WO1999026960A2

WO1999026960A2 - Genes encoding frizzled-like proteins

Info

Publication number: WO1999026960A2
Application number: PCT/US1998/025132
Authority: WO
Inventors: Andrew W. Shyjan
Original assignee: Millennium Pharmaceuticals, Inc.
Priority date: 1997-11-25
Filing date: 1998-11-24
Publication date: 1999-06-03
Also published as: AU1535899A

Description

GENES ENCODING FRIZZLED-LIKE PROTEINS

Background of the Invention The frizzled ( fz) gene of Drosophila melanoguster plays an essential role in the development of tissue polarity. Fz encodes a protein that is thought to direct both cell autonomous and cell non-autonomous functions (Krasnow and Adler, Development 120:1883, 1994) . A second member of the frizzled family in Drosophila, Dfz2, is thought to encode the receptor for wingless protein, a member of the Vint family of ligands (Bhanot et al . , Nature 382 :225-30, 1996) .

A human homologue of the Drosophila fz gene has been identified (Zhao et al . , Genomics 27:370, 1995; GenBank Accession No. L37882). The amino acid sequence of this human homologue (FZD2) is 56% identical to Drosophila fz protein. Expression of FZD2 is developmentally regulated, exhibiting high level expression in fetal kidney and lung and adult colon and ovary.

Homologues of fz have been identified in rat, chicken, zebrafish, sea urchin, and CaeπorhaJb i s elegans (Wang et al . , J. Biol . Chem . 271:4468-76 , 1996).

Identified members of the frizzled gene family encode receptors having a cysteine-rich extracellular domain tethered by a variable linker region to seven membrane spanning α-helices which are followed by an intracellular domain (Wang et al . , supra) .

Summary of the Invention The invention described herein relates to the discovery and characterization of hfz-4 and mfz-9, cDNAs that encode homologues of the Drosophila fz gene. The hfz-4 gene is thought to be the human homologue of the Drosophila frizzled gene, mfz-4. The mfz-9 gene is a murine gene .

Like other members of the frizzled gene family, hfz- 4 and mfz-9 are expected to encode receptors having a cysteine-rich extracellular domain tethered by a variable linker region to seven membrane spanning α-helices which are followed by an intracellular domain. The proteins encoded by hfz-4 and mfz-9 are likely receptors for proteins encoded by one or more members of the wnt gene family. Certain members of the wnt gene family, e . g. , murine Wnt -l OB and Wnt-1 and human Wnt - 2 , are thought to be involved in tumorigenesis (Hardiman et al . , Gene 172:199-205, 1996). Accordingly, the hfz-4 and mfz-9 genes described herein may be useful in the diagnosis of certain cancers, e . g. , breast cancer and colorectal cancer. Moreover, agonist ligands which bind to the proteins encoded by these genes may be useful for the treatment of certain cancers. The invention features isolated nucleic acid molecules (i.e., a nucleic acid molecule that is separated from the 5' and 3' coding sequences with which it is immediately contiguous in the naturally occurring genome of an organism, also referred to as a recombinant nucleic acid molecule) that encodes a Hfz-4 or Mfz-9 polypeptide. Within the invention are polypeptides having the sequence of SEQ ID NO: 2 or SEQ ID NO : 4 , or encoded by nucleic acid molecules having the sequence shown in SEQ ID NO:l or SEQ ID NO : 3. However, the invention is not limited to nucleic acid molecules and polypeptides that are identical to those SEQ ID Nos. For example, the invention includes nucleic acid molecules which encode splice variants, allelic variants or mutant forms of hfz-4 or mfz-9 as well as the proteins encoded by such nucleic acid molecules.

Also within the invention are nucleic acid molecules that hybridize under stringent conditions to a nucleic acid molecule having the sequence of SEQ ID NO : 1 or SEQ ID NO:3. Such molecules include, for example, the human homologue of mfz-9. As described further below, molecules that are substantially identical to those of SEQ ID Nos. 1-4 are also encompassed by the invention. As used herein, the term "hybridizes under stringent conditions" is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% (65%, 70%, 75%, 80%, 85%, or more than 95%) identical to each other typically remain hybridized to each other. Such stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. A preferred, non- limiting example of stringent hybridization conditions are hybridized in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2 X SSC, 0.1% SDS at 50-65°C. Preferably, an isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequence of SEQ ID NO:l or 2, or a complement thereof, corresponds to a naturally-occurring nucleic acid molecule. As used herein, a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein) .

In addition to naturally-occurring allelic variants of a nucleic acid molecule of the invention sequence that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation thereby leading to changes in the amino acid sequence of the encoded protein, without altering the biological activity of the protein. For example, one can make nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues. A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence without altering the biological activity, whereas an "essential" amino acid residue is required for biological activity. For example, amino acid residues that are not conserved or only semi -conserved among homologues of various species may be non-essential for activity and thus would be likely targets for alteration. Alternatively, amino adid residues that are conserved among the homologues of various species (e.g., murine and human) may be essential for activity and thus would not be likely targets for alteration.

The term "substantially pure" as used herein in reference to a given compound ( e . g. , a Hfz-4 or Mfz-9 polypeptide) means that the compound is substantially free from other compounds, such as those in cellular material, viral material, or culture medium, with which the compound may have been associated ( e . g. , in the course of production by recombinant DNA techniques or before purification from a natural biological source) . When chemically synthesized, a compound of the invention is substantially pure when it is substantially free from the chemical compounds used in the process of its synthesis. Polypeptides or other compounds of interest are substantially free from other compounds when they are within preparations that are at least 60% by weight (dry weight) the compound of interest. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight the compound of interest. Purity can be measured by any appropriate standard method, for example, by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

Where a particular polypeptide or nucleic acid molecule is said to have a specific percent identity (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) to a reference polypeptide or nucleic acid molecule of a defined length, the percent identity is relative to the reference polypeptide or nucleic acid molecule. Thus, a peptide that is 50% identical to a reference polypeptide that is 100 amino acids long can be a 50 amino acid polypeptide that is completely identical to a 50 amino acid long portion of the reference polypeptide. It might also be a 100 amino acid long polypeptide which is 50% identical to the reference polypeptide over its entire length. Of course, many other polypeptides will meet the same criteria. The same rule applies for nucleic acid molecules. For polypeptides, the length of the reference polypeptide sequence will generally be at least 16 amino acids, preferably at least 20 amino acids, more preferably at least 25 amino acids, and most preferably 35 amino acids, 50 amino acids, or 100 amino acids. For nucleic acids, the length of the reference nucleic acid sequence will generally be at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably at least 100 nucleotides ( e . g. , 150, 200, 250, or 300 nucleotides) .

In the case of polypeptide sequences that are less than 100% identical to a reference sequence, the non- identical positions are preferably, but not necessarily, conservative substitutions for the reference sequence. Conservative substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine, and leucine; aspartic acid and glutamic acid; asparagine and glut mine; serine and threonine; lysine and arginine; and phenylalanine and tyrosine.

Sequence identity can be measured using sequence analysis software (e.g., the Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705 with the default parameters as specified therein.

The BLAST programs, provided as a service by the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov), are very useful for making sequence comparisons. The programs are described in detail by Karlin et al . ( Proc . Natl . Acad . Sci . USA _.87:2264-68, 1990 and 9_0:5873-7, 1993) and Altschul et al . (Nucl . Acids Res . 25:3389-3402, 1997) and are available on the internet at: http://www.ncbi.nlm.nih.gov.

The invention also features a host cell that includes an isolated nucleic acid molecule encoding Hfz-4 or Mfz-9 (either alone or in conjunction with a heterologous polypeptide, such as a detectable marker) , or a nucleic acid vector that contains a sequence encoding Hfz-4 or Mfz-9 (again, with or without a heterologous polypeptide) . The vector can be an expression vector, and can include a regulatory element. An antibody that specifically binds a Hfz-4 or Mfz-9 polypeptide is also within the scope of the present invention and is useful, for example, to detect Hfz-4 or Mfz-9 in a biological sample, or to alter the activity of Hfz-4 or Mfz-9. For example, Hfz-4 or Mfz-9 can be detected in a biological sample by contacting the sample with an antibody that specifically binds Hfz-4 or Mfz-9 under conditions that allow the formation of a Hfz-4 or Mfz- 9-antibody complex and detecting the complex, if present, as an indication of the presence of Hfz-4 or Mfz- 9 in the sample. The use of an antibody in a treatment regime, where it can alter the activity of Hfz- 4 or Mfz-9, is discussed further below.

An antibody of the invention can be a monoclonal, polyclonal, or engineered antibody that specifically binds Hfz-4 or Mfz-9 (as described more fully below) . An antibody that "specifically binds" to a particular antigen, for example, a Hfz-4 or Mfz- 9 polypeptide of the invention, will not substantially recognize or bind to other molecules in a sample, such as a biological sample, that includes Hfz-4 or Mfz-9.

Given that an object of the present invention is to alter the expression or activity of Hfz-4 or Mfz-9 in vivo, a pharmaceutical composition containing, for example, an isolated nucleic acid molecule encoding Hfz-4 or Mfz-9 (or a fragment thereof) , a nucleic acid molecule that is antisense to hfz-4 or mfz-9 ( i . e . , that has a sequence that is the reverse and complement of a portion of the coding strand of a Hfz-4 or Mfz-9 gene) , a Hfz-4 or Mfz- 9 polypeptide, or an antibody, small molecule, or other compound that specifically binds a Hfz-4 or Mfz-9 polypeptide is also a feature of the invention.

The discovery and characterization of hfz-4 and mfz- 9 and the polypeptides they encode makes it possible to determine whether a given disorder is associated with aberrant expression of hfz-4 or mfz- 9 (meaning expression at the level of gene transcription or mRNA translation) or activity of Hfz-4 or Mfz-9. For example, one can diagnose a patient as having a disorder associated with aberrant expression of hfz-4 or mfz-9 by measuring hfz-4 or mfz-9 expression in a biological sample obtained from the patient. An increase or decrease in hfz-4 or mfz-9 expression in the biological sample, compared with hfz-4 or mfz-9 expression in a control sample (e.g., a sample of the same tissue collected from one or more healthy individuals) indicates that the patient has a disorder associated with aberrant expression of hfz-4 or mfz-9. Similarly, one can diagnose a patient as having a disorder associated with aberrant activity of Hfz-4 or Mfz-9 by measuring Hfz-4 or Mfz-9 activity in a biological sample obtained from the patient. An increase or decrease in Hfz-4 or Mfz-9 activity in the biological sample, compared with Hfz-4 or Mfz- 9 activity in a control sample, indicates that the patient has a disorder associated with aberrant activity of Hfz-4 or Mfz-9. The techniques required to measure gene expression or polypeptide activity are well known to those of ordinary skill in the art.

Thus, the invention includes a number of methods for detection and diagnosis. On one aspect, a method for detecting Hfz-4 in a biological sample, comprising: (a) contacting the sample with an antibody that specifically binds Hfz-4 under conditions that allow the formation of Hfz-4 -antibody complexes; and (b) detecting the complexes, if any, as an indication of the presence of Hfz-4 in said sample. A similar method can be applied to Mfz-4.

The invention also features a method for diagnosing a patient as having a disorder associated with expression of an isoform of Hfz-4, comprising isolating hfz-4 mRNA or Hfz-4 polypeptide from said patient and determining the sequence of the mRNA or polypeptide, a difference in the sequence, as compared to the nucleotide sequence of SEQ ID N0:1 or the polypeptide sequence encoded by SEQ ID NO:l, respectively, indicating expression of an isoform of hfz-4. A similar method can be applied to Mfz-4.

The invention further features a method for diagnosing a patient as having a disorder associated with aberrant activity of Hfz-4, comprising measuring Hfz-4 activity in a biological sample obtained from the patient, wherein increased or decreased Hfz-4 activity in said biological sample, compared with Hfz-4 activity in a control sample, indicates that said patient has a disorder associated with aberrant activity of Hfz-4. A similar method can be applied to mfz-4

In addition to diagnostic methods, such as those described above, the present invention encompasses methods and compositions for typing and evaluating the prognosis, appropriate treatment, and treatment effectiveness of disorders associated with inappropriate expression of hfz-4 or mfz- 9 or inappropriate activity of Hfz-4 or Mfz-9. For example, the nucleic acid molecules of the invention can be used as probes to classify cells in terms of their level of hfz-4 or mfz-9 expression, or as primers for diagnostic PCR analysis in which mutations, allelic variations, and regulatory defects in the hfz-4 or mfz-9 gene can be detected. Similarly, those of ordinary skill in the art can use routine techniques to identify inappropriate activity of Hfz-4 or Mfz-9, which can be observed in a variety of forms. For example, inappropriate activity can take the form of an alteration signal transduction mediated Hfz-4 or Mfz-9. Diagnostic kits for the practice of such methods are also provided.

The invention further encompasses transgenic animals that express Hfz-4 or Mfz-9 and recombinant "knock-out" animals that fail to express Hfz-4 or Mfz-9. These animals can serve as new and useful models of disorders in which hfz-4 or mfz-9 is misexpressed.

The invention also features antagonists and agonists of Hfz-4 or Mfz- 9 that can inhibit or enhance, respectively, one or more of the biological activities of Hfz-4 or Mfz-9. Suitable antagonists can include small molecules ( i . e . , molecules with a molecular weight below about 500), large molecules (i.e., molecules with a molecular weight above about 500) , antibodies that specifically bind and "neutralize" Hfz-4 or Mfz-9 (as described below) , and nucleic acid molecules that interfere with transcription or translation of hfz-4 or mfz-9 (e.g., antisense nucleic acid molecules and ribozymes) . Agonists of Hfz-4 or Mfz-9 also include small and large molecules, and antibodies other than neutralizing antibodies.

The invention also features molecules that can increase or decrease the expression of hfz-4 or mfz-9 (e.g., by altering transcription or translation) . Small molecules (as defined above) , large molecules (as defined above), and nucleic acid molecules (e.g., antisense and ribozyme molecules) can be used to inhibit the expression of hfz-4 or mfz-9. Other types of nucleic acid molecules (e.g., molecules that bind to hfz-4 or mfz-9 negative transcriptional regulatory sequences) can be used to increase the expression of hfz-4 or mfz-9.

Compounds that modulate the expression of hfz-4 or mfz- 9 in a cell can be identified by comparing the level of expression of hfz-4 or mfz-9 in the presence of a selected compound with the level of expression of hfz-4 or mfz-9 in the absence of that compound. A difference in the level of hfz-4 or mfz- 9 expression indicating that the selected compound modulates the expression of hfz-4 or mfz-9 in the cell. A comparable test for compounds that modulate the activity of Hfz-4 or Mfz-9 can be carried out by comparing the level of Hfz-4 or Mfz- 9 activity in the presence and absence of the compound.

Patients who have a disorder mediated by abnormal hfz-4 or mfz- 9 activity can be treated by administration of a compound that alters the expression of hfz-4 or mfz- 9 or the activity of Hfz-4 or Mfz-9. When the objective is to decrease expression or activity, the compound administered can be a hfz-4 or mfz- 9 antisense oligonucleotide or an antibody, such as a neutralizing antibody, that specifically binds Hfz-4 or Mfz-9, respectively.

The preferred methods and materials are described below in examples which are meant to illustrate, not limit, the invention. Skilled artisans will recognize methods and materials that are similar or equivalent to those described herein, and that can be used in the practice or testing of the present invention.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.

Other features and advantages of the invention will be apparent from the detailed description, and from the claims . Brief Description of the Drawings Fig. 1 is a depiction of a partial human hfz-4 nucleotide sequence (clone j thdb045c07 ; SEQ ID NO:l) that encodes a portion of Hfz-4. Fig. 2 is a depiction of a partial murme mfz- 9 nucleotide sequence (clone j fπrj f037e02tl ; SEQ ID NO: 2) that encodes a portion of Mfz-9.

Fig. 3 is a potential translation of tne partial human hfz-4 cDNA sequence of SEQ ID NO:l (SEQ ID NO: 3) . Fig. 4 is a potential translation of the partial murme mfz-9 cDNA sequence of SEQ ID NO: 2 (SEQ ID NO: 4) .

Detailed Description As described above, the nucleic acid molecules of the invention and the polypeptides they encode (e.g., Hfz-4 or Mfz-9 polypeptides or fragments thereof) can be used directly as diagnostic and therapeutic agents, or they can be used to generate antibodies or identify small molecules that, m turn, are clinically useful. In addition, hfz-4 or mfz-9 nucleic acid molecules are useful genetic mapping, to identify the chromosomal location of hfz-4 or rnfz-9, and as tissue-specific markers. Accordingly, expression vectors containing the nucleic acid molecules of the invention, cells transfected with these vectors, the polypeptides expressed, and antibodies generated, against either the entire polypeptide or an antigenic fragment thereof, are among the preferred embodiments. These embodiments and some of their clinical application are described further below. The cDNA clone, j thdb045c07 , (Figure 1; SEQ ID NO:l) encoding a portion of Hfz-4 was isolated from a human endothelial cell library. A potential translation of this cDNA clone is depicted in Figure 3 (SEQ ID NO: 3) . The cDNA clone, fπrj f037e02tl, (SEQ ID NO: 2) encoding a portion of Mfx-9 was isolated from a murme choroid plexus library. A potential translation of this cDNA clone is depicted in Figure 4 (SEQ ID NO:4) .

I. Nucleic Acid Molecules Encoding Hfz-4 or Mfz- 9

The hfz-4 or mfz-9 nucleic acid molecules of the invention can be cDNA, genomic DNA, synthetic DNA, or RNA, and can be double-stranded or single-stranded. In the event the nucleic acid molecule is single-stranded, it can be either a sense or an antisense strand. Fragments of these molecules are also considered within the scope of the invention, and can be produced, for example, by the polymerase chain reaction (PCR) , or generated by treating a longer fragment (e.g., a full- length hfz-4 or mfz-9 gene sequence) with one or more restriction endonucleases . Similarly, a full-length hfz- 4 or mfz- 9 RNA molecule, or a fragment thereof, can be produced by in vi tro transcription. The isolated nucleic acid molecule of the invention can encode a fragment of Hfz-4 or Mfz- 9 that is not found as such in the natural state. Although nucleic acid molecules encoding any given fragment of Hfz-4 or Mfz-9 are within the scope of the invention, fragments that retain the biological activity of Hfz-4 or Mfz-9 are preferred.

The nucleic acid molecules of the invention encompass recombinant molecules, such as those in which a nucleic acid molecule (e.g., an isolated nucleic acid molecule encoding Hfz-4 or Mfz-9, or a fragment thereof) is incorporated: (1) into a vector (e.g., a plasmid or viral vector), (2) into the genome of a heterologous cell, or (3) into the genome of a homologous cell, at a position other than the natural chromosomal location. Recombinant nucleic acid molecules, transgenic animals, and uses therefor are discussed further below.

The nucleic acid molecules of the invention can contain naturally occurring sequences, or sequences that differ from those that occur naturally, but, due to the degeneracy of the genetic code, encode the same polypeptide. In addition, the nucleic acid molecules of the invention are not limited to those that encode the amino acid residues of the Hfz-4 or Mfz-9 polypeptide encoded by SEQ ID NOs : 1 and 2, respectively; they can also include some or all of the non-coding sequences that lie upstream or downstream from a hfz-4 or mfz-9 coding sequence, a heterologous regulatory element, or a sequence encoding a heterologous polypeptide (e.g., a reporter gene) . Regulatory elements and reporter genes are discussed further below.

The nucleic acid molecules of the invention can be synthesized (for example, by phosphoramidite-based synthesis) or obtained from a biological cell, such as the cell of a mammal. Thus, the nucleic acids can be those of a human, mouse, rat, guinea pig, cow, sheep, goat, horse, pig, rabbit, monkey, dog, or cat. Combinations or modifications of the nucleotides within these types of nucleic acid molecules are also encompassed. In the event the nucleic acid molecules of the invention encode or act as antisense molecules, they can be used, for example, to regulate translation of hfz-4 or mfz- 9 mRNA. Techniques associated with detection of nucleic acid sequences or regulation of their expression are well known to persons of ordinary skill in the art, and can be used in the context of the present invention to diagnose or treat disorders associated with aberrant hfz-4 or mfz-9 expression. However, aberrant expression of hfz-4 or mfz-9 (or aberrant activity of Hfz-4 or Mfz- 9) is not a prerequisite for treatment according to the methods of the invention; the molecules of the invention (including the nucleic acid molecules described here) are expected to be useful in improving the symptoms associated with a variety of medical conditions regardless of whether or not the expression of hfz-4 or mfz-9 (or the activity of Hfz-4 or Mfz-9) is detectably aberrant . Nucleic acid molecules are discussed further below in the context of their clinical utility.

The invention also encompasses nucleic acid molecules that encode other members of the hfz-4 or mfz- 9 family (e.g., the human homologue of murine mfz-9) . Such nucleic acid molecules will be readily identified by the ability to hybridize under stringent conditions to a nucleic acid molecule encoding a Hfz-4 or Mfz-9 polypeptide ( e . g. , nucleic acid molecules having the sequence of SEQ ID NOs : 1 or 2 ) . The cDNA sequences described herein (SEQ ID NOs : 1 and 2) can be used to identify these nucleic acids, which include, for example, nucleic acids that encode homologous polypeptides in other species, splice variants of the hfz-4 or mfz-9 gene in humans or other mammals, allelic variants of the hfz-4 or mfz- 9 gene in humans or other mammals, and mutant forms of the hfz-4 or mfz- 9 gene in humans or other mammals .

The preferred class of nucleic acid molecules that hybridize to SEQ ID NOs : 1 and 2 are nucleic acid molecules that encode human allelic variants of hfz-4 or mfz-9. There are two major classes of such variants: active allelic variants, naturally occurring variants that have the biological activity of Hfz-4 or Mfz-9 and non-active allelic variants, naturally occurring allelic variants that lack the biological function of Hfz-4 or Mfz-9. Active allelic variants can be used as an equivalent for a hfz-4 or mfz-9 protein having the amino acid sequence encoded by SEQ ID NOs : 1 or 2 as described herein whereas nonactive allelic variants can be used in methods of disease diagnosis and as a therapeutic target.

The invention features methods of detecting and isolating such nucleic acid molecules. Using these methods, a sample (e.g., a nucleic acid library, such as a cDNA or genomic library) is contacted (or "screened") with a hfz-4 or mfz-9-specific probe (e.g., a fragment of SEQ ID N0:1 that is at least 17 nucleotides long) . The probe will selectively hybridize to nucleic acids encoding related polypeptides (or to complementary sequences thereof). The term "selectively hybridize" is used to refer to an event in which a probe binds to nucleic acid molecules encoding Hfz-4 or Mfz-9 (or to complementary sequences thereof) to a detectably greater extent than to nucleic acids encoding other polypeptides, particularly other types of transporter molecules (or to complementary sequences thereof) . The probe, which can contain at least 17 nucleotides (e.g., 18, 20, 25, 50, 100, 150, or 200 nucleotides) can be produced using any of several standard methods (see, e.g., Ausubel et al . , "Current Protocols in Molecular Biology, Vol. I," Green Publishing Associates, Inc., and John Wiley & Sons, Inc., NY, 1989) . For example, the probe can be generated using PCR amplification methods in which oligonucleotide primers are used to amplify a hfz-4 or mfz-9-specific nucleic acid sequence (for example, a nucleic acid encoding one of the transmembrane domains) that can be used as a probe to screen a nucleic acid library and thereby detect nucleic acid molecules (within the library) that hybridize to the probe.

One single-stranded nucleic acid is said to hybridize to another if a duplex forms between them. This occurs when one nucleic acid contains a sequence that is the reverse and complement of the other (this same arrangement gives rise to the natural interaction between the sense and antisense strands of DNA in the genome and underlies the configuration of the double helix) . Complete complementarity between the hybridizing regions is not required in order for a duplex to form; it is only necessary that the number of paired bases is sufficient to maintain the duplex under the hybridization conditions used. Typically, hybridization conditions initially used to identify related genes are of low to moderate stringency. These conditions favor specific interactions between completely complementary sequences, but allow some non-specific interaction between less than perfectly matched sequences to occur as well. After hybridization, the nucleic acids can be "washed" under moderate or high conditions of stringency to dissociate duplexes that are bound together by some non-specific interaction (the nucleic acids that form these duplexes are thus not completely complementary) .

As is known in the art, the optimal conditions for washing are determined empirically, often by gradually increasing the stringency. The parameters that can be changed to affect stringency include, primarily, temperature and salt concentration. In general, the lower the salt concentration and the higher the temperature, the higher the stringency. Washing can be initiated at a low temperature (e.g., room temperature) using a solution containing a salt concentration that is equivalent to or lower than that of the hybridization solution. Subsequent washing can be carried out using progressively warmer solutions having the same salt concentration. As alternatives, the salt concentration can be lowered and the temperature maintained in the washing step, or the salt concentration can be lowered and the temperature increased. Additional parameters can also be altered. For example, use of a destabilizing agent, such as formamide, alters the stringency conditions .

In reactions where nucleic acids are hybridized, the conditions used to achieve a given level of stringency will vary. There is not one set of conditions, for example, that will allow duplexes to form between all nucleic acids that are 85% identical to one another; hybridization also depends on unique features of each nucleic acid. The length of the sequence, the composition of the sequence (e.g., the content of purine- like nucleotides versus the content of pyrimidine-like nucleotides) and the type of nucleic acid (e.g., DNA or RNA) affect hybridization. An additional consideration is whether one of the nucleic acids is immobilized (e.g., on a filter) .

An example of a progression from lower to higher stringency conditions is the following, where the salt content is given as the relative abundance of SSC (a salt solution containing sodium chloride and sodium citrate; 2X SSC is 10-fold more concentrated than 0.2X SSC). Nucleic acid molecules are hybridized at 42°C in 2X SSC/0.1% SDS (sodium dodecylsulfate; a detergent) and then washed in 0.2X SSC/0.1% SDS at room temperature (for conditions of low stringency); 0.2X SSC/0.1% SDS at 42°C (for conditions of moderate stringency); and 0. IX SSC at 68 °C (for conditions of high stringency) . Washing can be carried out using only one of the conditions given, or each of the conditions can be used (for example, washing for 10-15 minutes each in the order listed above) . Any or all of the washes can be repeated. As mentioned above, optimal conditions will vary and can be determined empirically.

A second set of conditions that are considered "stringent conditions" are those in which hybridization is carried out at 50°C in Church buffer (7% SDS, 0.5% NaHP0₄, 1 M EDTA, 1% BSA) and washing is carried out at 50°C in 2X SSC.

Preferably, nucleic acid molecules of the invention that are defined by their ability to hybridize with nucleic acid molecules having the sequence shown in SEQ ID Nos. 1 or 2 under stringent conditions will have additional features in common with hfz-4 or mfz-9. For example, the nucleic acid molecules identified by hybridization may have a similar, or identical, expression profile as the hfz-4 or mfz-9 molecules described herein, or may encode a polypeptide having one or more of the biological activities possessed by Hfz-4 or Mfz-9. Once detected, the nucleic acid molecules can be isolated by any of a number of standard techniques (see, e.g., Sambrook et al . , "Molecular Cloning, A Laboratory Manual," 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) .

In as much as SEQ ID NOs : 1 and 2 represent portions of complete hfz-4 and mfz-9 genes, the above-described hybridization methods can be used to identify cDNAs encoding the remainder of Hfz-4 and Mfz-9 as well as splice variants of hfz-4 and mfz-9.

The invention also encompasses: (a) expression vectors that contain any of the foregoing hfz-4 or mfz-9- related coding sequences and/or their complements (i.e., "antisense" sequence) and fragments thereof; (b) expression vectors that contain any of the foregoing hfz-4 or mfz-9-related sequences operatively associated with a regulatory element (examples of which are given below) that directs the expression of the coding sequences; (c) expression vectors containing, in addition to sequences encoding a Hfz-4 or Mfz- 9 polypeptide, nucleic acid sequences that are unrelated to nucleic acid sequences encoding Hfz-4 or Mfz-9, such as molecules encoding a reporter or marker; and (d) genetically engineered host cells that contain any of the foregoing expression vectors, and thereby express the nucleic acid molecules of the invention in the host cell . The regulatory elements referred to above include, but are not limited to, inducible and non-inducible promoters, enhancers, operators and other elements, which are known to those skilled in the art, and which drive or otherwise regulate gene expression. Such regulatory elements include but are not limited to the cytomegalovirus hCMV immediate early gene, the early or late promoters of SV40 adenovirus, the lac system, the trp system, the TAC system, the TRC system, the major operator and promoter regions of phage λ, the control regions of fd coat protein, the promoter for 3 -phosphoglycerate kinase, the promoters of acid phosphatase, and the promoters of the yeast α-mating factors.

Additionally, the hfz-4 or mfz-9 encoding nucleic acid molecules of the present invention can form part of a hybrid gene encoding additional polypeptide sequences, for example, sequences that function as a marker or reporter. Examples of marker or reporter genes include /3-lactamase, chloramphenicol acetyltransferase (CAT), adenosine deaminase (ADA) , aminoglycoside phosphotransferase (neo^r, G418^r) , dihydrofolate reductase (DHFR) , hygromycin-B-phosphotransferase (HPH) , thymidine kinase (TK) , lacZ (encoding 3-galactosidase) , and xanthine guanine phosphoribosyltransferase (XGPRT) . As with many of the standard procedures associated with the practice of the invention, skilled artisans will be aware of additional useful reagents, for example, additional sequences that can serve the function of a marker or reporter. Generally, a chimeric or hybrid polypeptide of the invention will include a first portion and a second portion; the first portion being a Hfz-4 or Mfz-9 polypeptide or a fragment thereof (preferably a biologically active fragment) and the second portion being, for example, the reporter described above or an immunoglobulin constant region.

The expression systems that can be used for purposes of the invention include, but are not limited to, microorganisms such as bacteria (e.g., E. coli and B . subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing the nucleic acid molecules of the invention; yeast (e.g., Saccharomyces and Pichia) transformed with recombinant yeast expression vectors containing the nucleic acid molecules of the invention (preferably containing a nucleic acid sequence encoding all or a portion of Hfz-4 or Mfz-9 (such as the sequence of SEQ ID NO:l or SEQ ID NO : 2 ) ; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing a nucleic acid molecule of the invention; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus (CaMV) and tobacco mosaic virus (TMV) ) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing hfz-4 or mfz- 9 nucleotide sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, VERO, HeLa, MDCK, WI38, and NIH 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., the metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter and the vaccinia virus 7.5K promoter) . In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the gene product being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions containing Hfz-4 or Mfz-9 polypeptides or for raising antibodies to those polypeptides, vectors that are capable of directing the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruther et al . , EMBO J. 2 : 1191 , 1983), in which the coding sequence of the insert may be ligated individually into the vector in frame with the lacZ coding region so that a fusion protein is produced; pIN vectors (Inouye and Inouye, Nucleic Acids Res . 13_.: 3101-3109 , 1985; Van Heeke and Schuster, J^". Biol . Chem. 264:5503-5509, 1989); and the like. pGEX vectors may also 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. The pGEX vectors are designed to include thrombm or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

In an insect system, Autographa calif ornica nuclear polyhidrosis virus (AcNPV) can be used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The coding sequence of the insert may be cloned individually into non-essential regions (e.g., the polyhedrm gene) of the virus and placed under control of an AcNPV promoter (e.g., the polyhedrm promoter) . Successful insertion of the coding sequence will result inactivation of the polyhedrm gene and production of non-occluded recombinant virus ( i . e . , virus lacking the protemaceous coat coded for by the polyhedrm gene) . These recombinant viruses are then used to infect Spodoptera frugiperda cells m which the inserted gene is expressed (e.g., see Smith et al . , J. Virol . 4_6:584, 1983; and Smith, U.S. Patent No. 4,215, 051) . In mammalian host cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, the nucleic acid molecule of the invention can be ligated to an adenovirus transcription/translation control complex, for example, the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vi tro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region El or E3) will result in a recombinant virus that is viable and capable of expressing a hfz-4 or mfz-9 gene product m infected hosts (e.g., see Logan and Shenk, Proc . Natl . Acad . Sci . USA _.81:3655-3659, 1984). Specific initiation signals may also be required for efficient translation of inserted nucleic acid molecules. These signals include the ATG initiation codon and adjacent sequences. In cases where a complete gene or cDNA, including its own initiation codon and adjacent sequences, is inserted into the appropriate expression vector, no additional translational control signals may be needed. However, in cases where only a portion of the coding sequence is inserted (e.g., the portion encoding the mature form of a hfz-4 or mfz-9 protein) translational control signals, including, perhaps, the ATG initiation codon, must be provided. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al . , Methods in Enzymol . 153 :516-544, 1987) .

In addition, a host cell strain may be chosen that modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products.

Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used. The mammalian cell types listed above are among those that could serve as suitable host cells.

For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express Hfz-4 or Mfz-9 can be engineered. Rather than using expression vectors that contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter sequences, enhancer sequences, transcription terminators, polyadenylation sites, etc.) , and a selectable marker. Following the introduction of the foreign DNA, engineered cells can be allowed to grow for 1-2 days in an enriched media, and then switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection, and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which, in turn, can be cloned and expanded into cell lines. This method can advantageously be used to engineer cell lines that express hfz-4 or mfz-9. Such engineered cell lines may be particularly useful in screening and evaluating compounds that affect the endogenous activity of the gene product (i.e., Hfz-4 or Mfz-9) .

A number of selection systems can be used. For example, the herpes simplex virus thymidine kinase

(Wigler, et al . , Cell 1_1:223, 1977), hypoxanthine-guanine phosphoribosyltransferase (Szybalska and Szybalski, Proc . Natl . Acad . Sci . USA 4_8:2026, 1962), and adenine phosphoribosyltransferase (Lowy, et al . , Cell 22:817, 1980) genes can be employed in tk^" , hgprt^" or aprt^" cells, respectively. Also, anti -metabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al . , Proc . Natl . Acad . Sci . USA T∑ : 3561 , 1980; O'Hare et al . , Proc . Na tl . Acad . Sci . USA 7.8:1527, 1981); gpt, which confers resistance to mycophenolic acid (Mulligan and Berg, Proc. Na tl . Acad . Sci . USA 18_ : 2012 , 1981); neo, which confers resistance to the aminoglycoside G-418 (Colberre-Garapin et al . , J^". Mol . Biol . 150 : 1, 1981); and hygro, which confers resistance to hygromycin (Santerre et al., Gene 1_: 147, 1984). Alternatively, any Hfz-4 or Mfz-9-containing fusion proteins can be readily purified utilizing an antibody specific for the fusion protein being expressed. For example, a system described by Janknecht et al . allows for the ready purification of non-denatured fusion proteins expressed in human cell lines ( Proc . Natl . Acad . Sci . USA 8:8972-8976, 1991). In this system, the gene of interest is subcloned into a vaccinia recombination plasmid such that the gene's open reading frame is translationally fused to an amino-terminal tag consisting of six histidine residues. Extracts from cells infected with recombinant vaccinia virus are loaded onto Ni^{2+ •} nitriloacetic acid-agarose columns and histidine- tagged proteins are selectively eluted with imidazole- containing buffers.

As implied by the descriptions above, a host cell is any cell into which (or into an ancestor of which) a nucleic acid encoding a polypeptide of the invention (e.g., a Hfz-4 or Mfz-9 polypeptide) has been introduced by means of recombinant DNA techniques.

II. Hfz-4 and Mfz- 9 Polypeptides

The Hfz-4 or Mfz-9 polypeptides described herein are those encoded by any of the nucleic acid molecules described above, and include fragments of Hfz-4 or Mfz- 9, mutant forms of Hfz-4 or Mfz-9, active and non-active allelic variants of Hfz-4 or Mfz-9, splice variants of hfz-4 or mfz-9, truncated forms of Hfz-4 or Mfz-9, and fusion proteins containing all or a portion of Hfz-4 or Mfz-9. These polypeptides can be prepared for a variety of uses including, but not limited to, the generation of antibodies, as reagents in diagnostic assays, for the identification of other cellular gene products or exogenous compounds that can modulate the activity or expression of Hfz-4 or Mfz-9, and as pharmaceutical reagents useful for the treatment of any disorder in which the associated symptoms are improved by altering the activity of Hfz-4 or Mfz-9.

The terms "protein" and "polypeptide" are used herein to describe any chain of amino acid residues, regardless of length or post-translational modification (e.g., modification by glycosylation or phosphorylation) . Thus, the term "Hfz-4 or Mfz- 9 polypeptide" includes full-length, naturally occurring Hfz-4 or Mfz-9 polypeptides (that can be purified from tissues in which they are naturally expressed, according to standard biochemical methods of purification) , as well as recombinantly or synthetically produced polypeptides that correspond either to a full-length, naturally-occurring Hfz-4 or Mfz-9 polypeptide or to particular domains or portions of such a polypeptide. The term also encompasses mature Hfz-4 or Mfz- 9 having an added amino- terminal methionine (useful for expression in prokaryotic cells) .

Preferred polypeptides are substantially pure Hfz-4 or Mfz-9 polypeptides that are at least 50% (e.g., 55%, 60%, or 65%), more preferably at least 70% (e.g., 72%, 75%, or 78%), even more preferably at least 80% (e.g., 80%, 85% or 90%), and most preferably at least 95% (e.g., 97% or even 99%) identical to the sequences encoded by SEQ ID N0:1 or SEQ ID NO : 2. Those of ordinary skill in the art are well able to determine the percent identity between two amino acid sequences. Thus, if a polypeptide is encoded by a nucleic acid that hybridizes under stringent conditions with the hfz-4 or mfz- 9 sequence disclosed herein and also encodes one or more of the conserved regions present in Hfz-4 or Mfz-9, it will be recognized as a Hfz-4 or Mfz- 9 polypeptide and thereby considered within the scope of the present invention.

The invention also encompasses polypeptides that are functionally equivalent to Hfz-4 or Mfz-9. These polypeptides are equivalent to Hfz-4 or Mfz-9 in that they are capable of carrying out one or more of the functions of Hfz-4 or Mfz-9 m a biological system. Polypeptides that are functionally equivalent to Hfz-4 or Mfz-9 can have 20%, 40%, 50%, 75%, 80%, or even 90% of one or more of the biological activities of the full- length, mature human form of Hfz-4 or Mfz-9. Such comparisons are generally based on an assay of biological activity m which equal concentrations of the polypeptides are used and compared. The comparison can also be based on the amount of the polypeptide required to reach 50% of the maximal biological activity obtainable .

Functionally equivalent proteins can be those, for example, that contain additional or substituted ammo acid residues. Substitutions may be made on the basis of similarity m polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. Ammo acids that are typically considered to provide a conservative substitution for one another are specified the Summary of the Invention.

Polypeptides that are functionally equivalent to Hfz-4 or Mfz- 9 can be made using random mutagenesis techniques well known to those of ordinary skill m the art (and the resulting mutant Hfz-4 or Mfz-9 polypeptides can be tested for activity) . It is more likely, however, that such polypeptides will be generated by site-directed mutagenesis (again using techniques well known to persons of ordinary skill in the art) . These polypeptides may have increased functionality or decreased functionality. To design functionally equivalent polypeptides, it is useful to distinguish between conserved positions and variable positions. This can be done by aligning the ammo acid sequences of Hfz-4 or Mfz-9 that are obtained from various organisms (e.g., Hfz-4 can be aligned with its murme homologue Mfz-4) . Skilled artisans will recognize that conserved ammo acid residues are more likely to be necessary for preservation of function. Thus , it is preferable that conserved residues are not altered. Alignment of Hfz-4 or Mfz-9 with other members of the frizzled family will reveal regions that are more highly conserved. Such regions are preferably not altered.

Mutations within the hfz-4 or mfz-9 coding sequence can be made to generate variant hfz-4 or mfz-9 genes that are better suited for expression in a selected host cell . For example, N-linked glycosylation sites can be altered or eliminated to achieve, for example, expression of a homogeneous product that is more easily recovered and purified from yeast hosts which are known to hyperglycosylate N-linked sites. To this end, a variety of amino acid substitutions at one or both of the first or third amino acid positions of any one or more of the glycosylation recognition sequences which occur (in N-X-S or N-X--), and/or an amino acid deletion at the second position of any one or more of such recognition sequences, will prevent glycosylation at the modified tripeptide sequence (see, e.g., Miyaj ima et al . , EMBO J. 5_:1193, 1986) .

The polypeptides of the invention can be expressed fused to another polypeptide, for example, a marker polypeptide or fusion partner. For example, the polypeptide can be fused to a hexa-histidine tag to facilitate purification of bacterially expressed protein or a hemagglutinin tag to facilitate purification of protein expressed in eukaryotic cells. In addition, a hfz-4 or mfz-9 polypeptide can be fused to GST. The polypeptides of the invention can be chemically synthesized (e.g., see Creighton, "Proteins: Structures and Molecular Principles," W.H. Freeman & Co., NY, 1983), or, perhaps more advantageously, produced by recombinant DNA technology as described herein. For additional guidance, persons of ordinary skill in the art may consult Ausubel et al . ( supra) , Sambrook et al . ("Molecular Cloning, A Laboratory Manual," Cold Spring Harbor Press, Cold Spring Harbor, NY, 1989), and, particularly for examples of chemical synthesis, Gait ("Oligonucleotide Synthesis," IRL Press, Oxford, 1984).

III. Transgenic animals Hfz-4 or Mfz-9 polypeptides can also be expressed in transgenic animals. Such transgenic animals represent model systems for the study of disorders that are either caused by or exacerbated by misexpression of hfz-4 or mfz- 9, or disorders that can be treated by altering the expression of hfz-4 or mfz- 9 or the activity of Hfz-4 or Mfz- 9 (even though the expression or activity is not detectably abnormal) . Transgenic animals can also be used for the development of therapeutic agents that modulate the expression of hfz-4 or mfz-9 or the activity of Hfz-4 or Mfz-9.

Transgenic animals can be farm animals (e.g., pigs, goats, sheep, cows, horses, rabbits, and the like) rodents (such as rats, guinea pigs, and mice), non-human primates (e.g., baboons, monkeys, and chimpanzees), and domestic animals (e.g., dogs and cats) . Transgenic mice are especially preferred.

Any technique known m the art can be used to introduce a hfz-4 or mfz- 9 transgene into animals to produce founder lines of transgenic animals. Such techniques include, but are not limited to, pronuclear microin ection (U.S. Pat. No. 4,873,191); retrovirus mediated gene transfer into germ lines (Van der Putten et al . , Proc . Natl . Acad . Sci . , USA 82 :6148, 1985); gene targeting into embryonic stem cells (Thompson et al . , Cell J5_6:313, 1989); and electroporation of embryos (Lo, Mol . Cell . Biol . 2:1803, 1983).

The present invention provides for transgenic animals that carry a hfz-4 or mfz-9 transgene m all of their cells, as well as animals that carry a transgene m some, but not all of their cells. For example, the invention provides for mosaic animals. The hfz-4 or mfz- 9 transgene can be integrated as a single transgene or in concatamers, for example, head-to-head tandems or head- to-tail tandems. The transgene can also be selectively introduced into, and activated in, a particular cell type (Lasko et al . , Proc . Na tl . Acad . Sci . USA 89_ : 6232 , 1992) . The regulatory sequences required for such a cell-type specific activation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art . When it is desired that a hfz-4 or mfz-9 transgene be integrated into the chromosomal site of an endogenous hfz-4 or mfz-9 gene, gene targeting is preferred. Briefly, when such a technique is to be used, vectors containing some nucleotide sequences homologous to an endogenous hfz-4 or mfz-9 gene are designed for the purpose of integrating, via homologous recombination with chromosomal sequences, into and disrupting the function of the nucleotide sequence of the endogenous gene. The transgene also can be selectively introduced into a particular cell type, thus inactivating the endogenous hfz-4 or mfz-9 gene in only that cell type (Gu et al . , Science 265 : 103 , 1984). The regulatory sequences required for such a cell-type specific inactivation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art. These techniques are useful for preparing "knock outs" having no functional hfz-4 or mfz- 9 gene.

Once transgenic animals have been generated, the expression of the recombinant hfz-4 or mfz-9 gene can be assayed utilizing standard techniques. Initial screening may be accomplished by Southern blot analysis or PCR techniques to determine whether integration of the transgene has taken place. The level of mRNA expression of the transgene in the tissues of the transgenic animals may also be assessed using techniques which include, but are not limited to, Northern blot analysis of tissue samples obtained from the animal, in si tu hybridization analysis, and RT-PCR. Samples of hfz-4 or mfz-9 gene- expressing tissue can also be evaluated immunocytochemically using antibodies specific for the Hfz-4 or Mfz-9 transgene product. For a review of techniques that can be used to generate and assess transgenic animals, those of ordinary skill in the art can consult Gordon (Intl. .Rev. Cytol . 115 : 171-229 , 1989), and may obtain additional guidance from, for example: Hogan et al . "Manipulating the Mouse Embryo" (Cold Spring Harbor Press, Cold Spring Harbor,

NY, 1986); Krimpenfort et al . , Bio /Technology 1:86 , 1991; Palmiter et al . , Cell 4_1:343, 1985; Kraemer et al . , "Genetic Manipulation of the Early Mammalian Embryo, " Cold Spring Harbor Press, Cold Spring Harbor, NY, 1985; Hammer et al . , Nature 315:680, 1985; Purcel et al . , Science 244 : 1281, 1986; Wagner et al., U.S. Patent No. 5,175,385; and Krimpenfort et al . , U.S. Patent No. 5, 175,384.

The transgenic animals of the invention can be used to determine the consequence of altering the expression of hfz-4 or mfz- 9 in the context of various disease states. For example, hfz-4 or mfz- 9 knock out mice can be generated using an established line of mice that serve as a model for a disease in which activity of the missing gene is impaired.

IV. Anti-Hfz-4 and Anti-Mfz-9 Antibodies

Hfz-4 or Mfz- 9 polypeptides (or immunogenic fragments or analogs thereof) can be used to raise antibodies useful in the invention; such polypeptides can be produced by recombinant techniques or synthesized (see, for example, "Solid Phase Peptide Synthesis," supra ; Ausubel et al . , supra) . In general, Hfz-4 or Mfz- 9 polypeptides can be coupled to a carrier protein, such as KLH, as described in Ausubel et al . , supra, mixed with an adjuvant, and injected into a host mammal. Antibodies produced in that animal can then be purified by peptide antigen affinity chromatography.

In particular, various host animals can be immunized by injection with a Hfz-4 or Mfz- 9 polypeptide or an antigenic fragment thereof. Commonly employed host animals include rabbits, mice, guinea pigs, and rats. Various adjuvants that can be used to increase the immunological response depend on the host species and include Freund's adjuvant (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol . Potentially useful human adjuvants include BCG (bacille Calmette-Guerin) and Corynebacterium parvum . Polyclonal antibodies are heterogeneous populations of antibody molecules that are contained in the sera of the immunized animals.

Antibodies within the invention therefore include polyclonal antibodies and, in addition, monoclonal antibodies, humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab')₂ fragments, and molecules produced using a Fab expression library. Monoclonal antibodies, which are homogeneous populations of antibodies to a particular antigen, can be prepared using the Hfz-4 or Mfz- 9 polypeptides described above and standard hybridoma technology (see, for example, Kohler et al . , Nature 256 :495, 1975; Kohler et al . , Eur. J. Immunol . 6.: 511, 1976; Kohler et al . , Eur . J. Immunol . 6_.: 292, 1976; Hammerling et al . , "Monoclonal Antibodies and T Cell Hybridomas," Elsevier, NY, 1981; Ausubel et al . , supra) .

In particular, monoclonal antibodies can be obtained by any technique that provides for the production of antibody molecules by continuous cell lines in culture such as described in Kohler et al . , Nature 256 :495 , 1975, and U.S. Patent No. 4,376,110; the human B-cell hybridoma technique (Kosbor et al . , Immunology Today 4_:72, 1983; Cole et al . , Proc . Natl . Acad. Sci . USA ___ : 2026 , 1983), and the EBV-hybridoma technique (Cole et al . , "Monoclonal Antibodies and Cancer Therapy," Alan R. Liss, Inc., pp. 77-96, 1983) . Such antibodies can be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof. The hybridoma producing the mAb of this invention may be cultivated in vi tro or in vivo . The ability to produce high titers of mAbs in vivo makes this a particularly useful method of production.

Once produced, polyclonal or monoclonal antibodies are tested for specific Hfz-4 or Mfz-9 recognition by Western blot or immunoprecipitation analysis by standard methods, for example, as described in Ausubel et al . , supra . Antibodies that specifically recognize and bind to Hfz-4 or Mfz- 9 are useful in the invention. For example, such antibodies can be used in an immunoassay to monitor the level of Hfz-4 or Mfz-9 produced by a mammal (e.g., to determine the amount or subcellular location of Hfz-4 or Mfz-9) .

There are two major classes of antibodies which are within the scope of the present invention. The first class are antibodies that selectively bind to Hfz-4 or Mfz- 9 polypeptide, not bind to other members of the frizzled family of proteins. The second class are antibodies that bind to more than one member of the frizzled family of proteins.

Preferably, hfz-4 or mfz-9 selective antibodies of the invention are produced using fragments of the Hfz-4 or Mfz-9 polypeptide that lie outside highly conserved regions and appear likely to be antigenic, by criteria such as high frequency of charged residues. Cross- reactive anti-hfz-4 or mfz-9 antibodies are produced using a fragment of hfz-4 or mfz-9 that is conserved amongst members of this family of proteins. In one specific example, such fragments are generated by standard techniques of PCR, and are then cloned into the pGEX expression vector (Ausubel et al . , supra) . Fusion proteins are expressed in E. coli and purified using a glutathione agarose affinity matrix as described in Ausubel, et al . , supra . In some cases it may be desirable to minimize the potential problems of low affinity or specificity of antisera. In such circumstances, two or three fusions can be generated for each protein, and each fusion can be injected into at least two rabbits. Antisera can be raised by injections in a series, preferably including at least three booster injections.

Antiserum is also checked for its ability to immunoprecipitate recombinant Hfz-4 or Mfz-9 polypeptides or control proteins, such as glucocorticoid receptor, CAT, or luciferase.

The antibodies can be used, for example, in the detection of Hfz-4 or Mfz-9 in a biological sample as part of a diagnostic assay or to reduce Hfz-4 or Mfz-9 activity as part of a therapeutic regime (e.g., to reduce an undesirable level of Hfz-4 or Mfz-9 activity) .

Antibodies also can be used in a screening assay to measure the effect of a candidate compound on expression or localization of Hfz-4 or Mfz-9. Additionally, such antibodies can be used in conjunction with the gene therapy techniques. For example, they may be used to evaluate the normal and/or engineered Hfz-4 or Mfz-9- expressing cells prior to their introduction into the patient .

In addition, techniques developed for the production of "chimeric antibodies" (Morrison et al . , Proc . Natl . Acad . Sci . USA 21=6851, 1984; Neuberger et al . , Nature 312 :604, 1984; Takeda et al . , Nature 214:452 , 1984) by splicing the genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region.

Alternatively, techniques described for the production of single chain antibodies (U.S. Patent Nos. 4,946,778, 4,946,778, and 4,704,692) can be adapted to produce single chain antibodies against a Hfz-4 or Mfz-9 polypeptide, or a fragment thereof. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide.

Antibody fragments that recognize and bind to specific epitopes can be generated by known techniques. For example, such fragments include but are not limited to F(ab')₂ fragments that can be produced by pepsin digestion of the antibody molecule, and Fab fragments that can be generated by reducing the disulfide bridges of F(ab')₂ fragments. Alternatively, Fab expression libraries can be constructed (Huse et al . , Science 246 : 1275 , 1989) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.

Antibodies can be humanized by methods known in the art. For example, monoclonal antibodies with a desired binding specificity can be commercially humanized (Scotgene, Scotland; Oxford Molecular, Palo Alto, CA) . Fully human antibodies, such as those expressed in transgenic animals are also features of the invention (Green et al . , Nature Genetics 2=13-21, 1994; see also U.S. Patent Nos. 5,545,806 and 5,569,825). The methods described herein, in which anti-Hfz-4 or Mfz- 9 antibodies are employed, can be performed, for example, by utilizing pre-packaged diagnostic kits comprising at least one specific antibody reagent described herein, which may be conveniently used, for example, in clinical settings, to diagnose patients exhibiting symptoms of the disorders associated with aberrant expression of hfz-4 or mfz-9. V. Antisense Nucleic Acid Molecules

Treatment regimes based on an "antisense" approach involve the design of oligonucleotides (either DNA or RNA) that are complementary to a portion of a selected mRNA. These oligonucleotides bind to complementary mRNA transcripts and prevent their translation. Absolute complementarity, although preferred, is not required. A sequence "complementary" to a portion of an RNA molecule, as referred to herein, is a sequence having sufficient complementarily to hybridize with the RNA, forming a stable duplex; in the case of double-stranded antisense nucleic acids, a single strand of the duplex DNA can be tested, or triplex formation can be assayed. The ability to hybridize will depend on both the degree of complementarily and the length of the antisense nucleic acid. Generally, the longer the hybridizing nucleic acid, the more base mismatches with an RNA it may contain and still form a stable duplex (or triplex, as the case may be) . One of ordinary skill in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex.

Oligonucleotides that are complementary to the 5' end of the message, for example, the 5' untranslated sequence up to and including the AUG initiation codon, should work most efficiently at inhibiting translation. However, sequences complementary to the 3' untranslated sequences of mRNAs recently have been shown to be effective at inhibiting translation of mRNAs as well (Wagner, Nature 372 :333 , 1984). Thus, oligonucleotides complementary to either the 5' or 3 ' non-translated, non- coding regions of a hfz-4 or mfz-9 gene, could be used in an antisense approach to inhibit translation of endogenous hfz-4 or mfz-9- mRNA. Oligonucleotides complementary to the 5' untranslated region of the mRNA should include the complement of the AUG start codon. Antisense oligonucleotides complementary to mRNA coding regions are less efficient inhibitors of translation but could be used in accordance with the invention. Whether designed to hybridize to the 5', 3', or coding region of hfz-4 or mfz-9 mRNA, antisense nucleic acids should be at least six nucleotides m length, and are preferably oligonucleotides ranging from 6 to about 50 nucleotides m length. In specific aspects, the oligonucleotide is at least 10 nucleotides, at least 17 nucleotides, at least 25 nucleotides, or at least 50 nucleotides.

Regardless of the choice of target sequence, as with other therapeutic strategies directed to hfz-4 or mfz-9, it is preferred that in vi tro studies are first performed to assess the ability of an antisense oligonucleotide to inhibit gene expression. If desired, the assessment can be quantitative. It is preferred that these studies utilize controls that distinguish between antisense gene inhibition and any nonspecific biological effect that an oligonucleotide may cause. It is also preferred that these studies compare levels of the target RNA or protein with that of an internal control RNA or protein. Additionally, it is envisioned that results obtained using an antisense oligonucleotide are compared with those obtained using a control oligonucleotide. Preferably, the control oligonucleotide is of approximately the same length as the test oligonucleotide, and the nucleotide sequence of the control oligonucleotide differs from that of the test antisense sequence no more than is necessary to prevent specific hybridization between the control oligonucleotide and the targeted RNA sequence.

The oligonucleotides can contain DNA or RNA, or they can contain chimeric mixtures, derivatives, or modified versions thereof that are either smgle-stranded or double-stranded. The oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, hybridization, etc. Modified sugar moieties can be selected from the group including, but not limited to, arabinose, 2-fluoroarabinose, xylulose, and hexose . A modified phosphate backbone can be selected from the group consisting of a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphor- amidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal, or an analog of any of these backbones.

The oligonucleotide can include other appended groups such as peptides (e.g., for disrupting the transport properties of the molecule in host cells in vivo) , or agents that facilitate transport across the cell membrane (as described, for example, in Letsinger et al . , Proc . Natl . Acad . Sci . USA 26=6553, 1989; Lemaitre et al . , Proc . Natl . Acad . Sci . USA 2i=648, 1987; PCT Publication No. WO 88/09810) or the blood-brain barrier (see, for example, PCT Publication No. WO 89/10134) , or hybridization-triggered cleavage agents (see, for example, Krol et al . , BioTechniques 2=958, 1988), or intercalating agents (see, for example, Zon, Pharm . Res . 2=539, 1988). To this end, the oligonucleotide can be conjugated to another molecule, for example, a peptide, a hybridization triggered cross- linking agent, a transport agent, or a hybridization- triggered cleavage agent.

An antisense oligonucleotide of the invention can comprise at least one modified base moiety that is selected from the group including, but not limited to, 5-fluoro-uracil , 5-bromouracil , 5-chlorouracil , 5-iodouracil , hypoxanthine , xantine, 4-acetylcytosine, 5- (carboxyhydroxyl -methyl) uracil, 5- carboxymethylaminomethyl-2 -thiouridine , 5-carboxymethyl - aminomethyluracil , dihydrouracil , beta-D- galactosylqueosine , inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2 , 2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3 -methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5 -methyl - aminomethyluracil , 5-methoxyaminomethyl-2-thiouracil , beta-D-mannosylqueosine, 5' -methoxycarboxymethyluracil , 5-methoxyuracil , 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v) , wybutoxosine, pseudouracil , queosine, 2-thiocytosine, 5-methyl-2-theouracil , 2- thiouracil, 4-thio-uracil , 5-methyluracil , uracil-5- oxyacetic acid methylester, uracil-5-oxyacetic acid (v) , 5-methyl-2-thiouracil , 2- (3-amino-3-N-2-carboxypropl) uracil, (acp3)w, and 2 , 6-diaminopurine .

In yet another embodiment, the antisense oligonucleotide is an α-anomeric oligonucleotide. An α-anomeric oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual jβ-units, the strands run parallel to each other (Gautier et al . , Nucl . Acids . Res . 12=6625, 1987). The oligonucleotide is a 2 ' -O-methylribonucleotide (Inoue et al . , Nucl . Acids Res . 12=6131, 1987), or a chimeric RNA-DNA analog (Inoue et al . , FEBS Lett . 215 :327, 1987). Antisense oligonucleotides of the invention can be synthesized by standard methods known in the art, for example, by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.) . As examples, phosphorothioate oligonucleotides can be synthesized by the method of Stein et al . (Nucl . Acids Res . 12=3209, 1988), and methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al . , Proc . Na tl . Acad . Sci . USA 22=7448, 1988).

For therapeutic application, antisense molecules of the invention should be delivered to cells that express hfz-4 or mfz- 9 in vivo . A number of methods have been developed for delivering antisense DNA or RNA to cells; for example, antisense molecules can be injected directly into the tissue site. Alternatively, modified antisense molecules, which are designed to target cells that express hfz-4 or mfz-9 (e.g., antisense molecules linked to peptides or antibodies that specifically bind receptors or antigens expressed on the target cell surface) can be administered systemically . However, it is often difficult to achieve intracellular concentrations of antisense molecules that are sufficient to suppress translation of endogenous mRNAs. Therefore, a preferred approach uses a recombinant DNA construct m which the antisense oligonucleotide is placed under the control of a strong pol III or pol II promoter. The use of such a construct to transfecc target cells m the patient will result m the transcription of sufficient amounts of single stranded RNAs that will form complementary base pairs with endogenous hfz-4 or mfz- 9 transcripts and thereby prevent translation of hfz-4 or mfz-9 mRNA. For example, a vector can be introduced απ vivo such a way that it is taken up by a cell and thereafter directs the transcription of an antisense RNA. Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA.

Vectors encoding a hfz-4 or mfz- 9 antisense sequence can be constructed by recombinant DNA technology methods that are standard practice the art . Suitable vectors include plasmid vectors, viral vectors, or other types of vectors known or newly discovered m the art . The criterion for use is only that the vector be capable of replicating and expressing the hfz-4 or mfz-9 antisense molecule m mammalian cells. Expression of the sequence encoding the antisense RNA can be directed by any promoter known m the art to act mammalian, and preferably human, cells. Such promoters can pe mducible or constitutively active and include, cut are not limited to: the SV40 early promoter region (Bernoist et al . , Nature 290:304, 1981); the promoter contained m che 3' long cerminai repeat: of Rous sarcoma virus (Yamamoto et al . , Cell 22:787-797 , 1988); the herpes thymidine kinase promoter (Wagner et al . , Proc. Natl . Acad . Sci . USA 78:1441, 1981); or the regulatory sequences of the metallothionem gene (Brmster et al . , Nature 296:39, 1988).

VI . Ribozymes

Ribozyme molecules designed to catalytically cleave hfz-4 or mfz- 9 mRNA transcripts also can be used to prevent translation of hfz-4 or mfz- 9 mRNA and expression of Hfz-4 or Mfz-9 polypeptides (see, for example, PCT Publication WO 90/11364; Saraver et al . , Science 247 : 1222 , 1990). While various ribozymes that cleave mRNA at site-specifIC recognition sequences can be used to destroy hfz-4 or mfz-9 mRNAs, the use of hammerhead ribozymes is preferred. Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA. The sole requirement is that the target mRNA have the following sequence of two bases: 5'-UG-3'. The construction and production of hammerhead ribozymes is well known m the art (Haseloff et al . , Nature 221=585, 1988). There are numerous examples of potential hammernead ribozyme cleavage sites withm the nucleotide sequence of human hfz-4 and mfz-9 cDNA. Preferably, the ribozyme is engineered so that the cleavage recognition site is located near the 5' end of the hfz-4 or mfz-9 mRNA, i . e . , to increase efficiency and minimize the intracellular accumulation of non-functional mRNA transcripts.

The ribozymes of the present invention also include RNA endoπbonucleases (hereinafter "Cech-type ribozymes"), such as the one that occurs naturally m Tetrahymena Thermophila (known as the IVS or L-19 IVS RNA) , and which has been extensively described by Cecn and his collaborators (Zaug et al . , Science 224 : 574 , 1984; Zaug et al . , Science 231 :470 , 1986; Zug et al . , Nature 324 :429 , 1986; PCT Application No. WO 88/04300; and Been et al . , Cell 4_7:207, 1986) . The Cech-type ribozymes have an eight base-pair sequence that hybridizes to a target RNA sequence, whereafter cleavage of the target RNA takes place. The invention encompasses those Cech-type ribozymes that target eight base-pair active site sequences present in hfz-4 or mfz-9.

As in the antisense approach, the ribozymes can be composed of modified oligonucleotides (e.g., for improved stability, targeting, etc.), and should be delivered to cells which express the hfz-4 or mfz-9 in vivo . A preferred method of delivery involves using a DNA construct "encoding" the ribozyme under the control of a strong constitutive pol III or pol II promoter, so that transfected cells will produce sufficient quantities of the ribozyme to destroy endogenous hfz-4 or mfz-9 messages and inhibit translation. Because ribozymes, unlike antisense molecules, are catalytic, a lower intracellular concentration is required for efficiency.

VII . Peptide Nucleic Acids Nucleic acid molecules encoding hfz-4 or mfz-9 (or a fragment thereof) can be modified at the base moiety, sugar moiety, cr phosphate backbone to improve, for example, the stability or solubility of the molecule or its ability to hybridize with other nucleic acid molecules. For example, the deoxyribose phosphate backbone of the nucleic acid can be modified to generate peptide nucleic acids (see Hyrup et al . , Bioorganic Med . Chem . 4_.: 5-23 (1996) . As used herein, the terms "peptide nucleic acids" or "PNAs" refer to nucleic acid mimics, for example, DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained. The neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as descriPed m Hyrup et al . , supra; Perry-O' Keefe et al . Proc . Natl . Acad . Sci . USA 22=14670-14675 (1996) .

PNAs of hfz-4 or mfz-9 can be used m therapeutic and diagnostic applications. For example, PNAs can be used as antisense or antigene agents for sequence- specific modulation of gene expression by, for example, inducing transcription or translation arrest or inhibiting replication. PNAs of hfz-4 or mfz-9 can also be used, for example, m the analysis of single base pair mutations in a gene by, for example, PNA-directed PCR clamping; as artificial restriction enzymes when used in combination with other enzymes, for example, SI nucleases (Hyrup et al . , supra) ; or as probes or primers for DNA sequence and hypndization (Hyrup et al . , supra ; Perry- O'Keefe, supra) .

In other embodiments, PNAs of hfz-4 or mfz-9 can be modified, for example, to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to the PNA, py the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known m the art. For example, PNA-DNA chimeras of hfz-4 or mfz- 9 can e generated that may compme the advantageous properties of PNA and DNA. Such cnimeras allow DNA recognition enzymes, for example, RNAse H and DNA polymerases, to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity. PNA-DNA chimeras can be linked using linkers of appropriate lengths selected m terms of base stacking, number of bonds between the nucleobases, and orientation (Hyrup et al . , supra) . The synthesis of PNA- DNA cnimeras can be performed as described Hyrup, supra , and Finn et al . , Nucl . Acids Res . 24:3357-3363 (1996) . For example, a DNA chain can be synthesized on a solid support: using standard phosphoramidite coupling chemistry and modified nucleoside analogs, e.g., 5'-(4- methoxytrityl) ammo-5 ' -deoxy-tnymidme phospnora idite , can be used between the PNA and the 5' end of DNA (Mag et al . , Nucl . Acids Res . 17=5973-5988, 1989). PNA monomers are then coupled m a stepwise manner to produce a chimeric molecule with a 5' PNA segment and a 3' DNA segment (Finn et al . , supra) . Alternatively, chimeric molecules can be synthesized with a 5' DNA segment and a 3' PNA segment (Peterser et al . , Bioorganic Med . Chem . Lett . 2=1119-11124 (1975).

In other embodiments, the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo) , or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al . , Proc . Natl . Acad . Sci . USA 22=6553-6556 (1989); Lemaitre et al . , Proc . Natl . Acad . Sci . USA 21=648-652 (1987); PCT Publication No.

WO 88/09810, published December 15, 1988) or the blood- brain barrier (see, e.g., PCT Publication No. WO 89/10134, published April 25, 1988). In addition, oligonucleotides can be modified with hybndization- triggered cleavage agents (see, e.g., Krol et al . ,

BioTech . 2=958-976 (1988)) or integrating agents (see, e.g., Zon, Pharm . Res . 2=539-549 (1988)). To this end, the oligonucleotide may be conjugated to another molecule, for example, a peptide, hybridization triggered corss-lmk g agent, transport agent, hybridization- triggered cleavage agent etc.

VIII. Proteins that Associate with Hfz-4 or Mfz-9

The invention also features methods for identifying polypeptides that can associate with Hfz-4 or Mfz- 9, as well as the isolated interacting protein. Any method that is suitable for detecting protem-prote interactions can be employed to detect polypeptides that associate with Hfz-4 or Mfz-9, whether these polypeptides associate with the transme brane , intracellular, or extracellular domains of Hfz-4 or Mfz-9. Among the traditional methods that can be employed are co-immuno- precipitation, crosslmkmg, and co-puπfication through gradients or chromatographic columns of cell lysates or proteins obtained from cell lysates and the use of Hfz-4 or Mfz-9 to identify proteins m the lysate that interact with Hfz-4 or Mfz-9. For these assays, the Hfz-4 or Mfz- 9 polypetide can be a full length Hfz-4 or Mfz- 9, an extracellular domain of Hfz-4 or Mfz-9, or some other suitable Hfz-4 or Mfz-9 polypeptide. Once isolated, such an interacting protein can be identified and cloned and then used, conjunction with standard techniques, to alter the activity of the Hfz-4 or Mfz-9 polypeptide with which it interacts. For example, at least a portion of the am o acid sequence of a protein that interacts with Hfz-4 or Mfz- 9 can be ascertained using techniques well known to those of skill m the art, such as via the Edman degradation tecnnique. The ammo acid sequence obtained can be used as a guide for the generation of oligonucleotide mixtures that can be used to screen for gene sequences encoding the interacting protein. Screening can be accomplished, for example, by standard hybridization or PCR techniques. Techniques for the generation of oligonucleotide mixtures and the screening are well-known (Ausubel, supra ; and "PCR Protocols: A Guide to Methoαs and Applications," Innis et al . , eds. Academic Press, Inc., NY, 1990).

Additionally, methods can be employed that result directly m the identification of genes that encode proteins that interact with Hfz-4 or Mfz- 9. These methods include, for example, screening expression libraries, in a manner similar to the well known technique of antibody probing of λgtll libraries, using labeled Hfz-4 or Mfz-9 polypeptide or a Hfz-4 or Mfz-9 fusion protein, for example, a Hfz-4 or Mfz-9 polypeptide or domain fuseα to a marker such as an enzyme, fluorescent dye, a luminescent protein, or to an IgFc domain. There are also methods available that can detect protem-prote interaction in vivo . A method which detects protein interactions απ vivo is the two-hybrid system (Chien et ai . , Proc . Na tl . Acad . Sci . USA 88 : 9578 , 1991) . A kit for practicing this method is available from Clontech (Palo Alto, CA) .

Briefly, utilizing such a system, plasmids are constructed that encode two nybπd proteins : one plasmid includes a nucleotide sequence encoding the DNA-bmdmg domain of a transcription activator protein fused to a nucleotide sequence encoding Hfz-4 or Mfz-9, a Hfz-4 or Mfz-9 polypeptide, or a Hfz-4 or Mfz-9 fusion protein, and the other plasmid includes a nucleotide sequence encoding the transcription activator protein' s activation domain fused to a cDNA encoding an unknown protein which has been recombined into this plasmid as part of a cDNA library. The DNA-bmdmg domain fusion plasmid and the cDNA library are transformed into a strain of the yeast Saccharomyces cerevisiae that contains a reporter gene (e.g., HBS or LacZ) wnose regulatory region contains the transcription activator's binding site. Either nybπd protein alone cannot activate transcription of the reporter gene: the DNA-bmdmg domain hybrid cannot oecause it does not provide activation function, and the activation domain hybrid cannot because it cannot localize to the activator's binding sites. Interaction of the two hybrid proteins reconstitutes the functional activator protein and results m expression of the reporter gene, which is detected by an assay for the reporter gene product.

The two-hybrid system or related methodology can be used to screen activation domain libraries for proteins that interact with the "bait" gene product. By way of example, and not by way of limitation, Hfz-4 or Mfz-9 may be used as the bait gene product. Total genomic or cDNA sequences are fused to the DNA encoding an activation domain. This library and a plasmid encoding a hybrid of bait Hfz-4 or Mfz-9 gene product fused to the DNA-bmdmg domain are co-transformed into a yeast reporter strain, and the resulting transformants are screened for those that express the reporter gene. For example, a bait hfz- 4 or mfz- 9 gene sequence, such as that encoding Hfz-4 or Mfz-9 or a domain of Hfz-4 or Mfz-9 can be cloned into a vector such that it is translationally fused to the DNA encoding the DNA-bmdmg domain of the GAL4 protein. These colonies are purified and the library plasmids responsible for reporter gene expression are isolated. DNA sequencing is then used to identify the proteins encoded by the library plasmids.

A cDNA library of the cell line from which proteins that interact with bait hfz-4 or mfz-9 gene product are to be detected can be made using methods routinely practiced the art. Accordmg to the particular system described herein, for example, the cDNA fragments can be inserted into a vector such that they are translationally fused to the transcπptional activation domain of GAL4. This library can be co-transformed along with the bait hfz-4 or mfz- 9 gene-GAL4 fusion plasmid into a yeast strain wnich contains a lacZ gene driven by a promoter which contains GAL4 activation sequence. A cDNA encoded protein, fused to GAL4 transcπptional activation domain, that interacts with bait hfz-4 or mfz-9 gene product will reconstitute an active GAL4 protein and thereby drive expression of the HIS3 gene. Colonies that express HIS3 can then be purified from these strains and used to produce and isolate the bait hfz-4 or mfz-9 gene- interacting protein using techniques routinely practiced m the art .

IX. Detection of Hfz-4 or Mfz-9 and Nucleic Acid Molecules Encoding Hfz-4 or Mfz-9 and Related Diagnostic Assays The invention encompasses methods for detecting the presence of Hfz-4 or Mfz-9 protein or nucleic acid m a biological sample as well as methods for measuring the level of Hfz-4 or Mfz-9 protein or nucleic acid m a biological sample. Such methods are useful for diagnosis of disorders associated with aberrant expression of hfz-4 or mfz- 9. An exemplary method for detecting the presence or absence of Hfz-4 or Mfz- 9 a biological sample involves obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting a Hfz-4 or Mfz- 9 polypeptide or a hfz-4 or mfz- 9 nucleic acid (e.g., mRNA or genomic DNA) . A preferred agent for detecting hfz-4 or mfz-9 mRNA or genomic DNA is a labeled nucleic acid probe capable of hybridizing to hfz-4 or mfz-9 mRNA or genomic DNA. The nucleic acid probe can be, for example, a full- length hfz-4 or mfz- 9 nucleic acid molecule, such as a nucleic acid molecule having the sequence of SEQ ID NO : 1 or SEQ ID NO : 2 , or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250, or 500 nucleotides m length and sufficient to specifically hybridize under stringent conditions to hfz-4 or mfz-9 mRNA or genomic DNA.

A preferred agent for detecting a Hfz-4 or Mfz-9 polypeptide is an antibody capaole of binding to an Hfz- 4 or Mfz- 9 polypeptide, preferably an antibody with a detectable label. Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab')₂) can be used. The term "labeled," with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotm such that it can be detected with fluorescently labeled streptavidin. The term "biological sample" s intended to include tissues, cells, and biological fluids isolated from a subject, as well as tissues, cells and fluids present withm a sup ect. That is, the detection method of the mvention can be used to detect hfz-4 or mfz- 9 mRNA, a

Hfz-4 or Mfz-9 polypeptide, or hfz-4 or mfz-9 genomic DNA in a biological sample απ vitro as well as απ vαvo. For example, m vi tro techniques for detection of hfz-4 or mfz- 9 mRNA include Northern hybridizations and in si tu hybridizations. In vi tro techniques for detection of a Hfz-4 or Mfz-9 polypeptide include enzyme linked immunosoroent assays (ELISAs) , Western blots, lmmunoprecipitations and immunofluorescence. In vi tro tecnniques for detection of hfz-4 or mfz-9 genomic DNA include Southern hybridizations. Furthermore, in vivo techniques for detection of a Hfz-4 or Mfz-9 polypeptide include introducing into a subject a labeled antι-hfz-4 or antι-mfz-9 antibody. For example, the antibody can be labeled with a radioactive marker whose presence and location m a subject can be detected by standard imaging techniques .

In one embodiment, the biological sample contains protem molecules from the test supject. Alternatively, the biological sample can contain mRNA molecules from the test subject or genomic DNA molecules from the test subj ect .

In another embodiment, the methods further involve obtaining a control biological sample from a control subject, contacting the control sample with a compound or agent capaDle of detecting a Hfz-4 or Mfz-9 polypeptide, hfz-4 or mfz-9 mRNA, or hfz-4 or mfz-9 genomic DNA, such that the presence of a Hfz-4 or Mfz-9 polypeptide, hfz-4 or mfz-9 mRNA, or hfz-4 or mfz- 9 genomic DNA is detected in the biological sample, and comparing the presence of Hfz-4 or Mfz-9 polypeptide, hfz-4 or mfz-9 mRNA, or genomic DNA in the control sample with the presence of Hfz-4 or Mfz-9 polypeptides, mRNA or genomic DNA a test sample.

The invention also encompasses Kits for detecting the presence of hfz-4 or mfz-9 nucleic acid molecules or 5 Hfz-4 or Mfz- 9 polypeptides m a biological sample. For example, the kit can contain a labeled compound or agent capable of detecting a Hfz-4 or Mfz-9 polypeptide or a hfz-4 or mfz- 9 mRNA molecule m a biological sample; means for determining the amount of Hfz-4 or Mfz- 9 m the lo sample; and means for comparing the amount of Hfz-4 or Mfz- 9 in the sample with a standard. The compound or agent can be packaged a suitable container. The kit can further contain instructions for using the kit to detect a Hfz-4 or Mfz-9 polypeptide or hfz-4 or mfz-9

_^5 nucleic acid molecule.

X. Prognostic Assays

The invention also encompasses prognostic assays that can be used to identify subjects having or at risk of developing a disease or disorder associated with

20 aberrant hfz-4 or mfz-9 expression or Hfz-4 or Mfz-9 activity. Thus, the present invention provides methods m which a test sample is obtained from a subject and the level, or presence, or allelic form hfz-4 or mfz-9 nucleic acid molecules or Hfz-4 or Mfz-9 polypeptides la

25 assessed. As used herein, a "test sample" refers to a biological sample obtained from a subject of interest. For example, a test sample can be a biological fluid (e.g., serumj , a cell sample, or tissue.

Furthermore, the prognostic assays described herein

30 can be used to determine whether a subject can be administered an agent (e.g., an agonist, antagonist, peptidomimetic, polypeptide, nucleic acid, small molecule or other drug candidate) to treat a disease or disorder associated with aberrant hfz-4 or tnfz-9 expression or

J5 Hfz-4 or Mfz-9 activity. For example, such methods can be used to determine wnether a subiect can be effectively treated with an agent that modulates hfz-4 or mfz- 9 expression and/or activity. Thus, the present invention provides methods for determining whether a supject can be effectively treated with an agent for a disorder associated with aoerrant hfz-4 or mfz-9 expression or Hfz-4 or Mfz-9 activity which a test sample is obtained and hfz-4 or mfz- 9 nucleic acids or Hfz-4 or Mfz-9 polypeptides are detected (e.g., wherein the presence of a particular level of Hfz-4 or Mfz-9 expression or a particular Hfz-4 or Mfz-9 allelic variant is diagnostic for a subject that can be administered an agent to treat a disorder associated with aberrant hfz-4 or mfz-9 expression or Hfz-4 or Mfz-9 activity) .

The methods of the mvention can also Pe used to detect genetic alterations a hfz-4 or mfz-9. In preferred embodiments, the methods include detecting, in a sample of cells from the subject, the presence or absence of a genetic alteration characterized by at least one alteration affecting the integrity of the gene encoding a Hfz-4 or Mfz- 9 polypeptide or the misexpression of the hfz-4 or mfz-9 gene. For example, such genetic alterations can be detected by ascertaining the existence of at least one of: (1) a deletion of one or more nucleotides from a hfz-4 or mfz-9 gene; (2) an addition of one or more nucleotides to a hfz-4 or mfz-9 gene; (3) a substitution of one or more nucleotides of a hfz-4 or mfz- 9 gene; (4) a chromosomal rearrangement of a hfz-4 or mfz-9 gene; (5) an alteration in the level of a messenger RNA transcript of a hfz-4 or mfz- 9 gene ; (6) aberrant modification of a hfz-4 or mfz- 9 gene, such as of the methylation pattern of the genomic DNA, (7) the presence of a non-wild type splicing pattern of a messenger RNA transcript of a hfz-4 or mfz-9 gene; and (10) inappropriate post-translational modification of a Hfz-4 or Mfz-9 polypeptide. As described herein, there are a large numper cf assay techniques known m the art which can be used for detecting alterations m a hfz-4 or mfz- 9 gene.

In certain embodiments, detection of the alteration involves the use of a probe/primer a polymerase chain reaction (PCR; see, e.g., U.S. Patent Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or alternatively, a ligation chain reaction (LCR; see, e.g., Landegran et al . , Sci ence 241:1077-1080, 1988; and Nakazawa et al . Proc . Na tl . Acad . Sci . USA 91 :360-364 , 1994), the latter of which can be particularly useful for detecting point mutations the hfz-4 or mfz- 9 gene (see Abavaya et al . , Nucl . Acids Res . 22=675-681, 1995). This method can include the steps of collecting a sample of cells from a patient, isolating nucleic acid (e.g., genomic DNA, mRNA, or both; from the cells of the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to a hfz-4 or mfz-9 gene under conditions such that hybridization and amplification of the hfz-4 or mfz-9 nucleic acid (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. It is anticipated that PCR and/or LCR may be desirable to use as a preliminary amplification step conjunction with any of the techniques used for detecting mutations described herein.

Alternative amplification methods include: self sustained sequence replication (Guatelli et al . , Proc . Natl . Acad . Sci USA 22 = 1874-1878, 1990), transcπptional amplification system (Kwoh et al . , Proc . Natl . Acad . Sci USA 26=1173-1177, 1989), Q-Beta Replicase (Lizardi et al . , Bio /Tecnnology 6_ : 1197 , 1988), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of ordinary skill m the art . These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present m very low number.

In an alternative embodiment, alterations in a hfz-4 or mfz- 9 gene from a sample cell can oe identified by identifying changes in a restriction enzyme cleavage pattern. For example, sample and control DNA is isolated, amplified (optionally) , digested with one or more restriction endonucleases , and fragment length sizes are determined by gel electrophoresis and compared. Differences in fragment length sizes between sample and control DNA indicates mutations m the sample DNA. Moreover, the use of sequence specific ribozymes (see, e.g., U.S. Patent No. 5,498,531) can oe used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site.

In other embodiments, alterations m hfz-4 or mfz-9 can be identified by hybridizing a sample and control nucleic acids, e.g., DNA or RNA, to high density arrays containing tens to thousands of oligonucleotide probes (Cromn et al . , Human Mutation 2=244-255, 1996); Kozal et al . , Na ture Medicine 2=753-759, 1996). For example, alterations hfz-4 or mfz-9 can be identified m two dimensional arrays containing light -generated DNA probes as described Cromn et al . , supra . Briefly, a first hybridization array of probes can be used to scan through long stretches of DNA m a sample and control to identify base changes between the sequences by making linear arrays of sequential overlapping probes. This step allows the identification of point mutations. This step is followed by a second hybridization array that allows the characterization of specific mutations by using smaller, specialized probe arrays complementary to all variants or mutations detected. Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene . In yet another embodiment, any of a variety of sequencing reactions known the art can be used to directly sequence the hfz-4 or mfz- 9 gene and detect mutations by comparing the sequence of the sample hfz-4 or mfz-9 with the corresponding wild-type (control) sequence. Examples of sequencing reactions include those based on techniques developed by Maxim and Gilbert (Proc. Na tl . Acad . Sci . USA 21=560 (1977)) or Sanger ( Proc . Natl . Acad . Sci . USA 21=5463) . It is also contemplated that any of a variety of automated sequencing procedures can be utilized when performing the diagnostic assays (Bio/Techniques 12=448, 1995) including sequencing by mass spectrometry (see, e . g . PCT International Publication No. WO 94/16101; Cohen et ai . Adv. Chro atogr. 22=127-162 1996; and Griff ec al . , Appl . Biochem . Biotechnol . 22=147-159, 1993) .

Other methods of detecting mutations m the hfz-4 or mfz- 9 gene include methods which protection from cleavage agents is used to detect mismatched bases m RNA/RNA or RNA/DNA heteroduplexes (Myers et al . Science 230 : 1242 1985) . In general, the art technique of "mismatch cleavage" starts by providing heteroduplexes formed by hybridizing (labeled) RNA or DNA containing the wild-type nfz-4 or mfz- 9 sequence with potentially mutant RNA or DNA obtained from a tissue sample. The double- stranded duplexes are treated with an agent which cleaves single-stranded regions of the duplex such as which will exist due to basepair mismatches between the control and smaple strands. For instance, RNA/DNA duplexes can be treated with RNase and DNA/DNA hybrids treated with SI nuclease to enzymatically digest the mismatched regions. In other embodiments, either DNA/DNA or RNA/DNA duplexes can be treated with hydroxvlamme or osmium tetroxide and with pipeπdme order to digest mismatched regions. After digestion of the mismatcned regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine the site of mutation. (see, for example, Cotton et al . , Proc . Natl . Acad. Sci . USA 22=4397 1988; Saleeba et al . , Methods Enzymol . 212=286-295 1992. In a preferred embodiment, the control DNA or RNA can be labeled for detection. 5 In still another embodiment, tne mismatcn cleavage reaction employs one or more proteins that recognize mismatched base pairs m double-stranded DNA so called "DNA mismatcn repair" enzymes) defined systems for detecting and mapping point mutations in hfz-4 or mfz-9 lo cDNAs obtained from samples of cells. For example, the mutY enzyme of E. coli cleaves A at G/A mismatches (Hsu et al . , Carcmogenesis 12=1657-1662 1994. Accordmg to an exemplary erαoodiment , a probe pased on a hfz-4 or mfz- 9 sequence is hybridized to a cDNA or other DNA product

_5 from a test cell or cells. The duplex is treated with a DNA mismatcn repair enzyme, and the cleavage products, if any, can be detected from electrophoresis protocols or the like. See, for example, U.S. Patent No. 5,459,039.

In other embodiments, alterations in electrophoretic

20 mobility can be used to identify mutations m hfz-4 or mfz- 9 genes. For example, single strand conformation polymorphism (SSCP) can be used to detect differences m electropnoretic mopility between mutant and wild type nucleic acids (Orita et al . , Proc . Natl . Acad . Sci . USA

25 22=2766, see also Cotton utat Res . 285 : 125-144 1993; and Hayashi Genet. Anal . Tech . Appl . 2=73-79 1992. Single- stranded DNA fragments of sample and control hfz-4 or mfz- 9 nucleic acids will be denatured and allowed to renature . The secondary structure of single-stranded

30 nucleic acids varies accordmg to sequence, the resulting alteration m electrophoretic mobility enables the detection of even a single base change. The DNA fragments may be labeled or detected with labeled probes. The sensitivity of the assay may be enhanced by using RNA

_5 (rather than DNA) , in wnich the secondary structure is more sensitive to a change sequence. In a preferred empodiment , tne τetnod utilizes neteroduoiex analysis to separate double stranded heteroduplex molecules on the basis of changes electrophoretic mobility (Kee et al . , Trends Genev . 2=5 1991.

In yet another empodiment , the movement of mutant or wild-type fragments m a polyacrylamide gel containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE; Myers et al . , Nature 212=495 1985. When DGGE is used as the method of analysis, DNA will be modified to insure that it does not completely denture, for example by adding a GC clamp of approximately 40 bp of high-meltmg GC-πch DNA by PCR. In a further embodiment, a temperature gradient is used in place of a denaturing gradient to identify differences m the mobility of control and sample DNA (Rosenoaum et al . Biophys . Chem . 265 :12753 , 1987.

Examples of other techniques for detecting point mutations include, but are not limited to, selective oligonucleotide hybridization, selective amplification, or selective primer extension. For example, oligonucleotide primers may be prepared m which the known mutation is placed centrally and then hybridized to target DNA under conditions which permit hybridization only if a perfect match is found (Saiki et al . Nature 324; 163 (1986); Saiki et al . , Proc . NAtl . Acad . Sci . USA 22=6230 (1989)). Such allele specific oligonucleotides are hybridized to PCR amplified target DNA or a number of different mutations when the oligonucleotides are attached to the hybridizing membrane and hybridized with labeled target DNA. Alternatively, allele specific amplification technology which depends on selective PCR amplification may be used conjunction with the instant invention. Oligonucleotides used as primers for specific amplification may carry the mutation of interest m the center of the molecule, so that amplification depends on differential hybridization (Gibbs et al . , Nucl . Acids Res . 17:2437-2448 (1989)) or at the extreme 3' end of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (Prossner, Tib/Tech ϋ:238 (1993)) . In addition it may be desirable to introduce a novel restriction site m the region of the mutation to create cleavage-based detection

(Gasparmi et al . , Mol . Cell Probes 2=1 (1992)). It is anticipated that m certain embodiments amplification may also be performed using Taq ligase for amplification (Barany, Proc . Natl . Acad. Sci . USA 22=89 (1991)). In such cases, ligation will occur only if there is a perfect match at the 3' end of the 5' sequence making it possible to detect the presence of a known mutation at a specific site by looking for the presence of absence of amplification. The methods described herein may be performed, for example, by utilizing pre-packaged diagnostic kits comprising at least one probe nucleic acid or antibody reagent described herein, which may be conveniently used, for example, in a clinical setting to diagnose patient exhibiting symptoms or a family history of a disease or disorder involving abnormal Hfz-4 or Mfz- 9 activity.

XI. Pharmacogenetics

Agents or modulators which have a stimulatory or inhibitory effect on Hfz-4 or Mfz-9 activity (including those that alter activity by altering hfz-4 or mfz-9 gene expression) , identified by a screening assay described herein, can be administered to individuals to treat, prophylactically or therapeutically, disorders associated with aberrant Hfz-4 or Mfz-9 activity. In conjunction with such treatment, the pharmacogenetics (i.e., the study of the relationship between an individual's genotype and that individual ' s response to a foreign compound or drug) of the individual may be considered. Thus, the pharmacogenetics of the individual permits tne selection of effective agents (e.g., drugs) for prophylactic or therapeutic treatments based on a consideration of the individual's genotype. Such pharmacogenetics can further be used to determine appropriate dosages and therapeutic regimens. Accordingly, the activity of Hfz-4 or Mfz-9 polypeptides, expression of hfz-4 or mfz-9 nucleic acids, or sequence of hfz-4 or mfz-9 genes an individual can be determined and used to thereby select an appropriate agent for therapeutic or prophylactic treatment of the individual . Pharmacogenetics deals with clinically significant hereditary variations the response to drugs due to altered drug disposition and abnormal action m affected persons (See, e.g., Eichelbaum, Clm . Exp . Pharmacol . Physiol . 22=983-985 1996 and L der, Clm . Chem . 43 :254- 266 1997) . In general, two types of pnarmacogenetic conditions can be differentiated. Genetic conditions transmitted as single factors altering the way drugs act on the body (altered drug action) or genetic conditions transmitted as single factors altering the way the body acts on drugs (altered drug metabolism) . These pharmacogenetic conditions can occur either as rare defects or as polymorphisms. For example, glucose- 6- phosphate dehydrogenase deficiency ^fG6PD) is a common inherited enzymopathy which the mam clinical complication is haemolysis after mgestion of oxidant drugs (anti-malaπals, sulfonamides , analgesics, nitrofurans) and consumption of fava beans.

As an illustrative embodiment, the activity of drug metabolizing enzymes is a major determinant of both the intensity and duration of drug action. The discovery of genetic polymorphisms of drug metabolizing enzymes (e.g. N-acetyltransferase (NAT2) and cytocnrome P450 enzymes CYP2D6 and CYP2C19) has provided an explanation as to why some patients do not obtain the expected drug effects or show exaggerated drug response and serious toxicity after taking the standard and safe dose of a drug. These polymorpnisms are expressed in two pnenotypes the population, the excessive metabolizer (EM) and poor metabolizer (PM) . The prevalence of PM is different among different populations. For example, the gene coding for CYP2D6 is highly polymorphic and several mutations have been identified m PM, which ail lead to the absence of functional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quite frequently experience exaggerated drug response and side effects when they receive standard doses. If a metabolite is the active therapeutic moiety, PM show no therapeutic response, as demonstrated for the analgesic effect of codeine mediated by its CYP2D6- formed metabolite morphine. The other extreme is the so called ultra-rapid metabolizers who do not respond to standard doses. Recently, the molecular basis of ultra-rapid metabolism has been identified to be due to CYP2D6 gene amplification.

Thus, the activity of Hfz-4 or Mfz-9 polypeptide, expression of hfz-4 or mfz-9 nucleic acid, or mutation content of a hfz-4 or mfz- 9 gene in an individual can be determined and used to select an appropriate agent for therapeutic or propnylactic treatment of the individual. In addition, pharmacogenetic studies can be used to apply genotyp g of polymorphic alleles encoding drug- metaboliz g enzymes to the identification of an individual's drug responsiveness phenotype. This knowledge, when applied to dosing or drug selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficiency when treating a subject with a Hfz-4 or Mfz-9 modulator, such as a modulator identified by one of the exemplary screening assays described herein.

XII. Monitoring of Clinical Trials

Monitoring tne influence of agents (e.g., drugs, compounds) on the expression of hfz-4 or mfz-9 or the activity of Hfz-4 or Mfz-9 can be applied not only basic drug screening, out also m clinical trials. For example, the effectiveness of an agent determined by a screening assay as described herein to increase hfz-4 or mfz-9 gene expression, increase Hfz-4 or Mfz-9 polypeptide levels, or upregulate Hfz-4 or Mfz- 9 activity, can be monitored m clinical trials of subjects exhibiting decreased hfz-4 or mfz-9 gene expression, decreased Hfz-4 or Mfz-9 polypeptide levels, or downregulated Hfz-4 or Mfz- 9 activity. Alternatively, the effectiveness of an agent determined by a screening assay to decrease hfz-4 or mfz-9 gene expression, decrease Hfz-4 or Mfz-9 polypeptide levels, or downregulate Hfz-4 or Mfz-9 activity, can be monitored m clinical trials of subjects exhibiting increased hfz-4 or mfz-9 gene expression, increased Hfz-4 or Mfz-9 polypeptide levels, or upregulated Hfz-4 or Mfz-9 activity. In such clinical trials, the expression of hfz-4 or mfz-9 or activity of Hfz-4 or Mfz-9 can be used as a measure of the responsiveness of a particular cell. For example, and not by way of limitation, genes, including hfz-4 or mfz- 9, that are modulated m cells by treatment with an agent (e.g., a compound, drug, or small molecule) that modulates Hfz-4 or Mfz-9 activity (e.g., identified m a screening assay as described herein) can be identified. Thus, to study the effect of agents on a given disorder, for example, in a clinical trial, the level or expression of hfz-4 or mfz-9 or other genes implicated the disorder can be measured. The levels of gene expression (i.e., a gene expression pattern) can be quantified by Northern blot analysis or RT-PCR, as described herein, or alternatively by measuring the amount of polypeptide produced, by one of the methods described herein, or by measuring the levels of activity of Hfz-4 or Mfz- 9 or other genes. In this way, the gene expression pattern can serve as an indicative marker of the physiological response of the cells to the agent. Accordingly, this response state can be determined before, and at various points during, treatment of the individual with the agent .

In a preferred embodiment, the present invention provides a method for monitoring the effectiveness of treatment of a subject with an agent (e.g., an agonist, antagonist, peptidomimetic, polypeptide, nucleic acid, small molecule, or other drug candidate identified by the screening assays described herein) comprising the steps of (1) obtaining a pre-admmistration sample from a subject prior to administration of the agent; (2) detecting the level of expression of a Hfz-4 or Mfz-9 polypeptide or hfz-4 or mfz-9 mRNA m the pre- admmistration sample, or the level or activity of Hfz-4 or Mfz-9; (3) obtaining one or more post-admmistration samples from the subject; (4) detecting the level of expression of Hfz-4 or Mfz-9 polypeptide or hfz-4 or mfz- 9 mRNA or the level or activity of the Hfz-4 or Mfz- 9 polypeptide in the post-admmistration sample; (5) comparing the level of expression of hfz-4 or mfz- 9 mRNA in the pre-admmistration sample with that in the post- adm istration sample, or comparing the level or activity of the Hfz-4 or Mfz-9 polypeptide m the pre- administration sample with that m the post- admmistration sample; and (6) altering the administration of the agent to the subject accordingly.

XIII . Screening Assays for Compounds that Modulate hfz-4 or mfz- 9 Expression or Activity

The invention also encompasses methods for identifying compounds that interact with Hfz-4 or Mfz- 9 (or a domain of Hfz-4 or Mfz-9) including, but not limited to, compounds that interfere with the interaction of Hfz-4 or Mfz-9 with transmemprane, extracellular, or intracellular proteins which regulate Hfz-4 or Mfz-9 activity and compounds which modulate Hfz-4 or Mfz-9 activity. Also encompasses are method for identifying compounds vnicn bind to hfz-4 or mfz- 9 gene regulatory sequences (e.g., promoter sequences) and wnich may modulate hfz-4 or mfz- 9 gene expression.

The compounds whicn may be screened accordance with the invention include, put are not limited to 5 peptides, antibodies and fragments thereof, and other organic compounds that bind to Hfz-4 or Mfz- 9 and increase or decrease activity.

Such compounds may include, but are not limited to, peptides such as, for example, soluble peptides, o including but not limited to members of random peptide libraries; (Lam et al . , Nature 354 : 82-84 , 1991; Hougnten et al . , Nature 354 : 84-86 , 1991), and combinatorial chemistry-derived molecular library made of D- and/or L configuration ammo acids, pnospnopeptides (including,

.5 but not limited to, members of random or partially degenerate, directed pnospnopeptide libraries; Songyang, et al., Cell 12:767-778, 1993), antibodies (including, but not limited to, polyclonal, monoclonal, humanized, anti-idiotypic , chimeric or single chain antibodies, and

20 FAb, F(ab')₂ and FAb expression library fragments, and epitope-b dmg fragments thereof) , and small organic or inorganic molecules.

Other compounds wnich can be screened in accordance with the invention include but are not limited to small

25 organic molecules that are able to gam entry into an appropriate cell and affect the expression of the hfz-4 or mfz-9 gene or activity of Hfz-4 or Mfz-9 protein.

Computer modelling and searching technologies permit identification of compounds, or the improvement of

30 already identified compounds, that can modulate hfz-4 or mfz-9 expression or activity. Having identified such a compound or composition, the active sites or regions are identified. Such active sites might typically be a binding for a natural modulator of activity. The active

_5 site can be identified using methods known m the art including, for example, from the am o acid sequences of peptides, from tne nucleotide sequences of nucleic aciαs, or from study of complexes of the relevant compound or composition with its natural ligand. In the latter case, chemical or X-ray crystallographic methods can be used to find the active site by finding where on the factor the modulator (or 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 mtra-molecular 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 modulator ⁽"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 modelling can be used to complete the structure or improve its accuracy. Any recognized modelling method may be used, including parameterized models specific to particular iopolymers 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 searcmng 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 Hfz-4 or Mfz- 9 modulating compounds.

Alternatively, these methods can be used to identify improved modulating compounds from a previously identified 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 modelling methods described aoove applied to tne 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 composition, such as by varying side groups, can be quickly evaluated to obtain modified modulating compounds or ligands of improved specificity or activity.

Examples of molecular modelling systems are the CHARMm and QUANTA programs (Polygen Corporation, altham, MA) . CHARMm performs the energy minimization and molecular dynamics functions. QUANTA performs the construction, graphic modelling and analysis of molecular structure. QUANTA allows interactive construction, modification, visualization, and analysis of the behavior of molecules with eacn other. A numoer of articles review computer modelling of drugs interactive with specific proteins, such as Rotivmen et al . , Acta Pharmaceutical Fennica 97 : 159-166 , 1993; Ripka, New Scαeπtαst 54-57 (June 16, 1988); McK aly and Rossmann, Annu . Rev. Pharmacol . Toxiciol . 22=111-122, 1989; Perry and Davies, OSAR: Quantitative Structure-Activity Relationships Drug Design, pp. 189-193 (Alan R. Liss, Inc. 1989); Lewis and Dean, 1989 Proc. R. Soc . Lond. 236:125-140 and 141-162, 1980; and, with respect to a model receptor for nucleic acid components, Askew et al . , J^". Am. Chem . Soc . 111:1082- 1090, 1989. Other computer programs that screen and graphically depict chemicals are available from companies such as BioDesign, Inc. (Pasadena, CA. ) , Allelix, Inc. (Mississauga, Ontario, Canada), and Hypercube, Inc. (Cambridge, Ontario) . Although these are primarily designed for application to drugs specific to particular proteins, they can be adapted to design of drugs specific to regions of DNA or RNA, once that region is identified.

Although described above with reference to design and generation of compounds which could alter binding, one could also screen libraries of known compounds, including natural products or synthetic chemicals, and biologically active materials, including proteins, for compounds which are inhibitors or activators of Hfz-4 or Mfz-9 activity

Compounds identified via assays such as those described herein may be useful, for example, in elaborating the biological function of hfz-4 or mfz-9 and for the treatment of disorders associated with aberrant Hfz-4 or Mfz-9 activity or expression. Assays for testing the eff ctiveness of compounds identified with the above-described techniques are discussed below. In vi tro systems may be designed to identify compounds capable of interacting with Hfz-4 or Mfz- 9 (or a domain of Hfz-4 or Mfz-9) . Compounds identified may be useful, for example, modulating the activity of wild type and/or mutant Hfz-4 or Mfz- 9; may be useful m elaborating the biological function Hfz-4 or Mfz- 9; may be utilized m screens for identifying compounds that disrupt normal Hfz-4 or Mfz- 9 interactions; or may m themselves disrupt such interactions. The principle of the assays used to identify compounds that bind to Hfz-4 or Mfz- 9 involves preparing a reaction mixture of Hfz-4 or Mfz-9 (or a domain thereof) and the test compound under conditions and for a time sufficient to allow the two components to interact and bind, thus forming a complex which can be removed and/or detected the reaction mixture. The Hfz-4 or Mfz-9 species used can vary depending upon the goal of the screening assay. In some situations it is preferable to employ a peptide corresponding to a domain of Hfz-4 or Mfz- 9 fused to a heterologous protein or polypeptide that affords advantages m the assay system (e.g., labeling, isolation of the resulting complex, etc.) can be utilized.

The screening assays can be conducted m a variety of ways. For example, one method to conduct sucn an assay involves anchoring Hfz-4 or Mfz-9 protein, polypeptide, peptide or fusion protein or the test substance onto a solid phase and detecting Hfz-4 or Mfz- 9/test compound complexes anchored on the solid phase at the end of the reaction. In one embodiment of such a method, the Hfz-4 or Mfz-9 reactant may be anchored onto a solid surface, and the test compound, wnich is not anchored, may be labeled, either directly or indirectly.

In practice, microtiter plates may conveniently be utilized as the solid phase. The anchored component may be immobilized by non-covalent or covalent attachments. Non-covalent attachment may be accomplished by simply coating the solid surface with a solution of the protein and drying. Alternatively, an immobilized antibody, preferably a monoclonal antibody, specific for the protein to be immobilized may be used to anchor the protein to the solid surface. The surfaces may be prepared m advance and stored.

In order to conduct the assay, the nonimmoPilized component is added to the coated surface containing the anchored component. After the reaction is complete, unreacted components are removed (e.g., by washing; under conditions sucn tnat any complexes formed will remain lmmobilized on the solid surface. The detection of complexes anchored on the solid surface can be accomplished m a number of ways. Where the previously non- immobilized component is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the previously non- lmmobilized component is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the previously non- immobilized component (the antibody, in turn, may be directly labeled or indirectly labeled with a labeled anti-Ig antibody) .

Alternatively, a reaction can be conducted in a liquid phase, the reaction products separated from unreacted components, and complexes detected; e.g., using an immopilized antibody specific for Hfz-4 or Mfz-9 protein, polypeptide, peptide or fusion protein or the test compound to anchor any complexes formed m solution, and a labeled antibody specific for the other component of the possible complex to detect anchored complexes. Alternatively, cell-based assays can be used to identify compounds that interact with Hfz-4 or Mfz-9. To this end, cell lines that express Hfz-4 or Mfz-9, or cell lines that have been genetically engineered to express Hfz-4 or Mfz-9 can be used.

XIV. Assays for Compounds that Interfere with the

Interaction Between Hfz-4 or Mfz-9 and a Protein Binding Partner

Proteins that interact with the Hfz-4 or Mfz-9 are referred to, for purposes of this discussion, as "binding partners" . Such binding partners can be involved m regulating Hfz-4 or Mfz-9 activity. Therefore, it is desirable to identify compounds that interfere with or disrupt the interaction of such binding partners with Hfz-4 or Mfz-9. Such compounds may be useful m regulating the activity of the Hfz-4 or Mfz-9 and treatmg disorders associated with aberrant Hfz-4 or Mfz- 9 activity.

The basic principle of the assay systems used to identify compounds that interfere with the interaction between the Hfz-4 or Mfz- 9 and binding partner or partners involves preparing a reaction mixture containing Hfz-4 or Mfz-9 protein, polypeptide, peptide or fusion protein and the binding partner under conditions and for a time sufficient to allow the two to interact and bind, thus forming a complex. In order to test a compound for inhibitory activity, the reaction mixture is prepared m the presence and absence of the test compound. The test compound may oe initially included the reaction mixture, or may be added at a time supsequent to the addition of the Hfz-4 or Mfz-9 moiety and its binding partner. Control reaction mixtures are incubated without the test compound or with a non-active control compound. The formation of any complexes between the Hfz-4 or Mfz-9 moiety and the binding partner is then detected. The formation of a complex in the control reaction, but not m the reaction mixture containing the test compound, indicates that the compound interferes with the interaction of Hfz-4 or Mfz-9 and the interactive binding partner. Additionally, complex formation withm reaction mixtures containing the test compound and normal Hfz-4 or Mfz- 9 protein may also be compared to complex formation with reaction mixtures containing the test compound and a mutant Hfz-4 or Mfz-9. This comparison may be important in those cases wherein it is desirable to identify compounds that disrupt interactions of mutant but not normal Hfz-4 or Mfz- 9.

The assay for compounds that interfere with the interaction of the Hfz-4 or Mfz-9 and a binding partner can be conducted m a heterogeneous or homogeneous format . Heterogeneous assays involve anchoring either the hfz-4 or mfz-9 protein, polypeptide, peptide, or fusion protein, or the binding partner onto a solid pnase and detecting complexes anchored on the solid phase at the end of the reaction. In homogeneous assays, the entire reaction is carried out m a liquid phase. In either approach, the order of addition of reactants can be varied to obtain different information about the compounds being tested. For example, test compounds that interfere with the interaction by competition can be identified by conducting the reaction m the presence of the test substance; i.e., by adding the test substance to the reaction mixture prior to or simultaneously with the Hfz-4 or Mfz-9 moiety and interactive binding partner. Alternatively, test compounds that disrupt preformed complexes, e.g., compounds with higher binding constants that displace one of the components from the complex, can be tested by adding the test compound to the reaction mixture after complexes have been formed. The various formats are described briefly below.

In a heterogeneous assay system, either the Hfz-4 or Mfz- 9 moiety or the interactive binding partner, is anchored onto a solid surface, while the non-anchored species is labeled, either directly or indirectly. In practice, microtiter plates are conveniently utilized. The anchored species may be immobilized by non-covalent or covalent attachments. Non-covalent attachment may be accomplished simply by coating the solid surface with a solution of Hfz-4 or Mfz-9 (or a domain thereof) or binding partner and drying. Alternatively, an immobilized antibody specific for the species to be anchored may be used to anchor the species to the solid surface. The surfaces may be prepared in advance and stored.

In order to conduct the assay, the partner of the immobilized species is exposed to the coated surface with or without the test compound. After the reaction is complete, mαreacted components are removed (e.g., by washing) and any complexes formed will remain immobilized on the solid surface The detection of complexes anchored on the solid surface can oe accomplished m a number of ways. Where the non-immobilized species is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the non-immobilized species is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface, e.g., using a directly or indirectly labeled antibody specific for the initially non- immobilized species. Depending upon the order of addition of reaction components, test compounds which inhibit complex formation or which disrupt preformed complexes can be detected.

Alternatively, the reaction can be conducted m a liquid phase m the presence or absence of the test compound, the reaction products separated from unreacted components, and complexes detected, e.g., using an immobilized antibody specific for one of the binding components to anchor any complexes formed n solution, and a labeled antibody specific for the other partner to detect anchored complexes. Again, depending upon the order of addition of reactants to the liquid phase, test compounds which inhibit complex or which disrupt preformed complexes can be identified.

In an alternate emoodiment cf the invention, a homogeneous assay can be used. In this approach, a preformed complex of the Hfz-4 or Mfz-9 moiety and the interactive binding partner is prepared m which either the Hfz-4 or Mfz-9 or its binding partners is labeled, but the signal generated by the label is quenched due to formation of the complex (see, e.g., U.S. Patent

No. 4,109,496 by Rubenstem which utilizes this approach for immunoassays) . The addition of a test substance that competes with and displaces one of the species from the preformed complex will result m the generation of a signal above background. In this way, test substances which disrupt Hfz-4 or Mfz-9/mtracellular binding partner interaction can be identified. In a particular embodiment, a Hfz-4 or Mfz- 9 fusion can be prepared for immobilization. For example, the Hfz-4 or Mfz- 9 or a peptide fragment thereof can be fused to a glutathione-S-transferase (GST) gene using a fusion Ξ vector, such as pGEX-5X-l, such a manner that its binding activity is maintained m the resulting fusion protein. The interactive binding partner can be purified and used to raise a monoclonal antibody, using methods routinely practiced in the art. This antibody can be

10 labeled with the radioactive isotope ¹²⁵I, for example, by methods routinely practiced m the art. In a heterogeneous assay, the GST-Hfz-4 or Mfz-9 fusion protein can pe anchored to glutathione-agarose beads. The interactive binding partner can then be added the

-5 presence or absence of the test compound m a manner that allows interaction and binding to occur. At the end of the reaction period, unbound material can be washed away, and the labeled monoclonal antibody can be added to the system and allowed to bind to the complexed components.

20 The interaction between Hfz-4 or Mfz-9 and the interactive binding partner can be detected by measuring the amount of radioactivity that remains associated with the glutathione-agarose beads. A successful inhibition of the interaction by the test compound will result m a

25 decrease in measured radioactivity.

Alternatively, the GST-Hfz-4 or Mfz-9 fusion protein and the interactive binding partner can be mixed together in liquid m the absence of the solid glutathione-agarose beads . The test compound can be added either during or

30 after the species are allowed to interact. This mixture can then be added to the glutathione-agarose beads and unbound material is wasned away. Again the extent of inhibition of the Hfz-4 or Mfz-9/bmdmg partner interaction can oe detected by adding the labeled

35 antibody and measuring the radioactivity associated with the beads . In another embodiment of the invention, these same techniques can be employed using peptide fragments that correspond to the binding domains of Hfz-4 or Mfz- 9 and/or the interactive or binding partner (in cases where the binding partner is a protein) , m place of one or both of the full length proteins. Any number of methods routinely practiced m the art can be used to identify and isolate the binding sites. These methods include, but are not limited to, mutagenesis of the gene encoding one of the proteins and screening for disruption of binding m a co-immunoprecipitation assay. Compensating mutations m the gene encoding the second species m the complex can then be selected. Sequence analysis of the genes encoding the respective proteins will reveal the mutations that correspond to the region of the protein involved interactive binding. Alternatively, one protein can be anchored to a solid surface using methods described above, and allowed to interact with and bind to its labeled binding partner, which has been treated with a proteolytic enzyme, such as trypsm. After washing, a short, labeled peptide comprising the binding domain may remain associated with the solid material, which can be isolated and identified by ammo acid sequencing. Also, once the gene coding for the intracellular binding partner is obtained, short gene segments can be engineered to express peptide fragments of the protein, which can then be tested for binding activity and purified or synthesized.

XV. Methods for Reducing hfz-4 or mfz -9 Expression Expression of hfz-4 or mfz- 9 can be reduced through the use of modulatory compounds identified througn the use of the screening methods described above. In addition, endogenous hfz-4 or mfz-9 gene expression can also oe reduced by inactivating or "knocKmg out" the hfz-4 or mfz- 9 gene or its promoter using targeted homologous recombination (see, for example, U.S. Patent No . 5,464,764) . For example, a mutant, non- functional hfz-4 or mfz- 9 (or a completely unrelated DNA sequence) flanked by DNA homologous to the endogenous hfz-4 or mfz- 9 gene (either the coding regions or regulatory regions of the hfz-4 or mfz-9 gene) can be used, with or without a selectable marker and/or a negative selectable marker, to transfect cells that express hfz-4 or mfz-9-3 in vivo . Insertion of the DNA construct, via targeted homologous recombination, results inactivation of the hfz-4 or mfz-9 gene. Such approaches are particularly suited for use in developing animal models to study the role of Hfz- 4 or Mfz-9; in this instance, modifications to ES (embryonic stem) cells can be used to generate animal offspring with an inactive hfz-4 or mfz-9 gene. However, a knock out approach can be adapted for use m humans, provided the recombinant DNA constructs are directly administered or targeted to the required site in vivo using appropriate viral vectors.

Alternatively, endogenous hfz-4 or mfz- 9 gene expression can be reduced by targeting deoxyribonucleotide sequences complementary to the regulatory region of the hfz-4 or mfz-9 gene (i.e., the hfz-4 or mfz-9 promoter and/or enhancers) to form triple helical structures that prevent transcription of the hfz- 4 or mfz-9 gene m target cells m the body (Helene, Aπticaπcer Drug Res . 2 = 569, 1981; Helene et al . , Ann. N. Y. Acad . Sci . 660 :27 , 1992; and Maher, Bioassays 14:807, 1992) .

In addition, as discussed above, anti-sense molecules, ribozymes, and peptide nucleic acids can be used to reduce hfz-4 or mfz- 9 expression. XVI. Assays for the Identification of Compounds that Ameliorate Disorders Associated with Aberrant hfz-4 or mfz- 9 Expression or Activity

Compounds, including, but not limited to, compounds identified via assay techniques such as those described above may be useful for the treatment of disorders associated with aberrant hfz-4 or mfz-9 expression or aberrant Hfz-4 or Mfz-9 activity.

While animal model -based assays are particularly useful for the identification of such therapeutic compounds, cell -based assay systems are also very useful, particularly m combination with animal -model based assays. Such cell -based systems can include, for example, recombinant or non-recomomant cells which express hfz-4 or mfz-9. The effect of a selected modulatory compound on hfz-4 or mfz- 9 expression can be measured using any of the above-described techniques for measuring Hfz-4 or Mfz-9 protein or hfz-4 or mfz-9 mRNA.

XVII. Effective Dose Toxicity and therapeutic efficacy of the polypeptides of the invention and the compounds that modulate their expression or activity can be determined by standard pharmaceutical procedures, using either cells in culture or experimental animals to determine the LD₅₀ (the dose lethal to 50% of the population) and the ED₅₀ (the dose therapeutically effective in 50% of the population) . The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD_S0/ED₅₀. Polypeptides or other compounds that exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue m order to minimize potential damage to unmfected cells and, thereby, reduce side effects. The data obtained from the cell culture assays and animal studies can Pe used m formulating a range of dosage for use m humans . The dosage of such compounds lies preferably withm a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The αosage may vary withm this range depending upon the dosage form employed and the route of administration utilized. For any compound used m the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated m animal models to achieve a circulating plasma concentration range that includes the IC₅₀ (that is, the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined m cell culture. Such information can be used to more accurately determine useful doses m humans . Levels m plasma may be measured, for example, by high performance liquid chromatography.

XVIII . Formulations and Use

Pharmaceutical compositions for use m accordance with the present invention can be formulated in a conventional manner using one or more physiologically acceptable carriers or excipients. Thus, the compounds and their pnysiologically acceptable salts and solvates may be formulated for administration by inhalation or insufflation (either through the mouth or the nose) or oral, buccal , parenteral or rectal administration. For oral administration, the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatmised maize starch, polyvmylpyrrolidone , or hydroxypropyl methylcellulose) ; fillers (e.g., lactose, microcrystallme cellulose, or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc, or silica); dismtegrants (e.g., potato starch or sodium starch glycolate) ; or wetting agents (e.g., sodium lauryl sulphate) . The tablets may be coated by methods well known m the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid) . The preparations may also contain buffer salts, flavoring, coloring and sweetening agents as appropriate.

Preparations for oral administration may be suitably formulated to give controlled release of the active compound .

For ouccal administration the compositions may take the form of tablets or lozenges formulated m conventional manner.

For administration by inhalation, the compounds for use accordmg to the present invention are conveniently delivered m the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, for example, dichlorodifluoromethane , tπchlorofluoromethane , dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, for example, gelatin for use m an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch. The compounds may be formulated for parenteral administration by injection, for example, by bolus injection or continuous infusion. Formulations for inj ection may be presented in unit dosage form, for example, in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient can be in powder form for constitution with a suitable vehicle, for example, sterile pyrogen-free water, before use. The compounds can also be formulated in rectal compositions such as suppositories or retention enemas, for example, containing conventional suppository bases such as cocoa butter or other glycerides .

In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (e.g., subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt .

The compositions can, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack can, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device can be accompanied by instructions for administration. The therapeutic compositions of the invention can also contain a carrier or excipient, many of which are known to persons of ordinary skill in the art. Excipients that can be used include buffers (e.g., citrate buffer, phosphate buffer, acetate buffer, and bicarbonate buffer), amino acids, urea, alcohols, ascorbic acid, phospholipids , proteins (e.g., serum albumin) , EDTA, sodium chloride, liposomes, mannitol, sorbitol, and glycerol .

The nucleic acids, polypeptides, antibodies, or other modulatory compounds of the invention ( i . e . , compounds that alter the expression of hfz-4 or mfz-9 or the activity of Hfz-4 or Mfz-9) can be administered by any standard route of administration. For example, administration can be parenteral , intravenous, subcutaneous, intramuscular, mtracranial , mtraorbital, opthalmic, mtraventπcular, mtracapsular, mtraspmal, mtracisternal, mtrapeπtoneal , transmucosal , or oral. The modulatory compound can be formulated in various ways, according to the corresponding route of administration. For example, liquid solutions can be made for mgestion or injection; gels or powders can be made for mgestion, inhalation, or topical application. Methods for making such formulations are well known and can be found in, for example, "Remington's Pharmaceutical Sciences." It is expected that the preferred route of administration will be intravenous.

XXI. Deposit Statement

The clones described herein as have been deposited with the American Type Culture Collection and assigned accession numbers , respectively. The above-noted cultures have been deposited under conditions that assure that access to the cultures will be available during the pendency of the patent application to one determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37 CFR 1.14 and 35 U.S.C. 122. The deposits are available as required by foreign patent laws m countries wherein counterparts of the subject application, or its progeny, are filed. However, t should be understood that the availability of a deposit does not constitute a license to practice the subject invention derogation of patent rights granted by governmental action. Further, the subject culture deposits will be stored and made available to the public in accord with the provisions of the Budapest Treaty for the Deposit of Microorganisms, i.e., they will be stored with all the care necessary to keep them viable and uncontaminated for a period of at least five years after the most recent request for the furnishing of a sample of the deposits, and in any case, for a period of at least 30 (thirty) years after the date of deposit or for the enforceable life of any patent which may issue disclosing the cultures plus five years after the last request for a sample from a deposit. The depositor acknowledges the duty to replace the deposits should the depository be unable to furnish a sample when requested, due to the condition of the deposits. All restrictions on the availability to the public of the subject culture deposits will be irrevocably removed upon the granting of a patent disclosing them.

Claims

What is claimed is:

1. An isolated nucleic acid molecule comprising a sequence encoding a Hfz-4 polypeptide having a sequence that is at least 60% identical the sequence encoded by SEQ ID NO:l.

2. The isolated nucleic acid molecule of claim 1, said Hfz-4 polypeptide having the sequence encoded by SEQ ID NO:l.

3. An isolated nucleic acid molecule which hybridizes under stringent conditions to a nucleic acid molecule having sequence of SEQ ID NO:l or the complement thereof .

4. An isolated nucleic acid molecule comprising the sequence of SEQ ID NO:l.

5. A nucleic acid vector comprising the nucleic acid molecule of claim 1.

6. A host cell comprising the vector of claim 5.

7. The nucleic acid vector of claim 5, wherein said vector is an expression vector.

8. A substantially pure polypeptide comprising a Hfz-4 polypeptide that has a sequence that is at least 60% identical to the sequence encoded by SEQ ID NO:l.

9. The polypeptide of claim 8, wherein said polypeptide comprises the sequence encoded by SEQ ID NO:l.

10. The polypeptide of claim 8, further comprising a detectable marker.

11. An antibody that selectively binds to a polypeptide having the sequence encoded by SEQ ID NO:l.

12. A method for detecting hfz-4 nucleic acid molecules in a biological sample, comprising:

(a) contacting said sample with a nucleic acid probe under conditions that allow said nucleic acid probe to hybridize with hfz-4 nucleic acid molecules; and

(b) detecting said hybrids, if any, as an indication of the presence of hfz-4 nucleic acid molecules in said sample .

13. A method for diagnosing a patient as having a disorder associated with aberrant expression of hfz-4, comprising measuring hfz-4 expression in a biological sample obtained from said patient, wherein increased or decreased hfz-4 expression in said biological sample, compared with hfz-4 or expression in a control sample, indicates that said patient has a disorder associated with aberrant expression of hfz-4.