Light-activated chimeric opsins and methods of using the same

Patent metadata

Field	Value
Publication number	US-2016237126-A1
Application number	US-201615147772-A
Country	US
Kind code	A1
Filing date	May 5, 2016
Priority date	Nov 5, 2010
Publication date	Aug 18, 2016
Grant date	—

Abstract

Provided herein are compositions comprising light-activated chimeric proteins expressed on plasma membranes and methods of using the same to selectively depolarize excitatory or inhibitory neurons.

First claim

What is claimed is:

1 . An animal cell comprising a light-activated protein expressed on the cell membrane, wherein the protein
is a chimeric protein derived from VChR1 from Volvox carteri and ChR1 from Chlamydomonas reinhardti , wherein the protein comprises the amino acid sequence of VChR1 having at least the first and second transmembrane helices replaced by the first and second transmembrane helices of ChR1; is responsive to light; and is capable of mediating a depolarizing current in the cell when the cell is illuminated. with light.

2 . The animal cell of claim 1 , wherein the protein further comprises a replacement within the intracellular loop domain located between the second and third transmembrane helices of the chimeric light responsive protein, wherein at least a portion of the intracellular loop domain is replaced by the corresponding portion from the ChR1.

3 . The animal cell of claim 2 , wherein the portion of the intracellular loop domain is replaced with the corresponding portion from the ChR1 extending to amino acid residue A145 of the ChR1.

4 . The animal cell of claim 2 , wherein the protein further comprises a replacement within the third transmembrane helix of the chimeric light responsive protein, Wherein at least a portion of the third transmembrane helix is replaced by the corresponding sequence of ChR1.

5 . The animal cell of claim 4 , wherein the portion of the intracellular loop domain is replaced with the corresponding portion from the Chill extending to amino acid residue W163 of the ChR1.

6 . The animal cell of claim 1 , wherein the chimeric protein comprises an amino acid sequence at least 95% identical to the amino acid sequence shown in SEQ ID NO:1.

7 . The animal cell of claim 6 , further comprising a mutation at amino acid residue(s) E122 and/or E162 of SEQ ID NO:1.

8 . The animal cell of claim 7 , wherein the mutation at amino acid E122 is to threonine.

9 . The animal cell of claim 7 , wherein the mutation at amino acid E162 is to threonine.

10 . The animal cell of claim 1 , wherein. the chimeric protein comprises the amino acid sequence of SEQ ID NO:3.

11 . The animal cell of claim 1 , wherein the chimeric protein comprises the amino acid sequence of SEQ NO:1.

12 . The animal cell of claim 1 , wherein the chimeric protein comprises the amino acid sequence of SEQ ID NO:7.

13 . The animal cell of any one of claims 1 - 12 , wherein the protein further comprises a C-terminal membrane trafficking signal.

14 . The animal cell of claim 13 , wherein the C-terminal membrane trafficking signal comprises the amino acid sequence KSRITSEGEYIPLDQIDINV.

15 . The animal cell of claim 13 or 14 , wherein the membrane trafficking signal is linked to the C-terminus of the protein by a linker.

16 . The animal cell of any one of claims 1 - 15 , wherein the protein further comprises a C-terminal fluorescent protein selected from the group consisting of EYFP, GFP, CEP, and RFP.

17 . The animal cell of any one of claims 1 - 16 , wherein the animal cell is selected from the group consisting of: a neuronal cell, a muscle cell, and a stein cell.

18 . The animal cell of any one of claims 1 - 17 , further comprising a second light-activated protein expressed on the cell membrane.

19 . The animal cell of claim 18 , wherein the second light-activated protein is a protein capable of mediating a hyperpolarizing current in the cell when the cell is illuminated with light.

20 . The animal cell of claim 19 , wherein the second light-activated protein is selected from the group consisting of: an NpHr, an eNpHr2.0, an eNpHr3.0, an eNpHr3.1 and a GtR3.

21 . A population of cells comprising the cell of any one of claims 1 - 20 .

22 . A non-human animal comprising a cell of any one of claims 1 - 20 .

23 . A brain tissue slice comprising a cell of any one of claims 1 - 20 .

24 . An isolated polynucleotide comprising a nucleotide sequence encoding a light activated protein expressed on the cell membrane, wherein the protein is a chimeric protein derived from VChR1 from Volvox carteri and ChR1 from Chlamydomonas reinhardti, wherein the protein comprises the amino acid sequence of VChR1 having at least the first and second transmembrane helices replaced by the first and second transmembrane helices of ChR1; is responsive to light; and is capable of mediating a depolarizing current in the cell when the cell is illuminated with light.

25 . The isolated polynucleotide of claim 24 , wherein the polynucleotide comprises a sequence at least 95% identical to a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:1.

26 . The isolated polynucleotide of claim 24 , wherein the polynucleotide comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:3.

27 . The isolated polynucleotide of claim 24 , wherein the polynucleotide comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:5.

28 . The isolated polynucleotide of claim 24 , wherein the polynucleotide comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:7.

29 . An expression vector comprising any of the polynucleotide of claims 25 - 28 .

30 . The expression vector of claim 29 , wherein the expression vector is a viral vector.

31 . The expression vector of claim 30 , wherein the viral vector is selected from the group consisting of: an AAV vector, a retroviral vector, an adenoviral vector, a HSV vector, and a lentiviral vector.

32 . A method of using the cell of any one of claims 1 - 20 , comprising activating the light-activated protein with light.

33 . A method of using the cell of claim 32 , comprising activating the light-activated protein with red light.

34 . A method of using the cell of claim 32 , comprising activating the light-activated protein with green light.

35 . A method of selectively depolarizing excitatory or inhibitory neurons residing in the same microcircuit, the method comprising: selectively depolarizing an excitatory neuron comprising a first light-activated protein, wherein the first light activated protein is depolarized when exposed to light having a first wavelength; or selectively depolarizing an inhibitory neuron comprising a second light-activated protein, wherein the second light activated protein is depolarized when exposed to light having a second wavelength:, wherein the first or the second light activated protein is a chimeric protein derived from VChR1 from Volvox carteri and ChR1 from Chlamydomonas reinhardti , wherein the protein comprises the amino acid sequence of VChR1 having at least the first and second transmembrane helices replaced by the first and second transmembrane helices of ChR1.

36 . A method of selectively depolarizing excitatory or inhibitory neurons residing in the same microcircuit, the method comprising: expressing a first light-activated protein in an excitatory neuron; and expressing a second light activated protein in an inhibitory neuron, wherein the first light activated protein is independently depolarized when exposed to light having a first wavelength and wherein the second light activated protein is independently depolarized when exposed to light hav

Assignees

• Univ Leland Stanford Junior

Inventors

• Deisseroth Karl
• Yizhar Ofer
• Fenno Lief
• Hegemann Peter
• Prigge Matthias

Classifications

• A61P25/18

  Antipsychotics, i.e. neuroleptics; Drugs for mania or schizophrenia · CPC title

• A61P25/28

  for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia · CPC title

• A61P25/00

  Drugs for disorders of the nervous system · CPC title

• C07K14/405 Primary

  from algae · CPC title

• G01N33/5058

  Neurological cells · CPC title