JP2008523068A - Use of organic compounds - Google Patents

Abstract

  The present invention relates to DEFESIN5 polypeptides for the manufacture of a medicament for use in the treatment of diseases or conditions associated with iron balance or iron transport.

Description

RELATED APPLICATIONS This invention claims priority to US 60 / 634,349, filed Dec. 8, 2004, which is hereby incorporated by reference in its entirety.

The present invention is directed to the manufacture of a medicament for the treatment of diseases or conditions associated with defensin 5 (DEF5), DEF5 fragments, DEF5 homologues and / or DEF5 variants, in particular iron balance or iron transport. It relates to the pharmaceutical use.

Background Defensins (DEFs) are a large family of broad-spectrum antimicrobial peptides that were first identified in rabbit and human leukocytes. Defensins have three disulfide bonds in the peptide and are similar but fall into two families that exhibit features based on α- and β-defensins. Defensins are cationic / polar peptides (30-35aa; 3-4 kDa), of which β-defensins are slightly larger. The genes encoding human α and β-defensins are assembled in the contact segment of chromosome 8 p23.

  In humans, 6 α-defensins and 2 β-defensins have been identified. α-Defensin is encoded by a gene designated DEFA1-6, while human β-defensin is encoded by the DEFB1 and DEFB2 genes. Defensins are synthesized as large precursor molecules with a putative amino-terminal signal sequence, followed by a bioactive antimicrobial peptide residue in the middle portion and the last carboxy-terminal sequence of the precursor.

  Defensins provide a broad spectrum of antibiotic activity, primarily destroying microbial cell membranes. Defensins are expressed by a variety of epithelial cells including phagocytic leukocytes and panate cells.

  Human α5 defensin is also known as defensin 5α, and α defensin 5 precursor or defensin 5 is synthesized as a 94 amino acid precursor (HDEFA5; HNP-5; DEF A5, DEF5, DEF5 (1-94)). Recently, DEF5 precursor / DEF5 propeptide has been shown to efficiently produce various DEF5 types upon trypsinization in Paneth cells (Ghosh et al., Nature Immunology 2002, 3 (6), 583- 590).

SUMMARY OF THE INVENTION Surprisingly, it has now been found that polypeptides related to DEF5 (1-94) affect key genes that control iron balance or iron transport.

  Thus, the present invention is directed to the manufacture of a medicament for use in the treatment of diseases or conditions associated with iron balance or iron transport: a) DEF5 (1-94) (SEQ ID NO: 1) or DEF5 (1- 94) a fragment of b); b) a biologically active polypeptide having at least 50% identity with the amino acid sequence of any one of the polypeptides of (a); or c) any of the polypeptides of (a) or (b) There is provided the use of a polypeptide selected from the group consisting of:

  In a further aspect, the invention provides for the treatment of a disease or condition comprising administering to a mammal, including a human having a disease or condition associated with iron balance or iron transport, an effective amount of the polypeptide described above. Concerning the method.

  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 the above polypeptide and a pharmaceutically acceptable carrier.

Detailed Description of the Invention The present invention is directed to the manufacture of a medicament for use in the treatment of diseases or conditions associated with iron balance or iron transport: a) DEF5 (1-94) (SEQ ID NO: 1) or DEF5 A fragment of (1-94); b) a biologically active polypeptide having at least 50% identity with any one amino acid sequence of the polypeptide of (a); or c) a polypeptide of (a) or (b) There is provided the use of a polypeptide selected from the group consisting of any one biologically active variant of a peptide.

  The term “polypeptide” as used herein means a protein, peptide, oligopeptide or synthetic oligopeptide. These terms are intended to be used interchangeably. Any one of the terms refers to a chain of two or more amino acids joined together by peptide or amide bonds, regardless of post-translational modifications such as glycosylation or phosphorylation. The polypeptide may also include two or more subunits, where each subunit is encoded by a separate DNA sequence.

  The polypeptide of the invention may comprise DEF5 (1-94) having the amino acid sequence of SEQ ID NO: 1. The polypeptides of the present invention also include polypeptide fragments of the DEF5 (1-94) polypeptides of the present invention. Such a polypeptide fragment means a polypeptide having an amino acid sequence which is the same as part but not all of the amino acid sequence of the polypeptide of the present invention. Such polypeptide fragments can be “free-standing” or can be part of a larger polypeptide, wherein such polypeptide fragments are most preferably single A part or one region is formed as a continuous region. Preferably such polypeptide fragments retain at least one biological activity of the corresponding polypeptide DEF5 (1-94).

  A fragment of DEF5 (1-94) may contain at least 10 amino acids, preferably at least 15, 20 or 25 amino acids. More preferably, the fragment of DEF5 (1-94) comprises at least 30 amino acids. Other preferred DEF5 (1-94) fragments comprise 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 amino acids in sequence of SEQ ID NO: 1.

  Such a polypeptide can also be a fragment of DEF5 (1-94) produced by a protease such as trypsin, such as a proteolytic cleavage product of DEF5 (1-94). The polypeptide or polypeptide fragment of the invention may comprise a C-terminal fragment of DEF5 (1-94). Such a C-terminal fragment is at least 10 amino acids at the C-terminus of DEF5 (1-94), preferably at least 20 or 25, even more preferably at least 30 amino acids or at least 65, 70 or 75. Of amino acids. The at least 10 amino acids may comprise most C-terminal at least 10 amino acids, such as, for example, the C-terminal portion of DEF5 (1-94), such as DEF5 (76-87) (SEQ ID NO: 9). It may also contain at least 10 amino acids therein. The polypeptide can comprise at least 32, 35, 39, 66, 72 or 75 most C-terminal amino acids of DEF5 (1-94), preferably it is 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 It may have the amino acid sequence of 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) can also include an internal fragment of DEF5 (1-94), eg, DEF5 (34-55) (SEQ ID NO: 8).

  As used herein, the term “biological activity” means a molecule that induces or affects a biological event. Such biological events may be associated with diseases or conditions associated with, for example, iron balance or iron transport. In a preferred embodiment, the biological activity level of DEF5 (1-94) or a fragment thereof is measured to detect the expression level of one or more genes listed in Table 1. Preferably, the expression of more than 10, 20, 40 or most of the genes in Table 1 is determined.

  “Bioactive polypeptides” of the present invention include 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) or DEF5 (1-94) fragments such as DEF5 (76-87) are included.

  Also included are homologs having an amino acid sequence having at least 50% identity with DEF5 (1-94) or fragments thereof, and variants of DEF5 (1-94) or DEF5 (1-94) fragments.

  The polypeptide may also be DEF5 (1-94) or, for example, DEF5 (20-94), DEF5 (23-94) or DEF5 (29-94), DEF5 (56-94), DEF5 (60-94), 95%, 97% or 99% identical to the amino acid sequence of any one of the above polypeptides such as DEF5 (63-94), DEF5 (34-55) or fragments thereof such as DEF5 (76-87) It may have an amino acid sequence having at least 50%, preferably at least 60%, more preferably at least 70% or 80%, most preferably at least 90% identity, such as gender.

  Amino acid residues are indicated herein by their standard one-letter or three-letter code: A (Ala) alanine; C (Cys) cysteine; D (Asp) aspartic acid; E (Glu) glutamic acid; F ( Phe) phenylalanine; G (Gly) glycine; H (His) histidine; I (Ile) isoleucine; K (Lys) lysine; L (Leu) leucine; M (Met) methionine; N (Asn) asparagine; P (Pro) Proline; Q (Gln) Glutamine; R (Arg) Arginine; S (Ser) Serine; T (Thr) Threonine; V (Val) Valine; W (Trp) Tryptophan; Y (Tyr) Tyrosine.

  The term “percent identity” used for comparison of a reference sequence and other sequences (ie, “candidate” sequences), or such terms, indicates that the candidate sequence indicated in an optimal alignment between the two sequences. It means that it is identical to the reference sequence at a number of positions in the subunit that correspond to the proportions, which are nucleotides for polynucleotide comparison or amino acids for polypeptide comparison. As used herein, “optimal alignment” of the sequences being compared is one that maximizes the match between subunits and minimizes the number of gaps used to construct the alignment. Percent identity is the commercial value of the algorithm described by Needleman and Wunsch, J. Mol. Biol. 48: 443-453 (1970) (“GAP” program of Wisconsin Sequence Analysis Package, Genetics Computer Group, Madison, WI). Can be determined using any available implementation. Smith and Waterman, Advances in Applied Mathematics 2: 482-489 (1981) include other software packages in the art for building alignments and measuring percent identity. A “BestFit” program based on the algorithm of (Wisconsin Sequence Analysis Package, Genetics Computer Group, Madison, Wis.) Is included. Percent identity can also be brought about by WU-BLAST-2 (Altschul et al., Methods in Enzymology 266: 460-480 (1996)). WU-BLAST-2 uses several search parameters, most of which are set to initial values.

  The adjustment parameters are set using the following values: overlap span = 1, overlap fraction = 0.125, word threshold (T) = 11. The% amino acid sequence identity value is determined by dividing the number of matching identical residues by the total number of residues in the alignment region. For example, to obtain a polypeptide having an amino acid sequence that is at least 95% identical to a reference amino acid sequence, up to 5% of the amino acid residues in the reference sequence are deleted or replaced with another amino acid. Many amino acids up to 5% of all amino acid residues in the reference sequence can be inserted into the reference sequence. These modifications of the reference sequence occur at the amino or carboxy terminal site of the reference amino acid sequence, or are individually interspersed between residues in the reference sequence, somewhere between those terminal sites, or reference It can occur at one or more adjacent groups in the sequence. In comparison to a reference sequence of the present invention, the candidate sequence can be a component or segment of a larger polypeptide or polynucleotide, and such a comparison for computational calculations of percent identity. Is to be implemented with respect to related components or segments.

  The invention also includes functionally conserved variants of the polypeptides or polypeptide fragments described herein. Such variants can be made using standard methods in the art, eg, by conservative amino acid substitutions. Typically, such substitutions are between Ala, Val, Leu and Ile; between Ser and Thr; between acidic residues Asp and Glu; between Asn and Gln; and between basic residues Lys and Arg. Or between the aromatic residues Phe and Tyr. Particularly preferred are variants in which some 5 to 10, 1 to 5, or 2 amino acids undergo substitutions, deletions or additions, or any combination thereof.

  In various other embodiments, the polypeptide or fragment thereof or polypeptide variant or homologue may be linear or branched, which may contain modified amino acids and is interrupted by non-amino acids. Well and / or can be assembled into a complex of two or more polypeptide chains. As is well understood in the art, a polypeptide may be naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other such as conjugation with a labeling component. It can be modified by manipulation or modification of In some embodiments, the polypeptide or polypeptide fragment includes one or more analogs of amino acids (eg, including unnatural amino acids, etc.), as well as other modifications known in the art.

  The polypeptides or polypeptide fragments of the present invention include isolated naturally occurring polypeptides. Preferably, such naturally occurring polypeptides are included in the selected population with a frequency of at least 5%, most preferably at least 10%. The selected population can be any research population recognized in the field of population genetics. Preferably, the selected population is white, black, or Asian. More preferably, the selected group is French, German, British, Spanish, Japanese, Chinese, Korean, Chinese Singaporean, Icelandic, North American, Israeli, Arab, Turkish Man, Greek, Italian, Polish, Pacific Islander, or Indian.

  Polypeptides of the invention or fragments thereof also include recombinantly produced polypeptides, synthetically produced polypeptides, and recombinants of such polypeptides of the invention, and fragments thereof. obtain. Methods for making such polypeptides are well understood in the art. For example, the polynucleotide fragments or polypeptides of the invention can be isolated from body fluids including, but not limited to, serum, urine and ascites fluid, or chemical or biological methods (eg, cell culture , Recombinant gene expression). Unless otherwise specified herein, “isolated” includes the meaning of “separation from coexisting materials”.

  The recombinant polypeptides of the present invention can be produced from genetically modified host cells containing expression systems by methods well known in the art. Accordingly, in a further aspect, the present invention provides for the production of polypeptides by recombinant techniques, expression systems comprising nucleic acids or nucleic acids encoding the polypeptides of the invention, host cells genetically modified in such expression systems, and poly It relates to a method for isolating peptides.

  Another aspect provides that the polypeptide of the invention is encoded by a nucleic acid that hybridizes under stringent conditions to the nucleotide sequence of SEQ ID NO: 10. In some embodiments, the nucleic acid is 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, It contains 210, 215, 220, 225, or at least 250, 260, 270, 280, or at least 285 nucleotides. The nucleic acid can 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 a nucleic acid has at least 36, 66, 96, 105, 117, 198, 216, 225 or 282 contiguous nucleotides of SEQ ID NO: 10, or nucleotides that can hybridize to SEQ ID NO: 10 under stringent conditions, and most Preferably, 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 a variant or homologue thereof.

  The term “nucleic acid” means a natural or semi-synthetic or synthetic or modified nucleic acid molecule. It refers to a nucleotide sequence, oligonucleotide or polynucleotide comprising deoxyribonucleic acid (DNA) and / or ribonucleic acid (RNA) and / or modified nucleotides. These terms are intended to be used interchangeably. RNA can be in the form of tRNA (transfer RNA), snRNA (small nuclear RNA), rRNA (ribosomal RNA), mRNA (messenger RNA), antisense RNA, and ribozyme. The DNA can be in the form of plasmid DNA, viral DNA, linear DNA, chromosomal or genomic DNA, cDNA, or derivatives of these groups. Furthermore, these DNAs and RNAs can be single stranded, double stranded, triple stranded or quadruplexed. The term also includes PNA (peptide nucleic acids), phosphorothioates, and other variants of the phosphate backbone of natural nucleic acids.

  “Stringent conditions” for hybridization reactions can be readily determined by those skilled in the art and are generally calculated experimentally depending on the length of the probe, the wash temperature, and the salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes require lower temperatures. Hybridization generally relies on the ability of denatured nucleic acids to reanneal when complementary strands are present below their melting temperature but near ambient temperature. The higher the degree of homology between the probe and a hybridizable sequence such as SEQ ID NO: 3 or 4, the higher the relative temperature that can be used. As a result, the higher the relative temperature, the more stringent the reaction conditions tend to be, and the lower the temperature, the lower the stringency. In addition, stringency is also inversely proportional to salt concentration. “Stringent conditions” include: (1) washing with 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate at low ionic strength and high temperature, eg, 50 ° C .; ) Denaturing agents such as formamide, eg, 0.1% fetal bovine serum albumin / 0.1% Ficoll / 0.1% polyvinylpyrrolidone / 50 mM sodium phosphate buffer pH 6.5, and 750 mM chloride at 42 ° C. Illustrated by the reaction conditions characterized by the use of 50% (vol / vol) formamide with sodium, 75 mM sodium citrate. Alternatively, stringent conditions are: 42 ° C., 50% formamide, 5 × SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate May be 5 × Denhart solution, sonicated salmon sperm DNA (50 μg / ml), 0.1% SDS, and 10% dextran sulfate. Washing at 42 ° C. in sodium acid) and 50% formamide, followed by washing with a high stringency wash consisting of 0.1 × SSC containing EDTA at 55 ° C. See Ausubel et al., Protocols in Molecular Biology (1995) for further details and explanation of the stringency of hybridization reactions.

Methods for Recombinant Production of DEF5 (1-94) and DEF5 (1-94) Fragments In order to induce the expression of the corresponding polypeptide in a suitable host cell, the nucleic acid described herein as SEQ ID NO: 10 In addition, it can be used for recombinant DNA molecules. Because of the degeneracy of the genetic code, other DNA sequences can encode equivalent amino acid sequences and can be used to clone and express DEF5 (1-94) or fragments thereof. Preferred codons for a particular host cell can be selected and substituted in the naturally occurring nucleotide sequence to increase expression rate and / or efficiency. DEF5 (20-94), DEF5 (23-94), DEF5 (29-94), DEF5 (56-94), DEF5 (60-94), DEF5 (63-94), DEF5 (34-55) or DEF5 A nucleic acid (eg, cDNA or genomic DNA) encoding a desired DEF5 (1-94) or DEF5 (1-94) fragment, such as (76-87) or a variant or homologue thereof, is cloned (amplified DNA And / or inserted into a replicable vector for expression.

Expression Systems Polypeptides 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 viral derived systems such as bacterial plasmids, bacteriophages, transposons, yeast episomes, insertion elements, yeast chromosomal elements, baculoviruses, papovaviruses, SV40, vaccinia viruses, adenoviruses, Vectors derived from viruses such as poultry poxvirus, pseudorabies virus and retrovirus, and vectors derived from combinations thereof such as those derived from plasmids and bacteriophage genetic elements such as cosmids and phagemids are included.

  Expression systems can include regulatory regions that control as well as cause expression. In general, any system or vector that can retain, propagate or express nucleic acids to produce a polypeptide in a host may be used. Appropriate nucleotide sequences can be obtained by any of a variety of well-known and conventional techniques, such as Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989). Can be inserted into the expression system. 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 replication origins, one or more marker genes, an enhancer component, a promoter, a signal or secretory sequence, and a transcription termination sequence.

  An expression vector can have two replication systems and can therefore be maintained in two organisms, such as mammalian or insect cells, for expression and maintained 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 that allows selection of transformed host cells. Selection genes are well known in the art and can vary depending on the host cell used. Expression and cloning vectors may typically contain a selection gene, also termed a selectable marker. Typical selection genes are (a) conferring resistance to antibiotics or other toxins such as ampicillin, neomycin, methotrexate, or tetracycline, (b) complement complement auxotrophy, or (c) essential It encodes a protein that supplies nutrients such as the D-alanine racemase gene.

  A promoter sequence encodes either a constitutive or inducible promoter. The promoter can be either a naturally occurring promoter or a hybrid promoter. Hybrid promoters that combine two or more promoter elements are also known in the art and are useful in the present invention. Furthermore, to integrate the expression vector, the expression vector contains at least one sequence homologous to the host cell genome, and preferably contains two homologous sequences connecting the expression constructs. An integrated vector can be directed to a specific locus in the host cell by insertion of the appropriate homologous sequence in the vector. The construction of integrated vectors is well known in the art.

  Appropriate secretion signals can be incorporated into the desired polypeptide that allows 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 can be, for example, a prokaryotic signal sequence selected from the group of alkaline phosphatase, penicillinase, lpp, or thermostable enterotoxin II leader. For yeast secretion, the signal sequence can be, for example, a yeast invertase leader, an alpha factor leader (including Saccharomyces and Klaberomyces a factor leader). In mammalian cell expression systems, mammalian signal sequences from secreted polypeptides of the same or related species, as well as viral secretory leaders, can be expressed as DEF5 (1-94), or 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 homologues thereof Can be used to induce secretion of DEF5 (1-94) fragments such as Suitable host cells include yeast, bacteria, archaea, fungi, and animal cells including insect cells and mammalian cells including, but not limited to, primary culture cells. Representative examples of suitable hosts include bacterial cells such as E. coli, streptococci, staphylococci, Streptomyces, and Bacillus subtilis; budding yeast, other yeast cells or fungal cells such as Aspergillus; Drosophila S2 and nematodes Insect cells such as Sf9 cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK293 and Bose 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 allow expression of the 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 that cleaves the “prepro” form of the polypeptide may also be important for correct insertion, folding, and / or function.

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

  Transformed host cells include recombinant bacteriophages, microorganisms such as bacteria transformed with plasmid or cosmid DNA expression vectors, yeast transformed with yeast expression vectors, and recombinant insect viruses (eg, baculoviruses). ) Infected insect cells, as well as mammalian expression systems.

  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) Appropriate conditions for expression of DEF5 (63-94), DEF5 (34-55) or DEF5 (76-87) may vary depending on the choice of expression vector and host cell and can be readily determined by one skilled in the art through routine experimentation. Can be confirmed. For example, the use of constitutive promoters in expression vectors requires optimization of host cell growth and proliferation, while the use of inducible promoters requires appropriate growth conditions for induction. Furthermore, in some embodiments, the timing of collection is important. For example, the baculovirus system used with insect cells is a lytic virus, so the choice of harvest time can be important for product yield.

  The desired DEF5 (1-94) or DEF5 (1-94) fragment can be made not only recombinantly directly, but also as a fusion polypeptide comprising a heterologous polypeptide. Such heterologous polypeptides are generally located at the amino terminus or carboxyl terminus of DEF5 (1-94) or a DEF5 (1-94) fragment thereof and provide an epitope tag to which an anti-tag antibody can selectively bind. obtain. Thus, such epitope tags allow DEF5 (1-94) or fragments thereof to be easily purified 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 tags. Alternatively, DEF5 (1-94) or a fragment thereof can be expressed, for example, in the form of a GST fusion protein. Suitable constructs are generally known in the art and include Invitrogen (San Diego, Calif.), Stratagene (La Jolla, Calif.), Gibco BRL (Rockville, Md.), Or Clontech (Palo Alto, Commercially available from commercial suppliers such as Calif.).

Assessment of gene expression Gene expression can be performed using appropriately labeled probes, for example, standard techniques known to those skilled in the art, such as Southern blotting for DNA detection, Northern blotting to determine mRNA transcription, It can be assessed directly in a sample based on the sequences provided herein by dot blotting (DNA or RNA) or in situ hybridization. Alternatively, antibodies can be used for the detection of nucleic acids such as specific double strands, including double stranded DNA, double stranded RNA, and double stranded DNA-RNA hybrids or double stranded DNA-proteins. Can be used in analysis. Such antibodies can be labeled and analyzed when the duplex is bound to the surface because the presence of an antibody that binds to the duplex can be detected by the formation of a duplex on the surface. Do. Alternatively, gene expression is measured by immunohistochemical staining of cell or tissue secretion and cell culture or body fluid analysis to directly assess the expression of DEF5 (1-94) or a DEF5 (1-94) fragment thereof. be able to. Antibodies useful for such immunoassays can be either monoclonal or polyclonal, and are directed against the native sequence DEF5 (1-94) or DEF5 (1-94) fragment based on the DNA sequences provided herein. Can be manufactured.

Purification of the expressed protein Expressed DEF5 (1-94) or DEF5 (1-94) fragment, eg 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) is purified or isolated after expression using any of a variety of methods known to those skilled in the art. can do. The appropriate technique may vary depending on the method of expression of DEF5 (1-94) or DEF5 (1-94) fragment. The polypeptide can be recovered, for example, from the culture medium in the form of a secreted protein or from the host cell lysate. Cells can be destroyed by various physical or chemical means, such as freeze-thaw cycles, sonication, mechanical disruption, or by the use of cell lysing agents, while seeding the bound polypeptide with a suitable detergent solution. Release from the membrane using (eg, Triton-X100) or by enzymatic cleavage. Appropriate techniques for polypeptide purification or isolation may also vary depending on the presence of other elements in the sample. The required degree of purification may also vary depending on the use of DEF5 (1-94) or a fragment thereof. Contaminant elements that are removed by isolation or purification are typically substances that can interfere with the diagnostic or therapeutic use of the polypeptide and can include enzymes, hormones, and other solutes. The purification step (s) selected can vary depending on, for example, the method of production used and the nature of the particular DEF5 (1-94) or DEF5 (1-94) fragment produced.

  Usually, isolated DEF5 (1-94) or a fragment thereof can be made by at least one purification step. Well-known methods of purification include ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, high performance liquid chromatography, hydroxyapatite chromatography and Lectin chromatography is included. Most preferably, affinity chromatography is used for purification. For example, 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) can be purified using standard anti-DEF5 C-terminal polypeptide antibody columns. Ultrafiltration and dialysis techniques related to protein concentration are also useful (see, eg, Scopes, R., Protein Purification, Springer-Verlag, New York, N.Y., 1982). Well-known techniques for protein refolding can be used to regenerate the active structure when the polypeptide is denatured during isolation and / or purification.

Labeling the expressed polypeptide The nucleic acids, proteins and antibodies of the invention can be labeled. As used herein, “label” means that the compound has at least one element, isotope or chemical compound that binds to allow detection of the compound. In general, labels are divided into three classes: a) isotope labels that can be radioisotopes or heavy isotopes; b) immunolabels that can be antibodies or antigens; and c) colored or fluorescent dyes. The label may be incorporated in any position in the compound that does not interfere with the biological activity or properties of the compound to be detected.

Chemical production of DEF5 (1-94) and DEF5 (1-94) fragments Polypeptides or fragments thereof are produced not only by recombinant methods, but also by chemical methods well known in the art. be able to. Solid phase peptide synthesis can be performed batchwise or continuously in a continuous flow process in which α-amino- and side chain-protected amino acid residues are added to an insoluble polymer support via a linker group. A linker group such as methylamine-derived polyethylene glycol is attached to poly (styrene-co-divinylbenzene) to form a support resin. The amino acid residue, N ^alpha protected by labile Fmoc (9-fluorenyl methoxy carbonyl) labile Boc (t-butyloxycarbonyl) or base to the acid. The carboxyl group of the protected amino acid is combined with the amine of the linker group to fix the residue to the solid support resin. Trifluoroacetic acid or piperidine is used for removal of protecting groups in the case of Boc or Fmoc, respectively. Each additional amino acid is added to the anchor gene using a coupling agent or a pre-activated amino acid derivative, and the resin is washed. Full-length peptides are synthesized sequentially by 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 give the peptide acid or amide (Novabiochem 1997/98 Catalog and Peptide Synthesis Handbook, San Diego Calif. Pp. S1-S20). Automated synthesis can also be performed on machines such as the ABI 431A peptide synthesizer (Applied Biosystems). The polypeptide or fragment thereof can be purified by preparative high performance liquid chromatography and its composition can be confirmed by amino acid analysis or sequencing (Creighton TE (1984) Proteins, Structures and Molecular Properties, WH Freeman, New York NY).

  Natural polypeptide variants are desirable in a variety of contexts. For example, unwanted side effects can be reduced by any variant, particularly when the side effect activity is associated with a portion of the polypeptide that is different from a portion of the desired activity. In some expression systems, the native polypeptide can be sensitive to degradation by proteases. In such cases, selected substitutions and / or deletions of amino acids that alter the sensitive sequence can significantly increase yield. Variants can also increase the storage time of proteins by increasing yields in purification methods and / or making amino acids insensitive to oxidation, acylation, alkylation, or other chemical modifications. Preferably, such variants are designed to (i) conformational neutral changes, i.e., they produce minimal changes in the tertiary structure of the variant polypeptide compared to the native polypeptide; And (ii) antigenically neutral changes, i.e., they are designed to produce minimal changes in the antigenic determinants of the variant polypeptide as compared to the native polypeptide.

  The above polypeptides may be used according to the present invention for the manufacture of a medicament for use in the treatment of diseases or conditions 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, said method comprising a) DEF5 (1) to a mammal, including a human having said disease or condition. -94) (SEQ ID NO: 1) or a fragment of DEF5 (1-94); b) a biologically active polypeptide having at least 50% identity with the amino acid sequence of any one of the polypeptides of (a); or c ) Administering an effective amount of a polypeptide selected from the group consisting of a biologically active variant of any one of the polypeptides of (a) or (b). Thus, the above polypeptides can be administered. The polypeptide is preferably DEF5 (1-94), or DEF5 (20-94), DEF5 (23-94), DEF5 (29-94), DEF5 (56-94), DEF5 (60-94), Includes fragments thereof such as DEF5 (63-94), DEF5 (34-55) or DEF5 (76-87).

  A “mammal” for treatment purposes refers to any animal classified as a mammal, including humans, farm animals and animals from zoos, competition animals or pets such as dogs, horses, cats, sheep, pigs, cattle, etc. means. Preferably the mammal is a human.

  The term “treatment” refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already having the disease as well as those in which the disease should be prevented.

  A “disease” or “condition” is any condition for which treatment with a DEF5 (1-94) or DEF5 (1-94) fragment as described above and further below may be effective. This includes both chronic and acute diseases or conditions, as well as pathological conditions that are likely to result in the disease or condition in question.

  As used herein, non-limiting examples of diseases or conditions to be treated include any condition resulting from altered iron balance or iron transport in the body, iron overload, anemia or erythropoiesis. Examples of diseases or conditions associated with iron balance or iron transport are those associated with hemochromatosis, hereditary hemochromatosis, juvenile hemochromatosis, thalassemia, iron overload, anemia, sickle cell anemia.

  In another aspect of the invention, DEF5 (1-94) or a fragment of DEF5 (1-94) is used to treat an effective amount of DEF5 (1-94) or a fragment thereof in a disease or condition associated with iron balance or iron transport. As a suitable therapeutic agent for the treatment of the disease comprising administration to mammals including humans having

  Accordingly, provided is a DEF5 (1-94) or fragment thereof of the invention as described above, which is a pharmaceutical composition for use in a disease or condition associated with iron balance or iron transport, and a pharmaceutically acceptable carrier. The The DEF5 (1-94) fragment is preferably 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 present invention is administered in an effective amount.

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

  An “effective amount” of DEF5 (1-94) or a fragment, compound, or pharmaceutical composition thereof prevents and / or prevents iron overload, anemia, or erythropoiesis, eg, hemochromatosis, hereditary hemochroma An amount sufficient to produce a beneficial or desired result, including clinical results such as prevention and / or prevention of tosis, juvenile hemochromatosis, thalassemia, anemia, and / or sickle cell anemia.

  Such amount will also depend on the particular condition being treated, the severity of the condition, individual patient parameters, including age, health status, size and weight, duration of treatment, nature of combination therapy (if any), identification of administration It can vary depending on the knowledge of the attending physician, such as the route, and factors within the technique. These factors are well known to those skilled in the art and can be addressed only by routine experimentation.

  An effective amount can be administered in one or more doses, and can or may not be achieved in combination with another drug, compound, or pharmaceutical composition. Thus, an “effective amount” can be considered as administering one or more therapeutic agents, and a single agent can achieve or achieve the desired result in combination with one or more other agents. Can be considered to be given in an effective amount.

  An effective amount of a DEF5 (1-94) or DEF5 (1-94) fragment of the invention or a pharmaceutical composition comprising the polypeptide, alone or in combination with another drug, compound, or pharmaceutical composition, is: It can be administered by any conventional route including injection or gradual infusion over time. Administration can be, for example, oral, intravenous, intraperitoneal, intramuscular, intracavity, subcutaneous, topical or transdermal.

  Upon administration, the pharmaceutical composition of the present invention is administered in a pharmaceutically acceptable formulation. The term “pharmaceutically acceptable carrier” as used herein means one or more compatible solid or liquid fillers, diluents or encapsulants suitable for administration to mammals, including humans. The term “carrier” means an organic or inorganic component, a natural or synthetic component, with which the active ingredients are combined to facilitate application.

  The term “pharmaceutically acceptable” means a non-toxic substance that does not interfere with the biological activity of the active ingredient. Such formulations usually include pharmaceutically acceptable concentrations of salts, buffers, preservatives, compatible carriers, adjuvants such as adjuvants and cytokines, and optionally other chemotherapeutic agents such as chemotherapeutic agents. A therapeutic agent may be included.

  When used in medicine, the salt should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts can be conveniently used to produce the pharmaceutically acceptable salts of the present invention. Not excluded from the scope.

  The pharmaceutical composition can include suitable buffers, including acetate; citrate; borate; and phosphate.

  Pharmaceutical compositions can also optionally include suitable preservatives such as benzalkonium chloride; chlorobutanol; parabens and thimerosal.

  The dose of the polypeptide or nucleic acid encoding the polypeptide to be administered to a subject can be selected according to different parameters, in particular according to the method of administration used and the condition of the subject. Other factors include the desired duration of treatment. Higher doses (or effective higher doses through different, more localized delivery routes) can tolerate patients if the response in the subject is insufficient with the initial dose applied It can be used within the range of

  The pharmaceutical composition may conveniently be present in unit dosage form and may be manufactured by any method well known in the pharmaceutical art. All methods include the step of using the active agent with a carrier that constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately using the active compound together with a liquid carrier, a finely divided solid carrier or both, and then, if necessary, shaping the product.

  Compositions suitable for oral administration may exist as discrete units, each containing a predetermined amount of active compound, such as capsules, tablets, or lozenges. Other compositions include suspensions in aqueous or non-aqueous liquids such as sprays, elixirs or emulsions.

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

  Suitable carrier formulations for oral, subcutaneous, intravenous, intramuscular administration and the like can be found in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA.

  Another aspect of the invention is to detect the level of DEF5 (1-94) or a fragment thereof in a biological sample taken from a subject for diagnosis (where the altered level is iron balance or iron A method for prognosis or diagnosis of a disease or condition associated with iron balance or iron transport, including a disease or condition associated with transport. One embodiment of the present invention provides (i) detecting the level of DEF5 (1-94) or a fragment thereof in a biological sample obtained from an elephant to provide a first value pair; and ( ii) comparing the first value to the level of DEF5 (1-94) or a fragment thereof from a patient who does not have the disease or condition, wherein DEF5 (1-94 in the sample from the subject without the disease Or a change in the level of the biological sample from the subject relative to the level of the fragment indicates a subject susceptible to or having a disease or condition associated with iron balance or iron transport). Methods for prognosis or diagnosis of a disease or condition associated with iron balance or iron transport in a subject are provided.

  Such biological samples include blood or plasma or tissue samples. Suitable tissue samples include whole blood, semen, saliva, tears, urine, stool, sweat, cheek cells, skin, and biological tissue of specific organ tissues such as muscle, brain or nerve tissue and hair. Most preferably, suitable biological samples include blood or plasma. Tissue samples also include cells and cell types isolated from such biological samples. Changes in levels of DEF5 (1-94) or fragments thereof that may be in accordance with a preferred embodiment of the present invention include DEF5 (1 found in individuals who do not have diseases or conditions associated with iron balance or iron transport in the present invention. -94) or increased plasma levels of DEF5 (1-94) or fragments thereof as compared to plasma levels of DEF5 (1-94) fragments. The increase is preferably at least 1.2 times, more preferably at least 1.5 times, 2 times, 3 times, 5 times or 10 times.

  In accordance with further aspects, the present invention provides i) detecting the expression level of at least one gene listed in Table 1 in a sample of suitable tissue obtained from a subject and providing a first value; and ii) Comparing the first value to the expression level of the gene from a subject without disease (where a higher or lower expression level in the subject sample compared to a sample from a subject without disease) A method for prognosis or diagnosis of a disease or condition associated with iron balance or iron transport, comprising a subject or predisposed to having or having a disease or condition associated with iron balance or iron transport. provide. Gene expression can be detected at the mRNA or protein level. Suitable tissues include, but are not limited to, the liver, heart, intestine such as the duodenum, spleen, and bone marrow. mRNA expression levels can be detected by any suitable technique, such as microarray analysis, Northern blot analysis, reverse transcription PCR and real-time quantitative PCR. Similarly, protein levels can be detected by any suitable technique, for example, by Western blot using a labeled probe specific for the protein.

  In a preferred embodiment of the above aspect, the gene (s) is selected from the gene (s) listed in Table 1 that are up-regulated or down-regulated. Preferably, the gene (s) are up-regulated 1.2 times or more, 1.3 times or more, or 1.5, 1.7, 1.8, 1.9 times or more as described in Table 1. The gene (s); or alternatively, selected from the gene (s) listed in Table 1 that are down-regulated to less than 0.8, 0.7, or 0.6. In yet another preferred embodiment of the above aspect, the expression of at least 1, 2, 3, 4, 5, 10, 20, 30 genes is measured. In another aspect of the invention, the expression of at least 40, 50 or 60 genes selected from Table 1 is measured. A further aspect of the invention is that the expression of most genes selected from Table 1, such as the expression of at least 65, 70, 80, 90, 100, or at least 110 genes is determined or It provides that the expression of all 1 genes is determined.

  A further aspect of the invention provides that i) a test compound under the sample conditions that permits at least one biological activity of DEF5 (1-94) or DEF5 (1-94) fragment, Contacting the fragment; ii) determining the level of the at least one biological activity of the DEF5 (1-94) / DEF5 (1-94) fragment; iii) controlling the level with the test compound A method of identifying a modulator of a disease or condition associated with iron balance or iron transport comprising comparing to the level of a sample is provided. In a preferred embodiment, the test compound that results in the change in level is DEF5 (1-94) or DEF5 (1) as a modulator for prophylactic and / or therapeutic treatment of diseases or conditions associated with iron balance or iron transport. -94) The fragments are selected for further testing. In preferred embodiments, the level of biological activity of DEF5 (1-94) or DEF5 (1-94) fragment is one or more, such as at least 61, 70 or 100 genes listed in Table 1, or a plurality of It is measured by detecting the expression level of the gene.

  Examples of modulators of diseases or conditions associated with iron balance or iron transport include, but are not limited to, antisense nucleotides, ribozymes, double stranded RNA and antagonists.

  As used herein, the term “antisense” includes, 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) means a nucleotide sequence complementary to a portion of an RNA expression product encoding a polypeptide of the invention. By “complementary” nucleotide sequence is meant a nucleotide sequence that can base pair according to standard Watson-Crick complementarity rules, such as, for example, the base paired with SEQ ID NO: 10. That is, purines can base pair with pyrimidines and form guanine: cytosine and adenine: thymine in the case of DNA or adenine: uracil in the case of RNA. Other rare bases such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine, etc. may be included in the hybridizing sequence and they will not interfere with pairing.

  When introduced into a host cell, an antisense nucleotide sequence can be attached to the gene (s) to inhibit expression of the encoded protein, eg, by inhibiting transcription and / or translation within the cell. It specifically hybridizes with the corresponding cellular mRNA and / or genomic DNA.

  An isolated nucleic acid molecule comprising an antisense nucleotide sequence can be used, for example, as an expression vector that, when transcribed in a cell, yields RNA that is complementary to at least a unique portion of the encoded mRNA of the gene (s), Can be delivered. Alternatively, an isolated nucleic acid molecule comprising an antisense nucleotide sequence is produced ex vivo and, when introduced into a cell, results in coding by hybridizing to the mRNA and / or genomic sequence of the gene (s). An oligonucleotide probe that inhibits the expression of the generated protein.

  Preferably, the oligonucleotide comprises an artificial internucleotide linkage that represents an antisense molecule that is resistant to exonucleases and endonucleases and is therefore stable in the cell. Examples of modified nucleic acid molecules for use as antisense nucleotide sequences include, for example, the phosphates described in US Pat. Nos. 5,176,996; 5,264,564; and 5,256,775. Analogs of amide, phosphate thio and methyl phosphate DNA. General methods for producing oligomers useful for antisense therapy are described, for example, 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 methods involve the production of either DNA or RNA oligonucleotides that are complementary to the encoded mRNA of a gene. Antisense oligonucleotides can hybridize to the encoded mRNA of the gene and inhibit translation. The ability of an antisense nucleotide sequence to hybridize with a desired nucleic acid can depend on the degree of complementarity and length of the antisense nucleotide sequence. Typically, increasing the length of a hybridizing nucleic acid increases the number of mismatched bases with RNA that can be included, but forms a stable duplex or triplex. One skilled in the art can determine the degree of mismatch tolerance by using conventional methods to determine the melting point of the hybridized complex.

  The antisense oligonucleotide is preferably designed to be complementary to the 5 ′ end of the mRNA, eg, to the mRNA start site, ie, to the region complementary to the AUG and to the 5 ′ untranslated sequence containing it. . However, an oligonucleotide sequence complementary to the 3 ′ untranslated sequence of mRNA may be effective for inhibiting translation of mRNA as described in, for example, Wagner, Nature, Vol. 372, pp. 333 (1994). It is shown. Antisense oligonucleotides can be designed to be complementary to the mRNA coding region, but such oligonucleotides are less effective inhibitors of translation.

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

  Antisense nucleotides can also include at least one modified base moiety, such as 3-methylcytosine, 5-methylcytosine, 7-methylguanine, 5-fluorouracil, 5-bromouracil, and at least one Modified sugar moieties such as arabinose, hexose, 2-fluoroarabinose, and xylulose.

  In another embodiment, the antisense nucleotide sequence is an α-anomeric nucleotide sequence. The α-anomeric nucleotide sequence forms a specific double-stranded hybrid with complementary RNA, which differs from the normal β unit, for example, Gautier et al., Nucl. Acids. Res., Vol. 15, pp. They are parallel to each other as described in 6625-6441 (1987).

  Antisense nucleotides are specific for receptors or antigens expressed on the cell surface by various techniques such as direct injection into the relevant tissue, encapsulation of the antisense nucleotide in liposomes. By administering a modified antisense nucleotide that targets the relevant cell by linking with a binding peptide or antibody, it can be delivered in vivo to a cell that expresses the described gene.

  The term “ribozyme” refers to an RNA molecule that specifically cleaves other single-stranded RNA in a manner similar to a DNA restriction endonuclease. By modifying the nucleotide sequence encoding RNA, ribozymes can be synthesized and recognize specific nucleotide sequences in the molecule, eg, Cech, J. Amer. Med. Assn., Vol. 260, p. 3030 ( Cut it as described in 1988). Thus, only mRNA with a specific sequence is cleaved and inactivated.

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

  Ribozymes can be delivered directly into cells in the form of RNA oligonucleotides containing ribozyme sequences, or introduced into cells as expression vectors encoding the desired ribozyme RNA. Ribozyme sequences can be modified in a manner substantially similar to that described for antisense nucleotides, for example, a ribozyme sequence can comprise a modified base moiety.

  For example, the term “double-stranded RNA”, ie sense-antisense RNA, corresponding to at least one nucleic acid encoding a polypeptide of the invention such as SEQ ID NO: 10, is also expressed at least of the disclosed gene. Can be used to prevent single expression. Inhibition of endogenous gene function and expression by double-stranded RNA is described for nematodes, for example, as described in Fire et al., Nature, Vol. 391, pp. 806-811 (1998); for Drosophila, for example , Kennerdell et al., Cell, Vol. 95, No. 7, pp. 1017-1026 (1998); and for mice, eg Wianni et al., Nat. Cell Biol., Vol. , No. 2, pp. 70-75 (2000). Such double stranded RNA can be synthesized by in vitro transcription of single stranded RNA read from both template orientation and in vitro annealing of the sense and antisense RNA strands. Double stranded RNA can also be synthesized from a cDNA vector construct, where the gene of interest is cloned in the reverse direction separated by inverted repeats. After cell infection, the RNA is transcribed and the complementary strand is reannealed.

  The term “antagonist” refers to DEF5 (1-94) or a fragment thereof, such as 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) when bound to a polypeptide of the present invention that reduces or inhibits at least one biological activity of the polypeptide. Means a molecule. Antagonists include, but are not limited to, peptides, proteins, carbohydrates, and small molecules.

  In particularly useful embodiments, the antagonist is 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 can also be conjugated with reagents such as chemotherapeutic agents, radionuclides, ricin A chain, cholera toxin, pertussis toxin, etc. and function as a target agent.

  In yet another embodiment, the antagonist is an iron balance or iron, such as a condition associated with hemochromatosis, hereditary hemochromatosis, juvenile hemochromatosis, thalassemia, iron overload, anemia, sickle cell anemia, etc. Useful as therapeutic agents for treating diseases or conditions associated with transport.

  In the case of treatment with antisense nucleotides, the method comprises therapeutic treatment of an isolated nucleic acid molecule comprising an antisense nucleotide sequence derived from SEQ ID NO: 10 or a nucleic acid that hybridizes to SEQ ID NO: 10 under stringent conditions. Administration of an effective amount.

  The term “isolated” nucleic acid molecule means that the nucleic acid molecule has been removed from its original environment (eg, the natural environment when it is naturally occurring). For example, a naturally occurring nucleic acid molecule has not been isolated, but the same nucleic acid molecule separated from some or all of the coexisting materials in the natural system may later be reintroduced into the natural system. Has also been isolated. Such nucleic acid molecules could be further isolated in that they are part of a vector, or part of a composition, and such a vector or composition is not part of its natural environment.

  For treatment with ribozymes or double stranded RNA molecules, the method includes administering a nucleotide sequence encoding a ribozyme, or a therapeutically effective amount of a double stranded RNA molecule, wherein the ribozyme / double stranded The nucleotide sequence encoding the RNA molecule has the ability to reduce transcription / translation of DEF5 (1-94) or fragments thereof.

  A “therapeutically effective amount” of an isolated nucleic acid molecule comprising an antisense nucleotide, a nucleotide sequence encoding a ribozyme, a double stranded RNA or an antagonist is, for example, hemochromatosis, hereditary hemochromatosis, juvenile hemochroma Mean sufficient amount of one of these therapeutics to treat a disease or condition associated with iron balance or iron transport, such as a condition associated with tosis, thalassemia, iron overload, anemia, sickle cell anemia To do.

  The determination of a therapeutically effective amount is well within the capability of those skilled in the art. For any therapeutic agent, a therapeutically effective dose can be estimated initially either in cell culture assays or in animal models that are usually mice, rabbits, dogs, or pigs. The animal model may also be used to determine the appropriate concentration range and route of administration. Thus, such information can be used to determine useful doses and routes for administration in humans.

  For therapeutic applications, antisense nucleotides, nucleotide sequences encoding ribozymes, double stranded RNA (encapsulated in liposomes or contained in viral vectors) and antibodies preferably contain one therapeutic agent. It is administered as a pharmaceutical composition comprising the above pharmaceutically acceptable carrier. The composition is administered alone or in conjunction with other agents such as at least one stabilizing compound, which may include any sterile, including but not limited to saline, buffered saline, dextrose and water. It can be administered in a biocompatible pharmaceutical carrier. The composition can be administered to a subject alone or with other drugs, drugs or hormones.

Experiment 1
Method DEF5 (60-94) is administered subcutaneously to male C57BL / 6 mice at doses of 300, 600 or 1000 μg / day for 7 to 14 days. At the end of the treatment period, samples from all organs are snap frozen at dissection and analyzed using GeneChip® expression profile analysis.

  Total RNA is extracted from these frozen tissues using TRIzol reagent (Life Technologies) according to the manufacturer's instructions. Total RNA is quantified at an absorbance at 260 nm (A260 nm) of 1 and the purity is estimated at the A260 nm / A280 nm ratio. Integrity is checked by denaturing gel electrophoresis. RNA is stored at −80 ° C. until analysis. Good quality total RNA is used to synthesize double stranded cDNA using the Superscript Choice system (Life Technologies). Thereafter, the cDNA is transcribed in vitro (MEGAscriptau T7 Kit, Ambion) to form a biotin-labeled cRNA. Next, 12 to 15 mg of labeled cRNA is hybridized with the Affymetrix mouse MOE430A expression probe array at 45 ° C. for 16 hours. The array is then washed according to the EukGE-WS2 protocol (Affymetrix) and stained with 10 mg / ml streptavidin-phycoerythrin complex (Molecular Probes). Signals were 2 mg / ml acetylated BSA (Life Technologies), 100 mM MES, 1 M [Na +], 0.05% Tween 20, 0.005% Antifoam (Sigma), 0.1 mg / ml goat IgG Antibody-amplify with 0.5 mg / ml biotinylated antibody and re-stain with streptavidin solution. After washing, the array is scanned twice using a Gene Array® scanner (Affymetrix).

  Expression levels are estimated by averaging the difference in signal intensity measured by oligonucleotide pairing of a given probe (AvgDiff value). The image acquisition and numerical conversion software used for this study is Affymetrix Microarray Suite version 5 (MAS5). To identify the genes affected by the treatment, the dataset is systematically low in values with high experimental noise (AvgDiff value is at least 50 in the number of experiments corresponding to the minimum number of replicates at any experimental point). First filtered to remove the first analysis wave of genes in the expression range. In the second stage of selection, the threshold t-test p-value (0.05) is calculated as a two-component error model using the multiple hypothesis test corrected reduction method (Benjamini and Hochberg's false discover rate), if possible. Identify genes that have different values between processing and non-processing based on (global error model).

  The selected gene list is then compared to the established gene list for pathways and cellular components using Fisher's exact test. Venn diagrams are used to identify common genetic changes between different organs. Relevant gene expression profiles are used to find genes with correlated changes at individual experimental points using several distance metrics (standard, Pearson).

  The decision that a particular gene is considered relevant is that the numerical changes identified by the preliminary filtering and statistical algorithms described above co-occur and other regulatory genes that exhibit a common biological theme. Based on relationships.

Results Table 1 shows that at the RNA level, DEF5 (60-94) affects genes involved in heme biosynthesis and metabolism, porphyrin biosynthesis and metabolism, and erythrocyte production. Furthermore, dietary iron balance, genes involved in iron transport are also specifically controlled by infusion of DEF5 (60-94). Most genes are upregulated by at least 1.5 times or more, or downregulated by at least 0.7 times or less.

  Examples of genes that are upregulated include, but are not limited to, ceruloplasmin, inhibitors of DNA binding 4, ect2 oncogene, serine active site containing 1, matrix metalloproteinase 9, and steroidogenic acute regulatory proteins. Of particular interest is the upregulation of erythropoiesis and iron homeostasis, such as ceruloplasmin (a copper protein with ferodixidase activity to convert Fe2 + to Fe3 +) in the presence of oxygen. Associated gene (see Sarkar et al. (2003) J. Biol. Chem, 278: 44018-44024).

Examples of genes that are down-regulated include coproporphyrin oxidase, hydroxymethyl bilan synthase, ferrochelase, transferrin receptor, glycophorin A, aminolevulinate delta-dehydratase, alpha thalassemia / mental retardation syndrome X-binding homolog (human), cruper Like factor 1 (red blood cells) and aquaporin 1 are included but not limited to. Of particular interest are those associated with iron transport such as transferrin receptor (Aisen, (2004), 36: 2137-2143); those involved in chromatin remodeling pathways such as ATRX (Gibbons et al. (2003) Nat. Genet. 34: 446-449); transcription modulators restricted by cells involved in hematopoiesis such as KLF-1 (Bieker, (2005) Mt. Sinai J. Med. 72: 333-338) Genes associated with hemodialysis such as aquaporin (Ruello et al. (2002) Nephron 92: 846-852); and genes associated with pathways that protect against toxicity such as ALAD (Kim et al. (2004) Environ. Health Perspect, 112: 538-541).

  (Not described in the original)

Claims (24)

For the manufacture of a medicament for use in the treatment of diseases or conditions associated with iron balance or iron transport,
a) DEF5 (1-94) (SEQ ID NO: 1) or a fragment of DEF5 (1-94);
b) a bioactive polypeptide having at least 50% identity with any one amino acid sequence of the polypeptide of (a);
c) Use of a polypeptide selected from the group consisting of biologically active variants of any one of the polypeptides of (a) or (b).   A disease or condition associated with iron balance or iron transport is selected from the group consisting of hemochromatosis, hereditary hemochromatosis, juvenile hemochromatosis, thalassemia, iron overload, anemia, sickle cell anemia The use of a polypeptide according to claim 1.   Use of the polypeptide according to claim 1 or 2, wherein the fragment of DEF5 (1-94) comprises a C-terminal fragment of DEF5 (1-94).   The use of a polypeptide according to any one of claims 1 to 3, wherein the C-terminal fragment of DEF5 (1-94) comprises at least 10 amino acids at the C-terminus of DEF5 (1-94).   The C-terminal fragment of DEF5 (1-94) has the 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. Use of the polypeptide according to any one of the above.   The use of a polypeptide according to claim 5, wherein the C-terminal fragment of DEF5 (1-94) has the amino acid sequence of SEQ ID NO: 6.   The use of a polypeptide according to any one of claims 1 to 6, wherein the polypeptide is encoded by a nucleic acid which hybridizes with SEQ ID NO: 10 under stringent conditions. a) DEF5 (1-94) (SEQ ID NO: 1) or a fragment of DEF5 (1-94);
b) a bioactive polypeptide having at least 50% identity with any one amino acid sequence of the polypeptide of (a);
c) administering an effective amount of a polypeptide selected from the group consisting of any one of the biologically active variants of the polypeptide of (a) or (b) to a mammal including a human having the disease. A method of treating a disease or condition associated with iron balance or iron transport.   A disease or condition associated with iron balance or iron transport is selected from the group consisting of hemochromatosis, hereditary hemochromatosis, juvenile hemochromatosis, thalassemia, iron overload, anemia and sickle cell anemia 9. The method of claim 8, wherein:   10. The method of claim 8 or 9, wherein an effective amount of the polypeptide is administered intravenously, intramuscularly, subcutaneously, orally or topically.   11. A method according to 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 according to any one of claims 8 to 11, wherein the C-terminal fragment of DEF5 (1-94) comprises at least 10 amino acids at the C-terminus of DEF5 (1-94).   The C-terminal fragment of DEF5 (1-94) has the 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. The method as described in any one of.   14. The method according to any one of claims 8 to 13, wherein the C-terminal fragment of DEF5 (1-94) has the 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 that hybridizes to SEQ ID NO: 10 under stringent conditions.   A pharmaceutical composition for use in a disease or condition associated with iron balance or iron transport, comprising an effective amount of the polypeptide of claims 1 and 3 to 7 and a pharmaceutically acceptable carrier.   17. The pharmaceutical composition according to claim 16, comprising an effective amount of a fragment (60-94) of DEF5 (SEQ ID NO: 6). (I) detecting the level of DEF5 (1-94) or a fragment thereof in a biological sample obtained from the subject to obtain a first value; and (ii) determining the first value as a disease or condition. A method for prognosing or diagnosing a disease or condition associated with iron balance or iron transport in a subject comprising the step of comparing the level of DEF5 (1-94) or a fragment thereof from a subject without A change in level in a biological sample from a subject, as compared to the level of DEF5 (1-94) or a fragment thereof in a sample from a subject that does not have, affects a disease or condition associated with iron balance or iron transport. A method that is easy or is indicative of a subject having it.   The method of claim 18, wherein the biological sample is plasma. (I) detecting the expression level of at least one gene listed in Table 1 in a biological sample obtained from the subject to obtain a first value; and (ii) determining the first value as a disease or condition A method for prognosing or diagnosing a disease or condition associated with iron balance or iron transport, comprising the step of comparing the expression level of the gene from a subject without In comparison, a method wherein a higher or lower expression level in a subject sample is susceptible to or has a disease or condition associated with iron balance or iron transport.   21. The method of claim 20, wherein the expression levels of a number of genes listed in Table 1 are detected. (I) contacting the test compound with DEF5 (1-94) or a fragment thereof under sample conditions that permit at least one biological activity of the DEF5 (1-94) or DEF5 (1-94) fragment;
(Ii) determining the level of the at least one biological activity of the DEF5 (1-94) or DEF5 (1-94) fragment;
(Iii) comparing the level to the level of a control sample lacking the test compound; and (iv) DEF5 for prophylactic and / or therapeutic treatment of diseases or conditions associated with iron balance or iron transport ( 1-94) A method of identifying a modulator of a disease or condition associated with iron balance or iron transport comprising selecting a test compound that alters the level for further testing as a modulator.   23. The method of claim 22, wherein the level of biological activity of the DEF5 (1-94) or DEF5 (1-94) fragment is measured by determining the expression level of one or more genes listed in Table 1. .   24. The method of claim 22 or 23, wherein the expression levels of a number of genes listed in Table 1 are detected.

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