# US 5,707,800 A · Retinoic acid response elements and assays employing same

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- Number: US 5,707,800 A
- Document ID: US5707800A
- Status: Granted
- Kind: A
- Application number: 08/346,342
- Filed: 1994-11-28
- Granted: 1998-01-13
- Earliest claimed: 1991-03-18
- Assignees: The Salk Institute for Biological Studies
- Inventors: David John Mangelsdorf; Ronald M. Evans; Kazuhiko Umesono; Steven A. Kliewer
- Web page: https://patentgrep.com/patent/US5707800A

## CPC

- C07K 14/70567: {Nuclear receptors, e.g. retinoic acid receptor [RAR], RXR, nuclear orphan receptors}
- C12N 15/85: for animal cells
- G01N 33/5008: {for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics}
- C12N 2830/008: cell type or tissue specific enhancer/promoter combination
- C12N 2830/85: mammalian
- G01N 2500/10: involving cells
- 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)
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

DNA segments have been discovered, and characterized by sequence, which are response elements operative to confer responsiveness to retinoic acid, or derivatives thereof, for the transcriptional activation of promoters in cells. By using transcriptional control regions comprising response elements of the present invention in combination with a functional promoter, it is now possible to provide recombinant DNA vectors containing a gene, the transcription (and, thereby, also expression) of which is under the control of a promoter, the transcriptional activity of which is responsive to (and increased by) retinoic acid or derivatives thereof.

## Claims

### Claim 1

A substantially pure DNA construct comprising:
1) a steroid hormone receptor response element having the sequence:
5'--NNNNNN--(N--NNNNNN).sub.x --3'
wherein each --NNNNNN-- is a half site, --N-- is a spacer nucleotide, and X is 4,
wherein nucleotides at positions 4, 5, and 6 in each half site are nucleotides T, C, and A, respectively, each half site has at least 5 nucleotides corresponding to the sequence --AGGTCA--, and at least two of the half sites are directly tandemly repeated, and
2) a heterologous promoter,
wherein the response element is operatively linked to said heterologous promoter.

### Claim 2

A substantially pure DNA construct comprising:
1) asteroid hormone receptor response element having the sequence:
5'--NNNNNN--(N--NNNNNN).sub.x --3'
wherein each --NNNNNN-- is a half site, --N-- is a spacer nucleotide, and X is 3,
wherein nucleotides at positions 4, 5, and 6 in each half site are nucleotides T, C, and A, respectively, each half site has at least 5 nucleotides corresponding to the sequence --AGGTCA--, and at least two of the half sites are directly tandemly repeated, and
2) a heterologous promoter,
wherein the response element is operatively linked to said heterologous promoter.

### Claim 3

A substantially pure DNA construct comprising:
1) asteroid hormone receptor response element having the sequence:
5'--NNNNNN--(N--NNNNNN).sub.x --3'
wherein each --NNNNNN-- is a half site, --N-- is a spacer nucleotide, and X is 2,
wherein nucleotides at positions 4, 5, and 6 in each half site are nucleotides T, C, and A, respectively, each half site has at least 5 nucleotides corresponding to the sequence --AGGTCA--, and at least two of the half sites are directly tanderaly repeated, and
2) a heterologous promoter,
wherein the response element is operatively linked to said heterologous promoter.

### Claim 4

A substantially pure DNA construct comprising:
1) asteroid hormone receptor response element having the sequence:
5'--NNNNNN--N--NNNNNN--3'
wherein each --NNNNNN-- is a half site, --N-- is a spacer nucleotide,
wherein nucleotides at positions 4, 5, and 6 in each half site are nucleotides T, C, and A, respectively, each half site has at least 5 nucleotides corresponding to the sequence --AGGTCA--, and said half sites are tandemly repeated, and
2) a heterologous promoter,
wherein the response element is operatively linked to said heterologous promoter.

### Claim 5

A substantially pure DNA construct comprising:
1) asteroid hormone receptor response element having the sequence:
5'--NNNNNN--(N--NNNNNN).sub.x --3'
wherein each --NNNNNN-- is a half site, --N-- is a spacer nucleotide, and X is 1-4,
wherein the half sites are selected from the group consisting of AGGTCA, AGTTCA, CTGTCA, ATTTCA, ATGTCA, CGGTCA, AGCTCA, AGGCCA, AGGTGA, AGGTTA, AGGTCC, and ATGTCG and
wherein at least two of the half sites are directly tandemly repeated, and
2) a heterologous promoter,
wherein the response element is operatively linked to said heterologous promoter.

### Claim 6

A substantially pure DNA construct comprising:
1) a response element having the nucleic acid sequence CTGTCA--C--AGGTCA--C--AGGTCA--C--AGGTCA--C--AGGTCA (SEQ ID NO:6) and
2) a heterologous promoter,
wherein the response element is operatively linked to said heterologous promoter.

### Claim 7 (depends on 1)

The DNA construct of claim 1, wherein the promoter is selected from the group consisting of delta-MTV promoter of mouse mammary tumor virus, SV40 early gene promoter, Herpes simplex virus thymidine kinase promoter, and Drosophila alcohol dehydrogenase promoter.

### Claim 8 (depends on 2)

The DNA construct of claim 2, wherein the promoter is selected from the group consisting of delta-MTV promoter of mouse mammary tumor virus, SV40 early gene promoter, Herpes simplex virus thymidine kinase promoter, and Drosophila alcohol dehydrogenase promoter.

### Claim 9 (depends on 3)

The DNA construct of claim 3, wherein the promoter is selected from the group consisting of delta-MTV promoter of mouse mammary tumor virus, SV40 early gene promoter, Herpes simplex virus thymidine kinase promoter, and Drosophila alcohol dehydrogenase promoter.

### Claim 10 (depends on 4)

The DNA construct of claim 4, wherein the promoter is selected from the group consisting of delta-MTV promoter of mouse mammary tumor virus, SV40 early gene promoter, Herpes simplex virus thymidine kinase promoter, and Drosophila alcohol dehydrogenase promoter.

### Claim 11 (depends on 5)

The DNA construct of claim 5, wherein the promoter is selected from the group consisting of delta-MTV promoter of mouse mammary tumor virus, SV40 early gene promoter, Herpes simplex virus thymidine kinase promoter, and Drosophila alcohol dehydrogenase promoter.

### Claim 12 (depends on 6)

The DNA construct of claim 6, wherein the promoter is selected from the group consisting of delta-MTV promoter of mouse mammary tumor virus, SV40 early gene promoter, Herpes simplex virus thymidine kinase promoter, and Drosophila alcohol dehydrogenase promoter.

### Claim 13 (depends on 1)

The DNA construct of claim 1 operatively linked to a gene encoding a protein for transcription.

### Claim 14 (depends on 2)

The DNA construct of claim 2 operatively linked to a gene encoding a protein for transcription.

### Claim 15 (depends on 3)

DNA construct of claim 3 operatively linked to a gene encoding a protein for transcription.

### Claim 16 (depends on 4)

The DNA construct of claim 4 operatively linked to a gene encoding a protein for transcription.

### Claim 17 (depends on 5)

The DNA construct of claim 5 operatively linked to a gene encoding a protein for transcription.

### Claim 18 (depends on 6)

The DNA construct of claim 6 operatively linked to a gene encoding a protein for transcription.

### Claim 19 (depends on 13)

The DNA construct of claim 13, wherein said gene is a reporter gene selected from the group consisting of luc, cat, and beta-gal.

### Claim 20 (depends on 14)

The DNA construct of claim 14, wherein said gene is a reporter gene selected from the group consisting of luc, cat, and beta-gal.

### Claim 21 (depends on 15)

The DNA construct of claim 15, wherein said gene is a reporter gene selected from the group consisting of luc, cat, and beta-gal.

### Claim 22 (depends on 16)

The DNA construct of claim 16, wherein said gene is a reporter gene selected from the group consisting of luc, cat, and beta-gal.

### Claim 23 (depends on 17)

The DNA construct of claim 17, wherein said gene is a reporter gene selected from the group consisting of luc, cat, and beta-gal.

### Claim 24 (depends on 18)

The DNA construct according to claim 18, wherein said reporter gene is selected from the group consisting of luc, cat, and beta-gal.

### Claim 25 (depends on 19)

The DNA construct according to claim 19, wherein said reporter gene is cat.

### Claim 26 (depends on 20)

The DNA construct according to claim 20, wherein said reporter gene is cat.

### Claim 27 (depends on 21)

The DNA construct according to claim 21, wherein said reporter gene is cat.

### Claim 28 (depends on 22)

The DNA construct according to claim 22, wherein said reporter gene is cat.

### Claim 29 (depends on 23)

The DNA construct according to claim 23, wherein said reporter gene is cat.

### Claim 30 (depends on 24)

The DNA construct according to claim 24, wherein said reporter gene is cat.

### Claim 31 (depends on 13)

A vector comprising the DNA construct of claim 13.

### Claim 32 (depends on 14)

A vector comprising the DNA construct of claim 14.

### Claim 33 (depends on 15)

A vector comprising the DNA construct of claim 15.

### Claim 34 (depends on 16)

A vector comprising the DNA construct of claim 16.

### Claim 35 (depends on 17)

A vector comprising the DNA construct of claim 17.

### Claim 36 (depends on 18)

A vector comprising the DNA construct of claim 18.

### Claim 37 (depends on 31)

A cell transformed with the vector of claim 31.

### Claim 38 (depends on 32)

A cell transformed with the vector of claim 32.

### Claim 39 (depends on 33)

A cell transformed with the vector of claim 33.

### Claim 40 (depends on 34)

A cell transformed with the vector of claim 34.

### Claim 41 (depends on 35)

A cell transformed with the vector of claim 35.

### Claim 42 (depends on 36)

A cell transformed with the vector of claim 36.

### Claim 43 (depends on 37)

A cell according to claim 37, wherein the cell is selected from the group consisting of mamalian, avian, and insect cells.

### Claim 44 (depends on 38)

A cell according to claim 38, wherein the cell is selected from the group consisting of mamalian, avian, and insect cells.

### Claim 45 (depends on 39)

A cell according to claim 39, wherein the cell is selected from the group consisting of mamalian, avian, and insect cells.

### Claim 46 (depends on 40)

A cell according to claim 40, wherein the cell is selected from the group consisting of mamalian, avian, and insect cells.

### Claim 47 (depends on 41)

A cell according to claim 41, wherein the cell is selected from the group consisting of mamalian, avian, and insect cells.

### Claim 48 (depends on 42)

A cell according to claim 42, wherein the cell is selected from the group consisting of mamalian, avian, and insect cells.

### Claim 49 (depends on 43)

A cell according to claim 43, wherein the cell is selected from the group consisting of CV-1, COS, F9, CHO, HeLa, NIH 3T3, HuTu80, Rat2 fibroblast, HT1080.T, chick embryo fibroblast, quail QT6, and Drosophila Schneider S2.

### Claim 50 (depends on 44)

A cell according to claim 44, wherein the cell is selected from the group consisting of CV-1, COS, F9, CHO, HeLa, NIH 3T3, HuTu80, Rat2 fibroblast, HT1080.T, chick embryo fibroblast, quail QT6, and Drosophila Schneider S2.

### Claim 51 (depends on 45)

A cell according to claim 45, wherein the cell is selected from the group consisting of CV-1, COS, F9, CHO, HeLa, NIH 3T3, HuTu80, Rat2 fibroblast, HT1080.T, chick embryo fibroblast, quail QT6, and Drosophila Schneider S2.

### Claim 52 (depends on 46)

A cell according to claim 46, wherein the cell is selected from the group consisting of CV-1, COS, F9, CHO, HeLa, NIH 3T3, HuTu80, Rat2 fibroblast, HT1080.T, chick embryo fibroblast, quail QT6, and Drosophila Schneider S2.

### Claim 53 (depends on 47)

A cell according to claim 47, wherein the cell is selected from the group consisting of CV-1, COS, F9, CHO, HeLa, NIH 3T3, HuTu80, Rat2 fibroblast, HT1080.T, chick embryo fibroblast, quail QT6, and Drosophila Schneider S2.

### Claim 54 (depends on 48)

A cell according to claim 48, wherein the cell is selected from the group consisting of CV-1, COS, F9, CHO, HeLa, NIH 3T3, HuTu80, Rat2 fibroblast, HT1080.T, chick embryo fibroblast, quail QT6, and Drosophila Schneider S2.

### Claim 55 (depends on 37)

A method for the controlled expression of a gene encoding a protein for transcription, said method comprising culturing the cells of claim 37 in the presence or absence of retinoic acid or derivatives thereof and the receptor therefor.

### Claim 56 (depends on 55)

A method according to claim 55, wherein said receptor is RXR.

### Claim 57 (depends on 38)

A method for the controlled expression of a gene encoding a protein for transcription, said method comprising culturing the cells of claim 38 in the presence or absence of retinoic acid or derivatives thereof and the receptor therefor.

### Claim 58 (depends on 57)

A method according to claim 57, wherein said receptor is RXR.

### Claim 59 (depends on 39)

A method for the controlled expression of a gene encoding a protein for transcription, said method comprising culturing the cells of claim 39 in the presence or absence of retinoic acid or derivatives thereof and the receptor therefor.

### Claim 60 (depends on 59)

A method according to claim 59, wherein said receptor is RXR.

### Claim 61 (depends on 40)

A method for the controlled expression of a gene encoding a protein for transcription, said method comprising culturing the cells of claim 40 in the presence or absence of retinoic acid or derivatives thereof and the receptor therefor.

### Claim 62 (depends on 61)

A method according to claim 61, wherein said receptor is RXR.

### Claim 63 (depends on 41)

A method for the controlled expression of a gene encoding a protein for transcription, said method comprising culturing the cells of claim 41 in the presence or absence of retinoic acid or derivatives thereof and the receptor therefor.

### Claim 64 (depends on 63)

A method according to claim 63, wherein said receptor is RXR.

### Claim 65 (depends on 42)

A method for the controlled expression of a gene encoding a protein for transcription, said method comprising culturing the cells of claim 42 in the presence or absence of retinoic acid or derivatives thereof and the receptor therefor.

### Claim 66 (depends on 65)

A method according to claim 65, wherein said receptor is RXR.

### Claim 67 (depends on 37)

A method for testing the activity of a test compound as an agonist of retinoid X receptor or a receptor that can be activated by retinoic acid or its derivatives, said method comprising:
(a) culturing a cell according to claim 37 in the presence of retinoid X receptor (RXR), and in the further presence or absence of the test compound, and thereafter
(b) comparing the amount of protein expressed during the culturing in the presence or in the absence of the test compound.

### Claim 68 (depends on 38)

A method for testing the activity of a test compound as an agonist of retinoid X receptor or a receptor that can be activated by retinoic acid or its derivatives, said method comprising:
(a) culturing a cell according to claim 38 in the presence of retinoid X receptor (RXR), and in the further presence or absence of the test compound, and thereafter
(b) comparing the amount of protein expressed during the culturing in the presence or in the absence of the test compound.

### Claim 69 (depends on 39)

A method for testing the activity of a test compound as an agonist of retinoid X receptor or a receptor that can be activated by retinoic acid or its derivatives, said method comprising:
(a) culturing a cell according to claim 39 in the presence of retinoid X receptor (RXR), and in the further presence or absence of the test compound, and thereafter
(b) comparing the amount of protein expressed during the culturing in the presence or in the absence of the test compound.

### Claim 70 (depends on 40)

A method for testing the activity of a test compound as an agonist of retinoid X receptor or a receptor that can be activated by retinoic acid or its derivatives, said method comprising:
(a) culturing a cell according to claim 40 in the presence of retinoid X receptor (RXR), and in the further presence or absence of the test compound, and thereafter
(b) comparing the amount of protein expressed during the culturing in the presence or in the absence of the test compound.

### Claim 71 (depends on 41)

A method for testing the activity of a test compound as an agonist of retinoid X receptor or a receptor that can be activated by retinoic acid or its derivatives, said method comprising:
(a) culturing a cell according to claim 41 in the presence of retinoid X receptor (RXR), and in the further presence or absence of the test compound, and thereafter
(b) comparing the amount of protein expressed during the culturing in the presence or in the absence of the test compound.

### Claim 72 (depends on 42)

A method for testing the activity of a test compound as an agonist of retinoid X receptor or a receptor that can be activated by retinoic acid or its derivatives, said method comprising:
(a) culturing a cell according to claim 42 in the presence of retinoid X receptor (RXR), and in the further presence or absence of the test compound, and thereafter
(b) comparing the amount of protein expressed during the culturing in the presence or in the absence of the test compound.