Drug-regulatable transcription factors

An engineered cell comprising an inducible transcription factor (ITF), wherein the ITF comprises:

• a first monomer, wherein the first monomer comprises a DNA binding domain and a first ligand binding domain;
• and a second monomer, wherein the second monomer comprises a transcriptional effector domain and a second ligand binding domain,
• wherein each monomer is oligomerizable via a cognate ligand that binds to each ligand binding domain,
• wherein ligand-induced oligomerization forms the ITF,
• wherein the cognate ligand is selected from the group consisting of:
• caffeine, a cannabidiol (CBD), a phytocannabinoid, nicotine, rapamycin, AP1903, AP20187, FK1012, derivatives thereof, and analogs thereof,
• optionally wherein the DNA binding domain comprises a zinc finger (ZF) protein domain,
• optionally wherein each ligand binding domain comprises a single-domain antibody, wherein the single-domain antibody comprises a VHH, and
• optionally wherein the ITF is capable of modulating transcription of a gene of interest operably linked to an ITF-responsive promoter.

An engineered cell comprising an inducible transcription factor (ITF), wherein the ITF comprises a DNA binding domain, a transcriptional effector domain, and a ligand binding domain,

• wherein the ITF undergoes nuclear localization upon binding of the ligand binding domain to a cognate ligand,
• wherein when localized to a cell nucleus, the ITF is capable of modulating transcription of a gene of interest operably linked to an ITF-responsive promoter,
• wherein the cognate ligand is mifepristone or a derivative thereof, optionally wherein the ligand binding domain comprises a progesterone receptor domain and the progesterone receptor domain comprises the amino acid sequence of SEQ ID NO:
• 68,
• optionally wherein the DNA binding domain comprises a zinc finger (ZF) protein domain,
• optionally wherein the ITF further comprises a peptide linker between the DNA binding domain and the transcriptional effector domain,
• optionally wherein the DNA binding domain binds to the ITF-responsive promoter, and
• optionally wherein the cell further comprises an expression cassette comprising the ITF-responsive promoter operably linked to an exogenous polynucleotide sequence encoding the gene of interest,
• optionally wherein the ITF-responsive promoter comprises an ITF-binding domain sequence and a core promoter sequence,
• optionally wherein the core promoter sequence comprises a minimal promoter and the minimal promoter is selected from the group consisting of:
• minP, minCMV, YB_TATA, minTATA, and minTK, or wherein the core promoter sequence is derived from a constitutive promoter and the core promoter sequence is derived from a constitutive promoter selected from the group consisting of:
• CMV, EFS, SFFV, SV40, MND, PGK, UbC, EF1a, hCAGG, hACTb, heIF4A1, hGAPDH, hGRP78, hGRP94, hHSP70, hKINb, and hUBIb.

An expression system comprising:

• a) a first expression cassette comprising a first promoter and an exogenous polynucleotide sequence encoding a first monomer, wherein the first monomer comprises a DNA binding domain and a first ligand binding domain;
• and a second expression cassette comprising a second promoter and an exogenous polynucleotide sequence encoding a second monomer, wherein the second monomer comprises a transcription effector domain and a second ligand binding domain, wherein each monomer is oligomerizable via a cognate ligand that binds to each ligand binding domain, wherein ligand-induced oligomerization of the first and the second monomer forms an inducible transcription factor (ITF), and wherein the cognate ligand is:
• selected from the group consisting of:
• caffeine, a cannabidiol (CBD), a phytocannabinoid, nicotine, rapamycin, AP1903, AP20187, FK1012, derivatives thereof, and analogs thereof;
• or
• b) a first promoter and an exogenous polynucleotide sequence encoding an inducible transcription factor (ITF), wherein the ITF comprises a DNA binding domain, a transcriptional effector domain, and a ligand binding domain, wherein the ITF undergoes nuclear localization upon binding of the ligand binding domain to a cognate ligand, wherein when localized to a cell nucleus, the ITF is capable of modulating transcription of a gene of interest operably linked to an ITF-responsive promoter, wherein the cognate ligand is mifepristone or a derivative thereof,
• optionally wherein the DNA binding domain comprises a zinc finger (ZF) protein domain,
• optionally wherein the ZF protein domain is modular in design and is composed of one or more zinc finger arrays (ZFA),
• optionally wherein the ZF protein domain comprises an array of one to ten zinc finger motifs, and
• optionally wherein the expression system further comprises a third expression cassette comprising an ITF-responsive promoter operably linked to a gene of interest, wherein the ITF-responsive promoter comprises a core promoter sequence and a sequence that binds to the DNA binding domain of the first engineered polypeptide.