Ligand activated transcriptional regulator proteins

Abstract

Fusion proteins for use as ligand-dependent transcriptional regulators are provided. The fusion proteins include a nucleotide binding domain operatively linked to a ligand-binding domain. They also can include a transcription regulating domain. The nucleotide binding domain is a zinc-finger peptide that binds to a targeted contiguous nucleotide sequence of from 3 to about 18 nucleotides are provided. The fusion proteins are used for gene therapy. Also provided are polynucleotides encoding the fusion proteins, expression vectors, and transfected cells.

1 . A method for regulating gene expression in a cell, comprising:
 a) introducing into a cell a fusion protein or a nucleic acid molecule that comprises a sequence of nucleotides that encodes the fusion protein, wherein:   a monomer of the fusion protein comprises a nucleotide binding domain (DBD) operatively linked to a modified ligand binding domain (LBD) from an intracellular receptor;   the fusion protein is a ligand activated transcriptional regulator;   the nucleotide binding domain is a polydactyl zinc-finger domain that contains at least three modular portions thereof;   each modular portion of the nucleotide binding domain interacts with a contiguous sequence of nucleotides of at least 3 nucleotides; and   the ligand specificity of the LBD is modified to change its ligand specificity compared to the ligand specificity of the ligand binding domain of the native intracellular receptor from which the LBD was derived, whereby ligands that activate the fusion protein are not the ligands that activate the receptor from which the LBD was derived; and   b) contacting the cell with a ligand that interacts with the binding domain in the fusion protein, whereby the fusion protein interacts with a target nucleic acid molecule to activate or repress transcription of a gene encoded by the fusion protein.   
     
     
         2 . The method of  claim 1 , wherein the fusion protein interacts with the target nucleic acid molecule as a dimer. 
     
     
         3 . The method of  claim 2 , wherein the dimer is a heterodimer. 
     
     
         4 . The method of  claim 2 , wherein the dimer is a homodimer 
     
     
         5 . The method of  claim 1 , wherein the fusion protein further comprises an operatively linked transcription regulating domain. 
     
     
         6 . The method of  claim 1 , wherein the intracellular receptor is a nuclear hormone receptor. 
     
     
         7 . The method of  claim 1 , wherein the modified ligand-binding domain is not activated by ligands that activate the receptor from which the LBD was derived. 
     
     
         8 . The method of  claim 1 , wherein a module of the zinc-finger peptide binds to a sequence of nucleotides of the formula (GNN)n, where G is guanidine, N is any nucleotide and n is an integer from 3 to 6. 
     
     
         9 . The method of  claim 1 , wherein the zinc-finger peptide is comprised of modular units from a C2H2 zinc-finger. 
     
     
         10 . The method of  claim 1 , wherein the fusion protein monomer comprises at least four zinc fingers or variants thereof. 
     
     
         11 . The method of  claim 6 , wherein the nuclear hormone receptor is selected from the group consisting of estrogen receptors, progesterone receptors, glucocorticoid-α receptors, glucocorticoid-β receptors, mineralocorticoid receptors, androgen receptors, thyroid hormone receptors, retinoic acid receptors, retinoid X receptors, Vitamin D receptors, COUP-TF receptors, ecdysone receptors, Nurr-1 receptors and orphan receptors. 
     
     
         12 . The method of  claim 1 , wherein the intracellular receptor is a steroid receptor. 
     
     
         13 . The method of  claim 6 , wherein the hormone receptor is a progesterone receptor variant or an estrogen receptor variant. 
     
     
         14 . The method of  claim 5 , wherein the transcription regulating domain comprises a transcription activation domain. 
     
     
         15 . The method of  claim 5 , wherein the transcription regulating domain comprises a transcription activation domain selected from the group consisting of VP16, VP64, TA2, STAT-6, p65 and derivatives, multimers and combinations thereof that have transcription activation activity. 
     
     
         16 . The method of  claim 14 , wherein the transcription regulating domain comprises a nuclear hormone receptor transcription activation domain or variant thereof that has transcription activation activity. 
     
     
         17 . The fusion protein of  claim 14 , wherein the transcription regulating domain comprises a steroid hormone receptor transcription activation domain or variant thereof. 
     
     
         18 . The method of  claim 14 , wherein the transcription regulating domain comprises a viral transcription activation domain or variant thereof that has transcription activation activity. 
     
     
         19 . The method of  claim 18 , wherein the transcription regulating domain comprises a VP16 transcription activation domain or variant thereof. 
     
     
         20 . The method of  claim 1 , wherein the fusion protein further comprises a transcription regulating domain that comprises a transcription repression domain. 
     
     
         21 . The method of  claim 20 , wherein the transcription repression domain is selected from the group consisting of ERD, KRAB, SID, Deacetylase, and derivatives, multimers and combinations thereof. 
     
     
         22 . The method of  claim 1 , wherein the monomer of the fusion protein comprises a sequence of amino acids encoded by the sequence of nucleotides set forth in any of SEQ ID NOS: 1-18. 
     
     
         23 . The method of  claim 1 , wherein the target nucleic acid molecule is endogenous to the cell. 
     
     
         24 . The method of  claim 1 , wherein the target nucleic acid molecule is introduced to the cell as part of an expression cassette. 
     
     
         25 . The method of  claim 24 , wherein the expression cassette and fusion protein or nucleic acid encoding the fusion protein are introduced at the same time. 
     
     
         26 . The method of  claim 24 , wherein the expression cassette and fusion protein or nucleic acid encoding the fusion protein are introduced sequentially. 
     
     
         27 . The method of  claim 1 , wherein the ligand is delivered to the cell after the fusion protein or nucleic acid molecule encoding the fusion protein is introduced into the cell. 
     
     
         28 . The method of  claim 1 , wherein the nucleic acid molecule encoding the fusion protein comprises a vector. 
     
     
         29 . The method of  claim 28 , wherein the vector is a viral vector. 
     
     
         30 . The method of  claim 1 , wherein the cell is in a mammal. 
     
     
         31 . The method of  claim 24 , wherein the expression cassette is contained in a vector. 
     
     
         32 . The method of  claim 31 , wherein the vector is a viral vector. 
     
     
         33 . The method of  claim 24 , wherein the cell is in a mammal. 
     
     
         34 . The method of  claim 1 , wherein a fusion protein is introduced. 
     
     
         35 . The method of  claim 1 , wherein a nucleic acid molecule that comprises a sequence of nucleotides encoding the fusion protein is introduced.