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WO2003006687A2 - Nouveaux genes cibles concernant des maladies cardiaques - Google Patents

Nouveaux genes cibles concernant des maladies cardiaques Download PDF

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Publication number
WO2003006687A2
WO2003006687A2 PCT/EP2002/007704 EP0207704W WO03006687A2 WO 2003006687 A2 WO2003006687 A2 WO 2003006687A2 EP 0207704 W EP0207704 W EP 0207704W WO 03006687 A2 WO03006687 A2 WO 03006687A2
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amino acid
seq
acid sequence
polypeptide
sequence
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PCT/EP2002/007704
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English (en)
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WO2003006687A3 (fr
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Birgit Reuner
Daniela Bunk
Thomas Henkel
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Medigene Ag
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Publication of WO2003006687A2 publication Critical patent/WO2003006687A2/fr
Publication of WO2003006687A3 publication Critical patent/WO2003006687A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention is based on the finding that a variety of genes is abnormally expressed in diseased heart tissue. Assessment of the expression level of these genes may be used for testing the predisposition of mammals and preferably humans for a heart disease or for an acute state of such a disease. Diseases that preferably relate to the invention are congestive heart failure, dilative cardiomyopathy, hypertrophic cardiomyopathy and ischemic cardiomyopathy.
  • the present invention further relates to methods of identifying compounds capable of normalizing the expression level of the aforementioned genes and of further genes affected by the abnormal expression. The identified compounds may be used for formulating compositions, preferably pharmaceutical compositions for preventing or treating diseases.
  • the invention may also be used as lead compounds for the development of medicaments having an improved efficiency, a longer half-life, a decreased toxicity etc. and to be employed in the treatment of heart diseases.
  • the invention include also somatic gene therapy methods comprising the introduction of at least one functional copy of any of the above-mentioned genes into a suitable cell.
  • the invention relates to non-human transgenic animals comprising at least one of the aforementioned genes in their germ line.
  • the transgenic animals of the invention may be used for the development of medicaments for the treatment of heart diseases.
  • Cardiovascular diseases like high blood pressure (50.0 mio), Coronary heart disease (12.4 mio), Myocardial infarction (7.3 mio), Angina pectoris (6.4 mio), Stroke (4.5 mio), Congenital cardiovascular defects (1.0 mio), and Congestive heart failure (4.7 mio).
  • the mortality was 949,619 in 1998 in the USA, which means that about 40 % of all deaths were caused by Cardiovascular diseases.
  • Cardiovascular diseases are the number one cause of death (1918 was an exception) with one death every 33 seconds on average. At present there is no causal treatment for congestive heart failure available.
  • the technical problem underlying the present invention was to provide a new generation of tools useful in diagnosis, prognosis, prevention and treatment of heart-related diseases.
  • the present invention relates to a method for identifying a subject at risk for a disease of the heart, comprising the step of quantitating the amount of at least one RNA in the heart tissue of a subject, whereby (a) said at least one RNA encodes an amino acid sequence selected from the group consisting of: (aa) the amino acid sequence of SEQ ID NO: 1 , the amino acid sequence of SEQ ID NO: 2, the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5, the amino acid sequence of SEQ ID NO: 6, the amino acid sequence of SEQ ID NO: 7, the amino acid sequence of SEQ ID NO: 8, the amino acid sequence of SEQ ID NO: 9 or the amino acid sequence of SEQ ID NO: 10;_(ab) an amino acid sequence that is at least 60%, preferably at least 80%, more preferably at least 85%, especially at least 90%, more especially at least 95%, advantageously at least 99% identical to the amino acid sequence of (aa); (
  • disease of the heart means, in accordance with the present invention, any disease that affects the normal function of the heart. This definition includes hereditary as well as acquired diseases such as diseases induced by a pathogen or diseases due to lack of exercise.
  • rheumatic fever/ rheumatic heart disease hypertensive heart disease
  • hypertensive heart and renal disease ischemic heart disease (coronary heart disease)
  • diseases of pulmonary circulation which include acute and chronic pulmonary heart disease
  • arrhythmias congenital heart disease, angina and congestive heart failure.
  • the term "quantitating the amount of at least one RNA” is intended to mean the determination of the amount of mRNA in heart tissue as compared to a standard value such as an internal standard.
  • the (internal) standard would advantageously be the amount of a corresponding RNA produced by a heart tissue not affected by a disease.
  • Said (internal) standard would also include a mean value obtained from a variety of heart tissues not affected by a disease.
  • a possible way to get samples of heart tissue is to take a biopsy (catheter) from the ventricular wall.
  • a standard would take into account the genetic background of the subject under investigation.
  • RNA is effected in comparison to the amount of RNA of one or a variety of samples of the same or a similar genetic background
  • a variable number of "non-failing" humans are compared with a variable number of patients that suffer a distinct heart disease like dilated cardiomyopathy.
  • the determination can be effected by any known technology of analysing the amount of RNA produced in a sample such as a tissue sample.
  • RNA may be prepared as described in the appended examples.
  • isoform means a derivative of a gene resulting from alternative splicing, alternative polyadenylation, alternative promoter usage or RNA editing. Isoforms can be detected by
  • RNA any type of hybridisation techniques (e.g. Northern blots, nuclease protection assays, microarrays) starting from RNA (as described in Higgins, S.J., Hames, D. RNA Processing: A practical approach Oxford University Press (1994), Vol. 1 and 2; Sambrook, Fritsch, Maniatis. Molecular Cloning, a laboratory manual. (1989) Cold Spring Harbor Laboratory Press).
  • hybridisation techniques e.g. Northern blots, nuclease protection assays, microarrays
  • Primers/probes for RT-PCR or hybridisation techniques are designed in a fashion that at least one of the primers/probes specifically recognizes one isoform. If differences in the molecular weight of isoforms are large enough to separate them by electrophoretic or chromatographic methods, it is also possible to detect multiple isoforms at once by employing primers/probes that flank the spliced regions. The isoforms are then sequenced and analysed as described in a).
  • DNA molecule the complementary strand of which hybridizes in 4xSSC, 0.1 % SDS at 65°C to the DNA molecule encoding the amino acid sequence of (a), (c) or (d) means that the two DNA molecules hybridize under these experimental conditions to each other. This term does not exclude that the two DNA sequences hybridize at higher stringency conditions such as 2xSSC, 0.1% SDS at 65°C nor does it exclude that lower stringency conditions such as 6xSSC, 0.1% SDS at 60°C allow a hybridization of the two DNA sequences.
  • hybridization conditions for each sequence may be established on well-known parameters such as temperature, composition of the nucleic acid molecules, salt conditions etc.; see, for example, Sambrook et al., “Molecular Cloning, A Laboratory Manual”; CSH Press, Cold Spring Harbor, 1989 or Higgins and Hames (eds.), "Nucleic acid hybridization, a practical approach", IRL Press, Oxford 1985, see in particular the chapter “Hybridization Strategy” by Britten & Davidson, 3 to 15.
  • degenerate variant refers to the degeneracy of the genetic code. Said degenerarcy is know in the art and described in several text books, e.g. Lewin, Genes V, Oxford University Press 1994, Chapter 7.
  • the invention is based upon the unexpected result that the certain genes coding for the protein sequences referred to above, preferred embodiments of which are given in examples 2 to 11 are deregulated in the comparison of one or more failing heart samples to one or more non-failing heart samples and lead to a downregulation of the described polypeptides measured by their respective mRNAs or cDNAs. The significant changes in gene expression levels suggest a causative role in congestive heart failure.
  • the diagnosis of a disease of the heart established by a different methodology may be corroborated.
  • the term "causative” is not limited to mean that the aberrant expression of one gene as identified above or which is a member of said protein cascade is the sole cause for the onset of the disease. Whereas this option is also within the scope of the invention, expression the invention also encompasses embodiments wherein said aberrant is one of a variety of causative events that lead to the onset of the disease.
  • RNA is used to monitor the progress of a disease of the heart (said variation also applies to the method described herein below).
  • This variation may be employed for assessing the efficacy of a medicament or to determine a time point when administration of a drug is no longer necessary or when the dose of a drug may be reduced and/or when the time interval between administrations of the medicament may be increased.
  • This variation of the method of the invention may successfully be employed in cases where an aberrant expression of any of the aforementioned genes/genes as members of protein cascades is causative of the disease. It is also useful in cases where the aberrant expression of the gene/genes is the direct or indirect result of said disease.
  • RNA levels When assessing the risk or the status of the disease, one or more of the RNA levels may be determined. Generally, the assessment of more than 1 , such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 different RNAs is expected to enhance the fidelity of the prognosis/diagnosis. However, the gain in fidelity would, as a rule, have to be weighted against the costs generated by such additional tests. Accordingly, it is preferred that one or two different RNA levels are determined for a first assessment. If deemed necessary or appropriate, further RNA levels may be determined.
  • DNA sequence of SEQ ID NO: 24 or the DNA sequence as depicted in Figure 4F or a degenerate variant thereof recited in the methods of the invention described herein comprise the sequence of SEQ ID NO: 17, depicted in Figure 4B.
  • nucleic acid sequence that encodes an amino acid sequence that is at least 60%, preferably at least 80%, more preferably at least 85%, especially at least 90%, more especially at least 95%, advantageously at least 99% identical to the amino acid sequence of (c);
  • nucleic acid probe comprising a sequence that specifically hybridizes under physiological conditions to the nucleotide sequence selected from the group consisting of: (i) the DNA sequence of the RNA transcribed from the DNA sequence of SEQ ID NO 11 , the DNA sequence of SEQ ID NO: 12 the DNA sequence of SEQ ID NO 13, the DNA sequence of SEQ ID NO: 14 the DNA sequence of SEQ ID NO 15, the DNA sequence of SEQ ID NO: 16, the DNA sequence of SEQ ID NO 17, the DNA sequence of SEQ ID NO: 18, the DNA sequence of SEQ ID NO 19, the " DNA sequence of SEQ ID NO: 20, the DNA sequence of SEQ ID NO 21 , the DNA sequence of SEQ ID NO: 22, the DNA sequence of SEQ ID NO: 23, the DNA sequence of SEQ ID NO: 24 or the DNA sequence as depicted in Figure 4F, the DNA sequence of SEQ ID NO: 25 or the DNA sequence as depicted in Figure 6B, the DNA sequence of SEQ ID NO: 26 or the DNA sequence as depicted in Figure 7B the DNA sequence of SEQ ID NO:
  • the nucleic acid sequence described herein above as well as the fragments thereof is/are preferably a DNA sequence(s) and is/are, preferably, detectably labeled.
  • Appropriate labels include radioactive labels, wherein the radioactivity conferring molecules may be, e.g., 32 P, 35 S or 3 H.
  • Appropriate labels further include fluorescent, phosphorescent or bioluminescent labels or nucleic acid sequences coupled to biotin or streptavidin in order to detect them via anti- biotin or anti-streptavidin antibodies.
  • any of the above mentioned probes specifically hybridizing to the aforementioned RNAs may be employed, it is preferred that fragments of the full length coding sequence such as oligomers of a length between 15 and 25 nucleotides are used. Examples of such oligomers are oligomers of 18, 21 or 24 nucleotides.
  • the double strand formed after hybridization can be detected by anti-double strand DNA specific antibodies or aptamers etc.
  • the probe of SEQ ID NO: 13 and the mentioned variants thereof are used for quantitating the RNA of SEQ ID NO: 1 , but not to any of the other mentioned RNAs.
  • appropriate pairs of RNAs and corresponding probes for assessing risks etc. of diseases of the heart are mentioned with the understanding that (i) appropriate variants of the probes as mentioned above may be used and (ii) said probes are specific for the corresponding RNA only but not for any of the other mentioned RNAs.
  • SEQ ID NO: 2/SEQ ID NO: 14 SEQ ID NO: 3/SEQ ID NO: 15; SEQ ID NO: 4/SEQ ID NO: 16; SEQ ID NO: 5/SEQ ID NO: 17; SEQ ID NO: 6/SEQ ID NO: 18; SEQ ID NO: 7/SEQ ID NO: 19; SEQ ID NO: 8/SEQ ID NO: 20, SEQ ID NO: 9/SEQ ID NO: 21 and SEQ ID NO: 10/SEQ ID NO: 22.
  • An appropriate pair of RNA and corresponding probe in the above described context is SEQ ID NO:7 and SEQ ID NO 25.
  • washing steps are performed in order to remove unspecific signals.
  • Appropriate washing conditions include 2 washing steps at 65°C with 2xSSC, 0,1% SDS for 30 min (50 ml) and finally two washing steps with 50 ml of a solution containing O.lxSSC, 0.1% SDS for 30 min.; see also Sambrook et al., loc. cit, Higgins and Hames, loc. cit.
  • the label is detected, depending on its nature.
  • a radioactive label may be detected by exposure to an X-ray film or by a phosphorimager.
  • biotinylated probes can be detected by fluorescence, e.g. by using SAPE (streptavidin- phycoerythrin) with subsequent detection of the signal by a laser scanner.
  • the invention provides a kit for identifying a subject at risk for a diseases of the heart or for monitoring the status or progression of a subject with a disease of the heart, comprising a means for detecting at least one RNA in a sample and means for detecting the level of RNA in the sample.
  • the kit comprises at least one nucleic acid probe as described above and instructions for hybridizing of the nucleic acid probe with the nucleic acid molecules in the sample.
  • the probe is detectably labeled, as discussed above.
  • the nucleic acid probe is bound to a solid substrate.
  • the invention relates to a method for identifying a subject at risk for a disease of the heart, comprising the step of quantitating the amount of a polypeptide selected from the group consisting of: (a) the polypeptide having the amino acid sequence of SEQ ID NO: 1 , the amino acid sequence of SEQ ID NO: 2, the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5, the amino acid sequence of SEQ ID NO: 6, the amino acid sequence of SEQ ID NO: 7, the amino acid sequence of SEQ ID NO: 8, the amino acid sequence of SEQ ID NO: 9 or the amino acid sequence of SEQ ID NO: 10; (b) a polypeptide having an amino acid sequence that is at least 60%, preferably at least 80%, more preferably at least 85%, especially at least 90%, more especially at least 95%, advantageously at least 99% identical to the amino acid sequence of (a); and (c) a polypeptide having the amino acid sequence of (a) with at least
  • This embodiment of the invention makes use of the option that detection may not only be at the level of the mRNA but also at the level of the polypeptide translated from the mRNA. Whereas it is not excluded that the level of mRNA strictly correlates with the level of polypeptide translated from the mRNA, this may not always be the case. Accordingly, it may be assessed whether the mRNA or the protein level, if different, is more appropriate to establish if the heart of a subject is prone to develop a disease of the heart. Factors that contribute to differences in the expression levels of mRNA and protein are well-known in the art and include differential mRNA-export to the protein-synthesis machinery as well as differences in the translation efficacy of different mRNA species.
  • the amount of the said polypeptide is quantitated using an antibody or an antigen-binding portion of said antibody that specifically binds a polypeptide selected from the group consisting of: (a) the polypeptide having the amino acid sequence of SEQ ID NO: 1 , the amino acid sequence of SEQ ID NO: 2, the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5, the amino acid sequence of SEQ ID NO: 6, the amino acid sequence of SEQ ID NO: 7, the amino acid sequence of SEQ ID NO: 8, the amino acid sequence of SEQ ID NO: 9 or the amino acid sequence of SEQ ID NO: 10; (b) a polypeptide having an amino acid sequence that is at least 60%, preferably at least 80%, more preferably at least 85%, especially at
  • the antibody used in accordance with the invention may be a monoclonal or a polyclonal antibody (see Harlow and Lane, "Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor, USA, 1988) or a derivative of said antibody which retains or essentially retains its binding specificity. Whereas particularly preferred embodiments of said derivatives are specified further herein below, other preferred derivatives of such antibodies are chimeric antibodies comprising, for example, a mouse or rat variable region and a human constant region.
  • the term "specifically binds" in connection with the antibody used in accordance with the present invention means that the antibody etc. does not or essentially does not cross-react with (poly)peptides of similar structures. Cross-reactivity of a panel of antibodies etc.
  • said antibody or antibody binding portion is or is derived from a human antibody or a humanized antibody.
  • humanized antibody means, in accordance with the present invention, an antibody of non-human origin, where at least one complementarity determining region (CDR) in the variable regions such as the CDR3 and preferably all 6 CDRs have been replaced by CDRs of an antibody of human origin having a desired specificity.
  • CDR complementarity determining region
  • the non-human constant region(s) of the antibody has/have been replaced by (a) constant region(s) of a human antibody.
  • the specifically binding antibody etc. may be detected by using, for example, a labeled secondary antibody specifically recognizing the constant region of the first antibody.
  • a labeled secondary antibody specifically recognizing the constant region of the first antibody.
  • the antibody, the binding portion or derivative thereof itself is detectably labeled.
  • Detectable labels include a variety of established labels such as radioactive ( 125 I, for example) or fluorescent labels (see, e.g. Harlow and Lane, loc. cit.). Binding may be detected after removing unspecific labels by appropriate washing conditions (see, e.g. Harlow and Lane, loc. cit.).
  • said derivative of said antibody is an scFv fragment.
  • scFv fragment single-chain Fv fragment
  • scFv fragment single-chain Fv fragment
  • the invention provides a kit for identifying a subject at risk for a diseases of the heart or for monitoring the status or progression of a subject with a disease of the heart, comprising a means for detecting at least one polypeptide as described above in a sample and means for detecting the level of the polypeptide in the sample.
  • the kit comprises at least one antibody, binding portion thereof, or derivative thereof that specifically binds to the polypeptide, as described above and instructions for binding the antibody, binding portion thereof, or derivative thereof with the polypeptide in the sample.
  • the antibody, binding portion thereof, or derivative thereof is detectably labeled.
  • the kit provides another reagent that specifically binds the antibody, binding portion thereof, or derivative thereof and that is detectably labeled, as discussed above.
  • the antibody, binding portion thereof, or derivative thereof is bound to solid substrate.
  • said RNA or polypeptide is obtained from heart tissue.
  • a suitable way would be to take a biopsy (catheter) from the ventricular wall. The decision to do this is clearly affected by the severity of the disease and the general constitution of the patient. The cardiologist and the patient have to drive the final decision.
  • said RNA or polypeptide is quantitated in heart tissue.
  • the method of the invention further comprises the step of normalizing the amount of RNA or polypeptide against a corresponding RNA or polypeptide from a healthy subject or cells derived from a healthy subject.
  • the term "healthy subject" in connection with the present invention means a subject without any indication for heart disease.
  • RNA or polypeptide against a corresponding RNA or polypeptide from a healthy subject or cells derived from a healthy subject means, in accordance with the present invention, that levels of mRNA or polypeptide from a comparative number of cells from the heart of said subject under investigation and from the heart of an individual not affected by a disease of the heart are compared.
  • cells from the heart of the subject under investigation may be compared in terms of the indicated mRNA or polypeptide levels with cells derived from the heart of a healthy individual which are kept in cell culture and optionally form a cell line.
  • different sources of cells such as from different individuals and/or different cell lines may be used for the generation of the standard against which the mRNA or polypeptide level of the subject under investigation is compared.
  • Affymetrix Chip technology there is also the possibility to use external standards (that are given separately to the hybridisation cocktail) in order to normalize the values of different oligonucleotide-chips.
  • the method of the invention further comprises the step of normalizing the amount of polypeptide against a corresponding polypeptide from a healthy subject or cells derived from a healthy subject.
  • the invention relates to a method for identifying a compound that increases the level in heart tissue of a polypeptide selected from the group consisting of: (a) the polypeptide having the amino acid sequence of SEQ ID NO: 1 , the amino acid sequence of SEQ ID NO: 2, the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5, the amino acid sequence of SEQ ID NO: 6, the amino acid sequence of SEQ ID NO: 7, the amino acid sequence of SEQ ID NO: 8, the amino acid sequence of SEQ ID NO: 9 or the amino acid sequence of SEQ ID NO: 10; (b) a polypeptide having an amino acid sequence that is at least 60%, preferably at least 80%, more preferably at least 85%, especially at least 90%, more especially at least 95%, advantageously at least 99% identical to the amino acid sequence of (a); and (c) a polypeptide having the amino acid sequence of (a) with at least one conservative amino acid substitution, said method comprising the steps of
  • the term "compound” in accordance with the present invention shall mean any biologically active substance that has an effect on heart tissue or a single heart cell, whereas such compound has a positive or negative influence upon such heart tissue or heart cell.
  • Preferred compounds are nucleic acids, preferably coding for a peptide, polypeptide, antisense RNA or a ribozyme or nucleic acids that act independently of their transcription respective their translation as for example an antisense RNA or ribozyme; natural or synthetic peptids, preferably with a relative molecular mass of about 1.000, especially of about 500, peptide analogs polypeptides or compositions of polypeptides, proteins, protein complexes, fusion proteins, preferably antibodies, especially murine, human or humanized antibodies, single chain antibodies, F a b fragments or any other antigen binding portion or derivative of an antibody, including modifications of such molecules as for example glycosylation, acetylation, phosphorylation, famesylation, hydroxylation, methylation or est
  • under conditions that would permit the translation of said polypeptide denotes any conditions that allow the in vitro or in vivo translation of the polypeptide of interest.
  • in vitro conditions translation may be effected in a cell-free system, as described, for example in Stoss, Schwaiger, Cooper and Stamm (1999). J. Biol. Chem. 274: 10951-10962, using the TNT-coupled reticulocyte lysate system (Promega).
  • physiological conditions such as conditions naturally occurring inside a cell are preferred.
  • the method of the invention allows the convenient identification or isolation of compounds that counteract such aberrant expression such that normal expression levels are restored or essentially restored.
  • the DNA encoding the polypeptide of interest would normally be contained in an expression vector.
  • the expression vectors may particularly be plasmids, cosmids, viruses or bacteriophages used conventionally in genetic engineering plasmids, cosmids, viruses and bacteriophages used conventionally in genetic engineering that comprise the aforementioned polynucleotide.
  • said vector is a gene transfer or targeting vector.
  • Expression vectors derived from viruses such as retroviruses, vaccinia virus, adeno-associated virus, herpes viruses, or bovine papilloma virus, may be used for delivery of the polynucleotides into targeted cell population.
  • the polynucleotides and vectors can be reconstituted into liposomes for delivery to target cells.
  • the vectors containing the polynucleotides can be transferred into the host cell by well-known methods, which vary depending on the type of cellular host.
  • calcium phosphate or DEAE-Dextran mediated transfection or electroporation may be used for eukaryotic cellular hosts; see Sambrook, supra.
  • Such vectors may comprise further genes such as marker genes which allow for the selection of said vector in a suitable host cell and under suitable conditions.
  • the polynucleotide is operatively linked to expression control sequences allowing expression in eukaryotic cells. Expression of said polynucleotide comprises transcription of the polynucleotide into a translatable mRNA. Regulatory elements ensuring expression in eukaryotic cells, preferably mammalian cells, are well known to those skilled in the art.
  • regulatory elements usually comprise regulatory sequences ensuring initiation of transcription and, optionally, a poly-A signal ensuring termination of transcription and stabilization of the transcript, and/or an intron further enhancing expression of said polynucleotide.
  • Additional regulatory elements may include transcriptional as well as translational enhancers, and/or naturally- associated or heterologous promoter regions.
  • Possible regulatory elements permitting expression in eukaryotic host cells are the AOX1 or GAL1 promoter in yeast or the CMV-, SV40-, RSV-promoter (Rous sarcoma virus), CMV-enhancer, SV40-enhancer or a globin intron in mammalian and other animal cells.
  • Beside elements which are responsible for the initiation of transcription such regulatory elements may also comprise transcription termination signals, such as the SV40- poly-A site or the tk-poly-A site, downstream of the polynucleotide.
  • transcription termination signals such as the SV40- poly-A site or the tk-poly-A site
  • leader sequences capable of directing the polypeptide to a cellular compartment or secreting it into the medium may be added to the coding sequence of the aforementioned polynucleotide and are well known in the art.
  • the leader sequence(s) is (are) assembled in appropriate phase with translation, initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein, or a portion thereof, into the periplasmic space or extracellular medium.
  • the heterologous sequence can encode a fusion protein including an C- or N-terminai identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.
  • suitable expression vectors are known in the art such as Okayama-Berg cDNA expression vector pcDVI
  • the expression control sequences will be eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells.
  • the vector used in the method of the present invention may also be a gene transfer or targeting vector.
  • Gene therapy which is based on introducing therapeutic genes into cells by ex-vivo or in-vivo techniques is one of the most important applications of gene transfer. Suitable vectors and methods for in-vitro or in-vivo gene therapy are described in the literature and are known to the person skilled in the art; see, e.g., Giordano, Nature Medicine 2 (1996), 534-539; Schaper, Circ. Res.
  • the polynucleotides and vectors may be designed for direct introduction or for introduction via liposomes, or viral vectors (e.g. adenoviral, retroviral) into the cell.
  • said cell is a germ line cell, embryonic cell, or egg cell or derived therefrom, most preferably said cell is a stem cell.
  • the vector comprising the DNA would be used to transform a suitable eukaryotic host cell.
  • the test compound Upon expression of the DNA, which may be constitutive or induced, the test compound would be contacted with the DNA. This can be done by introducing the test compound into the cell.
  • the test compound is a (poly)peptide
  • introduction may be effected by transfection of the corresponding DNA, optionally comprised in a suitable expression vector.
  • the introduction into the cell may be effected by direct administration, optionally in conjunction with DMSO for hydrophobic compounds, especially liposomal transfer.
  • the method of the invention is carried out in vitro, for example, in a cell-free system, then introduction into a cell would not be necessary. Rather, the test compound would be admixed to the in vitro expression system and the effect of said admixture observed.
  • the effect of the contact of the DNA of interest with the test compound on the protein level may be assessed by any technology that measures changes in the quantitative protein level.
  • Such technologies include Western blots, ELISAs, RIAs and other techniques referred to herein above.
  • the change in protein level, if any, as a result of the contact of said DNA and said test compound is compared against a standard. This standard is measured applying the same test system but omits the step of contacting the compound with the DNA.
  • the standard may consist of the expression level of the polypeptide after no compound has been added.
  • the DNA may be contacted with a compound that has previously been demonstrated to have an influence on the expression level.
  • the invention relates to a method for identifying a compound that specifically binds to a polypeptide having an amino acid sequence selected from the group consisting of (a) the amino acid sequence of SEQ ID NO: 1 , the amino acid sequence of SEQ ID NO: 2, the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5, the amino acid sequence of SEQ ID NO: 6, the amino acid sequence of SEQ ID NO: 7, the amino acid sequence of SEQ ID NO: 8, the amino acid sequence of SEQ ID NO: 9 or the amino acid sequence of SEQ ID NO: 10; (b) a polypeptide having an amino acid sequence that is at least 60%, preferably at least 80%, more preferably at least 85%, especially at least 90%, more especially at least 95%, advantageously at least 99% identical to the amino acid sequence of of (a); and (c) a polypeptide having the amino acid sequence of (a) with at least one conservative amino acid substitution; said method comprising the steps of (1) providing
  • said binding results in activation of said polypeptide.
  • Said activation may be, for example, an enzymatic activity (as described herein below) and/or the initiation of a signal cascade.
  • a cell based assay can be developed to identify potential activators.
  • the protein under investigation is expressed in cardiomyocytes (e. g. by infection with recombinant adenovirus).
  • the expression of these proteins leads to characteristic morphological alterations. Reversal or reduction of these morphological alterations can be used e.g. in an HTS assay to identifiy compounds which act as activators of these proteins.
  • the system can be automated by use of digital image analysis systems.
  • Another possibility is to identify first proteins which are binding partners of the described proteins. This is especially important for structural proteins or adaptor proteins in signal transduction pathways.
  • Methods to identifiy compounds capable of binding include affinity chromatography with immobilised target protein and subsequent elution of bound proteins (e. g. by acid pH), co-immunoprecipitation and chemical crosslinking with subsequent analysis on SDS-PAGE.
  • optical spectroscopy e. g. fluorescence or surface plasmon resonance
  • calorimetry isothermal titration microcalorimetry
  • NMR nuclear magnetic resonance
  • the intrinsic protein fluorescence may change (in intensity and/or wavelength of emission maximum) upon complex formation with the binding compound or the fluorescence of a covalently attached fluorophore may change upon complex formation.
  • the claimed protein or its identified binding partner may be labelled on e. g. cysteine or lysine residues with a fluorophore (for a collection of fluorophores see catalogues of Molecular Probes or Pierce Chemical Company) which changes its optical properties upon binding.
  • These changes in the intrinsic or extrinsic fluorescence may be applied for use in a HTS assay to identifiy compounds capable of inhibiting or activating the mentioned protein-protein interaction.
  • the activation of this activity may be monitored by using labelled (fluorescently, radioactively or immunologically) derivates of the substrate.
  • This activity assay which is based on labelled substrates can be used for development of a HTS assay to identifiy compounds acting as activators.
  • the invention relates to a monoclonal antibody or derivative thereof that specifically binds to polypeptide having an amino acid sequence selected from the group consisting of the amino acid sequence of SEQ ID NO: 1 , the amino acid sequence of SEQ ID NO: 2, the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5, the amino acid sequence of SEQ ID NO: 6, the amino acid sequence of SEQ ID NO: 7, the amino acid sequence of SEQ ID NO: 8, the amino acid sequence of SEQ ID NO: 9 or the amino acid sequence of SEQ ID NO: 10.
  • the invention further comprises kits comprising the monoclonal antibody or derivative thereof.
  • the invention relates to a method for identifying a compound that increases the level in heart tissue of an mRNA encoding a polypeptide selected from the group consisting of: (a) the polypeptide having the amino acid sequence of SEQ ID NO: 1, the amino acid sequence of SEQ ID NO: 2, the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 4, the amin ⁇ acid sequence of SEQ ID NO: 5, the amino acid sequence of SEQ ID NO: 6, the amino acid sequence of SEQ ID NO: 7, the amino acid sequence of SEQ ID NO: 8, the amino acid sequence of SEQ ID NO: 9 or the amino acid sequence of SEQ ID NO: 10; (b) a polypeptide having an amino acid sequence that is at least 60%, preferably at least 80%, more preferably at least 85%, especially at least 90%, more especially at least 95%, advantageously at least 99% identical to the amino acid sequence of (a); and (c) a polypeptide having the amino acid sequence of (a) with at least one conservative amino acid substitution
  • the invention relates to a transgenic non-human mammal whose somatic and germ cells comprise at least one gene encoding a functional polypeptide selected from the group consisting of: (a) the polypeptide having the amino acid sequence of SEQ ID NO: 1, the amino acid sequence of SEQ ID NO: 2, the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5, the amino acid sequence of SEQ ID NO: 6, the amino acid sequence of SEQ ID NO: 7, the amino acid sequence of SEQ ID NO: 8, the amino acid sequence of SEQ ID NO: 9 or the amino acid sequence of SEQ ID NO: 10; (b) a polypeptide having an amino acid sequence that is at least 60%, preferably at least 80%, more preferably at least 85%, especially at least 90%, more especially at least 95%, advantageously at least 99% identical to the amino acid sequence of (a); and (c) a polypeptide having the amino acid sequence of (a) with at least one conservative amino acid substitution,
  • a method for the production of a transgenic non-human animal comprises introduction of the aforementioned polynucleotide or targeting vector into a germ cell, an embryonic cell, stem cell or an egg or a cell derived therefrom.
  • the non-human animal can be used in accordance with a screening method of the invention described herein. Production of transgenic embryos and screening of those can be performed, e.g., as described by A. L. Joyner Ed., Gene Targeting, A Practical Approach (1993), Oxford University Press.
  • the DNA of the embryonal membranes of embryos can be analyzed using, e.g., Southern blots with an appropriate probe; see supra.
  • transgenic non-human animals A general method for making transgenic non-human animals is described in the art, see for example WO 94/24274.
  • ES cells embryonal stem cells
  • Murine ES cells such as AB-1 line grown on mitotically inactive SNL76/7 cell feeder layers (McMahon and Bradley, Cell 62:1073-1085 (1990)) essentially as described (Robertson, E. J. (1987) in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach. E. J. Robertson, ed. (Oxford: IRL Press), p. 71-112) may be used for homologous gene targeting.
  • ES lines include, but are not limited to, the E14 line (Hooper et al., Nature 326:292-295 (1987)), the D3 line (Doetschman et al., J. Embryol. Exp. Morph. 87:27-45 (1985)), the CCE line (Robertson et al., Nature 323:445-448 (1986)), the AK-7 line (Zhuang et al., Cell 77:875-884 (1994)).
  • E14 line Hooper et al., Nature 326:292-295 (1987)
  • D3 line Doetschman et al., J. Embryol. Exp. Morph. 87:27-45 (1985)
  • the CCE line Robot et al., Nature 323:445-448 (1986)
  • the AK-7 line Zhuang et al., Cell 77:875-884 (1994)
  • the blastocysts containing the injected ES cells are allowed to develop in the uteri of pseudopregnant nonhuman females and are born as chimeric mice.
  • the resultant transgenic mice are chimeric for cells having either the recombinase or reporter loci and are backcrossed and screened for the presence of the correctly targeted transgene (s) by PCR or Southern blot analysis on tail biopsy DNA of offspring so as to identify transgenic mice heterozygous for either the recombinase or reporter locus/loci.
  • the transgenic non-human animals may, for example, be transgenic mice, rats, hamsters, dogs, monkeys, rabbits, pigs, or cows.
  • said transgenic non-human animal is a mouse.
  • transgenic non-human mammal of the invention said functional or disrupted gene was introduced into the non-human mammal or an ancestor thereof, at an embryonic stage.
  • the modification is inactivation or suppression of said gene(s) or leads to the reduction of the synthesis of the corresponding protein(s).
  • This embodiment allows for example the study of the interaction of various mutant forms of the aforementioned polypeptides on the onset of the clinical symptoms of a disease related to disorders in the heart.
  • All the applications that have been herein before discussed with regard to a transgenic animal also apply to animals carrying two, three or more transgenes for example encoding different aforementioned nucleic acid molecules.
  • a suitable inducible system is for example tetracycline-regulated gene expression as described, e.g., by Gossen and Bujard (Proc. Natl. Acad. Sci. 89 USA (1992), 5547-5551) and Gossen et al. (Trends Biotech. 12 (1994), 58-62). Similar, the expression of the mutant protein(s) may be controlled by such regulatory elements.
  • the invention also relates to a transgenic non-human animal, preferably mammal and cells of such animals which cells contain (preferably stably integrated into their genome) at least one of the aforementioned nucleic acid molecule(s) or part thereof, wherein the transcription and/or expression of the nucleic acid molecule or part thereof leads to reduction of the synthesis of (a) corresponding protein(s).
  • the reduction is achieved by an anti-sense, sense, ribozyme, co-suppression and/or dominant mutant effect.
  • Antisense and “antisense nucleotides” means DNA or RNA constructs which block the expression of the naturally occurring gene product.
  • nucleic acid molecule encoding the antisense-RNA is preferably of homologous origin with respect to the animal species used for transformation.
  • nucleic acid molecules which display a high degree of homology to endogenously occurring nucleic acid molecules encoding such a protein.
  • the homology is preferably higher than 60%, preferably at least 80%, more preferably at least 85%, especially at least 90%, more especially at least 95%, advantageously at least 99%.
  • the invention relates to a method for identifying a compound that increases the expression of a polypeptide in heart tissue selected from the group consisting of: (a) the polypeptide having the amino acid sequence of SEQ ID NO: 1 , the amino acid sequence of SEQ ID NO: 2, the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5, the amino acid sequence of SEQ ID NO: 6, the amino acid sequence of SEQ ID NO: 7, the amino acid sequence of SEQ ID NO: 8, the amino acid sequence of SEQ ID NO: 9 or the amino acid sequence of SEQ ID NO: 10; (b) a polypeptide having an amino acid sequence that is at least 60%, preferably at least 80%, more preferably at least 85%, especially at least 90%, more especially at least 95%, advantageously at least 99% identical to the amino acid sequence of (a); and (c) a polypeptide having the amino acid sequence of (a) with at least one conservative amino acid substitution, said method comprising the steps
  • test compound which has preferably been tested beforehand for essentially lacking toxicity for the animal can be administered to the animal by any convenient route suitable for administration. These routes include injection, topical and oral administration. Intervals and doses of administration may vary and will be decided upon by the physician/researcher on a case-by-case basis.
  • Detection may be effected by a variety of means.
  • increase of polypeptide production may be assessed as described, for example, in EP 95 94 1424.4 or in EP 99 12 4640.6.
  • blood of the non-human transgenic animal may be assessed for the changing quantity of the protein. It is preferred in such a case that the gene encoding the polypeptide of interest carries an inducible promoter.
  • the non-human transgenic animal will have to be sacrificed in order to assess whether a change in the level of polypeptide expression has occurred.
  • heart tissue may be removed from the sacrificed animal and assessed, using standard technologies, for the expression level of the protein.
  • an antibody specific for the polypeptide may be contacted with the heart tissue and the test developed with a second labeled antibody that is directed to the first antibody.
  • the first antibody itself may be labeled.
  • Heart tissue of a non-human transgenic animal that has been contacted with the test compound would be compared with heart tissue of a non- human transgenic animal that has not been contacted with said test compound.
  • the transgenic animal may carry more than one of the aforementioned nucleic acid molecules. Accordingly, the effect of a test compound on the expression level of any of these transgenes may be assessed. In addition, a variety of test compounds may be tested, at the same time, for the effect on one or a variety of said transgenes.
  • test compound that has proven to be effective in increasing the level of the polypeptide of interest and/or in decreasing the turnover of the polypeptide of interest may be either directly formulated into a medicament (if, for example, its structure is suitable for administration and if it has proven to be non-toxic) or may serve as a lead compound for downstream developments, the results of which may then be formulated into pharmaceutical compositions.
  • test compound prevents or ameliorates a disease of the heart in said transgenic non-human mammal.
  • the effect of the test compound may be assessed by observing the disease state of the transgenic animal.
  • this test compound is a prime candidate for the development of a medicament useful also in humans.
  • the compound could also inhibit disease establishment by treatment in advance.
  • a further embodiment of the invention is a method for identifying one or a pluratiy of isogenes of a gene coding for a polypeptide selected from the group consisting of: the polypeptide having the amino acid sequence of SEQ ID NO: 1 , the amino acid sequence of SEQ ID NO: 2, the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5, the amino acid sequence of SEQ ID NO: 6, the amino acid sequence of SEQ ID NO: 7, the amino acid sequence of SEQ ID NO: 8, the amino acid sequence of SEQ ID NO: 9 or the amino acid sequence of SEQ ID NO: 10; in heart tissue comprising the steps of (1) providing nucleic acid coding for said polypeptide or a part thereof; and (2) identifying a second nucleic acid from the same species that (i) has a homology of 60%, preferably at least 80%, more preferably at least 85%, especially at least 90%, more especially at least 95%, advantageously at least 99% or (i
  • isogenes shall mean genes that are considered to be generated by gene duplication. They can be identified by comparing the homology of the DNA-, RNA-, or protein-sequence of interest with other DNA, RNA or protein-sequences of the same species. There might be strong differences in the degree of homology between isogenes of the same species. This may be dependent on the time-point, when the gene duplication event took place in evolution and the degree of conservation during evolution.
  • Isogenes can be identified and cloned by RT-PCR as has been demonstrated by Screaton et al. (1995) EMBO J. 14:4336-4349 or Huang et al. (1998) Gene 211 : 49-55. Isogenes can also be identified and cloned by colony hybridisation or plaque hybridization as described in Sambrook, Fritsch, Maniatis (1989), Molecular Cloning. Cold Spring Harbor Laboratory Press. In a first step, either a genomic or a cDNA library e.g. in bacteria or phages is generated. In order to identify isogenes, colony hybridisation or plaque hybridization is slightly modified in a way that cross- hybridizations are detectable under conditions of lower stringency.
  • a low-stringency washing condition may include 2 washing steps at a temperature between 45°C and 65°C with 4xSSC, 0,1% SDS for 30 min (50 ml) and finally two washing steps with 50 ml of a solution containing 2xSSC, 0.1% SDS for 30 min. After detection, signal intensity of colonies containing an isogene is dependent on the homology of a gene and its isogene(s).
  • the invention relates to a method for identifying one or a plurality of genes whose expression in heart tissue is modulated by inhibiting or reducing the expression of a polypeptide selected from the group consisting of: (a) the polypeptide having the amino acid sequence of SEQ ID NO: 1 , the amino acid sequence of SEQ ID NO: 2, the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5, the amino acid sequence of SEQ ID NO: 6, the amino acid sequence of SEQ ID NO: 7, the amino acid sequence of SEQ ID NO: 8, the amino acid sequence of SEQ ID NO: 9 or the amino acid sequence of SEQ ID NO: 10; (b) a polypeptide having an amino acid sequence that is at least 60%, preferably at least 80%, more preferably at least 85%, especially at least 90%, more especially at least 95%, advantageously at least 99% identical to the amino acid sequence of (a); and (c) a polypeptide having the amino acid sequence of (a) with
  • gene expression profile shall mean all expressed genes of a cell or a tissue. Such profile can be assessed using methods well known in the art, for example isolation of total RNA, isolation of poly(A) RNA from total RNA, suppression subtractive hybridization, differential display, preparation of cDNA libraries or quantitative dot blot analysis, as for example described in Example 1 of this specification.
  • This embodiment of the method of the invention is particularly suitable for identifying further genes the expression level of which is directly affected by the aberrant expression of any of the aforementioned genes.
  • this embodiment of the method of the invention allows the identification of genes involved in the same protein cascade as the aberrantly expressed gene.
  • the method of the invention will be a method performed in cell culture.
  • the method of the invention allows for the design of further medicaments that use other targets than the aberrantly expressed gene. For example, if a potential target downstream of the aberrantly expressed gene is indeed targeted by a medicament, the negative effect of the aberrantly expressed gene may be efficiently counterbalanced. Compounds modulating other genes in the cascade may have to be refined or further developed prior to administration as a medicament as described elsewhere in this specification.
  • the invention relates to a method for identifying one or a plurality of genes whose expression in heart tissue is modulated by the inhibition or reduction of the expression of a polypeptide selected from the group consisting of: (a) the polypeptide having the amino acid sequence of SEQ ID NO: 1 , the amino acid sequence of SEQ ID NO: 2, the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5, the amino acid sequence of SEQ ID NO: 6, the amino acid sequence of SEQ ID NO: 7, the amino acid sequence of SEQ ID NO: 8, the arnino acid sequence of SEQ ID NO: 9 or the amino acid sequence of SEQ ID NO: 10; (b) a polypeptide having an amino acid sequence that is at least 60%, preferably at least 80%, more preferably at least 85%, especially at least 90%, more especially at least 95%, advantageously at least 99% identical to the amino acid sequence of (a); and (c) a polypeptide having the amino acid sequence of (
  • this embodiment of the method of the invention compares the expression profiles of cells from a healthy subject and a subject suffering from a heart disease.
  • the term "cells derived from a heart” includes cells that are held in cell culture or even cell lines that autonomously grow in cell culture and that were originally derived from heart tissue.
  • differences in expression levels of genes involved in the disease of the heart may be identified.
  • these genes may be part of a cascade involving the aberrantly expressed gene. Examples of such cascades are signaling cascades.
  • the method of the invention further comprises the steps of (3) determining at least one gene that is expressed at a lower or higher level in the presence of said compound; and (4) identifying a further compound that is capable of raising or lowering the expression level of said at least one gene.
  • This preferred embodiment of the invention requires that one of the genes the expression of which may directly or indirectly be lowered or increased by the expression of the aberrant gene is identified. Then, a further panel of test compounds may be tested for the capacity to increase or decrease the expression of said further gene. Compounds that are successfully tested would be prime candidates for the development of medicaments for the prevention or treatment of a disease of the heart.
  • the method of the invention further comprises the steps of (3) determining at least one gene that is expressed at a lower or higher level in said heart tissue cells from or derived from a heart of a subject suffering from a disease of the heart; and (4) identifying a further compound that is capable of raising or lowering the expression level of said at least one gene.
  • this embodiment requires that at least one gene is identified by comparing the expression profiles of tissue or cells derived from a healthy subject and from a subject suffering from a disease of the heart. Subsequently, at least one compound is identified that is capable of increasing or decreasing the expression of said gene.
  • the invention relates to a method for identifying proteins or a plurality of proteins in heart tissue whose activity is modulated by a polypeptide having the amino acid sequence selected from the group consisting of the amino acid sequence of SEQ ID NO: 1 , the amino acid sequence of SEQ ID NO: 2, the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5, the amino acid sequence of SEQ ID NO: 6, the amino acid sequence of SEQ ID NO: 7, the amino acid sequence of SEQ ID NO: 8, the amino acid sequence of SEQ ID NO: 9 or the amino acid sequence of SEQ ID NO: 10; said method comprising the steps of (1) providing said polypeptide and (2) identifying a further protein that is capable of interacting with said polypeptide.
  • the invention relates to a method of refining a compound identified by the method as described herein above, said method comprising the steps of (1) identification of the binding sites of the compound and the DNA or mRNA molecule by site-directed mutagenesis or chimeric protein studies; (2) molecular modeling of both the binding site of the compound and the binding site of the DNA or mRNA molecule; and (3) modification of the compound to improve its binding specificity for the DNA or mRNA.
  • This method in a preferred embodiment comprises the identification steps of the method described herein above, prior to the refinement steps.
  • Steps (1) and (2) can be carried out according tor conventional protocols.
  • a protocol for site directed mutagenesis is described in Ling MM, Robinson BH. (1997) Anal. Biochem. 254: 157-178.
  • the use of homology modelling in conjunction with site-directed mutagenesis for analysis of structure-function relationships is reviewed in Szklarz and Halpert (1997) Life Sci.
  • Chimeric proteins are generated by ligation of the corresponding DNA fragments via a unique restriction site using the conventional cloning techniques described in Sambrook, Fritsch, Maniatis. Molecular Cloning, a laboratory manual. (1989) Cold Spring Harbor Laboratory Press.
  • a fusion of two DNA fragments that results in a chimeric DNA fragment encoding a chimeric protein can also be generated using the gateway-system (Life technologies), a system that is based on DNA fusion by recombination.
  • gateway-system Life technologies
  • a prominent example of molecular modelling is the structure-based design of compounds binding to HIV reverse transcriptase that is reviewed in Mao, Sudbeck, Venkatachalam and Uckun (2000). Biochem. Pharmacol. 60: 1251-1265.
  • identification of the binding site of said drug by site-directed mutagenesis and chimerical protein studies can be achieved by modifications in the (poly)peptide primary sequence that affect the drug affinity; this usually allows to precisely map the binding pocket for the drug.
  • step (2) the following protocols may be envisaged: Once the effector site for drugs has been mapped, the precise residues interacting with different parts of the drug can be identified by combination of the information obtained from mutagenesis studies (step (1)) and computer simulations of the structure of the binding site provided that the precise three-dimensional structure of the drug is known (if not, it can be predicted by computational simulation). If said drug is itself a peptide, it can be also mutated to determine which residues interact with other residues in the polypeptide of interest.
  • the drug can be modified to improve its binding affinity or ist potency and specificity. If, for instance, there are electrostatic interactions between a particular residue of the polypeptide of interest and some region of the drug molecule, the overall charge in that region can be modified to increase that particular interaction.
  • Identification of binding sites may be assisted by computer programs.
  • appropriate computer programs can be used for the identification of interactive sites of a putative inhibitor and the polypeptide by computer assisted searches for complementary structural motifs (Fassina, Immunomethods 5 (1994), 114-120).
  • Further appropriate computer systems for the computer aided design of protein and peptides are described in the prior art, for example, in Berry, Biochem. Soc. Trans. 22 (1994), 1033-1036; Wodak, Ann. N. Y. Acad. Sci. 501 (1987), 1-13; Pabo, Biochemistry 25 (1986), 5987-5991.
  • Modifications of the drug can be produced, for example, by peptidomimetics and other inhibitors can also be identified by the synthesis of peptidomimetic combinatorial libraries through successive chemical modification and testing the resulting compounds. Methods for the generation and use of peptidomimetic combinatorial libraries are described in the prior art, for example in Ostresh, Methods in Enzymology 267 (1996), 220- 234 and Dorner, Bioorg. Med. Chem. 4 (1996), 709-715.
  • the three- dimensional and/or crystallographic structure of activators of the expression of the polypeptide of the invention can be used for the design of peptidomimetic activators, e.g., in combination with the (poly)peptide of the invention (Rose, Biochemistry 35 (1996), 12933-12944; Rutenber, Bioorg. Med. Chem. 4 (1996), 1545-1558).
  • said compound is further refined by peptidomimetics.
  • the invention furthermore relates to a method of modifying a compound identified or refined by the method of the invention as described herein above as a lead compound to achieve (i) modified site of action, spectrum of activity, organ specificity, and/or (ii) improved potency, and/or (iii) decreased toxicity (improved therapeutic index), and/or (iv) decreased side effects, and/or (v) modified onset of therapeutic action, duration of effect, and/or (vi) modified pharmakinetic parameters (resorption, distribution, metabolism and excretion), and/or (vii) modified physico-chemical parameters (solubility, hygroscopicity, color, taste, odor, stability, state), and/or (viii) improved general specificity, organ/tissue specificity, and/or (ix) optimized application form and route by (i) esterification of carboxyl groups, or (ii) esterification of hydroxyl groups with carbon acids, or (iii) esterification of hydroxyl groups to, e.g.
  • the invention additionally relates to a method for inducing a disease of the heart in a non-human mammal, comprising the step of contacting the heart tissue of said mammal with a compound that inhibits or reduces/decreases the expression and/or activity of a polypeptide selected from the group consisting of: (a) the polypeptide having the amino acid sequence of SEQ ID NO: 1 , the amino acid sequence of SEQ ID NO: 2, the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ1D NO: 4, the amino acid sequence of SEQ ID NO: 5, the amino acid sequence of SEQ ID NO: 6, the amino acid sequence of SEQ ID NO: 7, the amino acid sequence of SEQ ID NO: 8, the amino acid sequence of SEQ ID NO: 9 or the amino acid sequence of SEQ ID NO: 10; (b) a polypeptide having an amino acid sequence that is at least 60%, preferably at least 80%, more preferably at least 85%, especially at least 90%, more especially at least 95%, advantageously at least 99% identical
  • This embodiment of the invention is particularly useful for mimicking factors/developments leading to the onset of the disease.
  • differences in the expression of a protein contributes to heart failure has been shown for phospholamban, for example. Mice over-expressing phospholamban develop heart failure. This effect is thought to be due to the inhibition of Serca. (Minamisawa et al. (1999) Cell, 99:313-322).
  • said compound that decreases or increases is a small molecule, an antibody or an aptamer that specifically binds said polypeptide.
  • the invention moreover relates to a method of producing a pharmaceutical composition
  • a method of producing a pharmaceutical composition comprising formulating the compound identified, refined or modified by the method as described herein above optionally with a pharmaceutically active carrier and/or diluent.
  • Said method of producing a pharmaceutical composition preferably comprises in accordance with the present invention (the) steps of the methods described herein above.
  • the pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier and/or diluent.
  • suitable pharmaceutical carriers include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc.
  • Compositions comprising such carriers can be formulated by well known conventional methods. These pharmaceutical compositions can be administered to the subject at a suitable dose. Administration of the suitable compositions may be effected by different ways, e.g., by intravenous, intraperitoneal, subcutaneous, intramuscular, topical, intradermal, intranasal or intrabronchial administration. The dosage regimen will be determined by the attending physician and clinical factors.
  • dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently.
  • a typical dose can be, for example, in the range of 0.001 to 1000 ⁇ g (or of nucleic acid for expression or for inhibition of expression in this range); however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors.
  • the regimen as a regular administration of the pharmaceutical composition should be in the range of 1 ⁇ g to 10 mg units per day. If the regimen is a continuous infusion, it should also be in the range of 1 ⁇ g to 10 mg units per kilogram of body weight per minute, respectively.
  • compositions of the invention may be administered locally or systemically. Administration will generally be parenterally, e.g., intravenously; DNA may also be administered directly to the target site, e.g., by biolistic delivery to an internal or external target site or by catheter to a site in an artery. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents examples include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like.
  • Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
  • the pharmaceutical composition of the invention may comprise further agents such as interteukins or interferons depending on the intended use of the pharmaceutical composition.
  • the invention also relates to a method for preventing or treating a disease of the heart in a subject in need of such treatment, comprising the step of increasing the level of a polypeptide in the heart tissue of a subject, said polypeptide being selected from the group consisting of: (a) the polypeptide having the amino acid sequence of SEQ ID NO: 1 , the amino acid sequence of SEQ ID NO: 2, the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5, the amino acid sequence of SEQ ID NO: 6, the amino acid sequence of SEQ ID NO: 7, the amino acid sequence of SEQ ID NO: 8, the amino acid sequence of SEQ ID NO: 9 or the amino acid sequence of SEQ ID NO: 10; (b) a polypeptide having an amino acid sequence that is at least 60%, preferably at least 80%, more preferably at least 85%, especially at least 90%, more especially at least 95%, advantageously at least 99% identical to the amino acid sequence of (a); and (c)
  • the invention relates to a method of preventing or treating a disease of the heart in a subject in need of such treatment comprising the step of increasing the level of mRNA encoding a polypeptide in the heart tissue of a subject, said polypeptide being selected from the group consisting of: (a) the polypeptide having the amino acid sequence of SEQ ID NO: 1 , the amino acid sequence of SEQ ID NO: 2, the amino acid sequence of SEQ ID NO: 3, the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 5, the amino acid sequence of SEQ ID NO: 6, the amino acid sequence of SEQ ID NO: 7, the amino acid sequence of SEQ ID NO: 8, the amino acid sequence of SEQ ID NO: 9 or the amino acid sequence of SEQ ID NO: 10; (b) a polypeptide having an amino acid sequence that is at least 60%, preferably at least 80%, more preferably at least 85%, especially at least 90%, more especially at least 95%, advantageously at least 99% identical to the amino acid sequence of (a)
  • the invention in a preferred embodiment relates to a method wherein such increase is effected by administering the pharmaceutical composition obtained by the method as described herein above.
  • such an increase is effected by introducing the nucleic acid sequence, preferably DNA sequence, recited herein above into the germ line or into somatic cells of a subject in need thereof. Technologies for effecting such an introduction have been described herein above.
  • the disease of the heart to be treated is congestive heart failure, dilative cardiomyopathy, hypertrophic cardiomyopathy, ischemic cardiomyopathy, specific heart muscle disease, rhythm and conduction disorders, syncope and sudden death, coronary heart disease, systemic arterial hypertension, pulmonary hypertension and pulmonary heart disease, valvular heart disease, congenital heart disease, pericardial disease or endocarditis.
  • the invention relates to a method for identifying subjects at risk for congestive heart failure comprising the step of detecting an decreased diphosphoinositol polyphosphate phosphodihydrolase type II beta (DIPP2 ⁇ ) activity in the heart tissue of a subject.
  • DIPP2 ⁇ diphosphoinositol polyphosphate phosphodihydrolase type II beta
  • the invention relates to a methocf identifying a subject at risk for congestive heart failure, said method comprising detecting increased levels of ⁇ - phosphorylated diphosphoinositol polyphosphates.
  • a method of detecting increased levels of ⁇ -phosphorylated diphosphoinositol polyphosphates would be the structural analysis of diphosphoinositol polyphosphates in biological systems, which relies largely on NMR analysis as described in Laussmann, Eujen, Weisshuhn, Thiel and Vogel (1996) Biochem. J. 315:715-720.
  • the invention additionally relates to a method for preventing or treating congestive heart failure in a subject, said method comprising the step of contacting the heart tissue of said subject with a compound that increases the expression and/or the activity of DIPP2 ⁇ or DIPP2 .
  • the invention as well relates to a method for preventing or treating congestive heart failure in a subject, said method comprising the step of increasing the removal of ⁇ -phosphate from diphosphoinositol polyphosphates in the heart tissue of said subject.
  • the invention in a preferred embodiment relates to a method wherein the expression of DIPP2 ⁇ is increased in said heart tissue.
  • the expression of DlPP2 is increased in said heart tissue.
  • the expression of an exogenous gene is increased in said heart tissue.
  • the expression of an endogenous gene is increased in said heart tissue.
  • the activity of DIPP2 ⁇ is increased in said heart tissue.
  • the activity of DIPP2 ⁇ is increased in said heart tissue.
  • the invention further relates to a method for preventing or treating congestive heart failure in a subject comprising the step of decreasing the amount of ⁇ - phosphorylated diphosphoinositol polyphosphates in the heart tissue of said subject.
  • the invention additionally relates to a method for identifying a compound for preventing or treating congestive heart failure, said method comprising the steps of: (a) contacting DIPP2 ⁇ or DIPP2 with a substrate for DIPP2 ⁇ or DIPP2 ⁇ and a test compound, and (b) determining whether the removal of ⁇ -phosphate from the substrate is increased in the presence of the test compound.
  • the invention as well relates to a method for identifying a compound for preventing or treating congestive heart failure, said method comprising the steps of: (a) contacting a heart tissue cell expressing DIPP2 ⁇ or DIPP2 ⁇ with a test compound; and (b) determining whether removal of ⁇ -phbsphate from diphosphoinositol polyphosphates is increased.
  • the invention relates to a method for preventing or treating congestive heart failure in a subject comprising the step of increasing the glucose level in heart tissue in said subject.
  • said glucose level is increased by administering glucose to said subject.
  • said glucose level is increased by increasing the amount of glycogen storage in said heart tissue.
  • glycogen storage is increased by increasing the expression and/or activity of a glucan branching enzyme (GBE).
  • GBE glucan branching enzyme
  • the activity of alpha-1,4-glucan branching enzyme can be measured as described in Satoh and Sato (1980). Anal. Biochem. 108: 16-24 or in Tolmasky and Krisman (1987) Eur. J. Biochem. 168: 393-397 and references therein.
  • the GBE is 1,4- ⁇ - GBE.
  • the invention relates to a method for preventing or treating congestive heart failure in a subject, comprising increasing the activity of DERP2.
  • the invention relates to a method for preventing or treating congestive heart failure in a subject, comprising increasing the DERP2-mediated signal transduction in the heart tissue of said subject.
  • the invention in addition relates to a method for preventing or treating congestive heart failure in a subject, comprising increasing DERP2-mediated cellular transport in the heart tissue of said subject.
  • the method comprises the step of increasing the expression of DERP2 on the plasma membrane of cells in said heart tissue.
  • the method comprises the step of contacting said heart tissue with a compound that is a DERP2 activator.
  • the invention relates to the use of a compound identified, refined or modified by the method as described herein above or a monoclonal antibody for the manufacture of a pharmaceutical composition for the prophylaxis or treatment of heart diseases, especially congestive heart failure.
  • Fig. 1 A shows the cDNA sequence of clone 40095.
  • Fig. 1 B shows the sequence of the EST clone AF047439. Start and stop codons are marked by bold letters, the sequence of 40095 is marked in italic letters. The putative polyadenylation signal is underlined.
  • Fig. 1 C shows the putative amino acid sequence AAC39890.
  • Fig.1 D shows a schematic alignment of the cDNA fragment 40095 identified in SSH with the homologous Genbank entree and the open reading frame of 300 amino acids (aa). Not to scale.
  • Fig. 1 E Two filters were hybridized sequentially with [ ⁇ - 33 P]UTP labeled T3 transcripts from cDNA libraries prepared from mRNA of five control and four DCM heart tissues as indicated. Experiments were normalized by adjusting the overall signal intensity of each hybridization to 100%, relative expression levels are given. Mean values and standard deviations were calculated from all NF and DCM samples, respectively. Asterisks mark samples used for SSH.
  • Fig. 2 A shows the cDNA sequence of clone 41237.
  • Fig. 2 B shows the sequence of 41237contig (EST sequences available from proprietary LEADSTM database by Compugen Ltd. have been assembled using
  • Fig. 2 C shows the amino acid sequence AAF68859.
  • the amino acid sequence of the beta isoform is given.
  • the deletion of Gln86 (underlined) within the type 2 alpha coding sequence results in a 20-50% increase in catalytic activity of the alpha isoform.
  • Fig.2 D Schematic alignment of the cDNA fragment 41237 identified in SSH with its overlapping contig of assembled ESTs according to LabOnWeb analysis
  • Fig. 2 E Two filters were hybridized sequentially with [ - 33 P]UTP labeled T3 transcripts from cDNA libraries prepared from mRNA of five control and four DCM heart tissues as indicated. Experiments were normalized by adjusting the overall signal intensity of each hybridization to 100%), relative expression levels are given.
  • Fig.3 A shows the cDNA sequence of clone 41444.
  • Fig. 3 B shows the sequence of the EST clone L07956. Start and stop codons are marked in bold letters, the sequence of 41444 is given in italic letters. Putative polyadenylation sites are underlined.
  • Fig. 3 C shows the amino acid sequence AAA58642.
  • Fig.3 D Schematic alignment of the cDNA fragment 41444 identified in SSH with its homologous Genbank entree and the open reading frame of 702 amino acids
  • Fig. 3 E Two filters were hybridized sequentially with [ - 33 P]UTP labeled T3 transcripts from cDNA libraries prepared from mRNA of five control and four DCM heart tissues as indicated. Experiments were normalized by adjusting the overall signal intensity of each hybridization to 100%, relative expression levels are given.
  • Fig. 3 F The figure shows the hybridization pattern of GBE on HG-U133A oligo arrays (Affymetrix) in different tissues samples and heart RNA mixture. Individual relative signal intensities as calculated using MAS5 software of Affymetirx and relative ratios between tissues are given.
  • Fig. 4 A shows the cDNA sequence of clone 41482.
  • Fig. 4 B shows the sequence of the EST clone AB009685. Start and stop codons are marked in bold letters, the sequence of 41482 is marked in italic letters.
  • Fig. 4 C shows the putative amino acid sequence BAA93049.
  • Fig.4 D Schematic alignment of the cDNA fragment 41482 identified in SSH with its homologous Genbank entrees and putative open reading frames of 345 and 319 amino acids (aa), respectively. Not to scale.
  • Fig. 4 E Two filters were hybridized sequentially with [ ⁇ - 33 P]UTP labeled T3 transcripts from cDNA libraries prepared from mRNA of five control and four DCM heart tissues as indicated. Experiments were normalized by adjusting the overall signal intensity of each hybridization to 100%, relative expression levels are given.
  • Fig. 4 F The figure shows the sequence of 41482contig, which has been assembled from AB009685 and the cDNA fragment identified by SSH.
  • Fig. 5 A shows the cDNA sequence of clone 52529.
  • Fig. 5 B shows the sequence of the EST clone NM_006854. Start and stop codons are marked by bold letters, the sequence of 52529 is marked in italic letters.
  • Fig. 5 C shows the putative amino acid sequence NPJ 06845.
  • Fig.5 D Schematic alignment of the cDNA fragment 52529 identified in SSH with its homologous Genbank entree and the open reading frame of 212 amino acids
  • Fig. 5 E Two filters were hybridized sequentially with [ - 33 P]UTP labeled T3 transcripts from cDNA libraries prepared from mRNA of five control and five DCM heart tissues as indicated. Experiments were normalized by adjusting the overall signal intensity of each hybridization to 100%, relative expression levels are given.
  • Fig. 6 A shows the cDNA sequence of clone 55253.
  • Fig. 6 B shows the sequence of the EST clone U06631. Start and stop codons are marked by bold letters, the sequence of 55253 is marked in italic letters. Two putative polyadenylation sites are underlined.
  • Fig. 6 C shows the amino acid sequence AAA16607.
  • Fig.6 D Schematic alignment of the cDNA fragment 55253 identified in SSH with its homologous Genbank entree and the open reading frame of 597 amino acids
  • Fig. 6 E Two filters were hybridized sequentially with [ - 33 P]UTP labeled T3 transcripts from cDNA libraries prepared from mRNA of five control and five DCM heart tissues as indicated. Experiments were normalized by adjusting the overall signal intensity of each hybridization to 100%, relative expression levels are given.
  • Fig. 7 A shows the cDNA sequence of clone 61024.
  • Fig. 7 B shows the sequence of the EST clone 61024cons. The stop codon is marked by bold letters, the start codon is not annotated so far. The sequence of
  • 61024 is marked in italic letters.
  • Fig. 7 C shows the sequence of the 5 ' end of 61024 elongated by 292 nt as a result of PCR. The start methionine was still not identified and seems to be further upstream.
  • Fig. 7 D shows the putative amino acid sequence 61024pep.
  • the protein sequence of BAA82992 could be elongated at the 5 ⁇ end by 98 amino acids as underlined.
  • Fig.7 E Schematic alignment of the cDNA fragment 61024 identified in SSH with the homologous Genbank entree.
  • the open reading frame identified so far comprises 642 amino acids (aa). Not to scale.
  • Fig. 7 F Two filters were hybridized sequentially with [ ⁇ - 33 P]UTP labeled T3 transcripts from cDNA libraries prepared from mRNA of five control and five DCM heart tissues as indicated. Experiments were normalized by adjusting the overall signal intensity of each hybridization to 100%, relative expression levels are given.
  • Fig. 8 A shows the cDNA sequence of clone 61119.
  • Fig. 8 B shows the sequence of the EST clone AF115509. Start and stop codons are marked by bold letters, the sequence of 61119 is marked in italic letters.
  • Fig. 8 C shows the putative amino acid sequence AAD41257.
  • Fig.8 D Schematic alignment of the cDNA fragment 61119 identified in SSH with its homologous Genbank entree and the open reading frame of 721 amino acids
  • Fig. 8 E Two filters were hybridized sequentially with [ - 33 P]UTP labeled T3 transcripts from cDNA libraries prepared from mRNA of five control and five DCM heart tissues as indicated. Experiments were normalized by adjusting the overall signal intensity of each hybridization to 100%, relative expression levels are given.
  • Fig. 9 A shows the cDNA sequence of clone 62139.
  • Fig. 9 B shows the sequence of 62139contig (EST sequences available from proprietary LEADSTM database by Compugen Ltd. have been assembled using
  • Fig. 9 C shows the putative amino acid sequence CAA65989.
  • Fig.9 D Schematic alignment of the cDNA fragment 62139 identified in SSH with its overlapping contig of assembled EST sequences according to LabOnWeb analysis (Compugen), homologous Genbank entree and the open reading frame of
  • Fig. 9 E Two filters were hybridized sequentially with [ ⁇ - 33 P]UTP labeled T3 transcripts from cDNA libraries prepared from mRNA of five control and five DCM heart tissues as indicated. Experiments were normalized by adjusting the overall signal intensity of each hybridization to 100%, relative expression levels are given.
  • Fig. 9 F Affymetrix arrays were hybridized with biotinylated RNA isolated from either 11 DCM-, 9 ICM- or 10 non-failing tissue-samples. Normalization and data analysis occurred using the Microarray Suite 4.0 (Affymetrix). The y-axis shows the average differences measured.
  • Fig. 9 G Affymetrix arrays were hybridized with biotinylated RNA isolated from either 11 DCM-, 9 ICM- or 10 non-failing tissue-samples (see FIG. 9 F). Mean values and standard deviations were calculated from all NF, DCM and ICM samples, respectively. The y-axis shows the average differences measured.
  • Fig. 10 A shows the cDNA sequence of clone 62140 and 58185.
  • Fig. 10 B shows the sequence of 62140contig (EST sequences available from proprietary LEADSTM database by Compugen Ltd. have been assembled using
  • Fig.10 C Schematic alignment of cDNA fragments 62140 and 58185 identified in
  • Fig. 10 D Two filters were hybridized sequentially with [ ⁇ - 33 P]UTP labeled T3 transcripts from cDNA libraries prepared from mRNA of five control and five DCM heart tissues as indicated. Experiments were normalized by adjusting the overall signal intensity of each hybridization to 100%, relative expression levels are given. Mean values and standard deviations were calculated from all NF and DCM samples, respectively. Asterisks mark samples used for SSH.
  • 0.5 g tissue were disrupted using a mortar and pestle and grinded under liquid nitrogen.
  • the suspension of tissue powder and liquid nitrogen was decanted into a cooled 50 ml polypropylene tube and nitrogen allowed to evaporate completely without thawing the sample.
  • RNA was precipitated with 1 vol isopropanol at -20 °C for at least one hour. After centrifugation at 10000g for 30 min at 4 °C the RNA pellet was redissolved in 5 ml solution D and precipitated again with 1 vol isopropanol as described. The pellet was washed with cold 75% EtOH and dried at RT for 15 min. To completely dissolve RNA 500 ⁇ l DEPC- treated water were added and the sample was incubated at 60 °C for 10 min, final storage was at -80 °C. An aliquot was used for quantification by A260 measurement and separation on a formaldehyde agarose gel (Sambrook et al.) to check integrity and size distribution. TABLE 1: Human heart samples
  • Poly(A) RNA was isolated from 300 ⁇ g total RNA (1. supra) using the PolyA Quick mRNA Isolation Kit (Stratagene) according to the manufacturers protocol. Purified mRNA was dissolved in 30 ⁇ l RNase-free water (Stratagene), quantified and analyzed on a formaldehyde agarose gel as described (1. supra).
  • SSH Suppression subtractive hybridization
  • 2 ⁇ g of tester mRNA and 2 ⁇ g of driver mRNA were used to construct a subtracted and normalized cDNA library using the PCR-Select cDNA Subtraction Kit and Advantage cDNA-Polymerase Mix (Clontech) according to the manufacturers protocol.
  • two libraries were constructed for each tester and driver combination, since only transcripts can be identified that are over-represented in the tester mRNA.
  • Both, the subtracted and non-subtracted cDNA population were analyzed on an agarose gel as described (Clontech) and transferred onto Zeta-Probe GT nylon membrane (BioRad) by capillary forces (Sambrook et al., supra).
  • the membrane was UV crosslinked in a Stratalinker 2400 (Stratagene).
  • the membrane was hybridized with a Digoxigenin-labeled probe synthesized from a housekeeping gene using the Dig- DNA Labeling and Detection Kit (Roche).
  • a 451 bp fragment of human GAPDH was amplified from 0.5-1 ⁇ g cDNA of a NF heart library (5.1.) in a 100 ⁇ l PCR reaction with the primer pair provided by the PCR-Select cDNA Subtraction Kit (Clontech). 100 ng of gel purified (QIAquick Gel Extraction Kit, Qiagen) GAPDH cDNA fragment then were used for Dig-labeling.
  • the hybridized membrane was exposed to a X-ray film (X OMAT AR, Kodak) for 15 min. Only subtractions, where the GAPDH signal intensity of the subtracted cDNA population was at least four fold lowered compared to the corresponding non-subtracted cDNA-population, were selected for further analysis. 17 ⁇ l of the subtracted sample were purified using a PCR Purification Kit (Qiagen) and eluted in 20 ⁇ l ddH 2 ⁇ (Gibco BRL).
  • Subtracted cDNA clones were grown over night at 37 °C in 96 well microplates filled with 100 ⁇ l LB medium (Sambrook et al. , supra.) and supplemented with 10 ⁇ g/ml Amp. 1 ⁇ l of the bacterial culture then was transferred into 99 ⁇ l PCR premix (1x PCR buffer, 2.5 U Taq DNA polymerase (APB), 0.2 mM dNTP) and directly amplified using the nested primer pair 1 and 2R provided by the PCR-Select cDNA Subtraction Kit (Clontech). Best results were obtained with 27 cycles and an annealing and polymerization temperature of 68 °C. The size distribution of PCR- products was analyzed on an 1 % agarose gel (Sambrook et al., supra). Bacterial cultures were mixed with glycerol to a final concentration of 20% and stored at -80 °C.
  • Total RNA (1.) was digested using the MessageClean-Kit (GeneHunter) according to the manufacturers protocol.
  • RNA-free RNA 200 ng was denatured for 5 min at 70 °C in the presence of 0.2 ⁇ M anchor primer [T7]-T ⁇ 2 MX and 20 U rRNasin (Promega). After addition of RT buffer (Gibco), 10 mM DTT, 25 ⁇ M dNTP and 200 U Superscriptll RTasell (Gibco) on ice, the reaction with a final volume of 20 ⁇ l was performed for 5 min at 42 °C and 1 h at 50 °C. The reaction was stopped by heating 15 min at 70 °C.
  • Resulting cDNAs (4.2.) were reamplified in the presence of the same anchor primer labeled with Cy5 and a second primer with 10 nt of arbitrary chosen sequence.
  • a 16 nt segment of the M13 universal reverse (-48) 24mer priming sequence (ACAATTTCACACAGCA) was incorporated in the arbitrary primer [M13r]-ARPX ⁇ o for direct sequencing.
  • the PCR sample (20 ⁇ l, 4.3.) was mixed with 6 ⁇ l gel loading dye (95% formamide, 20 mM EDTA, 0.005% BPB), denatured for 2 min at 80 °C and separated on a standard sequencing gel (6% polyacrylamide/8.3 M urea) at 55 W for 3 h.
  • the gel was dried on Whatman 3MM paper and fluorescence signals read at 635 nm on a Storm fluorimager (Molecular Dynamics). Data analysis was performed using ImageQuant Software (Molecular Dynamics) as described below (7.3).
  • a 40 ⁇ l PCR was set up on ice with 3 ⁇ l template (4.5.), 1x PCR buffer, 1.5 mM MgCI 2 , 20 ⁇ M dNTP, 0.2 ⁇ M T7 primer, 0.2 ⁇ M M13r(-48) primer and 2 U Taq polymerase (Qiagen) and run as described above (4.3.).
  • RNA 50 ⁇ g total RNA was purified using RNeasy Mini columns (Qiagen) as described by the manufacturer. 5 ⁇ g of purified total RNA was used for synthesis of first and second strand cDNA and double stranded cDNA then used to synthesize the biotinylated cRNA probe as recommended by Affymetrix. 15 /g of fragmented labeled cRNA was hybridized to the human genome U95A array and stained by streptavidin-phycoerythrin (SAPE). Signal intensities were amplified using a biotinylated anti-streptavidin antibody and a second SAPE staining step. Data were analyzed by means of Microarray Suite 4.0 (Affymetrix) and Data Mining Tool software provided by Affymetrix. Fold changes in gene expression were analyzed comparing average intensity values of DCM and ICM heart samples with that of normal controls, respectively.
  • SAPE streptavidin-phycoerythrin
  • 200 ⁇ g plasmid DNA were digested with Xhol over night at 37 °C in a volume of 250 ⁇ l to linearize the plasmid at the 3' end of the insert.
  • the sample was controlled for complete digestion on an agarose gel, treated with 10 ⁇ g/ ⁇ l Proteinase K (Roche) at 37 °C for 30 min, extracted once with TE saturated phenol (pH 7.5-8) and once with chloroform/isoamylalcohol (24/1) and precipitated in the presence of 0.1 vol 3 M NaOAc (pH 5.2) and 3 volume EtOH.
  • the pellet was washed with 500 ⁇ l 75% ethanol, dried at RT for 10 min, dissolved in 150 ⁇ l DEPC- treated water and quantified.
  • RNA 1 ⁇ g of linearized plasmid was used for an in vitro transcription as described (6.3), omitting the radioactive labeled nucleotide and adding UTP to a final concentration of 10 mM.
  • the RNA was extracted with phenol/chloroform/isoamylalcohol (24/23/1), precipitated with EtOH and dissolved in 15 ⁇ l DEPC-treated water. The yield was in the range of 15-22 ⁇ g RNA.
  • 1.5 ⁇ l RNA were separated on a formaldehde agarose gel. A smear of transcripts was visible between 0.5 kb and 10 kb with a peak at about 1 kb.
  • RNA Transcription Kit (Stratagene) 1 ⁇ g of linearized template (6.2) was incubated in the presence of 1x transcription buffer, 10 M ATP, 10 mM CTP, 10 mM GTP, 1 mM UTP, 70 ⁇ Ci [ - 33 P]UTP (APB), 0.75 M DTT, 20 U rRNasin. (Promega) and 25 U T3 RNA polymerase for 30 min at 37 °C. After addition of 5 U RNase-free DNasel (Roche) the sample was incubated for 15 min at 37 °C. 25 ⁇ l STE-buffer (APB) was added to the probe and the reaction purified using G50 Micro Columns (APB) according to the manufacturers protocol.
  • 1x transcription buffer 10 M ATP, 10 mM CTP, 10 mM GTP, 1 mM UTP, 70 ⁇ Ci [ - 33 P]UTP (APB), 0.75 M DTT, 20 U rRNasin. (Pro
  • RNA was prehybridized to cot1-DNA. 213 ⁇ l DEPC-treated water, 100 ⁇ l 20x SSC, 2 ⁇ l 20%
  • PCR product 3.2.
  • gene-specific control cDNA fragments were mixed with 140 ⁇ l 0.4 M NaOH/10 mM EDTA pH 8.0 in 96 well microplates and denatured 10 min at 95 °C.
  • 50 ⁇ l of each PCR-fragment (at least 100 ng cDNA) were transferred on a nylon membrane (11.4x7.5 cm, BioRad) using a 384 hole vacuum apparatus (Keutz, custom-made). 50 ⁇ l 0.4 M NaOH were added to each position and transferred.
  • the membrane was washed in 2x SSC, dried for at least 1 h at RT and fixed by UV crosslinking (Strataiinker 2400, Stratagene). For each experiment two identical membranes were prepared in parallel.
  • the cDNA filter was soaked in 2x SSC and transferred into a hybridization flask.
  • the membrane was hybridized with 10 ml hybridization solution (6x SSC, 5x Denhardts, 0.2 % SDS, 0.2 % sodium pyrophosphate) supplemented with 50 ⁇ g/ml denatured salmon sperm DNA (Typ III, Sigma) at 65 °C for 2 h in an Unitherm 6/12 hybridization oven (UniEquip).
  • the prehybridization mix was poured off. 200-400 ⁇ l of cot 1 -hybridized probe (6.4) were added to 8 ml of hybridization solution (including salmon sperm DNA) preheated to 65 °C. Dot blots were hybridized over night at 65 °C. For washing of cDNA filters all solutions were heated to 65 °C. The membrane was washed twice with 50 ml wash solution 1 (2x SSC, 0.1 % SDS) for 30 min, then twice with 50 ml wash solution 2 (0.1x SSC, 0.1 % SDS) for 30 mjn and wrapped in a keep-fresh foil. The filter was exposed to a phosphor screen for two days and scanned at 450 nm using the Storm Phosphoimager (Molecular Dynamics).
  • DCM patients were treated by a number of drugs prior to heart transplantation. It was laid down that the regulation has to be consistent in at least two DCM patients and more or less homogenous in all but one non-failing patient.
  • Plasmids were isolated using the Plasmid Mini Kit (Qiagen) and sequenced.
  • PCR-fragments were then purified by agarose gel-electrophoresis followed by gel elution using the gel purification kit from Qiagen. PCR-fragments were finally cloned into P201-DONOR (Life Technologies) or pTOP02.1 (Invitrogen). The cloned cDNAs were verified by sequencing. In addition, in vitro translations were performed using the TNT Quick Coupled Transcription/Translation Systems (Promega) in order to verify the correct molecular weight of the proteins encoded by a given cDNA.
  • EST 40095 (FIG. 1A) was identified by suppression subtractive hybridization comparing transcript levels of heart tissue explanted from normal control h92 with one from DCM patient h97 (see TABLE 1). The fragment was found to be over- represented in control tissue.
  • the identified cDNA fragment is a part of the EST clone AF047439 (FIG. 1 B), which encodes the amino acid sequence AAC39890 (FIG. 1C).
  • the EST clone 40095 is identical to the coding region of a human mRNA of unknown function, which was identified to be expressed in CD34(+) hematopoietic stem and progenitor cells (HSPC001 , Mao et al., 1998). The respective gene is coded on chromosome 1 map P32.
  • Transcript levels are significantly downregulated by a factor 2.7 in four DCM patients compared to five NF controls (FIG. 1 E).
  • the probability of type 1 error is less than 5% as determined in a t-test and Wilcoxon test.
  • Significant downregulation of 40095 expression in heart tissue of four DCM patients compared to five normal controls indicates that a decreased expression of 40095 is associated with dilated cardiomyopathy. Therefore, the protein is expected to play a causative role in congestive heart failure.
  • the function of the protein is not known so far but since the sequence is highly conserved from mouse to human the protein seems to play an important role.
  • EST 41237 (FIG. 2 A) was identified by suppression subtractive hybridization comparing transcript levels of heart tissue explanted from normal control h92 with one from DCM patient h97 (see TABLE 1). The fragment was found to be over- represented in the control tissue.
  • the identified cDNA fragment was found to be a part of the 41237contig ( FIG. 2 B, assembled of AF191652 and AF191653), which encodes the amino acid sequence AAF68859 given in FIG. 2 C (schematic alignment see FIG. 2 D).
  • 41237 is identical to a part of the 3' untranslated region of human diphosphoinositol polyhosphate phosphohydrolase type 2 (NUDT4) of both, alpha (hDIPP2alpha) and beta (hDIPP2beta) isoform distinguished from each other solely by hDIPP2beta possessing one additional amino acid (Caffrey et al., 2000).
  • NUDT4 belongs to the MutT motif protein (or nudix hydrolase) family. Downregulation upon DCM was confirmed by quantitative dot blot analysis. The relative expression level of 41237 is reduced by a factor of 2.2 upon disease (FIG. 2E). The probability of type 1 error is less than 5% as determined in a t-test and Wilcoxon test.
  • Diphosphoinositol polyhosphate phosphohydrolase removes the beta- phosphate from all known diphosphoinositol polyphosphates.
  • Diphosphoinositol polyphosphates are members of the inositol-based cell signaling family and comprise a group of highly phosphorylated compounds which have a rapid rate of metabolic turnover through tightly-regulated kinase/phosphohydrolase substrate cycles.
  • Cellular levels are regulated by cAMP and cGMP in a protein kinase- independent manner. These inositides can also sense a specific mode of intracellular Ca 2+ pool depletion.
  • the phosphohydrolases occur as multiple isoforms, the expression of which is apparently carefully controlled (Safrany et al., 1999).
  • the enzymes are believed to eliminate toxic nucleotide derivatives from the cell and regulate the levels of important signalling nucleotides and their metabolites. Therefore, 41237 can serve as a CHF marker and a specific molecular target for drug development.
  • EST 41444 (FIG. 3 A) was identified by suppression subtractive hybridization comparing transcript levels of heart tissue explanted from normal control h92 with one from DCM patient h97 (see TABLE 1). The fragment was found to be over- represented in the control tissue.
  • the identified cDNA fragment was found to be a part of the EST clone L07956 (FIG. 3 B), which encodes the amino acid sequence AAA58642 (FIG. 3 C; schematic alignment see FIG. 3 D).
  • This amino acid sequence codes for the human 1,4-alpha glucan branching enzyme GBE (Thon et al., 1993).
  • GBE 1,4-alpha glucan branching enzyme
  • the two major glycogen storage sites are muscle and liver, which provide an energy reservoir for strenous muscular activity. Branching increases the rate of glycogen synthesis and degradation through a higher number of terminal residues. Downregulation upon DCM was confirmed by quantitative dot blot analysis.
  • the relative expression level of 41444 is reduced by a factor of 5 (FIG. 3 E).
  • the probability of type 1 error is less than 1% as determined in a t-test and Wilcoxon test.
  • Type IV glycogenosis (polyglucosan body disease) is a rare congenital autosomal recessive inherited disorder, caused by lack of the branching enzyme (amylo-1 ,4- 1 ,6 transglucosidase). This deficiency leads to storage of abnormal glycogen (polyglucosan bodies) in the liver and other tissues.
  • the clinical onset of the diseases is insidious with non-specific gastrointestinal symptoms followed by progressive hepatic failure. Usually patients die due to hepatic cirrhosis within 4 years. Sometimes myopathy of the heart and skeletal muscle is also present. This corresponds to our results and supports the finding that a decreased expression of 41444 is associated with cardiomyopathies (Nase et. al. 1995). Comparing expression levels of seven tissues the transcript was identified to be expressed at higher levels in the heart (Fig. 3F). Due to its expression predominantly in the heart, GBE can serve as a specific molecular target for drug development.
  • EST 41482 (FIG. 4 A) was identified by suppression subtractive hybridization comparing transcript levels of heart tissue explanted from normal control h92 with one from DCM patient h97 (see TABLE 1). The fragment was found to be over- represented in the control tissue.
  • DERP2 human dermal papilla derived gene
  • cDNA sequence AB009685, FIG. 4 B and included in FIG. 4 F codes for the amino acid sequence BAA93049 (FIG. 4 C).
  • Amino acid 1-298 of DERP2 are identical to human PTD010 (cDNA sequence AF078863; amino acid sequence AAD44495), an EST sequence isolated from pituitary tumor (Song et al., 1999).
  • the last 47 amino acids of DERP2 as well as the 3' end of 41482 clearly differ from PTD010.
  • the function of DERP2 is still unknown.
  • a schematic alignment of the different sequences is given in figure 4 D.
  • the protein is located in the plasma membrane and consists of five membrane-spanning regions (129-145, 161-177, 200-216, 248-264, 275-291).
  • the N-terminus is predicted to be extracellular and the C-terminal side to be cytoplasmic.
  • Two ER membrane retention signals were identified, a XXRR-like motif in the N-terminus (LAAR) and a KKXX-like motif in the C-terminus (GNRK).
  • LAAR XXRR-like motif in the N-terminus
  • GNRK KKXX-like motif in the C-terminus
  • Significant downregulation of 41482 expression in heart tissue of five DCM patients compared to four normal controls indicates that a decreased expression of 41482 is associated with dilated cardiomyopathy. Therefore, the protein is expected to play a causative role in congestive heart failure.
  • the predicted plasma membrane location indicates a role of 41482 in transport processes or signal transduction. This finding supports the idea that 41482 can be used
  • EST 52529 (FIG. 5 A) was identified by suppression subtractive hybridization comparing transcript levels of heart tissue explanted from normal control KN2 with one from DCM patient DHZM3 (see TABLE 1). The fragment was found to be over- represented in the control tissue.
  • the identified cDNA fragment was found to be a part of the coding region and 3' non-coding region of the EST clone NM_006845 (FIG. 5 B), which codes for the amino acid sequence NP_006845 given in FIG. 5 C (schematic alignment see FIG. 5 D), which encodes the human KDEL (Lys-Asp-Glu-Leu) endoplasmatic reticulum protein retention receptor 2 (KDELR2). Retention of luminal endoplasmic reticulum (ER) proteins is mediated via the conserved carboxy-terminal tetrapeptide KDEL that serves as a signal for their retrieval from subsequent compartments of the secretory pathway.
  • KDEL Lys-Asp-Glu-Leu
  • KDELR2 endoplasmatic reticulum protein retention receptor 2
  • KDELR2 also referred to as ELP-1
  • ELP-1 is a novel human homolog of ERD-2 retrieval receptor (Lewis and Pelham, 1992). Both receptors are proposed to return escaped ER resident proteins from the Golgi. Thus, these receptors may provide signals that regulate retrograde traffic between the Golgi and the ER (Hsu et al., 1992). Downregulation upon DCM was confirmed by quantitative dot blot analysis. The relative expression level of 52529 is reduced by a factor of 7.7 upon disease (FIG. 5 E). The probability of type 1 error is less than 5% as determined in a t-test and Wilcoxon test.
  • 52529 is a novel retrieval receptor proposed to return escaped ER resident proteins from the Golgi and regulate retrograde traffic between the Golgi and the ER. Significant downregulation of 52529 may interfere with the retrieval of KDEL proteins from later stages of the secretory pathway. Thus, minimizing fruitless recycling of secretory proteins and interfering with retrograde transport processes 52529 is expected to play a causative role in congestive heart failure. Upregulation of protein expression by gene therapeutic intervention, compensatory molecules or specific activators seems to be a very promising therapeutic tool to treat heart diseases.
  • EST 55253 (FIG. 6 A) was identified by suppression subtractive hybridization comparing transcript levels of heart tissue explanted from normal control KN5 with one from DCM patient 52 (see TABLE 1). The fragment was found to be over- represented in the control tissue.
  • EST 61024 (FIG. 7 A) was identified by suppression subtractive hybridization comparing transcript levels of heart tissue explanted from normal control KN4 with one from DCM patient h94 (see TABLE 1). The fragment was found to be over- represented in the control tissue. 61024 is identical to a new cDNA clone (AB028963, see FIG. 7 B) of unknown function (KIAA1040, Kikuno et al., 1999), which could be elongated by PCR at the
  • FIG. 7 C This EST clone was identified to code for a large protein in the brain with the predicted amino acid sequence 61024pep (FIG. 7 D; schematic alignment see FIG. 7 E). The respective gene is coded on chromosome
  • the probability of type 1 error is less than 5% as determined in a t-test and
  • EST 61119 (FIG. 8 A) was identified by suppression subtractive hybridization comparing transcript levels of heart tissue explanted from normal control KN4 with one from DCM patient 94 (see TABLE 1). The fragment was found to be over- represented in the control tissue.
  • 61119 is identical to a part of the EST clone AF115509 (FIG. 8 B), the coding region of leucine rich repeat of fligthless I (LRR FLI-I) interacting protein 2 (LRRFIP2) encoded by the amino acid sequence AAA16607 (FIG. 8 C, schematic alignment see FIG. 8 D).
  • LRR FLI-I leucine rich repeat of fligthless I
  • LRRFIP2 is a novel gene that shares sequence homology with LRRFIP1 and FLAP-1.
  • LRRFIP1 and LRRFIP2 both express alternative splice variants in heart and skeletal muscle tissue. Both genes are related and seem to arose from gene duplication. Downregulation upon DCM was confirmed by quantitative dot blot analysis. The relative expression level of 61119 is reduced by a factor of 2 upon disease (FIG. 8 E). The probability of type 1 error is less than 5% as determined in a t-test and Wilcoxon test.
  • Flightless-I is a novel member of the gelsolin family that is important for actin organization during Drosophila embryogenesis and myogenesis (Liu and Yin, 1998).
  • Drosophila flil and the human homolog FLI both contain the classic gelsolin 6-fold segmental repeats and an amino-terminal extension of 16 tandem leucine- rich repeats (LRR).
  • LRR 16 tandem leucine- rich repeats
  • FLI is involved in structural reorganization in heart and skeletal muscle and LRRFIP2 directly interacts with FLI LRRFIP2 is expected to play a causative role in congestive heart failure.
  • the LRR domain is highly homologous to those of three proteins involved in Ras-mediated signaling: S. cerevisiae adenylyl cyclase, C. elegans SUR-8, and mammalian RSP-1 (Goshima et al., 1999).
  • FLI-1 may be involved in regulation of the actin cytoskeleton through Ras. This supports the idea that 61119 plays a central role in muscle degeneration throughout disease and therefore can be used as a specific molecular target for drug development. Upregulation of protein expression by gene therapeutic intervention, compensatory molecules or specific activators seems to be a very promising therapeutic tool to treat heart diseases.
  • EST 62139 (FIG. 9 A) was identified by suppression subtractive hybridization comparing transcript levels of heart tissue explanted from normal control KN4 with one from DCM patient h94 (see TABLE 1). The fragment was found to be over- represented in the control tissue.
  • 62139 was found to be a part of 62139contig (FIG. 9 B), assembled of X97324 (and further fragments), which codes for the amino acid sequence CAA65989 (FIG. 9 C; schematic alignment see FIG. 9 D).
  • This amino acid sequence encodes the human adipophilin (ADFP), a specific marker for adipocyte cell differentiation and lipid accumulation in a variety of cells.
  • ADFP human adipophilin
  • Most mammalian cells package neutral lipids into droplets that are surrounded by a monolayer of phospholipids and a specific set of proteins including adipophilin, which is found in a wide array of cell types.
  • the 50-kD membrane-associated protein is associated with the surface of lipid droplets and was revealed as a possible new marker for the identification of specialized differentiated cells containing lipid droplets and for diseases associated with fat-accumulating cells (Heid et al., 1998).
  • Transcript levels are significantly downregulated by a factor 2.1 in five DCM patients compared to five normal controls (FIG. 9 E).
  • the probability of type 1 error is less than 5% as determined in a t-test and Wilcoxon test.
  • a downregulation of 62139 upon DCM has also been found using the Affymetrix array technology.
  • Transcript levels ' are significantly downregulated by a factor 1.91 in 11 DCM patients compared to ten normal controls (FIG. 9 F).
  • the probability of type 1 error for DCM is 5.8% and 3.5% as determined in a t-test and Mann- Whitney test.
  • transcript levels are downregulated by a factor of 1.55 in 9 ICM patients compared to ten normal controls (FIG. 9 F).
  • the probability of type 1 is 13.2% and 68.7% as determined in a t-test and Mann-Whitney test, respectively.
  • the comparison of the mean values of DCM patients, ICM patients and normal controls is shown in FIG. 9 G.
  • ADFP is a specific marker of lipid accumulation in diverse cell types and diseases.
  • the diseased heart uses fatty acids as main energy source instead of glucose (Paolisso et al., 1994, Lommi et al., 1998).
  • a lowered expression of 62139 by a factor of 2 upon DCM therefore may directly correspond to a decreased amount of lipids stored in lipid droplets. Therefore 62139 can be used as a specific marker for CHF and may also be a molecular target for drug development. Upregulation of protein expression by gene therapeutic intervention, compensatory molecules or specific activators seems to be a very promising therapeutic tool to treat heart diseases.
  • EST 62140 (FIG. 10 A) was identified by suppression subtractive hybridization comparing transcript levels of heart tissue explanted from normal control KN4 with one from DCM patient h94 (see TABLE 1). The fragment was found to be over- represented in the control tissue.
  • the identified cDNA fragment was found to be a part of the 62140contig (FIG. 10 B), which was assembled of AW952950 and AI653533 beside/and other ESTs (schematic alignment see FIG. 10 C).
  • 62140contig sequence 1373 nt as well as for the homologous EST sequences annotated in Genbank no clear open reading frame could be identified.
  • Downregulation upon DCM was confirmed by quantitative dot blot analysis.
  • the relative expression level of 62140 is reduced by a factor of 2.1 upon disease (FIG. 10 D).
  • the probability of type 1 error is less than 5% as determined in a t-test and Wilcoxon test.
  • H326 is a human gene homologous to murine PC326 that is ubiquitously expressed, and has a murine homologue that is also ubiquitously expressed (Unpublished).

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Abstract

L'invention concerne une variété de gènes présentant une expression anormale dans le tissu cardiaque, ainsi que des fragments de ces gènes. On peut utiliser l'évaluation du niveau d'expression de ces gènes afin de vérifier la prédisposition de mammifères et, de préférence, d'humains à une maladie cardiaque ou à un état aigu de ce type de maladie. Les maladies indiquées à titre préférentiel consistent en l'insuffisance cardiaque globale, la cardiomyopathie avec dilatation, la cardiomyopathie hypertrophique et la cardiomyopathie ischémique. L'invention concerne, de plus, des procédés servant à identifier des composés pouvant normaliser le niveau d'expression de ces gènes ou d'autres gènes affectés par cette expression anormale. On peut utiliser les composés identifiés afin de formuler des compositions, de préférence, des compositions pharmaceutiques permettant de traiter ces maladies à titre prophylactique ou thérapeutique. On peut également les utiliser en tant que composés précurseurs pour le développement de médicaments présentant une efficacité améliorée, une demi-vie prolongée, une toxicité limitée et les mettre en application pour traiter des maladies cardiaques. L'invention concerne également des procédés de thérapie génique somatique comparant l'introduction d'au moins une copie fonctionnelle d'un gène parmi les gènes mentionnés dans une cellule appropriée. Elle concerne enfin des animaux transgéniques non humains comprenant au moins un de ces gènes dans leur lignée germinale. On peut utiliser ces animaux transgéniques afin de développer des médicaments servant à traiter des maladies cardiaques.
PCT/EP2002/007704 2001-07-10 2002-07-10 Nouveaux genes cibles concernant des maladies cardiaques WO2003006687A2 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008053358A3 (fr) * 2006-07-25 2008-11-20 Deutsches Krebsforsch Signature d'une expression de gène commune dans une cardiomyopathie dilatée
US7608458B2 (en) 2004-02-05 2009-10-27 Medtronic, Inc. Identifying patients at risk for life threatening arrhythmias
US7622303B2 (en) 2004-02-05 2009-11-24 Medtronic, Inc. Methods for identifying patients at risk for life threatening arrhythmias
US8027791B2 (en) 2004-06-23 2011-09-27 Medtronic, Inc. Self-improving classification system
US8335652B2 (en) 2004-06-23 2012-12-18 Yougene Corp. Self-improving identification method

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000053753A2 (fr) * 1999-03-08 2000-09-14 Genentech, Inc. Activation ou inhibition de l'angiogenese et de la cardiovascularisation
US6610480B1 (en) * 1997-11-10 2003-08-26 Genentech, Inc. Treatment and diagnosis of cardiac hypertrophy
WO1999036522A1 (fr) * 1998-01-19 1999-07-22 Shanghai Second Medical University Gene a faible similitude avec la tropomyosine (cbcadb07)
AU4167499A (en) * 1998-06-26 2000-01-17 Protegene Inc. Human proteins having hydrophobic domains and dnas encoding these proteins
WO2000011942A1 (fr) * 1998-09-01 2000-03-09 Gene Logic, Inc. Identification d'un adn complementaire associe a l'ischemie dans le tissu cardiaque humain
ATE458050T1 (de) * 1998-12-01 2010-03-15 Genentech Inc Promotion oder inhibition von angiogenese und kardiovaskularisation

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7608458B2 (en) 2004-02-05 2009-10-27 Medtronic, Inc. Identifying patients at risk for life threatening arrhythmias
US7622303B2 (en) 2004-02-05 2009-11-24 Medtronic, Inc. Methods for identifying patients at risk for life threatening arrhythmias
US8027791B2 (en) 2004-06-23 2011-09-27 Medtronic, Inc. Self-improving classification system
US8335652B2 (en) 2004-06-23 2012-12-18 Yougene Corp. Self-improving identification method
WO2008053358A3 (fr) * 2006-07-25 2008-11-20 Deutsches Krebsforsch Signature d'une expression de gène commune dans une cardiomyopathie dilatée

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