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WO1994019461A1 - PROCEDE POUR IDENTIFIER DES COMPOSES INDUISANT UN NIVEAU ACCRU D'ARNm DU FACTEUR DE CROISSANCE NERVEUSE - Google Patents

PROCEDE POUR IDENTIFIER DES COMPOSES INDUISANT UN NIVEAU ACCRU D'ARNm DU FACTEUR DE CROISSANCE NERVEUSE Download PDF

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Publication number
WO1994019461A1
WO1994019461A1 PCT/US1994/000818 US9400818W WO9419461A1 WO 1994019461 A1 WO1994019461 A1 WO 1994019461A1 US 9400818 W US9400818 W US 9400818W WO 9419461 A1 WO9419461 A1 WO 9419461A1
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WIPO (PCT)
Prior art keywords
ngf
promoter
reporter gene
cell
compound
Prior art date
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Ceased
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PCT/US1994/000818
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English (en)
Inventor
Eric Hoffman
Susan Carswell
Michael E. Lewis
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Cephalon LLC
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Cephalon LLC
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Filing date
Publication date
Application filed by Cephalon LLC filed Critical Cephalon LLC
Publication of WO1994019461A1 publication Critical patent/WO1994019461A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • 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
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • 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
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/48—Nerve growth factor [NGF]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/001—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
    • C12N2830/002—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/80—Vector systems having a special element relevant for transcription from vertebrates
    • C12N2830/85—Vector systems having a special element relevant for transcription from vertebrates mammalian
    • 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
    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/44—Vectors comprising a special translation-regulating system being a specific part of the splice mechanism, e.g. donor, acceptor

Definitions

  • NGF is a neurotrophic agent for basal forebrain cholinergic neurons (Whittemore et al. Brain Res . Rev. 12:439-464 (1987)) leading to the suggestion that neurotrophic factor therapy might retard or prevent the loss of basal forebrain cholinergic neurons.
  • Cholinergic neurons express the NGF receptor, enabling them to respond to the beneficial effects of NGF (Hefti et al., Neurosci Lett . 69:37-41 (1986)).
  • the results of animal model studies demonstrate that administration of exogenous NGF can prevent the death of cholinergic neurons after axotomy (Hefti, J. Neurosci 6:2155-2162 (1986); Williams et al., Proc . Natl . head . Sci . USA 83:9231-9235 (1986)) and can enhance the function of remaining cholinergic neurons in aging animals (Fischer et al., Nature 329:65-68 (1987))
  • the promoter of the mouse NGF gene contains sequences characteristic of eukaryotic gene regulatory elements (Zheng et al., Mol . Brain Res . 3:133 -140 (1988)). These include consensus CAAT and TATA box elements, known to be essential for basal level gene transcription, as well as an AP-1 element which acts as a binding site for the transcription factor composed of the Fos and Jun proteins. Factors that stimulate the production of the Fos and Jun proteins also elevate the expression of some genes whose promoters contain AP-1 elements (Curran et al., Cell 55:395-397 (1988)).
  • the invention also features a method of treating Parkinson's disease in a mammal bearing a cellular transplant.
  • the method involves administering to the mammal a compound that increases the level of NGF protein in or around the transplant of the mammal.
  • the cellular transplant may contain neurons or adrenal cells.
  • the compound is identified by the NGF promoter transcription assay described herein.
  • the DNA molecule contains sequences for the stable maintenance and replication of the DNA molecule in the cell, as described by the art (Ausubel et al. eds. Current Protocols in Molecular Biology. John Wiley & Sons, publ. NY. 1987, 1989, hereby incorporated by reference; Sambrook et al. (1989), Molecular Cloning: A Laboratory Manual (2d ed.), CSH Press, hereby incorporated by reference) .
  • the DNA molecule is preferably stably integrated into the chromosomal DNA of the cell, but would also be functional for the purposes stated herein in the form of an extrachromosomal DNA element.
  • this modified CAT vector contains an RNA splice acceptor site, derived from the SV40 virus genome, that mates with the RNA splice donor site provided by the cloned NGF promoter DNA fragment. Inclusion of the acceptor site facilitates the correct splicing of the CAT mRNA and permits the inclusion of the NGF AP-l site in the promoter-reporter constructions. If applicants had not included the acceptor site, potential aberrant splicing of the CAT mRNA caused by the presence of a splice donor site and the absence of a corresponding acceptor site would likely have invalidated the results of the promoter studies presented herein (Huang et al., Mol . Cell .
  • the NGF promoter fragment was inserted into it, and the resulting constructions was transfected into mouse L929 cells (A.T.C.C. #CCL1) .
  • Applicants have tested the effectiveness and sensitivity of the assay by conducting a positive control.
  • Promoter activity is measured in transient expression assays by quantitating the levels of CAT enzymatic activity present in cellular extracts.
  • Cells containing the NGF promoter-reporter plasmid were treated with the phorbol ester TPA, resulting in an increase in CAT activity.
  • Increased CAT activity was not seen in similarly treated cells containing a promoterless CAT plasmid. Therefore TPA induced reporter-specific mRNA levels in this assay by an NGF promoter-specific mechanism.
  • Applicants have thereby demonstrated the utility of this system for the identification of compounds that induce NGF mRNA levels by acting on NGF promoter sequences.
  • This method can provide compounds useful as novel therapeutics for the treatment of pathologies that exhibit cholinergic neuronal dysfunction or atrophy. Such pathologies include, for example, Alzheimer's disease.
  • the method can further provide compounds useful as novel therapeutics for treating sensory peripheral neuropathies as well as for promoting the survival of NGF- sensitive transplanted neurons or adrenal cells.
  • the CAT reporter plasmid pCAT- BASIC (Promega) was digested with the enzyme EcoRI to produce 2.97 and 1.5 kb fragments.
  • the 1.5 kb fragment was replaced with a 700 bp EcoRI fragment from the plasmid pL- LPA (Carswell et al. 1989 supra) to form the construct pCBE-1.
  • This fragment exchange replaced the SV40 virus large T antigen splice signals and the SV40 early polyadenylation signal with the SV40 late polyadenylation signal.
  • pCBE-1 was digested with the restriction enzyme Xbal and the 5' overhangs were blunt-ended with the Klenow fragment of DNA polymerase.
  • a 41 bp Hindi/Ncol restriction fragment containing the 19S splice acceptor signal from the SV40 viral genome was also blunt-ended by Klenow enzyme treatment and ligated to the Xbal linear pCBE-1 vector.
  • the resulting plasmid construct is designated pCBE-36.
  • pCBE-36 was sequenced by the chain termination reaction method to confirm the orientation of the inserted DNA fragments and sequence composition.
  • the pCBE-36 plasmid DNA was digested with the restriction enzyme Sail. The 5' overhangs were blunt-ended with Klenow enzyme treatment.
  • Six DNA restriction fragments derived from the NGF promoter region present in the plasmid pBU2 were isolated and blunt-ended by Klenow treatment. These fragments include: a) an 855 bp Sphl/Xhol fragment; b) a 597 bp EcoRV/XhoI fragment; c) a 445 bp
  • Each DNA fragment was ligated to a Sail linear pCBE-36 plasmid DNA molecule in six separate reactions to yield six NGF promoter deletion-CAT reporter plasmids.
  • the promoter region of each of the six plasmids is illustrated in Fig. 2.
  • the clones were characterized by restriction enzyme mapping to confirm the orientation of the inserted DNA fragments.
  • Each plasmid DNA was purified by column chromatography (Qiagen, Chatsworth, CA) .
  • the plasmid DNA molecules were introduced into mouse L929 fibroblast cells.
  • L929 cells (5 x 10 5 cells per 100 mm dish) were seeded 48 hrs prior to transfection. Cells were washed with serum-free MEM-alpha medium (Gibco, Gaithersburg, MD) immediately prior to Lipofectin (Gibco/BRL)-mediated transfection (Feigner et al., Proc . Natl . Acad. Sci . USA 84:7413-7417 (1987)).
  • Plasmid DNA (2.5 ⁇ g) was combined with 55.0 ⁇ g Lipofectin reagent in a final volume of 110 ⁇ l and added to cells in 7.0 ml serum-free MEM-alpha medium. Cells were incubated for 16 hrs at 37° C in an atmosphere of 5% C0 2 . Sixteen hours after transfection, 3.3 ml of MEM-alpha medium containing 30% horse serum was added to each culture dish and the cells were incubated an additional 24 hours. Cells were harvested 48 hours after transfection by scraping the cells into a solution of 2.5 ml phosphate- buffered saline.
  • the cells were pelleted by centrifugation, resuspended in 50 ⁇ l 250 mM Tris-HCl pH 7.5, and lysed by three rounds of freezing and thawing. Cellular debris was removed from the extract by centrifugation. Protein content in the supernatant was determined by the Bradford assay (Bio-Rad, Hercules, CA) . Aliquots of cell extract (100 ⁇ g per reaction) were assayed for CAT enzymatic activity by the method of Gorman et al. (Mol . Cell . Biol . 2:1044-1051 (1982), hereby incorporated by reference) . Each of the six NGF promoter constructions.
  • the transient expression assays were repeated using the promoter construct pl21-CAT and the promoterless plasmid pCBE-36. Following transfection, cells were treated with serum containing a final concentration of 100 ng/ml TPA. Control reactions were treated with serum alone. As shown in Fig. 5, CAT activity in cells transfected with pl21-CAT increased approximately two-fold in the presence of TPA compared to untreated controls. Cells transfected with the promoterless construct were unaffected by TPA treatment. The results indicate that the isolated NGF promoter sequences contain a TPA-responsive element. This element most likely correlates with the intronic AP-1 binding site (D'Mello et al., supra) .
  • luciferase When luciferase is used as the reporter gene (Promega, Madison, WI) , the cells hosting the recombinant DNA molecule are lysed, adenosine triphosphate (ATP) and luciferin are added to the lysate in a liminometer, and the resulting light emission is correlated with the amount of luciferase present in the lysate (Nordeen, BioTechniques 6:454-457 (1988)). Beta- galactosidase (An et al., Mol . Cell . Bio .
  • the method of the invention is used as an in vitro screening procedure to identify compounds that increase NGF mRNA levels, and thereby identify compounds that would increase the level of NGF in a mammal, e.g., in a human patient.
  • the compounds positively identified in the screening method can be re-tested in an additional in vivo assay designed to confirm that increased NGF mRNA levels by the compound results in higher NGF levels in a mammal.
  • Examples of appropriate in vivo assays include a method of measuring endogenous NGF mRNA levels in rat brains in response to chemical induction (Fabrazzo et al., Mol . Pharm .
  • a compound of the invention to reduce peripheral neuropathy in sensory nerves is carried out by administering said compound to said patient in an effective dosing schedule and then measuring sensory nerve activity according to methods which are well known to those versed in the art, e.g., a combination of electrophysiological and neurological examination testing methods such as are described in DeAngelis et al., Cancer 67:2241-2246 (1991).

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  • Health & Medical Sciences (AREA)
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  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Biomedical Technology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Toxicology (AREA)
  • Medicinal Chemistry (AREA)
  • Plant Pathology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention se rapporte à un procédé in vitro pour identifier un composé capable d'accroître le niveau du facteur de croissance nerveuse (NGF) dans une cellule de mammifère. Ce procédé consiste: (a) à introduire un ADN de plasmide contenant le promoteur de NGF dans une cellule, où le promoteur est lié fonctionnellement à un gène reporteur; (b) à exposer ces cellules aux composés; et (c) à mesurer le niveau du produit génique reporteur obtenu par exposition desdites cellules au composé, un accroissement du produit génique reporteur indiquant une augmentation de l'activité du promoteur de NGF, ce qui indique aussi que le composé est capable d'accroître le niveau de NGF dans le mammifère en question. Ce procédé est utile dans le traitement des maladies associées à une atrophie des neurones cholinergique, telles que la maladie d'Alzheimer, ou dans le traitement des neuropathies périphériques sensitives.
PCT/US1994/000818 1993-02-17 1994-01-25 PROCEDE POUR IDENTIFIER DES COMPOSES INDUISANT UN NIVEAU ACCRU D'ARNm DU FACTEUR DE CROISSANCE NERVEUSE Ceased WO1994019461A1 (fr)

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US1875593A 1993-02-17 1993-02-17
US08/018,755 1993-02-17

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998035027A3 (fr) * 1997-02-06 1998-12-03 Hoechst Marion Roussel Inc Promoteurs de l'exon 1 et de l'exon 3 du facteur humain de croissance du tissu nerveux
US5958703A (en) * 1996-12-03 1999-09-28 Glaxo Group Limited Use of modified tethers in screening compound libraries
WO2000067033A1 (fr) * 1999-05-03 2000-11-09 Evotec Biosystems Ag Procedes permettant de diagnostiquer ou traiter la maladie d'alzheimer
US6309889B1 (en) 1999-12-23 2001-10-30 Glaxo Wellcome Inc. Nano-grid micro reactor and methods
WO2022105788A1 (fr) * 2020-11-17 2022-05-27 泰伦基国际有限公司 Procédé et médicament pour augmenter le taux de bdnf

Non-Patent Citations (10)

* Cited by examiner, † Cited by third party
Title
BIOTECHNIQUES, Volume 6, issued 1988, S.K. NORDEEN, "Luciferase Reporter Gene Vectors for Analysis of Promoters and Enhancers", pages 454-457. *
FEBS LETTERS, Volume 247, Number 2, issued April 1989, FURUKAWA et al., "Catecholamines Increase Nerve Growth Factor mRNA Content in Both Mouse Astroglial Cells and Fibroblast Cells", pages 463-467. *
FEBS LETTERS, Volume 262, Number 1, issued March 1990, WION et al., "Phorbol 12-Myristate 13-Acetate (PMA) Increases the Expression of the Nerve Growth Factor (NGF) Gene in Mouse L-929 Fibroblasts", pages 42-44. *
JOURNAL OF NEUROSCIENCE RESEARCH, Volume 28, issued 1991, WION et al., "1,25-Dihydroxyvitamin D-3 is a Potent Inducer of Nerve Growth Factor Synthesis", pages 110-114. *
MOLECULAR AND CELLULAR BIOLOGY, Volume 2, Number 12, issued December 1982, AN et al., "Expression of Bacterial Beta-Galactosidase in Animal Cells", pages 1628-1632. *
MOLECULAR AND CELLULAR BIOLOGY, Volume 2, Number 9, issued September 1982, GORMAN et al., "Recombinant Genomes which Express Chloramphenicol Acetyltransferase in Mammalian Cells", pages 1044-1051. *
MOLECULAR AND CELLULAR BIOLOGY, Volume 7, Number 9, issued September 1987, SELBY et al., "Mouse Nerve Growth Factor Gene: Structure and Expression", pages 3057-3064. *
MOLECULAR BRAIN RESEARCH, Volume 11, issued 1991, D'MELLO et al., "Structural and Functional Identification of Regulatory Regions and Cis Elements Surrounding the Nerve Growth Factor Gene Promoter", pages 255-264. *
MOLECULAR BRAIN RESEARCH, Volume 15, issued 1992, CARSWELL et al., "Induction of NGF by Isoproterenol, 4-Methylcatechol and Serum Occurs by Three Distinct Mechanisms", pages 145-150. *
MOLECULAR BRAIN RESEARCH, Volume 3, issued 1988, ZHENG et al., "Structural and Functional Analysis of the Promoter Region of the Nerve Growth Factor Gene", pages 133-140. *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5958703A (en) * 1996-12-03 1999-09-28 Glaxo Group Limited Use of modified tethers in screening compound libraries
US6309842B1 (en) 1996-12-03 2001-10-30 Glaxo Wellcome Inc. Use of modified tethers in screening compound libraries
WO1998035027A3 (fr) * 1997-02-06 1998-12-03 Hoechst Marion Roussel Inc Promoteurs de l'exon 1 et de l'exon 3 du facteur humain de croissance du tissu nerveux
WO2000067033A1 (fr) * 1999-05-03 2000-11-09 Evotec Biosystems Ag Procedes permettant de diagnostiquer ou traiter la maladie d'alzheimer
US6309889B1 (en) 1999-12-23 2001-10-30 Glaxo Wellcome Inc. Nano-grid micro reactor and methods
WO2022105788A1 (fr) * 2020-11-17 2022-05-27 泰伦基国际有限公司 Procédé et médicament pour augmenter le taux de bdnf

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