WO1998021363A1 - Compositions et therapies utilisables contre le diabete de type ii lie au hnf-4 - Google Patents

Compositions et therapies utilisables contre le diabete de type ii lie au hnf-4 Download PDF

Info

Publication number
WO1998021363A1
WO1998021363A1 PCT/US1997/020759 US9720759W WO9821363A1 WO 1998021363 A1 WO1998021363 A1 WO 1998021363A1 US 9720759 W US9720759 W US 9720759W WO 9821363 A1 WO9821363 A1 WO 9821363A1
Authority
WO
WIPO (PCT)
Prior art keywords
hnfl
gene
hnf
protein
sequence
Prior art date
Application number
PCT/US1997/020759
Other languages
English (en)
Inventor
Alexandra M. Glucksman
Jeffrey Thomas
Original Assignee
Millennium Pharmaceuticals, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Millennium Pharmaceuticals, Inc. filed Critical Millennium Pharmaceuticals, Inc.
Priority to AU52571/98A priority Critical patent/AU5257198A/en
Publication of WO1998021363A1 publication Critical patent/WO1998021363A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • 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
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/027—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a retrovirus

Definitions

  • Type I diabetes insulin dependent diabetes mellitus or IDDM
  • IDDM insulin dependent diabetes mellitus
  • Type II diabetes non-insulin dependent diabetes mellitus or NIDDM
  • NIDDM non-insulin dependent diabetes mellitus
  • loci have been linked to rare early-onset forms of Type II diabetes that is associated with chronic hyperglycemia and monogenic inheritance (i.e. maturity onset diabetes of the young (MODY) loci)
  • MODY loci maturity onset diabetes of the young loci
  • glucokinase (GCK) gene on human chromosome 7 The defects in the glucokinase (GCK) gene on human chromosome 7 have been found to be responsible for the relatively rare MODY2 phenotype. (Froguel, P. et al. (1992) Nature, 356:162-164).
  • the invention features compounds that are agonists of a normal (functional) HNF bioactivity and their use in treating diabetes.
  • a gene therapeutic comprising a gene encoding a functional HNF protein
  • a protein therapeutic comprising a functional HNF protein
  • the invention features compounds that are antagonists of a disease causing HNF bioactivity and their use in treating diabetes.
  • a therapeutically effective amount of an antisense, ribozyme or triple helix molecule to reduce or prevent gene expression, as described herein may be administered to the subject.
  • a therapeutically effective amount of an antagonist compound e.g.
  • a therapeutically effective amount of an anti-HNF antibody may be administered to the subject.
  • the invention provides assays, e.g., for screening test compounds to identify antagonists (e.g. inhibitors), or alternatively, agonists (e.g. potentiators), of an interaction between an HNF protein and, for example, a protein or nucleic acid that binds to the HNF protein.
  • antagonists e.g. inhibitors
  • agonists e.g. potentiators
  • An exemplary method includes the steps of (i) combining an HNF polypeptide or bioactive fragments thereof, an HNF target molecule (such as an HNF ligand or nucleic acid), and a test compound, e.g., under conditions wherein, but for the test compound, the HNF protein and HNF target molecule are able to interact; and (ii) detecting the formation of a complex which includes the HNF protein and the target molecule either by directly quantitating the complex or by measuring inductive effects of the HNF protein.
  • an HNF target molecule such as an HNF ligand or nucleic acid
  • a statistically significant change, such as a decrease, in the interaction of the HNF and HNF target molecule in the presence of a test compound (relative to what is detected in the absence of the test compound) is indicative of a modulation (e.g., inhibition or potentiation of the interaction between the HNF protein and the target molecule).
  • a modulation e.g., inhibition or potentiation of the interaction between the HNF protein and the target molecule.
  • Yet another aspect of the present invention concerns methods for modulating the transcription of certain genes in a cell by modulating HNF bioactivity, (e.g., by potentiating or disrupting an HNF bioactivity).
  • the method comprises treating the cell with an effective amount of an HNF therapeutic (agonist or antagonist of an HNF bioactivity) so as to alter, relative to the cell in the absence of treatment, the level of transcription of certain genes.
  • an HNF therapeutic agonist or antagonist of an HNF bioactivity
  • the method can be carried out with HNF therapeutics such as peptide and peptidomimetics or other molecules identified in the above-referenced drug screens which agonize or antagonize the effects of an HNF bioactivity (e.g. transcription) of a gene which is regulated by an HNF protein.
  • HNF therapeutics include antisense constructs for inhibiting expression of
  • HNF proteins and dominant negative mutants of HNF proteins which competitively inhibit interactions between ligands (e.g. proteins) and nucleic acids upstream and downstream of the wild-type HNF protein.
  • a further aspect of the present invention provides methods for determining whether a subject is at risk for developing type II diabetes.
  • the method can include detecting, in a tissue of the subject, the presence or absence of a genetic lesion characterized by at least one of (i) a mutation of a gene encoding an HNF protein; (ii) the mis-expression of an HNF gene; or (iii) an error or mutation in the promoter regulating an HNF gene that may lead to aberrant expression.
  • detecting the genetic lesion includes ascertaining the existence of at least one of: a deletion of one or more nucleotides from an HNF gene; an addition of one or more nucleotides to an HNF gene, a substitution of one or more nucleotides of an HNF gene, a gross chromosomal rearrangement of an HNF gene; an alteration in the level of a messenger RNA transcript of an HNF gene; the presence of a non- wild type splicing pattern of a messenger RNA transcript of an HNF gene; a non-wild type level of an HNF protein; and/or an aberrant level of an HNF protein.
  • detecting the genetic lesion can include (i) providing a probe/primer comprised of an oligonucleotide which hybridizes to a sense or antisense sequence of an HNF gene or naturally occurring mutants thereof, or 5' or 3' flanking sequences naturally associated with the HNF gene; (ii) contacting the probe/primer with an appropriate nucleic acid containing sample; and (iii) detecting, by hybridization of the probe/primer to the nucleic acid, the presence or absence of the genetic lesion; e.g. wherein detecting the lesion comprises utilizing the probe/primer to determine the nucleotide sequence of the HNF gene and, optionally, of the flanking nucleic acid sequences.
  • the primer can be employed in a polymerase chain reaction (PCR) or in a ligase chain reaction (LCR).
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • the level of an HNF protein is detected in an immunoassay using an antibody which is specifically immunoreactive with the HNF protein.
  • Figure 1 is a graph showing the results of multipoint nonparametric linkage analysis of NIDDM with markers in the MODYl region of chromosome 20q. On the x-axis, the genetic distance between markers is presented in cM.
  • the present invention is based, at least in part, on the finding that the MODY 1 locus can predispose a subject to development of NIDDM.
  • studies that failed to find linkage of NIDDM with the MODYl region (Baroni, MG et al, (1992) Diabetes 41: 1640-1643; Dow, E et al., (1994) Diabet. Med. 11: 856-861 ; Chuang, LM et al., (1995) Diabetolgia 35:1490-1491), as reported in the following Example 5.6
  • the instant study involved extended pedigrees in which NIDDM was transmitted in a manner consistent with an autosomal dominant disorder.
  • the present invention is also based, at least in part, on the interaction between human hepatic nuclear factor 1 (HNF1), which is encoded by a gene that maps to a critical region of chromosome 12 linked to type II diabetes and hepatic nuclear factor 4 (HNF4), which is encoded by a gene that maps within the MODYl interval.
  • HNF4 regulates transcription of HNF1; and HNF1 regulates transcription of HNF4 (Zhong, W.. et al., (1994) Mol. & Cell. Bio. 7 :7276-7284).
  • HNF1 human hepatic nuclear factor 1
  • HNF4 hepatic nuclear factor 4 alone or in conjunction with hepatic nuclear factor 1 are involved in biochemical pathways defects, which can result in diabetes.
  • the present invention relates to HNF agonists and antagonists and their use in treating diabetes.
  • nucleic acid molecules encoding functional HNF proteins;
  • anti-HNF antibodies and preparations of such compositions are disclosed herein.
  • the invention provides drug discovery assays for identifying additional agents that agonize or antagonize the biological function of an HNF bioactivity (e.g. transcription) of a gene which is regulated by an HNF protein.
  • HNF therapeutics include antisense constructs for inhibiting expression of HNF proteins and dominant negative mutants of HNF protein, which competitively inhibit interactions between HNF and proteins of nucleic acids upstream or downstream of a wildtype HNF.
  • present invention provides assays for diagnosing whether a subject has or has a predisposition towards developing type II diabetes (including MODY3).
  • Cells “host cells” or “recombinant host cells” are terms used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
  • a “chimeric protein” or “fusion protein” is a fusion of a first amino acid sequence encoding one of the subject HNF polypeptides with a second amino acid sequence defining a domain (e.g. polypeptide portion) foreign to and not substantially homologous with any domain of one of the HNF proteins.
  • a chimeric protein may present a foreign domain which is found (albeit in a different protein) in an organism which also expresses the first protein, or it may be an "interspecies", “intergenic”, etc. fusion of protein structures expressed by different kinds of organisms.
  • a fusion protein in general, can be represented by the general formula X-HNF-Y, wherein HNF represents a portion of the protein which is derived from one of the HNF proteins, and X and Y are independently absent or represent amino acid sequences which are not related to one of the HNF sequences in an organism, including naturally occurring mutants.
  • Complementary sequences as used herein refer to sequences which have sufficient complementarity to be able to hybridize, forming a stable duplex.
  • a “delivery complex” shall mean a targeting means (e.g. a molecule that results in higher affinity binding of a gene, protein, polypeptide or peptide to a target cell surface and/or increased cellular uptake by a target cell).
  • targeting means include: sterols (e.g. cholesterol), lipids (e.g. a cationic lipid, virosome or liposome), viruses (e.g. adenovirus, adeno-associated virus, and retrovirus) or target cell specific binding agents (e.g. ligands recognized by target cell specific receptors).
  • Preferred complexes are sufficiently stable in vivo to prevent significant uncoupling prior to internahzation by the target cell. However, the complex is cleavable under appropriate conditions within the cell so that the gene, protein, polypeptide or peptide is released in a functional form.
  • genes for a particular polypeptide may exist in single or multiple copies within the genome of an individual. Such duplicate genes may be identical or may have certain modifications, including nucleotide substitutions, additions or deletions, which all still code for polypeptides having substantially the same activity.
  • the term "DNA sequence encoding an HNF polypeptide" may thus refer to one or more genes within a particular individual.
  • certain differences in nucleotide sequences may exist between individual organisms, which are called alleles. Such allelic differences may or may not result in differences in amino acid sequence of the encoded polypeptide yet still encode a protein with the same biological activity.
  • Diabetes is a term used to refer to disorders, which relate to alterations in glucose homeostasis. In the mildest forms of diabetes, this alteration is detected only after challenge with a carbohydrate load, while in moderate to severe forms of disease, hyperglycemia is always present.
  • Type I diabetes insulin dependent diabetes mellitus or IDDM
  • IDDM insulin dependent diabetes mellitus
  • IDDM insulin dependent diabetes mellitus
  • NIDDM non-insulin dependent diabetes mellitus
  • NIDDM non-insulin dependent diabetes mellitus
  • Type II diabetes that develops during the age of 20-30 years old and is associated with chronic hyperglycemia and monogenic inheritance is referred to as maturity onset diabetes of the young (MODY).
  • MODY maturity onset diabetes of the young
  • Other forms of Type II diabetes develop in an individual sometime after 20-30 years of age (e.g. late-onset NIDDM).
  • hepatic nuclear factor (HNF) gene or “recombinant HNF gene” refers to a nucleic acid molecule comprising an open reading frame encoding one of the HNF polypeptides of the present invention, including both exon and (optionally) intron sequences.
  • a “recombinant gene” refers to nucleic acid encoding an HNF polypeptide and comprising an HNF-encoding exon sequences, though it may optionally include intron sequences which are either derived from a chromosomal HNF gene or from an unrelated chromosomal gene.
  • intron refers to a DNA sequence present in a given HNF gene which is not translated into protein and is generally found between exons.
  • HNF polypeptide refers to a hepatic nuclear factor and functional fragments thereof.
  • Hepatic Nuclear Factor 1 (HNF-1, also named HP-1, LFB1 or APF) is a transcription factor that binds to the promoters or enhancers of a variety of genes, particularly genes expressed exclusively in the liver, although HNF- 1 has also been found to be expressed in kidney, intestine, stomach, pancreas, spleen and testis.
  • Variant HNF1 vHNFl, also known as HNFl ⁇ or LFB3
  • HNF-1 is a nuclear factor that binds the same sequence elements in numerous liver specific genes as is bound by HNF-1 (Ringeisen, F. et al., (1993) J. Bio.
  • vHNFl-A GenBank Accession No. S 15342
  • vHNFl-B vHNFl-A
  • vHNFl-C vHNFl-C
  • HNF-1 is intended to encompass HNF-l-A, -B, and -C, as well as vHNFl-A, -B, and -C.
  • HNF3 is another liver enriched transcription factor that is apparently important in liver - specific gene expression.
  • HNF4 is yet another liver enriched transcription factor.
  • cDNA encoding rat HNF4 is described in International Patent Application WO 92/11365.
  • Human hepatocyte nuclear factor 4 isoforms e.g. HNF4 ⁇ -2, a splice variant of HNF4 ⁇ -4; and HNF4 ⁇
  • Homology refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An "unrelated" or “non-homologous" sequence shares less than 40 % identity, though preferably less than 25 % identity, with one of the HNF sequences of the present invention.
  • interact as used herein is meant to include detectable interactions (e.g. biochemical interactions) between molecules, such as can be detected using, for example, a yeast two hybrid assay.
  • interact is also meant to include "binding" interactions between molecules. Interactions may, for example, be protein-protein or protein- nucleic acid in nature.
  • isolated as used herein with respect to nucleic acids, such as DNA or RNA, refers to molecules separated from other DNAs, or RNAs, respectively, that are present in the natural source of the macromolecule.
  • an isolated nucleic acid encoding one of the subject HNF polypeptides preferably includes no more than 10 kilobases (kb) of nucleic acid sequence which naturally immediately flanks the HNF gene in genomic DNA, more preferably no more than 5kb of such naturally occurring flanking sequences, and most preferably less than 1.5kb of such naturally occurring flanking sequence.
  • isolated as used herein also refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.
  • isolated nucleic acid is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state.
  • isolated is also used herein to refer to polypeptides which are isolated from other cellular proteins and is meant to encompass both purified and recombinant polypeptides.
  • modulation refers to both upregulation (i.e., activation or stimulation) and downregulation (i.e. inhibition or suppression) of a response.
  • non-human animals include mammalians such as rodents, non-human primates, sheep, dog, cow, chickens, amphibians, reptiles, etc.
  • Preferred non-human animals are selected from the rodent family including rat and mouse, most preferably mouse, though transgenic amphibians, such as members of the Xenopus genus, and transgenic chickens can also provide important tools for understanding and identifying agents which can affect, for example, embryogenesis and tissue formation.
  • transgenic amphibians such as members of the Xenopus genus
  • transgenic chickens can also provide important tools for understanding and identifying agents which can affect, for example, embryogenesis and tissue formation.
  • chimeric animal is used herein to refer to animals in which the recombinant gene is found, or in which the recombinant gene is expressed in some but not all cells of the animal.
  • tissue- specific chimeric animal indicates that one of the recombinant HNF genes is present and/or expressed or disrupted in some tissues but not others.
  • nucleic acid refers to polynucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA).
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • the term should also be understood to include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs, and, as applicable to the embodiment being described, single (sense or antisense) and double-stranded polynucleotides.
  • promoter means a DNA sequence that regulates expression of a selected DNA sequence operably linked to the promoter, and which effects expression of the selected DNA sequence in cells.
  • tissue specific i.e. promoters, which effect expression of the selected DNA sequence only in specific cells (e.g. cells of a specific tissue).
  • leaky so-called “leaky” promoters, which regulate expression of a selected DNA primarily in one tissue, but cause expression in other tissues as well.
  • the term also encompasses non-tissue specific promoters and promoters that constitutively express or that are inducible (i.e. expression levels can be controlled).
  • protein protein
  • polypeptide peptide
  • recombinant protein refers to a polypeptide of the present invention which is produced by recombinant DNA techniques, wherein generally, DNA encoding an HNF polypeptide is inserted into a suitable expression vector which is in turn used to transform a host cell to produce the heterologous protein.
  • the phrase "derived from”, with respect to a recombinant HNF gene is meant to include within the meaning of "recombinant protein” those proteins having an amino acid sequence of a native HNF protein, or an amino acid sequence similar thereto which is generated by mutations including substitutions and deletions (including truncation) of a naturally occurring form of the protein.
  • the term “specifically hybridizes” or “specifically detects” refers to the ability of a nucleic acid molecule of the invention to hybridize to at least approximately 6, 12, 20, 30, 50, 100, 150, 200, 300, 350, 400 or 425 consecutive nucleotides of a vertebrate, preferably HNF gene.
  • Transcriptional regulatory sequence is a generic term used throughout the specification to refer to DNA sequences, such as initiation signals, enhancers, and promoters, which induce or control transcription of protein coding sequences with which they are operably linked.
  • transcription of one of the recombinant mammalian HNF gene is under the control of a promoter sequence (or other transcriptional regulatory sequence) which controls the expression of the recombinant gene in a cell-type in which expression is intended. It will also be understood that the recombinant gene can be under the control of transcriptional regulatory sequences which are the same or which are different from those sequences which control transcription of the naturally-occurring forms of HNF protein.
  • the term “transfection” means the introduction of a nucleic acid, e.g., an expression vector, into a recipient cell by nucleic acid-mediated gene transfer.
  • "Transformation" refers to a process in which a cell's genotype is changed as a result of the cellular uptake of exogenous DNA or RNA, and, for example, the transformed cell expresses a recombinant form of an HNF polypeptide or, in the case of anti-sense expression from the transferred gene, the expression of a naturally-occurring form of the HNF protein is disrupted.
  • transgene means a nucleic acid sequence (encoding, e.g., one of the HNF polypeptides, or pending an antisense transcript thereto), which is partly or entirely heterologous, i.e., foreign, to the transgenic animal or cell into which it is introduced, or, is homologous to an endogenous gene of the transgenic animal or cell into which it is introduced, but which is designed to be inserted, or is inserted, into the animal's genome in such a way as to alter the genome of the cell into which it is inserted (e.g., it is inserted at a location which differs from that of the natural gene or its insertion results in a knockout).
  • a transgene can include one or more transcriptional regulatory sequences and any other nucleic acid, such as introns, that may be necessary for optimal expression of a selected nucleic acid.
  • a "transgenic animal” refers to any animal, preferably a non-human mammal, bird or an amphibian, in which one or more of the cells of the animal contain heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques well known in the art.
  • the nucleic acid is introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus.
  • transgenic animal also includes those recombinant animals in which gene disruption of one or more HNF gene is caused by human intervention, including both recombination and antisense techniques.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • One type of preferred vector is an episome, i.e., a nucleic acid capable of extra-chromosomal replication.
  • Preferred vectors are those capable of autonomous replication and/or expression of nucleic acids to which they are linked.
  • Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as "expression vectors”.
  • expression vectors of utility in recombinant DNA techniques are often in the form of "plasmids" which refer generally to circular double stranded DNA loops which, in their vector form are not bound to the chromosome.
  • plasmid and "vector” are used interchangeably as the plasmid is the most commonly used form of vector.
  • vector is intended to include such other forms of expression vectors which serve equivalent functions and which become known in the art subsequently hereto.
  • One aspect of the invention pertains to the administration of isolated nucleic acids encoding HNF1 and/or 4 polypeptides, and/or equivalents of such nucleic acids to treat a diabetic subject.
  • the term equivalent is understood to include nucleotide sequences that are not identical to the hHNFl and/or 4 nucleic acid sequence described in Bach, I. et al., (1990)
  • Genomics 5:155-164 but that encode polypeptides having an activity of the human HNFl and/or 4 as described herein.
  • Equivalent nucleotide sequences will include sequences that differ by one or more nucleotide substitutions, additions or deletions, such as allelic variants of the reported sequence.
  • Preferred nucleic acids are obtained from vertebrates. Particularly preferred vertebrate HNFl and/or 4 nucleic acids are mammalian. Regardless of species, particularly preferred HNFl and/or 4 nucleic acids encode polypeptides that are at least about 90% similar to an amino acid sequence of a vertebrate HNFl and/or 4. Preferred nucleic acids encode an HNFl and/or 4 polypeptide comprising an amino acid sequence, which is at least about 90% homologous and more preferably 94% homologous with an amino acid sequence of hHNFl and/or 4.
  • Appropriate stringency conditions for identifying hHNFl and/or 4 homologs for example, 6.0 x sodium chloride/sodium citrate (SSC) at about 45°C, followed by a wash of 2.0 x SSC at 50°C, are known to those skilled in the art or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
  • the salt concentration in the wash step can be selected from a low stringency of about 2.0 x SSC at 50°C to a high stringency of about 0.2 x SSC at 50°C.
  • the temperature in the wash step can be increased from low stringency conditions at room temperature, about 22°C, to high stringency conditions at about 65°C.
  • Both temperature and salt may be varied, or either the temperature or the salt concentration may be held constant while the other variable is changed.
  • Nucleic acids having a sequence that differs from the reported hHNFl and/or 4 nucleotide sequence due to degeneracy in the genetic code are also within the scope of the invention. Such nucleic acids encode functionally equivalent peptides (i.e., a peptide having a biological activity of human HNFl and/or 4 polypeptide) but differ in sequence from the sequence shown in the sequence listing due to degeneracy in the genetic code. For example, a number of amino acids are designated by more than one triplet. Codons that specify the same amino acid, or synonyms (for example, CAU and CAC each encode histidine) may result in "silent" mutations which do not affect the amino acid sequence of an HNFl and/or 4 polypeptide.
  • Codons that specify the same amino acid, or synonyms for example, CAU and CAC each encode histidine
  • DNA sequence polymorphisms that do lead to changes in the amino acid sequences of the subject HNFl and/or 4 polypeptides will exist among mammalians.
  • these variations in one or more nucleotides (e.g., up to about 3-5% of the nucleotides) of the nucleic acids encoding polypeptides having an activity of an HNFl and/or 4 polypeptide may exist among individuals of a given species due to natural allelic variation.
  • HNFl and/or 4 protein-encoding nucleic acids can be obtained from mRNA present in any of a number of eukaryotic cells. It should also be possible to obtain nucleic acids encoding HNFl and/or 4 polypeptides of the present invention from genomic DNA from both adults and embryos. For example, a gene encoding an HNFl and/or 4 protein can be cloned from either a cDNA or a genomic library in accordance with protocols described herein, as well as those generally known to persons skilled in the art. Examples of tissues and/or libraries suitable for isolation of the subject nucleic acids include liver, pancreatic islet cells and lymphoblasts, among others.
  • a cDNA encoding an HNFl and/or 4 protein can be obtained by isolating total mRNA from a cell, e.g. a vertebrate cell, a mammalian cell, or a human cell, including embryonic cells. Double stranded cDNAs can then be prepared from the total mRNA, and subsequently inserted into a suitable plasmid or bacteriophage vector using any one of a number of known techniques.
  • the gene encoding an HNFl and/or 4 protein can also be cloned using established polymerase chain reaction techniques in accordance with the nucleotide sequence information provided by the invention.
  • the nucleic acid of the invention can be DNA or RNA.
  • the HNFl and/or 4 encoding gene is administered to a subject in an expression vector, i.e. a nucleic acid encoding an HNFl and/or 4 polypeptide. operably linked to at least one transcriptional regulatory sequence.
  • an expression vector i.e. a nucleic acid encoding an HNFl and/or 4 polypeptide.
  • operably linked is intended to mean that the nucleotide sequence is linked to a regulatory sequence in a manner which allows expression of the nucleotide sequence.
  • Regulatory sequences are art- recognized and are selected to direct expression of the subject HNFl and/or 4 proteins. Accordingly, the term “transcriptional regulatory sequence” includes promoters, enhancers and other expression control elements. Such regulatory sequences are described in Goeddel;
  • the expression vector includes a recombinant gene encoding a peptide having an agonistic activity of a subject HNFl and/or 4 polypeptide, or alternatively, encoding a peptide which is an antagonistic form of the HNFl and/or 4 protein.
  • Such expression vectors can be used to transfect cells and thereby produce polypeptides, including fusion proteins, encoded by nucleic acids as described herein.
  • the gene constructs of the present invention can also be used as a part of a gene therapy protocol to deliver nucleic acids encoding either an agonistic or antagonistic form of one of the subject HNFl and/or 4 proteins.
  • another aspect of the invention features expression vectors for in vivo or in vitro transfection and expression of an HNFl and/or 4 polypeptide in particular cell types so as to reconstitute the function of, or alternatively, abrogate the function of HNFl and/or 4-induced signaling in a tissue.
  • This could be desirable, for example, when the naturally-occurring form of the protein is misexpressed; or to deliver a form of the protein which alters differentiation of tissue.
  • Expression vectors may also be employed to inhibit neoplastic transformation.
  • non-viral methods can also be employed to cause expression of a subject HNFl and/or 4 polypeptide in the tissue of an animal.
  • Most nonviral methods of gene transfer rely on normal mechanisms used by mammalian cells for the uptake and intracellular transport of macromolecules.
  • non-viral targeting means of the present invention rely on endocytic pathways for the uptake of the subject HNFl and/or 4 polypeptide gene by the targeted cell.
  • Exemplary targeting means of this type include liposomal derived systems, poly-lysine conjugates, and artificial viral envelopes.
  • the present invention also makes available HNFl and/or 4 polypeptides which are isolated from, or otherwise substantially free of other cellular proteins, especially other signal transduction factors and/or transcription factors which may normally be associated with the HNFl and/or 4 polypeptide.
  • substantially free of other cellular proteins especially other signal transduction factors and/or transcription factors which may normally be associated with the HNFl and/or 4 polypeptide.
  • contaminating proteins also referred to herein as "contaminating proteins”
  • substantially pure or purified preparations are defined as encompassing preparations of HNFl and/or 4 polypeptides having less than about 20% (by dry weight) contaminating protein, and preferably having less than about 5% contaminating protein.
  • Functional forms of the subject polypeptides can be prepared, for the first time, as purified preparations by using a cloned gene as described herein.
  • purified it is meant, when referring to a peptide or DNA or RNA sequence, that the indicated molecule is present in the substantial absence of other biological macromolecules, such as other proteins.
  • purified as used herein preferably means at least 80% by dry weight, more preferably in the range of 95-99% by weight, and most preferably at least 99.8% by weight, of biological macromolecules of the same type present (but water, buffers, and other small molecules, especially molecules having a molecular weight of less than 5000, can be present).
  • pure as used herein preferably has the same numerical limits as “purified” immediately above. "Isolated” and “purified” do not encompass either natural materials in their native state or natural materials that have been separated into components (e.g., in an acrylamide gel) but not obtained either as pure (e.g.
  • purified HNFl and/or 4 preparations will lack any contaminating proteins from the same animal from which HNFl and/or 4 is normally produced, as can be accomplished by recombinant expression of, for example, a human HNFl and/or 4 protein in a non-human cell.
  • Full length proteins or fragments corresponding to one or more particular motifs and/or domains or to arbitrary sizes, for example, at least 5, 10, 25, 50, 75, 100, 125, 150 amino acids in length are within the scope of the present invention.
  • isolated HNFl and/or 4 polypeptides can include all or a portion of an amino acid sequence corresponding to an hHNFl and/or 4 polypeptide described in Bach, I. et al., (1990) Genomics 5:155-164.
  • Isolated peptidyl portions of HNFl and/or 4 proteins can be obtained by screening peptides recombinantly produced from the corresponding fragment of the nucleic acid encoding such peptides.
  • fragments can be chemically synthesized using techniques known in the art such as conventional Merrifield solid phase f-Moc or t-Boc chemistry.
  • an HNFl and/or 4 polypeptide of the present invention may be arbitrarily divided into fragments of desired length with no overlap of the fragments, or preferably divided into overlapping fragments of a desired length.
  • the fragments can be produced (recombinantly or by chemical synthesis) and tested to identify those peptidyl fragments which can function as either agonists or antagonists of a wild-type (e.g., "authentic") HNFl and/or 4 protein.
  • HNFl and/or 4 proteins are at least 92% homologous and more preferably 94% homologous and most preferably 95% homologous and preferably at least 98-99% homologous with the amino acid sequence described in Bach, I. et al., (1990) Genomics 5:155-164.
  • the HNFl and/or 4 protein is functional.
  • the function of HNF 1 and/or 4 is related to insulin regulation, glucose tolerance, or other biochemical activities, defects in which, result in symptoms of diabetes.
  • a polypeptide has biological activity if it is a specific agonist or antagonist of a naturally-occurring form of an HNFl and/or 4 protein.
  • the present invention further pertains to methods of producing the subject HNFl and/or 4 polypeptides.
  • a host cell transfected with a nucleic acid vector directing expression of a nucleotide sequence encoding the subject polypeptides can be cultured under appropriate conditions to allow expression of the peptide to occur.
  • the cells may be harvested, lysed and the protein isolated.
  • a cell culture includes host cells, media and other byproducts. Suitable media for cell culture are well known in the art.
  • the recombinant HNFl and/or 4 polypeptide can be isolated from cell culture medium, host cells, or both using techniques known in the art for purifying proteins including ion-exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, and immunoaffmity purification with antibodies specific for such peptide.
  • the recombinant HNFl and/or 4 polypeptide is a fusion protein containing a domain which facilitates its purification, such as GST fusion protein or poly(His) fusion protein.
  • Homologs of each of the subject HNFl and/or 4 proteins can be generated by mutagenesis, such as by discrete point mutation(s), or by truncation. For instance, mutation can give rise to homologs which retain substantially the same, or merely a subset, of the biological activity of the HNFl and/or 4 polypeptide from which it was derived.
  • antagonistic forms of the protein can be generated which are able to inhibit the function of the naturally occurring form of the protein, such as by competitively binding to a downstream or upstream member of the biochemical pathway, which includes the HNFl and/or 4 protein.
  • agonistic forms of the protein may be generated which are constitutively active.
  • the HNFl and/or 4 protein and homologs thereof provided by the subject invention may be either positive or negative regulators of gene expression.
  • the recombinant HNFl and/or 4 polypeptides of the present invention also include homologs of the authentic (wildtype) HNFl and/or 4 proteins, such as versions of those protein which are resistant to proteolytic cleavage, as for example, due to mutations which alter ubiquitination or other enzymatic targeting associated with the protein.
  • HNFl and/or 4 polypeptides may also be chemically modified to create HNFl and/or 4 derivatives by forming covalent or aggregate conjugates with other chemical moieties, such as glycosyl groups, lipids, phosphate, acetyl groups and the like.
  • Covalent derivatives of HNFl and/or 4 proteins can be prepared by linking the chemical moieties to functional groups on amino acid sidechains of the protein or at the N-terminus or at the C- terminus of the polypeptide.
  • Modification of the structure of the subject HNFl and/or 4 polypeptides can be for such purposes as enhancing therapeutic or prophylactic efficacy, stability (e.g., ex vivo shelf life and resistance to proteolytic degradation in vivo), or post-translational modifications (e.g., to alter phosphorylation pattern of protein).
  • Such modified peptides when designed to retain at least one activity of the naturally-occurring form of the protein, or to produce specific antagonists thereof, are considered functional equivalents of the HNFl and/or 4 polypeptides described in more detail herein.
  • Such modified peptides can be produced, for instance, by amino acid substitution, deletion, or addition.
  • Whether a change in the amino acid sequence of a peptide results in a functional HNFl and/or 4 homolog can be readily determined by assessing the ability of the variant peptide to produce a response in cells in a fashion similar to the wild-type protein, or competitively inhibit such a response. Polypeptides in which more than one replacement has taken place can readily be tested in the same manner.
  • This invention further contemplates a method for generating sets of combinatorial mutants of the subject HNFl and/or 4 proteins as well as truncation mutants, and is especially useful for identifying potential variant sequences (e.g. homologs) that are functional in modulating gene expression.
  • the purpose of screening such combinatorial libraries is to generate, for example, novel HNFl and/or 4 homologs which can act as either agonists or antagonist, or alternatively, possess novel activities all together.
  • HNFl and/or 4 homologs can be generated by the present combinatorial approach to selectively inhibit gene expression. For instance, mutagenesis can provide HNFl and/or 4 homologs which are able to bind other signal pathway proteins (or DNA) yet prevent propagation of the signal, e.g. the homologs can be dominant negative mutants. Moreover, manipulation of certain domains of HNFl and/or 4 by the present method can provide domains more suitable for use in fusion proteins.
  • the variegated library of HNFl and/or 4 variants is generated by combinatorial mutagenesis at the nucleic acid level, and is encoded by a variegated gene library.
  • a mixture of synthetic oligonucleotides can be enzymatically ligated into gene sequences such that the degenerate set of potential HNFl and/or 4 sequences are expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g. for phage display) containing the set of HNFl and/or 4 sequences therein.
  • a library of coding sequence fragments can be provided for an HNFl and/or 4 clone in order to generate a variegated population of HNFl and/or 4 fragments for screening and subsequent selection of bioactive fragments.
  • a variety of techniques are known in the art for generating such libraries, including chemical synthesis.
  • a library of coding sequence fragments can be generated by (i) treating a double stranded PCR fragment of an HNFl and/or 4 coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule; (ii) denaturing the double stranded DNA; (iii) renaturing the DNA to form double stranded DNA which can include sense/anti sense pairs from different nicked products; (iv) removing single stranded portions from reformed duplexes by treatment with SI nuclease; and (v) ligating the resulting fragment library into an expression vector.
  • an expression library can be derived which codes for N-terminal, C-terminal and internal fragments of various sizes.
  • a wide range of techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a certain property. Such techniques will be generally adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of HNFl and/or 4 homologs.
  • the most widely used techniques for screening large gene libraries typically comprises cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates relatively easy isolation of the vector encoding the gene whose product was detected.
  • Each of the illustrative assays described below are amenable to high through-put analysis as necessary to screen large numbers of degenerate HNFl and/or 4 sequences created by combinatorial mutagenesis techniques.
  • Combinatorial mutagenesis has a potential to generate very large libraries of mutant proteins, e.g., in the order of 10 26 molecules. Combinatorial libraries of this size may be technically challenging to screen even with high throughput screening assays.
  • recrusive ensemble mutagenesis REM
  • REM recrusive ensemble mutagenesis
  • REM is an algorithm which enhances the frequency of functional mutants in a library when an appropriate selection or screening method is employed (Arkin and Yourvan, 1992, PNAS USA 89:7811-7815; Yourvan et al., 1992, Parallel Problem Solving from Nature, 2., In Maenner and Manderick, eds., Elsevir Publishing Co., Amsterdam, pp. 401-410; Delgrave et al., 1993, Protein Engineering 6(3):327-331).
  • the invention also provides for reduction of the vertebrate HNFl and/or 4 proteins to generate mimetics, e.g. peptide or non-peptide agents, which are able to disrupt binding of a vertebrate HNFl and/or 4 polypeptide of the present invention with either upstream or downstream components.
  • mimetics e.g. peptide or non-peptide agents
  • Such mutagenic techniques as described above are also useful to map the determinants of the HNFl and/or 4 proteins which participate in protein-protein interactions involved in, for example, binding of the subject HNFl and/or 4 polypeptide to proteins which may function upstream (including both activators and repressors of its activity) or to proteins or nucleic acids which may function downstream of the HNFl and/or 4 polypeptide, whether they are positively or negatively regulated by it.
  • the critical residues of a subject HNFl and/or 4 polypeptide which are involved in molecular recognition of a component upstream or downstream of an HNFl and/or 4 protein can be determined and used to generate HNFl and/or 4-derived peptidomimetics which competitively inhibit binding of the authentic HNFl and/or 4 protein with that moiety.
  • peptidomimetic compounds can be generated which mimic those residues of the HNFl and/or 4 protein which facilitate the interaction.
  • mimetics may then be used to interfere with the normal function of an HNFl and/or 4 protein.
  • non-hydrolyzable peptide analogs of such residues can be generated using benzodiazepine (e.g., see Freidinger et al. in
  • This invention also pertains to a host cell transfected to express a recombinant form of the subject HNFl and/or 4 polypeptides.
  • the host cell may be any prokaryotic or eukaryotic cell.
  • a nucleotide sequence derived from the cloning of vertebrate HNFl and/or 4 proteins, encoding all or a selected portion of the full-length protein can be used to produce a recombinant form of an HNFl and/or 4 polypeptide via microbial or eukaryotic cellular processes.
  • a gene construct such as an expression vector
  • transforming or transfecting into hosts either eukaryotic (yeast, avian, insect or mammalian) or prokaryotic (bacterial cells) are standard procedures used in producing other well-known proteins, e.g. MAP kinase, p53, WT1, PTP phosphotases, SRC, and the like. Similar procedures, or modifications thereof, can be employed to prepare recombinant HNFl and/or 4 polypeptides by microbial means or tissue-culture technology in accord with the subject invention.
  • the recombinant HNFl and/or 4 genes can be produced by ligating nucleic acid encoding an HNFl and/or 4 protein, or a portion thereof, into a vector suitable for expression in either prokaryotic cells, eukaryotic cells, or both.
  • Expression vectors for production of recombinant forms of the subject HNFl and/or 4 polypeptides include plasmids and other vectors.
  • suitable vectors for the expression of an HNFl and/or 4 polypeptide include plasmids of the types: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids and pUC-derived plasmids for expression in prokaryotic cells, such as E. coli.
  • YEP24, YIP5, YEP51, YEP52, pYES2, and YRP17 are cloning and expression vehicles useful in the introduction of genetic constructs into S. cerevisiae (see, for example.
  • a HNFl and/or 4 polypeptide is produced recombinantly utilizing an expression vector generated by sub-cloning the coding sequence of one of the HNFl and/or 4 genes represented in SEQ ID Nos: 1 and 3.
  • the preferred mammalian expression vectors contain both prokaryotic sequences, to facilitate the propagation of the vector in bacteria, and one or more eukaryotic transcription units that are expressed in eukaryotic cells.
  • the pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples of mammalian expression vectors suitable for transfection of eukaryotic cells.
  • vectors are modified with sequences from bacterial plasmids, such as pBR322, to facilitate replication and drug resistance selection in both prokaryotic and eukaryotic cells.
  • derivatives of viruses such as the bovine papillomavirus (BPV-1), or Epstein-Barr virus (pHEBo, pREP-derived and p205) can be used for transient expression of proteins in eukaryotic cells.
  • BBV-1 bovine papillomavirus
  • pHEBo Epstein-Barr virus
  • the various methods employed in the preparation of the plasmids and transformation of host organisms are well known in the art.
  • suitable expression systems for both prokaryotic and eukaryotic cells, as well as general recombinant procedures see Molecular Cloning A Laboratory Manual, 2nd Ed., ed.
  • baculovirus expression systems include pVL-derived vectors (such as pVL1392, pVL1393 and pVL941), pAcUW-derived vectors (such as pAcUWl), and pBlueBac-derived vectors
  • an HNFl and/or 4 protein such as a form lacking a portion of the N-terminus, i.e. a truncation mutant which lacks the signal peptide
  • ATG start codon
  • a methionine at the N-terminal position can be enzymatically cleaved by the use of the enzyme methionine aminopeptidase (MAP).
  • MAP has been cloned from E. coli (Ben-Bassat et al. (1987) J. Bacteriol.
  • transgenic animals described in more detail below could be used to produce recombinant proteins.
  • the coding sequences for the polypeptide can be incorporated as a part of a fusion gene including a nucleotide sequence encoding a different polypeptide.
  • This type of expression system can be useful under conditions where it is desirable to produce an immunogenic fragment of an HNFl and/or 4 protein.
  • the VP6 capsid protein of rotavirus can be used as an immunologic carrier protein for portions of the HNFl and/or 4 polypeptide, either in the monomeric form or in the form of a viral particle.
  • the nucleic acid sequences corresponding to the portion of a subject HNFl and/or 4 protein to which antibodies are to be raised can be incorporated into a fusion gene construct which includes coding sequences for a late vaccinia virus structural protein to produce a set of recombinant viruses expressing fusion proteins comprising HNF 1 and/or 4 epitopes as part of the virion. It has been demonstrated with the use of immunogenic fusion proteins utilizing the Hepatitis B surface antigen fusion proteins that recombinant Hepatitis B virions can be utilized in this role as well.
  • chimeric constructs coding for fusion proteins containing a portion of an HNFl and/or 4 protein and the poliovirus capsid protein can be created to enhance immunogenicity of the set of polypeptide antigens (see, for example, EP Publication No: 0259149; and Evans et al. (1989) Nature 339:385; Huang et al.
  • the Multiple Antigen Peptide system for peptide-based immunization can also be utilized to generate an immunogen, wherein a desired portion of an HNFl and/or 4 polypeptide is obtained directly from organo-chemical synthesis of the peptide onto an oligomeric branching lysine core (see, for example, Posnett et al. (1988) JBC 263:1719 and Nardelli et al. (1992) J. Immunol. 148:914).
  • Antigenic determinants of HNFl and/or 4 proteins can also be expressed and presented by bacterial cells.
  • fusion proteins can also facilitate the expression of proteins, and accordingly, can be used in the expression of the HNFl and/or 4 polypeptides of the present invention.
  • HNFl and/or 4 polypeptides can be generated as glutathione-S-transferase (GST-fusion) proteins.
  • GST-fusion proteins can enable easy purification of the HNFl and/or 4 polypeptide, as for example by the use of glutathione-derivatized matrices (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al. (N.Y.: John Wiley & Sons, 1991)).
  • a fusion gene coding for a purification leader sequence such as a poly-(His)/enterokinase cleavage site sequence at the N-terminus of the desired portion of the recombinant protein, can allow purification of the expressed fusion protein by affinity chromatography using a Ni2+ metal resin.
  • the purification leader sequence can then be subsequently removed by treatment with enterokinase to provide the purified protein (e.g., see Hochuli et al. ( 1987) J. Chromatography 411 :177; and Janknecht et al. PNAS 88:8972). Techniques for making fusion genes are known to those skilled in the art.
  • the joining of various DNA fragments coding for different polypeptide sequences is performed in accordance with conventional techniques, employing blunt-ended or stagger- ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.
  • the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
  • PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).
  • Antisense, Ribozyme and Triplex Therapeutics Another aspect of the invention relates to nucleic acids that are effective antisense, ribozyme and triplex antagonists of mutant or otherwise defective (e.g overexpressed) HNFl and/or 4 nucleic acids.
  • antisense refers to administration or in situ generation of oligonucleotide molecules or their derivatives which specifically hybridize (e.g. bind) under cellular conditions, with the cellular mRNA and/or genomic DNA encoding one or more of the subject HNFl and/or 4 proteins so as to inhibit expression of that protein, e.g. by inhibiting transcription and/or translation.
  • binding may be by conventional base pair complementarity, or, for example, in the case of binding to DNA duplexes, through specific interactions in the major groove of the double helix.
  • antisense therapy refers to the range of techniques generally employed in the art, and includes any therapy which relies on specific binding to oligonucleotide sequences.
  • an antisense construct of the present invention can be delivered, for example, as an expression plasmid which, when transcribed in the cell, produces RNA which is complementary to at least a unique portion of the cellular mRNA which encodes a functional HNFl and/or 4 protein.
  • the antisense construct is an oligonucleotide probe which is generated ex vivo and which, when introduced into the cell causes inhibition of expression by hybridizing with the mRNA and/or genomic sequences of an HNF 1 and/or 4 gene.
  • oligonucleotide probes are preferably modified oligonucleotides which are resistant to endogenous nucleases, e.g.
  • nucleic acid molecules for use as antisense oligonucleotides are phosphoramidate, phosphothioate and methylphosphonate analogs of DNA (see also U.S. Patents 5,176,996; 5,264,564; and 5,256,775). Additionally, general approaches to constructing oligomers useful in antisense therapy have been reviewed, for example, by Van der Krol et al. (1988) Biotechniques 6:958-976; and Stein et al. (1988) Cancer Res 48:2659-
  • oligodeoxyribonucleotides derived from the translation initiation site e.g ⁇ , between the -10 and -MO regions of the HNFl and/or 4 nucleotide sequence of interest, are preferred.
  • Antisense approaches involve the design of oligonucleotides (either DNA or RNA) that are complementary to HNFl and/or 4 mRNA.
  • the antisense oligonucleotides will bind to the HNFl and/or 4 mRNA transcript (e.g. a mutant transcript) and prevent translation.
  • Absolute complementarity although preferred, is not required, a sequence "complementary" to a portion of an RNA, as referred to herein, means a sequence having sufficient complementarity to be able to hybridize with the RNA, forming a stable duplex: in the case of double-stranded antisense nucleic acids, a single strand of the duplex DNA may thus be tested, or triplex formation may be assayed.
  • the ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid. Generally, the longer the hybridizing nucleic acid, the more base mismatches with an RNA it may contain and still form a stable duplex (or triplex, as the case may be). One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex.
  • Oligonucleotides that are complementary to the 5' end of the message should work most efficiently at inhibiting translation.
  • sequences complementary to the 3' untranslated sequences of mRNAs have recently been shown to be effective at inhibiting translation of mRNAs as well. (Wagner, R. 1994. Nature 372:333). Therefore, oligonucleotides complementary to either the 5' or 3' untranslated, non-coding regions of an HNFl and/or 4 gene could be used in an antisense approach to inhibit translation of endogenous HNFl and/or 4 mRNA.
  • Oligonucleotides complementary to the 5' untranslated region of the mRNA should include the complement of the AUG start codon.
  • Antisense oligonucleotides complementary to mRNA coding regions are less efficient inhibitors of translation but could be used in accordance with the invention. Whether designed to hybridize to the 5'. 3' or coding region of an HNFl and/or 4 mRNA, antisense nucleic acids should be at least six nucleotides in length, and are preferably oligonucleotides ranging from
  • the oligonucleotide is at least 10 nucleotides, at least 17 nucleotides, at least 25 nucleotides, or at least 50 nucleotides.
  • in vitro studies are first performed to quantitate the ability of the antisense oligonucleotide to quantitate the ability of the antisense oligonucleotide to inhibit gene expression. It is preferred that these studies utilize controls that distinguish between antisense gene inhibition and nonspecific biological effects of oligonucleotides. It is also preferred that these studies compare levels of the target RNA or protein with that of an internal control RNA or protein. Additionally, it is envisioned that results obtained using the antisense oligonucleotide are compared with those obtained using a control oligonucleotide.
  • control oligonucleotide is of approximately the same length as the test oligonucleotide and that the nucleotide sequence of the oligonucleotide differs from the antisense sequence no more than is necessary to prevent specific hybridization to the target sequence.
  • the oligonucleotides can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded.
  • the oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, hybridization, etc.
  • the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci. U.S.A.
  • the oligonucleotide may be conjugated to another molecule, e.g.. a peptide, hybridization triggered cross-linking agent, transport agent, hybridization- triggered cleavage agent, etc.
  • the antisense oligonucleotide may comprise at least one modified base moiety which is selected from the group including but not limited to 5-fluorouracil, 5-bromouracil, 5- chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6- isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine
  • the antisense oligonucleotide may also comprise at least one modified sugar moiety selected from the group including but not limited to arabinose, 2-fluoroarabinose, xylulose, and hexose.
  • the antisense oligonucleotide comprises at least one modified phosphate backbone selected from the group consisting of a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal or analog thereof.
  • the antisense oligonucleotide is an ⁇ -anomeric oligonucleotide.
  • An ⁇ -anomeric oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual ⁇ -units, the strands run parallel to each other (Gautier et al, 1987, Nucl. Acids Res. 15:6625-6641).
  • the oligonucleotide is a 2'-0- methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res. 15:6131-6148), or a chimeric
  • RNA-DNA analogue (Inoue et al., 1987, FEBS Lett. 215:327-330).
  • Oligonucleotides of the invention may be synthesized by standard methods known in the art, e.g. by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.).
  • an automated DNA synthesizer such as are commercially available from Biosearch, Applied Biosystems, etc.
  • phosphorothioate oligonucleotides may be synthesized by the method of Stein et al. (1988, Nucl. Acids Res. 16:3209)
  • methylphosphonate olgonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:7448-7451). etc.
  • antisense nucleotides complementary to the HNFl and/or 4 coding region sequence could be used, those complementary to the transcribed untranslated region are most preferred.
  • the antisense molecules should be delivered to cells which express the HNFl and/or 4 in vivo.
  • a number of methods have been developed for delivering antisense DNA or RNA to cells; e.g., antisense molecules can be injected directly into the tissue site, or modified antisense molecules, designed to target the desired cells (e.g., antisense linked to peptides or antibodies that specifically bind receptors or antigens expressed on the target cell surface) can be administered systematically.
  • a preferred approach utilizes a recombinant DNA construct in which the antisense oligonucleotide is placed under the control of a strong pol III or pol II promoter.
  • the use of such a construct to transfect target cells in the patient will result in the transcription of sufficient amounts of single stranded RNAs that will form complementary base pairs with the endogenous HNFl and/or 4 transcripts and thereby prevent translation of the HNFl and/or 4 mRNA.
  • a vector can be introduced in vivo such that it is taken up by a cell and directs the transcription of an antisense RNA.
  • Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA.
  • Such vectors can be constructed by recombinant DNA technology methods standard in the art.
  • Vectors can be plasmid, viral, or others known in the art, used for replication and expression in mammalian cells.
  • Expression of the sequence encoding the antisense RNA can be by any promoter known in the art to act in mammalian, preferably human cells. Such promoters can be inducible or constitutive.
  • Such promoters include but are not limited to: the SV40 early promoter region (Bernoist and Chambon, 1981, Nature 290:304-310), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et al.,
  • any type of plasmid, cosmid, YAC or viral vector can be used to prepare the recombinant DNA construct which can be introduced directly into the tissue site; e.g., the choroid plexus or hypothalamus.
  • viral vectors can be used which selectively infect the desired tissue; (e.g., for brain, herpesvirus vectors may be used), in which case administration may be accomplished by another route
  • Ribozyme molecules designed to catalytically cleave HNFl and/or 4 mRNA transcripts can also be used to prevent translation of HNFl and/or 4 mRNA and expression of functional HNFl and/or 4 protein.
  • ribozymes that cleave mRNA at site specific recognition sequences can be used to destroy HNF 1 and/or 4 mRNAs
  • the use of hammerhead ribozymes is preferred.
  • Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA.
  • hammerhead ribozymes are well known in the art and is described more fully in Haseloff and Gerlach, 1988, Nature, 334:585- 591.
  • the ribozyme is engineered so that the cleavage recognition site is located near the 5' end of the HNFl and/or 4 mRNA; i.e., to increase efficiency and minimize the intracellular accumulation of non- functional mRNA transcripts.
  • the ribozymes of the present invention also include RNA endoribonucleases
  • Cech-type ribozymes such as the one which occurs naturally in Tetrahymena thermophila (known as the IVS, or L-19 IVS RNA) and which has been extensively described by Thomas Cech and collaborators (Zaug, et al., 1984, Science, 224:574-578; Zaug and Cech, 1986, Science, 231 :470-475; Zaug, et al., 1986, Nature, 324:429-433; published
  • the Cech-type ribozymes have an eight base pair active site which hybridizes to a target RNA sequence whereafter cleavage of the target RNA takes place.
  • the invention encompasses those Cech-type ribozymes which target eight base-pair active site sequences that are present in HNFl and/or 4.
  • the ribozymes can be composed of modified oligonucleotides (e.g. for improved stability, targeting, etc.) and should be delivered to cells which express the HNFl and/or 4 in vivo e.g., hypothalamus and/or the choroid plexus.
  • a preferred method of delivery involves using a DNA construct "encoding" the ribozyme under the control of a strong constitutive pol III or pol II promoter, so that transfected cells will produce sufficient quantities of the ribozyme to destroy endogenous HNFl and/or 4 messages and inhibit translation. Because ribozymes unlike antisense molecules, are catalytic, a lower intracellular concentration is required for efficiency.
  • Endogenous mutant HNFl and/or 4 gene expression can also be reduced by inactivating or "knocking out" the HNFl and/or 4 gene or its promoter using targeted homologous recombination, (e.g , see Smithies et al., 1985, Nature 317:230-234; Thomas & Capecchi, 1987, Cell 51 :503-512; Thompson et al., 1989 Cell 5:313-321 ; each of which is incorporated by reference herein in its entirety).
  • targeted homologous recombination e.g , see Smithies et al., 1985, Nature 317:230-234; Thomas & Capecchi, 1987, Cell 51 :503-512; Thompson et al., 1989 Cell 5:313-321 ; each of which is incorporated by reference herein in its entirety).
  • a wildtype, functional HNFl and/or 4 flanked by DNA homologous to the endogenous mutant HNF 1 and/or 4 gene (either the coding regions or regulatory regions of the HNFl and/or 4 gene) can be used, with or without a selectable marker and/or a negative selectable marker, to transfect cells that express mutant HNFl and/or 4 in vivo. Insertion of the DNA construct, via targeted homologous recombination, results in inactivation of the mutant HNFl and/or 4 gene.
  • Such approaches are particularly suited for generating transgenic animals, where modifications to ES (embryonic stem) cells can be used to generate animal offspring with an inactive HNFl and/or 4 (e.g., see Thomas & Capecchi 1987 and
  • the recombinant DNA constructs are directly administered or targeted to the required site in vivo using appropriate viral vectors, e.g., herpes virus vectors for delivery to brain tissue; e.g., the hypothalamus and/or choroid plexus.
  • appropriate viral vectors e.g., herpes virus vectors for delivery to brain tissue; e.g., the hypothalamus and/or choroid plexus.
  • endogenous (mutant or wildtype) HNFl and/or 4 gene expression can be reduced by targeting deoxyribonucleotide sequences complementary to the regulatory region of the HNFl and/or 4 gene (i.e., the HNFl and/or 4 promoter and/or enhancers) to form triple helical structures that prevent transcription of the HNFl and/or 4 gene in target cells in the body.
  • deoxyribonucleotide sequences complementary to the regulatory region of the HNFl and/or 4 gene i.e., the HNFl and/or 4 promoter and/or enhancers
  • the antisense constructs of the present invention by antagonizing the normal biological activity of one of the HNFl and/or 4 proteins, can be used in the manipulation of tissue, e.g. tissue differentiation, both in vivo and for ex vivo tissue cultures.
  • anti-sense techniques e.g. microinjection of antisense molecules, or transfection with plasmids whose transcripts are anti-sense with regard to an HNFl and/or 4 mRNA or gene sequence
  • Such techniques can be utilized in cell culture, but can also be used in the creation of transgenic animals, as detailed below.
  • Ribozymes are 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 an endonucleolytic cleavage.
  • the composition of ribozyme molecules must include one or more sequences complementary to the target gene mRNA, and must include the well known catalytic sequence responsible for mRNA cleavage. For this sequence, see U.S. Pat. No. 5,093,246, which is incorporated by reference herein in its entirety.
  • engineered hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of RNA sequences encoding HNFl and/or 4 proteins.
  • ribozyme cleavage sites within any potential RNA target are initially identified by scanning the molecule of interest for ribozyme cleavage sites which include the following sequences, GUA, GUU and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site may be evaluated for predicted structural features, such as secondary structure, that may render the oligonucleotide sequence unsuitable. The suitability of candidate sequences may also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using ribonuclease protection assays
  • Nucleic acid molecules to be used in triple helix formation for the inhibition of transcription are preferably single stranded and composed of deoxyribonucleotides.
  • the base composition of these oligonucleotides should promote triple helix formation via Hoogsteen base pairing rules, which generally require sizable stretches of either purines or pyrimidines to be present on one strand of a duplex.
  • Nucleotide sequences may be pyrimidine-based, which will result in TAT and CGC triplets across the three associated strands of the resulting triple helix.
  • the pyrimidine-rich molecules provide base complementarity to a purine-rich region of a single strand of the duplex in a parallel orientation to that strand.
  • nucleic acid molecules may be chosen that are purine-rich, for example, containing a stretch of G residues. These molecules will form a triple helix with a DNA duplex that is rich in GC pairs, in which the majority of the purine residues are located on a single strand of the targeted duplex, resulting in CGC triplets across the three strands in the triplex.
  • the potential sequences that can be targeted for triple helix formation may be increased by creating a so called "switchback" nucleic acid molecule.
  • Switchback molecules are synthesized in an alternating 5'-3', 3'-5' manner, such that they base pair with first one strand of a duplex and then the other, eliminating the necessity for a sizable stretch of either purines or pyrimidines to be present on one strand of a duplex.
  • Antisense RNA and DNA, ribozyme, and triple helix molecules of the invention may be prepared by any method known in the art for the synthesis of DNA and RNA molecules. These include techniques for chemically synthesizing oligodeoxyribonucleotides and oligoribonucleotides well known in the art such as for example solid phase phosphoramidite chemical synthesis.
  • RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding the antisense RNA molecule.
  • DNA sequences may be incorporated into a wide variety of vectors which incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters.
  • RNA polymerase promoters such as the T7 or SP6 polymerase promoters.
  • antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into cell lines.
  • nucleic acid molecules may be introduced as a means of increasing intracellular stability and half-life. Possible modifications include but are not limited to the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5' and/or 3' ends of the molecule or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the oligodeoxyribonucleotide backbone .
  • Another aspect of the invention pertains to antibodies or antigen binding agents, which are specifically reactive with an HNFl and/or 4 protein.
  • immunogens derived from an HNFl and/or 4 protein e.g. based on the cDNA sequences
  • anti-protein/anti-peptide antisera or monoclonal antibodies can be made by standard protocols (See, for example, Antibodies: A Laboratory Manual ed. by Harlow and Lane (Cold Spring Harbor Press: 1988)).
  • a mammal such as a mouse, a hamster or rabbit can be immunized with an immunogenic form of the peptide (e.g., an HNFl and/or 4 polypeptide or an antigenic fragment which is capable of eliciting an antibody response, or a fusion protein as described above).
  • an immunogenic form of the peptide e.g., an HNFl and/or 4 polypeptide or an antigenic fragment which is capable of eliciting an antibody response, or a fusion protein as described above.
  • Techniques for conferring immunogenicity on a protein or peptide include conjugation to carriers or other techniques well known in the art.
  • An immunogenic portion of an HNFl and/or 4 protein can be administered in the presence of adjuvant. The progress of immunization can be monitored by detection of antibody titers in plasma or serum. Standard
  • the subject antibodies are immunospecif ⁇ c for antigenic determinants of an HNFl and/or 4 protein of a mammal, e.g. antigenic determinants of a protein represented in Bach, I. et al., (1990) Genomics 5: 155-164 or closely related homologs (e.g. at least 92% homologous, and more preferably at least 94% homologous).
  • anti-HNF 1 and/or 4 antisera can be obtained and, if desired, polyclonal anti-HNF 1 and/or 4 antibodies isolated from the serum.
  • antibody-producing cells lymphocytes
  • immortalizing cells such as myeloma cells.
  • Hybridoma cells can be screened immunochemically for production of antibodies specifically reactive with an HNFl and/or 4 polypeptide of the present invention and monoclonal antibodies isolated from a culture comprising such hybridoma cells.
  • antibody as used herein is intended to include fragments thereof which are also specifically reactive with one of the subject HNFl and/or 4 polypeptides.
  • Antibodies can be fragmented using conventional techniques and the fragments screened for utility in the same manner as described above for whole antibodies. For example, F(ab)2 fragments can be generated by treating antibody with pepsin. The resulting F(ab)2 fragment can be treated to reduce disulfide bridges to produce Fab fragments.
  • the antibody of the present invention is further intended to include bispecific and chimeric molecules having affinity for an HNFl and/or 4 protein conferred by at least one CDR region of the antibody.
  • Antibodies which specifically bind HNF 1 and/or 4 epitopes can also be used in immunohistochemical staining of tissue samples in order to evaluate the abundance and pattern of expression of each of the subject HNFl and/or 4 polypeptides.
  • Anti-HNF 1 and/or 4 antibodies can be used diagnostically in immuno-precipitation and immuno-blotting to detect and evaluate HNFl and/or 4 protein levels in tissue as part of a clinical testing procedure. For instance, such measurements can be useful in predictive valuations of the onset or progression of prohferative disorders.
  • the ability to monitor HNFl and/or 4 protein levels in an individual can allow determination of the efficacy of a given treatment regimen for an individual afflicted with such a disorder.
  • the level of HNFl and/or 4 polypeptides may be measured from cells in bodily fluid, such as in samples of cerebral spinal fluid or amniotic fluid, or can be measured in tissue, such as produced by biopsy.
  • Diagnostic assays using anti-HNF 1 and/or 4 antibodies can include, for example, immunoassays designed to aid in early diagnosis of diabetes.
  • anti-HNF 1 and/or 4 antibodies of the present invention is in the immunological screening of cDNA libraries constructed in expression vectors such as ⁇ gtl l, ⁇ gtl8-23, ⁇ ZAP, and ⁇ ORF8.
  • Messenger libraries of this type having coding sequences inserted in the correct reading frame and orientation, can produce fusion proteins.
  • ⁇ gtl l will produce fusion proteins whose amino termini consist of ⁇ - galactosidase amino acid sequences and whose carboxy termini consist of a foreign polypeptide.
  • Antigenic epitopes of an HNFl and/or 4 protein e.g.
  • HNFl and/or 4 protein or other paralogs from the same species can then be detected with antibodies, as, for example, reacting nitrocellulose filters lifted from infected plates with anti-HNF 1 and/or 4 antibodies. Positive phage detected by this assay can then be isolated from the infected plate.
  • HNFl and/or 4 homologs can be detected and cloned from other animals, as can alternate isoforms (including splicing variants) from humans.
  • HNFl and/or 4 therapeutic whether an antagonist or agonist of wild type HNFl and/or 4 activity, can be, as appropriate, any of the preparations described above, including isolated polypeptides, gene therapy constructs, antisense, ribozyme or triplex molecules, peptidomimetics or agents identified in the drug assays provided herein.
  • the invention features compounds that are agonists of a normal (functional) HNFl and/or 4 bioactivity and their use in treating diabetes.
  • a gene therapeutic comprising a gene encoding a functional HNFl and/or 4 protein
  • a protein therapeutic comprising a functional HNFl and/or 4 protein
  • the invention features compounds that are agonists of a wildtype HNFl and/or 4 bioactivity or antagonists of a disease causing HNFl and/or 4 bioactivity and their use in treating diabetes.
  • a gene therapeutic comprising a gene encoding a functional HNFl and/or 4 protein
  • a protein therapeutic comprising a functional HNFl and/or 4 protein
  • a therapeutically effective amount of an antisense, ribozyme or triple helix molecule to reduce or prevent gene expression, as described herein, may be administered to the subject.
  • a therapeutically effective amount of a compound e.g.
  • a therapeutically effective amount of an anti-HNF 1 and/or 4 antibody, as described herein, may be administered to the subject.
  • Compounds that increase or decrease HNFl and/or 4 gene expression or protein activity can be administered to a subject at a therapeutically effective dose to treat or ameliorate diabetes.
  • Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD 50 (the dose lethal to 50% of the population) and the ED 50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LO 50 fED 50 .
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC 50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture.
  • IC 50 i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms
  • levels in plasma may be measured, for example, by high performance liquid chromatography.
  • compositions for use in accordance with the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers or excipients.
  • the compounds and their physiologically acceptable salts and solvates may be formulated for administration by, for example, injection, inhalation or insufflation (either through the mouth or the nose) or oral, buccal, parenteral or rectal administration.
  • the oligomers of the invention can be formulated for a variety of loads of administration, including systemic and topical or localized administration. Techniques and formulations generally may be found in Remmington's Pharmaceutical Sciences, Meade Publishing Co., Easton, PA.
  • systemic administration injection is preferred, including intramuscular, intravenous, intraperitoneal, and subcutaneous.
  • the oligomers of the invention can be formulated in liquid solutions, preferably in physiologically compatible buffers such as Hank's solution or Ringer's solution.
  • the oligomers may be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also included.
  • the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl me hylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g.. magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate).
  • binding agents e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl me hylcellulose
  • fillers e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate
  • lubricants e.g.. magnesium stearate, talc or silica
  • disintegrants e.
  • Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use.
  • Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl- p-hydroxybenzoates or sorbic acid).
  • the preparations may also contain buffer salts. flavoring, coloring and sweetening agents as appropriate.
  • Preparations for oral administration may be suitably formulated to give controlled release of the active compound.
  • compositions may take the form of tablets or lozenges formulated in conventional manner.
  • the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of e.g. gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
  • the compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
  • the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • the compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
  • the compounds may also be formulated as a depot preparation.
  • Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.
  • the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration bile salts and fusidic acid derivatives.
  • detergents may be used to facilitate permeation.
  • Transmucosal administration may be through nasal sprays or using suppositories.
  • the oligomers of the invention are formulated into ointments, salves, gels, or creams as generally known in the art.
  • the gene delivery systems for the therapeutic HNFl and/or 4 gene can be introduced into a patient by any of a number of methods, each of which is familiar in the art.
  • a pharmaceutical preparation of the gene delivery system can be introduced systemically, e.g. by intravenous injection, and specific transduction of the protein in the target cells occurs predominantly from specificity of transfection provided by the gene delivery vehicle, cell-type or tissue-type expression due to the transcriptional regulatory sequences controlling expression of the receptor gene, or a combination thereof.
  • initial delivery of the recombinant gene is more limited with introduction into the animal being quite localized.
  • the gene delivery vehicle can be introduced by catheter (see U.S. Patent 5,328,470) or by stereotactic injection (e.g.
  • HNFl and/or 4 gene, or a sequence homologous thereto can be delivered in a gene therapy construct by electroporation using techniques described, for example, by Dev et al. ((1994) Cancer Treat Rev 20:105-115).
  • Gene therapy vectors comprised of viruses that provide specific effective and highly localized treatment of eye diseases are described in Published International Patent Application No. WO 95/34580 to U. Eriksson et al..
  • the pharmaceutical preparation of the gene therapy construct can consist essentially of the gene delivery system in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded.
  • the pharmaceutical preparation can comprise one or more cells which produce the gene delivery system.
  • compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient.
  • the pack may for example comprise metal or plastic foil, such as a blister pack.
  • the pack or dispenser device may be accompanied by instructions for administration.
  • the invention features probes and primers for use in a prognostic or diagnostic assay.
  • the present invention also provides a probe/primer comprising a substantially purified oligonucleotide, which oligonucleotide comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least approximately 12, preferably 25, more preferably 40, 50 or 75 consecutive nucleotides of sense or anti- sense sequence of hHNFl and/or 4, including 5' and/or 3 1 untranslated regions.
  • the probe further comprises a label group attached thereto and able to be detected, e.g. the label group is selected from amongst radioisotopes, fluorescent compounds, enzymes, and enzyme co-factors.
  • the present invention also provides methods for determining whether a subject is at risk for developing Type II diabetes.
  • the methods can be characterized as comprising detecting, in a sample of cells from the subject, the presence or absence of a genetic lesion characterized by at least one of (i) an alteration affecting the integrity of a gene encoding an HNFl and/or 4 protein, or (ii) the mis-expression of the HNFl and/or 4 gene.
  • such genetic lesions can be detected by ascertaining the existence of at least one of (i) a deletion of one or more nucleotides from an HNFl and/or 4 gene, (ii) an addition of one or more nucleotides to an HNFl and/or 4 gene, (iii) a substitution of one or more nucleotides of an HNFl and/or 4 gene, (iv) a gross chromosomal rearrangement of an HNFl and/or 4 gene, (v) a gross alteration in the level of a messenger RNA transcript of an HNFl and/or 4 gene, (vii) aberrant modification of an HNFl and/or 4 gene, such as of the methylation pattern of the genomic DNA, (vii) the presence of a non-wild type splicing pattern of a messenger RNA transcript of an HNFl and/or 4 gene, (viii) a non- wild type level of an HNFl and/or 4 protein, (ix) allelic loss of an HNFl and
  • a nucleic acid composition comprising a (purified) oligonucleotide probe including a region of nucleotide sequence which is capable of hybridizing to a sense or antisense sequence of an hHNF 1 and/or 4 gene or naturally occurring mutants thereof, or 5' or 3' flanking sequences or intronic sequences naturally associated with the subject hHNFl and/or 4 genes or naturally occurring mutants thereof.
  • the nucleic acid of a cell is rendered accessible for hybridization, the probe is exposed to nucleic acid of the sample, and the hybridization of the probe to the sample nucleic acid is detected.
  • Such techniques can be used to detect lesions at either the genomic or mRNA level, including deletions, substitutions, etc., as well as to determine mRNA transcript levels.
  • one aspect of the present invention relates to diagnostic assays for determining, in the context of cells isolated from a patient, if one or more mutation exists in an HNFl and/or 4 gene of the sample cells.
  • the present method provides a method for determining if a subject is at risk for type II diabetes.
  • the method can be generally characterized as comprising detecting, in a sample of cells from the subject, the presence or absence of a genetic lesion characterized by an alteration affecting the integrity of a gene encoding an HNFl and/or 4 gene.
  • such genetic lesions can be detected by ascertaining the existence of at least one of (i) a deletion of one or more nucleotides from an HNFl and/or 4 gene, (ii) an addition of one or more nucleotides to an
  • the present invention provides a large number of assay techniques for detecting lesions in HNFl and/or 4 genes, and importantly, provides the ability to discern between different molecular causes underlying HNFl and/or 4 dependent aberrant cell growth, proliferation and/or differentiation.
  • detection of the lesion comprises utilizing the probe/primer in a polymerase chain reaction (PCR) (see, e.g. U.S. Patent Nos. 4.683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligase chain reaction (LCR) (see, e.g., Landegran et al. (1988) Science 241 :1077-1080; and Nakazawa et al. (1994) PNAS 91 :360-364), the latter of which can be particularly useful for detecting point mutations in the HNFl and/or 4 gene (see Abravaya et al. (1995) Nuc Acid Res 23:675-682).
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • the method includes the steps of (i) collecting a sample of cells from a patient, (ii) isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, (iii) contacting the nucleic acid sample with one or more primers which specifically hybridize to an HNF 1 and/or 4 gene under conditions such that hybridization and amplification of the HNFl and/or 4 gene (if present) occurs, and (iv) detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. It is anticipated that PCR and/or LCR may be desirable to use as a preliminary amplification step in conjunction with any of the techniques used for detecting mutations described herein.
  • nucleic acid e.g., genomic, mRNA or both
  • Alternative amplification methods include: self sustained sequence replication
  • mutations in an HNFl and/or 4 gene from a sample cell are identified by alterations in restriction enzyme cleavage patterns.
  • sample and control DNA is isolated, amplified (optionally), digested with one or more restriction endonucleases, and fragment length sizes are determined by gel electrophoresis.
  • sequence specific ribozymes see, for example, U.S.
  • Patent No. 5,498,531 can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site.
  • any of a variety of sequencing reactions known in the art can be used to directly sequence the HNFl and/or 4 gene and detect mutations by comparing the sequence of the sample HNFl and/or 4 with the corresponding wild-type (control) sequence.
  • Exemplary sequencing reactions include those based on techniques developed by Maxim and Gilbert (Proc. Natl Acad Sci USA (1977) 74:560) or Sanger (Sanger et al (1977) Proc. Nat. Acad. Sci 74:5463).
  • any of a variety of automated sequencing procedures may be utilized when performing the subject assays (Biotechniques (1995) 19:448), including sequencing by mass spectrometry (see, for example PCT publication WO 94/16101 ; Cohen et al. (1996) Adv Chromatogr 36:127-162; and Griffin et al. (1993) Appl Biochem Biotechnol 38:147-159).
  • sequencing by mass spectrometry see, for example PCT publication WO 94/16101 ; Cohen et al. (1996) Adv Chromatogr 36:127-162; and Griffin et al. (1993) Appl Biochem Biotechnol 38:147-159.
  • the occurence of only one, two or three of the nucleic acid bases need be determined in the sequencing reaction. For instance, A-tract or the like, e.g., where only one nucleic acid is detected, can be carried out.
  • protection from cleavage agents can be used to detect mismatched bases in RNA/RNA or RNA DNA heteroduplexes (Myers, et al. (1985) Science 230:1242).
  • RNA/DNA duplexes can be treated with RNase and DNA/DNA hybrids treated with SI nuclease to enzymatically digesting the mismatched regions.
  • either DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine or osmium tetroxide and with piperidine in order to digest mismatched regions. After digestion of the mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine the site of mutation. See, for example, Cotton et al (1988) Proc. Natl Acad Sci USA 85:4397; Saleeba et al (1992) Methods Enzymod.
  • control DNA or RNA can be labeled for detection.
  • the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called "DNA mismatch repair" enzymes) in defined systems for detecting and mapping point mutations in HNFl and/or 4 cDNAs obtained from samples of cells.
  • DNA mismatch repair enzymes
  • the mutY enzyme of E. coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from FleLa cells cleaves T at G/T mismatches (Hsu et al. (1994) Carcinogenesis 15:1657-1662).
  • a probe based on an HNFl and/or 4 sequence e.g., a wild-type HNFl and/or 4 sequence
  • a cDNA or other DNA product from a test cell(s).
  • the duplex is treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be detected from electrophoresis protocols or the like. See, for example, U.S. Patent No. 5,459,039.
  • alterations in electrophoretic mobility will be used to identify mutations in HNFl and/or 4 genes.
  • SSCP single strand conformation polymorphism
  • Single-stranded DNA fragments of sample and control HNFl and/or 4 nucleic acids will be denatured and allowed to renature.
  • the secondary structure of single-stranded nucleic acids varies according to sequence, the resulting alteration in electrophoretic mobility enables the detection of even a single base change.
  • the DNA fragments may be labelled or detected with labelled probes.
  • the sensitivity of the assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence.
  • the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility (Keen et al. (1991) Trends Genet 7:5).
  • DGGE denaturing gradient gel electrophoresis
  • a temperature gradient is used in place of a denaturing agent gradient to identify differences in the mobility of control and sample DNA (Rosenbaum and
  • oligonucleotide primers may be prepared in which the known mutation is placed centrally and then hybridized to target DNA under conditions which permit hybridization only if a perfect match is found (Saiki et al. (1986) Nature 324: 163); Saiki et al (1989) Proc. Natl Acad. Sci USA 86:6230).
  • Such allele specific oligonucleotide hybridization techniques may be used to test one mutation per reaction when oligonucleotides are hybridized to PCR amplified target DNA or a number of different mutations when the oligonucleotides are attached to the hybridizing membrane and hybridized with labelled target DNA.
  • Oligonucleotides used as primers for specific amplification may carry the mutation of interest in the center of the molecule (so that amplification depends on differential hybridization) (Gibbs et al (1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3' end of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (Prossner (1993) Tibtech 11 :238.
  • amplification may also be performed using Taq ligase for amplification (Barany (1991) Proc. Natl. Acad. Sci USA 88:189). In such cases, ligation will occur only if there is a perfect match at the 3' end of the 5' sequence making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification.
  • RNA is initially isolated from available tissue and reverse-transcribed, and the segment of interest is amplified by PCR. The products of reverse transcription PCR are then used as a template for nested PCR amplification with a primer that contains an RNA polymerase promoter and a sequence for initiating eukaryotic translation.
  • the unique motifs incorporated into the primer permit sequential in vitro transcription and translation of the PCR products.
  • the appearance of truncated polypeptides signals the presence of a mutation that causes premature termination of translation.
  • DNA as opposed to RNA is used as a PCR template when the target region of interest is derived from a single exon.
  • nucleic acid composition comprising a (purified) oligonucleotide probe including a region of nucleotide sequence which is capable of hybridizing to a sense or antisense sequence of an HNFl and/or 4 gene, or naturally occurring mutants thereof, or 5' or 3' flanking sequences or intronic sequences naturally associated with the subject HNFl and/or 4 genes or naturally occurring mutants thereof.
  • the nucleic acid of a cell is rendered accessible for hybridization, the probe is exposed to nucleic acid of the sample, and the hybridization of the probe to the sample nucleic acid is detected.
  • Such techniques can be used to detect lesions at either the genomic or mRNA level, including deletions, substitutions, etc., as well as to determine mRNA transcript levels.
  • Such oligonucleotide probes can be used for both predictive and therapeutic evaluation of allelic mutations which might be manifest in, for example, type II diabetes.
  • the methods described herein may be performed, for example, by utilizing pre-packaged diagnostic kits comprising at least one probe nucleic acid or antibody reagent described herein, which may be conveniently used, e.g., in clinical settings to diagnose patients exhibiting symptoms or family history of a disease or illness involving an HNFl and/or 4 gene.
  • any cell type or tissue, preferably hepatocytes, in which the HNFl and/or 4 is expressed may be utilized in the diagnostics described below.
  • a subject's bodily fluid e.g. blood
  • nucleic acid tests can be performed on dry samples (e.g. hair or skin).
  • Fetal nucleic acid samples can be obtained from maternal blood as described in International Patent Application No. W091/07660 to Bianchi.
  • amniocytes or chorionic villi may be obtained for performing prenatal testing.
  • Diagnostic procedures may also be performed in situ directly upon tissue sections (fixed and/or frozen) of patient tissue obtained from biopsies or resections, such that no nucleic acid purification is necessary.
  • Nucleic acid reagents may be used as probes and/or primers for such in situ procedures (see, for example, Nuovo, G.J., 1992, PCR in situ hybridization: protocols and applications, Raven Press, NY).
  • Fingerprint profiles may be generated, for example, by utilizing a differential display procedure, Northern analysis and/or RT-PCR.
  • Antibodies directed against wild type or mutant HNF 1 and/or 4 proteins may also be used in disease diagnostics and prognostics. Such diagnostic methods, may be used to detect abnormalities in the level of HNFl and/or 4 protein expression, or abnormalities in the structure and/or tissue, cellular, or subcellular location of HNFl and/or 4 protein. Structural differences may include, for example, differences in the size, electronegativity, or antigenicity of the mutant HNFl protein relative to the normal HNF 1 and/or 4 protein. Protein from the tissue or cell type to be analyzed may easily be detected or isolated using techniques which are well known to one of skill in the art, including but not limited to western blot analysis.
  • the antibodies (or fragments thereof) useful in the present invention may, additionally, be employed histologically, as in immunofluorescence or immunoelectron microscopy, for in situ detection of HNFl and/or 4 proteins.
  • In situ detection may be accomplished by removing a histological specimen from a patient, and applying thereto a labeled antibody of the present invention.
  • the antibody (or fragment) is preferably applied by overlaying the labeled antibody (or fragment) onto a biological sample.
  • a solid phase support or carrier is used as a support capable of binding an antigen or an antibody.
  • supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbros, and magnetite.
  • the nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention.
  • the support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to an antigen or antibody.
  • the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod.
  • the surface may be flat such as a sheet, test strip, etc.
  • Preferred supports include polystyrene beads.
  • EIA enzyme immunoassay
  • Enzymes which can be used to detectably label the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5- steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate, dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase.
  • the detection can be accomplished by colorimetric methods which employ a chromogenic substrate for the enzyme. Detection may also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.
  • Detection may also be accomplished using any of a variety of other immunoassays.
  • a radioimmunoassay RIA
  • the radioactive isotope can be detected by such means as the use of a gamma counter or a scintillation counter or by autoradiography.
  • fluorescent labeling compounds fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.
  • the antibody can also be detectably labeled using fluorescence emitting metals such as 3 Eu, or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).
  • DTPA diethylenetriaminepentacetic acid
  • EDTA ethylenediaminetetraacetic acid
  • the antibody also can be detectably labeled by coupling it to a chemiluminescent compound.
  • the presence of the chemiluminescent-tagged antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction.
  • chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
  • Bioluminescence is a type of chemiluminescence found in biological systems in, which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin.
  • any of the above methods for detecting alterations in an HNFl and/or 4 gene or gene product can be used to monitor the course of treatment or therapy.
  • the present invention facilitates the development of assays which can be used to screen for drugs, including HNFl and/or 4 homologs, which are either agonists or antagonists of the normal cellular function of the subject HNFl and/or 4 polypeptides, or of their role in the pathogenesis of cellular differentiation and/or proliferation and disorders related thereto.
  • the assay evaluates the ability of a compound to modulate binding between an HNFl and/or 4 polypeptide and a molecule, be it protein or DNA, that interacts either upstream or downstream of the HNFl and/or 4 polypeptide in an HNFl and/or 4 signaling pathway.
  • a compound to modulate binding between an HNFl and/or 4 polypeptide and a molecule, be it protein or DNA, that interacts either upstream or downstream of the HNFl and/or 4 polypeptide in an HNFl and/or 4 signaling pathway.
  • the compound of interest is contacted with proteins which may function upstream (including both activators and repressors of its activity) or to proteins or nucleic acids which may function downstream of the HNFl and/or 4 polypeptide, whether they are positively or negatively regulated by it.
  • proteins which may function upstream including both activators and repressors of its activity
  • proteins or nucleic acids which may function downstream of the HNFl and/or 4 polypeptide, whether they are positively or negatively regulated by it.
  • To the mixture of the compound and the upstream or downstream element is then added a composition containing an HNFl and/or 4 polypeptide.
  • Detection and quantification of complexes of HNFl and/or 4 with it's upstream or downstream elements provide a means for determining a compound's efficacy at inhibiting (or potentiating) complex formation between HNFl and/or 4 and the HNFl and/or 4 binding elements.
  • the efficacy of the compound can be assessed by generating dose response curves from data obtained using various concentrations of the test compound.
  • a control assay can also be performed to provide a baseline for comparison.
  • isolated and purified HNFl and/or 4 polypeptide is added to a composition containing the HNFl and/or 4 binding element, and the formation of a complex is quantitated in the absence of the test compound.
  • Complex formation between the HNFl and/or 4 polypeptide and an HNFl and/or 4 binding element may be detected by a variety of techniques.
  • Modulation of the formation of complexes can be quantitated using, for example, detectably labeled proteins such as radiolabeled, fluorescently labeled, or enzymatically labeled HNFl and/or 4 polypeptides, by immunoassay, or by chromatographic detection.
  • detectably labeled proteins such as radiolabeled, fluorescently labeled, or enzymatically labeled HNFl and/or 4 polypeptides
  • immunoassay or by chromatographic detection.
  • HNFl and/or 4 or its binding protein can be immobilized to facilitate separation of complexes from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay.
  • Binding of HNFl and/or 4 to an upstream or downstream element, in the presence and absence of a candidate agent, can be accomplished in any vessel suitable for containing the reactants. Examples include microtitre plates, test tubes, and micro-centrifuge tubes.
  • a fusion protein can be provided which adds a domain that allows the protein to be bound to a matrix.
  • glutathione-S-transferase/HNFl and/or 4 (GST/HNF1) fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtitre plates, which are then combined with the cell lysates, e.g. an 35 S- labeled, and the test compound, and the mixture incubated under conditions conducive to complex formation, e.g. at physiological conditions for salt and pH, though slightly more stringent conditions may be desired. Following incubation, the beads are washed to remove any unbound label, and the matrix immobilized and radiolabel determined directly (e.g.
  • the complexes can be dissociated from the matrix, separated by SDS-PAGE, and the level of HNF 1 and/or 4-binding protein found in the bead fraction quantitated from the gel using standard electrophoretic techniques such as described in the appended examples.
  • HNFl and/or 4 or its cognate binding protein can be immobilized utilizing conjugation of biotin and streptavidin.
  • biotinylated HNFl and/or 4 molecules can be prepared from biotin-NHS (N-hydroxysuccinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, IL), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).
  • antibodies reactive with HNFl and/or 4 but which do not interfere with binding of upstream or downstream elements can be derivatized to the wells of the plate, and HNFl and/or 4 trapped in the wells by antibody conjugation.
  • preparations of an HNFl and/or 4 binding protein and a test compound are incubated in the HNFl and/or 4 presenting wells of the plate, and the amount of complex trapped in the well can be quantitated.
  • Exemplary methods for detecting such complexes include immunodetection of complexes using antibodies reactive with the HNFl and/or 4 binding element, or which are reactive with
  • the enzyme can be chemically conjugated or provided as a fusion protein with the HNFl and/or 4-BP.
  • the HNFl- and/or 4-BP can be chemically cross-linked or genetically fused with horseradish peroxidase, and the amount of polypeptide trapped in the complex can be assessed with a chromogenic substrate of the enzyme, e.g. 3,3'-diamino-benzadine terahydrochloride or 4-chloro-l-napthol.
  • fusion protein comprising the polypeptide and glutathione-S-transferase can be provided, and complex formation quantitated by detecting the GST activity using l-chloro-2,4-dinitrobenzene (Habig et al (1 74) J Biol Chem 249:7130).
  • the protein to be detected in the complex can be "epitope tagged" in the form of a fusion protein which includes, in addition to the HNFl and/or 4 sequence, a second polypeptide for which antibodies are readily available (e.g. from commercial sources).
  • the GST fusion proteins described above can also be used for quantification of binding using antibodies against the GST moiety.
  • Other useful epitope tags include myc-epitopes (e.g., see Ellison et al.
  • a transcriptional control assay can be used to detect agonists or antagonists of HNF- 1 and/or HNF -4 which can be used for treatment of diabetes.
  • cells can be engineered to express HNF-1 or HNF -4 genes and a second gene construct containing an HNF response element in operative linkage with a reporter gene construct, such as luciferase or chloramphenicol acetyl transferase, or other reporter gene known in the art.
  • HNF ligands will cause transcriptional activation of the reporter gene as compared to that seen in control cells in the absence of ligand or in the absence of the recombinant HNF or HNF response element- reporter gene construct.
  • cells can be contacted with an agonist prior to being contacted with test compounds and an inhibition of reporter gene transcription or product can be detected.
  • a DNA footprinting assay can be used to detect agonists or antagonists of HNFl and/or HNF4 for the treatment of Diabetes.
  • DNase I footprinting may be used to detect compounds which alter the binding of HNFl or HNF4 to nucleic acids (see for example, Zhong et al. 1994. Mol Cell. Biol. 14:7276).
  • cell based assays In addition to cell-free assays, such as described above, the readily available source of HNFl and/or 4 proteins provided by the present invention also facilitates the generation of cell-based assays for identifying small molecule agonists/antagonists and the like. For example, cells can be caused to overexpress a recombinant HNFl and/or 4 protein in the presence and absence of a test agent of interest, with the assay scoring for modulation in HNFl and/or 4 responses by the target cell mediated by the test agent. As with the cell- free assays, agents which produce a statistically significant change in HNFl and/or 4- dependent responses (either inhibition or potentiation) can be identified.
  • the expression or activity of an HNFl and/or 4 is modulated in embryos or cells and the effects of compounds of interest on the readout of interest (such as tissue differentiation, proliferation, tumorigenesis) are measured.
  • the expression of genes which are up- or down-regulated in response to an HNFl and/or 4 dependent signal cascade can be assayed.
  • the regulatory regions of such genes e.g., the 5' flanking promoter and enhancer regions, are operably linked to a detectable marker
  • Exemplary cell lines may include non-recombinant monocyte cell lines, such as U937 (ATCC# CRL-1593), THP-1 (ATCC# TIB-202), and P388D1 (ATCC# TIB-63); endothelial cells such as HUVEC's and bovine aortic endothelial cells (BAEC's); as well as generic mammalian cell lines such as HeLa cells and COS cells, e.g., COS-7 (ATCC# CRL- 1651). Further, the transgenic animals discussed herein may be used to generate cell lines, containing one or more cell types involved in cardiovascular disease, that can be used as cell culture models for this disorder.
  • monocyte cell lines such as U937 (ATCC# CRL-1593), THP-1 (ATCC# TIB-202), and P388D1 (ATCC# TIB-63)
  • endothelial cells such as HUVEC's and bovine aortic endothelial cells (BAEC's
  • HUVECs can be treated with test compounds or transfected with genetically engineered HNFl and/or 4 genes. The HUVECs can then be examined for phenotypes associated with diabetes, including, but not limited to changes in insulin level and/or glucose tolerance.
  • HNFl and/or 4 proteins themselves, or in complexes with other proteins, are capable of binding DNA and modifying transcription of a gene
  • a transcriptional based assay could be used, for example, in which an HNFl and/or 4 responsive regulatory sequence is operably linked to a detectable marker gene.
  • Monitoring the influence of compounds on cells may be applied not only in basic drug screening, but also in clinical trials. In such clinical trials, the expression of a panel of genes may be used as a "read out" of a particular drug's therapeutic effect.
  • the subject HNFl and/or 4 polypeptides can be used in a "two hybrid" assay (see, for example, U.S. Patent No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J Biol Chem 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696; and Brent
  • HNFl and/or 4-binding proteins for isolating coding sequences for other cellular proteins which bind to or interact with HNFl and/or 4
  • HNFl and/or 4-binding proteins for isolating coding sequences for other cellular proteins which bind to or interact with HNFl and/or 4
  • HNFl and/or 4-binding proteins for isolating coding sequences for other cellular proteins which bind to or interact with HNFl and/or 4
  • HNFl and/or 4-binding proteins for isolating coding sequences for other cellular proteins which bind to or interact with HNFl and/or 4
  • the two hybrid assay relies on reconstituting in vivo a functional transcriptional activator protein from two separate fusion proteins.
  • the method makes use of chimeric genes which express hybrid proteins.
  • a first hybrid gene comprises the coding sequence for a DNA-binding domain of a transcriptional activator fused in frame to the coding sequence for an HNFl and/or 4 polypeptide.
  • the second hybrid protein encodes a transcriptional activation domain fused in frame to a sample gene from a cDNA library. If the bait and sample hybrid proteins are able to interact, e.g., form an HNFl and/or 4 dependent complex, they bring into close proximity the two domains of the transcriptional activator. This proximity is sufficient to cause transcription of a reporter gene which is operably linked to a transcriptional regulatory site responsive to the transcriptional activator, and expression of the reporter gene can be detected and used to score for the interaction of the HNFl and/or 4 and sample proteins.
  • the cell- and animal-based model systems may be used to further characterize HNFl and/or 4 genes and proteins.
  • assays may be utilized as part of screening strategies designed to identify compounds which are capable of ameliorating disease symptoms.
  • the animal- and cell-based models may be used to identify drugs, pharmaceuticals, therapies and interventions which may be effective in treating disease.
  • transgenic animals which are comprised of cells (of that animal) which contain a transgene of the present invention and which preferably (though optionally) express an exogenous HNF 1 and/or 4 protein in one or more cells in the animal.
  • An HNFl and/or 4 transgene can encode the wild-type form of the protein, or can encode homologs thereof, including both agonists and antagonists, as well as antisense constructs.
  • the expression of the transgene is restricted to specific subsets of cells, tissues or developmental stages utilizing, for example, cis-acting sequences that control expression in the desired pattern.
  • such mosaic expression of an HNFl and/or 4 protein can be essential for many forms of lineage analysis and can additionally provide a means to assess the effects of, for example, lack of HNF 1 and/or 4 expression which might grossly alter development in small patches of tissue within an otherwise normal embryo.
  • tissue-specific regulatory sequences and conditional regulatory sequences can be used to control expression of the transgene in certain spatial patterns.
  • temporal patterns of expression can be provided by, for example, conditional recombination systems or prokaryotic transcriptional regulatory sequences.
  • target sequence refers to a nucleotide sequence that is genetically recombined by a recombinase.
  • the target sequence is flanked by recombinase recognition sequences and is generally either excised or inverted in cells expressing recombinase activity.
  • Recombinase catalyzed recombination events can be designed such that recombination of the target sequence results in either the activation or repression of expression of one of the subject HNFl and/or 4 proteins.
  • excision of a target sequence which interferes with the expression of a recombinant HNFl and/or 4 gene such as one which encodes an antagonistic homolog or an antisense transcript, can be designed to activate expression of that gene.
  • This interference with expression of the protein can result from a variety of mechanisms, such as spatial separation of the HNFl and/or 4 gene from the promoter element or an internal stop codon.
  • the transgene can be made wherein the coding sequence of the gene is flanked by recombinase recognition sequences and is initially transfected into cells in a 3' to 5' orientation with respect to the promoter element. In such an instance, inversion of the target sequence will reorient the subject gene by placing the 5' end of the coding sequence in an orientation with respect to the promoter element which allow for promoter driven transcriptional activation.
  • the transgenic animals of the present invention all include within a plurality of their cells a transgene of the present invention, which transgene alters the phenotype of the "host cell" with respect to regulation of cell growth, death and/or differentiation.
  • transgenic organisms of the invention utilizing one or more of the transgene constructs described herein, a general description will be given of the production of transgenic organisms by referring generally to exogenous genetic material. This general description can be adapted by those skilled in the art in order to incorporate specific transgene sequences into organisms utilizing the methods and materials described below.
  • crelloxP recombinase system of bacteriophage PI (Lakso et al. (1992) PNAS 89:6232-6236; Orban et al. (1992) PNAS 89:6861-6865) or the FLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al. (1991) Science 251 :1351-1355; PCT publication WO 92/15694) can be used to generate in vivo site-specific genetic recombination systems. Cre recombinase catalyzes the site-specific recombination of an intervening target sequence located between loxP sequences.
  • loxP sequences are 34 base pair nucleotide repeat sequences to which the Cre recombinase binds and are required for Cre recombinase mediated genetic recombination.
  • the orientation of loxP sequences determines whether the intervening target sequence is excised or inverted when Cre recombinase is present (Abremski et al. (1984) J Biol. Chem. 259:1509-1514); catalyzing the excision of the target sequence when the loxP sequences are oriented as direct repeats and catalyzes inversion of the target sequence when loxP sequences are oriented as inverted repeats.
  • genetic recombination of the target sequence is dependent on expression of the Cre recombinase.
  • Expression of the recombinase can be regulated by promoter elements which are subject to regulatory control, e.g., tissue-specific, developmental stage-specific, inducible or repressible by externally added agents. This regulated control will result in genetic recombination of the target sequence only in cells where recombinase expression is mediated by the promoter element.
  • the activation expression of a recombinant HNFl and/or 4 protein can be regulated via control of recombinase expression.
  • crelloxP recombinase system to regulate expression of a recombinant HNFl and/or 4 protein requires the construction of a transgenic animal containing transgenes encoding both the Cre recombinase and the subject protein. Animals containing both the Cre recombinase and a recombinant HNFl and/or 4 gene can be provided through the construction of "double" transgenic animals. A convenient method for providing such animals is to mate two transgenic animals each containing a transgene, e.g., an HNFl and/or 4 gene and recombinase gene.
  • transgenic animals containing an HNFl and/or 4 transgene in a recombinase-mediated expressible format derives from the likelihood that the subject protein, whether agonistic or antagonistic, can be deleterious upon expression in the transgenic animal.
  • a founder population in which the subject transgene is silent in all tissues, can be propagated and maintained. Individuals of this founder population can be crossed with animals expressing the recombinase in, for example, one or more tissues and/or a desired temporal pattern.
  • a founder population in which, for example, an antagonistic HNF 1 and/or 4 transgene is silent will allow the study of progeny from that founder in which disruption of HNFl and/or 4 mediated induction in a particular tissue or at certain developmental stages would result in, for example, a lethal phenotype.
  • prokaryotic promoter sequences which require prokaryotic proteins to be simultaneous expressed in order to facilitate expression of the HNFl and/or 4 transgene.
  • Exemplary promoters and the corresponding trans-activating prokaryotic proteins are given in U.S. Patent No. 4,833,080.
  • conditional transgenes can be induced by gene therapy-like methods wherein a gene encoding the trans-activating protein, e.g. a recombinase or a prokaryotic protein, is delivered to the tissue and caused to be expressed, such as in a cell-type specific manner.
  • a gene encoding the trans-activating protein e.g. a recombinase or a prokaryotic protein
  • an HNFl and/or 4 transgene could remain silent into adulthood until "turned on” by the introduction of the trans-activator.
  • the "transgenic non-human animals" of the invention are produced by introducing transgenes into the germline of the non-human animal. Embryonal target cells at various developmental stages can be used to introduce transgenes.
  • the specific line(s) of any animal used to practice this invention are selected for general good health, good embryo yields, good pronuclear visibility in the embryo, and good reproductive fitness.
  • the haplotype is a significant factor.
  • strains such as C57BL/6 or FVB lines are often used (Jackson Laboratory, Bar Harbor, ME).
  • Preferred strains are those with H-2 b , H-2 d or H-21 haplotypes such as C57BL/6 or DBA/1.
  • the line(s) used to practice this invention may themselves be transgenics, and/or may be knockouts (i.e., obtained from animals which have one or more genes partially or completely suppressed) .
  • the transgene construct is introduced into a single stage embryo.
  • the zygote is the best target for micro-injection.
  • the male pronucleus reaches the size of approximately 20 micrometers in diameter which allows reproducible injection of 1-2 pi of DNA solution.
  • the use of zygotes as a target for gene transfer has a major advantage in that in most cases the injected DNA will be incorporated into the host gene before the first cleavage (Brinster et al. (1985) PNAS 82:4438-4442). As a consequence, all cells of the transgenic animal will carry the incorporated transgene. This will in general also be reflected in the efficient transmission of the transgene to offspring of the founder since 50% of the germ cells will harbor the transgene.
  • the nucleotide sequence comprising the transgene is introduced into the female or male pronucleus as described below. In some species such as mice, the male pronucleus is preferred. It is most preferred that the exogenous genetic material be added to the male DNA complement of the zygote prior to its being processed by the ovum nucleus or the zygote female pronucleus.
  • ovum nucleus or female pronucleus release molecules which affect the male DNA complement, perhaps by replacing the protamines of the male DNA with histones, thereby facilitating the combination of the female and male DNA complements to form the diploid zygote.
  • the exogenous genetic material be added to the male complement of DNA or any other complement of DNA prior to its being affected by the female pronucleus.
  • the exogenous genetic material is added to the early male pronucleus, as soon as possible after the formation of the male pronucleus, which is when the male and female pronuclei are well separated and both are located close to the cell membrane.
  • the exogenous genetic material could be added to the nucleus of the sperm after it has been induced to undergo decondensation.
  • Sperm containing the exogenous genetic material can then be added to the ovum or the decondensed sperm could be added to the ovum with the transgene constructs being added as soon as possible thereafter.
  • transgene nucleotide sequence into the embryo may be accomplished by any means known in the art such as, for example, microinjection, electroporation, or lipofection.
  • the embryo may be incubated in vitro for varying amounts of time, or reimplanted into the surrogate host, or both. In vitro incubation to maturity is within the scope of this invention.
  • a zygote is essentially the formation of a diploid cell which is capable of developing into a complete organism.
  • the zygote will be comprised of an egg containing a nucleus formed, either naturally or artificially, by the fusion of two haploid nuclei from a gamete or gametes.
  • the gamete nuclei must be ones which are naturally compatible, i.e., ones which result in a viable zygote capable of undergoing differentiation and developing into a functioning organism.
  • a euploid zygote is preferred.
  • the number of chromosomes should not vary by more than one with respect to the euploid number of the organism from which either gamete originated.
  • physical ones also govern the amount (e.g., volume) of exogenous genetic material which can be added to the nucleus of the zygote or to the genetic material which forms a part of the zygote nucleus. If no genetic material is removed, then the amount of exogenous genetic material which can be added is limited by the amount which will be absorbed without being physically disruptive. Generally, the volume of exogenous genetic material inserted will not exceed about 10 picoliters.
  • the physical effects of addition must not be so great as to physically destroy the viability of the zygote.
  • the biological limit of the number and variety of DNA sequences will vary depending upon the particular zygote and functions of the exogenous genetic material and will be readily apparent to one skilled in the art, because the genetic material, including the exogenous genetic material, of the resulting zygote must be biologically capable of initiating and maintaining the differentiation and development of the zygote into a functional organism.
  • the number of copies of the transgene constructs which are added to the zygote is dependent upon the total amount of exogenous genetic material added and will be the amount which enables the genetic transformation to occur. Theoretically only one copy is required; however, generally, numerous copies are utilized, for example, 1,000-20,000 copies of the transgene construct, in order to insure that one copy is functional. As regards the present invention, there will often be an advantage to having more than one functioning copy of each of the inserted exogenous DNA sequences to enhance the phenotypic expression of the exogenous DNA sequences.
  • exogenous genetic material is preferentially inserted into the nucleic genetic material by microinjection. Microinjection of cells and cellular structures is known and is used in the art.
  • Reimplantation is accomplished using standard methods. Usually, the surrogate host is anesthetized, and the embryos are inserted into the oviduct. The number of embryos implanted into a particular host will vary by species, but will usually be comparable to the number of off spring the species naturally produces. Transgenic offspring of the surrogate host may be screened for the presence and/or expression of the transgene by any suitable method. Screening is often accomplished by Southern blot or Northern blot analysis, using a probe that is complementary to at least a portion of the transgene. Western blot analysis using an antibody against the protein encoded by the transgene may be employed as an alternative or additional method for screening for the presence of the transgene product.
  • DNA is prepared from tail tissue and analyzed by Southern analysis or PCR for the transgene.
  • the tissues or cells believed to express the transgene at the highest levels are tested for the presence and expression of the transgene using Southern analysis or PCR, although any tissues or cell types may be used for this analysis.
  • Alternative or additional methods for evaluating the presence of the transgene include, without limitation, suitable biochemical assays such as enzyme and/or immunological assays, histological stains for particular marker or enzyme activities, flow cytometric analysis, and the like. Analysis of the blood may also be useful to detect the presence of the transgene product in the blood, as well as to evaluate the effect of the transgene on the levels of various types of blood cells and other blood constituents.
  • suitable biochemical assays such as enzyme and/or immunological assays, histological stains for particular marker or enzyme activities, flow cytometric analysis, and the like.
  • Analysis of the blood may also be useful to detect the presence of the transgene product in the blood, as well as to evaluate the effect of the transgene on the levels of various types of blood cells and other blood constituents.
  • Progeny of the transgenic animals may be obtained by mating the transgenic animal with a suitable partner, or by in vitro fertilization of eggs and/or sperm obtained from the transgenic animal.
  • the partner may or may not be transgenic and/or a knockout; where it is transgenic, it may contain the same or a different transgene, or both.
  • the partner may be a parental line.
  • in vitro fertilization is used, the fertilized embryo may be implanted into a surrogate host or incubated in vitro, or both. Using either method, the progeny may be evaluated for the presence of the transgene using methods described above, or other appropriate methods.
  • the transgenic animals produced in accordance with the present invention will include exogenous genetic material.
  • the exogenous genetic material will, in certain embodiments, be a DNA sequence which results in the production of an HNFl and/or
  • the sequence will be attached to a transcriptional control element, e.g., a promoter, which preferably allows the expression of the transgene product in a specific type of cell.
  • Retroviral infection can also be used to introduce transgene into a non-human animal.
  • the developing non-human embryo can be cultured in vitro to the blastocyst stage.
  • the blastomeres can be targets for retroviral infection (Jaenich, R. (1976) PNAS 73:1260-1264).
  • Efficient infection of the blastomeres is obtained by enzymatic treatment to remove the zona pellucida (Manipulating the Mouse Embryo, Hogan eds. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1986).
  • the viral vector system used to introduce the transgene is typically a replication-defective retrovirus carrying the transgene (Jahner et al.
  • the founder may contain various retroviral insertions of the transgene at different positions in the genome which generally will segregate in the offspring.
  • transgenes into the germ line by intrauterine retroviral infection of the midgestation embryo (Jahner et al. (1982) supra).
  • ES cells are obtained from pre-implantation embryos cultured in vitro and fused with embryos (Evans et al. (1981) Nature 292:154-156; Bradley et al. (1984) Nature 309:255-258;
  • Transgenes can be efficiently introduced into the ES cells by DNA transfection or by retrovirus-mediated transduction. Such transformed ES cells can thereafter be combined with blastocysts from a non-human animal. The ES cells thereafter colonize the embryo and contribute to the germ line of the resulting chimeric animal. For review see Jaenisch, R.
  • gene targeting which is a method of using homologous recombination to modify an animal's genome, can be used to introduce changes into cultured embryonic stem cells.
  • the gene targeting procedure is accomplished by introducing into tissue culture cells a DNA targeting construct that includes a segment homologous to a target HNFl and/or 4 locus, and which also includes an intended sequence modification to the HNFl and/or 4 genomic sequence (e.g., insertion, deletion, point mutation).
  • the treated cells are then screened for accurate targeting to identify and isolate those which have been properly targeted.
  • Gene targeting in embryonic stem cells is in fact a scheme contemplated by the present invention as a means for disrupting an HNFl and/or 4 gene function through the use of a targeting transgene construct designed to undergo homologous recombination with one or more HNFl and/or 4 genomic sequences.
  • the targeting construct can be arranged so that, upon recombination with an element of an HNF 1 and/or 4 gene, a positive selection marker is inserted into (or replaces) coding sequences of the targeted gene.
  • the inserted sequence functionally disrupts the HNFl and/or 4 gene, while also providing a positive selection trait.
  • Exemplary HNFl and/or 4 targeting constructs are described in more detail below.
  • the embryonic stem cells (ES cells ) used to produce the knockout animals will be of the same species as the knockout animal to be generated.
  • mouse embryonic stem cells will usually be used for generation of knockout mice.
  • Embryonic stem cells are generated and maintained using methods well known to the skilled artisan such as those described by Doetschman et al. (1985) J. Embryol. Exp. Morphol 87:27-45). Any line of ES cells can be used, however, the line chosen is typically selected for the ability of the cells to integrate into and become part of the germ line of a developing embryo so as to create germ line transmission of the knockout construct. Thus, any ES cell line that is believed to have this capability is suitable for use herein.
  • One mouse strain that is typically used for production of ES cells is the 129J strain.
  • Another ES cell line is murine cell line D3 (American Type Culture Collection, catalog no. CKL 1934)
  • Still another preferred ES cell line is the WW6 cell line (Ioffe et al. (1995) PNAS 92:7357-7361).
  • the cells are cultured and prepared for knockout construct insertion using methods well known to the skilled artisan, such as those set forth by Robertson in: Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E.J. Robertson, ed. IRL Press, Washington,
  • Insertion of the knockout construct into the ES cells can be accomplished using a variety of methods well known in the art including for example, electroporation, microinjection, and calcium phosphate treatment.
  • a preferred method of insertion is electroporation .
  • Each knockout construct to be inserted into the cell must first be in the linear form. Therefore, if the knockout construct has been inserted into a vectoi (described infra), linearization is accomplished by digesting the DNA with a suitable restriction endonuclease selected to cut only within the vector sequence and not within the knockout construct sequence.
  • the knockout construct is added to the ES cells under appropriate conditions for the insertion method chosen, as is known to the skilled artisan. Where more than one construct is to be introduced into the ES cell, each knockout construct can be introduced simultaneously or one at a time.
  • the ES cells are to be electroporated, the ES cells and knockout construct
  • DNA are exposed to an electric pulse using an electroporation machine and following the manufacturer's guidelines for use. After electroporation, the ES cells are typically allowed to recover under suitable incubation conditions. The cells are then screened for the presence of the knockout construct . Screening can be accomplished using a variety of methods. Where the marker gene is an antibiotic resistance gene, for example, the ES cells may be cultured in the presence of an otherwise lethal concentration of antibiotic. Those ES cells that survive have presumably integrated the knockout construct. If the marker gene is other than an antibiotic resistance gene, a Southern blot of the ES cell genomic DNA can be probed with a sequence of DNA designed to hybridize only to the marker sequence Alternatively, PCR can be used.
  • the marker gene is a gene that encodes an enzyme whose activity can be detected
  • the enzyme substrate can be added to the cells under suitable conditions, and the enzymatic activity can be analyzed.
  • suitable conditions e.g., ⁇ -galactosidase
  • the enzyme substrate can be added to the cells under suitable conditions, and the enzymatic activity can be analyzed.
  • markers e.g., ⁇ -galactosidase
  • the knockout construct may integrate into several locations in the ES cell genome, and may integrate into a different location in each ES cell's genome due to the occurrence of random insertion events.
  • the desired location of insertion is in a complementary position to the DNA sequence to be knocked out, e.g., the HNFl and/or 4 coding sequence, transcriptional regulatory sequence, etc.
  • the DNA sequence to be knocked out e.g., the HNFl and/or 4 coding sequence, transcriptional regulatory sequence, etc.
  • less than about 1-5 % of the ES cells that take up the knockout construct will actually integrate the knockout construct in the desired location.
  • total DNA can be extracted from the ES cells using standard methods.
  • the DNA can then be probed on a Southern blot with a probe or probes designed to hybridize in a specific pattern to genomic DNA digested with particular restriction enzyme(s).
  • the genomic DNA can be amplified by PCR with probes specifically designed to amplify DNA fragments of a particular size and sequence (i.e., only those cells containing the knockout construct in the proper position will generate DNA fragments of the proper size).
  • the cells can be inserted into an embryo. Insertion may be accomplished in a variety of ways known to the skilled artisan, however a preferred method is by microinjection. For microinjection, about 10-30 cells are collected into a micropipet and injected into embryos that are at the proper stage of development to permit integration of the foreign ES cell containing the knockout construct into the developing embryo. For instance. as the appended examples describe, the transformed ES cells can be microinjected into blastocytes.
  • the suitable stage of development for the embryo used for insertion of ES cells is very species dependent, however for mice it is about 3.5 days.
  • the embryos are obtained by perfusing the uterus of pregnant females. Suitable methods for accomplishing this are known to the skilled artisan, and are set forth by, e.g., Bradley et al. (supra).
  • preferred embryos are male.
  • the preferred embryos also have genes coding for a coat color that is different from the coat color encoded by the ES cell genes.
  • the offspring can be screened easily for the presence of the knockout construct by looking for mosaic coat color (indicating that the ES cell was incorporated into the developing embryo).
  • the embryo selected will carry genes for black or brown fur.
  • the embryo may be implanted into the uterus of a pseudopregnant foster mother for gestation. While any foster mother may be used, the foster mother is typically selected for her ability to breed and reproduce well, and for her ability to care for the young. Such foster mothers are typically prepared by mating with vasectomized males of the same species.
  • the stage of the pseudopregnant foster mother is important for successful implantation, and it is species dependent. For mice, this stage is about 2-3 days pseudopregnant.
  • Offspring that are bora to the foster mother may be screened initially for mosaic coat color where the coat color selection strategy (as described above, and in the appended examples) has been employed.
  • DNA from tail tissue of the offspring may be screened for the presence of the knockout construct using Southern blots and/or PCR as described above. Offspring that appear to be mosaics may then be crossed to each other, if they are believed to carry the knockout construct in their germ line, in order to generate homozygous knockout animals.
  • Homozygotes may be identified by Southern blotting of equivalent amounts of genomic DNA from mice that are the product of this cross, as well as mice that are known heterozygotes and wild type mice.
  • Northern blots can be used to probe the mRNA for the presence or absence of transcripts encoding either the gene knocked out, the marker gene, or both.
  • Western blots can be used to assess the level of expression of the HNF 1 and/or 4 gene knocked out in various tissues of the offspring by probing the Western blot with an antibody against the particular HNFl and/or 4 protein, or an antibody against the marker gene product, where this gene is expressed.
  • in situ analysis such as fixing the cells and labeling with antibody
  • FACS fluorescence activated cell sorting
  • knock-out or disruption transgenic animals are also generally known. See, for example, Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).
  • Recombinase dependent knockouts can also be generated, e.g. by homologous recombination to insert target sequences, such that tissue specific and/or temporal control of inactivation of an HNFl and/or 4 gene can be controlled by recombinase sequences (described infra).
  • Animals containing more than one knockout construct and/or more than one transgene expression construct are prepared in any of several ways.
  • the preferred manner of preparation is to generate a series of mammals, each containing one of the desired transgenic phenotypes. Such animals are bred together through a series of crosses, backcrosses and selections, to ultimately generate a single animal containing all desired knockout constructs and/or expression constructs, where the animal is otherwise congenic (genetically identical) to the wild type except for the presence of the knockout construct(s) and/or transgene(s) .
  • IDDM insulin, oral hypoglycemic agents, or treatment with diet or exercise
  • Genotyping was conducted using 14 to 24 microsatellite markers on chromosome 12q. The markers were those identified and described in Dib et al, Nature 380:152 (1996); and Hudson et al, Science 270, 5244 (22 December 1995). Microsatellite regions were PCR amplified from genomic DNA from the family members. Since microsatellite polymorphisms alter PCR product length, fluorescently labeled PCR products were sized on the ABI 377 DNA sequencing machines (Applied Biosystems, Irvine, CA). Electropherogram traces were evaluated for peaks corresponding to PCR products. The products were sized relative to internal DNA fragment size standards.
  • Genotype-alleles for several markers were analyzed for co-segregation within the families. Combinations of genotype-alleles, or haplotypes, were observed to segregate in a fashion consistent with the laws of Mendelian segregation. Linkage analysis
  • Haplotype analysis also provided evidence for crossing over during meiosis. Such crossovers helped to localize the diabetes gene by exclusion of portions of chromosome 12q. Crossovers reduced the number of microsatellite marker-alleles shared in common by affected members of a family.
  • the human MODY3 gene had previously been mapped to a 7cM region of chromosome 12q between marker D12S86 and D12S342 by Vaxillaire et al. (Vaxillaire, M. et al. (1995) Nature Genetics 9:418-423). S. Menzel et al. further localized the gene to a 5cM region between D12S86 and D12S807/D12S820. On average, 5cM corresponds to approximately 5 megabases in the human genome. This level of resolution is unsatisfactory for gene identification. Further, this region of chromosome 12q was not sufficiently characterized so as to be certain of exact marker location or order.
  • phenotypes were assigned according to the criteria described above. Families with diabetes caused by mutations in the MODY3 gene were identified by linkage analysis. 4 families with MODY3 were identifed,
  • JOS310, JOS312, JOS314 and JOS324 with linkage on chromosome 12.
  • a highly unlikely double-crossover observed in individual 60181 from family JOS303 led to the correct placement of marker D12S807 to lie between D12S1349 and GGAT1E2.
  • the MODY3 genetic interval was narrowed by determining the minimal common region of the disease-gene bearing chromosome in each family. Marker-genotypes defined the locations of recombinations defining the telomeric and centomeric boundaries of the MODY3 genetic interval. Recombinations observed in individual 60628 in family JOS310 and 60540 in family JOS324 defined the centromeric boundary to be located at AFMa82za5. Recombinations observed in individuals 60717 in family JOS314 and 60523 in family JOS324 defined the telomeric boundary to lie between D12S837 and AFM165ybl2.
  • the genetic interval between AFMa82za5 and AFM165ybl2 is 3cM. On average in the human genome, such a genetic distance corresponds to approximately 3 megabases.
  • YAC yeast artificial chromosomes maps were constructed using the publicly available markers in the region in an attempt to confirm the order of published YACs and markers.
  • the YAC libraries were screened by PCR amplification of a DNA pool representing the libraries. A description of the screening protocol can be found in Research Genetics Catalog # 95001).
  • a bubble vectorette and IRS were used to both clone the ends of the YACs and generate more markers throughout the region (Current Protocols in Human Genetics First Edition, (1994) John Wiley & Co., N.C. Dracopoli et al., eds).
  • Primers were designed from these sequences and used to rescreen the YAC library by PCR to identify adjacent YACs.
  • BACs bacterial artificial chromosome
  • PACs PI artificial chromosomes
  • Random sequencing of BamHI/Blgll restricted libraries of the BACs and PACs was also used to generate more STSs.
  • Creation of over 400 PCR markers enabled the creation of a high resolution sequence ready physical map consisting of a minimal tiling path of 23 BAC and PAC clones with markers spaced approximately every 5kb.
  • the BAC and PAC that make up the minimal tiling path were sized by pulse gel electrophoresis and range in size from 40 to 230 kb. Sizing all the clones in the interval between the markers AFMa82za5 and GGAT1E2 determined the size of the region to be approximately 2.4 Mb.
  • Random sheared libraries were made from all the BAC and PAC clones within the defined genetic interval by sonicating the DNA. Fragments in the size range of 3-5 kb were cloned into the vector pCDNAII (Invitrogen, San Diego, CA). Approximately 4000 subclones were sequenced with vector primers in order to generate 8-fold sequence coverage of each BAC or PAC clone. All sequences were processed through an automated sequence analysis pipeline that assessed quality, removed vector sequences, masked repetitive sequences. The sequences that passed through the automated sequence analysis pipeline were then aligned to public DNA and protein databases using BLAST algorithms (Altschul et al., 1990). Both known and novel genes were identified in the cited region. Exon trapping
  • Exon trapping was carried out by isolating internal exons from individual PI and BAC clones carrying mouse genomic DNA using the exon trapping vector D-pSPL3 as described in the Gibco BRL Exon Trapping System Instruction Manual (Cat. No. 18449-
  • D-pSPL3 was derived from the splicing vector pSPL3 (Gibco BRL) by deletion of the Ndel (I l l9)-Nhel (1976) fragment in the HIV tat intron to eliminate the cryptic splice-donor site at position 1134 in the pSPL3 sequence.
  • the exon trapping procedure involves: subcloning the Pl/Bac clone into D-pSPL3, plating of the resulting D-pSPL3 library and preparation of DNA. transfection of library DNA into COS-7 cells, RNA isolation after transient expression, first strand cDNA synthesis using a vector-specific primer by reverse transcriptase polymerase chain reaction (RT-PCR), cDNA amplification by an initial round of PCR, BstXI digestion of primary PCR products to eliminate vector-vector and cryptic splicing products, secondary amplification with dUMP-containing primers, and cloning of secondary PCR products into the phagemid vector pAMPIO (Gibco BRL) using uracil DNA glycosylase (UDG).
  • RT-PCR reverse transcriptase polymerase chain reaction
  • PI /BAC DNA was prepared from overnight cultures (1000 ml LB/kanamycin
  • Stocks of if ⁇ mHI-cut and Pstl-cut D-pSPL3 DNA were prepared by digesting 50-100 ⁇ g DNA with the corresponding enzyme and dephosphorylating the linearized vector with calf intestine alkaline phosphatase as specified by the manufacturers (New England Biolabs and Boehringer Mannheim, respectively).
  • the linearized vector was purified away from uncut plasmid DNA by agarose gel electrophoresis and electroelution and assayed to assess the level of uncut and self-ligated vector as described elsewhere (Pulido and Duyk, In Current Protocols in Human Genetics First Edition. (1994) John Wiley & Co., N.C.
  • Insert-vector ligation reactions contained 200 ng vector DNA, 20 ng insert DNA, 4 ⁇ l of 5x ligation buffer (Gibco BRL), and 0.6 units of T4 DNA ligase (Gibco BRL) in a total volume of 20 ⁇ l, and were incubated for 1-2 hours at room temperature. 2 ⁇ l of the ligation reaction was transformed into Escherichia coli XL- IB cells (New England Biolabs) by electroporation (Bio-Rad Instruction Manual Cat. No. 165-2098). Routinely, 0.1 and 0.01 ml of each transformation was plated on LB/carbenicillin (100 ⁇ g/ml) plates to obtain the library titer.
  • sequencing primers were: SPL3A (forward primer; 5'-CAT GCT CCT TGG GAT GT -3'; SEQ ID NO. 1 Operon Technologies) and SPL3C (reverse primer; 5'-TGA GGA TTG CTT AAA GA-3'; SEQ ID NO. 2 Operon Technologies).
  • Each PI /BAC library was plated on three 150-mm LB/carbenicillin plates at a density of 7x10- ⁇ colonies/plate (for a total of 2.1x10 ⁇ colonies) and grown overnight at 37° C. The colonies were resuspended and pooled in a total of 20 ml LB medium. DNA was prepared from the pooled cell suspension using a Qiagen-tip 500 column as specified by the manufacturer (Qiagen Plasmid Handbook).
  • RNA from transiently transfected COS-7 cells was isolated using TRIzol reagent (Gibco BRL) as described in the Gibco BRL Exon Trapping System Instruction Manual (Cat. No. 18449-017).
  • RT-PCR reverse transcriptase polymerase chain reaction
  • RNA template was degraded by addition of 1 ⁇ l RNase H (Gibco BRL) and incubation for 10 min at 55° C.
  • the primary PCR reaction contained in a total volume of 40 ⁇ l: 8 ⁇ l of RT-
  • PCR mixture 2 ⁇ l of 20 ⁇ M oligonucleotide SA2, 2 ⁇ l of 20 ⁇ M oligonucleotide SD6 (5'- TCT GAG TCA CCT GGA CAA CC-3'; SEQ ID NO. 4 Gibco BRL), 0.8 ⁇ l of 10 mM dNTP mix. 4 ⁇ l of lOx GeneAmp PCR buffer (Perkin Elmer) and 22.7 ⁇ l deionized water. The mixture was overlaid with 50 ⁇ l mineral oil, placed in a Perkin Elmer Cetus 480 thermal cycler preheated to 94° C, and incubated for 5 minutes.
  • the temperature was reduced to 80° C, and 0.5 ⁇ l (2.5 units) of AmpliTaq DNA polymerase (Perkin Elmer) was added per tube.
  • Six cycles of PCR amplification were performed as follows: 94° C for 1 minute, 60° C for 1 minute, and 72° C for 5 minutes, with a final extension at 72° C for 10 minutes.
  • the secondary PCR reaction contained in a total volume of 50 ⁇ l: 5 ⁇ l of /is XI-treated primary PCR product, 1 ⁇ l of 20 ⁇ M secondary amplification primer mix [20 ⁇
  • PCR buffer Perkin Elmer
  • 38 ⁇ l deionized water The mixture was overlaid with 50 ⁇ l mineral oil, placed in a Perkin Elmer Cetus 480 thermal cycler preheated to 94° C, and incubated for 5 minutes. The temperature was reduced to 80° C, and 0.5 ⁇ l (2.5 units) of AmpliTaq DNA polymerase (Perkin Elmer) was added per tube. 30 cycles of PCR amplification were performed as follows: 94° C for 1 minute, 60° C for 1 minute, and 72° C for 3 minutes, with a final extension at 72° C for 10 minutes.
  • the UDG cloning reaction contained: 6 ⁇ l of secondary PCR product, 2 ⁇ l of pAMPIO cloning vector (Gibco BRL), 1 ⁇ l of lOxGeneAmp PCR buffer (Perkin Elmer), and 1 ⁇ l (1 unit) of uracil DNA glycosylase (Gibco BRL). The reaction was incubated at 37° C for 30 minutes. 2 ⁇ l of the reaction was transformed into DHl lS cells (Gibco BRL) by electroporation (Bio-Rad). Transformants were selected on LB/carbenicillin plates and evaluated by colony PCR.
  • the colony PCR reaction contained in 25 ⁇ l total volume: colony resuspended in 10 ⁇ l deionized water, 0.5 ⁇ l of 20 ⁇ M secondary amplification primer mix (see above), 0.5 ⁇ l of 10 mM dNTP mix, 2.5 ⁇ l of lOx GeneAmp PCR buffer (Perkin Elmer), 0.25 ⁇ l (1.25 units) AmpliTaq DNA Polymerase (Perkin Elmer), and 11.3 ⁇ l deionized water. PCR reactions were performed in the Perkin-Elmer Cetus GeneAmp PCR System 9600.
  • the amplification parameters were: 94° C for 5 min, followed by 30 cycles of: 94° C for 45 seconds, 55° C for 30 seconds, and 72° C for 1 minute, with a final extension at 72° C for 10 minutes.
  • the colony PCR products were analyzed in 2% agarose gels. Clones with insert sizes greater than 177 bp were sequenced using Ml 3 forward and reverse primers.
  • cDNA selection was used as an additional method for gene identification of transcribed sequences over large regions of the genome. Through a combination of characterizations including physical mapping and RNA hybridization, the selected cDNAs were arranged into transcription units. The cDNA selection technique was carried out as described by J.M. Rommens et al. transcription mapping
  • sample sequencing, exon trapping and cDNA selection were arranged into tentative transcription units which provided the framework for a detailed transcription map of the genomic region of interest.
  • HNFl and/or 4 was one of the genes. Based on the fact that the reduced abundance and binding activity of HNFl and/or 4 has been found to correlate with the decreased albumin gene transcription characterisic of diabetes mellitus (Barrera-Hernandez, G., et al.. (1996) J. Bio. Chem. 271:9969-9975), the HNFl gene was determined to be implicated in the Mody 3 form of type II diabetes.
  • the Diabetes Gene is Expressed in Pancreatic Islet Cells RT-PCR with primers at the 5' end of the gene was carried out to determine if the gene was expressed in pancreatic islets.
  • 30 ⁇ g of total RNA were treated with DNAse- free RNAse (Boehringer Mannhiem, Indianapolis, Indiana) and incubated for 30 min. at 37 C. After incubation the product was treated with phenol/chloroform and ethanol precipitated.
  • First-strand synthesis was carried out with 6 ⁇ g of DNAse-treated RNA, oligo dT and superscript reverse transcriptase (Life Technologies, Germantown, Maryland). The second strand was synthesized with a primer unique to the gene with Vent polymerase (New England Biolab, Beverly, MA) to increase fidelity during synthesis.
  • a DNA sequence encoding the mutant HNFl and/or 4 gene can be obtained based on PCR amplification with the following primers: Table 1 Exon sizes and primers
  • the group of NIDDM probands was divided according to family history of diabetes. Almost one half of the patients had one parent with diabetes and 8% had two parents with diabetes. Within the group of patients having only one diabetic parent, 280 families were identified in which the patient had at least one sibling with NIDDM. This group of families was evaluated further with regard to their suitability for genetic studies. In 75 families, diabetes was reported to occur on one side of the family and in at least two generations with large numbers of family members available for study. A total of 26 families (all Caucasian) have been examined out of those originally identified. The protocol for this study was approved by the Human Subjects Committee of the Joslin Diabetes Center.
  • the forward PCR primer was end-labeled with (32 ⁇ )-P-ATP by T4 polynucleotide kinase (Promega, Madison, WI) according to the manufacturer's protocol. PCR was performed with a 10 ⁇ l volume in a
  • PCR reaction contained 50 ng of genomic DNA, lOpmol of each primer, 25 ⁇ M dNTPs, 0.3-0.5mM MgCl 2 and 0.2 units of
  • PCR reactions ended with 5 minutes of final incubation at 72°C. After PCR amplification, 2 volumes of sequencing stop solution was added to the PCR reaction, the mixture was heated at 95°C for 5 minutes, 2 ⁇ l of the denatured PCR product was loaded onto 5% polyacrylamide DNA sequencing gels and resolved electrophoretically in 0.6 x TBE at 100- W constant current.
  • Consistency between different gels and between different lanes of the same gel was maintained by using either standard PCR products of known genotype or size standards from Research Genetics (Huntsville, AL). Dried gels were exposed to X-ray films for 12 hours without using the intensifying screen. Autoradiograms were scored by two independent observers. PCR primers were purchased from Research Genetics (Huntsville, AL). Primer sequences were available from Genome Data Base (Johns Hopkins University). Allele frequencies of markers were estimated from 80 unrelated non-diabetic individuals (the majority being spouses of NIDDM patients).
  • the parametric (lod score) method of linkage analysis was performed using LINKAGE (Lathrop, GM et al., (1984) Proc. Natl. Acad. Sci. USA 57:3443-3446). Because the families in this study were selected for an apparent autosomal dominant pattern of inheritance of NIDDM, linkage was investigated assuming an autosomal dominant model with age-dependent penetrance.
  • the susceptible genotypes in this model have an 85% lifetime penetrance (10% susceptible allele frequency) that is 5% under the age of 40 years and is maximum at age 70 years.
  • the models also incorporate a 7% sporadic rate.
  • Nonparametric linkage analysis was performed using the GENEHUNTER program (Kruglyak, L., et al., Am. J. Hum. Genet. 55:1347-63), which performs multipoint analysis among all affected family members at each location along chromosome 20 that is spanned by D20S107 and D20S100. This approach calculates the nonparametric lod score (NPL) at each position, comparing the observed IBD sharing among all families members to that expected under the null hypothesis of no linkage.
  • NPL nonparametric lod score
  • TDT transmission disequilibrium test
  • the TDT determines the frequency that an allele is transmitted from a heterozygous parent to an affected offspring, treating all offspring of a mating as independent observations (Schman, R.S., et al. (1993), Am. J. Hum. Genet. 52:506-516). Unlike strict linkage tests, TDT only detects linkage in the presence of allelic association.
  • NIDDM pedigrees that were ascertained through the following three characteristics: 1) a proband with NIDDM diagnosed between age 30 and 59 years, 2) NIDDM present in at least one sibling of the proband, and 3) one and only one parent having diabetes according to the information available to the proband; were examined.
  • the 449 individuals examined ranged from 15-95 years of age.
  • Table 1 shows the distribution of examined individuals in families ranked according to the mean age at diagnosis of NIDDM. On average, 5 NIDDM individuals in one or two generations were examined in each family (range from 2 to 9). In addition, an average of 12 non-diabetic individuals (usually in 2 or 3 generations) were examined in each family (range 3 to 29). The mean age of the non-diabetic individuals at the time of examination was similar to the age at onset of NIDDM in their relatives. The non-diabetic individuals were selected because they were siblings of NIDDM individuals or were children or grandchildren of deceased NIDDM individuals. On examining the families more carefully, we found four of them to be bilineal, whereas in the others diabetes occurred on only one side of family in at least two generations. Table 1 Characteristics of NIDDM families enrolled in the study according to mean age at diagnosis of NIDDM
  • markers D20S176 and D20S178 exhibited strong evidence of linkage with NIDDM, as did D20S197 but not D20S176. Additional markers (D20S107 and ADA8PR) also showed linkage with NIDDM in this analysis.
  • the results of robust sib-pair analysis did not change after adjustment for the age at onset of NIDDM (data not shown).
  • Multipoint linkage analysis was employed to incorporate information from all chromosome 20 markers that were evaluated in the MODYl region in order to infer more effectively the probability distribution of IBD status at each point in the interval of interest (Figure 2).
  • the peak lod scores occur for markers D20S119 (Z score 1.8) and D20S197/D20S178 (Z score 1.8). From previous parametric linkage results, there was clear evidence for linkage heterogeneity; thus, multipoint analyses were repeated in those families that suggested linkage to the MODYl region and in those that suggested absence of linkage to this region.
  • Evidence for association between NIDDM and D20S119 was determined by use of the transmission disequilibrium test (TDT). Using the TDT, there were 195 transmission from a heterozygous parent. There was no significant evidence for deviation of transmitted alleles from that expected, suggesting that while there was evidence for linkage (based upon both sib-pair and linkage analyses) there was no support for association with alleles at D20S119.
  • NIDDM patients in these and the other families did not differ with regard to age at onset or treatment of NIDDM, or % IBW.
  • ADDRESSEE FOLEY, HOAG & ELIOT LLP
  • MOLECULE TYPE other nucleic acid
  • MOLECULE TYPE other nucleic acid
  • MOLECULE TYPE other nucleic acid
  • MOLECULE TYPE other nucleic acid
  • DESCRIPTION: /desc "primer”
  • MOLECULE TYPE other nucleic acid
  • MOLECULE TYPE other nucleic acid
  • MOLECULE TYPE other nucleic acid
  • MOLECULE TYPE other nucleic acid
  • MOLECULE TYPE other nucleic acid
  • MOLECULE TYPE other nucleic acid
  • MOLECULE TYPE other nucleic acid
  • MOLECULE TYPE other nucleic acid
  • MOLECULE TYPE other nucleic acid
  • MOLECULE TYPE other nucleic acid
  • MOLECULE TYPE other nucleic acid
  • MOLECULE TYPE other nucleic acid

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Toxicology (AREA)
  • Marine Sciences & Fisheries (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Immunology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

Sous l'un de ses aspects, l'invention concerne, d'une part des composés agissant en agonistes de l'activité biologique du facteur HNF (facteur nucléaire hépatique) normal (fonctionnel), et d'autre part l'utilisation de ces composés en thérapie contre le diabète. Ainsi, pour améliorer les symptômes cliniques liés à une insuffisance de l'expression d'un gène HNF et/ou à un niveau inapproprié de l'activité biologique du HNF fonctionnel chez un sujet, il est possible d'appliquer une thérapie génique (utilisant un gène codant une protéine HNF fonctionnelle) ou d'appliquer une thérapie protéique (utilisant une protéine du HNF fonctionnel. Sous un autre aspect, l'invention concerne, d'une part des composés agissant comme antagonistes d'une affection provoquant une activité biologique du HNF, et d'autre part l'utilisation de ces composés en thérapie contre le diabète. Ainsi, pour améliorer les symptômes d'une affection liée à l'expression d'un gène HNF mutant, ou à l'hyperexpression d'un gène HNF normal chez un sujet, il est possible d'administrer à ce sujet une quantité thérapeutiquement efficace d'une molécule antisens, ribozyme ou triple hélice de façon à diminuer l'expression génique déjà évoquée. Selon une autre réalisation, pour améliorer les symptômes cliniques liés à la régulation via une protéine HNF d'un élément amont ou aval d'un canal biochimique à médiation HNF (par exemple une transduction de signal), il est possible d'induire un effet thérapeutique au moyen d'une quantité thérapeutiquement efficace d'un composé antagoniste (tel qu'une petite molécule, un peptide ou un peptidomimétique) capable d'empêcher la liaison de la protéine HNF sauvage. En outre, pour améliorer les symptômes cliniques liés à une protéine HNF (non fonctionnelle) mutante, il est possible d'administrer au sujet considéré une quantité thérapeutiquement efficace d'un anticorps anti-HNF déjà évoqué.
PCT/US1997/020759 1996-11-15 1997-11-14 Compositions et therapies utilisables contre le diabete de type ii lie au hnf-4 WO1998021363A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU52571/98A AU5257198A (en) 1996-11-15 1997-11-14 Compositions and methods for treating type ii diabetes involving hnf-4

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US74943096A 1996-11-15 1996-11-15
US08/749,430 1996-11-15

Publications (1)

Publication Number Publication Date
WO1998021363A1 true WO1998021363A1 (fr) 1998-05-22

Family

ID=25013733

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1997/020759 WO1998021363A1 (fr) 1996-11-15 1997-11-14 Compositions et therapies utilisables contre le diabete de type ii lie au hnf-4

Country Status (2)

Country Link
AU (1) AU5257198A (fr)
WO (1) WO1998021363A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6187533B1 (en) 1996-09-10 2001-02-13 Arch Development Corporation Mutations in the diabetes susceptibility genes hepatocyte nuclear factor (HNF) 1 alpha (α), HNF1β and HNF4α
WO2001075123A1 (fr) * 2000-02-29 2001-10-11 Fudan University Nouveau polypeptide, facteur nucleolaire hepatique 12, et polynucleotide codant pour ce polypeptide
WO2002072874A1 (fr) * 2001-03-14 2002-09-19 Fujisawa Pharmaceutical Co., Ltd. Procede de criblage d'un agoniste hnf4$g(a)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992011365A1 (fr) * 1990-12-21 1992-07-09 The Rockefeller University Facteur de transcription enrichi par extraits hepatiques
WO1996032489A1 (fr) * 1995-04-14 1996-10-17 Institut National De La Sante Et De La Recherche Medicale Vecteur viral recombinant inductible par le glucose

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992011365A1 (fr) * 1990-12-21 1992-07-09 The Rockefeller University Facteur de transcription enrichi par extraits hepatiques
WO1996032489A1 (fr) * 1995-04-14 1996-10-17 Institut National De La Sante Et De La Recherche Medicale Vecteur viral recombinant inductible par le glucose

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
BARRERA-HERNANDEZ ET AL.: "Phlorizin or vanadate treatment reverses impaired expression of albumin and hepatocyte nuclear factor 1 in diabetic rats", DIABETES, vol. 45, no. 9, September 1996 (1996-09-01), pages 1217 - 1222, XP002059717 *
BELL G I ET AL: "GENE FOR NON-INSULIN-DEPENDENT DIABETES MELLITUS (MATURITY-ONSET DIABETES OF THE YOUNG SUBTYPE) IS LINKED TO DNA POLYMORPHISM ON HUMAN CHROMOSOME 20Q", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, vol. 88, February 1991 (1991-02-01), pages 1484 - 1488, XP002051546 *
CUIF ET AL: "Exploration of a liver-specific, glucose/insulin-responsive promoter in transgenic mice", J BIOLOGICAL CHEMISTRY, vol. 268, no. 19, 1993, pages 13769 - 13772, XP002059715 *
DREWES ET AL.: "Human hepatocyte nuclear factor 4 isoforms are encoded by distinct and differentially expressed genes", MOLECULAR CELLULAR BIOLOGY, vol. 16, no. 3, March 1996 (1996-03-01), pages 925 - 931, XP002059901 *
KRITIS A A ET AL: "Isolation and characterization of a third isoform of human hepatocyte nuclear factor 4", GENE, vol. 2, no. 173, 16 September 1996 (1996-09-16), pages 275-280, XP004043230 *
NAKAJIMA ET AL.: "Hepatocyte nuclear factor 4 alpha gene mutations in Japanese non-insulin dependent diabetes mellitus (NIDDM) patients", RES COMM MOLECUL. PATHOL. PHARMACOL., vol. 94, no. 3, December 1996 (1996-12-01), pages 327 - 330, XP002059714 *
POLONSKY ET AL.: "Non-insulin-dependent diabetes mellitus - a genetically programmed failure of the beta cell to compensate for insulin resistance", N ENGL. J. MED., vol. 334, no. 12, March 1996 (1996-03-01), pages 777 - 783, XP002059716 *
YAMAGATA ET AL.: "Mutations in the hepatocyte nuclear factor-4alpha gene in maturity onset diabetes of the young (MODY1)", NATURE, vol. 384, no. 6608, 5 December 1996 (1996-12-05), pages 458 - 460, XP002059713 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6187533B1 (en) 1996-09-10 2001-02-13 Arch Development Corporation Mutations in the diabetes susceptibility genes hepatocyte nuclear factor (HNF) 1 alpha (α), HNF1β and HNF4α
WO2001075123A1 (fr) * 2000-02-29 2001-10-11 Fudan University Nouveau polypeptide, facteur nucleolaire hepatique 12, et polynucleotide codant pour ce polypeptide
WO2002072874A1 (fr) * 2001-03-14 2002-09-19 Fujisawa Pharmaceutical Co., Ltd. Procede de criblage d'un agoniste hnf4$g(a)

Also Published As

Publication number Publication date
AU5257198A (en) 1998-06-03

Similar Documents

Publication Publication Date Title
US5800998A (en) Assays for diagnosing type II diabetes in a subject
AU6952498A (en) Novel human delta3 compositions and therapeutic and diagnostic uses therefor
US5807708A (en) Conservin nucleic acid molecules and compositions
US5885776A (en) Glaucoma compositions and therapeutic and diagnositic uses therefor
US6143491A (en) Therapeutic compositions and methods and diagnostic assays for type II diabetes involving HNF-1
US6399760B1 (en) RP compositions and therapeutic and diagnostic uses therefor
US6087107A (en) Therapeutics and diagnostics for congenital heart disease based on a novel human transcription factor
US6593104B1 (en) Macular degeneration diagnostics and therapeutics
US6403307B1 (en) Glaucoma therapeutics and diagnostics
US6518411B1 (en) RGS compositions and therapeutic and diagnostic uses therefor
CA2271235A1 (fr) Proteine associee au glaucome, acide nucleique correspondant et leurs utilisations diagnostiques et therapeutiques
WO1998046748A1 (fr) Compositions therapeutiques et dosages de diagnostic pour des affections liees a la trbp
AU766890B2 (en) Glaucoma therapeutics and diagnostics based on a novel human transcription factor
US6008014A (en) Method of making lipid metabolic pathway compositions
US6271026B1 (en) Glaucoma compositions
WO1998021363A1 (fr) Compositions et therapies utilisables contre le diabete de type ii lie au hnf-4
US6956103B2 (en) Glaucoma therapeutics and diagnostics
WO1998009979A9 (fr) Compositions relatives a la voie metabolique lipidique et utilisations therapeutiques et diagnostiques de telles compositions
US6306586B1 (en) Methods and compositions for the diagnosis and treatment of cataracts
US20050123960A1 (en) Glaucoma therapeutics and diagnostics
WO1998040495A1 (fr) Doublin, gene participant au developpement neuronal et utilisations de ce gene
AU3556402A (en) Glaucoma-associated protein and corresponding nucleic acid and their therapeutic and diagnostic uses

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU CA JP

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA