Diagnostic methods for neural disorders

• US 10,613,079 B2
• Filed: 09/13/2018
• Issued: 04/07/2020
• 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:
  US16/130,048
• Publication Number:
  US 20190025291A1
• Time in Patent Office: 572 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 comprising:

a) editing, ex vivo, an endogenous human gene within a human cell;

b) introducing an optical reporter of membrane electrical potential into the cell;

c) stimulating the cell; and

d) receiving a signal from the optical reporter in the cell.

2. The method of claim 1, wherein the editing step comprises use of a Cas endonuclease and a guide RNA.

3. The method of claim 1, further comprising:

comparing the signal to a second signal obtained from a control cell that is isogenic to the genetically-edited cell but for a mutation in the endogenous gene introduced by the editing step.

4. The method of claim 1, wherein the editing step comprises use of a nuclease selected from the group consisting of a zinc finger nuclease, a transcription activator-like effector nuclease (TALEN), and a clustered regularly interspaced short palindromic repeat (CRISPR) nuclease.

5. The method of claim 1, wherein the optical reporter comprises a microbial rhodopsin protein.

6. The method of claim 5, wherein the introducing step comprises introducing a plasmid to the cell, wherein the plasmid includes a gene for the microbial rhodopsin protein.

7. The method of claim 1, wherein the gene is associated with a neurological disorder or condition.

8. The method of claim 7, wherein the neurological condition or disorder is epilepsy.

9. The method of claim 8, wherein the epilepsy is Dravet syndrome.

10. The method of claim 8, wherein the endogenous gene is selected from the group consisting of ARX, BRAT1, CARS2, CDKL5, GABRG2, KCNC1, KCNQ2, KCNQ3, NAV1.1, NIPA2, PCDH19, PRRT2, SCN1A, SCN2A, SCN2B, SCN9A, SPTAN, STX1B, and WWOX.

11. The method of claim 8, wherein the neurological condition or disorder is amyotrophic lateral sclerosis.

12. The method of claim 11, wherein the endogenous gene is selected from the group consisting of ALS2, C9orf72, FUS, SETX, SOD1, TARDBP, and UBQL2.

13. The method of claim 1, further comprising exposing the cell to a compound prior to stimulating the cell.

14. The method of claim 13, further comprising:

comparing the signal to a second signal obtained from a control cell that is isogenic to the genetically-edited cell but for a mutation in the endogenous gene.

15. The method of claim 14, wherein the cell is electrically active.

16. The method of claim 15, wherein the cell is a neuron.

17. The method of claim 16, wherein the gene is selected from the group consisting of ARX, BRAT1, CARS2, CDKL5, GABRG2, KCNC1, KCNQ2, KCNQ3, NAV1.1, NIPA2, PCDH19, PRRT2, SCN1A, SCN2A, SCN2B, SCN9A, SPTAN, STX1B, and WWOX.

18. The method of claim 16, wherein the neurological condition or disorder is amyotrophic lateral sclerosis.

19. The method of claim 18, wherein the endogenous gene is selected from the group consisting of ALS2, C9orf72, FUS, SETX, SOD1, TARDBP, and UBQL2.

20. The method of claim 13, wherein the endogenous gene is associated with a neurological disorder or condition.

21. The method of claim 20, wherein the neurological condition or disorder is epilepsy.

22. The method of claim 21, wherein the epilepsy is Dravet syndrome.