Novel Substitution 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 nuclear receptors comprising a substitution mutation and their use in a nuclear receptor-based inducible gene expression system and methods of modulating the expression of a gene within a host cell using this inducible gene expression system.

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 polypeptide comprising:
 i) a transactivation domain; 
 ii) a DNA-binding domain that recognizes a response element associated with a gene whose expression is to be modulated; and 
 iii) a Group B nuclear receptor ligand binding domain comprising a substitution mutation, and 
 b) a second gene expression cassette that is capable of being expressed in a host cell comprising a polynucleotide that encodes a second polypeptide comprising a nuclear receptor ligand binding domain selected from the group consisting of an ecdysone receptor, a ubiquitous receptor, an orphan receptor 1, a steroid hormone nuclear receptor 1, a retinoid X receptor interacting protein 15, a liver X receptor β, a steroid hormone receptor like protein, a liver X receptor, a liver X receptor α, a farnesoid X receptor, a receptor interacting protein 14, and a farnesol receptor. 
 
     
     
         2 . 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 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 nuclear 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 polypeptide comprising:
 i) a transactivation domain; and 
 ii) a nuclear receptor ligand binding domain 
   wherein one of the nuclear receptor ligand binding domains is a Group B nuclear receptor ligand binding domain comprising a substitution mutation.   
     
     
         3 . The gene expression modulation system according to  claim 1 , wherein the Group B nuclear receptor ligand binding domain is selected from the group consisting of a retinoid X receptor, an H-2 region II binding protein (H-2REIBP), a Nuclear Receptor co-regulator-1 (RCoR-1), an ultraspiracle protein, a 2C1 nuclear receptor, and a chorion factor 1 (CF-1). 
     
     
         4 . The gene expression modulation system according to  claim 3 , wherein the Group B nuclear receptor ligand binding domain is encoded by a polynucleotide comprising a codon mutation that results in a substitution of an amino acid residue, wherein the amino acid residue is at a position equivalent to or analogous to a) 401 or 429 of SEQ TD NO: 1, b) 401 and 429 of SEQ ED NO: 1, c) 337, 344, 355, 385, 431, 442, 462, 470, 472, 473, 495, 500, 511, 516, or 528 of SEQ ED NO: 2, d) 321, 322, and 323 of SEQ ID NO: 2, e) 450, 451, and 452 of SEQ ED NO: 2, f) 455, 456, 457, and 458 of SEQ TD NO: 2, g) 470, 472, and 473 of SEQ ED NO: 2, h) 475, 476, 477, 478, and 479 of SEQ ED NO: 2, and i) 481, 482, and 483 of SEQ ED NO: 2. 
     
     
         5 . The gene expression modulation system according to  claim 4 , wherein the Group B nuclear receptor ligand binding domain comprising a substitution mutation is a retinoid X receptor ligand binding domain. 
     
     
         6 . The gene expression modulation system according to  claim 5 , wherein the substitution mutation is selected from the group consisting of a) E401D or G429S of SEQ ID NO: 1, b) E401D and G429S of SEQ ED NO: 1, c) T337S, D344N, K355R, S385A, M431L, R442K, V462L, S470A, E471D, T473E, A495S, E500S, K511R, T516V, or A528S of SEQ ED NO: 2, d) G321L, P322R, and G323V of SEQ ED NO: 2, e) D450E, A451V, and K452R of SEQ ED NO: 2, f) S455K, N456S, P457A, and S458Q of SEQ ED NO: 2, g) S470A, E472D, and T473Y of SEQ TD NO: 2, h) C475T, K476R, Q477T, K478T, and Y479H of SEQ ED NO: 2, and i) E481D, Q482E, and Q483P of SEQ ED NO: 2. 
     
     
         7 . The gene expression modulation system according to  claim 1 , wherein the DNA-binding domain is selected from the group consisting of an ecdysone receptor DNA-binding domain, a GAL4 DNA-binding domain, and a LexA DNA-binding domain. 
     
     
         8 . The gene expression modulation system according to  claim 1 , wherein the transactivation domain is selected from the group consisting of an ecdysone receptor transactivation domain, a VP16 transactivation domain, a B42 acidic activator transactivation domain, and a p65 transactivation domain. 
     
     
         9 . A gene expression cassette comprising a polynucleotide that encodes a polypeptide selected from the group consisting of
 a) a polypeptide comprising a transactivation domain, a DNA-binding domain, and a Group B nuclear receptor ligand binding domain comprising a substitution mutation,   b) a polypeptide comprising a DNA-binding domain and a Group B nuclear receptor ligand binding domain comprising a substitution mutation, and   c) a polypeptide comprising a transactivation domain and a Group B nuclear receptor ligand binding domain comprising a substitution mutation.   
     
     
         10 . An isolated polynucleotide encoding a Group B nuclear receptor ligand binding domain comprising a substitution mutation, wherein the isolated polynucleotide comprises a codon mutation that results in a substitution of an amino acid residue at a position equivalent to or analogous to a) 401 or 429 of SEQ TD NO: 1, b) 401 and 429 of SEQ TD NO: 1, c) 337, 344, 355, 385, 431, 442, 462, 470, 472, 473, 495, 500, 511, 516, or 528 of SEQ ED NO: 2, d) 321, 322, and 323 of SEQ ED NO: 2, e) 450, 451, and 452 of SEQ ID NO: 2, f) 455, 456, 457, and 458 of SEQ ED NO: 2, g) 470, 472, and 473 of SEQ ED NO: 2, h) 475, 476, 477, 478, and 479 of SEQ ID NO: 2, and i) 481, 482, and 483 of SEQ ID NO: 2. 
     
     
         11 . The isolated polynucleotide according to  claim 10 , wherein the codon mutation results in a substitution mutation selected from the group consisting of a) E401D or G429S of SEQ ED NO: 1, b) E401D and G429S of SEQ ED NO: 1, c) T337S, D344N, K355R, S385A, M431L, R442K, V462L, S470A, E471D, T473E, A495S, E500S, K511R, T516V, or A528S of SEQ ID NO: 2, d) G321L, P322R, and G323V of SEQ ED NO: 2, e) D450E, A451V, and K452R of SEQ TD NO: 2, f) S455K, N456S, P457A, and S458Q of SEQ ED NO: 2, g) S470A, E472D, and T473Y of SEQ ED NO: 2, h) C475T, 5 K476R, Q477T, K478T, and Y479H of SEQ ED NO: 2, and i) E481D, Q482E, and Q483P of SEQ ED NO: 2. 
     
     
         12 . An expression vector comprising the isolated polynucleotide of  claim 10 , operatively linked to a transcription regulatory element. 
     
     
         13 . A host cell comprising the expression vector of  claim 12 , wherein the transcription regulatory element is operative in the host cell. 
     
     
         14 . An isolated polypeptide encoded by the isolated polynucleotide according to  claim 10 . 
     
     
         15 . An isolated polypeptide comprising a Group B nuclear receptor ligand binding domain comprising a substitution mutation, wherein the substitution mutation is at a position equivalent to or analogous to a) 401 or 429 of SEQ ED NO: 1, b) 401 and 429 of SEQ ID NO: 1, c) 337, 344, 355, 385, 15 431, 442, 462, 470, 472, 473, 495, 500, 511, 516, or 528 of SEQ ID NO: 2, d) 321, 322, and 323 of SEQ TD NO: 2, e) 450, 451, and 452 of SEQ ED NO: 2, f) 455, 456, 457, and 458 of SEQ D NO: 2, g) 470, 472, and 473 of SEQ ED NO: 2, h) 475, 476, 477, 478, and 479 of SEQ ID NO: 2, and i) 481, 482, and 483 of SEQ ID NO: 2. 
     
     
         16 . The isolated polypeptide according to  claim 15 , wherein the Group B nuclear receptor 20 ligand binding domain comprising a substitution mutation is a retinoid X receptor ligand binding domain. 
     
     
         17 . The isolated polypeptide according to  claim 16 , wherein the substitution mutation is selected from the group consisting of a) E401D or G429S of SEQ ID NO: 1, b) E401D and G429S of SEQ ID NO: 1, c) T337S, D344N, K355R, S385A, M431L, R442K, V462L, S470A, E471D, T473E, A495S, E500S, K511R, T516V, or A528S of SEQ ID NO: 2, d) G321L, P322R, and G323V of SEQ ID 25 NO: 2, e) D450E, A451V, and K452R of SEQ ED NO: 2, f) S455K, N456S, P457A, and S458Q of SEQ ID NO: 2, g) S470A, E472D, and T473Y of SEQ ID NO: 2, h) C475T, K476R, Q477T, K478T, and Y479H of SEQ ED NO: 2, and i) E481D, Q482E, and Q483P of SEQ TD NO: 2. 
     
     
         18 . 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; 
 ii) a promoter that is activated by the transactivation domain; 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. 
     
     
         19 . The method according to  claim 18 , wherein the ligand is
 a) 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  i sH, 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 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, or 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; 
 
         b) an ecdysone, 20-hydroxyecdysone, ponasterone A, or muristerone A; 
         c) 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; or 
         d) a 9-cis-retinoic acid, 4-(1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)-ethenyl) benzoic acid (3-methyl-TTEB), ((E)-2)2-(5,6,7,8 tetrahydro-3,5,5,8,8-pentamethyl-2-naphthyl)propen-1-yl)-4-thiophenecarboxylic acid), 2-(5,6,7,8-tetra-hydro-3,5,5,8,8-tetramethy-2-naphthyl)-2-(carboxyphenyl)-1,3-dioxolane, 4-(5H-2,3-(2,5-dimethyl-2,5-hexano)-5-methyl-dibenzo (b,e) (1,4)(diazepin-11-yl)-benzoic acid (HX600) or thiadiazepin analogs thereof, 3,7,11,15-tetramethyl hexadeconoic acid (phytanic acid), 6-(1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)cyclopropyl)nicotinic acid, 2-(4-caroxyphenyl)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1,3-dithiane, or 4-(2-methyl)-1-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-napthalenyl)propenyl)benzoic acid. 
       
     
     
         20 . The method according to  claim 18 , wherein the method further comprises 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. 
     
     
         21 . A host cell comprising the gene expression modulation system according to  claim 1 . 
     
     
         22 . A non-human organism comprising the host cell of  claim 21 .