EP1824506A2 - Use of organic compounds - Google Patents

Abstract

The invention relates to DEFESIN 5 polypeptides for the manufacture of a medicament for use in the treatment of a disease or condition associated with iron imbalance or iron transport.

Description

Use of Organic Compounds

Related Applications

This application claims priority to US 60/634,349 filed December 8, 2004, which is herein incorporated by reference in its entirety.

Field of the Invention

The invention relates to the medical use of Defensin 5(DEF5), DEF5 fragments, DEF5 homologs and/or DEF5 variants, in particular for the manufacture of a medicament for the treatment of diseases or conditions associated with iron balance or iron transport.

Background

Defensins (DEF) are a large family of broad-spectrum antimicrobial peptides, identified originally in leukocytes of rabbits and humans. Defensins are classified into two families designated alpha- and beta-defensins - based on distinctive, although similar, tri-disulfide linkages in the peptides. Defensins are cationic/polar peptides (30-35 aa; 3-4 kDa), of which beta-defensins are slightly larger. The genes encoding human alpha and beta - defensins are clustered in a contiguous segment of chromosome 8p23.

In humans, six alpha-defensin and two beta-defensins have been identified. Alpha- defensins are encoded by genes designated DEFA1-6, whereas human beta-defensins are encoded by the DEFB1 and DEFB2 genes. Defensins are synthesized as larger precursor molecules with a putative amino-terminal signal sequence, followed by a middle segment, and the bioactive antimicrobial peptide resides in the final carboxy terminal sequence of the precursor.

Defensins effect a broad spectrum of antibiotic activity, primarily by disrupting microbial cell membranes. Defensins are expressed by phagocytic leukocytes and by various epithelial cells, including Paneth cells.

Human alpha 5 defensin is also known as defensin 5 alpha, alpha defensin 5 precursor or defensin 5 is synthesized as a 94 amino acid precursor (HDEFA5; HNP-5; DEFA5, DEF5, DEF5(1-94)). Recently it has been shown that DEF5 precursor / DEF5 propeptide is efficiently processed by trypsin in Paneth cells to yield various DEF5 forms (Ghosh et al., Nature Immunology 2002, 3(6), 583 - 590). Summary of the invention

Surprisingly, it has now been found that polypeptides relating to DEF5(1-94) affect key genes controlling iron balance or iron transport.

The present invention thus provides the use of a polypeptide for the manufacture of a medicament for use in the treatment of a disease or condition associated with iron balance or iron transport, wherein the polypeptide is selected from the groups consisting of a) DEF5(1-94) (SEQ. ID No: 1) or a fragment of DEF5(1-94); b) a bioactive polypeptide having a percentage of identity of at least 50% with the amino acid sequence of any one of the polypeptides of (a); or c) a bioactive variant of any one of the polypeptides of (a) or (b). In a further aspect, the present invention relates to a method for the treatment of a disease or condition associated with iron balance or iron transport comprising administering an effective amount of a polypeptide as defined above to a mammal including a human suffering from the disease or condition.

In another aspect, the present invention relates to a pharmaceutical composition for use in a disease or condition associated with iron balance or iron transport comprising an effective amount of a polypeptide as defined above and a pharmaceutically-acceptable carrier.

Detailed description of the invention The present invention provides for the use of a polypeptide for the manufacture of a medicament for use in the treatment of a disease or condition associated with iron balance or iron transport, wherein the polypeptide is selected from the groups consisting of a) DEF5(1-94) (SEQ. ID No: 1) or a fragment of DEF5(1-94); b) a bioactive polypeptide having a percentage of identity of at least 50% with the amino acid sequence of any one of the polypeptides of (a); or c) a bioactive variant of any one of the polypeptides of (a) or (b).

The term "polypeptide" as used herein, refers to a protein, peptide, oligopeptide or synthetic oligopeptide. These terms are intended to be used interchangeably. Any one of said terms refers to a chain of two or more amino acids which are linked together with peptide or amide bonds, regardless of post-translational modification such as glycosylation or phosphorylation. The polypeptides may also comprise more than one subunit, where each subunit is encoded by a separate DNA sequence. The polypeptide according to the invention may comprise DEF5(1-94) having the amino acid sequence of SEQ ID NO: 1. A polypeptide of the invention also includes a polypeptide fragment of a DEF5(1-94) polypeptide of the invention. Such polypeptide fragment is meant to be a polypeptide having an amino acid sequence that entirely is the same in part, but not in all, of the amino acid sequence of a polypeptide of the invention. Such polypeptide fragment may be "free-standing," or may be part of a larger polypeptide of which such polypeptide fragment forms a part or region, most preferably as a single continuous region. Preferably such polypeptide fragment retains at least one biological activity of the corresponding polypeptide DEF5(1-94). A fragment of DEF5(1-94) may comprise at least 10 amino acids, preferably at least

15, 20, or 25 amino acids. More preferably a fragment of DEF5(1-94) comprises at least 30 amino acids. Another preferred fragment of DEF5(1-94) comprises at least 40, 50, 60, 65, 70 or 75 amino acids . Most preferably the DEF5(1-94) fragment comprises 12, 22, 32, 35, 39, 66, 72 or 75 consecutively amino acids of SEQ ID NO: 1. Such polypeptide may also be a fragment of DEF5(1-94) such as a proteolytic cleavage product of DEF5(1-94) e.g. generated by proteases such as for example by trypsin. A polypeptide or a polypeptide fragment according to the invention may comprise a C-terminal fragment of DEF5(1-94)..Such C-terminal fragment may comprise at least 10 amino acids of the C-terminus of DEF5(1-94), preferably at least 20 or 25, even more preferred at least 30 amino acids or at least 65, 70 or 75 amino acids. The at least 10 amino acids may comprise the most C-terminal at least 10 amino acids, it may also comprise the at least 10 amino acids within the C-terminal part of DEF5(1-94) such as for example DEF5(76- 87) (SEQ. ID No: 9). The polypeptide may comprise the at least 32, 35, 39, 66, 72 or 75 most C-terminal amino acids of DEF5(1-94) and preferably it may have the amino acid sequence of DEF5(20-94) (SEQ. ID No: 2), DEF5(23-94) (SEQ. ID No: 3), DEF5(29-94) (SEQ. ID No: 4), DEF5(56-94) (SEQ. ID No: 5), DEF5(60-94) (SEQ. ID No: 6) or DEF5(63- 94) (SEQ. ID No: 7). Preferably the polypeptide is a DEF5(1-94) fragment comprising DEF5(20-94), most preferably it comprises DEF5(60-94). A fragment of DEF5(1-94) may also comprise an internal fragment of DEF5(1-94), such as for example DEF5(34-55) (SEQ. ID No: 8).

The term "bioactive", as used herein, refers to a molecule that elicits or affects a biological event. Such biological event may for example be related to a disease or condition associated with iron balance or iron transport. In a preferred embodiment, the level of biological activity of DEF5(1-94) or a fragment thereof is measured by detecting the level of expression of one or more genes set forth in Table 1. Preferably, the expression of more than 10, 20, 40 or of the majority of genes of Table 1 is determined.

A "bioactive polypeptide" of the invention includes DEF5(1-94), and fragments of DEF5(1-94) such as the DEF5(20-94), DEF5(23-94) or DEF5(29-94), DEF5(56-94), DEF5(60-94), DEF5(63-94), DEF5(34-55) or DEF5(76-87).

Also included are homologs which have an amino acid sequence having a percentage of identity of at least 50% to DEF5(1-94) or fragments thereof and variants of DEF5(1-94) or of DEF5(1-94) fragments. The polypeptide may also have an amino acid sequence having a percentage of identity of at least 50%, preferably at least 60%, more preferred at least 70% or 80%, and most preferably at least 90% such as 95%, 97%, or 99% identity with the amino acid sequence of any one of the aforementioned polypeptides such as DEF5(1-94) or fragments thereof such as for example such as the DEF5(20-94), DEF5(23-94) or DEF5(29-94), DEF5(56-94), DEF5(60-94), DEF5(63-94), DEF5(34-55) or DEF5(76-87).

Amino acid residues are referred to herein by their standard single-letter or three-letter notations: A (Ala) alanine; C (Cys) cysteine; D (Asp) aspartic acid; E (GIu) glutamic acid; F (Phe) phenylalanine; G (GIy) glycine; H (His) histidine; I (lie) isoleucine; K (Lys) lysine; L (Leu) leucine; M (Met) methionine; N (Asn) asparagine; P (Pro) proline; Q (GIn) glutamine; R (Arg) arginine; S (Ser) serine; T (Thr) threonine; V (VaI) valine; W (Trp) tryptophan; Y (Tyr) tyrosine.

The term "percentage (%) of identity", or like term, used in respect of the comparison of a reference sequence and another sequence (i.e. a "candidate" sequence), means that in an optimal alignment between the two sequences, the candidate sequence is identical to the reference sequence in a number of subunit positions equivalent to the indicated percentage, the subunits being nucleotides for polynucleotide comparisons or amino acids for polypeptide comparisons. As used herein, an "optimal alignment" of sequences being compared is one that maximizes matches between subunits and minimizes the number of gaps employed in constructing an alignment. Percent identities may be determined with commercially available implementations of algorithms described by Needleman and Wunsch, J. MoI. Biol. 48: 443-453 (197O)("GAP" program of Wisconsin Sequence Analysis Package, Genetics Computer Group, Madison, Wl). Other software packages in the art for constructing alignments and calculating percentage identity or other measures of similarity include the "BestFit" program, based on the algorithm of Smith and Waterman, Advances in Applied Mathematics 2: 482-489 (1981) (Wisconsin Sequence Analysis Package, Genetics Computer Group, Madison, Wl). The percentage of identity may also be generated by WU- BLAST-2 (Altschul et al., Methods in Enzymology 266: 460-480 (1996)). WU-BLAST-2 used several search parameters, most of which are set to the default values. The adjustable parameters are set with the following values: overlap span = 1 , overlap fraction = 0.125, word threshold (T) = 11. A % amino acid sequence identity value is determined by the number of matching identical residues divided by the total number of residues in the aligned region. For example, to obtain a polypeptide having an amino acid sequence at least 95% identical to a reference amino acid sequence, up to five percent of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to five percent of the total amino acid residues in the reference sequence may be inserted into the reference sequence. These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence of in one or more contiguous groups with in the references sequence. It is understood that in making comparisons with reference sequences of the invention that candidate sequence may be a component or segment of a larger polypeptide or polynucleotide and that such comparisons for the purpose computing percentage identity is to be carried out with respect to the relevant component or segment.

The invention also includes functionally preserved variants of the polypeptides or polypeptide fragments described herein. Such variants may be made using methods standard in the art, for example, by conservative amino acid substitutions. Typically such substitutions are among Ala, VaI, Leu and lie; among Ser and Thr; among the acidic residues Asp and GIu; among Asn and GIn; and among the basic residues Lys and Arg; or aromatic residues Phe and Tyr. Particularly preferred are variants in which several, 5 to 10, 1 to 5, or 2 amino acids are substituted, deleted or added, in any combination.

In various other embodiments, the polypeptide or fragment thereof or polypeptide variant or homolog may be linear or branched, it may comprise modified amino acids, it may be interrupted by non-amino acids, and/or it may be assembled into a complex of more than one polypeptide chain. As is well understood in the art, a polypeptide may be modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. In some embodiments, polypeptides or polypeptide fragments contain one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. A polypeptide or a polypeptide fragment of the invention includes isolated naturally occurring polypeptides. Preferably, such a naturally occurring polypeptide has a frequency in a selected population of at least five percent, and most preferably, of at least ten percent. The selected population may be any recognized population of study in the field of population genetics. Preferably, the selected population is Caucasian, Negroid, or Asian. More preferably, the selected population is French, German, English, Spanish, Swiss, Japanese, Chinese, Korean, Singaporean of Chinese ancestry, Icelandic, North American, Israeli, Arab, Turkish, Greek, Italian, Polish, Pacific Islander, or Indian.

A polypeptide or fragment thereof of the invention may also include recombinantly produced polypeptides, synthetically produced polypeptides and a combination of such polypeptides of the invention, and fragments thereof. Means for preparing such polypeptides are well understood in the art. For instance, a polynucleotide fragment or a polypeptide of the invention can be isolated from body fluids including, but not limited to, serum, urine, and ascites, or synthesized by chemical or biological methods (for example, cell culture, recombinant gene expression). "Isolated", if not otherwise specified herein includes the meaning "separated from coexisting material".

Recombinant polypeptides of the present invention may be prepared by processes well known in the art from genetically engineered host cells comprising expression systems. Accordingly, in a further aspect, the present invention relates to the production of polypeptides by recombinant techniques, to expression systems which comprise a nucleic acid or nucleic acids encoding the polypeptides of the present invention, to host cells which are genetically engineered with such expression systems, and to methods to isolate the polypeptides.

Another embodiment provides that a polypeptide of the invention is encoded by a nucleic acid which hybridizes under stringent conditions to nucleotide sequence of SEQ. ID No: 10. In some embodiments, the nucleic acid comprises at least 30, 40, 50, 65, or at least 95, 100, 105, 110, 115, 120, 130, 140, or at least 150, 160, 170, 180, 195, 210, 215, 220, 225, or at least 250, 260, 270, 280 or at least 285 nucleotides. The nucleic acid may also comprise at least 300, or at least 400 or at least 500 nucleotides. Preferably the nucleic acid comprises at least 36, 66, 96, 105, 117, 198, 216, 225 or 282 nucleotides. Such nucleic acids may comprise at least 36, 66, 96, 105, 117, 198, 216, 225 or 282 contiguous nucleotides of SEQ. ID No: 10 or nucleotides able to hybridize to SEQ. ID No: 10 under stringent conditions, and most preferably encoding DEF5(1-94), DEF5(20-94), DEF5(23-94), DEF5(29-94), DEF5(56-94), DEF5(60-94), DEF5(63-94), DEF5(34-55) or DEF5(76-87) or variants or homologs thereof.

The term "nucleic acid" means natural or semi-synthetic or synthetic or modified nucleic acid molecules. It refers to nucleotide sequences, oligonucleotides or polynucleotides including deoxyribonucleic acid (DNA) and/or ribonucleic acid (RNA) and/or modified nucleotides. These terms are intended to be used interchangeably. RNA may be in the form of an tRNA (transfer RNA), snRNA (small nuclear RNA), rRNA (ribosomal RNA), mRNA (messenger RNA), anti-sense RNA, and ribozymes. DNA may be in form of plasmid DNA, viral DNA, linear DNA, chromosomal or genomic DNA, cDNA, or derivatives of these groups. In addition these DNAs and RNAs may be single, double, triple, or quadruple stranded. The term also includes PNAs (peptide nucleic acids), phosphorothioates, and other variants of the phosphate backbone of native nucleic acids.

"Stringent conditions" of hybridization reactions is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation dependent upon probe length, washing temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures. Hybridization generally depends upon the ability of a denatured nucleic acid to reanneai when complementary strands are present in an environment near but below their melting temperature. The higher the degree of homology between the probe and the hybridizable sequence such as SEQ. ID No: 3 or 4, the higher the relative temperature which can be used. As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so. Moreover, stringency is also inversely proportional to salt concentrations. "Stringent conditions" are exemplified by reaction conditions characterized by: (1) low ionic strength and high temperature for washing, for example 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50°C; (2) the use of a denaturing agent, such as formamide, for example, 50% (vol/vol) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42°C. Alternatively, stringent conditions can be: 50% formamide, 5x SSC (0.75 M NaCI, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5x Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42°C, with washes at 42⁰C in 0.2x SSC (sodium chloride/sodium citrate) and 50% formamide at 55⁰C, followed by a high-stringency wash consisting of 0.1 x SSC containing EDTA at 55°C. For additional details and explanation of stringency of hybridization reactions, see Ausubel et al., Protocols in Molecular Biology (1995).

Recombinant Manufacture ofDEF5(1-94) and DEF5(1-94) Fragments The nucleic acids described herein above such as SEQ. ID No: 10 may be used in recombinant DNA molecules to direct the expression of the corresponding polypeptides in appropriate host cells. Because of the degeneracy in the genetic code, other DNA sequences may encode the equivalent amino acid sequence, and may be used to clone and express DEF5(1-94) or fragments thereof. Codons preferred by a particular host cell may be selected and substituted into the naturally occurring nucleotide sequences, to increase the rate and/or efficiency of expression. The nucleic acid (e.g., cDNA or genomic DNA) encoding the desired DEF5(1-94) or DEF5(1-94) fragments such as DEF5(20-94), DEF5(23-94), DEF5(29-94), DEF5(56-94), DEF5(60-94), DEF5(63-94), DEF5(34-55) or DEF5(76-87) or variants or homologs thereof may be inserted into a replicable vector for cloning (amplification of the DNA), and/or for expression.

Expression Systems

The polypeptide can be expressed recombinantly in any of a number of expression systems according to methods known in the art (Ausubel, et al., editors, Current Protocols in Molecular Biology, John Wiley Sons, New York, 1990). Such expression systems include chromosomal, episomal and virus-derived systems, e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids.

The expression systems may contain control regions that regulate as well as engender expression. Generally, any system or vector which is able to maintain, propagate or express a nucleic acid to produce a polypeptide in a host may be used. The appropriate nucleotide sequence may be inserted into an expression system by any of a variety of well-known and routine techniques, such as, for example, those set forth in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. (1989). In general, DNA is inserted into an appropriate restriction endonuclease site using techniques known in the art.

Vector components generally include, but are not limited to, one or more of an origin of replication, one or more marker genes, an enhancer element, a promoter, a signal or secretion sequence, and a transcription termination sequence: The expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example in mammalian or insect cells for expression and in a prokaryotic host for cloning and amplification. Such sequences are well known for a variety of bacteria, yeast strains, and viruses.

Preferably, the expression vector contains a marker gene to allow the selection of transformed host cells. Selection genes are well known in the art and will vary with the host cell used. Expression and cloning vectors will typically contain a selection gene, also termed a selectable marker. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients e.g., the D-alanine racemase gene.

Promoter sequences encode either constitutive or inducible promoters. The promoters may be either naturally occurring promoters or hybrid promoters. Hybrid promoters, which combine elements of more than one promoter, are also known in the art, and are useful in the present invention. Further, for integrating expression vectors, the expression vector contains at least one sequence homologous to the host cell genome, and preferably, two homologous sequences which flank the expression construct. The integrating vector may be directed to a specific locus in the host cell by insertion of the appropriate homologous sequence in the vector. Constructs for integrating vectors are well known in the art.

An appropriate secretion signal may be incorporated into the desired polypeptide to allow secretion of the polypeptide into the lumen of the endoplasmic reticulum, the periplasmic space or the extracellular environment. These signals may be endogenous to the polypeptide or they may be heterologous signals. The signal sequence may be a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin Il leaders. For yeast secretion the signal sequence may be, e.g., the yeast invertase leader, the alpha factor leader (including Saccharomyces and Kluyveromyces a-factor leaders). In mammalian cell expression systems, mammalian signal sequences from secreted polypeptides of the same or related species, as well as viral secretory leaders may be used to direct secretion of DEF5(1-94) or DEF5(1-94) fragments such as DEF5(20-94), DEF5(23-94), DEF5(29-94), DEF5(56-94), DEF5(60-94), DEF5(63-94), DEF5(34-55) or DEF5(76-87), variants or homologs thereof. Appropriate host cells include yeast, bacteria, archebacteria, fungi, and insect and animal cells, including mammalian cells, for example primary cells, including but not limited to stem cells. Representative examples of appropriate hosts include bacterial cells, such as E coli, Streptococci, Staphylococci, Streptomyces, and Bacillus subtilis; fungal cells, such as Saccharomyces cerevisiae, other yeast cells or Aspergillus; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293 and Bowes melanoma cells; and plant cells.

A host cell strain may be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed polypeptide in the desired fashion. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation. Post-translational processing, which cleaves a "prepro" form of the polypeptide, may also be important for correct insertion, folding and/or function.

DEF5(1-94) or DEF5(1-94) fragments such as DEF5(20-94), DEF5(23-94), DEF5(29- 94), DEF5(56-94), DEF5(60-94), DEF5(63-94), DEF5(34-55) or DEF5(76-87) may be produced by culturing a host cell transformed with an expression vector containing a nucleic acid encoding DEF5(1-94) or fragments thereof under the appropriate conditions to induce or cause expression of the protein or polypeptide. In a preferred embodiment of the invention a host cell is provided which is stably or transiently transfected with a nucleic acid of SEQ. ID No: 10 or transfected with a nucleic acid which hybridizes under stringent conditions to SEQ. ID No: 10. According to another embodiment of the invention said host cell is cultured to allow expression of DEF5(1-94) or of an DEF5(1-94) fragment, and the polypeptide is isolated from the cell culture.

Transformed host cells include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmic DNA expression vectors, yeast transformed with yeast expression vectors, and insect cells infected with a recombinant insect virus (such as baculovirus), and mammalian expression systems.

The appropriate conditions for expression of DEF5(1-94) or DEF5(1-94) fragments such as for example DEF5(20-94), DEF5(23-94), DEF5(29-94), DEF5(56-94), DEF5(60-94), DEF5(63-94), DEF5(34-55) or DEF5(76-87) will vary with the choice of the expression vector and the host cell, and will be easily ascertained by one skilled in the art through routine experimentation. For example, the use of constitutive promoters in the expression vector will require optimizing the growth and proliferation of the host cell, while the use of an inducible promoter requires the appropriate growth conditions for induction. In addition, in some embodiments, the timing of the harvest is important. For example, the baculoviral systems used together with insect cells are lytic viruses, and thus harvest time selection can be crucial for product yield.

The desired DEF5(1-94) or a DEF5(1-94) fragment may be produced recombinantly not only directly, but also as a fusion polypeptide with a heterologous polypeptide. Such heterologous polypeptide is generally placed at the amino- or carboxyl-terminus of DEF5(1- 94) or of an DEF5(1-94) fragment and may provide for an epitope tag to which an anti-tag antibody can selectively bind. Accordingly, such epitope tag enables DEF5(1-94) or a fragment thereof to be readily purified by using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag. Examples of epitope tags are 6xHis or c-myc tag. Alternatively DEF5(1-94) or a fragment thereof may be expressed in the form of e.g. an GST-fusion protein. Appropriate constructs are generally known in the art and are available from commercial suppliers such as Invitrogen (San Diego, Calif.), Stratagene (La JoIIa, Calif.), Gibco BRL (Rockville, Md.) or Clontech (Palo Alto, Calif.).

Evaluation of Gene Expression Gene expression may be evaluated in a sample directly, for example, by standard techniques known to those of skill in the art, e.g., Southern blotting for DNA detection, Northern blotting to determine the transcription of mRNA, dot blotting (DNA or RNA), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein. Alternatively, antibodies may be used in assays for detection of nucleic acids, such as specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. Such antibodies may be labeled and the assay carried out where the duplex is bound to a surface, so that upon the formation of duplex on the surface, the presence of antibody bound to the duplex can be detected. Gene expression, alternatively, may be measured by immunohistochemical staining of cells or tissue sections and assay of cell culture or body fluids, to directly evaluate the expression of DEF5(1-94) or of an DEF5(1-94) fragment. Antibodies useful for such immunological assays may be either monoclonal or polyclonal, and may be prepared against a native sequence DEF5(1-94) or DEF5(1-94) fragments based on the DNA sequences provided herein.

Purification of Expressed Protein

Expressed DEF5(1-94) or DEF5(1-94) fragments such as foe example DEF5(20-94), DEF5(23-94), DEF5(29-94), DEF5(56-94), DEF5(60-94), DEF5(63-94), DEF5(34-55) or DEF5(76-87) may be purified or isolated after expression, using any of a variety of methods known to those skilled in the art. The appropriate technique will vary depending upon the way of expression of DEF5(1-94) or an DEF5(1-94) fragment. The polypeptide may for example be recovered from culture medium in the form of a secreted potein or from host cell lysates. Cells can be disrupted by various physical or chemical means, such as freeze-thaw cycling, sonication, mechanical disruption, or by use of cell lysing agents, whereas membrane-bound polypeptides may be released from the membrane using a suitable detergent solution (e.g. Triton-X 100) or by enzymatic cleavage. The appropriate technique for polypeptide purification or isolation will also vary depending upon what other components are present in the sample. The degree of purification necessary will also vary depending on the use of DEF5(1-94) or a fragment thereof. Contaminant components that are removed by isolation or purification are materials that would typically interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other solutes. The purification step(s) selected will depend, for example, on the nature of the production process used and the particular DEF5(1-94) or DEF5(1-94) fragment produced. Ordinarily, isolated DEF5(1-94) or a fragment thereof will be prepared by at least one purification step. Well-known methods for purification include ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, high performance liquid chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, affinity chromatography is employed for purification. For example, the

DEF5(1-94) or a fragment thereof such as DEF5(20-94), DEF5(23-94), DEF5(29-94), DEF5(56-94), DEF5(60-94), DEF5(63-94), DEF5(34-55) or DEF5(76-87) may be purified using a standard anti-DEF5 C-terminal polypeptide antibody column. Ultrafiltration and dialysis techniques, in conjunction with protein concentration, are also useful (see, for example, Scopes, R., Protein Purification, Springer-Verlag, New York, N.Y., 1982). Well- known techniques for refolding proteins may be employed to regenerate active conformation when the polypeptide is denatured during isolation and or purification Labeling of Expressed Polypeptide

The nucleic acids, proteins and antibodies of the invention may be labeled. By labeled herein is meant that a compound has at least one element, isotope or chemical compound attached to enable the detection of the compound. In general, labels fall into three classes: a) isotopic labels, which may be radioactive or heavy isotopes; b) immune labels, which may be antibodies or antigens; and c) colored or fluorescent dyes. The labels may be incorporated into the compound at any position that does not interfere with the biological activity or characteristic of the compound which is being detected.

Chemical Manufacture of DEF5(1-94) and DEF5(1-94) Fragments

Polypeptides or fragments thereof may be produced not only by recombinant methods, but also by using chemical methods well known in the art. Solid phase peptide synthesis may be carried out in a batchwise or continuous flow process which sequentially adds alpha- ami no- and side chain-protected amino acid residues to an insoluble polymeric support via a linker group. A linker group such as methylamine-derivatized polyethylene glycol is attached to poly(styrene-co-divinylbenzene) to form the support resin. The amino acid residues are N^alpha-protected by acid labile Boc (t-butyloxycarbonyl) or base-labile Fmoc (9- fluorenylmethoxycarbonyl). The carboxyl group of the protected amino acid is coupled to the amine of the linker group to anchor the residue to the solid phase support resin. Trifluoroacetic acid or piperidine are used to remove the protecting group in the case of Boc or Fmoc, respectively. Each additional amino acid is added to the anchored residue using a coupling agent or pre-activated amino acid derivative, and the resin is washed. The full length peptide is synthesized by sequential deprotection, coupling of derivatized amino acids, and washing with dichloromethane and/or N, N-dimethylformamide. The peptide is cleaved between the peptide carboxy terminus and the linker group to yield a peptide acid or amide. (Novabiochem 1997/98 Catalog and Peptide Synthesis Handbook, San Diego Calif. pp. S1-S20). Automated synthesis may also be carried out on machines such as the ABI 431 A peptide synthesizer (Applied Biosystems). A polypeptide or a fragment thereof may be purified by preparative high performance liquid chromatography and its composition confirmed by amino acid analysis or by sequencing (Creighton T.E. (1984) Proteins, Structures and Molecular Properties, W H Freeman, New York N.Y.).

Variants of the natural polypeptide may be desirable in a variety of circumstances. For example, undesirable side effects might be reduced by certain variants, particularly if the side effect activity is associated with a different part of the polypeptide from that of the desired activity. In some expression systems, the native polypeptide may be susceptible to degradation by proteases. In such cases, selected substitutions and/or deletions of amino acids which change the susceptible sequences can significantly enhance yields. Variants may also increase yields in purification procedures and/or increase shelf lives of proteins by eliminating amino acids susceptible to oxidation, acylation, alkylation, or other chemical modifications. Preferably, such variants include alterations that are conformational^ neutral, i.e. they are designed to produce minimal changes in the tertiary structure of the variant polypeptides as compared to the native polypeptide, and (ii) antigenically neutral, i.e. they are designed to produce minimal changes in the antigenic determinants of the variant polypeptides as compared to the native polypeptide.

The aforementioned polypeptides may according to the invention be used for the manufacture of a medicament for use in the treatment of a disease or condition associated with iron balance or iron transport. A further aspect of the invention provides a method for the treatment of a disease or condition associated with iron balance or iron transport is provided said method comprises administering an effective amount of a polypeptide to a mammal including a human suffering from the disease or condition, wherein the polypeptide is selected from the groups consisting of a) DEF5(1-94) (SEQ. ID No: 1) or a fragment of DEF5(1-94); b) a bioactive polypeptide having a percentage of identity of at least 50% with the amino acid sequence of any one of the polypeptides of (a); or c) a bioactive variant of any one of the polypeptides of (a) or (b). Accordingly, a polypeptide as described above may be administered. The polypeptide preferably comprises DEF5(1-94), or a fragment thereof such as DEF5(20-94), DEF5(23- 94), DEF5(29-94), DEF5(56-94), DEF5(60-94), DEF5(63-94), DEF5(34-55) or DEF5(76-87). "Mammal" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and from animals, and zoo, sports, or pet animals, such as dogs, horses, cats, sheep, pigs, cattle, etc. Preferably, the mammal is human. The term "treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented.

A "disease" or a "condition" is any condition that would benefit from treatment with DEF5(1-94) or a fragment of DEF5(1-94) as defined above and further below. This includes both chronic and acute diseases or conditions, as well as those pathological conditions which predispose to the disease or condition in question.

Non-limiting examples of diseases or conditions to be treated herein include any condition which results from altered iron balance or iron transport in the body, leading to iron overload diseases, anemia or deficient red blood cell production. Examples of diseases or conditions associated with iron balance or iron transport are hemochromatosis, hereditary hemochromatosis, juvenile hemochromatosis, thalassemia, conditions related to iron- overload, anemia, sickle cell anemia.

In another aspect of the invention, DEF5(1-94) or a fragment of DEF5(1-94) is provided as suitable therapeutics for the treatment of a disease or condition associated with iron balance or iron transport comprising administering an effective amount of an DEF5(1- 94) or a fragment thereof to a mammal, including a human, suffering from said disease.

Accordingly, a pharmaceutical composition for use in a disease or condition associated with iron balance or iron transport DEF5(1-94) or a fragment thereof according to the invention as described above and a pharmaceutically-acceptable carrier is provided. The DEF5(1-94) fragment preferably comprises DEF5(20-94), DEF5(23-94), DEF5(29-94), DEF5(56-94), DEF5(60-94), DEF5(63-94), DEF5(34-55) or DEF5(76-87). The composition of the invention is administered in effective amounts.

The pharmaceutical composition may be used in the foregoing methods of treatment. Such compositions are preferably sterile and contain an effective amount of DEF5(1-94) or a fragment thereof or a nucleic acid encoding the polypeptide or fragment for inducing the desired response in a unit of weight or volume suitable for administration to a patient.

An "effective amount" of DEF5(1-94) or fragment thereof, compound, or pharmaceutical composition is an amount sufficient to effect beneficial or desired results including clinical results such as preventing and/or inhibiting-iron overload diseases, anemia or deficient red blood cell production, e.g. preventing and/or inhibiting hemochromatosis, hereditary hemochromatosis, juvenile hemochromatosis, thalassemia, anemia, and/or sickle cell anemia.

Such amounts will also depend on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation.

An effective amount can be administered in one or more administrations and may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition. Thus, an "effective amount" may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if, in conjunction with one or more other agents, a desirable result may be or is achieved. An effective amount of DEF5(1-94) or an DEF5(1-94) fragment or the pharmaceutical composition comprising the polypeptide of the invention, alone or in conjunction with another drug, compound, or pharmaceutical composition can be administered by any conventional route, including injection or by gradual infusion over time. The administration may, for example, be oral, intravenous, intraperitoneal, intramuscular, intracavity, subcutaneous, topical or transdermal.

When administered, the pharmaceutical composition of the present invention is administered in pharmaceutically acceptable preparations. The term "pharmaceutically- acceptable carrier" as used herein means one or more compatible solid or liquid fillers, diluents or encapsulating substances which are suitable for administration into a mammal including humans. The term "carrier" denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application.

The term "pharmaceutically acceptable" means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients. Such preparations may routinely contain pharmaceutically acceptable concentrations of salts, buffering agents, preservatives, compatible carriers, supplementary immune potentiating agents such as adjuvants and cytokines and optionally other therapeutic agents, such as chemotherapeutic agents. When used in medicine, the salts should be pharmaceutically acceptable, but non- pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically- acceptable salts thereof and are not excluded from the scope of the invention.

The pharmaceutical compositions may contain suitable buffering agents, including: acetic acid in a salt; citric acid in a salt; boric acid in a salt; and phosphoric acid in a salt.

The pharmaceutical compositions also may contain, optionally, suitable preservatives, such as: benzalkonium chloride; chlorobutanol; parabens and thimerosal.

The doses of polypeptide or nucleic acid encoding said polypeptide administered to a subject can be chosen in accordance with different parameters, in particular in accordance with the mode of administration used and the state of the subject. Other factors include the desired period of treatment. In the event that a response in a subject is insufficient at the initial doses applied, higher doses (or effectively higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance permits.

The pharmaceutical compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well-known in the art of pharmacy. All methods include the step of bringing the active agent into association with a carrier which constitutes, one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the active compound into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product. Compositions suitable for oral administration may be presented as discrete units, such as capsules, tablets, lozenges, each containing a predetermined amount of the active compound. Other compositions include suspensions in aqueous liquids or non-aqueous liquids such as a syrup, elixir or an emulsion.

Compositions suitable for parenteral administration conveniently comprise a sterile aqueous or non-aqueous preparation of a polypeptide or nucleic acid encoding the polypeptide, which is preferably isotonic with the blood of the recipient. This preparation may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation also may be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1 ,3-butane diol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or di-glycerides. In addition, fatty acids such as oleic acid may be used in the preparation of injectables.

Carrier formulation suitable for oral, subcutaneous, intravenous, intramuscular, etc. administrations can be found in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA.

Another aspect of the invention provides a method for the prognosis or diagnosis of a disease or condition associated with iron balance or iron transport comprising detecting the level of DEF5(1-94) or a fragment thereof in a biological sample taken from a subject to be diagnosed , wherein an altered level is indicative of a disease or condition associated with iron balance or iron transport. One embodiment of the invention provides a method for the prognosis or diagnosis of a disease or condition associated with iron balance or iron transport in a subject comprising the steps of (i)detecting the level of DEF5(1-94) or a fragment thereof in a biological sample obtained from the subject to provide a first value; and step (ii) comparing the first value with a level of DEF5(1-94) or fragment thereof from a disease- or condition-free subject, wherein an alteration in the level in the biological sample from the subject compared to the level of DEF5(1-94) or fragment thereof in the sample from the disease-free subject is indicative of the subject being predisposed to or having a disease or condition associated with iron balance or iron transport.

Such biological sample includes a blood or a plasma or a tissue sample. Suitable tissue samples include whole blood, semen, saliva, tears, urine, fecal material, sweat, buccal smears, skin, and biopsies of specific organ tissues, such as muscle, brain or nerve tissue and hair. Most preferably, a suitable biological sample comprises blood or plasma. Tissue samples also include cells and cell types isolated from such biological sample. An alteration in the level of DEF5(1-94) or a fragment of thereof may according to a preferred embodiment of the invention comprise an increased plasma level of DEF5(1-94) or a fragment thereof within the context of the present invention is relative to the DEF5(1-94) or a DEF5(1-94) fragment plasma level as found in individuals which do not suffer of a disease or condition associated with iron balance or iron transport. The increase is preferably at least 1.2 fold, more preferably at least 1.5 fold, 2 fold, 3 fold, 5 fold or 10 fold. According to a further aspect, the present invention provides a method for the prognosis or diagnosis of a disease or condition associated with iron balance or iron transport comprising i) detecting a level of expression of at least one gene identified in Table 1 in a sample of a suitable tissue obtained from the subject to provide a first value; and ii) comparing the first value with a level of expression of said gene from a disease-free subject, wherein a greater or smaller expression level in the subject sample as compared to the sample from the disease-free subject is indicative of the subject being predisposed to or having a disease or condition associated with iron balance or iron transport. Gene expression may be detected on mRNA or protein level. Suitable tissues include, but are not limited to liver, heart, intestines such as duodenum, spleen, bone marrow. The mRNA expression level may be detected by any suitable technique, such as for instance Microarray analysis, Northern blot analysis, reverse transcription PCR and real time quantitative PCR. Likewise, the protein level may be detected by any suitable technique, such as for instance through western blotting by utilizing a labeled probe specific for the protein.

In a preferred embodiment of the above aspects, the gene(s) are selected from the gene(s) set forth in Table 1 which are upregulated or downregulated. Preferably, the gene(s) are selected from the gene(s) set forth in Table 1 which are upregulated 1.2 fold or more, 1.3 fold or more, or 1.5, 1.7, 1.8, 1.9 fold or more; or from the gene(s) selected from the genes set forth in Table 1 are downregulated 0.8 fold or less, 0.7 fold or less, or 0.6 fold or less. In yet another preferred embodiment of the above aspects, the expression of at least 1, 2, 3, 4, 5, 10, 20, 30, is measured. In another embodiment of the invention, the expression of at least 40, 50, or 60 genes selected from Table 1 are measured. A still further embodiment of the invention provides that the expression of a majority of the genes selected from Table 1 is determined such as the expression of at least 65, 70, 80, 90, 100, or at least 110 or the expression of all genes of Table 1 is determined.

A still further aspect of the present invention provides a method of identifying a modulator of a disease or condition associated with iron balance or iron transport comprising the steps of i) contacting a test compound with DEF5(1-94) or a fragment of thereof, under sample conditions permissive for at least one biological activity of DEF5(1-94) or DEF5(1-94) fragment; ii) determining the level of said at least one DEF5(1-94) / DEF5(1-94) fragment biological activity; Hi) comparing said level to that of a control sample lacking said test compound. In a preferred embodiment said test compound, which causes said level to change, is selected for further testing as a DEF5(1-94) or DEF5(1-94) fragment modulator for the prophylactic and/or therapeutic treatment of a disease or condition associated with iron balance or iron transport. In a preferred embodiment, the level of biological activity of DEF5(1-94) or of DEF5(1-94) fragment is measured by detecting the level of expression of one or more or a of plurality of genes such as at least 61, 70 or 100 genes set forth in Table 1.

Examples of a modulator of a disease or condition associated with iron balance or iron transport include, but are not limited to, antisense nucleotides, ribozymes, double-stranded RNAs and antagonists.

As used herein, the term "antisense" refers to nucleotide sequences that are complementary to a portion of an RNA expression product encoding a polypeptide of the invention such as for example DEF5(1-94) (SEQ. ID No: 1), DEF5(20-94) (SEQ. ID No: 2), DEF5(23-94) (SEQ. ID No: 3), DEF5(29-94) (SEQ. ID No: 4), DEF5(56-94) (SEQ. ID No: 5), DEF5(60-94) (SEQ. ID No: 6), or DEF5(63-94) (SEQ. ID No: 7), DEF5(35-55) (SEQ. ID No: 8) or DEF5(76-87) (SEQ. ID No: 9). "Complementary" nucleotide sequences refer to nucleotide sequences that are capable of base-pairing according to the standard Watson- Crick complementarity rules such as for example base pairing with SEQ. ID No: 10. That is, purines will base- pair with pyrimidine to form combinations of guaninexytosine and adenine:thymine in the case of DNA, or adenine:uracil in the case of RNA. Other less common bases, e.g., inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine and others may be included in the hybridizing sequences and will not interfere with pairing.

When introduced into a host cell, antisense nucleotide sequences specifically hybridize with the cellular mRNA and/or genomic DNA corresponding to the gene(s) so as to inhibit expression of the encoded protein, e.g., by inhibiting transcription and/or translation within the cell.

The isolated nucleic acid molecule comprising the antisense nucleotide sequence can be delivered, e.g., as an expression vector, which when transcribed in the cell, produces RNA which is complementary to at least a unique portion of the encoded mRNA of the gene(s). Alternatively, the isolated nucleic acid molecule comprising the antisense nucleotide sequence is an oligonucleotide probe which is prepared ex vivo and, which, when introduced into the cell, results in inhibiting expression of the encoded protein by hybridizing with the mRNA and/or genomic sequences of the gene(s).

Preferably, the oligonucleotide contains artificial internucleotide linkages which render the antisense molecule resistant to exonucleases and endonucleases, and thus are stable in the cell. Examples of modified nucleic acid molecules for use as antisense nucleotide sequences are phosphoramidate, phosporothioate and methylphosphonate analogs of DNA as described, e.g., in U.S. Patent No. 5,176,996; 5,264,564; and 5,256,775. General approaches to preparing oligomers useful in antisense therapy are described, e.g., in Van der Krol, BioTechniques, Vol. 6, pp. 958-976 (1988); and Stein et al., Cancer Res., Vol. 48, pp. 2659-2668 (1988). Typical antisense approaches, involve the preparation of oligonucleotides, either DNA or RNA, that are complementary to the encoded mRNA of the gene. The antisense oligonucleotides will hybridize to the encoded mRNA of the gene and prevent translation. The capacity of the antisense nucleotide sequence to hybridize with the desired nucleic acid will depend on the degree of complementarity and the length of the antisense nucleotide sequence. Typically, as the length of the hybridizing nucleic acid increases, the more base mismatches with an RNA it may contain and still form a stable duplex or triplex. One skilled in the art can determine a tolerable degree of mismatch by use of conventional procedures to determine the melting point of the hybridized complexes.

Antisense oligonucleotides are preferably designed to be complementary to the 5' end of the mRNA, e.g., the 5' untranslated sequence up to and including the regions complementary to the mRNA initiation site, i.e., AUG. However, oligonucleotide sequences that are complementary to the 3' untranslated sequence of mRNA have also been shown to be effective at inhibiting translation of mRNAs as described, e.g., in Wagner, Nature, Vol. 372, pp. 333 (1994). While antisense oligonucleotides can be designed to be complementary to the mRNA coding regions, such oligonucleotides are less efficient inhibitors of translation.

Regardless of the mRNA region to which they hybridize, antisense oligonucleotides are generally from about 15 to about 25 nucleotides in length.

The antisense nucleotide can also comprise at least one modified base moiety, e.g., 3- methylcytosine, 5,-methylcytosine, 7-methylguanine, 5-fluorouracil, 5-bromouracil, and may also comprise at least one modified sugar moiety, e.g., arabinose, hexose, 2-fluorarabinose, and xylulose.

In another embodiment, the antisense nucleotide sequence is an alpha-anomeric nucleotide sequence. An alpha-anomeric nucleotide sequence forms specific double stranded hybrids with complementary RNA, in which, contrary to the usual beta-units, the strands run parallel to each other as described e.g., in Gautier et al., Nucl. Acids. Res., Vol. 15, pp. 6625-6641 (1987). Antisense nucleotides may be delivered to cells which express the described genes in vivo by various techniques, e.g., injection directly into the relevant tissue, entrapping the antisense nucleotide in a liposome, by administering modified antisense nucleotides which are targeted to the relevant cells by linking the antisense nucleotides to peptides or antibodies that specifically bind receptors or antigens expressed on said cell surface.

The term "ribozyme" refers to RNA molecules that specifically cleave other single- stranded RNA in a manner similar to DNA restriction endonucleases. By modifying the nucleotide sequences encoding the RNAs, ribozymes can be synthesized to recognize specific nucleotide sequences in a molecule and cleave it as described, e.g., in Cech, J. Amer. Med. Assn., Vol.260, p. 3030 (1988). Accordingly, only mRNAs with specific sequences are cleaved and inactivated.

Two basic types of ribozymes include the "hammerhead"-type as described for example in Rossie et al., Pharmac. Ther., Vol. 50, pp. 245-254 (1991); and the hairpin ribozyme as described, e.g., in Hampel et al., Nucl. Acids Res., Vol. 18, pp. 299-304 (1999) and U.S. Patent No. 5,254,678. Intracellular expression of hammerhead and hairpin ribozymes targeted to mRNA corresponding to at least one of the disclosed genes can be utilized to inhibit protein encoded by the gene.

Ribozymes may either be delivered directly to cells, in the form of RNA oligonucleotides incorporating ribozyme sequences, or introduced into the cell as an expression vector encoding the desired ribozymal RNA. Ribozyme sequences can be modified in essentially the same manner as described for antisense nucleotides, e.g., the ribozyme sequence can comprise a modified base moiety.

The term "double-stranded RNA", i.e., sense-antisense RNA, corresponding to at least one nucleic acid encoding a polypeptide of the invention such as for example SEQ ID No.: 10, can also be utilized to interfere with expression of at least one of the disclosed genes. Interference with the function and expression of endogenous genes by double-stranded RNA has been shown in various organisms such as. C. elegans as described, e.g., in Fire et al., Nature, Vol. 391 , pp. 806-811 (1998); drosophilia as described, e.g., in Kennerdell et al., Cell, Vol. 95, No. 7, pp. 1017-1026 (1998); and mouse embryos as described, e.g., in Wianni et al., Nat. Cell Biol., Vol. 2, No. 2, pp. 70-75 (2000). Such double-stranded RNA can be synthesized by in vitro transcription of single-stranded RNA read from both directions of a template and in vitro annealing of sense and antisense RNA strands. Double-stranded RNA can also be synthesized from a cDNA vector construct in which the gene of interest is cloned in opposing orientations separated by an inverted repeat. Following cell transfection, the RNA is transcribed and the complementary strands reanneal.

The term "antagonist" refers to a molecule which, when bound to a polypeptide of the inventions such as DEF5(1-94) or a fragment thereof such as for example DEF5(20-94), DEF5(23-94) or DEF5(29-94), DEF5(56-94), DEF5(60-94), DEF5(63-94), DEF5(34-55) or DEF5(76-87) reduces or inhibits at least one biological activity of said polypeptide. Antagonists can include, but are not limited to, peptides, proteins, carbohydrates, and small molecules. In a particularly useful embodiment, the antagonist is an antibody specific for DEF5(1-

94), DEF5(20-94), DEF5(23-94) or DEF5(29-94), DEF5(56-94), DEF5(60-94), DEF5(63-94), DEF5(34-55) and/or DEF5(76-87). The antibody may also be conjugated to a reagent such as a chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc., and serve as a target agent. In yet another embodiment, the antagonist useful as a therapeutic for treating adisease or condition associated with iron balance or iron transport such as for example hemochromatosis, hereditary hemochromatosis, juvenile hemochromatosis, thalassemia, conditions related to iron-overload, anemia, sickle cell anemia.

In the case of treatment with an antisense nucleotide, the method comprises administering a therapeutically effective amount of an isolated nucleic acid molecule comprising an antisense nucleotide sequence derived from SEQ ID No: 10 or from a nucleic acid which hybridizes under stringent conditions to SEQ. ID No: 10.

The term "isolated" nucleic acid molecule means that the nucleic acid molecule is removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally occurring nucleic acid molecule is not isolated, but the same nucleic acid molecule, separated from some or all of the co-existing materials in the natural system, is isolated, even if subsequently reintroduced into the natural system. Such nucleic acid molecules could be part of a vector or part of a composition and still be isolated, in that such vector or composition is not part of its natural environment. With respect to treatment with a ribozyme or double-stranded RNA molecule, the method comprises administering a therapeutically effective amount of a nucleotide sequence encoding a ribozyme, or a double-stranded RNA molecule, wherein the nucleotide sequence encoding the ribozyme/doub!e-stranded RNA molecule has the ability to decrease the transcription/translation DEF5(1-94) or a fragment thereof.

A "therapeutically effective amount" of an isolated nucleic acid molecule comprising an antisense nucleotide, nucleotide sequence encoding a ribozyme, double-stranded RNA, or antagonist, refers to a sufficient amount of one of these therapeutic agents to treat a disease or condition associated with iron balance or iron transport such as for example hemochromatosis, hereditary hemochromatosis, juvenile hemochromatosis, thalassemia, conditions related to iron-overload, anemia, sickle cell anemia.

The determination of a therapeutically effective amount is well within the capability of those skilled in the art. For any therapeutic, the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, usually mice, rabbits, dogs, or pigs. The animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans. For therapeutic applications, the antisense nucleotides, nucleotide sequences encoding ribozymes, double-stranded RNAs (whether entrapped in a liposome or contained in a viral vector) and antibodies are preferably administered as pharmaceutical compositions containing the therapeutic agent in combination with one or more pharmaceutically acceptable carriers. The compositions may be administered alone or in combination with at least one other agent, such as stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water. The compositions may be administered to a subject alone, or in combination with other agents, drugs or hormones.

Experiment 1

Methods

DEF5(60-94) is administered subcutaneously to male C57BL/6 mice for 7 to 14 days at a dose of 300, 600 or 1000 microg/day. At the end of the treatment period samples from all organs are subjected to snap freezing at necropsy and are analyzed with GeneChip® expression profiling. Total RNA is extracted from these frozen tissues using TRIzol reagent (Life Technologies) according to the manufacturer's instructions. Total RNA is quantified by the absorbance at λ = 260 nm (A260nm), and the purity is estimated by the ratio A260nm/A280nm. Integrity is checked by denaturing gel electrophoresis. RNA is stored at - 80⁰C until analysis. Good quality total RNA is used to synthesize double-stranded cDNA using the Superscript Choice System (Life Technologies). The cDNA is then in vitro transcribed (MEGAscript™ T7 Kit, Ambion) to form biotin labeled cRNA. Next, 12 to 15 mg of labeled cRNA is hybridized to the Affymetrix Mouse MOE430A expression probe arrays for 16 hours at 45°C. Arrays are then washed according to the EukGE-WS2 protocol (Affymetrix), and stained with 10 mg/ml of streptavidin-phycoerythrin conjugate (Molecular Probes). The signal is antibody-amplified with 2 mg/ml acetylated BSA (Life Technologies), 100 mM MES, 1 M [Na+], 0.05 % Tween 20, 0.005 % Antiofoam (Sigma), 0.1 mg/ml goat IgG and 0.5 mg/ml biotinylated antibody and re-stained with the streptavidin solution. After washing, the arrays are scanned twice with the Gene Array® scanner (Affymetrix). The expression level is estimated by averaging the differences in signal intensity measured by oligonucleotide pairs of a given probe (AvgDiff value). The image acquisition and numerical translation software used for this study is the Affymetrix Microarray Suite version 5 (MAS5). To identify genes that are impacted by treatment, the dataset is initially filtered to exclude in a first wave of analysis genes whose values are systematically in the lower expression ranges where the experimental noise is high (at least an AvgDiff value of 50 in a number of experiments corresponding to the smallest number of replicas of any experimental point). In a second round of selection a threshold t-test p-value (0.05) identifies genes with different values between treated and non-treated based on a two component error model (Global Error Model) and, where, possible, with a stepdown correction for multi- hypothesis testing (Benjamini and Hochberg false discovery rate).

The selected genelists are then compared with established genelists for pathways and cellular components using Fisher's exact test. Venn diagrams are used to identify the gene changes that are in common between the different organs. Expression profiles of highly relevant genes are used to find genes with correlated changes at individual experimental points, using several distance metrics (standard, Pearson).

The decision to consider a specific gene relevant is based on a conjunction of numerical changes identified by exploratory filtering and statistical algorithms as described above and the relationship to other modulated genes that point to a common biological theme.

Results

Tables 1 shows that at the RNA level DEF5(60-94) affects genes that are involved in heme biosynthesis and metabolism, porphyrin biosynthesis and metabolism and erythropoiesis. Additionally, genes involved in dietary iron balance, iron transport are also differentially regulated upon injection of DEF5(60-94). Most genes are up-regulated more than at least 1.5 fold or down-regulated at least less than 0.7 fold.

Table 1 Gene expression alteration upon DEF5(60-94) injection

Examples of genes that are up-regulated include, but are not limited to, Ceruplasmin, inhibitor of DNA binding 4, ect2 oncogene, serine active site containing 1, matrix metalloproteinase 9, and steroidogenic acute regulatory protein. Of particular interest are those genes that are up-regulated and involved in erythropoiesis and iron homeostasis such as, Ceruplasmin, a copper protein with ferrodixidase activity for converting Fe2+ to Fe3+ in the presence of oxygen. (See Sarkar et al. (2003) J. Biol. Chem, 278: 44018-44024).

Examples of genes that are down-regulated include, but are not limited to, coproporphyrinogen oxidase, hydroxymethylbilane synthase, ferrochelatase, transferring receptor, glycophorin A, aminolevulinate delta dehydratase, alpha thalassemia/mental retardation syndrome X-linked homolog (human), Kruppel-like factor 1 (erythroid), and aqυaporin 1. Of particular interest are those associated with iron delivery such as transferrin receptor (Aisen, (2004), 36: 2137-2143); those involved in the chromatic remodelling pathway such as ATRX (Gibbons et al. (2003) Nat. Genet. 34: 446-449); cell-restricted transcriptional modulators that are involved in hematopoiesis such as KLF- 1 (Bieker, (2005) Mt. Sinai J. Med. 72: 333-338); genes that are associated with hemaodialysis such as aquaporins (Ruello et al. (2002) Nephron 92: 846-852); and genes associated with pathways that are protective against toxicity such as ALAD (Kim et al. (2004) Environ. Health Perspect, 112: 538-541).

Claims

Claims

1. Use of a polypeptide for the manufacture of a medicament for use in the treatment of a disease or condition associated with iron balance or iron transport wherein the polypeptide is selected from the groups consisting of a) DEF5(1-94) (SEQ. ID No: 1) or a fragment of DEF5(1-94); b) a bioactive polypeptide having a percentage of identity of at least 50% with the amino acid sequence of any one of the polypeptides of (a); c) a bioactive variant of any one of the polypeptides of (a) or (b).

2. Use of a polypeptide according to claim 1 , wherein the disease or condition associated with iron balance or iron transport is selected from the group consisting of hemochromatosis, hereditary hemochromatosis, juvenile hemochromatosis, thalassemia, conditions related to iron-overload, anemia, sickle cell anemia.

3. Use of a polypeptide according to claim 1 or 2, wherein the fragment of DEF5(1-94) comprises a C-terminal fragment of DEF5(1-94).

4. Use of a polypeptide according to any one of claims 1 to 3, wherein the C-terminai fragment of DEF5(1-94) comprises at least 10 amino acids of the C-terminus of DEF5(1-94).

5. Use of a polypeptide according to any one of claims 1 to 4, wherein the C-terminai fragment of DEF5(1-94) has an amino acid sequence of SEQ ID No: 2, SEQ ID No: 3, SEQ ID No: 4, SEQ ID No: 5, SEQ ID No: 6, SEQ ID No: 7 or SEQ ID No: 9.

6. Use of a polypeptide according to claim 5, wherein the C-terminal fragment of DEF5(1-94) has an amino acid sequence of SEQ ID No: 6.

7. Use of a polypeptide according to any one of claims 1 to 6, wherein the polypeptide is encoded by a nucleic acid which hybridizes under stringent conditions to SEQ. ID No: 10.

8. A method for the treatment of a disease or condition associated with iron balance or iron transport comprising administering an effective amount of a polypeptide to a mammal including a human suffering from the disease, wherein the polypeptide is selected from the groups consisting of a) DEF5(1-94) (SEQ. ID No: 1) or a fragment of DEF5(1-94); b) a bioactive polypeptide having a percentage of identity of at least 50% with the amino acid sequence of any one of the polypeptides of (a); c) bioactive variant of any one of the polypeptides of (a) or (b).

9. The method of claim 8, wherein the disease or condition associated with iron balance or iron transport is selected from the group consisting of hemochromatosis, hereditary hemochromatosis, juvenile hemochromatosis, thalassemia, conditions related to iron- overload, anemia, and sickle cell anemia.

10. The method of claim 8 or 9 in which the effective amount of the polypeptide is administered intravenously, intramuscularly, subcutaneously, orally or topically.

11. The method of any one of claims 8 to 10, wherein the fragment of DEF5(1-94) comprises a C-terminal fragment of DEF5(1-94).

12. The method of any one of claims 8 to 11 , wherein the C-terminal fragment of

DEF5(1-94) comprises at least 10 amino acids of the C-terminus of DEF5(1-94).

13. The method of any one of claims 8 to 12, wherein the C-terminal fragment of

DEF5(1-94) has an amino acid sequence of SEQ ID No: 2, SEQ ID No: 3, SEQ ID No: 4, SEQ ID No: 5, SEQ ID No: 6, SEQ ID No: 7 or SEQ ID No: 9.

14. The method of any one of claims 8 to 13, wherein the C-terminal fragment of DEF5(1-94) has an amino acid sequence of SEQ ID No: 6.

15. The method of any one of claims 8 to 14, wherein the polypeptide is encoded by a nucleic acid which hybridizes under stringent conditions to SEQ. ID No: 10.

16. A pharmaceutical composition for use in a disease or condition associated with iron balance or iron transport comprising an effective amount of a polypeptide as defined in claims 1 , and 3 to 7 and a pharmaceutically-acceptable carrier.

17. The pharmaceutical composition of claim 16 comprising an effective amount of a fragment of DEF5(60-94) (SEQ ID No: 6).

18. A method for the prognosis or diagnosis of a disease or condition associated with iron balance or iron transport in a subject comprising (i) detecting the level of DEF5(1-94) or a fragment thereof in a biological sample obtained from the subject to provide a first value; and

(ii) comparing the first value with a level of DEF5(1-94) or fragment thereof from a disease- or condition-free subject, wherein an alteration in the level in the biological sample from the subject compared to the level of DEF5(1-94) or fragment thereof in the sample from the disease-free subject is indicative of the subject being predisposed to or having a disease or condition associated with iron balance or iron transport.

19. The method of claim 18, wherein the biological sample is plasma.

20. A method for the prognosis or diagnosis of a disease or condition associated with iron balance or iron transport comprising

(i) detecting a level of expression of at least one gene identified in Table 1 in a biological sample obtained from the subject to provide a first value; and (ii) comparing the first value with a level of expression of the said gene from a disease- or condition-free subject, wherein a greater or smaller expression level in the subject sample compared to the sample from the disease-free subject is indicative of the subject being predisposed to or having a disease or condition associated with iron balance or iron transport.

21. The method of claim 20, wherein the level of expression of a majority of genes identified in Table 1 is detected.

22. A method of identifying a modulator of a disease or condition associated with iron balance or iron transport comprising

(i) contacting a test compound with DEF5(1-94) or a fragment thereof under sample conditions permissive for at least one biological activity of DEF5(1-94) or DEF5(1 -94) fragment;

(ii) determining the level of said at least one biological activity of DEF5(1-94) or DEF5(1 -94) fragment;

(iii) comparing said level to that of a control sample lacking said test compound; and

(iv) selecting a test compound which causes said level to change for further testing as a DEF5(1-94) modulator for the prophylactic and/or therapeutic treatment of a disease or condition associated with iron balance or iron transport.

23. The method according to claim 22, wherein the level of biological activity of DEF5(1-94) or a DEF5(1-94) fragment is measured by determining the level of expression of one or more genes set forth in Table 1.

24. The method of claim 22 or 23, wherein the level of expression of a majority of genes identified in Table 1 is detected.