WO1996001316A1 - NOVEL GROWTH OR DIFFERENTIATION FACTOR OF THE TGF-β FAMILY - Google Patents

Abstract

The invention concerns a protein of the TGF-β family, the DNA which codes for it and a pharmaceutical composition containing such a protein.

Description

New growth / differentiation factor of the TGF _/ 3 family

description

The present invention relates to a new growth / differentiation factor of the TGF / 3 family and DNA sequences coding therefor.

The TGF-jß family of growth factors include the BMP, TGF and activin / inhibin related proteins (Roberts and Sporn, Handbook of Experimental Pharmacology 95, 419-472 (1990)). They are relevant for a wide range of medical treatment methods and applications. These factors are useful in procedures related to wound healing and tissue repair. Furthermore, several members of the TGF-? Family tissue growth, such as bone growth.

Wozney (Progress in Growth Factor Research 1 (1989), 267-280) and Vale et al. (Handbuch of Experimental Pharmacology 95 (1990), 211-248) describe various growth factors, such as those related to the BMP and activin / inhibin groups. The members of this group have significant structural similarities. The precursor of the protein consists of an amino-terminal signal sequence, a propeptide and a carboxy-terminal sequence of 110 to 140 amino acids, which is cleaved from the precursor and represents the mature protein. Furthermore, their members are defined by an amino acid sequence homology. The mature protein contains the most conserved sequences, particularly seven cysteine residues, which are conserved among family members. The TGF-type proteins are multifunctional, hormonally active growth factors. They also exhibit related biological activities, such as chemotactic attraction of cells, promoting cell differentiation, and tissue-induced skills. EP 0 222 491 AI discloses sequences of inhibin alpha and beta chains.

Overall, the proteins of the TGF-ß family show differences in their structure, which leads to considerable variations in their exact biological function. Furthermore, they are found in a wide range of different tissue types and stages of development. As a result, they may differ in their exact function, e.g. the required cellular physiological environment, their lifespan, their destinations, their requirements for auxiliary factors and their resistance to degradation. Although numerous proteins that show tissue-inductive potential have been described, their natural functions in the organism and, more importantly, their medical relevance still need to be researched in detail. The presence of as yet unknown members of the TGF-ß family, which are important for the differentiation / induction of different types of tissue, is assumed with great probability. A major difficulty in isolating these new TGF- -like proteins, however, is that their functions cannot yet be described precisely enough for the development of a distinctive bioassay. On the other hand, the expected nucleotide sequence homology to known members of the family is too low to allow screening by classic nucleic acid hybridization techniques. Nevertheless, the further isolation and characterization of new TGF-β-like proteins is urgently required in order to provide further induction and differentiation proteins which meet all the desired medical requirements. These factors could find medical application in the healing of damage and the treatment of degenerative diseases of different tissues.

Patent application PCT / EP93 / 00350 specifies a nucleotide and amino acid sequence for the TGF-β protein MP121, with a large part of the sequence corresponding to the mature peptide is specified. The full sequence of the MP121 propeptide is not disclosed.

The object on which the present invention is based is to provide DNA sequences which code for new members of the TGF-β protein family with mitogenic and / or differentiation-inductive potential. In particular, the object of the present invention is to provide the complete DNA and amino acid sequence of the TGF protein MP121.

This object is achieved by a DNA molecule which codes for a protein of the TGF-ß family and

(a) the portion coding for the mature protein and, if appropriate, further functional portions of the portion shown in SEQ ID NO. 1 nucleotide sequence shown,

(b) a nucleotide sequence corresponding to the sequence from (a) in the context of the degeneration of the genetic code,

(c) a nucleotide sequence corresponding to an allelic derivative of one of the sequences from (a) and (b), or

(d) a sequence which differs from sequence (a) due to its origin from other vertebrates,

(e) a sequence which hybridizes with one of the sequences from (a), (b), (c) or (d), provided that a DNA molecule according to (e) at least corresponds to a mature protein of the TGF contains β-family coding portion.

Further embodiments of the present invention relate to the subject matter of claims 2 to 10. Other features and advantages of the invention emerge from the description of the preferred embodiments. The sequence listings and drawings are now briefly described.

SEQ ID NO. 1 shows the complete nucleotide sequence of the DNA coding for the human TGF-β protein MP121. The ATG start codon begins with nucleotide 128. The start of the fully mature protein particularly preferably begins with nucleotide 836.

SEQ ID NO.2 shows the complete amino acid sequence of the prepro protein of the human TGF-β protein MP121, which was derived from the nucleotide sequence shown in SEQ ID NO.l. The start of the mature protein is preferably in the range of amino acids 217-240, particularly preferably at amino acid 236 or 237, most preferably at amino acid 237.

SEQ ID NO.3 shows the complete nucleotide sequence of the DNA coding for the TGF-β protein MP121 from mouse. The coding region begins at the ATG start codon with nucleotide 131 and ends at the stop codon beginning with position 1187. The start of the preferred mature protein begins with nucleotide 839. In the genomic DNA there is an approximately 5.5 kb between position 446 and 447 big intron.

SEQ ID NO.4 shows the complete amino acid sequence of the prepro protein of the TGF-ß protein MP121 from mouse, which was derived from the nucleotide sequence shown in SEQ ID NO.3. The beginning of the mature protein is analogous to the human MP121 in SEQ ID NO.2 in the range of amino acids 217-240. Most preferably, the mature protein begins at amino acid 237, so that the mature portion, like the human MP121, consists of 116 amino acids. Members of the TGF-ß family are often cut behind an RXXR cleavage site to separate the mature portion from the precursor (see Özkaynak et al., J.Biol.Chem. 267, 25220-25227 (1992) and the literature cited therein). In the case of MP121 from mouse, it is therefore also conceivable for the mature protein to begin at least in part with amino acid 236.

SEQ ID NO.5 shows the nucleotide sequence of the human MP121 gene at the exon / intron junctions. The nucleotides from both exons are identified by upper case letters, those of the intron by lower case letters. Figure 1 shows a comparison of the amino acid sequence of human MP121 with some members of the TGF-ß family (inhibin α and ß chains) starting at the first of the seven conserved cysteine residues. * means that the amino acid is the same in all compared proteins; + means that the amino acid in at least one of the proteins matches in comparison to human MP121.

Figure 2 shows the nucleotide sequences of the oligonucleotide primers used in the present invention and a comparison of these sequences with known members of the TGF-β family. M means A or C, S means C or G, R means A or G and K means G or T. 2a shows the sequence of the primer OD, 2b shows the sequence of the primer OID.

Figure 3 shows a schematic Western blot with chicken antibodies against human MP121.

FIG. 4 shows the expression of MP121 in comparison to activin β _A and β _B in different mouse tissues.

FIG. 5 shows a positive influence on the survival of dopaminergic neurons by treatment with partially cleaned MP121.

In the context of the present invention, the term "mature protein" also includes functional subregions of the total protein which have essentially the same biological activity and preferably those subregions which contain at least the region of the seven cysteines conserved in the TGF-β family include. In particular, it is possible here that the N-terminus of the mature proteins is slightly modified, that is to say deviates from the sequences shown in SEQ ID NO.2 and 4. Additional amino acids that do not influence the functionality of the protein can be present here as well as amino acids missing, as far as the functionality is not compromised in this case either. is limited. However, it is preferred that the human and the mouse protein contain all amino acids from amino acid 237 of the amino acid sequence shown in SEQ ID NO.2 and SEQ ID NO.4. It is already known from other family members of the TGF-β family that the addition of additional amino acids to the N-terminus of the mature protein does not impair the activity, with 6 additional histidines being attached to the N-terminus, among other things.

The present invention thus includes the portion coding for the mature protein as defined above and optionally further functional portions of the nucleotide sequence shown in SEQ ID NO.1 as well as sequences which correspond to this sequence in the context of the degeneration of the genetic code and all derivatives of such sequences . Furthermore, the present invention also encompasses DNA sequences which code for a protein of the TGF-β family which have been obtained from other mammals and which have a sequence which differs to a small extent on account of their origin, but for proteins with the same biological function and in principle also code only slightly different sequence. Such sequences have very large correspondences with one another, as can be seen from a comparison of SEQ ID NO.1 and NO.3.

Furthermore, the present invention also comprises sequences hybridizing with such sequences, provided that such a DNA molecule completely contains at least the portion coding for a mature protein of the TGF-β family (according to the above definition) and that the biological activity is retained.

The term “functional part” in the sense of the present invention means a protein part which is capable of acting, for example, as a signal peptide, propeptide or mature protein part, ie performing at least one of the biological functions of the natural parts of MP121. In the case of the preferred human MP121, the region coding for the mature portion of the protein preferably extends from nucleotide 836 to the stop codon, which begins at nucleotide 1184 of the sequence shown in SEQ ID No. 1. Optionally, the DNA molecule can also comprise further functional parts of the sequence shown in SEQ ID NO.1, namely the nucleotide sequences coding for the signal and / or propeptide part. The DNA molecule particularly preferably comprises the sequence for the signal and propeptide portion and the portion of the mature protein, ie the nucleotides 128 to 1184 of the sequence shown in SEQ ID NO.1. In the case of the preferred mouse MP121, the region coding for the mature portion of the protein preferably ranges from nucleotide 839 to the stop codon starting from position 1187 of the sequence shown in SEQ ID NO.3. If necessary, the DNA molecule can also comprise further functional parts of the sequence shown in SEQ ID NO.3, namely nucleotide sequences coding for signal and / or propeptide parts.

On the other hand, in addition to the portion coding for the mature protein, the DNA molecules can also contain functional signal and / or propeptide portions of other proteins, for example proteins with cystine knot motif (Cell, Vol. 73 (1993), pp. 421-424) and in particular of other proteins of the TGF-ß family, for example the activin / inhibin or BMP proteins mentioned above, in particular also MP52 (see PCT / EP94 / 02630). The corresponding nucleotide sequences can be found in the above-mentioned references, the disclosure of which is hereby incorporated by reference. It is important that the correct reading frame for the mature protein is preserved. Depending on the host cells in which the expression takes place, the presence of another signal sequence and / or a different propeptide portion could have a positive influence on the expression. The exchange of propeptide portions by corresponding portions of other proteins is, for example, in Mol. Endocrinol. 5 (1991), 149-155 and Proc. Natl. Acad. Be. USA 90 (1993), 2905-2909. Although the allelic, degenerate, other vertebrate and hybridizing sequences encompassed by the present invention have structural differences due to slight changes in the nucleotide or / and amino acid sequence, the proteins encoded by such sequences still have essentially the same useful ones Properties that enable their use in basically the same medical applications.

According to the present invention, the term "hybridization" means customary hybridization conditions, preferably conditions with a salt concentration of 6 × SSC at 62 to 66 ° C., followed by a one-hour wash with 0.6 × SSC, 0.1% SDS at 62 to 66 ° C.

Preferred embodiments of the present invention are DNA sequences, as defined above, which are obtainable from vertebrates, preferably mammals, such as pigs, cows and rodents, such as rats or mice, and in particular from primates, such as humans, or corresponding sequences are reproduced.

A particularly preferred embodiment of the present invention are those in SEQ ID NO. 1 and 3 and designated as human or mouse MP121 sequences. The transcripts of MP121 were obtained from liver tissue and code for a protein which shows a considerable amino acid homology to the mature part of the inhibin / activin-like proteins (see FIG. 1). The protein sequences of human a-inhibin, inhibin ß _A

(Activin ß _A ) and inhibin ß _B (activin ß ^ are in Mason et al.

(Biochem. Biophys. Res. Comm. 135, 957-964 (1986)). Some typical sequence homologies specific to known inhibin sequences have also been found in the propeptide part of MP121, while other parts of the propeptide of MP121 show significant differences from inhibin propeptides. So far, however, the expression patterns of MP121 and the activins differ. While activins are mainly expressed in the gonads (activin ß _A in ovaries and activin ß _u in testes and ovaries), MP121 is mainly expressed in the liver. However, the sensitivity in the previous experiments is not yet sufficient to detect even low expression. For the activins, for example, it is described in the literature that expression outside the gonadal expression in various rat tissues both in adult animals (Meunier et al., Proc.Natl. Acad.Sci.USA 85, 247-251 (1988)) as is also detectable during embryonic development (Roberts et al., Endocrinology 128, 3122-3129 (1991)). It is therefore also possible for MP121 that expression in other tissues is still detected.

Another object of the present invention is a vector which contains at least one copy of a DNA molecule according to the invention. In such a vector, the DNA sequence according to the invention is preferably operatively linked to an expression control sequence. Such vectors are suitable for the production of TGF-β-like proteins in stably or transiently transformed cells. Various animal, plant, fungus and bacterial systems can be used for transformation and subsequent cultivation. The vectors according to the invention preferably contain sequences necessary for replication in the host cell and can be replicated autonomously. Furthermore, the use of vectors which contain selectable marker genes is preferred, as a result of which the transformation of a host cell can be detected.

Another object of the invention is a host cell which is transformed with a DNA or a vector according to the invention. Examples of suitable host cells include various eukaryotic and prokaryotic cells such as E.coli, insect cells, plant cells, mammalian cells and fungi such as yeast. Another object of the invention is a protein of the TGF-ß family, which is encoded by a DNA sequence according to claim 1. The protein according to the invention preferably has the amino acid sequence shown in SEQ ID NO.2 or SEQ ID NO.4 or, if appropriate, functional parts thereof (as defined above) and exhibits biological properties, such as tissue inductive capabilities, which may be relevant for therapeutic use are. The above characteristics of the protein may vary depending on the formation of homodimers or heterodimers with other proteins with cystine knot motif and especially TGF-ß proteins. Such structures can also prove to be suitable for clinical applications and therefore also form an object of the present application. Preferred heterodimers include heterodimers composed of a monomer of the protein according to the invention and monomers of the a, βA or βB inhibin chains. The properties resulting from heterodimer formation can be shifted more towards the properties of activin or inhibins. If, for example, a heterodimer is formed with inhibin α proteins or with other inhibin β proteins, it is assumed that the MP 121 / inhibin (α chain) or MP 121 / activin (β _A or β g chain) -Heterodimer could inhibit or activate the production of follicle stimulating hormone (FSH). MP 121 / Aktivin heterodimers could also influence mesoderm development, for example. It can also be expected that heterodimeric forms with a member of the BMP group of TGF-β proteins lead to an increase in BMP-like activities, such as the ability to induce bone formation, cartilage formation or formation of connective tissue or to promote.

The invention therefore furthermore relates to heterodimeric proteins of a protein of the TGF-β family according to the invention, which is encoded by a DNA sequence according to claim 1, with a monomer of a protein with cystine knot motif, preferably another member of the TGF-β- Family. Similar heterodi- Other proteins are described in WO93 / 09229, EP 0 626 451 A2 and J. Biol. Chem. 265 (1990), 13198-13205.

Another object of the invention are chimeric proteins, the functional derivatives or portions of a protein of the invention encoded by a DNA sequence, as shown preferably in SEQ ID NO.2 or SEQ ID NO.4, in particular functional portions of the mature Have protein and also portions of another protein. The other protein can in turn be a protein with cystine knot motif, which preferably also belongs to the TGF-ß family, e.g. especially MP 52 (PCT / EP94 / 02630). However, parts of a completely different protein may also be present, e.g. Receptor-binding domains of proteins which give the original MP 121 protein a different specificity.

The biological properties of the proteins according to the invention, preferably MP121, can e.g. in assays according to Wrana et al. (Cell 71, 1003-1014 (1992)) Ling et al. (Proc. Natl. Acad. Of Science, 82, 7217-7221 (1985)), Takuwa et al. (Am. J. Physiol., 257, E797-E803 (1989)), Fann and Patterson (Proc. Natl. Acad. Of Science, 91, 43-47 (1994)), Broxmeyer et al. (Proc. Natl. Acad. Of Science, 85, 9052-9056 (1988)), Green et al. (Cell, 71, 731-739 (1992)), Partridge et al. (Endocrinology, 108, 213-219 (1981)) or Krieglstein et al. (EMBO J.14, 736-742 (1995)).

Survival-promoting effects on dopaminergic neurons in vitro have been described for activin A and TGF-β 1, -2 and -3 (Krieglstein et al., EMBO J.14, 736-742 (1995) and Krieglstein et al., Neuroscience 63, 1189 -1196 (1994)). For partially purified MP121 it could be shown that the survival of dopaminergic neurons in an 8-day culture is promoted beyond the influence of the control supernatant (FIG. 5).

Another object of the present invention is a method for producing a protein of the TGF-ß family, which is characterized in that a host cell transformed with a DNA or a vector according to the invention is cultivated and the TGF-β protein is obtained from the cell and / or from the culture supernatant. Such a method comprises culturing the transformed host cell in a suitable culture medium and purifying the TGF-β-like protein produced. In this way, the method enables a sufficient amount of the desired protein to be produced for use in medical treatment or in applications using cell culture techniques in which growth factors are required. The host cell can be a bacterium such as Bacillus or E. coli, a fungus such as yeast, a plant cell such as tobacco, potato or Arabidopsis or an animal cell, in particular a vertebrate cell line such as Mo-, Cos - or CHO cell lines or an insect cell line. Using the baculovirus system, expression can also take place in insect larvae. When produced in bacteria, it can happen that the protein according to the invention is produced in the form of inclusion bodies. These inclusion bodies are then renatured according to methods known per se and the protein is then obtained in an active form (see, for example, Jaenicke, R. and Rudolph, R., Protein Structure, ed. Creighton, TE, IRL Press, Chapter 9). For the production of heterodimeric proteins with other members of the TGF-ß family, both protein monomers are expressed either in the same cell or separately, a common renaturation also appearing suitable in the case of forms of inclusion bodies. When coexpressing in the same cell, viral systems such as the baculovirus system or the vaccina virus system are particularly suitable. The production of heterodimeric proteins is known in principle to the person skilled in the art and is described, for example, in WO93 / 09229 and EP 0 626 451 A2.

The production of chimeric proteins with other protein proportions requires a corresponding change at the DNA level, which is familiar to the person skilled in the art and carried out by him (EMBO J. 10 (1991), 2105-2110; Cell 69 (1992), 329-341; J. Neurosci. 39 (1994), 195-210).

Another object of the present invention is to provide pharmaceutical compositions which contain a pharmaceutically effective amount of a TGF-ß-like protein according to the invention as an active ingredient. Such a composition optionally comprises a pharmaceutically acceptable carrier, auxiliary, diluent or filler. Such a pharmaceutical composition can be used in wound healing and tissue restoration alone or in combination with other active ingredients, e.g. other proteins of the TGF-ß family or growth factors such as EGF (epidermal growth factor) or PDGF (platelet derived growth factor) can be used. Such a pharmaceutical composition can also be used in disease prevention.

Further subjects are pharmaceutical compositions which contain heterodimeric proteins and / or chimeric proteins according to the invention.

The pharmaceutical composition according to the invention is preferably used for the treatment and prevention of bone, cartilage, connective tissue, skin, mucous membrane, endothelial, epithelial, neuronal, brain, renal or tooth damage, for use in dental implants , for use in wound healing or tissue restoration processes, as a morphogen for use for inducing liver tissue growth, inducing the proliferation of progenitor cells or bone marrow cells, for maintaining a state of differentiation and for treating fertility disorders or for contraception. Furthermore, the pharmaceutical composition according to the invention can be used in the treatment of metabolic diseases such as diseases of the digestive system or diseases which affect the blood sugar level. Another possible clinical application of the TGF-β-like protein according to the invention is the use as a suppressor of the immune reaction to avoid rejection of organ transplants or an application in connection with angiogenesis. Furthermore, the protein according to the invention can be used for increasing fertility or contraception. The pharmaceutical composition according to the invention can also be used prophylactically or in cosmetic surgery. Furthermore, the use of the composition is not restricted to humans, but can also include animals, in particular domestic and farm animals.

In addition, with heterodimeric proteins and chimeric proteins, the possible use and specificity can also be varied as desired by the proportion of the other protein or other monomer.

In general, diseases which are related to the expression of MP 121 can be treated with the proteins according to the invention, on the one hand by increasing the amount or the activity of MP 121 present, and on the other hand by suppressing the MP 121 activity. Another object of the invention is the production of antisense nucleic acids and ribozymes which inhibit the translation of MP 121. This inhibition can take place either by masking the mRNA with an antisense nucleic acid or by cleavage with a ribozyme.

The production of antisense nucleic acids is known (Weintraub, HM, Scientific American 262: 40 (1990)). The antisense nucleic acids hybridize with the corresponding mRNA and form a double-stranded molecule, which can then no longer be translated. The use of antisense nucleic acids is, for example, from Marcus-Sekura, CJ, Anal. Biochem. 172 (1988), pp. 289-295. Ribozymes are RNA molecules that have the ability to specifically cleave other single-stranded RNA molecules similar to DNA restriction endonucleases. The production of ribozymes is described in Cech, J. Amer. Med. Assn. 260 (1988), p. 3030.

According to the invention, it is also possible to transfect suitable vectors with the DNA sequence according to the invention in vitro or in vivo in patient cells or to transfect the vectors in vitro in cells and then implant them in a patient. MP 121 antisense polynucleotides can also be introduced into cells which show an undesired expression of MP 121.

The MP 121 activity can also be suppressed by binding molecules to the MP 121 receptors which, in contrast to MP 121, do not trigger signal transmission.

The MP 121 receptors on cells are therefore also of interest in the context of the invention. To find receptors, different cell lines can first be tested for their binding behavior by radioactively labeled MP121 ( ¹⁵ J-MP121) with subsequent cross-linking. A cDNA library can subsequently be created from cells that bind MP121 in an expression vector (available from InVitrogen). Cells transfected with receptor cDNA can then be selected by binding radiolabelled MP121. These are methods known to the person skilled in the art, such as those used for the isolation of activin (Mathews, LS & Vale, WW, Cell 65 (1991), 973-982) and TGF-β receptors type II (Lin, HY et al. , Cell 68 (1992), 775-785) were used. In analogy to the known activin receptors, it can be assumed that the MP121 receptor is also a receptor complex belonging to this family, so that other methods known to the person skilled in the art, such as, for example, can be used to find parts of the heteromeric complex PCR with degenerate oligonucleotides can be used. This method is e.g. also used for type I activin and TGF-ß receptors (Tsuchida et al., Proc. Natl. Acad. Sci. USA 90 (1993), 11242-11246; Attisano et al., Cell 75 ( 1993), 671-680; Franzen et al., Cell 75 (1993), 681-692).

Finally, another object of the present invention is an antibody that can bind specifically to the proteins according to the invention, or such an antibody fragment (e.g. Fab or Fab '). Methods for producing such a specific antibody or antibody fragment are well within the ordinary skill in the art. Such an antibody is preferably a monoclonal antibody. Such antibodies or antibody fragments could also be suitable for diagnostic methods.

The following examples further illustrate the invention.

Example 1 Isolation of MP121

1.1 Total RNA was obtained from human liver tissue (40-year-old man) using the Chirgwin et al. (Biochemistry, 18, 5294-5299 (1979)). Poly (A +) RNA was separated from the total RNA by oligo (dT) chromatography according to the manufacturer's instructions (Stratagene Poly (A) Quick columns).

1.2 For the reverse transcription reaction, 1 to 2.5 μg of poly (A +) RNA were heated to 65 ° C. for 5 minutes and quickly cooled on ice. The reaction mixture contained 27 U RNA-Guard (Pharmacia), 2.5 μg oligo (dT) _12-18 (Pharmacia), 5 x buffer (250 mmol / 1 Tris / HCl pH 8.5, 50 mmol / 1 MgCl ₂ , 50 mmol / 1 DTT, 5 mmol / 1 of each dNTP, 600 mmol / 1 KC1) and 20 U AMV reverse transcriptase (Boehringer Mannheim) per μg poly (A +) RNA. The reaction mixture (25 μl) was incubated at 42 ° C. for 2 hours. The cDNA pool was kept at -20 ° C. 1.3 The deoxynucleotide primers OD and OID shown in FIG. 2 were produced on an automatic DNA synthesizer (biosearch). The purification was carried out by denaturing polyacrylamide gel electophoresis and isolation of the main band from the gel by isotachophoresis. The oligonucleotides were designed by comparing the nucleic acid sequences of known members of the TGF-ß family and selecting regions with high conservation. A comparison of this region is shown in Figure 2. To facilitate the cloning, both oligonucleotides contained Eco R1 sites and OD additionally contained an Nco I restriction site at its 5 'terminus.

1.4 20 ng of poly (A +) RNA corresponding cDNA (see 1.2) was used as starting material in the PCR reaction. The reaction was carried out in a volume of 50 μl and contained 1 × PCR buffer (16.6 mmol / 1 (NH ₄ ) ₂ SO ₄ , 67 mmol / 1 Tris / HCl pH 8.8, 2 mmol / 1 MgCl ₂ , 6.7 μmol / 1 EDTA, 10 πvmol / 1 ß-mercaptoethanol, 170 μg / ml bovine serum albumin (Gibco), 200 μmol / 1 of each dNTP (Pharmacia), 30 pmol of each oligonucleotide (OD and OID) and 1.5 U Taq polymerase (AmpliTaq, Perkin Elmer Cetus). The reaction mixture was coated with paraffin and 40 PCR cycles were carried out. The products of the PCR reaction were purified by phenol / chloroform extraction and concentrated by ethanol precipitation ¬ trated.

1.5 Half of the PCR reaction products were cleaved with the restriction enzymes SphI (Pharmacia) and AlwNI (Biolabs) according to the manufacturer's instructions. The second half was cut in a series of reactions with Ava I (BRL), AlwN I (Biolabs) and Tfi I (Biolabs). The

Restrictions were carried out in 100 μl with 8 U enzyme at 37 ° C. for 2 to 12 hours (except for Tfi I at 65 ° C.).

1.6 The products of the restriction cleavage were fractionated by agarose gel electrophoresis. After staining with Ethidium bromide, uncleaved amplification products were cut out of the gel and isolated by phenol extraction. The DNA obtained was then purified twice by phenol / chloroform extraction.

1.7 After ethanol precipitation, a quarter or a fifth of the isolated DNA was reamplified using the same conditions as for the primary amplification, except that the number of cycles was reduced to 13. The reamplification products were purified, cut with the same enzymes as above, and the uncut products were isolated from agarose gels as explained above for the amplification products. The reamplification step was repeated.

1.8 After the last isolation from the gel, the amplification products were cleaved by 4 U Eco RI (Pharmacia) under the conditions recommended by the manufacturer. A quarter of the restriction mixture was ligated into the vector pBluescript SK + (Stratagene) digested with Eco RI. After ligation, 24 clones of each enzyme combination were further analyzed by sequencing. The approach, which was cut with AlwN I and Sph I, contained no new sequences, only BMP6 and inhibin ßA sequences. 19 identical new sequences, called MP121, were found in the approaches cut with Ava I, AlwN I and Tfi I. These plasmids were called pSK-MP121 (OD / OID). One sequence differed from this mainly found sequence on two nucleotides. The ligation and transformation in E. coli was carried out as in Sambrook et al. , Molecular cloning: A laboratory manual (1989).

The clone was completed at the 3 'end of the cDNA according to the method described in detail by Frohmann (published by Perkin-Elmer Corp., Amplifications, 5, 11-15 (1990)). The same liver mRNA used to isolate the first MP121 fragment was reversed as described above transcribed using Oligo dT (16mer) connected to the adapter primer (AGAATTCGCATGCCATGGTCGACGAAGC -T ₁₆ ). The amplification was carried out with the adapter primer (AGAATTCGCATGCCATGGTCGACG) and an internal primer (GGCTACGCCATGAACTTCTGCATA), prepared according to the MP121 sequence. The amplification products were reamplified with a further internal primer (ACATAGCAGGCATGCCTGGTATTG), produced according to the MP-121 sequence, and the adapter primer. After restriction with Sph I, the reamplification products were cloned into the vector pT7 / T3 U19 (Pharmacia) which had also been cut and sequenced. The clones were characterized by their sequence overlap with the already known part of the MP121 sequence. A clone called pl21Lt 3 'MP13 was used to isolate an Nco I (blunted with T4 polymerase) / Sph I fragment. This fragment was cloned into one of the pSK-MP121 (OD / OID) vectors mentioned above, the OD primer sequence of which was oriented to the T7 primer of the pSK multiple cloning site. For this, the vector was restricted with SphI and Smal. The construct was named pMP121DFus6. It contains the MP121 sequence from position 922 to 1360 as shown in SEQ ID NO.1.

1.9 A Dde I fragment from pMP121DFus6, which extends from position 931 to 1304 in SEQ ID NO. 1, was used to screen a human liver cDNA library (Clontech, # HL3006b, lot 36223), as described in detail by Ausubel et al. (Current Protocols in Molecular Biology, published by Greene Publishing Associates and Wiley-Interscience (1989)). 24 mixed plaques were picked from 8.1 x 10 ⁵ phages and separated. From this, 10 clones which gave a positive signal with PCR using primers L02 (ACATAGCAGGCATGCCTGGTATTG) and LOH (CTGCAGCTGTGTTGGCCTTGAGA) were selected and separated. The cDNA was isolated from the phages via an EcoRI restriction and cloned into the pBluescript SK vector which had also been cleaved with EcoRI. Sequencing of one of the resulting plasmids, SK121L9.1, showed that the start codon begins at position 128 of SEQ ID NO.1 because three stop codons are in-frame in front of this start codon at positions 62, 77 and 92. The start of the mature MP121 is at position 836 of SEQ ID NO.1. If one bases the sequence analogy on other TGF-ß proteins, which corresponds to amino acid 237 in SEQ NO.2. The stop codon begins at position 1184 of SEQ ID NO.l.

The plasmid SK121L9.1 was deposited with the DSM on April 26, 1994 under the accession number 9177.

1.10 Isolation of the MP121 cDNA and Genomic DNA from Mouse: The sequence information from the human MP121 sequence was used to isolate the MP121 sequence from the mouse. The methods used for this are all known to the person skilled in the art and e.g. in Current Protocols in Molecular Biology (Ausubel et al., Greene Publishing Associates and Wiley-Interscience, Wiley & Sons, 1987-1995) or in Molecular Cloning (Sambrook et al., second edition, Cold Spring Harbor Laboratory Press 1989).

First, the primers ACGAATTCCGACGAGGCATCGACTGC and GCGTCGACTACCATGTCAGGTATGTC were synthesized from the human MP121 sequence with additional restriction sites at the 5 'end (EcoR I or Sal I). These primers were used for amplification on mouse genomic DNA. The resulting 0.35 kb fragment was subcloned into the ^" Bluescript vector (Stratagene) and used as a radioactive probe. Both a λ bank with genomic mouse DNA and a bank with cDNA were screened according to standard methods. The cDNA was derived from RNA derived from Mouse liver was isolated, synthesized and cloned in λgtlO provided with EcoR I / Not I linkers.

MP121 clones were isolated from both the genomic and the cDNA library. A cDNA containing the entire coding sequence was subcloned into the EcoRI interface of the vector Bluescript SK (Stratagene) and the resulting plasmid SKMP121 mouse was deposited with the DSM on May 10, 1995 (DSM 9964). Complete sequencing gave the sequence shown in SEQ ID NO.3. The start codon begins at position 131 in SEQ ID NO.3 and ends with the stop codon starting at position 1187. The protein derived from the sequence is shown in SEQ ID NO.4.

Subcloning and analysis of the clones from the genomic library containing MP121 showed that the MP121 sequence contains an intron of approximately 5.5 kb in the propeptide portion. This intron is located between positions 446 and 447 in SEQ ID NO.3. The exon / intron junctions are shown in SEQ ID NO.5.

Example 2 Expression of MP121

The expression of MP121 is possible in both eukaryotic and prokaryotic systems.

Only the mature portion of MP121 was used for expression in prokaryotes. After purification, the mature MP121 protein expressed as a monomer in E. coli can then be folded back into a dimer. In order to simplify the purification of the MP121, 6 histidines can additionally be attached to the N-terminus of the mature protein, which facilitate the purification of the protein by binding to nickel chelate columns.

As an example, the mature portion of human MP121 (amino acids 237 to 352 in SEQ ID NO.2) with 13 additional amino acids including 6 histidines at the N-terminus (MHHHHHHKLEFAM) was expressed in the prokaryotic vector pBP4. This vector is a pBR322 derivative with tetracycline resistance which additionally contains the T7 promoter from the pBluescript II SK plasmid (Stratagene). Furthermore, the vector after the T7 promoter contains a ribosomal binding site and a start codon followed by 6 codons for Histidine. A terminator (T0) follows behind several singular restriction interfaces such as Eco RI, Xho I, Sma I and Apa I for the insertion of inserts and stop codons in all three reading frames. To obtain the cDNA for the mature part of MP121, a PCR with the two oligonucleotides GAATTCGCCATGGGCATCGACTGCCAAGGAGG and

CCGCTCGAGAAGCTTCAACTGCACCCACAGGC on plasmid SK121L9.1 (DSM accession number: 9177). Both oligonucleotides contain restriction sites added at the ends (Eco RI and Nco I or Xho I and Hind III). The resulting 377 bp fragment was blunt-end cloned into the vector pBluescript II SK (Stra¬ tagene) restricted with Eco RV. A clone with the orientation of the 5 'end of MP121 to the T7 promoter was restricted with Eco RI and the resulting insert (0.38 kb) was cloned into the pBP4 vector which was also restricted with Eco RI. The correct orientation of the insert in the resulting plasmid p-BP4MP121His was determined by restriction analysis and sequencing. The plasmid pBP4MP121His was deposited with the DSM on January 30, 1995 (accession number: 9704).

MP121 protein can be expressed by simultaneously providing T7 RNA polymerase. The T7-RNA polymerase can be provided by various methods, such as a second plasmid with a gene for T7 RNA polymerase or by infection with phages which code for the T7 RNA polymerase or by special bacterial strains which carry the gene for T7 Have integrated RNA polymerase. Using the bacterial strain BL21 (DE3) pLysS (Nova¬ gen, # 69451-1) and inducing T7 RNA polymerase expression according to the manufacturer's instructions with IPTG, the mature MP121 protein with His tag (MP121His) is formed in inclusion bodies. The protein in SDS-polyacrylamide gels (15%) has an apparent molecular weight of almost 16 kD (theoretical molecular weight: 14.2 kD), as is shown representatively in the Western blot of FIG. 3. The bacteria transformed with pBP4 as controls each show no staining of specific bacteria. the. Because of the His tag, this protein can be obtained via nickel chelating columns as described, for example, in Hochuli et al. (BIO / Technology Vol. 6, 1321-1325 (1988)). Additional cleaning is possible using a reversed phase HPLC. A reversed phase column (Nucleosil 300-7C4 from Macherey-Nagel, Art. 715023) with a flow rate of 2 ml / min and an acetonitrile gradient in 0.1% TFA from 0 to 90% in 100 min was used. Under these conditions, MP121His elutes from approx. 40% acetonitrile.

The proof that it is human MP121 protein was carried out in each case via Western blot analysis with MP121-specific antibodies. Polyclonal antibodies to MP121 were raised in both chickens and rabbits. To obtain the antigen for the immunization, part of the mature portion of MP121 (amino acid 260 to 352 in SEQ ID NO.2) was fused with the first 98 amino acids of the polymerase of the MS2 bacteriophage and expressed in E. coli. After isolation of the inclusion bodies, the fusion protein (MS2-MP121) was separated on polyacrylamide gels and after copper staining by electroelution (Tessmer, U. & Dernick, R., IBL (1990) 8-13) for the immunization. Antibodies from both chickens and rabbits make it possible to specifically detect expression of MP121. For the schematic western blot in FIG. 3, chicken antibodies were used which via PEG precipitation (Thalley BS and Carroll, SB, BIO / Technology Vol. 8, 934-938 (1990)) and via membrane-bound antigen (Fusion protein (MS2-MP121)) (18.17 in Sambrook et al. Molecular Cloning, second edition, Cold Spring Harbor Laboratory Press 1989). Anti-Chicken IgG with coupled alkaline phosphatase (Sigma A9171) was used as the second antibody. Detection was carried out using the Tropix Western-Light Protein Detection Kit (Serva # WL10RC) according to the manufacturer's instructions.

To obtain biologically active material, the monomeric MP121 expressed and purified in E. coli can be combined into one dimeric MP121 can be folded back. This can be done using methods such as those described by Jaenicke, R. & Rudolph, R. (Protein structure, ed. Creighton, TE, IRL Press, chapter 9).

For the expression in eukaryotic cells, the vaccinia virus expression system was used, as described in detail in the Current Protocols in Molecular Biology (Ausubel et al., Greene Publishing Associates and Wiley-Interscience, Wiley & Sons) , hereinafter abbreviated to CP, is described under Chapter 16 Unit 16.15-16.18. The system is based on the fact that foreign DNA can be integrated into the vaccinia virus genome using certain vectors by homologous recombination. For this purpose, the vector used contains the TK (thymidine kinase) gene from the vaccinia genome. To enable selection for recombinant viruses, the vector further contains the E. coli xanthine guanine phosphoribosyl transferase gene (gpt) (Falkner, FG & Moss, B., J. of Virol. 62 (1988), 1849-1854). The cDNA with the entire coding region for MP121 was cloned into this vector.

In order to shorten the 5 'and 3' untranslated regions from the original plasmid SK121L9.1 (DSM, accession number: 9177) and to insert unique restriction interfaces at the ends, PCR reactions and intermediate cloning were necessary. All ^'PCR reactions were performed with the plasmid SK121L9.1: leads durchge (DSM Deposit No. 9177). To shorten the 5 'untranslated end, the primer CCCGGATCCGCTAGCACCATGACCTCCTCATTGCTTCTG with an inserted Barn HI and Nhel restriction site was used in a PCR with an internal primer (CCCTGTTGTCCTCTAGAAGTG). The fragment obtained was intermediate cloned in Bluescript SK (Stratagene), sequenced and checked for agreement with the sequence shown in SEQ ID NO.1. For a shortened 3 'untranslated end, the Sph I / Eco RI fragment (0.22 kb) from the plasmid pBP4MP121His used.

The two end fragments of human MP121 were cut via internal restriction cuts (Xba I and Sph I) with the missing middle DNA sequence from the plasmid SK121L9.1 (DSM deposit number: 9177) according to standard methods (Sambrook et al. Molecular Cloning, second edition, Cold Spring Harbor Laboratory Press 1989). The shortened cDNA thus obtained with the complete reading frame for MP121 (nucleotide 128 to nucleotide 1184 in SEQ ID No. 1) could be cloned into the vector pBPl, which had also been cut, by using the restriction sections Barn HI and Eco RI. The resulting plasmid pBPlMP121 was deposited on January 12, 1995 with the DSM (accession number: 9665).

The plasmid pBPlMP121 was used for the production of recombinant vaccinia viruses. For this purpose, 80% confluent 143B cells (HuTk, ATCC CRL 8303) in 35 mm culture dishes were infected with vaccinia wild-type virus in 1 ml PBS for 30 minutes at room temperature with occasional shaking (1 virus per 10 cells). After aspirating the supernatant and adding 2 ml of culture medium (MEM, Gibco BRL # 041-01095 with penicillin diluted 1: 500 and streptomycin Gibco BRL # 043-05140), the mixture was incubated at 37 ° C. for 2 hours. The medium was then removed and the transformation of these cells with 100 ng pBPlMP121, 2 μg carrier DNA (calf thymus, treated with ultrasound, Boehringer Mannheim # 104175) and 10 μl lipofectin (Gibco BRL # 18292-011) in 1 ml MEM for approx 15 h at 37 ° C. After adding 1 ml of MEM with 20% FCS (Sigma # F-7524), the mixture was incubated for a further 24 hours at 37 ° C. and the lysed cells were then frozen.

The gpt selection for the xanthine guanine phosphoribosyl transferase and the isolation and amplification of individual recombinant viruses was carried out essentially as in unit 16.17 of the CP described, with the difference that RK13 cells (ATCC CCL 37) were used.

The integration of the MP121 cDNA into the virus genome was confirmed by dot blot analysis (CP Unit 16.18). A recombinant virus from transfection with pBPMP121 and the wild-type virus were used for expression analyzes in the cell lines 143B

(HuTk-, ATCC CRL 8303, human) and NIH-3T3 (DSM ACC 59, swiss mouse embryo). The cells were cultivated according to the information provided by the distributors. The confluent cells were infected with three times the number of viruses for 30 minutes at 37 ° C. and then the corresponding culture medium with 10% FCS and penicillin / streptomycin (1: 500, Gibco BRL # 043-05140) was added. After 6 hours at 37 ° C the medium was removed, the cells were washed twice with e.g. HBSS (Gibco BRL # 14180-046) washed and production medium (MEM for HuTk or DMEM with 4.5 g / 1 glucose and NEAA (Gibco BRL # 11140-035) for NIH-3T3 each mixed with aprotinin (Fluka # 10820, 50 U / ml) and penicillin / streptomycin) added without FCS. After 20 to 22 hours of production, the cell supernatant was collected. Expression was analyzed by Western blots using standard methods (CP Unit 10.8). For this purpose, the proteins were precipitated from 1 to 3 ml of cell culture supernatant by adding the equivalent volume of acetone and incubating on ice for at least one hour and centrifuging. After the pellet had been resuspended in application buffer (7 M urea, 1% SDS, 7 mM sodium dihydrogen phosphate, 0.01% bromophenol blue and optionally 1% β-mercaptoethanol), it was separated in 15% polyacrylamide gels. A pre-stained protein molecular weight standard was used as the marker protein

(Gibco BRL # 6041-020). The transfer to PVDF membrane (Immobilon # IPVH00010) and the blocking of the membrane were carried out using standard methods.

A representative schematic drawing of the Western blot results in FIG. 3 shows that MP121-specific bands occur in the cells infected with recombinant viruses. The expression of MP121 in NIH-3T3 cells leads to a secreted protein with a molecular weight of approximately 18 kD appearing in the gel under non-reducing conditions (expected theoretical molecular weight: 25 kD). Under reducing conditions, the protein runs in the gel at approximately 15 kD (expected theoretical molecular weight: 12.5 kD). These results show that, as expected, MP121 is expressed as a dimeric mature protein. The relatively little slower running behavior of the dimeric MP121 protein compared to the monomeric MP121 protein is probably due to a globular structure. The processing of the precursor protein to the mature protein could also be demonstrated in HuTK cells. In cells infected with wild-type viruses (without integrated foreign DNA) (HuTK- or NIH-3T3), no bands appeared in the Western blot.

The vaccinia expression system is suitable for cotransfection with recombinant vaccinia viruses that code for different members of the TGF-ß family, especially for the formation of heterodimers. Affinity columns with specific antibodies against the individual TGF-ß family members then make it possible to separate heterodimers from homodimers. Of particular interest are the inhibins, as well as the βA and βB chains.

Example 3

Investigation of the expression of MP121 in different

Mouse tissues

Total RNA from various tissues (brain, heart, kidney, liver, lung, spleen, muscle, ovary, testis) from 6-week-old mice and from embryonic stem cells was isolated using standard methods. 10 μg total RNA was used in an RNAse Protection Assay (RPA) from Ambion (RPA II Kit, # 1410) according to the manufacturer's instructions. In order to obtain specific samples for activin ß _A and activin ß _B , mouse genomic DNA (129Sv) was used with correspondingly specific Primers from the mature portion of the proteins, amplified. To facilitate cloning, EcoR I and / or BamH I or Hind III restriction sites were inserted at the ends of the primers. For activin ß _A , the primers were derived from the mRNA from rats (GenBank Accession # M37482):

GGATCCGAATTCGGCTTGGAGTGTGATGGCAAGG and GGATCCGAATTCCTCTGGGACCTGGCAACTCTAG.

For activin ßg, degenerate primers were derived from the human sequence (Mason et al., Molecular Endocrinology 3, 1352-1358 (1989):

GAGAATTCCA (GA) CA (GA) TT (TC) TT (CT) AT and GCAAGCTTT (GA) TA (TC) TC (GA) TC (GA) TC.

The resulting PCR fragments were subcloned into the vector pGEM-4 (Promega) and checked. The activin-specific and thus protected in RPA sequences have the fragment size of 369 bp for activin ß _A and 254 bp for activin ß _ß . In MP121 the protected fragment comprises the sequence from position 887 to position 1164 in SEQ ID NO.3. The fragments cloned in pGEM-4 were transcribed in vitro to produce the radioactively labeled antisense RNA samples. This was done according to the manufacturer's instructions (Promega, Riboprobe Gemini Systems) using 100 μm CTP and at the same time α ³² P-CTP (800 Ci / mmol, Amersham). Likewise, as a control, a radioactively labeled RNA was synthesized from the plasmid pTri-GAPDH (Ambion # 7431) linearized with Dde I, but using 1 mM CTP. After isolation of the 4 antisense RNA samples from polyacrylamide gels, these were incubated with the respective tissue RNA from mouse (10 μg total RNA per sample with lxl0 ⁵ cpm) in the same mixture overnight at 42 ° C. The analysis was carried out in a denaturing gel according to standard methods with subsequent autoradiography over 4 days. Example 4

Partial cleaning of MP121 and investigation of the activity of partially cleaned MP121

MP121 protein, which was obtained by expression in the vaccinia system (see Example 2), could be partially purified using two columns.

For the production of MP121, confluent NIH-3T3 cells (DSM ACC 59, Swiss mouse embryo) were infected with the same number of recombinant viruses for 30 minutes at 37 ° C. and then the corresponding culture medium with 10% FCS and penicillin / streptomycin was added.

After 4 hours at 37 ° C., the medium was removed, the cells were washed twice and production medium (see Example 2) without FCS was added. After 20 to 22 hours of production, the cell supernatant was collected and centrifuged to remove the viruses (40,000 x g for 30 minutes at 4 ° C.) and filtered (0.1 μm pore size, Millex W, Millipore # SLW25LS). The control supernatant (wt) after infection by wild-type vaccinia viruses was obtained in a comparable manner. The expression of MP121 was checked by Western blot analysis and estimated to be 50-100 μg / l.

The protease inhibitor PMSF (1 μM) was added to the cell culture supernatant with MP121 (1.1 1), brought to a final concentration of 1 M (NH ₄ ) ₂ SO ₄ , 20 mM Tris pH 8.0 and to a phenyl Sepharose (Fast Flow (high sub) Pharmacia # 17-0973-05) column (5 ml bed), equilibrated in buffer A (1 M (NH ₄ ) ₂ SO ₄ , 20 mM Tris pH 8.0), loaded. The loaded column was washed with 15 column volumes of buffer A and 10 column volumes of buffer B (20 mM Tris pH 8.0) and with a linear gradient to 100% buffer C (20 mM Tris pH 8.0, 80% ethylene glycol) at a flow rate of 1 ml / min eluted within 50 min (5 ml per fraction). Western blot analysis showed that MP121 elutes between 50 and 80% ethylene glycol. Aliquots of these fractions were made in silver-colored 15% poly acrylamide gels (Silver Stain-II, Daiichi # SE140000) checked and the fractions containing MP121 pooled. The comparable fractions after purification of the control supernatant were also pooled after analysis in gels stained with silver.

The pooled fractions were further purified using a reversed phase HPLC. For this purpose, a C8 column (Aquapore RP300, Applied Biosystems, particle size: 7μm, pore size: 300Ä) was equilibrated with buffer A (0.1% trifluoroacetic acid / water). After the column had been loaded with the pooled fractions of the phenyl-Sepharose column containing MP121, the mixture was washed extensively with buffer A. The bound protein was eluted at a flow rate of 0.2 ml / min with a linear gradient of 1.5% buffer B (90% acetonitrile, 0.1% trifluoroacetic acid) per minute. Fractions of 600 μl were collected and analyzed both in the Western blot and in silver-stained gels. The MP121 protein elutes under the chosen conditions from approximately 55% acetonitrile. The MP121 fractions were pooled. The same was done with the corresponding fractions from the cleaning of the control supernatant. Analysis in silver gel showed that MP121 was still contaminated with other proteins. In order to obtain cleaned MP121, further purification steps are necessary.

Other methods known to the person skilled in the art are also conceivable for further purifications, e.g. Gel sieve columns, ion exchange columns, affinity columns or metal chelate columns.

It was estimated by Western blot analysis that approximately 8 μg of partially purified MP121 were isolated from 1 l cell culture supernatant. The partially purified protein was stored lyophilized at -80 ° C.

To check the influence of MP121 on dopaminergic neurons, neurons from the midbrain of 14-day-old rat embryos (E14) were described according to a method by Shimoda et al. (Brain Res. 586, 319-331 (1992)). The cells were separated and cultivated as described in Krieglstein et al. (Neuroscience 63, 1189-1196 (1994)). The cell density is 200,000 cells / cm ² on cover slips coated with poly ornithine / laminin. After 24 hours of cultivation and then every three days, two thirds of the medium (500 μl) were removed and replaced by fresh medium with the corresponding additives. The partially purified MP121 lyophilized after the phenyl Sepharose and reversed phase HPLC was dissolved in 50% acetonitrile and added to the medium. The final concentration of MP121 in the medium is 20 ng / ml (final concentration of acetonitrile is 0.3%). A comparable amount of the comparable purified control supernatant (wt) was dissolved in 50% acetonitrile and used. The medium control also contains 0.3% acetonitrile. After eight days, the cultures were fixed in 4% paraformaldehyde for 10 min at room temperature, the cells were permeabilized with acetone (10 min, -20 ° C.) and washed with PBS (phosphate buffered saline). After treatment with 1% H ₂ 0 ₂ in PBS, washing and blocking with horse serum, an immunocytochemical staining was carried out. Tyrosine hydroxylase (TH) is a limiting enzyme in the biosynthesis of dopamine and other catecholamines, so that TH can be used as a marker for dopa-inert neurons in the present cultures (cells containing noradrenaline are not isolated). TH was demonstrated by incubation for one hour at 37 ° C with a mouse monoclonal antibody against rats TH (diluted 1: 200, Boehringer Mannheim) and subsequent detection with the Vectastain ABC kit (Vecto Labs). The TH-positive cells were counted in an area of 0.12 cm ² . It can be seen from FIG. 5 that MP121 has a positive influence on the survival of dopaminergic neurons.

FIG. 5 shows the number of surviving TH-immunoreactive dopaminergic neurons after isolation from the midbrain of rat embryos (E14) and 8 days of cultivation. The effect of 20 ng / ml partially cleaned MP121 was tested in equal to the equivalent amount of partially purified control supernatant (wt) and untreated neurons (control: medium with 0.3% acetonitrile). The mean ± SEM from a triple determination is shown.

Detailed description of Figures 3 to 5

Figure 3: Schematic Western blot with chicken antibodies against MP121

1: E. coli cells transformed with pBP4MP121His under reducing conditions (1% ß-mercaptoethanol) 2: Cell culture supernatant from NIH-3T3 cells after infection with recombinant viruses (with inserted MP121 cDNA) under reducing conditions (1% ß-mercaptoethanol) 3: Cell culture supernatant from NIH-3T3 cells after infection with recombinant viruses (with inserted MP121 cDNA) under non-reducing conditions

M: pre-colored protein molecular weight marker with the indicated apparent molecular weights (Gibco BRL # 26041-020)

Figure 4: Autoradiogram after gel analysis of an RNAse protection assay with specific samples against activin ß _A (ß _A ), activin ^ (ß _B ), MP121 and as a control against GAPDH.

Total RNA was tested isolated from various mouse tissues (1: brain, 2: heart, 3: kidney, 4: liver, 5: lung, 6: muscle, 9: ovary, 10: spleen, 11: testis), from embryonic Stem cells (12: CJ7) and as a control from yeast (lane 13). In lane 14 no RNA was used as a control. The unprotected antisense RNA samples used in the hybridization are plotted in lanes 8 and 15 and marked in brackets on the right with the expected fragment size. The bands for the protected fragments are marked on the left edge. As a marker (lane 7), pBR322 was restricted with Msp I (Biolabs # 303) and end-labeled with γ- ³² P-ATP (Amersham).

FIG. 5 shows the number of surviving TH-immunoreactive dopaminergic neurons after isolation from the midbrain of rat embryos (E14) and 8 days of culture. activation. The effect of 20 ng / ml of partially purified MP121 was tested in comparison to the equivalent amount of partially purified control supernatant (wt) and untreated neurons (control: medium with 0.3% acetonitrile). The mean value j + SEM from a triple determination is shown.

SEQUENCE LOG

(1. GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: Biopharm Society for Biotechnological

Development of pharmaceuticals mhH

(B) ROAD: Czemyring 22

(C) LOCATION: Heidelberg

(E) COUNTRY: Germany

(F) POSTAL NUMBER: 69115

(ii) DESCRIPTION OF THE INVENTION: New growth / differentiation factor of the TGF-beta family

(iii) NUMBER OF SEQUENCES: 6

(iv) COMPUTER READABLE VERSION:

(A) DISK: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS / MS-DOS

(D) SOFTWARE: Patentin Release # 1.0, Version # 1.30 (EPA)

(v) DATE OF REGISTRATION:

REGISTRATION NUMBER: WO PCT / EP95 / 02552

(2) INFORMATION ON SEQ ID NO: 1:

(i) SEQUENCE LABEL:

(A) LENGTH: 2272 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: double strand

(D) TOPOLOGY: linear (vi) ORIGINAL ORIGIN:

(A) ORGANISM: Homo sapiens

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

CAAGGAGCCA TGCCAGCTGG ACACACACTT CTTCCAGGGC CTCTGGCAGC CAGGACAGAG 60

TTGAGACCAC AGCTGTTGAG ACCCTGAGCC CTGAGTCTGT ATTGCTCAAG AAGGGCCTTC 120

CCCAGCAATG ACCTCCTCAT TGCTTCTGGC CTTTCTCCTC CTGGCTCCAA CCACAGTGGC 180

CACTCCCAGAGCTGGCGGTC AGTGTCCAGC ATGTGGGGGG CCCACCTTGG AACTGGAGAG 240

CCAGCGGGAG CTGCTTCTTG ATCTGGCCAA GAGAAGCATC TTGGACAAGC TGCACCTCAC 300

CCAGCGCCCAACACTGAACC GCCCTGTGTC CAGAGCTGCT TTGAGGACTG CACTGCAGCA 360

CCTCCACGGG GTCCCACAGG GGGCACTTCT AGAGGACAAC AGGGAACAGG AATGTGAAAT 420

CATCAGCTTT GCTGAGACAG GCCTCTCCAC CATCAACCAGACTCGTCTTG ATTTTCACTT 480

CTCCTCTGAT AGAACTGCTG GTGACAGGGA GGTCCAGCAG GCCAGTCTCA TGTTCTTTGT 540

GCAGCTCCCT TCCAATACCA CTTGGACCTT GAAAGTGAGA GTCCTTGTGC TGGGTCCACA 600

TAATACCAAC CTCACCTTGG CTACTCAGTA CCTGCTGGAG GTGGATGCCAGTGGCTGGCA 660

TCAACTCCCC CTAGGGCCTG AAGCTCAAGC TGCCTGCAGC CAGGGGCACC TGACCCTGGA 720

SPARE BLADE (RULE 26) GCTGGTACTT GAAGGCCAGG TAGCCCAGAG CTCAGTCATC CTGGGTGGAG CTGCCCATAG 780

GCCTTTTGTG GCAGCCCGGG TGAGAGTTGG GGGCAAACAC CAGATTCACC GACGAGGCAT 840

CGACTGCCAA GGAGGGTCCA GGATGTGCTG TCGACAAGAG TTTTTTGTGGACTTCCGTGA 900

GATTGGCTGG CACGACTGGA TCATCCAGCC TGAGGGCTAC GCCATGAACT TCTGCATAGG 960

GCAGTGCCCA CTACACATAG CAGGCATGCC TGGTATTGCT GCCTCCTTTC ACACTGCAGT 1020

GCTCAATCTT CTCAAGGCCAACACAGCTGC AGGCACCACT GGAGGGGGCT CATGCTGTGT 1080

ACCCACGGCC CGGCGCCCCC TGTCTCTGCT CTATTATGAC AGGGACAGCAACATTGTCAA 1140

GACTGACATA CCTGACATGG TAGTAGAGGC CTGTGGGTGC AGTTAGTCTA TGTGTGGTAT 1200

GGGCAGCCCAAGGTTGCATG GGAAAACACG CCCCTACAGAAGTGCACTTC CTTGAGAGGA 1260

GGGAATGACC TCATTCTCTG TCCAGAATGT GGACTCCCTC TTCCTGAGCATCTTATGGAA 1320

ATTACCCCAC CTTTGACTTGAAGAAACCTT CATCTAAAGC AAGTCACTGT GCCATCTTCC 1380

TGACCACTAC CCTCTTTCCTAGGGCATAGT CCATCCCGCT AGTCCATCCC GCTAGCCCCA 1440

CTCCAGGGAC TCAGACCCAT CTCCAACCAT GAGCAATGCC ATCTGGTTCC CAGGCAAAGA 1500

CACCCTTAGC TCACCTTTAATAGACCCCAT AACCCACTAT GCCTTCCTGT CCTTTCTACT 1560

CAATGGTCCC CACTCCAAGATGAGTTGACA CAACCCCTTC CCCCAATTTT TGTGGATCTC 1620

CAGAGAGGCC CTTCTTTGGATTCACCAAAG TTTAGATCAC TGCTGCCCAAAATAGAGGCT 1680

TACCTACCCC CCTCTTTGTT GTGAGCCCCT GTCCTTCTTA GTTGTCCAGG TGAACTACTA 1740

AAGCTCTCTT TGCATACCTT CATCCATTTT TTGTCCTTCT CTGCCTTTCT CTATGCCCTT 1800

AAGGGGTGAC TTGCCTGAGC TCTATCACCT GAGCTCCCCT GCCCTCTGGC TTCCTGCTGA 1860

GGTCAGGGCATTTCTTATCC CTGTTCCCTC TCTGTCTAGG TGTCATGGTT CTGTGTAACT 1920

GTGGCTATTC TGTGTCCCTA CACTACCTGG CTACCCCCTT CCATGGCCCC AGCTCTGCCT 1980

ACATTCTGAT TTTTTTTTTT TTTTTTTTTT TGAAAAGTTAAAAATTCCTT AATTTTTTAT 2040

TCCTGGTACC ACTACCACAATTTACAGGGC AATATACCTG ATGTAATGAAAAGAAAAAGA 2100

AAAAGACAAA GCTACAACAGATAAAAGACC TCAGGAATGT ACATCTAATT GACACTACAT 2160

TGCATTAATC AATAGCTGCA CTTTTTGCAAACTGTGGCTA TGACAGTCCT GAACAAGAAG 2220

GGTTTCCTGT TTAAGCTGCA GTAACTTTTC TGACTATGGA TCATCGTTCC TT 2272 (2) INFORMATION ON SEQ ID NO: 2:

(i) SEQUENCE LABEL:

(A) LENGTH: 352 amino acids

(B) TYPE: amino acid

(C) STRAND FORM:

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

REPLACEMENT BLAIT (RULE 26) (vi) ORIGINAL ORIGIN:

(A) ORGANISM: Homo sapiens

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Met Thr Ser Ser Leu Leu Leu Ala Phe Leu Leu Leu Ala Pro Thr Thr 1 5 10 15

Val Ala Thr Pro Arg Ala Gly Gly Gin Cys Pro Ala Cys Gly Gly Pro 20 25 30

Thr Leu Glu Leu Glu Ser Gin Arg Glu Leu Leu Leu Asp Leu Ala Lys 35 40 45

Arg Ser Ile Leu Asp Lys Leu His Leu Thr Gin Arg Pro Thr Leu Asn 50 55 60

Arg Pro Val Ser Arg Ala Ala Leu Arg Thr Ala Leu Gin His Leu His 65 70 75 80

Gly Val Pro Gin Gly Ala Leu Leu Glu Asp Asn Arg Glu Gin Glu Cys

85 90 95

Glu Ile Ile Ser Phe Ala Glu Thr Gly Leu Ser Thr Ile Asn Gin Thr 100 105 110

Arg Leu Asp Phe His Phe Ser Ser Asp Arg Thr Ala Gly Asp Arg Glu 115 120 125

Val Gin Gin Ala Ser Leu Met Phe Phe Val Gin Leu Pro Ser Asn Thr 130 135 140

Thr Trp Thr Leu Lys Val Arg Val Leu Val Leu Gly Pro His Asn Thr 145 150 155 160

Asn Leu Thr Leu Ala Thr Gin Tyr Leu Leu Glu Val Asp Ala Ser Gly

165 170 175

Trp His Gin Leu Pro Leu Gly Pro Glu Ala Gin Ala Ala Cys Ser Gin 180 185 190

Gly His Leu Thr Leu Glu Leu Val Leu Glu Gly Gin Val Ala Gin Ser 195 200 205

Ser Val Ile Leu Gly Gly Ala Ala His Arg Pro Phe Val Ala Ala Arg 210 215 220

Val Arg Val Gly Gly Lys His Gin Ile His Arg Arg Gly Ile Asp Cys 225 230 235 240

Gin Gly Gly Ser Arg Met Cys Cys Arg Gin Glu Phe Phe Val Asp Phe

245 250 255

Arg Glu Ile Gly Trp His Asp Trp Ile Ile Gin Pro Glu Gly Tyr Ala 260 265 270

Met Asn Phe Cys Ile Gly Gin Cys Pro Leu His Ile Ala Gly Met Pro 275 280 285

Gly Ile Ala Ala Ser Phe His Thr Ala Val Leu Asn Leu Leu Lys Ala

ERSÄΓZBLAΓT (RULE 26) 290 295 300

Asn Thr Ala Ala Gly Thr Thr Gly Gly Gly Ser Cys Cys Val Pro Thr 305 310 315 320

Ala Arg Arg Pro Leu Ser Leu Leu Tyr Tyr Asp Arg Asp Ser Asn Ile

325 330 335

Val Lys Thr Asp Ile Pro Asp Met Val Val Glu Ala Cys Gly Cys Ser 340 345 350

(2) INFORMATION ON SEQ ID NO: 3:

(i) SEQUENCE LABEL:

(A) LENGTH: 1558 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: double strand

(D) TOPOLOGY: linear (vi) ORIGINAL ORIGIN:

(A) ORGANISM: Mouse

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

AAGGAGTCAT GCCAGTCGGA GGTCAGTCAC ATTCCTCCCA GGGTCCCTGG TGCCCAGGAC 60

AGAGTTGAAG CACTCCCGTT GAGACCCTGAATATAGGCTT TGGGTCCTTT AAGGAGGCTA 120

TCCTCCAGCAATGGCCTCCT CCTTGCTCCT GGCTCTTCTG TTCCTGACTC CAACCACAGT 180

AGTGAACCCC AAAACTGAGG GTCCATGCCC AGCATGTTGG GGTGCCATCT TTGACCTGGA 240

GAGCCAGCGG GAGCTGCTTC TCGATTTGGC CAAGAAAAGT ATCCTGGACAAGCTGCACCT 300

CAGCCAGCGC CCCATACTCAGTCGGCCAGT GTCCAGAGGG GCTCTCAAGA CCGCGCTGCA 360

GCGCCTCCGC GGGCCTCGAC GGGAAACCCT GTTGGAGCAT GACCAGAGAC AAGAAGAATA 420

TGAGATCATC AGCTTTGCTG ACACAGACCT CTCCAGCATC AACCAGACCC GGCTCGAGTT 480

CCACTTCTCT GGTAGAATGG CCAGTGGCAT GGAGGTCCGG CAGACCCGCT TCATGTTCTT 540

CGTGCAGTTC CCCCACAATG CCACCCAGAC CATGAATATAAGAGTTCTTG TGCTAAGACC 600

ATATGACACC AACCTCACCT TGACAAGTCA GTACGTGGTG CAGGTGAATG CCAGTGGCTG 660

GTACCAGCTT CTCCTGGGAC CTGAAGCTCAAGCTGCTTGC AGCCAGGGAC ACCTTACTCT 720

GGAGCTGGTA CCAGAAAGCC AGGTGGCCCA CAGTTCCTTG ATCCTGGGCT GGTTTTCCCA 780

CAGGCCTTTT GTGGCAGCCC AGGTAAGGGT TGAGGGCAAG CATCGGGTTC GCCGGCGAGG 840

TATCGATTGC CAGGGGGGGT CCAGGATGTG CTGTCGACAA GAGTTTTTTG TAGACTTCCG 900

TGAGATTGGC TGGAATGACT GGATCATCCA GCCTGAAGGC TATGCCATGAACTTCTGCAC 960

TGGGCAGTGC CCACTACATG TGGCAGGCAT GCCTGGCATC TCTGCCTCCT TTCACACTGC 1020

AGTGCTGAAT CTGCTCAAAG CCAACGCAGC TGCTGGCACC ACTGGCAGGG GCTCGTGCTG 1080

CGTGCCTACA TCTCGGCGCC CTCTGTCTTT GCTCTACTAT GACAGGGACA GCAACATTGT 1140

REPLACEMENT BLAΓΓ (RULE 26) CAAGACGGAT ATACCTGACATGGTGGTCGA GGCCTGCGGG TGTAGTTAGC TTATGGGTGA 1200

TACAGGCTGC CTGAGGTAGAATGGCCTTCC TCAGGAAGGG AAACTCTGTT CCCACTTCTG 1260

TCCAGAATGG AAACACCTTT CTAAGCATGC AGACATCCCT CTGTGGACTT CAGGGGATCC 1320

ACCTCTAAAG AGAGTCACTAGTGACCAACA GCCTTTCTCT CTCCTGGGAC ATGGTTGACC 1380

CAGTACACCC ATCCTCAGCC TTAAGTTAGA GGCTAATCGA CTCCTACATA TATATGTCAT 1440

TTTGTCCTAG CAAACACCCC TTAGCTCCCC TTAGTCAACT ATGTAATCTA CTCTGCCTCC 1500

CTGACCCTGC CACCGGAAGG TTCCTATTCC ACGATGATAT GCCTTAGTGT CTCCCCTT 1558 (2) INFORMATION ABOUT SEQ ID NO: 4:

(i) SEQUENCE LABEL:

(A) LENGTH: 352 amino acids

(B) TYPE: amino acid

(C) STRAND FORM:

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (vi) ORIGINAL ORIGIN:

(A) ORGANISM: Mouse

(i) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

Met Ala Ser Ser Leu Leu Leu Ala Leu Leu Phe Leu Thr Pro Thr Thr 1 5 10 15

Val Val Asn Pro Lys Thr Glu Gly Pro Cys Pro Ala Cys Trp Gly Ala 20 25 30

Ile Phe Asp Leu Glu Ser Gin Arg Glu Leu Leu Leu Asp Leu Ala Lys 35 40 45

Lys Ser Ile Leu Asp Lys Leu His Leu Ser Gin Arg Pro Ile Leu Ser 50 55 60

Arg Pro Val Ser Arg Gly Ala Leu Lys Thr Ala Leu Gin Arg Leu Arg 65 70 75 80

Gly Pro Arg Arg Glu Thr Leu Leu Glu His Asp Gin Arg Gin Glu Glu

85 90 95

Tyr Glu Ile Ile Ser Phe Ala Asp Thr Asp Leu Ser Ser Ile Asn Gin 100 105 110

Thr Arg Leu Glu Phe His Phe Ser Gly Arg Met Ala Ser Gly Met Glu 115 120 125

Val Arg Gin Thr Arg Phe Met Phe Phe Val Gin Phe Pro His Asn Ala 130 135 140

Thr Gin Thr Met Asn Ile Arg Val Leu Val Leu Arg Pro Tyr Asp Thr 145 150 155 160

Asn Leu Thr Leu Thr Ser Gin Tyr Val Val Gin Val Asn Ala Ser Gly

165 170 175

SPARE BLADES (RULE 26) Trp Tyr Gin Leu Leu Leu Gly Pro Glu Ala Gin Ala Ala Cys Ser Gin 180 185 190

Gly His Leu Thr Leu Glu Leu Val Pro Glu Ser Gin Val Ala His Ser 195 200 205

Ser Leu Ile Leu Gly Trp Phe Ser His Arg Pro Phe Val Ala Ala Gin 210 215 220

Val Arg Val Glu Gly Lys His Arg Val Arg Arg Arg Gly Ile Asp Cys 225 230 235 240

Gin Gly Gly Ser Arg Met Cys Cys Arg Gin Glu Phe Phe Val Asp Phe

245 250 255

Arg Glu Ile Gly Trp Asn Asp Trp Ile Ile Gin Pro Glu Gly Tyr Ala 260 265 270

Met Asn Phe Cys Thr Gly Gin Cys Pro Leu His Val Ala Gly Met Pro 275 280 285

Gly Ile Ser Ala Ser Phe His Thr Ala Val Leu Asn Leu Leu Lys Ala 290 295 300

Asn Ala Ala Ala Gly Thr Thr Gly Arg Gly Ser Cys Cys Val Pro Thr 305 310 315 320

Ser Arg Arg Pro Leu Ser Leu Leu Tyr Tyr Asp Arg Asp Ser Asn Ile

325 330 335

Val Lys Thr Asp Ile Pro Asp Met Val Val Glu Ala Cys Gly Cys Ser 340 345 350

(2) INFORMATION ON SEQ ID NO: 5:

(i) SEQUENCE LABEL:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: double strand

(D) TOPOLOGY: linear (vi) ORIGINAL ORIGIN:

(A) ORGANISM: Homo sapiens (ix) FEATURE:

(A) NAME / KEY: exon

(B) LOCATION: 1..3 (ix) FEATURE:

(A) NAME / KEY: intron

(B) LOCATION: 4..18

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: CAGGTAGGTC CATGGTCG 18

(2) INFORMATION ON SEQ ID NO: 6:

(i) SEQUENCE LABEL:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: double strand

(D) TOPOLOGY: linear (vi) ORIGINAL ORIGIN:

(A) ORGANISM: Homo sapiens (ix) FEATURE:

(A) NAME / KEY: intron

(B) LOCATION: 1..15 (ix) FEATURE:

(A) NAME / KEY: exon

(B) LOCATION: 16..18

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: CTTGATTTTT AACAGACC 18

ERSÄΓZBLAΓT (RULE 26)

Claims

 Claims

DNA molecule coding for a protein of the TGF-ß family and

(a) the portion coding for the mature protein and optionally further functional portions of the nucleotide sequence shown in SEQ ID NO.1,

(b) a nucleotide sequence corresponding to the sequence from (a) in the context of the degeneration of the genetic code,

(c) a nucleotide sequence corresponding to an allelic derivative of one of the sequences from (a) and (b), or

(d) a sequence which differs from sequence (a) due to its origin from other vertebrates,

(e) one with one of the sequences from (a), (b), (c) or

(d) Hybridizing nucleotide sequence includes provided that a DNA molecule according to (d) contains at least the portion coding for a mature protein of the TGF-ß family.

:. DNA molecule according to claim 1, characterized in that, in addition to one of the parts (a) to (e) of claim 1, it has a nucleic acid sequence which codes for at least part of another protein and is arranged in such a way that after expression gives a fusion protein.

;. Vector, characterized in that it has at least one copy of a DNA molecule

Claim 1 or 2 contains.

4. host cell, characterized in that it is transformed with a DNA according to claim 1 or 2 or a vector according to claim 3.

5. Host cell according to claim 4, characterized in that it is a bacterium, a fungus, a plant or an animal cell.

6. Protein of the TGF-ß family, which is encoded by a DNA sequence according to claim 1 or 2.

7. Protein according to claim 6, characterized in that it has the amino acid sequence shown in SEQ ID NO.2 or SEQ ID NO.4 or optionally functional parts thereof.

8. Chimeric protein, characterized in that it contains a protein according to claim 6 or 7 and at least part of another protein.

9. heterodimeric protein, characterized in that it consists of a monomer of a protein according to claim 6, 7 or 8 and a monomer of another protein from the superfamily with "cystine knot motif".

10. A method for producing a protein according to any one of claims 6 to 8, characterized in that cultivates a host cell according to claim 4 or 5 and the protein from the cell and / or the culture supernatant is obtained.

11. A method for producing a heterodimeric protein according to claim 9, characterized in that the two monomers are co-expressed in a host cell.

12. A method for producing a heterodimeric protein according to claim 9, characterized in that one brings about a common renaturation of inclusion bodies of both monomers.

13. Pharmaceutical composition, characterized in that it contains at least one protein according to one of claims 6 to 9 as an active ingredient, optionally together with pharmaceutically customary carriers, auxiliaries, diluents or fillers.

14. Pharmaceutical composition according to claim 13 for the treatment or prevention of bone, cartilage, connective tissue, skin, mucous membrane, endothelium, epithelial, neuronal, brain, renal or tooth damage, for use in dental implants , for use in wound healing or tissue restoration processes, as a morphogen for use for inducing liver tissue growth, inducing the proliferation of precursor cells or bone marrow cells, for maintaining a differentiated state and for treating fertility disorders or for contraception, or for treating metabolic disorders .

15. Anti-sense RNA, characterized in that it is complementary to part of a DNA molecule according to claim 1.

16. Ribozyme, characterized in

ERSÄΓZBLÄΓT (RULE 26) that it specifically cleaves an RNA molecule obtained after transcription of a DNA molecule according to claim 1.

17. Use of an antisense RNA according to claim 15 or a ribozyme according to claim 16 for blocking the expression of a protein according to one of claims 6 and 7.

18. Use of a DNA sequence according to claim 1 or 2 or a vector according to claim 3 for in vitro or in vivo transfection of patient cells.

19. Antibodies or antibody fragments, characterized in that they bind to a protein according to one of claims 6 to 9.

20th Receptor, characterized in that a protein according to one of claims 6 and 7 binds specifically to it.