Transcription biomarkers of biological responses and methods of use

Inventors: Shelley Force Aldred (Hayward, CA); Nathan D. Trinklein (Redwood City, CA); Michael Rose (Walnut Creek, CA); Patrick Collins (Millbrae, CA)

Assignee: ACTIVE MOTIF, INC.

G01N33/5011 C12N15/1037 C12Q1/6876 C12Q1/6883 C12Q1/6886 C12Q2600/136 C12Q2600/158

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Quick Facts

Patent No.: US 10,663,455
App. No.: 15/457,999
Granted: May 26, 2020
Kind: B2

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Abstract

This invention provides transcription regulatory control sequences, the activity of which function as biomarkers for a variety of biological responses. This invention also provides expression constructs in which a biomarker transcription regulatory sequence is operably linked with a sequence for a reporter. Cells that comprise these expression constructs can be used in assays to identify conditions that modulate activity of the biological response.

Claims (66)

1. A method for determining whether a test compound activates cholesterol biosynthesis in a cell comprising:

a) exposing a plurality of no more than 100 collections of cells to a test compound, wherein the cells in each collection comprise an expression construct comprising a promoter operatively linked with a heterologous sequence encoding a reporter, wherein the promoter within each collection is the same and each collection has a different promoter, wherein a plurality of the promoters are cholesterol biomarker promoters of different genes and are selected from the group consisting of:

(1) SEQ ID NO: 61 (LSS); SEQ ID NO: 63 (LDLR); SEQ ID NO: 64 (INSIG1); SEQ ID NO: 67; SEQ ID NO: 70 (FASN); SEQ ID NO: 76 (MVD); SEQ ID NO: 77 (SQLE); and SEQ ID NO: 78 (IDI1);

(2) a sequence of at least 500 nucleotides having at least 98% sequence identity to a sequence of (1); and

(3) a fragment of at least 500 nucleotides of a sequence of (1);

and the collections include a promoter from each of at least three different genes from the group;

b) measuring expression of the reporter; and

c) correlating the measurement with cholesterol biosynthesis, wherein increased expression of the reporter compared to a control, indicates that the test compound induces cholesterol biosynthesis and decreased expression of the reporter compared with a control indicates that the test compound inhibits cholesterol biosynthesis.

2. The method of claim 1 , wherein (a)(2) is a sequence and (a)(3) is a fragment of at least 700 nucleotides.

3. The method of claim 1 , wherein (a)(2) is a sequence and (a)(3) is a fragment of at least 900 nucleotides.

4. The method of claim 1 , wherein the sequence identity is at least 99.5%.

5. The method of claim 1 , wherein the plurality is no more than 50 collections of cells.

6. The method of claim 1 further comprising

i) measuring the activity of the reporter in the absence of the test compound or under a control compound;

ii) measuring the activity of the reporter in the presence of the test compound;

iii) measuring the difference or ratio of reporter activity between the cells exposed to the test compound and cells that were not; and

iv) correlating the difference or ratio between treated and untreated measurements with an cholesterol-mediated response.

7. The method of claim 1 wherein the test compound is a small organic molecule having a size up to about 5000 Da.

8. The method of claim 1 wherein the test compound is a nucleic acid derivative selected from a small interfering RNA, a micro RNA mimic and a micro RNA inhibitor.

9. The method of claim 1 wherein the expression construct is comprised in a plasmid, a virus, a transposon vector, or an artificial chromosome vector.

10. The method of claim 1 wherein the expression construct is integrated into a chromosome in the cell.

11. The method of claim 1 wherein the reporter is a light-emitting reporter, a fluorescent reporter or a colorimetric reporter.

12. The method of claim 1 wherein the reporter is luciferase.

13. The method of claim 1 wherein one of the cholesterol biomarker promoters is selected from:

(I) SEQ ID NO: 61 (LSS);

(II) a sequence of at least 500 nucleotides having at least 98% sequence identity to a sequence of (I); and