AU8438598A - Heart and skeleton muscle specific nucleic acid, the production and use thereof - Google Patents

Description

MediGene Aktiengesellschaft M25519PC BO Myocardium- and skeletal muscle-specific nucleic acid, 5 its preparation and use The invention relates to a nucleic acid which is expressed in human myocardium and skeletal muscle, to its preparation and use as diagnostic aid, medicinal 10 product and test for identifying functional interactors. The heart is a muscular hollow organ which has the task of keeping the bloodstream in the vessels in motion by alternating contraction (systole) and 15 relaxation (diastole) of atria and ventricles. The muscle of the heart, the myocardium, is composed of specialized striped muscle cells between which there is connective tissue. Each cell has a central nucleus, is bounded by the plasma membrane, the 20 sarcolemma, and contains numerous contractile myofibrils which are separated irregularly by sarcoplasm. The contractile substance of the heart is formed by long parallel myofibrils. Each myofibril is divided into several identical structural and 25 functional units, the sarcomeres. The sarcomeres in turn are composed of the thin filaments which mainly consist of actin, tropomyosin and troponin, and the thick filaments which mainly consist of myosin. The molecular mechanism of muscle contraction 30 is based on a cyclic attachment and detachment of the globular myosin heads by the F actin filaments. On electrical stimulation of the myocardium, Ca 2 + is released from the sarcoplasmic reticulum which influences, through an allosteric reaction, the 35 troponin complex and tropomyosin, and thus opens the way for contact of the actin filament with the myosin -2 head. The attachment causes a conformational change in the myosin which thus pulls the actin filament along on itself. ATP is needed to reverse this conformational change and return to the start of a contraction cycle. 5 Short-term adjustment of the activity of the myocardium to the particular perfusion requirement, that is to say blood flow requirement, of the body is possible by nervous and hormonal regulation measures. It is thus possible to increase both the force of 10 contraction and the rate of contraction. Long-term overstrain results in physiological transformation processes in the myocardium, which are characterized mainly by an increase in myofibrils (myocyte hypertrophy) . 15 If the myocardium is damaged, the originally physiological adaptation mechanisms often lead in the long term to pathophysiological states which develop into chronic cardiac insufficiency, that is to say cardiac weakness, and usually end with acute heart 20 failure. In cases of severe chronic insufficiency, the heart may no longer respond appropriately to changed output requirements, and even slight physical exertion leads to exhaustion and shortness of breath. Damage to the myocardium results from 25 ischaemia, that is to say depletion of blood, caused by coronary disease, bacterial or viral infections, toxins, metabolic abnormalities, autoimmune diseases or genetic defects. Therapeutic measures are currently aimed at strengthening the force of contraction and 30 controlling the compensatory neuronal and hormonal compensation mechanisms. Despite this treatment, the mortality from this disease remains high (35-50% in the first 5 years after diagnosis) . Cardiac insufficiency is the main cause of death in the world. The only 35 causal therapy is a heart transplant. The molecular changes in chronic cardiac insufficiency are only inadequately known. In

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-3 particular, the genetic changes underlying cardiac insufficiency are substantially unknown. The question of why secondary damage by toxins or viruses leads to cardiac insufficiency in some people but not in others 5 also remains unanswered. The present invention is thus based on the object of identifying and isolating genes which are at least partly responsible for, if not in fact the causes of, genetically related cardiac disorders. 10 Surprisingly, a gene has now been found, in a human cardiac tissue cDNA bank, which is expressed more strongly in insufficient cardiac tissue than in healthy cardiac tissue and thus is causally connected with a genetically related cardiac insufficiency. A so-called 15 EST (expressed sequence tag) already exists for this gene, although it is faulty and no function at all can be assigned to it (Tanaka, T. et al. (1996) Genomics, 35, 231-235; EMBL AC:C04498; clone 3NHC3467). One aspect of the invention is therefore a 20 nucleic acid coding for a polypeptide having an amino acid sequence as shown in Fig. 4 or a functional variant thereof, and parts thereof having at least 8 nucleotides, preferably at least 10 nucleotides, in particular at least 15 nucleotides, especially at least 25 20 nucleotides, except a nucleic acid having the sequence: 1 GCCAACACGC ANTCCGACGA CAGTGCAGCC ATGGTCATTG CAGAGATGCN TCAAAGTCAA 61 TGAGCACATC ACCAACGTAA ACGTCGAGTC CAACTTCATA ACGGGAAAGG GGATCCTGGC 121 CATCATGAGA GCTCTCCAGC ACAACACGGT GCTCACGGAG CTGCGTTTCC ATAACCAGAG 181 GCACATCATG GGCAGCCAGG TGGAAATGGA GATTGTCAAG CTNCTGAAGG AGAACACGAC 241 GCTNCTGAGG CTGGGNTACC ATTTTNAACT CCCAGGACC in which N denotes A, T, G or C. The nucleic acid according to the invention was 30 isolated from a human cardiac tissue cDNA bank and sequenced. For this, firstly complete RNA was isolated by standard methods from a healthy and insufficient cardiac sample and transcribed with the aid of a -4 3'anchor primer mixture, for example a 5'-T 12 ACN-3' primer, in which N denotes any deoxyribonucleotide, and reverse transcriptase into c-DNA. The cDNA was then amplified with a method based on the so-called 5 differential display method of Liang and Pardee (Liang, P. & Pardee, A. (1992) Science 257, 967-970) under specific PCR conditions with the aid of a 3' primer, for example a T 12 ACN primer, and of an arbitrarily selected 5'-decamer primer, for example a 10 5'-CCTTCTACCC-3' decamer primer. It was possible thereby to amplify a 321 base pair (bp)-long DNA fragment which is surprisingly present not in the healthy heart sample but distinctly in the insufficient heart sample. This was surprising because the 15 conventional methods such as the differential display method or else subtractive cDNA gene banks are associated with the problem of redundancy, of under representation and of false-positive clones. In particular, it is possible to identify the gene 20 products of weakly expressed genes only under special conditions. It is therefore also not astonishing that the hit rate is generally very low (10-20%) and, for example in the differential display method, also depends on the chosen PCR conditions, the primer length 25 or, for example in the production of subtractive banks, on the hybridization temperature. The complete gene was then isolated from a cDNA gene bank with the aid of the found DNA fragment and sequenced. In every case it is necessary to find out by 30 further methods whether the found cDNA can be assigned to an active and/or tissue-specific gene. Hence mRNAs from various human tissues were hybridized with the found DNA fragment in a so-called Northern blot, and the amount of bound m-RNA was determined, for example, 35 via the radiolabelling of the DNA fragment. This experiment led to detection of the corresponding RNA in particular in striped muscle, that is to say myocardial ,YC - -5 and skeletal muscle tissue, and very weakly in prostate tissue. In a further experiment comparing between healthy and insufficient cardiac tissues, increased expression was detected, for example expression of the 5 RNAs increased by about 35%, in insufficient tissue by comparison with healthy tissue. It was possible to demonstrate in particular that a relatively small RNA species preferentially shows increased expression in insufficient tissue by comparison with healthy tissue. 10 The increased expression of the relatively small RNA species is readily evident for example in the Northern blot in the form of a double band (see Fig. 5b). Comparison of the derived polypeptide sequence with a protein database additionally revealed a certain 15 relationship (homology) with the protein tropomodulin (see Fig. 4) . Tropomodulin is known to be a polypeptide which in chicken cardiomyocytes has an influence on the development of the myofibrils and the contractility of the cells (Gregorio et al. (1995) Nature 377, 83-86) . 20 This protein binds on the one hand to tropomyosin, and on the other hand to the actin filaments, but is not itself regulated in its activity. The derived polypeptide according to the invention likewise has some of the structural features of tropomodulins, such 25 as, for example, a tropomyosin binding domain. In contrast to tropomodulin, the polypeptide according to the invention has additional structural features indicating regulation of the activity of the polypeptide by so-called tyrosine kinases (see Fig. 4). 30 The term "functional variant" therefore means for the purpose of the present invention polypeptides which are functionally related to the polypeptide according to the invention, that is to say can likewise be referred to as a regulable modulator of the 35 contractility of myocardial cells, are expressed in striped muscle, preferably in myocardial, skeletal muscle and/or prostate tissue, especially in myocardial -6 and/or skeletal muscle and, in particular, in myocardial cells, have structural features of tropomodulin, such as, for example, one or more tropomyosin binding domains, and/or whose activity can 5 be regulated by tyrosine kinases. Examples of functional variants are the corresponding polypeptides derived from other organisms than humans, preferably from non-human mammals such as, for example, monkeys. In the wider sense, the term ,,functional 10 variant" includes polypeptides which have a sequence homology, in particular a sequence identity, of about 70%, preferably about 80%, in particular about 90%, especially about 95%, with the polypeptide having the amino acid sequence shown in Fig. 4. These include, for 15 example, polypeptides encoded by a nucleic acid which is isolated from non-heart-specific tissue, for example skeletal muscle tissue, but which has, after expression in a heart-specific cell, the identified function(s). These furthermore include deletions of the polypeptide 20 in the region of about 1-60, preferably of about 1-30, in particular of about 1-15, especially of about 1-5, amino acids. For example, the first amino acid methionine can be absent with negligible alteration in the function of the polypeptide. These also include 25 fusion proteins which comprise the above-described polypeptides according to the invention, it being possible for the fusion proteins themselves to have -the function of a regulable modulator of the contractility of myocardial cells, or to acquire the specific 30 function only after elimination of the fusion portion. They particularly include fusion proteins with a content of, in particular, non-heart-specific sequences of about 1-200, preferably about 1-150, in particular about 1-100, especially about 1-50, amino acids. 35 Examples of non-heart-specific peptide sequences are prokaryotic peptide sequences which may be derived, for example, from the galactosidase of E. coli.

-7 The nucleic acid according to the invention is generally a DNA or RNA, preferably a DNA. Preferred for expression of the relevant gene is in general a double stranded DNA and for use as probe is a single-stranded 5 DNA. Particular preference is given to a double- or single-stranded DNA having a nucleic acid sequence as shown in Fig. 1, 2 or 3 and the parts thereof described in detail above, with the DNA region coding for the polypeptide being particularly preferred. This region 10 starts with the nucleic acids "ATG" coding for methionine at position 89 to "TAG" coding for "amber" (stop) at position 1747. The nucleic acid according to the invention can, for example, be chemically synthesized on the 15 basis of the sequences disclosed in Figs. 1-3 or on the basis of the polypeptide sequence disclosed in Fig. 4 by use of the genetic code, for example by the phosphotriester method (see, for example, Uhlmann, E. & Peyman, A. (1990) Chemical Reviews, 90, 543-584, 20 No. 4) . Another possibility for obtaining the nucleic acid according to the invention is isolation from a suitable gene bank, for example from a heart-specific gene bank, using a suitable probe (see, for example, J. Sambrook et al., (1989), Molecular Cloning. A 25 Laboratory Manual 2 nd edn., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) . Suitable as probe are, for example, single-stranded DNA fragments with a length of about 100-1000 nucleotides, preferably with a length of about 200-500 nucleotides, in particular with 30 a length of about 300-400 nucleotides, whose sequence can be derived from the nucleic acid sequences shown in Figs. 1-3. One example of a probe is the DNA fragment of Example 1, which is 321 bp in size and corresponds to the underlined region in Fig. 1, using which the 35 nucleic acid according to the invention has already been isolated successfully from human cardiac tissue (see Example 2).

-8 The nucleic acid according to the invention is normally present in a vector, preferably in an expression vector or vector effective for gene therapy. The vector effective for gene therapy preferably 5 contains heart-specific regulatory sequences such as, for example, the troponin C (cTNC) promoter (see, for example, Parmacek, M.S. et al. (1990) J. Biol. Chem. 265 (26) 15970-15976 and Parmacek, M.S. et al. (1992) Mol. Cell Biol. 12(5), 1967-1976), which is 10 functionally connected to the nucleic acid according to the invention. The expression vectors may be prokaryotic or eukaryotic expression vectors. Examples of prokaryotic expression vectors for expression in E. coli are, for 15 example, the vectors pGEM or pUC derivatives, and of eukaryotic expression vectors for expression in Saccharomyces cerevisiae are, for example, the vectors p426Met25 or p426GAL1 (Mumberg et al. (1994) Nucl. Acids Res., 22, 5767-5768) for expression in insect 20 cells are, for example, baculovirus vectors as disclosed in EP-B1 0 127 839 or EP-B1 0 549 721, and for expression in mammalian cells are, for example, the vectors Rc/CMV and Rc/RSV or SV40 vectors, which are all generally available. 25 The expression vectors generally also contain promoters suitable for the particular host cell, such as, for example, the trp promoter for expression in E. coli (see, for example, EP-B1 0 154 133), the ADH2 promoter for expression in yeasts (Russell et al. 30 (1983), J. Biol. Chem. 258, 2674-2682), the baculovirus polyhedrin promoter for expression in insect cells (see, for example, EP-B1 0 127 839) or the SV40 early promoter or LTR promoters, for example of MMTV (mouse mammary tumour virus; Lee et al. (1981) Nature 214, 35 228-232). Examples of vectors effective for gene therapy are virus vectors, preferably adenovirus vectors, in -9 particular replication-deficient adenovirus vectors, or adeno-associated virus vectors, for example an adeno associated virus vector which consists exclusively of two inverted terminal repeats (ITR). 5 An adenovirus vector and, in particular, a replication-deficient adenovirus vector are particularly preferred for the following reasons. The human adenovirus belongs to the class of double-stranded DNA viruses with a genome of about 36 10 kilobase pairs (kb) . The viral DNA codes for about 2700 different gene products, a distinction being made between early ("early genes") and late ("late genes "). The "early genes" are divided into four transcriptional units El to E4. The late gene products code for the 15 capsid proteins. It is possible to distinguish immunologically at least 42 different adenoviruses and subgroups A to F, all of which are suitable for the present invention. A precondition for transcription of the viral genes is expression of the El region which 20 codes for a transactivator of adenoviral gene expression. This dependence of the expression of all subsequent viral genes on the El transactivator can be utilized to construct adenoviral vectors not capable of replication (see, for example, McGrory, W.J. et al. 25 (1988) Virol. 163, 614-617 and Gluzman, Y. et al. (1982) in "Eukaryotic Viral Vectors" (Gluzman, Y. ed.) 187 - 192, Cold Spring Harbor Press, Cold Spring Harbor, New York) . In adenoviral vectors, especially of type 5 (for sequence, see Chroboczek, J. et al. (1992) 30 Virol. 186, 280-285) and especially of subgroup C, in general the El gene region is replaced by a foreign gene with its own promoter or by the nucleic acid construct according to the invention. Replacement of the El gene region which is a precondition for 35 expression of the downstream adenoviral genes results in an adenovirus not capable of replication. These - 10 viruses are then able to replicate only in a cell line which replaces the missing El genes. Replication-deficient adenoviruses are therefore generally formed by homologous recombination 5 in the so-called 293 cell line (human embryonic kidney cell line) which has a copy of the El region stably integrated into the genome. For this purpose, the nucleic acid according to the invention is cloned into recombinant adenoviral plasmids under the control of 10 its own promoter, for example the troponin C promoter mentioned above. Homologous recombination then takes place with an El-deficient adenoviral genome such as, for example, d1327 or de11324 (adenovirus 5) in the 293 helper cell line. Where recombination is successful, 15 viral plaques are harvested. The replication-deficient viruses produced in this way are employed in high titres (for example 109 to 10" plaque forming units) for infecting the cell culture or for somatic gene therapy. 20 The exact site of insertion of the nucleic acid according to the invention into the adenoviral genome is in general not critical. It is, for example, also possible to clone the nucleic acid according to the invention in place of the deleted E3 gene (Karlsson, S. 25 et al. (1986), EMBO J. 5, 2377 - 2385). However, it is preferred for the El region or parts thereof, for example the ElA or ElB region (see, for example, WO 95/00655), to be replaced by the nucleic acid according to the invention, especially 30 when the E3 region is also deleted. However, the present invention is not confined to the adenoviral vector system; on the contrary, adeno-associated virus vectors are also particularly suitable in combination with the nucleic acid according 35 to the invention for the following reasons. The AAV virus belongs to the family of parvoviruses. These are distinguished by an - 11 icosahedral, non-enveloped capsid which has a diameter of 18 to 30 nm and which contains a linear, single stranded DNA of about 5 kb. For efficient replication of AAV, coinfection of the host cell with helper 5 viruses is necessary. Examples of suitable helpers are adenoviruses (Ad5 or Ad2), herpesviruses and vaccinia viruses (Muzyczka, N. (1992) Curr. Top. Microbiol. Immunol. 158, 97-129) . In the absence of a helper virus, AAV passes into a latency state where the virus 10 genome is able to integrate stably into the host cell genome. The property of AAV integrating into the host genome makes it particularly interesting as transduction vector for mammalian cells. Generally sufficient for the vector functions are the two 15 inverted terminal repeats (ITR: see, for example, WO 95/23867) which are about 145 bp long. They carry the signals necessary in "cis" for replication, packaging and integration into the host cell genome. For packaging into recombinant vector particles, a vector 20 plasmid which harbours the genes for non-structural proteins (rep proteins) and for structural proteins (cap proteins) is transfected into adenovirus-infected cells. After a few days, a cell-free lysate containing, besides the recombinant AAV particles, also 25 adenoviruses is prepared. The adenoviruses can advantageously be removed by heating at 56 0 C or by banding in a caesium chloride gradient. It is possible with this cotransfection method to achieve rAAV titres of 105 to 106 IE/ml. Contamination by wild-type viruses 30 is below the detection limit if the packaging plasmid and the vector plasmid have no overlapping sequences (Samulski, R.J. (1989) J. Virol. 63, 3822 - 3828) . Transfer of the nucleic acid according to the invention into somatic cells can be effected by AAV 35 into resting, differentiated cells, which is particularly advantageous for gene therapy of the heart. The ability to integrate which has been 71 - 12 mentioned also ensures long-lasting gene expression in vivo, which in turn is particularly advantageous. A further advantage of AAV is that the virus is not pathogenic for humans and is relatively stable in vivo. 5 Cloning of the nucleic acid according to the invention into the AAV vector or parts thereof takes place by methods known to the skilled person, as described, for example, in WO 95/23867, in Chiorini, J.A. et al. (1995), Human Gene Therapy 6, 1531-1541 or Kotin, R.M. 10 (1994), Human Gene Therapy 5, 793-801. Vectors effective for gene therapy can also be obtained by complexing the nucleic acid according to the invention with liposomes, because it is possible thereby to achieve a very high transfection efficiency, 15 in particular of myocardial cells (Felgner, P.L. et al. (1987), Proc. Natl. Acad. Sci USA 84, 7413-7417) . In lipofection, small unilamellar vesicles of cationic lipids are prepared by ultrasound treatment of the liposome suspension. The DNA is bound ionically to the 20 surface of the liposomes, specifically in a ratio such that a positive net charge remains and the plasmid DNA is 100% complexed by the liposomes. Besides the lipid mixtures DOTMA (1, 2 -dioleoyloxypropyl-3-trimethyl ammonium bromide) and DOPE (dioleoylphosphatidyl 25 ethanolamine) employed by Felgner et al. (1987, supra), numerous new lipid formulations have now been synthesized and tested for their efficiency in transfecting various cell lines (Behr, J.P. et al. (1989), Proc. Natl. Acad. Sci. USA 86, 6982-6986; 30 Felgner, J.H. et al. (1994) J. Biol. Chem. 269, 2550-2561; Gao, X. & Huang, L. (1991), Biochim. Biophys. Acta 1189, 195-203) . Examples of the novel lipid formulations are DOTAP N-[1-(2,3 dioleoyloxy) propyl] -N, N, N-trimethylammonium ethyl 35 sulphate or DOGS (TRANSFECTAM; dioctadecyl amidoglycylspermine). One example of the preparation of DNA-liposome complexes and successful use thereof in

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- 13 heart-specific transfection is described in DE 44 11 402. For use of the nucleic acid according to the invention in gene therapy, it is also advantageous if 5 the part of the nucleic acid which codes for the polypeptide contains one or more noncoding sequences, including intron sequences, preferably between the promoter and the start codon of the polypeptide, and/or a polyA sequence, in particular the naturally occurring 10 polyA sequence or an SV40 virus polyA sequence, especially at the 3' end of the gene, because this makes it possible to stabilize the mRNA in the myocardial cell (Jackson, R.J. (1993) Cell 74, 9-14 and Palmiter, R.D. et al. (1991) Proc. Natl. Acad. Sci. USA 15 88, 478-482). The present invention further relates to the polypeptide itself having an amino acid sequence as shown in Fig. 4 or a functional variant thereof, and parts thereof having at least 6 amino acids, preferably 20 having at least 12 amino acids, in particular having at least 15 amino acids and especially having at least 164 amino acids, except a polypeptide having the sequence: PTRNPTTVQPWSLQRCIKVNEHITNVNVESNFITGKGILAIMRALQ 10 20 30 40 HNTVLTELRFHNQRHIMGSQVEMEIVKLLKENTTLLRLGYHFKLPG 50 60 70 80 90 25 The polypeptide is prepared, for example, by expression of the nucleic acid according to the invention in a suitable expression system as described above using methods generally known to the skilled person. Examples of suitable host cells are the E. coli 30 strains DH5, HB101 or BL21, the yeast strain Saccharomyces cerevisiae, the lepidopteran insect cell line for example Spodoptera frugiperda, or the animal - 14 cells COS, Vero, 293 and HeLa, all of which are generally obtainable. The said parts of the polypeptide can also be synthesized by classical synthesis (Merrifield 5 technique). They are particularly suitable for obtaining antisera which can be used to screen suitable gene expression banks in order thus to obtain further functional variants of the polypeptide according to the invention. 10 The present invention therefore relates also to antibodies which react specifically with the polypeptide having an amino acid sequence as shown in Fig. 4 or a functional variant thereof, and parts thereof having at least 6 amino acids, preferably 15 having at least 12 amino acids, in particular having at least 15 amino acids and especially having at least 164 amino acids, the abovementioned parts of the polypeptide either themselves being immunogenic or being able to be made immunogenic, or have their 20 immunogenicity increased, by coupling to suitable carriers such as, for example, bovine serum albumin. The antibodies are either polyclonal or monoclonal. The preparation, to which the present invention also relates, takes place, for example, by 25 generally known methods, by immunizing a mammal, for example a rabbit, with the said polypeptide or the said parts thereof, where appropriate in the presence of, for example, Freund's adjuvant and/or aluminium hydroxide gels (see, for example, Diamond, B.A. et al. 30 (1981) The New England Journal of Medicine, 1344-1349) . The polyclonal antibodies raised in the animal on the basis of an immunological response can then easily be isolated from the blood by generally known methods and purified, for example, by column chromatography. It was 35 thus possible, for example, to produce in rabbits a polyclonal antiserum against a polypeptide which had amino acids 1-90 according to the invention, as shown

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- 15 in Fig. 4, which was expressed as fusion protein in bacteria and purified by affinity chromatography. The antibodies according to the invention specifically recognized the corresponding protein of about 80 kD in 5 extracts of human heart tissue. Monoclonal antibodies can be prepared, for example, by the known method of Winter & Milstein (Winter, G. & Milstein, C. (1991) Nature, 349, 293-299). 10 The present invention also relates to a medicinal product which contains a nucleic acid coding for a polypeptide having an amino acid sequence as shown in Fig. 4 or a functional variant thereof and the abovementioned parts thereof having at least 8 15 nucleotides, or a polypeptide having an amino acid sequence as shown in Fig. 4 or a functional variant thereof and the abovementioned parts thereof having at least 6 amino acids and, where appropriate, suitable additives or excipients, and to a process for producing 20 a medicinal product for treating cardiac disorders, in particular cardiac insufficiency, in which a said nucleic acid or a said polypeptide is formulated with a pharmaceutically acceptable carrier. One example of the use of nucleic acid 25 fragments as therapeutic agent is the use of DNA fragments in the form of antisense oligonucleotides (Uhlmann, E. & Peyman, A. (1990) Chemical Reviews, 90, 543-584, No. 4) . A particularly suitable medicinal product for 30 use for human gene therapy is one which contains the said nucleic acid in naked form or in the form of one of the vectors effective for gene therapy which are described above, or in a form complexed with liposomes. The pharmaceutical carrier is, for example, a 35 physiological buffer solution, preferably with a. pH of about 6.0-8.0, preferably of about 6.8-7.8, in particular of about 7.4 and/or an osmolarity of about - 16 200-400 milliosmol/litre, preferably of about 290-310 milliosmol/litre. The pharmaceutical carrier may additionally contain suitable stabilizers such as, for example, nuclease inhibitors, preferably complexing 5 agents such as EDTA and/or other excipients known to the skilled person. The said nucleic acid is normally administered intravenously, for example with the aid of a catheter, where appropriate in the form of the virus vectors 10 described in detail above or as liposome complexes. It is advantageous, for example, to infuse the nucleic acid according to the invention directly into the patient's coronary arteries (so-called percutaneous coronary gene transfer, PCGT), in particular in the 15 form of recombinant adenovirus vectors or adeno associated virus vectors. Administration with the aid of a balloon catheter is particularly preferred because it is possible thereby to confine the transfection not only to the heart but also to the injection site within 20 the heart (see, for example, Feldman, L.J. et al. (1994) JACC 235A, 906-934). It is also possible to administer the polypeptide itself intravenously or with the aid of a catheter or balloon catheter, where appropriate with 25 suitable additives or excipients, such as, for example, physiological saline, stabilizers, proteinase inhibitors etc., in order to influence the function of the heart immediately and directly. The present invention further relates to a 30 diagnostic aid containing a nucleic acid, a polypeptide or antibody according to the present invention and, where appropriate, suitable additives or excipients and to a process for producing a diagnostic aid for diagnosing cardiac disorders, in particular cardiac 35 insufficiency, in which a nucleic acid, a polypeptide or antibody according to the present invention is mixed with suitable additives or excipients.

- 17 It is possible, for example, according to the present invention to produce on the basis of the said nucleic acid a diagnostic aid based on the polymerase chain reaction (PCR diagnosis, for example as disclosed 5 in EP-0 200 362) or on a Northern blot as described in detail in Example 3 using the 321 bp DNA fragment according to the invention as probe. These tests are based on the specific hybridization of said nucleic acids with the complementary strand, normally of the 10 corresponding mRNA. The nucleic acid may also in this case be modified as described, for example, in EP 0 063 879. A DNA fragment, in particular the DNA fragment described in Example 1, is preferably labelled using suitable reagents, for example radioactively with 15 a- 32 P-dCTP or non-radioactively with biotin, by generally known methods and incubated with isolated RNA, which has preferably been pre-bound to suitable membranes made of, for example, cellulose or nylon. It is additionally advantageous, before the hybridization 20 and binding to a membrane, for the isolated RNA to be fractionated according to size, for example by agarose gel electrophoresis. With the same amount of investigated RNA from each tissue sample, it is thus possible to determine the amount of mRNA specifically 25 labelled by the probe. It is thus possible by using the diagnostic aid according to the invention also to measure a cardiac tissue sample in vitro specifically for the strength of expression of the corresponding gene in order to be 30 able to diagnose reliably possible cardiac insufficiency (see Example 1). A cDNA having a sequence as shown in Fig. 1 is particularly suitable for diagnosing a possible cardiac insufficiency (see Example 2). 35 A further diagnostic aid contains the polypeptide according to the present invention or the immunogenic parts thereof described above in detail.

- 18 The polypeptide or the parts thereof, which are preferably bound to a solid phase, for example made of nitrocellulose or nylon, can, for example, be brought into contact in vitro with the body fluid to be 5 investigated, for example blood, in order to react for example with autoimmune antibodies. The antibody peptide complex can then be detected for example by means of labelled antihuman IgG or antihuman IgM antibodies. The label is, for example, an enzyme such 10 as peroxidase which catalyses a colour reaction. The presence and the amount of autoimmune antibody present can thus be detected easily and rapidly by the colour reaction. Another diagnostic aid contains the antibodies 15 according to the invention themselves. These antibodies can be used, for example, for investigating a cardiac tissue sample easily and quickly to find whether the relevant polypeptide is present in an increased amount, in order thus to obtain information about possible 20 cardiac insufficiency. In this case, the antibodies according to the invention are labelled for example with an enzyme, as already described above. The specific antibody-peptide complex can thus be detected easily and equally quickly by an enzymatic colour 25 reaction. The present invention also relates to a test for identifying functional interactors containing a nucleic acid according to the invention coding for a polypeptide having an amino acid sequence as shown in 30 Fig. 4 or a functional variant thereof and the abovementioned parts thereof having at least 8 nucleotides, a polypeptide having the amino acid sequence as shown in Fig. 4 or a functional variant thereof, and the abovementioned parts thereof having at 35 least 6 amino acids or the antibodies according .to the invention and, where appropriate, suitable additives or excipients.

- 19 A suitable test for identifying functional interactors is, for example, the so-called two-hybrid system (Fields, S. & Sternglanz, R. (1994) Trends in Genetics, 10, 286-292). 5 In this test, a cell, for example a yeast cell, is transformed or transfected with one or more expression vectors which express a fusion protein which contains a polypeptide according to the present invention and a DNA binding domain of a known protein, 10 for example of Gal4 or LexA from E. coli, and/or expresses a fusion protein which contains an unknown polypeptide and a transcription activating domain, for example of Gal4, herpes virus VP16 or B42. The cell additionally contains a reporter gene, for example the 15 lacZ gene from E. coli, green fluorescence protein or the yeast amino acid biosynthesis genes His3 or Leu2, which is controlled by regulatory sequences, such as, for example, the LexA promoter/operator or by a so called upstream activation sequence (UAS) of yeast. The 20 unknown polypeptide is encoded, for example, by a DNA fragment which is derived from a gene bank, for example from a human cardiac tissue-specific gene bank. Normally a cDNA gene bank is produced directly, using the expression vectors described, in yeast so that the 25 test can be carried out immediately thereafter. For example, a nucleic acid according to the present invention is cloned into a yeast expression vector in a functional unit with the nucleic acid coding for the LexA DNA binding domain, so that a 30 fusion protein consisting of the polypeptide according to the invention and the LexA DNA binding domain is expressed in the transformed yeast. In another yeast expression vector, cDNA fragments from a cDNA gene bank are cloned in a functional unit with the nucleic acid 35 coding for the Gal4 transcription activating domain, so that a fusion protein consisting of an unknown polypeptide and the Gal4 transcription activating In, - 20 domain is expressed in the transformed yeast. The yeast which is transformed with the two expression vectors and is, for example, Leu2~ additionally contains a nucleic acid which codes for Leu2, and is controlled by 5 the LexA promoter/operator. In the event of a functional interaction between the polypeptide according to the invention and the unknown polypeptide, the Gal4 transcription activating domain binds via the LexA DNA binding domain to the LexA promoter/operator, 10 whereby the latter is activated and the Leu2 gene is expressed. The result of this is that the Leu2~ yeast is able to grow on minimal medium which contains no leucine. On use of the lacZ or green fluorescence 15 protein reporter gene in place of an amino acid biosynthesis gene, activation of transcription can be detected by the formation of blue or green-fluorescing colonies. The blue or fluorescent coloration can also be quantified easily in a spectrophotometer, for 20 example at 585 nm in the case of a blue coloration. Thus, it is possible to screen expression gene banks easily and quickly for polypeptides which interact with a polypeptide according to the present invention. It is then possible for the novel peptides 25 found to be isolated and further characterized. Another possible use of the two-hybrid system is the influence on the interaction between a polypeptide according to the present invention and a known or unknown polypeptide by other substances such 30 as, for example, chemical compounds. Thus, it is also possible to find easily novel and valuable active substances which can be chemically synthesized and can be employed as therapeutic agent for treating a cardiac disorder. The present invention is therefore not 35 restricted to a method for finding polypeptide-like interactors, but also extends to a method for finding substances which are able to interact with the protein- - 21 protein complex described above. Such polypeptide-like, as well as chemical interactors are therefore referred to as functional interactors for the purpose of the present invention. 5 The surprising advantage of the present invention is thus the possibility of using the subject matters according to the invention for specific and reliable diagnosis and therapy of cardiac disorders, especially cardiac insufficiency. However, other 10 valuable therapeutic and diagnostic possibilities also emerge. For example, the functional interactors which can be easily identified using the described test methods are so advantageous because it is possible with their aid in the form of suitable medicinal products to 15 influence deliberately the activity of the polypeptide according to the invention in its natural environment in the myocardium and thus also the contractility of the myocardial cells, in particular since the activity of this polypeptide can be regulated as already 20 described in detail above. The following figures and examples are intended to illustrate the invention in detail without restricting it. 25 Description of the figures Fig. 1 shows a 1936 nucleotide-long heart specific DNA sequence. The region which codes for the corresponding polypeptide is shown in bold. The DNA fragment from Example 1 is underlined. 30 Fig. 2 shows a 2080 nucleotide-long heart specific DNA sequence which has an extension at the 5' end of the DNA sequence from Fig. 1. The region which codes for the corresponding polypeptide is once again shown in bold. 35 Fig. 3 shows a 2268 nucleotide-long heart specific DNA sequence which has an extension at the 5' end of the DNA sequence from Fig. 1 or Fig. 2. The - 22 region which codes for the corresponding polypeptide is likewise shown in bold. Fig. 4 shows a 552 amino acid-long polypeptide sequence encoded by one of the DNA sequences shown in 5 Figs. 1-3. The regions homologous with human tropomodulin are shown in bold. The sequence motifs which indicate regulation of the polypeptide by tyrosine kinase signal transduction pathways are underlined. 10 Figs. 5a and 5b show Northern blots of mRNAs which correspond to the nucleic acid sequences shown in Figs. 1-3 for detecting expression in various human tissues (Fig. 5a) and for detecting expression in healthy and insufficient human cardiac tissue (Fig. 15 5b). Examples 1. Isolation of a DNA fragment from human insufficient cardiac tissue 20 Complete RNA was initially isolated by standard methods (Chomczynski & Sacchi (1987), Anal. Biochem, 162 (1), 156-159) from a healthy and an insufficient cardiac tissue sample. The RNA was then treated with DNAse in order to remove DNA contamination. An aliquot 25 of this RNA (0.2 pag) was then incubated in a 20 pl reaction mix with 1 x RT buffer (Gibco Y00121), 10 mM DTT, 20 pM dNTP mix, 1 U/pl RNAsin (Promega N2511), 1 pM 3' anchor primer mixture of the 5'-T 1 2 ACN-3' type, where N can be any deoxynucleotide, and 10 U/pl 30 SuperScript RNAse H~ reverser transcriptase at 37 0 C for 60 min and thus transcribed into cDNA. A cDNA aliquot was then subjected to a 20 pl PCR in 1 x PCR buffer (Perkin-Elmer) which, besides 1 pm 3' primer T 12 AC and 1 pM 5'-decamer primer (5'-CCTTCTACCC-3'), contains 35 10 pCi of a- 3 2 P-dCTP, 2 pM dNTP mix and 1 U of AmpliTaq (Perkin Elmer) . The mixture was incubated firstly at 94 0 C for 1 min, then 40 cycles each of 30 s at 94 0

C,

- 23 40*C for 2 min and 720C for 30 s and finally at 720C for 10 min. The resulting DNA fragment mixture was then fractionated on a 6% polyacrylamide gel and autoradiographed. A DNA fragment which is 321 bp in 5 length and which is not present in the healthy heart sample but is distinctly present in the insufficient heart sample is thus prepared. This fragment was then cut out of the gel on the basis of the X-ray film and was reamplified by PCR under the conditions already 10 described. The resulting fragment was then cloned into an appropriate vector, and the DNA sequence was determined. A fragment prepared in this way contains nucleotides 1627-1936 of the sequence according to Claim 1 and the 12 thymine nucleotides from the 3' 15 anchor primer. 2. Isolation of heart-specific nucleic acids A plaque hybridization was carried with a cDNA gene bank from cardiac tissue under standard conditions 20 (see Sambrook, J., Frisch, E.F. & Maniatis, T. (1989) Molecular Cloning, A Laboratory Manual, ch. 8-10) using an cc- 32 P-dCTP-labelled DNA fragment from Example 1 which comprises the nucleotides from position 1627-1936 in Fig. 1. The cDNAs found were then isolated and 25 sequenced. The sequences are shown in Figs. 1-3. It emerged from this that the cDNA having the sequence shown in Fig. 1 could be isolated with greater probability from insufficient cardiac tissue than the cDNA having the sequence shown in Fig. 2 or 3, which 30 could be isolated with greater probability from healthy cardiac tissue. 3. Detection of the strength of expression of the heart-specific gene in various human tissues by 35 means of Northern blots. The DNA fragment 321 bp in length already described in Examples 1 and 2 and Fig. 1 was firstly - 24 radiolabelled with a- P-dCTP by the random primer labelling method (Feinberg, A.P. & Vogelstein, B. (1983) Anal. Biochem., 132, 6). The RTS RadPrime DNA labelling system (GibcoBRL 10387-017) was used for this 5 purpose. The hybridization of blots with poly A+ RNA from human tissues (see Figs. 5a and 5b) took place at 68 0 C for 1 hour in accordance with the manufacturer's instructions (Multiple Tissue Northern Blots I & II, Clontech Laboratories GmbH, Heidelberg, #7760-1, 10 #7759-1) in ExpressHyb hybridization solution (Clontech #8015-1) . The blots were then washed with 2 x SSC and 0.05% SDS for 30 minutes and thereafter with 0.1 x SSC and 0.1% SDS for 1 hour and autoradiographed. It emerged that the probe 321 bp in length hybridizes 15 strongly with a polyA* RNA of about 2400 bp strongly in cardiac tissue and skeletal muscle, very weakly in prostate tissue and not in leucocytes, large intestinal, small intestinal, ovarian, testicular, thymus, splenic, renal, hepatic, lung, placental and 20 brain tissue (Fig. 5a). Expression of the corresponding RNAs in healthy and insufficient cardiac tissue was also investigated. Complete RNA was isolated from various human cardiac tissue samples for this purpose (Chomczynski & Sacchi 25 (1987), Anal. Biochem. 162, 156-159). Subsequently in each case 10 pg of RNA were fractionated using a 1% formaldehyde agarose gel and transferred by the capillary method to a charged nylon membrane (Zeta Probe GT BioRad #162-0197). The membrane was briefly 30 washed with 2 x SSC and then baked at 800C for 30 minutes. The membranes were incubated with prehybridization solution (0.5 M Na 2

HPO

4 , pH 7.2; 7% SDS) at 65 0 C for at least 1 hour. The solution was then replaced by a fresh solution, and the radioactive, 35 heat-denatured probe was added. The hybridizatiQn was carried out at 650C for 15 hours. The membranes were then washed firstly with 40 mM Na 2 HP0 4 pH 7.2; 5% SDS - 25 at 65 0 C for 15 hours and then with 40 mM Na 2

HPO

4 , pH 7.2; 1% SDS at 65*C for 2 x 30 minutes, and subsequently autoradiographed. It emerged that various RNA species having a length from about 2200 to 2400 bp 5 were fractionated in 1% agarose gels. These different species correspond well with the sizes of the three cDNAs found, including an average polyA tail 150 bp long (see Figs. 1-3) . In particular, the smallest RNA species was more clearly detectable in diseased tissue 10 than in healthy tissue. Quantification of the blot using a PhosphorImager and the ImageQuant software (Molecular Dynamics GmbH, Krefeld), taking into account a control hybridization with 04-thymosin and actin, revealed an approximately 35% increased expression of 15 the detected RNAs in insufficient cardiac tissue by comparison with healty tissue.

Claims (15)

1. Nucleic acid coding for a polypeptide having an amino acid sequence as shown in Fig. 4 or a functional variant thereof, and parts thereof having at least 8 nucleotides, except a nucleic acid having the sequence: 10 1 GCCAACACGC ANTCCGACGA CAGTGCAGCC ATGGTCATTG CAGAGATGCN TCAAASTZA 61 TGAGCACATC ACCAACGTAA ACGTCGAGTC CAACTTCATA ACGGGAAAGG GGAT :'t:3C 121 :ATCATGAGA GCTCTCCAGC ACAACACGGT GCTCACGGAG CTGCGTTTCC ATAA::AGAG 111 GCACATCATG GGCAGCCAGG TGGkATGGA GATTGTCAAG CTNC-GAAGG AGAA:C.: :C 241 GCTNCTGAGG CTGGGNTACC ATTTTNAACT CCCAGGACC

2. Nucleic acid according to Claim 1, characterized in that the nucleic acid is a DNA or RNA, 15 preferably a DNA, in particular a double-stranded DNA.

3. Nucleic acid according to Claim 1 or 2, characterized in that the nucleic acid contains a DNA having a nucleic acid sequence as shown in Fig. 1, 2 or 3. 20

4. Nucleic acid according to any of Claims 1-3, characterized in that the nucleic acid is present in a vector, preferably in an expression vector or vector effective for gene therapy.

5. Nucleic acid according to any of Claims 1-4, 25 characterized in that the part of the nucleic acid which codes for the polypeptide contains one or more noncoding sequences and/or a polyA sequence.

6. Process for the preparation of a nucleic acid according to any of Claims 1-5, characterized in that 30 the nucleic acid is chemically synthesized or isolated from a gene bank using a probe.

7. Polypeptide having an amino acid sequence as shown in Fig. 4 or a functional variant thereof, and - 38 parts thereof having at least 6 amino acids, except a polypeptide having the sequence: PTRNPTTVQPWSLQRCIKVNEHITNVNVESNFITGKGILAIMRALQ 10 20 30 40 HNTVLTELRFHNQRHIMGSQVEMEIVKLLKENTTLLRLGYHFKLPG 50 60 70 80 90 5

8. Process for the preparation of a polypeptide according to Claim 7, characterized in that a nucleic acid according to any of Claims 1-3 is expressed in a suitable host cell.

9. Antibody against a polypeptide having an amino 10 acid sequence as shown in Fig. 4 or a functional variant thereof, and parts thereof having at least 6 amino acids.

10. Process for the preparation of an antibody according to Claim 9, characterized in that a mammal is 15 immunized with a polypeptide having an amino acid sequence as shown in Fig. 4 or a functional variant thereof, and parts thereof having at least 6 amino acids, and the resulting antibodies are isolated.

11. Medicinal product containing a nucleic acid 20 coding for a polypeptide having an amino acid sequence as shown in Fig. 4 or a functional variant thereof, and parts thereof having at least 8 nucleotides, or a polypeptide having an amino acid sequence as shown in Fig. 4 or a functional variant thereof, and parts 25 thereof having at least 6 amino acids, and, where appropriate, a pharmaceutically acceptable carrier.

12. Process for the preparation of a medicinal product for treating cardiac disorders, characterized in that a nucleic acid coding for a polypeptide having 30 an amino acid sequence as shown in Fig. 4 or a functional variant thereof, and parts thereof having at least 8 nucleotides, or a polypeptide having an amino acid sequence as shown in Fig. 4 or a functional - 39 variant thereof, and parts thereof having at least 6 amino acids, is formulated with a pharmaceutically acceptable carrier.

13. Diagnostic aid containing a nucleic acid coding 5 for a polypeptide having an amino acid sequence as shown in Fig. 4 or a functional variant thereof, and parts thereof having at least 8 nucleotides, a polypeptide having an amino acid sequence as shown in Fig. 4 or a functional variant thereof, and parts 10 thereof having at least 6 amino acids, or an antibody according to Claim 9 and, where appropriate, suitable additives or excipients.

14. Process for the preparation of a diagnostic aid for diagnosing cardiac disorders, characterized in that 15 a nucleic acid coding for a polypeptide having an amino acid sequence as shown in Fig. 4 or a functional variant thereof, and parts thereof having at least 8 nucleotides, a polypeptide having an amino acid sequence as shown in Fig. 4 or a functional variant 20 thereof, and parts thereof having at least 6 amino acids, or an antibody according to Claim 9, is mixed with a pharmaceutically acceptable carrier.

15. Test for identifying functional interactors containing a nucleic acid coding for a polypeptide 25 having an amino acid sequence as shown in Fig. 4 or a functional variant thereof, and parts thereof having at least 8 nucleotides, or a polypeptide having an amino acid sequence as shown in Fig. 4 or a functional variant thereof, and parts thereof having at least 6 30 amino acids, and, where appropriate, suitable additives or excipients.