Novel Ecdysone Receptor/Invertebrate Retinoid X Receptor-Based Inducible Gene Expression System

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 ecdysone receptor/invertebrate retinoid X receptor-based 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 organisms.

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 that encodes a first hybrid polypeptide comprising:
 i) a DNA-binding domain that recognizes a response element associated with a gene whose expression is to be modulated; and 
 ii) an ecdysone receptor ligand binding domain; and 
   b) a second gene expression cassette that is capable of being expressed in the host cell comprising a polynucleotide that encodes a second hybrid polypeptide comprising:
 i) a transactivation domain; and ii) an invertebrate retinoid X receptor ligand binding domain. 
   
     
     
         2 . The gene expression modulation system according to  claim 1 , further comprising a third gene expression cassette comprising:
 i) a response element recognized by the DNA-binding domain of the first hybrid polypeptide binds;   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.   
     
     
         3 . The gene expression modulation system according to  claim 1 , wherein the ecdysone receptor ligand binding domain (LBD) of the first hybrid polypeptide is selected from the group consisting of a spruce budworm  Choristoneura fumiferana  EcR (“CfEcR”) LBD, a beetle  Tenebrio molitor  EcR (“TmEcR”) LBD, a  Manduca sexta  EcR (“MsEcR”) LBD, a  Heliothies virescens  EcR, (“HvEcR”) LBD, a midge  Chironomus tentans  EcR (“CfEcR”) LBD, a silk moth  Bombyx mori  EcR, (“BmEcR”) LBD, a fruit fly  Drosophila melanogaster  EcR (“DmEcR”) LBD, a mosquito  Aedes aegypti  EcR (“AaEcR”) LBD, a blowfly  Lucilia capitata  (“LcEcR”) LBD, a blowfly  Lucilia cuprina  EcR (“LucEcR”) LBD, a Mediterranean fruit fly  Ceratitis capitata  EcR (“CcEcR”) LBD, a locust  Locusta migratoria  EcR (“LmEcR”) LBD, an aphid  Myzus persicae  EcR (“MpEcR”) LBD, a fiddler crab  Celuca pugilator  EcR (“CpEcR”) LBD, an ixodid tick  Amblyomma americanum  EcR (“AmaEcR”) LBD, a whitefly  Bamecia argentifoli  EcR (“BaEcR”) LBD, and a leafhopper  Nephotetix cincticeps  EcR (“NcEcR”) LBD. 
     
     
         4 . The gene expression modulation system according to  claim 1 , wherein the ecdysone receptor ligand binding domain of the first hybrid polypeptide is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 53 and SEQ ID NO: 45. 
     
     
         5 . The gene expression modulation system according to  claim 1 , wherein the ecdysone receptor ligand binding domain of the first hybrid polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO 43, and SEQ ID NO: 59. 
     
     
         6 . The gene expression modulation system according to  claim 1 , wherein the invertebrate retinoid X receptor ligand binding domain of the second hybrid polypeptide is encoded by a polynucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, 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. 
     
     
         7 . The gene expression modulation system according to  claim 1 , wherein the invertebrate retinoid X receptor ligand binding domain of the second hybrid polypeptide comprises an amino 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, SEQ ID NO: 30. SEQ ID NO: 31, and SEQ ID NO: 32. 
     
     
         8 . The gene expression modulation system according to  claim 1 , wherein the first gene expression cassette comprises a polynucleotide that encodes a first hybrid polypeptide comprising a DNA-binding domain selected from the group consisting of a GAL4 DNA-binding domain and a LexA DNA-binding domain, and an ecdysone receptor ligand binding domain. 
     
     
         9 . The gene expression modulation system according to  claim 1 , wherein the second gene expression cassette comprises a polynucleotide that encodes a second hybrid polypeptide comprising a transactivation domain selected from the group consisting of a VP16 transactivation domain and a B42 acidic activator transactivation domain, and an invertebrate retinoid X receptor ligand binding domain. 
     
     
         10 . The gene expression modulation system according to  claim 1 , wherein the second gene expression cassette comprises a polynucleotide that encodes a second hybrid polypeptide comprising a transactivation domain encoded by a polynucleotide comprising a nucleic acid sequence selected from the group consisting of a VP16 AD (SEQ ID NO: 37) and a B42 AD (SEQ ID NO: 39), and an invertebrate retinoid X receptor ligand binding domain encoded by a polynucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, 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. 
     
     
         11 . The gene expression modulation system according to  claim 1 , wherein the second gene expression cassette comprises a polynucleotide that encodes a second hybrid polypeptide comprising a transactivation domain comprising an amino acid sequence selected from the group consisting of a VP16 AD (SEQ ID NO: 38) and a B42 AD (SEQ ID NO: 40), and an invertebrate retinoid X receptor ligand binding domain comprising an amino 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, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32. 
     
     
         12 . 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 that encodes a first hybrid polypeptide comprising:
 i) a DNA-binding domain that recognizes a response element associated with a gene whose expression is to be modulated; and 
 ii) an invertebrate retinoid X receptor ligand binding domain; and 
   b) a second gene expression cassette that is capable of being expressed in the host cell comprising a polynucleotide that encodes a second hybrid polypeptide comprising:
 i) a transactivation domain; and 
 ii) an ecdysone receptor ligand binding domain. 
   
     
     
         13 . The gene expression modulation system according to  claim 12 , further comprising a third gene expression cassette comprising:
 i) a response element recognized by the DNA-binding domain of the first hybrid polypeptide binds;   ii) 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.   
     
     
         14 . The gene expression modulation system according to  claim 12 , wherein the invertebrate retinoid X receptor ligand binding domain of the first hybrid polypeptide is encoded by a polynucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, 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. 
     
     
         15 . The gene expression modulation system according to  claim 12 , wherein the invertebrate retinoid X receptor ligand binding domain of the first hybrid polypeptide comprises an amino 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, SEQ ID NO: 30, SEQ ID NO 31, and SEQ ID NO: 32. 
     
     
         16 . The gene expression modulation system according to  claim 12 , wherein the ecdysone receptor ligand binding domain of the second hybrid polypeptide is selected from the group consisting of a spruce budworm  Choristoneura fumiferana  EcR (“CfEcR”) LBD, a beetle  Tenebrio molitor  EcR (“TmEcR”) LBD, a  Manduca sexta  EcR (“MsEcR”) LBD, a  Heliothies virescens  EcR (“HvEcR”) LBD, a midge  Chironomus tentans  EcR (“CfEcR”) LBD, a silk moth  Bombyx mori  EcR (“BmEcR”) LBD, a fruit fly  Drosophila melanogaster  EcR (“DmEcR”) LBD, a mosquito  Aedes aegypti  EcR (“AaEcR”) LBD, a blowfly  Lucilia capitata  (“LcEcR”) LBD, a blowfly  Lucilia cuprina  EcR (“LucEcR”) LBD, a Mediterranean fruit fly  Ceratitis capitata  EcR (“CcEcR”) LBD, a locust  Locusta migratoria  EcR (“LmEcR”) LBD, an aphid  Myzus persicae  EcR (“MpEcR”) LBD, a fiddler crab  Celuca pugilator  ErR (“CpEcR”) LBD, an ixodid tick  Amblyomma americanum  EcR (“AmaEcR”) LBD, a whitefly  Bamecia argentifoli  EcR (“BaEcR”) LBD, and a leafhopper  Nephotetix cincticeps  EcR (“NcEcR”) LBD. 
     
     
         17 . The gene expression modulation system according to  claim 12 , wherein the ecdysone receptor ligand binding domain of the second hybrid polypeptide is encoded by a polynucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 53 and SEQ ID NO: 45. 
     
     
         18 . The gene expression modulation system according to  claim 12 , wherein the ecdysone receptor ligand binding domain of the second hybrid polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 43, and SEQ ID NO: 59. 
     
     
         19 . The gene expression modulation system according to  claim 12 , wherein the first gene expression cassette comprises a polynucleotide that encodes a first hybrid polypeptide comprising a DNA-binding domain selected from the group consisting of a GAL4 DNA-binding domain and a LexA DNA-binding domain, and an invertebrate retinoid X receptor ligand binding domain. 
     
     
         20 . The gene expression modulation system according to  claim 12 , wherein the first gene expression cassette comprises a polynucleotide that encodes a first hybrid polypeptide comprising a DNA-binding domain encoded by a polynucleotide comprising a nucleic acid sequence selected from the group consisting of a GAL4 DBD (SEQ ID NO: 33) or a LexA DBD (SEQ ID NO: 35) and an invertebrate retinoid X receptor ligand binding domain encoded by a polynucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO 10, 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. 
     
     
         21 . The gene expression modulation system according to  claim 12 , wherein the first gene expression cassette comprises a polynucleotide that encodes a first hybrid polypeptide comprising a DNA-binding domain comprising an amino acid sequence selected from the group consisting of a GAL4 DBD (SEQ ID NO: 34) and a LexA DBD (SEQ ID NO: 36), and an invertebrate retinoid X receptor ligand binding domain comprising an amino 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, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32. 
     
     
         22 . The gene expression modulation system according to  claim 12 , wherein the second gene expression cassette comprises a polynucleotide that encodes a second hybrid polypeptide comprising a transactivation domain selected from the group consisting of a VP 16 transactivation domain and a B42 acidic activator transactivation domain, and an ecdysone receptor ligand binding domain. 
     
     
         23 .- 39 . (canceled) 
     
     
         40 . An isolated polynucleotide encoding a truncated invertebrate retinoid X receptor ligand binding domain, wherein the polynucleotide comprises a nucleic acid sequence selected from the group consisting of 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. 
     
     
         41 . An isolated polypeptide encoded by the isolated polynucleotide according to  claim 40 . 
     
     
         42 . An isolated truncated invertebrate retinoid X receptor ligand binding domain comprising an amino acid sequence selected from the group consisting of SEQ ID NO 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32. 
     
     
         43 . A method of modulating the expression of a gene in a host cell 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;   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.   
     
     
         44 . The method according to  claim 43 , 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, 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 2 OH, CH 2 OMe, CH 2 CN, 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 3 , OCF 2 CF 2 H, COEt, cyclopropyl, CF 2 CF 3 , CH═CHCN, allyl, azido, OCF 3 , 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 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 2 , CH 2 F, CH 2 Cl, CH 2 OH, CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, OMe, OEt, SMe, or SEt. 
 
     
     
         45 . The method according to  claim 43 , 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. 
     
     
         46 . A method of modulating the expression of a gene in a host cell comprising the steps of
 a) introducing into the host cell the gene expression modulation system of  claim 12 ; 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. 
   
     
     
         47 . The method according to  claim 46 , 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, 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 , CH 2 F, CH 2 Cl, CH 2 OH, CH 2 OMe, CH 2 CN, 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 3 , OCF 2 CF 2 H, COEt, cyclopropyl, CF 2 CF 3 , CH═CHCN, allyl, azido, OCF 3 , 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 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 2 , CH 2 F, CH 2 Cl, CH 2 OH, CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, OMe, OEt, SMe, or SEt. 
       
     
     
         48 . The method according to  claim 46 , 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. 
     
     
         49 . An isolated host cell comprising the gene expression modulation system according to  claim 1 . 
     
     
         50 . The isolated host cell according to  claim 49 , wherein the host cell is selected from the group consisting of a bacterial cell, a fungal cell, a yeast cell, an animal cell, and a mammalian cell. 
     
     
         51 . (canceled) 
     
     
         52 . An isolated host cell comprising the gene expression modulation system according to  claim 12 . 
     
     
         53 . The isolated host cell according to  claim 52 , wherein the host cell is selected from the group consisting of a bacterial cell, a fungal cell, a yeast cell, an animal cell, and a mammalian cell. 
     
     
         54 . (canceled) 
     
     
         55 . A non-human organism comprising the host cell of  claim 52 . 
     
     
         56 . The non-human organism according to  claim 55 , wherein the non-human organism is selected from the group consisting of a bacterium, a fungus, a yeast, an animal, and a mammal. 
     
     
         57 .- 60 . (canceled)