# US 5,700,640 A · Inducers of gamma globin gene expression and screening assays therefor

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- Number: US 5,700,640 A
- Document ID: US5700640A
- Status: Granted
- Kind: A
- Application number: 08/308,461
- Filed: 1994-09-16
- Granted: 1997-12-23
- Earliest claimed: 1994-09-16
- Assignees: BASF SE
- Inventors: Jeffrey W. Voss; Connie Caron
- Web page: https://patentgrep.com/patent/US5700640A

## CPC

- A61K 31/19: Carboxylic acids, e.g. valproic acid (salicylic acid A61K31/60)
- C12Q 1/6897: involving reporter genes operably linked to promoters
- Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
- 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)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

Methods for stimulating gamma globin gene expression in a mammalian cell comprising contacting the cell with a compound selected from valeric acid and certain isomers, derivatives or salts thereof, including isovaleric acid, 4-pentynoic acid and methylthioacetic acid, or an inhibitor of a short chain fatty acyl CoA dehydrogenase, or an activator of protein kinase C are disclosed. The methods of the invention are particularly useful for ameliorating .beta.-globin disorders, such as sickle cell anemia or .beta.-thalassemia. The method of the invention can also be used to prevent or ameliorate malaria in a mammal. The compounds of the invention can also be used to stimulate differentiation of a cell. Pharmaceutical compositions of the active compounds of the invention are also disclosed. Screening assays for identifying agents that stimulate gamma globin gene expression in an erythroid cell, stimulate cell differentiation or stimulate transport of a short chain fatty acid, or derivative or salt thereof, into cells are also disclosed.

## Claims

### Claim 1

A method for identifying an agent that stimulates gamma globin gene expression in a mammalian cell, comprising:
a) contacting a mammalian cell with an agent to be tested, wherein a nucleic acid molecule comprising a regulatory region of a gamma globin gene operatively linked to a reporter gene has been introduced into the mammalian cell; and
b) measuring reporter gene activity in the mammalian cell, wherein an increase in reporter gene activity in the presence of the agent, relative to reporter gene activity in the absence of the agent, is indicative that the agent stimulates gamma globin gene expression.

### Claim 2 (depends on 1)

The method of claim 1, wherein the nucleic acid molecule introduced into the mammalian cell comprises a human gamma globin gene promoter operatively linked upstream of the reporter gene.

### Claim 3 (depends on 1)

The method of claim 1, wherein the reporter gene encodes a gene product selected from the group consisting of luciferase, chloramphenicol acetyltransferase, alkaline phosphatase and .beta.-galactosidase.

### Claim 4 (depends on 1)

The method of claim 1, wherein the mammalian cell is an erythroid cell.

### Claim 5 (depends on 4)

The method of claim 4, wherein the mammalian cell is a human erythroleukemia cell.

### Claim 6

An isolated nucleic acid molecule comprising a human gamma globin gene promoter operatively linked upstream of a reporter gene, wherein the human gamma globin gene promoter has a 5' terminus at position -140 relative to the start site of transcription of the human gamma globin gene, wherein sequences in said nucleic acid molecule upstream of said position -140 are not human gamma globin promoter sequences.

### Claim 7 (depends on 6)

The nucleic acid molecule of claim 6, wherein the reporter gene encodes a gene product selected from the group consisting of luciferase, chloramphenicol acetyltransferase, alkaline phosphatase and .beta.-galactosidase.

### Claim 8 (depends on 6, 7)

A plasmid comprising the nucleic acid molecule of claim 6 or claim 7.

### Claim 9 (depends on 8)

A mammalian host cell into which the plasmid of claim 8 has been introduced.

### Claim 10 (depends on 9)

The mammalian host cell of claim 9, which is an erythroid cell.

### Claim 11 (depends on 9)

The mammalian host cell of claim 9, which is a human erythroleukemia cell.

### Claim 12

An isolated nucleic acid molecule comprising a human gamma globin gene promoter operatively linked upstream of a reporter gene, wherein the human gamma globin gene promoter has a 5' terminus at position -1200 relative to the start site of transcription of the human gamma globin gene, wherein sequences in said nucleic acid molecule upstream of said position -1200 are not human gamma globin promoter sequences.

### Claim 13 (depends on 12)

The nucleic acid molecule of claim 12, wherein the reporter gene encodes a gene product selected from the group consisting of luciferase, chloramphenicol acetyltransferase, alkaline phosphatase and .beta.-galactosidase.

### Claim 14 (depends on 12, 13)

A plasmid comprising the nucleic acid molecule of claim 12 or claim 13.

### Claim 15 (depends on 14)

A mammalian host cell into which the plasmid of claim 14 has been introduced.

### Claim 16 (depends on 15)

The mammalian host cell of claim 15, which is an erythroid cell.

### Claim 17 (depends on 15)

The mammalian host cell of claim 15, which is a human erythroleukemia cell.

### Claim 18

An isolated nucleic acid molecule comprising a human gamma globin gene 3' enhancer operatively linked downstream of a reporter gene.

### Claim 19 (depends on 18)

The nucleic acid molecule of claim 18, which further comprises a human gamma globin gene promoter operatively linked upstream of the reporter gene.

### Claim 20 (depends on 18)

The nucleic acid molecule of claim 18, wherein the reporter gene encodes a gene product selected from the group consisting of luciferase, chloramphenicol acetyltransferase, alkaline phosphatase and .beta.-galactosidase.

### Claim 21 (depends on 18, 19, 20)

A plasmid comprising the nucleic acid molecule of claim 18 or claim 19 or claim 20.

### Claim 22 (depends on 21)

A mammalian host cell into which the plasmid of claim 21 has been introduced.

### Claim 23 (depends on 22)

The mammalian host cell of claim 22, which is an erythroid cell.

### Claim 24 (depends on 22)

The mammalian host cell of claim 22, which is a human erythroleukemia cell.

### Claim 25

A method for identifying an agent that stimulates gamma globin gene expression in a mammalian cell, comprising:
a) contacting a mammalian cell with an agent to be tested, wherein a nucleic acid molecule comprising a human gamma globin gene 3' enhancer operatively linked downstream of a reporter gene has been introduced into the mammalian cell; and
b) measuring reporter gene activity in the mammalian cell, wherein an increase in reporter gene activity in the presence of the agent, relative to reporter gene activity in the absence of the agent, is indicative that the agent stimulates gamma globin gene expression.

### Claim 26 (depends on 25)

The method of claim 25, wherein the nucleic acid molecule further comprises a human gamma globin gene promoter operatively linked upstream of the reporter gene.

### Claim 27 (depends on 25)

The method of claim 25, wherein the reporter gene encodes a gene product selected from the group consisting of luciferase, chloramphenicol acetyltransferase, alkaline phosphatase and .beta.-galactosidase.

### Claim 28 (depends on 25)

The method of claim 25, wherein the mammalian cell is an erythroid cell.

### Claim 29 (depends on 25)

The method of claim 25, wherein the mammalian cell is a human erythroleukemia cell.

### Claim 30 (depends on 25)

The method of claim 25, wherein reporter gene activity is measured by assaying the enzymatic activity of the gene product encoded by the reporter gene.

### Claim 31

A method for identifying an agent that enhances stimulation of gamma globin gene expression by a short chain fatty acid, comprising:
a) contacting a mammalian cell with an agent to be tested and with a short chain fatty acid, wherein a nucleic acid molecule comprising a regulatory region of a gamma globin gene operatively linked to a reporter gene has been introduced into the mammalian cell; and
b) measuring reporter gene activity in the mammalian cell, wherein an increase in reporter gene activity in the presence of the agent, relative to reporter gene activity in the absence of the agent, is indicative that the agent enhances stimulation of gamma globin gene expression by a short chain fatty acid.

### Claim 32 (depends on 31)

The method of claim 31, wherein the short chain fatty acid is selected from the group consisting of butyric acid, valeric acid, isovaleric acid, 4-pentynoic acid, methylthioacetic acid and salts thereof.

### Claim 33 (depends on 31)

The method of claim 31, wherein the reporter gene encodes a gene product selected from the group consisting of luciferase, chloramphenicol acetyltransferase, alkaline phosphatase and .beta.-galactosidase.

### Claim 34 (depends on 31)

The method of claim 31, wherein the mammalian cell is an erythroid cell.

### Claim 35 (depends on 31)

The method of claim 31, wherein the mammalian cell is a human erythroleukemia cell.

### Claim 36 (depends on 31)

The method of claim 31, wherein reporter gene activity is measured by assaying the enzymatic activity of the gene product encoded by the reporter gene.

### Claim 37 (depends on 1)

The method of claim 1, wherein reporter gene activity is measured by assaying the enzymatic activity of the gene product encoded by the reporter gene.