# US 10,307,492 B2 · Modified MGLUR6 promoter and methods of use

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- Number: US 10,307,492 B2
- Document ID: US10307492B2
- Status: Granted
- Kind: B2
- Application number: 15/125,066
- Filed: 2015-03-11
- Published: 2017-01-26
- Granted: 2019-06-04
- Earliest claimed: 2014-03-11
- Assignees: Wayne State University
- Applicants: Wayne State University
- Inventors: Zhuo-Hua Pan; Qi Lu; Tushar H. Ganjawala; JrGang Cheng
- Web page: https://patentgrep.com/patent/US10307492B2

## CPC

- A61K 48/0058: {Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct}
- A61K 9/0048: {Eye, e.g. artificial tears}
- A61K 38/1787: {for neuromediators, e.g. serotonin receptor, dopamine receptor}
- A61K 48/0075: {characterised by an aspect of the delivery route, e.g. oral, subcutaneous}
- A61P 27/02: Ophthalmic agents
- C07K 14/70571: {for neuromediators, e.g. serotonin receptor, dopamine receptor}
- C12N 15/67: General methods for enhancing the expression
- C12N 15/85: for animal cells
- C12N 15/86: Viral vectors
- C12N 2750/14143: viral genome or elements thereof as genetic vector
- C12N 2830/008: cell type or tissue specific enhancer/promoter combination
- C12N 2830/42: being an intron or intervening sequence for splicing and/or stability of RNA
- 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)
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The invention provides nucleic acids and nucleic acid expression vectors containing optimized mGluR6 promoters for expression of transgenes in the retina. The compositions and methods of the invention are useful for expression of gene products to preserve, improve, or restore phototransduction or vision.

## Claims

### Claim 1

An isolated nucleic acid molecule comprising
a. an mGluR6 enhancer or a variant thereof at least 70% identical to SEQ ID NO: 1 or SEQ ID NO: 2;
b. an mGluR6 promoter or a variant thereof at least 70% identical to SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6;
c. an intron 4 of the mGluR6 gene or a variant thereof at least 70% identical to SEQ ID NO: 7 or SEQ ID NO. 8; and
d. an intron 3 of the mGluR6 gene or a variant thereof at least 70% identical to SEQ ID NO: 9 or SEQ ID NO. 10.

### Claim 2 (depends on 1)

The isolated nucleic acid molecule of claim 1, wherein said mGluR6 enhancer comprises the nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, wherein said mGluR6 promoter comprises the nucleic acid sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6, wherein said intron 4 of the mGluR6 gene comprises the nucleic acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8, and wherein said intron 3 of the mGluR6 gene comprises the nucleic acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10.

### Claim 3 (depends on 1)

The isolated nucleic acid molecule of claim 1, wherein the intron 4 of the mGluR6 gene is upstream of the intron 3 of the mGluR6 gene, wherein the intron 3 of the mGluR6 gene is upstream of the mGluR6 enhancer, and wherein the mGluR6 enhancer is upstream of the mGluR6 promoter.

### Claim 4 (depends on 1)

A nucleic acid expression vector comprising the nucleic acid molecule of claim 1 operably linked to at least one transgene.

### Claim 5 (depends on 4)

The nucleic acid expression vector of claim 4, wherein said mGluR6 enhancer comprises the nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, wherein said mGluR6 promoter comprises the nucleic acid sequence of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6, wherein said intron 4 of the mGluR6 gene comprises the nucleic acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8, and wherein said intron 3 of the mGluR6 gene comprises the nucleic acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10.

### Claim 6 (depends on 4)

The nucleic acid expression vector of claim 4, wherein the intron 4 of the mGluR6 gene is upstream of the intron 3 of the mGluR6 gene, wherein the intron 3 of the mGluR6 gene is upstream of the mGluR6 enhancer, and wherein the mGluR6 enhancer is upstream of the mGluR6 promoter.

### Claim 7 (depends on 4)

The nucleic acid expression vector of claim 4, wherein the vector is an adeno-associated virus vector or a recombinant adeno-associated virus (rAAV) vector.

### Claim 8 (depends on 7)

The nucleic acid expression vector of claim 7, wherein the vector is a recombinant AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12 vector.

### Claim 9 (depends on 4)

The nucleic acid expression vector of claim 4, wherein the vector comprises a capsid protein comprising at least one mutation.

### Claim 10 (depends on 9)

The nucleic acid expression vector of claim 9, wherein said at least one mutation is selected from the group consisting of:
a tyrosine (Y) to phenylalanine (F) at amino acid position 444;
a tyrosine (Y) to phenylalanine (F) at amino acid position 730;
a tyrosine (Y) to phenylalanine (F) at amino acid positions 272, 444, 500, and 730;
a threonine (T) to valine (V) at amino acid position 491; and
a tyrosine (Y) to phenylalanine (F) at amino acid positions 272, 444, 500, 730, and a threonine (T) to valine (V) at amino acid position 491,
wherein the amino acid positions correspond to amino acid positions of the AAV capsid protein.

### Claim 11 (depends on 9)

The nucleic acid expression vector of claim 9, wherein the capsid protein comprises a peptide insert selected from the group consisting of SEQ ID NO: 35, SEQ ID NO: 36, and SEQ ID NO: 37.

### Claim 12 (depends on 4)

The nucleic acid expression vector of claim 4, wherein the transgene is an opsin gene.

### Claim 13 (depends on 12)

The nucleic acid expression vector of claim 12, wherein said opsin gene is selected from the group consisting of channelrhodopsin, melanopsin, pineal opsin, photopsins, halorhodopsin, bacteriorhodopsin and proteorhodopsin.

### Claim 14 (depends on 4)

The nucleic acid expression vector of claim 4, wherein the transgene encodes a gene product that increases light sensitivity, increases light detection, increases photosensitivity, increases visual evoked potential, or restores vision in a retina.

### Claim 15 (depends on 4)

A pharmaceutical composition comprising the nucleic acid expression vector of claim 4 and a pharmaceutically acceptable excipient.

### Claim 16 (depends on 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14)

A method for expressing a transgene in the eye comprising introducing into the eye the nucleic acid expression vector of any one of claims 4-14.

### Claim 17 (depends on 15)

A method for expressing a transgene in the eye comprising introducing into the eye the pharmaceutical composition of claim 15.