# US 10,478,499 B2 · Engineered light-activated anion channel proteins and methods of use thereof

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- Number: US 10,478,499 B2
- Document ID: US10478499B2
- Status: Granted
- Kind: B2
- Application number: 16/057,639
- Filed: 2018-08-07
- Published: 2018-12-06
- Granted: 2019-11-19
- Earliest claimed: 2014-03-28
- Assignees: The Board of Trustees of the Leland Stanford Junior University
- Applicants: The Board of Trustees of the Leland Stanford Junior University
- Inventors: Karl A. Deisseroth; Soo Yeun Lee; Charu Ramakrishnan; Andre Berndt
- Web page: https://patentgrep.com/patent/US10478499B2

## CPC

- A61K 41/0042: {Photocleavage of drugs in vivo, e.g. cleavage of photolabile linkers in vivo by UV radiation for releasing the pharmacologically-active agent from the administered agent; photothrombosis or photoocclusion}
- A61N 5/0622: {Optical stimulation for exciting neural tissue}
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C07K 14/405: from algae
- C07K 14/47: from mammals
- A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00)
- C07K 2319/01: containing a localisation/targetting motif
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61N: ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY (measurement of bioelectric currents A61B; surgical instruments, devices or methods for transferring non-mechanical forms of energy to or from the body A61B18/00; anaesthetic apparatus in general A61M; incandescent lamps H01K; infrared radiators for heating H05B)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)

## Abstract

Aspects of the disclosure include compositions, devices, systems and methods for optogenetic modulation of action potentials in target cells. The subject devices include light-generating devices, control devices, and delivery devices for delivering light-responsive polypeptides, or nucleic acids encoding same, to target cells. The subject compositions and systems include light-activated polypeptides, nucleic acids comprising nucleotide sequences encoding these polypeptides, as well as expression systems that facilitate expression of these polypeptides in target cells. Also provided are methods of using the subject devices and systems to optogenetically manipulate action potentials in target cells, e.g., to treat a neurological or psychiatric condition in a human or animal subject.

## Claims

### Claim 1

A light-activated polypeptide comprising an amino acid sequence that is at least 95% identical to SEQ ID NO:23, wherein the polypeptide functions as a light-activated anion channel, and wherein the polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions selected from A59S, E90S, E101S, E123S, Q117K, H134R, V242K, T246N and N258Q, relative to the amino acid sequence of ChR2 (SEQ ID NO:79).

### Claim 2 (depends on 1)

The polypeptide of claim 1, wherein the polypeptide comprises an amino acid sequence having at least 98% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:23.

### Claim 3 (depends on 1)

The polypeptide of claim 1, comprising an endoplasmic reticulum (ER) export polypeptide.

### Claim 4 (depends on 3)

The polypeptide of claim 3, wherein the ER export polypeptide comprises the amino acid sequence FXYENE (SEQ ID NO:92), where X is any amino acid.

### Claim 5 (depends on 1)

The polypeptide of claim 1, comprising a membrane trafficking polypeptide.

### Claim 6 (depends on 5)

The polypeptide of claim 5, wherein the membrane trafficking polypeptide comprises the amino acid sequence KSRITSEGEYIPLDQIDINV (SEQ ID NO:83).

### Claim 7 (depends on 5)

The light-activated polypeptide of claim 5, wherein the polypeptide further comprises a C128T, C128A, or C128S substitution, relative to the amino acid sequence of ChR2 (SEQ ID NO:79).

### Claim 8 (depends on 1)

The light-activated polypeptide of claim 1, wherein the polypeptide further comprises a D156A or a D156N substitution, relative to the amino acid sequence of ChR2 (SEQ ID NO:79).

### Claim 9

A nucleic acid comprising a nucleotide sequence encoding a light-activated polypeptide comprising an amino acid sequence that is at least 95% identical to SEQ ID NO:23, wherein the polypeptide functions as a light-activated anion channel, and wherein the polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions selected from A59S, E90S, E101S, E123S, Q117K, H134R, V242K, T246N and N258Q, relative to the amino acid sequence of ChR2 (SEQ ID NO:79).

### Claim 10 (depends on 9)

A recombinant expression vector comprising the nucleic acid of claim 9.

### Claim 11 (depends on 10)

A pharmaceutical composition comprising:
a) the recombinant expression vector of claim 10; and
b) a pharmaceutically acceptable carrier.

### Claim 12 (depends on 9)

A cell comprising the nucleic acid of claim 9.

### Claim 13 (depends on 10)

A cell comprising the recombinant expression vector of claim 10.

### Claim 14 (depends on 10)

A system for modulating the membrane potential of a cell, the system comprising:
a) the recombinant vector of claim 10; and
b) a device configured to illuminate a target location with light.

### Claim 15 (depends on 14)

The system of claim 14, wherein the device is configured to illuminate the target location with light having a wavelength ranging from about 350 nm to about 750 nm, or from about 450 nm to about 500 nm.

### Claim 16 (depends on 14)

The system of claim 14, wherein the device is configured to:
a) constantly illuminate the target location with light;
b) illuminate the target location with pulses of light;
c) modulate the wavelength and/or the intensity of the light;
d) modulate the frequency and/or the duration of the pulses of light; or
e) illuminate the target location in response to a user input.

### Claim 17 (depends on 14)

The system of claim 14, wherein the device is configured to illuminate the target location in response to a user input, and wherein the user input comprises: the wavelength of light, the intensity of light, the duration of a light pulse, the frequency of a light pulse, and/or the target location.

### Claim 18 (depends on 14)

The system of claim 14, wherein the device is adapted to be implanted in a subject.

### Claim 19 (depends on 14)

The system of claim 14, wherein the target location is: a cell, a portion of a cell, a plurality of cells, a bundle of nerve fibers, a neuromuscular junction, a central nervous system (CNS) tissue, a peripheral nervous system (PNS) tissue, or an anatomical region.

### Claim 20 (depends on 10)

A method for modulating the membrane potential of a cell in response to light, the method comprising exposing a cell to light of an activating wavelength, wherein the cell is genetically modified with the recombinant vector of claim 10.

### Claim 21 (depends on 20)

The method of claim 20, wherein the cell is a neuron.