EP0850250A1 - Enzymes de conjugaison d'ubiquitine presentant une activite de repression de transcription - Google Patents

Enzymes de conjugaison d'ubiquitine presentant une activite de repression de transcription

Info

Publication number
EP0850250A1
EP0850250A1 EP96929829A EP96929829A EP0850250A1 EP 0850250 A1 EP0850250 A1 EP 0850250A1 EP 96929829 A EP96929829 A EP 96929829A EP 96929829 A EP96929829 A EP 96929829A EP 0850250 A1 EP0850250 A1 EP 0850250A1
Authority
EP
European Patent Office
Prior art keywords
protein
activity
amino acid
hubc
transcriptional repressor
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP96929829A
Other languages
German (de)
English (en)
Inventor
Thomas F. Deuel
Thomas Shenk
Zhao-Yi Wang
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Barnes Hospital
Princeton University
Original Assignee
Barnes Hospital
Princeton University
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 Barnes Hospital, Princeton University filed Critical Barnes Hospital
Publication of EP0850250A1 publication Critical patent/EP0850250A1/fr
Withdrawn legal-status Critical Current

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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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • C07K14/4702—Regulators; Modulating activity
    • 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
    • C12N15/635—Externally inducible repressor mediated regulation of gene expression, e.g. tetR inducible by tetracyline
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • FIG. 12A and 12B show the results of transient co-transfection assays using a 5xUAS pSV CAT reporter vector.
  • Fig. 12A shows the relative level of expression of the reporter vector for assays where a GAL4/hUBC-9 fusion protein (pSGhUBC-9) (0 or 10 ⁇ g) , TBP (0, 0.5 or 2.5 ⁇ g) and/or TFIIB (5 ⁇ g) were co-expressed in 293 cells in varying combinations.
  • pSGhUBC-9 fusion protein pSGhUBC-9 fusion protein
  • TBP 0., 0.5 or 2.5 ⁇ g
  • TFIIB 5 ⁇ g
  • a “substantially purified” protein means that the protein is separated from a majority of host cell proteins normally associated with it or that the protein is synthesized in substantially purified form, such synthesis including expression of the protein in a host cell from a nucleic acid polymer exogenously introduced into the cell by any suitable gene-therapy delivery means.
  • a “substantially isolated” nucleic acid polymer means that the mixture which comprises the nucleic acid polymer of interest is essentially free of a majority of other nucleic acid polymers normally associated with it.
  • a “nucleic acid polymer” includes a polymer of nucleotides or nucleotide derivatives or analogs, including for example deoxyribonucleotides, ribonucleotides, etc.
  • UBCs have, in addition to their ubiquitin conjugating activity, a transcriptional repression activity.
  • the repression activity is independent of the conjugating activity, as demonstrated by data showing that yUBC-9-m, a yUBC-9 C 93 S mutant which lacks ubiquitin conjugating activity, functions efficiently as transcription repressor when fused to a DNA binding domain.
  • yUBC-9-m a yUBC-9 C 93 S mutant which lacks ubiquitin conjugating activity
  • the nucleic acid composition comprises a pharmaceutically effective amount of the nucleic acid and a pharmaceutically acceptable gene therapy delivery means.
  • the amount of nucleic acid required will vary depending on the type of cell, the effect being sought and on the delivery system used to introduce the nucleic acid polymer into a target cell.
  • the amount of nucleic acid polymer is preferably an amount sufficient to, upon expression in the target cell, result in an amount of protein sufficient to regulate or modulate or repress transcription of the target gene.
  • the amount is sufficient to increase the concentration of the protein in the cell of the target gene being regulated by a factor ranging from about 1% to about 1000% relative to the amount of the protein which is endogenous to the cell of the gene being regulated.
  • compositions of the present invention can be used in a variety of pharmaceutical and non-pharmaceutical applications.
  • gene transcription in cells can be regulated, enhanced or repressed, by controlling the concentration of UBC-9 and/or of TBP to which a target gene is exposed or in which a target gene comes in contact.
  • repression of transcription can be carried out in a gene- specific manner by positioning the UBC enzymes near the promoter regions of various genes, for example, by fusion of a UBC-9 repressor domain with a gene-specific DNA binding domain, or alternatively, by protein-protein interactions between UBC-9 and proteins associated with the promoter region or involved with transcription initiation, such as WTl, TBP, or others.
  • Extracts were made from 2xl0 6 293 (human embryonic kidney cell) cells transfected with CMV promoter driven expression vectors encoding full length and the WT1 ⁇ 1-84, WT1 ⁇ 1-294, and WT1 ⁇ 297-429 domains of WTl.
  • a yUBC-9-m/Gal4 expression vector, pSG-yUBC-9-m was constructed by digestion of pUC19-yUBC9-m plasmid with Hindlll, blunted with Klenow fragment, and then digested by EcoRI and cloned into EcoRI-Smal digested pSG424 plasmid.
  • the reporter plasmids, depicted in Figure 7A, were as obtained described above (Example 8) .

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Medicinal Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • General Chemical & Material Sciences (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Toxicology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Immobilizing And Processing Of Enzymes And Microorganisms (AREA)

Abstract

Enzyme de conjugaison d'ubiquitine humaine, appelée hUBC-9, sa séquence d'acides aminés complète et polymères d'acides nucléiques codant ce hUBC-9. Outre son activité de conjugaison fonctionnelle de l'ubiquitine, cette enzyme présente une activité de répression de transcription indépendante de son activité de conjugaison. L'activité de conjugaison du hUBC-9 favorise la transcription par la dégradation de protéines réprimant la transcription telles que WT1, et peut-être de hUBC-9 elle-même. L'activité de répression de hUBC-9 empêche la transcription des gènes, sans doute par l'interruption du complexe de départ de transcription, par des interactions spécifiques avec la région de liaison d'ADN de la protéine de liaison de l'antigène TATA (TOP). En pratique, hUBC-9, yUBC-9 et d'autres enzymes de conjugaison d'ubiquitine présentant une activité de répression peuvent être fusionnées avec des protéines présentant un domaine de liaison d'ADN, par exemple Gal4, ou utilisés en association avec de répresseurs tels que le gène suppresseur associé à la tumeur de Wilm, WT1. Ces enzymes et les polymères d'acide nucléique les codant peuvent être utilisés pour réguler la transcription d'un gène cible dans des applications pharmaceutiques et non pharmaceutiques.
EP96929829A 1995-08-30 1996-08-30 Enzymes de conjugaison d'ubiquitine presentant une activite de repression de transcription Withdrawn EP0850250A1 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
US299595P 1995-08-30 1995-08-30
US1804096P 1996-05-21 1996-05-21
US18040 1996-05-21
PCT/US1996/014013 WO1997008195A1 (fr) 1995-08-30 1996-08-30 Enzymes de conjugaison d'ubiquitine presentant une activite de repression de transcription
US2995 1998-01-05

Publications (1)

Publication Number Publication Date
EP0850250A1 true EP0850250A1 (fr) 1998-07-01

Family

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Family Applications (1)

Application Number Title Priority Date Filing Date
EP96929829A Withdrawn EP0850250A1 (fr) 1995-08-30 1996-08-30 Enzymes de conjugaison d'ubiquitine presentant une activite de repression de transcription

Country Status (5)

Country Link
EP (1) EP0850250A1 (fr)
JP (1) JP2000516081A (fr)
AU (1) AU720755B2 (fr)
CA (1) CA2230689A1 (fr)
WO (1) WO1997008195A1 (fr)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU9664598A (en) * 1997-09-23 1999-04-12 Incyte Pharmaceuticals, Inc. Human ubiquitin-conjugating enzymes
CA2316036A1 (fr) 1999-08-27 2001-02-27 Keqiang Wu Regulation de l'expression genetique chez des vegetaux
CN103091498B (zh) * 2013-01-08 2014-12-31 中国科学院遗传与发育生物学研究所 植物体外泛素蛋白降解系统及其应用

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ATE193123T1 (de) * 1994-01-04 2000-06-15 Mitotix Inc Ubiquitin-konjugierende enzyme

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9708195A1 *

Also Published As

Publication number Publication date
CA2230689A1 (fr) 1997-03-06
JP2000516081A (ja) 2000-12-05
WO1997008195A1 (fr) 1997-03-06
AU720755B2 (en) 2000-06-08
AU7695196A (en) 1997-03-19

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