Method of modulating gene expression using an ecdysone receptor-based inducible gene expression system

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

a) introducing into the host cell a gene expression modulation system comprising

i) a first gene expression cassette that is capable of being expressed in a host cell comprising a polynucleotide sequence that encodes a first hybrid polypeptide comprising:

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

(B) an ecdysone receptor ligand binding domain; and

ii) a second gene expression cassette that is capable of being expressed in the host cell comprising a polynucleotide sequence that encodes a second hybrid polypeptide comprising:

(A) a transactivation domain; and

(B) a chimeric retinoid X receptor ligand binding domain, comprising

(i) helices 1-6 of a vertebrate retinoid X receptor and helices 7-12 of an invertebrate retinoid X receptor,

(ii) helices 1-7 of a vertebrate retinoid X receptor and helices 8-12 of an invertebrate retinoid X receptor or

(iii) helices 1-8 of a vertebrate retinoid X receptor and helices 9-12 of an invertebrate retinoid X receptor; and

b) introducing into the host cell a ligand; wherein the gene to be modulated is a component of a gene expression cassette comprising:

i) a response element recognized by the DNA binding domain from the first hybrid polypeptide;

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

iii) a gene whose expression is to be modulated; whereby upon introduction of the ligand into the host cell, expression of the gene of b)iii) is modulated.

2. The method according to claim 1 , wherein the ligand a compound of the formula:

wherein:

E is a (C 4 -C 6 )alkyl containing a tertiary carbon or a cyano(C 3 -C 5 )alkyl containing a tertiary carbon;

R 1 is H, Me, Et, i-Pr, F, formyl, CF 3 , CHF 2 , CHCl 2 , CH 2 F, CH 2 Cl, CH 3 OH, CH 2 OMe, CH 2 CN, CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, OH, OMe, OEt, cyclopropyl, CF 2 CF 3 , CH═CHCN, allyl, azido, SCN, or SCHF 2 ;

R 2 is H, Me, Et, n-Pr, i-Pr, formyl, CF 3 , CHF 2 , CHCl 2 , CH 2 F, CH 2 Cl, CH 3 OH, CH 3 OMe, CH 2 N, CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, Ac, F, Cl, OH, OMe, OEt, O-n-Pr, OAc, NMe 2 , NEt 2 , SMe, SEt, SOCF 1 , OCF 7 CF 2 H, COEt, cyclopropyl, CF 2 CF 3 , CH═CHCN, allyl, azido, OCF 3 , OCHF 2 , O-i-Pr, SCN, SCHF 7 , SOMe, NH—CN, or joined with R 3 and the phenyl carbons to which R 2 and R 3 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;

R 3 is H, Et, or joined with R 2 and the phenyl carbons to which R 2 and R 3 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;

R 4 , R 5 , and R 6 are independently H, Me, Et, F, Cl, Br, formyl, CF 3 , CHF 2 , CHCl 7 , CH 2 F, CH 2 Cl, CH 2 OH, CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, OMe, OEt, SMe, or SEt.

3. The method according to claim 1 , further comprising introducing into the host cell a second ligand, wherein the second ligand is 9-cis-retinoic acid or a synthetic analog of a retinoic acid.

4. The method according to claim 1 , wherein said gene expression modulation system further comprises a third gene expression cassette comprising:

i) a response element recognized by the DNA-binding domain of the first hybrid polypeptide;

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

iii) a gene whose expression is to be modulated.

5. The method according to claim 1 , wherein the ecdysone receptor ligand binding domain of the first hybrid polypeptide is a spruce budworm Choristoneura fumiferana EcR ligand binding domain or a fruit fly Drosophila melanogaster EcR ligand binding domain.

6. The method according to claim 1 , wherein the ecdysone receptor ligand binding domain of the first hybrid polypeptide is encoded by a polynucleotide comprising SEQ ID NO: 65 (CfEcR-DEF), SEQ ID NO: 59 (CfEcR-CDEF) and SEQ ID NO: 67 (DmEcR-DEF).

7. The method according to claim 1 , wherein the ecdysone receptor ligand binding domain of the first hybrid polypeptide comprises the amino acid sequence of SEQ ID NO: 57 (CfEcR-DEF), SEQ ID NO: 58 (DmEcR-DEF) and SEQ ID NO: 70 (CfEcR-CDEF).

8. The method according to claim 1 , wherein the chimeric retinoid X receptor ligand binding domain of the second hybrid polypeptide is encoded by a polynucleotide comprising a nucleic acid sequence selected from

a) nucleotides 1-348 of SEQ ID NO: 13 and nucleotides 268-630 of SEQ ID NO: 21,

b) nucleotides 1-408 of SEQ ID NO: 13 and nucleotides 337-630 of SEQ ID NO: 21, and

c) nucleotides 1-465 of SEQ ID NO: 13 and nucleotides 403-630 of SEQ ID NO: 21.

9. The method according to claim 1 , wherein the chimeric retinoid X receptor ligand binding domain of the second hybrid polypeptide comprises an amino acid sequence selected from

a) amino acids 1-116 of SEQ ID NO: 19 and amino acids 90-210 of SEQ ID NO: 27,

b) amino acids 1-136 of SEQ ID NO: 19 and amino acids 113-210 of SEQ ID NO: 27, and

c) amino acids 1-155 of SEQ ID NO: 19 and amino acids 135-210 of SEQ ID NO: 27.

10. The method according to claim 1 , wherein the first gene expression cassette comprises a polynucleotide sequence that encodes the first hybrid polypeptide comprising a DNA-binding domain selected from a GAL4 DNA-binding domain, a LexA DNA-binding domain, and an ecdysone receptor ligand binding domain.

11. The method according to claim 1 , wherein the second gene expression cassette comprises a polynucleotide that encodes the second hybrid polypeptide comprising a transactivation domain selected from VP16 transactivation domain, a B42 acidic activator transactivation domain, and a chimeric retinoid X receptor ligand binding domain.

12. The method of claim 1 , wherein the gene expression modulation system exhibits increased sensitivity to a non-steroid ligand than a gene expression modulation system that contains a vertebrate retinoid X receptor ligand binding domain.

13. The method of claim 1 , wherein said cell is selected from the group consisting of a bacterial cell, a fungal cell, a yeast cell, an animal cell and a mammalian cell.

14. The method of claim 13 , wherein said mammalian cell is selected from the group consisting of a murine cell, a monkey cell, a mouse a rat cell, a rabbit cell, a cat cell, a dog cell, a bovine cell, a goat cell, a pig cell, a horse cell, a sheep cell, a monkey cell, a chimpanzee cell and a human cell.

15. The method of claim 14 , wherein said mammalian cell a human.

16. The method of claim 1 , wherein said gene expression modulation system is contained in a vector.

17. The method of claim 16 , wherein said vector is a plasmid.

18. The method of claim 16 , wherein said vector is an expression plasmid.

19. The method of claim 16 , wherein said vector is a viral vector.

20. The method of claim 16 , wherein said vector is an adenovirus vector.

21. The method according to claim 1 , wherein the ecdysone receptor ligand binding domain is selected from the group consisting of a spruce bud worm Choristoneura fumiferana ecdysone receptor ligand binding domain, a beetle Tenebrio molitor ecdysone receptor ligand binding domain, a Manduca sexta ecdysone receptor ligand binding domain, a Heliothies virescens ecdysone receptor ligand binding domain, a midge Chironomus tentans ecdysone receptor ligand binding domain, a silk moth Bombyx mori ecdysone receptor ligand binding domain, a fruit fly Drosophila melanogaster ecdysone receptor ligand binding domain, a mosquito Aedes aegypti ecdysone receptor ligand binding domain, a blowfly Lucilia capitata ecdysone receptor ligand binding domain, a blowfly Lucilia cuprina ecdysone receptor ligand binding domain, a Mediterranean fruit fly Ceratitis capitata ecdysone receptor ligand binding domain, a locust Locusta migratoria ecdysone receptor ligand binding domain, an aphid Myzus persicae , a fiddler crab Celuca pugilator ecdysone receptor ligand binding domain, a whitefly Bamecia argentifoli , a leafhopper Nephotetix cincticeps ecdysone receptor ligand binding domain, and an ixodid tick Amblyomma americanum ecdysone receptor ligand binding domain.

22. The method according to claim 21 , Therein the ecdysone receptor ligand binding domain is a spruce budworm Choristoneura fumiferana ecdysone receptor ligand binding domain.

23. The method according to claim 1 , wherein the invertebrate retinoid X receptor helices are non-Dipteran/non-Lepidopteran helices.

24. The method according to claim 23 , wherein the invertebrate retinoid X receptor helices are locust Locusta migratoria ultraspiracle helices.

25. The method according to claim 1 , wherein vertebrate retinoid X receptor helices are Homo sapiens helices.

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

a) introducing into the host cell a gene expression modulation system comprising

i) a first gene expression cassette that is capable of being expressed in a host cell comprising a polynucleotide sequence that encodes a first hybrid polypeptide comprising:

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

(B) a chimeric retinoid X receptor ligand binding domain, comprising

(i) helices 1-6 of a vertebrate retinoid X receptor and helices 7-12 of an invertebrate retinoid X receptor,

(ii) helices 1-7 of a vertebrate retinoid X receptor and helices 8-12 of an invertebrate retinoid X receptor, or

(iii) helices 1-8 of a vertebrate retinoid X receptor and helices 9-12 of an invertebrate retinoid X receptor; and)

ii) second gene expression cassette that is capable of being expressed in the host cell comprising a polynucleotide sequence that encodes a second hybrid polypeptide comprising:

(A) a transactivation domain; and

(B) an ecdysone receptor ligand binding domain; and

b) introducing into the host cell a ligand;

wherein the gene to be modulated is a component of a gene expression cassette comprising:

i) a response element recognized by the DNA binding domain from the first hybrid polypeptide;

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

iii) a gene whose expression is to be modulated;

whereby upon introduction of the ligand into the host cell, expression of the gene of b)iii) is modulated.

27. The method according to claim 26 , wherein the ligand is a compound of the formula:

wherein:

E is a (C 4 -C 6 )alkyl containing a tertiary carbon or a cyano(C 3 -C 5 )alkyl containing a tertiary carbon;

R 1 is H, Me, Et, i-Pr, F, formyl, CF 3 , CHF 2 , CHCl 2 , CH 2 F, CH 2 Cl, CH 2 OH, CH 2 OMe, CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, OH, OMe, OEt, cyclopropyl, CF 2 CF 3 , CH═CHCN, allyl, SCN, or SCHF 2 ;

R 2 is H, Me, Et, n-Pr, i-Pr, formyl, CF 3 , CHF 2 , CHCl 2 , CH 2 F, CH 2 Cl, CH 2 OH, CH 2 OMe, CH 2 N, CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, Ac, F, Cl, OH, OMe, OEt, CH═CHCN, allyl, azido, OCHF 2 , O-i-Pr, SCN, SCHF 2 , SOMe, NH—CN, or joined with R 3 and the phenyl carbons to which R 2 and R 3 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;

R 3 is H, Et, or joined with R 2 and the phenyl carbons to which R 2 and R 3 are attached to form an ethylenedioxy, a dihydrofuryl ring with the oxygen adjacent o phenyl carbon, or a dihydropyryl ring with the oxygen adjacent to a phenyl carbon;

R 4 , R 5 and R 6 are independently H, Me, Et, F, Cl, Br, formyl, CF 3 CHF 2 , CHCl 2 , CH 2 F, CH 2 Cl, CH 2 OH, CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, OMe, OEt, SMe, or SEt.

28. The method according to claim 26 , further comprising introducing into the is host cell a second ligand, wherein the second ligand is 9-cis-retinoic acid or a synthetic analog of a retinoic acid.

29. The method according to claim 26 , wherein said gene expression modulation system further comprises a third gene expression cassette comprising:

i) a response element that recognizes the DNA-binding domain of the first hybrid polypeptide;

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

iii) a gene whose expression is to be modulated.

30. The method according to claim 26 , wherein the chimeric retinoid X receptor ligand binding domain of the first hybrid polypeptide is encoded by a polynucleotide comprising a nucleic acid sequence selected from

a) nucleotides 1-348 of SEQ ID NO: 13 and nucleotides 268-630 of SEQ ID NO: 21,

b) nucleotides 1-408 of SEQ ID NO: 13 and nucleotides 337-630 of SEQ ID NO: 21, and

c) nucleotides 1-465 of SEQ ID NO: 13 and nucleotides 403-630 of SEQ ID NO: 21.

31. The method according to claim 26 , wherein the chimeric retinoid X receptor ligand binding domain of the first hybrid polypeptide comprises an amino acid sequence selected from

a) amino acids 1-116 of SEQ ID NO: 19 and amino acids 90-210 of SEQ ID NO: 27,

b) amino acids 1-136 of SEQ ID NO: 19 and amino acids 113-210 of SEQ ID NO: 27, and

c) amino acids 1-155 of SEQ ID NO: 19 and amino acids 135-210 of SEQ ID NO: 27.

32. The method according to claim 20 wherein the ecdysone receptor ligand binding domain of the second hybrid polypeptide is encoded by a polynucleotide comprising a nucleic acid sequence selected from SEQ ID NO: 65 (CfEcR-DEF), SEQ ID NO 59 (CfEcR-CDEF) and SEQ ID NO 67 (DmEcR-DEF).

33. The method according to claim 26 , wherein the ecdysone receptor ligand binding domain of the second hybrid polypeptide comprises an amino acid sequence selected from SEQ ID NO: 57 (CfEcR-DEF), SEQ ID NO: 58 (DmEcR-DEF) and SEQ ID NO: 70 (CfEcR-CDEF).

34. The method according to claim 26 , wherein the first gene expression cassette comprises a polynucleotide that encodes the first hybrid polypeptide comprising a DNA-binding domain selected from a GAL4 DNA-binding domain and a LexA DNA-binding domain, and a chimeric retinoid X receptor ligand binding domain.

35. The method according to claim 26 , wherein the second gene expression cassette comprises a polynucleotide that encodes the second hybrid polypeptide comprising a transactivation domain selected from a VP16 transactivation domain and a B42 acidic activator transactivation domain, and an ecdysone receptor ligand binding domain.

36. The method of claim 26 , wherein the gene expression modulation system exhibits increased sensitivity to a non-steroid ligand than a gene expression modulation system that contains a vertebrate retinoid X receptor ligand binding domain.

37. The method of claim 26 , wherein said cell is selected from the group consisting of a bacterial cell, fungal cell, a yeast cell, an animal cell and a mammalian cell.

38. The method of claim 37 , wherein said mammalian cell is selected from the group consisting of a murine cell, a monkey cell, a mouse cell, a rat cell, a rabbit cell, a cat cell, a dog cell, a bovine cell, a goat cell, a pig cell, a horse cell, a sheep cell, a monkey cell, a chimpanzee cell and a human cell.

39. The method of claim 38 , wherein said mammalian cell a human cell.

40. The method of claim 26 , wherein said gene expression modulation system is contained in a vector.

41. The method of claim 40 , wherein said vector a plasmid.

42. The method of claim 40 , wherein said vector is an expression plasmid.

43. The method of claim 42 , wherein said vector is a viral vector.

44. The method of claim 43 , wherein said vector is an adenovirus vector.

45. The method according to claim 26 , wherein the ecdysone receptor ligand binding domain is selected from the group consisting of a spruce budworm Choristoneura fumiferana ecdysone receptor ligand binding domain, a beetle Tenebrio molitor ecdysone receptor ligand binding domain, a Manduca sexta ecdysone receptor ligand binding domain, a Heliothies virescens ecdysone receptor ligand binding domain, a midge Chironomus tentans ecdysone receptor ligand binding domain, a silk moth Bombyx mori ecdysone receptor ligand binding domain, a fruit fly Drosophila melanogaster ecdysone receptor ligand binding domain, a mosquito Aedes aegypti ecdysone receptor ligand binding domain, a blowfly Lucilia capitata ecdysone receptor ligand binding domain, a blowfly Lucilia cuprina ecdysone receptor ligand binding domain, a Mediterranean fruit fly Ceratitis capitata ecdysone receptor ligand binding domain, a locust Locusta migratoria ecdysone receptor ligand binding domain, an aphid Myzus persicae , a fiddler crab Celuca pugilator ecdysone receptor ligand binding domain, a whitefly Bamecia argentifoli , a leafhopper Nephotetix cincticeps ecdysone receptor ligand binding domain, and an ixodid tick Amblyomma americanum ecdysone receptor ligand binding domain.

46. The method according to claim 45 , Wherein the ecdysone receptor ligand binding domain is a spruce budworm Choristoneura fumiferana ecdysone receptor ligand binding domain.

47. The method according to claim 26 , wherein the invertebrate retinoid X receptor helices are non-Dipteran/non-Lepidopteran helices.

48. The method according to claim 47 , wherein the invertebrate retinoid X receptor helices are locust Locusta migratoria ultraspiracle helices.

49. The method according to claim 26 , wherein the vertebrate retinoid X receptor helices are Homo sapiens helices.