Novel ecdysone receptor-based inducible gene expression system

• US 20020119521A1
• Filed: 09/26/2001
• Published: 08/29/2002
• Est. Priority Date: 03/22/2000
• Status: Active Grant

• Current Assignee: Intrexon Corporation
• Original Assignee: Intrexon Corporation
• Inventors: Kapitskaya, Marianna Zinovjevna, Cress, Dean Ervin, Palli, Subba Reddy
• Granted Patent: US 7,091,038 B2
• Time in Patent Office: Days
• US Class Current: 435/69.1
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A61K 38/00 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 Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: C07K 14/70567 Nuclear receptors, e.g. retinoic acid receptor [RAR], RXR, nuclear orphan receptors
• CPC Class Codes: C07K 14/721 Steroid/thyroid hormone superfamily, e.g. GR, EcR, androgen receptor, oestrogen receptor
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C12N 15/1055 Protein x Protein interaction, e.g. two hybrid selection

Abstract

This invention relates to the field of biotechnology or genetic engineering. Specifically, this invention relates to the field of gene expression. More specifically, this invention relates to a novel inducible gene expression system and methods of modulating gene expression in a host cell for applications such as gene therapy, large scale production of proteins and antibodies, cell-based high throughput screening assays, functional genomics and regulation of traits in transgenic plants and animals.

Claims

1. A gene expression modulation system comprising:

a) a first gene expression cassette that is capable of being expressed in a host cell comprising a polynucleotide encoding a first polypeptide comprising;

i) a DNA-binding domain that recognizes a response element associated with a gene whose expression is to be modulated;

i) a ligand binding domain comprising a ligand binding domain from a nuclear receptor;

b) a second gene expression cassette that is capable of being expressed in the host cell comprising a polynucleotide encoding a second polypeptide comprising;

i) a transactivation domain; and

ii) a ligand binding domain comprising a ligand binding domain from a nuclear receptor other than ultraspiracle (USP).

2. The gene expression modulation system according to claim 1, further comprising a third gene expression cassette comprising:

i) a response element to which the DNA-binding domain of the first polypeptide binds;

ii) a promoter that is activated by the transactivation domain of the second polypeptide; and

iii) the gene whose expression is to be modulated.

3. The gene expression modulation system according to claim 1, wherein the ligand binding domain of the first polypeptide is an ecdysone receptor polypeptide.

4. The gene expression modulation system according to claim 1, wherein the ligand binding domain of the second polypeptide is a retinoid X receptor polypeptide.

30. A method of modulating the expression of a gene in a host cell comprising the gene to be modulated comprising the steps of:

a) introducing into the host cell the gene expression modulation system according to claim 1;

and

b) introducing into the host cell a ligand that independently combines with the ligand binding domains of the first polypeptide and the second polypeptide;

wherein the gene to be expressed is a component of a chimeric gene comprising;

i) a response element to which the DNA binding domain from the first polypeptide binds;

ii) a promoter that is activated by the transactivation domain of the second polypeptide; and

iii) a gene whose expression is to be modulated,

whereby expression of the gene is modulated in the host cell.

31. The method according to claim 30, wherein the ligand is either

a) a compound of the formula:

wherein;

E is a (C₄-C₆)alkyl containing a tertiary carbon or a cyano(C₃-C₅)alkyl containing a tertiary carbon;

R¹ is H, Me, Et, i-Pr, F, formyl, CF₃, CHF₂, CHCl₂, CH₂F, CH₂Cl, CH₂OH, CH₂OMe, CH₂CN, CN, C^oCH, 1-propynyl, 2-propynyl, vinyl, OH, OMe, OEt, cyclopropyl, CF₂CF₃, CH═

CHCN, allyl, azido, SCN, or SCHF₂;

R² is H, Me, Et, n-Pr, i-Pr, formyl, CF₃, CHF₂, CHCl₂, CH₂F, CH₂Cl, CH₂OH, CH₂OMe, CH₂CN, CN, C^oCH, 1-propynyl, 2-propynyl, vinyl, Ac, F, Cl, OH, OMe, OEt, O-n-Pr, OAc, NMe₂, NEt₂, SMe, SEt, SOCF₃, OCF₂CF₂H, COEt, cyclopropyl, CF₂CF₃, CH═

CHCN, allyl, azido, OCF₃, OCHF₂, O-i-Pr, SCN, SCHF₂, SOMe, NH—

CN, or joined with R³ and the phenyl carbons to which R² and R³ are attached to form an ethylenedioxy, a dihydrofuryl ring with the oxygen adjacent to a phenyl carbon, or a dihydropyryl ring with the oxygen adjacent to a phenyl carbon;

R³ is H, Et, or joined with R² and the phenyl carbons to which R² and R³ are attached to form an ethylenedioxy, a dihydrofuryl ring with the oxygen adjacent to a phenyl carbon, or a dihydropyryl ring with the oxygen adjacent to a phenyl carbon;

R⁴, R⁵, and R⁶ are independently H, Me, Et, F, Cl, Br, formyl, CF₃, CHF₂, CHCl₂, CH₂F, CH₂Cl, CH₂OH, CN, C^oCH, 1-propynyl, 2-propynyl, vinyl, OMe, OEt, SMe, or SEt, or

b) an ecdysone, 20-hydroxyecdysone, ponasterone A, muristerone A, an oxysterol, a 22(R) hydroxycholesterol, 24(S) hydroxycholesterol, 25-epoxycholesterol, T0901317, 5-alpha-6-alpha-epoxycholesterol-3-sulfate (ECHS), 7-ketocholesterol-3-sulfate, farnesol, a bile acid, a 1,1-biphosphonate ester, or Juvenile hormone III.

32. The method according to claim 31, wherein the method further comprises a second ligand, wherein the second ligand is 9-cis-retinoic acid or a synthetic analog of retinoic acid.

33. An isolated host cell into which the gene expression modulation system according to claim 1 has been introduced.

34. The isolated host cell according to claim 33, wherein the host cell is selected from the group consisting of a bacterial cell, a fungal cell, a yeast cell, a nematode cell, an insect cell, a fish cell, a plant cell, an avian cell, an animal cell, and a mammalian cell.

35. A non-human organism comprising a host cell into which the gene expression modulation system according to claim 1 has been introduced.

36. The non-human organism according to claim 35, wherein the non-human organism is selected from the group consisting of a bacterium, a fungus, a yeast, a nematode, an insect, a fish, a plant, a bird, an animal, and a mammal.

5. A gene expression modulation system comprising:

a) a first gene expression cassette that is capable of being expressed in a host cell comprising a polynucleotide encoding a first polypeptide comprising;

i) a DNA-binding domain that recognizes a response element associated with a gene whose expression is to be modulated; and

ii) a ligand binding domain comprising a ligand binding domain from an ecdysone receptor; and

b) a second gene expression cassette that is capable of being expressed in the host cell comprising a polynucleotide encoding a second polypeptide comprising;

i) a transactivation domain; and

ii) a ligand binding domain comprising a ligand binding domain from a retinoid X receptor.

6. The gene expression modulation system according to claim 5, further comprising a third gene expression cassette comprising:

i) a response element to which the DNA-binding domain of the first polypeptide binds;

ii) a promoter that is activated by the transactivation domain of the second polypeptide; and

iii) the gene whose expression is to be modulated.

7. The gene expression modulation system according to claim 5, wherein the ligand binding domain of the first polypeptide is encoded by a polynucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:

1, SEQ ID NO;

2, SEQ ID NO;

3, SEQ ID NO;

4, SEQ ID NO;

5, SEQ ID NO;

6, SEQ ID NO;

7, SEQ ID NO;

8, SEQ ID NO;

9, and SEQ ID NO;

10.

8. The gene expression modulation system according to claim 5, wherein the ligand binding domain of the first polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:

11, SEQ ID NO;

12, SEQ ID NO;

13, SEQ ID NO;

14, SEQ ID NO;

15, SEQ ID NO;

16, SEQ ID NO;

17, SEQ ID NO;

18, SEQ ID NO;

19, and SEQ ID NO;

20.

9. The gene expression modulation system according to claim 5, wherein the ligand binding domain of the second polypeptide is encoded by a polynucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:

21, SEQ ID NO;

22, SEQ ID NO;

23, SEQ ID NO;

24, SEQ ID NO;

25, SEQ ID NO;

26, SEQ ID NO;

27, SEQ ID NO;

28, SEQ ID NO;

29, and SEQ ID NO;

30.

10. The gene expression modulation system according to claim 5, wherein the ligand binding domain of the second polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:

31, SEQ ID NO;

32, SEQ ID NO;

33, SEQ ID NO;

34, SEQ ID NO;

35, SEQ ID NO;

36, SEQ ID NO;

37, SEQ ID NO;

38, SEQ ID NO;

39, and SEQ ID NO;

40.

11. A gene expression modulation system comprising:

a) a first gene expression cassette that is capable of being expressed in a host cell comprising a polynucleotide encoding a first polypeptide comprising;

i) a DNA-binding domain that recognizes a response element associated with a gene whose expression is to be modulated; and

ii) a ligand binding domain comprising a ligand binding domain from a retinoid X receptor; and

b) a second gene expression cassette that is capable of being expressed in the host cell comprising a polynucleotide encoding a second polypeptide comprising;

i) a transactivation domain; and

ii) a ligand binding domain comprising a ligand binding domain from an ecdysone receptor.

12. The gene expression modulation system according to claim 11, further comprising a third gene expression cassette comprising:

i) a response element to which the DNA-binding domain of the first polypeptide binds;

ii) a promoter that is activated by the transactivation domain of the second polypeptide; and

iii) the gene whose expression is to be modulated.

13. The gene expression modulation system according to claim 11, wherein the ligand binding domain of the first polypeptide is encoded by a polynucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:

21, SEQ ID NO;

22, SEQ ID NO;

23, SEQ ID NO;

24, SEQ ID NO;

25, SEQ ID NO;

26, SEQ ID NO;

27, SEQ ID NO;

28, SEQ ID NO;

29, and SEQ ID NO;

30.

14. The gene expression modulation system according to claim 11, wherein the ligand binding domain of the first polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:

31, SEQ ID NO;

32, SEQ ID NO;

33, SEQ ID NO;

34, SEQ ID NO;

35, SEQ ID NO;

36, SEQ ID NO;

37, SEQ ID NO;

38, SEQ ID NO;

39, and SEQ ID NO;

40.

15. The gene expression modulation system according to claim 11, wherein the ligand binding domain of the second polypeptide is encoded by a polynucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:

1, SEQ ID NO;

2, SEQ ID NO;

3, SEQ ID NO;

4, SEQ ID NO;

5, SEQ ID NO;

6, SEQ ID NO;

7, SEQ ID NO;

8, SEQ ID NO;

9, and SEQ ID NO;

10.

16. The gene expression modulation system according to claim 11, wherein the ligand binding domain of the second polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:

11, SEQ ID NO;

12, SEQ ID NO;

13, SEQ ID NO;

14, SEQ ID NO;

15, SEQ ID NO;

16, SEQ ID NO;

17, SEQ ID NO;

18, SEQ ID NO;

19, and SEQ ID NO;

20.

17. A gene expression cassette comprising a polynucleotide encoding a hybrid polypeptide comprising a DNA-binding domain or a transactivation domain, and an ecdysone receptor ligand binding domain, wherein the DNA binding domain or the transactivation domain is from a nuclear receptor other than an ecdysone receptor.

18. A gene expression cassette comprising a polynucleotide encoding a hybrid polypeptide comprising a DNA-binding domain or a transactivation domain, and a retinoid X receptor ligand binding domain, wherein the DNA binding domain or the transactivation domain is from a nuclear receptor other than a retinoid X receptor.

19. A gene expression cassette comprising a polynucleotide encoding a hybrid polypeptide comprising either a) a DNA-binding domain encoded by a polynucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:

41, SEQ ID NO;

43, and SEQ ID NO;

45, or b) a transactivation domain encoded by a polynucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO;

47, SEQ ID NO;

49, SEQ ID NO;

51, and SEQ ID NO;

53, and a ligand binding domain encoded by a polynucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO;

1, SEQ ID NO;

2, SEQ ID NO;

3, SEQ ID NO;

4, SEQ ID NO;

5, SEQ ID NO;

6, SEQ ID NO;

7, SEQ ID NO;

8, SEQ ID NO;

9, SEQ ID NO;

10, SEQ ID NO;

21, SEQ ID NO;

22, SEQ ID NO;

23, SEQ ID NO;

24, SEQ ID NO;

25, SEQ ID NO;

26, SEQ ID NO;

27, SEQ ID NO;

28, SEQ ID NO;

29, and SEQ ID NO;

30.

20. A gene expression cassette comprising a polynucleotide encoding a hybrid polypeptide comprising either a) a DNA-binding domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:

42, SEQ ID NO;

44, and SEQ ID NO;

46, or b) a transactivation domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO;

48, SEQ ID NO;

50, SEQ ID NO;

52, and SEQ ID NO;

54, and a ligand binding domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO;

11, SEQ ID NO;

12, SEQ ID NO;

13, SEQ ID NO;

14, SEQ ID NO;

15, SEQ ID NO;

16, SEQ ID NO;

17, SEQ ID NO;

18, SEQ ID NO;

19, SEQ ID NO;

20, SEQ ID NO;

31, SEQ ID NO;

32, SEQ ID NO;

33, SEQ ID NO;

34, SEQ ID NO;

35, SEQ ID NO;

36, SEQ ID NO;

37, SEQ ID NO;

38, SEQ ID NO;

39, and SEQ ID NO;

40.

21. An isolated polynucleotide encoding an ecdysone receptor polypeptide or a retinoid X receptor polypeptide comprising a truncation mutation, wherein the truncation mutation reduces ligand binding activity of the ecdysone receptor polypeptide or the retinoid X receptor polypeptide.

22. An isolated polynucleotide encoding an ecdysone receptor polypeptide or a retinoid X receptor polypeptide comprising a truncation mutation, wherein the truncation mutation enhances ligand binding activity of the ecdysone receptor polypeptide or the retinoid X receptor polypeptide.

25. The isolated polynucleotide according to claim 24, wherein the dimerization partner is an ecdysone receptor polypeptide.

27. An isolated polypeptide encoded by the isolated polynucleotide according to claim 26.

23. An isolated polynucleotide encoding a retinoid X receptor polypeptide comprising a truncation mutation, wherein the truncation mutation increases ligand sensitivity of the retinoid X receptor polypeptide.

24. An isolated polynucleotide encoding a retinoid X receptor polypeptide comprising a truncation mutation, wherein the truncation mutation increases ligand sensitivity of a heterodimer, wherein the heterodimer comprises said retinoid X receptor polypeptide and a dimerization partner.

26. An isolated polynucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:

1, SEQ ID NO;

2, SEQ ID NO;

3, SEQ ID NO;

4, SEQ ID NO;

5, SEQ ID NO;

6, SEQ ID NO;

7, SEQ ID NO;

8, SEQ ID NO;

9, SEQ ID NO;

10, SEQ ID NO;

21, SEQ ID NO;

22, SEQ ID NO;

23, SEQ ID NO;

24, SEQ ID NO;

25, SEQ ID NO;

26, SEQ ID NO;

27, SEQ ID NO;

28, SEQ ID NO;

29, and SEQ ID NO;

30.

28. An isolated truncated ecdysone receptor polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:

11, SEQ ID NO;

12, SEQ ID NO;

13, SEQ ID NO;

14, SEQ ID NO;

15, SEQ ID NO;

16, SEQ ID NO;

17, SEQ ID NO;

18, SEQ ID NO;

19, and SEQ ID NO;

20.

29. An isolated truncated retinoid X receptor polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:

31, SEQ ID NO;

32, SEQ ID NO;

33, SEQ ID NO;

34, SEQ ID NO;

35, SEQ ID NO;

36, SEQ ID NO;

37, SEQ ID NO;

38, SEQ ID NO;

39, and SEQ ID NO;

40.