WO2001057208A2

WO2001057208A2 - Mlp gene, nucleic acids, polypeptides and the utilization thereof

Info

Publication number: WO2001057208A2
Application number: PCT/EP2001/001042
Authority: WO
Inventors: Ralph KNÖLL
Original assignee: Schering Aktiengesellschaft
Priority date: 2000-02-03
Filing date: 2001-02-01
Publication date: 2001-08-09
Also published as: WO2001057208A3; DE10004857A1; AU2001233727A1; EP1252310A2; JP2003523743A

Abstract

The invention concerns mutated MLP sequences, whereby mutation occurs in base No. 10 of exon 2 of the translated sequence or in the third position of codon 112 in exon 4 of the translated sequence. The invention also concerns a muscle-specific promoter and the utilizations thereof.

Description

MLP gene, nucleic acids, polypeptides and their use

description

The present invention relates to nucleic acids coding for MLP, a polypeptide encoded thereby, probes for these nucleic acids, methods for the detection of and / or screening for myocardial diseases, kits for the detection of the nucleic acids and the polypeptide, regulatory nucleic acids, these comprehensive vectors, these comprehensive Cells and this comprehensive medication.

Dilated cardiomyopathy (DCM) is a myocardial disease of unknown origin, which is associated with reduced contractility (systolic ventricular dysfunction) and accompanying disturbed relaxation of the left and / or right ventricle (diastolic ventricular dysfunction). Cardiomegaly with dilatation of both ventricles, reduced ejection fraction and high end-diastolic volume is typical. The cardiac output is reduced (forward failure) and there is backflow of blood in front of the heart (backward failure) (Isselbacher K. et al; Haπson ^'s Principles of Intemal Medicine (McGraw Hill, New York, 1994)).

The incidence of DCM in the western industrialized countries is around 5 / 100,000 inhabitants / year. It thus represents the most frequently occurring primary cardiomyopathy. In recent studies, a family cluster has been described in about 30% (Towbin, The role of cytoskeletal proteins in cardiomyopathies 10, 13-139 (1998)). For example, the cost of this disease in the United States is $ 10-40 billion, i.e. extrapolated, there are likely to be DM 5 to 20 billion in Germany.

However, the etiology of the disease is still unclear. In about 30% of cases, enteroviral (flushing M. et al .; Circulation 99 (7), 889-895 (1999)) or adenoviral (flushing, M. et al .; Circulation 99 (10), 1348-1354 (1999)) DNAs and RNAs in the myocardium. A possible pathomechanism was suggested by Badorff (Badorff, C. et al .; Nature medicine 5, 320 - 326 (1999)). A viral genesis can therefore be considered probable. In addition, antibodies have been detected, for example, against beta receptors and the adenine non-nucleotide translocator (ANT), which is why autoimmune mechanisms are also being discussed as a pathogenetic principle (Schultheiss, HP et al; Circ Res. 76, 64-72 (1995)). However, DCM is also increasingly considered to be a genetic cause, not least because of the family clusters. The best documented are mutations in the dystrophin gene, e.g. in Duchenneschen and Becker's muscular dystrophy (Muntoni, F. et al .; N Engl J Med 329, 921-5 (1993)), which in addition to muscular dystrophy also lead to DCM , In addition, two mutations in the cardiac actin gene have been described which lead to DCM in the corresponding families (Olsen, T. et al .; Science 280, 750 - 752 (1998)). A mutation was also found in the metavinculin gene, which most likely led to cardiomyopathy in the affected patient (Maedam, M. et al .; Circulation 95, 17-20 (1997)). In addition, a mutation in the delta sarkoglycan gene was found to be the cause of the cardiomyopathy of the Syrian hamster (Nigro, V. et al .; Hum Mol Genet 6, 601-607 (1997)). These genes all code for proteins of the cytoskeleton, which is why it is assumed that mutations in cytoskeletal proteins lead to DCM, in contrast to the proteins of the sarcomere, in which mutations obviously lead to hypertrophic cardiomyopathy (HCM) (Towbin, J. et al .; Nature Medicine 5, 266-267 (1999) and Chen, J. et al .; J Clin Invest 103, 1483-1485 (1999)).

Since the MLP is counted among the cytoskeletal proteins, the MLP knock out, which is associated with a DCM, fits well into the thought model mentioned above (Arber et al .; Cell 88, 393-403 (1997)). The MLP was first described in 1994 using a subtractive cloning technique as a positive regulator of myogenesis (Arber S. et al .; Cell 79, 221-31 (1994)) and gained considerable importance in cardiology in 1997 when it became known (Arber et al .; Cell 88, 393-403 (1997)) that the MLP knock out in a mouse model is accompanied by a DCM. This was also the first genetically engineered organism to develop this clinical picture. However, it is still completely unclear in the prior art whether mutations in this gene also lead to DCM in humans.

As the name suggests, the MLP itself belongs to the LIM protein group. This group was named about 10 years ago after the initial letters of its first 3 members (lin 11, islet 1, mec3) and has essentially three subgroups: 1. The LIM kinases, d. H. Proteins with the LIM motif and kinase activity, 2. LIM homeobox genes, d. H. LIM proteins, which act as transcription factors, and 3. “LIM only” proteins, that is, LIM proteins that only carry LIM motifs. The MLP belongs to the group of “LIM only” proteins (Morgan, M. et al .; Biochem Biophys Res Com 212, 840-846 (1995)).

The protein itself consists of 194 amino acids and forms two LIM double zinc fingers, each followed by a glycine-rich amino acid sequence. The protein probably acts due to its location on the Z band as a cytoskeletal protein, where it binds to f-actin structures (Arber, S. et al .; Genes Dev 10, 289-300 (1996)).

The present invention has for its object to provide an agent that allows preventive treatment of dilated cardiomyopathy. The remedy is also intended to demonstrate the disposition of an individual to develop the clinical picture of the dilated Allow cardiomyopathy. Finally, the agent to be provided should enable the diagnosis of dilated cardiomyopathy.

Furthermore, it is an object of the invention to provide a method for the detection of myocardial diseases, for screening for myocardial diseases and for the detection of the disposition for the development of a myocardial disease, in particular in each case of dilated cardiomyopathy.

Finally, it is an object of the present invention to provide a kit which can be used in the context of the aforementioned methods.

In a further aspect, the invention is based on the object of providing a regulatory nucleic acid which allows the tissue-specific, in particular muscle-specific, expression of nucleic acids.

Furthermore, it is an object of the present invention to provide agents for the treatment and / or prevention of diseases which can be treated or prevented by tissue-specific expression.

According to the invention, the object is achieved by a nucleic acid which codes for an MLP and comprises a sequence according to SEQ ID NO: 1, the sequence having a mutation at base no. 10 of exon 2 of the translated sequence.

In a preferred embodiment, the mutation is an exchange from T to C.

Furthermore, the object is achieved by a nucleic acid which codes for an MLP and comprises a sequence according to SEQ ID NO: 1, the sequence having a mutation at the third position of codon 112 in exon 4 of the translated sequence. In a preferred embodiment, the mutation is an exchange from A to G.

In further preferred embodiments of the nucleic acids according to the invention, the nucleic acid only comprises the sequences coding for an amino acid sequence.

Furthermore, the object is achieved according to the invention by a nucleic acid which codes for an MLP and which comprises the sequence from positions 58 to 639 of SEQ ID NO: 1, a mutation being present at position 67.

In a preferred embodiment, the mutation is a mutation from T to C.

The object is also achieved by a nucleic acid which codes for an MLP and comprises the sequence from positions 58 to 639 of SEQ ID NO: 1, a mutation being present at position 393.

In a preferred embodiment, the mutation is a mutation from A to G.

It should be noted that in connection with the position information given above, these relate to the nucleic acid sequence according to SEQ ID NO: 1.

Furthermore, the object is achieved according to the invention by a nucleic acid coding for MLP, the sequence would correspond to a nucleic acid according to the invention without the degeneracy of the genetic code. Finally, the object is achieved according to the invention by a nucleic acid coding for MLP, the nucleic acid hybridizing with a nucleic acid according to the invention.

In a further aspect, the object is achieved according to the invention by an amino acid sequence which is encoded by a nucleic acid according to the invention or parts thereof.

In a preferred embodiment, the amino acid sequence according to the invention is encoded by the sequence (s) of the nucleic acid according to the invention or parts thereof, which one or both of the aforementioned mutations, i.e. at Base No. 10 of exon 2 of the translated sequence or at the third position of codon 112 in exon 4 of the translated sequence.

In a further aspect, the object is achieved according to the invention by an MLP comprising an amino acid sequence which is encoded by a nucleic acid according to the invention or a part thereof, or by the MLP comprising an amino acid sequence according to the invention.

In a still further aspect, the object is achieved according to the invention by a probe for a nucleic acid according to the invention, the probe comprising a sequence according to SEQ ID NO: 4.

In a preferred embodiment, the probe is used for the detection and / or amplification of the nucleic acid according to the invention.

In a further aspect, the object is achieved according to the invention by a probe for a nucleic acid according to the invention, the probe comprising the sequence according to SEQ ID NO: 5. In a preferred embodiment, the probe is used for the detection and / or the amplification of the nucleic acid according to the invention.

In a further aspect, the nucleic acids and / or the probes according to the invention, individually or in combination, are used to diagnose and / or screen for myocardial diseases.

In a preferred embodiment, myocardial disease is dilated cardiomyopathy.

In a further aspect, the object is achieved according to the invention by a method for the detection of and / or screening for myocardial diseases, a nucleic acid according to the invention and / or a probe according to the invention being used.

In a preferred embodiment, it is provided that the myocardial disease is dilated cardiomyopathy.

In a further aspect, the object is achieved according to the invention by a method for the detection of and / or screening for myocardial diseases, wherein a sample comprising a sequence coding for MLP is digested with a restriction enzyme and the presence of a mutated coding coding for MLP by the occurrence a restriction enzyme digestion pattern changed compared to the restriction enzyme digestion pattern of a non-mutated sequence coding for MLP is detected.

In one embodiment it is provided that the myocardial disease is dilated cardiomyopathy.

In a further embodiment of the method according to the invention, the method is an embodiment of the method according to the invention for the detection of and / or screening for myocardial diseases, wherein one nucleic acid according to the invention and / or a probe according to the invention is used.

In a further embodiment of the method according to the invention, the sequence coding for MLP is amplified by means of PCR before digestion.

In a further embodiment, the detection or screening is directed to a nucleic acid according to the invention.

In a particularly preferred embodiment, the amplification is carried out with a primer, the primer comprising a sequence according to SEQ ID NO: 13 and / or SEQ ID NO: 14.

In a particularly preferred embodiment it is provided that the restriction enzyme digestion is carried out by Nci I.

In a preferred embodiment it is provided that in the case of a non-mutated sequence the PCR product has a length of approximately 308 bp and in the case of a mutation at base no. 10 of exon 2 or a mutation at position 67 of the nucleic acid sequence of SEQ ID NO: 1, where the mutation is an exchange from T to C, two PCR products with approximately 205 and approximately 103 bp occur.

In the method according to the invention it can be provided that the detection or screening is directed to a nucleic acid according to the invention, preferably to a nucleic acid which has a mutation at the third position of codon no. 112 in exon 4 of the translated sequence from SEQ ID NO: 1 has.

In one embodiment of the method according to the invention, the amplification is carried out with a primer, the primer comprising the sequence according to SEQ ID NO: 15 and / or SEQ ID NO: 16. In a preferred embodiment, the restriction enzyme is digested by Cvi Rl.

In the method according to the invention for the detection of and / or screening for myocardial diseases, in particular dilated cardiomyopathy, in which a probe according to the invention is used, it can be provided that for the detection of or the screening for a nucleic acid sequence according to the invention, a hybridization of one to be examined Sample is carried out with a probe, the probe comprising a sequence according to SEQ ID NO: 4.

In a preferred embodiment it is provided that the hybridization takes place at 60 ° C.

In the method according to the invention for the detection of and / or screening for myocardial diseases, in particular dilated cardiomyopathy, using a probe according to the invention, it can be provided that for the detection of or the screening for a nucleic acid sequence according to the invention a hybridization of a sample to be examined done with a probe, the probe comprising a sequence according to SEQ ID NO: 5.

In a further aspect, the object of the invention is achieved by a method for the detection of and / or screening for myocardial diseases, an amino acid sequence according to the invention or an MLP according to the invention being detected.

In a preferred embodiment, the myocardial disease is dilated cardiomyopathy. In a further preferred embodiment it is provided that the amino acid sequence or the MLP is detected by means of an antibody.

In a preferred embodiment it is provided that the antibody is a monoclonal antibody.

In a further aspect of the invention, the object is achieved by a kit for detecting a nucleic acid according to the invention, it being provided that the kit comprises at least one nucleic acid according to the invention and / or at least one probe according to the invention.

In a further aspect, the object is achieved according to the invention by a kit for detecting an MLP according to the invention or for detecting an amino acid sequence according to the invention coding for an MLP, the kit comprising at least one antibody against the MLP according to the invention or against an amino acid sequence according to the invention.

In a further aspect, the object is achieved by a regulatory nucleic acid sequence which comprises approximately 500 base pairs, the 500 base pairs being those which are arranged upstream of the first base of the first exon of the human genomic sequence coding for MLP.

In one embodiment of the regulatory nucleic acid according to the invention it is provided that the regulatory nucleic acid comprises approximately 1000 base pairs, the 1000 base pairs being those which are arranged upstream of the first base of the first exon of the human genomic sequence coding for MLP.

In a further aspect, the object is achieved by a regulatory nucleic acid which can be represented on the basis of human genomic DANN using two primers in a PCR reaction, the upstream primer comprising SEQ ID NO: 9 and the downstream primer SEQ ID NO: 10.

In a further aspect, the object is achieved by a regulatory nucleic acid sequence comprising a sequence according to SEQ ID NO: 8.

In one embodiment of the regulatory nucleic acid according to the invention it is provided that the regulatory nucleic acid is a promoter.

In a further aspect, the object is achieved according to the invention by a vector comprising a regulatory sequence according to the invention.

In a preferred embodiment it is provided that the regulatory sequence is contained in the vector pAdCMVssTnl, the CMV promoter in this vector being replaced by the regulatory sequence according to the invention.

In a preferred embodiment, the vector according to the invention is an adenoviral vector.

In a further embodiment it is provided that the vector according to the invention comprises a coding sequence which is under the control of the regulatory nucleic acid according to the invention.

In yet another embodiment it is provided that the coding sequence is selected from the group comprising hsp70 and the "slow skeletal troponin I". In a still further aspect, the object is achieved by a cell comprising a regulatory nucleic acid according to the invention and / or a vector according to the invention.

In a preferred embodiment of the cell according to the invention, the cell is a eukaryotic cell.

In a more preferred embodiment, the cell is a mammalian cell.

In a very preferred embodiment, the cell is a human cell.

In a further aspect, the object is achieved according to the invention by a medicament comprising a regulatory nucleic acid according to the invention, a vector according to the invention and / or a cell according to the invention.

In a preferred embodiment, the medicament is used in the context of gene therapy.

In a further preferred embodiment of the medicament according to the invention it is provided that the medicament is one for the treatment and / or prevention of cardiovascular diseases.

In a preferred embodiment it is provided that the cardiovascular diseases are those in which a point mutation in a gene leads to a clinical picture.

In a preferred embodiment of the medicament according to the invention it is provided that the point mutation lies in genes which code for proteins of the sarcomere, the dystrophin and cardiac actin.

In very particularly preferred embodiments of the medicament according to the invention it is provided that the cardiovascular disease is selected from the group comprising hypertrophic cardiomyopathy, long QT syndrome and dilated cardiomyopathy.

The invention is based on the surprising finding that genetic causes for the development or disposition for the development of dilated cardiomyopathy in humans are of crucial importance. This finding is based on the discovery by the inventor that variants which are associated with dilated cardiomyopathy can be found in various exons of the human MLP gene.

The present genomic human sequence of the MLP gene is organized into six exons, all exons having the classic intron-exon transitions and elements which are characteristic of a promoter being found at the 5 ^' end. The position of the individual exons in the human genomic sequence of the MLP gene can be described as follows:

Exon 1 from 12983 - 1301 1 (total 28 bp) Exon 2 from 22480 - 22620 (total 140 bp) Exon 3 from 26653 - 26823 (total 170 bp)

Exon 4 of 2861 1 - 28746 (135 bp total) Exon 5 of 29912 - 30005 (total 93 bp) Exon 6 of 3221 1 - 32923 (total 712 bp)

The entire gene thus extends to approximately 20,000 bp.

Exon 1 of the gene encodes only a 5 ^' untranslated region; the translated areas are coded on exons 2 to 6. A computer-aided promoter analysis showed that there is a classic TATA box in front of the 1st exon. A large number of patients with familial DCM were analyzed and it was surprisingly found that point mutations occur in the coding sequence of the genomic human sequence of the MLP gene, and thus also in the corresponding mRNA or the derived cDNA.

A patient from such a family showed a base pair exchange (T-> C) in exon 2 which leads to an amino acid exchange from tryptophan to arginine at position 4 (W4R). The mutation causing this exchange occurs more precisely at base no. 10 of exon 2 of the translated human genomic sequence for MLP. The position corresponds to position 67 of the sequence according to SEQ ID No. 1. The mutated sequence can be found here as SEQ ID No. 2

The variant in the MLP gene discovered by the inventor leads to a new restriction interface, which is why large patient populations can be analyzed quickly. On the basis of this result, a further patient collective, the members of which suffer from DCM, was examined, the exon 2 being amplified from genomic DNA from patients with DCM and digested with the corresponding enzyme. In this way, six more patients with the variant could be detected from a collective of 500 patients. As a result, family trees were created and, as far as possible, family members were summoned to take blood. On the basis of these studies, two families have so far been found in which the disease is genegosegregated.

Without wishing to be determined in the following, the following considerations regarding the variant (mutant) of the nucleic acid sequence coding for MLP described above should be made.

The amino acid tryptophan present in the wild type belongs to the heterocyclic amino acids, which is exchanged for this, ie in the O 01/57208 _-j e> _ PCT / EP01 / 01042

Mutant nucleic acid present arginine is the most basic amino acid. So it is a structurally disruptive exchange. The amino acid exchange is in a highly conserved area of the protein, whereby not only the tryptophan is highly conserved at this point between the MLPs of different species (human, pig, rat, mouse), but also the neighboring amino acids at the amino terminal end of the protein. This applies not only to the MLP itself, but also to the most closely related proteins (CRPI and CRP2). It can be concluded from this that the amino-terminal region of the protein is needed to transport the MLP to its destination in the myocyte ("docking", such sequences are the same for many proteins and therefore occur ubiquitously). If so, could the modified MLP could no longer be transported to its destination and would have no biological effect. A computer-aided analysis showed that the amino-terminal 10 amino acids only have significant homology to the MLP, CRP1 and CRP2. After the exchange W4R there was no significant homology a known protein The amino-terminal end of the protein can therefore not be a frequently occurring, typical transporter sequence, and the variant W4R likewise does not result in homology to known proteins.

From the above it can be concluded that the variant in exon 2 has a pathogenetically important function in the genesis of a DCM in the corresponding patients with a probability bordering on certainty.

In addition to the point mutation described above, another has been found that is related to DCM. This is a mutation in exon 4 of the genomic human MLP gene, more precisely a base exchange at the third position of codon 112, with an exchange from G to A. This exchange does not change anything Primary sequence of the MLP gene product encoded thereby. The position in the translated sequence corresponding to said genomic position as shown in SEQ. ID. No1, is position 393. The mutated sequence is found here as SEQ ID No. 3

This mutated form of the MLP gene or its gene product was found in a patient who had severe dilated cardiomyopathy and had to undergo a heart transplant. The genesis of the disease was unclear. However, it could be shown that this patient had a significantly reduced MLP mRNA and that this patient had a homozygous base pair exchange from A to G in exon 4 in codon 112 at position 3. It seems that this variant leads to a reduced half-life of the mRNA or e.g. B. leads via an altered differential processing to an mRNA with different properties. It can therefore be assumed that the homozygous base pair exchange in codon 112 also has a pathogenetically important effect.

The nucleic acids disclosed herein are important in many ways. By correlating the disclosed mutated MRL sequences with heart diseases, in particular dialatative cardiomyopathy, examinations can be carried out on biological material in order to check whether the sample and thus the person from whom the sample originates has or at least has heart disease due to the genetic makeup carries the risk of developing this heart disease. The disclosed nucleic acids can thus be used both for the purposes of prevention and for diagnosis. In addition to an examination tailored to the individual, a patient collective can also be the subject of investigations that use the nucleic acids disclosed or make use of them. The sequences claimed here are not only intended to be the sequences according to SEQ ID No. 2 and SEQ ID No. 3, but also all other sequences derived therefrom, as long as the mutations specifically disclosed herein are still present. In particular, those derived sequences are to be understood here which have further mutations in addition to the two special mutations disclosed, in particular also those sequences which, despite these mutations, still code for an MLP. Such mutations can be further point mutations, but also a deletion or addition of a nucleotide or an entire nucleotide sequence. It is also conceivable that several deletions or additions are present in such a sequence. These additions and deletions can. at different points in the sequence. The nucleic acids disclosed herein also include those which contain the sequences according to SEQ ID No. 2 or SEQ ID No. 3, parts of each of the two sequences being separated by a nucleotide or a sequence of several nucleotides. An example of such a sequence is the genomic MLP sequence which has said mutations and whose coding regions are separated from one another by introns.

The representation of the genomic human MLP sequence is described in the examples herein.

The probes disclosed herein are allele-specific oligonucleotides that are complementary to those regions of the nucleic acids that carry the mutations disclosed. These probes can be used in particular in the polymerase chain reaction as primers for the amplification of the nucleic acid and allow detection even with small sample amounts. The probes can be used either after the polymerase reaction or directly for the detection of the nucleic acids containing the mutations. For this purpose, the probes can be in labeled form. Such markings can include radioactive or fluorescent markers. O 01/57208 _ _η g_ PCT / EP01 / 01042

The sequences of the probes or primers for detecting the mutation in exon 2 are:

AGA TGC CAA ACC GGG GCG

and is as SEQ ID No. 4 described herein, and

for the detection of the mutation in exon 4:

TTC ACT GCG AAG TTT GGA

and is referred to herein as SEQ ID No. 5 described.

In the case of exon 2, the sequence of the probes or primers for detecting the wild-type sequence corresponding to the respective mutations is:

AGA TGC CAA ACT GGG GCG

and is referred to herein as SEQ ID No. 6, and

in the case of exon 4:

TTC ACT GCA AAG TTT GGA

and is referred to herein as SEQ ID No. 7 described.

In the methods according to the invention, the sample preparation steps customary for the detection of nucleic acids or gene products are used performed that are known to those skilled in the art. For example, blood or biopsy material can be used as the starting material.

If the method according to the invention is based on the detection of the mutation via the generation of an additional interface for a restriction enzyme which is not present in the wild-type sequence, be it now in the genomic sequence, the mRNA or a cDNA, the nucleic acid obtained Fragments are usually separated on an agarose gel. However, other separation techniques are also conceivable, e.g. Capillary electrophoresis. If an agarose gel is used, the various nucleic acid fragments can be made visible by staining with, for example, ethidium bromide or as a result of a radioactive marker.

If the gene product of one of the nucleic acids disclosed herein is detected in one of the methods according to the invention, all methods suitable for the detection of peptides or proteins can be used for this. One possibility is to use antibodies, especially monoclonal antibodies.

It will be apparent to those skilled in the art that when detecting the peptide or protein encoded by a nucleic acid that has the mutation in exon 4 that changes from position 3 to position 3 of codon 112, other methods of detection are used which must not be based on the difference in the gene product as a result of the mutation, since this mutation is a silent mutation which does not manifest itself on the level of the primary sequence, ie on the amino acid sequence. As stated above, however, this mutation, in particular when there is a homozygous base pair exchange, ie both alleles have this mutation, leads to a markedly reduced MLP mRNA, so that on the level of the gene product the detection of the mutation by quantifying the mRNA or of the gene product can take place. The kits according to the invention comprise at least one of the nucleic acids, probes or a gene product of said nucleic acids. The kit according to the invention particularly preferably comprises at least one probe which is specific for one of the two mutations described. The probe corresponding to the wild type can be included as a negative control. The nucleic acids according to the invention can be contained in the kit as a positive and / or negative control. As an alternative or in addition to the probe, a restriction enzyme can be contained in the kit, which cuts at the interface newly created by the mutation and thus allows restriction digestion of the mutated sequence compared to the non-mutated sequence and thus to a different pattern of the - cut - Nucleic acid, ie leads to a restriction length polymorphism. The nucleic acids, probes or gene products can be present in a suitable buffer in the kit. According to the invention, the kit can also comprise a carrier on which a nucleic acid or a polypeptide or protein can be immobilized.

In a further aspect, the invention relates to a regulatory nucleic acid, in particular a promoter.

The terms regulatory nucleic acid, promoter structure and promoter are used synonymously here.

In the investigations carried out by the inventor, it was recognized that a sequence arranged in front of the 1st exon of the genomic human MLP sequence and having a length of approximately 2000 bp appears to be of considerable importance for muscle-specific expression. In particular, both a TATA box and a CAAT box are found before the first exon, both of which are important for the expression of the gene. In addition, AP-1 cis regulatory elements have been found that are known to be important for muscle-specific genes. The one described WO 01/57208 _2 <| _ PCT / EP01 / 01042

regulatory nucleic acid sequence also leads to the fact that the expression of the MLP is stress-inducible. The regulatory nucleic acid sequence thus represents a tissue-specific promoter structure which can also be induced by stress.

The above-mentioned tissue specificity of the regulatory nucleic acid results from the fact that the MLP gene only occurs in muscle tissue (i.e. striated and smooth muscles); it is therefore a muscle-specific promoter. The gene product or the mRNA is relatively easy to detect using various techniques (eg Northern blot, PCR), which suggests a strong promoter. The first exon encodes only about 30 base pairs of the 5 'untranslated region. About 20-30 base pairs before the first exon there are several TATA box elements, i.e. exactly the areas to which the DNA-dependent RNA polymerase II must bind in order to copy the gene. In addition, there are several transcription initiation sequences about 10 to 20 base pairs before the first exon. In addition, several AP-1 sequences are present immediately before the first exon. These sequences bind the so-called "leucine zipper" transcription factors, which have important functions, including in the stress inducibility of genes. The presence of these sequences underpins the stress inducibility of the MLP gene found by the inventor.

A whole series of AP-1 sequences are found "upstream" in the first 1000 base pairs. A CAAT box is found around 500 base pairs upstream. As a rule, these elements are found 80-120 base pairs in front of the gene, but an important function of this element in the promoter discovered by the inventor cannot be ruled out. However, it must be noted that there are also promoters, especially tissue-specific ones, without CAAT boxes, so that it is not an absolutely necessary element. In summary, it can be stated that up to about 1000 base pairs before the first exon AP-1 and CAAT sequences are found. The region of approximately 1000 base pairs before the first exon thus appears to be of considerable importance for the expression of the gene. In any case, the core region will be the area of around 500 base pairs (i.e. starting from the CAAT box) before the first exon.

The various elements of the regulatory nucleic acid disclosed herein are shown in FIG. 2.

In a further embodiment, the regulatory sequence according to the invention can also be determined by the sequence according to SEQ ID No. 8 are shown.

The regulatory sequence or promoter structure according to the invention can also be represented by amplifying the corresponding sequence from human genomic DNA with the aid of the polymerase chain reaction with two primers. For this purpose, the primer gP1 with the sequence is used as an upstream (“upstream”) primer

ATC TGATGT GAA GGC TGG

used as it is also in the sequence listing as SEQ ID No. 9 is specified.

As a downstream primer, the primer gP2 with the sequence

CCT GCC TGT GTG GAC TCC

used as it is also in the sequence listing as SEQ ID No. 10 is specified. The regulatory sequence according to the invention can also be integrated in a vector. Such vectors can be both plasmid and viral vectors. Typically, vectors are nucleic acid molecules that serve as a recipient or carrier for — preferably foreign — nucleic acids. Vectors generally have an origin of replication ("origin") and genetic markers which allow the vector to be detected in host cells. In addition, the vector can comprise further elements which control the translation and / or transcription of the recorded or in the vector contained nucleic acid serve.

Both the regulatory sequence according to the invention and the vector according to the invention can be contained in one cell. In principle, any cell or cell line can be used for this. The cell can be selected from the group comprising prokaryotic and eukaryotic cells. Within the group of eukaryotic cells, the cell can in turn be selected from the group which in particular comprises yeast cells, insect cells and mammalian cells. In the case of the mammalian cells in particular, it can be provided that they are primary cells, primary cell cultures or established cell cultures.

There are a multitude of possible uses for the regulatory sequences and the various biological systems containing them, which are based on the following considerations and the fact that the regulatory nucleic acid disclosed herein is a tissue-specific and in particular muscle-specific expression of nucleic acid sequences under the control of said regulatory sequence allowed.

A variety of human diseases can be attributed to genetic defects. Much of these clinical pictures, in turn, will triggered by monogenic diseases, ie mutations in a single gene. With knowledge of these molecular changes, gene therapy can repair the molecular defect.

A number of diseases are known in the cardiovascular system in which point mutations in a single gene lead to serious clinical pictures. These include mutations in the genes that encode sarcomere proteins and lead to hypertrophic cardiomyopathy (Towbin. The role of cytoskeletal proteins in cardiomyopathies. 10, 131-139 (1998)), long QT syndrome (Jerve Lange Nielson syndrome and Romano Ward syndrome) (Neyround, N. et al .; Circ Res 84, 290-297 (1999)) and mutations in dystrophin (Muntni F. et al .; N Engl J Med 329, 921 -5 (1993)) or cardiac actin gene (Olsen, T. et al .; Science 280, 750 - 752 (1998)), which lead to dilated cardiomyopathy. In principle, these diseases can be treated causally by applying the correct gene.

Recently, various authors have reported on the successful application of adenoviruses in cardiomyocytes in vitro and in vivo (Hajjar, R. et al .; Proc Natl Acad Sei USA 95, 5251-5256 (1998); Donahue J. et al .; Proc Natl Acad Sei USA 94, 4664-4668 (1997) and Westfall, M. et al .; Proc Natl Acad Sei USA 94, 5444-5449 (1997)). However, the vectors used hitherto have the disadvantage that they are controlled by a cytomegalovirus (CMV) promoter. They are therefore not only expressed in the myocardium or muscle tissue, but also in every other body cell. However, the clinical pictures listed above only affect cardiomyocytes. Expression of the gene to be transferred into cells other than cardiomyocytes makes no sense and only increases the dangers of gene therapy, namely the occurrence of further mutations or undesirable interactions in the sense of immune reactions. In contrast to the ubiquitously expressed CMV promoter, the regulatory nucleic acid according to the invention is only expressed in muscle tissue and therefore, and because of its strength, it is ideal for gene therapy in or of muscle tissue.

In the production of the regulatory nucleic acid according to the invention, in general and particularly if the representation is carried out using the two primers described above, appropriate sequences recognizable by a restriction enzyme (for example Eco Rl or any other enzyme) can be attached at the ends of the cloning via its interfaces should take place).

In the production of a vector according to the invention, the procedure can be such that from the shuttle vector pAdCMVssTnl, as described in Westfall et al. (Westfall, M. et al .; Proc Natl Acad Sei USA 94, 5444-5449 (1997)), with the corresponding enzyme, cut out the CMV promoter and the MLP promoter (ie a regulatory nucleic acid according to the invention) into the shuttle plasmid pAdCMVssTnl is installed. The resulting plasmid pAdMLPssTnl can together pJM17 via the calcium phosphate method, as in Westfall et al. (Westfall, M. et al .; Proc Natl Acad Sei USA 94, 5444-5449 (1997)), co-transfected in HEK 293 cells and an adenovirus with MLP promoter obtained via homologous recombination, which is expressed in a tissue-specific manner. As a special case, this virus would express the so-called "slow skeletal troponin I". However, any desired gene product can be cloned in a known manner in front of the MLP promoter in a suitable vector, for example a shuttle vector, and brought to expression. For example, the hsp70 gene, which has protective functions on the myocardium, could be connected upstream (Yellon, D. et al .; J Mol Cell Card 24, 113-124 (1992)). Only classic cloning methods are required, which are known to experts in the field and are described, for example, in Maniatis, Fritsch & Sambrook. Molecular cloning. Cold Spring Harbor Laboratories, 1989. In summary, it can be stated that the promoter according to the invention can be cloned in front of any desired gene product and the promoter according to the invention can thus express the nucleic acid under the control of the promoter in a tissue-specific manner with any desired virus or vector system. To this extent, the promoter disclosed herein can be used in particular in the diseases mentioned above and disclosed herein, including in the gene therapy treatment of DCM. In addition to the diseases explicitly mentioned here, it is also possible to treat all those diseases using the regulatory sequence according to the invention which require tissue-specific and in particular muscle-specific expression of a nucleic acid.

The invention is further explained on the basis of the following examples, the figures and the sequence listing, from which further features and advantages of the invention may result.

It shows

1 shows a restriction analysis of exon 2 of the genomic human MLP gene; and

2 shows the sequence of the regulatory nucleic acid according to the invention with the promoter-specific elements.

EXAMPLES

1. Representation of the human MLP cDNA

The human MLP cDNA was produced using the polymerase chain reaction ( ^: PCR) using the two primers KMLP1 and KLMP2. The sequence of KMLP1 is shown as SEQ ID No.11 and is as follows:

GGC AGA CTT GAC CTT GAC CA

The sequence of KMLP2 is shown as SEQ ID No.12 and is as follows:

GAG GTG CGC CGT TTC TCA GA

The cDNA thus obtained as the product of the PCR has a length of approximately 650 bp and contains the entire coding region of the mRNA.

2. Representation of the human genomic MLP sequence

Starting from the cDNA described in Example 1, the human genomic MLP sequence was shown by screening a human genomic library using the cDNA. For this purpose, the cDNA used as a probe was radioactively marked (Feinberg, A. et al .; Anal Biochem 132, 6-13 (1983)) and hybridized with a gene bank cloned into the bacteriophage P1 (loannou, PA et al .; Nature Genetics 6 , 84-89 (1994)). The screening was carried out using the usual procedures known to those skilled in the art, e.g. are described in Maniatis, Fritsch & Sambrook. Molecular Cloning, Cold Spring Harbor Laboratories, (1989).

The MLP gene itself is located on chromosome 11 p15.1 (Fung, Y.W. et al .; Genomics 28, 602-603 (1995)).

The clone of the human genomic MLP sequence thus obtained was then sequenced using an automatic DNA sequencer. The positive clone comprised a total of 80,000 base pairs and contains the entire human MLP gene, which is organized into a total of six exons, as already disclosed above.

3. Detection of the mutations in exon 2 and in exon 4 of the human genomic MLP sequence

As stated above, the two mutations found by the inventor in the MLP sequence are those at base 10 of exon 2 of the translated human genomic MLP sequence, with an exchange from T to C, and one at the third position of codon 112 of exon 4 of the translated sequence, with an exchange from A to G. Both mutations find their correspondence in the mRNA or the cDNA derived therefrom, the position of the mutation in base 67 corresponding to the mRNA or cDNA in the case of the mutation in exon 2 and the position of the mutation in the case of the mutation in exon 4 base 393 of the mRNA or cDNA, as also shown in SEQ ID No. 1, corresponds.

3.1 Isolate Genomic DNA

Production of the "buffv coats"

Whole blood is centrifuged for about 10 minutes at 3300 g and room temperature. After centrifugation, three phases are obtained, the upper clear phase corresponds to the plasma, the thin white phase corresponds to the leukocytes and the lower red phase corresponds to the erythrocytes. The leukocytes can now be easily removed with a pipette. They correspond to the "buffy coat".

Isolation of genomic DNA The genomic DNA was isolated using a "Qiagen Blood Kit" from 1997 and a standard "Eppendorf benchtop centrifuge" as follows:

a.) About 200 μl of the buffy coat was placed in a 1.5 ml Eppendorf tube. 25 μl of Proteinase K and 200 μl of a buffer AL were added and vortexed (shaken vigorously, mixed) for 15 seconds, b.) This mixture was heated in a water bath at 70 ° C. for 10 minutes. c.) 210 μl of ethanol (100%) were added to this mixture and mixed vigorously again. d.) A Qiagen column was placed in a 2 ml Eppendorf tube and the

Mixture of c) applied to the column. Centrifugation was carried out for 1 minute at 6000 g and the column was placed on a new 2 ml Eppendorf tube. The centrifuged liquid was discarded, the genomic DNA had bound to the column, e.) 500 μl of buffer AW were added to the column and centrifuged again at 6000 g for 1 minute. The centrifuged liquid was discarded again and the column was placed in a new 2 ml Eppendorf tube. f.) Another 500 μl of AW buffer were added to the column and at the highest speed (about 15,000 revolutions at one

Centrifuge) for 3 minutes. The centrifuged

Liquid was discarded again. g.) The column was now placed on a 1.5 ml Eppendorf tube and 200 μl of pure water, previously heated to 70 ° C., were added to the column. There was incubation for one minute at room temperature and centrifugation at 6000 g for one minute. The genomic DNA was in the centrifuged liquid, the eluate, and was used for further purposes. The concentration was determined photometrically in a further step. About 50 ng of the genomic DNA was used for the polymerase chain reaction.

Amplification of exon 2 of the human genomic MLP sequence

To amplify exon 2, the primers GMLP 3 with the sequence

ACT CTTAGA GAT TGG TTC ACT CC

according to SEQ ID No. 13 and

GMLP 4 with the sequence

ACC ACA CTATGA GAA CCA CTG GC

according to SEQ ID No. 14 used.

The following amplification conditions were used:

1. 94 ° C for 4 minutes

Then a total of 36 cycles begin

2. 94 ° C for 45 seconds 3. 62 ° C for 45 seconds

4. 72 ° C for 1 minute

After the 36 cycles:

5. 72 ° C for 5 minutes

3.2 Restriction digestion of exon 2 of the human genomic MLP sequence O 01/57208 -, 3,, 1- PCT / EP01 / 01

Some of the PCR products described and obtained under 3.1 were separated on an agarose gel and analyzed for their correct size. If the sizes were correct, which was usually the case, another part was digested with the restriction enzyme Nci I at 37 ° C. overnight and separated again on an agarose gel the next morning.

The PCR for the amplification of exon 2 results in a PCR product with a size of 308 base pairs. If a base pair exchange from T to C has occurred in position 1 in codon 4, the restriction enzyme Nci I cuts and 2 fragments with a size of 103 base pairs and 205 base pairs are formed.

In the homozygous case, one would not find a PCR product with a size of 308 base pairs, but only the two fragments. In the heterozygous case, i.e. there is a healthy one, i.e. an allele not mutated with respect to the special base, and an allele mutated with respect to the special base, one finds the PCR product with 308 base pairs in addition to the two smaller fragments.

The result of this restriction analysis is shown in FIG. 1, FIG. 1 showing in more detail the result of a restriction digest of amplified exon 2 of the human genomic MLP sequence, which was applied to an agarose gel and separated. The two outer traces of the agarose gel each show a molecular weight standard. The lane labeled "wt" shows exon 2 in its wild type, with no restriction enzyme being added to the applied restriction approach. The lane labeled "wt + E" shows exon 2 in its wild type, with the restriction enzyme Nci I being added to the applied restriction approach has been. As a result of the lack of the mutation disclosed here on base 10 of exon 2 in the human genomic wild-type MLP sequence, there is no interface for Nci I in exon 2, so that there is none compared to the restriction approach with the wild-type sequence without restriction enzyme changed pattern. The lane labeled "M" shows a restriction approach which was carried out with the mutated form of exon 2 of the human genomic MLP sequence without restriction enzyme. No additional bands are found in the gel without addition of the restriction enzyme, so that, as well as from the Comparison with the approach labeled "wt" reveals that the size of the nucleic acid fragment comprising exon 2 has not changed as a result of the mutation. If one carries out a restriction approach with the exon 2 mutated on base 10 and the restriction enzyme Nci I is added, the mutation-related formation of an interface for the said restriction enzyme leads to a cutting of the nucleic acid of exon 2 and formation of the two predicted nucleic acid fragments, such as this is also shown in FIG. 1 in the track above "M + E".

Using this technique, around 500 patients have been examined for the occurrence of the variant discovered by the inventor. Six patients with the variant were found.

3.3 Restriction digestion of exon 4 of the human genomic MLP sequence

In principle, the same procedure as for exon 2 was used to analyze exon 4. However, the primers gMLP7.2 with the sequence

CAA CAA CGC TAT gAg AAA gAC ACC

according to SEQ ID No. 15 and

gMLP8.2 with the sequence

ggA gCT AgA gAg AAT gAC AgC TgC according to SEQ ID No. 16 used under the following PCR conditions:

1. 94 ° C for 4 minutes

Then a total of 36 cycles begin

2. 94 ° C for 45 seconds

3. 60 ° C for 45 seconds

4. 72 ° C for 1 minute After the 36 cycles then:

5. 72 ° C for 5 minutes

Some of the PCR products obtained were separated on an agarose gel and analyzed for their correct size. With correct sizes, which was usually the case, another part was digested with the restriction enzyme Cvi Rl at 37 ° C. overnight and separated again on an agarose gel the next morning. Regarding the incubation, it should be noted that the restriction batch could have been incubated for 30 to 60 minutes at room temperature. The overnight incubation was only for reasons of care.

3.4 Detection of mutations in exon 2 or exon 4 using allele-specific oligonucleotides

In addition to carrying out restriction analyzes, the variants or mutations in exon 2 and exon 4 of the genomic human MLP sequence or the corresponding cDNA probes (so-called allele-specific oligonucleotides, ASO), which are disclosed herein, can be produced. For the detection of the variant or mutation in exon 2, a probe with the following sequence can be used, the SEQ ID No. 4 corresponds to:

AGA TGC CAAACC GGG GCG

To detect the wild-type form of exon 2, a probe with the following sequence, SEQ ID No. 6 corresponds to:

AGA TGC CAAACT GGG GCG

The melting temperature when detecting the mutation using the sequence according to SEQ ID No. 4 is 60 ° C, the melting temperature when detecting the wild type using the sequence according to SEQ ID No. 6 is approx. 58 ° C.

For the detection of the variant or mutation in exon 4, a probe with the following sequence can be used, the SEQ ID No. 5 corresponds to:

TTC ACT GCG AAG TTT GGA

To detect the wild-type form of exon 4, a probe with the following sequence, SEQ ID No. 7 corresponds to:

TTC ACT GCA AAG TTT GGA

The melting temperature when detecting the mutation using the sequence according to SEQ ID No. 5 is 52 ° C, the melting temperature when detecting the wild type using the sequence according to SEQ ID No. 7 is approx. 50 ° C. - o-

With the primers listed, exons 2 and 4 of all carriers of the mutation (s) determined by means of the restriction analysis and the probe analysis were also sequenced using standard techniques. The sequence analysis confirmed the result of the restriction and probe analysis in all cases. Thus, the sequencing of exons 2 or 4 or the corresponding cDNA or the corresponding cDNA of the genomic human MLP gene or the genomic MLP gene, in particular using the probes described herein, can also be used as a method according to the invention for the detection of and / or screening for myocardial diseases, in particular dilated cardiomyopathy, can be used.

3.5 Structure of the regulatory nucleic acid (sequence)

The in SEQ ID No. The specified regulatory nucleic acid is shown in FIG. 2 together with the labeled promoter-specific elements. This regulatory nucleic acid is isolated using the primers gP1 and gP2 described above, corresponding to SEQ ID No. 9 and SEQ ID No. 10, possible. The regulatory nucleic acid sequence or the corresponding MLP promoter (1000 bp) can be amplified using the two primers mentioned.

With reference to FIG. 2, the following should also be noted here:

The last sequence underlined at the 3 ^' end corresponds to the first exon.

The AP-1 sequences are marked by underlining, with a maximum of two mismatches being accepted from the consensus sequence TGAG / CTCA.

CAAT sequences are marked in bold, it should be noted in this regard that at the beginning of the promoter an AP-1 and a CAAT sequence overlap.

TATA boxes are marked in bold and underlined.

The disclosure of the various references cited herein is hereby fully incorporated by reference.

The features of the invention disclosed in the above description, the claims and the drawing can be essential both individually and in any combination for the implementation of the invention in its various embodiments.

Claims

Expectations

1. Nucleic acid coding for an MLP and comprising a sequence according to SEQ ID NO: 1, the sequence having a mutation at base no. 10 of exon 2 of the translated sequence.

2. Nucleic acid according to claim 1, characterized in that the mutation is an exchange of T to C.

3. Nucleic acid coding for an MLP and comprising a sequence according to SEQ ID NO: 1, the sequence having a mutation at the third position of codon 112 in exon 4 of the translated sequence.

4. Nucleic acid according to claim 3, characterized in that the mutation is an exchange from A to G.

5. Nucleic acid according to one of the preceding claims, characterized in that it comprises only the sequences coding for an amino acid sequence.

6. Nucleic acid coding for an MLP and comprising the sequence from positions 58 to 639 of SEQ ID NO: 1, characterized in that a mutation is present at position 67, in particular a mutation from T to C.

7. Nucleic acid coding for an MLP and comprising the sequence from positions 58 to 639 of SEQ ID NO: 1, characterized in that a mutation is present at position 393, in particular a mutation from A to G.

8. Nucleic acid coding for MLP, characterized in that the sequence would correspond to a nucleic acid according to one of claims 1 to 7 without the degeneracy of the genetic code.

9. Nucleic acid coding for MLP, characterized in that the nucleic acid hybridizes with a nucleic acid according to one of claims 1 to 8.

10. amino acid sequence, encoded by a nucleic acid according to one of claims 1 to 9 or parts thereof, in particular the sequences or parts thereof, which carry the mutation (s).

11. MLP comprising an amino acid sequence encoded by a nucleic acid according to one of claims 1 to 9 or a part thereof or an amino acid sequence according to claim 10.

12. Probe for a nucleic acid according to one of claims 1 to 9, in particular for its detection or amplification, comprising the sequence of SEQ ID NO: 4.

13. A probe for a nucleic acid according to one of claims 1 to 9, in particular for its detection or amplification, comprising the sequences according to SEQ ID NO: 5.

14. Use of a nucleic acid according to one of claims 1 to 9 and / or a probe according to claim 12 or 13 for the diagnosis of and / or screening for myocardial diseases, in particular dilated cardiomyopathy.

15. A method for the detection of and / or screening for myocardial diseases, in particular dilated cardiomyopathy, characterized in that a nucleic acid according to one of claims 1 to 9 and / or a probe according to claim 12 or 13 is used.

16. A method for the detection of and / or screening for myocardial diseases, in particular dilated cardiomyopathy, in particular according to claim 15, characterized in that

a sample comprising a sequence coding for MLP is digested with a restriction enzyme and

the presence of a mutant sequence coding for MLP is demonstrated by the occurrence of a restriction enzyme digest pattern which has been changed compared to the restriction enzyme digestion pattern of a non-mutated sequence coding for MLP.

17. The method according to claim 16, characterized in that the sequence coding for MLP is amplified by means of PCR before digestion.

18. The method according to claim 16 or 17, characterized in that the detection or screening for a nucleic acid according to one of claims 1 to 9 is directed.

19. The method according to claim 17 or 18, characterized in that the amplification is carried out with a primer, the primer comprising a sequence according to SEQ ID NO: 13 and / or SEQ ID NO: 14.

20. The method according to any one of claims 16 to 19, characterized in that the restriction enzyme digestion is carried out by Nci I.

21. The method according to claim 20, characterized in that in the case of a non-mutated sequence the PCR product has a length of approximately 308 bp and in the case of a mutation at base no. 10 of exon 2 or a mutation at position 67 of the nucleic acid sequence of SEQ ID NO: 1, where the mutation there is an exchange from T to C, two PCR products with approximately 205 and approximately 103 bp occur.

22. The method according to any one of claims 15 to 17, characterized in that the detection or screening for a nucleic acid according to one of claims 3 to 9 is directed.

23. The method according to claim 22, characterized in that the amplification is carried out with a primer, the primer comprising the sequence according to SEQ ID NO: 15 and / or SEQ ID NO: 16.

24. The method according to claim 22 or 23, characterized in that the restriction enzyme digestion is carried out by Cvi Rl.

25. The method according to claim 15, characterized in that for the detection of or screening for a nucleic acid sequence according to one of claims 1 to 9, a hybridization of a sample to be examined with a probe comprising a sequence according to SEQ ID NO: 4 is carried out.

26. The method according to claim 25, characterized in that the hybridization takes place at 60 ° C.

27. The method according to claim 15, characterized in that for the detection of or screening for a nucleic acid sequence according to one of claims 3 to 9, a hybridization of a sample to be examined with a probe comprising a sequence according to SEQ ID NO: 5 is carried out.

28. A method for the detection of and / or screening for myocardial diseases, in particular dilated cardiomyopathy, characterized in that an amino acid sequence according to claim 10 or an MLP according to claim 11 is detected.

29. The method according to claim 28, characterized in that the amino acid sequence or the MLP is detected by means of an antibody, preferably a monoclonal antibody.

30. Kit for detecting a nucleic acid according to one of claims 1 to 9, characterized in that it comprises at least one nucleic acid according to one of claims 1 to 9 and / or at least one probe according to claim 12 or 13.

31. Kit for the detection of an MLP according to claim 11 or an amino acid sequence coding for an MLP according to claim 10 comprising at least one antibody against an MLP according to claim 11 or against an amino acid sequence according to claim 10.

32. Regulatory nucleic acid comprising about 500 base pairs upstream of the first base of the first exon of the human genomic sequence coding for MLP.

33. Regulatory nucleic acid according to claim 32 comprising about 1000 base pairs upstream of the first base of the first exon of the human genomic sequence coding for MLP.

34. Regulatory nucleic acid, characterized in that, starting from human genomic DNA using two primers, the regulatory nucleic acid can be prepared in a PCR reaction, the upstream primer being SEQ ID NO: 9 and the downstream primer being SEQ ID NO: 10 includes.

35. Regulatory nucleic acid comprising a sequence according to SEQ ID NO: 8.

36. Regulatory nucleic acid according to one of claims 32 to 35, characterized in that the regulatory nucleic acid is a promoter.

37. Vector comprising a regulatory sequence according to one of claims 32 to 36.

38. Vector according to claim 37, characterized in that the regulatory sequence is contained in the vector pAdCMVssTnl, with this vector the CMV promoter being replaced by the regulatory sequence according to one of claims 32 to 36.

39. Vector according to claim 37, characterized in that it is an adenoviral vector.

40. Vector according to one of claims 37 to 39, comprising a coding sequence which is under the control of the regulatory nucleic acid according to one of claims 32 to 36.

41. Vector according to claim 40, characterized in that the coding sequence is selected from the group comprising hsp70 and the "slow skeletal troponin I".

42. Cell comprising a regulatory nucleic acid according to one of claims 32 to 36 and / or a vector according to one of claims 37 to 40.

43. Cell according to claim 42, characterized in that the cell is a eukaryotic cell, preferably a mammalian cell and most preferably a human cell.

44. Medicament comprising a regulatory nucleic acid according to one of claims 32 to 36, a vector according to claim 37 to 41 or a cell according to one of claims 42 or 43.

45. Medicament according to claim 44, characterized in that the medicament is used in the context of a gene therapy.

46. Medicament according to claim 44 or 45, characterized in that the medicament is one for the treatment and / or prevention of cardiovascular diseases, in particular cardiovascular diseases, in which a point mutation in a gene leads to a clinical picture.

47. Medicament according to claim 46, characterized in that the point mutation lies in genes which code for proteins of the sarcomere, dystrophin and cardiac actin.

48. Medicament according to claim 46 or 47, characterized in that the cardiovascular disease is selected from the group comprising hypertrophic cardiomyopathy, long QT syndrome and dilated cardiomyopathy.