Pharmacogenomics and idenitfication of drug targets by reconstruction of signal transduction pathways based on sequences of accessible regions

• US 20030190664A1
• Filed: 05/08/2003
• Published: 10/09/2003
• Est. Priority Date: 04/28/2000
• Status: Active Grant

• Current Assignee: Alan Wolffe, Brian Johnstone, Dmitry Guschin, Fyodor Urnov, Trevor Collingwood, Xiao-Yong Li
• Original Assignee: Alan Wolffe, Brian Johnstone, Dmitry Guschin, Fyodor Urnov, Trevor Collingwood, Xiao-Yong Li
• Inventors: Urnov, Fyodor, Collingwood, Trevor, Johnstone, Brian, Wolffe, Alan, Guschin, Dmitry, Li, Xiao-Yong
• Granted Patent: US 7,097,978 B2
• Time in Patent Office: Days
• US Class Current: 435/6.13
• CPC Class Codes: C07K 14/4702 Regulators; Modulating activity
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/02 containing a signal sequence
• CPC Class Codes: C07K 2319/09 containing a nuclear localisation signal
• CPC Class Codes: C07K 2319/24 containing a MBP (maltose binding protein)-tag
• CPC Class Codes: C07K 2319/43 containing a FLAG-tag
• CPC Class Codes: C07K 2319/71 containing domain for transcriptional activaation, e.g. VP16
• CPC Class Codes: C07K 2319/75 containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones
• CPC Class Codes: C07K 2319/81 containing a Zn-finger domain for DNA binding
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12N 15/1034 Isolating an individual clone by screening libraries
• CPC Class Codes: C12N 15/1072 Differential gene expression library synthesis, e.g. subtracted libraries, differential screening
• CPC Class Codes: C12N 15/62 DNA sequences coding for fusion proteins
• CPC Class Codes: C12N 15/63 Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• CPC Class Codes: C12N 15/66 General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
• CPC Class Codes: C12N 15/67 General methods for enhancing the expression
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Q 2522/101 Single or double stranded nucleic acid binding proteins
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: G16B 30/00 : ICT specially adapted for sequence analysis involving nucleotides or amino acids
• CPC Class Codes: G16B 30/10 : Sequence alignment; Homology search
• CPC Class Codes: G16B 50/00 : ICT programming tools or database systems specially adapted for bioinformatics

Abstract

Methods for the identification of a drug target, wherein the drug target is a component of a signal transduction pathway, are provided. The methods are based, in part, on selection of a gene of therapeutic interest and determination of regulatory DNA sequences which regulate expression of the gene in a cell of interest, followed by reconstruction of signal transduction pathways which act on molecules which bind to the regulatory sequence. Also provided are methods for assessing potential pharmacological effects and/or side effects of an agent, based on the effect of the agent on the distribution and properties of accessible regions in cellular chromatin.

Claims

1. A method for identifying a drug that affects accessible regions of cellular chromatin relative to a gene of interest, the method comprising:

(a) providing cellular chromatin having known accessible regions;

(b) exposing the cellular chromatin of step (a) to the drug under conditions that allow the drug to affect cellular chromatin;

(c) comparing the nature of accessible regions of the cellular chromatin of step (b) with the accessible regions of the cellular chromatin of step (a) to determine the effect, if any, of the drug on the accessible regions of the cellular chromatin.

2. The method of claim 1, wherein step (c) comprises reacting the cellular chromatin with an antibody against acetylated or phosphorylated histones.

3. The method of claim 1, wherein step (c) comprises mapping the accessible regions of the cellular chromatin.

4. The method of claim 3, wherein mapping accessible regions relative to a gene of interest comprises:

(a) reacting cellular chromatin with a chemical or an enzymatic probe to generate chromatin-associated DNA fragments, wherein the DNA fragments comprise, at their termini, sites of probe reaction and identify accessible regions of cellular chromatin;

(b) treating the DNA fragments to obtain blunt ends;

(c) ligating an adapter polynucleotide to at least one end of the DNA fragments to generate adapter-ligated fragments;

(d) amplifying the adapter-ligated fragments in the presence of a first primer that is complementary to the adapter and a second primer that is complementary to a segment of the gene of interest to form an amplified product, wherein the size of the amplified product is a measure of the distance from the segment of the gene to which the second primer binds and the site of probe reaction.

5. The method of claim 4, wherein probe is an enzymatic probe.

6. The method of claim 5, wherein the enzymatic probe is a nuclease.

7. The method of claim 6, wherein the nuclease is selected from the group consisting of DNase I and micrococcal nuclease.

8. The method of claim 4, wherein the probe is a chemical probe.

9. The method of claim 1, wherein the drug comprises a protein.

10. The method of claim 1, wherein the drug is a small molecule.

11. The method of claim 1, wherein the accessible regions are provided as one or more collections of polynucleotide sequences, each collection comprising a plurality of polynucleotide sequences corresponding to accessible regions.

12. A method for identifying a drug target, said method comprising:

(a) identifying an accessible region in cellular chromatin related to a gene of interest;

(b) examining the sequence of the accessible region to identify one or more binding sites for a transcriptional regulatory molecule; and

(c) identifying one or more members of a signal transduction pathway that regulate the activity of the transcriptional regulatory molecule;

wherein the member of the signal transduction pathway identified in step (c) comprises the drug target.

13. The method of claim 12, wherein the accessible region is identified by DNAse hypersensitivity.

14. The method of claim 12, wherein the signal transduction pathway includes a G-protein coupled receptor.

15. The method of claim 12, wherein the cellular chromatin is derived from a human cell.

16. The method of claim 12, wherein the cellular chromatin is derived from an animal cell.

17. The method of claim 12, wherein the cellular chromatin is derived from a plant cell.

18. The method of claim 12, wherein the cellular chromatin is derived from a microorganism.

19. The method of claim 18, wherein the microorganism is a pathogen.

20. The method of claim 19, wherein the pathogen is a human, animal or plant pathogen.

21. The method of claim 12, wherein the drug target comprises a nucleotide sequence.

22. The method of claim 12, wherein the drug target comprises a protein.

23. The method of claim 12, wherein the accessible regions are identified from one or more collections of polynucleotide sequences, each collection comprising a plurality of polynucleotide sequences corresponding to accessible regions.

24. A method of identifying a molecule that modulates expression of a gene comprising:

(a) identifying a member of a signal transduction pathway that affects expression of the gene according to the method of claim 12;

and

(b) adding a molecule that affects the expression of the gene via affecting the member of the signal transduction pathway identified in step (a).

25. The method of claim 24, wherein the molecule is a protein.

26. The method of claim 25, wherein the molecule affects a transcription factor.

27. The method of claim 24, wherein the molecule is small molecule.

28. The method of claim 24, wherein the molecule augments expression of the gene.

29. The method of claim 24, wherein the molecule inhibits expression of the gene.

30. The method of claim 24, wherein the gene is involved in pathogen resistance.

31. The method of claim 24, wherein the gene is involved in increasing cell growth.

32. The method of claim 24, wherein the gene is involved in inhibiting cell growth.

33. The method of claim 24, wherein the gene is involved in a disease state or condition.

34. The method of claim 32, wherein the diseases state or condition is cancer.

35. The method of claim 32, wherein the disease state or condition is osteoporosis.

36. The method of claim 24 wherein the accessible regions are provided as one or more collections of polynucleotide sequences, each collection comprising a plurality of polynucleotide sequences corresponding to accessible regions.

37. The method of claim 24, wherein the signal transduction pathway affects processes selected from the group consisting of tissue engineering, transplantation, response to a pathogen, inhibition of cell growth, activation of cell growth and apoptosis.

38. The method of claim 37, wherein the signal transduction pathway is present in human cells.

39. The method of claim 37, wherein the signal transduction pathway is present in animal cells.

40. The method of claim 37, wherein the signal transduction pathway is present in plant cells.

41. The method of claim 40, wherein the signal transduction pathway is involved in herbicide response, pathogen response, growth, yield, biochemical properties, production of chemical or biochemical.

42. The method of claim 37, wherein the signal transduction pathway is present in microbial cells.

43. The method of claim 42, wherein the signal transduction pathway is involved in production of a product.

44. The method of claim 43, wherein product is selected from the group consisting of a chemical, a biochemical, a protein or a pharmaceutical.

45. The method of claim 37, wherein the microorganism is a pathogen.

46. The method of claim 45, wherein the signal transduction pathway is involved in ability of pathogen to replicate and/or produce toxins.

52. The method of claim 44, wherein the disease or condition is osteoporosis.

47. A method for pharmacogenomically selecting an appropriate drug therapy to administer to a first individual having a disease or condition, comprising

(a) determining the location and nature of the regulatory accessible regions associated with drug response in the first individual;

and

(b) correlating the location and nature of the regulatory accessible regions associated with drug response in the first individual to known locations and natures of regulatory accessible regions associated with drug response, thereby selecting an appropriate drug therapy to be administered to the first individual.

48. The method of claim 47, wherein the regulatory accessible regions are associated with genes involved in drug metabolism.

49. The method of claim 48, wherein the gene involved in drug metabolism is a cytochrome P450 gene.

50. The method of claim 47, wherein the disease or condition is cancer.

51. The method of claim 47, wherein the disease or condition is cardiovascular disease.