Human cellular models with biosensors

• US 20160178635A1
• Filed: 02/26/2016
• Published: 06/23/2016
• Est. Priority Date: 09/05/2013
• Status: Active Grant

• Current Assignee: Tempo Biosciences Inc.
• Original Assignee: Tempo Biosciences Inc.
• Inventors: Huang, Angela
• Granted Patent: US 9,442,118 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C07K 14/00 Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
• CPC Class Codes: C07K 2319/03 containing a transmembrane segment
• CPC Class Codes: C07K 2319/04 containing an ER retention signal such as a C-terminal HDEL motif
• CPC Class Codes: C07K 2319/06 containing a lysosomal/endosomal localisation signal
• CPC Class Codes: C07K 2319/09 containing a nuclear localisation signal
• CPC Class Codes: C07K 2319/60 containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
• CPC Class Codes: C12N 9/16 acting on ester bonds (3.1)
• CPC Class Codes: G01N 2400/00 Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
• CPC Class Codes: G01N 2500/04 Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
• CPC Class Codes: G01N 2500/10 involving cells
• CPC Class Codes: G01N 33/5091 for testing the pathological state of an organism
• CPC Class Codes: G01N 33/5308 for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: G01N 33/5735 co-enzymes or co-factors, e.g. NAD, ATP
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: G01N 33/72 involving blood pigments, e.g. haemoglobin, bilirubin or other porphyrins; involving occult blood
• CPC Class Codes: G01N 33/721 Haemoglobin

Abstract

This invention provides biosensors, cell models, and methods of their use for monitoring heme, oxygen or ATP. Biosensors can include targeting domains, sensing domains and reporting domains. Biosensors can be introduced into cells reprogrammed to represent experimental or pathologic cells of interest. Model cells expressing the biosensors can be contacted with putative bioactive agents to determine possible activities.

Claims

1. An oxygen sensor peptide construct comprising:

A binding domain which binds to heme; and

,

a fluorescent reporter domain, wherein the fluorescent reporter is adapted to fluoresce at wavelengths in the range from 500 nm to 1400 nm;

wherein the fluorescent reporter domain is adapted to change fluorescent emissions characteristics in response to binding of the heme-binding domain to heme.

2. The sensor of claim 1 wherein the binding domain binds to amino acid residues 1-205 of heme.

3. The sensor of claim 1 wherein the binding domain comprises a sequence at least 80% identical to:

4. The sensor of claim 1 wherein the fluorescent reporter fluoresces in the 605 nm to 635 nm (excitation/emission) range.

5. The sensor of claim 1 wherein the fluorescent reporter has the sequence of Seq. Id. No. 2 or 6.

6. A nucleic acid construct encoding the sensor of claim 1.

7. The nucleic acid construct of claim 6, further comprising a tag sequence selected from the group consisting of:

a NLS tag, a lipid membrane tag, an ER tag, a golgi tag, an endosome tag, a mitochondrial tag, a synapsin tag, a cellular compartmental tag, a synaptic vesicle tag, and a ciliary tag.

8. A cell transformed with the nucleic acid construct of claim 6.

9. The cell of claim 8 which is a human cell.

10. The cell of claim 9 which is an iPSC derived cell, or is derived from a fibroblast or a blood cell.

20. A vector including the construct of claim 6.

11. An ATP sensor peptide construct comprising:

a binding domain which binds to ATP; and

,

a fluorescent reporter domain, wherein the fluorescent reporter is adapted to fluoresce at wavelengths in the range from 500 nm to 1400 nm;

wherein the fluorescent reporter domain is adapted to change fluorescent emissions characteristics in response to binding of the binding domain to ATP.

12. The sensor of claim 11 wherein the binding domain comprises a sequence at least 80% identical to:

13. The sensor of claim 11 wherein the fluorescent reporter fluoresces in the 605 nm to 635 nm (excitation/emission) range.

14. The sensor of claim 11 wherein the fluorescent reporter has the sequence of SEQ. ID. No. 2 or 6.

15. A nucleic acid construct encoding the sensor of claim 11.

16. The nucleic acid construct of claim 15, further comprising a tag sequence selected from the group consisting of:

a NLS tag, a lipid membrane tag, an ER tag, a golgi tag, an endosome tag, a mitochondrial tag, a synapsin tag, a cellular compartmental tag, a synaptic vesicle tag, and a ciliary tag.

17. A cell transformed with the nucleic acid construct of claim 15.

18. The cell of claim 17 which is a human cell.

19. The cell of claim 18 which is an iPSC derived cell, or is derived from a fibroblast or a blood cell.