Cell line, system and method for optical control of secondary messengers

• US 8,729,040 B2
• Filed: 05/29/2009
• Issued: 05/20/2014
• Est. Priority Date: 05/29/2008
• 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: Deisseroth, Karl, Airan, Raag D.
• Primary Examiner(s): Long, Scott
• Assistant Examiner(s): Leonard, Arthur S
• Application Number:
  US12/993,605
• Publication Number:
  US 20110112179A1
• Time in Patent Office: 1,817 Days
• US Class Current: 514/44.R
• CPC Class Codes: A61B 5/0059 using light, e.g. diagnosis by transillumination, diascopy, fluorescence A61B5/0093, A61B5/1455, A61B5/02416 and A61B5/0261 take precedence
• CPC Class Codes: A61B 5/4848 Monitoring or testing the effects of treatment, e.g. of medication
• CPC Class Codes: A61K 41/00 Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
• CPC Class Codes: A61K 48/0058 Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct
• CPC Class Codes: A61K 49/00 Preparations for testing in vivo
• CPC Class Codes: A61N 5/062 Photodynamic therapy, i.e. excitation of an agent
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07K 14/705 Receptors; Cell surface antigens; Cell surface determinants tumour specific antigens C07K14/4748
• CPC Class Codes: C07K 14/70571 for neuromediators, e.g. serotonin receptor, dopamine receptor
• CPC Class Codes: C07K 14/723 G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C12N 13/00 Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 5/0619 Neurons
• CPC Class Codes: G01N 2333/726 G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH
• CPC Class Codes: G01N 33/5035 on sub-cellular localization

Abstract

A variety of methods, devices and compositions are implemented for light-activated molecules. One such method is implemented for generating secondary messengers in a cell. A nucleotide sequence for expressing a chimeric light responsive membrane protein (e.g., rhodopsin) is modified with one or more heterologous receptor subunits {e.g., an adrenergic receptor (alpha1, Beta2)}. The light responsive membrane protein is expressed in a cell for producing a secondary messenger in response to light.

Claims

1. A method for generating secondary messengers in a cell, the method comprising:

expressing in the cell a chimeric light-responsive fusion protein comprising a light-responsive rhodopsin-based membrane protein and a heterologous alpha-1 adrenergic receptor, wherein said expression provides for production of a secondary messenger in response to light, and wherein the chimeric light-responsive fusion protein comprises an amino acid sequence having at least 85% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO;

4;

wherein the cell expresses a secondary messenger-targeted cation channel that is responsive to the secondary messenger; and

visualizing resultant activity using a cation-sensitive dye.

2. The method of claim 1, wherein the secondary messenger is inositol trisphosphate/inositol 1,4,5-trisphosphate/triphosphoinositol (IP₃).

3. The method of claim 1, wherein the step of expressing is accomplished in vivo.

4. The method of claim 1, wherein the step of expressing is accomplished in vitro.

5. The method of claim 1, further including the step of optically stimulating the expressed light responsive membrane protein.

7. The method of claim 1, wherein the amino acid sequence has at least 90% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:

4.

8. The method of claim 1, wherein the chimeric light-responsive fusion protein is encoded by a nucleotide sequence that is operably linked to a cell type-specific promoter.

9. The method of claim 1, wherein the cell is a mammalian cell.

10. The method of claim 1, wherein the cell is a neuron.

11. The method of claim 8, wherein the cell type-specific promoter is a neuron-specific promoter.

12. The method of claim 10, wherein the promoter is a synapsin-1 promoter.

13. The method of claim 5, wherein the step of optically stimulating the expressed light responsive chimeric membrane protein comprises applying light through an optical fiber.

14. The method of claim 5, wherein the step of optically stimulating the expressed light responsive chimeric membrane protein comprises applying a pulsatile optical stimulus.

15. The method of claim 1, wherein the amino acid sequence has at least 95% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:

4.

6. A method for assessing the efficacy of a treatment regimen relating to intracellular messengers, the method comprising:

expressing a chimeric light-responsive fusion protein comprising a light-responsive rhodopsin-based membrane protein and a heterologous alpha-1 adrenergic receptor in a mammalian cell, wherein said expression provides for production of a secondary messenger in response to light, and wherein the chimeric light-responsive fusion protein comprises an amino acid sequence having at least 85% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO;

4;

wherein the cell expresses a secondary messenger-targeted cation channel that is responsive to the secondary messenger;

exposing the chimeric light-responsive fusion protein to light; and

assessing the effects of the treatment by visualizing resultant activity using a cation-sensitive dye.

16. The method of claim 6, wherein the secondary messenger is inositol trisphosphate/inositol 1,4,5-trisphosphate/triphosphoinositol (IP₃).

17. The method of claim 6, wherein the amino acid sequence has at least 90% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:

4.

18. The method of claim 6, wherein the amino acid sequence has at least 95% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:

4.

19. The method of claim 6, wherein the chimeric light-responsive fusion protein is encoded by a nucleotide sequence that is operably linked to a cell type-specific promoter.

20. The method of claim 6, wherein the cell is a neuron.

21. The method of claim 19, wherein the cell type-specific promoter is a neuron-specific promoter.

22. The method of claim 21, wherein the promoter is a synapsin-1 promoter.