Diagnostic methods for neural disorders

• US 9,594,075 B2
• Filed: 04/21/2015
• Issued: 03/14/2017
• 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:
  US14/692,242
• Publication Number:
  US 20150301029A1
• Time in Patent Office: 693 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 for determining an effect of a compound on a neurological condition, the method comprising:

presenting a compound selected for treating a neurological condition to a cell sample exhibiting a genotype associated with the neurological condition, the cell sample comprising a plurality of neurons, wherein at least one of the plurality of neurons expresses an optical reporter of membrane electrical potential;

optically stimulating an optogenetic actuator in the sample following presentation of the compound, wherein the optogenetic actuator is a light gated ion channel;

generating an optical signal in response to the optical stimulation of the light gated ion channel using the optical reporter;

receiving, via a microscopy system, the optical signal generated by the optical reporter;

analyzing the optical signal generated by the optical reporter with respect to a control signal obtained from a control cell sample not exhibiting a genotype associated with the neurological condition; and

identifying the compound as a candidate for treatment of the neurological condition based on the analysis of said optical signal with respect to the control signal.

2. The method of claim 1, wherein a plurality of samples are exposed to a plurality of different compounds.

3. The method of claim 1, 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.

4. The method of claim 3, 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 any one of autism, epilepsy, Alzheimer'"'"'s, amyotrophic lateral sclerosis, or tuberous sclerosis.

5. The method of claim 1, 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.

6. The method of claim 5, 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.

7. The method of claim 1, 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.

8. The method of claim 1, further comprising performing the presenting, optically stimulating, generating, and receiving steps on a control sample, wherein the neurons in the cell sample are isogenic with cells in the control sample but for a mutation.

9. The method of claim 1, 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.

10. The method of claim 9, wherein the computer isolates the signal by performing an independent component analysis and identifying a spike train produced by the neuron.

11. The method of claim 10, further comprising using the microscopy system to obtain an image of a plurality of clusters of cells.