WO2006008008A2 - Diagnostics and therapeutics for diseases associated with hepatocyte nuclear factor 4, alpha (hnf4a) - Google Patents

Abstract

The invention provides a human HNF4A which is associated with the gastrointestinal and liver diseases, cancer disorders, cardiovascular disorders, metabolic diseases, inflammatory diseases, muscle-skeleton disorders, respiratory diseases, reproduction disorders and urological disorders.. The invention also provides assays for the identification of compounds useful in the treatment or prevention of gastrointestinal and liver diseases, cancer disorders, cardiovascular disorders, metabolic diseases, inflammatory diseases, muscle-skeleton disorders, respiratory diseases, reproduction disorders and urological disorders.. The invention also features compounds which bind to and/or activate or inhibit the activity of HNF4A as well as pharmaceutical compositions comprising such compounds.

Description

Diagnostics and Therapeutics for Diseases Associated with Hepatocvte Nuclear Factor 4, Alpha (ΗNF4A)

Technical field of the invention

The present invention is in the field of molecular biology, more particularly, the present invention relates to nucleic acid sequences and amino acid sequences of a human HNF4A and its regulation for the treatment of gastrointestinal and liver diseases, cancer disorders, cardiovascular disorders, metabolic diseases, inflammatory diseases, muscle-skeleton disorders, respiratory diseases, reproduction disorders and urological disorders, in mammals.

Background of the invention

Nuclear Receptors

HNF4A belongs to the superfamily of nuclear receptors (NR) [US2002001823, US6025196, WO200157190, Chartier et al. (1994), Thomas et al. (2001), Tirona et al. (2003), Parviz et al. (2003), Ribeiro et al. (1999), Li et al. (2000)] which forms a related but diverse array of ligand- modulated transcription factors that control diverse aspects of growth, development and homeostasis [Olefsky & Jerrold, 2001]. The family comprises the nuclear hormone receptors for which hormonal ligands have been identified, and orphan receptors with still unknown ligands.

The ligands of NRs are generally lipophilic because they traverse the plasma membrane to bind to their cognate receptors that are located in the cytoplasm or in the nucleus.

Six structural domains A-F (from N- to C-terminus) of NR are known [Bourguet et al. 2000]. All of the NRs have common structural features which include a central DNA binding domain (DBD, domain C) responsible for targeting the receptor to a specific DNA sequence, a ligand binding domain (LBD, domain E), that binds to specific ligands and activates transcription and a hinge region (domain D) connecting DBD and LBD. The N-terminus is formed by the A/B domain comprising a ligand-independent activation domain. The C-terminal F domain, the function of which is poorly understood, is not present in all receptors.

NRs activate transcription as homo- or heterodimers by binding to specific response elements (REs). The REs exist of two half-sites with different spacings, forming inverted or direct repeats. Based on their mode of action four different classes of NRs have been defined: class 1 is formed by the known steroid hormone receptors, that act as homodimers and bind to inverted repeats; class 2 includes receptors forming heterodimers with RXR; class 3 contains homodimeric NRs binding to direct repeats and class 4 is formed by NRs that bind as monomers to single half-sites. Classes 3 and 4 contain only orphan receptors.

NRs act as transcription activators in complex with co-activators. Ligand binding to NRs induces a conformational change which allows recruitment of specific co-activators. In the absence of ligand some receptors also act as transcriptional repressors in complex with co-repressors.

The fact that steroid hormone receptors belong to the family of nuclear receptors indicates that these receptors have an established, proven history as therapeutic targets. Examples for marketed pharmaceuticals based on steroid receptor ligands comprise preparations containing oestrogens, coticosteroids or mineralocorticoids. Clearly, there is a need for identification and characterization of further receptors which can play a role in preventing, ameliorating, or correcting dysfunctions or diseases including, but not limited to, infections such as bacterial, fungal, protozoan, and viral infections, particularly those caused by HIV viruses, cancers, allergies including asthma, cardiovascular diseases including acute heart failure, hypotension, hypertension, angina pectoris, myocardial infarction, hematological diseases, genito-urinary diseases including urinary incontinence and benign prostate hyperplasia, osteoporosis, peripheral and central nervous system disorders including pain, Alzheimer's disease and Parkinson's disease, respiratory diseases, metabolic diseases, inflammatory diseases, gastroenterological diseases, diseases of the endocrine system, dermatological diseases, diseases of muscles or the skeleton, immunological diseases, developmental diseases or diseases of the reproductive system.

TaqMan-Technology / expression profiling

TaqMan is a recently developed technique, in which the release of a fluorescent reporter dye from a hybridisation probe in real-time during a polymerase chain reaction (PCR) is proportional to the accumulation of the PCR product. Quantification is based on the early, linear part of the reaction, and by determining the threshold cycle (CT), at which fluorescence above background is first detected.

Gene expression technologies may be useful in several areas of drug discovery and development, such as target identification, lead optimization, and identification of mechanisms of action. The TaqMan technology can be used to compare differences between expression profiles of normal tissue and diseased tissue. Expression profiling has been used in identifying genes, which are up- or downregulated in a variety of diseases. An interesting application of expression profiling is temporal monitoring of changes in gene expression during disease progression and drug treatment or in patients versus healthy individuals. The premise in this approach is that changes in pattern of gene expression in response to physiological or environmental stimuli (e.g., drugs) may serve as indirect clues about disease-causing genes or drug targets. Moreover, the effects of drugs with established efficacy on global gene expression patterns may provide a guidepost, or a genetic signature, against which a new drug candidate can be compared.

HNF4A

The nucleotide sequence of HNF4A is accessible in public databases by the accession number

NM_000457 and is given in SEQ ID NO:1. The amino acid sequence of HNF4A is depicted in SEQ ID NO:2.

Cell specificity is based on differential gene expression, which is in turn determined, at least in part, by a particular set of transcription factors present and active in a given cell at a certain time. Isoforms of a transcription factor can be expressed at different stages of cell differentiation. Many transcription factors have been identified and characterized, particularly in the liver where there is a wide range of transcriptionally controlled genes. The extinction of many hepatic functions and their reexpression are correlated with the extinction and expression of hepatocyte nuclear factor 4 (HNF4). Moreover, HNF4 has a key role in a transcriptional hierarchy since it also controls the expression of the transcription factor HNFl (TCFl), which is important in the expression of several hepatic genes. Chartier et al. (1994) demonstrated that there are 2 isoforms of HNF4 in human liver, a situation comparable to that in the rat. The 2 isoforms differ by an extra peptide of 10 amino acids located in the C-terminal part of the protein. The gene is also symbolized TCF 14

Thomas et al. (2001) identified an alternative promoter of the HNF4A gene, P2, which is 46 kb 5- prime to the previously identified Pl promoter of the human gene. Based on RT-PCR, this distant upstream P2 promoter represents the major transcription site in pancreatic beta-cells, and is also used in hepatic cells. Transfection assays with various deletions and mutants of the P2 promoter revealed functional binding sites for HNFlA, HNFlB, and IPFl, the other transcription factors known to encode MODY genes. In a large MODY family, a mutated IPFl binding site in the P2 promoter of the HNF4A gene cosegregated with diabetes (lod score 3.25). The authors proposed a regulatory network of the 4 MODY transcription factors interconnected at the distant upstream P2 promoter of the HNF4A gene

Tirona et al. (2003) showed that HNF4A is critically involved in the PXR- and CAR-mediated transcriptional activation of CYP3 A4. They identified a specific cis-acting element in the CYP3 A4 gene enhancer that confers HNF4-alpha binding and thereby permits PXR- and CAR-mediated gene activation. Fetal mice with conditional deletion of Hnf4-alpha had reduced or absent expression of CYP3A. Furthermore, adult mice with conditional hepatic deletion of the gene had reduced basal and inducible expression of CYP3A. These data identified HNF4-alpha as an important regulator of coordinate nuclear receptor-mediated response to xenobiotics. To elucidate how differentiated cells form tissues and organs, Parviz et al. (2003) studied liver organogenesis because the cell and tissue architecture of this organ is well defined. Approximately 60% of the adult liver consists of hepatocytes that are arranged as single-cell anastomosing plates extending from the portal region of the liver lobule toward the central vein. The basal surface of the hepatocytes is separated from adjacent sinusoidal endothelial cells by the space of Disse, where the exchange of substances between serum and hepatocytes takes place. The apical surface of the hepatocytes forms bile canaliculi that transport bile to the hepatic ducts. Parviz et al. (2003) reported that hepatocyte nuclear factor 4-alpha is essential for morphologic and functional differentiation of hepatocytes, accumulation of hepatic glycogen stores, and generation of a hepatic epithelium. They showed that HNF4A is a dominant regulator of the epithelial phenotype because its ectopic expression in fibroblasts induces a mesenchymal-to-epithelial transition. The morphogenetic parameters controlled by HNF4A in hepatocytes are essential for normal liver architecture, including the organization of the sinusoidal endothelium.

By cotransfection in COS-I cells, Ribeiro et al. (1999) showed that mammalian HNF4 synergized with USF2a in the transactivation of the APOA2 promoter. HNF4 and USF2a bound to the enhancer cooperatively, which Ribeiro et al. (1999) suggested may account for the transcriptional synergism observed.

To study the contribution of HNF4A to hepatic development and differentiation, Li et al. (2000) used a technique in which Hnf4a -/- mouse embryos were complemented with wildtype visceral endoderm to counteract early embryonic lethality. By histologic analyses, the authors found that specification and early development of the liver and liver morphology did not require Hnf4a. In addition, the expression of many liver genes was unaffected in these mice. However, RT-PCR analysis showed that Hnf4a -/- fetal livers failed to express a large array of genes whose expression in differentiated hepatocytes is essential for a functional hepatic parenchyma, including apolipoproteins (e.g., APOAl), metabolic proteins (e.g., aldolase B), transferrin, retinol-binding protein (e.g., RBP4), and erythropoietin. The lack of Hnf4a did not affect the expression of most transcription factors but did significantly reduce the levels of Hnfla (TCFl) and the pregnane X receptor (NR 112), suggesting that HNF4A acts upstream of at least these 2 transcription factors, which are also important in hepatocyte gene expression

HNF4A is published in US2002001823, US6025196, WO200157190.

HNF4A shows the highest homology (48%) to the human mixed lineage kinase MLKl as shown in example 1. Summary of the invention

The invention relates to novel disease associations of HNF4A polypeptides and polynucleotides. The invention also relates to novel methods of screening for therapeutic agents for the treatment of gastrointestinal and liver diseases, cancer disorders, cardiovascular disorders, metabolic diseases, inflammatory diseases, muscle-skeleton disorders, respiratory diseases, reproduction disorders and urological disorders, in a mammal. The invention also relates to pharmaceutical compositions for the treatment of gastrointestinal and liver diseases, cancer disorders, cardiovascular disorders, metabolic diseases, inflammatory diseases, muscle-skeleton disorders, respiratory diseases, reproduction disorders and urological disorders, in a mammal comprising a HNF4A polypeptide, a HNF4A polynucleotide, or regulators of HNF4A or modulators of HNF4A activity. The invention further comprises methods of diagnosing gastrointestinal and liver diseases, cancer disorders, cardiovascular disorders, metabolic diseases, inflammatory diseases, muscle-skeleton disorders, respiratory diseases, reproduction disorders and urological disorders, in a mammal.

Brief Description of the Drawings

Fig. 1 shows the nucleotide sequence of a HNF4A polynucleotide (SEQ ID NO:1).

Fig. 2 shows the amino acid sequence of a HNF4A polypeptide (SEQ ID NO:2).

Fig. 3 shows the nucleotide sequence of a primer useful for the invention (SEQ ID NO:3).

Fig. 4 shows the nucleotide sequence of a primer useful for the invention (SEQ ID NO:4).

Fig. 5 shows a nucleotide sequence useful as a probe to detect proteins of the invention (SEQ ID NO: 5).

Detailed description of the invention

Definition of terms

An "oligonucleotide" is a stretch of nucleotide residues which has a sufficient number of bases to be used as an oligomer, amplimer or probe in a polymerase chain reaction (PCR). Oligonucleotides are prepared from genomic or cDNA sequence and are used to amplify, reveal, or confirm the presence of a similar DNA or RNA in a particular cell or tissue. Oligonucleotides or oligomers comprise portions of a DNA sequence having at least about 10 nucleotides and as many as about 35 nucleotides, preferably about 25 nucleotides. "Probes" may be derived from naturally occurring or recombinant single- or double-stranded nucleic acids or may be chemically synthesized. They are useful in detecting the presence of identical or similar sequences. Such probes may be labeled with reporter molecules using nick translation, Klenow fill-in reaction, PCR or other methods well known in the art. Nucleic acid probes may be used in southern, northern or in situ hybridizations to determine whether DNA or

RNA encoding a certain protein is present in a cell type, tissue, or organ.

A "fragment of a polynucleotide" is a nucleic acid that comprises all or any part of a given nucleotide molecule, the fragment having fewer nucleotides than about 6 kb, preferably fewer than about 1 kb.

"Reporter molecules" are radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents which associate with a particular nucleotide or amino acid sequence, thereby establishing the presence of a certain sequence, or allowing for the quantification of a certain sequence.

"Chimeric" molecules may be constructed by introducing all or part of the nucleotide sequence of this invention into a vector containing additional nucleic acid sequence which might be expected to change any one or several of the following HNF4A characteristics: cellular location, distribution, ligand-binding affinities, interchain affinities, degradation/turnover rate, signaling, etc.

"Active", with respect to a HNF4A polypeptide, refers to those forms, fragments, or domains of a HNF4A polypeptide which retain the biological and/or antigenic activity of a HNF4A polypeptide.

"Naturally occurring HNF4A polypeptide" refers to a polypeptide produced by cells which have not been genetically engineered and specifically contemplates various polypeptides arising from post-translational modifications of the polypeptide including but not limited to acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation.

"Derivative" refers to polypeptides which have been chemically modified by techniques such as ubiquitination, labeling (see above), pegylation (derivatization with polyethylene glycol), and chemical insertion or substitution of amino acids such as ornithine which do not normally occur in human proteins.

"Conservative amino acid substitutions" result from replacing one amino acid with another having similar structural and/or chemical properties, such as the replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine.

"Insertions" or "deletions" are typically in the range of about 1 to 5 amino acids. The variation allowed may be experimentally determined by producing the peptide synthetically while systematically making insertions, deletions, or substitutions of nucleotides in the sequence using recombinant DNA techniques.

A "signal sequence" or "leader sequence" can be used, when desired, to direct the polypeptide through a membrane of a cell. Such a sequence may be naturally present on the polypeptides of the present invention or provided from heterologous sources by recombinant DNA techniques.

An "oligopeptide" is a short stretch of amino acid residues and may be expressed from an oligonucleotide. Oligopeptides comprise a stretch of amino acid residues of at least 3, 5, 10 amino acids and at most 10, 15, 25 amino acids, typically of at least 9 to 13 amino acids, and of sufficient length to display biological and/or antigenic activity.

"Inhibitor" is any substance which retards or prevents a chemical or physiological reaction or response. Common inhibitors include but are not limited to antisense molecules, antibodies, and antagonists.

"Standard expression" is a quantitative or qualitative measurement for comparison. It is based on a statistically appropriate number of normal samples and is created to use as a basis of comparison when performing diagnostic assays, running clinical trials, or following patient treatment profiles.

"Animal" as used herein may be defined to include human, domestic (e.g., cats, dogs, etc.), agricultural (e.g., cows, horses, sheep, etc.) or test species (e.g., mouse, rat, rabbit, etc.).

A "HNF4A polynucleotide", within the meaning of the invention, shall be understood as being a nucleic acid molecule selected from a group consisting of

(i) nucleic acid molecules encoding a polypeptide comprising the amino acid sequence of

SEQ ID NO: 2,

(ii) nucleic acid molecules comprising the sequence of SEQ ID NO: 1,

(iii) nucleic acid molecules having the sequence of SEQ ID NO: 1 ,

(iv) nucleic acid molecules the complementary strand of which hybridizes under stringent conditions to a nucleic acid molecule of (i), (ii), or (iii); and

(v) nucleic acid molecules the sequence of which differs from the sequence of a nucleic acid molecule of (iii) due to the degeneracy of the genetic code;

wherein the polypeptide encoded by said nucleic acid molecule has HNF4A activity. A "HNF4A polypeptide", within the meaning of the invention, shall be understood as being a polypeptide selected from a group consisting of

(i) polypeptides having the sequence of SEQ E) NO: 2,

(ii) polypeptides comprising the sequence of SEQ ID NO: 2,

(iii) polypeptides encoded by HNF4A polynucleotides; and

(iv) polypeptides which show at least 99%, 98%, 95%, 90%, or 80% homology with a polypeptide of (i), (ii), or (iii);

wherein said polypeptide has HNF4A activity.

The nucleotide sequences encoding a HNF4A (or their complement) have numerous applications in techniques known to those skilled in the art of molecular biology. These techniques include use as hybridization probes, use in the construction of oligomers for PCR, use for chromosome and gene mapping, use in the recombinant production of HNF4A, and use in generation of antisense DNA or RNA, their chemical analogs and the like. Uses of nucleotides encoding a HNF4A disclosed herein are exemplary of known techniques and are not intended to limit their use in any technique known to a person of ordinary skill in the art. Furthermore, the nucleotide sequences disclosed herein may be used in molecular biology techniques that have not yet been developed, provided the new techniques rely on properties of nucleotide sequences that are currently known, e.g., the triplet genetic code, specific base pair interactions, etc.

It will be appreciated by those skilled in the art that as a result of the degeneracy of the genetic code, a multitude of HNF4A - encoding nucleotide sequences may be produced. Some of these will only bear minimal homology to the nucleotide sequence of the known and naturally occurring

HNF4A. The invention has specifically contemplated each and every possible variation of nucleotide sequence that could be made by selecting combinations based on possible codon choices. These combinations are made in accordance with the standard triplet genetic code as applied to the nucleotide sequence of naturally occurring HNF4A, and all such variations are to be considered as being specifically disclosed.

Although the nucleotide sequences which encode a HNF4A, its derivatives or its variants are preferably capable of hybridizing to the nucleotide sequence of the naturally occurring HNF4A polynucleotide under stringent conditions, it may be advantageous to produce nucleotide sequences encoding HNF4A polypeptides or its derivatives possessing a substantially different codon usage. Codons can be selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic expression host in accordance with the frequency with which particular codons are utilized by the host. Other reasons for substantially altering the nucleotide sequence encoding a HNF4A polypeptide and/or its derivatives without altering the encoded amino acid sequence include the production of RNA transcripts having more desirable properties, such as a greater half-life, than transcripts produced from the naturally occurring sequence.

Nucleotide sequences encoding a HNF4A polypeptide may be joined to a variety of other nucleotide sequences by means of well established recombinant DNA techniques. Useful nucleotide sequences for joining to HNF4A polynucleotides include an assortment of cloning vectors such as plasmids, cosmids, lambda phage derivatives, phagemids, and the like. Vectors of interest include expression vectors, replication vectors, probe generation vectors, sequencing vectors, etc. In general, vectors of interest may contain an origin of replication functional in at least one organism, convenient restriction endonuclease sensitive sites, and selectable markers for one or more host cell systems.

Another aspect of the subject invention is to provide for HNF4A-specific hybridization probes capable of hybridizing with naturally occurring nucleotide sequences encoding HNF4A. Such probes may also be used for the detection of similar Nuclear Receptors encoding sequences and should preferably show at least 40% nucleotide identity to HNF4A polynucleotides. The hybridization probes of the subject invention may be derived from the nucleotide sequence presented as SEQ E) NO: 1 or from genomic sequences including promoter, enhancers or introns of the native gene. Hybridization probes may be labelled by a variety of reporter molecules using techniques well known in the art.

It will be recognized that many deletional or mutational analogs of HNF4A polynucleotides will be effective hybridization probes for HNF4A polynucleotides. Accordingly, the invention relates to nucleic acid sequences that hybridize with such HNF4A encoding nucleic acid sequences under stringent conditions.

"Stringent conditions" refers to conditions that allow for the hybridization of substantially related nucleic acid sequences. For instance, such conditions will generally allow hybridization of sequence with at least about 85% sequence identity, preferably with at least about 90% sequence identity, more preferably with at least about 95% sequence identity. Hybridization conditions and probes can be adjusted in well-characterized ways to achieve selective hybridization of human- derived probes. Stringent conditions, within the meaning of the invention are 65°C in a buffer containing 1 mM EDTA, 0.5 M NaHPO₄ (pH 7.2), 7 % (w/v) SDS. Nucleic acid molecules that will hybridize to HNF4A polynucleotides under stringent conditions can be identified functionally. Without limitation, examples of the uses for hybridization probes include: histochemical uses such as identifying tissues that express HNF4A; measuring mRNA levels, for instance to identify a sample's tissue type or to identify cells that express abnormal levels of HNF4A; and detecting polymorphisms of HNF4A.

PCR provides additional uses for oligonucleotides based upon the nucleotide sequence which encodes HNF4A. Such probes used in PCR may be of recombinant origin, chemically synthesized, or a mixture of both. Oligomers may comprise discrete nucleotide sequences employed under optimized conditions for identification of HNF4A in specific tissues or diagnostic use. The same two oligomers, a nested set of oligomers, or even a degenerate pool of oligomers may be employed under less stringent conditions for identification of closely related DNAs or RNAs.

Rules for designing polymerase chain reaction (PCR) primers are now established, as reviewed by PCR Protocols. Degenerate primers, i.e., preparations of primers that are heterogeneous at given sequence locations, can be designed to amplify nucleic acid sequences that are highly homologous to, but not identical with HNF4A. Strategies are now available that allow for only one of the primers to be required to specifically hybridize with a known sequence. For example, appropriate nucleic acid primers can be ligated to the nucleic acid sought to be amplified to provide the hybridization partner for one of the primers. In this way, only one of the primers need be based on the sequence of the nucleic acid sought to be amplified.

PCR methods for amplifying nucleic acid will utilize at least two primers. One of these primers will be capable of hybridizing to a first strand of the nucleic acid to be amplified and of priming enzyme-driven nucleic acid synthesis in a first direction. The other will be capable of hybridizing the reciprocal sequence of the first strand (if the sequence to be amplified is single stranded, this sequence will initially be hypothetical, but will be synthesized in the first amplification cycle) and of priming nucleic acid synthesis from that strand in the direction opposite the first direction and towards the site of hybridization for the first primer. Conditions for conducting such amplifications, particularly under preferred stringent hybridization conditions, are well known.

Other means of producing specific hybridization probes for HNF4A include the cloning of nucleic acid sequences encoding HNF4A or HNF4A derivatives into vectors for the production of mRNA probes. Such vectors are known in the art, are commercially available and may be used to synthesize RNA probes in vitro by means of the addition of the appropriate RNA polymerase as T7 or SP6 RNA polymerase and the appropriate reporter molecules. - l i ¬

lt is possible to produce a DNA sequence, or portions thereof, entirely by synthetic chemistry. After synthesis, the nucleic acid sequence can be inserted into any of the many available DNA vectors and their respective host cells using techniques which are well known in the art. Moreover, synthetic chemistry may be used to introduce mutations into the nucleotide sequence. Alternately, a portion of sequence in which a mutation is desired can be synthesized and recombined with longer portion of an existing genomic or recombinant sequence.

HNF4A polynucleotides may be used to produce a purified oligo-or polypeptide using well known methods of recombinant DNA technology. The oligopeptide may be expressed in a variety of host cells, either prokaryotic or eukaryotic. Host cells may be from the same species from which the nucleotide sequence was derived or from a different species. Advantages of producing an oligonucleotide by recombinant DNA technology include obtaining adequate amounts of the protein for purification and the availability of simplified purification procedures.

Quantitative determinations of nucleic acids

An important step in the molecular genetic analysis of human disease is often the enumeration of the copy number of a nucleis acid or the relative expression of a gene in particular tissues.

Several different approaches are currently available to make quantitative determinations of nucleic acids. Chromosome-based techniques, such as comparative genomic hybridization (CGH) and fluorescent in situ hybridization (FISH) facilitate efforts to cytogenetically localize genomic regions that are altered in tumor cells. Regions of genomic alteration can be narrowed further using loss of heterozygosity analysis (LOH), in which disease DNA is analyzed and compared with normal DNA for the loss of a heterozygous polymorphic marker. The first experiments used restriction fragment length polymorphisms (RFLPs) [Johnson, (1989)], or hypervariable minisatellite DNA [Barnes, 2000]. In recent years LOH has been performed primarily using PCR amplification of microsatellite markers and electrophoresis of the radio labelled [Jeffreys, (1985)] or fluorescently labelled PCR products [Weber, (1990)] and compared between paired normal and disease DNAs.

A number of other methods have also been developed to quantify nucleic acids [Gergen, (1992)]. More recently, PCR and RT-PCR methods have been developed which are capable of measuring the amount of a nucleic acid in a sample. One approach, for example, measures PCR product quantity in the log phase of the reaction before the formation of reaction products plateaus

[Thomas, (1980)]. A gene sequence contained in all samples at relatively constant quantity is typically utilized for sample amplification efficiency normalization. This approach, however, suffers from several drawbacks. The method requires that each sample has equal input amounts of the nucleic acid and that the amplification efficiency between samples is identical until the time of analysis. Furthermore, it is difficult using the conventional methods of PCR quantitation such as gel electrophoresis or plate capture hybridization to determine that all samples are in fact analyzed during the log phase of the reaction as required by the method.

Another method called quantitative competitive (QC)-PCR, as the name implies, relies on the inclusion of an internal control competitor in each reaction [Piatak, (1993), BioTechniques]. The efficiency of each reaction is normalized to the internal competitor. A known amount of internal competitor is typically added to each sample. The unknown target PCR product is compared with the known competitor PCR product to obtain relative quantitation. A difficulty with this general approach lies in developing an internal control that amplifies with the same efficiency than the target molecule.

5' Fluorogenic Nuclease Assays

Fluorogenic nuclease assays are a real time quantitation method that uses a probe to monitor formation of amplification product. The basis for this method of monitoring the formation of amplification product is to measure continuously PCR product accumulation using a dual-labelled fluorogenic oligonucleotide probe, an approach frequently referred to in the literature simply as the "TaqMan method" [Piatak,(1993), Science; Heid, (1996); Gibson, (1996); Holland. (1991)].

The probe used in such assays is typically a short (about 20-25 bases) oligonucleotide that is labeled with two different fluorescent dyes. The 5' terminus of the probe is attached to a reporter dye and the 3' terminus is attached to a quenching dye, although the dyes could be attached at other locations on the probe as well. The probe is designed to have at least substantial sequence complementarity with the probe binding site. Upstream and downstream PCR primers which bind to flanking regions of the locus are added to the reaction mixture. When the probe is intact, energy transfer between the two fluorophors occurs and the quencher quenches emission from the reporter. During the extension phase of PCR, the probe is cleaved by the 5 ' nuclease activity of a nucleic acid polymerase such as Taq polymerase, thereby releasing the reporter from the oligonucleotide-quencher and resulting in an increase of reporter emission intensity which can be measured by an appropriate detector.

One detector which is specifically adapted for measuring fluorescence emissions such as those created during a fluorogenic assay is the ABI 7700 or 4700 HT manufactured by Applied Biosystems, Inc. in Foster City, Calif. The ABI 7700 uses fiber optics connected with each well in a 96-or 384 well PCR tube arrangement. The instrument includes a laser for exciting the labels and is capable of measuring the fluorescence spectra intensity from each tube with continuous monitoring during PCR amplification. Each tube is re-examined every 8.5 seconds.

Computer software provided with the instrument is capable of recording the fluorescence intensity of reporter and quencher over the course of the amplification. The recorded values will then be used to calculate the increase in normalized reporter emission intensity on a continuous basis. The increase in emission intensity is plotted versus time, i.e., the number of amplification cycles, to produce a continuous measure of amplification. To quantify the locus in each amplification reaction, the amplification plot is examined at a point during the log phase of product accumulation. This is accomplished by assigning a fluorescence threshold intensity above background and determining the point at which each amplification plot crosses the threshold (defined as the threshold cycle number or Ct). Differences in threshold cycle number are used to quantify the relative amount of PCR target contained within each tube. Assuming that each reaction functions at 100% PCR efficiency, a difference of one Ct represents a two-fold difference in the amount of starting template. The fluorescence value can be used in conjunction with a standard curve to determine the amount of amplification product present.

Non-Probe-Based Detection Methods

A variety of options are available for measuring the amplification products as they are formed. One method utilizes labels, such as dyes, which only bind to double stranded DNA. In this type of approach, amplification product (which is double stranded) binds dye molecules in solution to form a complex. With the appropriate dyes, it is possible to distinguish between dye molecules free in solution and dye molecules bound to amplification product. For example, certain dyes fluoresce only when bound to amplification product. Examples of dyes which can be used in methods of this general type include, but are not limited to, Syber Green.TM. and Pico Green from Molecular Probes, Inc. of Eugene, Oreg., ethidium bromide, propidium iodide, chromomycin, acridine orange, Hoechst 33258, Toto-1, Yoyo-1, DAPI (4',6-diamidino-2-phenylindole hydrochloride).

Another real time detection technique measures alteration in energy fluorescence energy transfer between fluorophors conjugated with PCR primers [Livak, (1995)]. Probe-Based Detection Methods

These detection methods involve some alteration to the structure or conformation of a probe hybridized to the locus between the amplification primer pair. In some instances, the alteration is caused by the template-dependent extension catalyzed by a nucleic acid polymerase during the amplification process. The alteration generates a detectable signal which is an indirect measure of the amount of amplification product formed.

For example, some methods involve the degradation or digestion of the probe during the extension reaction. These methods are a consequence of the 5'-3' nuclease activity associated with some nucleic acid polymerases. Polymerases having this activity cleave mononucleotides or small oligonucleotides from an oligonucleotide probe annealed to its complementary sequence located within the locus.

The 3' end of the upstream primer provides the initial binding site for the nucleic acid polymerase. As the polymerase catalyzes extension of the upstream primer and encounters the bound probe, the nucleic acid polymerase displaces a portion of the 5' end of the probe and through its nuclease activity cleaves mononucleotides or oligonucleotides from the probe.

The upstream primer and the probe can be designed such that they anneal to the complementary strand in close proximity to one another. In fact, the 3' end of the upstream primer and the 5' end of the probe may abut one another. In this situation, extension of the upstream primer is not necessary in order for the nucleic acid polymerase to begin cleaving the probe. Li the case in which intervening nucleotides separate the upstream primer and the probe, extension of the primer is necessary before the nucleic acid polymerase encounters the 5' end of the probe. Once contact occurs and polymerization continues, the 5 '-3' exonuclease activity of the nucleic acid polymerase begins cleaving mononucleotides or oligonucleotides from the 5' end of the probe. Digestion of the probe continues until the remaining portion of the probe dissociates from the complementary strand.

In solution, the two end sections can hybridize with each other to form a hairpin loop. In this conformation, the reporter and quencher dye are in sufficiently close proximity that fluorescence from the reporter dye is effectively quenched by the quencher dye. Hybridized probe, in contrast, results in a linearized conformation in which the extent of quenching is decreased. Thus, by monitoring emission changes for the two dyes, it is possible to indirectly monitor the formation of amplification product. Probes

The labeled probe is selected so that its sequence is substantially complementary to a segment of the test locus or a reference locus. As indicated above, the nucleic acid site to which the probe binds should be located between the primer binding sites for the upstream and downstream amplification primers.

Primers

The primers used in the amplification are selected so as to be capable of hybridizing to sequences at flanking regions of the locus being amplified. The primers are chosen to have at least substantial complementarity with the different strands of the nucleic acid being amplified. When a probe is utilized to detect the formation of amplification products, the primers are selected in such that they flank the probe, i.e. are located upstream and downstream of the probe.

The primer must have sufficient length so that it is capable of priming the synthesis of extension products in the presence of an agent for polymerization. The length and composition of the primer depends on many parameters, including, for example, the temperature at which the annealing reaction is conducted, proximity of the probe binding site to that of the primer, relative concentrations of the primer and probe and the particular nucleic acid composition of the probe. Typically the primer includes 15-30 nucleotides. However, the length of the primer may be more or less depending on the complexity of the primer binding site and the factors listed above.

Labels for Probes and Primers

The labels used for labeling the probes or primers of the current invention and which can provide the signal corresponding to the quantity of amplification product can take a variety of forms. As indicated above with regard to the 5' fiuorogenic nuclease method, a fluorescent signal is one signal which can be measured. However, measurements may also be made, for example, by monitoring radioactivity, colorimetry, absorption, magnetic parameters, or enzymatic activity. Thus, labels which can be employed include, but are not limited to, fluorophors, chromophores, radioactive isotopes, electron dense reagents, enzymes, and ligands having specific binding partners (e.g., biotin-avidin).

Monitoring changes in fluorescence is a particularly useful way to monitor the accumulation of amplification products. A number of labels useful for attachment to probes or primers are commercially available including fluorescein and various fluorescein derivatives such as FAM,

HEX, TET and JOE (all which are available from Applied Biosystems, Foster City, Calif.); lucifer yellow, and coumarin derivatives. Labels may be attached to the probe or primer using a variety of techniques and can be attached at the 5' end, and/or the 3' end and/or at an internal nucleotide. The label can also be attached to spacer arms of various sizes which are attached to the probe or primer. These spacer arms are useful for obtaining a desired distance between multiple labels attached to the probe or primer.

In some instances, a single label may be utilized; whereas, in other instances, such as with the 5' fluorogenic nuclease assays for example, two or more labels are attached to the probe. In cases wherein the probe includes multiple labels, it is generally advisable to maintain spacing between the labels which is sufficient to permit separation of the labels during digestion of the probe through the 5'-3' nuclease activity of the nucleic acid polymerase.

Patients Exhibiting Symptoms of Disease

A number of diseases are associated with changes in the copy number of a certain gene. For patients having symptoms of a disease, the real-time PCR method can be used to determine if the patient has copy number alterations which are known to be linked with diseases that are associated with the symptoms the patient has.

HNF4A expression

HNF 4 A fusion proteins

Fusion proteins are useful for generating antibodies against HNF4A polypeptides and for use in various assay systems. For example, fusion proteins can be used to identify proteins which interact with portions of HNF4A polypeptides. Protein affinity chromatography or library-based assays for protein-protein interactions, such as the yeast two-hybrid or phage display systems, can be used for this purpose. Such methods are well known in the art and also can be used as drug screens.

A HNF4A fusion protein comprises two polypeptide segments fused together by means of a peptide bond. The first polypeptide segment can comprise at least 54, 75, 100, 125, 139, 150, 175, 200, 225, 250, or 275 contiguous amino acids of SEQ ID NO: 2 or of a biologically active variant, such as those described above. The first polypeptide segment also can comprise full-length HNF4A.

The second polypeptide segment can be a full-length protein or a protein fragment. Proteins commonly used in fusion protein construction include, but are not limited to β galactosidase, β- glucuronidase, green fluorescent protein (GFP), autofluorescent proteins, including blue fluorescent protein (BFP), glutathione-S-transferase (GST), luciferase, horseradish peroxidase (HRP), and chloramphenicol acetyltransferase (CAT). Additionally, epitope tags are used in fusion protein constructions, including histidine (His) tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. Other fusion constructions can include maltose binding protein (MBP), S-tag, Lex a DNA binding domain (DBD) fusions, GAL4 DNA binding domain fusions, herpes simplex virus (HSV) BP 16 protein fusions and G-protein fusions (for example G(alpha)16, Gs, Gi). A fusion protein also can be engineered to contain a cleavage site located adjacent to the HNF4A.

Preparation of Polynucleotides

A naturally occurring HNF4A polynucleotide can be isolated free of other cellular components such as membrane components, proteins, and lipids. Polynucleotides can be made by a cell and isolated using standard nucleic acid purification techniques, or synthesized using an amplification technique, such as the polymerase chain reaction (PCR), or by using an automatic synthesizer.

Methods for isolating polynucleotides are routine and are known in the art. Any such technique for obtaining a polynucleotide can be used to obtain isolated HNF4A polynucleotides. For example, restriction enzymes and probes can be used to isolate polynucleotide fragments which comprise HNF4A nucleotide sequences. Isolated polynucleotides are in preparations which are free or at least 70, 80, or 90% free of other molecules.

HNF4A cDNA molecules can be made with standard molecular biology techniques, using HNF4A mRNA as a template. HNF4A cDNA molecules can thereafter be replicated using molecular biology techniques known in the art. An amplification technique, such as PCR, can be used to obtain additional copies of polynucleotides of the invention, using either human genomic DNA or cDNA as a template.

Alternatively, synthetic chemistry techniques can be used to synthesize HNF4A polynucleotides. The degeneracy of the genetic code allows alternate nucleotide sequences to be synthesized which will encode HNF4A having, for example, an amino acid sequence shown in SEQ DD NO: 2 or a biologically active variant thereof.

Extending Polynucleotides

Various PCR-based methods can be used to extend nucleic acid sequences encoding human HNF4A, for example to detect upstream sequences of HNF4A gene such as promoters and regulatory elements. For example, restriction-site PCR uses universal primers to retrieve unknown sequence adjacent to a known locus. Genomic DNA is first amplified in the presence of a primer to a linker sequence and a primer specific to the known region. The amplified sequences are then subjected to a second round of PCR with the same linker primer and another specific primer internal to the first one. Products of each round of PCR are transcribed with an appropriate RNA polymerase and sequenced using reverse transcriptase.

Inverse PCR also can be used to amplify or extend sequences using divergent primers based on a known region. Primers can be designed using commercially available software, such as OLIGO

4.06 Primer Analysis software (National Biosciences Inc., Plymouth, Minn.), to be 22-30 nucleotides in length, to have a GC content of 50% or more, and to anneal to the target sequence at temperatures about 68-72⁰C. The method uses several restriction enzymes to generate a suitable fragment in the known region of a gene. The fragment is then circularized by intramolecular ligation and used as a PCR template.

Another method which can be used is capture PCR, which involves PCR amplification of DNA fragments adjacent to a known sequence in human and yeast artificial chromosome DNA. In this method, multiple restriction enzyme digestions and ligations also can be used to place an engineered double-stranded sequence into an unknown fragment of the DNA molecule before performing PCR.

When screening for full-length cDNAs, it is preferable to use libraries that have been size-selected to include larger cDNAs. Randomly-primed libraries are preferable, in that they will contain more sequences which contain the 5' regions of genes. Use of a randomly primed library may be especially preferable for situations in which an oligo d(T) library does not yield a full-length cDNA. Genomic libraries can be useful for extension of sequence into 5' non-transcribed regulatory regions.

Commercially available capillary electrophoresis systems can be used to analyze the size or confirm the nucleotide sequence of PCR or sequencing products. For example, capillary sequencing can employ flowable polymers for electrophoretic separation, four different fluorescent dyes (one for each nucleotide) which are laser activated, and detection of the emitted wavelengths by a charge coupled device camera. Output/light intensity can be converted to electrical signal using appropriate equipment and software (e.g., GENOTYPER and Sequence NAVIGATOR, Perkin Elmer), and the entire process from loading of samples to computer analysis and electronic data display can be computer controlled. Capillary electrophoresis is especially preferable for the sequencing of small pieces of DNA which might be present in limited amounts in a particular sample. Obtaining Polypeptides

HNF4A can be obtained, for example, by purification from human cells, by expression of HNF4A polynucleotides, or by direct chemical synthesis.

Protein Purification

HNF4A can be purified from any human cell which expresses the receptor, including those which have been transfected with expression constructs which express HNF4A. A purified HNF4A is separated from other compounds which normally associate with HNF4A in the cell, such as certain proteins, carbohydrates, or lipids, using methods well-known in the art. Such methods include, but are not limited to, size exclusion chromatography, ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography, and preparative gel electrophoresis.

Expression ofHNF4A Polynucleotides

To express HNF4A, HNF4A polynucleotides can be inserted into an expression vector which contains the necessary elements for the transcription and translation of the inserted coding sequence. Methods which are well known to those skilled in the art can be used to construct expression vectors containing sequences encoding HNF4A and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination.

A variety of expression vector/host systems can be utilized to contain and express sequences encoding HNF4A. These include, but are not limited to, microorganisms, such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors, insect cell systems infected with virus expression vectors (e.g., baculovirus), plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g. , Ti or pBR322 plasmids), or animal cell systems.

The control elements or regulatory sequences are those non-translated regions of the vector - enhancers, promoters, 5¹ and 3¹ untranslated regions — which interact with host cellular proteins to carry out transcription and translation. Such elements can vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, can be used. For example, when cloning in bacterial systems, inducible promoters such as the hybrid lacZ promoter of the

BLUESCRIPT phagemid (Stratagene, LaJoIIa, Calif.) or pSPORTl plasmid (Life Technologies) and the like can be used. The baculovirus polyhedrin promoter can be used in insect cells. Promoters or enhancers derived from the genomes of plant cells (e.g., heat shock, RUBISCO, and storage protein genes) or from plant viruses (e.g., viral promoters or leader sequences) can be cloned into the vector. In mammalian cell systems, promoters from mammalian genes or from mammalian viruses are preferable. If it is necessary to generate a cell line that contains multiple copies of a nucleotide sequence encoding HNF4A, vectors based on SV40 or EBV can be used with an appropriate selectable marker.

Bacterial and Yeast Expression Systems

In bacterial systems, a number of expression vectors can be selected. For example, when a large quantity of HNF4A is needed for the induction of antibodies, vectors which direct high level expression of fusion proteins that are readily purified can be used. Such vectors include, but are not limited to, multifunctional E. coli cloning and expression vectors such as BLUESCRIPT (Stratagene). In a BLUESCRIPT vector, a sequence encoding HNF4A can be ligated into the vector in frame with sequences for the amino-terminal Met and the subsequent 7 residues of β- galactosidase so that a hybrid protein is produced. pIN vectors or pGEX vectors (Promega, Madison, Wis.) also can be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. Proteins made in such systems can be designed to include heparin, thrombin, or factor Xa protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety at will.

Plant and Insect Expression Systems

If plant expression vectors are used, the expression of sequences encoding HNF4A can be driven by any of a number of promoters. For example, viral promoters such as the 35S and 19S promoters of CaMV can be used alone or in combination with the omega leader sequence from TMV. Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters can be used. These constructs can be introduced into plant cells by direct DNA transformation or by pathogen-mediated transfection.

An insect system also can be used to express HNF4A. For example, in one such system Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae. Sequences encoding HNF4A can be cloned into a non-essential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter. Successful insertion of HNF4A will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein. The recombinant viruses can then be used to infect S. frugiperda cells or Trichoplusia larvae in which HNF4A can be expressed.

Mammalian Expression Systems

A number of viral-based expression systems can be used to express HNF4A in mammalian host cells. For example, if an adenovirus is used as an expression vector, sequences encoding HNF4A can be ligated into an adenovirus transcription/translation complex comprising the late promoter and tripartite leader sequence. Insertion in a non-essential El or E3 region of the viral genome can be used to obtain a viable virus which is capable of expressing HNF4A in infected host cells

[Engelhard, 1994)]. If desired, transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer, can be used to increase expression in mammalian host cells.

Human artificial chromosomes (HACs) also can be used to deliver larger fragments of DNA than can be contained and expressed in a plasmid. HACs of 6M to 1OM are constructed and delivered to cells via conventional delivery methods {e.g., liposomes, polycationic amino polymers, or vesicles). Specific initiation signals also can be used to achieve more efficient translation of sequences encoding HNF4A. Such signals include the ATG initiation codon and adjacent sequences. In cases where sequences encoding HNF4A, its initiation codon, and upstream sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a fragment thereof, is inserted, exogenous translational control signals (including the ATG initiation codon) should be provided. The initiation codon should be in the correct reading frame to ensure translation of the entire insert. Exogenous translational elements and initiation codons can be of various origins, both natural and synthetic.

Host Cells

A host cell strain can be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed HNF4A in the desired fashion. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing which cleaves a "prepro" form of the polypeptide also can be used to facilitate correct insertion, folding and/or function. Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38), are available from the American Type

Culture Collection (ATCC; 10801 University Boulevard, Manassas, VA 20110-2209) and can be chosen to ensure the correct modification and processing of the foreign protein. Stable expression is preferred for long-term, high-yield production of recombinant proteins. For example, cell lines which stably express HNF4A can be transformed using expression vectors which can contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells can be allowed to grow for 1-2 days in an enriched medium before they are switched to a selective medium. The purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced HNF4A sequences. Resistant clones of stably transformed cells can be proliferated using tissue culture techniques appropriate to the cell type. Any number of selection systems can be used to recover transformed cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase [Logan, (1984)] and adenine phosphoribosyltransferase [Wigler, (1977)] genes which can be employed in tk^~ or aprf cells, respectively. Also, antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection. For example, dhfr confers resistance to methotrexate [Lowy, (1980)], npt confers resistance to the aminoglycosides, neomycin and G-418 [Wigler, (1980)], and als and pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively [Colbere-Garapin, 1981]. Additional selectable genes have been described. For example, trpB allows cells to utilize indole in place of tryptophan, or hisD, which allows cells to utilize histinol in place of histidine. Visible markers such as anthocyanins, β- glucuronidase and its substrate GUS, and luciferase and its substrate luciferin, can be used to identify transformants and to quantify the amount of transient or stable protein expression attributable to a specific vector system

Detecting Polypeptide Expression

Although the presence of marker gene expression suggests that a HNF4A polynucleotide is also present, its presence and expression may need to be confirmed. For example, if a sequence encoding HNF4A is inserted within a marker gene sequence, transformed cells containing sequences which encode HNF4A can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a sequence encoding HNF4A under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of HNF4A polynucleotide.

Alternatively, host cells which contain a HNF4A polynucleotide and which express HNF4A can be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassay or immunoassay techniques which include membrane, solution, or chip-based technologies for the detection and/or quantification of nucleic acid or protein. For example, the presence of a polynucleotide sequence encoding HNF4A can be detected by DNA-DNA or DNA-RNA hybridization or amplification using probes or fragments or fragments of polynucleotides encoding HNF4A. Nucleic acid amplification-based assays involve the use of oligonucleotides selected from sequences encoding HNF4A to detect transformants which contain a HNF4A polynucleotide.

A variety of protocols for detecting and measuring the expression of HNF4A, using either polyclonal or monoclonal antibodies specific for the polypeptide, are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS). A two-site, monoclonal-based immunoassay using monoclonal antibodies reactive to two non-interfering epitopes on HNF4A can be used, or a competitive binding assay can be employed.

A wide variety of labels and conjugation techniques are known by those skilled in the art and can be used in various nucleic acid and amino acid assays. Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding HNF4A include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide. Alternatively, sequences encoding HNF4A can be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are commercially available, and can be used to synthesize RNA probes in vitro by addition of labeled nucleotides and an appropriate RNA polymerase such as T7, T3, or SP6. These procedures can be conducted using a variety of commercially available kits (Amersham Pharmacia Biotech, Promega, and US Biochemical). Suitable reporter molecules or labels which can be used for ease of detection include radionuclides, enzymes, and fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like.

Expression and Purification of Polypeptides

Host cells transformed with HNF4A polynucleotides can be cultured under conditions suitable for the expression and recovery of the protein from cell culture. The polypeptide produced by a transformed cell can be secreted or contained intracellularly depending on the sequence and/or the vector used. As will be understood by those of skill in the art, expression vectors containing

HNF4A polynucleotides can be designed to contain signal sequences which direct secretion of soluble HNF4A through a prokaryotic or eukaryotic cell membrane or which direct the membrane insertion of membrane-bound HNF4A.

As discussed above, other constructions can be used to join a sequence encoding HNF4A to a nucleotide sequence encoding a polypeptide domain which will facilitate purification of soluble proteins. Such purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp., Seattle, Wash.). Inclusion of cleavable linker sequences such as those specific for Factor XA or enterokinase (Invitrogen, San Diego, CA) between the purification domain and HNF4A also can be used to facilitate purification. One such expression vector provides for expression of a fusion protein containing HNF4A and 6 histidine residues preceding a thioredoxin or an enterokinase cleavage site. The histidine residues facilitate purification by IMAC (immobilized metal ion affinity chromatography) Maddox, (1983)], while the enterokinase cleavage site provides a means for purifying HNF4A from the fusion protein [Porath, ( 1992)] .

Chemical Synthesis

Sequences encoding HNF4A can be synthesized, in whole or in part, using chemical methods well known in the art. Alternatively, HNF4A itself can be produced using chemical methods to synthesize its amino acid sequence, such as by direct peptide synthesis using solid-phase techniques. Protein synthesis can either be performed using manual techniques or by automation.

Automated synthesis can be achieved, for example, using Applied Biosystems 43 IA Peptide Synthesizer (Perkin Elmer). Optionally, fragments of HNF4A can be separately synthesized and combined using chemical methods to produce a full-length molecule.

The newly synthesized peptide can be substantially purified by preparative high performance liquid chromatography. The composition of a synthetic HNF4A can be confirmed by amino acid analysis or sequencing. Additionally, any portion of the amino acid sequence of HNF4A can be altered during direct synthesis and/or combined using chemical methods with sequences from other proteins to produce a variant polypeptide or a fusion protein.

Production of Altered Polypeptides

As will be understood by those of skill in the art, it may be advantageous to produce HNF4A polynucleotides possessing non-naturally occurring codons. For example, codons preferred by a particular prokaryotic or eukaryotic host can be selected to increase the rate of protein expression or to produce an RNA transcript having desirable properties, such as a half-life which is longer than that of a transcript generated from the naturally occurring sequence.

The nucleotide sequences referred to herein can be engineered using methods generally known in the art to alter HNF4A polynucleotides for a variety of reasons, including but not limited to, alterations which modify the cloning, processing, and/or expression of the polypeptide or mRNA product. DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides can be used to engineer the nucleotide sequences. For example, site- directed mutagenesis can be used to insert new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, introduce mutations, and so forth.

Antibodies

Any type of antibody known in the art can be generated to bind specifically to an epitope of HNF4A.

"Antibody" as used herein includes intact immunoglobulin molecules, as well as fragments thereof, such as Fab, F(ab')₂, and Fv, which are capable of binding an epitope of HNF4A. Typically, at least 6, 8, 10, or 12 contiguous amino acids are required to form an epitope.

However, epitopes which involve non-contiguous amino acids may require more, e.g., at least 15, 25, or 50 amino acid. An antibody which specifically binds to an epitope of HNF4A can be used therapeutically, as well as in immunochemical assays, such as Western blots, ELISAs, radioimmunoassays, immunohistochemical assays, immunoprecipitations, or other immunochemical assays known in the art. Various immunoassays can be used to identify antibodies having the desired specificity. Numerous protocols for competitive binding or immunoradiometric assays are well known in the art. Such immunoassays typically involve the measurement of complex formation between an immunogen and an antibody which specifically binds to the HNF4A immunogen.

Typically, an antibody which specifically binds to HNF4A provides a detection signal at least 5-,

10-, or 20-fold higher than a detection signal provided with other proteins when used in an immunochemical assay. Preferably, antibodies which specifically bind to HNF4A do not detect other proteins in immunochemical assays and can immunoprecipitate HNF4A from solution.

HNF4A can be used to immunize a mammal, such as a mouse, rat, rabbit, guinea pig, monkey, or human, to produce polyclonal antibodies. If desired, HNF4A can be conjugated to a carrier protein, such as bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin. Depending on the host species, various adjuvants can be used to increase the immunological response. Such adjuvants include, but are not limited to, Freund's adjuvant, mineral gels (e.g., aluminum hydroxide), and surface active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol). Among adjuvants used in humans, BCG (bacilli Calmette-Gueriή) and Corynebacterium parvum are especially useful. Monoclonal antibodies which specifically bind to HNF4A can be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These techniques include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique [Roberge, (1995)].

In addition, techniques developed for the production of "chimeric antibodies", the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity, can be used. Monoclonal and other antibodies also can be "humanized" to prevent a patient from mounting an immune response against the antibody when it is used therapeutically. Such antibodies may be sufficiently similar in sequence to human antibodies to be used directly in therapy or may require alteration of a few key residues. Sequence differences between rodent antibodies and human sequences can be minimized by replacing residues which differ from those in the human sequences by site directed mutagenesis of individual residues or by grating of entire complementarity determining regions. Antibodies which specifically bind to HNF4A can contain antigen binding sites which are either partially or fully humanized, as disclosed in U.S. 5,565,332.

Alternatively, techniques described for the production of single chain antibodies can be adapted using methods known in the art to produce single chain antibodies which specifically bind to HNF4A. Antibodies with related specificity, but of distinct idiotypic composition, can be generated by chain shuffling from random combinatorial immunoglobin libraries. Single-chain antibodies also can be constructed using a DNA amplification method, such as PCR, using hybridoma cDNA as a template. Single-chain antibodies can be mono- or bispecific, and can be bivalent or tetravalent. Construction of tetravalent, bispecific single-chain antibodies is taught. A nucleotide sequence encoding a single-chain antibody can be constructed using manual or automated nucleotide synthesis, cloned into an expression construct using standard recombinant DNA methods, and introduced into a cell to express the coding sequence, as described below.

Alternatively, single-chain antibodies can be produced directly using, for example, filamentous phage technology.

Antibodies which specifically bind to HNF4A also can be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents. Other types of antibodies can be constructed and used therapeutically in methods of the invention. For example, chimeric antibodies can be constructed as disclosed in WO 93/03151. Binding proteins which are derived from immunoglobulins and which are multivalent and multispecific, such as the "diabodies" described in WO 94/13804, also can be prepared. Antibodies according to the invention can be purified by methods well known in the art. For example, antibodies can be affinity purified by passage over a column to which HNF4A is bound. The bound antibodies can then be eluted from the column using a buffer with a high salt concentration.

Antisense Oligonucleotides

Antisense oligonucleotides are nucleotide sequences which are complementary to a specific DNA or RNA sequence. Once introduced into a cell, the complementary nucleotides combine with natural sequences produced by the cell to form complexes and block either transcription or translation. Preferably, an antisense oligonucleotide is at least 11 nucleotides in length, but can be at least 12, 15, 20, 25, 30, 35, 40, 45, or 50 or more nucleotides long. Longer sequences also can be used. Antisense oligonucleotide molecules can be provided in a DNA construct and introduced into a cell as described above to decrease the level of HNF4A gene products in the cell.

Antisense oligonucleotides can be deoxyribonucleotides, ribonucleotides, or a combination of both. Oligonucleotides can be synthesized manually or by an automated synthesizer, by covalently linking the 5' end of one nucleotide with the 3' end of another nucleotide with non-phosphodiester internucleotide linkages such alkylphosphonates, phosphorothioates, phosphorodithioates, alkylphosphonothioates, alkylphosphonates, phosphoramidates, phosphate esters, carbamates, acetamidate, carboxymethyl esters, carbonates, and phosphate triesters.

Modifications of HNF4A gene expression can be obtained by designing antisense oligonucleotides which will form duplexes to the control, 5', or regulatory regions of the HNF4A gene.

Oligonucleotides derived from the transcription initiation site, e.g., between positions -10 and +10 from the start site, are preferred. Similarly, inhibition can be achieved using "triple helix" base- pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or chaperons. Therapeutic advances using triplex DNA have been described in the literature

[Nicholls, (1993)]. An antisense oligonucleotide also can be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.

Precise complementarity is not required for successful complex formation between an antisense oligonucleotide and the complementary sequence of a HNF4A polynucleotide. Antisense oligonucleotides which comprise, for example, 2, 3, 4, or 5 or more stretches of contiguous nucleotides which are precisely complementary to a HNF4A polynucleotide, each separated by a stretch of contiguous nucleotides which are not complementary to adjacent HNF4A nucleotides, can provide sufficient targeting specificity for HNF4A mRNA. Preferably, each stretch of complementary contiguous nucleotides is at least 4, 5, 6, 7, or 8 or more nucleotides in length. Non-complementary intervening sequences are preferably 1, 2, 3, or 4 nucleotides in length. One skilled in the art can easily use the calculated melting point of an antisense-sense pair to determine the degree of mismatching which will be tolerated between a particular antisense oligonucleotide and a particular HNF4A polynucleotide sequence. Antisense oligonucleotides can be modified without affecting their ability to hybridize to a HNF4A polynucleotide. These modifications can be internal or at one or both ends of the antisense molecule. For example, internucleoside phosphate linkages can be modified by adding cholesteryl or diamine moieties with varying numbers of carbon residues between the amino groups and terminal ribose. Modified bases and/or sugars, such as arabinose instead of ribose, or a 3', 5 '-substituted oligonucleotide in which the 3' hydroxyl group or the 5' phosphate group are substituted, also can be employed in a modified antisense oligonucleotide. These modified oligonucleotides can be prepared by methods well known in the art.

Ribozymes

Ribozymes are RNA molecules with catalytic activity [Uhlmann, (1987)]. Ribozymes can be used to inhibit gene function by cleaving an RNA sequence, as is known in the art. The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Examples include engineered hammerhead motif ribozyme molecules that can specifically and efficiently catalyze endonucleolytic cleavage of specific nucleotide sequences. The coding sequence of a HNF4A polynucleotide can be used to generate ribozymes which will specifically bind to mRNA transcribed from a HNF4A polynucleotide. Methods of designing and constructing ribozymes which can cleave other RNA molecules in trans in a highly sequence specific manner have been developed and described in the art. For example, the cleavage activity of ribozymes can be targeted to specific RNAs by engineering a discrete "hybridization" region into the ribozyme. The hybridization region contains a sequence complementary to the target RNA and thus specifically hybridizes with the target RNA.

Specific ribozyme cleavage sites within a HNF4A RNA target can be identified by scanning the target molecule 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 RNA containing the cleavage site can be evaluated for secondary structural features which may render the target inoperable. Suitability of candidate HNF4A RNA targets also can be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays. The nucleotide sequences shown in SEQ ID NO: 1 and its complement provide sources of suitable hybridization region sequences. Longer complementary sequences can be used to increase the affinity of the hybridization sequence for the target. The hybridizing and cleavage regions of the ribozyme can be integrally related such that upon hybridizing to the target RNA through the complementary regions, the catalytic region of the ribozyme can cleave the target.

Ribozymes can be introduced into cells as part of a DNA construct. Mechanical methods, such as microinjection, liposome-mediated transfection, electroporation, or calcium phosphate precipitation, can be used to introduce a ribozyme-containing DNA construct into cells in which it is desired to decrease HNF4A expression. Alternatively, if it is desired that the cells stably retain the DNA construct, the construct can be supplied on a plasmid and maintained as a separate element or integrated into the genome of the cells, as is known in the art. A ribozyme-encoding DNA construct can include transcriptional regulatory elements, such as a promoter element, an enhancer or UAS element, and a transcriptional terminator signal, for controlling transcription of ribozymes in the cells (U.S. 5,641,673). Ribozymes also can be engineered to provide an additional level of regulation, so that destruction of mRNA occurs only when both a ribozyme and a target gene are induced in the cells.

Screening / Screening Assays

Regulators

Regulators as used herein, refer to compounds that affect the activity of a HNF4A in vivo and/or in vivo. Regulators can be agonists and antagonists of a HNF4A polypeptide and can be compounds that exert their effect on the HNF4A activity via the expression, via post-translational modifications or by other means. Agonists of HNF4A are molecules which, when bound to

HNF4A, increase or prolong the activity of HNF4A. Agonists of HNF4A include proteins, nucleic acids, carbohydrates, small molecules, or any other molecule which activate HNF4A. Antagonists of HNF4A are molecules which, when bound to HNF4A, decrease the amount or the duration of the activity of HNF4A. Antagonists include proteins, nucleic acids, carbohydrates, antibodies, small molecules, or any other molecule which decrease the activity of HNF4A.

The term "modulate", as it appears herein, refers to a change in the activity of HNF4A polypeptide. For example, modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of

HNF4A. As used herein, the terms "specific binding" or "specifically binding" refer to that interaction between a protein or peptide and an agonist, an antibody, or an antagonist. The interaction is dependent upon the presence of a particular structure of the protein recognized by the binding molecule (i.e., the antigenic determinant or epitope). For example, if an antibody is specific for epitope "A" the presence of a polypeptide containing the epitope A, or the presence of free unlabeled A, in a reaction containing free labeled A and the antibody will reduce the amount of labeled A that binds to the antibody.

The invention provides methods (also referred to herein as "screening assays") for identifying compounds which can be used for the treatment of gastrointestinal and liver diseases, cancer disorders, cardiovascular disorders, metabolic diseases, inflammatory diseases, muscle-skeleton disorders, respiratory diseases, reproduction disorders and urological disorders.. The methods entail the identification of candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other molecules) which bind to HNF4A and/or have a stimulatory or inhibitory effect on the biological activity of HNF4A or its expression and then determining which of these compounds have an effect on symptoms or diseases regarding the gastrointestinal and liver diseases, cancer disorders, cardiovascular disorders, metabolic diseases, inflammatory diseases, muscle-skeleton disorders, respiratory diseases, reproduction disorders and urological disorders, in an in vivo assay.

Candidate or test compounds or agents which bind to HNF4A and/or have a stimulatory or inhibitory effect on the activity or the expression of HNF4A are identified either in assays that employ cells which express HNF4A on the cell surface (cell-based assays) or in assays with isolated HNF4A (cell-free assays). The various assays can employ a variety of variants of HNF4A (e.g., full-length HNF4A, a biologically active fragment of HNF4A, or a fusion protein which includes all or a portion of HNF4A). Moreover, HNF4A can be derived from any suitable mammalian species (e.g., human HNF4A, rat HNF4A or murine HNF4A). The assay can be a binding assay entailing direct or indirect measurement of the binding of a test compound or a known HNF4A ligand to HNF4A. The assay can also be an activity assay entailing direct or indirect measurement of the activity of HNF4A. The assay can also be an expression assay entailing direct or indirect measurement of the expression of HNF4A mRNA or HNF4A protein. The various screening assays are combined with an in vivo assay entailing measuring the effect of the test compound on the symptoms of gastrointestinal and liver diseases, cancer disorders, cardiovascular disorders, metabolic diseases, inflammatory diseases, muscle-skeleton disorders, respiratory diseases, reproduction disorders and urological disorders.. Determining the ability of the test compound to modulate the activity of HNF4A can be accomplished, for example, by determining the ability of HNF4A to bind to or interact with a target molecule. The target molecule can be a molecule with which HNF4A binds or interacts with in nature, for example, a molecule from the extracellular milieu able to travers the cytoplasmic membrane or a cytoplasmic molecule. The target molecule can be a component of a signal transduction pathway which facilitates transduction of an extracellular signal (e.g., a signal generated by another cell) through the cell membrane and into the cell. The target HNF4A molecule can be, for example, a second intracellular protein which has catalytic activity,a protein which facilitates the association of downstream signaling molecules with HNF4A or the target DNA of the nuclear receptor.

Nuclear receptor assays

In general two modes exist to determine ligand dependent NR activity in cell based assays. Cells that endogenously express a certain NR can be transfected with a reporter construct expressing a reporter gene (e.g. firefly luciferase) under control of a responsive promoter (Gutendorf et al., 2001). Alternatively cells lacking an endogenous NR are co-transfected with a reporter construct and a NR expressing vector (Berger et al., 1992).

In a second approach a fusion protein of the NRs LBD and a non-mammalian DBD, that targets a corresponding promoter with a downstream reporter gene, are used. DBD from the bacterial LexA repressor or the yeast Gal4 transcription activator are described (Lee et al.,1998; Charles et al., 2000). The corresponding reporter constructs are formed by a minimal promoter containing binding sites for LexA or Gal4 respectively.

Both systems are induced by addition of the receptor activating ligands and the expression level of the reporter reflects transcription activation by the NR.

Binding of ligands to NRs can also be detected in competition-binding assays with purified NRs or NR LBDs. A specific radiolabeled ligand binds to NRs in the presence of unlabeled test compounds. The amount of receptor-bound radioactivity gives information about the affinity of the test compounds. In the scintillation proximity assay (SPA) biotinylated NR LBDs are linked to streptavidine coated beads impregnated with scintillant (Nichols et al., 1998). Radioactive counts are observed only when the receptor-ligand complex is captured on the surface of the bead, eliminating the need to separate bound from free ligand. Therefore SPA is fully automatable and compatible to high throughput. The homogenous fluorescence polarization-based ligand-binding assay utilizes His- and GST- tagged LBDs of steroid NR and a selective high affinity fluorescent-labeled ligand (Rosen et al., 2003). The binding affinity of a test compound is determined by displacement of the fluorescent steroid, accompanied by a change in the polarization value.

The competition assays are sensitive tools to identify NR ligands but cannot distinguish between agonists or antagonists, because only the affinity not the potential to induce conformational changes is measured.

The present invention also includes cell-free assays. Such assays involve contacting a form of HNF4A (e.g., full-length HNF4A, a biologically active fragment of HNF4A, or a fusion protein comprising all or a portion of HNF4A) with a test compound and determining the ability of the test compound to bind to HNF4A. Binding of the test compound to HNF4A can be determined either directly or indirectly as described above, hi one embodiment, the assay includes contacting HNF4A with a known compound which binds HNF4A to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with HNF4A, wherein determining the ability of the test compound to interact with HNF4A comprises determining the ability of the test compound to preferentially bind to HNF4A as compared to the known compound.

The cell-free assays of the present invention are amenable to use of either a membrane-bound form of HNF4A or a soluble fragment thereof. In the case of cell-free assays comprising the membrane- bound form of the polypeptide, it may be desirable to utilize a solubilizing agent such that the membrane-bound form of the polypeptide is maintained in solution. Examples of such solubilizing agents include but are not limited to non-ionic detergents such as n-octylglucoside, n- dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-N-methylgluc- amide, Triton X-100, Triton X-114, Thesit, Isotridecypoly(ethylene glycol ether)n, 3-[(3-chol- amidopropyl)dimethylamminio]-l -propane sulfonate (CHAPS), 3-[(3-cholamidopropyl)dimethyl- amminio]-2-hydroxy-l -propane sulfonate (CHAPSO), or N-dodecyl=N,N-dimethyl-3-ammonio-l- propane sulfonate.

In various embodiments of the above assay methods of the present invention, it may be desirable to immobilize HNF4A (or a HNF4A target molecule) to facilitate separation of complexed from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Binding of a test compound to HNF4A, or interaction of HNF4A with a target molecule in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtitre plates, test tubes, and micro- centrifuge tubes. In one embodiment, a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix. For example, glutathione-S-transferase (GST) fusion proteins or glutathione-S-transferase 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 test compound or the test compound and either the non-adsorbed target protein or HNF4A, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtitre plate wells are washed to remove any unbound components and complex formation is measured either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of binding or activity of HNF4A can be determined using standard techniques.

Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the invention. For example, either HNF4A or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated polypeptide of the invention or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals; Rockford, 111.), and immobilized in the wells of streptavidin-coated plates (Pierce Chemical). Alternatively, antibodies reactive with HNF4A or target molecules but which do not interfere with binding of the polypeptide of the invention to its target molecule can be derivatized to the wells of the plate, and unbound target or polypeptide of the invention trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with HNF4A or target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with HNF4A or target molecule.

The screening assay can also involve monitoring the expression of HNF4A. For example, regulators of expression of HNF4A can be identified in a method in which a cell is contacted with a candidate compound and the expression of HNF4A protein or mRNA in the cell is determined. The level of expression of HNF4A protein or mRNA the presence of the candidate compound is compared to the level of expression of HNF4A protein or mRNA in the absence of the candidate compound. The candidate compound can then be identified as a regulator of expression of

HNF4A based on this comparison. For example, when expression of HNF4A protein or mRNA protein is greater (statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of HNF4A protein or mRNA expression. Alternatively, when expression of HNF4A protein or mRNA is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of HNF4A protein or mRNA expression. The level of HNF4A protein or mRNA expression in the cells can be determined by methods described below.

Binding Assays

For binding assays, the test compound is preferably a small molecule which binds to and occupies the active site of HNF4A polypeptide, thereby making the ligand binding site inaccessible to substrate such that normal biological activity is prevented. Examples of such small molecules include, but are not limited to, small peptides or peptide-like molecules. Potential ligands which bind to a polypeptide of the invention include, but are not limited to, the natural ligands of known HNF4ANuclear Receptors and analogues or derivatives thereof.

In binding assays, either the test compound or the HNF4A polypeptide can comprise a detectable label, such as a fluorescent, radioisotopic, chemiluminescent, or enzymatic label, such as horseradish peroxidase, alkaline phosphatase, or luciferase. Detection of a test compound which is bound to HNF4A polypeptide can then be accomplished, for example, by direct counting of radioemmission, by scintillation counting, or by determining conversion of an appropriate substrate to a detectable product. Alternatively, binding of a test compound to a HNF4A polypeptide can be determined without labeling either of the interactants. For example, a microphysiometer can be used to detect binding of a test compound with a HNF4A polypeptide. A microphysiometer (e.g., Cytosensor™) is an analytical instrument that measures the rate at which a cell acidifies its environment using a light-addressable potentiometric sensor (LAPS). Changes in this acidification rate can be used as an indicator of the interaction between a test compound and HNF4A [Haseloff, (1988)].

Determining the ability of a test compound to bind to HNF4A also can be accomplished using a technology such as real-time Bimolecular Interaction Analysis (BIA) [McConnell, (1992); Sjolander, (1991)]. BIA is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore™). Changes in the optical phenomenon surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules.

In yet another aspect of the invention, a HNF4A-like polypeptide can be used as a "bait protein" in a two-hybrid assay or three-hybrid assay [Szabo, (1995); U.S. 5,283,317), to identify other proteins which bind to or interact with HNF4A and modulate its activity. The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. For example, in one construct, polynucleotide encoding HNF4A can be fused to a polynucleotide encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct a DNA sequence that encodes an unidentified protein ("prey" or "sample") can be fused to a polynucleotide that codes for the activation domain of the known transcription factor. If the "bait" and the "prey" proteins are able to interact in vivo to form an protein- dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ), which is operably linked to a transcriptional regulatory site responsive to the transcription factor.

Expression of the reporter gene can be detected, and cell colonies containing the functional transcription factor can be isolated and used to obtain the DNA sequence encoding the protein which interacts with HNF4A.

It may be desirable to immobilize either the HNF4A (or polynucleotide) or the test compound to facilitate separation of the bound form from unbound forms of one or both of the interactants, as well as to accommodate automation of the assay. Thus, either the HNF4A-like polypeptide (or polynucleotide) or the test compound can be bound to a solid support. Suitable solid supports include, but are not limited to, glass or plastic slides, tissue culture plates, microtiter wells, tubes, silicon chips, or particles such as beads (including, but not limited to, latex, polystyrene, or glass beads). Any method known in the art can be used to attach HNF4A-like polypeptide (or polynucleotide) or test compound to a solid support, including use of covalent and non-covalent linkages, passive absorption, or pairs of binding moieties attached respectively to the polypeptide

(or polynucleotide) or test compound and the solid support. Test compounds are preferably bound to the solid support in an array, so that the location of individual test compounds can be tracked. Binding of a test compound to HNF4A (or a polynucleotide encoding for HNF4A) can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and microcentrifuge tubes.

In one embodiment, HNF4A is a fusion protein comprising a domain that allows binding of HNF4A to a solid support. For example, glutathione-S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtiter plates, which are then combined with the test compound or the test compound and the non-adsorbed HNF4A; the mixture is then incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components. Binding of the interactants can be determined either directly or indirectly, as described above. Alternatively, the complexes can be dissociated from the solid support before binding is determined.

Other techniques for immobilizing proteins or polynucleotides on a solid support also can be used in the screening assays of the invention. For example, either HNF4A (or a polynucleotide encoding HNF4A) or a test compound can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated HNF4A (or a polynucleotide encoding biotinylated HNF4A) or test compounds can be prepared from biotin-NHS (N-hydroxysuccinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Hl.) and immobilized in the wells of streptavidin-coated plates (Pierce Chemical). Alternatively, antibodies which specifically bind to HNF4A, polynucleotide, or a test compound, but which do not interfere with a desired binding site, such as the active site of HNF4A, can be derivatized to the wells of the plate. Unbound target or protein can be trapped in the wells by antibody conjugation.

Methods for detecting such complexes, in addition to those described above for the GST- immobilized complexes, include immunodetection of complexes using antibodies which specifically bind to HNF4A polypeptide or test compound, enzyme-linked assays which rely on detecting an activity of HNF4A polypeptide, and SDS gel electrophoresis under non-reducing conditions.

Screening for test compounds which bind to a HNF4A polypeptide or polynucleotide also can be carried out in an intact cell. Any cell which comprises a HNF4A polypeptide or polynucleotide can be used in a cell-based assay system. A HNF4A polynucleotide can be naturally occurring in the cell or can be introduced using techniques such as those described above. Binding of the test compound to HNF4A or a polynucleotide encoding HNF4A is determined as described above.

Test Compounds

Suitable test compounds for use in the screening assays of the invention can be obtained from any suitable source, e.g., conventional compound libraries. The test compounds can also be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the "one-bead one-compound" library method; and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds [Lam, (1997)]. Examples of methods for the synthesis of molecular libraries can be found in the art. Libraries of compounds may be presented in solution or on beads, bacteria, spores, plasmids or phage. Modeling of Regulators

Computer modeling and searching technologies permit identification of compounds, or the improvement of already identified compounds, that can modulate HNF4A expression or activity.

Having identified such a compound or composition, the active sites or regions are identified. Such active sites might typically be ligand binding sites, such as the interaction domain of the ligand with HNF4A. The active site can be identified using methods known in the art including, for example, from the amino acid sequences of peptides, from the nucleotide sequences of nucleic acids, or from study of complexes of the relevant compound or composition with its natural ligand.

Li the latter case, chemical or X-ray crystallographic methods can be used to find the active site by finding where on the factor the complexed ligand is found.

Next, the three dimensional geometric structure of the active site is determined. This can be done by known methods, including X-ray crystallography, which can determine a complete molecular structure. On the other hand, solid or liquid phase NMR can be used to determine certain intramolecular distances. Any other experimental method of structure determination can be used to obtain partial or complete geometric structures. The geometric structures may be measured with a complexed ligand, natural or artificial, which may increase the accuracy of the active site structure determined.

If an incomplete or insufficiently accurate structure is determined, the methods of computer based numerical modeling can be used to complete the structure or improve its accuracy. Any recognized modeling method may be used, including parameterized models specific to particular biopolymers such as proteins or nucleic acids, molecular dynamics models based on computing molecular motions, statistical mechanics models based on thermal ensembles, or combined models. For most types of models, standard molecular force fields, representing the forces between constituent atoms and groups, are necessary, and can be selected from force fields known in physical chemistry. The incomplete or less accurate experimental structures can serve as constraints on the complete and more accurate structures computed by these modeling methods.

Finally, having determined the structure of the active site, either experimentally, by modeling, or by a combination, candidate modulating compounds can be identified by searching databases containing compounds along with information on their molecular structure. Such a search seeks compounds having structures that match the determined active site structure and that interact with the groups defining the active site. Such a search can be manual, but is preferably computer assisted. These compounds found from this search are potential HNF4A modulating compounds. Alternatively, these methods can be used to identify improved modulating compounds from an already known modulating compound or ligand. The composition of the known compound can be modified and the structural effects of modification can be determined using the experimental and computer modeling methods described above applied to the new composition. The altered structure is then compared to the active site structure of the compound to determine if an improved fit or interaction results. In this manner systematic variations in composition, such as by varying side groups, can be quickly evaluated to obtain modified modulating compounds or ligands of improved specificity or activity.

Therapeutic Indications and Methods

It was found by the present applicant that HNF4A is expressed in various human tissues.

Cardiovascular Disorders

Heart failure is defined as a pathophysiological state in which an abnormality of cardiac function is responsible for the failure of the heart to pump blood at a rate commensurate with the requirement of the metabolizing tissue. It includes all forms of pumping failures such as high-output and low- output, acute and chronic, right-sided or left-sided, systolic or diastolic, independent of the underlying cause.

Myocardial infarction (Ml) is generally caused by an abrupt decrease in coronary blood flow that follows a thrombotic occlusion of a coronary artery previously narrowed by arteriosclerosis. MI prophylaxis (primary and secondary prevention) is included as well as the acute treatment of MI and the prevention of complications.

Ischemic diseases are conditions in which the coronary flow is restricted resulting in a perfusion which is inadequate to meet the myocardial requirement for oxygen. This group of diseases includes stable angina, unstable angina and asymptomatic ischemia.

Arrhythmias include all forms of atrial and ventricular tachyarrhythmias, atrial tachycardia, atrial flutter, atrial fibrillation, atrio-ventricular reentrant tachycardia, preexitation syndrome, ventricular tachycardia, ventricular flutter, ventricular fibrillation, as well as bradycardic forms of arrhythmias.

Hypertensive vascular diseases include primary as well as all kinds of secondary arterial hypertension, renal, endocrine, neurogenic, others. The genes may be used as drug targets for the treatment of hypertension as well as for the prevention of all complications arising from cardiovascular diseases.

Peripheral vascular diseases are defined as vascular diseases in which arterial and/or venous flow is reduced resulting in an imbalance between blood supply and tissue oxygen demand. It includes chronic peripheral arterial occlusive disease (PAOD), acute arterial thrombosis and embolism, inflammatory vascular disorders, Raynaud's phenomenon and venous disorders.

Atherosclerosis is a cardiovascular disease in which the vessel wall is remodeled, compromising the lumen of the vessel. The atherosclerotic remodeling process involves accumulation of cells, both smooth muscle cells and monocyte/macrophage inflammatory cells, in the intima of the vessel wall. These cells take up lipid, likely from the circulation, to form a mature atherosclerotic lesion.

Although the formation of these lesions is a chronic process, occurring over decades of an adult human life, the majority of the morbidity associated with atherosclerosis occurs when a lesion ruptures, releasing thrombogenic debris that rapidly occludes the artery. When such an acute event occurs in the coronary artery, myocardial infarction can ensue, and in the worst case, can result in death.

The formation of the atherosclerotic lesion can be considered to occur in five overlapping stages such as migration, lipid accumulation, recruitment of inflammatory cells, proliferation of vascular smooth muscle cells, and extracellular matrix deposition. Each of these processes can be shown to occur in man and in animal models of atherosclerosis, but the relative contribution of each to the pathology and clinical significance of the lesion is unclear.

Thus, a need exists for therapeutic methods and agents to treat cardiovascular pathologies, such as atherosclerosis and other conditions related to coronary artery disease.

Cardiovascular diseases include but are not limited to disorders of the heart and the vascular system like congestive heart failure, myocardial infarction, ischemic diseases of the heart, all kinds of atrial and ventricular arrhythmias, hypertensive vascular diseases, peripheral vascular diseases, and atherosclerosis.

Too high or too low levels of fats in the bloodstream, especially cholesterol, can cause long-term problems. The risk to develop atherosclerosis and coronary artery or carotid artery disease (and thus the risk of having a heart attack or stroke) increases with the total cholesterol level increasing. Nevertheless, extremely low cholesterol levels may not be healthy. Examples of disorders of lipid metabolism are hyperlipidemia (abnormally high levels of fats (cholesterol, triglycerides, or both) in the blood, may be caused by family history of hyperlipidemia), obesity, a high-fat diet, lack of exercise, moderate to high alcohol consumption, cigarette smoking, poorly controlled diabetes, and an underactive thyroid gland), hereditary hyperlipidemias (type I hyperlipoproteinemia (familial hyperchylomicronemia), type II hyperlipoproteinemia (familial hypercholesterolemia), type in hyperlipoproteinemia, type IV hyperlipoproteinemia, or type V hyperlipoproteinemia), hypolipoproteinemia, lipidoses (caused by abnormalities in the enzymes that metabolize fats),

Gaucher's disease, Niemann-Pick disease, Fabry's disease, Wolman's disease, cerebrotendinous xanthomatosis, sitosterolemia, Refsum's disease, or Tay-Sachs disease.

Kidney disorders may lead to hypertension or hypotension. Examples for kidney problems possibly leading to hypertension are renal artery stenosis, pyelonephritis, glomerulonephritis, kidney tumors, polycistic kidney disease, injury to the kidney, or radiation therapy affecting the kidney.

Excessive urination may lead to hypotension.

The human HNF4A is highly expressed in the following cardiovascular related tissues: fetal liver, liver, liver, liver liver cirrhosis, liver lupus disease, liver tumor, adipose, fetal kidney, kidney, kidney, kidney. Expression in the above mentioned tissues demonstrates that the human HNF4A or mRNA can be utilized to diagnose of cardiovascular diseases. Additionally the activity of the human HNF4A can be modulated to treat cardiovascular diseases.

Gastrointestinal and Liver Diseases

Gastrointestinal diseases comprise primary or secondary, acute or chronic diseases of the organs of the gastrointestinal tract which may be acquired or inherited, benign or malignant or metaplastic, and which may affect the organs of the gastrointestinal tract or the body as a whole. They comprise but are not limited to 1) disorders of the esophagus like achalasia, vigoruos achalasia, dysphagia, cricopharyngeal incoordination, pre-esophageal dysphagia, diffuse esophageal spasm, globus sensation, Barrett's metaplasia, gastroesophageal reflux, 2) disorders of the stomach and duodenum like functional dyspepsia, inflammation of the gastric mucosa, gastritis, stress gastritis, chronic erosive gastritis, atrophy of gastric glands, metaplasia of gastric tissues, gastric ulcers, duodenal ulcers, neoplasms of the stomach, 3) disorders of the pancreas like acute or chronic pancreatitis, insufficiency of the exocrinic or endocrinic tissues of the pancreas like steatorrhea, diabetes, neoplasms of the exocrine or endocrine pancreas like 3.1) multiple endocrine neoplasia syndrome, ductal adenocarcinoma, cystadenocarcinoma, islet cell tumors, insulinoma, gastrinoma, carcinoid tumors, glucagonoma, Zollinger-Ellison syndrome, Vipoma syndrome, malabsorption syndrome, 4) disorders of the bowel like chronic inflammatory diseases of the bowel, Crohn's disease, ileus, diarrhea and constipation, colonic inertia, megacolon, malabsorption syndrome, ulcerative colitis, 4.1) functional bowel disorders like irritable bowel syndrome, 4.2) neoplasms of the bowel like familial polyposis, adenocarcinoma, primary malignant lymphoma, carcinoid tumors, Kaposi's sarcoma, polyps, cancer of the colon and rectum.

Liver diseases comprise primary or secondary, acute or chronic diseases or injury of the liver which may be acquired or inherited, benign or malignant, and which may affect the liver or the body as a whole. They comprise but are not limited to disorders of the bilirubin metabolism, jaundice, syndroms of Gilbert's, Crigler-Najjar, Dubin-Johnson and Rotor; intrahepatic cholestasis, hepatomegaly, portal hypertension, ascites, Budd-Chiari syndrome, portal-systemic encephalopathy, fatty liver, steatosis, Reye's syndrome, liver diseases due to alcohol, alcoholic hepatitis or cirrhosis, fibrosis and cirrhosis, fibrosis and cirrhosis of the liver due to inborn errors of metabolism or exogenous substances, storage diseases, syndromes of Gaucher' s, Zellweger's,

Wilson's - disease, acute or chronic hepatitis, viral hepatitis and its variants, inflammatory conditions of the liver due to viruses, bacteria, fungi, protozoa, helminths; drug induced disorders of the liver, chronic liver diseases like primary sclerosing cholangitis, alphai-antitrypsin- defϊciency, primary biliary cirrhosis, postoperative liver disorders like postoperative intrahepatic cholestasis, hepatic granulomas, vascular liver disorders associated with systemic disease, benign or malignant neoplasms of the liver, disturbance of liver metabolism in the new-born or prematurely born.

The human HNF4A is highly expressed in the following tissues of the gastroenterological system: stomach tumor, colon, colon tumor, small intestine, ileum, rectum, rectum tumor, fetal liver, liver, liver, liver liver cirrhosis, liver lupus disease, liver tumor, HEP G2 cells. The expression in the above mentioned tissues and in particular the differential expression between diseased tissue liver liver cirrhosis and healthy tissue liver, between diseased tissue liver lupus disease and healthy tissue liver demonstrates that the human HNF4A or mRNA can be utilized to diagnose of gastroenterological disorders. Additionally the activity of the human HNF4A can be modulated to treat gastroenterological disorders.

Musculoskeletal Diseases

Components of the musculoskeletal system are skeleton, muscles, tendons, ligaments, and other components of joints. Disorders of the musculoskeletal system often cause chronic pain and physical disability. They range from injures, infections, inflammation or other types of disorders. Examples of musculoskeletal disorders are presented in the following.

Examples are osteoporosis, postmenopausal osteoporosis, senile osteoporosis, secondary osteoporosis, idiopathic juvenile osteoporosis, Paget's disease of the bone, osteochondromas (osteocartilaginous exostoses), tumors of the bone (benign chondromas, chondroblastomas, chondromyxoid fibromas, osteoid osteomas, giant cell tumors of the bone, multiple myeloma, osteosarcoma (osteogenic sarcoma), fibrosarcomas and malignant fibrous histiocytomas, chondrosarcomas, Ewing's tumor (Ewing's sarcoma), malignant lymphoma of bone (reticulum cell sarcoma, metastatic tumors of the bone), osteoarthritis, and gout and Pseudogout.

Examples of disorders of joints and connective tissue are rheumatoid arthritis, psoriatic arthritis, discoid lupus erythematosus, systemic lupus erythematosus, scleroderma (systemic sclerosis), Sjogren's syndrome, connective tissue disease, polymyositis and dermatomyositis, relapsing polychondritis, vasculitis, polyarteritis nodosa, polymyalgia rheumatica, temporal arteritis, Wegener's granulomatosis, Reiter's syndrome, Behcet's syndrome, ankylosing spondylitis, or Charcot's joints (neuropathic joint disease).

Examples for bone and joint infections are osteomyelitis, and infectious arthritis.

Examples of disorders of muscles, bursas, and tendons are spasmodic torticollis, fibromyalgia syndromes (myofascial pain syndromes, fibromyositis), bursitis, tendinitis and tenosynovitis.

Foot problems are, for example ankle sprain, foot fractures, heel spurs, Sever's disease, posterior achilles tendon bursitis, anterior achilles tendon bursitis, posterior tibial neuralgia, pain in the ball of the foot (caused by damage to the nerves between the toes or to the joints between the toes and foot), onychomycosis, or nail discoloration.

The human HNF4A is highly expressed in the following muscle/skeleton tissues: adipose. The expression in muscle/skleleton tissues demonstrates that the human HNF4A or mRNA can be utilized to diagnose of diseases of the muscle/skeleton system. Additionally the activity of the human HNF4A can be modulated to treat those diseases.

Cancer Disorders

Cancer disorders within the scope of this definition comprise any disease of an organ or tissue in

I¹ mammals characterized by poorly controlled or uncontrolled multiplication of normal or abnormal cells in that tissue and its effect on the body as a whole. Cancer diseases within the scope of the definition comprise benign neoplasms, dysplasias, hyperplasias as well as neoplasms showing metastatic growth or any other transformations like e.g. leukoplakias which often precede a breakout of cancer. Cells and tissues are cancerous when they grow more rapidly than normal cells, displacing or spreading into the surrounding healthy tissue or any other tissues of the body described as metastatic growth, assume abnormal shapes and sizes, show changes in their nucleocytoplasmatic ratio, nuclear polychromasia, and finally may cease. Cancerous cells and tissues may affect the body as a whole when causing paraneoplastic syndromes or if cancer occurs within a vital organ or tissue, normal function will be impaired or halted, with possible fatal results. The ultimate involvement of a vital organ by cancer, either primary or metastatic, may lead to the death of the mammal affected. Cancer tends to spread, and the extent of its spread is usually related to an individual's chances of surviving the disease. Cancers are generally said to be in one of three stages of growth: early, or localized, when a tumor is still confined to the tissue of origin, or primary site; direct extension, where cancer cells from the tumour have invaded adjacent tissue or have spread only to regional lymph nodes; or metastasis, in which cancer cells have migrated to distant parts of the body from the primary site, via the blood or lymph systems, and have established secondary sites of infection. Cancer is said to be malignant because of its tendency to cause death if not treated. Benign tumors usually do not cause death, although they may if they interfere with a normal body function by virtue of their location, size, or paraneoplastic side effects. Hence benign tumors fall under the definition of cancer within the scope of this definition as well. In general, cancer cells divide at a higher rate than do normal cells, but the distinction between the growth of cancerous and normal tissues is not so much the rapidity of cell division in the former as it is the partial or complete loss of growth restraint in cancer cells and their failure to differentiate into a useful, limited tissue of the type that characterizes the functional equilibrium of growth of normal tissue. Cancer tissues may express certain molecular receptors and probably are influenced by the host's susceptibility and immunity and it is known that certain cancers of the breast and prostate, for example, are considered dependent on specific hormones for their existence. The term "cancer" under the scope of the definition is not limited to simple benign neoplasia but comprises any other benign and malign neoplasia like 1) Carcinoma, 2) Sarcoma, 3) Carcinosarcoma, 4) Cancers of the blood-forming tissues, 5) tumors of nerve tissues including the brain, 6) cancer of skin cells. Cancer according to 1) occurs in epithelial tissues, which cover the outer body (the skin) and line mucous membranes and the inner cavitary structures of organs e.g. such as the breast, lung, the respiratory and gastrointestinal tracts, the endocrine glands, and the genitourinary system. Ductal or glandular elements may persist in epithelial tumors, as in adenocarcinomas like e.g. thyroid adenocarcinoma, gastric adenocarcinoma, uterine adenocarcinoma. Cancers of the pavement-cell epithelium of the skin and of certain mucous membranes, such as e.g. cancers of the tongue, lip, larynx, urinary bladder, uterine cervix, or penis, may be termed epidermoid or squamous-cell carcinomas of the respective tissues and are in the scope of the definition of cancer as well. Cancer according to 2) develops in connective tissues, including fibrous tissues, adipose (fat) tissues, muscle, blood vessels, bone, and cartilage like e.g. osteogenic sarcoma; liposarcoma, fibrosarcoma, synovial sarcoma. Cancer according to 3) is cancer that develops in both epithelial and connective tissue. Cancer disease within the scope of this definition may be primary or secondary, whereby primary indicates that the cancer originated in the tissue where it is found rather than was established as a secondary site through metastasis from another lesion. Cancers and tumor diseases within the scope of this definition may be benign or malign and may affect all anatomical structures of the body of a mammal. By example but not limited to they comprise cancers and tumor diseases of I) the bone marrow and bone marrow derived cells (leukemias), H) the endocrine and exocrine glands like e.g. thyroid, parathyroid, pituitary, adrenal glands, salivary glands, pancreas IH) the breast, like e.g. benign or malignant tumors in the mammary glands of either a male or a female, the mammary ducts, adenocarcinoma, medullary carcinoma, comedo carcinoma, Paget's disease of the nipple, inflammatory carcinoma of the young woman, IV) the lung, V) the stomach, VI) the liver and spleen, VII) the small intestine, Vπi) the colon, EX) the bone and its supportive and connective tissues like malignant or benign bone tumour, e.g. malignant osteogenic sarcoma, benign osteoma, cartilage tumors; like malignant chondrosarcoma or benign chondroma; bone marrow tumors like malignant myeloma or benign eosinophilic granuloma, as well as metastatic tumors from bone tissues at other locations of the body; X) the mouth, throat, larynx, and the esophagus, XT) the urinary bladder and the internal and external organs and structures of the urogenital system of male and female like ovaries, uterus, cervix of the uterus, testes, and prostate gland, XII) the prostate, XIQ) the pancreas, like ductal carcinoma of the pancreas; XIV) the lymphatic tissue like lymphomas and other tumors of lymphoid origin, XV) the skin, XVI) cancers and tumor diseases of all anatomical structures belonging to the respiration and respiratory systems including thoracal muscles and linings, XVIT) primary or secondary cancer of the lymph nodes XVIH) the tongue and of the bony structures of the hard palate or sinuses, XVIV) the mouth, cheeks, neck and salivary glands, XX) the blood vessels including the heart and their linings, XXI) the smooth or skeletal muscles and their ligaments and linings, XXII) the peripheral, the autonomous, the central nervous system including the cerebellum, XXITf) the adipose tissue.

The human HNF4A is highly expressed in the following cancer tissues: stomach tumor, colon tumor, rectum tumor, liver tumor, HEP G2 cells. The expression in the above mentioned tissues and in particular the differential expression between diseased tissue colon tumor and healthy tissue colon, between diseased tissue rectum tumor and healthy tissue rectum, between diseased tissue liver tumor and healthy tissue liver, between diseased tissue HEP G2 cells and healthy tissue liver demonstrates that the human HNF4A or mRNA can be utilized to diagnose of cancer. Additionally the activity of the human HNF4A can be modulated to treat cancer.

Inflammatory Diseases

Inflammatory diseases comprise diseases triggered by cellular or non-cellular mediators of the immune system or tissues causing the inflammation of body tissues and subsequently producing an acute or chronic inflammatory condition. Examples for such inflammatory diseases are hypersensitivity reactions of type I - IV, for example but not limited to hypersensitivity diseases of the lung including asthma, atopic diseases, allergic rhinitis or conjunctivitis, angioedema of the lids, hereditary angioedema, antireceptor hypersensitivity reactions and autoimmune diseases, Hashimoto's thyroiditis, systemic lupus erythematosus, Goodpasture's syndrome, pemphigus, myasthenia gravis, Grave's and Raynaud's disease, type B insulin-resistant diabetes, rheumatoid arthritis, psoriasis, Crohn's disease, scleroderma, mixed connective tissue disease, polymyositis, sarcoidosis, glomerulonephritis, acute or chronic host versus graft reactions.

The human HNF4A is highly expressed in the following tissues of the immune system and tissues responsive to components of the immune system as well as in the following tissues responsive to mediators of inflammation: liver liver cirrhosis, lung lupus disease. The expression in the above mentioned tissues and in particular the differential expression between diseased tissue liver liver cirrhosis and healthy tissue liver demonstrates that the human HNF4A or mRNA can be utilized to diagnose of inflammatory diseases. Additionally the activity of the human HNF4A can be modulated to treat inflammatory diseases.

Disorders Related to Pulmology

Asthma is thought to arise as a result of interactions between multiple genetic and environmental factors and is characterized by three major features: 1) intermittent and reversible airway obstruction caused by bronchoconstriction, increased mucus production, and thickening of the walls of the airways that leads to a narrowing of the airways, 2) airway hyperresponsiveness, and 3) airway inflammation. Certain cells are critical to the inflammatory reaction of asthma and they include T cells and antigen presenting cells, B cells that produce IgE, and mast cells, basophils, eosinophils, and other cells that bind IgE. These effector cells accumulate at the site of allergic reaction in the airways and release toxic products that contribute to the acute pathology and eventually to tissue destruction related to the disorder. Other resident cells, such as smooth muscle cells, lung epithelial cells, mucus-producing cells, and nerve cells may also be abnormal in individuals with asthma and may contribute to its pathology. While the airway obstruction of asthma, presenting clinically as an intermittent wheeze and shortness of breath, is generally the most pressing symptom of the disease requiring immediate treatment, the inflammation and tissue destruction associated with the disease can lead to irreversible changes that eventually make asthma a chronic and disabling disorder requiring long-term management.

Chronic obstructive pulmonary (or airways) disease (COPD) is a condition defined physiologically as airflow obstruction that generally results from a mixture of emphysema and peripheral airway obstruction due to chronic bronchitis [Botstein, 1980]. Emphysema is characterised by destruction of alveolar walls leading to abnormal enlargement of the air spaces of the lung. Chronic bronchitis is defined clinically as the presence of chronic productive cough for three months in each of two successive years. In COPD, airflow obstruction is usually progressive and is only partially reversible. By far the most important risk factor for development of COPD is cigarette smoking, although the disease does also occur in non-smokers.

The human HNF4A is highly expressed in the following tissues of the respiratory system: lung lupus disease. The expression in the above mentioned tissues demonstrates that the human HNF4A or mRNA can be utilized to diagnose of respiratory diseases. Additionally the activity of the human HNF4A can be modulated to treat those diseases.

Disorders Related to Urology

Genitourinary disorders comprise benign and malign disorders of the organs constituting the genitourinary system of female and male, renal diseases like acute or chronic renal failure, immunologically mediated renal diseases like renal transplant rejection, lupus nephritis, immune complex renal diseases, glomerulopathies, nephritis, toxic nephropathy, obstructive uropathies like benign prostatic hyperplasia (BPH), neurogenic bladder syndrome, urinary incontinence like urge-, stress-, or overflow incontinence, pelvic pain, and erectile dysfunction.

The human HNF4A is highly expressed in the following urological tissues: bladder, fetal kidney, kidney, kidney, kidney. The expression in the above mentioned tissues demonstrates that the human HNF4A or mRNA can be utilized to diagnose of urological disorders. Additionally the activity of the human HNF4A can be modulated to treat urological disorders.

Diseases of the Reproductive System

Disorders of the male reproductive system include but are not limited to balanoposthitis, balanitis xerotica obliterans, phimosis, paraphimosis, erythroplasia of Queyrat, skin cancer of the penis, Bowen's and Paget's diseases, syphilis, herpes simplex infections, genital warts, molluscum contagiosum, priapism, Peyronie's disease, benign prostatic hyperplasia (BPH), prostate cancer, prostatitis, testicular cancer, testicular torsion, inguinal hernia, epididymo-orchitis, mumps, hydroceles, spermatoceles, or varicoceles.

Impotence (erectile dysfunction) may results from vascular impairment, neurologic disorders, drugs, abnormalities of the penis, or psychologic problems.

Examples of disorders of the female reproductive include premature menopause, pelvic pain, vaginitis, vulvitis, vulvovaginitis, pelvic inflammatory disease, fibroids, menstrual disorders (premenstrual syndrome (PMS), dysmenorrhea, amenorrhea, primary amenorrhea, secondary amenorrhea, menorrhagia, hypomenorrhea, polymenorrhea, oligomenorrhea, metrorrhagia, menometrorrhagia, Postmenopausal bleeding), bleeding caused by a physical disorder, dysfunctional uterine bleeding, polycystic ovary syndrome (Stein-Leventhal syndrome), endometriosis, cancer of the uterus, cancer of the cervix, cancer of the ovaries, cancer of the vulva, cancer of the vagina, cancer of the fallopian tubes, or hydatidiform mole.

Infertility may be caused by problems with sperm, ovulation, the fallopian tubes, and the cervix as well as unidentified factors.

Complications of pregnancy include miscarriage and stillbirth, ectopic pregnancy, anemia, Rh incompatibility, problems with the placenta, excessive vomiting, preeclampsia, eclampsia, and skdn rashes (e.g. herpes gestationis, urticaria of pregnancy) as well as preterm labor and premature rupture of the membranes.

Breast disorders may be noncancerous (benign) or cancerous (malignant). Examples of breast disorders are but are not limited to breast pain, cysts, fibrocystic breast disease, fibrous lumps, nipple discharge, breast infection, breast cancer (ductal carcinoma, lobular carcinoma, medullary carcinoma, tubular carcinoma, and inflammatory breast cancer), Paget's disease of the nipple or Cystosarcoma phyllodes.

The human HNF4A is highly expressed in the following tissues of the reproduction system: breast. The expression in the above mentioned tissues demonstrates that the human HNF4A or mRNA can be utilized to diagnose of reproduction disorders. Additionally the activity of the human

HNF4A can be modulated to treat reproduction disorders.

Metabolic Disorders

Metabolic diseases are defined as conditions which result from an abnormality in any of the chemical or biochemical transformations and their regulating systems essential to producing energy, to regenerating cellular constituents, to eliminating unneeded products arising from these processes, and to regulate and maintain homeostasis in a mammal regardless of whether acquired or the result of a genetic transformation. Depending on which metabolic pathway is involved, a single defective transformation or disturbance of its regulation may produce consequences that are narrow, involving a single body function, or broad, affecting many organs, organ-systems or the body as a whole. Diseases resulting from abnormalities related to the fine and coarse mechanisms that affect each individual transformation, its rate and direction or the availability of substrates like amino acids, fatty acids, carbohydrates, minerals, cofactors, hormones, regardless whether they are inborn or acquired, are well within the scope of the definition of a metabolic disease according to this application.

Metabolic diseases often are caused by single defects in particular biochemical pathways, defects that are due to the deficient activity of individual enzymes or molecular receptors leading to the regulation of such enzymes. Hence in a broader sense disturbances of the underlying genes, their products and their regulation lie well within the scope of this definition of a metabolic disease. For example, but not limited to, metabolic diseases may affect 1) biochemical processes and tissues ubiquitous all over the body, 2) the bone, 3) the nervous system, 4) the endocrine system, 5) the muscle including the heart, 6) the skin and nervous tissue, 7) the urogenital system, 8) the homeostasis of body systems like water and electrolytes. For example, but not limited to, metabolic diseases according to 1) comprise obesity, amyloidosis, disturbances of the amino acid metabolism like branched chain disease, hyperaminoacidemia, hyperaminoaciduria, disturbances of the metabolism of urea, hyperammonemia, mucopolysaccharidoses e.g. Maroteaux-Lamy syndrom, storage diseases like glycogen storage diseases and lipid storage diseases, glycogenosis diseases like Cori's disease, malabsorption diseases like intestinal carbohydrate malabsorption, oligosaccharidase deficiency like maltase-, lactase-, sucrase-insufficiency, disorders of the metabolism of fructose, disorders of the metabolism of galactose, galactosaemia, disturbances of carbohydrate utilization like diabetes, hypoglycemia, disturbances of pyruvate metabolism, hypolipidemia, hypolipoproteinemia, hyperlipidemia, hyperlipoproteinemia, carnitine or carnitine acyltransferase deficiency, disturbances of the porphyrin metabolism, porphyrias, disturbances of the purine metabolism, lysosomal diseases, metabolic diseases of nerves and nervous systems like gangliosidoses, sphingolipidoses, sulfatidoses, leucodystrophies, Lesch-Nyhan syndrome. For example, but not limited to, metabolic diseases according to 2) comprise osteoporosis, osteomalacia like osteoporosis, osteopenia, osteogenesis imperfecta, osteopetrosis, osteonecrosis, Paget's disease of bone, hypophosphatemia. For example, but not limited to, metabolic diseases according to 3) comprise cerebellar dysfunction, disturbances of brain metabolism like dementia, Alzheimer's disease, Huntington's chorea, Parkinson's disease, Pick's disease, toxic encephalopathy, demyelinating neuropathies like inflammatory neuropathy, Guillain-Barre syndrome. For example, but not limited to, metabolic diseases according to 4) comprise primary and secondary metabolic disorders associated with hormonal defects like any disorder stemming from either an hyperfunction or hypofunction of some hormone-secreting endocrine gland and any combination thereof. They comprise Sipple's syndrome, pituitary gland dysfunction and its effects on other endocrine glands, such as the thyroid, adrenals, ovaries, and testes, acromegaly, hyper- and hypothyroidism, euthyroid goiter, euthyroid sick syndrome, thyroiditis, and thyroid cancer, over- or underproduction of the adrenal steroid hormones, adrenogenital syndrome, Cushing's syndrome, Addison's disease of the adrenal cortex, Addison's pernicious anemia, primary and secondary aldosteronism, diabetes insipidus, carcinoid syndrome, disturbances caused by the dysfunction of the parathyroid glands, pancreatic islet cell dysfunction, diabetes, disturbances of the endocrine system of the female like estrogen deficiency, resistant ovary syndrome. For example, but not limited to, metabolic diseases according to 5) comprise muscle weakness, myotonia, Duchenne's and other muscular dystrophies, dystrophia myotonica of Steinert, mitochondrial myopathies like disturbances of the catabolic metabolism in the muscle, carbohydrate and lipid storage myopathies, glycogenoses, myoglobinuria, malignant hyperthermia, polymyalgia rheumatica, dermatomyositis, primary myocardial disease, cardiomyopathy. For example, but not limited to, metabolic diseases according to 6) comprise disorders of the ectoderm, neurofibromatosis, scleroderma and polyarteritis, Louis-Bar syndrome, von Hippel-Lindau disease, Sturge-Weber syndrome, tuberous sclerosis, amyloidosis, porphyria. For example, but not limited to, metabolic diseases according to 7) comprise sexual dysfunction of the male and female. For example, but not limited to, metabolic diseases according to 8) comprise confused states and seizures due to inappropriate secretion of antidiuretic hormone from the pituitary gland, Liddle's syndrome, Bartter's syndrome, Fanconi's syndrome, renal electrolyte wasting, diabetes insipidus.

The human HNF4A is highly expressed in the following metabolic disease related tissues: fetal liver, liver, liver, liver liver cirrhosis, liver lupus disease, HEP G2 cells and adipose. The expression in the above mentioned tissues and in particular the differential expression between diseased tissue liver liver cirrhosis and healthy tissue liver, between diseased tissue liver lupus disease and healthy tissue liver demonstrates that the human HNF4A or mRNA can be utilized to diagnose of metabolic diseases. Additionally the activity of the human HNF4A can be modulated to treat metabolic diseases.

Applications

The present invention provides for both prophylactic and therapeutic methods for gastrointestinal and liver diseases, cancer disorders, cardiovascular disorders, metabolic diseases, inflammatory diseases, muscle-skeleton disorders, respiratory diseases, reproduction disorders and urological disorders..

The regulatory method of the invention involves contacting a cell with an agent that modulates one or more of the activities of HNF4A. An agent that modulates activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally-occurring cognate ligand of the polypeptide, a peptide, a peptidomimetic, or any small molecule. In one embodiment, the agent stimulates one or more of the biological activities of HNF4A. Examples of such stimulatory agents include the active HNF4A and nucleic acid molecules encoding a portion of HNF4A. In another embodiment, the agent inhibits one or more of the biological activities of HNF4A. Examples of such inhibitory agents include antisense nucleic acid molecules and antibodies. These regulatory methods can be performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g, by administering the agent to a subject). As such, the present invention provides methods of treating an individual afflicted with a disease or disorder characterized by unwanted expression or activity of HNF4A or a protein in the HNF4A signaling pathway. In one embodiment, the method involves administering an agent like any agent identified or being identifiable by a screening assay as described herein, or combination of such agents that modulate say upregulate or downregulate the expression or activity of HNF4A or of any protein in the HNF4A signaling pathway. In another embodiment, the method involves administering a regulator of HNF4A as therapy to compensate for reduced or undesirably low expression or activity of HNF4A or a protein in the HNF4A signaling pathway.

Stimulation of activity or expression of HNF4A is desirable in situations in which activity or expression is abnormally low and in which increased activity is likely to have a beneficial effect.

Conversely, inhibition of activity or expression of HNF4A is desirable in situations in which activity or expression of HNF4A is abnormally high and in which decreasing its activity is likely to have a beneficial effect.

This invention is further illustrated by the following examples which should not be construed as limiting. The contents of all references, patents and published patent applications cited throughout this application are hereby incorporated by reference.

Pharmaceutical Compositions

This invention further pertains to novel agents identified by the above-described screening assays and uses thereof for treatments as described herein.

The nucleic acid molecules, polypeptides, and antibodies (also referred to herein as "active compounds") of the invention can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier. As used herein the language "pharma¬ ceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharma¬ ceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

The invention includes pharmaceutical compositions comprising a regulator of HNF4A expression or activity (and/or a regulator of the activity or expression of a protein in the HNF4A signaling pathway) as well as methods for preparing such compositions by combining one or more such regulators and a pharmaceutically acceptable carrier. Also within the invention are pharmaceutical compositions comprising a regulator identified using the screening assays of the invention packaged with instructions for use. For regulators that are antagonists of HNF4A activity or which reduce HNF4A expression, the instructions would specify use of the pharmaceutical composition for treatment of gastrointestinal and liver diseases, cancer disorders, cardiovascular disorders, metabolic diseases, inflammatory diseases, muscle-skeleton disorders, respiratory diseases, reproduction disorders and urological disorders.. For regulators that are agonists of HNF4A activity or increase HNF4A expression, the instructions would specify use of the pharmaceutical composition for treatment of gastrointestinal and liver diseases, cancer disorders, cardiovascular disorders, metabolic diseases, inflammatory diseases, muscle-skeleton disorders, respiratory diseases, reproduction disorders and urological disorders..

An antagonist of HNF4A may be produced using methods which are generally known in the art. In particular, purified HNF4A may be used to produce antibodies or to screen libraries of pharmaceutical agents to identify those which specifically bind HNF4A. Antibodies to HNF4A may also be generated using methods that are well known in the art. Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric, single chain antibodies, Fab fragments, and fragments produced by a Fab expression library. Neutralizing antibodies like those which inhibit dimer formation are especially preferred for therapeutic use.

In another embodiment of the invention, the polynucleotides encoding HNF4A, or any fragment or complement thereof, may be used for therapeutic purposes. In one aspect, the complement of the polynucleotide encoding HNF4A may be used in situations in which it would be desirable to block the transcription of the mRNA. In particular, cells may be transformed with sequences complementary to polynucleotides encoding HNF4A. Thus, complementary molecules or fragments may be used to modulate HNF4A activity, or to achieve regulation of gene function. Such technology is now well known in the art, and sense or antisense oligonucleotides or larger fragments can be designed from various locations along the coding or control regions of sequences encoding HNF4A. Expression vectors derived from retroviruses, adenoviruses, or herpes or vaccinia viruses, or from various bacterial plasmids, may be used for delivery of nucleotide sequences to the targeted organ, tissue, or cell population. Methods which are well known to those skilled in the art can be used to construct vectors which will express nucleic acid sequence complementary to the polynucleotides of the gene encoding HNF4A. These techniques are described, for example, in [Scott and Smith

(1990) Science 249:386-390].

Any of the therapeutic methods described above may be applied to any subject in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.

An additional embodiment of the invention relates to the administration of a pharmaceutical composition containing HNF4A in conjunction with a pharmaceutically acceptable carrier, for any of the therapeutic effects discussed above. Such pharmaceutical compositions may consist of HNF4A, antibodies to HNF4A, and mimetics, agonists, antagonists, or inhibitors of HNF4A. The compositions may be administered alone or in combination with at least one other agent, such as a stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier including, but not limited to, saline, buffered saline, dextrose, and water. The compositions may be administered to a patient alone, or in combination with other agents, drugs or hormones.

A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EM™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, a pharmaceutically acceptable polyol like glycerol, propylene glycol, liquid polyetheylene glycol, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin. Sterile injectable solutions can be prepared by incorporating the active compound (e.g., a polypeptide or antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof.

Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed.

Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring. For administration by inhalation, the compounds are delivered in the form of an aerosol spray from a pressurized container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. 4,522,811.

It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals. The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration. For pharmaceutical compositions which include an antagonist of HNF4A activity, a compound which reduces expression of HNF4A, or a compound which reduces expression or activity of a protein in the HNF4A signaling pathway or any combination thereof, the instructions for administration will specify use of the composition for gastrointestinal and liver diseases, cancer disorders, cardiovascular disorders, metabolic diseases, inflammatory diseases, muscle-skeleton disorders, respiratory diseases, reproduction disorders and urological disorders.. For pharmaceutical compositions which include an agonist of HNF4A activity, a compound which increases expression of HNF4A, or a compound which increases expression or activity of a protein in the HNF4A signaling pathway or any combination thereof, the instructions for administration will specify use of the composition for gastrointestinal and liver diseases, cancer disorders, cardiovascular disorders, metabolic diseases, inflammatory diseases, muscle-skeleton disorders, respiratory diseases, reproduction disorders and urological disorders..

Diagnostics

In another embodiment, antibodies which specifically bind HNF4A may be used for the diagnosis of disorders characterized by the expression of HNF4A, or in assays to monitor patients being treated with HNF4A or agonists, antagonists, and inhibitors of HNF4A. Antibodies useful for diagnostic purposes may be prepared in the same manner as those described above for therapeutics. Diagnostic assays for HNF4A include methods which utilize the antibody and a label to detect HNF4A in human body fluids or in extracts of cells or tissues. The antibodies may be used with or without modification, and may be labeled by covalent or non-covalent joining with a reporter molecule. A wide variety of reporter molecules, several of which are described above, are known in the art and may be used.

A variety of protocols for measuring HNF4A, including ELISAs, RIAs, and FACS, are known in the art and provide a basis for diagnosing altered or abnormal levels of HNF4A expression.

Normal or standard values for HNF4A expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, preferably human, with antibody to HNF4A under conditions suitable for complex formation The amount of standard complex formation may be quantified by various methods, preferably by photometric means. Quantities of HNF4A expressed in subject samples from biopsied tissues are compared with the standard values.

Deviation between standard and subject values establishes the parameters for diagnosing disease.

In another embodiment of the invention, the polynucleotides encoding HNF4A may be used for diagnostic purposes. The polynucleotides which may be used include oligonucleotide sequences, complementary RNA and DNA molecules, and PNAs. The polynucleotides may be used to detect and quantitate gene expression in biopsied tissues in which expression of HNF4A may be correlated with disease. The diagnostic assay may be used to distinguish between absence, presence, and excess expression of HNF4A, and to monitor regulation of HNF4A levels during therapeutic intervention.

Polynucleotide sequences encoding HNF4A may be used for the diagnosis of gastrointestinal and liver diseases, cancer disorders, cardiovascular disorders, metabolic diseases, inflammatory diseases, muscle-skeleton disorders, respiratory diseases, reproduction disorders and urological disorders, associated with expression of HNF4A. The polynucleotide sequences encoding HNF4A may be used in Southern, Northern, or dot-blot analysis, or other membrane-based technologies; in

PCR technologies; in dipstick, pin, and ELISA assays; and in microarrays utilizing fluids or tissues from patient biopsies to detect altered HNF4A expression. Such qualitative or quantitative methods are well known in the art.

In a particular aspect, the nucleotide sequences encoding HNF4A may be useful in assays that detect the presence of associated disorders, particularly those mentioned above. The nucleotide sequences encoding HNF4A may be labelled by standard methods and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybridization complexes. After a suitable incubation period, the sample is washed and the signal is quantitated and compared with a standard value. If the amount of signal in the patient sample is significantly altered from that of a comparable control sample, the nucleotide sequences have hybridized with nucleotide sequences in the sample, and the presence of altered levels of nucleotide sequences encoding HNF4A in the sample indicates the presence of the associated disorder. Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials, or in monitoring the treatment of an individual patient.

In order to provide a basis for the diagnosis of gastrointestinal and liver diseases, cancer disorders, cardiovascular disorders, metabolic diseases, inflammatory diseases, muscle-skeleton disorders, respiratory diseases, reproduction disorders and urological disorders, associated with expression of HNF4A, a normal or standard profile for expression is established. This may be accomplished by combining body fluids or cell extracts taken from normal subjects, either animal or human, with a sequence, or a fragment thereof, encoding HNF4A, under conditions suitable for hybridization or amplification. Standard hybridization may be quantified by comparing the values obtained from normal subjects with values from an experiment in which a known amount of a substantially purified polynucleotide is used. Standard values obtained from normal samples may be compared with values obtained from samples from patients who are symptomatic for a disorder. Deviation from standard values is used to establish the presence of a disorder.

Another technique for drug screening which may be used provides for high throughput screening of compounds having suitable binding affinity to the protein of interest as described in published PCT application WO84/03564. Li this method, large numbers of different small test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. The test compounds are reacted with HNF4A, or fragments thereof, and washed. Bound HNF4A is then detected by methods well known in the art. Purified HNF4A can also be coated directly onto plates for use in the aforementioned drug screening techniques. Alternatively, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.

In another embodiment, one may use competitive drug screening assays in which neutralizing antibodies capable of binding HNF4A specifically compete with a testcompound for binding HNF4A. In this manner, antibodies can be used to detect the presence of any peptide which shares one or more antigenic determinants with HNF4A.

Another technique for drug screening which may be used provides for high throughput screening of compounds having suitable binding affinity to the protein of interest as described in published PCT application WO84/03564. In this method, large numbers of different small test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. The test compounds are reacted with HNF4A, or fragments thereof, and washed. Bound HNF4A is then detected by methods well known in the art. Purified HNF4A can also be coated directly onto plates for use in the afore-mentioned drug screening techniques. Alternatively, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.

In another embodiment, one may use competitive drug screening assays in which neutralizing antibodies capable of binding HNF4A specifically compete with a testcompound for binding HNF4A. In this manner, antibodies can be used to detect the presence of any peptide which shares one or more antigenic determinants with HNF4A.

Determination of a Therapeutically Effective Dose

The determination of a therapeutically effective dose is well within the capability of those skilled in the art. A therapeutically effective dose refers to that amount of active ingredient which increases or decreases HNF4A activity relative to HNF4A activity which occurs in the absence of the therapeutically effective dose. For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, usually mice, rabbits, dogs, or pigs. The animal model also can be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.

Therapeutic efficacy and toxicity, e.g., ED₅₀ (the dose therapeutically effective in 50% of the population) and LD₅₀ (the dose lethal to 50% of the population), can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. The dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD₅₀ZED₅₀. Pharmaceutical compositions which exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use. The dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration. The exact dosage will be determined by the practitioner, in light of factors related to the subject that requires treatment. Dosage and administration are adjusted to provide sufficient levels of the active ingredient or to maintain the desired effect. Factors which can be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long-acting pharmaceutical compositions can be administered every 3 to 4 days, every week, or once every two weeks depending on the half-life and clearance rate of the particular formulation.

Normal dosage amounts can vary from 0.1 micrograms to 100,000 micrograms, up to a total dose of about 1 g, depending upon the route of administration. Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art. Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc. If the reagent is a single-chain antibody, polynucleotides encoding the antibody can be constructed and introduced into a cell either ex vivo or in vivo using well- established techniques including, but not limited to, transferrin-polycation-mediated DNA transfer, transfection with naked or encapsulated nucleic acids, liposome-mediated cellular fusion, intracellular transportation of DNA-coated latex beads, protoplast fusion, viral infection, electroporation, "gene gun", and DEAE- or calcium phosphate-mediated transfection.

If the expression product is mRNA, the reagent is preferably an antisense oligonucleotide or a ribozyme. Polynucleotides which express antisense oligonucleotides or ribozymes can be introduced into cells by a variety of methods, as described above. Preferably, a reagent reduces expression of HNF4A gene or the activity of HNF4A by at least about 10, preferably about 50, more preferably about 75, 90, or 100% relative to the absence of the reagent. The effectiveness of the mechanism chosen to decrease the level of expression of HNF4A gene or the activity of HNF4A can be assessed using methods well known in the art, such as hybridization of nucleotide probes to HNF4A-specific mRNA, quantitative RT-PCR, immunologic detection of HNF4A, or measurement of HNF4A activity.

In any of the embodiments described above, any of the pharmaceutical compositions of the invention can be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy can be made by one of ordinary skill in the art, according to conventional pharmaceutical principles. The combination of therapeutic agents can act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects. Any of the therapeutic methods described above can be applied to any subject in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.

Nucleic acid molecules of the invention are those nucleic acid molecules which are contained in a group of nucleic acid molecules consisting of (i) nucleic acid molecules encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, (ii) nucleic acid molecules comprising the sequence of SEQ ID NO: 1, (iii) nucleic acid molecules having the sequence of SEQ ID NO: 1,

(iv)nucleic acid molecules the complementary strand of which hybridizes under stringent conditions to a nucleic acid molecule of (i), (ii), or (iii); and (v) nucleic acid molecules the sequence of which differs from the sequence of a nucleic acid molecule of (iii) due to the degeneracy of the genetic code, wherein the polypeptide encoded by said nucleic acid molecule has HNF4A activity.

Polypeptides of the invention are those polypeptides which are contained in a group of polypeptides consisting of (i) polypeptides having the sequence of SEQ ID NO: 2, (ii) polypeptides comprising the sequence of SEQ ID NO: 2, (iii) polypeptides encoded by nucleic acid molecules of the invention and (iv) polypeptides which show at least 99%, 98%, 95%, 90%, or 80% homology with a polypeptide of (i), (ii), or (iii), wherein said purified polypeptide has HNF4A activity.

An object of the invention is a method of screening for therapeutic agents useful in the treatment of a disease comprised in a group of diseases consisting of gastrointestinal and liver diseases, cancer disorders, cardiovascular disorders, metabolic diseases, inflammatory diseases, muscle- skeleton disorders, respiratory diseases, reproduction disorders and urological disorders, in a mammal comprising the steps of (i) contacting a test compound with a HNF4A polypeptide, (ii) detect binding of said test compound to said HNF4A polypeptide. E.g., compounds that bind to the HNF4A polypeptide are identified potential therapeutic agents for such a disease.

Another object of the invention is a method of screening for therapeutic agents useful in the treatment of a disease comprised in a group of diseases consisting of gastrointestinal and liver diseases, cancer disorders, cardiovascular disorders, metabolic diseases, inflammatory diseases, muscle-skeleton disorders, respiratory diseases, reproduction disorders and urological disorders, in a mammal comprising the steps of (i) determining the activity of a HNF4A polypeptide at a certain concentration of a test compound or in the absence of said test compound, (ii) determining the activity of said polypeptide at a different concentration of said test compound. E.g., compounds that lead to a difference in the activity of the HNF4A polypeptide in (i) and (ii) are identified potential therapeutic agents for such a disease.

Another object of the invention is a method of screening for therapeutic agents useful in the treatment of a disease comprised in a group of diseases consisting of gastrointestinal and liver diseases, cancer disorders, cardiovascular disorders, metabolic diseases, inflammatory diseases, muscle-skeleton disorders, respiratory diseases, reproduction disorders and urological disorders, in a mammal comprising the steps of (i) determining the activity of a HNF4A polypeptide at a certain concentration of a test compound, (ii) determining the activity of a HNF4A polypeptide at the presence of a compound known to be a regulator of a HNF4A polypeptide. E.g., compounds that show similar effects on the activity of the HNF4A polypeptide in (i) as compared to compounds used in (ii) are identified potential therapeutic agents for such a disease.

Other objects of the invention are methods of the above, wherein the step of contacting is in or at the surface of a cell.

Other objects of the invention are methods of the above, wherein the cell is in vitro.

Other objects of the invention are methods of the above, wherein the step of contacting is in a cell- free system.

Other objects of the invention are methods of the above, wherein the polypeptide is coupled to a detectable label.

Other objects of the invention are methods of the above, wherein the compound is coupled to a detectable label. Other objects of the invention are methods of the above, wherein the test compound displaces a ligand which is first bound to the polypeptide.

Other objects of the invention are methods of the above, wherein the polypeptide is attached to a solid support.

Other objects of the invention are methods of the above, wherein the compound is attached to a solid support.

Another object of the invention is a method of screening for therapeutic agents useful in the treatment of a disease comprised in a group of diseases consisting of gastrointestinal and liver diseases, cancer disorders, cardiovascular disorders, metabolic diseases, inflammatory diseases, muscle-skeleton disorders, respiratory diseases, reproduction disorders and urological disorders, in a mammal comprising the steps of (i) contacting a test compound with a HNF4A polynucleotide, (ii) detect binding of said test compound to said HNF4A polynucleotide. Compounds that, e.g., bind to the HNF4A polynucleotide are potential therapeutic agents for the treatment of such diseases.

Another object of the invention is the method of the above, wherein the nucleic acid molecule is

RNA.

Another object of the invention is a method of the above, wherein the contacting step is in or at the surface of a cell.

Another object of the invention is a method of the above, wherein the contacting step is in a cell- free system.

Another object of the invention is a method of the above, wherein the polynucleotide is coupled to a detectable label.

Another object of the invention is a method of the above, wherein the test compound is coupled to a detectable label.

Another object of the invention is a method of diagnosing a disease comprised in a group of diseases consisting of gastrointestinal and liver diseases, cancer disorders, cardiovascular disorders, metabolic diseases, inflammatory diseases, muscle-skeleton disorders, respiratory diseases, reproduction disorders and urological disorders, in a mammal comprising the steps of (i) determining the amount of a HNF4A polynucleotide in a sample taken from said mammal, (ii) determining the amount of HNF4A polynucleotide in healthy and/or diseased mammal. A disease is diagnosed, e.g., if there is a substantial similarity in the amount of HNF4A polynucleotide in said test mammal as compared to a diseased mammal.

Another object of the invention is a pharmaceutical composition for the treatment of a disease comprised in a group of diseases consisting of gastrointestinal and liver diseases, cancer disorders, cardiovascular disorders, metabolic diseases, inflammatory diseases, muscle-skeleton disorders, respiratory diseases, reproduction disorders and urological disorders, in a mammal comprising a therapeutic agent which binds to a HNF4A polypeptide.

Another object of the invention is a pharmaceutical composition for the treatment of a disease comprised in a group of diseases consisting of gastrointestinal and liver diseases, cancer disorders, cardiovascular disorders, metabolic diseases, inflammatory diseases, muscle-skeleton disorders, respiratory diseases, reproduction disorders and urological disorders, in a mammal comprising a therapeutic agent which regulates the activity of a HNF4A polypeptide.

Another object of the invention is a pharmaceutical composition for the treatment of a disease comprised in a group of diseases consisting of gastrointestinal and liver diseases, cancer disorders, cardiovascular disorders, metabolic diseases, inflammatory diseases, muscle-skeleton disorders, respiratory diseases, reproduction disorders and urological disorders, in a mammal comprising a therapeutic agent which regulates the activity of a HNF4A polypeptide, wherein said therapeutic agent is (i) a small molecule, (ii) an RNA molecule, (iii) an antisense oligonucleotide, (iv) a polypeptide, (v) an antibody, or (vi) a ribozyme.

Another object of the invention is a pharmaceutical composition for the treatment of a disease comprised in a group of diseases consisting of gastrointestinal and liver diseases, cancer disorders, cardiovascular disorders, metabolic diseases, inflammatory diseases, muscle-skeleton disorders, respiratory diseases, reproduction disorders and urological disorders, in a mammal comprising a HNF4A polynucleotide.

Another object of the invention is a pharmaceutical composition for the treatment of a disease comprised in a group of diseases consisting of gastrointestinal and liver diseases, cancer disorders, cardiovascular disorders, metabolic diseases, inflammatory diseases, muscle-skeleton disorders, respiratory diseases, reproduction disorders and urological disorders, in a mammal comprising a HNF4A polypeptide.

Another object of the invention is the use of regulators of a HNF4A for the preparation of a pharmaceutical composition for the treatment of a disease comprised in a group of diseases consisting of gastrointestinal and liver diseases, cancer disorders, cardiovascular disorders, metabolic diseases, inflammatory diseases, muscle-skeleton disorders, respiratory diseases, reproduction disorders and urological disorders, in a mammal.

Another object of the invention is a method for the preparation of a pharmaceutical composition useful for the treatment of a disease comprised in a group of diseases consisting of gastrointestinal and liver diseases, cancer disorders, cardiovascular disorders, metabolic diseases, inflammatory diseases, muscle-skeleton disorders, respiratory diseases, reproduction disorders and urological disorders, in a mammal comprising the steps of (i) identifying a regulator of HNF4A, (ii) determining whether said regulator ameliorates the symptoms of a disease comprised in a group of diseases consisting of gastrointestinal and liver diseases, cancer disorders, cardiovascular disorders, metabolic diseases, inflammatory diseases, muscle-skeleton disorders, respiratory diseases, reproduction disorders and urological disorders, in a mammal; and (iii) combining of said regulator with an acceptable pharmaceutical carrier.

Another object of the invention is the use of a regulator of HNF4A for the regulation of HNF4A activity in a mammal having a disease comprised in a group of diseases consisting of gastrointestinal and liver diseases, cancer disorders, cardiovascular disorders, metabolic diseases, inflammatory diseases, muscle-skeleton disorders, respiratory diseases, reproduction disorders and urological disorders..

The uses, methods or compositions of the invention are useful for each single disease comprised in a group of diseases consisting of gastrointestinal and liver diseases, cancer disorders, cardiovascular disorders, metabolic diseases, inflammatory diseases, muscle-skeleton disorders, respiratory diseases, reproduction disorders and urological disorders..

The expression of human HNF4A 1 in cardiovascular diseases and urological related tissues (as described above) suggests a particular - but not limited to - utilization of HNF4A for diagnosis and modulation cardiovascular disorders and urological diseases. Furthermore the above described expression suggest a - but not limited to - utilization of HNF4A to gastrointestinal and liver diseases, cancer disorders, metabolic diseases, inflammatory diseases, muscle-skeleton disorders, respiratory diseases, reproduction disorders.

The examples below are provided to illustrate the subject invention. These examples are provided by way of illustration and are not included for the purpose of limiting the invention. Examples

Example 1: Search for homologous sequences in public sequence data bases

The degree of homology can readily be calculated by known methods. Preferred methods to determine homology are designed to give the largest match between the sequences tested. Methods to determine homology are codified in publicly available computer programs such as

BestFit, BLASTP, BLASTN, and FASTA. The BLAST programs are publicly available from NCBI and other sources in the internet.

For HNF4A the following hits to known sequences were identified by using the BLAST algorithm [Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ; Nucleic Acids Res 1997 Sep 1; 25(17): 3389-402] and the following set of parameters: matrix = BLOSUM62 and low complexity filter. The following databases were searched: NCBI (non-redundant database) and DERWENT patent database (Geneseq).

The following hits were found:

>emb|CQ727128.1| Sequence 13062 from Patent WO02068579 Length = 1441, Score = 1980 bits (999), Expect = 0.0, Identities = 999/999 (100%)

>emb|AX676870.1| Sequence 63 from Patent WO02103028 Length = 1441, Score = 1980 bits (999), Expect = 0.0, Identities = 999/999 (100%)

>dbj|BD 176753.11 Method of screening HNF4-alpha agonist Length = 1441, Score = 1980 bits (999), Expect = 0.0, Identities = 999/999 (100%)

>gb|AR129495.1|AR129495 Sequence 78 from patent US 6187533

Length = 1441, Score = 1980 bits (999), Expect = 0.0, Identities = 999/999 (100%)

>emb|X76930.1|HSHNF4 H.sapiens HNF4 mRNA for hepatocyte nuclear factor 4 Length = 1441, Score = 1980 bits (999), Expect = 0.0, Identities = 999/999 (100%)

>ref]NM_l 78850.11 Homo sapiens hepatocyte nuclear factor 4, alpha (HNF4A), transcriptvariant 3, mRNA

Length = 1600, Score = 1980 bits (999), Expect = 0.0, Identities = 999/999 (100%)

>ref]NM_l 78849.11 Homo sapiens hepatocyte nuclear factor 4, alpha (HNF4A), transcriptvariant l. mRNA

Length = 3209, Score = 1980 bits (999), Expect = 0.0, Identities = 999/999 (100%) >reflNM_000457.3| Homo sapiens hepatocyte nuclear factor 4, alpha (HNF4A), transcriptvariant 2, mRNA Length = 3239, Score = 1980 bits (999), Expect = 0.0, Identities = 999/999 (100%)

>emb|X87872.1|HSHNF4CGN H.sapiens mRNA for hepatocyte nuclear factor 4c Length = 1614, Score = 1966 bits (992), Expect = 0.0, Identities = 999/1000 (99%), Gaps =

1/1000 (0%)

>emb|X87871.1|HSHNP4BGN H.sapiens mRNA for hepatocyte nuclear factor 4b Length = 1635, Score = 1959 bits (988), Expect = 0.0, Identities = 998/1000 (99%), Gaps = 1/1000 (0%)

>emb|X87870.1|HSHNF4AGN H.sapiens mRNA for hepatocyte nuclear factor 4a

Length = 2288, Score = 1959 bits (988), Expect = 0.0, Identities = 998/1000 (99%), Gaps = 1/1000 (0%)

>emb|Z49825.1|HSHNF4A H.sapiens mRNA for hepatocyte nuclear factor 4 alpha Length = 2289, Score = 1770 bits (893), Expect = 0.0, Identities = 893/893 (100%)

>gb|AY318752.11 Bos taurus hepatocyte nuclear factor 4alpha mRNA, complete cds

Length = 4515, Score = 1429 bits (721), Expect = 0.0, Identities = 925/993 (93%)

>dbj|AB037379.1| Tamias sibricus HNF-4 mRNA for hepatocyte nuclear factor 4, complete cds Length = 1440, Score = 1407 bits (710), Expect = 0.0, Identities = 920/990 (92%)

>dbj|BD 142440.11 Agent for inhibiting tumor cell-proliferation Length = 691 , Score = 1362 bits (687), Expect = 0.0, Identities = 690/691 (99%)

>emb|X57133.1|RNHNF4 Rat mRNA for hepatocyte nuclear factor 4 (HNF4) Length = 1758, Score = 1211 bits (611), Expect = 0.0, Identities = 899/995 (90%)

>ref]NM_022180.1| Rattus norvegicus hepatocyte nuclear factor 4, alpha (Hnf4a), mRNA Length = 1446, Score = 1211 bits (611), Expect = 0.0, Identities = 899/995 (90%)

>dbj|D10554.1|RATHNF4CL Rattus norvegicus mRNA for hepatocyte nuclear factor 4, complete cds Length = 1446, Score = 1211 bits (611), Expect = 0.0, Identities = 899/995 (90%)

>gb|AR269263.1| Sequence 1 from patent US 6500672 Length = 1758, Score = 1195 bits (603), Expect = 0.0, Identities = 897/995 (90%). Example 2: Expression profiling

Total cellular RNA was isolated from cells by one of two standard methods: 1) guanidine isothiocyanate/Cesium chloride density gradient centrifugation [Kellogg, (1990)]; or with the Tri- Reagent protocol according to the manufacturer's specifications (Molecular Research Center, Inc., Cincinatti, Ohio). Total RNA prepared by the Tri-reagent protocol was treated with DNAse I to remove genomic DNA contamination.

For relative quantitation of the mRNA distribution of HNF4A, total RNA from each cell or tissue source was first reverse transcribed. 85 μg of total RNA was reverse transcribed using 1 μmole random hexamer primers, 0.5 mM each of dATP, dCTP, dGTP and dTTP (Qiagen, Hilden, Germany), 3000 U RnaseQut (Invitrogen, Groningen, Netherlands) in a final volume of 680 μl.

The first strand synthesis buffer and Omniscript reverse transcriptase (2 u/μl) were from (Qiagen, Hilden, Germany). The reaction was incubated at 37°C for 90 minutes and cooled on ice. The volume was adjusted to 6800 μl with water, yielding a final concentration of 12.5 ng/μl of starting RNA.

For relative quantitation of the distribution of HNF4A mRNA in cells and tissues the Applied

Biosystems 7900 HT Sequence Detection system or Biorad iCycler was used according to the manufacturer's specifications and protocols. PCR reactions were set up to quantitate HNF4A and the housekeeping genes HPRT (hypoxanthine phosphoribosyltransferase), GAPDH (glyceralde- hyde-3 -phosphate dehydrogenase), β-actin, and others. Forward and reverse primers and probes for HNF4A were designed using the Perkin Elmer ABI Primer Express™ software and were synthesized by TibMolBiol (Berlin, Germany). The HNF4A forward primer sequence was: Primerl (SEQ ID NO: 3). The HNF4A reverse primer sequence was Primer2 (SEQ ID NO: 4). Probe 1 (SEQ ID NO: 5), labelled with FAM (carboxyfluorescein succinimidyl ester) as the reporter dye and TAMRA (carboxytetramethylrhodamine) as the quencher, is used as a probe for HNF4A. The following reagents were prepared in a total of 25 μl : Ix TaqMan buffer A, 5.5 mM

MgCl₂, 200 nM of dATP, dCTP, dGTP, and dUTP, 0.025 U/μl AmpliTaq Gold™, 0.01 U/μl AmpErase and Probe 1 (SEQ ID NO: 5), HNF4A forward and reverse primers each at 200 nM, 200 nM HNF4A FAM/TAMRA-labelled probe, and 5 μl of template cDNA. Thermal cycling parameters were 2 min at 50⁰C, followed by 10 min at 95°C, followed by 40 cycles of melting at 95°C for 15 sec and annealing/extending at 60⁰C for 1 min. Calculation of corrected CT values

The CT (threshold cycle) value is calculated as described in the "Quantitative determination of nucleic acids" section. The CF-value (factor for threshold cycle correction) is calculated as follows :

1. PCR reactions were set up to quantitate the housekeeping genes (HKG) for each cDNA sample.

2. CT_HKG-values (threshold cycle for housekeeping gene) were calculated as described in the "Quantitative determination of nucleic acids" section.

3. CT_HKG-mean values (CT mean value of all HKG tested on one cDNAs) of all HKG for each cDNA are calculated (n = number of HKG) :

CTn_KG-_n-mean value = (CTuKGi-value + CT_HKG₂-value +... + CTπK_G-_n-value) / n

4. CT_pannei mean value (CT mean value of all HKG in all tested cDNAs) =

(CT_HKGi-mean value + CT_HKG₂-mean value +...+ CTmc_G-y-mean value) / y

(y = number of cDNAs)

5. CF_cDNA._n (correction factor for cDNA n) = CT_pannei-mean value - CT_HKG-_n~^mean value

6. CT_cDNA-n (CT value of the tested gene for the cDNA n) + CF_cDNA-n (correction factor for cDNA n) = CT_0Or-CDN_A-_I, (corrected CT value for a gene on cDNA n)

Calculation of relative expression

Definition : highest CT_cor-cDNA-_n ≠ 40 is defined as CT_cor-cDNA [high]

Relative Expression = ₂ ^(CΪC0™^mh^^{φ] "} cτ^eor-^cDN _A._n)

Tissues

The expression of HNF4A was investigated in the tissues listed in table 1.

Expression profile

The results of the the mRNA-quantification (expression profiling) is shown in Table 1. Table 1: Relative expression of HNF 4 A in various human tissues.

T-cells peripheral blood CD4+ 0

T-cells peripheral blood CD4+ Dl 17 II virus 0 infected

T-cells peripheral blood CD4+ D34 virus 0 infected monocytes 6 monocytes HIV-I infected 0

fetal heart 0 heart 0 heart 23 heart 20 heart myocardial infarction 0 heart myocardial infarction 28 heart myocardial infarction 25 pericardium 0 heart atrium (right) 0 heart atrium (right) 14 heart atrium (left) 0 heart ventricle (left) 0 heart ventricle (left) 25 heart ventricle (right) 0 heart ventricle (right) 18 heart apex 0

Purkinje fibers 0 interventricular septum 0 fetal aorta 22 aorta 1 aorta 0 arcus aorta 0 aorta valve 39 artery 0 coronary artery 0 pulmonary artery 0 carotid artery 2 mesenteric artery 0 arteria radialis 0 vein 6 pulmonic valve 3 vein (saphena magna) 0

(caval) vein 0 coronary artery endothel cells 208 coronary artery smooth muscle primary cells 0 aortic smooth muscle cells 0 pulmonary artery smooth muscle cells 0 aortic endothel cells 0

HUVEC cells 0 pulmonary artery endothel cells 0 iliac artery endothel cells 0

skin

adrenal gland 0 thyroid 0 thyroid tumor 0 pancreas 140 pancreas liver cirrhosis 685

esophagus 0 esophagus tumor 0 stomach 449 stomach tumor 13216 colon 9675 colon tumor 8964 small intestine 6039 ileum 28133 ileum tumor 0 ileum chronic inflammation 0 rectum 4390 rectum tumor 1046 fetal liver 4012 liver 16498 liver 1898 liver 508 liver liver cirrhosis 2721 liver lupus disease 20595 liver tumor 18179

HEP G2 cells 130167

leukocytes (peripheral blood) 0

Jurkat (T-cells) 0

Raji (B-cells) 0 bone marrow 0 erythrocytes 0 lymphnode 0 thymus 0 thrombocytes 0 bone marrow stromal cells 0 bone marrow CD71+ cells 0 bone marrow CD33+ cells 0 bone marrow CD34+ cells 0 bone marrow CD 15+ cells 0 cord blood CD71+ cells 2 cord blood CD34+ cells 0 neutrophils cord blood 3

T-cells peripheral blood CD8+ 0 monocytes peripheral blood CD 14+ 0

B-cells peripheral blood CD 19+ 0 neutrophils peripheral blood 0 spleen 0 spleen liver cirrhosis 0

skeletal muscle 0 cartilage 0 bone connective tissue 0 adipose 0 adipose 2304 adipose 10 fetal adipose 13 adipose (subcutaneous) BMI 21.74 0 adipose (subcutaneous) BMI 35.04 0

brain 7 cerebellum 0 cerebral cortex 0 frontal lobe 8 occipital lobe 0 parietal lobe 0 temporal lobe 0 substantia nigra 0 caudatum 0 corpus callosum 26 nucleus accumbens 0 putamen 0 hippocampus 0 thalamus 9 posteroventral thalamus 0 dorsalmedial thalamus 2 hypothalamus 0 dorsal root ganglia 2 spinal cord 6 spinal cord (ventral horn) 0 spinal cord (dorsal horn) 0 glial tumor H4 cells 21 astrocytes 0 retina 0

fetal lung 25 fetal lung fibroblast IMR-90 cells 0 fetal lung fibroblast MRC-5 cells 0 lung 4 lung 0 lung right upper lobe 21 lung right mid lobe 39 lung right lower lobe 48 lung lupus disease 1951 lung tumor 54 lung COPD 3 trachea 3 primary bronchia 0 secondary bronchia 2 bronchial epithelial cells 0 bronchial smooth muscle cells 0 small airway epithelial cells 12

cervix 0 testis 51

HeLa cells (cervix tumor) 0 placenta 3 uterus 5 uterus tumor 31 ovary 131 ovary tumor 0 breast 34636 breast tumor 0 mammary gland 12

prostate 8 prostate 0 prostate 2 prostate BPH 0 prostate tumor 0 bladder 2 bladder 7434 bladder 0 ureter 7 penis 0 corpus cavemosum 0 fetal kidney 7332 kidney 11747 kidney 3541 kidney 17560 kidney tumor 609 renal epithelial cells 0

HEK 293 cells 0

Example 3: Antisense Analysis

Knowledge of the correct, complete cDNA sequence coding for HNF4A enables its use as a tool for antisense technology in the investigation of gene function. Oligonucleotides, cDNA or genomic fragments comprising the antisense strand of a polynucleotide coding for HNF4A are used either in vitro or in vivo to inhibit translation of the mRNA. Such technology is now well known in the art, and antisense molecules can be designed at various locations along the nucleotide sequences. By treatment of cells or whole test animals with such antisense sequences, the gene of interest is effectively turned off. Frequently, the function of the gene is ascertained by observing behavior at the intracellular, cellular, tissue or organismal level (e.g., lethality, loss of differentiated function, changes in morphology, etc.).

In addition to using sequences constructed to interrupt transcription of a particular open reading frame, modifications of gene expression is obtained by designing antisense sequences to intron regions, promoter/enhancer elements, or even to trans-acting regulatory genes.

Example 4: Expression of HNF4A

Expression of HNF4A is accomplished by subcloning the cDNAs into appropriate expression vectors and transfecting the vectors into expression hosts such as, e.g., E. coli. In a particular case, the vector is engineered such that it contains a promoter for β-galactosidase, upstream of the cloning site, followed by sequence containing the amino-terminal Methionine and the subsequent seven residues of β-galactosidase. Immediately following these eight residues is an engineered bacteriophage promoter useful for artificial priming and transcription and for providing a number of unique endonuclease restriction sites for cloning.

Induction of the isolated, transfected bacterial strain with Isopropyl-β-D-thiogalactopyranoside

(IPTG) using standard methods produces a fusion protein corresponding to the first seven residues of β-galactosidase, about 15 residues of "linker", and the peptide encoded within the cDNA. Since cDNA clone inserts are generated by an essentially random process, there is probability of 33% that the included cDNA will lie in the correct reading frame for proper translation. If the cDNA is not in the proper reading frame, it is obtained by deletion or insertion of the appropriate number of bases using well known methods including in vitro mutagenesis, digestion with exonuclease in or mung bean nuclease, or the inclusion of an oligonucleotide linker of appropriate length.

The HNF4A cDNA is shuttled into other vectors known to be useful for expression of proteins in specific hosts. Oligonucleotide primers containing cloning sites as well as a segment of DNA (about 25 bases) sufficient to hybridize to stretches at both ends of the target cDNA is synthesized chemically by standard methods. These primers are then used to amplify the desired gene segment by PCR. The resulting gene segment is digested with appropriate restriction enzymes under standard conditions and isolated by gel electrophoresis. Alternately, similar gene segments are produced by digestion of the cDNA with appropriate restriction enzymes. Using appropriate primers, segments of coding sequence from more than one gene are ligated together and cloned in appropriate vectors. It is possible to optimize expression by construction of such chimeric sequences.

Suitable expression hosts for such chimeric molecules include, but are not limited to, mammalian cells such as Chinese Hamster Ovary (CHO) and human 293 cells., insect cells such as Sf9 cells, yeast cells such as Saccharomyces cerevisiae and bacterial cells such as E. coli. For each of these cell systems, a useful expression vector also includes an origin of replication to allow propagation in bacteria, and a selectable marker such as the β-lactamase antibiotic resistance gene to allow plasmid selection in bacteria. In addition, the vector may include a second selectable marker such as the neomycin phosphotransferase gene to allow selection in transfected eukaryotic host cells. Vectors for use in eukaryotic expression hosts require RNA processing elements such as 3¹ polyadenylation sequences if such are not part of the cDNA of interest.

Additionally, the vector contains promoters or enhancers which increase gene expression. Such promoters are host specific and include MMTV, SV40, and metallothionine promoters for CHO cells; trp, lac, tac and T7 promoters for bacterial hosts; and alpha factor, alcohol oxidase and PGH promoters for yeast. Transcription enhancers, such as the rous sarcoma virus enhancer, are used in mammalian host cells. Once homogeneous cultures of recombinant cells are obtained through standard culture methods, large quantities of recombinantly produced HNF4A are recovered from the conditioned medium and analyzed using chromatographic methods known in the art. For example, HNF4A can be cloned into the expression vector pcDNA3, as exemplified herein. This product can be used to transform, for example, HEK293 or COS by methodology standard in the art. Specifically, for example, using Lipofectamine (Gibco BRL catalog no. 18324-020) mediated gene transfer. Example 5: Isolation of Recombinant HNF4A

HNF4A is expressed as a chimeric protein with one or more additional polypeptide domains added to facilitate protein purification. Such purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals [Appa Rao, 1997] and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp., Seattle, Washington). The inclusion of a cleavable linker sequence such as Factor Xa or enterolάnase (Invitrogen, Groningen, The Netherlands) between the purification domain and the HNF4A sequence is useful to facilitate expression of HNF4A.

Example 6: Production of HNF4A Specific Antibodies

Two approaches are utilized to raise antibodies to HNF4A, and each approach is useful for generating either polyclonal or monoclonal antibodies. In one approach, denatured protein from reverse phase HPLC separation is obtained in quantities up to 75 mg. This denatured protein is used to immunize mice or rabbits using standard protocols; about 100 μg are adequate for immunization of a mouse, while up to 1 mg might be used to immunize a rabbit. For identifying mouse hybridomas, the denatured protein is radioiodinated and used to screen potential murine B- cell hybridomas for those which produce antibody. This procedure requires only small quantities of protein, such that 20 mg is sufficient for labeling and screening of several thousand clones.

In the second approach, the amino acid sequence of an appropriate HNF4A domain, as deduced from translation of the cDNA, is analyzed to determine regions of high antigenicity. Oligopeptides comprising appropriate hydrophilic regions are synthesized and used in suitable immunization protocols to raise antibodies. The optimal amino acid sequences for immunization are usually at the C-terminus, the N-terminus and those intervening, hydrophilic regions of the polypeptide which are likely to be exposed to the external environment when the protein is in its natural conformation.

Typically, selected peptides, about 15 residues in length, are synthesized using an Applied

Biosystems Peptide Synthesizer Model 43 IA using fmoc-chemistry and coupled to keyhole limpet hemocyanin (KLH; Sigma, St. Louis, MO) by reaction with M-maleimidobenzoyl-N- hydroxysuccinimide ester, MBS. If necessary, a cysteine is introduced at the N-terminus of the peptide to permit coupling to KLH. Rabbits are immunized with the peptide-KLH complex in complete Freund's adjuvant. The resulting antisera are tested for antipeptide activity by binding the peptide to plastic, blocking with 1% bovine serum albumin, reacting with antisera, washing and reacting with labeled (radioactive or fluorescent), affinity purified, specific goat anti-rabbit IgG. Hybridomas are prepared and screened using standard techniques. Hybridomas of interest are detected by screening with labeled HNF4A to identify those fusions producing the monoclonal antibody with the desired specificity. In a typical protocol, wells of plates (FAST; Becton- Dickinson, Palo Alto, CA) are coated during incubation with affinity purified, specific rabbit anti- mouse (or suitable antispecies 1 g) antibodies at 10 mg/ml. The coated wells are blocked with 1% bovine serum albumin, (BSA), washed and incubated with supernatants from hybridomas. After washing the wells are incubated with labeled HNF4A at 1 mg/ml. Supernatants with specific antibodies bind more labeled HNF4A than is detectable in the background. Then clones producing specific antibodies are expanded and subjected to two cycles of cloning at limiting dilution. Cloned hybridomas are injected into pristane-treated mice to produce ascites, and monoclonal antibody is purified from mouse ascitic fluid by affinity chromatography on Protein A. Monoclonal antibodies with affinities of at least

10⁸ M^'1, preferably 10⁹ to 10¹⁰ M^"1 or stronger, are typically made by standard procedures.

Example 7: Diagnostic Test Using HNF4A Specific Antibodies

Particular HNF4A antibodies are useful for investigating signal transduction and the diagnosis of infectious or hereditary conditions which are characterized by differences in the amount or distribution of HNF4A or downstream products of an active signaling cascade.

Diagnostic tests for HNF4A include methods utilizing antibody and a label to detect HNF4A in human body fluids, membranes, cells, tissues or extracts of such. The polypeptides and antibodies of the present invention are used with or without modification. Frequently, the polypeptides and antibodies are labeled by joining them, either covalently or noncovalently, with a substance which provides for a detectable signal. A wide variety of labels and conjugation techniques are known and have been reported extensively in both the scientific and patent literature. Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent agents, chemiluminescent agents, chromogenic agents, magnetic particles and the like.

A variety of protocols for measuring soluble or membrane-bound HNF4A, using either polyclonal or monoclonal antibodies specific for the protein, are known in the art. Examples include enzyme- linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescent activated cell sorting (FACS). A two-site monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on HNF4A is preferred, but a competitive binding assay may be employed. Example 8: Purification of Native HNF4A Using Specific Antibodies

Native or recombinant HNF4A is purified by immunoaffϊnity chromatography using antibodies specific for HNF4A. In general, an immunoaffϊm^'ty column is constructed by covalently coupling the anti-TRH antibody to an activated chromatographic resin.

Polyclonal immunoglobulins are prepared from immune sera either by precipitation with ammonium sulfate or by purification on immobilized Protein A (Pharmacia LKB Biotechnology, Piscataway NJ.)- Likewise, monoclonal antibodies are prepared from mouse ascites fluid by ammonium sulfate precipitation or chromatography on immobilized Protein A. Partially purified immunoglobulin is covalently attached to a chromatographic resin such as CnBr-activated Sepharose (Pharmacia LKB Biotechnology). The antibody is coupled to the resin, the resin is blocked, and the derivative resin is washed according to the manufacturer's instructions.

Such immunoaffinity columns are utilized in the purification of HNF4A by preparing a fraction from cells containing HNF4A in a soluble form. This preparation is derived by solubilization of whole cells or of a subcellular fraction obtained via differential centrifugation (with or without addition of detergent) or by other methods well known in the art. Alternatively, soluble HNF4A containing a signal sequence is secreted in useful quantity into the medium in which the cells are grown.

A soluble HNF4A-containing preparation is passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of HNF4A (e.g., high ionic strength buffers in the presence of detergent). Then, the column is eluted under conditions that disrupt antibody/protein binding (e.g., a buffer of pH 2-3 or a high concentration of a chaotrope such as urea or thiocyanate ion), and HNF4A is collected.

Example 9: Drug Screening

This invention is particularly useful for screening therapeutic compounds by using HNF4A or binding fragments thereof in any of a variety of drug screening techniques. As HNF4A is a

Nuclear Receptor any of the methods commonly used in the art may potentially be used to identify

HNF4A ligands. For example, the activity of a Nuclear Receptor such as HNF4A can be measured using any of a variety of appropriate functional assays in which activation of the receptor results in an observable change in the level of a reportergene which is transcriptionally activated by the Nuclear Receptor.Alternatively, the polypeptide or fragment employed in such a test is either free in solution, affixed to a solid support, borne on a cell surface or located intracellularly. One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant nucleic acids expressing the polypeptide or fragment. Drugs are screened against such transformed cells in competitive binding assays. Such cells, either in viable or fixed form, are used for standard binding assays.

Measured, for example, is the formation of complexes between HNF4A and the agent being tested. Alternatively, one examines the diminution in complex formation between HNF4A and a ligand caused by the agent being tested.

Thus, the present invention provides methods of screening for drug canditates, drugs, or any other agents which affect signal transduction. These methods, well known in the art, comprise contacting such an agent with HNF4A polypeptide or a fragment thereof and assaying (i) for the presence of a complex between the agent and HNF4A polypeptide or fragment, or (ii) for the presence of a complex between HNF4A polypeptide or fragment and the cell. In such competitive binding assays, the HNF4A polypeptide or fragment is typically labeled. After suitable incubation, free HNF4A polypeptide or fragment is separated from that present in bound form, and the amount of free or uncomplexed label is a measure of the ability of the particular agent to bind to HNF4A or to interfere with the HNF4A-agent complex.

Another technique for drug screening provides high throughput screening for compounds having suitable binding affinity to HNF4A polypeptides. Briefly stated, large numbers of different small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. The peptide test compounds are reacted with HNF4A polypeptide and washed. Bound HNF4A polypeptide is then detected by methods well known in the art. Purified HNF4A are also coated directly onto plates for use in the aforementioned drug screening techniques. In addition, non-neutralizing antibodies are used to capture the peptide and immobilize it on the solid support.

This invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of binding HNF4A specifically compete with a test compound for binding to HNF4A polypeptides or fragments thereof. In this manner, the antibodies are used to detect the presence of any peptide which shares one or more antigenic determinants with HNF4A.

Example 10: Rational Drug Design

The goal of rational drug design is to produce structural analogs of biologically active polypeptides of interest or of small molecules with which they interact, agonists, antagonists, or inhibitors. Any of these examples are used to fashion drugs which are more active or stable forms of the polypeptide or which enhance or interfere with the function of a polypeptide in vivo. In one approach, the three-dimensional structure of a protein of interest, or of a protein-inhibitor complex, is determined by x-ray crystallography, by computer modeling or, most typically, by a combination of the two approaches. Both the shape and charges of the polypeptide must be ascertained to elucidate the structure and to determine active site(s) of the molecule. Less often, useful information regarding the structure of a polypeptide is gained by modeling based on the structure of homologous proteins. In both cases, relevant structural information is used to design efficient inhibitors. Useful examples of rational drug design include molecules which have improved activity or stability or which act as inhibitors, agonists, or antagonists of native peptides.

It is also possible to isolate a target-specific antibody, selected by functional assay, as described above, and then to solve its crystal structure. This approach, in principle, yields a pharmacore upon which subsequent drug design is based. It is possible to bypass protein crystallography altogether by generating anti-idiotypic antibodies (anti-ids) to a functional, pharmacologically active antibody. As a mirror image of a mirror image, the binding site of the anti-ids is expected to be an analog of the original receptor. The anti-id is then used to identify and isolate peptides from banks of chemically or biologically produced peptides. The isolated peptides then act as the pharmacore.

By virtue of the present invention, sufficient amount of polypeptide are made available to perform such analytical studies as X-ray crystallography. In addition, knowledge of the HNF4A amino acid sequence provided herein provides guidance to those employing computer modeling techniques in place of or in addition to x-ray crystallography.

Example 11 : Identification of Other Members of the Signal Transduction Complex

The inventive purified HNF4A is a research tool for identification, characterization and purification of other signal transduction pathway proteins. Radioactive labels are incorporated into a selected HNF4A domain by various methods known in the art and used in vitro to capture interacting molecules. A preferred method involves labeling the primary amino groups in HNF4A with ¹²⁵I BoI ton-Hunter reagent. This reagent has been used to label various molecules without concomitant loss of biological activity.

Labeled HNF4A is useful as a reagent for the purification of molecules with which it interacts. In one embodiment of affinity purification, membrane-bound HNF4A is covalently coupled to a chromatography column. Cell-free extract derived from synovial cells or putative target cells is passed over the column, and molecules with appropriate affinity bind to HNF4A. HNF4A- complex is recovered from the column, and the HNF4A-binding ligand disassociated and subjected to N-terminal protein sequencing. The amino acid sequence information is then used to identify the captured molecule or to design degenerate oligonucleotide probes for cloning the relevant gene from an appropriate cDNA library.

In an alternate method, antibodies are raised against HNF4A, specifically monoclonal antibodies. The monoclonal antibodies are screened to identify those which inhibit the binding of labeled HNF4A. These monoclonal antibodies are then used therapeutically.

Example 12: Use and Administration of Antibodies, Inhibitors, or Antagonists

Antibodies, inhibitors, or antagonists of HNF4A or other treatments and compunds that are limiters of signal transduction (LSTs), provide different effects when administered therapeutically. LSTs are formulated in a nontoxic, inert, pharmaceutically acceptable aqueous carrier medium preferably at a pH of about 5 to 8, more preferably 6 to 8, although pH may vary according to the characteristics of the antibody, inhibitor, or antagonist being formulated and the condition to be treated. Characteristics of LSTs include solubility of the molecule, its half-life and antigenicity/immunogenicity. These and other characteristics aid in defining an effective carrier. Native human proteins are preferred as LSTs, but organic or synthetic molecules resulting from drug screens are equally effective in particular situations.

LSTs are delivered by known routes of administration including but not limited to topical creams and gels; transmucosal spray and aerosol; transdermal patch and bandage; injectable, intravenous and lavage formulations; and orally administered liquids and pills particularly formulated to resist stomach acid and enzymes. The particular formulation, exact dosage, and route of administration is determined by the attending physician and varies according to each specific situation.

Such determinations are made by considering multiple variables such as the condition to be treated, the LST to be administered, and the pharmacokinetic profile of a particular LST. Additional factors which are taken into account include severity of the disease state, patient's age, weight, gender and diet, time and frequency of LST administration, possible combination with other drugs, reaction sensitivities, and tolerance/response to therapy. Long acting LST formulations might be administered every 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate of the particular LST.

Normal dosage amounts vary from 0.1 to 10⁵ μg, up to a total dose of about 1 g, depending upon the route of administration. Guidance as to particular dosages and methods of delivery is provided in the literature; see U.S. Pat. Nos. 4,657,760; 5,206,344; or 5,225,212. Those skilled in the art employ different formulations for different LSTs. Administration to cells such as nerve cells necessitates delivery in a manner different from that to other cells such as vascular endothelial cells.

It is contemplated that abnormal signal transduction, trauma, or diseases which trigger HNF4A activity are treatable with LSTs. These conditions or diseases are specifically diagnosed by the tests discussed above, and such testing should be performed in suspected cases of viral, bacterial or fungal infections, allergic responses, mechanical injury associated with trauma, hereditary diseases, lymphoma or carcinoma, or other conditions which activate the genes of lymphoid or neuronal tissues.

Example 13: Production of Non-human Transgenic Animals

Animal model systems which elucidate the physiological and behavioral roles of the HNF4A are produced by creating nonhuman transgenic animals in which the activity of the HNF4A is either increased or decreased, or the amino acid sequence of the expressed HNF4A is altered, by a variety of techniques. Examples of these techniques include, but are not limited to: 1) Insertion of normal or mutant versions of DNA encoding a HNF4A, by microinjection, electroporation, retroviral transfection or other means well known to those skilled in the art, into appropriately fertilized embryos in order to produce a transgenic animal or 2) homologous recombination of mutant or normal, human or animal versions of these genes with the native gene locus in transgenic animals to alter the regulation of expression or the structure of these HNF4A sequences. The technique of homologous recombination is well known in the art. It replaces the native gene with the inserted gene and hence is useful for producing an animal that cannot express native

HNF4As but does express, for example, an inserted mutant HNF4A, which has replaced the native HNF4A in the animal's genome by recombination, resulting in underexpression of the transporter. Microinjection adds genes to the genome, but does not remove them, and the technique is useful for producing an animal which expresses its own and added HNF4A, resulting in overexpression of the HNF4A.

One means available for producing a transgenic animal, with a mouse as an example, is as follows: Female mice are mated, and the resulting fertilized eggs are dissected out of their oviducts. The eggs are stored in an appropriate medium such as cesiumchloride M2 medium. DNA or cDNA encoding HNF4A is purified from a vector by methods well known to the one skilled in the art. Inducible promoters may be fused with the coding region of the DNA to provide an experimental means to regulate expression of the transgene. Alternatively or in addition, tissue specific regulatory elements may be fused with the coding region to permit tissue-specific expression of the transgene. The DNA, in an appropriately buffered solution, is put into a microinjection needle (which may be made from capillary tubing using a piper puller) and the egg to be injected is put in a depression slide. The needle is inserted into the pronucleus of the egg, and the DNA solution is injected. The injected egg is then transferred into the oviduct of a pseudopregnant mouse which is a mouse stimulated by the appropriate hormones in order to maintain false pregnancy, where it proceeds to the uterus, implants, and develops to term. As noted above, microinjection is not the only method for inserting DNA into the egg but is used here only for exemplary purposes.

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Claims

Claims

1. A method of screening for therapeutic agents useful in the treatment of a disease comprised in a group of diseases consisting of gastrointestinal and liver diseases, cancer disorders, cardiovascular disorders, metabolic diseases, inflammatory diseases, muscle-skeleton disorders, respiratory diseases, reproduction disorders and urological disorders, in a mammal comprising the steps of

i) contacting a test compound with a HNF4A polypeptide, ii) detect binding of said test compound to said HNF4A polypeptide.

2. A method of screening for therapeutic agents useful in the treatment of a disease comprised in a group of diseases consisting of gastrointestinal and liver diseases, cancer disorders, cardiovascular disorders, metabolic diseases, inflammatory diseases, muscle-skeleton disorders, respiratory diseases, reproduction disorders and urological disorders, in a mammal comprising the steps of

i) determining the activity of a HNF4A polypeptide at a certain concentration of a test compound or in the absence of said test compound, ii) determining the activity of said polypeptide at a different concentration of said test compound.

3. A method of screening for therapeutic agents useful in the treatment of a disease comprised in a group of diseases consisting of gastrointestinal and liver diseases, cancer disorders, cardiovascular disorders, metabolic diseases, inflammatory diseases, muscle-skeleton disorders, respiratory diseases, reproduction disorders and urological disorders, in a mammal comprising the steps of

i) determining the activity of a HNF4A polypeptide at a certain concentration of a test compound, ii) determining the activity of a HNF4A polypeptide at the presence of a compound known to be a regulator of a HNF4A polypeptide.

4. The method of any of claims 1 to 3, wherein the step of contacting is in or at the surface of a cell.

5. The method of any of claims 1 to 3, wherein the cell is in vitro.

6. The method of any of claims 1 to 3, wherein the step of contacting is in a cell-free system.

7. The method of any of claims 1 to 3, wherein the polypeptide is coupled to a detectable label.

8. The method of any of claims 1 to 3, wherein the compound is coupled to a detectable label.

9. The method of any of claims 1 to 3, wherein the test compound displaces a ligand which is first bound to the polypeptide.

10. The method of any of claims 1 to 3, wherein the polypeptide is attached to a solid support.

11. The method of any of claims 1 to 3, wherein the compound is attached to a solid support.

12. A method of screening for therapeutic agents useful in the treatment of a disease comprised in a group of diseases consisting of gastrointestinal and liver diseases, cancer disorders, cardiovascular disorders, metabolic diseases, inflammatory diseases, muscle-skeleton disorders, respiratory diseases, reproduction disorders and urological disorders, in a mammal comprising the steps of

i) contacting a test compound with a HNF4A polynucleotide, ii) detect binding of said test compound to said HNF4A polynucleotide.

13. The method of claim 12 wherein the nucleic acid molecule is RNA.

14. The method of claim 12 wherein the contacting step is in or at the surface of a cell.

15. The method of claim 12 wherein the contacting step is in a cell-free system.

16. The method of claim 12 wherein polynucleotide is coupled to a detectable label.

17. The method of claim 12 wherein the test compound is coupled to a detectable label.

18. A method of diagnosing a disease comprised in a group of diseases consisting of gastrointestinal and liver diseases, cancer disorders, cardiovascular disorders, metabolic diseases, inflammatory diseases, muscle-skeleton disorders, respiratory diseases, reproduction disorders and urological disorders, in a mammal comprising the steps of

i) determining the amount of a HNF4A polynucleotide in a sample taken from said mammal, ii) determining the amount of HNF4A polynucleotide in healthy and/or diseased mammals.

19. A pharmaceutical composition for the treatment of a disease comprised in a group of diseases consisting of gastrointestinal and liver diseases, cancer disorders, cardiovascular disorders, metabolic diseases, inflammatory diseases, muscle-skeleton disorders, respiratory diseases, reproduction disorders and urological disorders, in a mammal comprising a therapeutic agent which binds to a HNF4A polypeptide.

20. A pharmaceutical composition for the treatment of a disease comprised in a group of diseases consisting of gastrointestinal and liver diseases, cancer disorders, cardiovascular disorders, metabolic diseases, inflammatory diseases, muscle-skeleton disorders, respiratory diseases, reproduction disorders and urological disorders, in a mammal comprising a therapeutic agent which regulates the activity of a HNF4A polypeptide.

21. A pharmaceutical composition for the treatment of a disease comprised in a group of diseases consisting of gastrointestinal and liver diseases, cancer disorders, cardiovascular disorders, metabolic diseases, inflammatory diseases, muscle-skeleton disorders, respiratory diseases, reproduction disorders and urological disorders, in a mammal comprising a therapeutic agent which regulates the activity of a HNF4A polypeptide, wherein said therapeutic agent is

i) a small molecule, ϋ) an RNA molecule, iii) an antisense oligonucleotide, iv) a polypeptide, v) an antibody, or vi) a ribozyme.

22. A pharmaceutical composition for the treatment of a disease comprised in a group of diseases consisting of gastrointestinal and liver diseases, cancer disorders, cardiovascular disorders, metabolic diseases, inflammatory diseases, muscle-skeleton disorders, respira¬ tory diseases, reproduction disorders and urological disorders, in a mammal comprising a HNF4A polynucleotide.

23. A pharmaceutical composition for the treatment of a disease comprised in a group of diseases consisting of gastrointestinal and liver diseases, cancer disorders, cardiovascular disorders, metabolic diseases, inflammatory diseases, muscle-skeleton disorders, respiratory diseases, reproduction disorders and urological disorders, in a mammal comprising a HNF4A polypeptide.

24. Use of regulators of a HNF4A for the preparation of a pharmaceutical composition for the treatment of a disease comprised in a group of diseases consisting of gastrointestinal and liver diseases, cancer disorders, cardiovascular disorders, metabolic diseases, inflammatory diseases, muscle-skeleton disorders, respiratory diseases, reproduction disorders and urological disorders, in a mammal.

25. Method for the preparation of a pharmaceutical composition useful for the treatment of a disease comprised in a group of diseases consisting of gastrointestinal and liver diseases, cancer disorders, cardiovascular disorders, metabolic diseases, inflammatory diseases, muscle-skeleton disorders, respiratory diseases, reproduction disorders and urological disorders, in a mammal comprising the steps of

i) identifying a regulator of HNF4A, ii) determining whether said regulator ameliorates the symptoms of a disease comprised in a group of diseases consisting of gastrointestinal and liver diseases, cancer disorders, cardiovascular disorders, metabolic diseases, inflammatory diseases, muscle-skeleton disorders, respiratory diseases, reproduction disorders and urological disorders, in a mammal; and iii) combining of said regulator with an acceptable pharmaceutical carrier.

26. Use of a regulator of HNF4A for the regulation of HNF4A activity in a mammal having a disease comprised in a group of diseases consisting of gastrointestinal and liver diseases, cancer disorders, cardiovascular disorders, metabolic diseases, inflammatory diseases, muscle-skeleton disorders, respiratory diseases, reproduction disorders and urological disorders..