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Title:
NUCLEIC ACID CODIFYING FOR A UTROPHIN TRANSCRIPTION SPECIFIC REGULATING PROTEIN, PROTEIN CODIFIED THEREBY AND USES THEREOF
Document Type and Number:
WIPO Patent Application WO/2009/044383
Kind Code:
A1
Abstract:
The present invention relates to a nucleic acid molecule comprising a nucleotide sequence codifying for a protein characterized in having at least four zinc finger domains and in modulating the transcription of the utrophin gene by binding to a nucleotidic sequence comprised in a utrophin gene untranscribed regulatory sequence. Protein codified thereby and uses thereof.

Inventors:
DI CERTO MARIA GRAZIA (IT)
PASSANANTI CLAUDIO (IT)
CORBI NICOLETTA (IT)
ONORI ANNALISA (IT)
Application Number:
PCT/IB2008/054089
Publication Date:
April 09, 2009
Filing Date:
October 06, 2008
Export Citation:
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Assignee:
CONSIGLIO NAZIONALE RICERCHE (IT)
DI CERTO MARIA GRAZIA (IT)
PASSANANTI CLAUDIO (IT)
CORBI NICOLETTA (IT)
ONORI ANNALISA (IT)
International Classes:
C12N15/12; C07K14/47; A61K38/17; A61K48/00; A61P21/00; C12N15/62; C12N15/85
Other References:
ONORI ANNALISA ET AL: "The artificial 4-zinc-finger protein Bagly binds human utrophin promoter A at the endogenous chromosomal site and activates transcription.", BIOCHEMISTRY AND CELL BIOLOGY = BIOCHIMIE ET BIOLOGIE CELLULAIRE JUN 2007, vol. 85, no. 3, June 2007 (2007-06-01), pages 358 - 365, XP002510672, ISSN: 0829-8211
BELTRAN ADRIANA ET AL: "Interrogating genomes with combinatorial artificial transcription factor libraries: asking zinc finger questions.", ASSAY AND DRUG DEVELOPMENT TECHNOLOGIES JUN 2006, vol. 4, no. 3, June 2006 (2006-06-01), pages 317 - 331, XP002510673, ISSN: 1540-658X
CORBI N ET AL: "The Artificial Zinc Finger Coding Gene Jazz Binds the Utrophin Promoter and Activates transcription", GENE THERAPY, MACMILLAN PRESS LTD., BASINGSTOKE, GB, vol. 7, no. 12, 1 June 2000 (2000-06-01), pages 1076 - 1083, XP002988476, ISSN: 0969-7128
SEGAL DAVID J ET AL: "Evaluation of a modular strategy for the construction of novel polydactyl zinc finger DNA-binding proteins.", BIOCHEMISTRY 25 FEB 2003, vol. 42, no. 7, 25 February 2003 (2003-02-25), pages 2137 - 2148, XP002510674, ISSN: 0006-2960
Attorney, Agent or Firm:
CAPASSO, Olga et al. (Via Vincenzo Bellini 20, Roma, IT)
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Claims:

CLAIMS

1. A nucleic acid molecule comprising a nucleotide sequence codifying for a protein characterized in having at least four zinc finger domains and in modulating the transcription of the utrophin gene by binding to a nucleotidic sequence comprised in a utrophin gene untranscribed regulatory sequence.

2. The nucleic acid molecule according to claim 1 wherein the protein comprises six zinc finger domains.

3. The nucleic acid molecule according to claim 1 or 2 wherein the zinc finger domains are selected among: SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID

NO. 5 or SEQ ID NO. 6 and may also be present in more than one sequence copy.

4. The nucleic acid molecule according to claim 3 wherein the zinc finger domains are SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6. 5. The nucleic acid molecule according to claim 4 codifying for a protein comprising the SEQ ID NO. 12.

6. The nucleic acid molecule according to claim 4 codifying for a protein having the SEQ ID NO. 12.

7. The nucleic acid molecule according to claim 6 having essentially the SEQ ID NO. 11. 8. The nucleic acid molecule according to claim 5 codifying for a protein having essentially the SEQ ID NO. 8 or SEQ ID NO. 10.

9. The nucleic acid molecule according to claim 8 having essentially the SEQ ID NO. 7 o SEQ ID NO. 9, respectively.

10. An expression vector comprising the nucleic acid molecule according to anyone of the preceding claims.

11. A recombinant host cell comprising the vector according to claim 10.

12. A protein characterized in having at least four zinc finger domains and in modulating the transcription of the utrophin gene by binding to a nucleotidic sequence comprised in a utrophin gene untranscribed regulatory sequence. 13. The protein according to claim 12 having six zinc finger domains.

14. The protein according to claim 12 or 13 wherein the nucleotidic sequence has essentially the sequence: GCT GCT GCG GGC TGG GAG (SEQ ID NO. 13).

15. The protein according to anyone of the claims 12 to 14 having essentially the SEQ ID NO. 12.

16. The protein according to anyone of the claims 12 to 14 having essentially the SEQ ID NO. 8 or SEQ ID NO. 10. 17. The protein according to anyone of the claims 12 to 16 for medical use.

18. The protein according to anyone of the claims 12 to 16 for use as an anti-muscular pathology agent.

19. The expression vector according to claim 10 for gene therapy.

20. Use of the protein according to anyone of the claims 12 to 16 for the preparation of a medicament for a muscular pathology.

21. The use according to claim 20 wherein the muscular pathology is the Duchenne (DMD) or the Becker (BMD) muscular dystrophy.

Description:

Nucleic acid codifying for a utrophin transcription specific regulating protein, protein codified thereby and uses thereof

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a nucleic acid comprising a nucleotidic sequence codifying for a protein regulating the utrophin transcription, called "UtroUp". UtroUp is able to bind, with very high affinity and specificity, a DNA target sequence of 18-nucleotide-long existing both in the human and murine promoter of "utrophin" gene and to modulate the transcription thereof.

STATE OF ART

Mutations in the dystrophin gene are responsible for myopathies linked to chromosome X, called Duchenne (DMD) and Becker (BMD) muscular dystrophies. The phenotype DMD is characterized by the complete absence of dystrophin, due to promoter defects or to mutations of nonsense type or deletions. DMD affects 1 out of 3500 borne males and it involves a progressive muscular weakening with lethal results. The phenotype BMD is less serious and more variable, as it is associated to a lowering of the expression levels of dystrophin mutated versions. The dystrophin gene extends for about 2.5 megabases of DNA, in the Xp21 locus, with a corresponding transcript of 14 kilobases (accession number BC127103) and a protein of 427 kilodalton (accession number AA127104) (Davies et al. 1983). In 1989, an autosomal homologous of the dystrophin gene was described for the first time (Love et al., 1989). Such protein, at first defined DRP (dystrophin related protein), was subsequently called utrophin. The utrophin gene maps in chromosome 6q24, it has a transcript of 13 kilobases (accession number NM 007124) and a protein product of 400 kilodalton (accession number NP 009055). Utrophin and dystrophin have strong structural and functional homologies: both act like a connection bridge between the actin cytoskeleton, the cell membrane and, at last, the extracell matrix by means of the proteins collectively designated as DAPs (dystrophin associated proteins) (Matsumura et al. 1992). In adult muscle, utrophin is localized in the neuromuscular junction, whereas dystrophin is placed along the whole sarco lemma. Studies on "mdx" mice (murine model of DMD), transgenic for the utrophin have demonstrated that an over-expression and relocalization of utrophin induces

a clear improvement of the dystrophic phenotype (Tinsley et al 1996, Rafael et al 1998). The increase in the utrophin expression levels is then currently one of the most promising therapeutic approaches for the DMD (Miura and Jasmin 2006) treatment. To this purpose, various pharmacological treatments have been proposed (Khurana and Davies 2003, Bogdanovich et al. 2004, Voisin and La Porte 2004).

The authors, in order to obtain utrophin over-expression have designed different synthetic zinc-finger based transcriptional factors able to bind and activate the utrophin gene transcription (Corbi et al. 2000, Corbi et al. 2004, Onori et al. 2007, Mattei et al. 2007). The affinity and specificity of binding to DNA is one of the critical points in the construction of synthetic zinc fingers. In a cellular context, a hypothetical transcriptional factor will recognize multiple DNA sequences with a different affinity level, by thus exerting an additional transcrictional regulation level. For natural transcriptional factors, the capability of recognizing specifically a determined target sequence has been accurately selected during the evolution, so that each regulating protein acts in the suitable pathway. Statistically, by assuming a stochastic distribution of the DNA bases, a 9-base long DNA sequence is present in the human genome (≡ 3.5 x 10 9 bp) about 1.3 x 10 4 times, a 12-base long sequence about 210 times, whereas a 16-base sequence is present one time only. In the present invention a new domain binding to DNA on zing finger base, called "UtroUp", has been synthesized and assayed. UtroUp has six zinc finger domains and it is able to bind, with very high affinity and specificity, a 18-nucleotide long target DNA sequence existing both in the human and murine promoter of the "utrophin" gene. The aim is to make available synthetic transcriptional factors having an action targeted at the exclusive utrophin over-regulation.

SUMMARY OF THE INVENTION

Therefore the object of the present invention is a nucleic acid molecule comprising a nucleotide sequence codifying for a protein characterized in having at least four zinc finger domains and in modulating the transcription of the utrophin gene by binding to a nucleotidic sequence comprised in a utrophin gene untranscribed regulatory sequence. Preferably, the protein comprises six zinc finger domains.

Still preferably, the zinc finger domains are selected among: SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5 or SEQ ID NO. 6 and may also be present in more than one sequence copy.

More preferably the zinc finger domains are SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO.

3, SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6.

Yet preferably the nucleic acid molecule of the invention codifies for a protein comprising the SEQ ID NO. 12. Still preferably it codifies for a protein having the SEQ ID NO. 12. Preferably the nucleic acid molecule of the invention has essentially the SEQ ID NO. 11.

Still preferably the nucleic acid molecule of the invention codifies for a protein having essentially the SEQ ID NO. 8 or SEQ ID NO. 10. Preferably the nucleic acid molecule has essentially the SEQ ID NO. 7 o SEQ ID NO. 9, respectively.

It is a further object of the invention an expression vector comprising the nucleic acid molecule as described above.

It is a further object of the invention a recombinant host cell comprising the vector of the inevntion.

It is a further object of the invention a protein characterized in having at least four zinc finger domains and in modulating the transcription of the utrophin gene by binding to a nucleotidic sequence comprised in a utrophin gene untranscribed regulatory sequence.

Preferably, the protein has six zinc finger domains. Preferably the nucleotidic sequence has essentially the sequence: GCT GCT GCG GGC TGG GAG (SEQ ID NO. 13).

Still preferably, the protein has essentially the SEQ ID NO. 12. More preferably, the protein has essentially the SEQ ID NO. 8 or SEQ ID NO. 10. It is an objet of the invention the protein as described above for medical use. Preferably for use as an anti-muscular pathology agent.

It is an objet of the invention the expression vector as described above for gene therapy.

It is an objet of the invention the use of the protein as described above for the preparation of a medicament for a muscular pathology. Preferably, the muscular pathology is the Duchenne (DMD) or the Becker (BMD) muscular dystrophy.

In the present invention zinc-finger based domain is defined as a synthetic aminoacidic domain able to bind specifically a DNA sequence inside the utrophin gene promoter.

The present invention will be now described by non-limiting examples thereof, with particular reference to the following figures: Figure 1 : A) Nucleotidic sequence of the utrophin "A" murine promoter. The DNA target sequence of UtroUp is underlined and in bolt. The bond sites of the main transcriptional factors are indicated. B) Aminoacidic sequence of the domain binding to UtroUp DNA.

The sequences of the six zinc finger domains are enumerated and underlined (SEQ ID NO.

1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6). C and D) Schemes of the synthetic B-UtroUp constructs for producing and purifying the protein in bacteria and V-UtroUp for assaying the transcriptional properties in mammal cells. Figure 2: A) Assay of electrophoretic mobility for determining the dissociation constant (Kd) of the B-UtroUp protein/ DNA target complex. The protein concentrations expressed in nanomoles from the first to the ninth lanes are: 1, 2, 4, 8, 12, 30, 60, 120, 0. B) Histogram related to the luciferase activity of the pXP constructs (containing a small portion of the utrophin promoter including the UtroUp bond site) and pGL2-Control. Both constructs have been cotransfected with UtroUp, containing only the DNA binding domain, or V-UtroUp containing the DNA binding domain fused to the activation domain of the Vp 16 transcription. The results represent the average of four independent experiments performed three times and are reported with respect to the basal activity (thereto the value 1 has been given) respectively of the pXP and pGL2-Control constructs.

MATERIALS AND METHODS

Sequences' construction

UtroUp comprises six zinc finger domains (Table 1) placed in tandem, and it has been generated with the aim of binding, with very high affinity and specificity, the 18- nucleotide-long DNA target sequence: 5'- GCT GCT GCG GGC TGG GAG- 3' (SEQ ID NO. 13), existing both in the human and murine promoter of the "utrophin" gene (Figure IA). Table 1. Nucleotidic sequence of the UtroUp domains (SEQ ID NO. 11)

5 ' TACGCCTGCCCTGTGGAATCTTGCGACCGCCGGTTCTCCCGCAGCGATAACCTGGT GCGGCACATCCGGATTCACACCGGCCAGAAACCTTTCCAGTGCAGGATCTGCATGAGA AATTTCTCCCGGTCCGACCACCTGACCACCCACAATAGGACCCACACCGGCGAGAAAC CCTTTGCCTGCGACATCTGCGGGAGAAAGTTCGCCGACCCCGGCCACCTGGTGAGACA CAATAGAATCCACACCGGTGAAAAGCCCTTCGCCTGTCCCGTGGAGAGCTGCGATCGC AGATTCAGCCGCAGCGACGAGCTGACAAGGCACATCAGAATCCACACCGGGCAGAAGC CTTTTCAGTGCCGGATCTGCATGAGGAACTTCAGCTCCCGGGACGTGCTGAGACGCCA CAATCGCACACACACCGGCGAAAAGCCCTTCGCCTGTGATATTTGCGGGCGGAAATTT GCCTCCAGAGATGTGCTGCGCCGCCACAACCGCATTCACCTGAGACAGAACGATCTCG AG 3'

Aminoacidic sequence of the UtroUp domains (SEQ ID NO. 12)

I LDRPYACPVESCDRRFSRSDHLVRHIRIHTG

QKPfQCRiCMRNFSRSDHLTTH^RTBTGEKFP ACDlCGRKFADPGHLVRBNRXHTGEKPfACPV ESCDBRFSRSDE LTRHlRIHTGQKPFQCRiCM RNFSSRDVLRRHMRTHTGEKPFACDICGRKFα

3RDVLRRHNR1HLRQMDLE

UtroUp has been fused to different protein domains (GST, accession number AAB03573, VP 16, accession number P04486) in order to be able to test its biochemical and functional properties.

Two different basic constructs have been generated: 1) B-UtroUp (Table 2 and 3)

B-UtroUp expresses in bacterial systems (Fig 1C). It is a fusion protein with the bacterial protein glutathione S-transferase (GST) which allows the expression and purification thereof in quantity sufficient such that its DNA binding properties (specificity and affinity) can be assayed. Table 2. Nucleotidic sequence of B-UtroUp (SEQ ID NO. 7)

5 ' ATGGCCATCATACGTTATATAGCTGACAAGCACAACATGTTGGGTGGTTGTCCA AAAGAGCGTGCAGAGATTTCAATGCTTGAAGGAGCGGTTTTGGATATTAGATACGG TGTTTCGAGAATTGCATATAGTAAAGACTTTGAAACTCTCAAAGTTGATTTTCTTA GCAAGCTACCTGAAATGCTGAAAATGTTCGAAGATCGTTTATGTCATAAAACATAT TTAAATGGTGATCATGTAACCCATCCTGACTTCATGTTGTATGACGCTCTTGATGT TGTTTTATACATGGACCCAATGTGCCTGGATGCGTTCCCAAAATTAGTTTGTTTTA AAAAACGTATTGAAGCTATCCCACAAATTGATAAGTACTTGAAATCCAGCAAGTAT ATAGCATGGCCTTTGCAGGGCTGGCAAGCCACGTTTGGTGGTGGCGACCATCCTCC AAAATCGGATCTGGTTCCGCGTGGATCCCCGGAATTCCCGGGGATCCTGGATCGCC CTTACGCCTGCCCTGTGGAATCTTGCGACCGCCGGTTCTCCCGCAGCGATAACCTG GTGCGGCACATCCGGATTCACACCGGCCAGAAACCTTTCCAGTGCAGGATCTGCAT GAGAAATTTCTCCCGGTCCGACCACCTGACCACCCACAATAGGACCCACACCGGCG AGAAACCCTTTGCCTGCGACATCTGCGGGAGAAAGTTCGCCGACCCCGGCCACCTG GTGAGACACAATAGAATCCACACCGGTGAAAAGCCCTTCGCCTGTCCCGTGGAGAG CTGCGATCGCAGATTCAGCCGCAGCGACGAGCTGACAAGGCACATCAGAATCCACA CCGGGCAGAAGCCTTTTCAGTGCCGGATCTGCATGAGGAACTTCAGCTCCCGGGAC GTGCTGAGACGCCACAATCGCACACACACCGGCGAAAAGCCCTTCGCCTGTGATAT TTGCGGGCGGAAATTTGCCTCCAGAGATGTGCTGCGCCGCCACAACCGCATTCACC TGAGACAGAACGATCTCGAGTAA 3 '

Table 3. Aminoacidic sequence of B-UtroUp (SEQ ID NO. 8)

5 'MAI IRYIADKHNMLGGCPKERAEI SMLEGAVLDIRYGVSRIAYSKDFETLKVDFLS KLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDWLYMDPMCLDAFPKLVCFKKR IEAI PQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLVPRGSPEFPGILDRPYACP VESCDRRFSRSDNLVRHIRIHTGQKPFQCRICMRNFSRSDHLTTHNRTHTGEKPFACD ICGRKFADPGHLVRHNRIHTGEKPFACPVESCDRRFSRSDELTRHIRIHTGQKPFQCR ICMRNFSSRDVLRRHNRTHTGEKPFACDICGRKFASRDVLRRHNRIHLRQNDLE 3 '

2) V-UtroUp (Table 4 and 5)

V-UtroUp has been constructed for the expression in mammal cells (figure ID) and it has been implemented by synthesizing and assembling various portions described herebelow: a) The construct includes the region promoting the CMV cytomegalovirus gene (GenBank ADDGENE VECDB 5506 4661, from nucleotide 15 to 591). b) The transcript V-UtroUp has been equipped with an intron coming from the precocious region of the SV40 virus (from the nucleotide 276 to 621 of the sequence AJO 12749). The presence of this intron and the consequent processing thereof increases the stability of the transcript V-UtroUp . c) In the carboxy-terminal region the following signal has been added (deriving from the SV40 virus), SEQ ID NO. 14:

5 ' -AACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCAC

AT -3' which allows a correct polyadenylation of the transcript. d) The protein region responsible for binding to DNA is of the zinc-finger type, it is completly synthetic and it comprises six zinc finger domains assembled in tandem and properly modified (Figure IB) for recognizing the target sequence: 5'-GCT GCT GCG GGC TGG GAG-3'(SEQ ID NO. 13) present both in the human and murine promoter of the "utrophin" gene. e) To the zinc-finger region was fused the strong transcription activation domain "Vp 16" deriving from the virus herpes simplex (P04486).

In the amino -terminal portion of the V-UtroUp protein two domains have been further added: f) A nuclear localization signal (NLS, MAPKKKRKV, SEQ ID NO. 15) originating from the Large T precocious protein of the SV40 virus. g) A sequence of 13 aminoacids (MEQKLISEEDLNE, SEQ ID NO. 16) specifically recognized by the monoclonal antibody 9E10 which allows detecting the protein presence.

Table 4. Nucleotidic sequence of V-UtroUp (SEQ ID NO. 9)

5'ATGGAGCAAAAGCTCATTTCTGAAGAGGACTTGAATGAAATGGAGCAAAAGCT

CATTTCTGAAGAGGACTTGAATGAAATGGAGCAAAAGCTCATTTCTGAAGAGGAC

TTGAATGAAATGGAGCAAAAGCTCATTTCTGAAGAGGACTTGAATGAAATGGAGC

AAAAGCTCATTTCTGAAGAGGACTTGAATGAAATGGAGAGCTTGGGCGACCTCAC

CATGGGCCCTAAAAAGAAGCGTAAAGTCGCCCCCCCGACCGATGTCAGCCTGGGG

GACGAGCTCCACTTAGACGGCGAGGACGTGGCGATGGCGCATGCCGACGCGCTAG

ACGATTTCGATCTGGACATGTTGGGGGACGGGGATTCCCCGGGTCCGGGATTTAC

CCCCCACGACTCCGCCCCCTACGGCGCTCTGGATATGGCCGACTTCGAGTTTGAG

CAGATGTTTACCGATGCCCTTGGAATTGACGAGTACGGTGGGGAATTCCCGGGGA

TCCTGGATCGCCCTTACGCCTGCCCTGTGGAATCTTGCGACCGCCGGTTCTCCCG

CAGCGATAACCTGGTGCGGCACATCCGGATTCACACCGGCCAGAAACCTTTCCAG

TGCAGGATCTGCATGAGAAATTTCTCCCGGTCCGACCACCTGACCACCCACAATA

GGACCCACACCGGCGAGAAACCCTTTGCCTGCGACATCTGCGGGAGAAAGTTCGC

CGACCCCGGCCACCTGGTGAGACACAATAGAATCCACACCGGTGAAAAGCCCTTC

GCCTGTCCCGTGGAGAGCTGCGATCGCAGATTCAGCCGCAGCGACGAGCTGACAA

GGCACATCAGAATCCACACCGGGCAGAAGCCTTTTCAGTGCCGGATCTGCATGAG

GAACTTCAGCTCCCGGGACGTGCTGAGACGCCACAATCGCACACACACCGGCGAA

AAGCCCTTCGCCTGTGATATTTGCGGGCGGAAATTTGCCTCCAGAGATGTGCTGC

GCCGCCACAACCGCATTCACCTGAGACAGAACGATCTCGAGTAA 3'

Table 5. Aminoacidic sequence of V-UtroUp (SEQ ID NO. 10)

5'MEQKLISEEDLNEMEQKLISEEDLNEMEQKLISEEDLNEMEQKLISEEDLNEMEQ KLISE

EDLNEMESLGDLTMGPKKKRKVAPPTDVSLGDELHLDGEDVAMAHADALDDFDLDML GDGD SPGPGFTPHDSAPYGALDMADFEFEQMFTDALGIDEYGGEFPGILDRPYACPVESCDRRF S RSDNLVRHIRIHTGQKPFQCRICMRNFSRSDHLTTHNRTHTGEKPFACDICGRKFADPGH L VRHNRIHTGEKPFACPVESCDRRFSRSDELTRHIRIHTGQKPFQCRICMRNFSSRDVLRR H NRTHTGEKPFACDICGRKFASRDVLRRHNRIHLRQNDLE 3'

Construction of B and V-UtroUp Construction of B-UtroUp

The synthetic gene B-UtroUp (Table 2) has been cloned in the bacterial expression vector pGEX-4Tl (Pharmacia) in the carboxy-terminal position with respect to the bacterial glutathione S-transferase (GST) protein. Construction of V-UtroUp

The synthetic gene UtroUp fused to the domain of strong activation of the "Vp 16" transcription originating from the virus herpes simplex has been cloned in the eucariotic expression vector pRK5 (Clonthec) under the control of the sequences regulating the cytomegalovirus (CMV). The final construct apart from epitope myc (in five copies), has a nuclear localization signal, an intron and a polyadenylation signal, all elements deriving from the SV40virus (Table 4). Expression in batcteria or mammal cells

B-UtroUp was expressed in the bacterial strain BL21 (strain of E. CoIi without protease (Invitrogen) and purified with resin glutathione Sepharose 4B (Pharmacia). The expression of the V-UtroUp transgene was controlled by using the monoclonal antibody anti myc 9E10 (Calbiochem) in experiments of Western Blot, performed with total and nuclear extracts deriving from HeLa cells transfected transiently. Assays of luciferase activity have been performed on cell extracts deriving from experiments of transient transfection in HeLa cells. The luciferase activity has been measured at the lumino meter, model Berthold LB9506. Experiments of electrophoretic delay (EMSA) In order to measure the dissociation constant (Kd) of B-UtroUp for the DNA target thereof (GCT GCT GCG GGC TGG GAG, SEQ ID No. 13) experiments of electrophoretic delay (EMSA) were performed. In the EMSA assay increasing quantities of B-UtroUp (concentrations from 1 to 120 nM), were placed to incubate with a fixed quantity (10000 cpm equal to about 0.5 ng) of the labelled oligonucleotide (double strand) containing 1 copy of the DNA target. After incubation, the reaction mix was loaded onto an acrylamide gel under non-denaturing conditions. The radioactive gel image was acquired with Phospholmager (Molecular Dinamics) and the band intensity was measured at the different protein concentrations with the ImageQuant programme. The data were analyzed with the KaleidaGraph (Abelbeck Sofware) programme, by assuming as Kd value the protein concentration expressed in nM subtracted of 50% of the radioactive signal.

RESULTS

The authors have designed, synthesized and assayed the biochemical properties a new zinc-finger-based DNA binding domain called "UtroUp" (Figure IB).

Binding to DNA target

The capability of B-UtroUp to bind DNA has been evaluated by means of electrophoretic delay experiments (EMSA), which have allowed calculating the dissociation constant thereof (Kd ) (Figure 2A). The dissociation constant of B-UtroUp results to be equal to 3,5 nM. This value shows an optimum level of affinity/specificity of B-UtroUp with respect to the DNA target thereof. The dissociation constant of B-UtroUp is comparable to the values obtained for other zinc finger natural proteins and it is significantly lower (more similar)

than the Kds determined for other zinc finger synthetic proteins produced by the authors (Corbi et al. 2000, Corbi et al. 2004, Libri et al. 2004).

Electrophoretic delay experiments performed with other properly mutagenized 18-bp sequence, similar to the DNA target sequence of UtroUP, show a strong descrease/absence of binding by UtropUp, confirming the high specificity thereof for binding to the DNA target.

It is important noting that UtroUp, comprising six zinc finger domains (three thereof deriving from Vpl6-Jazz (nucleotidic sequence of Jazz: AJ243577, aminoacidic sequence of Jazz: CAB52142, Corbi et al. 2000), shows an affinity of about ten times higher than Vpl6-Jazz containing only zinc finger domains (patent application RM 2005 A 000493 of 30/09/ 2005) (Corbi et al. 2000). The affinity to DNA increases proportionally upon increasing the number of zinc finger domains and upon increasing the number of DNA/protein contacts (Beltran et al. 2006, Onori et al. 2007).

Transcription

The capability of the protein V-UtroUp (Figure ID) to modulate the transcription of the luciferase reporter gene under the control of the utrophin promoter, which includes the

DNA target sequence of UtroUp (pXP) was verified.

In the histogram shown in figure 2B, V-UtroUp displays a high capability of activating the transcription.

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