WO2002036620A9 - Promoteur specifique de compose relie au facteur de transcription ets, et transactivateurs associes - Google Patents

Promoteur specifique de compose relie au facteur de transcription ets, et transactivateurs associes

Info

Publication number
WO2002036620A9
WO2002036620A9 PCT/EP2001/012662 EP0112662W WO0236620A9 WO 2002036620 A9 WO2002036620 A9 WO 2002036620A9 EP 0112662 W EP0112662 W EP 0112662W WO 0236620 A9 WO0236620 A9 WO 0236620A9
Authority
WO
WIPO (PCT)
Prior art keywords
nucleic acid
gene
transcription factor
ets
fusion protein
Prior art date
Application number
PCT/EP2001/012662
Other languages
English (en)
Other versions
WO2002036620A2 (fr
WO2002036620A3 (fr
Inventor
Markus A Rueegg
Alexandre Briguet
Original Assignee
Myocontract Pharmaceutical Res
Markus A Rueegg
Alexandre Briguet
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Myocontract Pharmaceutical Res, Markus A Rueegg, Alexandre Briguet filed Critical Myocontract Pharmaceutical Res
Priority to AU2002219070A priority Critical patent/AU2002219070A1/en
Priority to EP01992719A priority patent/EP1366072A2/fr
Publication of WO2002036620A2 publication Critical patent/WO2002036620A2/fr
Publication of WO2002036620A9 publication Critical patent/WO2002036620A9/fr
Publication of WO2002036620A3 publication Critical patent/WO2002036620A3/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4707—Muscular dystrophy
    • C07K14/4708—Duchenne dystrophy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
    • C12N2710/16622—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the promoter/reporter construct may be prepared by insertion of the promoter into firefly luciferase encoding plasmids, e.g. pGL2-basicTM (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.
  • the following transfection protocol is used:
  • the firefly and Renilla luciferase reporter activities contained in 20 ⁇ l 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 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 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)).
  • NCBI National Center for Biotechnology Information
  • the well-known Smith-Waterman algorithm can also be used for the determination of homologies.
  • 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.
  • the transcription factor is preferably a member of the GABP-family of transcription factors.
  • the transcription factor is GABP ⁇ .
  • 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 ⁇ is constitutively active by means of fusing the amino-terminal portion GABP ⁇ 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.
  • the human AChR ⁇ subunit gene promoter sequence has been deposited under GenBank accession numbers Z84811 and GI1922319. The transcription rate of this AChR ⁇ subunit gene promoter/luciferase reporter construct in cotransfection experiments with the C2C12 myogenic cell line has been compared. In addition to the AChR ⁇ subunit gene promoter/luciferase reporter construct cells were either transfected with GABP ⁇ _VP16 construct or with the NLS-LacF construct for control (Briguet, A.; R ⁇ egg, M.A. J. Neurosci. 20:5989-5996 (2000)).
  • 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 GABP ⁇ _VP16. 24 h later the proliferation medium containing 20% fetal calf serum was replaced with differentiation medium containing 5% horse serum.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicinal Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Toxicology (AREA)
  • Wood Science & Technology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Virology (AREA)
  • Plant Pathology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne des moyens de traitement de maladies, en particulier de maladies musculaires. Elle concerne aussi un acide nucléique pouvant réguler l'expression d'un gène hétérologue et être sélectivement activé par un composé relié au facteur de transcription Ets. Elle concerne encore des constructions, des cellules hôtes comprenant cet acide nucléique, des procédés d'expression recombinante de gènes hétérologues, ainsi que des procédés de criblage et d'identification de composés candidats, possédant une activité régulatrice de transcription, les utilisant. L'invention concerne enfin une protéine de fusion destinée à activer spécifiquement cet acide nucléique, ainsi qu'un nécessaire comprenant l'acide nucléique et la protéine de fusion aux fins d'expression recombinante d'un gène hétérologue.
PCT/EP2001/012662 2000-11-02 2001-10-31 Promoteur specifique de compose relie au facteur de transcription ets, et transactivateurs associes WO2002036620A2 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU2002219070A AU2002219070A1 (en) 2000-11-02 2001-10-31 Ets-transcription factor related compound specific promoter and transactivators thereof
EP01992719A EP1366072A2 (fr) 2000-11-02 2001-10-31 Promoteur specifique de compose relie au facteur de transcription ets, et transactivateurs associes

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP00123842.7 2000-11-02
EP00123842 2000-11-02

Publications (3)

Publication Number Publication Date
WO2002036620A2 WO2002036620A2 (fr) 2002-05-10
WO2002036620A9 true WO2002036620A9 (fr) 2002-09-19
WO2002036620A3 WO2002036620A3 (fr) 2003-06-05

Family

ID=8170271

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2001/012662 WO2002036620A2 (fr) 2000-11-02 2001-10-31 Promoteur specifique de compose relie au facteur de transcription ets, et transactivateurs associes

Country Status (3)

Country Link
EP (1) EP1366072A2 (fr)
AU (1) AU2002219070A1 (fr)
WO (1) WO2002036620A2 (fr)

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE4340116A1 (de) * 1993-11-25 1995-06-01 Boehringer Ingelheim Int Neue Transkriptionsfaktor-Mutanten und ihre Verwendung
JP4364941B2 (ja) * 1995-04-24 2009-11-18 メディカル リサーチ カウンシル ウトロフィン遺伝子プロモーター
CA2331266A1 (fr) * 1998-06-16 1999-12-23 The Regents Of The University Of California Les exons 4 et 7 codent pour des domaines distincts de transactivation et de localisation de la chromotine dans le facteur esx
WO2000035474A1 (fr) * 1998-12-11 2000-06-22 Khurana Tejvir S Facteurs de croissance derives de la neurite pour le traitement de dystrophies musculaires
GB9923423D0 (en) * 1999-10-04 1999-12-08 Isis Innovation Promoting gene expression

Also Published As

Publication number Publication date
AU2002219070A1 (en) 2002-05-15
WO2002036620A2 (fr) 2002-05-10
WO2002036620A3 (fr) 2003-06-05
EP1366072A2 (fr) 2003-12-03

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