EP1366072A2

EP1366072A2 - Ets-transcription factor related compound specific promoter and transactivators thereof

Info

Publication number: EP1366072A2
Application number: EP01992719A
Authority: EP
Inventors: Markus A. RÜEGG; Alexandre Briguet
Original assignee: MYOCONTRACT AG
Current assignee: Santhera Pharmaceuticals Schweiz GmbH
Priority date: 2000-11-02
Filing date: 2001-10-31
Publication date: 2003-12-03
Also published as: WO2002036620A9; WO2002036620A3; WO2002036620A2; AU2002219070A1

Abstract

The present invention relates to novel means for the treatment of diseases, in particular muscular diseases. The present invention relates to a nucleic acid capable of controlling the expression of a heterologous gene and of being selectively activated by an Ets-transcription factor related compound. The invention further relates to constructs, host cells comprising said nucleic acid, methods for recombinant expression of heterologous genes and methods for screening and identifying of candidate compounds having transcription regulatory activity using them. The invention additionally relates to a fusion protein for specifically activating said nucleic acid. The invention also relates to a kit comprising the nucleic acid and the fusion protein for recombinant expression of a heterologous gene.

Description

ETS-TRANSCRIPTION FACTOR RELATED COMPOUND SPECIFIC PROMOTER AND TRANSACTIVATORS THEREOF

The present invention relates to nucleic acids capable of controlling the expression of a gene and of being activated by a novel Ets-Transcription Factor Related Compound, methods for recombinant expression using them, and methods for screening of candidate activator compounds. Furthermore, the present invention relates to a fusion protein capable of binding to said nucleic acid and methods for inducing gene expression using said fusion protein. Additionally, the present invention relates to a system for expression of a gene.

Duchenne Muscular Dystrophy (DMD) is an inherited muscle-wasting disease caused by the absence of the muscle cytoskeleton protein dystrophin. Young patients experience retardation in the achievement of motor milestones during infancy and early childhood when distal limb muscle mass is lost and replaced by connective and fatty tissue. Most DMD patients become wheelchair-bound as teenagers and die of respiratory or cardiac failure around the early age of 20 to 25. There is currently no effective treatment by medicaments available that would prevent the fatal outcome of this disease or at least contribute to the quality of life of the patient. The principal cause of DMD is the absence of functional "dystrophin" protein (Koenig et al., Cell 53 (1988), 219-226), a cytoskeleton protein that contributes to the stability of muscle fibers during cycles of contraction and relaxation. A similar, but milder form of muscle wasting called "Becker muscular dystrophy (BMD)" also is a consequence of dystrophin gene mutations.

Possible strategies to prevent muscle cell deterioration in dystrophic muscles are based on the observation that even in diseased muscle tissue a protein is expressed that shares structural and functional similarities with the lacking dystrophin. This "dystrophin- related protein" (Love et al., Nature 339 (1989), 55-58, later called "utrophin" is encoded on chromosome 6 in humans and is not affected in DMD patients. The cDNA sequence encoding for human and mouse dystrophin have been deposited under gene bank accessions nos. Gl 6005937 and Gl 1934962, respectively.

Experimental evidence supports the scientific opinion that elevated levels of utrophin expression in DMD muscle might be of therapeutic relevance. This is based on the finding that in dystrophic muscle from DMD patients as well as in a rodent model for DMD, the MDX mouse, utrophin is already found at elevated levels, in addition, when experimentally overexpressed in transgenic mice that lack dystrophin but show elevated levels of utrophin message and protein. The characteristic phenotype of muscle damage is significantly ameliorated (Tinsley et al., Nature 384 (1996), 349-353 and WO 97/22696). One strategy for DMD treatment involves elevated transcription from the endogenous utrophin gene and any pharmacological induction of utrophin gene transcription is of potential therapeutic interest.

Generally, utrophin can be transcribed from two distinct promoters. The first described promoter (called UTR-promoter (A)) is located within the CpG island at the 5' end of the locus (Dennis et al., Nucleic Acid Research 24 (1996), 1646-1652). The second utrophin promoter which provides an alternative target for therapeutic up-regulation of utrophin is located in the intron 2 of the utrophin gene, driving the expression of a unique first exon that splices into the 13 kb utrophin mRNA (Burton et al., PNAS USA, 96 (1999), 14025- 14030). Due to this different start of mRNA, the utrophin protein derived from the second utrophin promoter, called "UTR-promoter (B)" hereinafter, has an N-terminal sequence that is distinct from utrophin protein derived from the UTR-promoter (A). It has been found that the B-utrophin is the most abundant form in the heart assessed on the basis of mRNA, whereas A-utrophin predominates in the kidney. Approximately, equal amounts of B-utrophin and A-utrophin RNA have been observed both in the brain and in skeletal muscle.

The present inventors have now concluded that a different organ-specific transcription of utrophin and due to this different transcription of mRNAs a different production of utrophin A versus utrophin B may facilitate the treatment of organ-specific diseases, in particular muscle diseases as e.g. Duchenne Muscle Dystrophy.

Furthermore, the use of a nucleic acid being selectively activated by organ-specific or tissue-specific distributed transcription factors and/or activators would allow a very fine tuned therapy of different diseases allowing the selective expression of genes in a tissue and/or organ-specific manner to affect treatment.

Consequently, one technical problem underlying the present invention in view of the prior art was to provide means useful in the treatment of muscle diseases such as nucleic acids and proteins. Further technical problems relate to the provision of methods for the recombinant expression of a gene and methods for screening of candidate compounds being capable of regulating transcription.

The technical problem is solved by the provision of a nucleic acid capable of controlling the expression of a gene and of being activated by an Ets-transcription factor related compound, wherein the nucleic acid is selected from the following:

(a) a nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO:1 ;

(b) a nucleic acid molecule capable of hybridizing with the nucleic acid molecule of (a);

(c) a nucleic acid molecule being degenerate to any of the nucleic acid molecules of (a) or (b) due to the genetic code;

(d) a nucleic acid molecule being at least 60% homologous to any of the nucleic acid molecules of (a) to c);

(e) a variant of any of the nucleic acid molecules of (a) to (d) wherein said variant differs from the nucleic molecules by at least one addition, deletion, insertion and/or inversion;

(f) a fragment of any of the nucleic aqid molecules of a) to e); and

(g) a combination of at least two nucleic acid molecules of (a) to (f), wherein the nucleic acid molecule of (a) to (g) comprises at least one Ets-transcription factor binding-site, preferably at least 2, more preferably at least 3 Ets-transcription factor binding-sites.

The term "an Ets-transcription factor related compound" means an Ets-transcription factor and any variant thereof capable of activating the transcription from the nucleic acid according to the present invention. The Ets-transcription factor related compound further comprises the Ets-related transcription factor complex GA-binding protein α/β, members of the YAN-, ELG-, PEA3-, ERF-, and TCF-subfamilies of the Ets-transcription factor- family (Wasylyk, B. et al., TIBS 23 (1998), 213-216).

"Cloning" means any of the cloning methods known in the art and which may be applied in the present context, none of which, however, are outlined in detail because they belong to the common general knowledge of the person skilled in the art. The term "recombinant expression in a suitable host cell" means any of the expression methods and expression systems known in the art which may be applied in the present context which are, however, not explained in detail, because they belong to common general knowledge of the person skilled in the art.

The term "inducibility" means the increase in the amount of transcription from a given nucleic acid due to the presence of a transcription factor, preferably GABP, more preferably GABP_VP16 (SEQ ID NO:2). The inducibility is determined by the ratio of transcriptional activity from a given nucleic acid in the presence versus absence of said transcription factor. In accordance with the present invention inducibility is defined by a ratio of at least two. The inducibility may be determined by promoter-reporter assay, northern blot, RT-PCR or Real-time PCR analysis. Preferably, the inducibility is determined as follows:

The promoter/reporter construct may be prepared by insertion of the promoter into firefly luciferase encoding plasmids, e.g. pGL2-basic™ (Promega) such that the firefly luciferase is expressed under the control of the promoter sequence. The generated promoter/luciferase-reporter construct is transfected into C2C12 muscle cells. Preferably, the following transfection protocol is used:

C2C12 myoblasts are seeded at a density of 10,000/well in 24 well-plates that are previously coated with gelatin. 24 h after seeding the myoblasts are transfected with 100 ng of the reporter construct, together with 10 ng of the standard pRL-TK™ vector (Promega) encoding Renilla luciferase under the control of the thymidine kinase promoter, and optionally the compound to be tested. 24 h later the proliferation medium containing 20 % fetal calf serum is replaced with differentiation medium containing 5 % horse serum. After 96 h in differentiation medium, the cells are lysed in 150 μl/well passive lysis buffer (Promega). The firefly and Renilla luciferase reporter activities contained in 20 μl lysate are then measured using the dual luciferase reporter assay (Promega). Luciferase activity produced by the promoter reporter construct is normalized to the activity derived from the cotransfected pRL-TK vector.

The term "basal transcriptional activity" means the transcription of a gene mediated by the nucleic acid having the nucleotide sequence set forth in SEQ ID NO:1 in the absence of additional or exogenous transcription factors and/or activators. The basal transcriptional activity may be determined by promoter-reporter assay, northern blot, RT- PCR or Real-time PCR analysis. Preferably, the basal transcriptional activity is determined as described above for the inducibility in the absence of any compound.

The term "transcription factor" means any compound capable of specifically binding to enhancers and/or promoters, in particular to the nucleic acid having the nucleotide sequence set forth in SEQ ID NO:1.

The term "transcription activator" means in the context of the present invention any substance capable of activating the transcription of a gene, in particular the utrophin gene (Pearce et al., Horn. Mol. Gene (1993), 1765-1772).

The present inventors were the first to demonstrate the selective activation of utrophin gene transcription from the nucleic acid according to the invention using a member of the Ets-transcription factor family. It was surprisingly found that utrophin gene transcription could selectively be activated via the utrophin B-promoter using the transcription factor according to the invention while no induction could be affected by the use of the same transcription factor and the utrophin A promoter. It should be noted that mere sequence analysis within promoters does not allow the reliable prediction of functional Ets- transcription factor binding sites within a given sequence.

The nucleic acid according to the present invention may be derived from genomic DNA, cDNA or synthetic DNA, wherein a synthetic DNA-sequence also comprises such and having modified nucleoside bonds. Furthermore, the nucleic acid may be an RNA sequence which may be necessary for the expression in recombinant vector systems. The nucleic acid according to (b) may e.g. be obtained by the use of a detectably labeled probe which at least partly corresponds to the sequence according to (a) or a fragment or the opposite strand thereof for screening of cDNA-libraries and genomic DNA-libraries, respectively, from eukaryotes, preferably mammalians, most preferably humans and mice. The identification of positive cDNA and genomic DNA clones, respectively, may be obtained using standard procedures; cf. Maniatis et al., Molecular Cloning (1989), Cold Spring Harbor Laboratory Press. In a preferred embodiment the hybridization according to step (b) is carried out under stringent conditions. Stringent hybridization conditions are e.g. incubation at 65 °C overnight in 7% SDS, 1% BSA, 1 mM EDTA, 250 mM sodium phosphate (pH 7.2) and subsequent washing at 65 °C with 2 x SSC; 0.1% SDS. In a preferred embodiment nucleic acids are provided which are at least 80% homologous to any of the nucleic acid molecules of (a) to (c), more preferably the nucleic acids are at least 90% and most preferably at least 95% homologous to the nucleic acid according to (a).

According to the present invention, the term "homology" means homology on the DNA level which may be determined using standard procedures, e.g., computer-based sequence alignments (basic local alignment search tool, S.F. Altschul et al., J. Mol. Biol. 215 (1990) 403-410).

The expression "homology", well-known to the skilled person, designates the degree of relatedness between two or more nucleic acid molecules, which is determined by the agreement between the sequences. The "percentage homology" is obtained from the percentage of identical regions in two or more sequences taking account of gaps or other sequence features.

The homology of mutually related nucleic acid molecules can be determined by means of known procedures. As a rule, special computer programs with the algorithms taking account of the special requirements are used.

Preferred procedures for the determination of homology firstly generate the greatest agreement between the sequences studied. Computer programs for determination of homology include, but are not limited to, the GCG program package, including GAP (Devereux, J., et al., Nucleic Acids Research 12 (12): 387 (1984); Genetics Computer Group University of Wisconsin, Madison, (Wl)); and BLASTP, BLASTN and FASTA (Altschul S., et al., J. Mol. Biol., 215: 403-410 (1990)). The BLASTX program can be obtained from the National Center for Biotechnology Information (NCBI) and from other sources (BLAST Handbook, Altschul S., et al., NCB NLM NIH Bethesda MD 20894; Altschul S., et al., J. Mol. Biol., 215: 403-410 (1990)). The well-known Smith-Waterman algorithm can also be used for the determination of homologies.

Preferred parameters for the nucleic acid sequence comparison include the following:

Algorithm: Needleman and Wunsch, J. Mol. Biol. 48: 443-453 (1970)

Comparison matrix: Matches = + 10

Mismatches = 0 Gap penalty 50 Gap length penalty: 3

The GAP program is also suitable for use with the above parameters. The above parameters are the default parameters for nucleic acid sequence comparisons.

Further examples of algorithms, gap opening penalties, gap extension penalties and comparison matrices including those named in the program handbook, Wisconsin package, Version 9, September 1997, can be used. The choice will depend on the comparison to be performed and further on whether the comparison is performed between sequence pairs, when GAP or Best Fit are preferred, or between one sequence and a large sequence database, when FASTA or BLAST are preferred.

An agreement of 60% determined with the aforesaid algorithm is described in the context of the present application as 60% homology. The same applies for higher degrees of homology.

In a preferred embodiment, the nucleic acid molecule of (d) to (g) exhibits at least 60%, preferably 80%, more preferably 90% of the inducibility of the nucleic acid having SEQ ID NO:1 by the transcription factor GABP and/or at least 60%, preferably 80%, more preferably 90% of the basal transcriptional activity of the nucleic acid having SEQ ID NO:1.

In a further preferred embodiment the Ets-transcription factor related compound is a GABP sequence. It is particularly preferred that the GAPB variant used to determine the inducibility of the nucleic acid is GAPB_VP 16 (SEQ ID NO:2).

The present invention further provides constructs comprising the nucleic acid according to claim 1 and at least one heterologous gene operably linked to the nucleic acid such that the heterologous gene is expressed under the control of the nucleic acid. The heterologous gene may be derived from any source. Preferably, the heterologous gene may be derived from a gene whose gene product has therapeutic potential such as e.g. cytokines, complement factors or hematopoietins.

Preferably, said construct may further comprise regulatory regions for transcription and/or replication. The above regulatory regions for transcription and/or replication may be derived from a vector selected from bacteriophages such as λ-derivatives, plasmids, adenoviruses, vaccinia viruses, baculoviruses, SV 40 viruses.

It is further preferred that the construct additionally comprises a signal peptide encoding nucleic acid sequence ensuring export of the expressed protein, wherein the signal peptide coding nucleic acid sequence preferably is directly 5' of the heterologous gene to be expressed.

According to a further preferred embodiment of the present invention, host cells are provided comprising the nucleic acid molecule and/or the construct and being capable of expressing the heterologous gene. From common general knowledge, numerous prokaryotic and eukaryotic expressions systems are known. The host cells may e.g. be selected from prokaryotic cells such as E. coli or B. subtilis from eukaryotic cells such as yeast cells, insect cells and yeast cells, e.g. CHO-cells, COS-cells or HeLa-cells and derivatives thereof.

In a preferred embodiment, the Ets-transcription factor binding-site is selected from the following consensus sequences: 5' (C/A) GGA (A/T) (A/G) 3' or in the reverse orientation 5' (C/T) (A/T) TCC (T/G) 3'. It has been found that the nucleic acid having the nucleotide sequence set forth in SEQ ID NO:1 (human utrophin B promoter) comprises three Ets- transcription factor binding-sites at positions 32, 145 and 294 with reference to SEQ ID NO:1. For comparison, the promoter sequence of the mouse utrophin gene has also been analyzed where two conserved sequences for the binding of Ets-transcription factors can be identified at equivalent positions.

In another embodiment the present invention provides a pharmaceutical composition comprising at least one nucleic acid according to the present invention and/or one construct according to the present invention. The medical indication for which said pharmaceutical composition is useful depends on the heterologous gene to be expressed under the control of the nucleic acid according to the present invention. It is therefore preferred that the heterologous gene is a gene encoding a protein having therapeutic activity. It is preferred that the heterologous gene encodes cytokine, a complement factor, a hematopoietin or a cytoskeletal protein. More preferably, the gene is utrophin. The usefulness of utrophin has been suggested when utrophin has been experimentally overexpressed in transgenic mice that lack dystrophin but show elevated levels of utrophin message and protein. The characteristic phenotype of muscle damage has been significantly ameliorated by overexpression of utrophin in said mice (Tinsley et al., Nature 384 (1996), 349-353). It is intended that the pharmaceutical compositions according to the invention are for enteral, oral, subcutaneous, parenteral, or intravenous administration. The pharmaceutical composition may further comprise pharmaceutically acceptable excipients, vehicles or carriers, and optionally other ingredients well-known in the art. The amount to be administered may be determined on the basis of common general knowledge by the physician and depends on the age and physical conditions of the human and/or animal to be treated.

The present invention further provides a method for recombinant expression of a gene comprising the following steps:

(a) providing a construct according to the invention;

(b) transfecting a suitable host cell with said construct; and

(c) expressing the gene under the control of said nucleic acid in the transfectant.

The person skilled in the art is aware of numerous methods for recombinant expression of nucleic acids; cf. Recombinant Gene Expression Protocols in Methods in Molecular Biology, volume 62, Humana Press Totowa, New Jersey, (1995). The expression may be constitutive or inducible, wherein suitable inducers such as IPTG and Zn²⁺ are known to the person skilled in the art. Optionally, the produced gene product may be purified by chromatography methods known in the art such as ion exchange, gel chromatography, hydrophobic chromatography and/or gel filtration chromatography. Preferably, recombinant expression of the gene is effected and/or facilitated by the provision of an Ets-transcription factor related compound to the transfectant. The Ets-transcription factor related compound may be administered either by a nucleic acid containing a construct wherein the nucleic acid encodes the Ets-transcription factor related compound or by the administration of the compound as such to the transfected cell.

In another embodiment the present invention relates to a method for screening and/or providing of candidate compounds being capable of regulating transcription comprising the step of bringing into contact the nucleic acid according to the invention with compounds to be screened and detecting the transcriptional activity in the presence and absence of said compounds and optionally purifying and/or synthesizing the positively tested compound. Preferably, the gene operably linked to the nucleic acid according to the present invention is a reporter gene. Suitable reporter genes are e.g. firefly luciferase or β-galactosidase. The determination of expression of the reporter gene is well-known in the art. The nucleic acid according to the present invention is capable of being selectively activated by an Ets-transcription factor related compound, in particular GABP or a variant thereof (GABP_VP16). Thus, the nucleic acid according to the present invention should be particularly useful in retrieving novel-compounds being capable of mimicking the effect of GABP. The method is useful for the screening of candidate compounds from synthetic libraries or from isolates from natural sources.

The present invention also encompasses the compounds obtainable by said method. The novel compounds obtained by said method may be identified by routine methods such as N-terminal sequencing and/or mass spectrometry well-known in the art.

In another embodiment the present invention provides a fusion protein capable of binding to a nucleic acid according to the invention, wherein the fusion protein comprises at least a part of a transcription factor domain and at least a part of a viral transcription activator domain. Generally, the present invention contemplates the use of any transcription factor domain for said fusion protein. Preferably, the transcription factor domain comprises at least a part of a DNA binding domain, a part of a domain required for heterodimerization with a DNA binding protein and/or a part of a domain for nuclear localization, or a combination thereof. Preferably, the transcription factor is selected from a member of the Ets-transcription factor family such as e.g. a member of the YAN-, ELG- , PEA3-, ERF- and TCF-subfamilies of Ets-transcription factors. More preferably, the transcription factor is GABP β or a fragment thereof. Particularly preferred is human GABP β having amino acids 1 to 330. GABP β comprises a notch/ankyrin repeat motif domain required for heterodimerization with the DNA-binding protein GABP α, a domain consisting of amino acids 243 to 330 responsible for nuclear localization, a leucine- zipper-like structure domain required for homodimerization and activation of transcription by GABP.

Surprisingly, it has now been found that the replacement of the natural transcription activation domain of GABP by the transactivation domain of the viral protein herpes simplex virus I VP16 renders GABP β constitutively active, whereas the natural GABP β does not exhibit said property. While the transcription activation domain of the mutant has been replaced, the mutant maintains the nuclear localization and heterodimerization properties of GABP β.

According to a preferred embodiment, the transcription activator domain present in the fusion protein has a stretch of at least 4 negatively charged amino acids, wherein said negatively charged amino acids may be consecutive or interrupted by non-negatively charged amino acids. More preferably, the negatively charged amino acids are ordered in the form of a negatively charged amphipathic helix. It has been postulated that a negatively charged amphipathic helix is a common structural theme and a synthetic construction thereof has been found to be active (Giniger and Ptashne, Nature 330 (1987), 670).

The transcription activator from which the activation domain is derived may be selected from herpes simplex virus I protein VP 16, yeast transcription factors GCN4 and GAL4.

Preferably, the transcription activator is herpes simplex virus I protein VP16 or a fragment thereof. The minimal transcription activator domain from VP16 useful as a viral transcription activator domain according to the present invention comprises position 436 to 447 of the VP16 sequence (Baron et al., Nucleic Acids Res. 25 (1997), 2723-2729), i.e. the transcription activator domain having the sequence PADALDDFDLDML (SEQ ID NO:3).

Most preferred, the fusion protein has the amino acid sequence (SEQ ID NO:2). Said fusion protein comprises as the N-terminal portion amino acids 1 to 330 of human GABP β, a 6 amino acid linker followed by the 78 amino acids transactivation domain of VP16, i.e. amino acids 336 to 414 of GABPβ_VP16. The GABP β part thereof maintains the domain required for heterodimerization with GABP α and the domain required for nuclear localization.

According to a preferred embodiment, the fusion protein is capable of activating the expression of a gene under the control of the nucleic acid according to the present invention.

As demonstrated in the exemplary part, the fusion protein according to the present invention is capable of specifically activating the expression of genes by binding to Ets- transcription factor binding-sites. Contrary to endogenous GABP β, the fusion protein according to the present invention exhibits constitutive activity by the transfer of the viral transcription activator domain.

The present invention further provides a method for inducing gene expression comprising administering a fusion protein according to the present invention to a cell. Preferably, the method represents an in vitro method. The method is considered to be useful for the promoter sequence specific activation of gene expression provided that the promoter sequence comprises at least one, preferably two or more Ets-transcription factor binding sites.

According to another embodiment, the present invention provides pharmaceutical compositions comprising at least one fusion protein. The pharmaceutical composition may further comprise pharmaceutically acceptable excipients, carriers or diluents. Said additives are well-known in the art. The pharmaceutical composition may be formulated for intravenous, subcutaneous, intramuscular, peritoneal, perenteral administration. The mode of administration can be determined by the physician.

Particularly, the pharmaceutical composition is used for inducing the gene expression of a gene selected from disease-relevant genes selected from cytokines, complement factors, hematopoietins and cytoskeletal proteins. Preferably, the gene is selected from the utrophin gene, IL-2, factor IX, CD18, thrombopoeitin and Apo-1/Fas (CD95). The involvement of GABP in the regulation of said genes has been demonstrated. Due to the involvement of said genes and gene products in various diseases the fusion protein according to the present invention is considered to be useful for the treatment of e.g. muscle diseases, preferably muscular dystrophy, haemophilia, immune deficiency and/or cancer.

In a further embodiment the present invention provides a kit for expression of a gene comprising:

the nucleic acid according to the invention being operably linked to a gene such that the gene is expressed under the control of a nucleic acid according to the invention; and

an Ets-transcription factor related compound. Having regard to the exemplary part it has been demonstrated that the fusion protein according to the present invention being specific for Ets-transcription factor binding sites specifically activated transcription of the utrophin B promoter while transcription from the utrophin A promoter has not been not activated.

Preferably, the gene is selected from utrophin, IL-2, factor IX, CD18, TPO and Apo- 1/Fas(CD95). It is preferred that the Ets-transcription factor related compound is GABP or a variant thereof. More preferably, the Ets-transcription factor related compound is human GABP β, most preferably, the fusion protein according to the present invention, comprising a domain of GABP β and the transactivation domain of VP16.

The kit is useful for the Ets-transcription factor related compound-controlled expression of a gene in a host cell.

The nucleic acid according to the invention being operably linked to a gene may be comprised in a construct as described above optionally further comprising regulatory regions for transcription and/or replication.

The Ets-transcription factor related compound is as defined above. Preferably, the Ets- transcription factor related compound is GABP or a variant thereof, particularly preferred, the GABP variant is the fusion protein comprising a domain of GABP β and the transcription activation domain of herpes simplex Virus I VP16. The present invention further provides a method for tissue-specific increase of utrophin- expression comprising the step of providing a fusion protein according to the present invention with said tissue. The tissue is selected from skeletal muscle, heart, brain and kidney.

Since it has been found that the fusion protein having SEQ ID NO:2 selectively activates the utrophin promoter (B), administration of the fusion protein selectively increases the production of utrophin B-protein which may have potential therapeutic implications. Tissue- and/or organ-specification administration by selective vehicles such as viruses, electroporation, „gene-gun" or naked DNA injection allows for site-controlled expression of utrophin B. The present invention further relates to a method for affecting the transcriptional activity of a promoter containing an Ets-transcription factor binding-site comprising the step of bringing into contact said promoter with a fusion protein according to the present invention.

Generally, any promoter having at least one Ets-transcription factor binding-site is considered to be useful. The promoter may be selected from e.g. the promoter of the acetylcholine receptor δ and ε subunits and IL-2 and CD18.

The present invention relates to the specific application consensus sequence for the binding of Ets-transcription factors located in the human UTR-promoter (B). The consensus sequence, is preferably selected from 5' (C/A) GGA (A/T) (A/G) 3' or 5' (C/T) (A/T) TCC (T/G) 3'. The consensus sequence is located at positions 32 and 145 and 294 in the human utrophin promoter (B).

The present invention further relates to the use of the specific application consensus sequence for the binding of Ets-transcription factors according to the present invention for the treatment of muscle diseases. The muscle diseases are preferably selected from muscular dystrophy as a consequence of dystrophin deficiency, such as Duchenne- and Becker-Type-Muscular Dystrophy.

The present invention further relates to the specific application of Ets-transcription factors and transcription factors of Ets-subfamilies for the activation of the UTR-promoter (B).

The transcription factor is preferably a member of the GABP-family of transcription factors. Preferably, the transcription factor is GABP β. Also, preferably the transcription factor is constitutively activated by any pharmacological or molecular manipulation of the transcription factor itself or the cellular environment. It is preferred that the GABP β is constitutively active by means of fusing the amino-terminal portion GABP β to any transcription activation domain. The transcription activation domain preferably consists of sequences derived from the herpes simplex virus I transcription factor VP16. The activated transcription factors are endogenous components of muscle cells. The activated transcription factors are introduced into the diseased muscle tissue by any means of manipulation. It is further preferred that the activated transcription factors are introduced in the diseased muscle tissue by means of viral transfection using any form of viral vehicle. The viral vehicle may be selected from a retrovirus or an adenovirus, or an adeno-associated virus carrying genetic information including unmodified or activated forms of said transcription factors.

The present invention further relates to a process for activation of the UTR-promoter (B) by using the constitutively active GABP β_VP16 factor.

The present invention further provides a pharmaceutical preparation by using consensus sequences according to the invention for use in a method of treating the human or animal body. The pharmaceutical preparation may be used in a method of treating Duchenne- and Becker-type muscular dystrophy and related forms of muscle wasting. The present invention also relates to the use of the specific application consensus sequences for the binding of Ets-transcription factors according to the present invention for the manufacture of pharmaceutical preparations for treating Duchenne- and Becker-type muscular dystrophy and related forms of muscle wasting.

The following examples illustrate the invention:

Figure 1

(A,B) Consensus sequence for the binding of Ets-related transcription factors in two possible orientations. (C) Alignment of the human (top) and mouse (bottom) region of the utrophin-promoter (B) region. The relative sequence positions refer to sequences published under GenBank accession number AJ250044 (human) and GenBank accession number AJ250045 (mouse). Gray boxes indicate putative binding sites for Ets-transcription factors. Note that two of these sites are conserved in human and mouse. An arrow indicates the location of the transcription start site. "M" indicated the translation start site.

Figure 2

Structure of GABPα and β subunits and expression in COS-7 cells. Schematic representation of the native α and β subunit of GABP (A) and the constitutively active mutant of GABPβ (GABPβ_VP16) derived form the GAPBβ^DN-deletion mutant (B). Indicated in black is the Ets-related DNA binding domain of GABPα located at the amino acid position 318-399. In GABPβ, the gray region (amino acids 1-130) represents four tandem repeats of a Notch/ankyrin motif required for heterodimerization with GABPα. The black bar indicates the approximate location of a nuclear localization sequence. The hatched region (amino acids 341-370) represents a leucine-zipper-like structure essential for homodimerization and activation of transcription by GABP. In the constitutively active mutant of GABPβ (GABPβ_VP16) used in this study, the last 52 amino acids of GABPβ were replaced by a 6 amino acids linker followed by the 78 amino acids transactivation domain of VP16. (C) Intracellular anti-myc staining of COS cells transfected with a myc- tagged GABPα construct (GABPα-myc). GABPα is localized in the cytoplasm (left). Upon cotransfection with GABPβ_VP16, GABPα accumulates in the cell nucleus (right). This indicates that the GABPβ_VP16 protein dimerizes with the GABPα subunit and translocates into the cell nucleus. Scale bar 50 μm. (D) Sequence of GABPβ_VP16. (amino acid sequence SEQ ID NO:2; nucleotide sequence SEQ ID NO:12). The 6 amino acids linker is shaded with gray. The EcoRI restriction site used for the fusion of the mouse GABPβ-sequence to the herpes simplex virus I VP16 sequence is indicated. The GABPβ-portion of the hybrid molecule is equivalent to previously published sequences (GenBank accession numbers GM 93384 for mouse and GI8051594 of the human sequence).

Figure 3

GABPβ_VP16 is capable of inducing target gene transcription that contains Ets- transcription factor binding-sites.

(A) Effect of GABPβ_VP16 on the wild-type (AChRε WT) and mutant (AChRε MT) AChRε subunit reporter constructs in C2C12 muscle cells. The activation of the AchRε subunit promoter by GABPβ_VP16 is strongly reduced by the 1 bp G-A mutation present in the Ets-transcription factor binding sequence located in the N-box of the AChRε MT sequence. The sequences of wildtype and mutated Ets-transcription factor binding-sites are indicated below the bar histogram. The relative luciferase activities are shown for each experimental group. The activities represent the mean (± standard deviation) of 6 (N=6) or 4 (N=4) experiments done in triplicate. All activities were measured 96 hours after induction of myoblast fusion. (B) Activation of AChRε WT by GABPβ_VP16 at different time-points after induction of myoblast fusion. The maximal activation of the AChRε-subunit gene promoter by transfection with the GABPβ_VP16 construct was observed 96 hours after induction of fusion and declined gradually at later time-points. Activity of the AChRε WT in the NLS-LacF transfected cell, on the contrary, increased slightly with time.

Figure 4

Effect of GABPβ_VP16 on selected promoter reporter constructs. The effect of GABPβ_VP16 on the UTR-promoter (A) and UTR-promoter (B) constructs (designated as UPA, UPB) as well as on the muscle creatine kinase reporter construct (MCK) and the N-CAM reporter construct (N-CAM) were measured 96 hours after induction of myoblast fusion. (A) Activity of the utrophin promoter/luciferase-reporter constructs (UPA and UPB) coexpressed with GABPβ_VP16 and NLS-LacF in C2C12 myotubes presented as relative luciferase activity. Data is mean ± standard deviation from triplicates. Activation of UPB was significantly induced by GABPβ_VP16 (p < 0.01). A schematic representation of the mouse UTR-promoter (A) [mUPA]/lucif erase reporter and human UTR-promoter (B) [hUPB]/luciferase reporter constructs is also shown. (B) GABPβ_VP16 actually repressed the activity of MCK-promoter/luciferase reporter activity, and had no effect on the activity of N-CAM-promoter/luciferase reporter. Data is mean ± standard deviation from triplicates. This experiment surprisingly demonstrates that GABPβ_VP16 specifically drives the UTR-promoter (B) presumably by interacting with the conserved Ets-transcription factor binding-sites described in Example 1.

Examples

Example 1

Sequence analysis reveals the presence of Ets-transcription factor binding sites within the UTR-promoter (B).

The UTR-promoter (B) has been analyzed for the presence of sequences related to the consensus sequence for the binding of Ets-transcription factors using the matrix recognition software Matlnspector (Quandt, K.; Freeh, M.J., Karas, H.; Wingender, E.; Werner, T. Nucleic Acid Res. 23: 4878-4884 (1995)). Three such consensus sequences were identified in the human sequence of utrophin starting at positions 32, 145, and 294 with reference to the published human utrophin sequence. For comparison the sequence of the mouse utrophin gene has also been analyzed where two conserved sequences for the binding of Ets-transcription factors can be identified at equivalent positions.

Example 2

Generation of a constitutively active GABPβ transcription factor. The constitutively active construct of GABPβ (designated as GABPβ-VP16 thereafter) was generated by in-frame fusion of a cDNA fragment encoding the 78 COOH-terminal amino acids of VP16 (Triezenberg, S.J.; Kingsbury, R.C; McKnight, S.L. Genes Dev. 2: 718-729 (1988)), preceded by a 6 amino acids linker, to the 3' end of a cDNA fragment encoding GABPβ^DN (Fig. 2). The transactivation domain of herpes simplex virus I, transcription factor VP16 (GenBank accession number GI330054), was amplified by PCR and inserted into pcDNAI (Invitrogen, Carlsbad, CA). The GABPβ^DN fragment was amplified by PCR using primers sGABPβ, 5' GGAATTCGAAGCTTTTCCAGATGT 3' (SEQ ID NO:4); asGABPβ^DN_EcoRI, 5' GGAATTCTTCTGCACATTCCACCC 3' (SEQ ID NO:5), and a previously described GABPβ construct as a template (Briguet, A.; Rϋegg, M.A. J. Neurosci. 20:5989-5996 (2000)). After digestion with EcoRI, the fragment was introduced into pcDNAI_VP16 opened with EcoRI. A schematic representation of the resulting GABPβ_VP16 where the terminal 52 amino acids of GABPβ have been replaced by the VP16 transactivation domain is shown in Fig. 2B. This constitutively active form of GABP is based on the GABPβ subunit that does not bind DNA on its own. Therefore, when expressed in cells, the DNA-binding specificity of GABPβ_VP16 is dictated by the endogenous GABPα subunit. Thus GABPβ_VP16 should retain the same target gene specificity as wild-type GABPβ. This prediction has been tested by cotransfection experiments using a myc-tagged GABPα^' As shown in Fig. 2C, it can be demonstrated that GABPβ_VP16 is able to dimerize with GABPα and is translocated into the cell nucleus. In conclusion, the replacement of the Leucine-Zipper-like domain of GABPβ with the VP16 transactivation domain renders the GABPβ_VP16 protein fully functional with regard to the ability to dimerize with GABPα and the accumulation to the nucleus of cells. Fig. 2D shows the ssequence of GABP_VP16.

Example 3

GABPβ_VP16 activation of the acetylcholine receptor (AChR) ε-subunit gene promoter is dependent on the Ets-transcription factor binding site present in this promoter region.

Further analysis demonstrated that the constitutively active GABPβ_VP16 when transfected into muscle cells is sufficient to drive the transcription of a target gene sequence containing a conserved Ets-binding site. For this, the potential of GABPβ_VP16 to transactivate a luciferase reporter gene placed under the control of a 533-bp fragment of the human AChRε subunit cis-regulator region containing the Ets- transcription factor binding site has been tested (Duclert, A.; Savatier, N.; Schaeffer, L.; Changuex, J.P. J. Biol. Chem. 271 :17433-17438 (1996)). The human AChRε subunit gene promoter sequence has been deposited under GenBank accession numbers Z84811 and GI1922319. The transcription rate of this AChRε subunit gene promoter/luciferase reporter construct in cotransfection experiments with the C2C12 myogenic cell line has been compared. In addition to the AChRε subunit gene promoter/luciferase reporter construct cells were either transfected with GABPβ_VP16 construct or with the NLS-LacF construct for control (Briguet, A.; Rϋegg, M.A. J. Neurosci. 20:5989-5996 (2000)).

The following transfection protocol was used: C2C12 myoblasts were seeded at a density of 10'000/well in 24-wells plates that were previously coated with gelatin. 24 h after seeding the myoblasts were transfected with 100 ng of either reporter constructs, together with 10 ng of the standard pRL-TK vector (Promega) encoding Renilla luciferase under the control of the thymidine kinase promoter, and 25 ng of NLS-LacF or GABPβ_VP16. 24 h later the proliferation medium containing 20% fetal calf serum was replaced with differentiation medium containing 5% horse serum. After 96 h in differentiation medium, the cells were lyzed in 150 μl/well passive lysis buffer (Promega). The firefly and Renilla luciferase activities contained in 20 μl lysate were then measured using the dual luciferase reporter assay (Promega). Luciferase activity produced by the promoter reporter constructs was normalized to the activity derived from the cotransfected pRL-TK vector.

Expression of the luciferase reporter was about 3 fold higher in myotubes cotransfected with GABPβ_VP16 compared to myotubes cotransfected with the control NLS-LacF construct (Fig. 3A left). This induction by GABPβ_VP16 critically depends upon the sequence of the Ets-transcription factor binding-site. AChRε subunit reporter construct with a single base-pair mutation (mutation G to A at a position of 94 base pairs upstream from the translation start site of the AChRε subunit gene) cannot be activated by GABPβ_VP16 (Fig. 3A right). This demonstrates the target sequence specificity of the GABPβ_VP16 construct.

Example 4

Activation of the UTR-promoter (B) by the constitutively active GABPβ_VP16.

Using utrophin-promoter/luciferase-reporter constructs transfected into C2C12 muscle cells the effect of constitutively active GABPβ_VP16 and the control NLS-LacF construct on the transcription of UTR-promoter (A) and UTR-promoter (B) and the promoters for muscle specific creatine kinase (MCK) and the cell adhesion molecule N-CAM has been compared. For this, the following constructs were used:

The UTR-promoter (A)-reporter construct was generated by PCR using primers s71mUP, 5' GGTCAGCACCAACACTATTTG 3' (SEQ ID NO:6); as1157 mUP, 5' GTGGAAAGCCCGACAAGATCC 3' (SEQ ID NO:7), and mouse genomic DNA as a template. The PCR product was inserted into pGL-2 basic (Promega) opened with Nhel. The UTR-promoter (B) reporter construct was generated by PCR using primers sUP2, 5' GATTGTGGTGATGGTTGTAGAA 3' (SEQ ID NO:8 asUP2, 5' GAGATGAGGAAAAAGATGTGGAG 3' (SEQ ID NO:9), and human genomic DNA as a template. The PCR product was inserted into pGL3-basic opened with Smal. A schematic representation of the UTR-promoter/luciferase-reporter constructs is shown in Fig. 4A.

The muscle creatine kinase MCK promoter reporter construct was generated by inserting a Hindlll fragment isolated form pBS-MCK construct into the Smal site of pGL3 (Promega). The mouse MCK gene promoter sequence has been deposited under GenBank accession number Gl 199087. The N-CAM promoter reporter construct (GenBank accession number: GI35004) was generated by PCR using primers NCAMs- 611, 5' CCTCTCGAGAATCGAAATGGAGGGATTT 3' (SEQ ID NO: 10), NCAMas-144, 5' GTAGATCTGTTTCTCGCCAGCCGAG 3' (SEQ ID NO: 11), and human genomic DNA as a template. The PCR product was digested with Xhol-Bgll and inserted into pGL3- basic opened with Xhol-Bgll.

The experiments performed indicate that transfection of myoblasts with GABPβ_VP16 did not increase the mouse UTR-promoter (A) transcription rate when compared to experiments where the NLS-LacF control plasmid was used for transfection (Fig. 4A). In contrast, the transcription rate of the human UTR-promoter (B) was increased several- fold following the transfection with GABPβ_VP16 compared to the control transfection. This experiment clearly demonstrates that GABPβ_VP16 is capable of specifically activating the UTR-promoter (B) presumably by binding to the Ets-transcription factor binding sites identified in this UTR-promoter (B) as described in Example 3. In addition, this promoter activation was specific since neither the UTR-promoter (A) nor the promoter for MCK or the promoter for N-CAM can be activated by GABPβ_VP16 (Fig. 4B). SEQUENCE LISTING

<110> Myocontract Pharmaceutical Research Ltd.

<120> Ets-Transcription Factor related compound specific promoter and transactivators thereof

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Gin Asn Gin lie Asn Thr Asn Pro Glu Ser Pro Asp Thr Val Thr lie 165 170 175

His Ala Ala Thr Pro Gin Phe lie He Gly Pro Gly Gly Val Val Asn 180 185 190

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Pro Ala Asp Ala Leu Asp Asp Phe Asp Leu Asp Met Leu 1 5 10

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Claims

1. A nucleic acid capable of controlling the expression of a gene and of being activated by an Ets-transcription factor related compound wherein the nucleic acid is selected from any of the following:

a) a nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO: 1 ;

b) a nucleic acid molecule capable of hybridizing with the nucleic acid molecule of (a);

c) a nucleic acid molecule being degenerate to any of the nucleic acid molecules of (a) or (b) due to the genetic code;

d) a nucleic acid molecule being at least 60 % homologous to any of the nucleic acid molecules of (a) to (c);

e) a variant of any of the nucleic acid molecules of (a) to (d), wherein said variant differs from the nucleic acid molecule by at least one addition, deletion, insertion and/or inversion;

f) a fragment of any of the nucleic acid molecules of (a) to (e); and

g) a combination of at least two nucleic acid molecules of (a) to (f),

wherein the nucleic acid molecule of (a) to (g) comprises at least one Ets-transcription factor binding-site, preferably at least two, more preferably at least three.

2. The nucleic acid according to claim 1 , wherein the nucleic acid molecule of (b) to (g) exhibits at least 60 %, preferably 80 %, more preferably 90 % of the inducibility of the nucleic acid having the nucleotide sequence set forth in SEQ ID NO:1 by GABP and/or at least 60 %, preferably 80 %, more preferably 90 % of the basal transcriptional activity of the nucleic acid having the nucleotide sequence set forth in SEQ ID NO:1.

3. The nucleic acid according to claim 1 or 2, wherein the Ets-transcription factor related protein comprises a GABP-sequence.

4. The nucleic acid according to any of claim 1 to 3, wherein the Ets-transcription factor binding site is selected from the consensus sequence 5'(C/A)GGA(A/T)(A G)3' or 5'(C/T)(A/T)TCC(T/G)3'.

5. A construct comprising at least one nucleic acid according to any of claims 1 to 4 and optionally , at least one gene operably linked to the nucleic acid such that the gene is expressed under the control of the nucleic acid.

6. A host cell comprising at least one nucleic acid according to any of claims 1 to 4 and/or at least one construct according to claim 5.

7. A pharmaceutical composition comprising at least one nucleic acid according to any of claims 1 to 4 being operably linked to a gene such that the gene is expressed under the control of the nucleic acid.

8. A method for recombinant expression of a gene comprising the following steps:

(a) providing a construct according to claim 5;

(b) transfecting a suitable host cell with said construct; and

(c) expressing the gene under the control of said nucleic acid.

9. The method according to claim 8, wherein the transfected cell is provided with an Ets-transcription factor related compound.

10. A method for screening and/or providing of candidate compounds being capable of regulating transcription comprising the step of bringing into contact the nucleic acid according to any of claims 1 to 4 with compounds to be screened and detecting the transcriptional activity from the nucleic acid in the presence and absence of said compounds and optionally purifying and/or synthesizing the positively tested compound.

11. Compound obtainable by the method according to claim 10.

12. A fusion protein capable of binding to the nucleic acid according to any of claims 1 to 4 wherein the fusion protein comprises at least a part of a transcription factor domain and at least a part of a viral transcription activator domain.

13. The fusion protein according to claim 12, wherein the transcription factor domain comprises at least a part of a DNA binding domain, a part of a domain for heterodimerization with a DNA binding protein and/or a part of a domain for nuclear localization, or a combination thereof.

14. The fusion protein according to claim 12 or 13 wherein the transcription factor is selected from a member of the Ets-transcription factor family such as e.g. a member of the YAN-, ELG-, PEA3-, ERF- and TCF-subfamilies.

15. The fusion protein according to claim 14 wherein the transcription factor is GABP beta or a fragment thereof.

16. The fusion protein according to any of claims 12 to 15 wherein the transcription activator domain has a stretch of at least four negatively charged amino acids which may be consecutive or interrupted by non-negatively charged amino acids.

17. The fusion protein according to any of claim 12 to 16 wherein the transcription activator is selected from herpes simplex virus I VP16 and yeast transcription activators GCN4 and GAL4.

18. The fusion protein according to claim 17 wherein the transcription activator is Herpes simplex virus I protein VP16 or a fragment thereof, preferably the fragment PADALDDFDLDML (SEQ ID NO:3).

19. The fusion protein according to any of claims 12 to 18 having the amino acid sequence of SEQ ID NO:2.

20. The fusion protein according to any of claims 12 to 19 capable of activating the expression of a gene under the control of the nucleic acid according to any of claims 1 to 4.

21. A method for inducing gene expression comprising administering a fusion protein according to any of claims 12 to 20 to a cell.

22. A pharmaceutical composition comprising at least one fusion protein according to any of claims 12 to 20.

23. Use of the fusion protein according to any of claims 12 to 20 for the preparation of a medicament for inducing gene expression

24. The use according to claim 23 wherein the gene is selected from utrophin gene, IL-2, factor IX, CD18, TPO, Fas and AchR δ and ε subunits.

25. The use according to claim 23 or 24 for the treatment of muscle diseases, preferably muscular dystrophy, hemophilia, immune deficiency, and/or cancer

26. Kit for expression of a gene comprising:

the nucleic acid according to any of claims 1 to 4 being operably linked to a gene such that the gene is expressed under the control of the nucleic acid; and

an Ets-transcription factor related compound.

27. The kit according to claim 26 wherein the gene is selected from utrophin gene, IL- 2, factor IX, CD18, TPO, Fas and AchR δ and ε subunits, preferably utrophin.

28. The system according to claim 26 or 27 wherein the Ets-transcription factor related compound is GABP or a variant thereof, preferably GABPβ_VP16 (SEQ ID NO:2).

29. Use of the kit according to any of claims 26 to 28 for controlled expression of a gene in a host cell.

30. A method for tissue specific increase of utrophin expression comprising the step of providing a fusion protein according to any of claims 12 to 20 with said tissue.

31. A method for affecting the transcriptional activity of a promoter containing an Ets- transcription factor binding-site comprising the step of bringing into contact said promoter with a fusion protein according to any of claims 12 to 20.