Non-Invasive localization of a light-emitting conjugate in a mammal

• US 20030113264A1
• Filed: 12/16/2002
• Published: 06/19/2003
• Est. Priority Date: 07/01/1994
• Status: Active Grant

• Current Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Original Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Inventors: Contag, Christopher H., Benaron, David A., Contag, Pamela R.
• Granted Patent: US 6,923,951 B2
• Time in Patent Office: Days
• US Class Current: 424/9.6
• CPC Class Codes: C07K 14/195 from bacteria
• CPC Class Codes: C12Q 1/02 involving viable microorganisms
• CPC Class Codes: C12Q 1/66 involving luciferase
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: Y10S 435/968 High energy substrates, e.g. fluorescent, chemiluminescent, radioactive
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine

Abstract

Methods and compositions for detecting and localizing light originating from a mammal are disclosed. Also disclosed are methods for targeting light emission to selected regions, as well as for tracking entities within the mammal. In addition, animal models for disease states are disclosed, as are methods for localizing and tracking the progression of disease or a pathogen within the animal, and for screening putative therapeutic compounds effective to inhibit the disease or pathogen.

Claims

1. A noninvasive method for detecting the localization of a biocompatible entity in a mammalian subject, said method comprising the steps:

(a) administering to the subject a conjugate of the entity and a light-generating moiety,

(b) after a period of time in which the conjugate can achieve localization in the subject, immobilizing the subject within the detection field of a photodetector device,

(c) maintaining the subject in an immobilized condition,

(d) during said maintaining, measuring photon emission from the light-generating moiety, localized in the subject, with the photodetector device until an image of photon emission can be constructed, and

(e) constructing such an image.

2. The method of claim 1, said method further comprising:

repeating steps (b) through (e) at selected intervals,

wherein said repeating is effective to track the localization of the entity in the subject over time.

3. The method of claim 1, wherein said measuring is carried out with an intensified charge-coupled photodetector device.

4. The method of claim 1, for detecting the localization of tumor cells in a mammalian subject, wherein said administering includes administering a conjugate that contains a tumor cell targeting moiety.

5. The method of claim 1, for detecting the localization of inflammation in a mammalian subject, wherein said administering includes administering a conjugate that contains an inflammation-targeting moiety.

6. The method of claim 1, for detecting the localization of infection by a pathogen in a mammalian subject, wherein said administering includes administering a conjugate that contains an infection-targeting moiety.

7. The method of claim 1, for detecting the localization of thrombotic plaques in a mammalian subject, wherein said administering includes administering a conjugate that contains a thrombotic plaque-targeting moiety.

8. The method of claim 1, wherein said administering includes administering a conjugate that is a particle containing a light-generating moiety.

9. The method of claim 1, for detecting the localization of infection by a pathogen in an animal model, wherein the bio-compatible entity is the pathogen.

10. The method of claim 9, wherein the pathogen is selected from the group consisting of Pseudomonas, Staphylococcus, Streptococcus, Enterococcus, Enterobacter, Citrobacter, Leginella, Helicobacter, Acinetobacter, Escherichia, Klebsiella and Serratia.

11. The method of claim 9, wherein the pathogen is Salmonella.

12. The method of claim 1, wherein the light-generating moiety is a light-generating protein.

13. The method of claim 12, wherein the protein is selected from the group consisting of luciferase, yellow fluorescent protein and ferredoxin IV.

14. The method of claim 12, wherein said administering includes administering a conjugate that is a fusion protein of an antibody fragment and a light-generating protein.

15. The method of claim 12, wherein the biocompatible entity is a transformed cell, and the light-generating moiety is a product of a heterologous gene expressed by the cell.

16. The method of claim 13, wherein expression of the heterologous gene is under the control of an activatable promoter.

17. A noninvasive method for detecting the level of a biocompatible entity in a mammalian subject over time, said method comprising the steps:

(a) administering to the subject a conjugate of the entity and a light-generating moiety,

(b) placing the subject within the detection field of a photodetector device,

(c) maintaining the subject in the detection field of the device,

(d) during said maintaining, measuring photon emission from the light-generating moiety, in the subject, with the photodetector device, and

(e) repeating steps (b) through (d) at selected intervals, wherein said repeating is effective to detect changes in the level of the entity in the subject over time.

18. A noninvasive method for detecting the integration of a transgene in a mammalian subject, said method comprising the steps:

(a) administering to the subject, a vector construct effective to integrate into mammalian cells and containing a transgene, a gene encoding a light-generating protein, and an activatable promoter, said gene encoding a light-generating protein being under the control of said promoter,

(b) after a period of time in which the construct can achieve integration, activating said promoter,

(c) placing the subject within the detection field of a photodetector device,

(d) maintaining the subject within the detection field of the photodetector device,

(e) during said maintaining, measuring the level of photon emission from expressed light-generating protein, in the subject, with the photodetector device, and

(f) confirming integration of the transgene if the level of photon emission is significantly higher than background.

19. A noninvasive method for detecting the localization of a promoter-induction event in an animal, said method comprising the steps:

(a) triggering the event in a transgenic animal having an inducible promoter responsive to such event and a heterologous gene encoding a light-generating protein under control thereof,

(b) placing the animal in the detection field of a photodetector device,

(c) maintaining the animal in an immobilized condition,

(d) during said maintaining, measuring photon emission from expressed light-generating protein, localized in the animal, with the photodetector device until an image of photon emission can be constructed, and

(e) constructing such an image.

20. Salmonella transformed with a gene expressing a light-generating protein.

21. A transgenic animal comprising a construct composed of a promoter upstream of a coding sequence of a reporter gene.

22. The transgenic animal of claim 21, wherein said reporter gene is a luciferase gene.

23. The transgenic animal of claim 21, wherein said promoter is an inducible promoter.

24. The transgenic animal of claim 21, wherein the reporter gene is a firefly luciferase gene.

25. The transgenic animal of claim 22, wherein said promoter comprises a regulatory portion of the HIV LTR.