Opsin polypeptides and methods of use thereof

Patent metadata

Field	Value
Publication number	US-9505817-B2
Application number	US-201615097925-A
Country	US
Kind code	B2
Filing date	Apr 13, 2016
Priority date	Dec 16, 2011
Publication date	Nov 29, 2016
Grant date	Nov 29, 2016

Abstract

The present disclosure provides opsins, including variant opsins with increased activity and/or increased trafficking to the plasma membrane. The opsins are useful in therapeutic and screening applications, which are also provided.

First claim

What is claimed is:

1. An isolated fusion polypeptide comprising:
a) an opsin polypeptide comprising an amino acid sequence having at least 98% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:31;
b) an endoplasmic reticulum (ER) export signal; and
c) a membrane trafficking signal.

2. The fusion polypeptide of claim 1 , wherein the ER export signal comprises the amino acid sequence VXXSL, where X is any amino acid; or FXYENE (SEQ ID NO: 4), where X is any amino acid.

3. The fusion polypeptide of claim 1 , wherein the polypeptide further comprises a fluorescent protein disposed between (a) and (b).

4. The fusion polypeptide of claim 1 , wherein the membrane trafficking signal comprises an amino acid sequence having at least 90% amino acid sequence identity to an amino acid sequence selected from:

MDYGGALSAVGRELLFVTNPVVVNGS;
(SEQ ID NO: 6)

MAGHSNSMALFSFSLLWLCSGVLGTEF;
(SEQ ID NO: 7)

MGLRALMLWLLAAAGLVRESLQG;
(SEQ ID NO: 8)

MRGTPLLLVVSLFSLLQD;
(SEQ ID NO: 9)

and

KSRITSEGEYIPLDQIDINV.
(SEQ ID NO: 10)

5. The fusion polypeptide of claim 1 , wherein the fusion polypeptide comprises, in order from N-terminus to C-terminus:
a) an opsin polypeptide comprising an amino acid sequence having at least 98% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:31;
b) a membrane trafficking signal comprising the amino acid sequence KSRITSEGEYIPLDQIDINV (SEQ ID NO: 10); and
c) an ER export signal comprising the amino acid sequence FCYENEV (SEQ ID NO:5).

6. An isolated variant opsin polypeptide comprising, in order from amino terminus to carboxyl terminus: a) an amino acid sequence having at least 98% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:31; and b) an endoplasmic reticulum (ER) export signal.

7. An isolated polynucleotide comprising a nucleotide sequence encoding the fusion polypeptide of claim 1 .

8. The polynucleotide of claim 7 , wherein the fusion polypeptide-encoding nucleotide sequence is operably linked to a promoter that provides for neuron-selective expression.

9. A recombinant expression vector comprising the isolated polynucleotide of claim 7 .

10. A method of modulating the voltage potential of a cell in response to a light stimulus, the method comprising exposing a cell that is genetically modified with the recombinant expression vector of claim 9 to a light stimulus, wherein the encoded variant opsin is expressed in the cell, and wherein in response to exposure to a light stimulus, the voltage potential of the cell is modulated.

11. The fusion polypeptide of claim 1 , wherein the opsin polypeptide comprises an amino acid sequence having at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:31.

12. The fusion polypeptide of claim 1 , wherein the opsin polypeptide comprises the amino acid sequence set forth in SEQ ID NO:31.

13. The fusion polypeptide of claim 1 , wherein the ER export signal comprises the amino acid sequence VKESL (SEQ ID NO: 1), VLGSL (SEQ ID NO: 2), or FCYENE (SEQ ID NO: 5).

14. The fusion polypeptide of claim 1 , wherein the membrane trafficking signal comprises the amino acid sequence KSRITSEGEYIPLDQIDINV (SEQ ID NO:10).

15. The polynucleotide of claim 7 , wherein the nucleotide sequence is codon optimized for expression in a mammalian cell.

16. The polynucleotide of claim 7 , wherein the nucleotide sequence is operably linked to a promoter.

17. The recombinant expression vector of claim 9 , wherein the recombinant vector is a lentiviral vector, an adenoviral vector, or an adeno-associated viral vector.

18. A genetically modified cell comprising the polynucleotide of claim 7 .

19. A genetically modified cell comprising the recombinant vector of claim 9 .

20. The genetically modified cell of claim 19 , wherein the cell is a mammalian cell.

21. The genetically modified cell of claim 20 , wherein the cell is a neuron.

Assignees

• Univ Leland Stanford Junior

Inventors

• Deisseroth Karl
• Zhang Feng
• Gradinaru Viviana

Classifications

• A61P43/00

  Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00 · CPC title

• C07K2319/60

  containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP] · CPC title

• C12N13/00

  Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves · CPC title

• A61K38/00

  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) · CPC title

• C07K14/415 Primary

  from plants · CPC title