MODULAR EXTRACELLULAR SENSOR ARCHITECTURE FOR CELL-BASED BIOSENSORS

• US 20140234851A1
• Filed: 08/03/2012
• Published: 08/21/2014
• Est. Priority Date: 08/05/2011
• Status: Active Grant

• Current Assignee: Northwestern University
• Original Assignee: Northwestern University
• Inventors: Leonard, Joshua N., Dudek, Rachel M., Daringer, Nichole M.
• Granted Patent: US 9,732,392 B2
• Time in Patent Office: Days
• US Class Current: 435/6.13
• CPC Class Codes: C12Q 1/6897 involving reporter genes operably linked to promoters
• CPC Class Codes: G01N 33/566 using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: G01N 33/6872 Intracellular protein regulatory factors and their receptors, e.g. including ion channels

Abstract

The present invention provides modular extracellular sensors, nucleic acids encoding such sensors, and cells expressing such sensors, and methods of employing such sensors and cells for detecting extracellular ligands. In certain embodiments, the extracellular sensors comprise a ligand binding domain, a transmembrane domain, a protease domain, a protease cleavage site, and a transcription factor. In other embodiments, a pair of extracellular receptors is provided where both receptors contain a ligand binding domain and transmembrane domain, and one receptor contains a protease cleavage site and a transcription factor and the other receptor contains a protease domain.

Claims

1. A composition comprising:

i) an exogenous extracellular sensor, and/or ii) a nucleic acid sequence encoding said exogenous extracellular sensor, wherein said exogenous extracellular sensor comprises;

a) a ligand binding domain,

b) a transmembrane domain,

c) a protease cleavage site, and

d) a functional domain.

2. The composition of claim 1, wherein said exogenous extracellular sensor further comprises a protease domain.

3. The composition of claim 1, wherein said exogenous extracellular sensor further comprises an extracellular spacer.

4. The composition of claim 1, wherein said exogenous extracellular sensor further comprises an intracellular spacer that is one, two, three, four, five, or six amino acids in length.

5. The composition of claim 1, wherein said functional domain is a transcription factor.

6. The composition of claim 5, further comprising a genetic construct, wherein said genetic construct is configured to express a gene in response to said transcription factor.

7. The composition of claim 6, wherein said gene is a reporter gene or a therapeutic gene.

8. The composition of claim 1, further comprising a cell, wherein said exogenous extracellular sensor is located in said cell.

9. The composition of claim 1, comprising said nucleic acid sequence.

10. The composition of claim 1, further comprising an additional exogenous extracellular sensor, wherein said additional exogenous extracellular sensor comprises:

a different ligand binding domain, a transmembrane domain, a protease cleavage site, and a different functional domain.

11. The composition of claim 10, further comprising a genetic construct, wherein said genetic construct is configured to express a reporter or therapeutic gene in response to said functional domain and said different functional domain.

12. The composition of claim 1, further comprising a third exogenous extracellular sensor, wherein third additional exogenous extracellular sensor comprises:

a third ligand binding domain, a transmembrane domain, a protease cleavage site, and a third functional domain.

13. The composition of claim 1, wherein said transmembrane domain is immediately adjacent to said protease cleavage site such that there is no intracellular spacer there between.

14. A composition comprising:

i) an exogenous extracellular sensor, and/or ii) a nucleic acid sequence encoding said exogenous extracellular sensor, wherein said exogenous extracellular sensor comprises;

a) a ligand binding domain,

b) a transmembrane domain,

c) a protease domain.

15. The composition of claim 14, wherein said protease domain comprises a protease that is at least partially auto-inhibited.

16. A composition comprising:

i) first and second exogenous extracellular sensors, and/or ii) one or more nucleic acid sequences encoding said first and second exogenous extracellular sensors, wherein said first exogenous extracellular sensor comprises;

a) a ligand binding domain,

b) a transmembrane domain,

c) a protease cleavage site, and

d) a functional domain;

and wherein said second exogenous extracellular sensor comprises;

e) a ligand binding domain,

f) a transmembrane domain, and

g) a protease domain.

17. The composition of claim 16, further comprising third and fourth exogenous extracellular sensors.

18. The composition of claim 16, further comprising a cell, wherein said first and second exogenous extracellular sensors are located in said cell.

19-22. -22. (canceled)