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US 12,559,530 B2Granted
Regulated synthetic gene expression systems
AssigneeTrustees of Boston University
InventorsAhmad S. Khalil, Wilson Wai Chun Wong, Divya Israni, Huishan Li
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Abstract
The technology described herein is directed to regulated synthetic gene expression systems. In one aspect described herein are synthetic transcription factors (synTFs) comprising a DNA binding domain, a transcriptional effector domain, and a regulator protein. In other aspects described herein are gene expression systems comprising said synTFs and methods of treating diseases and disorders using said synTFs.
Claims23
A synthetic transcription factor (synTF) comprising;
- a. at least one DNA binding domain (DBD), wherein the DBD comprises an engineered zinc-finger binding domain which binds to a DNA-binding motif (DBM),
- b. a transcriptional effector domain (ED),
- c. at least one cytosolic sequestering protein, wherein the cytosolic sequestering protein is an estrogen ligand binding domain (ERT) or a variant thereof, or comprises at least a portion of the estrogen receptor (ER), and
- wherein the ED is directly or indirectly coupled or linked to the DBD, and
- wherein the cellular localization of the ED is regulated by the cytosolic sequestering protein.
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A system for controlling gene expression, comprising:
- a. at least one synthetic transcription factor (synTF) comprising at least one DNA binding domain (DBD), a transcriptional effector domain (ED), and at least one cytosolic sequestering protein,
- wherein the ED is directly or indirectly coupled or linked to the DBD, wherein the cellular localization of the ED is regulated by the cytosolic sequestering protein, and wherein the cytosolic sequestering protein is an estrogen ligand binding domain (ERT) or a variant thereof, or comprises at least a portion of the estrogen receptor (ER),
- wherein the DBD can bind to a target DNA binding motif (DBM) located upstream of a promoter operatively linked to a gene,
- b. a nucleic acid construct comprising:
- i. at least one target DNA binding motif (DBM) comprising a target nucleic acid for binding of the at least one DBD of the synTF, and
- ii. a promoter sequence located 3′ of the at least one DBM, and
- iii. a gene of interest operatively linked to the promoter sequence,
- wherein for synTFs where the cellular localization of the ED linked to the DBD is regulated by the at least one cytosolic sequestering protein;
- in the presence of a ligand for the at least one cytosolic sequestering protein, the ED coupled to the DBD of the synTF is not sequestered in the cytosol, enabling the DBD to bind to the DNA binding motif (DBM) and enabling the transcriptional effector domain (ED) to be in proximity to the promoter sequence to control the expression of the gene of interest (“ED-on”), or
- in the absence of the ligand for the at least one cytosolic sequestering protein, the ED coupled to the DBD of the synTF is sequestered in the cytosol, preventing the DBD of the synTF from binding to the DBM, and preventing the effector domain (ED) from being in proximity to the promoter sequence, preventing expression of the gene of interest (“ED-off”).
7 more elements
Dependencies are inferred from the claim text.