Mutant Receptors and Their Use in a Nuclear 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 novel substitution mutant receptors and their use in a nuclear receptor-based inducible gene expression system and methods of modulating the expression of a gene 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 .- 21 . (canceled) 
     
     
         22 . A recombinant gene expression modulation system comprising a recombinant gene expression cassette that is capable of being expressed in a host cell, the gene expression cassette comprising a polynucleotide that encodes a first polypeptide comprising:
 (a) a transactivation domain;   (b) a DNA-binding domain that recognizes a response element associated with a gene whose expression is to be modulated; and   (c) a nuclear receptor ligand binding domain that is capable of binding a diacylhydrazine,   wherein said nuclear receptor ligand binding domain comprises either an amino acid residue substitution mutation at amino residue 128 of SEQ ID NO: 1, or an amino acid residue substitution mutation at each of amino acid residues 110 and 128 of SEQ ID NO: 1.   
     
     
         23 . The recombinant gene expression modulation system according to  claim 22 , wherein the substitution mutation of SEQ ID NO: 1 is either V128F or V128F/A110P. 
     
     
         24 . The recombinant gene expression modulation system according to  claim 22 , further comprising a second polynucleotide that encodes a second polypeptide, said second polypeptide comprising a second nuclear receptor ligand binding domain which is a vertebrate retinoid X receptor ligand binding domain, an invertebrate retinoid X receptor ligand binding domain, an ultraspiracle protein ligand binding domain, or a chimeric ligand binding domain comprising a first polypeptide fragment and a second polypeptide fragment, wherein said first polypeptide fragment is different from said second polypeptide fragment and each of the said first and said second fragments is a vertebrate retinoid X receptor ligand binding domain, an invertebrate retinoid X receptor ligand binding domain, or an ultraspiracle protein ligand binding domain. 
     
     
         25 . The recombinant gene expression modulation system according to  claim 22 , wherein said nuclear receptor ligand binding domain that comprises an amino acid residue substitution mutation is an ecdysone receptor ligand binding domain, a ubiquitous receptor ligand binding domain, an orphan receptor 1 ligand binding domain, a NER-1 ligand binding domain, a steroid hormone nuclear receptor 1 ligand binding domain, a retinoid X receptor interacting protein-15 ligand binding domain, a liver X receptor β ligand binding domain, a steroid hormone receptor like protein ligand binding domain, a liver X receptor ligand binding domain, a liver X receptor α ligand binding domain, a farnesoid X receptor ligand binding domain, a receptor interacting protein 14 ligand binding domain, or a farnesol receptor ligand binding domain. 
     
     
         26 . A vector comprising the gene expression modulation system of  claim 22 . 
     
     
         27 . A host cell comprising the vector of  claim 26 . 
     
     
         28 . A recombinant gene expression modulation system comprising:
 (a) a first recombinant gene expression cassette that is capable of being expressed in a host cell comprising a polynucleotide that encodes 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 first nuclear receptor ligand binding domain; and 
   (b) a second recombinant gene expression cassette that is capable of being expressed in the host cell comprising a polynucleotide that encodes a second polypeptide comprising:
 (i) a transactivation domain; and 
 (ii) a second nuclear receptor ligand binding domain, 
   wherein one of said first and said second nuclear receptor ligand binding domains is capable of binding a diacylhydrazine, and comprises either an amino acid residue substitution mutation at amino residue 128 of SEQ ID NO: 1, or an amino acid residue substitution mutation at each of amino acid residues 110 and 128 of SEQ ID NO: 1.   
     
     
         29 . The recombinant gene expression modulation system according to  claim 28 , wherein the substitution mutation of SEQ ID NO: 1 is either V128F or V128F/A110P. 
     
     
         30 . The recombinant gene expression modulation system according to  claim 28 , wherein said nuclear receptor ligand binding domain that is capable of binding a diacylhydrazine is an ecdysone receptor ligand binding domain, a ubiquitous receptor ligand binding domain, an orphan receptor 1 ligand binding domain, a NER-1 ligand binding domain, a steroid hormone nuclear receptor 1 ligand binding domain, a retinoid X receptor interacting protein-15 ligand binding domain, a liver X receptor β ligand binding domain, a steroid hormone receptor like protein ligand binding domain, a liver X receptor ligand binding domain, a liver X receptor α ligand binding domain, a farnesoid X receptor ligand binding domain, a receptor interacting protein 14 ligand binding domain, or a farnesol receptor ligand binding domain. 
     
     
         31 . The recombinant gene expression modulation system according to  claim 28 , wherein the other of said first and second nuclear receptor ligand binding domains is a vertebrate retinoid X receptor ligand binding domain, an invertebrate retinoid X receptor ligand binding domain, an ultraspiracle protein ligand binding domain, or a chimeric ligand binding domain comprising a first polypeptide fragment and a second polypeptide fragment, wherein said first polypeptide fragment is different from said second polypeptide fragment and each of the said first and said second fragments is a vertebrate retinoid X receptor ligand binding domain, an invertebrate retinoid X receptor ligand binding domain, or an ultraspiracle protein ligand binding domain. 
     
     
         32 . The recombinant gene expression modulation system according to  claim 28 , wherein the DNA-binding domain is an ecdysone receptor DNA-binding domain, a GAL4 DNA-binding domain, or a LexA DNA-binding domain. 
     
     
         33 . The recombinant gene expression modulation system according to  claim 28 , wherein the transactivation domain is an ecdysone receptor transactivation domain, a VP 16 transactivation domain, a B42 acidic activator transactivation domain, or a p65 transactivation domain. 
     
     
         34 . A vector comprising the gene expression modulation system of  claim 28 . 
     
     
         35 . The vector of  claim 34 , wherein said vector is an expression vector. 
     
     
         36 . The vector of  claim 34 , wherein said vector is a plasmid. 
     
     
         37 . The vector of  claim 34 , wherein said vector is a viral vector. 
     
     
         38 . The vector of  claim 37 , wherein said vector is an adenoviral vector. 
     
     
         39 . The gene expression modulation system of  claim 28 , wherein said nuclear receptor ligand binding domain that comprises an amino acid residue substitution mutation is an ecdysone receptor ligand binding domain. 
     
     
         40 . The gene expression modulation system of  claim 39 , wherein said ecdysone receptor ligand binding domain is an invertebrate ecdysone receptor ligand binding domain, an Arthropod ecdysone receptor ligand binding domain, a Lepidopteran ecdysone receptor ligand binding domain, a Dipteran ecdysone receptor ligand binding domain, an Orthopteran ecdysone receptor ligand binding domain, a Homopteran ecdysone receptor ligand binding domain, a Hemipteran ecdysone receptor ligand binding domain, 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 squinting bush brown  Bicyclus anynana  ecdysone receptor ligand binding domain, a buckeye  Junonia coenia  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 blowfly  Calliphora vicinia  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, an ixodid tick  Amblyomma americanum  ecdysone receptor ligand binding domain, a whitefly  Bamecia argentifoli  ecdysone receptor ligand binding domain or a leafhopper  Nephotetix cincticeps  ecdysone receptor ligand binding domain. 
     
     
         41 . The gene expression modulation system of  claim 40 , wherein said ecdysone receptor ligand binding domain is a spruce budworm  Choristoneura fumiferana  ecdysone receptor ligand binding domain. 
     
     
         42 . The gene expression modulation system of  claim 28 , wherein said gene expression modulation system further comprising a gene expression cassette comprising:
 (a) a response element recognized by the DNA binding domain;   (b) a promoter that is activated by the transactivation domain; and   (c) a gene whose expression is to be modulated   
     
     
         43 . A method of modulating the expression of a gene in a host cell, said method comprising:
 (a) introducing into the host cell the gene expression modulation system of  claim 42 ; and   (b) introducing into the host cell a ligand;   whereby upon introduction of the ligand into the host cell, expression of the gene whose expression is to be modulated is induced, compared to the expression of said gene by a gene expression modulation system in which the same nuclear receptor ligand binding domain that is capable of binding a diacylhydrazine does not contain said mutation.   
     
     
         44 . The method according to  claim 43 , wherein the ligand is selected from the group consisting of:
 a) a compound of the formula:   
       
         
           
           
               
               
           
         
       
       wherein:
 E is a branched (C 4 -C 12 )alkyl or branched (C 4 -C 12 )alkenyl containing a tertiary carbon or a cyano(C 3 -C 12 )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 CI, 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 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 O, CN, C≡CH, 1-propynyl, 2-propynyl, vinyl, OMe, OEt, SMe, or SEt; 
 b) a compound of the formula: 
 
       
         
           
           
               
               
           
         
         wherein: 
         R 1  is CH 2 CH 3  or CH 3 ; 
         R 2  is OCH 3 , CH 2 CH 3  or i-Pr; and 
         R 3  and R 4  are CH 3 ; 
         c) a compound of the formula: 
       
       
         
           
           
               
               
           
         
         wherein: R 1  and R 2  are F; and 
         R 3  is 3-F-4-CH 3 —Ph or 3-CH 3 -4-F-Ph; or 
         d) 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, 7-ketocholesterol-3-sulfate, farnesol, a bile acid, a 1,1-biphosphonate ester, or a Juvenile hormone III. 
       
     
     
         45 . The method according to  claim 44 , 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 . An isolated polynucleotide encoding a nuclear receptor ligand binding domain that is capable of binding a diacylhydrazine,
 wherein said nuclear receptor ligand binding domain comprises either an amino acid residue substitution mutation at amino residue 128 of SEQ ID NO: 1, or an amino acid residue substitution mutation at each of amino acid residues 110 and 128 of SEQ ID NO: 1.   
     
     
         47 . The isolated polynucleotide according to  claim 46 , wherein the substitution mutation of SEQ ID NO: 1 is either V128F or V128F/A110P. 
     
     
         48 . A vector comprising the isolated polynucleotide of  claim 46 . 
     
     
         49 . The vector of  claim 48 , wherein said vector is an expression vector. 
     
     
         50 . The vector of  claim 48 , wherein said vector is a plasmid. 
     
     
         51 . The vector of  claim 48 , wherein said vector is a viral vector. 
     
     
         52 . The vector of  claim 51 , wherein said vector is an adenoviral vector. 
     
     
         53 . The isolated polynucleotide of  claim 46 , wherein said nuclear receptor ligand binding domain that comprises an amino acid residue substitution mutation is an ecdysone receptor ligand binding domain. 
     
     
         54 . The isolated polynucleotide of  claim 53 , wherein said ecdysone receptor ligand binding domain is selected from the group consisting of: an invertebrate ecdysone receptor ligand binding domain, an Arthropod ecdysone receptor ligand binding domain, a Lepidopteran ecdysone receptor ligand binding domain, a Dipteran ecdysone receptor ligand binding domain, an Orthopteran ecdysone receptor ligand binding domain, a Homopteran ecdysone receptor ligand binding domain, a Hemipteran ecdysone receptor ligand binding domain, 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 squinting bush brown  Bicyclus anynana  ecdysone receptor ligand binding domain, a buckeye  Junonia coenia  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 blowfly  Calliphora vicinia  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, an ixodid tick  Amblyomma americanum  ecdysone receptor ligand binding domain, a whitefly  Bamecia argentifoli  ecdysone receptor ligand binding domain and a leafhopper  Nephotetix cincticeps  ecdysone receptor ligand binding domain. 
     
     
         55 . The isolated polynucleotide of  claim 54 , wherein said ecdysone receptor ligand binding domain is a spruce budworm  Choristoneura fumiferana  ecdysone receptor ligand binding domain.