DIAGNOSTIC METHODS FOR NEURAL DISORDERS

• US 20150301029A1
• Filed: 04/21/2015
• Published: 10/22/2015
• Est. Priority Date: 04/22/2014
• Status: Active Grant

• Current Assignee: Quelltx Incorporated
• Original Assignee: Q-State Biosciences, Inc.
• Inventors: Eggan, Kevin C., Cohen, Adam, Kralj, Joel, Kiskinis, Evangelos
• Granted Patent: US 9,594,075 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12N 2502/081 neurons
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 5/0619 Neurons
• CPC Class Codes: G01N 21/6486 Measuring fluorescence of biological material, e.g. DNA, RNA, cells G01N21/6428 takes precedence
• CPC Class Codes: G01N 2333/705 Assays involving receptors, cell surface antigens or cell surface determinants
• CPC Class Codes: G01N 2800/2835 Movement disorders, e.g. Parkinson, Huntington, Tourette
• CPC Class Codes: G01N 2800/2842 Pain, e.g. neuropathic pain, psychogenic pain
• CPC Class Codes: G01N 2800/302 Schizophrenia
• CPC Class Codes: G01N 33/48728 Investigating individual cells, e.g. by patch clamp, voltage clamp investigating individual particles in general G01N15/10
• CPC Class Codes: G01N 33/502 for testing non-proliferative effects
• CPC Class Codes: G01N 33/5023 on expression patterns
• CPC Class Codes: G01N 33/5058 Neurological cells
• CPC Class Codes: G01N 33/5091 for testing the pathological state of an organism
• CPC Class Codes: G01N 33/6872 Intracellular protein regulatory factors and their receptors, e.g. including ion channels

Abstract

The invention generally relates to optical methods for the diagnosis of neuronal condition by converting a cell from a patient into a neuron and optically evaluating action potentials of that cell in vitro. The cell is transformed with an optical reporter and exhibits an optical signature in response to neural stimulation. Using genome-editing, a control cell can be made that is isogenic but-for a known mutation and a control signature obtained from the control cell. Thus, methods of the invention reveal potential neurodegenerative effects of a mutation as manifested in a patient'"'"'s genetic context. The optical signature of the cell, or the difference between the signature and the control signature, is correlated to a diagnosis of the neurodegenerative disease

Claims

1. A method of diagnosing a condition, the method comprising:

obtaining an accessible cell from a person suspected of having the condition;

causing the cell to express an optical reporter of electrical activity;

observing a signature generated by the optical reporter in response to a stimulation of the cell; and

comparing the observed signature to an expected signature, wherein a difference between the observed signature and the expected signature corresponds to a positive diagnosis of the condition.

2. The method of claim 1, further comprising causing the cell to express an optical actuator that initiates an action potential in response to optical stimulation.

3. The method of claim 2, wherein the stimulation of the cell comprises illuminating the optical actuator.

4. The method of claim 1, wherein obtaining the cell comprises obtaining a somatic cell from the subject and converting the somatic cell into an electrically active cell type.

5. The method of claim 4, wherein the somatic cell is converted into a neuron.

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

direct lineage conversion; and

through an iPS intermediary.

7. The method of claim 4, further comprising obtaining the expected signal by obtaining a control cell suspected of not having the condition and observing a control signal generated by a control optical reporter in the control cell.

8. The method of claim 7, wherein obtaining the control cell comprises editing a genome from the subject such that the control cell and the cell are isogenic but for a mutation.

9. The method of claim 1, wherein causing the cell to express the optical reporter comprises transforming the cell with a vector bearing a genetically encoded fluorescent voltage reporter.

10. The method of claim 9, wherein the vector also comprises a genetically encoded optical voltage actuator.

11. The method of claim 1, wherein observing 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.

12. The method of claim 11, wherein the computer isolates the signal by performing an independent component analysis and identifying a spike train associated with the cell.

13. The method of claim 1, wherein the condition comprises a disease of the nervous system.

14. The method of claim 13, wherein the condition comprises a neurodegenerative disease.

15. The method of claim 13, wherein the condition comprises one selected from the list 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, and spinal and bulbar muscular atrophy.

16. The method of claim 13, wherein the condition comprises amyotrophic lateral sclerosis.

17. The method of claim 1, wherein the subject has a mutation in a SOD1 gene.

18. The method of claim 17, wherein the mutation is SOD1A4V.

19. The method of claim 1, wherein the difference between the observed signal and the expected signal comprises a modified probability of a voltage spike in response to the stimulation of the cell relative to a control.

20. The method of claim 1, wherein the difference between the observed signal and the expected signal comprises a decreased probability of a voltage spike in response to the stimulation of the cell relative to a control and an increased probability of a voltage spike during periods of no stimulation of the cell relative to a control

21. A method for determining an effect of a compound a neurological condition, the method comprising:

presenting a compound to a sample comprising a plurality of neurons, wherein at least one of the plurality of neurons expresses an optical reporter of membrane electrical potential;

receiving, via a microscopy system, an optical signal generated by the optical reporter in response to optical stimulation of a light gated ion channel in the sample following presentation of said compound; and

identifying the compound as a candidate for treatment of the neurological condition based on said optical signal.

22. The method of claim 21, wherein a plurality of samples are exposed to a plurality of different compounds.

23. The method of claim 21, wherein the light gated ion channel comprises an algal channelrhodopsin being expressed by a second neuron in synaptic communication with the at least one of the plurality of neurons, and the optical reporter of membrane potential comprises a microbial rhodopsin with between 1 and 10 amino acid substitutions relative to a wild type form of the microbial rhodopsin.

24. The method of claim 23, wherein the at least one of the plurality of neurons also expresses a genetically-encoded indicator of intracellular calcium level, and

wherein the receive optical signal includes a signal from the genetically-encoded indicator of intracellular calcium level, and further wherein the neurological condition is one of autism, epilepsy, Alzheimer'"'"'s, amyotrophic lateral sclerosis, and tuberous sclerosis.

25. The method of claim 21, wherein each of the plurality of neurons is caused to express both the optical reporter of membrane electrical potential and the light gated ion channel.

26. The method of claim 25, further comprising transforming the neurons with a vector that includes a nucleic acid encoding the optical reporter of membrane electrical potential and the light gated ion channel.

27. The method of claim 21, wherein the neurological condition 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 21, further comprising performing the steps on a control sample, wherein the neurons in the sample are isogenic with cells in the control sample but for a mutation.

29. The method of claim 21, wherein receiving the optical signal comprises observing a cluster of cells with a microscope and using a computer to isolate a signal generated by the optical reporter from among a plurality of signals from the cluster of cells.

30. The method of claim 29, wherein the computer isolates the signal by performing an independent component analysis and identifying a spike train produced by the neuron.

31. The method of claim 30, further comprising using the microscopy system to obtain an image of a plurality of clusters of cells.