CN-122029294-A - Multiple reporter gene system for chemical screening

Abstract

Described herein are systems and methods for rapid screening of large numbers of compounds that modulate the function of a given signaling pathway or cell. In certain aspects, the systems and methods described herein include multiplex assays or multimodal reporter genes.

Inventors

• KURT FISHER
• Galen Luowen
• Srilam Kusuli

Assignees

• 奥科坦特公司

Dates

Publication Date

20260512

Application Date

20240718

Priority Date

20230720

Claims (20)

1. 1. A system comprising a plurality of engineered cell lines in a partition, wherein the plurality of engineered cell lines comprises a first engineered cell line and a second engineered cell line, wherein the first engineered cell line comprises a first reporter construct and the second engineered cell line comprises a second reporter construct, wherein, The first reporter construct and the second reporter construct are selected from the group consisting of: a) A constitutive promoter operably coupled to the reporter gene; b) A first inducible promoter operably coupled to the reporter gene, or C) A second inducible promoter operably coupled to the reporter gene; wherein the first reporter construct is different from the second reporter construct, and wherein the first reporter construct and the second reporter construct are independently readable.
2. 2. The system of claim 1, wherein the plurality of engineered cell lines further comprises a third engineered cell line, wherein the third engineered cell line comprises a third reporter construct different from the first reporter construct and the second reporter construct, wherein the third reporter construct is selected from the group consisting of: a) A constitutive promoter operably coupled to the reporter gene; b) A first inducible promoter operably coupled to the reporter gene, or C) A second inducible promoter operably coupled to the reporter gene.
3. 3. The system of claim 1 or 2, wherein one or more of the first engineered cell line, the second engineered cell line, the third engineered cell line, or a combination thereof further comprises a first heterologous polypeptide.
4. 4. The system of claim 3, wherein one or more of the first engineered cell line, the second engineered cell line, the third engineered cell line, or a combination thereof further comprises a second heterologous polypeptide, wherein the second heterologous polypeptide comprises at least one amino acid change relative to the first heterologous polypeptide.
5. 5. The system of claim 4, wherein the second heterologous polypeptide comprises less than 10, less than 5, less than 3, or less than 2 amino acid changes relative to the first heterologous polypeptide.
6. 6. The system of claim 4, wherein the second heterologous polypeptide comprises more than 10, more than 20, more than 50, more than 100, more than 500, or more than 1000 amino acid changes relative to the first heterologous polypeptide.
7. 7. The system of any one of claims 3-6, wherein the first heterologous polypeptide, the second heterologous polypeptide, or both are coupled to a transcription factor.
8. 8. The system of claim 7, wherein the transcription factor comprises one or more of aGal4, PPR1, lac9, zinc finger, or LexA DNA binding domain.
9. 9. The system of claim 7 or 8, wherein the transcription factor comprises one or more of aVP64, p65, roTev, or Rta DNA activation domains.
10. 10. The system of any one of claims 1 to 9, wherein the first reporter construct and the second reporter construct are independently readable.
11. 11. The system of any one of claims 2 to 10, wherein the third reporter construct is independently readable from the first reporter construct and/or the second reporter construct reporter.
12. 12. The system of any one of claims 3-11, wherein the first inducible promoter, the second inducible promoter, or both are configured to be activated by the first heterologous polypeptide, a signal from the first heterologous polypeptide, a transcription factor coupled to the first heterologous polypeptide, or any combination thereof.
13. 13. The system of any one of claims 1 to 12, wherein the first, second or third engineered cell line comprises an additional reporter construct selected from the group consisting of: a) A constitutive promoter operably coupled to the reporter gene; b) A first inducible promoter operably coupled to the reporter gene, or C) A second inducible promoter operably coupled to the reporter gene, Wherein the additional reporter construct is different from the first reporter construct and the second reporter construct.
14. 14. The system of any one of claims 1 to 13, wherein one or more of the first, second, or third reporter constructs is integrated into the genome of the cell line.
15. 15. The system of any one of claims 1 to 14, wherein any one or more of the first, second, or third engineered cell lines is a eukaryotic cell line.
16. 16. The system of claim 15, wherein the eukaryotic cell line is a mammalian cell line.
17. 17. The system of claim 16, wherein the mammalian cell line is a human cell line.
18. 18. The system of any one of claims 1 to 17, wherein the reporter gene encodes a fluorescent protein or a luciferase protein.
19. 19. The system of any one of claims 1 to 17, wherein the reporter gene encodes a barcoded RNA sequence.
20. 20. The system of any one of claims 1 to 19, wherein the reporter gene encodes a fluorescent protein and a barcoded RNA sequence or a luciferase protein and a barcoded RNA sequence.

Description

Multiple reporter gene system for chemical screening Cross reference The application claims the benefit of U.S. provisional application No. 63/528,030, filed on 7.20 at 2023, which provisional application is incorporated herein by reference in its entirety. Background Understanding signaling pathways and cellular functions and identifying test agents that modulate a given signaling pathway or cellular function are important targets for pharmaceutical science and drug discovery. Disclosure of Invention The methods and systems described herein provide cell-based screening assays. These systems and methods include some improvements over previous screening methods. The systems and methods described herein may allow multiplexing to screen multiple signaling pathways or cell functions in parallel. These systems and methods allow for the identification of off-target effects or the identification of compounds that agonize or antagonize specific target variants, signaling pathways, and/or biological functions. The systems and methods described herein include multiple indices to allow for improved statistical significance. Methods and systems also allow for the identification or avoidance of non-specific or off-target effects by allowing screening for molecules that agonize or antagonize the same target but signal through different downstream effectors. In general, the methods and systems described herein increase the efficiency of drug screening and understanding of biological systems. The methods and systems described herein allow for simultaneous or substantially simultaneous determination of certain test agent activity on a target (e.g., a heterologous polypeptide), variants of the target, different downstream promoters that may be activated by the target, and simultaneous off-target effects of the test agents in a single reaction vessel (e.g., a well of a multi-well plate). The methods described herein are capable of determining thousands of compounds at the same time. Described herein are methods and systems for screening and identifying test agents that modulate the activity of a target, either positively (as agonists) or negatively (as antagonists). Described herein is a system comprising a plurality of engineered cell lines in a partition, wherein the plurality of engineered cell lines comprises a first engineered cell line and a second engineered cell line, wherein the first engineered cell line comprises a first reporter construct and the second engineered cell line comprises a second reporter construct, wherein the first reporter construct and the second reporter construct are selected from (a) a constitutive promoter operably coupled to a reporter gene, (b) a first inducible promoter operably coupled to a reporter gene, or (c) a second inducible promoter operably coupled to a reporter gene, wherein the first reporter construct is different from the second reporter construct, and wherein the first reporter construct and the second reporter construct are independently readable. In certain embodiments, the plurality of engineered cell lines further comprises a third engineered cell line, wherein the third engineered cell line comprises a third reporter construct different from the first reporter construct and the second reporter construct, wherein the third reporter construct is selected from (a) a constitutive promoter operably coupled to the reporter gene, (b) a first inducible promoter operably coupled to the reporter gene, or (c) a second inducible promoter operably coupled to the reporter gene. In certain embodiments, one or more of the first engineered cell line, the second engineered cell line, the third engineered cell line, or a combination thereof further comprises a first heterologous polypeptide. In certain embodiments, one or more of the first engineered cell line, the second engineered cell line, the third engineered cell line, or a combination thereof further comprises a second heterologous polypeptide, wherein the second heterologous polypeptide comprises at least one amino acid change relative to the first heterologous polypeptide. In certain embodiments, the second heterologous polypeptide comprises less than 10, less than 5, less than 3, or less than 2 amino acid changes relative to the first heterologous polypeptide. In certain embodiments, the second heterologous polypeptide comprises more than 10, more than 20, more than 50, more than 100, more than 500, or more than 1000 amino acid changes relative to the first heterologous polypeptide. In certain embodiments, the first heterologous polypeptide, the second heterologous polypeptide, or both are coupled to a transcription factor. In certain embodiments, the transcription factor comprises one or more of aGal, PPR1, lac9, zinc finger, or LexA DNA binding domains. In certain embodiments, the transcription factor comprises one or more of aVP, p65, roTev, or Rta DNA activating domains. In certain embodiments, the first reporter constru