Title of Invention	A RECOMBINANT ADENOVIRAL VECTOR
Abstract	The present invention relates to a recombinant adenoviral vector consisting of one polynucleotide of the following groups: a polynucleotide having a sequence as specified in SEQ ill No.5; a polynucleotide, which is 95% identical to the polynucleotide of SEQ ill No.5; a polynucleotide, which is at least of the same length as the polynucleotide of SEQ ill No.5 and which hybridizes to a polynucleotide of SEQ ill No.5 when applying highly stringent hybridization conditions. This invention also relates to the above recombinant adenoviral vector comprising a polynucleotide sequence encoding a protein ofSEQ ill No.2

Title of Invention

A RECOMBINANT ADENOVIRAL VECTOR

Abstract

The present invention relates to a recombinant adenoviral vector consisting of one polynucleotide of the following groups: a polynucleotide having a sequence as specified in SEQ ill No.5; a polynucleotide, which is 95% identical to the polynucleotide of SEQ ill No.5; a polynucleotide, which is at least of the same length as the polynucleotide of SEQ ill No.5 and which hybridizes to a polynucleotide of SEQ ill No.5 when applying highly stringent hybridization conditions. This invention also relates to the above recombinant adenoviral vector comprising a polynucleotide sequence encoding a protein ofSEQ ill No.2

Full Text	Jse of EDG2 receptor in an animal model of heart failure The invention refers to a transient transformed mammal which is useful as animai model for heart failure. 3 protein-coupled receptors (GPCRs) play a central role in a multiplicity of physiological processes. It is assumed that in the human genome about 1000 genes code for this receptor family. Approximately 60 % of the pharmaceuticals presently available through prescription act as GPCR agonists or-antagonists. This underlines the importance of this receptor class fcr the pharmaceutical research industry. Owing to the size and importance of said protein family and in view of the fact that physiological ligands are still unknown for many GPCRs (orphan GPCRs), it can be assumed that this receptor class will be one of the most important reservoirs for suitable target proteins in the search for novel medicinal substances in the future. GPCRs are a family of integral membrane proteins which are located on cell surfaces They receive signals from extracellular signaling substances (e.g. hormones, neurotransmitters, peptides, lipids) and transfer these signals into the cell interior via a family of guanine nucleotide-binding proteins, the "G proteins". Dependin on the receptor specificity,.the G protein activated and the cell type, these receptors induce various signal transduction pathways. All GPCR polypeptide chains fold into seven a-helices which span across the phospholipid bilayer of the cell membrane. The seven membrane passages result in the formation of extra- and intracellular loops which allow extracellular ligand binding and intracellular coupling of G proteins. For this reason, GPCRs are alsp denoted seven-pass transmembrane receptors. All G protein-coupled receptors act according to a common basic pattern: binding of = extracellular ligand leads to a conformational change in the receptor protein which enables the receptor protein to contact a G protein, G protein-mediated signal transduction cascades in the cell finally lead to a biological response of the ceil. G proteins are heterotrimeric proteins which consist of the subunits c, 8 and y. They are located on the inside of the cell membrane via lipid anchors. Coupling of activated GPCRs to G proteins induces a GDP/GTP exchange at the Ga subunit and dissociation of the heterotrimeric G protein into an a and a βy subunit. Both the activated a subunit and the βy complex are able to interact with intracellular effector proteins. Activation of membrane-bound adenylate cyclase (AC) by Gas-type G proteins, for example, leads to an increase in the intracellular cAMP level or, in the case of activation by Gai-type G proteins, to the decrease therein. Gq-type G proteins activate phospholipase C (PLC) which catalyzes the formation of inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG). These molecules lead to the release of Ca2+ from intracellular storage organelles or to activation of proteinkinase C (PKC). The polynucleotide sequence and the amino acid sequence of the human EDG2 (Endothelial Differentiation Gene 2) has been made available to the public. The sequence is available for example from NCBI (Accession: NM_001401). The protein sequence is available from Swiss Prot (Accession: Q 92633). Cloning of the receptor from a human lung cDNA library was published in "An et al., Biochem. Biophys. Res. Commun. 24t231 (1997)". The full length sequence encodes a 359 amino acid protein which belongs to the superfamily of guanine nucleotide-binding protein-coupled receptors (GPCR). Human EDG2 rnRNA is widely distributed in human tissues with the highest abundance in brain. HEK293 cells expressing the human EDG2 protein showed an elevated response to lysophosphatidic acid (LPA) in a serum response element reporter gene assay, which was LPA concentration dependent and specific to LPA. » The mouse counterpart of EDG2 protein was also identified as a receptor for LPA. Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) are potent phospholipid mediators with diverse biological activities. Their appearance and functional properties suggest possible roles in development, wound healing, and tissue regeneration. The growth-stimulating and other complex biological activities of LPA and S1P are attributable in part to the activation of multiple G protein-mediated intracellular signaling pathways. Several heterotrimeric G proteins, as well as Ras- and Rho-dependent pathways play central roies in the cellular responses to LPA and St P. Within the scope of this invention and in all cases used and without any exemption mammal shall not encompass the human species (Homosapiens) or an Individual of Homosapiens of part of a body of a human. The invention refers-to a myocardial ceil of a mammal which ceil contains an adenoviral vector sequence for simultaneous expression of a G protein coupled receptor EDG2 and GFP (Green Fluorescent Protein). The adenoviral vector sequence consists preferably of a recombinant E 1/E 3 deficient adenovirus which expresses the G protein coupled receptor EDG2 and GFP under control of two independent promoters. Such promoters could be two CMV promoters. The myocardial cell of a mammal which contains an adenoviral vector sequence as aforementioned expresses the G protein coupled receptor EDG2 and GFP and contains therefore protein of the G protein coupled receptor EDG2 and protein of GFP. The myocardial cell, which contains an adenoviral vector sequence is preferably the cell of a rabbit, a mouse or a rat. ; The invention refers also to production of a myocardial cell which cells contains an adenoviral vector sequence for simultaneous expression of G protein coupled receptor EDG2 and GFP wherein a] the heart"of a mammal is removed by state of the art veterinary medicine ) operative techniques, b] the heart is perfused and digested with collagenase, c] the isolated cardiomyocytes are Infected with an adenoviral vector consisting of a recombinant E1/E3 deficient adenovirus which allows for expression of the G protein coupled receptor EDG2 and GFP under control of two independent promoters. Such promoters are preferably two CMV promoters. Furthermore the invention refers to a mammal having a myocardium which contains an adenoviral vector for simultaneous expression of a G protein coupled receptor EDG2 and GFP. This adenoviral vector sequence of the mammals consists preferably of a recombinant E1/E3 deficient adenovirus which allows for expression of the G protein coupled receptor EDG2 and GFP under control of two independent promoters. Such two independent promoters are preferably two CMV promoters." The invention refers also to a mammal having a myocardium which contains a protein of G protein coupled receptor EDG2 and a protein of GFP. Such a mammal having a myocardium with an adenoviral vector for simultaneous expression of a G protein coupled receptor EDG2 and GFP and/or having a myocardium with protein of G protein coupled receptor EDG2 and protein of GFP is preferably a rabbit, a mouse, or a rat. Furthermore the invention refers tc production of a mammal having a myocardium with an adenoviral vector for simultaneous expression of a G protein coupled receptor EDG2 and GFP and/or having a myocardium with protein of G protein coupled receptor EDG2 and protein of GFP wherein a] an adenoviral vector sequence for simultaneous expression of G protein coupled receptor EDG2 and GFP is provided, b] a mammal is provided, c] the adenoviral vector system from a] is transferred into the myocardium of the mammal from b] by means of a catheter. The invention concerns also use of a mamma! having a myocardium with an adenoviral vector for simultaneous expression of a G protein coupled receptor EDG2 and GFP and/or having a myocardium with protein of G coupled receptor EDG2 and protein of GFP for producing myocardial cells which can be taken for a method for Identification of a compound which modifies the activity of G protein coupled receptor EDG2. The invention refers to a method fcr identification of a compound which modifies the activity of receptor EDG2 wherein a] a transformed cell from a heart muscle which expresses the receptor EDG2 or a fusion protein comprising the receptor EDG2 is provided, b] possibly a treatment of the cell from a] is performed by use of Isoproterenoi and/or iyophQSphatidic acid, c] a chemical compound is provided, d] the cell from a] or b] is brought in contact with the chemical compound from c], e] the contractility of a cell from d] is determined and is brought in relation to the contractility of a ceil which has the same characteristics as a ceil from a] but which has not brought in contact with a chemical compound from c] and wherein a relative enhancement or reduction of contractility of the cell which has brought in contact with a chemical compound according to d] by this compound demonstrates the ability of such compound to modify the activity of receptor EDG2. The invention refers furthermore to a method for identification of a compound which modifies the activity of receptor EDG2, wherein a] a transformed cell from a heart muscle which expresses the receptor EDG2 or a fusion protein comprising the receptor EDG2 is provided, b] possibly a treatment of the ceil from a] is performed by use of proterenol and/or lysophosphatidic acid, c] a chemical compound is provided, d] the cell from a] or b] is brought in contact with the chemical compound from c], e] the contractility of a cell from d] is determined and is brought in relation to contractility of a cell of same cell type as a cell according to a] but which does not express a receptor EDG2 or a fusion protein comprising a receptor EDG2 wherein a relative enhancement or reduction of contractility of the cell which expresses a receptor EDQ2 or a fusion protein comprising a receptor EDG2 by a compound demonstrates :he ability of such compound to modify the activity c-f receptor EDG2. The invention refers furthermore to an adenoviral vector consistinc of one polynucleotide of the following groups: a] a polynucleotide having a sequence as specified in SEQ ID NO. 5, b] a polynucleotide which is 95 % identical to the-polynucleotide of SEQ ID NO. 5, c] a polynucleotide which is a: least of the same length as the polynucleotide of SEQ ID NO. 5 and which hybridizes to a polynucleotide of SEQ ID NO. 5 when applying highly stringent hybridization conditions. The adenoviral vector sequence encompasses preferably a polynucleotide sequence which is encoding a protein of SEC ID NO. 2. Hybridization means assembly of two single polynucleotide strains which have complementary sequences to double stands. Hybridization might occur between two DNA-strand, one DNA- and one RNA-strand as well as between two RNA-strands. Forming of hybrid polynucleotide strands may start from a solution which contains double stranded polynucleotide molecules by heating this solution to separate the double strands in single stranded polynucleotides. The heating step could consist of boiling in a water bath during 1G tc 20 minutes. When the solution is slowly cooled down to room temperature after it was heated the hybridization to double stranded molecules will occur. Under experimental conditions the hybridization is commonly carried out by means of hybridization filters which polynucleotides have,been fixed upon by blotting or electrophoresis. Hybridization might be visualized by use of complementary polynucleotide molecules which carry a radioactive or fluorescenic » label. Stringency describes the degree of correspondence under certain conditions. The demands with respect to correspondence are higher under high stringent conditions. Under circumstance of hybridization of nucleic acids the stringency conditions are adjusted in dependence of participating nucleic acids as well as use and objective. The conditions for a highly stringent hybridization are such that oniy very well fitting complementary molecules are able to hybridize. A very well itting complementary polynucleotide exhibits for example a degree of identity :f 90. 31, 92, 93, 94, 95, 96, 97, 98, 99 % with respect to the complementary partner molecule. Under low stringency hybridization occurs also between polynucleotide molecules which are only complementary within certain segments of the molecule or which have large sections with mismatched or unpaired base pairs. A hybridization condition of high stringency could be a hybridization wherein the hybridization step in presence of a labeled probe will be carried out in an aqueous 2XSSC solution at 68°C during at least 2 hours, and the following washing steps consist of a first washing in 2XSSC/0,1 % SDS at room temperature for 5 minutes, a second washing in 1 XSSC/0,1 % SDS at 68°C for 1 hour, and a third washing in 0,2 % SSC/0,1 % SDS at 68°C for another hour. A 2XSSC-, 1XSSC-, or 0,2 XSSC solution is obtained by dilution of a 20XSSC solution. A 20XSSC solution consists of 3 mol/l NaCI and 0,3 mol/l Na-Citrate. The skilled person is well known of other standard methods for hybridization of polynucleotides under stringent conditions. Advice is given to him in particular by textbooks as "Current Protocols in Molecular Biology (Wiley interscience; ISBN: 0-471-50338-X; eds.: F.M. Ansubel, R. Brant, R.R. Kingston, D.J. Moore, J.G. Seidmann, K. Struhl). ! The invention consists further of use of an adenoviral vector consisting of one polynucleotide of tine following groups: a] a polynucleotide having a sequence as specified in SEQ ID NO. 5,. b] a polynucleotide which is 95 % identical to the polynucleotide of SEQ ID NO. 5, a polynucleotide which is at least of the same length as the polynucleotide of SEQ ID NO. 5 and which hybridizes to a polynucleotide of SEQ ID'NC. 5 whs-. applying highly stringent hybridization conditions for constructing of transgenic mammals wherein the Q protein coupled recepic: EDG2 is transiently or permanently expressed in at leas: one tissue. Such a tissue is preferably a part'of the near: of the mammal. "Tissue could aiso cons;s: of a part of brain, muscle, fat, liver, kidney or other organs of a mamma!. General technical aspects of the invention will be further explained within the following chapters Adenoviruses can infect a wide variety of ceil types and Tissues in coin dividing and non-dividing ceils. This characteristic, together with their relative ease of preparation and purification, has led to their extensive use as gene vectors. The virus can incorporate only about 2 kb of foreign DNA withou; significant affects on its stability or its infectivitv. The introduction of ionger seauences therefore reouires the removai of some or alI of the virus aenes. There are a range of techniques for oonstructing recombinant adenoviruses. vectors can be utilized for (amongst other things): (i) cancer therapy to deliver genes that will lead to tumor suppression and elimination: (ii) gene therapy, i.e. to deliver genes to tissues to augment defective aenes; (iii) supplementarv me^apy to deliver genes, expression of which will combat disease processes. in the first generation of vectors, the E1 and/or E2 gene casssttes were removed, allowing the introduction of up to 6.5 kb of foreign DNA, often under-ins-'Contro: of a heterologous promoter, in the case of the E1 deletions, care was taken to ensure the retention of the ITR and the packaging sequences. Removal of the E1 region had the additional apparent advantage of impairing the transcription of the E2 genes (which are E1 dependent) and Consequently the renlication of virus DNA and the production of the virus capsid proteins. The defective El viruses could be propagated by infection of 293 cells, which provide the E1 gene products in trans. Although many cf the initial studies :n vitro provided much promise, it scon became evident that the expression cf the transgene in vivo was only transient and was depressed because cf the overwhelming :mmune response, mounted mainly against the virus capsid antigens as well as "he expressed transgene. One of the reason for this was the observation that many cells harbored E1-iike proteins that allowed the E2 genes to function, albeit at reduced levels, in turn, this facilitated virus DNA replication and the synthesis of the lata structural antigens and the production of replication-competent adenovirus (RCA), it also became evident that, at higher rn.o.i, the E1 dependence of E2 gene transcription could be ablated. The next approach was to construct vectors (using suitable complementing cell lines) with some or ail of the E2 genes excised and hence with the capacity to repiicate virus DNA and to produce RCAs removed- Generation cf RCAs could also be prevented by constructing cell lines that do not contain adenovirus sequences that overlap those in the vector. Nevertheless, the host immune response was still a major impediment to achieving persistent transgene expression and was particularly evident when repeated infections were attempted. A number of studies confirmed that the infecting recombinant virus itself was sufficient to induce the immune response, perhaps not surprising in view of the early activation of signaling cascades noted above and the potent antigenicity of the capsid components. Other, rather more sophisticated vectors (third generation) have been constructed by deleting other virus genes (Amalfitano et al. 1988) and the latest of these have all or nearly all of the virus genes removed. These so-called 'gutless' vectors (Hardy et aL, 1997) originally retained only the ITR and packaging sequences and required helper virus and appropriate complementing cells for propagation, followed by careful purification. Nevertheless, there were problems associated with these techniques, mainly due to contaminating helper virus and vector instability. A further development, which prevented the packaging of the helper virus, involved the use of the Cre-!ox helper-dependent system. expressed in the host cell by Western blotting of ceil lysates of transfected ceils in combination with an immunological detection method. For this too, the required laboratory protocols can be found by the skilled worker in the manuals mentioned above. Specific antibodies for immunodetection cf GPCR receptors, which are suitable xr carrying out the method cf the invention, are commercially available. A chemical compound is provided in particular by chemical synthesis or isolation of :remicai substances from biological material The skilled worker may use routine methods for chemical synthesis of a compound or isolation of a substance from cells. Such methods are available to the skilled worker in textbooks such as Organic Synthesis Workbook: 1995; John Wiley & Sens; ISBN 3-527-30137-9, The Organic Chemistry of Drug Synthesis; 1998; John Wiley & Sons; ISSN 0-471-24510-0, or Bioactive Compounds from Natural Sources; 2001; Taylor & Francis; ISBN 0-748443890-8. The compounds obtained by synthesis or isolation may be dissolved in a suitable solvent. Suitable solvents may contain water, buffer substances (e.g. Tris, HEPES, MOPS, etc.), monovalent and/or divalent ions (e.g. K", Na, Mg2\ Ca2+, etc.), acids (e.g. HCi, H2SO4, formic acid, acetic acid, etc.), bases (e.g. NaOH, etc.), alcohol (e.g. methanol, ethanol, glycerol), detergents (e.g. Na dodecyi sulfate, etc.), organic solvents (e.g. formamide, acetone, dimethyl sulfoxide, etc.) and other components, in particular solubiiizers and stabilizers. The skilled worker can contact the chemical compound with said cell tine by using laboratory routine methods. Contacting may take place, for example, in Erlenmeyer vessels, tubes, Eppendorf vessels or on microtiter plates. Temperture-controlled incubators for which a constant temperature of, for example, 30C or 37°C and fixed CO2 or humidity conditions can be set may be used for said contacting. Contacting may in particular also be carried out in laboratory rcbot devices provided therefore 'FUPR). Contacting is possible for different periods cf time, from a few seconds to minutes and up to several hours. The conditions to be chosen in each case depend on the receptor, the cell line and the chemical compound. The final form of a pharmaceutical relates to the final formulation, for example, as tablet granules, spray, solution, ointment, tincture or other formulation forms. Processing to the final form refers to the preparation of the particular formulation in generally, the daily dose is in the range from 0.3 mg to 100 mg (typically from 3 mg to 50 mg) per day and per kilogram of body weight, for example, 3-10 mg/kg/day. An intravenous does may be, for example, in the range from 0.3 mg to 1.0 mg/kg and can most suitably be administered as in infusion of from 10 ng to 100 ng per kilogram and per minute. Suitable infusion solutions for these purposes may contain, for example,from 0.1 ng to 10 mg, typically from 1 ng to 10 mg, per milliliter. Single doses may contain, for example, from 1 mg to 10 g of the active substance. It is thus possible for ampoules for injections to contain, for example, from 1 mg to 100 mg, and for single-dose formulations which can be administered orally, such as, for example, tablets or capsules, to contain, for example, from 1.0 to 1000 mg, typically from 10 to 600 mg. Examples The following examples disclose the invention without restricting it to the scope of these examples. Description of the experimental animals The animals'employed for the studies are ten-week-old female New Zealand white rabbits supplied by Asamhof, Kissing (Germany). At the start of the study the animals have a body weight of between 2.7 and 3.3 kg. Rearing and housing conditions of the experimental animals The young are weaned at 30 days. To minimize .veaning stress the dam is separated from the young so that the litter initially remains together. After about 10-14 days the young are housed in pairs in fattening cages (I x b x h = 28.5 x 60 x 34 cm) and then after about two weeks they are housed singly. The fatteners are introduced and removed in accordance with the "All in - All cut" system tc facilitate cleaning and cisinfecticn. The feed is produced in the in-house agricultural plant and is available tc the rabbits ad ibitum, as is drinking water. Climate: The ventilation rate is 10,000 rrr/h in summer and 3000 m2/h in winter. Particular attention is paid to avoiding drafts, in cold weather the temperature is maintained at about 15°C; overheating of the animal house in summer is prevented as far as possible. Young weaners are kept at abcut"19°C. Ammonia in the animal house is to be kept below 30 ppm, relative humidity below 70 %. The breeding house is i!uminated for 16 hours at an intensity of about 20 lux. Study animal housing The rabbits are brought in an air conditioned vehicle directly to the company, where they are allowed an adaptation period of 2 to 3 days to acclimatize themselves to the new diet and environment. The rabbits are kept in conventional cages. The cage material consists of stainless steel with PVC inserts; the cage floor has an area of 4040 cm2 and takes the form of a perforated bottom plate. The feces trays are cleaned daily and the cages are washed end hot-air-sterilized weekly. They are kept under constant conditions at a room temperature between 18 and 21°C and a relative humiditv of 55 ± 5 %. As the animal rouse has windows the illumination corresponds to the natural night-day cycle with an intensity of at least 100 lux. Anesthesia and preparation for surgery On the first day of the study, feed and water are available ad libitum to the rabbits until the start of anesthesia. Then the designated animal is weighed and undergoes a clinical examination, particularly of the cardiovascular system and the respiratory tract. The animals are provided with double intravenous access via indwelling catheters in the left and right lateral auricular veins (Venflon™ 08. x 25 mm), through which anesthesia is then induced with 1 % propofol (Disoprivan, Fresenius AG, Bad Homburg) in a dose of 7 mg/kg body weight i.v. Eye ointment (Vitamin A Dispersa, Ciba Vision®, Grossostheim) is applied to the cornea immediately after induction of anesthesia. After disappearance of the righting reflex the rabbits are shaved ventrally on the neck and on the chest between the elbows and the last rib and are then incubated by advancing a Magili tube with a cuff (internal diameter 2.5 to 3.5 mm, Rusch AG, Waiblingen) into the trachea during inspiration. To maintain anesthesia during the operation, the animals are given 2 % propofol (Disoprivan 2 %, Zeneca. Italy) i.v. in a dose of 12 to 14 ml/h via an infusion apparatus (Perfusor®, ED1-3Q0, B. Braun, Melsungen AG). As analgesic the rabbits are given fentanyi (Fentanyi-Janssen 0.5 rng, Janssen-Cilag GmbH, Neuss) in a dose of 0.01 mg/kg i.v. immediately after intubation and then as required during the operation, roughly every 30 minutes, to maintain the surgical tolerance stage. Using a small-animal ventiiator (Anesthesia Workstation, Halloweli EMC, Volker GmbH, Kaltenkirchen), the rabbits are ventilated with 100 % oxygen at a breathing pressure of about 10 mm Hg, a respiratory volume of 8 to 12 ml/kg body weight and a respiratory rate of 29 to 32 breaths per minute, giving a C02 partial pressure of about 35 mm Hg in the expired air. Cardiovascular function is monitored intraoperativeiy via an EGG (Medtronic®, 9790 Programmer, Vitatron Medical B.VM Dieren, Netherlands). Respiration and circulation are monitored by pulse oximetry and capnometry. intended for single-cell isolation are briefly stored in a sterile tube of cold heparinized saline at about 4°C pending immediate processing. For determination of GFP fluorescence, frozen sections are prepared. Freshly removed hearts intended for ceil microscopy are similarly washed with sterile saline, dried with cellulose, and then deep-frozen in a test tube of liquid nitrogen (-196°C) and stored at-80°C until further processing. The animals are autopsied in accordance with veterinary college guidelines, paying particular attention to evaluation of the extent of typical heart failure symptoms: ascites, pleural effusion, heart weight heart shape, liver congestion and liver weight Disposal of the carcasses After collection in a deep-freeze cabinet at -20°C, the carcasses are fetched by the carcass disposal unit for disposal. Construction and Purification of Recombinant Adenovirus: A human EDG2 receptor was cloned by using a PCR-based strategy on the basis of the coding sequence of the human EDG2 receptor. Recombinant (E1/E3-deficient) flag-tagged adenoviruses for this receptor (Ad-EDG2-GFP) were generated, expressing the transgene and green fluorescence protein (GFP) under control"of two independent CMV promoters. As a control, Ad-GFP without further transgenes was used. Large virus stocks were prepared and adenoviral titers were determined using plaque titration and GFP expression titration in non E1-expressing cells. Cloning of EDG2 The DNA of the EDG2 receptor was amplified from cDNA from human brain by PCR using the forward primer 5'-gcggggggtaccaccatggctgccatctctacttccatcc-3' (SEQ ID NO. 5) and the reverse primer 5,-gcggggctcgagtcacttgtcgtcgtcgtccttatagtcaaccacagagtgatcattgct-31 (SEQ ID NO. 6). The PCR reaction was performed at 58°C annealing for 1 min and 72°C amplification temperature for 1 min over 20 cycles with the Expand High Fidelity PCR System (Roche Molecular Biochemicais, Mannheim, Germany), Within the PCR reaction, a HA-tag Epitope of 9 amino acids from hernaglutinin of the human influenza A virus) was generated in-frame at the 3T-errd of the gene. The PCR fragment was cloned into :he plasmid pAd-Shuttle (Q Biogene. Heidelberg, Germany) by using the restriction sites for Kpni and Xhcl and the sequence of resulting pAd Track-CMV-EDG2 was checked by sequencing (MediGenomix. Mariinsried, Germany). SEQ ID NO. 1 discioses the polynucleotide sequence of EDG2 comprising the coding region of a HA-tag within the 30 nucleotides of the 3'-end. SEQ ID NO. 2 refers to the amino acid sequence of EDG2 comprising the 9 amino acid HA-tag of the C-terminus. In SEQ ID NO. 8 the polynucleotide sequence of EDG2 having a 5'-Hind[ir and 3'Xhol site is disclosed. This EDG2 gene has been cloned into Hindlll/Xbai sites of pcDNA 3.1 (invitrogen). Such a vector construct is also suitable for amplifying the EDG2 gene. Construction of recombinant flag-tagged adenovirus (pAD easy 1-EDG2-HA-GFP) The piasmid pADTrack CMV-EDG2 c-HA was linearized with Pmel (New England Biolabs, Beverly, MA) overnight, dephosphorylated and purified (GFX DNA and Ge1 Purification Kit; Amersham Pharmacia Biotech, Uppsala, Sweden). For homologous recombination, electro competent E. coll BJ5183 (Stratagene, La Jolla, California) were cotransformed with 1 pg of the linearized piasmid pADTrack CMV EDG2 c-HA and 0.1 pg pAdeasyl at 2500 V, 200 W and 25 pFD (E. coli-pulser; Biorad, Heidelberg, Germany), plated and incubated overnight at 37°C. The resulting vector, "pAdEasyl-edg2-cHA-GFP, contained the full recombinant adenoviral DNA for Transfection. The full DNA sequence is shown in SEQ ID NO. 5. The colonies were checked-after niniprsparation of the plasmid DNA with Pacl and the positive clones were rerransformed into E. coli DH5a. For transfection (Effectene Transaction reagent; Qiagen, Hilden, Germany) of 293 cei!sT piasmid DNA was digested with Feci. The cells were cultured for 7 days and harvested by scraping and centnfugation. The peiiet was resuspended in Duibecco's PBS and the ceiis were !ysed by four repetitive freezing (-80°C) and thawing (37°C) cycies. Ceil debris was removed by cantrifugation and the lysare stored at -80cC. For plaque selection of recombinant virus, 293 cells were infected in Duibecco's PBS for 1 hour at room temperature under gentle agitation with different serial dilutions of lysate from transfection. Following the infection, the cells were overiayed with growth medium containing 0.5 % agarose (1:1 mix of modified Eagles medium 2 x, Gibco Life technologies #21935, supplemented with 20 % Serum, 2x penicillin/streptomycin, 2x L-glutamin and agarose in water 1 %, Seacam). 5-14 days post infection the cell layer was monitored for formation of plaques which were picked using a pasteur pipette, resuspended in 0,5 ml Dulbeccos ?3S and stored at-8O C. The plaques were used for further amplification rounds on 293 cells. Model of Heart Failure New Zealand White rabbits were treated by rapid pacing at 350 beats/min after pacemaker implantation. Under this protocol, a tachycardia-induced heart failure (HF) develops reproducibly over two weeks. The average +dp/dtmax-vaiue in failing heans was 2200±320 mmHg/sec (vs. 32GG±230 mmHg/sec in healthy controls; p Adenoviral Qene Transfer to Rabbit Myocardium Before the start of rapid pacing, ail raboits received catheter-based adenoviral gene transfer (4x1010 pfu) to the myocardium. For the inten/ention, the rabbits were anesthetized with fentanyl and propofol. The efficacy of gene transfer was assessed in all hearts after the end of the experiments by investigating transverse freeze-cut sections for expression of GFP by fluorescence microscopy. (Morphological changes were assessed after fixation with 4 % paraformaldehyde. Gene transfer led to reproducible transgene expression in -50 % of cardiomyocytes. Shortening measurements in isolated cardiomyocytes Contractility of infected cardiomyocytes was measured by an electro-optical monitoring system connected to online digitalized assessment of amplitude and velocity of shortening and of relaxation. Transgene-positive cardiomyocytes were identified by co-expression of GFP under fluorescent Sight After the contraction amplitude reached stability, increasing concentrations of isoproterenol were applied at constant concentrations of lysophosphatidic acid (LPA; 10"5 moi/I); or increasing LPA concentrations were added to constant concentrations of isoproterenol (10-3 mol/l). Single cell contraction In order to investigate the effects of EDG2 on cardiomyocyte contractility, fractional shortening and velocity of shortening in single, isolated cardiomyocytes from failing hearts after ex vivo gene transfer was measured- At a concentration of Iysophosphatidic acid (LPA) of 10"5 moi/I which does not alter basal contractility, increasing concentrations of isoproterenol had a significantly lower positively inotropic effect in EDG2-overexpressing cardiomyocytes; (Fig. 1). After prestimulation with a low concentration of isoproterenol (10T* mol/I), increasing concentrations of LPA showed a significant negatively inotropic effect in EDG2-overexpressing cardiomyocytes whereas no effect was observed in the control GFP group (Fig. 2). In the absence of prestimulation with isoproterenol, LPA has no effect on the contractility of cardiomyocytes. Western blot of infected cardiomyocytes Cardiomyocytes were harvested 48 hours after adenoviral infection. The cells were homogenized and cytosolic extracts were then used for western blotting with antibodies against the HA tag or against EDG2. Horse radish peroxidase-coupled goat anti-rabbit antibodies by Dianova, Germany, were used as second antibodies. In vivo adenoviral delivery of transgene to failing heart Overexpression of all transgenes was investigated by studying the co-expression of GFP in the hearts after in vivo gene transfer, since all transgenes were expressed together with GFP. A macroscopic slice of a rabbit heart Infected with Ad-EDG 2-GFP showed GFP co-expression occurring throughout the left ventricle when determined by anti-GFP antibody staining. Transgene expression assessed by Western biotting Western blotting documented the expression of EDG2 by means of an antibody directed against the HA tag or by a specific antibody against EDG2 in cardiornyocytes. Preparation and culture of adult ventricular cardiornyocytes and adenovirus infections Single calcium-tolerant ventricular cardiornyocytes were isolated from failing White New Zealand rabbit hearts. Briefly, the hearts were perfused and digested with coliagenase. The isolated cardiornyocytes were cultured in modified M199 on laminin-precoated dishes (5-10 jjg/cm2) at a density of 1.5x10° cells per cm2 (at 5 % C02 and 37CC). For contraction experiments, the ce!is were infected with adenovirus (multiplicity of infection (rnoi) 1 pfu/cell) 5 hours after plating. 50-60 % of the infected cardiornyocytes expressed the transgene at this titer. Myocardial Contractility Measurement by Echocardiography and intraventricular Tip Catheter Left ventricular contractility was examined by echocardiography before the initiation of rapid pacing, after 1 week and after two weeks after the start of pacing. Tip catheter measurements were performed after 2 weeks of pacing. The rabbits were anesthetized; ECG was monitored continuously. For echocardiography, a 7.5 MHz probe was fixed on a tripcc. Szancarz sections were recorded, which were well reproducible. For tip catheter measurements, a Miliar 3F tip catheter connected to a differentiating device was placed in the left ventricle via a sheath placed in the carotid artery. After definition of basai contractility 2nd left ventricular pressure, 200 uL of NaCl (0.9 %) was injected as a negative :ontrci. Isoproterenol and lysophosphatidic acid (LPA) were infused intravenously at increasing doses. After a 20 min equilibration period, tip catheter measurements were carried out Deterioration of LV dysfunction in pacing-induced heart failure Fig. 3 shows tip catheterization measurements after 2 weeks of rapid pacing in rabbits suffering from severe heart failure (NYHA IV). in the EDG2-expression croup, the first derivatives of LV pressure (dp/dt max) were significantly lower than in the Ad-GFP-infected control group at basal conditions and at increasing doses of'LPA. This was also true for the increases in systolic LV pressure (Fig. 4). Echocardiography showed a marked hypertrophy of the myocardium after 2 weeks in the EDG2-overexpressing hearts, which was also evidenced by decreases in systolic and diastolic diameters. The mean thicknesses of the posterior wall and of the septum of the LV were significantly greater in EDG2~overexpressing hearts compared to the GFP controls. The time course of LV fractional shortening (FS) was assessed by serial echocardiography during the two week-observation period. In both groups, FS declined gradually during the time period of rapid pacing. Description of Figures: Fig. 1: Contraction amplitude of single cardiomyocytes isolated from failing hearts. The cardiomyocytes were infected ex vivo with either Ad-GFP or Ad-EDG2-GFP. Fractional shortening (FS) was determined in response :c increasing concentrations of isoproterenol after prestimuiation with 10 uM of LPA. Data represent means ± SEM. Fig. 2: Contraction amplitude of single cardiomyocytes isoiatedfrom failing hearts. Similar to the experiments shown in Figure 1, FS was compared in cardiomyocytes after gene transfer with either Ad-GFP or Ac-EDG2~GF? in vitro. Fractional shortening was determined in response to increasing LFA concentrations after prestimuiation with 10 μM of isoproterenol. Data represent means ± SEM. Fig. 3: Maximum first derivative of left ventricular pressure (LV dp/dt max) at baseline and in response to increasing doses of LPA as determined by tip catheterization. In rabbits with terminal heart failure due to rapid pacing and after two weeks after gene transfer of either GFP or EDG2. Data represent means i SEM. Ail measurements were done in 8 animals in triplicates p Fig. 4: Left ventricular systolic pressure at baseline and in response to increasing doses of ) LPA as determined by tip catheterization in rabbits with terminal heart failure due to rapid pacing and after two weeks after gene transfer of either GFP or EDG2. Data represent means ± SEM. All measurements were done in 8 animals in triplicates. p Claims Claim 1 Myocardial cel! of a mammal which ceil contains an adenoviral vector sequence for simultaneous expression of G protein coupled receptor EDG2 and GFP. Claim 2 Myocardial cell of a mammal as claimed in claim 1 wherein the adenoviral vector sequence consists of a recombinant E1/E3 deficient adenovirus which expresses the G protein coupled receptor EDG2 and GFP under control of two independent promoters. Claim 3 Myocardial cell of a mammal as claimed in claim 2 wherein the two independent promoters are two CMV promoters. Claim 4 Myocardial cell of a. mammal as claimed in one of claims 1 to 3 which contains protein of G protein coupled receptor EDG2 and protein of GFP. Claim 5 Myocardial cell of a mammal as claimed in one of claims 1 to 4 wherein the mammal is a rabbit, mouse or rat Claim 6 Production of a myocardial cell as claimed in.claims 1 to 5 wherein a] the heart of a mammal is -emcved by state of a veterinary medicine operative techniques, . . b] the heart is perfused and cigested with coilagenase, c] the isolated cardiomyocytes are infected with an adenoviral vector consisting of a recombinant E1/E3 deficient adenovirus which expresses the G protein coupled receptor EDG2 and GF? under control of two independent promoters. Claim 7 Mammal having a myocardium which contains an adenoviral vector for simultaneous expression of a G protein coupled receptor EDG2 and GFP. Claim 8 Mammal as claimed in claim 7 wherein the adenoviral vector sequence consists of a recombinant E1/E3 deficient adenovirus which expresses the G protein coupled receptor EDG2 and GFP under control of two independent promoters. Claim 9 Mammal as claimed in claim 3 wherein the two independent promoters are two CMV promoters. Claim 10 Mammal as claimed in one of claims 7 tc S which myocardium contains protein of G protein coupled receptor EDG2 and protein of GFP. Claim 11 Mammal as claimed in one of claims 7 to 10 being a rabbit, a mouse, or a rat Claim 12 Production of a mammal as claimed in claims 7 tc 11 wherein 3] an adenoviral vector sequence for simultaneous expression of G protein coupled receptor EDG2 and GFP is provided, 3] a mammal is provided, z] the adenoviral vector sequence from a] is transferred into the myocardium of the mamma! from b] by means of a catheter. claim 13 Jse of a mammal of claims for producing a myocardial cell as claimed in claims 1 to 6 for performing of claims 14 and 15. claim 14 Vlethod for identification of a compound, which modifies the activity of G protein :oupled receptor EDG2, wherein 3] a transformed cell from a heart muscle which expresses the receptor EDG2 or a fusion protein comprising the receptor EDG2 is provided, b] possibly a treatment of the cell from a] is performed by use of isoproterenol and/or lysophosphatidic acid, c] a chemical compound is provided, d] the cell from a] or b] is brought in contact with the chemical compound from c], ej the contractility of a ceil from d] is determined and is brought in relation to contractility of a cell which has the same .characteristics as a cell from aj but has not brought in contact with a chemical compound from c] wherein a relative enhancement or reduction of contractility cf the cell which has brought in contact with a chemical compound according to d] by this compound demonstrates the ability of such compound to modify the activity of receptor EDG2. claim 15 Method for identification of a compound which modifies the activity of G protein ;oupied receptor EDG2, wherein 3] a transformed cell from a heart muscle which expresses the receptor EDG2 or a fusion protein comprising the receptor EDG2 is provided, D] possibly a treatment of the cell from a] is performed by use of isoproterenol and/or lysophosphatidic acid, :] a chemical compound is provided, j] the cell from a] or b] us brought in contact with the chemical compound from c]T 5] the contractility of a cell from d] is determined and is brought in relation to contractility of a ceil of same cell type as a cell according to a] but which does not express a receptor EDG2 or a fusion protein comprising a receptor EDG2 wherein a relative enhancement or reduction of contractility of the cell which expresses a receptor EDG2 or a fusion protein comprising a receptor EDG2 by a compound demonstrates the ability of such compound to modify the activity of receptor EDG2. Claim 16 Recombinant adenoviral vector consisting of one polynucleotide of the following groups: a] a polynucleotide having a sequence as specified in SEQ ID NO. 5, b] a polynucleotide, which is 95 % identical to the polynucleotide of SEQ ID NO. 5, :] a polynucleotide, which is at feast of the same length as the polynucleotide of SEQ ID NO. 5 and which hybridizes to a polynucleotide of SEQ ID NO. 5 when applying highly stringent hybridization conditions. cIaim 17 Recombinant adenoviral vector as claimed in claim 16 comprising a polynucleotide sequence which is encoding a protein of SEQ ID NO. 2. cIaim 18 Jse of an adenoviral vector of claims 16 and 17 for constructing of transgenic mammals wherein the G protein coupled receptor EDG2 is transiently or permanently sxpressed in at least one tissue. Claim 19 Use of an adenoviral vector of claim 18, wherein the tissue is part of the heart.

Full Text

Jse of EDG2 receptor in an animal model of heart failure
The invention refers to a transient transformed mammal which is useful as animai model for heart failure.
3 protein-coupled receptors (GPCRs) play a central role in a multiplicity of physiological processes. It is assumed that in the human genome about 1000 genes code for this receptor family. Approximately 60 % of the pharmaceuticals presently available through prescription act as GPCR agonists or-antagonists. This underlines the importance of this receptor class fcr the pharmaceutical research industry. Owing to the size and importance of said protein family and in view of the fact that physiological ligands are still unknown for many GPCRs (orphan GPCRs), it can be assumed that this receptor class will be one of the most important reservoirs for suitable target proteins in the search for novel medicinal substances in the future.
GPCRs are a family of integral membrane proteins which are located on cell surfaces They receive signals from extracellular signaling substances (e.g. hormones, neurotransmitters, peptides, lipids) and transfer these signals into the cell interior via a family of guanine nucleotide-binding proteins, the "G proteins". Dependin on the receptor specificity,.the G protein activated and the cell type, these receptors induce various signal transduction pathways.
All GPCR polypeptide chains fold into seven a-helices which span across the phospholipid bilayer of the cell membrane. The seven membrane passages result in the formation of extra- and intracellular loops which allow extracellular ligand binding and intracellular coupling of G proteins. For this reason, GPCRs are alsp denoted seven-pass transmembrane receptors.
All G protein-coupled receptors act according to a common basic pattern: binding of = extracellular ligand leads to a conformational change in the receptor protein which

enables the receptor protein to contact a G protein, G protein-mediated signal transduction cascades in the cell finally lead to a biological response of the ceil. G proteins are heterotrimeric proteins which consist of the subunits c, 8 and y. They are located on the inside of the cell membrane via lipid anchors. Coupling of activated GPCRs to G proteins induces a GDP/GTP exchange at the Ga subunit and dissociation of the heterotrimeric G protein into an a and a βy subunit. Both the activated a subunit and the βy complex are able to interact with intracellular effector proteins.
Activation of membrane-bound adenylate cyclase (AC) by Gas-type G proteins, for example, leads to an increase in the intracellular cAMP level or, in the case of activation by Gai-type G proteins, to the decrease therein. Gq-type G proteins activate phospholipase C (PLC) which catalyzes the formation of inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG). These molecules lead to the release of Ca2+ from intracellular storage organelles or to activation of proteinkinase C (PKC).
The polynucleotide sequence and the amino acid sequence of the human EDG2 (Endothelial Differentiation Gene 2) has been made available to the public. The sequence is available for example from NCBI (Accession: NM_001401). The protein sequence is available from Swiss Prot (Accession: Q 92633). Cloning of the receptor from a human lung cDNA library was published in "An et al., Biochem. Biophys. Res. Commun. 24t231 (1997)".
The full length sequence encodes a 359 amino acid protein which belongs to the superfamily of guanine nucleotide-binding protein-coupled receptors (GPCR). Human EDG2 rnRNA is widely distributed in human tissues with the highest abundance in brain. HEK293 cells expressing the human EDG2 protein showed an elevated response to lysophosphatidic acid (LPA) in a serum response element reporter gene assay, which was LPA concentration dependent and specific to LPA. » The mouse counterpart of EDG2 protein was also identified as a receptor for LPA.
Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) are potent phospholipid mediators with diverse biological activities. Their appearance and functional properties suggest possible roles in development, wound healing, and

tissue regeneration. The growth-stimulating and other complex biological activities of LPA and S1P are attributable in part to the activation of multiple G protein-mediated intracellular signaling pathways. Several heterotrimeric G proteins, as well as Ras- and Rho-dependent pathways play central roies in the cellular responses to LPA and St P.
Within the scope of this invention and in all cases used and without any exemption mammal shall not encompass the human species (Homosapiens) or an Individual of Homosapiens of part of a body of a human.
The invention refers-to a myocardial ceil of a mammal which ceil contains an adenoviral vector sequence for simultaneous expression of a G protein coupled receptor EDG2 and GFP (Green Fluorescent Protein).
The adenoviral vector sequence consists preferably of a recombinant E 1/E 3 deficient adenovirus which expresses the G protein coupled receptor EDG2 and GFP under control of two independent promoters. Such promoters could be two CMV promoters.
The myocardial cell of a mammal which contains an adenoviral vector sequence as aforementioned expresses the G protein coupled receptor EDG2 and GFP and contains therefore protein of the G protein coupled receptor EDG2 and protein of GFP.
The myocardial cell, which contains an adenoviral vector sequence is preferably the cell of a rabbit, a mouse or a rat.
; The invention refers also to production of a myocardial cell which cells contains an adenoviral vector sequence for simultaneous expression of G protein coupled receptor EDG2 and GFP wherein
a] the heart"of a mammal is removed by state of the art veterinary medicine ) operative techniques,
b] the heart is perfused and digested with collagenase,

c] the isolated cardiomyocytes are Infected with an adenoviral vector consisting of a recombinant E1/E3 deficient adenovirus which allows for expression of the G protein coupled receptor EDG2 and GFP under control of two independent promoters. Such promoters are preferably two CMV promoters.
Furthermore the invention refers to a mammal having a myocardium which contains an adenoviral vector for simultaneous expression of a G protein coupled receptor EDG2 and GFP. This adenoviral vector sequence of the mammals consists preferably of a recombinant E1/E3 deficient adenovirus which allows for expression of the G protein coupled receptor EDG2 and GFP under control of two independent promoters. Such two independent promoters are preferably two CMV promoters."
The invention refers also to a mammal having a myocardium which contains a protein of G protein coupled receptor EDG2 and a protein of GFP. Such a mammal having a myocardium with an adenoviral vector for simultaneous expression of a G protein coupled receptor EDG2 and GFP and/or having a myocardium with protein of G protein coupled receptor EDG2 and protein of GFP is preferably a rabbit, a mouse, or a rat. Furthermore the invention refers tc production of a mammal having a myocardium with an adenoviral vector for simultaneous expression of a G protein coupled receptor EDG2 and GFP and/or having a myocardium with protein of G protein coupled receptor EDG2 and protein of GFP wherein
a] an adenoviral vector sequence for simultaneous expression of G protein coupled receptor EDG2 and GFP is provided,
b] a mammal is provided,
c] the adenoviral vector system from a] is transferred into the myocardium of the mammal from b] by means of a catheter.
The invention concerns also use of a mamma! having a myocardium with an adenoviral vector for simultaneous expression of a G protein coupled receptor EDG2 and GFP and/or having a myocardium with protein of G coupled receptor EDG2 and protein of GFP for producing myocardial cells which can be taken for a method for

Identification of a compound which modifies the activity of G protein coupled receptor EDG2.
The invention refers to a method fcr identification of a compound which modifies the activity of receptor EDG2 wherein
a] a transformed cell from a heart muscle which expresses the receptor EDG2 or a fusion protein comprising the receptor EDG2 is provided,
b] possibly a treatment of the cell from a] is performed by use of Isoproterenoi and/or iyophQSphatidic acid,
c] a chemical compound is provided,
d] the cell from a] or b] is brought in contact with the chemical compound from c],
e] the contractility of a cell from d] is determined and is brought in relation to the contractility of a ceil which has the same characteristics as a ceil from a] but which has not brought in contact with a chemical compound from c] and wherein a relative enhancement or reduction of contractility of the cell which has brought in contact with a chemical compound according to d] by this compound demonstrates the ability of such compound to modify the activity of receptor EDG2.
The invention refers furthermore to a method for identification of a compound which modifies the activity of receptor EDG2, wherein
a] a transformed cell from a heart muscle which expresses the receptor EDG2 or a fusion protein comprising the receptor EDG2 is provided,
b] possibly a treatment of the ceil from a] is performed by use of proterenol and/or lysophosphatidic acid,
c] a chemical compound is provided,
d] the cell from a] or b] is brought in contact with the chemical compound from c],
e] the contractility of a cell from d] is determined and is brought in relation to contractility of a cell of same cell type as a cell according to a] but which does not express a receptor EDG2 or a fusion protein comprising a receptor EDG2

wherein a relative enhancement or reduction of contractility of the cell which expresses a receptor EDQ2 or a fusion protein comprising a receptor EDG2 by a compound demonstrates :he ability of such compound to modify the activity c-f receptor EDG2.
The invention refers furthermore to an adenoviral vector consistinc of one polynucleotide of the following groups:
a] a polynucleotide having a sequence as specified in SEQ ID NO. 5,
b] a polynucleotide which is 95 % identical to the-polynucleotide of SEQ ID NO. 5,
c] a polynucleotide which is a: least of the same length as the polynucleotide of SEQ ID NO. 5 and which hybridizes to a polynucleotide of SEQ ID NO. 5 when applying highly stringent hybridization conditions.
The adenoviral vector sequence encompasses preferably a polynucleotide sequence which is encoding a protein of SEC ID NO. 2.
Hybridization means assembly of two single polynucleotide strains which have complementary sequences to double stands. Hybridization might occur between two DNA-strand, one DNA- and one RNA-strand as well as between two RNA-strands. Forming of hybrid polynucleotide strands may start from a solution which contains double stranded polynucleotide molecules by heating this solution to separate the double strands in single stranded polynucleotides. The heating step could consist of boiling in a water bath during 1G tc 20 minutes. When the solution is slowly cooled down to room temperature after it was heated the hybridization to double stranded molecules will occur. Under experimental conditions the hybridization is commonly carried out by means of hybridization filters which polynucleotides have,been fixed upon by blotting or electrophoresis. Hybridization might be visualized by use of complementary polynucleotide molecules which carry a radioactive or fluorescenic » label. Stringency describes the degree of correspondence under certain conditions. The demands with respect to correspondence are higher under high stringent conditions. Under circumstance of hybridization of nucleic acids the stringency

conditions are adjusted in dependence of participating nucleic acids as well as use and objective. The conditions for a highly stringent hybridization are such that oniy very well fitting complementary molecules are able to hybridize. A very well itting complementary polynucleotide exhibits for example a degree of identity :f 90. 31, 92, 93, 94, 95, 96, 97, 98, 99 % with respect to the complementary partner molecule. Under low stringency hybridization occurs also between polynucleotide molecules which are only complementary within certain segments of the molecule or which have large sections with mismatched or unpaired base pairs.
A hybridization condition of high stringency could be a hybridization wherein the hybridization step in presence of a labeled probe will be carried out in an aqueous 2XSSC solution at 68°C during at least 2 hours, and the following washing steps consist of a first washing in 2XSSC/0,1 % SDS at room temperature for 5 minutes, a second washing in 1 XSSC/0,1 % SDS at 68°C for 1 hour, and a third washing in 0,2 % SSC/0,1 % SDS at 68°C for another hour.
A 2XSSC-, 1XSSC-, or 0,2 XSSC solution is obtained by dilution of a 20XSSC solution. A 20XSSC solution consists of 3 mol/l NaCI and 0,3 mol/l Na-Citrate. The skilled person is well known of other standard methods for hybridization of polynucleotides under stringent conditions. Advice is given to him in particular by textbooks as "Current Protocols in Molecular Biology (Wiley interscience; ISBN: 0-471-50338-X; eds.: F.M. Ansubel, R. Brant, R.R. Kingston, D.J. Moore, J.G. Seidmann, K. Struhl).
! The invention consists further of use of an adenoviral vector consisting of one polynucleotide of tine following groups:
a] a polynucleotide having a sequence as specified in SEQ ID NO. 5,.
b] a polynucleotide which is 95 % identical to the polynucleotide of SEQ ID NO. 5,

a polynucleotide which is at least of the same length as the polynucleotide of SEQ ID NO. 5 and which hybridizes to a polynucleotide of SEQ ID'NC. 5 whs-. applying highly stringent hybridization conditions
for constructing of transgenic mammals wherein the Q protein coupled recepic: EDG2 is transiently or permanently expressed in at leas: one tissue. Such a tissue is preferably a part'of the near: of the mammal. "Tissue could aiso cons;s: of a part of brain, muscle, fat, liver, kidney or other organs of a mamma!.
General technical aspects of the invention will be further explained within the following chapters
Adenoviruses can infect a wide variety of ceil types and Tissues in coin dividing and non-dividing ceils. This characteristic, together with their relative ease of preparation and purification, has led to their extensive use as gene vectors. The virus can incorporate only about 2 kb of foreign DNA withou; significant affects on its stability or its infectivitv. The introduction of ionger seauences therefore reouires the removai of some or alI of the virus aenes. There are a range of techniques for oonstructing recombinant adenoviruses.
vectors can be utilized for (amongst other things): (i) cancer therapy to deliver genes that will lead to tumor suppression and elimination: (ii) gene therapy, i.e. to deliver genes to tissues to augment defective aenes; (iii) supplementarv me^apy to deliver genes, expression of which will combat disease processes.
in the first generation of vectors, the E1 and/or E2 gene casssttes were removed, allowing the introduction of up to 6.5 kb of foreign DNA, often under-ins-'Contro: of a heterologous promoter, in the case of the E1 deletions, care was taken to ensure the retention of the ITR and the packaging sequences. Removal of the E1 region had the additional apparent advantage of impairing the transcription of the E2 genes (which are
E1 dependent) and Consequently the renlication of virus DNA and the production of the
virus capsid proteins.

The defective El viruses could be propagated by infection of 293 cells, which provide the E1 gene products in trans. Although many cf the initial studies :n vitro provided much promise, it scon became evident that the expression cf the transgene in vivo was only transient and was depressed because cf the overwhelming :mmune response, mounted mainly against the virus capsid antigens as well as "he expressed transgene. One of the reason for this was the observation that many cells harbored E1-iike proteins that allowed the E2 genes to function, albeit at reduced levels, in turn, this facilitated virus DNA replication and the synthesis of the lata structural antigens and the production of replication-competent adenovirus (RCA), it also became evident that, at higher rn.o.i, the E1 dependence of E2 gene transcription could be ablated.
The next approach was to construct vectors (using suitable complementing cell lines) with some or ail of the E2 genes excised and hence with the capacity to repiicate virus DNA and to produce RCAs removed- Generation cf RCAs could also be prevented by constructing cell lines that do not contain adenovirus sequences that overlap those in the vector. Nevertheless, the host immune response was still a major impediment to achieving persistent transgene expression and was particularly evident when repeated infections were attempted. A number of studies confirmed that the infecting recombinant virus itself was sufficient to induce the immune response, perhaps not surprising in view of the early activation of signaling cascades noted above and the potent antigenicity of the capsid components.
Other, rather more sophisticated vectors (third generation) have been constructed by deleting other virus genes (Amalfitano et al. 1988) and the latest of these have all or nearly all of the virus genes removed. These so-called 'gutless' vectors (Hardy et aL, 1997) originally retained only the ITR and packaging sequences and required helper virus and appropriate complementing cells for propagation, followed by careful purification. Nevertheless, there were problems associated with these techniques, mainly due to contaminating helper virus and vector instability. A further development, which prevented the packaging of the helper virus, involved the use of the Cre-!ox helper-dependent system.

expressed in the host cell by Western blotting of ceil lysates of transfected ceils in combination with an immunological detection method. For this too, the required laboratory protocols can be found by the skilled worker in the manuals mentioned above. Specific antibodies for immunodetection cf GPCR receptors, which are suitable xr carrying out the method cf the invention, are commercially available.
A chemical compound is provided in particular by chemical synthesis or isolation of :remicai substances from biological material
The skilled worker may use routine methods for chemical synthesis of a compound or isolation of a substance from cells. Such methods are available to the skilled worker in textbooks such as Organic Synthesis Workbook: 1995; John Wiley & Sens; ISBN 3-527-30137-9, The Organic Chemistry of Drug Synthesis; 1998; John Wiley & Sons; ISSN 0-471-24510-0, or Bioactive Compounds from Natural Sources; 2001; Taylor & Francis; ISBN 0-748443890-8.
The compounds obtained by synthesis or isolation may be dissolved in a suitable solvent. Suitable solvents may contain water, buffer substances (e.g. Tris, HEPES, MOPS, etc.), monovalent and/or divalent ions (e.g. K", Na*, Mg2\ Ca2+, etc.), acids (e.g. HCi, H2SO4, formic acid, acetic acid, etc.), bases (e.g. NaOH, etc.), alcohol (e.g. methanol, ethanol, glycerol), detergents (e.g. Na dodecyi sulfate, etc.), organic solvents (e.g. formamide, acetone, dimethyl sulfoxide, etc.) and other components, in particular solubiiizers and stabilizers.
The skilled worker can contact the chemical compound with said cell tine by using laboratory routine methods. Contacting may take place, for example, in Erlenmeyer vessels, tubes, Eppendorf vessels or on microtiter plates. Temperture-controlled incubators for which a constant temperature of, for example, 30*C or 37°C and fixed CO2 or humidity conditions can be set may be used for said contacting. Contacting may in particular also be carried out in laboratory rcbot devices provided therefore 'FUPR). Contacting is possible for different periods cf time, from a few seconds to

minutes and up to several hours. The conditions to be chosen in each case depend on the receptor, the cell line and the chemical compound.
The final form of a pharmaceutical relates to the final formulation, for example, as tablet granules, spray, solution, ointment, tincture or other formulation forms.
Processing to the final form refers to the preparation of the particular formulation in generally, the daily dose is in the range from 0.3 mg to 100 mg (typically from 3 mg to 50 mg) per day and per kilogram of body weight, for example, 3-10 mg/kg/day. An intravenous does may be, for example, in the range from 0.3 mg to 1.0 mg/kg and can most suitably be administered as in infusion of from 10 ng to 100 ng per kilogram and per minute. Suitable infusion solutions for these purposes may contain, for example,from 0.1 ng to 10 mg, typically from 1 ng to 10 mg, per milliliter. Single doses may contain, for example, from 1 mg to 10 g of the active substance. It is thus possible for ampoules for injections to contain, for example, from 1 mg to 100 mg, and for single-dose formulations which can be administered orally, such as, for example, tablets or capsules, to contain, for example, from 1.0 to 1000 mg, typically from 10 to 600 mg.
Examples
The following examples disclose the invention without restricting it to the scope of these examples.
Description of the experimental animals
The animals'employed for the studies are ten-week-old female New Zealand white rabbits supplied by Asamhof, Kissing (Germany). At the start of the study the animals have a body weight of between 2.7 and 3.3 kg.

Rearing and housing conditions of the experimental animals
The young are weaned at 30 days. To minimize .veaning stress the dam is separated from the young so that the litter initially remains together. After about 10-14 days the young are housed in pairs in fattening cages (I x b x h = 28.5 x 60 x 34 cm) and then after about two weeks they are housed singly. The fatteners are introduced and removed in accordance with the "All in - All cut" system tc facilitate cleaning and cisinfecticn.
The feed is produced in the in-house agricultural plant and is available tc the rabbits ad ibitum, as is drinking water.
Climate: The ventilation rate is 10,000 rrr/h in summer and 3000 m2/h in winter. Particular attention is paid to avoiding drafts, in cold weather the temperature is maintained at about 15°C; overheating of the animal house in summer is prevented as far as possible. Young weaners are kept at abcut"19°C. Ammonia in the animal house is to be kept below 30 ppm, relative humidity below 70 %. The breeding house is i!uminated for 16 hours at an intensity of about 20 lux.
Study animal housing
The rabbits are brought in an air conditioned vehicle directly to the company, where they are allowed an adaptation period of 2 to 3 days to acclimatize themselves to the new diet and environment.
The rabbits are kept in conventional cages. The cage material consists of stainless steel with PVC inserts; the cage floor has an area of 4040 cm2 and takes the form of a perforated bottom plate. The feces trays are cleaned daily and the cages are washed end hot-air-sterilized weekly. They are kept under constant conditions at a room temperature between 18 and 21°C and a relative humiditv of 55 ± 5 %. As the animal rouse has windows the illumination corresponds to the natural night-day cycle with an intensity of at least 100 lux.

Anesthesia and preparation for surgery
On the first day of the study, feed and water are available ad libitum to the rabbits until the start of anesthesia. Then the designated animal is weighed and undergoes a clinical examination, particularly of the cardiovascular system and the respiratory tract. The animals are provided with double intravenous access via indwelling catheters in the left and right lateral auricular veins (Venflon™ 08. x 25 mm), through which anesthesia is then induced with 1 % propofol (Disoprivan, Fresenius AG, Bad Homburg) in a dose of 7 mg/kg body weight i.v. Eye ointment (Vitamin A Dispersa, Ciba Vision®, Grossostheim) is applied to the cornea immediately after induction of anesthesia. After disappearance of the righting reflex the rabbits are shaved ventrally on the neck and on the chest between the elbows and the last rib and are then incubated by advancing a Magili tube with a cuff (internal diameter 2.5 to 3.5 mm, Rusch AG, Waiblingen) into the trachea during inspiration.
To maintain anesthesia during the operation, the animals are given 2 % propofol (Disoprivan 2 %, Zeneca. Italy) i.v. in a dose of 12 to 14 ml/h via an infusion apparatus (Perfusor®, ED1-3Q0, B. Braun, Melsungen AG). As analgesic the rabbits are given fentanyi (Fentanyi-Janssen 0.5 rng, Janssen-Cilag GmbH, Neuss) in a dose of 0.01 mg/kg i.v. immediately after intubation and then as required during the operation, roughly every 30 minutes, to maintain the surgical tolerance stage.
Using a small-animal ventiiator (Anesthesia Workstation, Halloweli EMC, Volker GmbH, Kaltenkirchen), the rabbits are ventilated with 100 % oxygen at a breathing pressure of about 10 mm Hg, a respiratory volume of 8 to 12 ml/kg body weight and a respiratory rate of 29 to 32 breaths per minute, giving a C02 partial pressure of about 35 mm Hg in the expired air. Cardiovascular function is monitored intraoperativeiy via an EGG (Medtronic®, 9790 Programmer, Vitatron Medical B.VM Dieren, Netherlands). Respiration and circulation are monitored by pulse oximetry and capnometry.

intended for single-cell isolation are briefly stored in a sterile tube of cold heparinized saline at about 4°C pending immediate processing. For determination of GFP fluorescence, frozen sections are prepared. Freshly removed hearts intended for ceil microscopy are similarly washed with sterile saline, dried with cellulose, and then

deep-frozen in a test tube of liquid nitrogen (-196°C) and stored at-80°C until further processing. The animals are autopsied in accordance with veterinary college guidelines, paying particular attention to evaluation of the extent of typical heart failure symptoms: ascites, pleural effusion, heart weight heart shape, liver congestion and liver weight
Disposal of the carcasses
After collection in a deep-freeze cabinet at -20°C, the carcasses are fetched by the carcass disposal unit for disposal.
Construction and Purification of Recombinant Adenovirus:
A human EDG2 receptor was cloned by using a PCR-based strategy on the basis of the coding sequence of the human EDG2 receptor. Recombinant (E1/E3-deficient) flag-tagged adenoviruses for this receptor (Ad-EDG2-GFP) were generated, expressing the transgene and green fluorescence protein (GFP) under control"of two independent CMV promoters. As a control, Ad-GFP without further transgenes was used. Large virus stocks were prepared and adenoviral titers were determined using plaque titration and GFP expression titration in non E1-expressing cells.
Cloning of EDG2
The DNA of the EDG2 receptor was amplified from cDNA from human brain by PCR using the forward primer 5'-gcggggggtaccaccatggctgccatctctacttccatcc-3' (SEQ ID NO. 5) and the reverse primer 5,-gcggggctcgagtcacttgtcgtcgtcgtccttatagtcaaccacagagtgatcattgct-31 (SEQ ID NO. 6).
The PCR reaction was performed at 58°C annealing for 1 min and 72°C amplification temperature for 1 min over 20 cycles with the Expand High Fidelity PCR System (Roche Molecular Biochemicais, Mannheim, Germany), Within the PCR reaction, a

HA-tag Epitope of 9 amino acids from hernaglutinin of the human influenza A virus) was generated in-frame at the 3T-errd of the gene.
The PCR fragment was cloned into :he plasmid pAd-Shuttle (Q Biogene. Heidelberg, Germany) by using the restriction sites for Kpni and Xhcl and the sequence of resulting pAd Track-CMV-EDG2 was checked by sequencing (MediGenomix. Mariinsried, Germany).
SEQ ID NO. 1 discioses the polynucleotide sequence of EDG2 comprising the coding region of a HA-tag within the 30 nucleotides of the 3'-end.
SEQ ID NO. 2 refers to the amino acid sequence of EDG2 comprising the 9 amino acid HA-tag of the C-terminus.
In SEQ ID NO. 8 the polynucleotide sequence of EDG2 having a 5'-Hind[ir and 3'Xhol site is disclosed. This EDG2 gene has been cloned into Hindlll/Xbai sites of pcDNA 3.1 (invitrogen). Such a vector construct is also suitable for amplifying the EDG2 gene.
Construction of recombinant flag-tagged adenovirus (pAD easy 1-EDG2-HA-GFP)
The piasmid pADTrack CMV-EDG2 c-HA was linearized with Pmel (New England Biolabs, Beverly, MA) overnight, dephosphorylated and purified (GFX DNA and Ge1 Purification Kit; Amersham Pharmacia Biotech, Uppsala, Sweden). For homologous recombination, electro competent E. coll BJ5183 (Stratagene, La Jolla, California) were cotransformed with 1 pg of the linearized piasmid pADTrack CMV EDG2 c-HA and 0.1 pg pAdeasyl at 2500 V, 200 W and 25 pFD (E. coli-pulser; Biorad, Heidelberg, Germany), plated and incubated overnight at 37°C. The resulting vector, "pAdEasyl-edg2-cHA-GFP, contained the full recombinant adenoviral DNA for Transfection. The full DNA sequence is shown in SEQ ID NO. 5.

The colonies were checked-after niniprsparation of the plasmid DNA with Pacl and the positive clones were rerransformed into E. coli DH5a.
For transfection (Effectene Transaction reagent; Qiagen, Hilden, Germany) of 293 cei!sT piasmid DNA was digested with Feci. The cells were cultured for 7 days and harvested by scraping and centnfugation. The peiiet was resuspended in Duibecco's PBS and the ceiis were !ysed by four repetitive freezing (-80°C) and thawing (37°C) cycies. Ceil debris was removed by cantrifugation and the lysare stored at -80cC. For plaque selection of recombinant virus, 293 cells were infected in Duibecco's PBS for 1 hour at room temperature under gentle agitation with different serial dilutions of lysate from transfection. Following the infection, the cells were overiayed with growth medium containing 0.5 % agarose (1:1 mix of modified Eagles medium 2 x, Gibco Life technologies #21935, supplemented with 20 % Serum, 2x penicillin/streptomycin, 2x L-glutamin and agarose in water 1 %, Seacam). 5-14 days post infection the cell layer was monitored for formation of plaques which were picked using a pasteur pipette, resuspended in 0,5 ml Dulbeccos ?3S and stored at-8O C. The plaques were used for further amplification rounds on 293 cells.
Model of Heart Failure
New Zealand White rabbits were treated by rapid pacing at 350 beats/min after pacemaker implantation. Under this protocol, a tachycardia-induced heart failure (HF) develops reproducibly over two weeks. The average +dp/dtmax-vaiue in failing heans was 2200±320 mmHg/sec (vs. 32GG±230 mmHg/sec in healthy controls; p Adenoviral Qene Transfer to Rabbit Myocardium
Before the start of rapid pacing, ail raboits received catheter-based adenoviral gene transfer (4x1010 pfu) to the myocardium. For the inten/ention, the rabbits were anesthetized with fentanyl and propofol. The efficacy of gene transfer was assessed in all hearts after the end of the experiments by investigating transverse freeze-cut sections for expression of GFP by fluorescence microscopy. (Morphological changes

were assessed after fixation with 4 % paraformaldehyde. Gene transfer led to reproducible transgene expression in -50 % of cardiomyocytes.
Shortening measurements in isolated cardiomyocytes
Contractility of infected cardiomyocytes was measured by an electro-optical monitoring system connected to online digitalized assessment of amplitude and velocity of shortening and of relaxation. Transgene-positive cardiomyocytes were identified by co-expression of GFP under fluorescent Sight After the contraction amplitude reached stability, increasing concentrations of isoproterenol were applied at constant concentrations of lysophosphatidic acid (LPA; 10"5 moi/I); or increasing LPA concentrations were added to constant concentrations of isoproterenol (10-3 mol/l).
Single cell contraction
In order to investigate the effects of EDG2 on cardiomyocyte contractility, fractional shortening and velocity of shortening in single, isolated cardiomyocytes from failing hearts after ex vivo gene transfer was measured- At a concentration of Iysophosphatidic acid (LPA) of 10"5 moi/I which does not alter basal contractility, increasing concentrations of isoproterenol had a significantly lower positively inotropic effect in EDG2-overexpressing cardiomyocytes; (Fig. 1). After prestimulation with a low concentration of isoproterenol (10T* mol/I), increasing concentrations of LPA showed a significant negatively inotropic effect in EDG2-overexpressing cardiomyocytes whereas no effect was observed in the control GFP group (Fig. 2). In the absence of prestimulation with isoproterenol, LPA has no effect on the contractility of cardiomyocytes.
Western blot of infected cardiomyocytes
Cardiomyocytes were harvested 48 hours after adenoviral infection. The cells were homogenized and cytosolic extracts were then used for western blotting with

antibodies against the HA tag or against EDG2. Horse radish peroxidase-coupled goat anti-rabbit antibodies by Dianova, Germany, were used as second antibodies.
In vivo adenoviral delivery of transgene to failing heart
Overexpression of all transgenes was investigated by studying the co-expression of GFP in the hearts after in vivo gene transfer, since all transgenes were expressed together with GFP. A macroscopic slice of a rabbit heart Infected with Ad-EDG 2-GFP showed GFP co-expression occurring throughout the left ventricle when determined by anti-GFP antibody staining.
Transgene expression assessed by Western biotting
Western blotting documented the expression of EDG2 by means of an antibody directed against the HA tag or by a specific antibody against EDG2 in cardiornyocytes.
Preparation and culture of adult ventricular cardiornyocytes and adenovirus infections
Single calcium-tolerant ventricular cardiornyocytes were isolated from failing White New Zealand rabbit hearts. Briefly, the hearts were perfused and digested with coliagenase. The isolated cardiornyocytes were cultured in modified M199 on laminin-precoated dishes (5-10 jjg/cm2) at a density of 1.5x10° cells per cm2 (at 5 % C02 and 37CC). For contraction experiments, the ce!is were infected with adenovirus (multiplicity of infection (rnoi) 1 pfu/cell) 5 hours after plating. 50-60 % of the infected cardiornyocytes expressed the transgene at this titer.
Myocardial Contractility Measurement by Echocardiography and intraventricular Tip Catheter
Left ventricular contractility was examined by echocardiography before the initiation of rapid pacing, after 1 week and after two weeks after the start of pacing. Tip catheter

measurements were performed after 2 weeks of pacing. The rabbits were anesthetized; ECG was monitored continuously.
For echocardiography, a 7.5 MHz probe was fixed on a tripcc. Szancarz sections were recorded, which were well reproducible. For tip catheter measurements, a Miliar 3F tip catheter connected to a differentiating device was placed in the left ventricle via a sheath placed in the carotid artery. After definition of basai contractility 2nd left ventricular pressure, 200 uL of NaCl (0.9 %) was injected as a negative :ontrci. Isoproterenol and lysophosphatidic acid (LPA) were infused intravenously at increasing doses. After a 20 min equilibration period, tip catheter measurements were carried out
Deterioration of LV dysfunction in pacing-induced heart failure
Fig. 3 shows tip catheterization measurements after 2 weeks of rapid pacing in rabbits suffering from severe heart failure (NYHA IV). in the EDG2-expression croup, the first derivatives of LV pressure (dp/dt max) were significantly lower than in the Ad-GFP-infected control group at basal conditions and at increasing doses of'LPA. This was also true for the increases in systolic LV pressure (Fig. 4).
Echocardiography showed a marked hypertrophy of the myocardium after 2 weeks in the EDG2-overexpressing hearts, which was also evidenced by decreases in systolic and diastolic diameters. The mean thicknesses of the posterior wall and of the septum of the LV were significantly greater in EDG2~overexpressing hearts compared to the GFP controls. The time course of LV fractional shortening (FS) was assessed by serial echocardiography during the two week-observation period. In both groups, FS declined gradually during the time period of rapid pacing.
Description of Figures:
Fig. 1:
Contraction amplitude of single cardiomyocytes isolated from failing hearts. The cardiomyocytes were infected ex vivo with either Ad-GFP or Ad-EDG2-GFP. Fractional

shortening (FS) was determined in response :c increasing concentrations of isoproterenol after prestimuiation with 10 uM of LPA. Data represent means ± SEM.
Fig. 2:
Contraction amplitude of single cardiomyocytes isoiatedfrom failing hearts. Similar to the experiments shown in Figure 1, FS was compared in cardiomyocytes after gene transfer with either Ad-GFP or Ac-EDG2~GF? in vitro. Fractional shortening was determined in response to increasing LFA concentrations after prestimuiation with 10 μM of isoproterenol. Data represent means ± SEM.
Fig. 3:
Maximum first derivative of left ventricular pressure (LV dp/dt max) at baseline and in response to increasing doses of LPA as determined by tip catheterization. In rabbits with terminal heart failure due to rapid pacing and after two weeks after gene transfer of either GFP or EDG2. Data represent means i SEM. Ail measurements were done in 8 animals in triplicates *p Fig. 4:
Left ventricular systolic pressure at baseline and in response to increasing doses of ) LPA as determined by tip catheterization in rabbits with terminal heart failure due to rapid pacing and after two weeks after gene transfer of either GFP or EDG2. Data represent means ± SEM. All measurements were done in 8 animals in triplicates. *p

Claims
Claim 1
Myocardial cel! of a mammal which ceil contains an adenoviral vector sequence for simultaneous expression of G protein coupled receptor EDG2 and GFP.
Claim 2
Myocardial cell of a mammal as claimed in claim 1 wherein the adenoviral vector sequence consists of a recombinant E1/E3 deficient adenovirus which expresses the G protein coupled receptor EDG2 and GFP under control of two independent promoters.
Claim 3
Myocardial cell of a mammal as claimed in claim 2 wherein the two independent promoters are two CMV promoters.
Claim 4
Myocardial cell of a. mammal as claimed in one of claims 1 to 3 which contains protein of G protein coupled receptor EDG2 and protein of GFP.
Claim 5
Myocardial cell of a mammal as claimed in one of claims 1 to 4 wherein the mammal is a rabbit, mouse or rat
Claim 6
Production of a myocardial cell as claimed in.claims 1 to 5 wherein

a] the heart of a mammal is -emcved by state of a veterinary medicine operative techniques, . .
b] the heart is perfused and cigested with coilagenase,
c] the isolated cardiomyocytes are infected with an adenoviral vector consisting of a recombinant E1/E3 deficient adenovirus which expresses the G protein coupled receptor EDG2 and GF? under control of two independent promoters.
Claim 7
Mammal having a myocardium which contains an adenoviral vector for simultaneous expression of a G protein coupled receptor EDG2 and GFP.
Claim 8
Mammal as claimed in claim 7 wherein the adenoviral vector sequence consists of a recombinant E1/E3 deficient adenovirus which expresses the G protein coupled receptor EDG2 and GFP under control of two independent promoters.
Claim 9
Mammal as claimed in claim 3 wherein the two independent promoters are two CMV promoters.
Claim 10
Mammal as claimed in one of claims 7 tc S which myocardium contains protein of G protein coupled receptor EDG2 and protein of GFP.
Claim 11
Mammal as claimed in one of claims 7 to 10 being a rabbit, a mouse, or a rat

Claim 12
Production of a mammal as claimed in claims 7 tc 11 wherein
3] an adenoviral vector sequence for simultaneous expression of G protein
coupled receptor EDG2 and GFP is provided, 3] a mammal is provided, z] the adenoviral vector sequence from a] is transferred into the
myocardium of the mamma! from b] by means of a catheter.
claim 13
Jse of a mammal of claims for producing a myocardial cell as claimed in claims 1 to 6 for performing of claims 14 and 15.
claim 14
Vlethod for identification of a compound, which modifies the activity of G protein :oupled receptor EDG2, wherein
3] a transformed cell from a heart muscle which expresses the receptor EDG2 or a fusion protein comprising the receptor EDG2 is provided,
b] possibly a treatment of the cell from a] is performed by use of isoproterenol and/or lysophosphatidic acid,
c] a chemical compound is provided,
d] the cell from a] or b] is brought in contact with the chemical compound from c],
ej the contractility of a ceil from d] is determined and is brought in relation to
contractility of a cell which has the same .characteristics as a cell from aj but has not brought in contact with a chemical compound from c] wherein a relative enhancement or reduction of contractility cf the cell which has brought in contact with a chemical compound according to d] by this compound

demonstrates the ability of such compound to modify the activity of receptor EDG2.
claim 15
Method for identification of a compound which modifies the activity of G protein ;oupied receptor EDG2, wherein
3] a transformed cell from a heart muscle which expresses the receptor EDG2 or a fusion protein comprising the receptor EDG2 is provided,
D] possibly a treatment of the cell from a] is performed by use of isoproterenol and/or lysophosphatidic acid,
:] a chemical compound is provided,
j] the cell from a] or b] us brought in contact with the chemical compound from c]T
5] the contractility of a cell from d] is determined and is brought in relation to
contractility of a ceil of same cell type as a cell according to a] but which does not express a receptor EDG2 or a fusion protein comprising a receptor EDG2 wherein a relative enhancement or reduction of contractility of the cell which expresses a receptor EDG2 or a fusion protein comprising a receptor EDG2 by a compound demonstrates the ability of such compound to modify the activity of receptor EDG2.
Claim 16
Recombinant adenoviral vector consisting of one polynucleotide of the following groups:
a] a polynucleotide having a sequence as specified in SEQ ID NO. 5,
b] a polynucleotide, which is 95 % identical to the polynucleotide of SEQ ID NO. 5,

:] a polynucleotide, which is at feast of the same length as the polynucleotide of SEQ ID NO. 5 and which hybridizes to a polynucleotide of SEQ ID NO. 5 when applying highly stringent hybridization conditions.
cIaim 17
Recombinant adenoviral vector as claimed in claim 16 comprising a polynucleotide sequence which is encoding a protein of SEQ ID NO. 2.
cIaim 18
Jse of an adenoviral vector of claims 16 and 17 for constructing of transgenic mammals wherein the G protein coupled receptor EDG2 is transiently or permanently sxpressed in at least one tissue.
Claim 19
Use of an adenoviral vector of claim 18, wherein the tissue is part of the heart.

Documents:

878-CHENP-2005 ABSTRACT.pdf

878-CHENP-2005 CLAIMS GRANTED.pdf

878-CHENP-2005 CORRESPONDENCE OTHERS.pdf

878-CHENP-2005 CORRESPONDENCE PO.pdf

878-CHENP-2005 FORM 1.pdf

878-CHENP-2005 FORM 18.pdf

878-CHENP-2005 FORM 2.pdf

878-CHENP-2005 PETITIONS.pdf

878-CHENP-2005 POWER OF ATTORNEY.pdf

878-chenp-2005-abstract.pdf

878-chenp-2005-claims.pdf

878-chenp-2005-correspondnece-others.pdf

878-chenp-2005-correspondnece-po.pdf

878-chenp-2005-description(complete).pdf

878-chenp-2005-drawings.pdf

878-chenp-2005-form 1.pdf

878-chenp-2005-form 13.pdf

878-chenp-2005-form 26.pdf

878-chenp-2005-form 3.pdf

878-chenp-2005-form 5.pdf

878-chenp-2005-pct.pdf

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Patent Number

229552

Indian Patent Application Number

878/CHENP/2005

PG Journal Number

13/2009

Publication Date

27-Mar-2009

Grant Date

18-Feb-2009

Date of Filing

10-May-2005

Name of Patentee

SANOFI-AVENTIS DEUTSCHLAND GMBH

Applicant Address

Brüningstrasse 50, D-65929 Frankfurt am Main,

Inventors:

#	Inventor's Name	Inventor's Address
1	KOSTENIS, Evi	Langwiesenweg 29, 36323 Grebenau,
2	WOHLFART, Paulus	Bertolt-Brecht-Ring 16c, 64625 Bensheim,
3	HUBER, Jochen	Speyererstrasse 3, 67133 Maxdorf,

PCT International Classification Number

C07K14/705

PCT International Application Number

PCT/EP03/12325

PCT International Filing date

2003-11-05

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	02025161.7	2002-11-11	EUROPEAN UNION