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 . A gene expression modulation system comprising a gene expression cassette that is capable of being expressed in a host cell, the gene expression cassette comprising a polynucleotide that encodes a 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 H nuclear receptor ligand binding domain comprising at least one mutation, wherein the mutation is at a position equivalent to or analogous to amino acid residues selected from the group consisting of: a) at least one of 48, 51, 52, 54, 92, 95, 96, 109, 110, 119, 120, 125, 128, 132, 219, 223, 234, and 238 of SEQ ID NO: 1, b) both 96 and 119 of SEQ ID NO: 1, c) both 110 and 128 of SEQ ID NO: 1, d) both 52 and 110 of SEQ ID NO: 1, e) both 107, 110, and 127 of SEQ ID NO: 1, f) both 52, 107 and 127 of SEQ ID NO: 1 and g) both 107, 127 and 259 of SEQ ID NO: 1, or any combination thereof.   
     
     
         2 . The gene expression modulation system according to  claim 1 , further comprising a second nuclear receptor ligand binding domain selected from the group consisting of a vertebrate retinoid X receptor ligand binding domain, an invertebrate retinoid X receptor ligand binding domain, an ultraspiracle protein ligand binding domain, and a chimeric ligand binding domain comprising two polypeptide fragments, wherein the first polypeptide fragment is from a vertebrate retinoid X receptor ligand binding domain, an invertebrate retinoid X receptor ligand binding domain, or an ultraspiracle protein ligand binding domain, and the second polypeptide fragment is from a different vertebrate retinoid X receptor ligand binding domain, invertebrate retinoid X receptor ligand binding domain, or ultraspiracle protein ligand binding domain. 
     
     
         3 . 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 H nuclear receptor ligand binding domain comprising at least one mutation, wherein the mutation is at a position equivalent to or analogous to amino acid residues selected from the group consisting of: a) at least one of 48, 51, 52, 54, 92, 95, 96, 109, 110, 119, 120, 125, 128, 132, 219, 223, 234 and 238 of SEQ ID NO: 1, b) both 96 and 119 of SEQ ID NO: 1, c) both 110 and 128 of SEQ ID NO: 1, d) both 52 and 110 of SEQ ID NO: 1, e) all three of 107, 110, and 127 of SEQ ID NO: 1, f) all three of 52, 107 and 127 of SEQ ID NO: 1 and g) all three of 107, 127 and 259 of SEQ ID NO: 1, or any combination thereof. 
     
     
         4 . The gene expression modulation system according to  claim 1 , wherein the Group H nuclear receptor ligand binding domain is from a Group H nuclear receptor selected from the group consisting of an ecdysone receptor, a ubiquitous receptor, an orphan receptor 1, a NER-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. 
     
     
         5 . The gene, expression modulation system according to  claim 4 , wherein the Group H nuclear receptor ligand binding domain is encoded by a polynucleotide comprising at least one mutation that results in at least one mutation of an amino acid residue, wherein the amino acid residue is at a position equivalent to or analogous to: a) at least one of amino acid residue 48, 51, 52, 54, 92, 95, 96, 109, 110, 119, 120, 125, 128, 132, 219, 223, 234, and 238 of SEQ ID NO: 1, b) both amino acid residues 96 and 119 of SEQ ID NO: 1, c) both amino acid residues 110 and 128 of SEQ ID NO: 1, d) both amino acid residues 52 and 110 of SEQ ID NO: 1, e) all three amino acid residues 107, 110, and 127 of SEQ ID NO: 1, f) all three amino acid residues 52, 107 and 127 of SEQ ID NO: 1 or g) all three amino acid residues 107, 127 and 259 of SEQ ID NO: 1, or any combination thereof. 
     
     
         6 . The gene expression modulation system according to  claim 5 , wherein the Group H nuclear receptor ligand binding domain comprising a substitution mutation is an ecdysone receptor ligand binding domain. 
     
     
         7 . The gene expression modulation system according to  claim 6 , wherein the mutation is selected from the group consisting of F48Y, F48W, F48L, F48N, F48R, F48K, I51M, I51N, I51L, T52M, T52V, T52L, T52E, T52P, T52R, T52W, T52G, M54W, M54T, M92L, M92E, R95H, R95M, R95W, V96L, V96W, V96S, V96E, F109W, F109P, F109L, F109M, F109N, A110E, A110N, A110W, N119F, Y120W, Y120M, M1125P, M125R, M125E, M125L, M125C, M125W, M125G, M125I, M125N, M125S, M125V, V128F, L132M, L132N, L132V, L132E, M219K, M219W, M219Y, M219A, L223K, L223R, L223Y, L234M, L234I, L234R, L234W, W238P, W238E, W238Y, W238M, W238L, N119F/V96T, V128F/A110P, T52V/A110P, V107I/Y127L/T52V, V107I/Y127E/G259 and V107I/Y127E/A110P of SEQ ID NO: 1. 
     
     
         8 . 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. 
     
     
         9 . 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. 
     
     
         10 . An isolated polynucleotide encoding a Group H nuclear receptor ligand binding domain comprising at least one mutation, wherein the isolated polynucleotide comprises at least one mutation that results in at least one mutation of an amino acid residue at a position equivalent to or analogous to a) at least one of amino acid residue 48, 51, 52, 54, 92, 95, 96, 109, 110, 119, 120, 125, 128, 132, 219, 223, 234, and 238 of SEQ ID NO: 1, b) both amino acid residues 96 and 119 of SEQ ID NO: 1, c) both amino acid residues 110 and 128 of SEQ ID NO: 1, d) both amino acid residues 52 and 110 of SEQ ID NO: 1, e) all three amino acid residues 107, 110, and 127 of SEQ ID NO: 1, f) all three amino acid residues 52, 107 and 127 of SEQ ID NO: 1 or g) all three amino acid residues 107, 127 and 259 of SEQ ID NO: 1 or any combination thereof. 
     
     
         11 . The isolated polynucleotide according to  claim 10 , wherein the mutation results in a mutation selected from the group consisting of F48Y, F48W, F48L, F48N, F48R, F48K, I51M, I51N, I51L, T52M, T52V, T52L, T52E, T52P, T52R, T52W, T52G, T52Q, M54W, M54T, M92L, M92E, R95H, R95M, R95W, V96L, V96W, V96S, V96E, F109W, F109P, F109L, F109M, F109N, A110E, A110N, A110W, N119F, Y120W, Y120M, M125P, M125R, M125E, M125L, M125C, M125W, M125G, M125I, M125N, M125S, M125V, V128F, L132M, L132N, L132V, L132E, M219K, M219W, M219Y, M219A, L223K, L223R, L223Y, L234M, L234I, L234R, L234W, W238P, W238E, W238Y, W238M, W238L, N119F/V96T, V128F/A110P, T52V/A110P, V107I/Y127E/T52V, V107I/Y127E/G259 and V107I/Y127E/A110P of SEQ ID NO: 1. 
     
     
         12 . An expression vector comprising the isolated polynucleotide of  claim 10  operatively linked to a transcription regulatory element. 
     
     
         13 . An isolated 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 H nuclear receptor ligand binding domain comprising at least one mutation, wherein the mutation is at a position equivalent to or analogous to a) at least one of amino acid residue 48, 51, 52, 54, 92, 95, 96, 109, 110, 119, 120, 125, 128, 132, 219, 223, 234, and 238 of SEQ ID NO: 1, b) both amino acid residues 96 and 119 of SEQ ID NO: 1, c) both amino acid residues 110 and 128 of SEQ ID NO: 1, d) both amino acid residues 52 and 110 of SEQ ID NO: 1, e) all three amino acid residues 107, 110, and 127 of SEQ ID NO: 1, f) all three amino acid residues 52, 107 and 127 of SEQ ID NO: 1 or g) all three amino acid residues 107, 127 and 259 of SEQ ID NO: 1, or any combination thereof. 
     
     
         16 . The isolated polypeptide according to  claim 15 , wherein the mutation is selected from the group consisting of F48Y, F48W, F48L, F48N, F48R, F48K, I51M, I51N, I51L, T52M, T52V, T52L, T52E, T52P, T52R, T52W, T52G, T52Q, M54W, M54T, M92L, M92E, R95H, R95M, R95W, V96L, V96W, V96S, V96E, F109W, F109P, F109L, F109M, F109N, A110E, A110N, A110W, N119F, Y120W, Y120M, M125P, M125R, M125E, M125L, M125C, M125W, M125G, M125I, M125N, M125S, M125V, V128F, L132N, L132V, L132V, L132E, M219K, M219W, M219Y, M219A, L223K, L223R, L223Y, L234M, L234I, L234R, L234W, W238P, W238E, W238Y, W238M, W238L, N119F/V96T, V128F/A110P, T52V/A110P, V107I/Y127E/T52V, V107I/Y127E/G259 and V107I/Y127E/A110P of SEQ ID NO: 1. 
     
     
         17 . 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.   
     
     
         18 . The method according to  claim 17 , 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 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 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; 
         b) a compound of the formula: 
       
       
         
           
           
               
               
           
         
         wherein: 
         R 1  is CH 2 CH 3 , 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; and 
         d) an ecdysone, 20-hydroxyeedysone, 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. 
       
     
     
         19 . The method according to  claim 18 , 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. 
     
     
         20 . An isolated host cell comprising the gene expression modulation system according to  claim 1 . 
     
     
         21 . A non-human organism comprising the host cell of  claim 20 .