US2003129623A1PendingUtilityA1

SR-BI regulatory sequences and therapeutic assays based on the same

Assignee: MILLENNIUM PHARM INCPriority: Mar 6, 1997Filed: Sep 13, 2002Published: Jul 10, 2003

Est. expiryMar 6, 2017(expired)· nominal 20-yr term from priority

A01K 2217/075A01K 2267/0393A01K 2267/0375C12N 2830/008C12N 15/85A01K 2217/05C12N 2830/85A01K 2217/00A01K 2227/105C07K 14/705A01K 2207/15C12N 15/8509A01K 2267/03C12N 2830/002A01K 2267/0362C12N 2800/30C12N 2800/204

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Claims

Abstract

The invention features nucleic acid molecules that are involved with (e.g. activate or regulate) human SR—BI receptor transcription, as well as complements thereto, and homologs thereof. In addition, drug discovery assays are provided for identifying agents which modulate SR—BI promoter activity and thereby modulate the expression of a gene regulated thereby. Such agents can be useful therapeutically for treating or preventing the development of a disease or condition that is caused or contributed to by an aberrant SR—BI activity. In a preferred embodiment, the disease or condition is characterized by inappropriate lipid transfer or metabolism (e.g., atherosclerosis or gallstone formation). Such agents can also be used to modulate expression of a specific gene under the control of the SR—BI promoter in gene therapy. Moreover, the present invention provides diagnostic assays and reagents for determining whether a subject has a disorder involving, for example, aberrant expression of SR—BI genes.

Claims

exact text as granted — not AI-modified

What is claimed is:

1 . A method for identifying a test compound that modulates transcription from an SR—BI promoter comprising the steps of:
(i) combining an SR—BI transcription factor with a test compound and a functional reporter construct comprised of a gene encoding a reporter molecule under the control of the SR—BI basic promoter and at least one SR—BI regulatory element, which is a binding site for the transcription factor; and
(ii) determining the level of specific binding of the at least one transcription factor to the at least one regulatory element; and
(iii) comparing the level obtained in step (ii) with the level of specific binding of the at least one transcription factor to the at least one SR—BI regulatory element in the absence of the test compound, wherein a statistically significant change in the level of specific binding in the presence of the test compound relative to in the absence of the test compound indicates that the test compound specifically modulates the binding of the at least one transcription factor to the SR—BI regulatory element.

2 . A method of claim 1 , wherein the SR—BI basic promoter or SR—BI regulatory element is human.

3 . A method of claim 1 , wherein the SR—BI basic promoter or SR—BI regulatory element comprises a nucleic acid shown in FIG. 1(SEQ ID NO: 1).

4 . A method of claim 3 , wherein the SR—BI basic promoter or SR—BI regulatory element comprises at least about 5 and less than about 40 consecutive nucleotides of the nucleic acid shown in FIG. 1 ( SEQ ID NO: 1).

5 . A method of claim 4 , wherein the SR—BI basic promoter or SR—BI regulatory element comprises at least about 15 and less than about 30 consecutive nucleotides of the nucleic acid sequence shown in FIG. 1 (SEQ ID NO: 1).

6 . A method of claim 1 , wherein the transcription factor is provided in a nuclear extract.

7 . A method of claim 6 , wherein the cellular extract is from a hepatic cell.

8 . The method of claim 1 , wherein the test compound is a small molecule.

9 . A method for identifying a test compound that modulates transcription form an SR-B1 promoter comprising the steps of:
(i) combining in a cell or cellular extract, the test compound and a reporter construct comprised of a reporter gene operably linked to an SR-B1 promoter; and (ii) detecting the level of expression of the reporter gene, wherein a statistically significant change in the level of expression relative to expression in the absence of the test compound indicates that the test compound modulates the activity of an SR—BI promoter.

10 . A method of claim 9 , wherein the SR-B1 basic promoter comprises a nucleic acid sequence shown in FIG. 1 (SEQ ID NO: 1).

11 . A method of claim 10 , wherein the SR-B1 basic promoter comprises at least about 5 and less than about 40 consecutive nucleotides of the nucleic acid sequence shown in FIG. 1 (SEQ ID NO: 1).

12 . A method of claim 11 , wherein the SR-B1 basic promoter comprises at least about 15 and less than about 30 consecutive nucleotides of the nucleic acid sequence shown in FIG. 1 (SEQ ID NO: 1).

13 . A method of claim 10 , wherein the cell or cellular extract is a hepatic cell or cellular extract from a hepatic cell.

14 . A method of claim 10 , wherein the reporter construct is additionally comprised of at least one SR-B1 regulatory element.

15 . A method of claim 10 , wherein the compound is selected from the group consisting of a nucleic acid, a peptide, and a small molecule.

16 . A method of claim 10 , wherein the reporter molecule is a luciferase.

17 . A method of claim 10 , which comprises the additional step of administering a test compound identified as modulating transcription from an SR-B1 promoter to a subject having a disease or condition that is caused or contributed to by an aberrant SR-B1 activity.

18 . A method of claim 17 , wherein the disease or condition is selected from the group consisting of atherosclerosis, gallstone formation, and abnormal lipoprotein levels.

19 . A method for identifying a test compound that modulates transcription from an SR-B1 promoter comprising the steps of:
(i) combining an SR-B1 transcription factor with a test compound and a functional reporter construct comprised of a gene encoding a reporter molecule under the control of the SR-B1 basic promoter and at least one SR-B1 regulatory element, which is a binding site for the transcription factor; and (ii) determining the level of specific biding of the at least one transcription factor to the at least one regulatory element; and (iii) comparing the level obtained in step (ii) with the level of specific binding of the at least one transcription factor to the at least one SR-B1 regulatory element in the absence of the test compound, wherein a statistically significant change in the level of specific binding in the presence of the test compound indicates that the test compound specifically modulates the binding of the at least one transcription factor to the SR-B1 regulatory element.

20 . A method of claim 19 , wherein the SR-B1 basic promoter or SR-B1 regulatory element is human.

21 . A method of claim 20 , wherein the SR-B1 basic promoter or SR-B1 regulatory element comprises a nucleic acid shown in FIG. 1 (SEQ ID NO: 1).

22 . A method of claim 21 , wherein the SR-B1 basic promoter or SR-B1 regulatory element comprises at least about 5 and less than about 40 consecutive nucleotides of the nucleic acid sequence shown in FIG. 1 (SEQ ID NO: 1).

23 . A method of claim 22 , wherein the SR-B1 basic promoter or SR-B1 regulatory element comprises at least about 15 and less than about 30 consecutive nucleotides of the nucleic acid sequence shown in FIG. 1 (SEQ ID NO: 1).

24 . A method of claim 19 , wherein the transcription factor is provided in a nuclear extract.

25 . A method of claim 24 , wherein the cellular extract is from a hepatitic cell.

26 . The method of claim 19 , wherein the test compound is a small molecule.

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