Systems, methods, and workflows for optogenetics analysis

• US 9,207,237 B2
• Filed: 06/12/2014
• Issued: 12/08/2015
• Est. Priority Date: 08/23/2010
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Cohen, Adam E., Kralj, Joel, Douglass, Adam D., Hochbaum, Daniel
• Primary Examiner(s): NAVARRO, ALBERT MARK
• Application Number:
  US14/303,178
• Publication Number:
  US 20140295413A1
• Time in Patent Office: 544 Days
• US Class Current: 1/1
• CPC Class Codes: A61K 49/0017 Fluorescence in vivo
• CPC Class Codes: A61K 49/0097 Cells, viruses, ghosts, red blood cells, viral vectors, used for imaging or diagnosis in vivo
• CPC Class Codes: G01N 2333/705 Assays involving receptors, cell surface antigens or cell surface determinants
• CPC Class Codes: G01N 33/54373 involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
• 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

Abstract

The invention provides methods for characterizing cellular physiology by incorporating into an electrically excitable cell an optical reporter of, and an optical actuator of, electrical activity. A signal is obtained from the optical reporter in response to a stimulation of the cell. Either or both of the optical reporter and actuator may be based on genetically-encoded rhodopsins incorporated into the cell. The invention provides all optical methods that may be used instead of, or as a complement to, traditional patch clamp technologies and that can provide rapid, accurate, and flexible assays of cellular physiology.

Claims

1. A method for characterizing a cell, the method comprising:

incorporating into an electrically excitable cell an optical actuator of, and an optical reporter of, electrical activity;

obtaining a signal from the optical reporter in response to a stimulation of the cell; and

evaluating the signal, thereby characterizing the cell.

2. The method of claim 1, wherein incorporating the actuator and reporter into the cell comprises transforming the electrically active cell with a vector that includes a nucleic acid encoding the optical actuator of, and the optical reporter of, electrical activity.

3. The method of claim 1, further comprising obtaining a somatic cell and converting the somatic cell into the electrically excitable cell.

4. The method of claim 3, wherein converting the somatic cell into the electrically active cell comprises one selected from the list consisting of:

direct conversion; and

via an iPS intermediary.

5. The method of claim 1 wherein the electrically excitable cell is derived from a human embryonic stem cell.

6. The method of claim 1, wherein the electrically excitable cell is one selected from the list consisting of a neuron, a cardiomyocyte, and a glial cell.

7. The method of claim 1, wherein the optical actuator initiates an action potential in response to the stimulation.

8. The method of claim 7, wherein the stimulation comprises illuminating the cell.

9. The method of claim 8, wherein illuminating the cell is done using spatially resolved light from a digital micromirror array.

10. The method of claim 8, wherein the excitation of, and the signal from, the optical reporter comprise light that does not stimulate the cell.

11. The method of claim 8, further comprising illuminating the cell and obtaining the signal simultaneously.

12. The method of claim 1, wherein the optical actuator comprises a modified rhodopsin.

13. The method of claim 1, wherein the optical actuator comprises CheRiff.

14. The method of claim 1, wherein the optical reporter comprises a rhodopsin that has been modified for voltage-sensitive fluorescence and absence of a steady-state photocurrent.

15. The method of claim 1, wherein the optical reporter comprises Arch D95N.

16. The method of claim 1, further comprising obtaining a control cell and observing a control signal generated by a control optical reporter in the control cell.

17. The method of claim 16, wherein obtaining the control cell comprises editing a genome from the cell such that the control cell and the cell are isogenic but for a mutation.

18. The method of claim 1, wherein obtaining the signal comprises observing a cluster of different cells with a microscope and using a computer to isolate the signal generated by the optical reporter from a plurality of signals from the different cells.

19. The method of claim 18, wherein the computer isolates the signal by performing an independent component analysis and identifying a spike train associated with the cell.

20. The method of claim 19, further comprising using the microscope to obtain an image of a plurality of clusters of cells.

21. The method of claim 1, wherein the observed signal comprises a probability of a voltage spike in response to the stimulation of the cell.

22. The method of claim 1, wherein the observed signal comprises a changed probability of a voltage spike in response to the stimulation of the cell relative to a control.

23. The method of claim 1, wherein the observed signal comprises a change in the waveform of a voltage spike.

24. The method of claim 1, wherein the observed signal comprises a sub-threshold increase in the membrane potential.

25. The method of claim 1, wherein the observed signal comprises a decrease in the membrane potential.

26. The method of claim 1, wherein characterizing the cell comprises diagnosing a disease.

27. The method of claim 26, wherein the disease is selected from the group consisting of Cockayne syndrome, Down Syndrome, Dravet syndrome, familial dysautonomia, Fragile X Syndrome, Friedreich'"'"'s ataxia, Gaucher disease, hereditary spastic paraplegias, Machado-Joseph disease, Phelan-McDermid syndrome (PMDS), polyglutamine (polyQ)-encoding CAG repeats, spinal muscular atrophy, Timothy syndrome, Alzheimer'"'"'s disease, frontotemporal lobar degeneration, Huntington'"'"'s disease, multiple sclerosis, Parkinson'"'"'s disease, spinal and bulbar muscular atrophy, and amyotrophic lateral sclerosis.

28. The method of claim 1, wherein characterizing the cell comprises evaluating a response of the cell to exposure to a compound.

29. The method of claim 1, wherein characterizing the cell further comprises measuring a concentration of an ion.

30. The method of claim 1, wherein characterizing the cell comprises determining progress of a treatment.

31. The method of claim 1, further comprising editing the genome of the electrically active cells.

32. A method for characterizing an interaction between cells, the method comprising:

incorporating into a first electrically excitable cell an optical actuator of electrical activity incorporating into a second electrically excitable cell an optical reporter of electrical activity;

culturing the first electrically excitable cell and the second electrically excitable cell in proximity to one another;

obtaining a signal from the optical reporter in response to a stimulation of the first electrically excitable cell; and

evaluating the signal, thereby characterizing an interaction between the first electrically excitable cell and the second electrically excitable cell.

33. The method of claim 32, wherein the first electrically excitable cell and the second electrically excitable cell are of the same cell type.

34. The method of claim 33, wherein the cell type is one selected from the list consisting of a neuron, a cardiomyocyte, and a glial cell.

35. The method of claim 32, wherein the first electrically excitable cell and the second electrically excitable cell are each of a different cell type.

36. The method of claim 32, wherein the characterized interaction comprises excitatory neurotransmission.

37. The method of claim 32, wherein the characterized interaction comprises inhibitory neurotransmission.

38. The method of claim 32, wherein characterizing the interaction comprises measurement of conduction velocity of cardiac action potential.

39. The method of claim 32, wherein incorporating the actuator into the first electrically excitable cell comprises transforming first electrically excitable cell with a vector that includes a nucleic acid encoding the optical actuator of electrical activity.

40. The method of claim 32, wherein incorporating the reporter into the second electrically excitable cell comprises transforming the second electrically excitable cell with a vector that includes a nucleic acid encoding the optical reporter of, electrical activity.

41. The method of claim 32, further comprising obtaining somatic cells and converting the somatic cells into the first electrically excitable cell and the second electrically excitable cell.

42. The method of claim 41, wherein converting the somatic cells comprises one selected from the list consisting of:

direct conversion; and

via an iPS intermediary.

43. The method of claim 32, wherein the first electrically excitable cell and the second electrically excitable cell are derived from a human embryonic stem cell.

44. The method of claim 32, wherein the optical actuator initiates an action potential in response to the stimulation.

45. The method of claim 44, wherein the stimulation comprises illuminating the first electrically excitable cell.

46. The method of claim 45, wherein the illuminating is done using spatially resolved light from a digital micromirror array.

47. The method of claim 45, wherein the excitation of, and the signal from, the optical reporter comprise light that does not stimulate the first electrically excitable cell.

48. The method of claim 47, wherein the illuminating and obtaining the signal are done simultaneously.

49. The method of claim 32, wherein the optical actuator comprises a modified rhodopsin.

50. The method of claim 32, wherein the optical actuator comprises CheRiff.

51. The method of claim 32, wherein the optical reporter comprises a rhodopsin that has been modified for voltage-sensitive fluorescence and absence of a steady-state photocurrent.

52. The method of claim 32, wherein the optical reporter comprises Arch D95N.

53. The method of claim 32, wherein obtaining the signal comprises observing a cluster of different cells with a microscope and using a computer to isolate the signal generated by the optical reporter from a plurality of signals from the different cells.

54. The method of claim 53, wherein the computer isolates the signal by performing an independent component analysis and identifying a spike train associated with the second electrically excitable cell.

55. The method of claim 54, further comprising using the microscope to obtain an image of a plurality of clusters of cells.

56. The method of claim 32, wherein the observed signal comprises a probability of a voltage spike in response to the stimulation of the cell.

57. The method of claim 32, wherein the observed signal comprises a changed probability of a voltage spike in response to the stimulation of the cell relative to a control.

58. The method of claim 32, wherein the observed signal comprises a change in the waveform of a voltage spike.

59. The method of claim 32, wherein the observed signal comprises a sub-threshold increase in the membrane potential.

60. The method of claim 32, wherein the observed signal comprises a decrease in the membrane potential.

61. The method of claim 32, wherein characterizing the interaction comprises diagnosing a disease.

62. The method of claim 32, wherein characterizing the interaction comprises evaluating a cellular response to exposure to a compound.

63. The method of claim 32, wherein characterizing the interaction comprises determining progress of a treatment.

64. The method of claim 32, further comprising editing the genome of the electrically active cells.