IP Library › Granted Patent US 12,559,530
Granted Patent B2
US 12,559,530 · App. 17/938,787 · Granted Feb 24, 2026

Regulated synthetic gene expression systems

Inventors: Ahmad S. Khalil (Lexington, MA); Wilson Wai Chun Wong (Brookline, MA); Divya Israni (Boston, MA); Huishan Li (Brookline, MA)
Assignee: Trustees of Boston University
C07K14/4702C07K2319/09C07K2319/20C07K2319/50C07K2319/715C07K2319/81
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Quick Facts
Patent No.
US 12,559,530
App. No.
17/938,787
Granted
Feb 24, 2026
Kind
B2
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.

Claims (54)

1 . 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.

2 . The synTF of claim 1 , wherein the transcriptional ED is a transcriptional activator (TA) domain or a transcriptional repressor (TR) domain.

3 . The synTF of claim 2 , wherein the TA is selected from the group consisting of: p65; Rta; miniVPR; full VPR; VP16; VP64; p300; p300 HAT Core; and a CBP HAT domain, or wherein the TR is selected from the group consisting of: KRAB; KRAB-MeCP2; Hp1a; DNA methyltransferase DNMT; EED; and HDAC4.

4 . The synTF of claim 3 , wherein the p65 comprises one of SEQ ID NOs: 69, 117-121, 193-197 or a protein having at least 85% sequence identity one of SEQ ID NOs: 69, 117-121, 193-197.

5 . The synTF of claim 3 , wherein the KRAB comprises one of SEQ ID NOs: 72, 97, or 214-215, or a protein having at least 85% sequence identity to one of SEQ ID NO: 72, 97, or 214-215.

6 . The synTF of claim 1 , wherein the ZF-binding domain comprises any one of: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or more ZF motifs arranged adjacent to each other in tandem to form a ZF array (ZFA).

7 . The synTF of claim 1 , wherein the ZF binding domain is selected from any of:

a. ZF 1-1, ZF 1-2, ZF 1-3, ZF 1-4, ZF 1-5, ZF 1-6, ZF 1-7, ZF 1-8, ZF 2-1, ZF 2-2, ZF 2-3, ZF 2-4, ZF 2-5, ZF 2-6, ZF 2-7, ZF 2-8, ZF 3-1, ZF 3-2, ZF 3-3, ZF 3-4, ZF 3-5, ZF 3-6, ZF 3-7, ZF 3-8, ZF 4-1, ZF 4-2, ZF 4-3, ZF 4-4, ZF 4-5, ZF 4-6, ZF 4-7, ZF 4-8, ZF 5-1, ZF 5-2, ZF 5-3, ZF 5-4, ZF 5-5, ZF 5-6, ZF 5-7, ZF 5-8, ZF 6-1, ZF 6-2, ZF 6-3, ZF 6-4, ZF 6-5, ZF 6-6, ZF 6-7, ZF 6-8, ZF 7-1, ZF 7-2, ZF 7-3, ZF 7-4, ZF 7-5, ZF 7-6, ZF 7-7, ZF 7-8, ZF 8-1, ZF 8-2, ZF 8-3, ZF 8-4, ZF 9-1, ZF 9-2, ZF 9-3, ZF 9-4, ZF 10-1 and ZF 11-1 or a ZF binding domain is selected from any of SEQ ID Nos: 1-3, 76, 101, 377, or 380; or

b. a ZF binding domain that specifically binds to a sequence comprising at least one of SEQ ID NOs: 181-191.

8 . The synTF of claim 1 , wherein the at least one DBD is selected from one or more of any of: SEQ ID NO: 221 or 222, 36-4 (SEQ ID NO: 223), 43-8 (SEQ ID NO: 224 or 225), 42-10 (SEQ ID NO: 226 or 227), 97-4 (SEQ ID NO: 228), or wherein the DBD binds to DNA binding motifs (DBM) comprising any of: SEQ ID NOs: 229-240.

9 . The synTF of claim 1 , wherein the cytosolic sequestering protein comprises a ligand binding domain (LBD), wherein in the presence of the ligand, the sequestering of the protein to the cytosol is inhibited.

10 . The synTF of claim 1 , wherein the cytosolic sequestering protein comprises a ligand binding domain and a nuclear localization signal (NLS), wherein in the absence of the ligand the NLS is inhibited thereby preventing translocation of the sequestering protein to the nucleus, and wherein in the presence of the ligand the nuclear localization signal is exposed enabling translocation of the sequestering protein to the nucleus.

11 . The synTF of claim 1 , wherein the cytosolic sequestering protein is selected from the group consisting of: ERT2, ERT, and ERT3.

12 . The synTF of claim 1 , wherein the ERT binds to one or more ligands selected from: tamoxifen, 4-hydroxytamoxifen (4OHT), endoxifen, Fulvestrant, wherein binding of the ligand to ERT exposes a NLS and results in nuclear translocation of the ERT.

13 . The synTF of claim 1 , wherein the cytosolic sequestering protein comprises the amino acid of SEQ ID NOs: 74, 335-337, or a homologue of at least 85% sequence identity to SEQ ID NOs: 74, 335-337.

14 . The synTF of claim 1 , wherein the cytosolic sequestering protein comprises a transmembrane receptor sequestering protein.

15 . The synTF of claim 1 , wherein the synTF comprises:

a N-terminal DBD, the cytosolic sequestering protein, and a C-terminal effector domain; or

a N-terminal effector domain, a DBD and a C-terminal cytosolic sequestering protein.

16 . The synTF of claim 1 , wherein synTF further comprises a Small molecule-Assisted Shutoff (SMASh) tag, wherein the SMASh tag is a N-terminal or C-terminal SMASh domain comprising a repressible protease, a partial protease helical domain and a cofactor domain.

17 . The synTF of claim 16 , wherein the SMASh tag is selected from:

a. a C-terminal SMASh domain comprising in a N-terminal to C-terminal order: a NS3 cleavage site, at least one linker, a NS3 domain, a NS3 partial helicase, a NS4A domain, wherein the SMASh tag is fused to the C-terminus of the effector domain of the synTF, or

b. a N-terminal SMASh domain comprising in a N-terminal to C-terminal order: at least one Linker, a NS3 domain, a NS3 partial helicase, a NS4 domain, and a NS3 cleavage site, wherein the SMASh tag is fused to the N-terminus of the synTF.

18 . The synTF of claim 16 , wherein in the absence of an inhibitor for the NS3 protease, the NS3 protease is active and self cleaves/uncouples from the synTF, thereby resulting in the SMASh tag targeted for degradation (“SMASh-degradation”, synTF-on/TA-on/RP-on), wherein the synTF is active in the presence of the ligand for the cytosolic sequestering protein and the absence of the inhibitor for the NS3 protease; and wherein in the presence of an inhibitor for NS3 protease, NS3 protease activity is inhibited thereby resulting in the SMASh tagged synTF targeted for degradation (“synTF-degradation”, synTF-OFF/TA-off/RP-off”), wherein the synTF is inactive in the absence of the ligand for the cytosolic sequestering protein and the presence of the inhibitor for the NS3 protease.

19 . 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”).

20 . The system of claim 19 , wherein the transcriptional effector domain (ED) is a transcriptional activator (TA), wherein

for synTFs where the cellular localization of the ED linked to the DBD is regulated by the at least one cytosolic sequestering protein;

i. 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 TA domain to be in proximity to the promoter sequence to turn on expression of the gene of interest (“TA-on”), or

ii. 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 from binding to the DBM, and preventing the TA domain from being in proximity to the promoter sequence, preventing expression of the gene of interest (“TA-off”).

21 . The system of claim 19 , wherein the ED is a transcriptional repressor (TR), wherein

for synTFs where the cellular localization of the ED linked to the DBD is regulated by the at least one cytosolic sequestering protein;

i. 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 repressor (TR) to be in proximity to the promoter sequence to turn off expression of the gene of interest (“TR-on” (no-expression)), or

ii. 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 from binding to the DBM, and preventing the transcriptional repressor (TR) from being in proximity to the promoter sequence, allowing expression of the gene of interest (“TR-off” (yes-expression)).

22 . The system of claim 19 , wherein the at least one synTF further comprises a N-terminal or C-terminal Small molecule-Assisted Shutoff (SMASh) domain, wherein SMASh domain comprises a self-cleaving SMASh protease, a partial protease helical domain and a cofactor domain,

wherein in the presence of an inhibitor to the SMASh protease, the SMASh protease activity is inhibited, resulting in the synTF being degraded and preventing the DBD of the synTF binding to the DBM and controlling the expression or repression of the gene of interest, wherein the synTF is inactive in the absence of a ligand for the cytosolic sequestering protein and the presence of the inhibitor for the SMASh protease (“synTF-degradation”; TA-off (no expression), TR-off (yes-expression)),

wherein in the absence of an inhibitor to the SMASh protease, the SMASh protease is active and self cleaves/uncouples from the synTF, resulting the SMASh domain being targeted for degradation and, in the presence of the ligand for the cytosolic sequestering protein, allowing the DBD of the synTF to bind to the DBM and the ED of synTF to control the expression of the gene of interest, wherein the synTF is active in the presence of the ligand for the cytosolic sequestering protein and the absence of the inhibitor for the NS3 protease (“SMASh-degradation, TA-on (yes-expression), TR-on (no-expression)).

23 . A cell comprising

a. a first nucleic acid sequence comprising at least one target DNA binding motif (DBM) comprising a target nucleic acid for binding of the at least one DBD of a synTF, a promoter sequence located 3′ of the at least one DBM, and a nucleic acid encoding a gene of interest (GOI) operatively linked to the promoter sequence, and

b. a second nucleic acid sequence comprising a nucleic acid encoding a synthetic transcription factor (synTF) according to claim 1 , operatively linked to an inducible or constitutive promoter.

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Oct 28, 2022
From: KHALIL, AHMAD S.; WONG, WILSON WAI CHUN; ISRANI, DIVYA; LI, HUISHAN
To: TRUSTEES OF BOSTON UNIVERSITY
Reel/Frame 061582/0261 →
Continuity (3)
Continuation 16875591 · May 15, 2020
Provisional Application 62848850 · May 16, 2019
Related Publication 20230159600A1 · May 25, 2023
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