Compositions and methods for detecting proteolytic activity

• US 7,378,078 B2
• Filed: 06/02/2003
• Issued: 05/27/2008
• Est. Priority Date: 12/13/2000
• Status: Expired due to Fees

• Current Assignee: Board of Regents of the University of Michigan (University of Michigan)
• Original Assignee: Board of Regents of the University of Michigan (University of Michigan)
• Inventors: Ross, Brian D., Rehemtulla, Alnawaz
• Primary Examiner(s): Woitach; Joseph
• Assistant Examiner(s): HIRIYANNA, KELAGINAMANE T
• Application Number:
  US10/452,184
• Publication Number:
  US 20040053332A1
• Time in Patent Office: 1,821 Days
• Field of Search: None
• US Class Current: 424/9.1
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: C12Q 1/37 involving peptidase or proteinase
• CPC Class Codes: G01N 2333/96466 Cysteine endopeptidases (3.4.22)
• CPC Class Codes: G01N 2500/10 involving cells

Abstract

The invention provides compositions and methods for non-invasive imaging of enzyme (e.g., protease) activity in cells, tissues and organs and entire bodies in vitro, in vivo and in situ. The invention provides a chimeric polypeptide having a bioluminescent or chemiluminescent polypeptide, or a heterologous kinase, and at least one silencing moiety, and a protease cleavage motif positioned between the first and second domains. The imaging can be by computer assisted tomography (CAT), magnetic resonance spectroscopy (MRS), magnetic resonance imaging (MRI), positron emission tomography (PET), single-photon emission computed tomography (SPECT), or bioluminescence imaging (BLI).

Claims

1. A method of imaging, comprising:

a) providing

i) cells,

ii) a mammalian subject, wherein said mammalian subject has endogenous expression of a target caspase,

iii) a genetic construct, wherein said genetic construct encodes a chimeric polypeptide, where said chimeric polypeptide comprises

1) an illumination domain

wherein said illumination domain comprises a bioluminescence imaging unit,

2) at least two silencing domains, wherein said at least two silencing domains comprise a receptor ligand binding unit, wherein said receptor ligand binding unit is a steroid hormone receptor ligand binding unit, and

3) a target caspase recognition site,

wherein said target caspase recognition site is configured to bind whith an endogenous target caspase expressed whithin said subject;

wherein said target caspase recongnition site comprises a caspase-3 recognition site, wherein said caspase-3 recognition site comprise DEVD; and

iv) an imaging agent, and

b) transfecting said cells with said genetic construct;

c) implanting said cells into said mammalian subject;

d) administering said imaging agent to said mammalian subject; and

e) measuring the activity of said endogenous target caspase by detecting signal from said illumination unit.

2. The method of claim 1, wherein said target caspase recognition site is positioned between said illumination domain and said at least two silencing domains.

3. The method of claim 1, wherein said cells are tumor cells.

4. The method of claim 3, wherein said tumor cells are mammalian tumor cells.

5. The method of claim 4, wherein said mammalian tumor cells are rat brain tumor cells.

6. The method of claim 1, wherein said imaging agent is luciferin.

7. The method of claim 1, wherein said bioluminescent imaging unit is a bioluminescent polypeptide.

8. The method of claim 7, wherein said bioluminescent polypeptide is luciferase.

9. The method of claim 1, wherein said bioluminescent imaging unit is a chemiluminescent polypeptide.

10. The method of claim 1, wherein said steroid hormone receptor ligand binding unit is an estrogen receptor regulatory domain (ER).

11. The method of claim 1, wherein said at least two silencing domains are capable of binding with said illumination domain.

12. The method of claim 10, wherein binding of said at least two silencing domains with said illumination domain results in inhibited illumination of said bioluminescent imaging unit.

13. The method of claim 1, wherein said target caspase is capable of binding and cleaving said target caspase recognition site.

14. The method of claim 12, wherein cleaving of said target caspase recognition site results in liberation of said bioluminescent imaging unit.

15. The method of claim 14, wherein said liberation of said bioluminescent imaging unit results in illumination of said bioluminescent imaging unit.

16. The method of claim 15, wherein said illumination of said bioluminescent imaging unit may be imaged.

17. The method of claim 15, wherein said illumination of said bioluminescent imaging unit is indicative of said target caspase activity.

18. The method of claim 17, wherein said target caspase activity is indicatiye of cellular apoptosis.

19. The method of claim 1, wherein said chimeric polypeptide comprises one illumination domain, two target caspase recognition sites, and two silencing domains.

20. The method of claim 19, wherein said illumination domain is luciferase.

21. The method of claim 19, wherein said two target caspase recognition sites comprise the amino acid sequence:

DEVD.

22. The method of claim 19, wherein said two silencing domains are estrogen receptors.

23. The method of claim 19, wherein said illumination domain is luciferase, wherein said two target caspase recognition sites comprise the amino acid sequence:

DEVD, wherein said two silencing domains are estrogen receptors.

24. The method of claim 1, wherein said mammalian subject is a rodent.