WO2002083728A2 - Splice variant - Google Patents

Abstract

This invention relates to a novel isoform of the EGLN3 gene and the product of this gene, that has been implicated in certain disease states. This novel isoform lacks an internal stretch of nucleotides form the wild type gene, probably due to an alternative splicing event. Both the full length EGLN3 protein and EGLN3 splice variant have been shown to be biologically active. Overexpression of both these isoforms reduce HIF-mediated gene expression through HRE reporters, thus demonstrating their role in the HIF signalling pathway. The suppression effect of the EGLN3 splice variant appears to be stronger than that of the full length EGLN3 protein.

Description

Splice variant

This invention relates to novel genes and gene products that are implicated in certain disease states.

All publications, patents and patent applications cited herein are incorporated in full by reference.

Over recent years, the so-called "genomics revolution" has allowed access to large portions of whole genomes, including the human genome. The amount of sequence information now available considerably facilitates the analysis of the results of experiments that aim to elucidate the functions of proteins that are expressed in the body. As this information increases in scope and becomes more readily available, the study of the molecular mechanism of disease, and the elucidation of techniques for combating these diseases will be considerably facilitated.

One particular physiological condition that has considerable relevance to human and other animal disease is the cellular response to hypoxia. The term "hypoxia" is intended to refer to an environment of reduced oxygen tension, as compared to the normal physiological environment for a particular organism, which is termed "normoxia".

In a variety of human diseases, cells are exposed to conditions of low oxygen tension, usually as a result of poor oxygen supply to the diseased area. For instance, tissue oxygenation plays a significant regulatory role in both apoptosis and in angiogenesis (Bouck et al, 1996, Adv. Cancer Res. 69:135-174; Bunn et al, 1996, Physiol. Rev. 76:839-885; Dor et al, 1997, Trends Cardiovasc. Med., 7:289-294; Carmeliet et al, 1998, Nature 394:485-490). Apoptosis (see Duke et al, 1996, Sci. American,, 80-87 for review) and growth arrest occur when cell growth and viability are reduced due to oxygen deprivation. Angiogenesis (i.e. blood vessel growth, vascularization), is stimulated when hypooxygenated cells secrete factors that stimulate proliferation and migration of endothelial cells in an attempt to restore oxygen homeostasis (for review see Hanahan et al, 1996, Cell, 86:353-364).

Ischaemic disease pathologies involve a decrease in the blood supply to a bodily organ, tissue or body part generally caused by constriction or obstruction of the blood vessels. For example, solid tumours typically have a disorganised blood supply, leading to hypoxic regions. One particular example of an ischaemic disease pathology is myocardial ischaemia, which encompasses several chronic and acute cardiac pathologies that involve the deprivation of the myocardium of its blood supply, usually through coronary artery occlusion. A key component of ischaemia is hypoxia. Following transient ischaemia, the affected tissue may be subjected to reperfusion and re-oxygenation, and this is of significance in its own right.

Ischaemia/reperfusion is well known to induce cell death in myocardial tissue by apoptosis, leading to impaired function of the myocardium and infarction. Many of the specific molecules required to execute the process of apoptosis are known, but not all of these molecules have been characterised in detail. Cell death may also proceed by a distinct process called necrosis, which unlike apoptosis, is not initiated and controlled by specific and dedicated cellular and biochemical mechanisms (see Nicotera et al, Biochem Soc Symp. 1999; 66:69-73). There is substantial evidence that apoptotic cell death occurs either during or after myocardial ischaemia (Kajstura et al, Lab Invest. 1996; 74(1):86-107; Cheng et al, Exp Cell Res. 1996; 226(2):316-27; Fliss and Gattinger, Circ Res. 1996; 79(5):949-56; Veinot et al, Hum Pathol. 1997; 28(4):485-92; Bialik et al, J Clin Invest. 1997; 100(6): 1363-72; Gottlieb et al, J Clin Invest. 1994; 94(4):1621-8; Gottlieb and Engler, Ann N Y Acad Sci. 1999; 874:412-26). In the laboratory, apoptosis is also induced by subjecting cardiac myocytes to hypoxia (Tanaka et al, Circ Res. 1994 Sep;75(3):426-33; Long et al, J Clin Invest. 1997 99(11): 2635-43).

Clearly, there is a significant clinical application were a successful method to inhibit apoptosis in ischaemic myocardial tissue to be devised. A specific and effective treatment requires identifying biochemical target(s), which are responsible for mediating apoptosis, specifically in ischaemic myocardial cells. One target which plays a common role in mediating apoptosis in many cell types, namely p53, is not involved in apoptosis resulting from myocardial ischaemia (Bialik et al, J Clin Invest. 1997; 100(6): 1363-72). Others have shown that inhibiting key mediators of apoptosis, caspases, provides protection against lethal reperfusion injury, following myocardial ischaemia in rat models (Mocanu et al, Br J Pharmacol. 2000; 130(2): 197-200; Yaoita et al, Circulation. 1998 97(3): 276-81; Holly et al, J Mol Cell Cardiol. 1999 31(9): 1709-15). However, this approach lacks specificity, since the caspases play a key role in mediating apoptosis in the majority of mammalian cell types, where it is usually beneficial. An approach that involves modulating the activity of molecules shown specifically to mediate apoptosis in ischaemic cardiac cells, would present a distinct advantage in both specificity and efficacy. Other disease conditions involving hypoxia include stroke, atherosclerosis, retinopathy, acute renal failure, myocardial infarction and diseases involving infection of the airways (such as cystic fibrosis). Therefore, apoptosis and angiogenesis as induced by the ischaemic condition are also considered to be involved in these disease states. It is generally considered that understanding the mechanism by which cells respond to these diseases may be the key to the disease pathology and thus relevant to disease treatment.

In a different but related approach, it is now recognised that angiogenesis is necessary for tumour growth and that retardation of this process provide a useful tool in controlling malignancy and retinopathies. For example, neoangiogenesis is seen in many forms of retinopathy and in tumour growth. The ability to be able to induce tumourigenic cells to undergo apoptosis is an extremely desirable goal; particularly in the cancer field, it has been observed that apoptosis and angiogenesis-related genes provide potent therapeutic targets. It has also been observed that hypoxia plays a critical role in the selection of mutations that contribute to more severe tumourigenic phenotypes (Graeber et al, 1996 Nature, 379(6560):88-91).

A number of genes have been identified that are implicated in the physiological response to hypoxia. Early in the history of this field it was discovered that a transcription factor, HIF- 1 alpha, is ubiquitously present in cells and is responsible for the induction of a number of genes in response to hypoxia. This protein is considered a master regulator of oxygen homeostasis (see, for example, Semenza, (1998) Curr. Op. Genetics and Dev. 8:588-594). Where HIF1 alpha is genetically knocked out, the hypoxia-inducible transcription of virtually all glycolytic enzymes has been shown to be inhibited. Glycolysis is an essential process which goes on in all mammalian cells. This finding is therefore consistent with previous work showing that when cells are exposed to conditions of hypoxia, they up-regulate glycolytic enzymes to enable ATP production, since oxidative phosphorylation is no longer feasible under conditions of low oxygen (Webster (1987) Mol.Cell.Biochem, 77: 19-28). Further support for a critical and general role of HIF1 alpha in the hypoxic response is demonstrated by the knockout mouse, which dies at day 10.5 of gestation. The same is true of the knockout of the ARNT protein, the dimerisation partner of HIF1 alpha.

Although HIF-lα is well known to mediate responses to hypoxia, other transcription factors are also known or suspected to be involved. These include a protein called endothelial PAS domain protein 1 (EPAS1) or HIF-2α which shares 48% sequence identity with HIF-lα (Tian et al, Genes Dev. 1997 Jan l;ll(l):72-82.). Evidence suggests that EPAS1 is especially important in mediating the hypoxia-response in certain cell types, and it is clearly detectable in human macrophages, suggesting a role in this cell type (Griffiths et al, 2000, Gene Ther., 7(3):255-62).

However, there remains a great need for the identification of other genes that are implicated in this condition, in order to develop a spectrum of diagnostic and therapeutic agents for use as tools in combating diseases in which hypoxia plays a role. Such genes and the proteins that they encode are candidate targets for antagonist or agonist agents that modulate human disease states. Furthermore, the identified genes are associated with regulatory elements that provide alternative and additional candidate targets for exploitation for the delivery of gene products in a cell-specific fashion. Any genes and regulatory elements identified as having a role in hypoxia may be used directly in therapeutic applications via gene therapy, via recombinant protein methods or via chemical mimetics or as targets for the development of agonists and antagonists such as antibodies, small chemical molecules, peptides, regulatory nucleic acids. According to the invention, a novel gene and its encoded protein are provided, that have been identified and functionally annotated for the first time.

One embodiment of the invention thus provides a substantially purified polypeptide, which polypeptide: i) comprises the amino acid sequence recited in SEQ ID No: 85a; ii) has an amino acid sequence encoded by a nucleic acid sequence recited in SEQ ID

No: 86a; iii) is a fragment of a polypeptide according to i) or ii), provided that said fragment retains a biological activity possessed by the full length polypeptide of i) or ii), or has an antigenic determinant in common with the polypeptide of i) or ii); or iv) is a functional equivalent of a polypeptide of i), ii) or (iii).

The polypeptide sequence recited in SEQ ID No: 85a was, prior to the present disclosure, was totally unknown in the literature and public sequence databases. Accordingly, until now, no biological function has been attributed to this polypeptide sequence. The inventors have now elucidated a biological function for this polypeptide, in that it has been found to be differentially regulated under physiological conditions of hypoxia. This polypeptide is also postulated to be active as a HIF proline hydroxylase.

The polypeptide sequence recited in SEQ ID No: 85a is a novel isoform of the polypeptide sequence recited in SEQ ID No: 85 (Protein accession number BAB 15101, encoded by Homo sapiens cDNA: FLJ21620 fis, clone COL07838 Nucleotide accession AK025273). The BAB15101 gene is now known as EGLN3. This gene was originally identified by the present inventors using Research Genetics Human GeneFilters arrays, which contain an EST corresponding to the gene (accession number R00332). The encoded protein sequence is referred to herein as SEQ ID No 85 and is presented below.

1 MPLGHI R D LΞKIALEYIV PCLHEVGFCY LDNFLGEWG DCVLΞRVKQ HCTGALRDGQ

61 LAGPRAGVSK RHLRGDQITW IGGNEEGCEA ISFLLS IDR LVLYCGSRLG KYYVKERSKA

121 MVACYPGNGT GYVRHVDNPW GDGRCITCIY Y NKNWDAK HGGILRIFPE GKSFIADVΞP

181 IFDRLLFFWS DRRNPHEVQP SYATRYAMTV YFDAEERAE AKKKFRN TR KTESALTED

Other public domain sequences corresponding to this gene include Homo sapiens cDNA: FLJ23265 fis, clone COL06456 Nucleotide accession AK026918.

A high degree of amino acid homology is observed between the protein encoded by this gene, and a rat protein called "Growth factor responsive smooth muscle protein" or "SM20" (Nucleotide accession U06713; Protein accession A53770). The cDNA sequence FLJ21620 fis (EGLN3) is thus considered to be a human equivalent of the rat gene SM20. Analysis of the human genome shows that there are in fact at least 5 human genes with similarity to rat SM-20 (Ensembl Protein Family ENSF00000003427). One of these human genes (from Chromosome 1) has been published, and was named Clorfl2, also known as EGLN1 (Dupuy et al, 2000, Genomics, 69(3):348-54). This gene has nucleotide accession AAG34568 (protein accession AAG34568) and is referred to herein as SEQ ID No 89). However, the gene corresponding to Homo sapiens cDNA: FLJ21620 fis, clone COL07838 (EGLN3) has not been sequenced and analysed by the human genome project (April 2001), and its exon / intron structure is therefore not in the public domain. From sequence tagged site information, the gene is thought to be on Chromosome 14.

An alignment of single letter amino acid sequences for rat SM20 and SEQ ID 85 (BAB 15101; EGLN3) is shown below. Over the highlighted region there is 97% amino acid similarity and 96% amino acid identity.

A53770 ( 1 ) MTLRSRRGFLSF PGLRPPRRWLRISKRGPPTSHWASPALGGRT HYSCR

BAB15101 ( 1 ) 51 100

A53770 ( 51 ) SQSGTPFSSEFQATFPAFAAKVARGPWLPQWEPPARLSASPLCVRSGQA

BAB15101 ( 1 )

101 150

A53770 ( 101 ) GACTLGVPR GSVSEMP GH1MRLDLEKIA EYIVPCLHEVGFCY DNF BAB15101 ( 1 ) MPLGHIMRLDLEKIA EYIVPCLHEVGFCYLDNF

151 200

A53770 (151) LGEWGDCV ERVKQLHYpALRDGQLAGPRAGVSKRHLRGDQITWIGGN

BAB15101 (35) LGEWGDCVLERVKQ HCTGALRDGQLAGPRAGVSKRH RGDQITWIGGN

201 __ __ 250 A53770 ( 201 ) EEGCEATJSfFLLSLIDR V YCGSR G TYVKERSKAMVACYPGNGTGYVR

BAB15101 ( 85 ) EEGCEAISFLLSLIDRLV YCGSRLGKYYV ERSKAMVACYPGNGTGYVR

251 300 A53770 ( 251 ) HVDKPNGDGRCITCIYYLNKN DAKLHGGVLRIFPEGKSFVADVEPIFDR

BAB15101 (135) HVDNPKGDGRCITCIYYLNKN DAKLHGGILRIFPEGKSFIADVEPIFDR 301 ^" _ _ 350

A53770 (301) iLLFSWSDRR PHEVQPSYATRYAMTV YFDAEERAEAKKRFRNLTRKTES

BAB15101 (185) LLFF SDRRNPHEVQPSYATRYAMTVWYFDAEERAEAKKKFRNLTRKTES 351

A53770 (351) ALAKD

BAB15101 (235) LTEΓJ

This high degree of amino acid similarity suggests that the human protein BAB 15101 has an equivalent biochemical function to the rat SM20 protein. Recent publications have shown that SM20 functions to promote apoptosis in neurons (Lipscomb et al, J Neurochem 1999; 73(l):429-32; Lipscomb et al, J Biol Chem 2000 Nov 1 ; [epub ahead of print]). Significantly, SM20 has been shown to be expressed at high levels in the heart (Wax et ai, J Biol Chem 1994; 269(17): 13041-7).

We have cloned and sequenced a novel cDNA isoform of FLJ21620 fis, clone COL07838, which lacks an internal stretch of nucleotides, probably due to an alternative splicing event. The nucleotide sequence of the sequenced portion of the new variant is shown below, and is referred to herein as SEQ ID No. 86a:

1 CTCGATTCTG CGGGCGAGAT GCCCCTGGGA CACATCATGA GGCTGGACCT

51 GGAGAAAATT GCCCTGGAGT ACATCGTGCC CTGTCTGCAC G*AGGCAATGG

101 TGGCTTGCTA TCCGGGAAAT GGAACAGGTT ATGTTCGCCA CGTGGACAAC

151 CCCAACGGTG ATGGTCGCTG CATCACCTGC ATCTACTATC TGAACAAGAA 201 TTGGGATGCC AAGCTACATG GTGGGATCCT GCGGATATTT CCAGAGGGGA

251 AATCATTCAT AGCAGATGTG GAGCCCATTT TTGACAGACT CCTGTTCTTC

301 TGGTCAGATC GTAGGAACCC ACACGAAGTG CAGCCCTCTT ACGCAACCAG

351 A ATGCTATG ACTGTCTGGT ACTTTGATGC TGAAGAAAGG GCAGAAGCCA

401 AAAAGAAATT CAGGAATTTA ACTAGGAAAA CTGAATCTGC CCTCACTGAA 451 GAC

The part of FLJ21620 fis, clone COL07838 which is missing in the above sequence at the position indicated by an asterisk, is shown below:

1 AGGTGGGCTT CTGCTACCTG GACAACTTCC TGGGCGAGGT GGTGGGCGAC

51 TGCGTCCTGG AGCGCGTCAA GCAGCTGCAC TGCACCGGGG CCCTGCGGGA

101 CGGCCAGCTG GCGGGGCCGC GCGCCGGCGT CTCCAAGCGA CACCTGCGGG

151 GCGACCAGAT CACGTGGATC GGGGGCAACG AGGAGGGCTG CGAGGCCATC

201 AGCTTCCTCC TGTCCCTCAT CGACAGGCTG GTCCTCTACT GCGGGAGCCG 251 GCTGGGCAAA TACTACGTCA AGGAGAGGTC TA At the protein level the new variant forms a functional protein, and retains the open reading frame, as shown below (SEQ ID No. :85a). This sequence starts and finishes with the same amino acid sequence but is missing an internal portion compared with the translation of

FLJ21620 fis, clone COL07838.

1 MPLGHIMR D EKIALEYIV PCLHEAMVAC YPGNGTGYVR HVDNPNGDGR

51 CITCIYYLNK N DAKLHGGI LRIFPEGKSF IADVEPIFDR LLFF SDRRN

101 PHEVQPSYAT RYAMTVWYFD AEERAEAKKK FRNLTRKTES ALTED

It has also been discovered that a polypeptide encoded by a gene identified from the EST recited in SEQ ID No 90, having the Protein accession number CAB81622, is regulated by hypoxia. The encoding human gene has been annotated in the UniGene database as "Similar to rat smooth muscle protein SM-20"; the nucleotide sequence is contained within the nucleotide accession AL117352. More recently, a longer fragment of this gene has been cloned, referred to above as Clorfl2 (Nucleotide accession AAG34568; Protein accession AAG34568; SEQ ID No 89 herein). This protein has been named EGLN1.

This distinct human gene, encoding a protein related to SM20 and to EGLN3 (BAB 15101), has been found by the inventors to be induced in response to hypoxia. This gene was identified using Research Genetics Human GeneFilters arrays, which contain an EST corresponding to the gene (accession number H56028). The protein sequence, SEQ ID No 89, is given below:

1 ANDSGGPGG PSPSERDRQY CELCGKMENL RCSRCRSSF YCCKEHQRQD WKKHKLVCQG

61 SEGALGHGVG PHQHSGPAPP AAVPPPRAGA REPRKAAARR DNASGDAAKG KVKAKPPADP

121 AAAASPCRAA AGGQGSAVAA EAEPGKEEPP ARSS FQEKA NLYPPSNTPG DALSPGGGLR 181 PNGQTKPLPA LKLALEYIVP CMNKHGICW DDFLGKETGQ QIGDEVRALH DTGKFTDGQL

241 VSQKSDSSKD IRGDKITWIE GKEPGCETIG LMSSMDDLI RHCNGKLGSY KINGRTKAMV

301 ACYPGNGTGY VRHVDNPNGD GRCVTCIYY NKD DAKVSG GILRIFPEGK AQFADIEPKF

361 DRLLFFWSDR RNPHEVQPAY ATRYAITVWY FDADERARAK VKYLTGEKGV RVE NKPSDS

421 VGKDVF

Independently to this, a fragment of this gene has been cloned from a cDNA library derived from hypoxic human cardiomyoblasts, and it has been shown that the gene is increased in expression in response to hypoxia in this cell type (see Table 1 herein; penultimate row). The nucleotide sequence of this cDNA fragment (SEQ ID No 90a) is: 1 ACCTCTACAG TTGTAAAAAG TATTAGATTC TACTATCTGT GGGTTGTGCT TGCCAGACAG

61 GTCTTAAATT GTATATTTTT TGGAAAAGTT TATATACTCT CTTAGGAATC ATTGTGAAAA

121 GATCAAGAAA TCAGGATGGC CATTTATTTA ATATCCATTC ATTTCATGTT AGTGGGACTA

181 TTAACTTGTC ACCAAGCAGG ACTCTATTTC AAACAAAATT TAAAACTGTT TGTGGCCTAT

241 ATGTGTTTAA TCCTGGTTAA AGATAAAGCT TCATAATGCT GTTTTTATTC AACACATTAA 301 CCAGCTGTAA AACACAGACC TT ATCAAGA GTAGGCAAAG ATTTTCAGGA TTCATATACA 361 GATAGACTAT AAAGTCATGT AATTTGAAAA GCAGTGTTTC ATTATGAAAG AGCTCTCAAG 421 TTGCTTGTAA AGCTAATCTA ATTAAAAAGA TGTATAAATG TTGTCAAAAA AAAAAAAAAA 481 AAAAGAAAAA AAGT

The data presented herein provide the first connection between these related genes and the physiological response to hypoxia. Recently published research papers have identified that the protein products of these genes can act as proline hydroxylases (see Bruick RK et al Science. 2001 294:1337-40 and Epstein AC et al Cell. 107:43-54). This is consistent with our observations that certain proline hydroxylases are induced in response to hypoxia and that the genes EGLN1 and EGLN3 are part of the hypoxia response. For example, two genes encoding proline hydroxylases have been identified as being increased in expression in response to hypoxia (proline 4-hydroxylase, alpha polypeptide 1; proline 4-hydroxylase, alpha polypeptide II; see co-pending International patent application PCT/GB01/05458). This identified a functional significance of proline hydroxylation as a response to hypoxia. A preferred embodiment of the invention thus includes methods for modulating the biological response to hypoxia by modulating the proline hydroxylase activity of the EGLN3 protein (BAB15101), the EGLN3 splice variant (SEQ ID No: 85a), the EGLN1 protein (clorfl2; AAG34568), and the CAB81622 and SM20 proteins.

Furthermore, a number of bacteria, such as moraxella, are thought to be involved in the initiation of inflammatory diseases. Many bacteria contain, within their genome, genes encoding proteins that share homology to the EGLN family of prolyl hydroxylases. We therefore propose that these bacterial genes may initiate a hypoxic like response at the site of infection thereby causing localised inflammation. The resulting inflammatory infiltrate could then cause the tissue to become hypoxic thereby continuing the cycle of hypoxia response. In the light of this novel discovery that these human equivalents of SM20 are induced by hypoxia, it is herein proposed that in cardiac ischaemia, the resulting apoptosis may be due at least in part, to increased expression of these genes. For example, the therapeutic modulation of the activity of EGLN3 (BAB 15101), EGLN3 splice variant (SEQ ID No: 85a), EGLN1 (clorfl2; AAG34568), CAB81622, SM20, and other equivalent proteins and encoding genes may therefore provide a novel means for the treatment of myocardial ischaemia, through the alteration of the propensity of myocardial cells to undergo apoptosis. For instance, a suitable treatment may involve altering the susceptibility of ischaemic myocardial tissue to subsequent reperfusion and re-oxygenation, or may involve modulating the susceptibility of chronic ischaemic myocardial tissue (including forms of angina) to later more severe ischaemia, which would result in myocardial infarction. It is submitted that, by way of analogy, cerebral ischaemia may be treated using the same principle.

These discoveries allow the development of regulators, such as small drug molecules, that affect the activity of these polypeptides, so allowing diseases and physiological conditions that are caused by hypoxia, or in which hypoxia has been implicated, to be treated. These discoveries also allow the development of diagnostic agents that are suitable for the detection of hypoxia in biological tissues and, through the identification of mutations and polymorphisms (such as SNPs) within genes coding for the proteins implicated herein, allows the assessment of an individual's risk of being susceptible to diseases and physiological conditions in which hypoxia is implicated.

The biological activity of polypeptides whose sequences are listed in SEQ ID Nos: 85, 85a and 89 has thus been found to be hypoxia-regulated. The expression of these polypeptides has been found to be induced under conditions of hypoxia. By "hypoxia-induced" is meant that the polypeptide is expressed at a higher level when a cell is exposed to hypoxia conditions as compared to its expression level under normoxic conditions. The term "hypoxia-repressed" as used herein is intended to mean that the polypeptide is expressed at a lower level when a cell is exposed to hypoxia conditions as compared to its expression level under normoxic conditions.

For the purposes of this document, the term "hypoxia" should be taken to mean an environment of oxygen tension such that the oxygen content is between about 5% and 0.1% (v/v). In most cases, hypoxic tissue will have an oxygen content that is less than or equal to about 2%. The term "normoxia" should be taken to mean conditions comprising a normal level of oxygen for the environment concerned. Normoxic tissue typically has an oxygen content above about 5%.

The polypeptide sequences whose amino acid sequence is presented in SEQ ID Nos 85 and 85a, or which are encoded by a nucleic acid sequence recited in SEQ ID Nos: 86 and 86a, were, prior to the present disclosure, unannotated in the literature and public sequence databases, meaning that until now, no biological function has been attributed to these polypeptide sequences. A biological function has now been attributed to the polypeptides that are encoded by genes incorporating cDNA and EST sequences that are set out above, in that these sequences have been found to be differentially regulated under physiological conditions of hypoxia. In the case of EST sequences, the sequences may not be part of the actual coding sequence for a gene, often representing regulatory regions of the gene, or regions that are transcribed, but not translated into polypeptide. Accordingly, this aspect of the invention also includes polypeptides that are encoded by a gene identified from the sequences recited in either of SEQ ID Nos: 86 or 86a.

Polypeptides of this aspect of the invention are intended to include fragments of polypeptides according to i) or ii) as defined above, provided that the fragment retains a biological activity that is possessed by the full length polypeptide of i) or ii), or has an antigenic determinant in common with the polypeptide of i) or ii). As used herein, the term "fragment" refers to a polypeptide having an amino acid sequence that is the same as part, but not all, of an amino acid sequence as recited in SEQ ID No: 85a, an amino acid sequence that is encoded by a nucleic acid sequence recited in SEQ ID No. 86a, or an amino acid sequence that is encoded by a gene that is linked to a nucleic acid sequence recited in SEQ ID No. 86a. The fragments should comprise at least n consecutive amino acids from the sequence and, depending on the particular sequence, n preferably is 7 or more (for example, 8, 10, 12, 14, 16, 18, 20 or more). Small fragments may form an antigenic determinant.

Such fragments may be isolated fragments, that are not part of or fused to other amino acids or polypeptides, or they may be comprised within a larger polypeptide, of which they form a part or region. When comprised within a larger polypeptide, a fragment of the invention most preferably forms a single continuous region. For instance, certain preferred embodiments relate to a fragment having a pre - and/or pro- polypeptide region fused to the amino terminus of the fragment and/or an additional region fused to the carboxyl terminus of the fragment. However, several fragments may be comprised within a single larger polypeptide.

The polypeptides of the present invention or their immunogenic fragments (comprising at least one antigenic determinant) can be used to generate ligands, such as polyclonal or monoclonal antibodies, that are immunospecific for the polypeptides. Such antibodies may be employed to isolate or to identify clones that express a polypeptide according to the invention or, for example, to purify the polypeptide by affinity chromatography. Such antibodies may also be employed as diagnostic or therapeutic aids, amongst other applications, as will be apparent to the skilled reader.

The term "immunospecific" means that an antibody has substantially greater affinity for a polypeptide according to the invention than their affinity for related polypeptides. As used herein, the term "antibody" is intended to include intact molecules as well as fragments thereof, such as Fab, F(ab')₂ and scFv, which are capable of binding to the antigenic determinant in question. The invention also includes functional equivalents of a polypeptide of i), ii) or (iii) as recited above. A functionally-equivalent polypeptide according to this aspect of the invention may be a polypeptides that is homologous to a polypeptide whose sequence is explicitly recited herein. Two polypeptides are said to be "homologous" if the sequence of one of the polypeptides has a high enough degree of identity or similarity to the sequence of the other polypeptide. "Identity" indicates that at any particular position in the aligned sequences, the amino acid residue is identical between the sequences. "Similarity" indicates that, at any particular position in the aligned sequences, the amino acid residue is of a similar type between the sequences. Degrees of identity and similarity can be readily calculated according to methods known in the art (see, for example, Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing. Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993).

Typically, greater than 50% identity between two polypeptides is considered to be an indication of functional equivalence, provided that either the biological activity of the polypeptide is retained or the polypeptides possess an antigenic determinant in common. Preferably, a functionally equivalent polypeptide according to this aspect of the invention exhibits a degree of sequence identity with a polypeptide sequence explicitly identified herein, or with a fragment thereof, of greater than 50%. More preferred polypeptides have degrees of identity of greater than 60%, 70%, 80%, 90%, 95%, 98% or 99%, respectively. The polypeptides EGLN3 (BAB15101), EGLNl (clorfl2; AAG34568), CAB81622 and SM20 are intended to be excluded from this aspect of the invention.

Functionally-equivalent polypeptides according to the invention are therefore intended to include natural biological variants (for example, allelic variants or geographical variations within the species from which the polypeptides are derived) and mutants (such as mutants containing amino acid substitutions, insertions or deletions) of the polypeptides whose sequences are explicitly recited herein. Such mutants may include polypeptides in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code. Typical such substitutions are among Ala, Val, Leu and He; among Ser and Thr; among the acidic residues Asp and Glu; among Asn and Gin; among the basic residues Lys and Arg; or among the aromatic residues Phe and Tyr.

Particularly preferred functionally-equivalent polypeptides are variants in which several, i.e. between 5 and 10, 1 and 5, 1 and 3, 1 and 2 or just 1 amino acids are substituted, deleted or added in any combination. Especially preferred are silent substitutions, additions and deletions, which do not alter the properties and activities of the protein. Also especially preferred in this regard are conservative substitutions. "Mutant" polypeptides also include polypeptides in which one or more of the amino acid residues include a substituent group.

According to a further aspect of the invention, there is provided a purified and isolated nucleic acid molecule that encodes a polypeptide according to any one of the aspects of the invention discussed above. Such a nucleic acid molecule may consist of the nucleic acid sequence as recited in SEQ ID No. 86a, or form a redundant equivalent or fragment thereof. This aspect of the invention also includes a purified nucleic acid molecule which hydridizes under high stringency conditions with a nucleic acid molecule as described above. Nucleic acid molecules that encode EGLN3 (BAB15101), EGLNl (clorf!2; AAG34568), CAB81622 and SM20 are specifically excluded from this aspect of the invention.

According to a further aspect of the invention, there is provided an expression vector that contains a purified and isolated nucleic acid molecule according to the aspects of the invention described above. The invention also incorporates a delivery vehicle, such as a liposome, comprising a nucleic acid according to the above-described aspects of the invention. Such vectors and delivery vehicles are especially useful for the expression of polypeptides that comprise a sequence as recited in SEQ ID No. 85a.

In a further aspect, the invention provides a host cell transformed with a vector of the above- described aspect of the invention. In a still further aspect, the invention provides a ligand that binds specifically to a polypeptide according to the above-described aspects of the invention. The ligand may be an antagonist ligand that inhibits the biological activity of the polypeptide, or may be an agonist ligand that activates the hypoxia-induced activity of the polypeptide to augment or potentiate a hypoxia- induced activity. In a still further aspect of the invention, there is provided a ligand which binds specifically to, and which preferably inhibits the hypoxia-induced activity of, a polypeptide according to any one of the above-described aspects of the invention. Such a ligand may, for example, be an antibody that is immunospecific for the polypeptide in question.

According to a further aspect, the invention provides a polypeptide whose amino acid sequence is recited in SEQ ID No. 85a, or which is encoded by a nucleic acid sequence recited in SEQ ID No.: 86a, for use in therapy or diagnosis of a disease or abnormal physiological condition. This aspect of the invention also provides the use of a nucleic acid molecule encoding such a polypeptide, or a vector that contains such a purified and isolated nucleic acid molecule, or a ligand that binds specifically to a polypeptide, for use in therapy or diagnosis of a disease or abnormal physiological condition.

Preferably, the disease or abnormal physiological condition is one that is affected by hypoxia; examples of such diseases include cancer, ischaemic conditions (such as stroke, coronary arterial disease, peripheral arterial disease), reperfusion injury, retinopathy, neonatal stress, preeclampsia, atherosclerosis, inflammatory conditions (including rheumatoid arthritis), wound healing, myocardial infarction and diseases involving infection of the airways (such as cystic fibrosis). The undesired cellular process involved in said diseases might include, but is not restricted to; tumourigenesis, angiogenesis, apoptosis, inflammation or erythropoiesis. The undesired biochemical processes involved in said cellular processes might include, but is not restricted to, glycolysis, gluconeogenesis, glucose transportation, catecholamine synthesis, iron transport or nitric oxide synthesis.

According to a further aspect of the invention, there is provided a substantially purified polypeptide, which polypeptide: i) comprises the amino acid sequence as recited in SEQ ID No: 85a; ii) has an amino acid sequence encoded by a nucleic acid sequence recited in SEQ ID No: 86a; iii) is a fragment of a polypeptide according to i) or ii), provided that said fragment retains a biological activity possessed by the full length polypeptide of i) or ii), or has an antigenic determinant in common with the polypeptide of i) or ii); or iv) is a functional equivalent of a polypeptide of i), ii) or (iii); for use in the diagnosis or therapy of tumourigenesis, angiogenesis, apoptosis, the biological response to hypoxia conditions, or a hypoxic-associated pathology. The invention also provides a purified and isolated nucleic acid molecule that encodes a polypeptide according to this aspect of the invention, for use in the diagnosis or therapy of tumourigenesis, angiogenesis, apoptosis, the biological response to hypoxia conditions, or a hypoxic-associated pathology. One such sequence is provided in SEQ ID No. 86a. As described above for the EST nucleic acid sequences annotated herein, this aspect of the invention includes redundant equivalents and fragments of the sequences explicitly recited in SEQ ID No.: 86a, and purified nucleic acid molecules which hybridize under high stringency conditions with such nucleic acid molecules, and vectors containing such nucleic acid molecules for use in the diagnosis or therapy of tumourigenesis, angiogenesis, apoptosis, the biological response to hypoxia conditions, or a hypoxic-associated pathology.

This aspect of the invention also includes ligands which bind specifically to, and which preferably inhibit the hypoxia-induced activity of, a polypeptide listed in SEQ ID No.: 85a, or encoded by a nucleic acid sequence recited in SEQ ID No: 86a, for use in the diagnosis or therapy of tumourigenesis, angiogenesis, apoptosis, the biological response to hypoxia conditions, or a hypoxic-associated pathology.

The invention also provides a pharmaceutical composition suitable for modulating the biological response to hypoxia and/or ischaemia, comprising a therapeutically-effective amount of a polypeptide, a nucleic acid molecule, vector or ligand as described above, in conjunction with a pharmaceutically-acceptable carrier.

The invention also provides a vaccine composition comprising a polypeptide, or a nucleic acid molecule as described above.

The invention also provides a method of treating a disease in a patient in need of such treatment by administering to a patient a therapeutically effective amount of a polypeptide, a nucleic acid molecule, vector, ligand or pharmaceutical composition as described above. For diseases in which the expression of the natural gene or the activity of the polypeptide is lower in a diseased patient when compared to the level of expression or activity in a healthy patient, the polypeptide, nucleic acid molecule, ligand, compound or composition administered to the patient should be an agonist. For diseases in which the expression of the natural gene or activity of the polypeptide is higher in a diseased patient when compared to the level of expression or activity in a healthy patient, the polypeptide, nucleic acid molecule, vector, ligand, compound or composition administered to the patient is an antagonist. By the term "agonist" is meant herein, any polypeptide, peptide, synthetic molecule or organic molecule that functions as an activator, by increasing the effective biological activity of a polypeptide, for example, by increasing gene expression or enzymatic activity. By the term "antagonist" is meant herein, any polypeptide, peptide, synthetic molecule or organic molecule that functions as an inhibitor, by decreasing the effective biological activity of the gene product, for example, by inhibiting gene expression of an enzyme or a pharmacological receptor. The invention also provides a polypeptide, nucleic acid molecule, vector, ligand or pharmaceutical composition according to any one of the above-described aspects of the invention, for use in the manufacture of a medicament for the treatment of a hypoxia-regulated condition. The invention also provides a method of monitoring the therapeutic treatment of disease or physiological condition in a patient, comprising monitoring over a period of time the level of expression or activity of polypeptide, nucleic acid molecule, vector or ligand in tissue from said patient, wherein altering said level of expression or activity over the period of time towards a control level is indicative of regression of said disease or physiological condition.

The invention also provides a method of providing a hypoxia regulating gene, an apoptotic or an angiogenesis regulating gene by administering directly to a patient in need of such therapy an expressible vector comprising expression control sequences operably linked to one or more of the nucleic acid molecules as described above. The invention also provides a method of diagnosing a hypoxia-regulated condition in a patient, comprising assessing the level of expression of a natural gene encoding a polypeptide according to any one of the aspects of the invention described above in tissue from said patient and comparing said level of expression or activity to a control level, wherein a level that is different to said control level is indicative of the hypoxia-related condition. Such a method of diagnosis may be carried out in vitro. One example of a suitable method comprises the steps of: (a) contacting a ligand as described above with a biological sample under conditions suitable for the formation of a ligand-polypeptide complex; and (b) detecting said complex.

A further example of a suitable method may comprises the steps of: a) contacting a sample of tissue from the patient with a nucleic acid probe under stringent conditions that allow the formation of a hybrid complex between a nucleic acid molecule whose sequence is recited in SEQ ID No.: 86a and the probe; b) contacting a control sample with said probe under the same conditions used in step a); and c) detecting the presence of hybrid complexes in said samples; wherein detection of levels of the hybrid complex in the patient sample that differ from levels of the hybrid complex in the control sample is indicative of the hypoxia-related condition.

A still further example of a suitable method may comprise the steps of: a) contacting a sample of nucleic acid from tissue of the patient with a nucleic acid primer under stringent conditions that allow the formation of a hybrid complex between a nucleic acid molecule whose sequence is recited in SEQ ID No.: 86a, and the primer; b) contacting a control sample with said primer under the same conditions used in step a); c) amplifying the sampled nucleic acid; and d) detecting the level of amplified nucleic acid from both patient and control samples; wherein detection of levels of the amplified nucleic acid in the patient sample that differ significantly from levels of the amplified nucleic acid in the control sample is indicative of the hypoxia- related condition.

A still further example of a suitable method may comprised the steps of: a) obtaining a tissue sample from a patient being tested for the hypoxia-related condition; b) isolating a nucleic acid molecule according to any one of the above-described aspects of the invention from said tissue sample; and c) diagnosing the patient for the hypoxia-related condition by detecting the presence of a mutation which is associated with the hypoxia-related condition in the nucleic acid molecule as an indication of the hypoxia-related condition. This method may comprise the additional step of amplifying the nucleic acid molecule to form an amplified product and detecting the presence or absence of a mutation in the amplified product.

Particular hypoxia-related conditions that may be diagnosed in this fashion include cancer, ischaemia, reperfusion, retinopathy, neonatal stress, preeclapmsia, atherosclerosis, rheumatoid arthritis, cardiac arrest or stroke, for example, caused by a disorder of the cerebral, coronary or peripheral circulation. In a further aspect, the invention provides a method for the identification of a compound that is effective in the treatment and/or diagnosis of a hypoxia-regulated condition, comprising contacting a polypeptide, nucleic acid molecule, or ligand according to any one of the above- described aspects of the invention with one or more compounds suspected of possessing binding affinity for said polypeptide, nucleic acid molecule or ligand, and selecting a compound that binds specifically to said nucleic acid molecule, polypeptide or ligand.

According to a still further aspect of the invention, there is provided a kit useful for diagnosing a hypoxia-regulated condition, comprising a first container containing a nucleic acid probe that hybridises under stringent conditions with a nucleic acid molecule according to any one of the aspects of the invention described above; a second container containing primers useful for amplifying said nucleic acid molecule; and instructions for using the probe and primers for facilitating the diagnosis of the hypoxia-regulated condition. The kit may additionally comprise a third container holding an agent for digesting unhybridised RNA.

To facilitate in the diagnosis of the hypoxia-regulated condition using one of the methods outlined above, in a further aspect, the invention provides an array of at least two nucleic acid molecules, wherein each of said nucleic acid molecules either corresponds to the sequence of, is complementary to the sequence of, or hybridises specifically to a nucleic acid molecule according to any one of the aspects of the invention described above. Such an array may contain nucleic acid molecules that either correspond to the sequence of, are complementary to the sequence of, or hybridise specifically to at least 1-4 or more of the nucleic acid molecules implicated in a hypoxia-regulated condition as recited above. The nucleic acid molecules on the array may consist of oligonucleotides of between twelve and fifty nucleotides, more preferably, between forty and fifty nucleotides. Alternatively, the nucleic acid molecules on the array may consist of PCR-amplified cDNA inserts where the nucleic acid molecule is between 300-2000 nucleotides.

In a related aspect, again useful for diagnosis, the invention provides an array of antibodies, comprising at least two different antibody species, wherein each antibody species is immunospecific with a polypeptide implicated in a hypoxia-regulated condition as described above. The invention also provides an array of polypeptides, comprising at least two polypeptide species as recited above, wherein each polypeptide species is implicated in a hypoxia-regulated condition, or is a functional equivalent variant or fragment thereof.

Kits useful in the diagnostic methods of the invention may comprise such nucleic acid, antibody and/or polypeptide arrays. According to the invention, a kit may also comprise one or more antibodies that bind to a polypeptide as recited above, and a reagent useful for the detection of a binding reaction between said antibody and said polypeptide.

According to a still further aspect of the invention, there is provided a genetically-modified non-human animal that has been transformed to express higher, lower or absent levels of a polypeptide according to any one of the aspects of the invention described above. Preferably, said genetically-modified animal is a transgenic or knockout animal.

The invention also provides a method for screening for a compound effective to treat a hypoxia-regulated condition, by contacting a non-human genetically-modified animal as described above with a candidate compound and determining the effect of the compound on the physiological state of the animal.

An alignment of the amino acid sequences of rat SM20 (Accession A53770), its human equivalent EGLN3 (Accession BAB15101; SEQ ID No: 85) and the distinct human ho ologue EGLNl (Accession CAB81622 or AAG34568; SEQ ID No: 89) is shown below:

l so

BAB15101 ( 1 )

A53770 ( 1 )

AAG34568 ( 1 ) MANDSGGPGGPSPSERDRQYCELCGKMENL RCSRCRSSFYCCKEHQRQD

Consensus (1) 51 100

BAB15101 (1

A53770 (1 tøTLRSRRGFLSFLPGLRPPRRWLRISKRGPPTSHWASP AL

AAG34568 (51 WKKHKLVCQGSEGALGHGVGPHQHSGPAPPAAVPPPRAGAREPRKAAARR Consensus (51 L G G PP P

101 150

BAB15101 (1

A53770 (41 GGRTLHYSCRSQSGTPFSSEFQATFPAFAAKVARGPW PQVVEPPA

AAG34568 (101 DNASGDAAKGKVKAKPPADPAAAASPCRAAAGGQGSAVAAEAEPGKEEPP Consensus (101 S A P A A P AA A G L EP

151 200

BAB15101 (1 MPLGHIMRLDLEKIA EYIVP'

A53770 (88 LSASPLCVRSGQALGACTLGVPRLGSVSEMPLGHI RliDLEKlALEYIVP

AAG34568 (151 AR^rSSLFQE AJSr YPPSNTPGDA SPGGG RPWGQTKPLPA K Ar.EYIVP Consensus (151 AS KA A G MP GHIMRLDLEKIA EYIVP

201 250

BAB15101 (22 C HEVGFCYLDNFLGEWGDCVLERVKQLHCTGALRDGQLAGPRAGVSKR

A53770 (138 CLHEVGFCYLDNFLGEWGDCVLERVKQLHYNGALRDGQ AGPRAGVSKR

AAG34568 (201 CMNKHGICWDDFLGKETGQQIGDEVRA HDTGKFTDGQLVSQΪΞS-DSS Consensus (201 CLHEVGFCY DNFLGEWGDCVLERVKQLH TGALRDGQLAGPRAGVSKR

251 300

BAB15101 (72 HLRGDQΪTWIGGNEEGCEATSFLLSLIDR VLYCGSRTjGKYYVKERS AM

A53770 (188 HLRGDQlT IGGNEΞGCEATNFLLfe IDFiVLYCGSRLGK YVKERSKAM

AAG34568 (250 D^RGDKITWrEGKE GCETIGLtMSSMDD^IRfeCNG LGSYKlNGRTKAM Consensus (251 HLRGDQIT IGGNEEGCΞAI FLLSLIDRLVLYCGSRLGKYYVKERSKAM 301 350

BAB15101 (122 VACYPGHGTGY/RHVDNPNGDGRCITCIYYLNKNWDAKLHGGIliRIFPEG

A53770 (238 VACYPGNGTGYVRHVDNPNGDGRCI-TCTYYLM NWDAKLHGGVLRIFPEG

AAG34568 (300 VACYPGNGTGYVRHVDNPNGDGRCVTCIYYLNKDWDAKVSGGipϋRIFPEG Consensus (301 VACYPGNGTGYVRHVDNPNGDGRCITCIYYLNK WDAKLHGGILRIFPEG 351 400

BAB15101 (172 KSFIADVEPIFDR FFWSDRRNPHEVQPSYATRYAMTV YFDAEERAE

A53770 (288 KSFVADVEPIFDR LFSWSDRHNPHEVQPSYATRYAMTVWYFDAEERAEA

AAG34568 (350 AQFADIEPKFDRLLFFWSDRRNPHEVQPAYATRYAITV YFDADERARA Consensus (351 KSFIADVEPIFDR LFFWSDRRNPHEVQPSYATRYA TVWYFDAEERAEA 401 427

BAB15101 (222 KKKFRNLTRKTESALTΞD

A53770 (338 KKKFRNLTRKTESALAKD

AAG34568 (400 KVKYLTGEfeVRVELNKPSDSVGKDVF Consensus (401 KKKFRNLTRKTESAL KD

From this sequence alignment, a highly conserved region of amino acid sequence may be noted, the consensus of which is as follows:

KA VACYPGNGTGYVRHVDNPNGDGRCITCIYYLNKNWDAKLHGGILRIFPEGKSFIADVEPI FDRLLFF SDRR PHEVQPSYATRYAMTVWYFDAEERAEAKKK

This is presumably a functional domain. The region of sequence lost in the splice variant, SEQ ID No. 85a, is directly upstream to the conserved domain. This consensus sequence, and variants thereof, may be used in the identification of other proteins that are implicated in the biological response to hypoxia. Neither this consensus domain nor any proteins that contain this domain have been previously associated with the cellular response to hypoxia/ischaemia. Searches of the public databases indicate that the human genome contains several genes that encode proteins that contain this consensus sequence. These proteins may have similar functions or may function in the same biochemical pathway, potentially with an antagonistic effect. The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA technology and immunology, which are within the skill of those working in the art.

Most general molecular biology, microbiology recombinant DNA technology and immunological techniques can be found in Sambrook et al, Molecular Cloning, A Laboratory Manual (2000) Cold Harbor-Laboratory Press, Cold Spring Harbor, N.Y. or Ausubel et al, Current protocols in molecular biology (1990) John Wiley and Sons, N.Y.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

A. Polypeptides The term "polypeptide" as used herein, refers to a chain (may be branched or unbranched) of two or more amino acids linked to each other by means of a peptide bond or modified peptide bond (isosteres). The term polypeptide encompasses but is not limited to oligopeptides, peptides and proteins. The polypeptide of the invention may additionally be either in a mature protein form or in a pre-, pro- or prepro-protein form that requires subsequent cleavage for formation of the active mature protein. The pre-, pro-, prepro- part of the protein is often a leader or secretory sequence but may also be an additional sequence added to aid protein purification (for example, a His tag) or to conform a higher stability to the protein.

A polypeptide according to the invention may also include modified amino acids, that is, amino acids other than those 20 that are gene-encoded. This modification may be a result of natural processes such as post-translational processing or by chemical modification. Examples of modifications include acetylation, acylation, amidation, ADP-ribosylation, arginylation, attachment of a lipid derivative or phosphatidylinositol, γ-carboxylation, covalent attachment of a flavin or haeme moiety, a nucleotide or nucleotide derivative, cyclisation, demethylation, disulphide bond formation, formation of covalent cross-links, formylation, glycosylation, GPI anchor formation, hydroxylation, iodination, lipid attachment, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemisation, selenoylation, sulphation, and ubiquitination. Modification of the polypeptide can occur anywhere within the molecule including the backbone, the amino acid side-chains or at the N- or C-terminals.

A polypeptide according to the invention may either be isolated from natural sources (for example, purified from cell culture), or be a recombinantly produced polypeptide, or a synthetically produced polypeptide or a combination of all the above.

Antibodies

A polypeptide according to the invention, its functional equivalents and/or any immunogenic fragments derived from the polypeptide may be used to generate ligands including immunospecific monoclonal or polyclonal antibodies, or antibody fragments. These antibodies can then be used to isolate or identify clones expressing the polypeptide of the invention or to purify the polypeptide by affinity chromatography. Further uses of these immunospecific antibodies may include, but are not limited to, diagnostic, therapeutic or general assay applications. Examples of assay techniques that employ antibodies are immunoassays, radioimmunoassays (RIA) or enzyme linked immunosorbent assay (ELISA). In these cases, the antibodies may be labelled with an analytically-detectable reagent including radioisotopes, a fluorescent molecule or any reporter molecule.

The term "immunospecific" as used herein refers to antibodies that have a substantially higher affinity for a polypeptide of this invention compared with other polypeptides. The term "antibody" as used herein refers to a molecule that is produced by animals in response to an antigen and has the particular property of interacting specifically with the antigenic determinant that induced its formation. Fragments of the aforementioned molecule such as Fab, F(ab')₂ and scFv, which are capable of binding the antigen determinant, are also included in the term "antibody". Antibodies may also be modified to make chimeric antibodies, where non-human variable regions are joined or fused to human constant regions (for example, Liu et al, PNAS, USA, 84, 3439 (1987)). Particularly, antibodies may be modified to make them less immunogenic to an individual in a process such as humanisation (see, for example, Jones et al, Nature, 321, 522 (1986); Verhoeyen et al, Science, 239, 1534 (1988); Kabat et al, J. Immunol., 147, 1709 (1991); Queen et al, PNAS, USA, 86, 10029 (1989); Gorman et al, PNAS, USA, 88, 34181 (1991) and Hodgson et al, Bio/Technology, 9, 421 (1991)). The term "humanised antibody", as used herein, refers to antibody molecules in which the amino acids of the CDR (complementarity-determining region) and selected other regions in the variable domains of the heavy and/or light chains of a non-human donor antibody have been substituted with the equivalent amino acids of a human antibody. The humanised antibody therefore closely resembles a human antibody, but has the binding ability of the donor antibody. Antibodies may also have a "bispecific" nature, that is, the antibody has two different antigen binding domains, each domain being directed against a different epitope.

Specific polyclonal antibodies may be made by immuno-challenging an animal with a polypeptide of this invention. Common animals used for the production of antibodies include the mouse, rat, chicken, rabbit, goat and horse. The polypeptide used to immuno-challenge the animal may be derived by recombinant DNA technology or may be chemically-synthesised. In addition, the polypeptide may be conjugated to a carrier protein. Commonly used carriers to which the polypeptides may be conjugated include, but are not limited to BSA (bovine serum albumin), thyroglobulin and keyhole limpet haemocyanin. Serum from the immuno-challenged animal is collected and treated according to known procedures, for example, by immunoaffinity chromatography.

Specific monoclonal antibodies can generally be made by methods known to one skilled in the art (see for example, Kohler, G. and Milstein, C, Nature 256, 495-497 (1975); Kozbor et al, Immunology Today 4: 72 (1983); Cole et al, 77-96 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985) and Roitt, I. et al, Immunology, 25.10, Mosby-Year Book Europe Limited (1993)). Panels of monoclonal antibodies produced against the polypeptides of the invention can be screened for various properties, i.e., for isotype, epitope, affinity, etc. against which they are directed. Alternatively, genes encoding the monoclonal antibodies of interest may be isolated from hybridomas, for instance using PCR techniques known in the art, and cloned and expressed in appropriate vectors.

Phage display technology may be utilised to select the genes encoding the antibodies that have exhibited an immunospecific response to the polypeptides of the invention (see McCafferty, J., et al, (1990), Nature 348, 552-554; Marks, J. et al, (1992) Biotechnology 10, 779-783).

Ligands

The polypeptides of the invention may also be used to search for interacting ligands. Methods for doing this include the screening of a library of compounds (see Coligan et al, Current Protocols in Immunology 1(2); Chapter 5 (1991), isolating the ligands from cells, isolating the ligands from a cell-free preparation or natural product mixtures. Ligands to the polypeptide may activate (agonise) or inhibit (antagonise) its activity. Alternatively, compounds may affect the levels of the polypeptide present in the cell, including affecting gene expression and/or mRNA stability. Ligands to the polypeptide form a further aspect of the invention, as discussed in more detail above. Preferred "antagonist" ligands include those that bind to the polypeptide of this invention and strongly inhibit any activity of the polypeptide. Preferred "agonist" ligands include those that bind to the polypeptide and strongly induce activity of the polypeptide of this invention or increases substantially the level of the polypeptide in the cell. As defined above, the term "agonist" is meant to include any polypeptide, peptide, synthetic molecule or organic molecule that functions as an activator, by increasing the effective biological activity of a polypeptide, for example, by increasing gene expression or enzymatic activity. The term "antagonist" is meant to include any polypeptide, peptide, synthetic molecule or organic molecule that functions as an inhibitor, by decreasing the effective biological activity of the gene product, for example, by inhibiting gene expression of an enzyme or a pharmacological receptor.

Ligands to a polypeptide according to the invention may come in various forms, including natural or modified substrates, enzymes, receptors, small organic molecules such as small natural or synthetic organic molecules of up to 2000Da, preferably 800Da or less, peptidomimetics, inorganic molecules, peptides, polypeptides, antibodies, structural or functional mimetics of the aforementioned.

B. Nucleic acid molecules

Preferred nucleic acid molecules of the invention are those which encode the polypeptide sequences recited in any one of SEQ ID Nos. 85, 85a or 89, or which encode polypeptides encoded by a nucleic acid sequence recited in any one of SEQ ID Nos: 86, 86a, 90 or 90a, or encoded by a gene identified from an EST recited in any one of these SEQ ID Nos. Examples of such nucleic acid molecules include those listed in SEQ ID Nos. 86, 86a, 90 and 90a, homologous nucleic acids and nucleic acids that are complementary to these nucleic acid molecules. Nucleic acid molecules of this aspect of the invention may be used in numerous methods and applications, as described generally herein. A nucleic acid molecule preferably comprises of at least n consecutive nucleotides from any one of the sequences disclosed in

SEQ ID Nos.: 86, 86a, 90 and 90a, where n is 10 or more. A nucleic acid molecule of the invention also includes sequences that are complementary to the nucleic acid molecule described above (for example, for antisense or probing purposes).

A nucleic acid molecule according to this aspect of the invention may be in the form of RNA, such as mRNA, DNA, such as cDNA, synthetic DNA or genomic DNA. The nucleic acid molecule may be double-stranded or single-stranded. The single-stranded form may be the coding (sense) strand or the non-coding (antisense) strand. A nucleic acid molecule may also comprise an analogue of DNA or RNA, including, but not limited to modifications made to the backbone of the molecule, such as, for example, a peptide nucleic acid (PNA). The term "PNA" as used herein, refers to an antisense molecule that comprises an oligonucleotide of at least five nucleotides in length linked to a peptide backbone of amino acid residues, preferably ending in lysine. The terminal lysine confers solubility to the composition. PNAs may be pegylated to extend their lifespan in a cell, where they preferentially bind complementary single-stranded DNA and RNA and stop transcript elongation (Nielsen, P.E. et al. (1993) Anticancer Drug Des. 8:53-63). A nucleic acid molecule according to this aspect of the invention can be isolated by cloning, purification or separation of the molecule directly from a particular organism, or from a library, such as a genomic or cDNA library. The molecule may also be synthesised, for example, using chemical synthetic techniques such as solid phase phosphoramidite chemical synthesis. RNA may be synthesized in vitro ox in vivo by transcription of the relevant DNA molecule. Due to the degeneracy of the genetic code, differing nucleic acid sequences may encode the same polypeptide (or mature polypeptide). Thus, nucleic acid molecules included in this aspect of the invention include any molecule comprising a variant of the sequence explicitly recited. Such variants may include variant nucleic acid molecules that code for the same polypeptide (or mature polypeptide) as that explicitly identified, that code for a fragment of the polypeptide, that code for a functional equivalent of the polypeptide or that code for a fragment of the functional equivalent of the polypeptide. Also included in this aspect of the invention, are variant nucleic acid molecules that are derived from nucleotide substitutions, deletions, rearrangements or insertions or multiple combinations of the aforementioned. Such molecules may be naturally occurring variants, such as allelic variants, non-naturally occurring variants such as those created by chemical mutagenesis, or variants isolated from a species, cell or organism type other than the type from which the sequence explicitly identified originated. Variant nucleic acid molecules may differ from the nucleic acid molecule explicitly recited in a coding region, non-coding region or both these regions.

Nucleic acid molecules may also include additional nucleic acid sequence to that explicitly recited, for example, at the 5' or 3' end of the molecule. Such additional nucleic acids may encode for a polypeptide with added functionality compared with the original polypeptide whose sequence is explicitly identified herein. An example of this would be an addition of a sequence that is heterologous to the original nucleic acid sequence, to encode a fusion protein. Such a fusion protein may be of use in aiding purification procedures or enabling techniques to be carried out where fusion proteins are required (such as in the yeast two hybrid system). Additional sequences may also include leader or secretory sequences such as those coding for pro-, pre- or prepro- polypeptide sequences. These additional sequences may also include non- coding sequences that are transcribed but not translated including ribosome binding sites and termination signals.

A nucleic acid molecule of the invention may include molecules that are at least 70% identical over their entire length to a nucleic acid molecule as explicitly identified herein in SEQ ID Nos.: 86, 86a, 90 or 90a. Preferably, a nucleic acid molecule according to this aspect of the invention comprises a region that is at least 80% identical over its entire length to a nucleic acid molecule as explicitly identified herein in these SEQ ID Nos., preferably at least 90%, more preferably at least 95% and most preferably at least 98% or 99% identical. Further preferred embodiments include nucleic acid molecules that encode polypeptides that retain substantially the same biological function or activity as the polypeptide explicitly identified herein. The nucleic acid molecules of the invention can also be engineered using methods generally known in the art. These methods include but are not limited to DNA shuffling; random or non- random fragmentation (by restriction enzymes or shearing methods) and reassembly of fragments; insertions, deletions, substitutions and rearrangements of sequences by site-directed mutagenesis (for example, by PCR). These alterations may be for a number of reasons including for ease of cloning (such as introduction of new restriction sites), altering of glycosylation patterns, changing of codon preferences, splice variants changing the processing, and/or expression of the gene product (the polypeptide) in general or creating fusion proteins (see above).

Hybridisation Nucleic acid molecules of the invention may also include antisense molecules that are partially complementary to a nucleic acid molecule as explicitly identified herein in SEQ ID Nos.: 86, 86a, 90 or 90a, and which therefore will hybridise to the encoding nucleic acid molecules. These antisense molecules, including oligonucleotides, can be designed to recognise, specifically bind to and prevent transcription of a target nucleic acid encoding a polypeptide of the invention, as will be known by those of ordinary skill in the art (see Cohen, J.S., Trends in Pharm. Sci., 10, 435 (1989), Okano, J. Neurochem. 56, 560 (1991); O'Connor, J. Neurochem 56, 560 (1991); Lee et al, Nucleic Acids Res 6, 3073 (1979); Cooney et al, Science 241, 456 (1988); Dervan et al, Science 251, 1360 (1991). The term "hybridisation" used herein refers to any process by which a strand of nucleic acid binds with a complementary strand of nucleic acid by hydrogen bonding, typically forming Watson-Crick base pairs. As carried out in vitro, one of the nucleic acid populations is usually immobilised to a surface, whilst the other population is free. The two molecule types are then placed together under conditions conducive to binding.

The phrase "stringency of hybridisation" refers to the percentage of complementarity that is needed for duplex formation. "Stringency" thus refers to the conditions in a hybridization reaction that favour the association of very similar molecules over association of molecules that differ. Conditions can therefore exist that allow not only nucleic acid strands with 99- 100% complementarity to hybridise, but sequences with lower complementarity (for example, 50%) to also hybridise. High stringency hybridisation conditions are defined herein as overnight incubation at 42°C in a solution comprising 50% formamide, 5XSSC (150mM NaCI, 15mM trisodium citrate), 50mM sodium phosphate (pH7.6), 5x Denhardts solution, 10% dextran sulphate, and 20 microgram/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1X SSC at approximately 65°C. Low stringency conditions involve the hybridisation reaction being carried out at 35°C (see Sambrook et al. [supra]). Preferably, the conditions used for hybridization are those of high stringency.

Some trans- and cw-acting factors that may affect the binding of two complementary strands include strand length, base composition (GC pairs have an extra hydrogen bond and are thus require more energy to separate than AT pairs) and the chemical environment. The presence of monovalent cations (such as Na⁺) stabilises duplex formation whereas chemical denaturants such as formamide and urea destabilise the duplex by disruption of the hydrogen bonds. Use of compounds such as polyethylene glycol (PEG) can increase reassociation speeds by increasing overall DNA concentration in aqueous solution by abstracting water molecules. Denhardt's reagent or BLOTTO are chemical agents often added to block non-specific attachment of the liquid phase to the solid support. Increasing the temperature will also increase the stringency of hybridisation, as will increasing the stringency of the washing conditions following hybridisation (Sambrook et al. [supra]).

Numerous techniques exist for effecting hybridisation of nucleic acid molecules. Such techniques usually involve one of the nucleic acid populations being labelled. Labelling methods include, but are not limited to radiolabelling, fluorescence labelling, chemiluminescent or chromogenic labelling or chemically coupling a modified reporter molecule to a nucleotide precursor such as the biotin-streptavidin system. This can be done by oligolabelling, nick-translation, end-labelling or PCR amplification using a labelled polynucleotide. Labelling of RNA molecules can be achieved by cloning the sequences encoding the polypeptide of the invention into a vector specifically for this purpose. Such vectors are known in the art and may be used to synthesise RNA probes in vitro by the addition of an appropriate RNA polymerase such as T7, T3 or SP6 and labelled nucleotides.

Various kits are commercially available that allow the labelling of molecules. Examples include those made by Pharmacia & Upjohn (Kalamazoo, MI); Promega (Madison WI); and the U.S. Biochemical Corp. (Cleveland, OH). Hybridisation assays include, but are not limited to dot-blots, Southern blotting, Northern blotting, chromosome in situ hybridisation (for example, FISH [fluorescence in situ hybridisation]), tissue in situ hybridisation, colony blots, plaque lifts, gridded clone hybridisation assays, DNA microarrays and oligonucleotide microarrays. These hybridisation methods and others, may be used by a skilled artisan to isolate copies of genomic DNA, cDNA, or RNA encoding homologous or orthologous proteins from other species.

The invention therefore also embodies a process for detecting a nucleic acid molecule according to the invention, comprising the steps of: (a) contacting a nucleic probe with a biological sample under hybridising conditions to form duplexes: and (b) detecting any such duplexes that are formed. The term "probe" as used herein refers to a nucleic acid molecule in a hybridisation reaction whose molecular identity is known and is designed specifically to identify nucleic acids encoding homologous genes in other species. Usually, the probe population is the labelled population, but this is not always the case, as for example, in a reverse hybridisation assay.

One example of a use of a probe is to find nucleic acid molecules with an equivalent function to those that are explicitly identified herein, or to identify additional family members in the same or other species. This can be done by probing libraries, such as genomic or cDNA libraries, derived from a source of interest, such as a human, a non-human animal, other eukaryote species, a plant, a prokaryotic species or a virus. The probe may be natural or artificially designed using methods recognised in the art (for example, Ausubel et al, [supra]). A nucleic acid probe will preferably possess greater than 15, more preferably greater than 30 and most preferably greater than 50 contiguous bases complementary to a nucleic acid molecule explicitly identified herein. In many cases, isolated DNA from cDNA libraries will be incomplete in the region encoding the polypeptide, normally at the 5' end. Methods available for subsequently obtaining full- length cDNA sequence include RACE (rapid amplification of cDNA ends) as described by Frohman et al, (Proc. Natl. Acad. Sci. USA 85, 8998-9002 (1988)), and restriction-site PCR, which uses universal primers to retrieve unknown nucleic acid sequence adjacent to a known locus (Sarkar, G. (1993) PCR Methods Applic, 2:318-322). "Inverse PCR" may also be used to amplify or to extend sequences using divergent primers based on a known region (Triglia, T. et al, (1988) Nucleic Acids Res. 16:8186). Another method which may be used is "capture PCR", which involves PCR amplification of DNA fragments adjacent to a known sequence in human and yeast artificial chromosome DNA (Lagerstrom, M. et al, (1991) PCR Methods Applic, 1:111-119). Another method which may be used to retrieve unknown sequences is that of Parker, J.D. et al, (1991); Nucleic Acids Res. 19:3055-3060). Additionally, one may use PCR, nested primers, and libraries, such as the PromoterFinder™ library (Clontech, Palo Alto, CA) to walk genomic DNA. This latter process avoids the need to screen libraries and is useful in finding intron/exon junctions.

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

In one embodiment of the invention, a nucleic acid molecule according to the invention may be used for chromosome localisation. In this technique, a nucleic acid molecule is specifically targeted to, and can hybridise with, a particular location on an individual human chromosome. The mapping of relevant sequences to chromosomes is an important step in the confirmatory correlation of those sequences with the gene-associated disease. Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found in, for example, McKusick, Mendelian Inheritance in Man (available on-line through Johns Hopkins University Welch Medical Library). The relationships between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheritance of physically adjacent genes). This provides valuable information to investigators searching for disease genes using positional cloning or other gene discovery techniques. Once the disease or syndrome has been crudely localised by genetic linkage to a particular genomic region, any sequences mapping to that area may represent associated or regulatory genes for further investigation. The nucleic acid molecule may also be used to detect differences in the chromosomal location due to translocation, inversion, etc. among normal, carrier, or affected individuals. Nucleic acid molecules of the present invention are also valuable for tissue localisation. Such techniques facilitate the determination of expression patterns of the polypeptide in tissues by detection of the mRNAs that encode them. These techniques include in situ hybridisation techniques and nucleotide amplification techniques, such as PCR. Results from these studies provide an indication of the normal functions of the polypeptide in the organism, as well as highlighting the involvement of a particular gene in a disease state or abnormal physiological condition.

In addition, comparative studies of the normal expression pattern of mRNAs with that of mRNAs encoded by a mutant gene provide valuable insights into the role of mutant polypeptides in disease. Such inappropriate expression may be of a temporal, spatial or quantitative nature.

Vectors

The nucleic acid molecules of the present invention may be incorporated into vectors for cloning (for example, pBluescript made by Stratagene) or expression purposes. Vectors containing a nucleic acid molecule explicitly identified herein (or a variant thereof) form another aspect of this invention. The nucleic acid molecule may be inserted into an appropriate vector by any variety of well known techniques such as those described in Sambrook et al. [supra]. Generally, the encoding gene can be placed under the control of a control element such as a promoter, ribosome binding site or operator, so that the DNA sequence encoding the desired polypeptide is transcribed into RNA in the transformed host cell. Vectors may be derived from various sources including, but not limited to bacterial plasmids, bacteriophage, transposons, yeast episomes, insertion elements, yeast chromosomal elements, viruses for example, baculoviruses and SV40 (simian virus), vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, or combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, including cosmids and phagemids. Human, bacterial and yeast artificial chromosomes (HACs, BACs and YACs respectively) may also be employed to deliver larger fragments of DNA than can be contained and expressed in a plasmid.

Examples of retroviruses include but are not limited to: murine leukaemia virus (MLV), human immunodeficiency virus (HIV), equine infectious anaemia virus (EIAV), mouse mammary tumour virus (MMTV), Rous sarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney murine leukaemia virus (Mo-MLV), FBR murine osteosarcoma virus (FBR MSV), Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukaemia virus (A-MLV), Avian myelocytomatosis virus-29 (MC29), and Avian erythroblastosis virus (AEV). A detailed list of retroviruses may be found in Coffin et al ("Retroviruses" 1997 Cold Spring Harbour Laboratory Press Eds: JM Coffin, SM Hughes, HE Varmus pp 758-763).

Lentiviruses can be divided into primate and non-primate groups. Examples of primate lentiviruses include but are not limited to: the human immunodeficiency virus (HIV), the causative agent of human auto-immunodeficiency syndrome (AIDS), and the simian immunodeficiency virus (SIV). The non-primate lentiviral group includes the prototype "slow virus" visna/maedi virus (VMV), as well as the related caprine arthritis-encephalitis virus (CAEV), equine infectious anaemia virus (EIAV) and the more recently described feline immunodeficiency virus (FIV) and bovine immunodeficiency virus (BIV).

A distinction between the lenti virus family and other types of retroviruses is that lentiviruses have the capability to infect both dividing and non-dividing cells (Lewis et al 1992 EMBO. J 11 : 3053-3058; Lewis and Emerman 1994 J. Virol. 68: 510-516). In contrast, other retroviruses - such as MLV - are unable to infect non-dividing cells such as those that make up, for example, muscle, brain, lung and liver tissue.

A vector may be configured as a split-intron vector. A split intron vector is described in PCT patent applications WO 99/15683 and WO 99/15684.

If the features of adenoviruses are combined with the genetic stability of retroviruses/lentiviruses then essentially the adenovirus can be used to transduce target cells to become transient retroviral producer cells that could stably infect neighbouring cells. Such retroviral producer cells engineered to express an antigen of the present invention can be implanted in organisms such as animals or humans for use in the treatment of angiogenesis and/or cancer.

Poxvirus vectors are also suitable for use in accordance with the present invention. Pox viruses are engineered for recombinant gene expression and for the use as recombinant live vaccines. This entails the use of recombinant techniques to introduce nucleic acids encoding foreign antigens into the genome of the pox virus. If the nucleic acid is integrated at a site in the viral DNA which is non-essential for the life cycle of the virus, it is possible for the newly produced recombinant pox virus to be infectious, that is to say to infect foreign cells and thus to express the integrated DNA sequence. The recombinant pox virus prepared in this way can be used as live vaccines for the prophylaxis and/or treatment of pathologic and infectious disease.

For vaccine delivery, preferred vectors are vaccinia virus vectors such as MVA or NYVAC. Most preferred is the vaccinia strain modified virus ankara (MVA) or a strain derived therefrom. Alternatives to vaccinia vectors include avipox vectors such as fowlpox or canarypox known as ALVAC and strains derived therefrom which can infect and express recombinant proteins in human cells but are unable to replicate.

Bacterial vectors may be also used, such as salmonella, listeria and mycobacteria.

Vectors containing the relevant nucleotide sequence may enter the host cell by a variety of methods well known in the art and described in many standard laboratory manuals (such as Sambrook et al, [supra], Ausubel et al, [supra], Davis et al, Basic Methods in Molecular Biology (1986)). Methods include calcium phosphate transfection, cationic lipid-mediated transfection, DEAE-dextran mediated transfection, electroporation, microinjection, scrape loading, transduction, and ballistic introduction or infection.

Host cells

The choice of host cells is often dependent on the vector type used as a carrier for the nucleic acid molecule of the present invention. Bacteria and other microorganisms are particularly suitable hosts for plasmids, cosmids and expression vectors generally (for example, vectors derived from the pBR322 plasmid), yeast are suitable hosts for yeast expression vectors, insect cell systems are suitable host for virus expression vectors (for example, baculovirus) and plant cells are suitable hosts for vectors such as the cauliflower mosaic virus (CaMV) and tobacco mosaic virus (TMV). Other expression systems include using animal cells (for example, with the LentiVectors™, Oxford BioMedica) as a host cell or even using cell-free translating systems. Some vectors, such as "shuttle vectors" may be maintained in a variety of host cells. An example of such a vector would be pEG 202 and other yeast two-hybrid vectors which can be maintained in both yeast and bacterial cells (see Ausubel et al, [supra] and Gyuris, J., Cell, 75, 791-803).

Examples of suitable bacterial hosts include Streptococci, Staphylococci, Escheήchia coli, Streptomyces and Bacillus subtilis cells. Yeast and fungal hosts include Saccharomyces cerevisiae and Aspergillus cells. Mammalian cell hosts include many immortalised cell lines available from the American Type Culture Collection (ATCC) such as CHO (Chinese Hamster Ovary) cells, HeLa cells, BHK (baby hamster kidney) cells, monkey kidney cells, C127, 3T3, BHK, HEK 293, Bowes melanoma and human hepatocellular carcinoma (for example, Hep G2) cells. Insect host cells that are used for baculovirus expression include Drosophila S2 and Spodoptera Sf9 cells. Plant host cells include most plants from which protoplasts be isolated and cultured to give whole regenerated plants. Practically, all plants can be regenerated from cultured cells or tissues, including but not limited to all major species of sugar cane, sugar beet, cotton, fruit and other trees, legumes and vegetables.

Expression systems

Also included in present invention are expression vectors that comprise a nucleic acid molecule as described above. Expression vectors and host cells are preferably chosen to give long term, high yield production and stable expression of the recombinant polypeptide and its variants.

Expression of a polypeptide can be effected by cloning an encoding nucleic acid molecule into a suitable expression vector and inserting this vector into a suitable host cell. The positioning and orientation of the nucleic acid molecule insert with respect to the regulatory sequences of the vector is important to ensure that the coding sequence is properly transcribed and translated. Alternatively, control and other regulatory sequences may be ligated onto the nucleic acid molecule of this invention prior to its insertion into the expression vector. In both cases, the sequence of the nucleic acid molecule may have to be adjusted in order to effect correct transcription and translation (for example, addition of nucleotides may be necessary to obtain the correct reading frame for translation of the polypeptide from its encoding nucleic acid molecule).

A nucleic acid molecule of the invention may comprise control sequences that encode signal peptides or leader sequences. These sequences may be useful in directing the translated polypeptide to a variety of locations within or outside the host cell, such as to the lumen of the endoplasmic reticulum, to the nucleus, to the periplasmic space, or into the extracellular environment. Such signals may be endogenous to the nucleic acid molecules of the invention, or may be a heterologous sequence. These leader or control sequences may be removed by the host during post-translational processing.

A nucleic acid molecule of the present invention may also comprise one or more regulatory sequences that allow for regulation of the expression of polypeptide relative to the growth of the host cell. Alternatively, these regulatory signals may be due to a heterologous sequence from the vector. Stimuli that these sequences respond to include those of a physical or chemical nature such as the presence or absence of regulatory compounds, changing temperatures or metabolic conditions. Regulatory sequences as described herein, are non- translated regions of sequence such as enhancers, promoters and the 5' and 3' untranslated regions of genes. Regulatory sequences interact with host cellular proteins that carry out translation and transcription. These regulatory sequences may vary in strength and specificity. Examples of regulatory sequences include those of constitutive and inducible promoters. In bacterial systems, an example of an inducible promoter is the hybrid lacZ promoter of the Bluescript phagemid (Stratagene, LaJolla, CA) or pSportlTM plasmid (Gibco BRL). The baculovirus polyhedrin promoter may be used in insect cells.

An example of a preferred expression system is the lentivirus expression system, for example, as described in International patent application WO98/17815.

Detection of uptake of vectors by the host organism

Various methods are known in the art to detect the uptake of a nucleic acid or vector molecule by a host cell and/or the subsequent successful expression of the encoded polypeptide (see for example Sambrook et al, [supra]).

Vectors frequently have marker genes that can be easily assayed. Thus, vector uptake by a host cell can be readily detected by testing for the relevant phenotype. Markers include, but are not limited to those coding for antibiotic resistance, herbicide resistance or nutritional requirements. The gene encoding dihydrofolate reductase (DHFR) for example, confers resistance to methotrexate (Wigler, M. et al. (1980) PNAS 77:3567-70) and the gene npt confers resistance to the aminoglycosides neomycin and G-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol. 150:1-14). Additional selectable genes have been described, examples of which will be clear to those of skill in the art.

Markers however, only indicate that a vector has been taken up by a host cell but does not distinguish between vectors that contain the desired nucleic acid molecule and those that do not. One method of detecting for the said nucleic acid molecule is to insert the relevant sequence at a position that will disrupt the transcription and translation of a marker gene. These cells can then be identified by the absence of a marker gene phenotype. Alternatively, a marker gene can be placed in tandem with a sequence encoding a polypeptide of the invention under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as well. More direct and definitive methods to detect the presence of the nucleic acid molecule of the present invention include DNA-DNA or DNA-RNA hybridisation with a probe comprising the relevant antisense molecule, as described above. More direct methods to detect polypeptide expression include protein bioassays for example, fluorescence activated cell sorting (FACS), immunoassay techniques such as ELISA or radioimmunoassays.

Alternative methods for detecting or quantitating the presence of the nucleic acid molecule or polypeptide of this invention include membrane, solution or chip-based technologies (see Hampton, R. et al, (1990) Serological Methods, a Laboratory Manual, APS Press, St Paul, MN) and Maddox, D.E. et al., (1983) J. Exp. Med, 158, 1211-1216).

Trails genie animals

In another embodiment of this invention, a nucleic acid molecule according to the invention may be used to create a transgenic animal, most commonly a rodent. The modification of the animal's genome may either be done locally, by modification of somatic cells or by germ line therapy to incorporate inheritable modifications. Such transgenic animals may be particularly useful in the generation of animal models for drug molecules effective as modulators of the polypeptides of the present invention.

Polypeptide purification

A polypeptide according to the invention may be recovered and purified from recombinant cell cultures by methods including, but not limited to cell lysis techniques, ammonium sulphate precipitation, ethanol precipitation, acid extraction, anion or cation chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography, high performance liquid chromatography (HPLC) or fast performance liquid chromatography (FPLC). The polypeptide may need refolding after purification or isolation and many well known techniques are available that will help regenerate an active polypeptide conformation.

Many expression vectors are commercially available that aid purification of the relevant polypeptide. These include vectors that join the sequence encoding the polypeptide to another expressed sequence creating a fused protein that is easier to purify. Ways in which these fused parts can facilitate purification of the polypeptide of this invention include fusions that can increase the solubility of the polypeptide, joining of metal chelating peptides (for example, histidine-tryptophan modules) that allow for purification with immobilised metals, joining of protein A domains which allow for purification with immobilised immunoglobulins and the joining of the domain that is utilised in the FLAGS extension/affinity purification system (Immunex Corp., Seattle, WA). Fusion of the polypeptide of this present invention with a secretion signal polypeptide may also aid purification. This is because the medium into which the fused polypeptide has been secreted can subsequently be used to recover and purify the expressed polypeptide.

If necessary, these extraneous polypeptides often comprise a cleavable linker sequence which allows the polypeptide to be isolated from the fusion. Cleavable linker sequences between the purification domain and the polypeptide of the invention include those specific for Factor Xa or for enterokinase (Invitrogen, San Diego, CA). One such expression vector provides for expression of a fusion protein containing the polypeptide of the invention fused to several histidine residues preceding a thioredoxin or an enterokinase cleavage site. The histidine residues facilitate purification by IMAC (immobilised metal ion affinity chromatography as described in Porath, J. et al. (1992), Prot. Exp. Purif. 3: 263-281), while the thioredoxin or enterokinase cleavage site provides a means for purifying the polypeptide from the fusion protein. A discussion of vectors that contain fusion proteins is provided in Kroll, D.J. et al. (1993; DNA Cell Biol. 12:441-453).

Assays

Another aspect of this invention includes assays that may be carried out using a polypeptide or nucleic acid molecule according to the invention. Such assays may be for many uses including the development of drug candidates, for diagnostic purposes or for the gathering of information for therapeutics.

If the polypeptide is to be expressed for use in screening assays, generally it is preferred that it be produced at the surface of the host cell in which it is expressed. In this event, the host cells may be harvested prior to use in the screening assay, for example using techniques such as fluorescence activated cell sorting (FACS) or immunoaffinity techniques. If the polypeptide is secreted into the medium, the medium can be recovered in order to recover and purify the expressed polypeptide. If polypeptide is produced intracellularly, the cells must first be lysed before the polypeptide is recovered.

The polypeptide of the invention can be used to screen libraries of compounds in any of a variety of drug screening techniques. Such compounds may activate (agonise) or inhibit

(antagonise) the level of expression of the gene or the activity of the polypeptide of the invention and form a further aspect of the present invention. Examples of suitable compounds are those which are effective to alter the expression of a natural gene which encodes a polypeptide of the invention or to regulate the activity of a polypeptide of the invention.

Agonist or antagonist compounds may be isolated from, for example, cells, cell-free preparations, chemical libraries or natural product mixtures. These agonists or antagonists may be natural or modified substrates, ligands, enzymes, receptors or structural or functional mimetics. For a suitable review of such screening techniques, see Coligan et al., Current Protocols in Immunology l(2):Chapter 5 (1991).

Potential agonists or antagonists include small organic molecules, peptides, polypeptides and antibodies that bind to the polypeptide of the invention and thereby modulate its activity. In this fashion, binding of the polypeptide to normal cellular binding molecules may be potentiated or inhibited, such that the normal biological activity of the polypeptide is enhanced or prevented.

The polypeptide of the invention that is employed in such a screening technique may be free in solution, affixed to a solid support, borne on a cell surface or located intracellularly. In general, such screening procedures may involve using appropriate cells or cell membranes that express the polypeptide that are contacted with a test compound to observe binding, or stimulation or inhibition of a functional response. The functional response of the cells contacted with the test compound is then compared with control cells that were not contacted with the test compound. Such an assay may assess whether the test compound results in a signal generated by activation of the polypeptide, using an appropriate detection system. Inhibitors of activation are generally assayed in the presence of a known agonist and the effect on activation by the agonist in the presence of the test compound is observed.

Alternatively, simple binding assays may be used, in which the adherence of a test compound to a surface bearing the polypeptide is detected by means of a label directly or indirectly associated with the test compound or in an assay involving competition with a labelled competitor. In another embodiment, competitive drug screening assays may be used, in which neutralising antibodies that are capable of binding the polypeptide specifically compete with a test compound for binding. In this manner, the antibodies can be used to detect the presence of any test compound that possesses specific binding affinity for the polypeptide. Assays may also be designed to detect the effect of added test compounds on the production of mRNA encoding the polypeptide in cells. For example, an ELISA may be constructed that measures secreted or cell-associated levels of polypeptide using monoclonal or polyclonal antibodies by standard methods known in the art, and this can be used to search for compounds that may inhibit or enhance the production of the polypeptide from suitably manipulated cells or tissues. The formation of binding complexes between the polypeptide and the compound being tested may then be measured.

Another technique for drug screening which may be used provides for high throughput screening of compounds having suitable binding affinity to the polypeptide of interest (see International patent application WO84/03564). In this method, large numbers of different small test compounds are synthesised on a solid substrate, which may then be reacted with the polypeptide of the invention and washed. One way of immobilising the polypeptide is to use non-neutralising antibodies. Bound polypeptide may then be detected using methods that are well known in the art. Purified polypeptide can also be coated directly onto plates for use in the aforementioned drug screening techniques.

A polypeptide according to the invention may be used to identify membrane-bound or soluble receptors, through standard receptor binding techniques that are known in the art, such as ligand binding and crosslinking assays in which the polypeptide is labelled with a radioactive isotope, is chemically modified, or is fused to a peptide sequence that facilitates its detection or purification, and incubated with a source of the putative receptor (for example, a composition of cells, cell membranes, cell supernatants, tissue extracts, or bodily fluids). The efficacy of binding may be measured using biophysical techniques such as surface plasmon resonance and spectroscopy. Binding assays may be used for the purification and cloning of the receptor, but may also identify agonists and antagonists of the polypeptide, that compete with the binding of the polypeptide to its receptor. Standard methods for conducting screening assays are well understood in the art.

A typical polypeptide-based assay might involve contacting the appropriate cell(s) or cell membrane(s) expressing the polypeptide with a test compound. In such assays, a polypeptide according to the invention may be free in solution, affixed to a solid support, borne on a cell surface or located intracellularly. Any response to the test compound, for example a binding response, a stimulation or inhibition of a functional response may then be compared with a control where the cell(s) or cell membrane(s) was/were not contacted with the test compound.

A binding response could be measured by testing for the adherence of a test compound to a surface bearing a polypeptide according to the invention. The test compound may aid polypeptide detection by being labelled, either directly or indirectly. Alternatively, the polypeptide itself may be labelled, for example, with a radioisotope, by chemical modification or as a fusion with a peptide or polypeptide sequence that will facilitate polypeptide detection. Alternatively, a binding response may be measured, for example, by performing a competition assay with a labelled competitor or vice versa. One example of such a technique is a competitive drug screening assay, where neutralising antibodies that are capable of specifically binding to the polypeptide compete with a test compound for binding. In this manner, the antibodies may be used to detect the presence of any test compound that possesses specific binding affinity for the polypeptide. Alternative binding assay methods are well known in the art and include, but are not limited to, cross-linking assays and filter binding assays. The efficacy of binding may be measured using biophysical techniques including surface plasmon resonance and spectroscopy. High throughput screening is a type of assay which enables a large number of compounds to be searched for any significant binding activity to the polypeptide of interest (see patent application WO84/03564). This is particularly useful in drug screening. In this scenario, many different small test compounds are synthesised on to a solid substrate. The polypeptide is then introduced to this substrate and the whole apparatus washed. The polypeptide is then immobilised by, for example, using non-neutralising antibodies. Bound polypeptide may then be detected using methods that are well known in the art. Purified polypeptide may also be coated directly onto plates for use in the aforementioned drug screening techniques.

Assay methods that are also included within the terms of the present invention are those that involve the use of the genes and polypeptides of the invention in overexpression or ablation assays. Such assays involve the manipulation of levels of these genes/polypeptides in cells and assessment of the impact of this manipulation event on the physiology of the manipulated cells. For example, such experiments reveal details of signaling and metabolic pathways in which the particular genes/polypeptides are implicated, generate information regarding the identities of polypeptides with which the studied polypeptides interact and provide clues as to methods by which related genes and proteins are regulated.

Another aspect of this invention provides for any screening kits that are based or developed from any of the aforementioned assays.

C. Pharmaceuticals

A further aspect of the invention provides a pharmaceutical composition suitable for modulating the biological response to hypoxia and/or ischaemia, comprising a therapeutically- effective amount of a polypeptide, a nucleic acid molecule, vector or ligand as described above, in conjunction with a pharmaceutically-acceptable carrier. A composition containing a polypeptide, nucleic acid molecule, ligand or any other compound of this present invention (herein known as X) is considered to be "substantially free of impurities" (herein known as Y) when X makes up more than 85% mass per mass of the total [X+Y] mass. Preferably X comprises at least 90% of the total X+Y mass. More preferably X comprises at least 95%, 98% and most preferably 99% of the total X+Y mass. Carriers

Carrier molecules may be genes, polypeptides, antibodies, liposomes or indeed any other agent provided that the carrier does not itself induce toxicity effects or cause the production of antibodies that are harmful to the individual receiving the pharmaceutical composition. Further examples of known carriers include polysaccharides, polylactic acids, polyglycolic acids and inactive virus particles. Carriers may also include pharmaceutically acceptable salts such as mineral acid salts (for example, hydrochlorides, hydrobromides, phosphates, sulphates) or the salts of organic acids (for example, acetates, propionates, malonates, benzoates). Pharmaceutically acceptable carriers may additionally contain liquids such as water, saline, glycerol, ethanol or auxiliary substances such as wetting or emulsifying agents, pH buffering substances and the like. Carriers may enable the pharmaceutical compositions to be formulated into tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions to aid intake by the patient. A thorough discussion of pharmaceutically acceptable carriers is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ. 1991).

Dosage The amount of component X in the composition should also be in therapeutically effective amounts. The phrase "therapeutically effective amounts" used herein refers to the amount of agent needed to treat, ameliorate, or prevent (for example, when used as a vaccine) a targeted disease or condition. An effective initial method to determine a "therapeutically effective amount" may be by carrying out cell culture assays (for example, using neoplastic cells) or using animal models (for example, mice, rabbits, dogs or pigs). In addition to determining the appropriate concentration range for X to be therapeutically effective, animal models may also yield other relevant information such as preferable routes of administration that will give maximum effectiveness. Such information may be useful as a basis for patient administration. A "patient" as used in herein refers to the subject who is receiving treatment by administration of X. Preferably, the patient is human, but the term may also include animals.

The therapeutically-effective dosage will generally be dependent on the patient's status at the time of administration. Factors that may be taken into consideration when determining dosage include the severity of the disease state in the patient, the general health of the patient, the age, weight, gender, diet, time and frequency of administration, drug combinations, reaction sensitivities and the patient's tolerance or response to the therapy. The precise amount can be determined by routine experimentation but may ultimately lie with the judgement of the clinician. Generally, an effective dose will be from 0.01 mg/kg (mass of drug compared to mass of patient) to 50 mg/kg, preferably 0.05 mg/kg to 10 mg/kg. Compositions may be administered individually to a patient or may be administered in combination with other agents, drugs or hormones.

Routes of administration

Uptake of a pharmaceutical composition of the invention by a patient may be initiated by a variety of methods including, but not limited to enteral, intra-arterial, intrathecal, intramedullary, intramuscular, intranasal, intraperitoneal, intravaginal, intravenous, intraventricular, oral, rectal (for example, in the form of suppositories), subcutaneous, sublingual, transcutaneous applications (for example, see WO98/20734) or transdermal means.

Gene guns or hyposprays may also be used to administer the pharmaceutical compositions of the invention. Typically, the therapeutic compositions may be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared. Direct delivery of the compositions can generally be accomplished by injection, subcutaneously, intraperitoneally, intravenously or intramuscularly, or delivered to the interstitial space of a tissue. The compositions can also be administered into a lesion. Dosage treatment may be a single dose schedule or a multiple dose schedule.

Inhibition of excessive activity

If a particular disease state is partially or completely caused by an inappropriate excess in the activity of a polypeptide according to the invention, several approaches are available for inhibiting this activity.

One approach comprises administering to a patient an inhibitor compound (antagonist) along with a pharmaceutically acceptable carrier in an amount effective to inhibit the function of the polypeptide, such as by blocking the binding of a ligand, substrate, enzyme, receptor, or by inhibiting a second signal, and thereby alleviating the abnormal condition. Such an antagonist molecule may, for example, be an antibody. Most preferably, such antibodies are chimeric and/or humanised to minimise their immunogenicity, as previously described.

In another approach, soluble forms of the polypeptide that retain binding affinity for the ligand, substrate, enzyme, receptor, in question, may be administered to the patient to compete with the biological activity of the endogenous polypeptide. Typically, the polypeptide may be administered in the form of a fragment that retains a portion that is relevant for the desired biological activity.

In an alternative approach, expression of the gene encoding the polypeptide can be inhibited using expression blocking techniques, such as by using antisense nucleic acid molecules (as described above), either internally generated or separately administered. Modifications of gene expression may be effected by designing complementary sequences or antisense molecules (DNA, RNA, or PNA) to the control, 5' or regulatory regions (signal sequence, promoters, enhancers and introns) of the gene encoding the polypeptide. Similarly, inhibition can be achieved using "triple helix" base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules. Recent therapeutic advances using triplex DNA have been described in the literature (Gee, J.E. et al. (1994) In: Huber, B.E. and B.I. Carr, Molecular and Immunologic Approaches, Futura Publishing Co., Mt. Kisco, NY). The complementary sequence or antisense molecule may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes. Such oligonucleotides may be administered or may be generated in situ from expression in vivo.

Gene silencing approaches may also be undertaken to down-regulate endogenous expression of a gene. RNA interference (RNAi) (Elbashir, SM et al., Nature 2001, 411, 494-498) is one method of sequence specific post-transcriptional gene silencing that may be employed. Short dsRNA oligonucleotides are synthesised in vitro and introduced into a cell. The sequence specific binding of these dsRNA oligonucleotides triggers the degradation of target mRNA, reducing or ablating target protein expression.

In addition, expression of a polypeptide according to the invention may be prevented by using a ribozyme specific to the encoding mRNA sequence for the polypeptide. Ribozymes are catalytically active RNAs that can be natural or synthetic (see for example Usman, N, et al, Curr. Opin. Struct. Biol (1996) 6(4), 527-33). Synthetic ribozymes can be designed to specifically cleave mRNAs at selected positions thereby preventing translation of the mRNAs into functional polypeptide. Ribozymes may be synthesised with a natural ribose phosphate backbone and natural bases, as normally found in RNA molecules. Alternatively the ribozymes may be synthesised with non-natural backbones, for example, 2'-O-methyl RNA, to provide protection from ribonuclease degradation and may contain modified bases. Efficacy of the gene silencing approaches assessed above may be assessed through the measurement of polypeptide expression (for example, by Western blotting), and at the RNA level using TaqMan-based methodologies.

RNA molecules may be modified to increase their intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends of the molecule or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the backbone of the molecule. This concept is inherent in the production of PNAs and can be extended in all of these molecules by the inclusion of non- traditional bases such as inosine, queosine and butosine, as well as acetyl-, methyl-, thio- and similarly modified forms of adenine, cytidine, guanine, thymine and uridine that are not as easily recognised by endogenous endonucleases.

Activation of a polypeptide activity

If a particular disease state is partially or completely due to a lowered level of biological activity from a polypeptide according to the invention, various methods may be used. An example of such a method includes administering a therapeutically effective amount of compound that can activate (i.e. an agonist) or cause increased expression of the polypeptide concerned. Administration of such a compound may be via any of the methods described previously.

Gene Therapy Another aspect of the present invention provides for gene therapy methods involving nucleic acid molecules identified herein. Gene therapy may be used to affect the endogenous production of the polypeptide of the present invention by relevant cells in a patient. For example, gene therapy can be used permanently to treat the inappropriate production of a polypeptide by replacing a defective gene with the corrected therapeutic gene. Treatment may be effected either in vivo or ex vivo. Ex vivo gene therapy generally involves the isolation and purification of the patient's cells, introduction of the therapeutic gene into the cells and finally, the introduction of the genetically-altered cells back into the patient. In vivo gene therapy does not require the isolation and purification of patient cells prior to the introduction of the therapeutic gene into the patient. Instead, the therapeutic gene can be packaged for delivery into the host. Gene delivery vehicles for in vivo gene therapy include, but are not limited to, non-viral vehicles such as liposomes, replication-deficient viruses (for example, adenovirus as described by Berkner, K.L., in Curr. Top. Microbiol. Immunol., 158, 39-66 (1992)) or adeno-associated virus (AAV) vectors as described by Muzyczka, N., in Curr. Top. Microbiol. Immunol., 158, 97-129 (1992) and U.S. Patent No. 5,252,479. Alternatively, "naked DNA" may be directly injected into the bloodstream or muscle tissue as a form of in vivo gene therapy.

One example of a strategy for gene therapy including a nucleic acid molecule of this present invention may be as follows. A nucleic acid molecule encoding a polypeptide of the invention is engineered for expression in a replication-defective retroviral vector. This expression construct may then be isolated and introduced into a packaging cell transduced with a retroviral plasmid vector containing RNA encoding the polypeptide, such that the packaging cell now produces infectious viral particles containing the gene of interest. These producer cells may be administered to a patient for engineering cells in vivo and expression of the polypeptide in vivo (see Chapter 20, Gene Therapy and other Molecular Genetic-based Therapeutic Approaches, (and references cited therein) in Human Molecular Genetics (1996), T Strachan and A P Read, BIOS Scientific Publishers Ltd).

Genetic delivery of antibodies that bind to polypeptides according to the invention may also be effected, for example, as described in International patent application WO98/55607.

Vaccines

A further embodiment of the present invention provides that the polypeptides or nucleic acid molecules identified may be used in the development of vaccines. Where the aforementioned polypeptide or nucleic acid molecule is a disease-causing agent, vaccine development can involve the raising of antibodies against such agents. Where the aforementioned polypeptide or nucleic acid molecule is one that is up-regulated, vaccine development can involve the raising of antibodies or T cells against such agents (as described in WO00/29428).

Vaccines according to the invention may either be prophylactic (i.e. prevents infection) or therapeutic (i.e. treats disease after infection). Such vaccines comprise immunising antigen(s), immunogen(s), polypeptide(s), protein(s) or nucleic acid, usually in combination with pharmaceutically-acceptable carriers as described above. Additionally, these carriers may function as immunostimulating agents ("adjuvants"). Furthermore, the antigen or immunogen may be conjugated to a bacterial toxoid, such as a toxoid from diphtheria, tetanus, cholera, H. pylori, and other pathogens. Vaccination processes may involve the use of heterologous vectors eg: prime with MVA and boost with DNA.

Since polypeptides may be broken down in the stomach, vaccines comprising polypeptides are preferably administered parenterally (for instance, subcutaneous, intramuscular, intravenous, or intradermal injection). Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions that may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the recipient, and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents.

The vaccine formulations of the invention may be presented in unit-dose or multi-dose containers. For example, sealed ampoules and vials and may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier immediately prior to use. The dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation.

The technology referred to as jet injection (see, for example, www.powderject.com) may also be useful in the formulation of vaccine compositions.

In accordance with this aspect of the present invention, polypeptides can be delivered by viral or non-viral techniques. Non-viral delivery systems include but are not limited ^• to DNA transfection methods. Here, transfection includes a process using a non-viral vector to deliver a antigen gene to a target mammalian cell. Typical transfection methods include electroporation, nucleic acid biolistics, lipid-mediated transfection, compacted nucleic acid- mediated transfection, liposomes, immunoliposomes, lipofectin, cationic agent-mediated, cationic facial amphiphiles (CFAs) (Nature Biotechnology 1996 14; 556), multivalent cations such as spermine, cationic Hpids or polylysine, 1, 2,-bis (oleoyloxy)-3-(trimethylammonio) propane (DOTAP)-cholesterol complexes (Wolff and Trubetskoy 1998 Nature Biotechnology 16: 421) and combinations thereof.

Viral delivery systems include but are not limited to adenovirus vectors, adeno-associated viral (AAV) vectors, herpes viral vectors, influenza, retroviral vectors, lentiviral vectors or baculoviral vectors, Venezuelan equine encephalitis virus (VEE), poxviruses such as: canarypox virus (Taylor et al 1995 Vaccine 13:539-549), entomopox virus (Li Y et al 1998 Xllth International Poxvirus Symposium pi 44. Abstract), penguine pox (Standard et al. J Gen Virol. 1998 79:1637-46) alphavirus, and alphavirus based DNA vectors.

In addition to the use of polypeptide-based vaccines, this aspect of the invention includes the use of genetically-based vaccines, for example, those vaccines that are effective through eliciting the expression of a particular gene (either endogenous or exogenously derived) in a cell, so targeting this cell for destruction by the immune system of the host organism. A number of suitable methods for vaccination and vaccine delivery systems are described in International patent application WO00/29428.

D. Diagnostics

Another aspect of the present invention provides for the use of a nucleic acid molecule identified herein as a diagnostic reagent.

For example, a nucleic acid molecule may be detected or isolated from a patient's tissue and used for diagnostic purposes. "Tissue" as defined herein refers to blood, urine, any matter obtained from a tissue biopsy or any matter obtained from an autopsy. Genomic DNA from the tissue sample may be used directly for detection of a hypoxia-related condition. Alternatively, the DNA may be amplified using methods such as polymerase chain reaction (PCR), the ligase chain reaction (LCR), strand displacement amplification (SDA), or other amplification techniques (see Saiki et al, Nature, 324, 163-166 (1986); Bej, et al, Crit. Rev. Biochem. Molec. Biol., 26, 301-334 (1991); Birkenmeyer et al, J. Virol. Meth., 35, 117-126 (1991) and Brunt, J., Bio/Technology, 8, 291-294 (1990)). Such diagnostics are particularly useful for prenatal and even neonatal testing.

A method of diagnosis of disease using a polynucleotide may comprise assessing the level of expression of the natural gene and comparing the level of encoded polypeptide to a control level measured in a normal subject that does not suffer from the disease or physiological condition that is being tested. The diagnosis may comprise the following steps: a) contacting a sample of tissue from the patient with a nucleic acid probe under stringent conditions that allow the formation of a hybrid complex between a nucleic acid molecule of the invention and the probe; b) contacting a control sample with said probe under the same conditions used in step a); and c) detecting the presence of hybrid complexes in said samples; wherein detection of differing levels of the hybrid complex in the patient sample compared to levels of the hybrid complex in the control sample is indicative of the dysfunction.

A further aspect of the invention comprises a diagnostic method comprising the steps of: a) obtaining a tissue sample from a patient being tested for disease; b) isolating a nucleic acid molecule according to the invention from said tissue sample; and c) diagnosing the patient for disease by detecting the presence of a mutation in the nucleic acid molecule which is associated with disease.

To aid the detection of nucleic acid molecules in the above-described methods, an amplification step, such as PCR, may be included. An example of this includes detection of deletions or insertions indicative of the dysfunction by a change in the size of the amplified product in comparison to the normal genotype. Point mutations can be identified by hybridising amplified DNA to labelled RNA of the invention or alternatively, labelled antisense DNA sequences of the invention. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by assessing differences in melting temperatures. The presence or absence of the mutation in the patient may be detected by contacting DNA with a nucleic acid probe that hybridises to the DNA under stringent conditions to form a hybrid double-stranded molecule, the hybrid double-stranded molecule having an unhybridised portion of the nucleic acid probe strand at any portion corresponding to a mutation associated with disease; and detecting the presence or absence of an unhybridised portion of the probe strand as an indication of the presence or absence of a disease-associated mutation in the corresponding portion of the DNA strand.

Point mutations and other sequence differences between the reference gene and "mutant" genes can be identified by other well-known techniques, such as direct DNA sequencing or single- strand conformational polymorphism, (see Orita et al, Genomics, 5, 874-879 (1989)). For example, a sequencing primer may be used with double-stranded PCR product or a single- stranded template molecule generated by a modified PCR. The sequence determination is performed by conventional procedures with radiolabelled nucleotides or by automatic sequencing procedures with fluorescent-tags. Cloned DNA segments may also be used as probes to detect specific DNA segments. The sensitivity of this method is greatly enhanced when combined with PCR. Further, point mutations and other sequence variations, such as polymorphisms, can be detected as described above, for example, through the use of allele- specific oligonucleotides for PCR amplification of sequences that differ by single nucleotides.

DNA sequence differences may also be detected by alterations in the electrophoretic mobility of DNA fragments in gels, with or without denaturing agents, or by direct DNA sequencing (for example, Myers et al, Science (1985) 230:1242). Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and SI protection or the chemical cleavage method (see Cotton et al, PNAS. USA (1985) 85: 4397-4401). In addition to conventional gel electrophoresis and DNA sequencing, mutations such as microdeletions, aneuploidies, trans! ocations, inversions, can also be detected by in situ analysis (see, for example, Keller et al, DNA Probes, 2nd Ed., Stockton Press, New York, N.Y., USA (1993)), that is, DNA or RNA sequences in cells can be analysed for mutations without need for their isolation and/or immobilisation onto a membrane. FISH is presently the most commonly applied method and numerous reviews of FISH have appeared (see, for example, Trachuck et al, Science, 250, 559-562 (1990), and Trask et al, Trends, Genet., 7, 149-154 (1991)).

Arrays In another embodiment of the invention, an array of oligonucleotide probes comprising a nucleic acid molecule according to the invention can be constructed to conduct efficient screening of genetic variants, mutations and polymorphisms. Array technology methods are well known and have general applicability and can be used to address a variety of questions in molecular genetics including gene expression, genetic linkage, and genetic variability (see for example: M.Chee et al, Science (1996), Vol 274, pp 610-613).

In one embodiment, the array is prepared and used according to the methods described in W095/11995 (Chee et al); Lockhart, D. J. et al. (1996) Nat. Biotech. 14: 1675-1680); and Schena, M. et al (1996) PNAS 93: 10614-10619). Oligonucleotide pairs may range from two to over one million. The oligomers are synthesized at designated areas on a substrate using a light-directed chemical process. The substrate may be paper, nylon or other type of membrane, filter, chip, glass slide or any other suitable solid support. In another aspect, an oligonucleotide may be synthesized on the surface of the substrate by using a chemical coupling procedure and an ink jet application apparatus, as described in PCT application W095/251116 (Baldeschweiler et al). In another aspect, a "gridded" array analogous to a dot (or slot) blot may be used to arrange and link cDNA fragments or oligonucleotides to the surface of a substrate using a vacuum system, thermal, UV, mechanical or chemical bonding procedures. An array, such as those described above, may be produced by hand or by using available devices (slot blot or dot blot apparatus), materials (any suitable solid support), and machines (including robotic instruments), and may contain 8, 24, 96, 384, 1536 or 6144 oligonucleotides, or any other number between two and over one million which lends itself to the efficient use of commercially-available instrumentation. Diagnostics using polypeptides or mRNA

In addition to the methods discussed above, diseases may be diagnosed by methods comprising determining, from a sample derived from a subject, an abnormally decreased or increased level of polypeptide or mRNA. Decreased or increased expression can be measured at the RNA level using any of the methods well known in the art for the quantitation of polynucleotides, such as, for example, nucleic acid amplification, for instance PCR, RT-PCR, RNase protection, Northern blotting and other hybridization methods.

Assay techniques that can be used to determine levels of a polypeptide of the present invention in a sample derived from a host are well-known to those of skill in the art and are discussed in some detail above (including radioimmunoassays, competitive-binding assays, Western Blot analysis and ELISA assays). One example of this aspect of the invention provides a diagnostic method which comprises the steps of: (a) contacting a ligand as described above with a biological sample under conditions suitable for the formation of a ligand-polypeptide complex; and (b) detecting said complex. Protocols such as ELISA, RIA, and FACS for measuring polypeptide levels may additionally provide a basis for diagnosing altered or abnormal levels of polypeptide expression. Normal or standard values for polypeptide expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, preferably humans, with antibody to the polypeptide under conditions suitable for complex formation The amount of standard complex formation may be quantified by various methods, such as by photometric means.

Antibodies which specifically bind to a polypeptide of the invention may be used for the diagnosis of conditions or diseases characterised by expression of the polypeptide, or in assays to monitor patients being treated with the polypeptides, nucleic acid molecules, ligands and other compounds of the invention. Antibodies useful for diagnostic purposes may be prepared in the same manner as those described above for therapeutics. Diagnostic assays for the polypeptide include methods that utilise the antibody and a label to detect the polypeptide in human body fluids or extracts of cells or tissues. The antibodies may be used with or without modification, and may be labelled by joining them, either covalently or non-covalently, with a reporter molecule. A wide variety of reporter molecules known in the art may be used, several of which are described above.

Quantities of polypeptide expressed in subject, control and disease samples from biopsied tissues are compared with the standard values. Deviation between standard and subject values establishes the parameters for diagnosing disease. Diagnostic assays may be used to distinguish between absence, presence, and excess expression of polypeptide and to monitor regulation of polypeptide levels during therapeutic intervention. Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal.studies, in clinical trials or in monitoring the treatment of an individual patient. Diagnostic kits

A diagnostic kit of the present invention may comprise:

(a) a nucleic acid molecule of the present invention;

(b) a polypeptide of the present invention; or

(c) a ligand of the present invention. In one aspect of the invention, a diagnostic kit may comprise a first container containing a nucleic acid probe that hybridises under stringent conditions with a nucleic acid molecule according to the invention; a second container containing primers useful for amplifying the nucleic acid molecule; and instructions for using the probe and primers for facilitating the diagnosis of disease. The kit may further comprise a third container holding an agent for digesting unhybridised RNA.

In an alternative aspect of the invention, a diagnostic kit may comprise an array of nucleic acid molecules, an array of antibody molecules, and/or an array of polypeptide molecules, as discussed in more detail above.

Such kits will be of use in diagnosing a disease or susceptibility to disease, particularly inflammation, oncology, or cardiovascular disease.

Various aspects and embodiments of the present invention will now be described in more detail by way of example, with particular reference to polypeptides regulated differentially under hypoxic conditions as opposed to normoxic conditions. It will be appreciated that modification of detail may be made without departing from the scope of the invention. Brief description of the Figures

Figure 1 shows a scatter plot, showing normalised signal intensities in hypoxia versus normoxia, with each dot representing a single gene.

Figure 2: Hypoxia responses amplified by HIF1 alpha overexpression. Data shown is the average of 4 repeat experiments. Values represent fold change as compared to untreated cells (condition 1). Error bars represent standard error of the mean. Figure 3: Hypoxia responses amplified by EPAS1 overexpression. Data shown is the average of 4 repeat experiments. Values represent fold change as compared to untreated cells (condition 1). Error bars represent standard error of the mean.

Figure 4: Hypoxia responses amplified by HIF1 alpha / EPAS1 overexpression. Data shown is the average of 4 repeat experiments. Values represent fold change as compared to untreated cells (condition 1). Error bars represent standard error of the mean.

Figure 5 shows genes that are induced by hypoxia to a greater degree in resting macrophages, as compared to activated macrophages. Error bars show the standard deviation from both repeat experiments and multiple exposures from single experiments. These data are not shown in table form. All bars are ratios of mRNA expression in hypoxia/ normoxia. These are calculated separately for resting (light bars) and activated (dark bars) macrophages, and do not illustrate differences resulting from activation in normoxia.

Figure 6 shows genes which are induced by hypoxia to a greater degree in activated macrophages, compared to resting macrophages. Figure 7 shows genes that are repressed by hypoxia to a greater degree in activated macrophages.

For Figures 8a and 8d, mRNA levels, determined from a custom gene array, of particular genes are shown on the Y-axis, expressed as a value as compared to the median expression level of this gene throughout all samples. Eleven primary human cell types as shown on the x-axis were cultured in normoxia (black), or exposed to hyopxia for 6hr (grey) or 18hr (white).

Figure 8a: EGL nine (C.elegans) homolog 3 (SeqID: 85/86)

Figure 8b: Gene expression profiles in macrophages with and without activation. mRNA levels, determined from a custom gene array, of clorfl2 are shown on the Y-axis, expressed as a value compared to the mean value of a set of control genes on each array (pre-chip normalisation). All cells were human macrophages, cultured either without cytokines or with IL-10 or with the combination of IFNγ and LPS in normoxia and hypoxia.

Figure 8c: Gene expression profiles in macrophages with and without activation. mRNA levels, determined from a custom gene array, of EGLN3 are shown on the Y-axis, expressed as a value compared to the mean value of a set of control genes on each array (pre-chip normalisation). All cells were human macrophages, cultured either without cytokines or with IL-10 or with the combination of IFNγ and LPS in normoxia and hypoxia.

Figure 8d: Clorfl2 (SeqID: 89.90) Figure 8e: The effect of EPAS/ HIF overexpression on expression of the gene Clorfl2 EGLN genes using a custom gene array. mRNA expression levels of the gene clORF12 as determined by the custom array, in response to hypoxia and adenoviral over-expression of HIF or EPAS are shown. Experimental conditions are as follows: #1 no adeno / normoxia; #2 empty adeno (low dose)/ normoxia; #3 empty adeno (high dose)/ normoxia; #4 empty adeno (low dose)/ hypoxia; #5 empty adeno (high dose)/ hypoxia; #6 HIF-1 adeno (low dose)/ hypoxia; #7 HIF-1 adeno (high dose)/ hypoxia; #8 EPAS adeno (low dose)/ hypoxia; #9 EPAS adeno (high dose)/ hypoxia. Error bars are the standard error of the mean.

Figure 8f: The effect of EPAS/ HIF overexpression on expression of the gene EGLN3 gene using a custom gene array. mRNA expression levels of the gene EGLN3 as determined by the custom array, in response to hypoxia and adenoviral over-expression of HIF or EPAS are shown. Experimental conditions are as follows: #1 no adeno / normoxia; #2 empty adeno (low dose)/ normoxia; #3 empty adeno (high dose)/ normoxia; #4 empty adeno (low dose)/ hypoxia; #5 empty adeno (high dose)/ hypoxia; #6 HIF-1 adeno (low dose)/ hypoxia; #7 HIF-1 adeno (high dose)/ hypoxia; #8 EPAS adeno (low dose)/ hypoxia; #9 EPAS adeno (high dose)/ hypoxia. Error bars are the standard error of the mean.

Figure 8g: The effect of EPAS/ HIF overexpression on expression of the EGLN3 gene using AffyMetrix Hu95 ver2 GeneChips. mRNA expression levels of the gene in response to hypoxia and adenoviral over-expression of HIF or EPAS are shown. Graphs show the mean of two replicate arrays, with error bars as standard deviation. Above each graph, data values are shown, including the normalised values and raw values (the AffyMetrix average difference parameter) and Present/ Absent flags.

Figure 8h: The effect of EPAS/ HIF overexpression on expression of the clorfl2 gene using AffyMetrix Hu95 ver2 GeneChips. mRNA expression levels of the gene in response to hypoxia and adenoviral over-expression of HIF or EPAS are shown. Graphs show the mean of two replicate arrays, with error bars as standard deviation. Above each graph, data values are shown, including the normalised values and raw values (the AffyMetrix average difference parameter) and Present/ Absent flags.

Figure 8i: Flag immunocytochemistry in HEK293T cells Figure 8j: Human Cardiomyocyte Caspase Activity after 72 hours transduction with EIAV- ELG9-Homolog 3

Figure 9: Qualitiative RT PCR of EGLN3 isoforms in various primary cell types. Cell types are as follows: "Adipocytes" (Clonetics CC-2568; derived from subcutaneous adult adipose tissue), "Cardiomyocyte" (Clonetics CC-2582; derived from fetal tissue; prior to experimentation cultured in minimal medium: DMEM, 4% Horse serum), "HUVEC" (TCS CellWorks ZHC-2101 human umbilical vein endothelial cells), "Dermal fibroblast" (Clonetics CC-2511 dermal fibroblasts derived from adult tissue), "Macrophage" (derived from human blood as described elsewhere in the specification), "Mammary epithelium" (Clonetics CC-2551; derived from adult tissue), "Monocyte" (derived from human blood as described elsewhere in the specification but without the 7 day differentiation culture period), "SHSY5Y" (neuroblastoma-derived cell line SH-SY5Y), "Renal epithelial" (Clonetics CC-2556; derived from fetal tissue),

"SKM" skeletal muscle myocyte (Clonetics CC-2561 ; derived from adult tissue).

"N" = cells growing in normoxia. "6hr H" = after exposure to 0.1 % oxygen for 6hr. "18hr H" = after exposure to 0.1% oxygen for 18hr. "+" = positive control RNA. "-" = no RNA negative control. Figure 10: Effects of prolyl hydroxylase inhibitors on EGLN3 and the EGLN3 splice variant. Abbreviations are as follows: DAU=daunorubicin; MIMO=L-mimosine; HM=HREluc, MIMO; HH=HIF,HREluc; SlH=SVFLl,HREluc; S1HM=S1H, MIMO; S2H=SVFL2,HREluc; S2HM=S2H, MIMO; SlHH^*=SVFLl,HIF,HREluc; S1HHM=S1HH, MIMO; S2HH=SVFL2,HIF,HREluc; S2HHM=S2HH, MIMO. Examples

Summary

Subtracted cDNA libraries were separately prepared for hypoxic macrophages and cardiomyoblasts. This involved harvesting RNA from cells both in normoxia and hypoxia, and preparing cDNA. Subtractive hybridization / suppression PCR was then performed to remove genes from the hypoxic cell cDNA, which are also present in cDNA from normoxic cells. Insert DNA from the libraries was PCR amplified and arrayed onto duplicate membranes. Quantitative hybridizations with pre-library cDNA material (normoxia and hypoxia) were done to identify clones in the libraries that actually contain hypoxia inducible genes. The insert DNA was then sequenced. This procedure was done independently for macrophage and cardiomyoblast. The hypoxia inducible genes identified from these different cell types differed widely, with only a minority of these genes being identified from both cell types. To characterise the differences between the two tissues further, arrays were produced containing all confirmed hypoxia-inducible genes from the macrophage library. Replicate arrays were hybridised with cDNA from normoxic and hypoxic cardiomyoblasts to allow quantitative evaluation of these genes in the cardiomyoblast. This revealed quantitative differences in the hypoxia induced activation these genes in the two cell types.

Example la: Comparison of the hypoxic-response between human macrophages and cardiomyoblasts by a subtraction cloning / array screening approach

Methods / Results

To isolate human macrophage, monocytes were derived from peripheral blood of healthy human donors. 100ml bags of buffy coat from the Bristol Blood Transfusion Centre were mixed with an equal volume of RPMI1640 medium (Sigma). This was layered on top of 10ml ficol-paque (Pharmacia) in 50ml centrifuge tubes and centrifuged for 25 min at 800 x g. The interphase layer was removed, washed in MACS buffer (phosphate buffered saline pH 7.2, 0.5% bovine serum albumin, 2mM EDTA) and resuspended at 80 microliter per 10n7 cells. To this 20 microliter CD14 Microbeads (Miltenyi Biotec) were added, and the tube incubated at 4 degrees for 15 min. Following this one wash was performed in MACS buffer at 400 x g and the cells were resuspended in 3 ml MACS buffer and separated on an LS+ MACS Separation Column (Miltenyi Biotec) positioned on a midi-MACS magnet (Miltenyi Biotec). The column was washed with 3 x 3ml MACS buffer. The column was removed from the magnet and cells were eluted in 5 ml MACS buffer using a syringe. Cells were washed in culture medium (AIM V (Sigma) supplemented with 2% human AB serum (Sigma), and resuspended at 2 x 10n5 cells per ml in the same medium and placed in large teflon-coated culture bags (Sud- Laborbedarf GmbH, 82131 Gauting, Germany) and transferred to a tissue culture incubator (37 degrees, 5% CO2) for 7-10 days. During this period monocytes spontaneously differentiate to macrophages. This is confirmed by examining cell morphology using phase contrast microscopy. Cells are removed from the bags by placing at 4 degrees for 30 min and emptying the contents. The cells are then washed and resuspended in culture medium at 5 x 105 cell/ml and plated out in Primeria 10 cm tissue culture petri dishes (Falcon Becton Dickinson) at 5 x 10n6 cells per dish. Culture is continued for 16-24hr to allow cell adherence, prior to experimentation involving hypoxia.

As an alternative primary cell type human cardiomyoblast cultures were established. Cells derived from the ventricular tissue of newborn or foetal hearts were purchased from BioWhittaker (CC-2582). Growth conditions were used to allow maximum expansion of the cells in vitro, by using a medium rich in growth factors. Under such conditions cardiomyoblast-like cells predominate (the developmental precursor of cardiomyocytes). This has been previously described by Goldman and Wurzel (In Vitro Cell Dev. Biol. 28A: 109-119 (1992)) and Goldman et al, (1996, Exp.Cell.Res. 228(2): 237-245). For these cultures, cells were seeded at lxlO⁶ per T150 flask in human smooth muscle growth medium (TCS CellWorks ZHM-3935) and were expanded in the same medium up to a maximum number of 4 passages. The growth medium is purchased pre-prepared, and includes in the formula, 5% fetal bovine serum, insulin, epidermal growth factor and fibroblast growth factor. Prior to experimentation involving hypoxia, cells were plated onto 10 cm tissue culture petri dishes and allowed to reach confluency.

For experimentation with hypoxia, for all cell types, an equal number of identical culture dishes were divided into two separate incubators: One at 37 degrees, 5% C02, 95% air (=Normoxia) and the other at 37 degrees, 5% C0₂, 94.9% Nitrogen, 0.1% Oxygen (=Hypoxia). After 6 hours culture under these conditions, the dishes were removed from the incubator, placed on a chilled platform, washed in cold PBS and total RNA was extracted using RNazol B (Tel-Test, Inc; distributed by Biogenesis Ltd) following the manufacturer's instructions. Polyadenylated mRNA was extracted from the total RNA using a commercial kit following the manufacturer's instructions (Promega; PolyATract mRNA isolation System IV).

The hypoxia period of 6 hr was previously determined to be sufficient to allow the induction of known hypoxia-regulated genes, as determined by RNase protection assays. During these preliminary studies it was noted that macrophages, cardiomyoblasts and an additional control cell type, Jurkat T-cells, showed different patterns of gene induction in response to hypoxia:

Known Hypoxia-inducible gene level of hypoxia-induced increase in mRNA levels

Macrophage Myoblast T-cell phosphoglycerate kinase-1 none none high

(PGK) vascular endothelial growth factor-A high low high

(VEGF) solute carrier family 2, member 1 high low high (Glut-1)

Separate subtracted cDNA populations were generated from mRNA extracted from hypoxic macrophages and hypoxic cardiomyoblasts, using a combination of two kits, purchased from Clontech Laboratories- SMART PCR cDNA synthesis kit and PCR Select cDNA subtraction kit. The manufacturer's instructions were followed for both kits. All diagnostic steps were followed as recommended by the manufacturers. All PCR reactions were done using an Applied Biosystems 9700 with 96-well block, using Applied Biosystems plastics. Driver and tester populations used for subtraction were as below:

subtracted cDNA tester driver

Subtracted macrophage macrophage (hypoxia) macrophage (normoxia)

Subtracted cardiomyoblast cardiomyoblast (hypoxia) cardiomyoblast (normoxia)

The final subtracted cDNA samples were evaluated by performing RT-PCR using the following primers for human beta actin: sense: TCACCCACACTGTGCCCATCTACGA antisense: CAGCGGAACCGCTCATTGCCAAATGG

This showed that an additional 5 cycles of PCR were required to achieve similar levels of beta actin product from subtracted compared to unsubtracted cDNA, indicating a significant reduction in the representation of a non-regulated gene in the subtracted cDNA. Glyceraldehyde 3-Phosphate dehydrogenase PCR primers, as contained in the kit, were not used.

The three subtracted cDNA populations were ligated into a plasmid vector (pCRII, Invitrogen) to generate libraries, which were transformed into E.coli (INVαF', Invitrogen) and plated out onto agar, supplemented with ampicillin and X-Gal, according to standard methods.

Colonies that are white indicate the presence of a recombinant plasmid, and these were picked into individual wells of 96-well plates containing 100 microliters LB-Ampicillin, and given 3-8 hr growth at 37 degrees. In this way, for each library, up to 15 x 96-well plates of clones were generated.

To screen clones for the presence of differentially expressed genes, replicate arrays of plasmid insert DNA were generated on nylon membranes: Firstly, PCR was performed using nested PCR primers 2R and 1, which flank the cDNA insert of each clone (sequence described in the PCR Select kit). The reaction mix also contains 200 uM d(A,T,C,G)TP, Advantage2 polymerase mix (Clontech Laboratories) and supplied lOx buffer. 40 ul reactions were set up in 96-well PCR reaction plates and inoculated with 0.5 ul bacteria from the library plates. 23 cycles of PCR were performed (95 degrees 10 sec; 68 degrees 2 min), and a selection of wells were checked on an agarose gel. In this manner a 96-well plate of insert DNA was generated for each 96-well plate of bacterial clones. Arrays of insert DNA were generated by transferring 4ul of each well to 384-well plates (Genetix), and denaturing the DNA by adding 4ul 0.4M NaOH and incubating at 37 degrees for 15 minutes. Bromophenol blue was added to the wells to allow visualisation of arraying. A 384-pin replicator (Genetix) was used to spot small volumes of denatured insert DNA onto dry nylon membranes (Hybond N+, AmershamPharmacia).

By repeating this operation from the same 384-well plate onto several membranes, matched pairs of membranes were produced, suitable for array screening. A fragment of the beta actin gene was spotted at specific positions of the arrays. Following spotting, the membranes were left at room temperature for 2 hr, re-denatured by placing on chromatography paper wetted with 0.3 M NaOH, neutralised by placing on chromatography paper wetted with 0.5 M Tris pH 7.5, dried at room temperature for 2 hr and crosslinked by exposing to 2000 joules UV radiation. Prior to hybridisation, residual salts were removed from the arrays, by washing in hot 0.5% SDS.

Matched pairs of membranes were hybridised with subtracted cDNA samples; from hypoxic and normoxic cells, to determine the abundance of the genes corresponding to each spotted clone in the cDNA samples. Because the cDNA probes were subtracted, large differences in the hybridisation signal for individual spots were apparent, which can be identified by eye. Prior to probe labelling, subtracted cDNA samples were digested with Rsal and run through Qiagen Qiaquick PCR purification columns to remove adapter sequences added during the PCR Select procedure. 25 ng cDNA was labelled with 33P using a commercial kit following the manufacturer's instructions (Promega, Prime-a-gene kit), and unincorporated label was removed using BioRad Biospin-6 columns following adding 2.5ug yeast tRNA carrier.

Pre-hybridisation, hybridisation and washes were performed essentially according to the Research Genetics GeneFilters protocol, but supplementing the hybridisation mixture with 10 ug of a cocktail of oligonucleotides complementary to the Clontech PCR Select nested PCR primers (equimolar mix of primers 1 and 2R and their reverse complements). Hybridized arrays were exposed to X-ray film or were exposed to a phosphorimager (Molecular Dynamics, Storm) and clones showing gross differences in the hybridization signals with hypoxic compared to normoxic cDNA probes were identified. This procedure was used to process all clones originally picked from the primary libraries and PCR amplified. The selected clones were grouped together onto a single array (referred to here as a secondary array), and were re-screened with cDNA probes which had not been subtracted, to allow a more quantitative though less sensitive, evaluation of the relative abundance of the genes in hypoxia vs. normoxia. In this case, probes were ds cDNA generated from the Clontech SMART cDNA synthesis kit (labelled using the Promega Prime-a-gene kit) or were total RNA (labelled according to the Research Genetics GeneFilters protocol), and hybridisations were done according to the Research Genetics GeneFilters protocol.

Hybridization signals were measured using a phosphorimager and were processed with ArrayVision (Imaging Research Inc) software using multiple beta-actin spots to normalise the quantitation and individual spot background correction. At this stage, the inserts of clones showing consistent up-regulation in hypoxia were sequenced using the 2R primer.

The identity of the genes were determined using BLAST at the NCBI (NLM, NIH) against the non-redundant data base collection. Where significant matches to human genes were not made, the human EST database was used. For both EST and non-EST hits, identifier numbers were also obtained from the UniGene database.

The above strategy was used independently for libraries derived from macrophages and from cardiomyoblasts. By screening a relatively large number of clones (several thousand per library), single genes were identified from multiple clones from any individual library. Multiple clones covered either the same or different regions of the genes.

In the above manner, certain hypoxia-inducible genes were identified from clones only derived from the cardiomyoblast library. These genes are listed in Table 1. Certain hypoxia-inducible genes were identified from clones only derived from the macrophage libraries. These genes are listed in Table 2. Certain hypoxia-inducible genes were identified from clones derived from both macrophage and myoblast libraries. These genes are listed in Table 3.

It can be seen that Table 3 contains many less genes than either Tables 1 and 2; demonstrating that these cell types have large differences in the genes induced by hypoxia. Importantly, the subtracted libraries for macrophage and cardiomyoblast were constructed in parallel. Therefore, major differences in the spectrum of genes isolated from these libraries are likely to be due to differences in the starting material, rather than due to technical differences in the production of the libraries. Importantly, the genes contained in these tables were confirmed to be hypoxia-regulated in the relevant cell type(s) by the described two-stage array hybridisation screening process. From Table 3 it is clear that although this subset of genes was found in subtracted libraries from both hypoxic macrophages and cardiomyoblasts, the fold-induction obtained between hypoxia and normoxia, for the different tissues differs widely. For the first 5 genes in this table, the hypoxia response is greater for macrophages, whereas for the last 2 genes it is greater for cardiomyoblasts.

To test whether genes isolated only in the macrophage-derived subtracted libraries are not responsive to hypoxia in cardiomyoblast, cardiomyoblast cDNA isolated from normoxic and hypoxic cells was hybridised to an array of macrophage-derived clones. These data are presented as a scatter plot, showing normalised signal intensities in hypoxia versus normoxia, with each dot representing a single gene on the array. This plot is presented in Figure 1. A gene that is not affected by hypoxia will localise around the y=x line, running diagonally through the centre of the graph. From the figure, it can be seen that most genes lie in this region, even though all the genes were responsive to hypoxia in the macrophage (Table 2). There is a subset of genes that lie beneath this region (x>y), representing induction of these genes by hypoxia in the cardiomyoblast.

Sequence data for the cDNA inserts of clones from our custom subtracted cDNA libraries is available. These are usually short fragments of 300-1000 bp. Some have been resequenced to obtain an accurate full insert sequence (see document "gene sequences/analysis").

Several of the genes presented in Tables 1-3 encode hypothetical proteins of unknown function and others have no database matches with protein coding sequence. The work presented here provides some functional annotation for these genes, and potential applications for the treatment of disease. Certain genes, in particular the glycolytic enzymes and transporters, have been hypothesised previously as forming part of the generic hypoxia response. The data provided herein provide solid, validating data for these hypotheses. It was surprising to note that cells from our cultures of human ventricle-derived cells, showing a cardiomyoblast-like phenotype, do not support significant induction of the following genes: Lactate dehydrogenase A„ Enolase 1, Phosphoglycerate kinase 1, Triosephosphate isomerase 1. These genes have been identified as being targets of the "ubiquitous" transcription factor HIF-1 alpha ("HIF-1: mediator of physiological and pathophsiological responses to hypoxia" JΛppLPhysiol 88: 1474-1480 (2000)).

Example lb: Preparation of custom array

To confirm the findings presented in Example la, and to obtain more accurate and additional data, both the subtracted cDNA library clones and the IMAGE clones identified from the Research Genetics Human GeneFilters have now been fabricated by the authors into an independently produced and verified gene array (referred to herein as the "custom gene array"), composed of PCR-amplified insert DNA. The methods used to produce this array are common in the art, but the key points are summarised below. Clones from the subtracted cDNA library were PCR amplified as defined in Example la. In many cases, there were multiple cDNA clones corresponding to different regions of the same gene, and all these were represented on the custom gene array. IMAGE clones were obtained from the UK MRC HGMP Resource Centre (Hinxton, Cambridge CB10 1SB, UK) and were re-isolated as individual colonies and sequenced to verify the correct identity of the clone. In the majority of cases, the same IMAGE clone identified from the Research Genetics Human GeneFilters was selected, but in some instances these clones were not available and alternatives were selected, corresponding to the same gene.

Additional genes, with well-defined roles in various disease processes relevant to hypoxia, were also represented on the array, as derived from IMAGE clones. It is well established in the literature that genes with similar functions are often co-regulated at the mRNA level, as determined by microarray data clustering methods (Iyer VR et al, Science. 1999 283(5398):83- 7; Eisen MB et al Proc Natl Acad Sci USA. 1998 95(25): 14863-8). This allows associations to be made between genes of unknown function (as present in the current specification) to genes of well defined function, in order to add significance to the former. Normalisation is a key issue in array analysis. The custom gene array is a single colour type array, and contains a selection of additional IMAGE clones corresponding to genes which were empirically determined not to be affected by hypoxia and which are highly expressed in a wide range of human tissues and cell types. During data analysis, spot intensities were divided by the mean of all the reference genes shown below, each of which was present in quadruplicate on each array.

Gene IMAGE clone Ace.

FLJ11102 fis clone PLACE1005646 AA464704 matrix Gla protein AA155913 guanine nucleotide binding protein alpha stimulating 1 R43581 DKFZp434A1319 W74725 cDNA FLJ23280 fis clone HEP07194 AA669443 beta actin (in house clone)

EF1 a-like protein AI817566 ribosomal protein L37a W91881 IMAGE clone plasmid miniprep DNA was prepared and PCR amplified with flanking vector primers of the sequences GTTTTCCCAGTCACGACGTTG and

TGAGCGGATAACAATTTCACACAG. This was then purified and concentrated by ethanol precipitation, and the presence of a single band and DNA concentration were determined by agarose gel electrophoresis and by digital imaging methods.

Purified PCR product corresponding to all the clones (IMAGE and non-IMAGE) were normalised to 0.5 mg/ ml by dilution. Arrays were fabricated onto Hybond N+ (Amersham) membranes using a BioRobotics TAS arrayer (Biorobotics, Cambridge CB37LW, UK) with a 500 micron pin tool. Using 384-well source plates and a 2x2 arraying format this array was relatively low density, thereby eliminating problems of spot-to-spot signal bleed. Also the large pin size and high source plate DNA concentration improves the sensitivity of detection. Post-arraying denaturation/ neutralisation was essentially as described by Bertucci F et al, 1999 (Oncogene 18: 3905-3912).

Total RNA was extracted from cells using RNeasy (Qiagen) and 7 micrograms RNA was labelled with 100 microCi 33P dCTP using 2 micrograms poly dT (10-20 mer) as primer in a reverse transcription reaction. First strand RNA was then degraded under alkaline contitions, and this was then neutralised with Tris HCI pH 8.0, and the labelled cDNA was purified using

BioRad BioSpin-6 chromatography columns. Pre-hybridisation was performed in 4 ml

Research Genetics MicroHyb solution supplemented with lOmicrograms poly dA (10-20 mer) and 10 micrograms Cot-1 DNA, at 45 degrees for 2-3 hours. The cDNA was then denatured by heating and added to the pre-hybridisation, which was continued for 18-20hr. Washing steps were done as follows: 2xSSC/ 1% SDS 2x20min at 50 degrees and 0.5xSSC/ 1% SDS lOmin at 55 degrees. Arrays were exposed to Amersham Low Energy phosphor screens for 24hr and scanned using a phosphorimager at 50 micron resolution. Image analysis was done using ArrayVision software (Imaging Research Inc). Tab delimited data files were exported and a full analysis performed using GeneSpring software (Silicon Genetics).

Using the described methodology a dynamic range of detection of 4 logs and a sensitivity of at least 1 / 50,000 is obtained, as determined by spike doping titration experiments. Having several technical differences compared to the Research Genetics Human GeneFilters as used in the initial filing, data from the custom gene array is expected to be quantitatively different.

Example lc: Hypoxia regulation of gene expression in macrophages by exposing cells to hypoxia +/- additional signal amplification. The transcription factor HIF-lα, is ubiquitously present in cells and is responsible for the induction of a number of genes in response to hypoxia. This protein is considered a master regulator of oxygen homeostasis (see, for example, Semenza, (1998) Curr. Op. Genetics and Dev. 8:588-594). Although HIF-la is well known to mediate responses to hypoxia, other transcription factors are also known or suspected to be involved. These include a protein called endothelial PAS domain protein 1 (EPASl) or HIF-2a, which shares 48% sequence identity with HIF-la (Tian H, et al. Genes Dev. 1997 11:72-82.). Evidence suggests that EPASl is especially important in mediating the hypoxia-response in certain cell types, and it is clearly detectable in human macrophages, suggesting a role in this cell type (Griffiths et al., 2000, Gene Ther., 7(3):255-62).

As supporting evidence for the hypoxic regulation of the genes contained within this specification, adenoviral vectors were used to overexpress HIF-la and EPASl in primary human macrophages prior to exposure to hypoxia, in order to amplify the response. Because the role of these transcription factors as mediators of the hypoxia response is very well established, any further increases in the inducibility of specific genes resulting from this approach represents credible supporting evidence that those genes are responsive to hypoxia.

A commercially available system was used herein to produce adenoviral particles involving the adenoviral transfer vector AdApt, the adenoviral genome plasmid AdEasy and the packaging cell line Per-c6 (Crucell, Leiden, The Netherlands). The standard manufacturer's instructions were followed. Three derivatives of the AdApt transfer vector have been prepared, named AdApt ires-GFP, AdApt HIF-la-ires-GFP and AdApt EPASl-ires-GFP. In these vectors, for convenience, AdApt was modified such that inserted genes (i.e. HIF-la or EPASl) expressed from the powerful cytomegalovirus (CMV) promoter were linked to the green fluorescent protein (gfp) marker, by virtue of an internal ribosome entry site (ires). Therefore presence of green fluorescence provides a convenient indicator of viral expression of HIF-la or EPASl in transduced mammalian cells. The control vector AdApt ires-GFP was used to allow discrimination between effects of the inserted genes (i.e. HIF-la or EPASl) to that of potential non-specific effects of adenoviral transduction or GFP expression. Standard subcloning methods were used to construct the adenoviral constructs as described in detail elsewhere (see co-pending, co-owned International patent application PCT/GB01/00758; Example 2).

The adenoviral transfer vectors AdApt HIF-la-ires-GFP and AdApt EPASl-ires-GFP, were verified prior to production of adenoviral particles, for their ability to drive expression of functionally active HIF-la or EPASl protein from the CMV promoter in mammalian cells. This was achieved by transient transfection luciferase-reporter assays as described (Boast K et al Hum Gene Ther. 1999 Sep 1;10:2197-208).

Using the aforementioned Introgene adenoviral system, caesium-banded, pure adenoviral particles were produced for each of the vectors AdApt ires-GFP, AdApt HIF-la-ires-GFP and AdApt EPASl-ires-GFP. Following the Introgene manual, adenoviral preparations were quantitated by spectrophotometry, yielding values of viral particles (VP) per milliliter.

Primary human macrophages isolated as described above, were washed and resuspended in DMEM (Gibco, Paisley, UK) supplemented with 4% fetal bovine serum (Sigma). 5xl0⁶ cells were plated into nine individual 10cm Primeria (Falcon) tissue culture dishes containing medium plus adenovirus as shown below (experimental design), to give a total volume of 10 ml per plate. Two doses of adenovirus were used; 5.3xl0⁸ viral particles / ml (low) and 1.6xl0⁹ viral particles / ml (high). These amounts were chosen following a series of titration experiments. Following culture for 16 hr, during which the macrophages adhere to the plate and are infected by the adenoviral particles, the medium was removed and replaced by IMDM medium (Gibco, Paisley, UK) supplemented with 2% human AB serum. A further 24 hr period of culture was allowed prior to experimentation, to allow gene expression from the transduced adenovirus. Gene transduction was verified by visually assessing gfp expression and expression of the viral HIF-la and EPASl genes was determined by real time quantitative RT- PCR using an ABI Prism 7700 TaqMan and CyberGreen protocol. For the high doses of virus, the total levels of HIF-la or EPASl mRNA present in the transduced cells were increased by 10-30 fold.

For experimentation with conditions of hypoxia, identical culture dishes were divided into two separate incubators: One at 37 degrees, 5% CO2, 95% air (=Normoxia; equivalent to 20% Oxygen) and the other at 37 degrees, 5% CO2, 94.9% Nitrogen, 0.1% Oxygen (=Hypoxia). After 6 hours culture under these conditions, the dishes were removed from the incubator, placed on a chilled platform, washed in cold PBS and total RNA was extracted using RNeasy (Qiagen) following the manufacturer's instructions. Experimental design

Condition Adenovirus Adenovirus Oxygen (type) amount (%)

(low= :5.3xl0⁸ vp/ml high= 1.6xl0⁹ vp/ml)

1 none none 20

2 AdApt ires-GFP low 20 3 AdApt ires-GFP high 20 4 AdApt ires-GFP low 0.1 5 AdApt ires-GFP high 0.1 6 AdApt HIF-la-ires-GFP low 0.1 7 AdApt HIF-la-ires-GFP high 0.1 AdApt EPASl-ires-GFP low 0.1 9 AdApt EPASl-ires-GFP high 0.1

RNA samples from the experimental conditions shown above were each hybridised to individual copies of the Custom gene array and processed as described earlier. To ensure reproducible data, this was repeated so each RNA sample was hybridised to 4 separate arrays. Therefore a total of 36 arrays were used for this experiment. Data analysis was done taking the mean signal of each spot from the four array replicates of each RNA sample. When displayed graphically, standard error of the mean is displayed as the error bar. Expression values were calculated so that they represent the fold-change ratio as compared to condition#l, i.e. untreated cells.

For genes shown in Table 4 it can be seen that in cells transduced by the control adenovirus AdApt ires-GFP there is a response to hypoxia (conditions 4,5) as compared to in normoxia (conditions 2,3). However this response is significantly greater when the natural hypoxia response is amplified by overexpression of HIF-1 alpha from the adenovirus AdApt HIF-la- ires-GFP (conditions 6,7). Furthermore, this effect is usually dependent on the amount of HIFlalpha overexpression (i.e. greater in condition 7 compared to 6). This same data is displayed graphically in Figure 2. It can be seen that these genes encode metallothionein proteins. One of these (Nucleotide Seq ID No. 84; Protein Seq ID No. 83) is a novel member of the matallothionein family. Several metallothionein genes are known in the art to be activated by hypoxia, supporting the usefulness of this data. For genes shown in Table 5 and Figure 3 it can be seen that in cells transduced by the control adenovirus AdApt ires-GFP there is a response to hypoxia (conditions 4,5) as compared to in normoxia (conditions 2,3). However this response is significantly greater when the natural hypoxia response is amplified by overexpression of EPASl from the adenovirus AdApt EPAS 1 -ires-GFP (conditions 8,9).

In the case of the protein encoded by Seq ID No. 24, results are available independently for two separate cDNA clones representing non-overlapping regions of the same full length gene.

In the case of the protein encoded by Seq ID No. 86 (EGL nine (C.elegans) homolog 3), additional evidence is described above in support of the function of this protein. Furthermore, real time quantitative RT-PCR analysis of this gene using an ABI Prism 7700 TaqMan and CyberGreen protocol, has been performed, to verify and more accurately quantitate the upregulation of EGL nine (C.elegans) homolog 3 in response to hypoxia and EPASl adenoviral overexpression. The main difference between the array-based and real time quantitative RT-PCR methodologies is that the latter is far more sensitive and therefore can detect expression in the off-state (here normoxia) for weakly expressed genes. This data has shown an induction ratio of 819-fold for EGL nine (C.elegans) homolog 3 in response to hypoxia with additional EPASl expression, from RNA generated from an independent experiment. This data was normalised to beta actin.

Example 2: Differences in the hypoxia responses of resting and activated macrophages. Macrophages accumulate at hypoxic areas in various disease states, including cancer, rheumatoid arthritis, atherosclerosis and wound healing. At these sites macrophages activation is liable to occur, such as in response to T-cell derived gamma interferon. For instance, in atherosclerotic plaques there is an accumulation of both T-cells and macrophages, and these are known to interact with one another (reviewed in Lusis AJ, Atherosclerosis. Nature. 2000 Sep 14;407(6801):233-41).

It is well established that the macrophage has a significant role in the pathology of the above diseases involving hypoxia, and that most functions of the macrophage (including inflammatory functions) are greatly increased following activation. Therefore any therapeutic strategy aimed at the hypoxic macrophage, needs to also consider the effects of macrophage activation and possible cross talk between the responses to macrophage activation and hypoxia. 2.1: Research Genetics Human GeneFilters

This work was carried out using Research Genetics Human GeneFilters, which contain DNA derived from clones of the IMAGE cDNA collection, representing genes of varying degrees of characterisation. A series of 6 arrays of human genes were used (GeneFilters GF200-205), potentially covering a total of 31,104 genes. Generally, single genes are represented only once in these arrays. However, sometimes IMAGE clones initially thought to represent separate genes, upon re-analysis were found to be different regions of the same gene. Here we have presented data for all clones individually, though they possess the same UniGene ID and gene name. An example is Hypothetical protein FLJ20037. The methodology for Research Genetics arrays is similar in principle to that described for the array screening of clones from subtracted libraries. There are several attributes to this method: Relatively small amounts of RNA can be labelled to make cDNA probes, in a single step reaction, and probes are labelled with the same chemical group (33P), so there are no errors introduced as a result of using different dyes, which may differ in stability etc. Using a Phosphorimager allows detection over a wide range of intensities (over 4 logs). Overall it is interesting to note a recent study, which has favourably re-evaluated the performance of the nylon based array, as compared with the glass-based microarray method (Bertucci F et al, Hum Mol Genet 8:1715-1722 (1999)).

Experiments were done essentially as described in the Research Genetics GeneFilters protocol. Duplicate copies of each array from the same production batch, were used and hybridised in parallel with labelled RNA isolated from normoxic and hypoxic primary human macrophages. Hybridised arrays were scanned twice using a Molecular Dynamics Storm phosphorimager, and both images were analysed to ensure reproducibility. Furthermore, the experiments were repeated using the same RNA samples, but with different array lot numbers, again to ensure reproducibility.

Analysis was performed using Research Genetics Pathways software, with normalisation using the 'all data points' option. Analyses were output as spreadsheets and filtered to remove data points where the signal intensity was less than 4-fold above the general background for the experimental condition with the higher signal (hypoxia or normoxia depending on whether hypoxia causes induction or repression). Sometimes expression in the lower state was not significantly above background, and the ratio will therefore be underestimated. Ratios were calculated by normalised signal intensity in hypoxia divided by normoxia. Changes were verified visually from the original array images. In this manner, comparisons were made between normoxia and hypoxia in resting macrophages. The whole procedure was then repeated for activated macrophages, to investigate possible differences in the response to hypoxia. It is possible that potential differences for certain genes could be correlated with changes in expression resulting from activation, prior to challenge with hypoxia. To explore this possibility, comparisons were made between resting and activated macrophages, both in normoxia. Since some of the genes we have identified as being activated by hypoxia have very low hybridisation signals in normoxia (for both resting and activated macrophages), this comparison was not possible.

We have found various patterns of gene expression changes occurring in response to hypoxia, related to the activation state of macrophages, which are presented below. Such a range of responses, specific to various subsets of genes, was not expected, and contradicts a view that the hypoxia response is a largely a generic mechanism.

Table 7 shows genes that are induced by hypoxia to a similar degree in resting and activated macrophages. Table 8 shows genes that are induced by hypoxia to a greater degree in resting macrophages, as compared to activated macrophages. These data are presented illustratively in Figure 5.

Data from Table 8/Figure 5 reveals several unexpected observations.

A) From the final column it can be seen that macrophage activation in the absence of hypoxia, causes induction of many of these genes. This suggests that the signalling pathways resulting from activation and hypoxia might converge to a single transcriptional regulator, causing macrophage activation to pre-empt the response to subsequent hypoxia. This is exemplified most strikingly for Interleukin 8, which is dramatically induced in response to macrophage activation, but shows no additional response to hypoxia. B) Genes in rows 11, 13 and 14 have no response to hypoxia following macrophage activation, though there is not a preceding large increase in expression in response to macrophage activation alone. This suggests that in the activated macrophage, the necessary signalling pathway or transcriptional regulator is not functional.

C) Although Table 8 was produced electronically, without selecting genes based on their names, it can be seen that genes encoding proteins of the metallothionein family feature strongly. Table 9 shows genes which are induced by hypoxia to a greater degree in activated macrophages, compared to resting macrophages. These data are presented illustratively in Figure 6.

In Table 7, there are several genes for which hypoxia/ normoxia ratios were only obtained for activated macrophages, such as Cox-2 (see row 47). For these genes, macrophage activation usually increases expression of the gene to detectable levels, thus allowing the study of subsequent changes in response to hypoxia. It is likely that these genes are not significantly expressed in resting macrophages irrespective of hypoxia, and therefore the hypoxia response is probably specific to activated macrophages. Certain genes respond to hypoxia by decreasing mRNA expression (repression), and these genes therefore have hypoxia/normoxia ratios of < 1.0. This phenomenon is known in the field of hypoxia, although the mechanism is obscure. Data is presented in tables 7-9, which unexpectedly shows that this hypoxia-induced repression for specific genes is not a generic process, but is dependent on the cellular context. In Table 10/ Figure 7, genes are presented that are hypoxia-repressed to a greater degree in activated (column 7) compared with resting (column 8) macrophages. Prior to any hypoxic challenge, these gene are induced to varying degrees, in response to macrophage activation (column 9), suggesting a shared mechanism for these separate responses. From Table 10, genes in rows 1-6 show that macrophage activation is necessary to obtain any response to hypoxia. In resting macrophages, these genes are not responsive to hypoxia at all.

Strikingly, Table 10/ Figure 7 shows that seven separate genes encoding chemokine proteins (Monocyte chemotactic protein 1, Macrophage inflammatory protein lb, Monocyte chemotactic protein 3 and Small inducible cytokine A3, Monocyte chemotactic protein 2, Macrophage inflammatory protein 2a and Macrophage inflammatory protein 2 precursor) are more strongly repressed in activated macrophages as compared to resting macrophages. These genes are also among the most inducible in response to activation alone, in normoxia (column 9). These findings are of potential utility in view of the great significance of chemokines to inflammatory disease. For example, macrophage chemotactic factor 1 (Table 10, row 19) is key to the pathological role of the macrophage in atherosclerosis ("Chemokines and atherosclerosis" Reape TJ and Groot PHE, Atherosclerosis 147: 213-225, 1999).

Genes in rows 20-30 of Table 10, were not detectably expressed in resting macrophages, irrespective of hypoxia. Table 11 shows other genes that were down-regulated in response to hypoxia in macrophages. Example 3: Tissue-specific hypoxia regulation of gene expression by an analysis of a series of primary human cell cultures.

Equivalent cultures of non-immortalised, non-transformed primary human cells of 10 distinct types, were cultured in either normoxia or were exposed to hypoxia for 6 hr and 18 hr, and gene expression changes were determined. To the inventors' knowledge, this is the first time that such a study has been reported. Moreover, unlike the vast majority of information in the public domain relating to genes responsive to hypoxia, all of these cells were human and were cultured without any modifications following isolation from the human donors. By using primary cells rather than cell lines or immortalised cultures, the findings of this work more accurately represents the situation in the human body.

Most cell types were obtained from Clonetics (distributed by BioWhittaker, Walkersville, MD) and cultured according to the manufacturer's recommendations, unless where otherwise shown. #l:adipocyte (Clonetics CC-2568; derived from subcutaneous adult adipose tissue), #2:cardiomyocyte (Clonetics CC-2582; derived from fetal tissue; prior to experimentation cultured in minimal medium: DMEM, 4% Horse serum), #3:endothelial (TCS CellWorks ZHC-2101 human umbilical vein endothelial cells), #4:fibroblast (Clonetics CC-2511 dermal fibroblasts derived from adult tissue), #5:hepatocyte (Clonetics CC-2591, derived from adult tissue), #6:macrophage (derived from human blood as described elsewhere in the specification), #7:mammary epithelial (Clonetics CC-2551; derived from adult tissue), #8:monocyte (derived from human blood as described elsewhere in the specification but without the 7 day differentiation culture period), #9:neuroblastoma (neuroblastoma-derived cell line SH-SY5Y), #10:renal epithelial (Clonetics CC-2556; derived from fetal tissue), #11 skeletal muscle myocyte (Clonetics CC-2561; derived from adult tissue). A non-primary cell type (#9) was used to represent neurons, since primary human neurons are difficult to source. Therefore a total of 11 cell types are compared. It should be noted that RNA from hepatocytes at the 16hr timepoint of hypoxia was not available for this work.

Genes which were induced or repressed preferentially in particular cell type(s) were identified by hybridisation of the RNA samples to the custom gene array, as described in Examples lb and lc. Each RNA sample was hybridised to duplicate or triplicate arrays, to ensure reproducible data, and was analysed using GeneSpring software. Data from replicate arrays were merged during analysis to generate mean values. Data normalisation was achieved per- array using the aforementioned list of control genes, such that differences in RNA labelling or hybridisation due to experimental variation were corrected by referencing each gene to the mean value of the reference genes on the same array. Also, for each gene, expression values were obtained which represent the value in each experimental condition (e.g. macrophages 6hr hypoxia) as compared to the median of value of that gene throughout the full range of experimental conditions (i.e. from all cell types). This transformation does not alter the relative values of any gene between the different experimental conditions, and is done since these is no obvious single reference experimental condition to create ratio values. This is common in microarray data analysis.

Table 12 shows the full dataset of this analysis. From this it can be seen that certain genes respond to hypoxia differently, depending on the particular cell type. This information is valuable in identifying biological targets for the development of therapeutic and diagnostic products. Not only does it indicate a particularly significant role for these genes in the specific cell type implicated in a disease, but it also identifies that any therapeutic product is less likely to produce problematic toxicological effects. Data shown in Table 12 and the derived figures, are reproducible, and are an accurate determination of mRNA expression levels. This may be confirmed by independent means, such as quantitative real time RT-PCR. Certain genes from Table 12 will be presented for illustration.

Genes with a greater response in hepatocytes

The dataset of Table 12 also contains genes which are induced preferentially in hepatocytes, in response to hypoxia. The results for the EGLN3 gene are presented in Figure 8a.

SeqID:85/86 EGL nine (C.elegans) homolog 3 As described above, it has been discovered that a polypeptide encoded by a gene identified from the EST recited in SEQ ID No 86, having the Protein accession number BAB 15101 (encoded by Homo sapiens cDNA: FLJ21620 fis, clone COL07838 Nucleotide accession AK025273) is regulated by hypoxia. Other public domain sequences corresponding to this gene include Homo sapiens cDNA: FLJ23265 fis, clone COL06456 Nucleotide accession AK026918. Accordingly, when referring in the present specification to the EST recited in SEQ ID No 86, it is intended that these gene and protein sequences are also embraced. This gene was identified using Research Genetics Human GeneFilters arrays, which contain an EST corresponding to the gene (accession number R00332). The gene is now termed EGL nine (C.elegans) homolog 3. There are no reports that describe the function of this human gene. However, a high degree of amino acid homology is observed between the protein encoded by this gene, and a rat protein called "Growth factor responsive smooth muscle protein" or "SM20" (Nucleotide accession U06713; Protein accession A53770). An alignment of single letter amino acid sequences is shown below. Over the highlighted region there is 97% amino acid similarity and 96% amino acid identity.

A53770 (1) MTLRSRRGFLSF PG RPPRRWLRISKRGPPTSHWASPA GGRTLHYSCR BAB15101 (1)

51 100

A53770 (51) SQSGTPFSSEFQATFPAFAAKVARGPWLPQWEPPAR SASPLCVRSGQA

BAB15101 (1)

101 _ __ _ 150 A53770 (101) LGACTLGVPRLGSVSEMP GHIMRLDLEKIALEYIVPC HEVGFCY DHF

BAB15101 (1) felPLGHIMR D EKIALEYIVPC HEVGFCYXiDNF

151 ^" 200

A53770 (151) LGEVVGDCVBERVKQ HYNGALRDGQLAGPRAGVSKRH RGDQI'PWIGGN

BAB15101 (35) LGEWGDCVLERVKQLHCTGA RDGQI/AGPRAGVSKRH RGD lTWtGGN 201 ^" 250

A53770 (201) EEGCEAINFLTJSIIΪDRLVLYCGSRLGKYWKERSKAMVACYPG^GTGYVR BABI51OI (85) EEGCEAISFLLSMDR VLYCGSR GKYWK SKAMVACYPGNGTGYVR

251 ^{" "} 300

A53770 (251) HVDNPNGDGRCITCIYY NiCN DAK HGGV RlFPEGKSFV DVEPIFDR BAB15101 (135) HVDrøNGDGRCITCiYYLNKlrøDAK HGGlLRI#PEGKSFlADVEPIFDR

301~ _ _^" 350

A53770 (301) LFSWSDRrøPHEVQPSYA*PRYAMT YFDABERAEAJKKFPαgLΦRK ES BAB15101 (185) LFFWSDRP^PHEVQPSYATRYAMTV YF AEEPAEAKKKFRJSLTRKTES_j 351 A53770 (351) ALAKD

BAB15101 (235) A TED'

The high degree of amino acid similarity suggests that the human protein BAB 15101 has an equivalent biochemical function to the rat protein A53770 ("Growth factor responsive smooth muscle protein" or "SM20"). Recent publications have shown that SM20 functions to promote apoptosis in neurons (Lipscomb et al, J Neurochem 1999; 73(l):429-32; Lipscomb et al, J Biol Chem 2000 Nov 1 ; [epub ahead of print]). Significantly, SM20 has been shown to be expressed at high levels in the heart (Wax et al, J Biol Chem 1994; 269(17): 13041-7).

It has also been discovered that a polypeptide encoded by a gene identified from the EST recited in SEQ ID No 90, having the Protein accession number CAB 81622, is regulated by hypoxia. The encoding human gene has been annotated in the UniGene database as "Similar to rat smooth muscle protein SM-20"; the nucleotide sequence is contained within the nucleotide accession AL117352. More recently, a longer fragment of this gene has been cloned, named clorfl2, or EGLNl (Nucleotide accession AAG34568; Protein accession AAG34568). Accordingly, when referring in the present specification to the EST recited in SEQ ID No 90, it is intended that these gene and protein sequences are also embraced.

This distinct human gene, encoding a protein related to SM20 and EGLN3 (BAB15101), is also induced in response to hypoxia. This gene was identified using Research Genetics Human GeneFilters arrays, which contain an EST corresponding to the gene (accession number H56028).

Independently to this, a fragment of this gene has been cloned from a cDNA library derived from hypoxic human cardiomyoblasts, and it has been shown that the gene is increased in expression in response to hypoxia in this cell type (see Table 1 herein; penultimate row). The nucleotide sequence of this cDNA fragment is referred to herein as SEQ ID No 90a.

In the light of this novel discovery reported herein that these human equivalents of SM20 are induced by hypoxia, it is herein proposed that in cardiac ischaemia, the resulting apoptosis is due at least in part, to increased expression of these genes. The therapeutic modulation of the activity of EGLN3 (BAB15101), clorfl2 (AAG34568), CAB81622, SM20 and other equivalent proteins and encoding genes therefore provides a novel means for the treatment of myocardial ischaemia, through the alteration of the propensity of myocardial cells to undergo apoptosis. For example, a suitable treatment may involve altering the susceptibility of ischaemic myocardial tissue to subsequent reperfusion and re-oxygenation, or may involve modulating the susceptibility of chronic ischaemic myocardial tissue (including forms of angina) to later more severe ischaemia, which would result in myocardial infarction. It is submitted that, by way of analogy, cerebral ischaemia may be treated using the same principle.

Although the Applicant does not wish to be bound by this theory, the downstream effects of SM20 and related genes such as EGLN3 (BAB 15101), clorfl2 (AAG34568), and CAB81622, namely, apoptosis and angiogenesis might be explained as follows. The apoptotic effect of NGF withdrawal may be mediated by regulation of the hypoxia pathway, but may be an aspect of the supposed involvement of the HIF protein in the stress response. HIFlα is induced by reactive oxygen species (see Richard et al. J Biol Chem 2000 Sep 1;275(35):26765-71). This could, in turn, be mediated by over-load of the proteosomal pathway for HIFlα degradation and the consequent accumulation of undegraded HIFlα. Accordingly, it is considered that modulation of SM20 and the related genes EGLN3 (BAB 15101), clorfl2 (AAG34568), and CAB 81622 may have applications in the treatment of diseases resulting from disturbances in proteosome function, such as prion diseases and other neuro-degenerative diseases.

These data provide the first connection between these related genes and the physiological response to hypoxia. Recently published research papers have identified that the protein products of these genes can act as proline hydroxylases (see Bruick RK et al Science. 2001 294:1337-40 and Epstein AC et al Cell. 107:43-54). This is consistent with our observations that certain proline hydroxylases are induced in response to hypoxia and the genes EGLNl and EGLN3 are part of the hypoxia response. For example, two genes encoding proline hydroxylases have been identified herein as being increased in expression in response to hypoxia (proline 4-hydroxylase, alpha polypeptide 1; SeqID: 231/232, proline 4-hydroxylase, alpha polypeptide II; SeqID: 349/ 350). This identified a functional significance of proline hydroxylation as a response to hypoxia.

Proline hydroxylase leads to degradation of HIFlα in normoxia (HIF regulates its own degradation - feedback). Hydroxylated HIFlα + VHL leads to ubquitination and consequent degradation of HIFlα by proteosome. The activity of the prolyl hydroxylase is 0₂-dependent, so under conditions of hypoxia, HIFlα is not hydroxylated efficiently and is stabilised. HIFlα protein thus accumulates to a high level. The hypoxia-induction of the prolyl hydroxylase ensures that when 0₂ concentration returns to normal, there is sufficient enzyme available to target this high level of HIFlα efficiently for rapid degradation.

Degradation of HIFlα is dependent on HIF1 -induced transcription (i.e. is hypoxia inducible). Berra et al (FEBS Lett 2001 Feb 23 ;491(1-2): 85-90) raises the specific hypothesis of an unknown hypoxia-inducible factor which targets HIFla for proteosomal degradation. It appears reasonable to propose that this factor will clearly be hypoxia-inducible, to ensure that a rapid and effective constraint on the hypoxic response would operate on return to normoxia. It now appears as if the genes EGLNl and EGLN3 form part of this mechanism.

It is also hypothesised that SM20 and the related genes EGLN3 (BAB15101), clorfl2 (AAG34568), and CAB81622 may act as tetramers. Known prolyl hydroxylases such as prolyl 4-hydroxylase (P4H) are known to act as tetramers of two alpha subunits and two beta subunits. SM20 and the related genes exhibits high similarity to the alpha subunit of P4H and it therefore seems likely that SM20 and the related genes are likely to have a binding partner that is equivalent to the beta subunit of P4H. SM20 has been shown to bind to the transcription factor HIFlα, and shares a low level homology with a p53 binding protein. P53 is a transcription factor that is known to be involved in apoptosis. Accordingly, it is proposed that in addition to binding to HIF1A, SM20 and the related genes EGLN3 (BAB15101), clorfl2 (AAG34568), and CAB81622 may also bind and modify other transcription factors that are involved in the hypoxic response such as EPAS and HIF3A, or other transcription factors such as p53 and thereby influencing apoptosis. This aspect of the invention thus provides dimer and tetrameric forms of the EGLN3 (BAB15101), clorfl2 (AAG34568), and CAB81622 proteins, preferably complexed with a protein selected from the group consisting of HIFlα, p53 and a protein binding partner that is equivalent to the beta subunit of P4H. Preferably, such dimers and tetramers are heterodimers/heterotetramers.

To provide further evidence that these related genes are a significant part of the hypoxia response additional expression data is presented here. Expression profiles for these two genes will be displayed with pre-chip normalisation to correct for differences in RNA labelling etc, but within each gene no further normalisation is done (per-gene normalisation), so the relative absolute expression levels of the two genes can be compared and Y-axis units between separate graphs from the same experiment are comparable. These graphs are presented as Figures 8b (clorfl2) and 8c (EGLN3). It can be seen from these Figures that both genes (clorfl2 and EGLN3) are inducible in response to hypoxia in macrophages whether activated by gamma interferon and lipopolysaccharide or if de-activated by treatment with interleukin-10. In macrophages the absolute expression level of Clorfl2 appears to be higher than EGLN3.

There is a prejudice in the art that the response to hypoxia is generic to all cell types. Contrary to this, we show herein that genes are regulated by hypoxia to a greater degree in certain cell types, substantiating their utility in designing specific therapeutic products for diseases involving those cell types.

From Figures 8a and 8d and the data presented below, differing expression profiles of the two related genes clORF12 and EGLN3 are apparent throughout the 11 tested cell types, though C 1 orf 12 is generally expressed at higher levels than EGLN3.

Cell type Oxygen mRNA expression mRNA expression

(C1ORF12 SeqID:89/90) (EGLN3 SeqID:85/86) adipocyte normoxia 0.0075 0.0033 adipocyte hypoxia 6hr 0.0091 0.0027 adipocyte hypoxia 18hr 0.0182 0.0025 cardiomyocyte normoxia 0.0067 0.0019 cardiomyocyte hypoxia 6hr 0.0381 0.0023 cardiomyocyte hypoxia 18hr 0.0201 0.0026 endothelial normoxia 0.0198 0.0019 endothelial hypoxia 6hr 0.0583 0.0033 endothelial hypoxia 18hr 0.0397 0.0026 fibroblast normoxia 0.0119 0.0032 fibroblast hypoxia 6hr 0.0260 0.0046 fibroblast hypoxia 18hr 0.0235 0.0040 hepatocyte normoxia 0.0075 0.0080 hepatocyte hypoxia 6hr 0.0074 0.0146 macrophage normoxia 0.0033 0.0008 macrophage hypoxia 6hr 0.0083 0.0018 macrophage hypoxia 18hr 0.0058 0.0021 mammary epithelial normoxia 0.0065 0.0014 mammary epithelial hypoxia 6hr 0.0137 0.0055 mammary epithelial hypoxia 18hr 0.0144 0.0065 monocyte normoxia 0.0027 0.0006 monocyte hypoxia 6hr 0.0084 0.0014 monocyte hypoxia 18hr 0.0080 0.0016 neuroblastoma normoxia 0.0344 0.0011 neuroblastoma hypoxia 6hr 0.1085 0.0013 neuroblastoma hypoxia 18hr 0.0551 0.0020 renal epithelial normoxia 0.0275 0.0046 renal epithelial hypoxia 6hr 0.0560 0.0046 renal epithelial hypoxia 18hr 0.0395 0.0096 skeletal myocyte normoxia 0.0088 0.0029 skeletal myocyte hypoxia 6hr 0.0277 0.0035 skeletal myocyte hypoxia 18hr 0.0245 0.0038

For instance, in the hypoxic hepatocyte (6hr) the normalised expression values of EGLN and clorfl2 are 0.015 and 0.0074 respectively, i.e. EGLN being the dominant gene. In contrast, in the neuroblastoma cell line SH-SY5Y, the normalised expression values of EGLN and clorfl2 after 6hr hypoxia are 0.0012 and 0.108 respectively, i.e. clorfl2 being the dominant gene by a large margin. This data demonstrates that clORF12 and EGLN3 are not constitutively expressed at an equal amount in different tissues indicating specificity of function. Therefore, it is considered that therapeutic products may be developed based on this data, with the goal of modulating proline hydroxylation of target proteins (such as HIFlalpha) in specific tissues, based on the differing expression profile of clORF12 and EGLN3 in those tissues.

In Example lb herein, genes were identified from a custom array, which give a greater induction in macrophages (by a factor of at least 1.5) when hypoxia is augmented by over- expression of HIFlalpha or EPAS from an adenovirus. The data from the HJF/ EPAS over- expression work is presented herein in Example lc, but specifically relating to clORF12 and EGLN3 is summarised in Figures 8e and 8f. From this data it is apparent that EGLN3/ FLJ21620 fis cl.COL07838 but not clORF12 is increased in expression by the transcription factor EPASl but not HIFlalpha. This is apparent by comparing experimental condition 9 (hypoxia with EPAS overexpression; expression value=3.48) to that of 5 (hypoxia without EPAS overexpression; expression value^***** 1.65). This adds valuable information about the mechanism of regulation of the gene encoding EGLN3.

To confirm this data the RNA samples for experimental conditions 1,3,5,7,9 (corresponding to the high dose of adenovirus) were also measured using a different array-based methodology- the AffyMetrix GeneChip. The results of this experiment are presented in Figures 8g and 8h.

Functional Characterisation of EGL nine (C.elegans) homolog 3 role in the induction of Cardiomyocyte apoptotic cell death

Human EGLN3 has been cloned into pONY8.1 and Smart2.IRES.GFP equine infectious anaemia virus (EIAV) vectors, and AdCMV.TRACK.GFP (AdenoQuest) adenoviral genome vectors (see co-owned co-pending International patent application PCT/GB01/00758). These vectors have been used in "gain-of-function" studies in which EGLN3 has been overexpressed in order to elucidate corresponding protein function. Human embryo kidney (HEK 293T) and dog osteosarcoma (D17) cell lines have been used in transient plasmid transfection experiments to confirm EGLN3 expression from viral vector genomes. Rat cardiomyocyte cell line (H9C2) and primary human neonatal cardiomyocytes (PHNC) (BioWhittaker, CC2582) have been used in viral transduction experiments to determine the biological activity of

EGLN3. In all cell types, expression of EGLN3 has been followed by combinations of immunofluorescence, Western blotting and TaqMan quantitative PCR. Immunofluorescence and Western blotting employ an antibody specific for the FLAG epitope engineered into the 3' terminus of EGL nine (C.elegans) homolog 3 (Sigma, F3165). TaqMan quantitative PCR utilises the SYBR Green method (Applied Biosystems).

Western blotting has confirmed the transient expression of EGLN3 from an EIAV genome construct in HEK 293T (expected size approx 717 bp, 26 Kda). Immunofluorescence has localised transient expression of EGL nine (C.elegans) homolog 3 from EIAV expression construct in HEK293T to the cytoplasm. Expression of EGL nine (C.elegans) homolog 3 is elevated after 4 hours exposure to hypoxic conditions (0.1% (v/v) oxygen), when compared to expression observed under normoxia (20% (v/v) oxygen) (see Figure 8i). TaqMan primers have been designed and optimised for the initial measurement of EGL nine (C.elegans) homolog 3 expression in EIAV or Adenovirus transduced H9C2 and PHNC (Forward: TCATCGACAGGCTGGTCCTC; Reverse: GTTCCATTTCCCGGATAGAA). All findings at the RNA level are corroborated by immunofluorescence and Western blotting analyses at the protein level.

EIAV transduction of H9C2 and PHNC has been optimised with constructs containing green fluorescence protein (GFP) and LacZ reporter genes, using the VSVg envelope and a range of MOI between 10 and 100. GFP results were scored by fluorescence microscopy, while LacZ transductants were identified through the assay of β-galactosidase activity. An MOI of 50 transduced approximately 50% of the cell population.

EGLN3 is predicted to have pro-apoptotic activity in cardiomyocytes. Early, Mid and late phase apoptosis are characterised by translocation of membrane phospholipid phosphatidylserine (PS) from the inner face of the plasma membrane to the cell surface, activation of specific proteases (caspases) and fragmentation of DNA, respectively (Martin, S.J., et al., J. Exp. Med. 1995, 182, 1545-1556; Alnemri, E.S., et al., J. Cell. Biochem. 1997, 64, 33-42; Wylie, A.H., et al., Int. Rev. Cytol. 1980, 68, 251-306). Translocation of PS has been identified through use of ApoAlert kit (Clontech; K2025-1), which employs FITC- labelled antibodies to detect surface expression of the PS, Annexin V. Caspase activity has been followed using the homogeneous fluorimetric caspase assay (Roche; 3005372) which allows the quantification of caspase activity through the cleavage of a fluorescent substrate. DNA fragmentation has been estimated using the nuclear stain Hoescht 33345 (Sigma, B2261; and fluorescence microscopy to locate areas of chromatin condensation. Total viability of cell population has been quantified through measurement of the ability of mitochondrial reductase to metabolise the fluorescent substrate MTT (Sigma, M2128)(Levitz S.M & Diamond, R.D. J. Infect. Dis. 1985 Nov; 152(5):938-45).

Conditions for early, mid and late stage apoptosis in H9C2 and PHNC have been defined using hypoxia and nutrient-depleted growth medium to mimic those ischaemic conditions found in vivo (Brar, B.K., et al., J. Biol. Chem. 2000, 275, 8508-8514). Transduction of PHNC with EIAV vectors containing EGLN3 is sufficient to cause an increase in caspase activity in cells cultured under normoxic conditions, confirming the role of EGLN3 in the induction of cardiomyocyte apoptosis. Using an MOI of 50, a 2-fold increase in caspase activity was seen in EGLN3 transduced cells, when compared to controls 48 hours post transduction (see Figure 8j). Increased expression of EGL nine (C.elegans) homolog 3 in transduced cells is confirmed by TaqMan, immunofluorescence and Western blotting. Similar experiments are performed to determine whether EGL nine (C.elegans) homolog 3 expression further sensitises H9C2 and PHNC to previously defined ischaemic insults. Staurosporine (Calbiochem; 569397) and Smart2.IRES.GFP EIAV vectors containing the Bax gene will be applied as chemical and viral pro-apoptotic controls, respectively (Yue, T-L., et al, J. Mol. Cell. Cardiol. 1998, 30, 495-507; Reed, J.C. J Cell Biol. 1994, 124(1-2): 1-6).

Gene silencing approaches may be undertaken to down-regulate endogenous expression of EGLN3 in PHNC to determine the degree of protection against apoptotic cell death provided by a reduction in EGLN3 activity. RNA interference (RNAi) (Elbashir, SM et al., Nature 2001, 411, 494-498) is one method of sequence specific post-transcriptional gene silencing that may be employed. Short dsRNA oligonucleotides are synthesised in vitro and introduced into a cell. The sequence specific binding of these dsRNA oligonucleotides triggers the degradation of target mRNA, reducing or ablating target protein expression. A Hammerhead ribozyme library, contained in EIAV expression vectors, may also be applied. Efficacy of both gene silencing approaches may be assessed initially through the measurement of EGLN3 expression, at the RNA level by TaqMan and at the protein level by Western blotting. Protection against previously described ischaemic insults provided by these methods of EGLN3 gene silencing may be assayed biologically as detailed above. Caspase inhibitors (caspase 3 inhibitor V, 2129002 and caspase inhibitor I, 627610, both Calbiochem) and Smart2.IRES.GFP EIAV vectors containing the Bcl-2 gene may be applied as chemical and viral anti-apoptotic controls, respectively (Kroemer, G. Nat Med. 1997, 3(6):614-20).

Similar "gain-of-function" and gene silencing approaches will be applied to the related gene, encoded by SEQ ID 90, named clofl2.

Example 4: PCR results for the EGLN3 splice variant (SEQ ID NO: 85a)

Initial work using RNase protection assays using a 32P-labelled riboprobe which spans the sequence of both isoforms validated the existence of the unexpected isoform (data not shown).

Qualitative RT-PCR was then used to estimate the relative abundance of the two splice forms of EGLN3 in a range of primary human cell types growing in normoxia and hypoxia, as described below. This method was also used to clone and sequence the splice isoform to demonstrate qualitative comparisons in the levels of the two isoforms. This data gives further proof that the splice isoform actually exists, but moreover demonstrates that the relative expression levels of the two isoforms varies between cell types. It is therefore clear that cell type differential alternative splicing occurs for this gene. This may allow the design of more specific inhibitors to the different splice isoforms, which may have significant implications for the treatment and diagnosis of human disease. There are many examples of the significance of splicing to human disease, where the products of different splice forms having distinct biochemical and biological activity.

Primary human cell types were obtained from Clonetics (distributed by BioWhittaker, Walkersville, MD) and cultured according to the manufacturer's recommendations, unless where otherwise shown.

RT-PCR was performed by reverse transcribing 2 ug total RNA with Superscript II reverse transcriptase (Invitrogen) in a 20ul reaction, lul of the resulting cDNA was used as template for PCR reactions using Clontech Advantage II polymerase. Primer nucleotide sequences were as follows: Sense ctcgattctgcgggcgagatgc

Antisense gtcttcagtgagggcagattcag

PCR cycling was performed using an Applied Biosystems 9700, using the following "touchdown" cycling parameters: (94° 1 min) x 1 (94° lOsec; 72° 2min) x 5

(94° lOsec; 70° 20sec; 72° 2min) x 5 (94° lOsec; 68° 20sec; 72° 2min) x 22

It should be noted that the combination of an accurate and processive reverse transcriptase enzyme (Superscript II) and highly stringent touchdown PCR cycling parameters as shown above, and a high fidelity hot-started enzyme (Advantage II polymerase) render these data highly specific and not liable to artefacts, as experienced with PCR amplifications performed under less stringent conditions.

Using these conditions, the expected ELGN3 PCR product of 735 bp is observed, though an additional band of 453 bp is also seen, representing the novel splice isoform described herein. This band was cut out, cloned and sequenced. The band has also been found in a variety of screened human cDNA libraries. Example 5: Evaluation of potential HIF prolyl hydroxylase inhibitors

As discussed above, the stability of HIF is controlled by proline hydroxylation, catalysed by the product of the EGLN3 gene which renders the HIF protein unstable. EGLN3 is only active in normoxia since the reaction that it catalyses requires oxygen. By this mechanism, in normoxia HIF is degraded and its target genes are not expressed (Epstein AC et al, Cell. 2001; 107(l):43-54). Analysis of the sequence of the novel splice isoform of EGLN3, contained in this specification, suggests that it retains an intact catalytic domain, and therefore should be active as a HEF proline hydroxylase. Here we have experimentally verified this.

Although endogenous HIF is unstable in normoxia, when it is overexpressed by experimental means, it is able to overcome the degradation mechanism and transcription of HIF target genes occurs. The commonly used human cell line 293T growing in conditions of normoxia was transiently transfected with a reporter plasmid (HRE-Luc), which provides a measure of HIF activity in units of Luciferase activity. This plasmid is described in Boast K et al Hum Gene Ther. 1999; 10(13):2197-208. Plasmids and conditions used were as follows:.

MIMO=L-mimosine; 0.4mM used. DAU=Daunorubicin; due to toxicity noted in a previous experiment, dosage of Daunorubicin used was 2.5μM. Plasmid CMV-SVFLl expresses full length EGLN3. Plasmid CMV-SVFL2 expresses the EGLN3 splice variant.

Three plasmid transfections were performed and pCIneo was used as the "stuffing'Vcontrol plasmid to fill the vacancies.

Transfected 293 cells were incubated under normoxia till the end of the experiment.

The results of this experiment are shown in Figure 10. The collagen prolyl hydroxylase (PH) inhibitors EDB and MIMO were found to work as HIF-PH inhibitors under conditions of normoxia, even when the full length EGLN3 protein or EGLN3 splice variant was over- expressed.

It can be seen that when a plasmid containing HIFlα expressed from the CMV promoter was co-transfected with HRE-Luc, HIFlα activity was detected as 20953 Luciferase units. Accordingly, overexpressing HIFlα increased the luciferase activity (compare H and HM in Figure 10). In a similar transfection, where a third plasmid containing full length EGLN3 (CMV-SVFLl) was also introduced, the units of luciferase produced were reduced to 918 (see col. S1HH in Figure 10). This reflects the fact that EGLN3 targets HIFlα, including over- expressed HIFlα, for destruction leading to a decreased reading for HIFlα activity (measured here in Luciferase units). When this experiment was performed with the novel splice isoform of EGLN3 (CMV-SVFL2), as described herein, HIFlα was similarly inhibited to an even greater degree, with a Luciferase reading of 773 (see col S2HH in Figure 10).

Over-expression of the full length EGLN3 protein or EGLN3 splice variant thus suppressed the effect of overexpression of HIFlα on luciferase. Both the full length EGLN3 protein and EGLN3 splice variant thus have been proven to possess biological activity. Overexpression of both these isoforms reduce HIF-mediated gene expression through HRE reporters, thus demonstrating their role in the HIF signalling pathway. The suppression effect of the EGLN3 splice variant appeared to be stronger than that of the full length EGLN3 protein. TABLES

TABLE 1: Hypoxia-inducible genes identified from clones only derived from the cardiomyoblast library

TABLE 2: Hypoxia-inducible genes identified from clones only derived from the macrophage libraries

The gene entitled "Jk-recombination signal binding protein" was found to be hypoxia- inducible using subtracted cDNA probes for hybridization, but with non-subtracted probes, where the hybridisation is quantitative, no signal was detected. This indicates that the gene is probably hypoxia-regulated but the absolute expression levels are very low.

TABLE 3: Hypoxia-inducible genes identified from clones derived from both macrophage and myoblast libraries.

TABLE 4: Hypoxia responses amplified by HIFlalpha overexpression

Legend: Data shown in the average of 4 repeat experiments. Experimental condition is as shown in the text. Values represent fold change as compared to untreated cells (condition 1). TABLE 5: Hypoxia responses amplified by EPASl overexpression

Legend: Data shown is the average of 4 repeat experiments. Experimental condition is as shown in the text. Values represent fold change as compared to untreated cells (condition 1).

TABLE 6. Negative hypoxia responses amplified by HIFlalpha / EPASl overexpression

Table 7: Genes induced by hypoxia (similar response +/- cell activation)

CO c

CD CO

m

CO

I m m

73 c m r

CO c

CD CO

m

CO

I m m

73 c m r

Legend

The last 3 columns show mRNA expression as a ratio between the conditions being compared. Of these three columns the first two show expression in hypoxia relative to normoxia, done separately in resting macrophages or activated macrophages. The final column shows expression in activated macrophages relative to resting macrophages (both in normoxia) as a ratio, n/d = not determined due to low signal intensities. IMAGE ID and accession descride the exact identity of the arrayed clones and do not describe full length cDNA sequence database entries.

CO c

CD CO

m

CO

I m m

73 c m r

Th Tas_{t 3 c}olons show mRNA expression as a ratio be_tween the conditions being compared. Of these three columns the first two show expressⁱon iJS S to n oxia, done separately in resting macrophages or activated macrophages. The final column sh^ows ^expr^ession ^activa^ted m cr_Onha_Ees n ative to reSng macrophages (both in normoxia) as a ratio, n/d = not deteπnihed due to low sⁱgnal ⁱntens^ities. IMAGE ID an^d ΞS ^£ ribe eΞ ity of thf arrayed clones and do describe full length cDNA sequence database entrⁱes.

TABLE 9:_Genes induced by hypoxia (greater response in activated cells)

CO c

CD CO

m

CO

I m m Legend

5 The last 3 columns show mRNA expression as a ratio between the conditions being compared. Of these three columns the first two show expression

73 in hypoxia relative to normoxia, done separately in resting macrophages or activated macrophages. The final column shows expression in activated c macrophages relative to resting macrophages (both in normoxia) as a ratio, n/d = not determined due to low signal intensⁱtⁱes. IMAGE ID and m r ' accession describe the exact identity of the arrayed clones and do not describe full length cDNA sequence database entrⁱes.

TABLE 10: Genes repressed by hypoxia (greater response in activated cells)

CO c

CD CO

m

CO

I m m

73 c m r

CO c

CD CO

m

CO

I m m

73 c m r

Legend to Table 30

The last 3 columns show mRNA expression as a ratio between the conditions being compared. Of these three columns the first two show exp e o in hypoxia relative to normoxia, done separately in resting macrophages or activated macrophages. The final column shows expressⁱon m actⁱvated macrophages relative to resting macrophages (both in normoxia) as a ratio, n/d = not determined due to low sⁱgnal ⁱntensⁱtⁱes. IMAGE ID and accession descride the exact identity f the arrayed clones and do hot describe full length cDNA sequence database entrⁱes.

TABLE 11: Other genes repressed by hypoxia in macrophages

CO c

CD CO

m

CO

I m m

73 c m r

CO c

CD CO

m

CO

I m m

73 c m r

CO c

CD CO

m

CO

I m m

73 c m r

CO c Legend

CO The last 3 columns show mRNA expression as a ratio between the conditions being compared. Of these three columns the first two show expression in hypoxia relative to normoxia, done separately in resting macrophages or activated macrophages. The final column shows expression in activated 5 macrophages relative to resting macrophages (both in normoxia) as a ratio, n/d = not determined due to low signal intensities. IMAGE ID and m accession descride the exact identity of the arrayed clones and do not describe full length cDNA sequence database entries.

CO

I m m

73 c m

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D1E16 0.5611.06 ).95 to h-90 fe.76 LOO 12.43 12.67 132 0.49 0.81 .01 .17 P.38 h.75 W

(8 bl05 .71 1.21 H.40 1.58 .81 h.66 12.13 1.9312.05 0.84 6 0.

079 078 075 0.80 0.98 047 tø 051 0.520.74 0

08 blD6 0.24 1.42. 12.51 028 061 0.93 -77 .57 H.85 025 11.35 2.82 034 052 061 1.49 (2.56 h.76 1.9 0.51 1.35 12.

70 p1E12 054 041 m 098 1.29 067 098 .47 .13 0.84 087 1.11 1.17 059 .01 .03 0.67 H.66 089 0.53 088 1

2 WEIO u m W.31 13.17 5.08 (2.27 .17 07611.03 hoi .06 075 0.740.74 079 0.91 h.62 1.31 079 0.78 0

4 blC21 B.75 bi 093 12.33 092 Ml 2.27 (1.45 091 0.96 095 0.41 033 (2.10 2.68 0.60 066 0.49 0.60 0.65 0

76 blDIO 0.45 088 h.49 0.60 .63 1.34 E.61 be 0.34 1.83065 1.21 0.36 049 .452J 2.94 035 b.35 b

78 bl013 fe.93 .83 2.94 j).80 3.69 1.10 (1.51 0891.59 1.30 0.67 0.64 0.50 075 082 O50 0.76 0.45 015 027

00 blE9 |1.54 0.89 (1.48 1.18 1.: 1.53068 082 0.60 ta 064 b.73 0921.62 (4.20 B.97 15.19 025 042 b.18 fe.33 B.63 1

I blFI 058 033 052 1.42 .19 1.14 1.64 1.70 1.49 0.54 037 062 051 091 to 030 034 053

34 blE7 1.20 121 11.63 012 018 022 1.40 .03 6.17 2.85 075 1.07 08712.06 1.32 075

1.09 Ϊ1.01 079 092 1.04 0.79 .04 .32 H.87 1.63 025 fe.97 034 0.74 085 05812.26 2.67 025 0.57

5. blE6 136 1.35 h

38 b2B1 1.51 097 (2.31 2.25 1.06 Ii .22 11.18 1.10 077 051 072 1.19 0.50 0.77 097 fi.60 165 0.87

BO blD14 0.67 0.821.640.60 B.42 .78 B.24 B.57 .07 2.332.11 .68 b 0.67 0..2299 b.74 0.52 058 k.75 b.72

&2 blD17 0.32 fe.30 B.77 D.46 B.08 1.47 0.41 0.60 P.43 1.05 0.92 0.91 .17 1.13 B.73 B.80 B.77 0.58 1.01 076 0.98 1.26 1.31 b.3

Q2 V>1P14 P.43 Q66 0.741.22 .60 1.14 .00 1.53 1.32 049 073 1.15 0.43 0.69 021 045 068 2.63 1.8 6.90 014 0.71 084 1.

B4 b!C24 0.92 088 0.53 1.76 1.65 .37 JO .09 089 .23 .07 085 0.68 .28 060 077 B.67 1.02 2.70 1.06 .66 088 .

B6 b!D3 0.621 I.56 I .13 .83 0.85 P.73 Q.09 B.43 029 0.52 0.71 0.15 028 .72 B.67 fe.ao B.79 0.49 5.80 B.32 B.26 B.79 P.

B8 blE14 1.09 1.92 0.89 1.93 1.46 096 .23 1.58 0.96 0.88 0.71 1.26 0.87 .68 0.67 089 2.54 1.20 B.33 2.55 0.38 0.74 1.03 0. loo WE20 063 0.61 1.12 .47 .34 b.99 .45 .94 .49 068 088 098 0.19 0.34 036 043 0.37 .33 1.44 0.65 023 0.43 0.33 0

02 b2A24 1.21 0.65 0.67 2.42 2.21 2.48 2.35 B.05 2.59 1.10 0.76 068 0.68 0.91 0.28 0.22 0.38 037 0.40 0.20 0,18 0.26 0.17 .

04 blE17 094 099 095 l.55_ 196 1.93 2.41 2.37 088 0.80 P.53 073 0.84 1.15 0.29 0.39 b.24 .96 2.03 2.06 1.

31E19 065 062 2.64 2.37 2.99 2.10 1.61 .77 0.61 0.40 P.64 0.42 .89 068 076 0.41 0.50 032 038 0.90 0.4 1. 7_ M2 101_ 185_ 152 165_ 1.11 0

108 }1E15 Li m 142 11 0.92 1.10 178 11 IK 172_ ii! K35 12

110 31E11 Ά 11 187 W 103 174 91 188 189 159 179 276 2 7 21184_ 134 164 UL 135_ 1

112 )1E23 1.19 165 1.55 124 ^lA 175_ 11 186 VJD9 135 140 131_ 136 188 102_ 155_ 166 1

114_ )1E21 0.85 4 2*1 189

2*28. vn_ 174 379 0.51 .04 148 167 1OT 135 134_ 121_ IK U 1 47 0.68 1.21 1.62 1.30 199 1.17 1.10 1.39 1.20 1.05 0.35 049 3.28 127 3.34 3.80 185 3.66 139 149 0.53 2. 116 )1023 3.95 P.

102

era cS ^

111

112

tlJ23 b.96 b.57 b.50 .05 .18 .38 b.73 b.01 b.70 -54 b.42 0.54 b.94 .33 jlθ.5 fe. 9 B.21 21.1 fe.3 28.6 B.69 4.3 B.43 fa bU21 P.75 P.68 b.96 H.16 tt.09 b.52 tt.61. pp h.15 b.96 b.79 .98 -BO .5β .66 D.35 .18 b.82 138 3.54 b.65

H52 blJ24 H.62 P.56 D.84 .98 .51 ,96 .56 2.04 .37 .05 P.81 P.52 b.56 b.62 .30 b.27 b.28 .05 ,90 160 1 .21 b.24 b.2

W54 blJ16 .55 2.31 .38 2.76 H.29 2.85 M.20 B.66 H.65 h.84 tt.78 b.53 b.76 tt.04 b.62 b.68 b.33 b.38 b.23 b.49 b.53 b.5

W56 bU2 b.77 P.76 b.37 h.57 b.75 b.51 2.52 B.12 2.29 b.38 b.28 b.45 b.72 h.14 b.63 b.30 b.43 tt.28 h-69 b.86 b.57 b.52 b.4

H58 bU9 b.73 P.45 b.38 h.22 .38 ,06 2.10 h.oo b.87 b.38 b.34 b.34 b.71 H.18 B.04 B.92 B.04 b.94 H.07 b.55 hl.36tf.8

W60 pUIO b.86 b.48 b.72 h.18 11.10 P.85 h.70 2.20 h.82 tt.20 b.92 b.26 b.32 b.62 b.48 b.39 b.78 b.97 b.64 .48 B.lfl b.8 m bUI P.97 P.70 P.68 .41 H.09 b.87 0.09 2.75 b.63 0.45 b.51 b.63 P.86 b.55 0.46 b.59 b.97 .90 P.78 b.41 .40 b.4 blJ5 b.94 b.74 E6.2 22,5 P6.6 .12 2.88 2.58 tt.22 b.79 b.83 h.06 .15 B.02 W7 h.06 b.53 b.98 b.47 b.61 .97 b.5

466 bUII b.95 P.57 b.30 i.71 0.51 P.60 b.95 b.70 P.70 b.37 P.22 b.18 .20 h.77 b.97 h.15 tt.60 2.41 h.21 b.99 h.52 b.8

^ββ bU8 .77 b.67 h.20 .73 b.98 b.86 .16 tt.28 H.70 tt.59 H.57 b.28 b.34 b.n b.08 b.06 b.55 b.87 .22 b.08 b.14 b.1

W bll20 2.84 h.42 h.67 .17 B.14 .41 P.99 K.46 .03 b.79 b.72 b.92 b.74 b.90 7.59 2.80 2.20 D.34 b.55 b.24 h.76 .8 .1

W72 bU3 P.70 b.39 b.64 .75 .28 .04 tt.24 .61 h.81 b.94 b.69 b.52 b.53 b.51 b.85 b.99 B.72 b.28 b.37 b.

M74 blJ12 P.58 μ.02 P.90 Ml h.83 P.84 .06 b.78 .06 b.90 .01 194 B.33 Ml m b.37 b.30 b.29 b.85 b.66 .

M76 bll23 .35 b.87 .71 H.36 H.43 .71 .03 P.70 b.βo tt.12 .23 b.41 184 2.67 .69 b.46 b.77 b-41 2.0 h5.2 26

TABLE 13 cross-references all protein and nucleotide sequences (SEQ I ) Nos.) that are referenced herein to accession numbers in public databases available as Of 8.12.00.

CO c

CD CO

m co m m

73 c m r n

CO c

CD CO

m co m m

73 c m r n

CO c

CD CO

m co m m

73 c m r cn

CO c

CD CO

m co m m

73 c m r cn

CO c

CD CO

m co m m

73 c m r cn

CO c

CD CO

m co m m

73 c m r cn

CO c

CD CO

m co m m

73

C m r cn

TABLE 14 cross-references all protein and nucleotide sequences (SEQ ID Nos.) that are referenced herein to accession numbers in public databases available as of 8.12.01.

CO c

CD CO

m co m m

73 c m r cn

CO c

CD CO

m co m m

73 c m r cn

CO c

CD CO

m co m m

73 c m to cn

CO c

CD CO

m co m m

73 c m to cn

CO c

CD CO

m co m m

73 c m r cn

CO c

CD CO

m co m m

73 c m r cn

CO c

CD CO

m co m m

73 c m r cn

CO c

CD CO

m co m m

73 c m r cn

CO c

CD CO

m co m m

73 c m r cn

Claims

1. A substantially purified polypeptide, which polypeptide: i) comprises the amino acid sequence as recited in SEQ ID No: 85a; ii) has an amino acid sequence encoded by a nucleic acid sequence recited in

SEQ ID No: 86a; iii) is a fragment of a polypeptide according to i) or ii), provided that said fragment retains a biological activity possessed by the full length polypeptide of i) or ii), or has an antigenic determinant in common with the polypeptide of i) or ii); or iv) is a functional equivalent of a polypeptide of i), ii) or (iii).

2. A polypeptide according to claim 1, wherein said biological activity is a hypoxia- regulated activity.

3. A polypeptide according to claim 2, wherein the expression of the polypeptide is hypoxia-induced.

4. A polypeptide which is a functional equivalent according to part iv) of any one of claims 1-3, is homologous to the amino acid sequence as recited in SEQ ID No: 85a or is homologous to the amino acid sequence encoded by a nucleic acid as recited in SEQ ID No: 86a, and has equivalent biological activity to that possessed by the full length polypeptide of i) or ii).

5. A fragment or functional equivalent according to any one of claims 1-4, which has greater than 50% sequence identity with the amino acid sequence as recited in SEQ ID No: 85a or with the amino acid sequence that is encoded by a nucleic acid as recited in SEQ ID No: 86a, or with fragments thereof, preferably greater than 60%, 70%, 80%, 90%, 95%, 98% or 99% sequence identity.

6. A fragment as recited in any one of claims 1-5, having an antigenic determinant in common with a polypeptide according to part i) of any one of claims 1-5, which consists of 7 or more (for example, 8, 10, 12, 14, 16, 18, 20 or more) amino acid residues from the amino acid sequence as recited in SEQ ID No: 85a or the amino acid sequence encoded by a nucleic acid as recited in SEQ ID No: 86a.

7. A purified and isolated nucleic acid molecule that encodes a polypeptide according to any one of claims 1-6.

8. A purified nucleic acid molecule according to claim 7, which consists of the nucleic acid sequence as recited in SEQ ID No.: 86a, or is a redundant equivalent or fragment thereof.

9. A purified nucleic acid molecule which hydridizes under high stringency conditions with a nucleic acid molecule according to claim 7 or claim 8.

10. A vector comprising a nucleic acid molecule as recited in any one of claims 7-9.

11. A delivery vehicle comprising a nucleic acid according to any one of claims 7-9, or a vector according to claim 10.

12. A host cell transformed with a vector according to claim 11.

13. An antagonist ligand which binds specifically to, and which inhibits the hypoxia- induced activity of, a polypeptide according to any one of claims 1-6.

14. An agonist ligand which binds specifically to, and which activates the hypoxia-induced activity of, a polypeptide according to any one of claims 1-6 to augment or potentiate a hypoxia-induced activity.

15. A ligand according to claim 13 or 14, which is an antibody.

16. A ligand according to claim 13 or 14, which is a peptide, a peptidomimetic, or a drug molecule, such as a small natural or synthetic organic molecule of up to 2000Da, preferably 800Da or less.

17. A pharmaceutical composition suitable for modulating the biological response to hypoxia and/or ischaemia, comprising a therapeutically-effective amount of a polypeptide as recited in any one of claims 1-6, a nucleic acid molecule as recited in any one of claims 7-9, a vector containing a nucleic acid molecule as recited in any one of claims 7-9 or a ligand that binds specifically to a polypeptide as recited in any one of claims 1-6, in conjunction with a pharmaceutically-acceptable carrier.

18. A vaccine composition comprising a polypeptide, nucleic acid molecule, vector or ligand as recited in claim 17.

19. A polypeptide as recited in any one of claims 1-6, a nucleic acid molecule as recited in any one of claims 7-9, a vector containing a nucleic acid molecule as recited in any one of claims 7-9 or a ligand that binds specifically to a polypeptide as recited in any one of claims 1-6, for use in therapy or diagnosis of disease.

20. A polypeptide, nucleic acid molecule, vector or ligand as recited in claim 19, wherein said disease is a hypoxia-regulated condition.

21. A polypeptide, nucleic acid molecule, vector or ligand as recited in claim 19, wherein said hypoxia-regulated condition is selected from the group consisting of myocardial ischaemia, tumourigenesis, angiogenesis, apoptosis, inflammation, erythropoiesis, the biological response to hypoxia conditions (including processes such as glycolysis, gluconeogenesis, glucose transportation, catecholamine synthesis, iron transport or nitric oxide synthesis), myocardial infarction, diseases involving infection of the airways (such as cystic fibrosis) and stroke.

22. A method of treating a disease in a patient in need of such treatment by administering to a patient a therapeutically effective amount of a polypeptide as recited in any one of claims 1-6, a nucleic acid molecule as recited in any one of claims 7-9, a vector containing a nucleic acid molecule as recited in any one of claims 7-9 or a ligand that binds specifically to a polypeptide as recited in any one of claims 1-6, in conjunction with a pharmaceutically-acceptable carrier, or a pharmaceutical composition according to claim 17.

23. A method of regulating tumourigenesis, angiogenesis, apoptosis, the biological response to hypoxia conditions, or a hypoxic-associated pathology in a patient in need of such treatment by administering to a patient a therapeutically effective amount of a polypeptide, a nucleic acid molecule, vector, ligand or pharmaceutical composition as recited in claim 22.

24. A method according to claim 23, wherein, for diseases in which the expression of the natural gene or the activity of the polypeptide is lower in a diseased patient when compared to the level of expression or activity in a healthy patient, the polypeptide, nucleic acid molecule, ligand, compound or composition administered to the patient is an agonist.

25. A method according to claim 23, wherein, for diseases in which the expression of the natural gene or activity of the polypeptide is higher in a diseased patient when compared to the level of expression or activity in a healthy patient, the polypeptide, nucleic acid molecule, vector, ligand, compound or composition administered to the patient is an antagonist.

26. A polypeptide, a nucleic acid molecule, vector, ligand or pharmaceutical composition as recited in claim 22, for use in the manufacture of a medicament for the treatment of a hypoxia-regulated condition.

27. A method of monitoring the therapeutic treatment of a disease or physiological condition in a patient, comprising monitoring over a period of time the level of expression or activity of a polypeptide, a nucleic acid molecule, vector or ligand as recited in claim 17 in tissue from said patient, wherein altering said level of expression or activity over the period of time towards a control level is indicative of regression of said disease.

28. A method of providing a hypoxia regulating gene, an apoptotic or an angiogenesis regulating gene by administering directly to a patient in need of such therapy an expressible vector comprising expression control sequences operably linked to one or more of the nucleic acid molecules that are recited in claims 7-9.

29. A method of diagnosing a hypoxia-regulated condition in a patient, comprising assessing the level of expression of a natural gene encoding a polypeptide as recited in claim 17, or assessing the activity of such a polypeptide, in tissue from said patient and comparing said level of expression or activity to a control level, wherein a level that is different to said control level is indicative of the hypoxia-related condition.

30. A method according to claim 29, that is carried out in vitro.

31. A method according to claim 29 or claim 30, which comprises the steps of: (a) contacting a ligand as recited in claim 17 with a biological sample under conditions suitable for the formation of a ligand-polypeptide complex; and (b) detecting said complex.

32. A method according to claim 29 or claim 30, comprising the steps of: a) contacting a sample of tissue from the patient with a nucleic acid probe under stringent conditions that allow the formation of a hybrid complex between a nucleic acid molecule as recited in claim 17 and the probe; b) contacting a control sample with said probe under the same conditions used in step a); and c) detecting the presence of hybrid complexes in said samples; wherein detection of levels of the hybrid complex in the patient sample that differ from levels of the hybrid complex in the control sample is indicative of the hypoxia- related condition.

33. A method according to claim 29 or claim 30, comprising the steps of: a) contacting a sample of nucleic acid from tissue of the patient with a nucleic acid primer under stringent conditions that allow the formation of a hybrid complex between a nucleic acid molecule as recited in claim 17 and the primer; b) contacting a control sample with said primer under the same conditions used in step a); c) amplifying the sampled nucleic acid; and d) detecting the level of amplified nucleic acid from both patient and control samples; wherein detection of levels of the amplified nucleic acid in the patient sample that differ significantly from levels of the amplified nucleic acid in the control sample is indicative of the hypoxia-related condition.

34. A method according to claim 29 or claim 30, comprising the steps of: a) obtaining a tissue sample from a patient being tested for the hypoxia-related condition; b) isolating a nucleic acid molecule as recited in claim 17 from said tissue sample; and c) diagnosing the patient for disease by detecting the presence of a mutation which is associated with the hypoxia-related condition in the nucleic acid molecule as an indication of the hypoxia-related condition.

35. The method of claim 34, further comprising amplifying the nucleic acid molecule to form an amplified product and detecting the presence or absence of a mutation in the amplified product.

36. A method according to any one of claims 29-35, wherein said disease is cancer, ischaemic conditions, reperfusion injury, retinopathy, neonatal stress, preeclapmsia, atherosclerosis, inflammatory conditions, wound healing, tumourigenesis, angiogenesis, apoptosis, inflammation or erythropoiesis, myocardial infarction, diseases involving infection of the airways (such as cystic fibrosis) or stroke.

37. A method according to claim 36, wherein said hypoxia or ischaemia-related tissue damage is due to a disorder of the cerebral, coronary or peripheral circulation.

38. A method according to any one of claims 29, 30 and 32-36, wherein the tissue is a cancer tissue.

39. A method for the identification of a compound that is effective in the treatment and/or diagnosis of disease, comprising contacting a polypeptide, or nucleic acid molecule as recited in claim 17 with one or more compounds suspected of possessing binding affinity for said polypeptide or nucleic acid molecule, and selecting a compound that binds specifically to said nucleic acid molecule or polypeptide.

40. A compound identified by a method according to claim 39.

41. A compound according to claim 40, which is a natural or modified substrate, an enzyme, a receptor, a small organic molecule, such as a small natural or synthetic organic molecule of up to 2000Da, preferably 800Da or less, a peptidomimetic, an inorganic molecule, a peptide, a polypeptide, an antibody, or a structural or functional mimetics of any of these compounds.

42. A kit useful for diagnosing disease comprising a first container containing a nucleic acid probe that hybridises under stringent conditions with a nucleic acid molecule as recited in claim 17; a second container containing primers useful for amplifying said nucleic acid molecule; and instructions for using the probe and primers for facilitating the diagnosis of disease.

43. The kit of claim 42, further comprising a third container holding an agent for digesting unhybridised RNA.

44. An array of at least two nucleic acid molecules, wherein each of said nucleic acid molecules either corresponds to the sequence of, is complementary to the sequence of, or hybridises specifically to a nucleic acid molecule as recited in claim 17.

45. An array according to claim 44, which contains nucleic acid molecules that either correspond to the sequence of, are complementary to the sequence of, or hybridise specifically to at least 2, 3 or 4 of the nucleic acid molecules implicated in a hypoxia- regulated condition as recited in claim 17.

46. An array according to any claim 44 or claim 45, wherein said nucleic acid molecules consist of between twelve and two thousand nucleotides.

47. An array of antibodies, comprising at least two different antibody species, wherein each antibody species is immunospecific with a polypeptide implicated in a hypoxia- regulated condition as recited in claim 17.

48. An array of polypeptides, comprising at least two polypeptide species as recited in claim 17, wherein each polypeptide species is implicated in a hypoxia-regulated condition, or is a functional equivalent variant or fragment thereof.

49. A kit comprising an array of nucleic acid molecules as recited in claim 17.

50. A kit comprising one or more antibodies that bind to a polypeptide as recited in claim 17; and a reagent useful for the detection of a binding reaction between said antibody and said polypeptide.

51. A transgenic or knockout non-human animal that has been transformed to express higher, lower or absent levels of a polypeptide as recited in claim 17.

52. A method for screening for a compound effective to treat disease, by contacting a non- human transgenic animal according to claim 51 with a candidate compound and determining the effect of the compound on the disease of the animal.