# US 5,707,803 A · DNA regulatory elements responsive to cytokines and methods for their use

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- Number: US 5,707,803 A
- Document ID: US5707803A
- Status: Granted
- Kind: A
- Application number: 08/410,780
- Filed: 1995-03-27
- Granted: 1998-01-13
- Earliest claimed: 1994-04-14
- Assignees: Ligand Pharmaceuticals Incorporated
- Inventors: Ian Peter Lamb; H. Martin Seidel
- Web page: https://patentgrep.com/patent/US5707803A

## CPC

- C12N 15/85: for animal cells
- C12Q 1/6811: Selection methods for production or design of target specific oligonucleotides or binding molecules
- C12Q 1/6813: Hybridisation assays
- C12Q 1/6876: Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q 1/701: {Specific hybridization probes}
- C12Q 2600/136: Screening for pharmacological compounds
- C12Q 2600/158: Expression markers
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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

## Abstract

The present invention provides oligonucleotide sequences comprising DNA regulatory elements comprising point mutations of Ly6E GAS element that bind activated transcriptional regulatory proteins in response to signaling molecules, such as cytokines. Further, the present invention also provides DNA constructs comprising the oligonucleotide sequences, cells transfected with the DNA constructs, and methods of using the DNA constructs and transfected cells to provide for the controlled expression of structural genes, for the detection and recovery of transcriptional regulatory proteins, and for measuring the ability of compounds to act as agonist and antagonists of gene transcription.

## Claims

### Claim 1

An isolated DNA molecule 13 to 200 nucleotides in length comprising at least one regulatory element that binds to an activated transcriptional regulatory protein, said regulatory element comprising a nucleotide sequence of TATTCCTGGAAGT (SEQ ID No. 1), TATTCCGGTAAGT (SEQ ID No. 2), TCTTCCTGTAAGT (SEQ ID No. 3), TATTCCCGTAAGT (SEQ ID NO. 6), or TATTCCTATAAGT (SEQ ID No. 7).

### Claim 2 (depends on 1)

The isolated DNA molecule according to claim 1, wherein the transcriptional regulatory protein comprises a STAT protein.

### Claim 3 (depends on 2)

The isolated DNA molecule according to claim 2, wherein the STAT protein is selected from the group consisting of STAT1.alpha. protein, STAT1.beta. protein, STAT2 protein, STAT3 protein, STAT4 protein, STAT5 protein and STAT6.

### Claim 4 (depends on 1)

The isolated DNA molecule according to claim 1, wherein the regulatory element binds to an activated transcriptional regulatory protein comprising STAT1.alpha. protein in response to a cytokine that activates the STAT1.alpha. protein.

### Claim 5 (depends on 1)

The isolated DNA molecule according to claim 1, wherein the regulatory element binds to an activated transcriptional regulatory protein comprising STAT1.alpha. protein or STAT3 protein in response to a cytokine that activates the STAT1.alpha. protein or the STAT3 protein.

### Claim 6 (depends on 5)

The isolated DNA molecule according to claim 5, said regulatory element comprising a nucleotide sequence of TATTCCCGTAAGT (SEQ ID No. 6), or TATTCCTATAAGT (SEQ ID No. 7).

### Claim 7 (depends on 1)

The isolated DNA molecule according to claim 1, wherein the regulatory element binds to activated transcriptional regulatory proteins comprising STAT1.alpha. protein, STAT3 protein, STAT5 protein or STAT6 protein in response to a cytokine that activates the STAT1.alpha. protein, STAT3 protein, STAT5 protein or STAT6 protein.

### Claim 8 (depends on 7)

The isolated DNA molecule according to claim 7, said regulatory element comprising a nucleotide sequence of TATTCCTGGAAGT (SEQ ID No. 1), TATTCCGGTAAGT (SEQ ID No. 2), or TCTTCCTGTAAGT (SEQ ID No. 3).

### Claim 9 (depends on 1)

The isolated DNA molecule according to claim 1, wherein activation of said activated transcriptional regulatory protein is in response to a cytokine selected from the group consisting of IFN.gamma., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15 GM-CSF, Oncostatin M, growth hormone, G-CSF, Epo, Tpo, EGF, PDGF, CNTF and LIF.

### Claim 10 (depends on 1)

The isolated DNA molecule according to claim 1 which is double stranded.

### Claim 11

A DNA construct comprising a heterologous gene that comprises at least one regulatory element and a promoter operably linked to a structural gene, wherein said structural gene is under the transcriptional control of the regulatory element and promoter, said regulatory element comprising a nucleotide sequence of TATTCCTGGAAGT (SEQ ID No. 1), TATTCCGGTAAGT (SEQ ID No. 2), TCTTCCTGTAAGT (SEQ ID No. 3), TATTCCCGTAAGT (SEQ ID NO. 6), or TATTCCTATAAGT (SEQ IDS No. 7).

### Claim 12 (depends on 11)

The DNA construct according to claim 11, wherein the promoter is selected from the group consisting of the gene promoter of the Herpes simplex virus thymidine kinase, adneovirus E1b and yeast alcohol dehydogenase.

### Claim 13 (depends on 11)

The DNA construct according to claim 11, wherein the heterologous gene comprises a structural gene for luciferase, chloramphenicol acetyl transferase, .beta.-galactosidase, secreted placental alkaline phosphatase, human growth hormone, t-PA, green fluorescent protein or inteferon.

### Claim 14 (depends on 11)

The DNA construct according to claim 11, wherein the heterologous gene comprises a multimer of at least one of the regulatory elements.

### Claim 15 (depends on 11)

The DNA construct according to claim 11, wherein the heterologous gene comprises the gene promoter of the Herpes simplex virus thymidine kinase and a structural gene for luciferase.

### Claim 16 (depends on 11)

A host cell transfected with the DNA construct of claim 11.

### Claim 17 (depends on 16)

A method for the controlled expression of a heterologous gene of interest comprising culturing the cells of claim 16 in the presence of a cytokine.

### Claim 18 (depends on 1)

A method for detecting the presence of a transcriptional regulatory protein in a sample comprising contacting the sample with the isolated DNA molecule according to claim 1 under conditions where the transcriptional regulatory protein is activated and binds with the isolated DNA molecule to form a complex, and detecting the presence of the complex in the sample.

### Claim 19 (depends on 18)

The method according to claim 18, further comprising, separating the complex from the sample and the isolated DNA molecule from the complex to yield the transcriptional regulatory protein.

### Claim 20 (depends on 16)

A method for measuring the ability of a compound to act as an agonist of gene transcription comprising:
(a) contacting the compound with the host cell according to claim 16 under conditions in which the heterologous gene is expressed in response to the compound; and
(b) comparing the level of gene expression in step (a) with the level of gene expression from the host cell in the absence of the compound, wherein the ability of the compound to act as an agonist of gene transcription is measured as the increase in the level of gene expression in step (a) compared to the level of gene expression from the host cell in the absence of the compound.

### Claim 21 (depends on 20)

The method according to claim 20, wherein the heterologous gene comprises a structural gene for luciferase, chloramphenicol acetyl transferase, .beta.-galactosidase, green fluorescent protein or secreted placental alkaline phosphatase.

### Claim 22 (depends on 16)

A method for measuring the ability of a compound to act as an antagonist of gene transcription comprising:
(a) contacting the compound with the host cell according to claim 16 in the presence of a predetermined amount of a signaling molecule under conditions in which the heterologous gene is expressed in response to the signaling molecule; and
(b) comparing the level of gene expression in step (a) with the level of gene expression from the host cell in the presence of the cytokine, but the absence of the compound, wherein the ability of the compound to act as an antagonist of gene transcription is measured as the amount of decrease in the level of gene expression in step (a) compared to the level of gene expression from the host cell in the presence of the cytokine and absence of the compound.

### Claim 23 (depends on 22)

The method according to claim 22, wherein the heterologous gene comprises a structural gene for luciferase, chloramphenicol acetyl transferase, .beta.-galactosidase, green fluorescent protein or secreted placental alkaline phosphatase.

### Claim 24

An isolated DNA molecule 13 to 200 nucleotides in length comprising at least one regulatory element, said regulatory element comprising a nucleotide sequence of TATTCCTGGAAGT (SEQ ID No. 1), TATTCCGGTAAGT (SEQ ID No. 2), TCTTCCTGTAAGT (SEQ ID No. 3), TATTCCCGTAAGT (SEQ ID NO. 6), or TATTCCTATAAGT (SEQ ID No. 7), or a complement thereof.

### Claim 25 (depends on 24)

The isolated DNA molecule according to claim 24, wherein the isolated DNA molecule comprises a multimer of at least one of the regulatory elements.

### Claim 26 (depends on 16)

A method for measuring the ability of a compound to agonize or antagonize the induction of STAT heterodimers comprising:
(a) contacting the compound with the host cell according to claim 16 under conditions in which the heterologous gene is expressed in response to the compound, wherein the host cell is transfected with a DNA construct comprising a single copy of said regulatory element that selectively binds to an activated STAT heterodimer; and
(b) comparing the level of gene expression in step (a) with the level of gene expression from the host cell in the absence of the compound, wherein the ability of the compound to act as an antagonist of the induction of STAT heterodimers is measured as the amount of decrease in the level of gene expression in step (a) compared to the level of gene expression from the host cell in the absence of the compound and wherein the ability of the compound to act as an agonist of the induction of STAT heterodimers is measured as the amount of increase in the level of gene expression in step (a) compared to the level of gene expression from the host cell in the absence of the compound.

### Claim 27 (depends on 26)

The method according to claim 26, wherein the STAT heterodimer comprises a STAT1.alpha./STAT3 heterodimer.