WO2009036976A1 - Utilisation du gène de photomédine-2/analogue à l'olfactomédine 2b (olfml2b), de ses variants et de sa protéine dans des approches diagnostiques et thérapeutiques des maladies cardiaques - Google Patents

Utilisation du gène de photomédine-2/analogue à l'olfactomédine 2b (olfml2b), de ses variants et de sa protéine dans des approches diagnostiques et thérapeutiques des maladies cardiaques Download PDF

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WO2009036976A1
WO2009036976A1 PCT/EP2008/007821 EP2008007821W WO2009036976A1 WO 2009036976 A1 WO2009036976 A1 WO 2009036976A1 EP 2008007821 W EP2008007821 W EP 2008007821W WO 2009036976 A1 WO2009036976 A1 WO 2009036976A1
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olfml2b
protein
gene
wild type
nucleic acid
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PCT/EP2008/007821
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Arne Pfeufer
Thomas Meitinger
Stefan KÄÄB
Mahmut Akyol
Moritz Sinner
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Helmholtz Zentrum München-Deutsches Forschungszentrum Für Gesundheit Und Umwelt (Gmbh)
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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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • C12Q2600/156—Polymorphic or mutational markers
    • 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 relates to a method for determining a predisposition for or an acute or chronic heart disease in a subject, comprising assaying a sample from the subject for a genetic variant of the OLFML2B gene or mRNA transcribed therefrom or a variant of the OLFML2B protein or for an alteration in the expression level of the OLFML2B gene, wherein the presence of said genetic variant, said variant of the protein or said alteration in the expression level is indicative of a predisposition for or an acute or chronic heart disease.
  • the invention relates to a nucleic acid molecule representative of the variant of the invention.
  • the invention also relates to a vector comprising the nucleic acid molecule of the present invention and a method for the production of a polypeptide encoded by the nucleic acid molecule or by the vector of the invention and culturing a host under conditions allowing the expression of the polypeptide and recovering the polypeptide of the present invention.
  • the invention discloses a polypeptide encoded by the nucleic acid molecule or produced by the method of the present invention and an antibody specifically recognizing said polypeptide.
  • the invention also envisages the use of OLFML2B protein or a functional fragment thereof in the manufacture of a pharmaceutical composition for the treatment of a heart disease, wherein the OLFML2B protein is the wild type protein, and moreover, gene therapies on the basis of nucleic acid molecules encoding the wild type protein.
  • the heart is a hollow, muscular organ responsible for pumping blood through the blood vessels by repeated, rhythmic contractions.
  • the cardiac muscle is myogenic, i.e. able to contract and relax on its own being modulated by a specific regulatory system containing the sinoatrial and atrioventricular nodes. In the light of its existential role in the maintenance of blood circulation the deleterious effect of a malfunctioning heart on the body can easily be envisioned.
  • a number of heart diseases are characterized by disturbances of repolarization leading to a prolongation or shortening of the QT-interval in the electrocardiogram (ECG) and are known to be associated with an increased risk of sudden cardiac death (SCD).
  • SCD is estimated to account for approximately 50 percent of all deaths from cardiovascular causes and believed to be triggered by changes in the frequency and efficiency of rhythmic contractions.
  • the heart diseases being caused by disturbances in cardiac repolarization are sudden cardiac death, ventricular tachyarrhythmia, bradyarrhythmia, long-QT Syndrome, Brugada Syndrome, dilative cardiomyopathy, or using "umbrella terms" congestive heart-failure, cardiac hypertrophy and/or arrhythmias.
  • DCM dilated cardiomyopathy
  • ARVC arrhythmogenic right ventricular cardiomyopathy
  • HCM hypertrophic cardiomyopathy
  • LQT long-QT syndrome
  • diLQT drug-induced long-QT syndrome
  • the technical problem underlying the present invention was to identify appropriate means that allow a prognosis of acute or chronic heart diseases or a predisposition therefore.
  • the present invention relates to a method for determining a predisposition for or an acute or chronic heart disease in a subject, comprising assaying a sample from the subject for a genetic variant of the OLFML2B gene or mRNA transcribed therefrom or a variant of the OLFM L2B protein or for an alteration in the expression level of the OLFML2B gene, wherein the presence of said genetic variant (i.e. of the gene or mRNA), said variant of the protein or said alteration in the expression level is indicative of a predisposition for or an acute or chronic heart disease.
  • composition for or an acute or chronic heart disease in a subject relates to a susceptibility, preferably to a genetic susceptibility to develop a heart disease.
  • heart disease encompasses both acute and chronic heart diseases.
  • Acute or chronic heart diseases include but are not limited to: congestive heart failure, dilative cardiomyopathy, left ventricular hypertrophy, septal hypertrophy, hypertrophic cardiomyopathy, arrhythmogenic right ventricular dysplasia, hypertensive heart disease, heart disease of the elderly, ischemic heart disease, myocardial infarction, cardiac fibrosis, restrictive cardiomyopathy, left ventricular noncompaction, Barth Syndrome, long-QT syndrome, Brugada syndrome, Andersen syndrome , ventricular tachycardia, ventricular fibrillation and sudden cardiac death.
  • sample in accordance with the invention relates to a biological sample, such as, for example, cells, tissues, or fluids (including serum, whole blood, cerebrospinal fluid, lymph, saliva, milk, pus) to be isolated from an individual or from cell culture constituents.
  • a biological sample such as, for example, cells, tissues, or fluids (including serum, whole blood, cerebrospinal fluid, lymph, saliva, milk, pus) to be isolated from an individual or from cell culture constituents.
  • tissue or liquid sample obtained from a patient and/or (human) subject can be used for the detection of a genetic variant of the OLFML2B gene or mRNA transcribed therefrom or a variant of the OLFML2B protein or to detect an alteration in the expression level of the OLFML2B gene.
  • the passage "genetic variant of the OLFML2B gene or mRNA transcribed therefrom or a variant of the OLFML2B protein" as used herein means that said OLFML2B gene differs from the wild type OLFML2B gene by way of nucleotide substitution(s), addition(s) and/or deletion(s) resulting in a different mRNA transcribed therefrom. Said nucleotide substitution(s), addition(s) and/or deletion(s) may result in an altered expression of the OLFML2B gene, and/or preferably produce altered OLFML2B protein compared to the corresponding wild type OLFML2B protein and are indicative of a predisposition for or an acute or chronic heart disease.
  • said genetic variants may additionally contain mutations not predisposing subjects to heart diseases or being implicated in the onset of or acute or chronic heart diseases.
  • the wild type OLFML2B gene is represented by SEQ ID NO.: 1 as well as various allelic variants thereof, which are not indicative of a predisposition for or indicative of an acute or chronic heart disease.
  • the wild type OLFML2B mRNA or the wild type OLFML2B protein, as used infra and supra, is represented by SEQ ID NO.:2 or by SEQ ID NO.: 4, respectively, as well as various allelic variants thereof, which are not indicative of a predisposition for or indicative of an acute or chronic heart disease.
  • wild type OLFML2B mRNA transcribed from said wild type OLFML2B gene may contain nucleotide substitution(s), addition(s) and/or deletion(s) which are not indicative of a predisposition for or an acute or chronic heart disease.
  • the genetic variants of the OLFML2B mRNA contain mutations which are indicative of a predisposition for or an acute or chronic heart disease and may further contain mutations not predisposing subjects to heart diseases or being implicated in the onset of or acute or chronic heart diseases.
  • Indicative and not indicative mutations towards a predisposition can be distinguished by studying large numbers of affected subjects and investigating the association between mutation and the disease. In addition functional studies of the mutant protein can help to resolve the question of indicative and not indicative mutations.
  • variant of the OLFML2B protein as used in accordance with the present invention relates to OLFML2B proteins, which differ from the wild type OLFML2B protein wherein said differences relative to the wild type sequence are indicative of a predisposition for or an acute or chronic heart disease. Further, the variant may contain other differences which are not indicative of a predisposition for or an acute or chronic heart disease.
  • the wild type OLFML2B protein may contain mutations not indicative of a predisposition for or an acute or chronic heart disease.
  • Alterations in the expression levels of the OLFML2B gene can be measured by any method known that can provide quantitative information regarding the levels to be measured.
  • the methods preferably are highly sensitive and provide reproducible results and are well-known to the person skilled in the art and described for example in "Analysing Gene Expression, A Handbook of Methods: Possibilities and Pitfalls” by Stefan Lorkowski, Paul M. Cullen (eds.); Wiley-VCH, Weinheim. Examples of such methods are flow-cytometry, immunoassays (e.g., ELISAs), affinity mass spectrometry, DNA- and protein-microarrays. Further, methods based on the detection of mRNA level alteration, which are described infra, are encompassed.
  • the method as disclosed in the present invention is an important step in the improvement of clinical diagnosis and subsequent treatment of patients with heart diseases. Moreover, it also provides the possibility of prospective diagnosis of persons at risk such as, for example, persons having affected family members. Additionally, the method of the present invention provides the basis for successful treatment, prevention, and/or delay of heart diseases as will be discussed in further embodiments in detail infra.
  • the present inventors investigated the OLFML2B locus, gene, mRNA and protein for its implication in heart diseases, for example in acute or chronic congestive heart-failure, cardiac hypertrophy, cardiac death and/or arrhythmias, screening for mutations in 762 selected patients with genetically unresolved familial and sporadic cases of cardiac arrhythmias with or without cardiomyopathy.
  • VT idiopathic ventricular tachycardia
  • VF ventricular fibrillation
  • LQT long QT Syndrome
  • SCD sudden death syndrome
  • the OLFML2B gene products can be contributing to an acute or chronic heart disease either in their wildtype or in their mutant forms.
  • the involved mechanisms can include interference with RNA processing and splicing in case of the RNA and in case of the protein accumulation or depletion in the cytosol, ER, Golgi or extracellular compartment as well as overtly secretion or nonsecretion into the medium, gain or loss of the capability of interaction with other proteins, alteration in redox status, or interaction with small molecular weight compounds.
  • the present inventors sought to define the involvement of the OLFML2B gene in cardiac repolarization by gene knockdown and mutant expression in Zebrafish, Danio rerio, as outlined infra in Example 3.
  • the morpholino-mediated knockdown of the OLFML2B gene resulted in a phenotype characterized in that morphologic parameters (enlarged LVESD (left ventricular endsystolic diameter) and LVEDD (left ventricular enddiastolic diameter)) as well as physiologic (reduced FS) and electrophysiological parameters were highly abnormal.
  • morphologic parameters enlarged LVESD (left ventricular endsystolic diameter) and LVEDD (left ventricular enddiastolic diameter)
  • physiologic reduced FS
  • the OLFML2B gene is encoded by the nucleotide sequence of SEQ ID NO.: 1.
  • the OLFML2B gene is a gene located on chromosome 1 in the human genome flanked by the ATF6 and NOS1AP gene. It consists of a total of 40663 bp, which are partitioned into 8 exons and 7 introns. Further information is displayed in Figure 2 and 3.
  • MYOC myocilin
  • OLFML2B, as well as myocilin (MYOC) and other OLF-domain proteins are glycoproteins targeted for the extracellular space and getting secreted into the medium.
  • POAG autosomal dominant primary open angle glaucoma
  • the OLFML2B gene is also known for example under the following aliases: Photomedin-2, RP11-227F8. 1 or MGC51337; the protein under DKFZP586L151 or DKFZP686F1561.
  • SEQ ID NO.: 1 corresponds to the sequence of the OLFML2B gene as provided by the Human March 2006 (hg18; NCBI Build 36.1) assembly maintained at the University of California Santa Cruz Genome Bioinformatics Site (world wide web at http://genome.ucsc.edu/index.html) of chromosome 1 , covering the range from 160219606 to 160260268. Further sequence or other information on the gene OLFML2B, its mRNA, cDNA or protein can readily be retrieved from the databases maintained at the National Center for Biotechnology Information (NCBI ;world wide web at http://www.ncbi.nlm.nih.gov/sites/entrez) using the following GenelD: 25903.
  • NCBI National Center for Biotechnology Information
  • database IDs may be useful such as, for example, 24558 (database maintained at the HUGO Gene Nomenclature Committee (HGNC) site); Q9Y3X6, Q86X11 , Q68BL8, Q5VU96, Q6NX46 (database maintained at the Iniversal Protein Resource (UniProt) site accessible via the world wide web), or ENSG00000162745 (database maintained at the Ensembl site accessible via the world wide web), NP_056256, NMJD15441 (database maintained at the National Center for Biotechnology Information (NCBI) site accessible via the world wide web).
  • NCBI National Center for Biotechnology Information
  • the genetic variant comprises at least one of the nucleotides selected from the group consisting of a T at position 1156, a G at position 1166, a C at position 1186, a T at position 4437, a T at position 4534, an A at position 26294, an A at position 26312, a T at position 26353, a T at position 26487, a T at position 26621 , an A at position 39678, an A at position 40390, an A at position 40435 or a G at position 40694 of SEQ ID NO: 1.
  • the present invention features genetic variants, which represent mutations indicative of a predisposition for or indicative of an acute or chronic heart disease, wherein preferred mutations are set forth in Table 1. Furthermore, said genetic variants may additionally contain mutations not predisposing subjects to heart diseases or being implicated in the onset of or acute or chronic heart diseases.
  • Table 1 Disease-producing mutations in the OLFML2B gene.
  • the present inventors detected 14 different nonsynonymous mutations of the OLFML2B gene in a total of 23 patients as summarized above in Table 1. These patients had various heart conditions (LQT: long-QT Syndrome, diLQT: drug induced long-QT Syndrome, AF: atrial fibrillation, DCM: dilative cardiomyopathy, CHF: heart failure, HCM, hypertrophic cardiomyopathy, LVH: left ventricular hypertrophy, VT: ventricular tachycardia, Rea : Status post cardiopulmonary reanimation, SSS: Sick sinus syndrome, LBBB: Left bundle branch block). All mutation carriers detected were heterozygotes consistent with an autosomal dominant disease pattern.
  • the genetic variant of the mRNA comprises at least one of the nucleotides selected from the group consisting of a U at position 458, a G at position 468, a C at position 488, a U at position 665, a U at position 762, an A at position 1464, an A at position 1482, a U at position 1523, a U at position 1657, a U at position 1791 , an A at position 2004, an A at position 2189, an A at position 2234 or a G at position 2493 of SEQ ID NO: 2.
  • the present invention features genetic variants of mRNA, which represent mutations indicative of a predisposition for or indicative of an acute or chronic heart disease, wherein preferred mutations are set forth in Table 2. Furthermore, said genetic variants of mRNA may additionally contain mutations not predisposing subjects to heart diseases or being implicated in the onset of or acute or chronic heart diseases.
  • Table 2 Disease-producing mutations in the OLFML2B mRNA.
  • the OLFML2B protein has the amino acid sequence of SEQ ID NO.: 4.
  • the variant of the OLFML2B protein comprises at least one of the amino acid residues selected from the group consisting of a serine residue at position 12, a glycine residue at position 15, an arginine residue at position 22, a leucine residue at position 81 , a leucine residue at position 113, a glutamine residue at position 347, a histidine residue at position 353, a tryptophan at position 367, a histidine residue at position 411 , a isoleucine residue at position 456, a glutamine residue at position 527, a asparagine residue at position 589, a methionine residue at position 604 or a serine residue at position 690 of SEQ ID NO: 4.
  • the present invention features variants of the wild type OLFML2B protein, which represent mutations indicative of a predisposition for or indicative of an acute or chronic heart disease, wherein preferred variants of the wild type OLFML2B protein are set forth in Table 3. Furthermore, said variants may additionally contain mutations not predisposing subjects to heart diseases or being implicated in the onset of or acute or chronic heart diseases.
  • Table 3 Disease-producing mutations in the OLFML2B protein.
  • the alteration of the expression level is detected on the basis of the OLFML2B mRNA level.
  • a suitable approach according to the invention is, for example, real-time PCR employing the relative quantification approach using GAPDH as housekeeping gene.
  • Further suitable methods are well-known to the skilled person, as for example, Northern blotting, and are described in, e.g. "Analysing Gene Expression, A Handbook of Methods: Possibilities and Pitfalls” by Stefan Lorkowski, Paul M. Cullen (eds.); Wiley-VCH, Weinheim.
  • the sample is blood, serum, amniotic fluid, saliva, lymph or tissue.
  • genomic DNA of individuals which harbours the individual genetic makeup of all genes, including the OLFML2B gene can easily be purified from individual blood samples.
  • a preferred sample to detect genetic variants of the invention is blood.
  • Methods for preparing the sample for genomic DNA extraction are well known in the art, and can be carried out using commercially available kits such as, for example, FlexiGene DNA Kit (Qiagen), or Qiagen Mini Prep Kit (Qiagen).
  • said assaying of the genetic variant of the OLFML2B gene or mRNA transcribed therefrom or the variant of the OLFML2B protein comprises PCR, melting curve analysis, direct sequencing, MALDI-TOF spectroscopy and/or immunohistochemistry of the sample.
  • PCR is well known in the art and is employed to make large numbers of copies of a target sequence. This is done on an automated cycler device, which can heat and cool containers with the reaction mixture in a very short time.
  • the PCR generally, consists of many repetitions of a cycle which consists of: (a) a denaturing step, which melts both strands of a DNA molecule and terminates all previous enzymatic reactions; (b) an annealing step, which is aimed at allowing the primers to anneal specifically to the melted strands of the DNA molecule; and (c) an extension step, which elongates the annealed primers by using the information provided by the template strand.
  • PCR can be performed for example in a 50 ⁇ l reaction mixture containing 5 ⁇ l of 10 x PCR buffer with 1.5 mM MgCI 2 , 200 ⁇ M of each deoxynucleoside triphosphate, 0.5 ⁇ l of each primer (10 ⁇ M), about 10 to 100ng of template DNA and 1 to 2.5 units of Taq Polymerase.
  • the primers for the amplification may be labeled or be unlabeled.
  • DNA amplification can be performed, e.g., with a model 2400 thermal cycler (Applied Biosystems, Foster City, CA): 2 min at 94°C, followed by 30 to 40 cycles consisting of annealing (e. g. 30 s at 50°C), extension (e. g.
  • Suitable polymerases for use with a DNA template include, for example, E. coli DNA polymerase I or its Klenow fragment, T4 DNA polymerase, Tth polymerase, Taq polymerase, a heat-stable DNA polymerase isolated from Thermus aquaticus, Vent, Amplitaq, Pfu and KOD, some of which may exhibit proof-reading function and/or different temperature optima.
  • reaction conditions as used in the present invention for PCR of the OLFML2B gene fitting the requirements of all exons are the following conditions: 1 :
  • PCR products can be differentiated by analysis of melting curves whose shape is a function of GC content, length, and sequence. Unlike gel electrophoresis, melting curve analysis can distinguish products of the same length but different GC/AT ratio. In addition, two products with the same length and GC content, but differing in their GC distribution (e.g., equally distributed vs. a GC clamp on one end) would have very different melting curves.
  • PCR is compatible with many dsDNA-specific dyes, including ethidium bromide and SYBR Green I
  • product specificity can be established via monitoring of fluorescence levels during heating of PCR product from a given temperature to 95 0 C. When heated, the DNA strands dissociate and the intercalated dye is released leading to a decrease in fluorescence.
  • PCR products with the same nucleotide sequence will melt at exactly the same temperature, whereas even single base pair differences will result in a different melting temperature, as described in principle supra.
  • other strategies to monitor the transition from double to single strand DNA or vice versa can be used which are well known to the person skilled in the art, such as, for example, fluorescence resonance energy transfer and hybridizing probes.
  • the melting curve analysis of the invention can be performed by any method known to the skilled person that can provide PCR product information regarding sequence differences of said PCR products.
  • Sequence analysis may be performed using several techniques well known to the person skilled in the art.
  • the dye termination method can be used.
  • the methods and mechanisms underlying the dye termination method (Sanger didesoxy chain termination) are well known in the art and described (F. Sanger et al., (1977), DNA sequencing with chain-terminating inhibitors; Proc Natl Acad Sci USA, 74:5463- 5467).
  • a primer extension assay a differential hybridization assay, an assay which detects allele-specific enzyme cleavage or preferably MALDI TOF (Matrix Assisted Laser Desorption/lonisation Time of Flight) may be used.
  • MALDI TOF Microx Assisted Laser Desorption/lonisation Time of Flight
  • Fixation can be achieved with standard formalin fixation using a solution, such as, for example, 4% paraformaldehyde in 0.1 M phosphate buffer, 2% paraformaldehyde with picric acid in 0.1 M phosphate buffer, PLP fixative (4% paraformaldehyde, 0.2% periodate and 1.2% lysine in 0.1 M phosphate buffer) or 4% paraformaldehyde with 0.05% glutaraldehyde.
  • tissues can be rapidly frozen in liquid nitrogen and cut with a cryostat with subsequent fixation with cold acetone or alcohol. In preparation for sectioning the tissue sample can be embedded with paraffin wax or, alternatively frozen tissue can used as stated above.
  • paraffin-embedded tissue sections can be pretreated with antigen retrieval reagents such as heat for varying lengths of time and retrieval solution such as, for example, citrate buffer pH 6.0, TRIS- EDTA pH 9.0 or EDTA pH 8.0.
  • antigen retrieval reagents such as heat for varying lengths of time and retrieval solution such as, for example, citrate buffer pH 6.0, TRIS- EDTA pH 9.0 or EDTA pH 8.0.
  • Proteolytic treatment with proteinase K, trypsin, chymotrypsin, pepsin, pronase and various other proteases- can also be used to restore immunoreactivity.
  • Triton X-100 is a widely used detergent.
  • Other methods to unmask antigens include repeated freezing and thawing or sodium borohydride (1 % in phosphate buffer).
  • Staining can be achieved by direct method involving a labeled antibody reacting directly with the antigen in tissue sections or an indirect method involving a unlabeled primary antibody which reacts with the antigen, and a labeled secondary antibody which reacts with the primary antibody.
  • the latter method providing the advantage of an amplification of the detectable signal due to binding of multiple secondary antibodies to one primary antibody.
  • Other methods include the peroxidase anti- peroxidase method, avidin-biotin complex method, labeled streptavidin biotin method and polymeric methods such as, for example, EnVision System (DakoCytomation), ImmPRESS polymerized reporter enzyme staining system (Vector).
  • CSA methods from DakoCytomation providing very high levels of sensitivity and electron microscopic immunohistochemical techniques can be employed to visusalize antibody-antigen binding.
  • region of the gene is amplified e.g. by PCR and then in a sequencing reaction process, e.g. Sanger didesoxy terminator capillary sequencing, the nucleotide sequence in the area of the mutation is determined. By comparison to a reference sequence the mutation is identified.
  • the present invention relates to a nucleic acid molecule comprising a nucleic acid molecule which (a) has a DNA sequence of the genetic variant of the invention, (b) is transcribed into an mRNA sequence of the genetic variant of the invention, (c) encodes a polypeptide having an amino acid sequence of the variant of OLFML2B protein of the invention.
  • sequences and their implication in the onset of or an acute or chronic heart disease have been described in detail supra.
  • the provided sequences can be used in a vector comprising said sequences, in a host cell comprising said vector or in a method for production of a polypeptide.
  • the nucleic acid molecule may be used for gene therapy.
  • the present invention relates to a vector comprising the nucleic acid molecule of the present invention.
  • the vector is a plasmid, cosmid, virus, bacteriophage or another vector used e.g. conventionally in genetic engineering.
  • the polynucleotide of the present invention may be inserted into several commercially available vectors.
  • Non-limiting examples include prokaryotic plasmid vectors, such as the pUC-series, pBluescript (Stratagene), the pET-series of expression vectors (Novagen) or pCRTOPO (Invitrogen) and vectors compatible with an expression in mammalian cells like pREP (Invitrogen), pcDNA3 (Invitrogen), pCEP4 (Invitrogen), pMCI neo (Stratagene), pXT1 (Stratagene), pSG5 (Stratagene), EBO-pSV2neo, pBPV-1 , pdBPVMMTneo, pRSVgpt, pRSVneo, pSV2-dhfr, plZD35, pLXIN, pSIR (Clontech), plRES-EGFP (Clontech), pEAK-10 (Edge Biosystems) pTriEx-Hygro (Novagen
  • the nucleic acid molecule of the present invention referred to above may also be inserted into vectors such that a translational fusion with another nucleic acid molecule is generated.
  • the other nucleic acid molecule may encode a protein which may e.g. increase the solubility and/or facilitate the purification of the fusion protein.
  • Non-limiting examples include pET32, pET41 , pET43.
  • the vectors may also contain an additional expressible polynucleotide coding for one or more chaperones to facilitate correct protein folding.
  • Suitable bacterial expression hosts comprise e. g.
  • vectors can contain one or more origin of replication (ori) and inheritance systems for cloning or expression, one or more markers for selection in the host, e. g., antibiotic resistance, and one or more expression cassettes.
  • origins of replication include, for example, the CoI E1 , the SV40 viral and the M 13 origins of replication.
  • the vector may further comprise nucleotide sequences encoding secretion signals as further regulatory elements.
  • sequences are well known to the person skilled in the art.
  • leader sequences capable of directing the expressed polypeptide to a cellular compartment may be added to the coding sequence of the polynucleotide of the invention.
  • Such leader sequences are well known in the art.
  • regulatory elements ensuring the initiation of transcription comprise the cytomegalovirus (CMV) promoter, SV40-promoter, RSV-promoter (Rous sarcome virus), the lacZ promoter, the gai10 promoter, human elongation factor 1 ⁇ - promoter, CMV enhancer, CaM-kinase promoter, the Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) polyhedral promoter or the SV40-enhancer.
  • CMV cytomegalovirus
  • SV40-promoter RSV-promoter
  • RSV-promoter Rousarcome virus
  • the lacZ promoter the lacZ promoter
  • the gai10 promoter human elongation factor 1 ⁇ - promoter
  • CMV enhancer CMV enhancer
  • CaM-kinase promoter the Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) polyhedral promoter or the SV40-en
  • transcription termination signals such as SV40-poly-A site or the tk-poly-A site or the SV40, lacZ and AcMNPV polyhedral polyadenylation signals, downstream of the polynucleotide.
  • the vector of the invention preferably comprises a selectable marker.
  • selectable markers include neomycin, ampicillin, and hygromycine, kanamycine resistance and the like.
  • Specifically-designed vectors allow the shuttling of DNA between different hosts, such as bacteria- fungal cells or bacteria-animal cells (e. g. the Gateway® system available at Invitrogen).
  • An expression vector according to this invention is capable of directing the replication, and the expression, of the polynucleotide and encoded enzyme of this invention.
  • Suitable expression vectors which comprise the described regulatory elements are known in the art such as Okayama-Berg cDNA expression vector pcDV1 (Pharmacia), pRc/CMV, pcDNAI , pcDNA3 (In-Vitrogene, as used, inter alia in the appended examples), pSPORTI (GIBCO BRL) or pGEMHE (Promega), or prokaryotic expression vectors, such as lambda gt11 , pJOE, the pBBR1-MCS -series, pJB861 , pBSMuL, pBC2, pUCPKS, pTACTI or, preferably, the pET vector (Novagen).
  • a typical mammalian expression vector contains the promoter element, which mediates the initiation of transcription of mRNA, the protein coding sequence, and signals required for the termination of transcription and polyadenylation of the transcript. Moreover, elements such as origin of replication, drug resistance gene, regulators (as part of an inducible promoter) may also be included.
  • the lac promoter is a typical inducible promoter, useful for prokaryotic cells, which can be induced using the lactose analogue isopropylthiol-b-D-galactoside. ("IPTG").
  • IPTG lactose analogue isopropylthiol-b-D-galactoside.
  • the polynucleotide of interest may be ligated between e.g.
  • PeIB leader signal which directs the recombinant protein in the periplasm and the gene III in a phagemid called pHEN4 (described in Ghahroudi et al, 1997, FEBS Letters 414:521-526). Additional elements might include enhancers, Kozak sequences and intervening sequences flanked by donor and acceptor sites for RNA splicing. Highly efficient transcription can be achieved with the early and late promoters from SV40, the long terminal repeats (LTRs) from retroviruses, e.g., RSV, HTLVI, HIVI, and the early promoter of the cytomegalovirus (CMV).
  • LTRs long terminal repeats
  • CMV cytomegalovirus
  • cellular elements can also be used (e.g., the human actin promoter).
  • Suitable expression vectors for use in practicing the present invention include, for example, vectors such as pSVL and pMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146) and pBC12MI (ATCC 67109).
  • Mammalian host cells that could be used include, human HeIa, 293, H9 and Jurkat cells, mouse NIH3T3 and C127 cells, Cos 1 , Cos 7 and CV1 , quail QC1-3 cells, mouse L cells and Chinese hamster ovary (CHO) cells.
  • the recombinant (poly)peptide can be expressed in stable cell lines that contain the gene construct integrated into a chromosome.
  • a selectable marker such as dhfr, gpt, neomycin, hygromycin allows the identification and isolation of the transfected cells.
  • the transfected nucleic acid can also be amplified to express large amounts of the encoded (poly)peptide.
  • the DHFR (dihydrofolate reductase) marker is useful to develop cell lines that carry several hundred or even several thousand copies of the gene of interest.
  • Another useful selection marker is the enzyme glutamine synthase (GS) (Murphy et al.1991 , Biochem J.
  • the mammalian cells are grown in selective medium and the cells with the highest resistance are selected.
  • the expression vectors will preferably include at least one selectable marker.
  • markers include dihydrofolate reductase, G418 or neomycin resistance for eukaryotic cell culture and tetracycline, kanamycin or ampicillin resistance genes for culturing in E. coli and other bacteria.
  • Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E.
  • coli Streptomyces and Salmonella typhimurium cells
  • fungal cells such as yeast cells
  • insect cells such as Drosophila S2 and Spodoptera Sf9 cells
  • animal cells such as CHO, COS, 293 and Bowes melanoma cells
  • plant cells Appropriate culture mediums and conditions for the above-described host cells are known in the art.
  • said vector is an expression vector and/or a gene transfer or targeting vector.
  • Expression vectors and gene targeting or transfer vectors are well-known in the art and can be adapted for specific purposes of the invention by the person skilled in the art.
  • expression vectors derived from viruses such as retroviruses, vaccinia viruses, adeno-associated virus, herpes virus, or bovine papilloma virus, may be used for delivery of the polynucleotides or vectors of the invention into targeted cell populations.
  • Methods which are well known to those skilled in the art can be used to construct recombinant viral vectors; see, for example, the techniques described in Sambrook, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N. Y. and Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N. Y. (1889).
  • the polynucleotides and vectors of the invention can be reconstituted into liposomes for delivery to target cells.
  • the vector of the invention comprises the nucleic acid molecule of the invention operatively linked to expression control sequences allowing expression in prokaryotes or eukaryotic cells.
  • Transcriptional regulatory elements parts of an expression cassette ensuring expression in prokaryotes or eukaryotic cells are well known to those skilled in the art. These elements comprise regulatory sequences ensuring the initiation of the transcription (e. g., translation initiation codon, promoters, enhancers, and/or insulators), internal ribosomal entry sites (IRES) (Owens, Proc. Natl. Acad. Sci. USA 98 (2001), 1471-1476) and optionally poly-A signals ensuring termination of transcription and stabilization of the transcript.
  • IRS internal ribosomal entry sites
  • Additional regulatory elements may include transcriptional as well as translational enhancers, and/or naturally-associated or heterologous promoter regions.
  • the expression control sequences linked to the nucleic acid molecule can e.g. be synthesized by standard methods, or isolated from natural sources. Ligation of the nucleic acid molecule to said transcriptional regulatory elements can be carried out using established methods.
  • the nucleic acid molecules of the invention may be designed for direct introduction or for introduction via liposomes, phage vectors or viral vectors (e.g. adenoviral, retroviral) into the cell. Additionally, baculoviral systems or systems based on Vaccinia Virus or Semliki Forest Virus can be used as eukaryotic expression system for the nucleic acid molecules of the invention.
  • the present invention furthermore relates to a host cell comprising a polynucleotide or a vector of the invention.
  • Said host cell may be a prokaryotic or eukaryotic cell; see supra.
  • the polynucleotide or vector of the invention which is present in the host cell may either be integrated into the genome of the host cell or it may be maintained extra-chromosomally.
  • the recombinant DNA molecule of the invention can be used for "gene targeting” and/or “gene replacement", for restoring a mutant gene or for creating a mutant gene via homologous recombination; see for example Mouellic, Proc. Natl, Acad. Sci. USA, 87 (1990), 4712-4716; Joyner, Gene Targeting, A Practical Approach, Oxford University Press.
  • the host cell is a mammalian cell, a fungal cell, a plant cell, an insect cell or a bacterial cell.
  • Preferred fungal cells are, for example, those of the genus Saccharomyces, in particular those of the species S. cerevisiae.
  • the term "prokaryotic" is meant to include all bacteria which can be transformed or transfected with a polynucleotide for the expression of the proteins of the present invention.
  • Prokaryotic hosts may include gram negative as well as gram positive bacteria such as, for example, E. coli, S. typhimurium, Serratia marcescens and Bacillus subtilis.
  • the transformed prokaryotic host can be grown in fermentors and cultured according to techniques known in the art to achieve optimal cell growth.
  • the polypeptides of the present invention can the be isolated from the grown medium, cellular lysates, or cellular membrane fractions.
  • the isolation or purification of the microbially or otherwise expressed polypeptides of the invention may be by any conventional means such as, for example, preparative chromatographic separations and immunological separations such as those involving the use of polyclonal or monoclonal antibodies.
  • the invention relates to a method for the production of a polypeptide encoded by the nucleic acid molecule of the invention comprising culturing the host of the invention under conditions allowing the expression of the polypeptide and recovering the polypeptide.
  • polypeptides or fusion proteins
  • the host is a unicellular organism such as a prokaryote, a mammalian or insect cell, the person skilled in the art can revert to a variety of culture conditions.
  • the produced protein is harvested from the culture medium, lysates of the cultured organisms or from isolated (biological) membranes by established techniques.
  • the host may be a cell which is part of or derived from a part of the organism, for example said host cell may be the harvestable part of a plant.
  • a preferred method involves the recombinant production of protein in hosts as indicated above.
  • nucleic acid sequences comprising the polynucleotide according to the invention can be synthesized by PCR, inserted into an expression vector. Subsequently a suitable host may be transformed with the expression vector. Thereafter, the host is cultured to produce the desired polypeptide(s), which is/are isolated and purified.
  • Suitable cell-free expression systems for use in accordance with the present invention include rabbit reticulocyte lysate, wheat germ extract, canine pancreatic microsomal membranes, E. coli S30 extract, and coupled transcription/translation systems such as the TNT-system (Promega). These systems allow the expression of recombinant polypeptides upon the addition of cloning vectors, DNA fragments, or RNA sequences containing coding regions and appropriate promoter elements.
  • fragments of the protein, the fusion protein or fragments of the invention may e.g. be produced by direct peptide synthesis using solid-phase techniques (cf. Stewart et al. (1969) Solid Phase Peptide Synthesis; Freeman Co, San Francisco; Merrifield, J. Am. Chem. Soc. 85 (1963), 2149-2154).
  • Protein isolation and purification can be achieved by any one of several known techniques; for example and without limitation, ion exchange chromatography, gel filtration chromatography and affinity chromatography, high pressure liquid chromatography (HPLC), reversed phase HPLC, and preparative disc gel electrophoresis.
  • Protein isolation/purification techniques may require modification of the proteins of the present invention using conventional methods. For example, a ' histidine tag can be added to the protein to allow purification on a nickel column. Other modifications may cause higher or lower activity, permit higher levels of protein production, or simplify purification of the protein.
  • the present invention relates to a polypeptide encoded by the nucleic acid molecule of the invention or produced by the method of the present invention.
  • the invention relates to an antibody specifically recognizing the polypeptide of the present invention, wherein the antibody specifically recognizes variants of OLFML2B protein encoded by the nucleic acid of the invention but does not recognize the wild type OLFML2B protein.
  • antibody refers to intact molecules as well as fragments thereof, such as Fab, F(ab') 2 , Fv, scFv, which are capable of binding an epitopic or antigenic determinant.
  • the antibodies may be polyclonal, but preferably monoclonal, such as Fab, Fv or scFv fragments.
  • specifically recognizing refers to the interaction between polypeptide and a binding molecule, such as agonist, an antagonist, or an antibody. The interaction is dependent upon presence of the aforementioned epitopic or antigenic determinant of the protein that is recognized by the binding molecule.
  • Antibodies against the variants of OLFML2B protein of the invention can be prepared by well known methods using a purified protein according to the invention or a (synthetic) fragment derived therefrom as an antigen.
  • Monoclonal antibodies can be prepared, for example, by the techniques as originally described in K ⁇ hler and Milstein, Nature 256 (1975), 495 - 497, and Galfre, Meth. Enzymol. 73 (1981), 3, which comprises the fusion of mouse myeloma cell to spleen cells derived from immunized mammals.
  • antibodies or fragments thereof to the aforementioned polypeptides can be obtained by using methods which are described, e.g., in Harlow and Lane “Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor, 1988. These antibodies can be used, for example, for immunohistochemistry and immunolocalization of the genetic variant OLFML2B proteins.
  • the antibody recognizes an epitope containing one or more amino acid substitution(s), deletion(s), addition(s) of the present invention and/or a combination thereof.
  • the term "specifically recognizing" as used in accordance with the present invention means that the antibody does not or essentially does not cross-react with an epitope of similar structure, wherein epitopes encompass the tertiary as well as primary structure of a variant of the OLFML2B protein.
  • a specifically binding antibody of the invention will only bind to an epitope which is different to the respective epitope of the wild type protein, i.e. the epitope of the variant of the OLFML2B protein contains a mutation which is indicative of a predisposition for or an acute or chronic heart disease. Also encompassed are indicative mutations leading to the formation of new epitopes due to changes in the tertiary structure relative to the wild type OLFML2B protein.
  • Cross-reactivity of antibodies may be tested, for example, by assessing binding of said antibodies under conventional conditions to the epitope of interest as well as to a number of more or less (structurally and/or functionally) closely related epitopes. Only those antibodies that bind to the epitope of interest in its relevant context (e.g. a specific motif in the structure of a protein) but do not or not essentially bind to any other epitope are considered specific for the epitope of interest and thus to be antibodies in accordance with this invention. Corresponding methods are described e.g. in Harlow and Lane, 1988 and 1999, loc cit.
  • the invention also relates to the use of (a) wild type OLFML2B protein or a functional fragment thereof or (b) wild type OLFML2B gene in the manufacture of a pharmaceutical composition for the treatment or prevention of a heart disease.
  • the invention relates to wild type OLFML2B protein or a functional fragment thereof (a) or wild type OLFML2B gene (b) for use in the treatment or prevention of a heart disease.
  • the term "functional fragment" of OLFML2B protein refers to fragments of the wild type OLFML2B protein, wherein the fragments are capable of exerting at least partially the function and at least 80%, such as at least 90%, and preferably at least 95% of the activity as said wild type protein. Preferred are functional fragments exhibiting the same function and activity as the unmodified wild type OLFML2B protein.
  • This pharmaceutical composition may conveniently be administered by any of the routes conventionally used for drug administration, for instance, orally, topically, parenterally or by inhalation.
  • the compound may be administered in conventional dosage forms prepared by combining the drugs with standard pharmaceutical carriers according to conventional procedures. These procedures may involve mixing, granulating and compressing or dissolving the ingredients as appropriate to the desired preparation. It will be appreciated that the form and character of the pharmaceutically acceptable character or diluent is dictated by the amount of active ingredient with which is to be combined, the route of administration and other well- known variables.
  • the carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
  • the pharmaceutical carrier employed may be, for example, either a solid or liquid.
  • solid carriers are lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid and the like.
  • the carrier of diluent may include time delay material well known to the art, such as glyceryl mono stearate or glycerol distearate alone or with a wax.
  • the dosage regimen will be determined by the attending physician and other clinical factors; preferably in accordance with any one of the above described methods. As is well known in the medical arts, dosages for any one patient depends upon may 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. Progress can be monitored by periodic assessment.
  • the use of the wild type OLFML2B gene in the manufacture of a composition for treatment of a heart disease is especially preferred in the case of gene therapy.
  • the use of an effective dose of nucleic acid encoding a functional and expressible wild type OLFML2B protein is intended for treating, preventing and/or delaying a disorder diagnosed by the method of the invention.
  • a gene encoding a functional and expressible OLFML2B protein can be introduced into cells which in turn produce the protein of interest.
  • Gene therapy which ⁇ s 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. 79 (1996), 911-919; Anderson, Science 256 (1992), 808-813; Isner, Lancet 348 (1996), 370-374; Muhlhauser, Circ. Res. 77 (1995), 1077-1086; Wang, Nature Medicine 2 (1996), 714-716; WO 94/29469; WO 97/00957 or Schaper, Current Opinion in Biotechnology 7 (1996), 653-640, and references cited therein.
  • the gene 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 nucleic acid is operatively linked to regulatory elements allowing for the expression and/or targeting of the OLFML2B protein to specific cells.
  • Suitable gene delivery systems may include liposomes, receptor- mediated delivery systems, naked DNA, and viral vectors such as herpes viruses, retroviruses, adenoviruses, and adeno-associated viruses, among others. Delivery of nucleic acids to a specific site in the body for gene therapy may also be accomplished using a biolistic delivery system, such as described by Williams (Proc. Natl. Acad. Sci. USA 88 (1991), 2726-2729).
  • Gene therapy may be carried out by directly administering the recombinant DNA molecule or vector of the invention ex vivo and infusing cells into the patient.
  • the invention relates to the above use or the wild type OLFML2B protein or a functional fragment thereof (a) or wild type OLFML2B gene (b), wherein the antibody of the invention is admixed to the OLFML2B protein or a functional fragment thereof.
  • the effect of the use of the present invention can be further increased by admixing antibodies specifically recognizing variants of OLFML2B protein. Said antibodies binding to the variants of OLFML2B protein are aimed at decreasing, preferably abolishing the effect of said variants of OLFML2B proteins on the heart function.
  • antisense RNA, siRNA or shRNA specifically hybridizing to mRNA sequence segments carrying a mutation indicative, causative for and/or contributory to a heart disease of the nucleic acid molecule of the invention is admixed to OLFML2B protein or a functional fragment thereof and optionally the antibody of the invention, wherein the nucleotide reflecting said mutation is contained in the double-stranded RNA region resulting upon hybridization.
  • hybridizing refers to a pairing of an RNA molecule to a complementary strand of another RNA molecule which thereby form a hybrid.
  • Said complementary strand RNA molecules are, e.g. the mRNA variants of the invention or parts thereof. Therefore said RNA molecules are useful, for example in mediating RNA interference.
  • antisense RNA molecules can directly bind and form said hybrids and block translation
  • siRNA molecules and shRNA molecules induce so- called RNA interference which mediates cleavage of mRNA.
  • the "dicer complex" a type lll-ribonuclease, cleaves double stranded RNA molecules into short (approx.
  • RNA- induced silencing complex RISC
  • shRNA molecules are identically processed into single stranded RNA molecules which mediate RNAi.
  • hybridization conditions he has to use in accordance with the present invention.
  • Such hybridization conditions are referred to in standard text books such as "Molecular Cloning A Laboratory Manual", Cold Spring Harbor Laboratory (1989) N.Y. or Higgins, SJ. , Hames, D. "RNA Processing: A practical approach", Oxford University Press (1994), Vol. 1 and 2.
  • the term "specifically” in accordance with the present invention relates to RNA molecules which are capable of hybridizing to the RNA molecules of the invention or parts thereof, under stringent hybridization conditions, i.e. which do not cross hybridize to unrelated RNA molecules, i.e. RNA molecules not containing mutations indicative, causative and/or contributory to a heart disease.
  • stringent hybridization conditions i.e. which do not cross hybridize to unrelated RNA molecules, i.e. RNA molecules not containing mutations indicative, causative and/or contributory to a heart disease.
  • the effect of the use or the wild type OLFML2B protein or a functional fragment thereof (a) or wild type OLFML2B gene (b) of the present invention can furthermore be increased by admixing or further admixing said antisense RNA, siRNA or shRNA molecules.
  • the deployment of said RNAi molecules is aimed at decreasing the amount of expression of variant OLFML2B protein thereby decreasing, preferably abolishing the effect of said variants of OLFML2B proteins on the heart function.
  • RNAi molecules according to this preferred embodiment of the invention which specifically hybridize to mRNA sequence segments carrying a mutation indicative, causative and/or contributory for a heart disease of the nucleic acid molecule of the invention may be used for the repression of expression of a gene comprising such a sequence segment. This is conceivable in cases in which the concentration of the mutated form in the tissue should be reduced.
  • the techniques underlying said repression of expression are well known in the art. Standard methods relating to RNAi technology have also been described (Lu et al., 2005, Adv Genet., 54:117-42; Sandy et al., 2005, Biotechniques, 39(2):215-24).
  • Said nucleic acid molecules may be chemically synthesized or transcribed by an appropriate vector containing a chimeric gene which allows for the transcription of said nucleic acid molecule in the cell.
  • the antisense RNA, a siRNA or a shRNA molecule has a sequence characterized in that it is complementary to an mRNA sequence which is itself complementary to the sequence segment carrying a mutation of the nucleic acid molecule described as a genetic variant of OLFML2B gene or complementary to the sequence segment carrying a mutation of an mRNA of the present invention and can selectively bind to said mRNA, said sequence being capable of inhibiting the synthesis of the protein encoded by said nucleic acid molecules.
  • RNAi molecules will form a double-stranded RNA region upon hybridization containing the nucleotide reflecting a mutation indicative, causative and/or contributory to a heart disease.
  • the person skilled in the art provided with the sequences of the nucleic acid molecules of the present invention will be in a position to produce and utilize the above described antisense RNAs, siRNA or shRNA.
  • said mRNA comprises at least one of the nucleotides selected from the group consisting of a U at position 458, a G at position 468, a C at position 488, a U at position 665, a U at position 762, an A at position 1464, an A at position 1482, a U at position 1523, a U at position 1657, a U at position 1791 , an A at position 2004, an A at position 2189, an A at position 2234 or a G at position 2493 of SEQ ID NO: 2, said nucleotides reflecting said mutations.
  • the invention relates to the use of an antibody specifically recognizing the wild type OLFML2B protein in the manufacture of a pharmaceutical composition for the treatment or prevention of a heart disease.
  • the invention relates to an antibody specifically recognizing the wild type OLFML2B protein for use in the treatment or prevention of a heart disease
  • the term "specifically recognizing the wild type OLFML2B protein" as used in accordance with the present invention means that the antibody does not or essentially does not cross-react with an epitope of similar structure, wherein epitopes encompass the tertiary as well as primary structure of the wild type OLFML2B protein.
  • a specifically binding antibody of the invention will only bind to an epitope which is different to the respective epitope of the variant OLFML2B protein, i.e. the epitope of the variant of the OLFML2B protein contains a mutation which is indicative of a predisposition for or an acute or chronic heart disease and thus different to the wild type OLFML2B protein epitope.
  • Cross-reactivity of antibodies may be tested, for example, by assessing binding of said antibodies under conventional conditions to the epitope of interest as well as to a number of more or less (structurally and/or functionally) closely related epitopes. Only those antibodies that bind to the epitope of interest in its relevant context (e.g. a specific motif in the structure of a protein) but do not or not essentially bind to any other epitope are considered specific for the epitope of interest and thus to be antibodies in accordance with this invention. Corresponding methods are described e.g. in Harlow and Lane, 1988 and 1999, loc cit.
  • a said antibody is conceivable, for example, in situations where the expression of the wild type OLFML2B protein is altered, for example more OLFML2B protein is expressed relative to a control sample, being indicative of a predisposition for or indicative of an acute or chronic heart disease. Reducing the amount of wild type OLFML2B protein is appropriate in preventing or treating a heart disease.
  • antisense RNA, siRNA or shRNA molecules specifically hybridizing to wild type OLFML2B mRNA sequences may be admixed to the antibody.
  • the heart disease is selected from the group consisting of congestive heart-failure, cardiac hypertrophy and arrhythmias.
  • All acute or chronic heart diseases may be influenced by OLFML2B, including but not limited to: congestive heart failure, dilative cardiomyopathy, left ventricular hypertrophy, septal hypertrophy, hypertrophic cardiomyopathy, arrhythmogenic right ventricular dysplasia, hypertensive heart disease, heart disease of the elderly, ischemic heart disease, myocardial infarction, cardiac fibrosis, restrictive cardiomyopathy, left ventricular noncompaction, Barth Syndrome, long-QT syndrome, Brugada syndrome,
  • Figure 1 shows pericardial and ventricular dilatation of the startcodon-specific morpholino-knockdown in zebrafisch (mo) compared to wildtype (wt).
  • mo startcodon-specific morpholino-knockdown in zebrafisch
  • ED diastole
  • Figure 2 shows the region of the OLFML2B and NOS1AP genes from the "Golden Path".
  • the "Golden Path” http://genome.ucsc.edu) displays the following region (700 kb, in hg17 coordinates: chr1 :158, 500,001-159,200, 000) around the OLFML2B gene at Chromosome 1q23.3: Among the other genes in the Region are ATF6 and NOS1AP (CAPON).
  • Figure 3 displays the "HapMap” (http://www.hapmap.org/) exhibiting the LD-structure of the region (500 kb, in hg17 coordinates: chr1 :158, 500,001-159, 000,000) around the OLFML2B gene at Chromosome 1q23.3:
  • the other genes in the Region are ATF6 and NOS1AP (CAPON).
  • the examples illustrate the invention.
  • Example 1 Screening of patients for mutations in the OLFML2B gene.
  • a total of 762 selected patients with genetically unresolved familial and sporadic cases of cardiac arrhythmias with or without cardiomyopathy were screened for mutations in the OLFML2B gene.
  • Val604Met in exon 8 of the OLFML2B gene was identified.
  • the patient was an 62 year old woman who had presented with drug induced long QT syndrome, survived sudden cardiac death with cardiopulmonal reanimation, a dilated cardiomyopathy, sick sinus syndrome and a history of supraventicular arrhythmias since early adulthood. She had an affected daughter, who also carried the mutation and a positive family history for SCD.
  • the mutation leads to a nonsynonymous amino acid exchange of valine to methionine in the corresponding protein (pOLFML2B-V604M).
  • the mutation is situated in a highly conserved region of the OLFML2B protein, within the typical Olfactomedin- characteristic beta-sheet protein domain.
  • a third patient carries the mutation Arg 527GIn in exon 7 of the gene. She was initially diagnosed with drug induced long-QT Syndrome and a thorough investigation also revealed a hypertrophied heart with a slightly reduced left ventricular function. Her father, who is also a mutation carrier, also has left ventricular hypertrophy and has suffered from superventricular arrhythmias since adulthood. Other mutations detected in similarly affected patients and their family members are Ala12Ser, Val15Gly and Ser22Arg, all three in exon 1 , Arg353His, Gln411 His and Thr456lle all three in exon 6 and Asn690Ser in exon 8 of the gene.
  • nonsynonymous mutations in the OLFML2B were detected in affected patients, which in large enough families also cosegregated with affection status. Thus, detection of nonsynonymous mutations in the OLFML2B is indicative of predisposition at least for congestive heart-failure, cardiac hypertrophy and/or arrhythmias.
  • no nonsynonymous mutation in OLFML2B was detected applying exactly the same mutation detection methods than in the patients.
  • a total of 14 different nonsynonymous mutations of the OLFML2B Gene were detected in 23 affected individuals (index patients and their relatives).
  • the mutation carrying patients had various heart conditions (Phenotype Column; LQT: long-QT Syndrome, diLQT: drug induced long-QT Syndrome, AF: atrial fibrillation, DCM: dilative cardiomyopathy, CHF: heart failure, HCM, hypertrophic cardiomyopathy, LVH: left ventricular hypertrophy, VT: ventricular tachycardia, Rea : Status post cardiopulmonary reanimation, SSS: Sick sinus syndrome, LBBB: Left bundle branch block). All mutation carrieres detected were heterozygotes consistent with an autosomal dominant disease pattern. Autosomal recessive inheritance may be rare but may still exist although not encountered here. All variants are nonsynonymous aminoacid exchanging Mutations (nsMut).
  • Table 4 Indicative mutations in the OLFML2B gene.
  • Example 2 Conditions and oligonucleotides for the amplification the coding regions of OLFML2B.
  • Exemplary reaction conditions for PCR for the OLFML2B gene fitting the requirements of all exons and of all cDNA amplicons is PCR with the following conditions: 1 : 95°C for 15 minutes; 2: 95°C for 20 seconds; 3: 70 0 C for 30 seconds and decrease of temperature 0,5 0 C per cycle; 4: 72°C for 1 minute; 5: repeat of 2 to 4 x 20; 6: 95°C for 20 seconds; 7: 62°C for 30 seconds; 72°C for 1 minute; 9: repeat of 6 to 8 X 40; 10: 72°C for 10 minutes; 11 : 4°C for 1 minute.
  • OLFML2B_g_Ex2_F 20 AATggAgAgCCTCCTTCTgg
  • OLFML2B_g_Ex5_R 19 ATCCAgCCCACTATggTCC
  • OLFML2B_g_Ex6_F1 20 TgCCATgTgCTCTTgTCATC
  • OLFML2B_g_Ex6_F2 19 AACCATgCCTCAgTgggAC
  • OLFML2B_g_Ex7_R 18 CAAAgAggCCTggCTgTg
  • OLFML2B_g_Ex8_F1 20 TCACTT AACCACATgggCTg
  • OLFML2B_g_Ex8_R1 20 TCCgCTggTTgTAgCTATCC
  • Example 3 OLFML2B knockdown in Zebrafish.
  • OLFML2B The evidence for an involvement of OLFML2B in the pathophysiology of heart diseases is substantiated by experiments also in cell culture. We have performed more detailed cell biology investigation. A single 3.2 kb transcript of OLFML2B and no alternative splice variants were detected in porcine as well as human myocardium upon northern blot.
  • Example 4 Isolation of genomic DNA, purification, MALDI TOF and sequencing of OLFML2B gene fragments.
  • Genomic DNA was amplified by the PCR process with the following reaction conditions for the OLFML2B gene fitting the requirements of all exons 1 : 95°C for 15 minutes; 2: 95°C for 20 seconds; 3: 70 0 C for 30 seconds and decrease of temperature 0,5 0 C per cycle; 4: 72°C for 1 minute; 5: repeat of 2 to 4 x 20; 6: 95°C for 20 seconds; 7: 62°C for 30 seconds; 72°C for 1 minute; 9: repeat of 6 to 8 X 40; 10: 72°C for 10 minutes; 11 : 4°C for 1 minute. Primer sequences are detailed in Example 2.
  • PCR-products were purified with both Exonuclease I and Shrimp Alkaline Phosphatase (MBI-Fermentas) to digest remaining primers and to reduce remaining dNTPs.
  • SNPs The detection of SNPs was performed by the analysis of primer extension products generated from previously amplified genomic DNA using a Sequenom chip-based MALDI-TOF mass spectrometry platform.
  • PCR and extension reactions were designed using MassARRAY design software, and were carried out using 2.5 ng of template DNA. Unincorporated nucleotides in the PCR product were deactivated using shrimp alkaline phosphatase.
  • the amplification of the SNP site was carried out using the MassExtend primer and involved the use of specific d/ddNTP termination mixes which were also determined using MassARRAY assay design software.
  • the primer extension products were then cleaned and spotted onto a SpectroChip.
  • the chips were scanned using a mass spectrometry workstation (Bruker) and the resulting spectra were analyzed and genotypes were determined using the Sequenom SpectroTYPER-RT software. All procedures were carried out according to the manufacturer ' s recommendations.

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Abstract

La présente invention concerne un procédé de détermination d'une prédisposition à une maladie cardiaque aiguë ou chronique chez un sujet, comprenant le dosage dans un échantillon provenant du sujet d'un variant génétique du gène OLML2B ou d'un ARNm transcrit à partir de celui-ci, ou d'un variant de la protéine OLFML2B ou d'une altération du niveau d'expression du gène OLFML2B, la présence du variant génétique, du variant de la protéine ou de l'altération du niveau d'expression indiquant une prédisposition à une maladie cardiaque aiguë ou chronique. En outre, l'invention concerne également une molécule d'acide nucléique représentative du variant de l'invention. L'invention concerne également un vecteur comprenant la molécule d'acide nucléique de la présente invention et un procédé de production d'un polypeptide codé par la molécule d'acide nucléique ou par le vecteur de l'invention, et la culture d'un hôte dans des conditions permettant l'expression du polypeptide et la récupération du polypeptide de la présente invention. De plus, l'invention concerne un polypeptide codé par la molécule d'acide nucléique ou produit par le procédé de la présente invention et un anticorps reconnaissant spécifiquement ledit polypeptide. L'invention concerne également l'utilisation de la protéine OLFML2B ou d'un de ses fragments fonctionnels dans la fabrication d'une composition pharmaceutique destinée au traitement d'une maladie cardiaque, la protéine OLFML2B étant la protéine sauvage, et aussi des thérapies géniques basées sur les molécules d'acide nucléique codant la protéine sauvage.
PCT/EP2008/007821 2007-09-18 2008-09-18 Utilisation du gène de photomédine-2/analogue à l'olfactomédine 2b (olfml2b), de ses variants et de sa protéine dans des approches diagnostiques et thérapeutiques des maladies cardiaques WO2009036976A1 (fr)

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US60/973,395 2007-09-18

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003040407A2 (fr) * 2001-11-09 2003-05-15 Max-Planck-Gesellschaft Nouveaux marqueurs pour cardiopathies dilatees
WO2006026074A2 (fr) * 2004-08-04 2006-03-09 Duke University Genes determinant le phenotype atherosclerotique et methodes d'utilisation

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003040407A2 (fr) * 2001-11-09 2003-05-15 Max-Planck-Gesellschaft Nouveaux marqueurs pour cardiopathies dilatees
WO2006026074A2 (fr) * 2004-08-04 2006-03-09 Duke University Genes determinant le phenotype atherosclerotique et methodes d'utilisation

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
FURUTANI YUTAKA ET AL: "Identification and characterization of photomedins: novel olfactomedin-domain-containing proteins with chondroitin sulphate-E-binding activity", BIOCHEMICAL JOURNAL, THE BIOCHEMICAL SOCIETY, LONDON, vol. 389, no. Pt 3, 1 August 2005 (2005-08-01), pages 675 - 684, XP002465216, ISSN: 0264-6021 *

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