BIOSENSORS FOR CHLORIDE IONS

• US 20190112597A1
• Filed: 04/10/2018
• Published: 04/18/2019
• Est. Priority Date: 04/12/2017
• Status: Active Grant

• Current Assignee: Tempo Biosciences Inc.
• Original Assignee: Tempo Biosciences Inc.
• Inventors: Huang, Angela
• Granted Patent: US 10,640,766 B2
• Time in Patent Office: Days
• CPC Class Codes: C07K 14/705 Receptors; Cell surface antigens; Cell surface determinants tumour specific antigens C07K14/4748
• CPC Class Codes: C07K 2319/03 containing a transmembrane segment
• CPC Class Codes: C07K 2319/033 containing a motif for targeting to the internal surface of the plasma membrane, e.g. containing a myristoylation motif
• CPC Class Codes: C07K 2319/05 containing a GOLGI retention signal
• CPC Class Codes: C07K 2319/60 containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
• CPC Class Codes: C12N 15/1086 Preparation or screening of expression libraries, e.g. reporter assays
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 2015/859 Animal models comprising reporter system for screening tests
• CPC Class Codes: C12N 5/0606 Pluripotent embryonic cells, e.g. embryonic stem cells [ES] embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696
• CPC Class Codes: C12N 5/0647 Haematopoietic stem cells; Uncommitted or multipotent progenitors
• CPC Class Codes: C12N 5/0656 Adult fibroblasts
• CPC Class Codes: C12N 5/0696 Artificially induced pluripotent stem cells, e.g. iPS
• CPC Class Codes: G01N 2333/705 Assays involving receptors, cell surface antigens or cell surface determinants
• CPC Class Codes: G01N 33/52 Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements immunological elements G01N33/54386; involving labelled immunochemicals G01N33/58; for haemoglobin or occult blood G01N33/72
• CPC Class Codes: G01N 33/68 involving proteins, peptides or amino acids involving lipoproteins G01N33/92
• CPC Class Codes: G01N 33/84 involving inorganic compounds or pH

Abstract

This invention provides biosensors, cell models, and methods of their use for monitoring chloride ion, where the 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, including as detectors of chloride ions, as TempoChloro™ accomplishes.

Claims

1. A chloride ion sensor peptide construct comprising:

a chloride ion binding domain having a sequence at least 70% identical to SEQ ID NO;

1 which binds to chloride ion; 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 chloride ion binding domain binding to chloride ion.

2. The sensor of claim 1 wherein the fluorescent reporter has a fluorescent emission range of 500 nm-700 nm and a fluorescent excitation range of 400 nm-700 nm.

3. The sensor of claim 1 wherein the fluorescent reporter has a sequence at least 70% identical to SEQ ID NO:

3.

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

5. The nucleic acid construct of claim 4 including SEQ ID NO:

2, and SEQ ID NO;

4.

6. The nucleic acid construct of claim 4, 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.

7. A cell transformed with the nucleic acid construct of claim 4.

8. The cell of claim 7 which is a human cell.

9. The cell of claim 7 which is a companion animal, livestock or other mammalian cell.

10. The cell of claim 7 which is an iPSC derived cell, or is derived from a fibroblast or a blood cell.

11. The cell of claim 7 which is an astrocyte.

12. A process of making a cell transformed with the nucleic acid construct of claim 4, comprising:

transfecting the nucleic acid construct of claim 4 into a eukaryotic cell; and

expressing the transfected nucleic acid construct by the cell.

13. The process of claim 11 wherein the cell is a human cell.

14. The process of claim 11 wherein the cell is an iPSC derived cell, or is derived from a fibroblast or a blood cell.

15. The process of claim 13 wherein the cell is an astrocyte.

16. A process of making a cell acting as a chloride ion biosensor, comprising:

transforming the cell with a first nucleic acid sequence encoding a peptide segment at least 70% identical to SEQ ID NO;

1; and

transforming the cell with a second nucleic acid sequence encoding a fluorescent reporter domain.

17. The process of claim 16 wherein the fluorescent reporter sequence is at least 70% identical to the sequence of SEQ ID NO:

3.

18. The process of claim 16 wherein the first nucleic acid sequence is SEQ ID NO:

2, and the second nucleic acid sequence is SEQ ID NO;

4.

19. The process of claim 16 wherein the cell is a human cell.

20. The process of claim 16 wherein the cell is a companion animal, livestock or other mammalian cell.

21. The process of claim 16 wherein the cell is an iPSC derived cell, or is derived from a fibroblast or a blood cell.

22. The process of claim 21 wherein the cell is an astrocyte.

23. The process of claim 16 further including the step of transforming the cell with a nucleic acid sequence encoding a tag sequence.

24. The process of claim 23 wherein the tag sequence has the sequence of one of SEQ ID NO:

5 to SEQ ID NO;

11.