Promoter-reporter cells for determining drug metabolism, drug interactions, and the effects of allotype variation

• US 20080152632A1
• Filed: 06/22/2006
• Published: 06/26/2008
• Est. Priority Date: 06/22/2005
• Status: Abandoned Application

• Current Assignee: CXR Biosciences Ltd. (Concept Life Sciences (Midco) Ltd.)
• Original Assignee: CXR Biosciences Ltd. (Concept Life Sciences (Midco) Ltd.), Roslin Institute
• Inventors: Clark, A. John, Wolf, C. Roland, Clark, Helen
• Application Number:
  US11/473,864
• Publication Number:
  US 20080152632A1
• Time in Patent Office: Days
• US Class Current: 424/93.21
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C12N 2500/30 Organic components metal chelators C12N2500/10; calcitonin C12N2500/14; transferrin C12N2500/24
• CPC Class Codes: C12N 2501/11 Epidermal growth factor [EGF]
• CPC Class Codes: C12N 2501/12 Hepatocyte growth factor [HGF]
• CPC Class Codes: C12N 2503/02 Drug screening
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 5/0606 Pluripotent embryonic cells, e.g. embryonic stem cells [ES] embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696
• CPC Class Codes: C12N 5/067 Hepatocytes

Abstract

This invention provides a system for rapid determination of pharmacologic effects on target tissue types in cell populations cultured in vitro. The cells contain a promoter-reporter construct that reflects a toxicologic or metabolic change caused by the agent being screened. The promoter is taken from a gene known to be up- or down-regulated according to the metabolic state of the cell, and linked to a reporter gene that provides an external signal for monitoring promoter activity. The promoter-reporter cells may be produced by placing these genetic alterations into a line of human embryonic stem cells, bulking up the cells to any extent desired, and then differentiating the cells into the desired tissue type. This disclosure explains some of the powerful features of the promoter-reporter cells of this invention, and shows various ways the skilled reader can use the invention for pharmaceutical development and testing, or to monitor graft survival.

Claims

1. A population of hepatocyte lineage cells, neural cells, cardiomyocyte lineage cells, or undifferentiated human embryonic stem (hES) cells, or a population of cells differentiated from hES cells, wherein the population comprises cells that have been genetically altered so that a promoter that responds to a metabolic or toxicologic change in the cell controls expression of a reporter gene.

2. The cell population of claim 1, wherein the promoter has one or more of the following features:

it responds to apoptosis, such as a promoter for a PUMA gene;

it responds to DNA damage, such as a promoter for a p21 OR p21/WAF1 gene;

it responds to hyperplasia, such as a promoter for a Ki-67 gene;

it responds to oxidative stress, such as a promoter for a heme oxygenase 1, superoxide dismutase, γ

-glutamyl cysteinyl ligase, or metallothionine gene;

it is a promoter for a transcription factor that reflects a metabolic or toxicologic change in the cell, such as a PXR, CAR, aryl hydrocarbon receptor (AhR), or Nrf2 gene;

it is a promoter for an androgen, such as a pPAG responsive gene, or a promoter for prostate specific antigen (PSA);

it is a promoter for a cytochrome P450 enzyme, such as a promoter for CYP3A4 or CYP1A1;

it is a promoter for a drug transporter gene, such as MDR1;

it is a promoter for a gene that affects the contraction rate or the QT interval of the hear, such as a calcium flux gene.

3. The cell population of claim 1, having one or more of the following features:

the reporter gene encodes a protein that produces a fluorescent or phosphorescent signal when expressed, such as an isoform of green fluorescent protein;

the promoter and the reporter gene are both heterologous to the cell population

a heterologous reporter gene has been placed under control of an endogenous promoter.

4. The cell population of claim 1, which has also been genetically altered so that a second promoter that is tissue specific controls expression of a second reporter gene.

5. The cell population of claim 4, wherein the second promoter is a promoter for a hepatocyte specific marker selected from albumin, α

1-antitrypsin, α

-fetoprotein, γ

-glutamyl tranpeptidase, glucose-6-phosphatase, catalase, and monooxygenase.

6. The cell population of claim 1, which has been genetically altered to express a gene encoding a variant of an endogenous drug target or metabolizing enzyme.

7. The cell population of claim 6, wherein the variant is an allelic variant of CYP3A4, CYP2D6, CYP2C9, CYP2C19, N-acetyl transferase, or thiopurine methyltransferase.

8. The cell population of claim 6, in which different cells have been genetically altered to express different variants of the same drug target or drug metabolizing enzyme.

9. The cell population of claim 8, wherein cells having different variants of the drug target or drug metabolizing enzyme also contain a promoter linked to a different reporter gene.

11. A system or kit for producing or maintaining promoter-reporter cells, comprising a differentiated cell population according to claim 1, along with undifferentiated hES cells from the same line used to derive said promoter-reporter cell population, for producing more of said promoter-reporter cells.

12. A method of drug testing, comprising combining a drug with a cell population according to claim 1, and determining whether expression of the reporter gene in the cells is affected thereby.

13. The method of claim 12, whereby the drug is selected for further development because it does not cause substantial upregulation of the reporter gene.

14. The method of claim 12, wherein the drug is selected for further development because it prevents or lowers expression of the reporter gene in the cells caused by the presence of a separate stress-inducing compound or culture condition.

15. A method of drug testing, comprising combining the drug with a cell population according to claim 4, and determining whether there is a change in expression of the first reporter gene in cells expressing the second reporter gene.

16. A method of drug testing, comprising combining the drug with a cell population according to claim 1 in the presence or absence of an RNAi for a drug target or drug metabolizing enzyme, and determining whether there is a difference in expression of the reporter gene in the presence of the drug with or without the RNAi.

17. A system or kit for drug testing according to the method of claim 12, comprising a cell population according to claim 1, and optionally comprising a compound known to change expression of the reporter gene in the cells.

18. The system or kit of claim 15, comprising a plurality of cell populations sharing the same genome, but genetically altered such that different promoters that respond to a metabolic or toxicologic change control expression of a reporter gene in each cell population.

19. The system or kit of claim 15, comprising a plurality of cell populations sharing the same genome, but differentiated into cells of different tissues.

20. The system or kit for determining the effect of drugs of different variants of a drug target or drug metabolizing enzyme, comprising a plurality of cell populations according to claim 1 sharing the same genome, but having different variants of said drug target or drug metabolizing enzyme.

21. The system or kit of claim 20, wherein the variants are allelic variants of CYP3A4, CYP2D6, CYP2C9, CYP2C19, N-acetyl transferase, or thiopurine methyltransferase.

22. A system or kit for validating a drug target according to the method of claim 14, comprising a cell population according to claim 1, and an RNAi for a gene encoding a drug target or metabolizing enzyme.

23. Isolated tissue prepared for transplantation and adapted for monitoring after engraftment into a subject, containing cells having a promoter-reporter construct according to claim 1.

24. The isolated tissue of claim 23, wherein the reporter gene in the promoter-reporter cell population encodes an excretable reporter, such as human chorionic gonadotropin (hCG).

25. A method grafting a subject with a tissue that can be monitored after engraftment, comprising engrafting the tissue of claim 24 into the subject.

26. A method for evaluating a tissue allograft containing a promoter-reporter cell population according to claim 21 following engraftment into a subject, comprising monitoring expression of said reporter gene in the graft.

10. A method for producing a differentiated cell population from a line of hES cells, comprising:

a) genetically altering cells from said hES cell line, thereby producing hES cells in which a promoter that responds to a metabolic or toxicologic change in the cell controls expression of a reporter gene;

b) proliferating the genetically altered hES cells to form a genetically altered hES cell population; and

then

c) differentiating the genetically altered hES cell population into a population of genetically altered differentiated cells, such as neural, cardiomyocyte, or hepatocyte lineage cells.