Chimeric Retinoid X Receptors and Their Use in a Novel Ecdysone 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/chimeric 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 sequence 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 sequence that encodes a second hybrid polypeptide comprising:
 i) a transactivation domain; and 
 ii) a chimeric retinoid X receptor ligand binding domain. 
   
     
     
         2 . (canceled) 
     
     
         3 . The gene expression modulation system according to  claim 1 , wherein the ecdysone receptor ligand binding domain of the first hybrid polypeptide is selected from the group consisting of a spruce budworm  Choristoneura fumiferana  ecdysone receptor ligand binding domain, a beetle  Tenebrio monitor  ecdysone receptor ligand binding domain, a  Manduca sexta  ecdysone receptor ligand binding domain, a  Heliothies virescens  ecdysone receptor ligand binding domain LBD, a midge  Chironomus tentans  ecdysone receptor ligand binding domain LBD, 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  ecdysone receptor ligand binding domain, a fiddler crab  Celuca pugilator  ecdysone receptor ligand binding domain, a whitefly  Bamecia argentifoli  ecdysone receptor ligand binding domain, a leafhopper  Nephotetix cincticeps  ecdysone receptor ligand binding domain, and an ixodid tick  Amblyomma americanum  ecdysone receptor ligand binding domain. 
     
     
         4 . (canceled) 
     
     
         5 . (canceled) 
     
     
         6 . The gene expression modulation system according to  claim 1 , wherein the chimeric retinoid X receptor ligand binding domain of the second hybrid polypeptide comprises at least two different retinoid X receptor ligand binding domain fragments selected from the group consisting of a vertebrate species retinoid X receptor ligand binding domain fragment, an invertebrate species retinoid X receptor ligand binding domain fragment, and a non-Dipteran/non-Lepidopteran invertebrate species retinoid X receptor homolog ligand binding domain fragment. 
     
     
         7 .- 13 . (canceled) 
     
     
         14 . 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 sequence 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) a chimeric 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 sequence that encodes a second hybrid polypeptide comprising:
 i) a transactivation domain; and 
 ii) an ecdysone receptor ligand binding domain. 
   
     
     
         15 . (canceled) 
     
     
         16 . The gene expression modulation system according to  claim 14 , wherein the chimeric retinoid X receptor ligand binding domain of the first hybrid polypeptide comprises at least two different retinoid X receptor ligand binding domain fragments selected from the group consisting of a vertebrate species retinoid X receptor ligand binding domain fragment, an invertebrate species retinoid X receptor ligand binding domain fragment, and a non-Dipteran/non-Lepidopteran invertebrate species retinoid X receptor homolog ligand binding domain fragment. 
     
     
         17 .- 25 . (canceled) 
     
     
         26 . A gene expression cassette comprising a polynucleotide encoding a hybrid polypeptide comprising a DNA-binding domain and a chimeric retinoid X receptor ligand binding domain. 
     
     
         27 . The gene expression cassette according to  claim 26 , wherein the chimeric retinoid X receptor ligand binding domain comprises at least two different retinoid X receptor ligand binding domain fragments selected from the group consisting of a vertebrate species retinoid X receptor ligand binding domain fragment, an invertebrate species retinoid X receptor ligand binding domain fragment, and a non-Dipteran/non-Lepidopteran invertebrate species retinoid X receptor homolog ligand binding domain fragment. 
     
     
         28 .- 31 . (canceled) 
     
     
         32 . A gene expression cassette comprising a polynucleotide encoding a hybrid polypeptide comprising a transactivation domain and a chimeric retinoid X receptor ligand binding domain. 
     
     
         33 .- 35 . (canceled) 
     
     
         36 . An isolated polynucleotide selected from the group consisting of:
 (a) a polynucleotide encoding a truncated chimeric retinoid X receptor ligand binding domain comprising a truncation mutation, wherein the truncation mutation reduces ligand binding activity of the truncated chimeric retinoid X receptor ligand binding domain;   (b) a polynucleotide encoding a truncated chimeric retinoid X receptor ligand binding domain comprising a truncation mutation, wherein the truncation mutation reduces steroid binding activity of the truncated chimeric retinoid X receptor ligand binding domain;   (c) a polynucleotide encoding a truncated chimeric retinoid X receptor ligand binding domain comprising a truncation mutation, wherein the truncation mutation reduces non-steroid binding activity of the truncated chimeric retinoid X receptor ligand binding domain;   (d) a polynucleotide encoding a truncated chimeric retinoid X receptor ligand binding domain comprising a truncation mutation, wherein the truncation mutation enhances ligand binding activity of the truncated chimeric retinoid X receptor ligand binding domain;   (e) a polynucleotide encoding a truncated chimeric retinoid X receptor ligand binding domain comprising a truncation mutation, wherein the truncation mutation enhances steroid binding activity of the truncated chimeric retinoid X receptor ligand binding domain;   (f) a polynucleotide encoding a truncated chimeric retinoid X receptor ligand binding domain comprising a truncation mutation, wherein the truncation mutation enhances non-steroid binding activity of the truncated chimeric retinoid X receptor ligand binding domain;   (g) a polynucleotide encoding a truncated chimeric retinoid X receptor ligand binding domain comprising a truncation mutation, wherein the truncation mutation increases ligand sensitivity of the truncated chimeric retinoid X receptor ligand binding domain; and   (h) a polynucleotide encoding a truncated chimeric retinoid X receptor ligand binding domain comprising a truncation mutation, wherein the truncation mutation increases ligand sensitivity of a heterodimer, wherein the heterodimer comprises the truncated chimeric retinoid X receptor ligand binding domain and a dimerization partner.   
     
     
         37 .- 44 . (canceled) 
     
     
         45 . An isolated polynucleotide encoding a chimeric retinoid X receptor ligand binding domain, wherein the polynucleotide comprises a nucleic acid sequence selected from the group consisting of
 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,   c) nucleotides 1-465 of SEQ ID NO: 13 and nucleotides 403-630 of SEQ ID NO: 21,   d) nucleotides 1-555 of SEQ ID NO: 13 and nucleotides 490-630 of SEQ ID NO: 21,   e) nucleotides 1-624 of SEQ ID NO: 13 and nucleotides 547-630 of SEQ ID NO: 21,   f) nucleotides 1-645 of SEQ ID NO: 13 and nucleotides 601-630 of SEQ ID NO: 21, and   g) nucleotides 1-717 of SEQ ID NO: 13 and nucleotides 613-630 of SEQ ID NO: 21.   
     
     
         46 . (canceled) 
     
     
         47 . An isolated chimeric retinoid X receptor polypeptide comprising an amino acid sequence selected from the group consisting of
 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,   c) amino acids 1-155 of SEQ ID NO: 19 and amino acids 135-210 of SEQ ID NO: 27,   d) amino acids 1-185 of SEQ ID NO: 19 and amino acids 164-210 of SEQ ID NO: 27,   e) amino acids 1-208 of SEQ ID NO: 19 and amino acids 183-210 of SEQ ID NO: 27,   f) amino acids 1-215 of SEQ ID NO: 19 and amino acids 201-210 of SEQ ID NO: 27, and   g) amino acids 1-239 of SEQ ID NO: 19 and amino acids 205-210 of SEQ ID NO: 27.   
     
     
         48 . 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;   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 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; 
   whereby upon introduction of the ligand into the host cell, expression of the gene of b)iii) is modulated.   
     
     
         49 . The method according to  claim 48 , 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 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 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. 
       
     
     
         50 . (canceled) 
     
     
         51 . 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 of  claim 14 ; 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.   
     
     
         52 . The method according to  claim 51 , 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 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 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. 
       
     
     
         53 . (canceled) 
     
     
         54 . An isolated host cell comprising the gene expression modulation system according to  claim 1 . 
     
     
         55 . (canceled) 
     
     
         56 . (canceled) 
     
     
         57 . An isolated host cell comprising the gene expression modulation system according to  claim 14 . 
     
     
         58 . (canceled) 
     
     
         59 . (canceled) 
     
     
         60 . A non-human organism comprising the host cell of  claim 54 . 
     
     
         61 . (canceled) 
     
     
         62 . (canceled) 
     
     
         63 . A non-human organism comprising the host cell of  claim 57 . 
     
     
         64 . (canceled) 
     
     
         65 . (canceled) 
     
     
         66 . The non-human organism of  claim 60 , wherein said non-human organism is a non-transgenic organism. 
     
     
         67 . The non-human organism of  claim 63 , wherein said non-human organism is a non-transgenic organism.