled
  • ATP synthase subunits can also be used to detect the presence of a complex.
  • These proteins can be labeled by covalently or non-covalently attaching a suitable reporter molecule or moiety, for example any of various enzymes, fluorescent materials, luminescent materials and radioactive materials.
  • suitable enzymes include, but are not limited to, horseradish peroxidase, biotin, alkaline phosphatase, ⁇ - galactosidase and acetylcholinesterase.
  • suitable fluorescent materials include, but are not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride and phycoerythrin.
  • Appropriate luminescent materials include luminol, and suitable radioactive materials include radioactive phosphorus [ 32 P], iodine [ l23 l or l31 I] or tritium [ H].
  • At least one ATP synthase subunit polypeptide as provided herein is combined with at least one detectably labeled IFl polypeptide (e.g., an epitope tagged IFl fusion protein) which interacts with the ATP synthase subunit under conditions and for a time sufficient to permit formation of an intermolecular complex.
  • detectably labeled IFl polypeptide e.g., an epitope tagged IFl fusion protein
  • a method for identifying an agent that alters the interaction between an IFl and an ATP synthase comprising comparing the level of IFl -ATP synthase binding in the presence of a candidate agent to the level of binding in the absence of such agent.
  • binding assays may employ direct or indirect detection of the presence of a binding complex, and are readily adaptable to high throughput screening formats with which those having ordinary skill in the art will be familiar.
  • ATP synthase catalytic activity (instead of binding interactions between IFl and ATP synthase) may be determined according to established methods, such that the influence of a candidate agent on the IFl effect upon ATP synthase catalytic activity may be observed.
  • THERAPEUTIC APPLICATIONS Therapeutic agents derived therefrom according to the above embodiments can be employed in combination with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for parenteral application which do not deleteriously react with the active compound.
  • suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohol, vegetable oils, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid monoglycerides and diglycerides, petroethral fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidone, etc.
  • the pharmaceutical preparations can be sterilized and if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavoring and/or aromatic substances and the like which do not deleteriously react with the active compounds.
  • auxiliary agents e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavoring and/or aromatic substances and the like which do not deleteriously react with the active compounds.
  • auxiliary agents e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavoring and/or aromatic substances and the like which do not deleteriously react with the active compounds.
  • particularly suitable vehicles consist of solutions
  • terapéuticaally effective amount for the pu ⁇ oses of the invention, refers to the amount of a therapeutic agent which is effective to achieve its intended pu ⁇ ose. While individual needs vary, determination of optimal ranges for effective amounts of a therapeutic agent is within the skill of the art. Human doses can be extrapolated from animal studies (Fingle and Woodbury, Chapter 1 in Goodman and Gilman's The Pharmacological Basis of Therapeutics, 5th Ed., MacMillan Publishing Co., New York (1975), pages 1-46). Generally, the dosage required to provide an effective amount of the composition, and which can be adjusted by one of ordinary skill in the art will vary, depending on the age, health physical condition, weight, extent of disease of the recipient, frequency of treatment and the nature and scope of the desired effect.
  • Therapeutic agents of the invention can be delivered to mammals via intermittent or continuous intravenous injection of one or more these compositions or of a liposome (Rahman and Schein, in Liposomes as Drug Carriers, Gregoriadis, ed., John Wiley, New York (1988), pages 381-400; Gabizon, A., in Drug Carrier Systems, Vol. 9, Roerdink et al., eds., John Wiley, New York, 1989, pp. 185-212) microparticle (Tice et al., U.S.
  • Patent 4,542,025) or a formulation comprising one or more of these compositions; via subdermal implantation of drug-polymer conjugates (Duncan, Anti- Cancer Drugs 3:175-210, 1992; via microparticle bombardment (Sanford et al., U.S. Patent 4,945,050); via infusion pumps (Blackshear and Rohde, in: Drug Carrier Systems, Vol. 9, Roerdink et al., eds., John Wiley, New York, 1989, pp. 293-310) or by other appropriate methods known in the art (see, generally, Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, PA, 1990).
  • Transgenic animals modified with regards to a nucleic acid of interest, may be prepared. Such animals are useful for developing animal models of human disease and for evaluating the safety and effectiveness of therapeutic agents of the invention.
  • transgenic animals are of four types: (i) "transgenic knockouts,” in which the animal's homologs of a gene of interest are disrupted or removed, with a resulting loss of function of the corresponding gene product; (ii) "constitutive transgenics,” in which the gene of interest in operably linked to a constitutive promoter, (iii) “regulatable transgenics,” in which the gene of interest is operably linked to an inducible promoter; and (iv) "replacement transgenics,” in which the animal's homolog of the gene of interest has been replaced with the human gene of interest, or with an alternate form, for example a mutated form, of the gene of interest, which may be expressed from an endogenous or inducible promoter.
  • the non-human transgenic animals of the invention comprise any animal that can be genetically manipulated to produce one or more of the above-described classes of transgenic animals.
  • Such non-human animals include vertebrates such as rodents, non-human primates, sheep, dog, cow, amphibians, reptiles, etc.
  • Preferred non-human animals are selected from non-human mammalian species of animals, including without limitation animals from the rodent family including but not limited to rats and mice, most preferably mice (see, e.g., U.S. Patents 5,675,060 and 5,850,001).
  • Other non-human transgenic animals that may be prepared include without limitation rabbits (U.S. Patent No. 5,792,902), pigs (U.S. Patent No.
  • mice such as mice or rats, that have identified IFl genes can be engineered such that the animal IFl is "knocked out" and replaced with the human version.
  • Such mice can be made using homologous recombination. These animals can be compared to their non-engineered counte ⁇ arts to evaluate the activity of the human IFl.
  • the transgenic animals of the invention are animals into which has been introduced by nonnatural means (i.e., by human manipulation), one or more genes that do not occur naturally in the animal, e.g., foreign genes, genetically engineered endogenous genes, etc.
  • the nonnaturally introduced genes known as transgenes, may be from the same species as the animal but not naturally found in the animal in the configuration and/or at the chromosomal locus conferred by the transgene, or they may be from a different species.
  • Transgenes may comprise foreign DNA sequences, i.e., sequences not normally found in the genome of the host animal.
  • transgenes may comprise endogenous DNA sequences that are abnormal in that they have been rearranged or mutated in vitro in order to alter the normal in vivo pattern of expression of the gene, or to alter or eliminate the biological activity of an endogenous gene product encoded by the gene.
  • Transgenes may be introduced into the genome by homologous recombination, whereby the transgene replaces the endogenous copy of the gene in the recipient animal's genome. Methods of generating and screening targeted gene replacements and the generation of transgenic animals carrying targeted gene replacements are described in U.S. Patent No. 5,814,300.
  • the transgenic non-human animals of the invention are produced by introducing transgenic constructs comprising sequences of interest, or the host animal's homologs thereof, into the germline of the non-human animal.
  • Embryonic target cells at various developmental stages are used to introduce the transgenes of the invention. Different methods are used depending on the stage of development of the embryonic target cell(s).
  • Microinjection of zygotes is the preferred method for inco ⁇ orating transgenes into animal genomes in the course of practicing the invention.
  • a zygote a fertilized ovum that has not undergone pronuclei fusion or subsequent cell division, is the preferred target cell for microinjection of transgenic DNA sequences.
  • the murine male pronucleus reaches a size of approximately 20 micrometers in diameter, a feature which allows for the reproducible injection of 1-2 picoliters of a solution containing transgenic DNA sequences.
  • transgenic allele demonstrates Mendelian inheritance: half of the offspring resulting from the cross of a transgenic animal with a non-transgenic animal will inherit the transgenic allele, in accordance with Mendel's rules of random assortment.
  • Viral integration can also be used to introduce the transgenes of the invention into an animal.
  • the developing embryos are cultured in vitro to the developmental stage known as a blastocyte.
  • the blastomeres may be infected with appropriate retroviruses (Jaenisch, Proc. Natl Sci. U.S.A. 73:1260-1264, 1976; Soriano and Jaenisch, Cell 46:19-29, 1986).
  • Infection of the blastomeres is enhanced by enzymatic removal of the zona pellucida (Hogan, et al., in Manipulating the Mouse Embryo, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1986).
  • Transgenes are introduced via viral vectors which are typically replication-defective but which remain competent for integration of viral-associated DNA sequences, including transgenic DNA sequences linked to such viral sequences, into the host animal's genome (Jahner et al., Proc. Natl. Acad. Sci. U.S.A. 52:6927-6931, 1985; Van der Putten et al., Proc. Natl Acad. Sci. U.S.A. 52:6148-6152, 1985). Transfection is easily and efficiently obtained by culture of blastomeres on a mono-layer of cells producing the transgene-containing viral vector (Van der Putten et al., Proc. Natl. Acad. Sci. U.S.A.
  • transgenic founder animals produced by viral integration will be mosaics for the transgenic allele; that is, the transgene is inco ⁇ orated into only a subset of all the cells that form the transgenic founder animal.
  • multiple viral integration events may occur in a single founder animal, generating multiple transgenic alleles which will segregate in future generations of offspring.
  • transgenes into germline cells by this method are possible but probably occurs at a low frequency (Jahner et al., Nature 295:623-628, 1982).
  • offspring may be produced in which the transgenic allele is present in all of the animal's cells, i.e., in both somatic and germline cells.
  • Embryonic stem (ES) cells can also serve as target cells for introduction of the transgenes of the invention into animals.
  • ES cells are obtained from pre- implantation embryos that are cultured in vitro (Evans et al., Nature 292:154-156, 1981; Bradley et al., N ⁇ twre 309:255-258, 1984; Gossler et al., Proc. Natl. Acad. Sci. U.S.A.
  • ES cells which are commercially available (from, e.g., Genome Systems, Inc., St.
  • Transformed ES cells can be combined with an animal blastocyst, whereafter the ES cells colonize the embryo and contribute to the germline of the resulting animal, which is a chimera (composed of cells derived from two or more animals) (Jaenisch, Science 240: 1468-1474, 1988; Bradley in: Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, Robertson, E.J., ed., IRL Press, Oxford 1987, pp. 1 13-151).
  • offspring may be produced in which the transgenic allele is present in all of the animal's cells, i.e., in both somatic and germline cells.
  • transgenes of the invention may be stably integrated into germ line cells and transmitted to offspring of the transgenic animal as Mendelian loci. In mosaic transgenic animals, some cells carry the transgenes and other cells do not. In mosaic transgenic animals in which germ line cells do not carry the transgenes, transmission of the transgenes to offspring does not occur. Nevertheless, mosaic transgenic animals are capable of demonstrating phenotypes associated with the transgenes.
  • Offspring that have inherited the transgenes of the invention are distinguished from littermates that have not inherited transgenes by analysis of genetic material from the offspring for the presence of biomolecules that comprise unique sequences corresponding to sequences of, or encoded by, the transgenes of the invention.
  • biomolecules that comprise unique sequences corresponding to sequences of, or encoded by, the transgenes of the invention.
  • biological fluids that contain polypeptides uniquely encoded by the transgenes of the invention may be immunoassayed for the presence of the polypeptides.
  • a more simple and reliable means of identifying transgenic offspring comprises obtaining a tissue sample from an extremity of an animal, e.g., a tail, and analyzing the sample for the presence of nucleic acid sequences corresponding to the DNA sequence of a unique portion or portions of the transgenes of the invention.
  • nucleic acid sequences may be determined by, e.g., hybridization ("Southern") analysis with DNA sequences corresponding to unique portions of the transgene, analysis of the products of PCR reactions using DNA sequences in a sample as substrates and oligonucleotides derived from the transgene's DNA sequence, etc.
  • Cloned animals, transgenic and otherwise, of the invention may also be prepared (for a review of mammalian cloning techniques, see Wolf et al., J. Assist. Reprod. Genet. 75:235-239, 1998).
  • Such cloned animals include, without limitation, ovine species such as sheep (Campbell et al., Nature 350:64-66, 1996; Wells et al., Biol. Reprod. 57:385-393, 1997) rodents such as mice (Wakayama et al., Nature 394:369- 374, 1998) and non-human primates such as rhesus monkeys (Meng et al., Biol. Reprod. 57:454-459, 1997).
  • the transgenic and cloned animals of the invention may be used as animal models of human disease states and to evaluate potential therapies for such disease states.
  • a first transgenic animal having a disease state (or one or more symptomatic components thereof) is given a known dose of a candidate therapeutic composition or exposed to a candidate therapeutic treatment, and a second (control) transgenic animal is given a placebo or not exposed to the candidate therapeutic treatment.
  • Symptoms and/or clinical end-points relevant to the disease state are measured in both animals over appropriate intervals of time, and the results are compared.
  • Therapeutic (desirable) compositions and treatments are identified as those which ameriolate, delay the onset of or eliminate such symptoms and end-points in the treated animal relative to the control animal.
  • compositions and treatments that aggravate or accelerate the disease state are identified as those which enhance the degree of such symptoms and end-points and/or hasten their onset. Because of their high degree of genetic identity, cloned transgenic animals are preferred in such methods.
  • the present invention provides a method to increase at least one mitochondrial function in cells, particularly ex vivo or in vivo.
  • the present invention is not limited to any particular cell type, disease or disorder.
  • the present invention increases at least one mitochondrial function in diabetic or prediabetic cells or subjects (diabetes type I or diabetes type II), particularly in insulin producing cells or glucose responsive cells.
  • Such increase in at least one mitochondrial function can preferably be accomplished by regulating the transcription, translation or activity of IFl .
  • the invention provides a method for treating diabetes that includes improving at least one mitochondrial function in cells in a subject in need thereof. This method can be accomplished in any number of ways, including providing appropriate stimuli, compounds or compositions, including small molecules, polypeptides, nucleic acid molecules, gene therapy constructs or organic molecules, compounds or compositions identified using a method of the present invention or combinations thereof.
  • Increasing or improving at least one mitochondrial function in a cell can be accomplished in any manner.
  • the mitochondrial function being improved is in functional (i.e., not uncoupled) mitochondria such that ATP production within the cell is increased.
  • mitochondria may according to certain embodiments be uncoupled to some degree, for example by uncoupling factors such as UCP's (Wu et al., Cell 98:1 15-124 (1999)).
  • mitochondrial function in increased such that ATP production within the cell is increased.
  • the increase in ATP production related to the increase in mitochondrial function in insulin producing cells results in an increase in insulin production and/or insulin secretion.
  • the increase in ATP production can increase the sensitivity of insulin sensitive cells to insulin.
  • the cells can be any cells within the subject, preferably insulin producing cells or insulin sensitive cells.
  • Preferred insulin producing cells are pancreatic cells, such as within the islets of Langerhans, preferably the beta cells.
  • Preferred insulin sensitive cells are those cells involved in glucose metabolism, homeostasis and/or storage, such as liver cells and/or muscle cells.
  • One additional benefit to increasing mitochondrial function in liver cells is that the activity of the liver can increase such that these cells can perform detoxification functions, such as for reducing the toxicity or increasing the solubility of compounds, including therapeutics such as antiviral compounds and antisense compounds.
  • subjects that have liver diseases or disorders, such as hepatitis, cirrhosis, toxic intake of compounds can have their liver function increased using the methods of the present invention.
  • the subject and/or the cells are treated with at least one agent that enhances at least one activity of an IFl gene or polypeptide.
  • Agents that increase the activity of an IFl gene are those that can directly or indirectly increase the transcription of such gene, modulate post- transcriptional modification or mRNA half-life. Examples of such compounds can include cold and caloric intake.
  • the cell or subject can include a nucleic acid molecule that can be induced to increase the transcription of endogenous or exogenous IFl genes.
  • constructs can include an IFl gene operably linked to an inducible or constitutive promoter such that IFl transcription can be increase in a regulated or non-regulated fashion.
  • IF 1 can be any IF 1 , such as a wildtype or mutated rat, mouse or human IFl .
  • An IFl can have at least one activity of an IFl, preferably binding to a subunit of an ATP synthase but without an inhibitory effect on ATP production, which can then lead to increased ATP synthesis.
  • Wildtype IF Is that bind to and inhibit ATP synthase catalytic activity are also useful according to the present invention, for example in screening assays for agents that interfere with these functional activities.
  • Various IFl nucleic acid sequences and amino acid sequences from a variety of biological sources are provided in SEQ ID NOs:12-16. These sequences or portions thereof or related sequences as described herein that include at least one activity of an IFl can be used in the present invention.
  • the present invention provides a method to screen for compounds that increase mitochondrial function, particularly ex vivo or in vivo.
  • the present invention is not limited to a particular mechanism cell type, disease state or disorder.
  • mitochondrial function is increased in cells that are prediabetic or diabetic in nature, particularly insulin producing cells, including glucose responsive cells (diabetes type I or diabetes type II).
  • Such increase in mitochondrial function can be accomplished by regulating the transcription, translation or activity of IF 1.
  • One embodiment of the present invention is a method for screening for identifying test compounds that influence the expression of a nucleic acid that encodes an IFl protein, that includes contacting at least one cell that includes a nucleic acid molecule that encodes an IFl protein with one or more test compounds; and measuring the expression of an IFl protein.
  • the cell used in the methods of the present invention can be any cell including, preferably a cell that is insulin producing or insulin sensitive, preferably cells in culture, such as continuous cell lines.
  • cells from whole organisms including cells in suspension or from a tissue or organ or fluid from an organism, such as Zucker diabetic fatty rats (ZDF's), preferably pancreatic cells such as beta cells can be used.
  • ZDF's Zucker diabetic fatty rats
  • pancreatic cells such as beta cells
  • beta cells preferably pancreatic cells such as beta cells
  • the rat cell line INS1 is preferred.
  • Other preferred cells include SY5Y cells, HEK293 cells, G7/V79 cells, rho° 3T3-L1, and rho° INS-1 cells, and 293 cells.
  • muscle cells or liver cells are preferred as they are known in the art, such as HEPG2 cells.
  • the nucleic acid molecules that encode an IFl can be endogenous to the genome of the cell or can be engineered into the genome such as by homologous recombination or by random integration (Whitney et al., WO98/13353, published April 2, 1998, Smith et al., WO 94/24301, published October 27, 1994).
  • the expression of the IFl can be enhanced using stimuli or compounds known or expected to enhance such expression.
  • such nucleic acid molecules can be operably linked to an endogenous regulatory element or an exogenous regulatory element that can be modulated in the presence of an inducer or repressor, such as 2XTetO 2 .
  • the IFl gene can be operably linked to a reporter gene, such as green fluorescent protein, beta-lactamase or luciferase, for example, or tag, such as FLAG, such that the expression of the IFl gene can be monitored by measuring the expression of the reporter gene or tag.
  • a reporter gene such as green fluorescent protein, beta-lactamase or luciferase, for example, or tag, such as FLAG, such that the expression of the IFl gene can be monitored by measuring the expression of the reporter gene or tag.
  • the genes can be operably linked to a regulatory element to form a construct that is extra-chromosomal, such as a plasmid.
  • the expression of the IFl gene can be modulated by a repressor or inducer of the regulatory element.
  • the IF 1 gene can be operably linked to a reporter gene, such as green fluorescent protein, beta-lactamase or luciferase, for example, or a tag, such as FLAG, such that the expression of the IFl gene can be monitored by measuring the expression of the reporter gene or tag in vitro, ex vivo or in vivo.
  • the IFl gene and, optionally, a second gene of interest, when provided together in the same cell, can be on the same or different extra-chromosomal elements.
  • Such general technology is known in the art (U.S. Patent NO. 5,298,429 to Evans issued March 29, 1994).
  • the expression of IFl can be measured using a variety of methods (in vitro, ex vivo or in vivo), including reporter genes or tags, such as immunological tags.
  • reporter genes or tags such as immunological tags.
  • other detection methods such as Northern blots or Southern blots can be used.
  • nucleic acid amplification methods such as PCR, such as quantitative PCR or RT-PCR can be used.
  • in situ hybridization methods or immunohistochemical or other receptor-ligand reactions can be used.
  • the activity of an IFl can be directly measured, such as IFl binding to at least one ATP synthase subunit or to an IFl -specific antibody, or by other methods known in the art.
  • Compounds that alter or modulate IFl activity can also presumptively influence mitochondrial biogenesis, ATP synthesis, insulin production or insulin secretion, among others.
  • the cells of the present invention can be contacted with one or more test chemicals.
  • the expression of IFl in the cells can be monitored and test compounds that increase such expression can be identified.
  • test compounds that increase the production of ATP, decrease the hydrolysis of ATP, increase the synthesis or secretion of insulin or increase the insulin sensitivity of the cell can be monitored using methods known in the art.
  • Test compounds having such activity can be identified and screened for other activities described herein.
  • Nucleic acid molecules of the present invention can be provided as part of an expression construct.
  • An expression construct is a nucleic acid molecule that includes expression control sequences, such as promoters, appropriate for the expression of a nucleic acid molecule in an appropriate expression system.
  • a nucleic acid molecule of the present invention is operably linked to an expression control sequence, such as a promoter, that is appropriate for a particular expression system, such as an in vitro expression system or a host cell, such as a bacterial or eukaryotic cell.
  • Operaably linked refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner.
  • a control sequence operably linked to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.
  • Control sequences refer to polynucleotide sequences that effect the expression of coding and non-coding sequences to which they are ligated. The nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequences; in eukaryotes, generally, such control sequences include promoters and transcription termination sequences.
  • the term control sequences is intended to include components whose presence can influence expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.
  • a nucleic acid molecule can be engineered into an expression construct, such as a plasmid or viral vector, using methods known in the art (Sambrook et al., supra, 1989).
  • the nucleic acid molecule is preferably inserted in-frame and in the proper orientation in the expression construct such that a polypeptide of appropriate amino acid sequence relative to the native polypeptide coded by the nucleic acid is produced upon expression thereof.
  • Such in-frame insertions can be inferred from the nucleotide sequence of a nucleic acid molecule and be confirmed using a variety of methods, including computer anlaysis of predicted amino acid sequences and the folding thereof, or by binding with antibodies that specifically bind with identified or o ⁇ han proteins, such as unidentified proteins or portions of proteins that do not have an identified function.
  • the nucleic acid molecules of the invention can be inserted into a host cell, such as a prokaryotic cell (such as a bacterium such as E coli) or a eukaryotic cell (such as a HeLa cell) using methods known in the art, such as electroporation or treatment with cold calcium solutions.
  • a host cell such as a prokaryotic cell (such as a bacterium such as E coli) or a eukaryotic cell (such as a HeLa cell) using methods known in the art, such as electroporation or treatment with cold calcium solutions.
  • the expression construct is preferably configured such that an expression control element, such as a promoter, is operably linked to a nucleic acid molecule of the present invention in- frame and in the proper orientation such that the native amino acid sequence encoded by the nucleic acid molecule of the present invention are expressed by the expression construct.
  • Expression constructs can be chosen such that the nucleic acid molecule of the present invention is expressed efficiently in a chosen host cell.
  • RNA transcripts are RNA molecules that are synthesized (“transcribed") by RNA polymerase using DNA as a template.
  • Another aspect of the present invention is a gene therapy construct that includes an expression vector that includes a promoter operably linked to at least one nucleic acid of the present invention.
  • the nucleic acid of the present invention is selected from a) a substantially pure nucleic acid molecule including at least one of SEQ ID NO:l through SEQ ID NO: 12 and reverse complements thereof, a cDNA molecule prepared by a method of the present invention and reverse complements thereof.
  • the gene therapy construct is preferably a viral vector, such as a retrovirus, adenovirus, adenoassociated virus, papilloma virus or other type of virus vector used in gene therapy systems or genetic manipulation of cells.
  • Preferred gene therapy constructs include those that can target insulin producing cells or insulin sensitive cells.
  • Coxsakievisus particular Coxsackievirus B and Coxsackievirus B4
  • Echoviruses such as Echo 1 1
  • certain adenoviral vectors and certain retroviruses such as C-type retroviruses
  • pancreatic cells such as beta cells (Ramsingh et al., Bioessays 19:793-800 (1997), Hyoty et al., Clin. Diagn. Virol. 9:77-84 (1998), Jenson et al., Lancet, 2(8190):354-358 (1980), Luppi et al., J. Biol. Regul. Homeost. Agents 13:14-24 (1999), Tsumura et al., Lab. Anim.
  • liposomes and lipid preparations can also be used as vectors.
  • a variety of these types of vectors are known in the art (see, for example: U.S. Patent No. 5,399,346 to Anderson et al., issued March 21, 1995; Bandara et al., DNA and Cell Biology, 1 1 :227-231 (1992); Berkner, Biotechniques 6:616-629 (1989); U.S. Patent No.
  • Appropriate viral vectors can be selected based on the route of administration and the target cell type or population. For example, retroviruses are preferred if the target cell type or population is actively proliferating and other viruses, such as lentivirus, adeno associated virus, adenoviruses, are preferred if the target cell type or population is not actively proliferating (see, for example, Larrick et al, Gene Therapy, Elsevier, New York (1991)). Different viruses have different specificity for different cell types and populations. Thus, viruses that infect a targeted cell type of population of cells can be selected.
  • the viral vector can be provided as a pharmaceutical composition in an appropriate pharmaceutically acceptable carrier, such as an exciptient, at an appropriate dose for an appropriate route of administration and regime.
  • the gene therapy construct can also be a naked DNA construct such as plasmids that are useful in a gene therapy treatment system (see, for example, U.S. Patent No. 5,580,859 to Feigner et al., issued December 3, 1996; U.S. Patent No. 5,703,055 to Feigner et al., issued December 30, 1997; U.S. Patent No. 5,846,946 to Huebner et al., issued December 8, 1998; and U.S. Patent No. 5,910,488 to Nabel et al., issued June 8, 1999).
  • a particular vector can be made with a particular target tissue, cell type or population of cells in mind.
  • particular regulatory elements such as control elements and promoters
  • the vector is preferably introduced into a subject via direct injection into the pathological location, such as the brain, but other methods of delivery, such as systemic or intra-tissue or organ administration distal from the pathological location, such as the muscle, may also be used.
  • These types of vectors can be provided as a pharmaceutical composition in an appropriate pharmaceutically acceptable carrier, such as an exipient, at an appropriate dose for an appropriate route of administration and regime.
  • the present invention also includes a variety of methods to identify biologically active agents that can modulate the activity of at least one function of a polypeptide of the present invention.
  • the functions can be in vitro (outside of a whole cell), ex vivo (within or on a cell but not in a whole organism such as samples from a whole organism or cells in culture) or in vivo (within a whole organism).
  • the present invention also includes biologically active agents identified by these methods.
  • Organism refers to a subject, such as a non-human animal (such as a test animal or transgenic animal) or a human.
  • modulation refers to the capacity to either enhance or inhibit a functional property of a biological activity or process, for example, enzyme activity or receptor binding. Such enhancement or inhibition may be contingent on the occurrence of a specific event, such as activation of a signal transduction pathway and/or may be manifest only in particular cell types.
  • modulator refers to a chemical (naturally occurring or non- naturally occurring), such as a biological macromolecule (for example, nucleic acid, protein, non-peptide or organic molecule) or an extract made from biological materials, such as prokaryotes, bacteria, eukaryotes, plants, fungi, multicellular organisms or animals, invertebrates, vertebrates, mammals and humans, including, where appropriate, extracts of: whole organisms or portions of organisms, cells, organs, tissues, fluids, whole cultures or portions of cultures, or environmental samples or portions thereof.
  • a biological macromolecule for example, nucleic acid, protein, non-peptide or organic molecule
  • an extract made from biological materials such as prokaryotes, bacteria, eukaryotes, plants, fungi, multicellular organisms or animals, invertebrates, vertebrates, mammals and humans, including, where appropriate, extracts of: whole organisms or portions of organisms, cells, organs, tissues, fluids, whole cultures or portions of cultures, or environmental samples
  • Modulators are typically evaluated for potential activity as inhibitors or activators (directly or indirectly) of a biological process or processes (for example, agonists, partial antagonists, partial agonists, antagonists, antineoplastic agents, cytotoxic agents, inhibitors of neoplastic transformation or cell proliferation, cell proliferation promoting agents, antiviral agents, antimicrobial agents, antibacterial agents, antibiotics, and the like) by inclusion in assays described herein.
  • a modulator may be known, unknown or partially known.
  • test compound refers to a chemical, compound, composition or extract to be tested by at least one method of the present invention to be a putative modulator.
  • a test compound or test chemical identified by the present invention is a "biologically active agent.”
  • Test compounds can include small molecules, such as drugs, proteins or peptides or active fragments thereof, such as antibodies, nucleic acid molecules such as DNA, RNA or combinations thereof, antisense molecules or ribozymes, or other organic or inorganic molecules, such as lipids, carboydrates, or any combinations thereof.
  • Test compounds that include nucleic acid molecules can be provided in a vector, such as a viral vector, such as a retrovirus, adenovirus or adeno-associated virus, a liposome, a plasmid or with a lipofection agent.
  • Test compounds once identified, can be agonists, antagonists, partial agonists or inverse agonists of a target.
  • a test compound is usually not known to bind to the target of interest.
  • Control test compound refers to a compound known to bind to the target (for example, a known agonist, antagonist, partial agonist or inverse agonist).
  • Test compound does not typically include a compound added to a mixture as a control condition that alters the function of the target to determine signal specificity in an assay.
  • control compounds or conditions include chemicals that (1) non-specifically or substantially disrupt protein structure (for example denaturing agents such as urea or guandium, sulfhydryl reagents such as dithiotritol and beta-mercaptoethanol), (2) generally inhibit cell metabolism (for example mitochondrial uncouplers) or (3) non- specifically disrupt electrostatic or hydrophobic interactions of a protein (for example, high salt concentrations or detergents at concentrations sufficient to non-specifically disrupt hydrophobic or electrostatic interactions).
  • test compound also does not typically include compounds known to be unsuitable for a therapeutic use for a particular indication due to toxicity to the subject. Usually, various predetermined concentrations of test compounds are used for determining their activity.
  • the concentration of test chemical used can be expressed on a weight to volume basis. Under these circumstances, the following ranges of concentrations can be used: between about 0.001 micrograms/ml and about 1 milligram/ml, preferably between about 0.01 micrograms/ml and about 100 micrograms/ml, and more preferably between about 0.1 micrograms/ml and about 10 micrograms/ml.
  • Test compounds that modulate the activity of the at least one in vitro or ex vivo function of a polypeptide of the present invention have presumptive therapeutic activity in modulating the activity of that in vivo function in a subject, including a human.
  • the present invention includes biologically active agents identified by a method of the present invention. Such biologically active agents can be provided as a pharmaceutical, such as with an excipient.
  • Another aspect of the invention involves a method for identifying biologically active agents, including: providing a sample that includes at least one polypeptide of the present invention; contacting the sample with at least one test chemical; detecting at least one in vitro function of the polypeptide; and identifying at least one test chemical that modulates (such as enhances or inhibits) the at least one in vitro function of the polypeptide.
  • this method is practiced in a high throughput format and device, such as described in WO 98/52047 to Stylli et al., published November 19, 1998.
  • a polypeptide of the present invention having at least one in vitro function that is detectable using a compound that provides a readout of the at least one in vitro function, such as an enzymatic substrate that changes at least one property, such as, for example, colormetric, spectrographic or fluorescent properties, upon the action of the at least one in vitro function upon the enzymatic substrate is provided.
  • a compound that provides a readout of the at least one in vitro function such as an enzymatic substrate that changes at least one property, such as, for example, colormetric, spectrographic or fluorescent properties, upon the action of the at least one in vitro function upon the enzymatic substrate.
  • Such enzymatic substrates are known in the art for a variety of activities, such as, for example, proteases and kinases (see, for example, WO 97/28261 to Tsien et al., published August 7, 1997; WO 98/02571 to Tsien et al., published January 22, 1998; and The Sigma Catalogue, Sigma Chemical Company, St. Louis, MO (1999)).
  • the polypeptide of the present invention having at least one in vitro function is contacted with a test chemical before or contemporaneously with being contacted with the compound that provides a readout for the at least one in vitro function.
  • the at least one in vitro function is monitored by monitoring the readout of that activity.
  • the results of these studies can be compared to an appropriate control to determine the ability of a test chemical to modulate the activity of the at least one in vitro function.
  • Appropriate controls are known in the art, such as performing the test in the absence of the test chemical.
  • the control can be performed at the same time as the test, but can also be performed at a time and place distant from the test. For example, standard curves or values can be obtained and provided for a particular test which can be used in the comparison.
  • EX VIVO FUNCTION Another aspect of the invention involves a method for identifying biologically active agents, including: providing a sample that includes at least one cell that includes at least one polypeptide of the present invention; contacting the sample with at least one test chemical; detecting at least one ex vivo function of the polypeptide; and identifying at least one test chemical that modulates (such as enhances or inhibits) the at least one ex vivo function of the polypeptide.
  • the polypeptide of the present invention is preferably within or associated with a cell and the test chemical is contacted with the cell.
  • this method is practiced in a high throughput format and device, such as described in WO 98/52047 to Stylli et al., published November 19, 1998.
  • the at least one cell can be from a sample from a subject, such as a test animal, transgenic animal, or human, or can be a cell in culture.
  • a polypeptide of the present invention having at least one ex vivo function that is detectable using a compound that provides a readout of the at least one in vitro function, such as an enzymatic substrate that changes at least one property, such as, for example, colormetric, spectrographic or fluorescent properties, upon the action of the at least one ex vivo function upon the enzymatic substrate is provided.
  • a compound that provides a readout of the at least one in vitro function such as an enzymatic substrate that changes at least one property, such as, for example, colormetric, spectrographic or fluorescent properties, upon the action of the at least one ex vivo function upon the enzymatic substrate.
  • Such enzymatic substrates are known in the art for a variety of activities, such as, for example, proteases, kinases (see, for example, WO 97/28261 to Tsien et al., published August 7, 1997; WO 98/02571 to Tsien et al., published January 22, 1998; and The Sigma Catalogue, Sigma Chemical Company, St. Louis, MO (1999)).
  • the cell that includes at least one polypeptide of the present invention having at least one ex vivo function is contacted with a test chemical before or contemporaneously with being contacted with the compound that provides a readout for the at least one ex vivo function.
  • the at least one ex vivo function is monitored by monitoring the readout of that activity.
  • Appropriate controls are known in the art, such as performing the test in the absence of the test chemical.
  • the control can be performed at the same time as the test, but can also be performed at a time and place distant from the test. For example, standard curves or values can be obtained and provided for a particular test which can be used in the comparison.
  • Another aspect of the invention involves a method for identifying biologically active agents, including: providing at least one subject that includes at least one polypeptide of the present invention; contacting the at least one subject with a test chemical; detecting at least one in vivo function of the polypeptide; and identifying at least one test chemical that modulates (such as enhances or inhibits) the at least one in vivo function of the polypeptide.
  • a polypeptide of the present invention having at least one in vivo function that is detectable using a compound that provides a readout of the at least one in vitro function, such as an enzymatic substrate that changes at least one property, such as, for example, colorimetric, spectrographic or fluorescent properties, upon the action of the at least one in vivo function upon the enzymatic substrate is provided.
  • a compound that provides a readout of the at least one in vitro function such as an enzymatic substrate that changes at least one property, such as, for example, colorimetric, spectrographic or fluorescent properties, upon the action of the at least one in vivo function upon the enzymatic substrate.
  • Such enzymatic substrates are known in the art for a variety of activities, such as, for example, proteases, kinases (see, for example, WO 97/28261 to Tsien et al., published August 7, 1997; WO 98/02571 to Tsien et al., published January 22, 1998; and The Sigma Catalogue, Sigma Chemical Company, St. Louis, MO (1999)).
  • the subject that includes at least one polypeptide of the present invention having at least one in vivo function is contacted with a test chemical before or contemporaneously with being contacted with the compound that provides a readout for the at least one in vivo function.
  • the at least one in vivo function is monitored by monitoring the readout of that activity.
  • the results of these studies can be compared to an appropriate control to determine the ability of a test chemical to modulate the activity of the at least one in vivo function.
  • Appropriate controls are known in the art, such as performing the test in the absence of the test chemical.
  • the control can be performed at the same time as the test, but can also be performed at a time and place distant from the test.
  • a preferred animal model is the non-obese diabetic (NOD) mouse.
  • NOD non-obese diabetic
  • the successful use of this animal model in diabetic drug discovery is reported in the literature (Yang et al., J. Autoimmun. 10:257-260 (1997), Akashi et al., Int. Immunol. 9:1 159-1164 (1997), Suri and Katz, Immunol. Rev. 169:55- 65 (1999), Pak et al., Autoimmunity 20:19-24 (1995), Toy ⁇ da and Formby, Bioessays 20:750-757 (1998), Cohen, Res. Immunol.
  • test compound The structure of a test compound can be determined or confirmed by methods known in the art, such as mass spectroscopy. For test compounds stored for extended periods of time under a variety of conditions, the structure, activity and potency thereof can be confirmed.
  • Identified test compounds can be evaluated for a particular activity using recognized methods and those disclosed herein. For example, if an identified test compound is found to have anticancer cell activity in vitro, then the test compound would have presumptive pharmacological properties as a chemotherapeutic to treat cancer. Such nexuses are known in the art for several disease states, and more are expected to be discovered over time. Based on such nexuses, appropriate confirmatory in vitro and in vivo models of pharmacological activity, and toxicology, can be selected and performed. The methods described herein can also be used to assess pharmacological selectivity and specificity, and toxicity.
  • test compounds can be evaluated for toxicological effects using known methods (see, Lu, Basic Toxicology, Fundamentals, Target Organs, and Risk Assessment, Hemisphere Publishing Co ⁇ ., Washington (1985); U.S. Patent Nos; 5,196,313 to Culbreth (issued March 23, 1993) and 5,567,952 to Benet (issued October 22, 1996)).
  • toxicology of a test compound can be established by determining in vitro toxicity towards a cell line, such as a mammalian, for example a human cell line.
  • Test compounds can be treated with, for example, tissue extracts, such as preparations of liver, such as microsomal preparations, to determine increased or decreased toxicological properties of the test compound after being metabolized by a whole organism.
  • tissue extracts such as preparations of liver, such as microsomal preparations
  • the toxicological properties of a test compound in an animal model can be determined using established methods (see, Lu, supra (1985); and Creasey, Drug Disposition in Humans, The Basis of Clinical Pharmacology, Oxford University Press, Oxford (1979)).
  • an animal model such as mice, rats, rabbits, dogs or monkeys
  • the toxicological properties of a test compound in an animal model can be determined using established methods (see, Lu, supra (1985); and Creasey, Drug Disposition in Humans, The Basis of Clinical Pharmacology, Oxford University Press, Oxford (1979)).
  • the skilled artisan would not be burdened to determine appropriate doses, LD 50 values, routes of administration and regimes that would be appropriate to determine the toxicological properties of the test compound.
  • test compound can be established using several art recognized methods, such as in vitro methods, animal models or human clinical trials (see, Creasey, supra (1979)). Recognized in vitro models exist for several diseases or conditions. For example, the ability of a test compound to extend the life-span of HIV- infected cells in vitro is recognized as an acceptable model to identify chemicals expected to be efficacious to treat HIV infection or AIDS (see, Daluge et al., Antimicro. Agents Chemother. 41 :1082-1093 (1995)).
  • CsA cyclosporin A
  • acceptable animal models can be used to establish efficacy of test compounds to treat various diseases or conditions.
  • the rabbit knee is an accepted model for testing agents for efficacy in treating arthritis (see, Shaw and Lacy, J. Bone Joint Surg. (Br.) 55:197-205 (1973)).
  • Hydrocortisone which is approved for use in humans to treat arthritis, is efficacious in this model which confirms the validity of this model (see, McDonough, Phys. Ther. 62:835-839 (1982)).
  • the selectivity of a test compound can be established in vitro by testing the toxicity and effect of a test compound on a plurality of cell lines that exhibit a variety of cellular pathways and sensitivities.
  • the data obtained form these in vitro toxicity studies can be extended to animal model studies, including human clinical trials, to determine toxicity, efficacy and selectivity of a test compound.
  • the selectivity, specificity and toxicology, as well as the general pharmacology, of a test compound can be often improved by generating additional test compounds based on the structure/property relationship of a test compound originally identified as having activity.
  • Test compounds can be modified to improve various properties, such as affinity, life-time in blood, toxicology, specificity and membrane permeability.
  • test compounds can be subjected to additional assays as they are known in the art or described herein.
  • Methods for generating and analyzing such compounds or compositions are known in the art, such as U.S. Patent No. 5,574,656 to Agrafiotis et al.
  • the present invention also encompasses a test compound in a pharmaceutical composition
  • a pharmaceutically acceptable carrier prepared for storage and preferably subsequent administration, which has a pharmaceutically effective amount of the test compound in a pharmaceutically acceptable carrier or diluent.
  • Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co., (A.R. Gennaro edit. (1985)).
  • Preservatives, stabilizers, dyes and even flavoring agents can be provided in the pharmaceutical composition.
  • sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid can be added as preservatives.
  • antioxidants and suspending agents can be used.
  • test compounds of the present invention can be formulated and used as tablets, capsules or elixirs for oral administration; suppositories for rectal administration; sterile solutions or suspensions or injectable administration; and the like.
  • injectables can be prepared in conventional forms either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Suitable excipients are, for example, water, saline, dextrose, mannitol, lactose, lecithin, albumin, sodium glutamate, cysteine hydrochloride and the like.
  • the injectable pharmaceutical compositions can contain minor amounts of nontoxic auxiliary substances, such as wetting agents, pH buffering agents and the like. If desired, abso ⁇ tion enhancing preparations, such as liposomes, can be used.
  • the pharmaceutically effective amount of a test compound required as a dose will depend on the route of administration, the type of animal or patient being treated, and the physical characteristics of the specific animal under consideration.
  • the dose can be tailored to achieve a desired effect, but will depend on such factors as weight, diet, concurrent medication and other factors which those skilled in the medical arts will recognize.
  • the pharmaceutical compositions can be used alone or in combination with one another, or in combination with other therapeutic or diagnostic agents. These products can be utilized in vivo, preferably in a mammalian patient, preferably in a human, or in vitro.
  • the pharmaceutical compositions can be administered to the patient in a variety of ways, including parenterally, intravenously, subcutaneously, intramuscularly, colonically, rectally, nasally or intraperiotoneally, employing a variety of dosage forms. Such methods can also be used in testing the activity of test compounds in vivo.
  • the useful in vivo dosage to be administered and the particular mode of administration will vary depending upon the age, weight and type of patient being treated, the particular pharmaceutical composition employed, and the specific use for which the pharmaceutical composition is employed.
  • the determination of effective dosage levels can be accomplished by one skilled in the art using routine methods as discussed above, and can be guided by agencies such as the USFDA or NIH.
  • human clinical applications of products are commenced at lower dosage levels, with dosage level being increased until the desired effect is achieved.
  • acceptable in vitro studies can be used to establish useful doses and routes of administration of the test compounds.
  • the dosage for the test compounds of the present invention can range broadly depending upon the desired affects, the therapeutic indication, route of administration and purity and activity of the test compound.
  • dosages can be between about 1 ng/kg and about 10 mg/kg, preferably between about 10 ng/kg and about 1 mg/kg, more preferably between about 100 ng/kg and about 100 micrograms/kg, and most preferably between about 1 microgram/kg and about 10 micrograms/kg.
  • the exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition (see, Fingle et al., in The Pharmacological Basis of Therapeutics (1975)). It should be noted that the attending physician would know how to and when to terminate, interrupt or adjust administration due to toxicity, organ dysfunction or other adverse effects. Conversely, the attending physician would also know to adjust treatment to higher levels if the clinical response were not adequate.
  • the magnitude of an administrated does in the management of the disorder of interest will vary with the severity of the condition to be treated and to the route of administration. The severity of the condition may, for example, be evaluated, in part, by standard prognostic evaluation methods.
  • the dose and perhaps dose frequency will also vary according to the age, body weight and response of the individual patient, including those for veterinary applications.
  • such pharmaceutical compositions can be formulated and administered systemically or locally. Techniques for formation and administration can be found in Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, PA (1990). Suitable routes of administration can include oral, rectal, transdermal, otic, ocular, vaginal, transmucosal or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.
  • the pharmaceutical compositions of the present invention can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution or physiological saline buffer.
  • physiologically compatible buffers such as Hanks' solution, Ringer's solution or physiological saline buffer.
  • penetrans appropriate to the barrier to be permeated are used in the formulation.
  • Such penetrans are generally known in the art.
  • Use of pharmaceutically acceptable carriers to formulate the pharmaceutical compositions herein disclosed for the practice of the invention into dosages suitable for systemic administration is within the scope of the invention.
  • the compositions of the present invention in particular, those formulation as solutions, can be administered parenterally, such as by intravenous injection.
  • compositions can be formulated readily using pharmaceutically acceptable carriers well known in the art into dosages suitable for oral administrations.
  • Such carriers enable the test compounds of the invention to be formulated as tables, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.
  • Agents intended to be administered intracellularly may be administered using techniques well known to those of ordinary skill in the art. For example, such agents may be encapsulated into liposomes, then administered as described above. Substantially all molecules present in an aqueous solution at the time of liposome formation are inco ⁇ orated into or within the liposomes thus formed. The liposomal contents are both protected from the external micro-environment and, because liposomes fuse will cell membranes, are efficiently delivered into the cell cytoplasm. Additionally, due to their hydrophobicity, small organic molecules can be directly administered intracellularly.
  • compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve its intended pu ⁇ ose. Determination of the effective amount of a pharmaceutical composition is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
  • these pharmaceutical compositions can contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active chemicals into preparations which can be used pharmaceutically.
  • the preparations formulated for oral administration may be in the form of tables, dragees, capsules or solutions.
  • compositions of the present invention can be manufactured in a manner that is itself known, for example by means of conventional mixing, dissolving, granulating, dragee-making, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • Pharmaceutical formulations for parenteral administration include aqueous solutions of active chemicals in water-soluble form.
  • suspensions of the active chemicals may be prepared as appropriate oily injection suspensions.
  • Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides or liposomes.
  • Aqueous injection suspensions may contain substances what increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran.
  • the suspension can also contain suitable stabilizers or agents that increase the solubility of the chemicals to allow for the preparation of highly concentrated solutions.
  • compositions for oral use can be obtained by combining the active chemicals with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tables or dragee cores.
  • suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone.
  • disintegrating agents can be added, such as the cross-linked polyvinyl pyrolidone, agar, alginic acid or a salt thereof such as sodium alginate.
  • Dragee cores can be provided with suitable coatings. Dyes or pigments can be added to the tablets or dragee coatings for identification or to characterize different combinations of active doses.
  • test compounds of the present invention and pharmaceutical compositions that include such test compounds are useful for treating a variety of ailments in a patient, including a human.
  • a patient in need of such treatment can be provided a test compound of the present invention, preferably in a pharmacological composition in an effective amount to reduce the symptoms, pathology or rate of progression of a disease or disorder in a patient.
  • the amount, dosage, route of administration, regime and endpoint can all be determined using the procedures described herein or by appropriate government agencies, such as the United Stated Food and Drug Administration.
  • Another aspect of the invention involves a method of treating diabetes by administering an effective amount of pharmaceutical composition of the present invention to a subject, such as a human patient, in need of treatment of diabetes.
  • the pharmaceutical composition is administered to the subject in an amount, route of administration and regime sufficient to have a therapeutic, palliative, prophylactic, impeditive effect to ameliorate the effects, reversing the course of, delaying the onset of or preventing diabetes.
  • the subject preferably is suspected of having or being at risk of developing diabetes.
  • an “effective amount” is the amount of a therapeutic reagent that when administered to a subject by an appropriate dose and regime results the desired result.
  • a "subject in need of treatment of diabetes” is a subject diagnosed with diabetes or is suspected of having diabetes.
  • a “therapeutic effect” is the reduction or elimination of a disease state or pathological condition.
  • a “palliative effect” is the alleviation of symptoms associated with a disease or pathological condition.
  • a "prophylactic effect” is the prevention of a disease state or pathological condition.
  • An “impeditive effect” is the reduction of the rate of progression of a disease state or pathological condition.
  • the therapeutic composition of the present invention includes at least one nucleic acid molecule of the present invention, preferably a nucleic.
  • the nucleic acids may be covalently or noncovalently conjugated or bound to other molecules, such as, but not limited to, proteins that may facilitate their delivery to the target tissue or tissues.
  • Small molecules such as folate may be conjugated to nucleic acid molecules to enhance transport across the blood-brain barrier (Wu, D. et al. (1999) Pharm. Res. 76: 415-19.)
  • the nucleic acid molecules can be complexed with cationic lipids, packaged within liposomes, inco ⁇ orated into hydrogels, cyclodextrins, biodegradable nanocapsules, or bioadhesive microspheres.
  • the pharmaceutical compostition may include carriers, thickeners, diluents, buffers, preservatives, surface active agents, and the like in addition to oligonucleotides.
  • Pharmaceutical compositions can also include one or more active ingredients such as antimicrobial agents, anti inflammatory agents, anesthetics, and the like in addition to oligonucleotides.
  • nucleic acid molecules cann be delivered directly or in the aforementioned compositions in sterile solution, which may also contain buffers, diluents, and other suitable additives.
  • Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders.
  • Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like can be necessary or desirable.
  • Nasal inhalation may be particularly effective for delivery of pharmaceutical compositions to the brain (Wang, Y. et al. (1998) Biopharm Drug Dispos. 19: 571-5) and/or cerebrospinal fluid (Sakane T. (1991) J. Pharm. Pharmacol. 43: 449-51).
  • Pharmaceutical compositions that include nucleic acid molecules can also include compounds that enhance abso ⁇ tion by nasal epithelial cells such as cationic compounds (Natsume, H. et al. (1999) Int. J. Pharm. 185: 1-12), cyclodextrins (Martin, et al., J. Drug Target. 6: 17-36), or other compounds that are known or may be later discovered to enhance nasal abso ⁇ tion.
  • Solutions containing nucleic acids for nasal delivery may be supplied in spray containers for aerosol inhalation.
  • Compositions for oral delivery include powders or granules, suspensions or solutions in water or nonaqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders may be desirable.
  • Optimum doses of pharmaceutical compositions that include nucleic acid molecules depends on a variety of factors, including the severity of the condition to be treated, the toxicity of the nucleic acid molecules being delivered, the route of administration, and the individual patient's response to the treatment. The skilled practitioner is able to determine the appropriate dose based on these factors and the effective dose derived from animal and clinical studies. In general, dosage is from 0.01 micrograms to 100 g per kg of body weight, and may be given once or more daily, weekly, monthly, or yearly, or even once every 2 to 20 years. Persons of ordinary skill in the art can estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues.
  • nucleic acids are administered in maintenance doses, ranging from 0.01 microgram to 100 g per kg of body weight once or more daily to once every 20 years.
  • Nucleic acid molecules may be administered by any appropriate route of administration, such as, for example, parenteral or intravenous injection. Nucleic acids may also be delivered intravenously through pump, stent, or drip. Nucleic acid molecules may be introduced into the cerebrospinal fluid by injection into the spinal column. For delivery into the brain, injection may be into the brain cavity via a canula. Other routes of delivery include oral delivery and topical application. Nasal inhalation of aerosols may be particularly effective for administering the nucleic acids of the invention and their formulations to the brain. Nucleic acids may also be encased in or applied to a polymer, solid support or fabric, or gel which is delivered locally. Such solid supports, fabrics, polymers, or gels may be biodegradable.
  • the dose regime is determined experimentally based on animal studies and clinical trials. Doses may be given once or more daily, weekly, monthly, or yearly, or even once every 2 to 20 years. Persons of ordinary skill in the art can estimate repetition rates based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the patient under maintenance therapy to prevent the recurrence of the disease state, wherein the oligonucliotide is administered in maintenance doses, ranging from 0.01 micorgrams to 100 grams per kg of body weight, once or more daily, to once every 20 years.
  • the progress of treatment for diabetes can be measured using methods known in the art. For example, blood glucose, urine glucose or blood or serum insulin levels can be monitored using established methods. These measurements can be taken at appropriate intervals, including before, during and after feeding or fasting. In this instance, the caloric intake and type of caloric intake, such as carbohydrates, should be noted.
  • Gene therapy constructs contain nucleic acids comprising a nucleic acid molecule of the present invention optionally operably linked to gene regulatory elements.
  • the nucleic acid molecule and gene regulatory elements may be in a plasmid or may be inco ⁇ orated into a vector, such as, but not limited to, a retroviral vector, an adenoviral vector, an adeno-associated viral vector, a vaccinia viral vector, a he ⁇ es viral vector, or other vectors as they are known or later developed in the art.
  • the gene therapy constructs may be administered as DNA, as viral particles, or in cells.
  • Gene therapy constructs that consist of nucleic acid molecules not inco ⁇ orated into vectors such as viruses may be delivered as free nucleic acids, or may be delivered covalently or noncovalently conjugated or bound to other molecules, such as, but not limited to, molecules that enhance their transport across the blood-brain barrier or that may facilitate their delivery to the target tissue or tissues.
  • Other DNA sequences such as adenovirus VA genes can be included in the administration medium and be co-transfected with the gene of interest. The presence of genes coding for the adenovirus VA gene product may significantly enhance the translation of mRNA transcribed from the plasmid.
  • Gene therapy constructs that are packaged in viruses may have proteins or other molecules or compounds, such as, but not limited to lipids, proteins, or polymers inco ⁇ orated into or associated with the virus to enhance delivery into cells.
  • the gene therapy constructs whether naked DNA or packaged vector constructs, may be complexed with cationic lipids, packaged within liposomes, inco ⁇ orated into hydrogels, cyclodextrins, biodegradable nanocapsules, or bioadhesive microspheres.
  • the pharmaceutical composition may include carriers, thickeners, diluents, buffers, preservatives, surface active agents, and the like in addition to oligonucleotides.
  • compositions may also include one or more active ingredients such as antimicrobial agents, antiinflammatory agents, anesthetics, and the like in addition to oligonucleotides. If administration is by injection or infusion, the gene therapy constructs may be delivered directly or in the aforementioned compositions in sterile solution, which may also contain buffers, diluents, and other suitable additives.
  • Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
  • Nasal inhalation may be particularly effective for delivery of pharmaceutical compositions to the brain (Wang, Y. et al. (1998) Biopharm Drug Dispos. 19: 571-5) and or cerebrospinal fluid (Sakane T. (1991) J. Pharm. Pharmacol 43: 449-51).
  • Pharmaceutical compositions that include gene therapy constructs may also include compounds that enhance abso ⁇ tion by nasal epithelial cells such as cationic compounds (Natsume, H. et al. (1999) Int. J. Pharm. 185: 1-12), cyclodextrins (Martin, et al., J. Drug Target. 6: 17-36), or other compounds that are known or may be later discovered to enhance nasal abso ⁇ tion. Solutions containing gene therapy constructs may be supplied in spray containers for aerosol inhalation.
  • compositions for oral delivery include powders or granules, suspensions or solutions in water or nonaqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders may be desirable. Nucleic acids may also be encased in or applied to a polymer, solid support or fabric, or gel which is delivered locally. Such solid supports, fabrics, polymers, or gels may be biodegradable.
  • Gene therapy constructs may also be delivered in cells.
  • Cells containing gene therapy constructs may be derived from the patient, another human being, or even an animal of another species.
  • Gene therapy constructs may be introduced into the cells ex vivo by viral transfection, electroporation, membrane fusion with liposomes, high velocity bombardment with DNA coated microprojectiles, incubation with calcium- phosphate-DNA precipitate, tansfection with DEAE-dextran, direct microinjection, or other methods known or later developed in the art.
  • the cells are then delivered to the patient by any of a variety of means, including implantation or injection.
  • the cells may express the gene therapy construct in vivo to obtain the therapeutic effect in the patient.
  • the cells containing the gene therapy construct may replicate and/or package the gene therapy construct such that endogenous cells in the patient may be infected, transformed, or transfected with the gene therapy construct and thereby express it.
  • Cells containing gene therapy constructs may be enclosed in structures composed of polymers or other materials to retain them at the instillation site or to protect them from the patient's cellular immunity mechanisms.
  • Optimum doses depend on the severity of the condition to be treated, the toxicity of the gene therapy construct being delivered, the route of administration, and the individual patient's response to the treatment. The skilled practitioner is able to determine the appropriate dose based on these factors and the effective dose derived from animal and clinical studies. In general, for naked DNA gene therapy constructs, the dosage is from 0.01 micrograms to 100 g per kg of body weight. For viral gene therapy constructs, an appropriate dose is in the range of 0.1 to 50 ml of 10 6 to 10" particle forming units per ml viral expression vectors.. For cells containing viral expression constructs, about 10 5 to about 10 8 cells may be delivered to an appropriate site.
  • DNA gene therapy constructs and viral gene therapy constructs may be delivered by intravenous or intraperitoneal injection, intratracheally, intrathecally parenterally, intraarticularly, intramuscularly, or introduced into the brain by injection via a cannula or injected into the spinal column for distribution within the cerebrospinal fluid.
  • Gene therapy constructs may be administered intravenously, by injection, catheter, pump, or drip.
  • Cells containing gene therapy constructs may be implanted surgically into the brain, or they may be delivered to another site in the body.
  • Cells may be administered topically, intraocularly, parenterally, intranasally, intratracheally, intrabronchially, intramuscularly, subcutaneously, or by any other means.
  • the dose regime is determined experimentally based on animal studies and clinical trials. Doses may be given once or more daily, weekly, monthly, or yearly, or even once every 2 to 20 years. Persons of ordinary skill in the art can estimate repetition rates based on measured residence times and concentrations of the gene product of the gene therapy vector in bodily fluids or tissues. Following successful treatment, it may be desirable to have the patient receive additional doses of the gene therapy vector if it is determined that levels of the gene product have declined below a level necessary to prevent disease progression, or if there are symptoms of disease progression.
  • the gene therapy construct or cells containing the gene therapy construct may be administered in maintenance doses, where the dose has been determined based on animal and clinical studies, and may be monitored by measuring the expression product of the gene therapy construct in the patient's bodily fluids.
  • the progress of treatment for diabetes can be measured using methods known in the art. For example, blood glucose, urine glucose or blood or serum insulin levels can be monitored using established methods. These measurements can be taken at appropriate intervals, including before, during and after feeding or fasting. In this instance, the caloric intake and type of caloric intake, such as carbohydrates, should be noted.
  • a therapeutic composition of the present invention can include at least one biologically active agent of the present invention. At least one biologically active agent of the present invention can optionally be covalently or noncovalently conjugated or bound to other molecules, such as, but not limited to, proteins that may facilitate their delivery to the target tissue or tissues. Small molecules such as folate may be conjugated to the biologically active agents of the invention to enhance transport across the blood-brain barrier (Wu, D. et al. (1999) Pharm. Res. 16: 415-19.).
  • the pharmaceutical composition may comprise a pharmaceutically acceptable carrier prepared for storage and preferably subsequent administration, which has a pharmaceutically effective amount of the biologically active agent in a pharmaceutically acceptable carrier or diluent.
  • Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington 's Pharmaceutical Sciences, Mack Publishing Co., (A.R. Gennaro edit. (1985)).
  • Preservatives, stabilizers, dyes and even flavoring agents can be provided in the pharmaceutical composition.
  • sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid can be added as preservatives.
  • antioxidants and suspending agents can be used.
  • the biologically active agents of the present invention can be formulated and used as tablets, capsules or elixirs for oral administration; suppositories for rectal administration; sterile solutions or suspensions for injectable administration; and the like.
  • injectables can be prepared in conventional forms either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions.
  • Suitable excipients are, for example, water, saline, dextrose, mannitol, lactose, lecithin, albumin, sodium glutamate, cysteine hydrochloride and the like.
  • the injectable pharmaceutical compositions can contain minor amounts of nontoxic auxiliary substances, such as wetting agents, pH buffering agents and the like. If desired, abso ⁇ tion enhancing preparations, such as liposomes, can be used.
  • the pharmaceutical composition may also include carriers, thickeners, diluents, buffers, preservatives, surface active agents, and the like in addition to one or more biologically active agents.
  • Pharmaceutical compositions may also include one or more active ingredients such as antimicrobial agents, anti inflammatory agents, anesthetics, and the like in addition to the biologically active agents of the invention.
  • Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders.
  • Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
  • Agents intended to be administered intracellularly may be administered using techniques well known to those of ordinary skill in the art. For example, such agents may be encapsulated into liposomes, then administered as described above. Substantially all organic molecules present in an aqueous solution at the time of liposome formation are inco ⁇ orated into or within the liposomes thus formed. The liposomal contents are both protected from the external micro-environment and, because liposomes fuse with cell membranes, are efficiently delivered into the cell cytoplasm. Additionally, due to their hydrophobicity, small organic molecules can be directly administered intracellularly. Nasal inhalation may be particularly effective for delivery of pharmaceutical compositions to the brain (Wang, Y. et al. (1998) Biopharm Drug Dispos.
  • compositions that include biologically active agents may also include compounds that enhance abso ⁇ tion by nasal epithelial cells such as cationic compounds (Natsume, H. et al. (1999) Int. J. Pharm. 185: 1-12), cyclodextrins (Martin, et al., J. Drug Target. 6: 17-36), or other compounds that are known or may be later discovered to enhance nasal abso ⁇ tion.
  • Solutions containing biologically active agents for nasal delivery may be supplied in spray containers for aerosol inhalation.
  • the pharmaceutically effective amount of a biologically active agent of the present invention required as a dose will depend on the route of administration and the physical characteristics of the specific animal under consideration. The dose can be tailored to achieve a desired effect, but will depend on such factors as weight, diet, concurrent medication and other factors which those skilled in the medical arts will recognize.
  • the pharmaceutical compositions can be used alone or in combination with one another, or in combination with other therapeutic or diagnostic agents. The skilled practitioner is able to determine the appropriate dose based on these factors and the effective dose derived from animal and clinical studies. The determination of effective dosage levels, that is the dose levels necessary to achieve the desired result, can be accomplished by one skilled in the art using routine methods.
  • the pharmaceutical compositions containing at least one biologically active agent of the present invention can be administered to the patient in a variety of ways, including, for example, parenterally, intravenously, subcutaneously, intramuscularly, colonically, rectally, nasally or intraperiotoneally, employing a variety of dosage forms.
  • Biologically active agents may be introduced into the cerebrospinal fluid by injection into the spinal column. For delivery into the brain, injection may be into the brain via cannula.
  • the progress of treatment for diabetes can be measured using methods known in the art. For example, blood glucose, urine glucose or blood or serum insulin levels can be monitored using established methods. These measurements can be taken at appropriate intervals, including before, during and after feeding or fasting. In this instance, the caloric intake and type of caloric intake, such as carbohydrates, should be noted.
  • INS-1 rat insulinoma cells were provided by Prof. Claes Wollheim, University Medical Centre, Geneva, Switzerland, and cultured at 37°C in a humidified 5% CO 2 environment in RPMI cell culture media (Gibco BRL, Gaithersburg, MD) supplemented with 10% fetal bovine serum (Irvine Scientific), 2 mM L-glutamine, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin, 10 mM HEPES, 1 mM sodium pyruvate and 50 ⁇ M ⁇ -mercaptoethanol.
  • RPMI cell culture media Gibco BRL, Gaithersburg, MD
  • Irvine Scientific 10% fetal bovine serum
  • 2 mM L-glutamine 100 U/ml penicillin
  • 100 ⁇ g/ml streptomycin 100 ⁇ g/ml streptomycin
  • 10 mM HEPES 1 mM sodium pyruvate
  • 50 ⁇ M ⁇ -mercaptoethanol 50
  • INS-1 cells were cultured for 3-60 days under conditions as described above except media were additionally supplemented with 50 ⁇ g/ml uridine and nucleoside analogs 2'3'-dideoxycytidine [ddC], 2'3'-dideoxyinosine [ddl] or 2'3'-didehydro-3-deoxythymidine [d4T] (all from Sigma) at varying concentrations (1-500 ⁇ M) diluted from 100X stock in PBS or a comparable dilution of PBS without. Media were replenished every two days. Cells were harvested at periodic intervals and assayed for insulin secretion and mtDNA content.
  • ddC 2'3'-dideoxycytidine
  • ddl 2'3'-dideoxyinosine
  • d4T 2'3'-didehydro-3-deoxythymidine
  • Total DNA was prepared from rat liver (for probing rat-derived cells) or the murine cell line 3T3 LI (for probing mouse-derived cells; see Green et al., Cell 3:127-133, 1974 and Cell 5:19-27, 1975) using DNAzolTM reagents (Molecular Research Center, Inc., Cincinnati, OH) and method essentially according to the manufacturer's instructions.
  • the template DNAs were examined by agarose gel electrophoresis and ethidium bromide staining and found to be roughly equivalent.
  • PCR polymerase chain reaction
  • oligonucleotide primers specific for the mitochondrially encoded cytochrome c oxidase subunit I (COX-I) gene, were used for reactions for either rat or mouse templates.
  • the pair of primers consisted of forward and reverse oligonucleotides having the following sequences:
  • the PCR reactions contained appropriate amounts of template DNA, primers, MgCl 2 , all four dNTPs, reaction buffer, and Taq polymerase, brought up to a volume of 50 ul using sterile water.
  • the reactions were incubated at 95°C for 10 seconds, followed by 30 cycles of 95°C for 1 minute, 60°C for 1 minute and 72°C for 1 minute, after which the reactions were incubated at 72°C for 4 minutes and then cooled to 4°C.
  • PCR reactions mixes were extracted with phenolxhloroform and, along with a series of molecular weight markers, electrophoresed on an agarose gel that was stained with ethidium bromide and visualized with ultraviolet light. For both reactions, a single band of the predicted size (i.e., about 1.2 kilobases) was observed.
  • the rat probe was radiolabeled with P using a Prime-a-Gene® random priming kit (Promega, Madison, WI) essentially according to the manufacturer's instructions.
  • INS-1 cells or p° INS- 1 cells generated using ddC as described above, were seeded into 12-well plates containing RPMI media supplemented as described above at 0.4 x 10 6 cells/well and cultured at 37°C, 5% CO 2 for 2 days. Cells (0.7 x 10 6 cells/well) were rinsed with PBS and total cellular DNA was extracted using DNAzol (Molecular Research Center, Inc., Cincinnati, Ohio) according to the manufacturer's instructions.
  • DNAzol Molecular Research Center, Inc., Cincinnati, Ohio
  • the membranes were rinsed in hybridization buffer (5X SSC, 0.1% N- laurylsarcosine, 0.02% SDS, 1% blocking solution, Boehringer Mannheim, Indianapolis, Indiana) and hybridized overnight in the same buffer at 42°C with the [ 32 P]-labeled rat COX I probe. Following hybridization, membranes were washed twice with 2X SSC/0.1% SDS and twice with 0.1X SSC/0.1% SDS and exposed to X-ray film. Mitochondrial DNA was quantified by densitometric scanning of the resulting autoradiographs. Incubation of INS-1 cells with ddC, ddl or d4T for seven days decreased mtDNA content in a dose-dependent fashion.
  • hybridization buffer 5X SSC, 0.1% N- laurylsarcosine, 0.02% SDS, 1% blocking solution, Boehringer Mannheim, Indianapolis, Indiana
  • the IC 50 for ddC was approximately 50 ⁇ M.
  • the decline in mtDNA content was time-dependent, with a t 1/2 of approximately three days; mtDNA was undetectable in these cells after 21 days.
  • INS-1 Cells Depleted of Mitochondrial DNA INS-1 cells, or p° INS-1 cells generated using ddC as described above, were seeded into 12-well plates containing RPMI media supplemented as described at 0.5 x 10 6 cells/well and cultured at 37°C, 5% CO 2 for 2 days.
  • ICN Biochemicals, Irvine, CA insulin-specific radioimmunoassay kit
  • INS-1 Cells Depleted of Mitochondrial DNA The ability of mitochondrially proficient and INS-1 cells that have been treated with ddC, and thus depleted of mtDNA, to respond to glucose in other ways was examined. Intracellular ATP levels were determined using an ATP bioluminescent assay kit (Sigma) for both types of cells in response to various doses of glucose. The results ( Figure 2 A) show that untreated INS-1 cells produce increasing amounts of ATP in response to increasing amounts of glucose. In contrast, INS-1 cells that have been substantially depleted of mtDNA, although able to maintain a basal level of ATP, do not show any substantial response to stimulation by glucose.
  • Lactate production was also determined for both types of cells in response to various doses of glucose.
  • Cells were grown in 35 mm dishes with various concentrations of glucose. Media were replenished about 16 hr before assay with normal culture media containing various amounts of glucose. The media were then collected, and lactate measured using a commercially available kit, in which lactate dehydrogenase is used to produce a fluorescent compound (Sigma, St. Louis, MO), essentially according to the manufacturer's instructions.
  • EXAMPLE 2 CONSTRUCTION OF IFl FUSION PROTEIN EXPRESSION CONSTRUCTS Two IFl -derived fusion proteins were constructed (see Example 3 for details) having structures that may be diagrammed as follows:
  • TAT.IF 1.Ma ( His tag H TAT H IF 1 )
  • TAT.IF1.FL ( His tag H TAT H mtOTS )-(IFl), wherein:
  • His tag denotes 6 histidine amino acid residues in contiguous order (SEQ ID NO:l);
  • TAT denotes a cellular targeting sequence (CTS) derived from HIV-1 (SEQ ID NO: 10);
  • mtOTS denotes a mitochondrial targeting sequence (SEQ ID NO: 14) derived from Rattus norvegicus IFl;
  • IFl denotes the IFl polypeptide derived from Rattus norvegicus (SEQ ID NO:13).
  • Rat Heart cDNA Library A cDNA library derived from total cellular RNA rat heart was prepared according to methods known in the art. In brief, rat hearts were dissected away from associated tissues and gently minced in a buffer containing 40 mM Trsi-HCl, pH 7.0, with surgical instruments that had been treated to remove any RNase or contaminating RNAs.
  • Rat IFl cDNAs were amplified from the rat heart cDNA library by polymerase chain reactions (PCR) in a thermal cycler using the following primers, AMPLITAQTM DNA Polymerase (Perkin-Elmer), and reagents and buffers supplied in a GENEAMPTM PCR Reagent Kit (Perkin-Elmer), according to the manufacturer's instructions.
  • PCR polymerase chain reactions
  • underlined nucleotides indicate sequences complementary to the 5 '-ends and 3 '-ends of the rat IFl cDNAs
  • double-underlined nucleotides indicate recognition sequences for the restriction enzymes S cl (recognition sequence: 5'-GAGCTC) and Hindlll (recognition sequence: 5'-AAGCTT)
  • S cl recognition sequence: 5'-GAGCTC
  • Hindlll recognition sequence: 5'-AAGCTT
  • TGA reverse complement of the stop codon
  • the sequence between the Sacl restriction enzyme site and the start codon for rat IFl encodes a Tat-derived cellular targeting sequence, underlined in the following representation thereof:
  • Rat IFl cDNAs were amplified from the rat heart cDNA library by PCR as in the preceding section, with the exception that the following primers were used.
  • underlined nucleotides indicate sequences complementary to the 5'-ends and 3'-ends of the rat IFl cDNAs
  • double- underlined nucleotides indicate recognition sequences for the restriction enzymes S ⁇ cl (recognition sequence: 5'-GAGCTC) and Hindlll (recognition sequence: 5'-AAGCTT), and the reverse complement of the rat IFl stop codon (TGA, having the reverse complement TCA) is emboldened.
  • the sequence between the Sad restriction enzyme site and the start codon for rat IFl encodes a Tat-derived cellular targeting sequence.
  • the PCR products were digested with Sad and Hindlll (Roche) and purified by horizontal agarose gel electrophoresis and band extraction using the UltraCleanTM GelSpin kit (Mo Bio Laboratories).
  • This vector contains the following elements operably linked in a 5' to 3' orientation: the inducible, but tightly regulatable, araBAD promoter; optimized E. coli translation initiation signals; an amino terminal polyhistidine (6xHis)-encoding sequence (also referred to as a "His-Tag”); an XPRESSTM epitope-encoding sequence; an enterokinase cleavage site which can be used to remove the preceding N-terminal amino acids following protein purification, if so desired; a multiple cloning site; and an in-frame termination codon.
  • 6xHis amino terminal polyhistidine
  • His-Tag also referred to as a "His-Tag”
  • XPRESSTM epitope-encoding sequence an enterokinase cleavage site which can be used to remove the preceding N-terminal amino acids following protein purification, if so desired
  • a multiple cloning site a multiple cloning site
  • an in-frame termination codon
  • Plasmid pBAD/His DNA was prepared by digestion with the restriction endonucleases Sad and Hindlll essentially according to the manufacturer's instructions and subjected to horizontal agarose gel electrophoresis and band extraction using the UltraCleanTM GelSpin kit (Mo Bio Laboratories). Restricted and purified IF.FL or IF. Ma DNAs were ligated with restricted expression vector DNA using T4 DNA ligase (New England Biolabs, Beverly, MA) using the manufacturer's reaction buffer and following the manufacturer's instructions. Competent recAl hsdR. endAlE.
  • coli cells (strain TOPI OF'; Invitrogen) were transformed with ligation mixtures containing the prokaryotic vector construct according to the manufacturer's instructions. Single colonies were selected and grown in 3-5 ml of LB broth (Sambrook, J., Fritsch, E.F., and Maniatis, T., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) containing 50 ⁇ g/ml ampicillin (Roche Molecular Biochemicals). Plasmid DNA was isolated from the bacterial cultures using the WIZARDTM Plus Series 9600 Miniprep Reagents System (Promega, Madison, WI).
  • TAT.IFl .ma and TAT.IFl.ma fusion proteins were examined as follows. Following overnight culture and dilution into fresh media, E. coli cells harboring pBAD/His.TAT.IFl .FL or pBAD/His.TAT.IFl .Ma were induced by treatment with L-arabinose at a concentration of 0.02% for about 4 hours. Cells were harvested, lysed and sonicated to prepare protein extracts. The protein content of the extracts was determined and equivalent amounts of protein were subject to Western analysis.
  • the His-tagged proteins were purified from induced cells using the ProBondTM Nickel-chelating resin (Invitrogen) essentially according to the manufacturer's instructions.
  • Nickel-affinity column purified IFl fusion proteins are shown in Figure 7, which depicts a Coomassie blue stained electrophoretogram of the expressed products of the indicated constructs, and which also shows western blot analysis of IFl fusion proteins detected using an antibody specific for the XpressTM epitope tag as described above.
  • Example were labeled by attaching a fluorescent moiety, Oregon GreenTM, using the Oregon GreenTM FluoReporter Protein Labeling Kit (Molecular Probes, Eugene, OR).
  • INS-1 cells were cultured as in Example 4, and purified TAT.IFl .Ma and TAT.IF1.FL polypeptides were added to separate sets of cells to a final concentration of 100 ug/ml. The cells were visually examined by fluorescent microscopy. Control
  • INS-1 cells to which unlabeled TAT.IF1.FL polypeptide was added, exhibited a slight diffuse fluroescence, as the cells naturally fluoresce to some degree, producing some background signal.
  • cells to which the labeled TAT.IFl.Ma polypeptide was added exhibited a diffuse pattern of fluorescence, which was not more intense than the background signal.
  • cells to which the labeled TAT.IF1.FL polypeptide was added demonstrated a punctate pattern of fluorescence, indicating organellar delivery thereof.
  • Synthetic polypeptides corresponding to portions of rat IFl were prepared using Fmoc chemistry according to methods known in the art. After synthesis was completed, protecting groups were removed and the polypeptide chains were cleaved from the resin in order to achieve their release therefrom.
  • IF1 22 . 46
  • IF1 consists of amino acids 22- 46 of the mature form of rat IFl (i.e., the protein remaining after cleavage and removal of the mitochondrial targeting sequence) and has the sequence: FGKREKAEEDRYFREKTREQLAALK (SEQ ID NO: 25 )
  • IF1 (42 . 58) consists of amino acids 42-58 of the mature form of rat IFl and has the sequence:
  • LAALKKHHEDEIDHHSK (SEQ ID NO: 26 )
  • F0-F1 ATPase and FI ATPase The complete mitochondrial ATP synthase complex is thought to comprise a membrane-bound portion (F0) and a "lollipop-shaped" portion (FI) that projects into the matrix.
  • FI -ATPase is an active, water-soluble subcomplex of ATPase that represents the portion of the mitochondrial ATPase that faces into the mitochondrial matrix.
  • FI ATPase can be isolated from F0, and retains some enzymatic activities as an isolated subcomplex. Isolated FI ATPase can be reassociated with the F0 subcomplex to reform the original membrane-bound structure (Kagawa and Racker, J. Biol. Chem. 247:2467, 1966).
  • F0-F1 ATPase complexes and FI ATPase subcomplexes were isolated from bovine cardiac samples essentially according to the method of Walker et al. (Methods in Enzymology 260:163-190, 1995), with the exception that, instead of preparing SMPs (submitochondrial particles) at neutral pH, ASMPs (alkaline submitochondrial particles; see Rouslin and Broge, Anal Biochem. 222:68-75, 1994) were prepared from bovine heart by sonication in a basic solution. The ASMPs were extracted with chloroform and used to prepare F0-F1 ATPase and FI ATPase according to the method of Walker et al. (Methods in Enzymology 260: 163 - 190, 1995).
  • IFl was isolated from bovine cardiac samples essentially according to the method of Rouslin and Broge (Anal. Biochem. 222:68-75, 1994) with the following exceptions. Following their preparation, SMPs heated in a solution having a pH of about 5.0. The filtrate was poured over a Dowex 50 column which was washed with ammonium sulfate, H20 and 7 M urea. Proteins were eluted by applying a solution of 7 M urea, 100 mM Tris-SO4, pH 7.3, to the column. Eluted fractions were pooled and IFl proteins were concentrated using a Centricon® concentrator (Millipore, Bedford, MA).
  • ASMPs alkaline submitochondrial particles
  • One assay for ATPase activity involves measuring ATP hydrolysis according to methods known in the art (Walker et al., Methods in Enzymology 260: 163-
  • Such assays can be performed using purified F0-F1 ATPase complexes or FI ATPase subcomplexes, or using alkaline submitochondrial particles (ASMPs).
  • ASMPs alkaline submitochondrial particles
  • Aurovertin-B was used as a positive control. This antibiotic binds to and inhibits the activity of bacterial and mitochondrial ATPases (van Raaij et al., Proc. Natl Acad. Sci. U.S.A. 93:14 6913-14 6917, 1996).
  • the purified Fl-ATPase described in the preceding Example was treated with varying concentrations of Aurovertin-B, and ATP hydrolyis was measured.
  • IC50 0.85 uM).
  • the partially purified IF 1 of the preceding Example was tested for its ability to inhibit the ATP hydrolytic activity of the purified Fl- ATPase.
  • pooled fractions 12-16 of the IFl preparation (filled circles) inhibited ATP hydrolysis by purified Fl-ATPase in a manner that was dependent upon the amount (volume) of the pooled fractions.
  • pooled fractions 8-1 1 (open circles in Figure 4) had essentially no effect on the Fl-ATPase, indicating that these fractions contained insubstantial amounts of IFl.
  • this result suggests that there may be two modes of action of IF 1.
  • the results even raise the possibility that there may be two separable ATPase binding sites in IF 1 , a first site that is contained in amino acids 22-46 of the mature IFl protein which is blocked by the F0 subunit, and a second site that is present in amino acids 42-58 of the mature IFl protein and which is not impacted by the presence of F0 in ATPase complexes.
  • the IF l ( 2-58) polypeptide was also tested for its ability to inhibit the F0- Fl -ATPase in rat ASMPs (alkaline submitochondrial particles) prepared as in the preceding Example.
  • the results ( Figure 6) show that the IF1 (42 _ 58) polypeptide inhibits the FO-Fl-ATPase in rat ASMPs in a dose-dependent fashion.
  • the IC50 of the IF1 (42 . 58) polypeptide in this experiment (about 2.5 uM) was somewhat different than that seen with the purified bovine FO-Fl-ATPase (0.18 uM, Figure 5), this may reflect a species difference between the bovine and rat FO-Fl-ATPases.
  • Figure 8 shows an inhibition curve that was generated when the indicated concentration of recombinant tat.lFI .fl (described above) was incubated with rat liver submitochondrial particles and ATP hydrolase activity was measured. ATP hydrolase activity was expressed as the percent of detectable activity in the absence of added IF 1 -containing fusion protein, and the level of oligomycin-inhibitable activity was also determined (Fig. 8).
  • the tat.lFI .fl fusion protein also enhanced GSIS in INS-1 cells using the assay conditions described above in Example 1 ( Figure 9). Briefly, INS-1 cells were incubated in the presence of varying amounts of tat.IFl .fi or vehicle control in RPMI at 37°C for 1 hr. Glucose-free RPMI was then added for 1 hr with the continued presence of the fusion protein (or vehicle control), media were collected and insulin concentrations were determined by ELISA. Fig. 9 shows insulin secretion expressed as the percentage of insulin secretion determined in the absence of glucose and IFl fusion protein.

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Abstract

La présente invention concerne des compositions et des méthodes pouvant modifier la production d'ATP mitochondrial, y compris des immunodétections et des dosages fonctionnels exploitant les interactions d'IF1 avec l'ATP-synthase. L'invention concerne aussi des méthodes utilisées dans des dosages de criblage pour un composé pouvant réduire l'hydrolyse de l'ATP mitochondrial et/ou augmenter la synthèse de l'ATP mitochondrial, ainsi que des une compositions pharmaceutiques identifiées par ces méthodes. L'invention concerne en outre des méthodes de traitement du diabète, notamment le diabète non insulino-dépendant, au moyen d'un agent identifié selon les méthodes de l'invention.
PCT/US2000/030862 1999-11-10 2000-11-10 Compositions et methodes pour reguler l'inhibiteur endogene de l'atp-synthase, et pour le traitement du diabete WO2001034833A2 (fr)

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CA002390646A CA2390646A1 (fr) 1999-11-10 2000-11-10 Compositions et methodes pour reguler l'inhibiteur endogene de l'atp-synthase, et pour le traitement du diabete
EP00980320A EP1230379A2 (fr) 1999-11-10 2000-11-10 Compositions et methodes pour reguler l'inhibiteur endogene de l'atp-synthase, et pour le traitement du diabete
JP2001536758A JP2003527835A (ja) 1999-11-10 2000-11-10 Ａｔｐシンターゼにおける調節内因性インヒビター
AU17599/01A AU1759901A (en) 1999-11-10 2000-11-10 Compositions and methods for regulating endogenous inhibitor of atp synthase, including treatment for diabetes

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US16462299P 1999-11-10 1999-11-10
US60/164,622 1999-11-10

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002024204A2 (fr) * 2000-09-20 2002-03-28 Mitokor Inhibition de l'antiport calcium/sodium mitochondrial
WO2002068680A2 (fr) * 2001-02-27 2002-09-06 Mitokor Compositions et procedes de regulation d'inhibiteur endogene d'atp synthase, y compris un traitement du diabete
WO2003020963A2 (fr) * 2001-09-05 2003-03-13 Pride Proteomics A/S Proteines impliquees dans le diabete de type 2
WO2022157548A1 (fr) * 2021-01-24 2022-07-28 Forrest Michael David Inhibiteurs d'utilisations cosmétiques et thérapeutiques d'atp synthase
US12059450B2 (en) 2018-10-02 2024-08-13 Korea University Research And Business Foundation Anticancer pharmaceutical composition containing IF1 as active ingredient

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR102239074B1 (ko) * 2018-09-14 2021-04-14 고려대학교 산학협력단 IF1 (ATPase inhibitory factor 1)을 유효성분으로 함유하는 비만의 예방 또는 치료용 약학 조성물
KR102239075B1 (ko) * 2018-10-02 2021-04-14 고려대학교 산학협력단 IF1 (ATPase inhibitory factor 1)을 유효성분으로 함유하는 근감소증의 예방 또는 치료용 약학 조성물
KR102276379B1 (ko) * 2018-10-02 2021-07-14 고려대학교 산학협력단 IF1 (ATPase inhibitory factor 1)을 유효성분으로 함유하는 골질환의 예방 또는 치료용 약학 조성물

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998033909A1 (fr) * 1997-01-31 1998-08-06 Incyte Pharmaceuticals, Inc. Nouvelle proteine humaine inhibitrice de l'atpase
US5804604A (en) * 1989-12-21 1998-09-08 Biogen, Inc. Tat-derived transport polypeptides and fusion proteins
WO2000028041A1 (fr) * 1998-11-05 2000-05-18 Microbiological Research Authority Apport de superoxyde-dismutase aux cellules neuronales
US6140067A (en) * 1999-04-30 2000-10-31 Mitokor Indicators of altered mitochondrial function in predictive methods for determining risk of type 2 diabetes mellitus

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5804604A (en) * 1989-12-21 1998-09-08 Biogen, Inc. Tat-derived transport polypeptides and fusion proteins
WO1998033909A1 (fr) * 1997-01-31 1998-08-06 Incyte Pharmaceuticals, Inc. Nouvelle proteine humaine inhibitrice de l'atpase
WO2000028041A1 (fr) * 1998-11-05 2000-05-18 Microbiological Research Authority Apport de superoxyde-dismutase aux cellules neuronales
US6140067A (en) * 1999-04-30 2000-10-31 Mitokor Indicators of altered mitochondrial function in predictive methods for determining risk of type 2 diabetes mellitus

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ROUSLIN WILLIAM ET AL: "Analysis of factors affecting functional assays for estimating IF-1, the mitochondrial ATPase inhibitor." ANALYTICAL BIOCHEMISTRY, vol. 222, no. 1, 1994, pages 68-75, XP002170981 ISSN: 0003-2697 cited in the application *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002024204A2 (fr) * 2000-09-20 2002-03-28 Mitokor Inhibition de l'antiport calcium/sodium mitochondrial
WO2002024204A3 (fr) * 2000-09-20 2002-08-22 Mitokor Inhibition de l'antiport calcium/sodium mitochondrial
WO2002068680A2 (fr) * 2001-02-27 2002-09-06 Mitokor Compositions et procedes de regulation d'inhibiteur endogene d'atp synthase, y compris un traitement du diabete
WO2002068680A3 (fr) * 2001-02-27 2003-10-16 Mitokor Compositions et procedes de regulation d'inhibiteur endogene d'atp synthase, y compris un traitement du diabete
WO2003020963A2 (fr) * 2001-09-05 2003-03-13 Pride Proteomics A/S Proteines impliquees dans le diabete de type 2
WO2003020963A3 (fr) * 2001-09-05 2004-03-25 Pride Proteomics As Proteines impliquees dans le diabete de type 2
US7470542B2 (en) 2001-09-05 2008-12-30 Pride Proteomics A/S Proteins in type 2 diabetes
US12059450B2 (en) 2018-10-02 2024-08-13 Korea University Research And Business Foundation Anticancer pharmaceutical composition containing IF1 as active ingredient
WO2022157548A1 (fr) * 2021-01-24 2022-07-28 Forrest Michael David Inhibiteurs d'utilisations cosmétiques et thérapeutiques d'atp synthase

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EP1230379A2 (fr) 2002-08-14
AU1759901A (en) 2001-06-06
JP2003527835A (ja) 2003-09-24
CA2390646A1 (fr) 2001-05-17
WO2001034833A3 (fr) 2002-01-17

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