Steroidal ligands and their use in gene switch modulation

Patent metadata

Field	Value
Publication number	US-9243263-B2
Application number	US-201313947631-A
Country	US
Kind code	B2
Filing date	Jul 22, 2013
Priority date	Mar 14, 2008
Publication date	Jan 26, 2016
Grant date	Jan 26, 2016

Abstract

The present invention relates to steroidal ligands for use in nuclear receptor-based inducible gene expression systems. The invention further relates to methods of modulating the expression of genes of interest with a system containing one or more nuclear receptor complexes and one or more steroidal ligands. Further aspects include ligand compositions including therapeutic compositions.

First claim

What is claimed is:

1. A composition comprising a plurality of individually operable recombinant gene switches, wherein each individually operable recombinant gene switch comprises:
a first recombinant cassette comprising first polynucleotide encoding a first polypeptide comprising:
a DNA-binding domain that recognizes a response element operatively linked with a gene of interest whose expression is to be modulated; and
a E274V/V390I/Y410E mutant of the Choristoneura fumiferana ecdysone receptor ligand binding domain; and

a second recombinant cassette comprising:
a response element capable of binding to said DNA binding domain;
a promoter that is activated by the transactivation domain; and
a gene of interest;

wherein the composition comprises a plurality of ligands, wherein at least one ligand is:
20-hydroxyecdysone-2-methyl ether;
20-hydroxyecdysone-3-methyl ether;
20-hydroxyecdysone-14-methyl ether;
20-hydroxyecdysone-2,22-dimethyl ether;
20-hydroxyecdysone-3,22-dimethyl ether;
20-hydroxyecdysone-14,22-dimethyl ether;
20-hydroxyecdysone-22,25-dimethyl ether;
20-hydroxyecdysone-2,3,14,22-tetramethyl ether;
20-hydroxyecdysone-22-n-propyl ether;
20-hydroxyecdysone-22-n-butyl ether;
20-hydroxyecdysone-22-allyl ether;
20-hydroxyecdysone-22-benzyl ether;
20-hydroxyecdysone-22-(28R,S)-2′-ethyloxiranyl ether;
ponasterone A-2-methyl ether;
ponasterone A-14-methyl ether;
ponasterone A-22-methyl ether;
ponasterone A-2,22-dimethyl ether;
ponasterone A-3,22-dimethyl ether;
ponasterone A-14,22-dimethyl ether;
dacryhainansterone-22-methyl ether;
25,26-didehydroponasterone A (iso-stachysterone C (Δ25(26)));
shidasterone (stachysterone D);
stachysterone C;
22-deoxy-20-hydroxyecdysone (taxisterone);
ponasterone A;
polyporusterone B;
22-dehydro-20-hydroxyecdysone;
20-hydroxyecdysone;
pterosterone;
(25R)-inokosterone;
(25S)-inokosterone;
pinnatasterone;
25-fluoroponasterone A;
24(28)-dehydromakisterone A;
24-epi-makisterone A;
makisterone A;
20-hydroxyecdysone-22-methyl ether;
20-hydroxyecdysone-25-methyl ether;
abutasterone;
22,23-di-epi-geradiasterone;
20,26-dihydroxyecdysone (podecdysone C);
24-epi-abutasterone;
geradiasterone;
29-norcyasterone;
ajugasterone B;
24(28)[Z]-dehydroamarasterone B;
amarasterone A;
makisterone C;
rapisterone C;
20-hydroxyecdysone-22-ethyl ether;
carthamosterone;
24(25)-dehydroprecyasterone;
leuzeasterone;
cyasterone;
24(28)[Z]-dehydro-29-hydroxymakisterone C;
20-hydroxyecdysone-22-acetate;
viticosterone E (20-hydroxyecdysone 25-acetate);
24-hydroxycyasterone;
ponasterone A 22-hemisuccinate;
22-acetoacetyl-20-hydroxyecdysone;
canescensterone;
20-hydroxyecdysone-22-hemisuccinate;
inokosterone-26-hemisuccinate;
20-hydroxyecdysone-22-benzoate;
20-hydroxyecdysone-22-β-D-glucopyranoside;
20-hydroxyecdysone-25-β-D-glucopyranoside;
sileneoside A (20-hydroxyecdysone-22α-galactoside);
3-deoxy-1β,20-dihydroxyecdysone (3-deoxyintegristerone A);
2-deoxyintegristerone A;
1-epi-integristerone A;
integristerone A;
sileneoside C (integristerone A 22α-galactoside);
2,22-dideoxy-20-hydroxyecdysone;
2-deoxy-20-hydroxyecdysone;
2-deoxy-20-hydroxyecdysone-3-acetate;
2-deoxy-20,26-dihydroxyecdysone;
2-deoxy-20-hydroxyecdysone-22-acetate;
2-deoxy-20-hydroxyecdysone-3,22-diacetate;
2-deoxy-20-hydroxyecdysone-22-benzoate;
ponasterone A 2-hemisuccinate;
20-hydroxyecdysone-2-acetate;
20-hydroxyecdysone-2-hemisuccinate;
20-hydroxyecdysone-2-βD-glucopyranoside;
2-dansyl-20-hydroxyecdysone;
ponasterone A 3β-D-xylopyranoside (limnantheoside B);
20-hydroxyecdysone-3-acetate;
20-hydroxyecdysone-3β-D-xylopyranoside (limnantheoside A);
20-hydroxyecdysone-3-β-D-glucopyranoside;
sileneoside D (20-hydroxyecdysone-3α-galactoside);
20-hydroxyecdysone 3β-D-glucopyranosyl-[1-3]-β-D-xylopyranoside (limnantheoside C);
cyasterone-3-acetate;
2-dehydro-3-epi-20-hydroxyecdysone;
3-epi-20-hydroxyecdysone (coronatasterone);
rapisterone D;
3-dehydro-20-hydroxyecdysone;
5β-hydroxy-25,26-didehydroponasterone A;
5β-hydroxystachysterone C;
25-deoxypolypodine B;
polypodine B;
25-fluoropolypodine B;
5β-hydroxyabutasterone;
26-hydroxypolypodine B;
29-norsengosterone;
sengosterone;
6β-hydroxy-20-hydroxyecdysone;
6α-hydroxy-20-hydroxyecdysone;
20-hydroxyecdysone-6-oxime;
ponasterone A 6-carboxymethyloxime;
20-hydroxyecdysone-6-carboxymethyloxime;
ajugasterone C;
rapisterone B;
muristerone A;
atrotosterone B;
atrotosterone A;
turkesterone-2-acetate;
punisterone (rhapontisterone);
turkesterone;
atrotosterone C;
25-hydroxyatrotosterone B;
25-hydroxyatrotosterone A;
paxillosterone;
turkesterone-2,22-diacetate;
turkesterone-22-acetate;
turkesterone-11α-acetate;
turkesterone-2,11α-diacetate;
turkesterone-11α-propionate;
turkesterone-11α-butanoate;
turkesterone-11α-hexanoate;
turkesterone-11α-decanoate;
turkesterone-11α-laurate;
turkesterone-11α-myristate;
turkesterone-11α-arachidate;
22-dehydro-12β-hydroxynorsengosterone;
22-dehydro-12β-hydroxycyasterone;
22-dehydro-12β-hydroxysengosterone;
14-deoxy(14α-H)-20-hydroxyecdysone;
14α-perhydroxy-20-hydroxyecdysone;
20-hydroxyecdysone 14,22-dimethyl ether;
(20S)-22-deoxy-20,21-dihydroxyecdysone;
22,25-dideoxyecdysone;
(22S)-20-(2,2′-dimethylfuranyl)ecdysone;
(22R)-20-(2,2′-dimethylfuranyl)ecdysone;
22-deoxyecdysone;
25-deoxyecdysone;
22-dehydroecdysone;
ecdysone;
22-epi-ecdysone;
24-methylecdysone (20-deoxymakisterone A);
ecdysone-22-hemisuccinate;
25-deoxyecdysone-22-β-D-glucopyranoside;
ecdysone-22-myristate;
22-dehydro-20-iso-ecdysone;
20-iso-ecdysone;
20-iso-22-epi-ecdysone;
2-deoxyecdysone;
sileneoside E (2-deoxyecdysone 3β-glucoside; blechnoside A);
2-deoxyecdysone-22-acetate;
2-deoxyecdysone-3,22-diacetate;
2-deoxyecdysone-22-β-D-glucopyranoside;
2-deoxyecdysone 25-β-D-glucopyranoside;
2-deoxy-21-hydroxyecdysone;
3-epi-22-iso-ecdysone;
3-dehydro-2-deoxyecdysone (silenosterone);
3-dehydroecdysone;
3-dehydro-2-deoxyecdysone-22-acetate;
ecdysone-6-carboxymethyloxime;
ecdysone-2,3-acetonide;
14-epi-20-hydroxyecdysone-2,3-acetonide;
20-hydroxyecdysone-2,3-acetonide;
20-hydroxyecdysone-20,22-acetonide;
14-epi-20-hydroxyecdysone-2,3,20,22-diacetonide;
paxillosterone-20,22-p-hydroxybenzylidene acetal;
poststerone;
(20R)-dihydropoststerone;
(20S)dihydropoststerone;
poststerone-20-dansylhydrazine;
(20S)-dihydropoststerone-2,3,20-tribenzoate;
(20R)-dihydropoststerone-2,3,20-tribenzoate;
(20R)-dihydropoststerone-2,3-acetonide;
(20S)-dihydropoststerone-2,3-acetonide;
(5α-H)-dihydrorubrosterone;
2,14,22,25-tetradeoxy-5α-ecdysone-5α-ketodiol;
bombycosterol;
2α,3α,22S,25-tetrahydroxy-5α-cholestan-6-one;
(5α-H)-2-deoxy-21-hydroxyecdysone;
castasterone;
24-epi-castasterone;
(5α-H)-2-deoxyintegristerone A;
(5α-H)-22-deoxyintegristerone A;
(5α-H)-20-hydroxyecdysone;
24,25-didehydrodacryhaininansterone;
25,26-didehydrodacryhainansterone;
5-deoxykaladasterone (dacryhainansterone);
(14α-H)-14-deoxy-25-hydroxydacryhainansterone;
25-hydroxydacryhainansterone;
rubrosterone;
(5β-H)-dihydrorubrosterone;
dihydrorubrosterone-17β-acetate;
sidisterone;
20-hydroxyecdysone-2,3,22-triacetate;
14-deoxy(14β-H)-20-hydroxyecdysone;
14-epi-20-hydroxyecdysone;
9α,20-dihydroxyecdysone;
malacosterone;
2-deoxypolypodine B-3-β-D-glucopyranoside;
ajugalactone;
cheilanthone B;
2β,3β,6α-trihydroxy-5β-cholestane;
2β,3β,6β-trihydroxy-5β-cholestane;
14-dehydroshidasterone;
stachysterone B;
2β,3β,9α,20R,22R,25-hexahydroxy-5β-cholest-7,14-dien-6-one;
kaladasterone;
(14-βH)-14-deoxy-25-hydroxydacryhainansterone;
4-dehydro-20-hydroxyecdysone;
14-methyl-12-en-shidasterone;
14-methyl-12-en-15,20-dihydroxyecdysone;
podecdysone B;
2β,3β,20R,22

Assignees

• Intrexon Corp

Inventors

• Hormann Robert E
• Lapenna Silvia
• Dinan Laurence Neil

Classifications

• C12N2830/002

  inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor · CPC title

• C07K14/70567

  Nuclear receptors, e.g. retinoic acid receptor [RAR], RXR, nuclear orphan receptors · CPC title

• A61K31/575

  substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol · CPC title

• C07J9/00 Primary

  Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane · CPC title

• C07K2319/715

  containing a domain for ligand dependent transcriptional activation, e.g. containing a steroid receptor domain · CPC title