Diagnostic methods for neural disorders

• US 10,107,796 B2
• Filed: 01/04/2017
• Issued: 10/23/2018
• 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
• Primary Examiner(s): Shen, Bin
• Application Number:
  US15/398,130
• Publication Number:
  US 20170115279A1
• Time in Patent Office: 657 Days
• 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 characterizing a neurological condition, the method comprising:

obtaining a human-derived cell having a mutation associated with a condition;

expressing an exogenous optical reporter and an exogenous optical voltage actuator in the cell;

illuminating the optical voltage actuator expressed by the cell or a neighboring cell, wherein the optical voltage actuator initiates an action potential in response to the illumination as an optical stimulation;

detecting an optical signature generated by the optical reporter in response to the optical stimulation of the cell; and

comparing the detected signature to an expected signature detected from control cell, wherein a difference between the observed signature and the expected signature provides information about the condition.

2. The method of claim 1, wherein the optical reporter of electrical activity is an exogenous fluorescent protein.

3. The method of claim 2, wherein the protein comprises at least a portion of a microbial rhodopsin.

4. The method of claim 3, wherein the protein includes the microbial rhodopsin operably linked to at least one additional fluorescent protein or chromophore.

5. The method of claim 1, further comprising editing a genome of the cell to include a mutation relative to a control genome.

6. The method of claim 1, further comprising obtaining the expected signature by obtaining a control cell suspected of not having the condition and observing a control signature generated by a control optical reporter in the control cell.

7. The method of claim 1, wherein obtaining the cell comprises differentiating a stem cell into a neuron or converting a somatic cell to a neuron.

8. The method of claim 7, wherein converting the somatic cell into the neuron comprises one selected from the list consisting of:

direct lineage conversion; and

conversion through an iPS intermediary.

9. The method of claim 1, 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, spinal and bulbar muscular atrophy, and amyotrophic lateral sclerosis.

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

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

obtaining a cell from a patient suspected of having the condition;

expressing an exogenous optical reporter and an exogenous optical voltage actuator in the cell;

illuminating the optical voltage actuator expressed by the cell or a neighboring cell, wherein the optical voltage actuator initiates an action potential in response to the illumination as an optical stimulation;

detecting an optical signature from the optical reporter in response to the optical stimulation of the cell; and

comparing the detected signature to an expected signature detected from control cell, wherein a difference between the observed signature and the expected signature corresponds to a diagnosis of the condition; and

generating a report that includes the diagnosis for the patient.

12. The method of claim 11, further comprising editing a genome of the cell to include a mutation relative to a control genome.

13. The method of claim 11, further comprising obtaining the expected signature by obtaining a control cell suspected of not having the condition and observing a control signature generated by a control optical reporter in the control cell.

14. The method of claim 13, wherein the control cell and the cell are isogenic but for a mutation.

15. The method of claim 11, wherein obtaining the cell comprises one selected from the group consisting of:

differentiating a stem cell into a neuron; and

direct lineage conversion of a somatic cell into a neuron.

16. The method of claim 11, wherein detecting an optical signature comprises detecting a cluster of different cells with a microscope and using a computer to isolate the signature generated by the optical reporter from a plurality of signature from the different cells.

17. The method of claim 11, 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, bulbar muscular atrophy, and amyotrophic lateral sclerosis.

18. The method of claim 11, wherein the difference between the observed signature and the expected signature comprises a modified probability of a voltage spike in response to the stimulation of the cell relative to a control.