Chiral diacylhydrazine ligands for modulating the expression of exogenous genes via an ecdysone receptor complex
Inventors
Hormann, Robert Eugene • Li, Bing
Assignees
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Publication Number
US-10633361-B2
Publication Date
2020-04-28
Expiration Date
Abstract
The present invention provides diacylhydrazine ligands and chiral diacylhydrazine ligands for use with ecdysone receptor-based inducible gene expression systems. Thus, the present invention is useful for applications such as gene therapy, large scale production of proteins and antibodies, cell-based screening assays, functional genomics, proteomics, metabolomics, and regulation of traits in transgenic organisms, where control of gene expression levels is desirable. An advantage of the present invention is that it provides a means to regulate gene expression and to tailor expression levels to suit the user's requirements.
Core Innovation
The invention provides methods for regulating endogenous or heterologous gene expression in a transgenic subject using an ecdysone receptor complex ligand. Cells within the subject contain a DNA binding sequence for the ecdysone receptor complex, and formation of an ecdysone receptor complex-ligand-DNA binding sequence complex induces expression of the gene. The ligand is selected from an enumerated group of (R)-configured diacylhydrazine hydrazides.
The ligand family includes enantiomerically enriched (R)-configured compounds such as (R)-3,5-dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazide and related benzoyl-substituted analogs, including N′-benzoyl, 2-methyl, 2-fluoro, 2-bromo, 3-methyl, 4-methyl, and 3-methoxy-2-methyl derivatives. The disclosed system uses an ecdysone receptor complex, an EcR response element, a DNA-binding domain, and a transactivation domain in a gene-expression modulation system.
The document also describes characterization and stereochemical determination for at least one named (R)/(S) configured hydrazide derivative, including analytical data and an x-ray crystal structure note. Example embodiments include use of enantiopure (R) ligand in transgenic subject regulation and transgene regulation contexts.
Claims Coverage
The consolidated claim coverage includes two independent claims. The inventive features center on an ecdysone receptor complex-ligand-DNA binding sequence complex that induces gene expression and on regulation of transgene expression in a transgenic subject using a recombinant ecdysone receptor complex and an effective amount of selected (R)-configured hydrazide compounds.
Ecdysone receptor complex-ligand-DNA binding sequence complex for gene induction
A method for regulating endogenous or heterologous gene expression in a transgenic subject by contacting a ligand with an ecdysone receptor complex within cells that contain a DNA binding sequence for the ecdysone receptor complex, wherein formation of an ecdysone receptor complex-ligand-DNA binding sequence complex induces expression of the gene.
Enantiomerically enriched (R)-configured diacylhydrazide ligand selection
The ligand is selected from an enumerated group of (R)-configured diacylhydrazide hydrazides, including (R)-3,5-dimethyl-benzoic acid N-(1-tert-butyl-butyl)-N′-(2-ethyl-3-methoxy-benzoyl)-hydrazide and related benzoyl-substituted analogs.
Recombinant ecdysone receptor complex for transgene regulation
A method of regulating transgene expression in a transgenic subject comprising a recombinant ecdysone receptor complex capable of regulating transgene expression, wherein the method comprises administering to the subject an effective amount of an enumerated (R)-configured hydrazide compound.
Overall, the claims cover regulation of gene or transgene expression in transgenic subjects through ligand-driven ecdysone receptor complex formation with a DNA binding sequence, using specified (R)-configured diacylhydrazide compounds and a recombinant ecdysone receptor complex in the transgene-regulation claim.
Stated Advantages
Reduced background gene expression.
Response at submicromolar ligand concentrations.
Enhanced in vivo gene expression with enantiopure (R) ligand.
Oral plasma exposure is stated for the example enantiopure (R) ligand/pharmaceutical composition.
Regulates endogenous or heterologous gene expression in a transgenic subject.
Regulates transgene expression in a transgenic subject.
Documented Applications
Inducible regulation of endogenous or heterologous gene expression in a transgenic subject using an EcR-based gene switch system.
Inducible regulation of transgene expression in a transgenic subject comprising a recombinant ecdysone receptor complex.
Regulation where the transgene encodes human interleukin-12.
Regulating endogenous or heterologous gene expression in a transgenic subject using an ecdysone receptor complex ligand to induce gene expression via an ecdysone receptor complex-ligand-DNA binding sequence complex.
Regulating transgene expression in a transgenic subject comprising a recombinant ecdysone receptor complex by administering an effective amount of an enumerated (R)-3,5-dimethyl-benzoic acid hydrazide compound.
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