Methods of determining chemicals that modulate transcriptionally expression of genes associated with cardiovascular disease

• US 5,580,722 A
• Filed: 02/07/1992
• Issued: 12/03/1996
• Est. Priority Date: 07/18/1989
• Status: Expired due to Term

• Current Assignee: Osi Pharmaceuticals Incorporated (Astellas Pharma Incorporated)
• Original Assignee: Oncogene Science, Inc. (Bayer AG)
• Inventors: Foulkes, J. Gordon, Case, Casey C., Pieler, Christian, Stephenson, John R., Liechtfried, Franz E.
• Primary Examiner(s): Zitomer, Stephanie W.
• Application Number:
  US07/832,905
• Time in Patent Office: 1,761 Days
• Field of Search: 435/6, 435/91, 435/91.1, 435/91.2, 935/77, 935/78
• US Class Current: 435/6.14
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: C07K 14/535 Granulocyte CSF; Granulocyte-macrophage CSF
• CPC Class Codes: C07K 14/61 Growth hormone [GH], i.e. somatotropin
• CPC Class Codes: C07K 14/775 Apolipopeptides
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C12N 15/00 Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor mutants or genetically engineered microorganisms, per se C12N1/00, C12N5/00, C12N7/00; new plants per se A01H; plant reproduction by tissue culture techniques A01H4/00; new animals per se A01K67/00; use of medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases, gene therapy A61K48/00
• CPC Class Codes: C12N 15/65 using markers enzymes used as markers C12N15/52
• CPC Class Codes: C12N 15/81 for yeasts
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 2800/108 episomal vectors
• CPC Class Codes: C12N 2830/008 cell type or tissue specific enhancer/promoter combination
• CPC Class Codes: C12N 2830/30 being an enhancer not forming part of the promoter region
• CPC Class Codes: C12N 2830/85 mammalian
• CPC Class Codes: C12N 2840/20 translation of more than one cistron
• CPC Class Codes: C12N 2840/44 being a specific part of the splice mechanism, e.g. donor, acceptor
• CPC Class Codes: C12N 9/0069 acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
• CPC Class Codes: C12Q 1/6897 involving reporter genes operably linked to promoters
• CPC Class Codes: C12Y 113/12007 Photinus-luciferin 4-monooxygenase (ATP-hydrolysing) (1.13.12.7), i.e. firefly-luciferase

Abstract

The invention provided for a method of directly and specifically transcriptionally modulating the expression of a gene encoding a protein of interest associated with treatment of one or more symptoms of a cardiovascular disease such as atherosclerosis, restenosis or hypertension.

Further provided is a method of determining whether a molecule not previously known to be a modulator of protein biosynthesis is capable of directly and specifically transcriptionally modulating the expression of a gene encoding a protein of interest associated with treatment of one or more symptoms of a cardiovascular disease.

Lastly, the invention provides a method of directly and specifically transcriptionally modulating in a human being the expression of a gene encoding a protein of interest associated with treatment of one or more symptoms of a cardiovascular disease, thus ameliorating the disease.

Claims

1. A method of determining whether a chemical not previously known to be a modulator of protein biosynthesis is capable of specifically transcriptionally modulating the expression of a gene encoding a protein of interest associated with the treatment of one or more symptoms of a cardiovascular disease which comprises:

(a) contacting a sample which contains a predefined number of identical eucaryotic cells with a predetermined concentration of the chemical to be tested, each such cell comprising a single DNA construct consisting essentially of in 5'"'"' to 3'"'"' order (i) a modulatable transcriptional regulatory sequence of the gene encoding the protein of interest, (ii) a promoter of the gene encoding the protein of interest, and (iii) a reporter gene which expresses a polypeptide capable of producing a detectable signal, coupled to, and under the control of, the promoter, under conditions such that the chemical if capable of acting as a transcriptional modulator of the gene encoding the protein of interest, causes a measurable detectable signal to be produced by the polypeptide expressed by the reporter gene;

(b) quantitatively determining the amount of the signal so produced; and

(c) comparing the amount so determined with the amount of produced signal detected in the absence of any chemical being tested and upon contacting the sample with other chemicals so as to thereby identify the chemical as a chemical which causes a change in the detectable signal produced by the polypeptide, and determining whether the chemical specifically transcriptionally modulates expression of the gene associated with the treatment of one or more symptoms of the cardiovascular disease.

4. A screening method according to any one of claims 1 or 2, which comprises separately contacting each of a plurality of identical samples, each sample containing a predefined number of identical cells under conditions wherein said contacting is effected with a predetermined concentration of each different chemical to be tested.

5. A screening method of claim 4, wherein the plurality of samples comprises more than about 10⁴ samples.

6. A screening method of claim 5, wherein the plurality of samples comprises more than about 5×

10⁴ samples.

7. A screening method of claim 4, where more than about 10³ samples per week are contacted with different chemicals.

2. A method of determining whether a chemical not previously known to be a modulator of protein biosynthesis is capable of specifically transcriptionally modulating the expression of a gene encoding a protein of interest associated with the treatment of one or more symptoms of a cardiovascular disease which comprises:

(a) contacting a sample which contains a predefined number of identical eucaryotic cells with a predetermined concentration of the chemical to be tested, each such cell comprising a single DNA construct consisting essentially of in 5'"'"' to 3'"'"' order (i) a modulatable transcriptional regulatory sequence of the gene encoding the protein of interest, (ii ) a promoter of the gene encoding the protein of interest, and (iii) a DNA sequence transcribable into mRNA coupled to, and under the control of, the promoter, under conditions such that the chemical if capable of acting as a transcriptional modulator of the gene encoding the protein of interest, causes a measurable difference in the amount of mRNA transcribed from the DNA sequence;

(b) quantitatively determining the amount the mRNA so produced; and

(c) comparing the amount so determined with the amount of mRNA detected in the absence of any chemical being tested and upon contacting the sample with other chemicals so as to thereby identify the chemical as a chemical which causes a change in the detectable mRNA amount, and determining whether the chemical specifically transcriptionally modulates expression of the gene at interest associated with the treatment of one or more symptoms of the cardiovascular disease.

3. A method of claim 2, wherein the amount of mRNA is determined by quantitative polymerase chain reaction.