Regulated synthetic gene expression systems

Abstract

The technology described herein is directed to a regulated synthetic gene expression system. One aspect described herein is a synthetic transcription factor (synTF) comprising a DNA binding domain, a transcription effector domain, and a regulatory protein. Other aspects described herein are gene expression systems comprising the synTF, and methods for treating diseases and disorders using the synTF. TIFF2022532236000196.tif173170

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. 62 / 848,850, filed May 16, 2019, the contents of which are incorporated herein by reference in their entirety.

[0002] government support This invention was made with government support under Grant No. D16AP00142 awarded by the Defense Advanced Research Projects Agency and Grant No. CCF-1522074 awarded by the National Science Foundation. The government has certain rights in this invention.

[0003] Sequence Listing This application contains a Sequence Listing, which has been submitted via EFS-Web in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on May 15, 2020, is titled 701586-095350_SL.txt and is 921,581 bytes in size.

[0004] Technical Field The technology described herein relates to regulated synthetic gene expression systems. [Background technology]

[0005] background Next-generation cell therapy seeks to create designer immune cells that can sense and respond to disease in sophisticated ways. Reaching this goal fundamentally requires engineered regulatory elements and circuitry that can be used to program the function of human cells by processing complex environmental inputs and mediating precisely regulated expression of therapeutic agents. Toward this goal, synthetic transcriptional programs can interface with sensing and response modules to provide a new layer of regulation in cells.

[0006] To advance immune cell therapy beyond a reliance on simple constitutive expression of therapeutic agents, programmable genetic components are needed that confer tunable and versatile regulatory profiles. Moreover, these components must themselves possess properties compatible with the human therapeutic context, including high specificity, low immunogenicity, and deliverability.

[0007] T cell immunotherapy has shown great promise for the treatment of cancer, including liquid tumors such as leukemia and lymphoma. However, alongside its remarkable efficacy, there are significant side effects, such as cytokine release syndrome (CRS) and neurotoxicity, which pose life-threatening risks to patients undergoing immunotherapy. It is becoming increasingly important to develop safe and effective sensor-response strategies that can control the activity of engineered T cells after infusion. Summary of the Invention

[0008] The technology described herein is directed to a method for controlling gene expression. Overall, the technology described herein relates to a synthetic transcription factor (synTF) comprising a regulatory protein (RP), where the regulatory protein regulates the activity of the synTF. In particular, the synTF according to the methods, systems, and compositions disclosed herein comprises (i) a DNA-binding domain (DBD) that binds to a target nucleic acid sequence (or target DNA-binding motif (DBM)) located 3' of a promoter that is operably linked to the nucleic acid of a gene of interest (GOI) to be expressed, (ii) an effector domain (ED) and a regulatory protein (RP), where the regulatory protein controls the coupling of the DNA-binding domain (DBD) with the effector domain (ED) or controls the cellular localization of the ED, so that when the ED and DBD are attached and / or located in the nucleus, the ED can function to recruit or repress the translation machinery to the promoter to regulate gene expression of the gene of interest. In some embodiments, the ED can be a transcriptional activator (TA), such that when the ED is present at the transcription start site of a gene of interest, it regulates gene expression; in some embodiments, the ED is a transcriptional repressor (TR), such that when the ED is present at the transcription start site of a gene of interest, gene expression is inhibited or repressed.

[0009] In some embodiments of the systems, compositions, and methods disclosed herein, the regulatory protein of SynTF is selected from a protease, a pair of inducible proximity domains (IPDs), or a translocation domain (i.e., a cytosolic sequestration protein), each of which is described in more detail herein and below.

[0010] In some embodiments of the systems, compositions, and methods disclosed herein, a synTF comprises a regulatory protein that is a self-cleaving protease; for example, one exemplary protease is NS3. SynTFs that comprise a self-cleaving protease are referred to herein as " Inhibitory protease SynTFIn such embodiments of the systems, compositions, and methods disclosed herein, the DBD is directly or indirectly linked (or coupled) to the effector domain, and a protease regulatory protein (typically located between the DBD and the ED) controls the coupling of the DBD to the ED. In such embodiments, in the presence of an agent that inhibits the regulatory protein (i.e., the NS3 protein), the DBD and ED remain coupled or intact (either directly or indirectly), and the effector domain can control gene expression from the promoter (i.e., turning on gene expression of the gene of interest (GOI) if the ED is a TA, or repressing gene expression if the ED is a TR) (see, e.g., Figures 5A-5C). In such an embodiment, in the absence of an agent that inhibits the regulatory protein (i.e., the NS3 protein), the connection between the DBD and the ED is disrupted or cleaved, such that the ED is no longer in close proximity to the transcription start site of the gene (or the ED dissociates from the start site), and as a result, the TA can no longer initiate gene expression of the GOI, and in return, the RP can no longer repress gene expression of the gene of interest.

[0011] In another embodiment of the systems, compositions, and methods disclosed herein, a synTF comprises a regulatory protein that is a pair of inducer proximal domains (referred to as an "IPD pair") located between a DBD and an ED, where each domain of the IPD comes together in the presence of an inducer substance, thereby linking the DBD and ED and controlling gene expression (see, e.g., Figures 3 and 7A). SynTFs that comprise an IPD pair are referred to herein as " Heterodimerization domain SynTF". For example, in such embodiments where the regulatory protein is an IPD pair, each domain of the IPD pair is attached to either the DBD or the ED, such that in the presence of an inducer agent, each domain of the IPD binds to the inducer agent, thereby indirectly coupling the DBD and ED, such that when the DBD binds to the promoter region, the ED can control gene expression from the promoter (i.e., if the ED is a TA, it turns on gene expression, or if the ED is a TR, it represses gene expression) (see, e.g., Figure 7A). In alternative embodiments where the RP is an IPD pair, in the absence of an inducer agent, the DBD and ED remain uncoupled, such that the ED is not in a position to regulate gene transcription from the transcription start site at the GOI. Exemplary IPD pairs and their inducers are disclosed herein.

[0012] In some embodiments of the systems, compositions, and methods disclosed herein, the synTF comprises a regulatory protein that is a translocation domain. In some embodiments, the translocation domain is a cytosolic sequestration protein, for example, one exemplary cytosolic sequestration protein is ERT2 and its variants. SynTFs that comprise a translocation domain, for example, a cytosolic sequestration protein, are referred to herein as " Transition domain SynTFIn such embodiments of the systems, compositions, and methods disclosed herein, the DBD is directly or indirectly linked (or coupled) to an effector domain, and a translocation domain, e.g., a cytosol sequestration protein regulatory protein (which can be attached to either the ED or the DBD, or can be located between the DBD and ED), controls the cellular localization of the DBD-ED-containing synTF (see, e.g., Figure 4). In such embodiments, in the absence of a ligand that binds to the cytosol sequestration protein, the cytosol sequestration protein sequesters the ED in the cytosol and couples to the DBD, such that the ED is no longer in proximity to the transcription start site of the gene (and the ED dissociates from the start site), such that the TA can no longer initiate gene expression of the GOI, and in return, the RP can no longer repress gene expression of the gene of interest. In contrast, in the presence of a ligand that binds to the cytosolic sequestration protein, the cytosolic sequestration protein is inhibited, allowing the DBD-ED of the synTF to translocate from the cytosol to the nucleus where the DBD can bind to the DNA-binding motif (DBM), and the effector domain (ED) can control gene expression from the promoter (i.e., turn on gene expression of the gene of interest (GOI) if the ED is a TA, or repress gene expression if the ED is a TR) (see, e.g., Figure 4).

[0013] Another aspect of the systems, compositions, and methods disclosed herein is a synTF that includes a small-molecule assisted shutoff (SMASh) domain, which may also be referred to herein as an "inducible degradation domain." Generally, the SMASh domain functions to target a polypeptide attached to the SMASh domain for degradation. In some embodiments, the SMASh domain is attached to a synTF that includes a regulatory protein that is an inducer proximity domain pair (IPD) or a cytosolic sequestration protein (see, e.g., Figure 6B). In alternative embodiments, the regulatory protein can be a SMASh domain (i.e., the SMASh domain replaces a self-cleaving protease regulatory protein in the synTF) (see, e.g., Figure 6A).

[0014] In all aspects of the methods, systems, and compositions disclosed herein, the SMASh domain comprises a self-cleaving protease and a degron domain. In some embodiments, the self-cleaving protease is the NS3 protease disclosed herein or a variant thereof. In some embodiments, the SMASh domain comprises an NS3 protease domain, a partial NS3 helical domain, and an NS4A domain, and can be fused to the N-terminus or C-terminus of a synTF described herein. Without being limited by theory, and merely illustrative, when a SMASh domain is attached to a synTF disclosed herein and an inhibitor of the SMASh domain self-cleaving protease is present, both the SMASh domain and the attached synTF are targeted for degradation. This is referred to as "SynTF-degradation," and results in the synTF being "SynTF-off," because the synTF is degraded and cannot bind to DBM or regulate the expression of a gene of interest, regardless of the type of effector domain present in the synTF. Conversely, in the absence of an inhibitor of the self-cleaving protease, the self-cleaving protease cleaves (or uncouples) the SMASh domain from the synTF, targeting only the SMASh domain for degradation, allowing the activity of the released synTF to be regulated via regulatory proteins. Thus, when the SMASh domain is attached to the synTF, in the absence of a protease inhibitor, it is referred to as "SMASh-degraded," resulting in the synTF being "SynTF-on," allowing it to be regulated by regulatory proteins. Depending on whether the ED is a transcriptional activator or repressor protein, gene expression can be either activated or repressed. Thus, the presence of the SMASh domain attached to the synTF allows a second level of control for the expression of the GOI, in addition to regulatory proteins.

[0015] In some embodiments, the SMASh domain itself serves as a regulatory protein for a synTF (i.e., the SMASh domain replaces a self-cleaving protease regulatory protein), and is referred to as an inducible degradation domain SynTF (see, e.g., FIG. 6A). In such embodiments in which the SMASh domain serves as a regulatory protein, the SMASh domain can be attached to either the ED or DBD of the synTF; in the absence of the NS3 protease, the NS3 protease is active and the SMASh domain uncouples from the synTF, thereby targeting only the SMASh domain for degradation, allowing the synTF containing the DBD and coupled ED to control gene expression from the promoter (i.e., the DBD binds to the DBM, bringing the ED into proximity with the promoter, turning on gene expression of the GOI if the ED is a TA, or repressing gene expression if the ED is a TR). In such an embodiment, in the absence of an agent that inhibits the regulatory protein (i.e., the NS3 protein), the connection between the DBD and the ED is disrupted or cleaved, such that the ED is no longer in close proximity to the transcription start site of the gene (or the ED dissociates from the start site), and as a result, the TA can no longer initiate gene expression of the GOI, and in return, the RP can no longer repress gene expression of the gene of interest.

[0016] In some embodiments where the SMASh domain is attached to a translocation domain synTF (e.g., where the regulatory protein is a sequestration protein) or a heterodimerization domain synTF (i.e., where the regulatory protein is a pair of inducible proximity domains (IPD pairs)), the SMASh domain can be referred to as a "SMASh tag" and can be attached to the C-terminus or N-terminus of the synTF (see, e.g., Figure 6B or Figure 16A). By way of example only, a C-terminal SMASh tag can be attached to the C-terminus of the ED, or the regulatory protein of a synTF can comprise, from N-terminus to C-terminus, an NS3 cleavage site, at least one linker, an NS3 domain, an NS3 partial helicase, and an NS4A domain, where the SMASh tag is fused to the C-terminus of the effector domain of a synTF. In some embodiments, and by way of example only, in which the SMASh tag is fused to the N-terminus of the synTF (referred to herein as an "N-terminal SMASh tag"), the SMASh tag comprises, in order from N-terminus to C-terminus, at least one linker, an NS3 domain, an NS3 partial helicase, an NS4 domain, and an NS3 cleavage site, wherein the SMASh tag is fused to the N-terminus of the synTF.

[0017] Another aspect of the technology disclosed herein relates to a system for controlling gene expression of a gene of interest (GOI), comprising a synTF as described herein and a nucleic acid construct comprising an element to which the synTF binds to regulate gene expression. In particular, in some aspects, the system comprises (i) at least one synthetic transcription factor (synTF) disclosed herein, and (ii) at least one nucleic acid construct, wherein the synTF comprises at least one DNA-binding domain (DBD), a transcriptional effector domain (ED), and at least one regulatory protein (RP), wherein the ED is coupled or linked, directly or indirectly, to the DBD, and the coupling is regulated by the at least one RP, or the cellular localization of the ED linked to the DBD is regulated by the at least one RP, and the at least one RP is regulated by an RP inducer, wherein the DBD is capable of binding to a target DNA-binding motif (DBM) located upstream of a promoter operably linked to a gene, and the nucleic acid construct comprises (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 (GOI) operably linked to the promoter sequence. In some embodiments (e.g., protease domain synTFs or inducible proximity domain synTFs) in which a regulatory protein of a synTF regulates coupling of the ED to the DBD, in the presence of an RP inducer, coupling of the ED to the DBD of the synTF is maintained, allowing binding of the DBD to the DNA-binding motif (DBM) to bring the ED into proximity with the promoter sequence, where the ED controls expression of the gene of interest ("ED-on"). In embodiments in which the ED is a transcriptional activator (TA), it results in gene expression being turned on ("TA-on" (expression)), while in embodiments in which the ED is a transcriptional repressor protein (TR), it inhibits or represses gene expression ("TR-on" (no expression)).In contrast, in the absence of an RP inducer, coupling between the ED and DBD of the synTF is dissociated, and binding of the DBD to the DNA-binding motif (DBM) prevents the ED from coming into close proximity with the promoter sequence, thereby preventing gene expression of the gene of interest ("ED-off"). In embodiments where the ED is a transcriptional activator (TA), this results in gene expression being turned off ("TA-off" (no expression)), whereas in embodiments where the ED is a transcriptional repressor protein (TR), it turns gene expression on ("TR-off" / repression off, allowing gene expression).

[0018] In embodiments in which a regulatory protein of a synTF regulates the cellular localization of the synTF (DBD and linked ED), when an RP inducer is present, the ED coupled to the DBD of the synTF is not sequestered in the cytosol, allowing the DBD to bind to the DNA-binding motif (DBM) and the transcriptional effector domain (ED) to be in close proximity to the promoter sequence, allowing expression of the gene of interest to be controlled ("ED-on"). In embodiments in which the ED is a transcriptional activator (TA), it results in gene expression being turned on ("TA-on" (expression)), whereas in embodiments in which the ED is a transcriptional repressor protein (TR), it inhibits or represses gene expression ("TR-on" (no expression)).

[0019] Furthermore, in the absence of an RP inducer, the ED coupled to the DBD of the synTF is sequestered in the cytosol, preventing binding of the DBD of the synTF to the DBM and preventing the proximity of the effector domain (ED) to the promoter sequence and preventing expression of the gene of interest ("ED-off"). In embodiments where the ED is a transcriptional activator (TA), this results in gene expression being turned off ("TA-off" (no expression)), whereas in embodiments where the ED is a transcriptional repressor protein (TR), it turns gene expression on ("TR-off" / repression off, allowing gene expression).

[0020] Table 17. Summary of the presence or absence of regulatory protein inducers on the ultimate expression of genes of interest. TIFF0007663513000001.tif87166

[0021] Therefore, whether gene expression of the GOI occurs depends on three levels of control, including but not limited to: (i) the type of regulatory protein in the synTF, (ii) the presence or absence of a regulatory protein inducer (RP inducer), and (iii) the type of effector domain.

[0022] Moreover, in some embodiments, systems for regulating gene expression can be configured for additional levels of control over gene expression depending on whether or not there is a SMASh domain attached to the synTFs disclosed herein. For example, attachment of a SMASh domain to a synTF is thought to result in the following: if an inhibitor of the SMASh protease is present, SMASh protease activity is inhibited, resulting in the synTF being degraded ("Syn-degradation") and preventing the DBD of the synTF from binding to the DBM and controlling expression or repression of the gene of interest ("synTF-degradation"; TA-off (no expression), TR-off (expression). In an alternative embodiment, if an inhibitor of the SMASh protease is not present, the SMASh protease is active and self-cleaves / uncouples from the synTF, resulting in the SMASh domain being targeted for degradation and allowing the DBD of the synTF to bind to the DBM and ED of the synTF, controlling expression of the gene of interest ("SMASh-degradation, TA-on (expression), TR-on (no expression)).

[0023] Another aspect of the technology described herein relates to cells that also contain a nucleic acid sequence disclosed herein for binding of a synTF to regulate expression of a gene of interest and a nucleic acid encoding a synthetic transcription factor. In some embodiments, the nucleic acid sequences are on separate constructs, and in some embodiments, they are the same construct as disclosed herein, referred to as a "single vector."

[0024] Other embodiments will be readily apparent from this disclosure. Aspects of the present invention teach certain advantages in construction and use, which result in the following exemplary benefits.

[0025] [The present invention 1001] a. at least one DNA binding domain (DBD); b. a transcription effector domain (ED); c. at least one regulatory protein (RP) A synthetic transcription factor (synTF) comprising: the ED is directly or indirectly coupled or linked to the DBD, and the coupling is regulated by at least one RP, or The cellular localization of the ED is regulated by at least one regulatory protein; The synTF. [The present invention 1002] 1001. The synTF of the present invention, wherein said transcription ED is a transcription activator (TA) domain. [The present invention 1003] 1002. The synTF of the present invention, wherein said TA is selected from the group consisting of p65; Rta; miniVPR; complete VPR; VP16; VP64; p300; p300 HAT core; and CBP HAT domain. [The present invention 1004] The synTF of the present invention 1002, wherein the TA is p65 or a variant thereof. [The present invention 1005] 1004. The synTF of the present invention, wherein the p65 comprises one of SEQ ID NOs: 69, 117-121, 193-197, or a protein having at least 85% sequence identity with one of SEQ ID NOs: 69, 117-121, 193-197. [The present invention 1006] 1001. The synTF of the present invention, wherein said transcription ED is a transcription repressor (TR) domain. [The present invention 1007] 1006. The synTF of the present invention, wherein said TR is selected from the group consisting of KRAB; KRAB-MeCP2; Hp1a; DNMT3B; EED; and HDAC4. [The present invention 1008] The synTF of the present invention 1006, wherein said TR is KRAB or a variant thereof. [The present invention 1009] 1008. The synTF of the present invention, 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 NOs: 72, 97, or 214-215. [The present invention 1010] 1001. The synTF of claim 10, wherein said at least one DBD is an engineered zinc finger (ZF) binding domain. [The present invention 1011] The synTF of the present invention 1010, wherein said ZF binding domain comprises two or more ZF motifs. [The present invention 1012] 1011. The synTF of the present invention, wherein the ZF binding domain comprises any one of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or more ZF motifs, and the ZF motifs are arranged in tandem adjacent to each other to form a ZF array (ZFA). [The present invention 1013] The ZF binding domain is selected from the group consisting of 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 Any one of synTFs 1001 to 1012 of the present invention, selected from any one of 11-1. [The present invention 1014] The synTF of any one of claims 1001 to 1013, wherein the ZF binding domain is selected from any one of SEQ ID NOs: 1 to 3, 76, 101, 377, or 380. [The present invention 1015] 1014. The synTF of the present invention, wherein the ZF binding domain specifically binds to a sequence comprising at least one of SEQ ID NOs: 181 to 191. [The present invention 1016] 10. The synTF of any of claims 1001 to 1015, wherein at least one DBD is selected from any one or more of SEQ ID NOs: 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 a DNA binding motif (DBM) comprising any of SEQ ID NOs: 229 to 240. [The present invention 1017] 1001. The synTF of the present invention, wherein said regulatory protein is located between said DBD and said transcription effector domain. [The present invention 1018] The synTF of any one of claims 1001 to 1017, wherein the regulatory protein is an inhibitory protease. [The present invention 1019] The synTF of any one of claims 1001 to 1018, wherein the regulatory protein is an NS3 protease protein. [The present invention 1020] The synTF of any one of claims 1001 to 1019, wherein the regulatory protein comprises the amino acids of SEQ ID NO: 82, 85, 91, 102, 241-255, 304-315, or a homolog having at least 85% sequence identity to SEQ ID NO: 82, 85, 91, 102, 241-255, 304-315. [The present invention 1021] The synTF of any one of claims 1001 to 1019, wherein in the presence of a protease inhibitor or an inhibitor of NS3, the NS3 protease protein is inhibited, thereby maintaining the coupling between the DBD and the effector domain. [The present invention 1022] Any of the synTFs of the present inventions 1001 to 1019, wherein in the absence of a protease inhibitor or an inhibitor against NS3, the NS3 protease protein is active and causes excision of the NS3 protease protein from the DBD, thereby uncoupling the connection between the DBD and the effector domain. [The present invention 1023] The synTF of the present invention 1021 or 1022, wherein the NS3 inhibitor is selected from any one of grazoprevir (GRZ / GZV), danoprevir, simeprevir, asunaprevir, cilprevir, boceprevir, sovaprevir, paritaprevir, ombitasvir, paritaprevir, ritonavir, dasabuvir, and telaprevir. [The present invention 1024] The at least one RP is a pair of induced proximity domains (IPD pair), the IPD pair comprising: The first induced proximity domain (IPD) A ), and at least a second complementary IPD (IPD B ) Including, wherein in the presence of an inducer substance or an inducer signal, the IPD A and IPD B together provide a link between the ED and the DBD of a synthetic TF; and in the absence of the inducer agent or inducer signal, the ED is not coupled or ligated to the DBD of the synthetic TF; The synTF of the present invention 1001. [The present invention 1025] the induced proximal domain pair (IPD pair) a. An IPD containing a GID1 domain or a fragment thereof, in which the GID1 domain and GAI domain bind to an inducer substance, gibberellin ester (GIB), A , and IPDs containing GAI domains B ; b. an IPD containing an FKBP domain or a fragment thereof, wherein the FKBP domain and the FRB domain bind to an inducer substance, rapalog (RAP); A , and IPDs containing FRB domains B ; c. An IPD containing a PYL domain or a fragment thereof, in which the PYL domain and the ABI domain bind to the inducer substance abscisic acid (ABA). A , and the IPD containing the ABI domain B ; d. IPD containing a light-inducible dimerization domain (LIDD) A wherein the LIDD is exposed to a light inducer signal of an appropriate wavelength to form a complementary LIDD (IPD B ) and the IPD A IPD selected from any one or more of A and IPD B 1024 synTFs of the present invention, comprising: [The present invention 1026] The induced proximal domain pair (IPD pair) is an IPD pair comprising a PYL domain or a fragment thereof and an ABI domain. A and IPD B wherein the PYL domain and the ABI domain bind to the inducer substance abscisic acid (ABA). [The present invention 1027] The synTF of the present invention 1024, wherein the LIDD is nMag or CIBN or a photochromic protein domain, wherein the nMag is capable of dimerizing with a complementary LIDD pMag upon exposure to a blue light inducer signal, and the CIBN is capable of dimerizing with a complementary CRY2 upon exposure to a blue light inducer light signal, and the photochromic protein is capable of dimerizing upon exposure to a blue light inducer light signal. [The present invention 1028] The synTF of the present invention 1024, wherein the light-inducible signal is a pulsed light signal. [The present invention 1029] The synTF of any of claims 1001 to 1017, wherein said at least one RP is a cytosol sequestration protein. [The present invention 1030] 1029. The synTF of the present invention, wherein said sequestration protein comprises a ligand binding domain (LBD), and in the presence of a ligand, sequestration of said protein into the cytosol is inhibited. [The present invention 1031] The synTF of the present invention 1029, wherein the cytosolic sequestration protein comprises a ligand binding domain and a nuclear localization signal (NLS), wherein in the absence of a ligand, the NLS is inhibited, thereby preventing the translocation of the sequestration protein to the nucleus, and in the presence of the ligand, the nuclear localization signal is exposed, allowing the translocation of the sequestration protein to the nucleus. [The present invention 1032] The synTF of any one of claims 1029 to 1031, wherein the sequestering protein comprises at least a portion of an estrogen receptor (ER). [The present invention 1033] The synTF of any one of claims 1029 to 1032, wherein the sequestering protein comprises an estrogen ligand binding domain (ERT) or a variant thereof selected from the group consisting of ERT2, ERT, and ERT3. [The present invention 1034] The synTF of the present invention 1033, wherein the ERT binds to one or more ligands selected from tamoxifen, 4-hydroxytamoxifen (4OHT), endoxifen, and fulvestrant, and the binding of the ligand to the ERT exposes the NLS and results in nuclear translocation of the ERT. [This invention 1035] The synTF of the present invention 1029, wherein said cytosolic sequestration protein comprises a transmembrane receptor sequestration protein. [The present invention 1036] The synTF of any one of claims 1001 to 1035, comprising an N-terminal DBD, said sequestration protein, and a C-terminal effector domain. [This invention 1037] The synTF of the present invention 1036, wherein the effector domain is a transcription activator (TA). [The present invention 1038] The synTF of any of claims 1001 to 1035, comprising an N-terminal effector domain, a DBD, and a C-terminal cytosol sequestration protein. [This invention 1039] The synTF of the present invention, wherein the effector domain is a transcription repressor (TR). [The present invention 1040] The synTF of any of claims 1001 to 1023, wherein the NS3 protein is part of a Small molecule-Assisted Shutoff (SMASh) domain, and the SMASh domain comprises the NS3 protein, a partial protease helical domain, and an NS4A domain. [The present invention 1041] Any of the synTFs of the present inventions 1001 to 1009 and 1024 to 1040, wherein the synTF further comprises a small molecule auxiliary shutoff (SMASh) tag, the SMASh tag being an N-terminal or C-terminal SMASh domain comprising an inhibitory protease, a partial protease helical domain, and a cofactor domain. [The present invention 1042] The synTF of the present invention 1041, wherein the SMASh tag is a C-terminal SMASh domain, which comprises, in order from the N-terminus to the C-terminus, an NS3 cleavage site, at least one linker, an NS3 domain, an NS3 partial helicase, and an NS4A domain, and the SMASh tag is fused to the C-terminus of the effector domain of the synTF. [This invention 1043] The synTF of the present invention 1041, wherein the SMASh tag is an N-terminal SMASh domain, which comprises, in order from the N-terminus to the C-terminus, at least one linker, an NS3 domain, an NS3 partial helicase, an NS4 domain, and an NS3 cleavage site, and the SMASh tag is fused to the N-terminus of the synTF. [This invention 1044] In the absence of an inhibitor to the NS3 protease, the NS3 protease is active and self-cleaves / uncouples from the synTF, thereby targeting the SMASh tag for degradation ("SMASh-degradation", synTF-on / TA-on / RP-on), and In the presence of an inhibitor of the NS3 protease, NS3 protease activity is inhibited, thereby resulting in the targeting of the SMASh-tagged synTF for degradation ("synTF-degradation", synTF-off / TA-off / RP-off"). Any one of synTFs 1040 to 1043 of the present invention. [This invention 1045] The synTF of the present invention 1044, wherein the inhibitor against NS3 protease is selected from any of grazoprevir (GRZ / GZV), danoprevir, simeprevir, asunaprevir, cilprevir, boceprevir, sovaprevir, paritaprevir, ombitasvir, paritaprevir, ritonavir, dasabuvir, and telaprevir. [The present invention 1046] Any of synTFs 1024 to 1045 of the present invention, comprising a SMASh tag and a cytosolic sequestration protein. [This invention 1047] A synTF of the present invention 1046, which is active in the presence of a ligand for the cytosol sequestration protein and in the absence of an inhibitor for the NS3 protease; and is inactive in the absence of a ligand for the cytosol sequestration protein and in the presence of an inhibitor for the NS3 protease. [This invention 1048] the synTF further comprises a linker peptide, which can be located between the DBD and the regulatory protein; between the regulatory protein and the effector domain; between the DBD and the effector domain; within the DBD, within the effector domain, or within the regulatory protein; or any combination thereof; TIFF0007663513000002.tif71153 The synTF of any one of 1001 to 1047 of the present invention may be [This invention 1049] c. at least one synthetic transcription factor (synTF) comprising at least one DNA binding domain (DBD), a transcription effector domain (ED), and at least one regulatory protein (RP); The ED is directly or indirectly coupled or linked to the DBD, and the coupling is regulated by at least one RP, or the cellular localization of the ED linked to the DBD is regulated by at least one RP; At least one RP is regulated by an RP inducer; The DBD is capable of binding to a target DNA binding motif (DBM) located upstream of a promoter operably linked to a gene, the synTF, at least one target DNA binding motif (DBM) comprising a target nucleic acid for binding of at least one DBD of the di-synTF; and ii. a promoter sequence located 3' of at least one DBM; and iii. A gene of interest operably linked to a promoter sequence a nucleic acid construct comprising: A system for controlling gene expression, comprising: For synTFs in which coupling between the ED and DBD is regulated by at least one RP, In the presence of an RP inducer, coupling between the ED and DBD of the synTF is maintained, allowing the ED to be in close proximity to the promoter sequence upon binding of the DBD to the DNA-binding motif (DBM), where the ED controls expression of the gene of interest ("ED-on"), or In the absence of an RP inducer, the coupling between the ED and DBD of the synTF is dissociated, preventing the ED from coming into close proximity with the promoter sequence upon binding of the DBD to the DNA-binding motif (DBM), thereby preventing gene expression of the gene of interest ("ED-off"); and For synTFs, the cellular localization of the DBD-linked ED is regulated by at least one regulatory protein. In the presence of an RP inducer, the ED coupled to the DBD of the synTF is not sequestered in the cytosol, allowing the DBD to bind to the DNA-binding motif (DBM), and thus allowing the transcriptional effector domain (ED) to be in close proximity to the promoter sequence and control expression of the gene of interest ("ED-on"); or In the absence of an RP inducer, the ED coupled to the DBD of the synTF is sequestered in the cytosol, preventing binding of the DBD of the synTF to the DBM and preventing proximity of the effector domain (ED) to the promoter sequence, thereby preventing expression of the gene of interest ("ED-off"); The system. [The present invention 1050] the transcription effector domain (ED) is a transcription activator (TA); c. For synTFs in which coupling between the ED and DBD is regulated by at least one RP, i. In the presence of an RP inducer, coupling between the ED and DBD of the synTF is maintained, allowing binding of the DBD to the DNA-binding motif (DBM) to bring the TA domain into proximity with the promoter sequence, where the TA turns on expression of the gene of interest ("TA-on"); or ii. In the absence of an RP inducer, coupling between the ED and DBD of the synTF is dissociated, preventing the TA domain from coming into close proximity with the promoter sequence upon binding of the DBD to the DNA-binding motif (DBM), thereby preventing expression of the gene of interest ("TA-off"); and d. For synTFs in which at least one RP regulates the cellular localization of the DBD-linked ED, i. In the presence of an RP inducer, the ED coupled to the DBD of the synTF is not sequestered in the cytosol, allowing the DBD to bind to the DNA-binding motif (DBM), and thus allowing the TA domain to come into close proximity with the promoter sequence and turn on expression of the gene of interest ("TA-on"); or ii. In the absence of an RP inducer, the ED coupled to the DBD of the synTF is sequestered in the cytosol, preventing binding of the DBD to the DBM and preventing the proximity of the TA domain to promoter sequences, thereby preventing expression of the gene of interest ("TA-off"); The system of the present invention 1049. [This invention 1051] ED is a transcriptional repressor (TR), c. For a synTF, in which coupling of the ED to the DBD is regulated by at least one regulatory protein, i. In the presence of an RP inducer, coupling between the ED and DBD of the synTF is maintained, allowing binding of the DBD to the DNA-binding motif (DBM) to bring the transcriptional repressor (TR) into close proximity with the promoter sequence, where the TR prevents expression of the gene of interest ("TR-on" (no expression) or ii. In the absence of an RP inducer, the coupling between the ED and DBD of the synTF is dissociated, and binding of the DBD to the DNA-binding motif (DBM) prevents the transcriptional repressor (TR) from approaching the promoter sequence, thereby allowing expression of the gene of interest ("TR-off" (expression present)); and d. For synTFs in which the cellular localization of the DBD-linked ED is regulated by at least one regulatory protein, i. In the presence of an RP inducer, the ED coupled to the DBD of the synTF is not sequestered in the cytosol, allowing the DBD to bind to the DNA-binding motif (DBM), and thus allowing the transcriptional repressor (TR) to come into close proximity with the promoter sequence and turn off expression of the gene of interest (either "TR-on" (no expression) or ii. In the absence of RP inducers, the ED coupled to the DBD of the synTF is sequestered in the cytosol, preventing binding of the DBD to the DBM and preventing the proximity of transcriptional repressors (TRs) to the promoter sequence, thereby allowing expression of the gene of interest (with "TR-off" expression); The system of the present invention 1049. [This invention 1052] The system of any one of claims 1049 to 1051, wherein the synTF is selected from any one of the synTFs of claims 1001 to 1048. [This invention 1053] the at least one synTF further comprises a small molecule auxiliary shutoff (SMASh) domain at the N-terminus or C-terminus, the SMASh domain comprising a self-cleaving SMASh protease, a partial protease helical domain, and a cofactor domain; wherein in the presence of an inhibitor of the SMASh protease, the SMASh protease activity is inhibited, resulting in the degradation of the synTF and preventing the binding of the DBD of the synTF to the DBM, thereby controlling the expression or repression of the gene of interest ("synTF-degradation"; TA-off (no expression), TR-off (expression)); In the absence of an inhibitor to the SMASh protease, the SMASh protease is active and self-cleaves / uncouples from the synTF, resulting in the SMASh domain being targeted for degradation, and thus allowing the DBD of the synTF to bind to the DBM and ED of the synTF and regulate expression of the gene of interest ("SMASh-degraded, TA-on (expressed), TR-on (no expression)"); Any of the systems of the present invention 1049 to 1052. [This invention 1054] Any of the systems of present inventions 1049 to 1053, wherein the promoter is selected from the group consisting of a mini-CMV promoter, a mini-TK promoter, a ybTATA promoter, a minSV40 promoter, a CMV53 promoter, a pJB42CAT5 promoter, an MLP promoter, a TATA promoter, a pSFFV promoter, a CMV promoter, a pUb / UbC promoter, an EF1a promoter, a PGK / pGK promoter, a CAG / CAGG promoter, an SV40 promoter, and a β-actin / ACTB promoter. [This invention 1055] a. a first nucleic acid sequence comprising at least one target DNA binding motif (DBM) comprising a target nucleic acid for binding of 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) operably linked to the promoter sequence; and b. A second nucleic acid sequence comprising a nucleic acid encoding a synthetic transcription factor (synTF) of any one of claims 1001 to 1048 of the present invention, operably linked to an inducible or constitutive promoter. including, cells. [The present invention 1056] a. a nucleic acid sequence encoding a gene of interest (GOI) in reverse orientation; b. a first promoter sequence in reverse orientation and operably linked to a nucleic acid encoding the GOI; c. a nucleic acid sequence comprising at least one target DNA binding motif (DBM) comprising a target nucleic acid for binding of at least one DBD of a synTF, wherein binding of the DBD of the synTF places an effector domain (ED) in proximity to the promoter sequence operably linked to the GOI; d. a second promoter sequence, and e. a nucleic acid sequence encoding a synthetic transcription factor (synTF) operably linked to the second promoter sequence, wherein the encoded synTF comprises at least one DBD that binds to at least one DBM of the nucleic acid sequence of (c). 1055. The cell of claim 1055, comprising a nucleic acid construct comprising, in a 5' to 3' direction: [This invention 1057] The cell of claim 1055 or 1056, wherein the promoter sequence operably linked to the GOI is selected from the group consisting of a mini-CMV promoter, a mini-TK promoter, a ybTATA promoter, a minSV40 promoter, a CMV53 promoter, a pJB42CAT5 promoter, an MLP promoter, and a TATA promoter. [This invention 1058] The cell of 1055 or 1056, wherein the promoter sequence operably linked to the nucleic acid encoding the synTF is a pSFFV promoter, a CMV promoter, a pUb / UbC promoter, an EF1a promoter, a PGK / pGK promoter, a CAG / CAGG promoter, an SV40 promoter, or a β-actin / ACTB promoter. [This invention 1059] A polynucleotide encoding any one of the synTFs of the present invention 1001 to 1048 or any one of the synTF systems of the present invention 1049 to 1054. [The present invention 1060] A vector comprising the polynucleotide of the present invention. [This invention 1061] Any of the synTFs of the present inventions 1001 to 1048, any of the synTF systems of the present inventions 1049 to 1054, any of the synTF cells of the present inventions 1055 to 1058, the polynucleotide of the present invention 1059, or the vector of the present invention 1060. A composition comprising: [This invention 1062] A synTF according to any one of 1001 to 1048 of the present invention, a synTF system according to any one of 1049 to 1054 of the present invention, a synTF cell according to any one of 1055 to 1058 of the present invention, a polynucleotide according to 1059 of the present invention, or a vector according to 1060 of the present invention; a pharmaceutically acceptable carrier; 10. A pharmaceutical composition comprising: [The present invention 1063] a. providing a population of cells according to any one of claims 1055 to 1058; and b. contacting said population of cells with an effective amount of a regulatory protein inducer. A method for regulating the activity of synTF, comprising: [The present invention 1064] a. providing a population of cells according to any one of claims 1055 to 1058; and b. contacting said population of cells with an effective amount of a regulatory protein inducer. A method for regulating the expression of a gene of interest, comprising: [This invention 1065] 1. A method of treating a subject in need of cell therapy, comprising: a. administering to the subject a population of cells according to any one of 1055 to 1058 of the present invention; and b. administering to said subject an effective amount of a regulatory protein inducer. The method comprises: [The present invention 1066] 1066. The method of any one of claims 1063 to 1065, wherein said population of cells comprises immune cells. [This invention 1067] 1065. The method of claim 105, wherein said population of immune cells comprises CD4+ T cells, CD8+ T cells, Tregs, or NK cells. [The present invention 1068] The method of any of claims 1065 to 1067, wherein said population of cells is administered simultaneously with said regulatory protein inducer. [The present invention 1069] The method of any of claims 1065 to 1067, wherein said regulatory protein inducer is administered after said population of cells is administered. Other features and advantages of aspects of the present invention will become apparent from the following more detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, the principles of aspects of the present invention. [Brief explanation of the drawings]

[0026] (Figure 1) Figures 1A-1H are a series of schematics and graphs showing the design and characterization of mammalian synTFs based on orthogonal ZF arrays. Figure 1A is a schematic showing the design of synTFs based on a 6-unit orthogonal ZF array. Figure 1B is a schematic showing the synTF and reporter system. Figure 1C is a bar graph showing that synTFs robustly activate corresponding integrated reporters across a library of cognate synTF / DBM pairs; note that mCherry expression occurs only in the presence of the specific ZF-p65 synTF, not in the absence of the TF or in the presence of GFP-p65. Figure 1D is a heatmap showing the coexpression of specific synTFs and reporters; note that the mCherry reporter is activated by the synthetic operator and its cognate synTF. Figures 1E-1G are a series of scatter plots showing transcriptome profiling of human cells expressing synTFs, including ZF1-p65 (Figure 1E), ZF3-p65 (Figure 1F), and ZF10-p65 (Figure 1G), revealing highly specific orthogonal regulation of the synTFs. Figure 1H is a bar graph showing transcriptome profiling of synTFs relative to Gal4 and TetR. Differentially regulated transcripts = Log2|fold change|>1; FDR<0.1 (for a total of three independent replicates). (Figure 2) Schematic diagram showing the small molecule-responsive synTF system. (FIG. 3) Schematic showing exemplary synTFs regulated by induced proximity. (Figure 4) Schematic showing exemplary synTFs regulated by cytosolic sequestration. (Figure 5) Figures 5A-5C are a series of schematic diagrams showing exemplary synTFs regulated by self-cleaving protease inhibition. Figure 5A is a schematic diagram showing a synTF regulated by NS3. GRZ indicates grazoprevir (a small molecule inhibitor of NS3). Figure 5B is a schematic diagram showing the system in the absence of a small molecule. Self-cleavage of the NS3 protease leads to decoupling of the ZF (zinc finger binding domain or DNA binding domain (DBD)) and ED (effector domain), thereby disallowing transcriptional regulatory activity. Figure 5C is a schematic diagram showing the system in the presence of a small molecule. Inhibition of NS3 protease activity allows local coupling of the ZF and ED, thereby allowing transcriptional regulatory activity. (FIG. 6) Figures 6A-6B are a series of schematic diagrams showing exemplary synTFs regulated by inducible degradation. Figure 6A is a schematic diagram showing a synTF regulated by SMASh. Figure 6B is a schematic diagram showing a regulatory transcription factor utilizing both ERT2 and SMASh domains. The SMASh domain was placed at the C-terminus of an ERT2-containing synTF ("C-terminal SMASh," top) and at the N-terminus of an ERT2-containing synTF ("N-terminal SMASh," bottom). (Figure 7) Figures 7A-7C are a series of schematic diagrams depicting exemplary heterodimerized synTFs and graphs showing that administration of a small molecule (ABA) led to the transient activation of fluorescent protein output from a ZF-responsive promoter in a Jurkat cell line. Figure 7A shows a schematic diagram of an inducible proximity domain (IPD) that couples the DBD (or ZF1) to the ED. In this exemplary embodiment, ABA serves as an inducer ligand to maintain the coupling of ZF1 to the ED. Figure 7A also shows the measured output fluorescence as a function of several different ABA treatment concentrations (dose response) as indicated. Figure 7B shows that administration of a small molecule (ABA) led to the transient activation of fluorescent protein output from a ZF-responsive promoter in a Jurkat cell line. This expression level was compared to an untreated cell line, which did not activate output expression. Figure 7C shows that removal of the small molecule (ABA) after 4 days led to the transient deactivation of fluorescent protein output from a ZF-responsive promoter in a Jurkat cell line. This expression level was compared to an untreated cell line that did not activate output expression. Measurement time points on the chart begin on the day of drug removal ("Day 0"). (Figure 8) Figures 8A-8C are a series of schematic diagrams depicting an exemplary cytosol-sequestered synTF and graphs showing that administration of a small molecule (4OHT) led to the transient activation of fluorescent protein output from a ZF-responsive promoter in a Jurkat cell line. Figure 8A shows an exemplary synTF with a DBD (or ZF3) fused to an ED (i.e., p63) along with the cytosol-sequestering protein ERT2. Figure 8A also shows the measured output fluorescence as a function of several different 4OHT treatment concentrations (dose response). Figure 8B shows that administration of a small molecule (4OHT) led to the transient activation of fluorescent protein output from a ZF-responsive promoter in a Jurkat cell line. This expression level was compared to an untreated cell line that did not activate output expression. Figure 8C shows that removal of the small molecule (4OHT) after 4 days led to the transient deactivation of fluorescent protein output from a ZF-responsive promoter in a Jurkat cell line. This expression level was compared to an untreated cell line that did not activate output expression. Measurement time points on the chart begin on the day of drug removal ("Day 0"). (Figure 9) Figures 9A-9C are a series of schematic diagrams of exemplary inhibitory protease synTFs with NS3 as the protease and graphs showing that administration of a small molecule (grazoprevir) led to transient activation of fluorescent protein output from a ZF-responsive promoter in Jurkat cell lines. Figure 9A shows an exemplary synTF with a DBD (or ZF10) coupled to a protease coupled to an ED (i.e., p65). Figure 9A also shows the measured output fluorescence as a function of several different grazoprevir treatment concentrations (dose response) indicated. Figure 9B shows that administration of a small molecule (grazoprevir) led to transient activation of fluorescent protein output from a ZF-responsive promoter in Jurkat cell lines. This expression level was compared to an untreated cell line, which did not activate output expression. Figure 9C shows that removal of the small molecule (grazoprevir) after 4 days led to transient deactivation of fluorescent protein output from a ZF-responsive promoter in Jurkat cell lines. This expression level was compared to an untreated cell line that did not activate output expression. Measurement time points on the chart begin on the day of drug removal ("Day 0"). (Figure 10) Figures 10A-10D are a series of schematic diagrams depicting exemplary synTFs and graphs showing that administration of small molecules (e.g., ABA, 4OHT, or grazoprevir) led to the transient activation of fluorescent protein output from ZF-responsive promoters in both HEK293 and Jurkat cell lines. Figure 10A is a schematic diagram depicting three exemplary synTFs: a heterodimerization domain synTF, a cytosolic sequestration domain synTF, and an inhibitory protease synTF, all of which are regulated by ABA, 4OHT, or grazoprevir, respectively. Figure 10B is a schematic diagram depicting the reporter system. Figures 10C-10D are a series of line graphs (left) depicting synTF activation of reporters in HEK293 cells (Figure 10C) or Jurkat cells (Figure 10D) at various times and small molecule doses (doses are indicated by shades of gray, with darker gray corresponding to the highest dose indicated). Figures 10C-10D also show a series of bar graphs (right) depicting reporter synTF activation in HEK293 cells (Figure 10C) or Jurkat cells (Figure 10D) at the highest doses of D4 and small molecules (ABA 1 mM; 4OHT 4 μM; GRZ 4 μM). Figures 10C-10D show the top graph corresponding to the ABA-regulated synTF, the middle graph corresponding to the 4OHT-regulated synTF, and the bottom graph corresponding to the grazoprevir-regulated synTF. In Figures 10C-10D, this increased expression level was compared to an untreated cell line that did not activate output expression. (Figure 11) Figures 11A-11B are a series of diagrams and graphs showing inducible synthetic transcriptional repression of a fluorescent protein in human cell lines. Figure 11A is a schematic diagram showing a reporter system containing a repressor as an effector domain and an inducible synTF. Figure 11B is a series of graphs showing that administration of small molecules (top graph: ABA, middle graph: 4OHT; bottom graph: GRZ) led to transient silencing of fluorescent protein output from a ZF-responsive promoter in an HEK293 cell line. This reduced expression level was compared to an untreated cell line, which did not silence output expression. Figure 11B shows a series of line graphs (left) showing synTF repression of the reporter in HEK293 cells at various times and a series of bar graphs (right) showing synTF repression of the reporter in HEK293 cells at day 8. (Figure 12) Figures 12A-12C are a series of diagrams and graphs showing inducible synthetic transcriptional repression of a fluorescent protein in human cell lines. Figure 12A is a schematic diagram showing a reporter system and inducible synTF containing a repressor as the effector domain. Figures 12B-12C are a series of graphs showing that administration of a small molecule (grazoprevir) led to transient silencing of fluorescent protein output from a ZF-responsive promoter in an HEK293 cell line (Figure 12B) and a Jurkat cell line (Figure 12C). This reduced expression level was compared to an untreated cell line, which did not silence output expression. Figures 12B-12C are a series of line graphs (left) showing synTF repression of the reporter in cells at various times and a series of bar graphs (right) showing synTF repression of the reporter in cells at day 8. (Figure 13) Figures 13A-13D are a series of diagrams and graphs showing that administration of a small molecule (grazoprevir) led to transient activation of fluorescently tagged chimeric antigen receptor (CD19-CAR) protein from a ZF-responsive promoter in CD4+ primary human T cells. Figure 13A is a schematic diagram showing the CD19-CAR expression system and inducible synTF. The inhibitory protease synTF, NS3, is shown; however, any previously described synTF can be used to regulate expression of the CD19-CAR GOI using the methods disclosed herein. Figure 13B shows a line graph (left) depicting synTF activation of CD-19 synTF expression in CD4+ primary T cells over various times and at several different grazoprevir treatment concentrations, and a bar graph (right) depicting synTF activation of CD-19 synTF expression in CD4+ primary T cells at day 3. This increased expression level was compared to an untreated cell line that did not activate output expression. Figures 13C-13D show that subsequent coculture of these primary cells with CD19 antigen-presenting target cells (CD19+ NALM cells) resulted in T cell activation, as measured by enhanced cytokine production. This increased cytokine production level was compared to an untreated cell line that did not activate cytokine expression. Figures 13C-13D are a series of bar graphs showing IFNγ expression (Figure 13C) and IL-2 expression (Figure 13D) at several different grazoprevir treatment concentrations at D1 or D2. (Figure 14) Figures 14A-14B are a series of schematic diagrams and graphs showing the regulation of CD19 CAR and IL-4 expression in primary human T cells. Figure 14A is a schematic diagram showing the CD19-CAR and IL-4 expression system and exemplary inducible synTFs, namely, an inhibitory protease synTF for regulating CD19-CAR and a cytosol-sequestered synTF for regulating IL4 expression. Figure 14B is a series of line graphs showing CD19-CAR expression (top graph) and IL-4 expression (bottom graph) in the presence or absence of the indicated GZV or 4OHT at various time points. Figure 14C is a bar graph showing CD19-CAR expression (light gray, left axis) and IL-4 expression (dark gray, right axis) in the presence or absence of the indicated GZV or 4OHT on day 5. (Figure 15) Figures 15A-15C are a series of schematic diagrams showing nucleic acid constructs for a system for controlling gene expression and a graph showing that administration of a small molecule (grazoprevir) led to transient activation of a cytokine (IL10) from a ZF-responsive promoter in a Jurkat T cell line. Figures 15A-15B are a series of schematic diagrams showing that this inducible activation can be achieved by delivery of a separate nucleic acid construct for NS3-synTF expression, which then controls IL10 production ("double lentiviral vector," Figure 15A), or by delivery of a single nucleic acid construct that not only controls expression of NS3-synTF but also regulates IL10 production ("single lentiviral vector," Figure 15B). Figure 15C is a bar graph showing IL-10 in the presence (dark gray) or absence (light gray) of 1 μM GZV over 2 days in the double-vector or single-vector system. (Figure 16) Figures 16A-16B are a series of diagrams and graphs showing inducible synthetic transcriptional activation and tunable deactivation of fluorescent proteins in human cell lines using cytosol-sequestered and inducible degradation synTFs containing ERT2 and SMASh domains, respectively. Figure 16A is a diagram showing N-terminal and C-terminal SMASh synTFs. Figure 16B is a line graph showing reporter expression and the presence or absence of grazoprevir after removal of 4OHT as indicated. (Figure 17) A series of heat maps showing tunable synthetic transcriptional activation of fluorescent proteins in human cell lines using ERT2 domains and SMASh domains with C-terminal SMASh synTFs (left heat map) and N-terminal SMASh synTFs (right heat map). (FIG. 18) A series of line graphs showing inducible synthetic transcriptional repression of fluorescent proteins in human cell lines using KRAB-ZF-ERT2-SMASh (top graph), HP1a-ZF-ERT2-SMASh (middle graph), and EED-ZF-ERT2-SMASh (bottom graph). "Always off (+GZV)" indicates the presence of grazoprevir and the absence of 4OHT. "Always off (-GZV)" indicates the absence of both grazoprevir and 4OHT. "Always on" indicates the absence of grazoprevir and the presence of 4OHT. (Figure 19) TIFF0007663513000003.tif81164 (Figure 20) TIFF0007663513000004.tif81163 (Figure 21) TIFF0007663513000005.tif55164 (Figure 22) TIFF0007663513000006.tif49164 (Figure 23) TIFF0007663513000007.tif55164 (Figure 24) TIFF0007663513000008.tif55164 (Figure 25) TIFF0007663513000009.tif68164 (Figure 26) TIFF0007663513000010.tif68164 (Figure 27) TIFF0007663513000011.tif61164 (Figure 28) TIFF0007663513000012.tif68164 (Figure 29) TIFF0007663513000013.tif68164 (Figure 30) TIFF0007663513000014.tif68164 (Figure 31) is a series of graphs showing the identification of synthetic transcriptional operator sequences orthogonal to the human genome. We evaluated the occurrence of operator sequences in the human genome using a bioinformatic string-matching algorithm called "Biostrings." Here, the x-axis represents the number of occurrences of a particular string in the hg19 ref assembly; note that the scale varies for different sets and panels. The top panel shows 11 "synthetic operators" and instances of either the correct sequence or all possible mismatch sequences. The algorithm showed that there were no correct sequences or sequences with only one or two mismatches for these operators, suggesting that the sequence composition can be considered genomically distant. Several classical TF operator sequences were also compared to the human genome. The synthetic operators described herein outperform these "heterologous" recognition sequences of similar or shorter length. DETAILED DESCRIPTION OF THE INVENTION

[0027] The following abbreviations used herein are defined as follows: synTF (synthetic transcription factor); ZF (zinc finger); HEK (human embryonic kidney 293 cells); ED (effector domain); AD (activator domain); RD (repressor domain); GOI (gene of interest); OUT (output); DBM (DNA-binding motif); pUb (ubiquitin C promoter); p65 (transcription factor P65); minCMV (minimal cytomegalovirus promoter); AAVS1 (adeno-associated virus integration site 1); chr19 (human chromosome 19); GFP (green fluorescent protein); TPM (transcripts per million; i.e., a normalized value for each individual RNA transcript across the entire pool of RNA transcripts for the sequenced sample); ABI (ABA-insensitive sex); PYL (PYR1-like, pyrabactin resistance 1-like); ABA (abscisic acid); ERT2 (estrogen receptor ligand-binding domain mutant variant), 4OHT (4-hydroxytamoxifen); NS3 (HCV nonstructural protein 3); GZV or GRZ (grazoprevir); SMASh (small molecule auxiliary shutoff); au (arbitrary units or relative luminescence intensity); IFNγ (interferon gamma); IL-2 (interleukin 2); IL-4 (interleukin 4); CD19 (cluster of differentiation 19); mCh (mCherry); pSFFV (silencing-prone spleen focus-forming virus promoter); minTK (minimal promoter fragment from HSV thymidine kinase (TK) promoter); Lenti (lentivirus).

[0028] Detailed Description Described herein are synTFs for use in the methods and compositions disclosed herein, wherein the synTFs comprise (i) a DNA-binding domain (DBD) that binds to a target nucleic acid sequence (or target DNA-binding motif (DBM)), (ii) an effector domain (ED) and a regulatory protein (RP), wherein the regulatory protein controls the coupling or linkage of the DNA-binding domain (DBD) to the effector domain (ED) (such coupling can also be referred to as a "mediator domain"), or controls the cellular localization of the ED such that when the ED and DBD are attached and / or located in the nucleus, the ED functions to recruit or repress the translation machinery to a promoter to regulate gene expression of a gene of interest.

[0029] In some embodiments of any of the aspects, regulatory proteins can be activated or inhibited by a variety of inputs, non-limiting examples of which include inducers (e.g., small molecules), light-induced regulation (e.g., dimerization, aggregation, localization), temperature, pH, phosphorylation, oxygen, lipids, magnetism, electricity, spatial mechanisms (e.g., intracellular and / or extracellular; e.g., synthetic receptors and / or soluble factors), endogenous ligands (e.g., biomarkers), cell cycle status, native signaling pathways, or disease and / or pathogenic conditions (e.g., aggregation, infection).

[0030] In some embodiments of the systems, compositions, and methods disclosed herein, the regulatory protein of SynTF is selected from a protease, a pair of inducible proximity domains (IPDs), a translocation domain (i.e., a cytosolic sequestration protein), or an inducible degradation domain, each of which is described herein and in more detail below.

[0031] Four overall frameworks for inducible or drug-controllable synthetic transcription factors are described herein: (1) synTFs containing an inhibitory protease, termed inhibitory protease synTFs; (2) synTFs containing an inducible proximity domain, termed inducible proximity domain synTFs; (3) synTF systems containing a cytosolic sequestration domain, termed cytosolic sequestration synTFs; and (4) synTFs containing an inducible degradation domain, termed inducible degradation domain synTFs. Polynucleotides and vectors encoding synTF polypeptides, cells expressing synTF polypeptides, pharmaceutical compositions containing synTF polypeptides, and methods of using synTF polypeptides are also described herein.

[0032] Described herein is a class of engineered transcription factor proteins (synTFs) and corresponding responsive artificial promoters capable of precisely controlling gene expression in a wide range of eukaryotic cells and organisms, including mammalian cells. These synTFs are specifically designed to have reduced or minimal binding capacity in the host genome (i.e., "orthogonal" activity to the host genome). The synTF proteins described herein contain a DNA-binding domain (DBD) based on an engineered zinc finger (ZF) array designed to target and bind to distinct, specific 18-20 nucleotide sequences distant from the host genome sequence when the synTF protein is used in a selected host. This strategy limits nonspecific interactions between the synTF protein and the host genome; such nonspecific interactions are not ideal and therefore undesirable.

[0033] The synTFs described herein are, in some aspects, designed according to the following parameters: (1) a targetable DNA sequence (also known as a ZF binding site) specifically designed to have reduced binding affinity for the ZF array is identified in the host genome; (2) the ZF array is designed and assembled; (3) the synTF is designed by coupling the engineered (i.e., covalently linked) ZF array with transcriptional and / or epigenetic effector domains; (4) a corresponding responsive promoter is designed by placing an instance of the targetable DNA sequence (i.e., a ZF binding site) upstream of a constitutive promoter. The targetable DNA sequence is operably linked to the promoter, such that the occupancy of the synTF on the targetable DNA sequence regulates promoter activity in gene expression. The combination of synTF and targetable DNA sequence-promoter forms an artificial, scalable, regulatable, and unique expression system for expression of an internally placed desired gene, which has no or minimal effect on endogenous gene expression, meaning there is no or minimal ex situ gene regulation of the endogenous gene.

[0034] The synTFs described herein have reduced or minimal functional binding capacity in the host genome, which in part provides the advantage of no or minimal out-of-site DNA targeting by the synTFs. Additionally, the synthetic ZF-based proteins (synTFs) described herein are derived from mammalian protein scaffolds, conferring minimal immunogenicity compared to other prokaryotic-derived domains. In contrast to other classes of programmable DNA targeting domains, these zinc finger-based regulatory proteins are significantly smaller (approximately 4-5 times) than TALE and dCas9 proteins and less repetitive than TALE repeat proteins, allowing them to be unconstrained by lentiviral packaging limitations and conveniently packaged into lentiviral delivery constructs, providing space for other desired regulatory elements.

[0035] I. Synthetic Transcription Factor Domains In several aspects, described herein are synTF polypeptides or synTF polypeptide systems comprising at least one of the following domains: a transcription effector domain, a DNA-binding domain, an inhibitory protease, an inducible proximity domain, a cytosolic sequestration domain, an inducible degradation domain, at least one regulatory protein selected from the group consisting of at least one linker peptide, at least one detectable marker, and / or a self-cleaving peptide, or any combination thereof. In some embodiments of any of the aspects, the synTF polypeptide or synTF polypeptide system collectively (i.e., the first polypeptide and / or the second polypeptide) comprises at least: a transcription effector domain, a DNA-binding domain, an inhibitory protease, an inducible proximity domain, a cytosolic sequestration domain, and / or an inducible degradation domain. In some embodiments of any of the aspects, the synTF polypeptide or system further comprises at least one linker peptide, or at least one detectable marker, and / or at least one self-cleaving peptide, or any combination thereof. The specific synTFs described herein should not be considered limiting. For example, the following combinations are contemplated herein (see, eg, Table 8):

[0036] Table 8: Exemplary combinations of domains in synTF polypeptides or synTF polypeptide systems. Each exemplary synTF polypeptide shown in Table 8 also contains a transcriptional effector domain and a DNA-binding domain. The domains can be in any order. "PRO" indicates inhibitory protease. "IPD" indicates inducible proximity domain. "CS" indicates cytosolic sequestration domain. "DD" indicates inducible degron domain. "LP" indicates linker peptide. "DM" indicates detectable marker. "SP" indicates self-cleaving peptide. TIFF0007663513000015.tif212167TIFF0007663513000016.tif138167

[0037] Transcriptional effector domain (ED) Described herein are synTFs that include a transcription effector domain (ED), which may also be referred to herein as an effector domain. In one embodiment of any aspect described herein, the transcription effector domain (ED) of the synTF is a transcription activation domain (TA) or a transcription repressor domain (also referred to herein as a transcription repressor (TR)). For example, the transcription effector domain is selected from the group consisting of a herpes simplex virus protein 16 (VP16) activation domain; an activation domain consisting of four tandem copies of VP16, a VP64 activation domain; the p65 activation domain of NFkB or a functional fragment thereof; an Epstein-Barr virus R transactivator (Rta) activation domain or a functional fragment thereof; a tripartite activator consisting of VP64, p65, and Rta activation domains, known as the VPR activation domain; mini-VPR; the histone acetyltransferase (HAT) core domain of human E1A-associated protein p300, known as the p300 HAT core activation domain; a CBP HAT domain; a Krüppel-associated box (KRAB) repression domain; KRAB-MeCP2; a DNA (cytosine-5)-methyltransferase 3B (DNMT3B) repressor domain; an HDAC4 domain; an HP1α repression domain; and an EED (embryonic ectoderm development) repressor domain.

[0038] In some embodiments of any of the aspects, a synTF polypeptide (or collectively, a synTF polypeptide system) described herein comprises one, two, three, four, or five transcription effector domains. In some embodiments of any of the aspects, a synTF polypeptide or system comprises one transcription effector domain. In embodiments comprising multiple transcription effector domains, the multiple transcription effector domains can be different individual transcription effector domains or multiple copies of the same transcription effector domain, or a combination of the above.

[0039] In some embodiments of any of the aspects, the transcriptional ED is a transcriptional activator (TA) domain. As used herein, the term "transcriptional activator" domain refers to an effector that increases gene expression. In some embodiments of any of the aspects, the TA is selected from the group consisting of p65; Rta; mini-VPR; complete VPR; VP16; VP64; p300; p300 HAT core; and CBP HAT domain. For example, US Patent No. 10,138,493; US Patent No. 10,590,182; Khalil et al., Cell Volume 150, Issue 3, 3 August 2012, Pages 647-658; Vora et al., Rational design of a compact CRISPR--Cas9 activator for AAV-mediated delivery, bioRxiv 2018 doi.org / 10.1101 / 298620; Chavez et al., Nat Methods. 2015 April, 12(4):326-328;Park et al., Cell. 2019 Jan 10, 176(1-2):227-238, e20;Hilton et al., Nature Biotechnology volume 33, pages 510-517(2015);Sajwan et al., Sci Rep. 2019; 9:18104, the contents of each of which are incorporated herein by reference in their entirety.

[0040] In some embodiments of any of the aspects, the TA is p65 or a functional fragment thereof. Transcription factor p65, also known as nuclear factor NF-κ-B p65 subunit, is a protein encoded by the RELA gene in humans. In some embodiments of any of the aspects, p65 comprises SEQ ID NO:69 or a protein having at least 85% sequence identity to SEQ ID NO:69. In some embodiments of any of the aspects, p65 comprises SEQ ID NO:117 or a protein having at least 85% sequence identity to SEQ ID NO:117. In some embodiments of any of the aspects, p65 comprises SEQ ID NO:118 or a portion of SEQ ID NO:118, e.g., residues 150-261, 100-261, 200-261, 1-200, 1-50, 1-100, or 50-100 of SEQ ID NO:118. In some embodiments of any of the aspects, p65 comprises one of SEQ ID NOs: 69, 117-121, 193-197, or a polypeptide comprising a sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 69, 117-121, 193-197 and maintains its function. In some embodiments of any of the aspects, p65 comprises SEQ ID NO:120 (p65 100-261), or a polypeptide comprising a sequence at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:120 and maintains the same function.

[0041] SEQ ID NO:69, p65 (amino acids 361-551 of NFkB) activation domain (191 aa) TIFF0007663513000017.tif17164SEQ ID NO: 117, p65 (complete sequence, 551 aa), transcription factor p65 isoform 1 [Homo sapiens], NCBI Reference Sequence: NP_068810.3 TIFF0007663513000018.tif158166TIFF0007663513000019.tif111165

[0042] In some embodiments of any of the aspects, the TA is Rta or a functional fragment thereof. Rta is the Epstein-Barr virus R transactivator (Rta) activation domain. In some embodiments of any of the aspects, Rta comprises SEQ ID NO:198 or a protein having at least 85% sequence identity to SEQ ID NO:198. In some embodiments of any of the aspects, Rta comprises a portion of SEQ ID NO:198, e.g., residues 75-190, 125-190, 50-175, 75-175, 100-175, or 125-175 of SEQ ID NO:198. In some embodiments of any of the aspects, Rta includes one of SEQ ID NOs:198-204, or a polypeptide comprising a sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs:198-204 and maintains its function. In some embodiments of any of the aspects, Rta includes SEQ ID NO:200 (Rta 125-190), or a polypeptide comprising a sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:200 and maintains the same function.

[0043] TIFF0007663513000020.tif131165

[0044] In some embodiments of any of the aspects, the TA is VPR or a functional fragment thereof. VPR is a tripartite activator consisting of VP64, p65, and Rta activation domains. In some embodiments of any of the aspects, the VPR comprises VP64 (e.g., SEQ ID NO:208), p65 (e.g., any one of SEQ ID NOs:69, 117-121, or 193-197, or a polypeptide having at least 85% sequence identity to any one of SEQ ID NOs:69, 117-121, or 193-197 and maintaining the same function), and Rta (e.g., any one of SEQ ID NOs:198-204, or a polypeptide having at least 85% sequence identity to any one of SEQ ID NOs:198-204 and maintaining the same function). In some embodiments of any of the aspects, the VPR comprises one of SEQ ID NOs:205, 206, or a polypeptide comprising a sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs:205 or 206 and maintains its function.

[0045] SEQ ID NO:205, a mini-VPR containing a p65 (100-261 aa; SEQ ID NO:120) truncation and an RTA (125-190 aa; SEQ ID NO:200) truncation; bold text indicates VP64 (SEQ ID NO:208); italic text indicates SV40 NLS (SEQ ID NO:65); bold italic text indicates p65 (100-261 aa; SEQ ID NO:120); double-underlined text indicates RTA (125-190 aa; SEQ ID NO:200) TIFF0007663513000021.tif31166.

[0046] SEQ ID NO:206, full VPR; bold text indicates VP64 (SEQ ID NO:208); italic text indicates SV40 NLS (SEQ ID NO:65); bold italic text indicates p65 (SEQ ID NO:118); double-underlined text indicates RTA (SEQ ID NO:198) TIFF0007663513000022.tif58166.

[0047] In some embodiments of any of the aspects, the TA comprises a herpes simplex virus protein 16 (VP16) activation domain. In some embodiments of any of the aspects, the TA comprises a VP64 activation domain containing four tandem copies of VP16. In some embodiments of any of the aspects, the TA comprises one of SEQ ID NOs:207, 208, or a polypeptide that maintains its function comprising a sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs:207 or 208.

[0048] SEQ ID NO:207, VP16(11aa)DALDDFDLDML.

[0049] SEQ ID NO:208, VP64 (53 aa), VP16 domain indicated by bold text TIFF0007663513000023.tif3146.

[0050] In some embodiments of any of the aspects, the TA comprises p300 or a functional fragment thereof. The adenovirus E1A-associated cellular p300 transcriptional coactivator protein functions as a histone acetyltransferase that regulates transcription through chromatin remodeling. In some embodiments of any of the aspects, p300 comprises SEQ ID NO:209 or a protein having at least 85% sequence identity to SEQ ID NO:209. In some embodiments of any of the aspects, p300 comprises a portion of SEQ ID NO:209, e.g., residues 1048-1664 of SEQ ID NO:209. In some embodiments of any of the aspects, the TA comprises a p300 HAT core activation domain. In some embodiments of any of the aspects, p300 comprises SEQ ID NO:210 or a protein having at least 85% sequence identity to SEQ ID NO:210. In some embodiments of any of the aspects, the TA comprises one of SEQ ID NOs:209, 210, or a polypeptide that maintains its function comprising a sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs:209 or 210.

[0051] SEQ ID NO:209, human acetyltransferase p300 (2414aa), bold text indicates core activation domain TIFF0007663513000024.tif185165TIFF0007663513000025.tif71165.

[0052] SEQ ID NO:210, p300 HAT core activation domain (617aa) TIFF0007663513000026.tif64165.

[0053] In some embodiments of any of the aspects, the TA comprises CBP or a functional fragment thereof. CBP (CREB (cyclic AMP-responsive element-binding protein) binding protein; CREBBP) is involved in transcriptional coactivation of many different transcription factors and has intrinsic histone acetyltransferase activity. In some embodiments of any of the aspects, the CBP is derived from Homo sapiens, Drosophila melanogaster, or any other organism expressing a homologous CBP protein. In some embodiments of any of the aspects, the TA comprises a CBP HAT core activation domain. In some embodiments of any of the aspects, the TA comprises one of SEQ ID NOs:211-213, or a polypeptide that maintains its function comprising a sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs:211-213.

[0054] SEQ ID NO: 211, Homo sapiens CBP, histone acetyltransferase (HAT) domain; residues 1342-1649 of CREB-binding protein isoform a [Homo sapiens], NCBI Reference Sequence: NP_004371.2; residues 1304-1611 of CREB-binding protein isoform b [Homo sapiens], NCBI Reference Sequence: NP_001073315.1 TIFF0007663513000027.tif31165.

[0055] SEQ ID NO: 212, Nejire, residues 1954-2267 of isoform E [Drosophila melanogaster], NCBI Reference Sequence: NP_001259387.1, HAT_KAT11, histone acetylation protein TIFF0007663513000028.tif31164.

[0056] SEQ ID NO: 213, aa 1696 to 2329 from Drosophila CBP (Nejire), NCBI reference sequence: NP_001259387.1, contains bromodomain, PHD domain, and HAT domain TIFF0007663513000029.tif64167.

[0057] In some embodiments of any of the aspects, the transcriptional ED is a transcriptional repressor (TR) domain. As used herein, the term "transcriptional repressor" domain refers to an effector that reduces gene expression. In some embodiments of any of the aspects, the TR is selected from the group consisting of KRAB; KRAB-MeCP2; Hp1a; DNMT3B; EED; and HDAC4. See, for example, U.S. Pat. No. 10,138,493; U.S. Pat. No. 10,590,182; Khalil et al., Cell Volume 150, Issue 3, 3 August 2012, Pages 647-658; Park et al., Cell. 2019 Jan 10, 176(1-2):227-238, e20; Yeo et al., Nature Methods volume 15, pages 611-616(2018); Bintu et al., Science. 2016 Feb 12;351(6274):720-724, the contents of each of which are incorporated herein by reference in their entirety.

[0058] In some embodiments of any of the aspects, the TR comprises KRAB or a functional fragment thereof. Krüppel-associated box (KRAB) domains are a class of transcriptional repression domains present in approximately 400 human zinc finger protein-based transcription factors (KRAB zinc finger proteins), which associate with other chromatin regulators that write or read H3K9me3. In some embodiments of any of the aspects, the TR comprises a KRAB-MeCP2, bipartite repressor domain. In some embodiments of any of the aspects, the KRAB domain comprises one of SEQ ID NOs: 72, 97, 214-215, or a polypeptide that maintains its function comprising a sequence at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs: 72, 97, or 214-215. In some embodiments of any of the aspects, the TR comprises a transcriptional repression domain (TRD) domain of MeCP2 or a functional fragment thereof. In some embodiments of any of the aspects, the transcriptional repression domain (TRD) domain of MeCP2 comprises SEQ ID NO:216 or a polypeptide that maintains its function comprising a sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:216.

[0059] SEQ ID NO: 72: KRAB repressor domain (96 aa) TIFF0007663513000030.tif11163.

[0060] SEQ ID NO:97: KRAB repressor domain (65 aa) TIFF0007663513000031.tif4168.

[0061] SEQ ID NO:214, KRAB-MeCP2 (382 aa), contains the KRAB domain (bold text), a glycine-serine-rich linker (unformatted text), and the transcriptional repression domain (TRD) domain of MeCP2 (italic text). TIFF0007663513000032.tif37165.

[0062] SEQ ID NO:215, KRAB repressor domain (74 aa) TIFF0007663513000033.tif10163.

[0063] SEQ ID NO:216, transcriptional repression domain (TRD) domain of MeCP2 (296 aa) TIFF0007663513000034.tif30165.

[0064] In some embodiments of any of the aspects, the TR comprises an Hp1a repressor domain or a functional fragment thereof. Heterochromatin protein 1 (HP1a in Drosophila) is a conserved eukaryotic chromosomal protein that prominently associates with pericentric heterochromatin and mediates simultaneous gene silencing. HP1a binds to H3K9me2 / 3 via its chromodomain and to SU(VAR)3-9, a histone methyltransferase that methylates histone H3 on K9, via its chromoshadow domain. In some embodiments of any of the aspects, the Hp1a repressor domain comprises SEQ ID NO:98 or a polypeptide that maintains its function comprising a sequence at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:98.

[0065] SEQ ID NO:98: Hp1a repressor domain (190 aa) TIFF0007663513000035.tif17165.

[0066] In some embodiments of any of the aspects, the TR comprises an EED repressor domain or a functional fragment thereof. EED (Embryonic Ectoderm Development) functions as part of Polycomb Repressive Complex 2 (PRC2), which methylates histone H3 at lysine 27 (H3K27me3). Members of the Polycomb family form multimeric protein complexes involved in maintaining the transcriptionally repressed state of genes across successive cell generations. In some embodiments of any of the aspects, the EED repressor domain comprises SEQ ID NO:99 or a polypeptide that maintains its function comprising a sequence at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:99.

[0067] SEQ ID NO:99:EED repressor domain (440 aa) TIFF0007663513000036.tif44165.

[0068] In some embodiments of any of the aspects, the TR comprises a DNA (cytosine-5)-methyltransferase 3B (DNMT3B) repressor domain or a functional fragment thereof. DNMT3B is involved in CpG methylation, an epigenetic modification important in embryonic development, imprinting, and X-chromosome inactivation. In some embodiments of any of the aspects, the DNMT3B repressor domain comprises SEQ ID NO:217 or a polypeptide that maintains its function comprising a sequence at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:217.

[0069] SEQ ID NO: 217, DNMT3B repressor domain (792 aa), Uniprot identifier Q9UBC3-5, DNA (cytosine-5)-methyltransferase DNM3B_HUMAN isoform 5 TIFF0007663513000037.tif84165.

[0070] In some embodiments of any of the aspects, the TR comprises a histone deacetylase 4 (HDAC4) repressor domain or a functional fragment thereof. HDAC4 removes acetyl groups from histones H3 and H4. In some embodiments of any of the aspects, the HDAC4 repressor domain comprises SEQ ID NO:218 or a polypeptide that maintains its function comprising a sequence at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:218.

[0071] SEQ ID NO: 218, HDAC4 domain, GenBank: AAD29046.1 (1084 aa) TIFF0007663513000038.tif111165.

[0072] In another embodiment of any aspect described herein in the synTF or ZF-containing fusion protein described herein, the transcriptional effector domain is an epigenetic effector domain. For example, at least one ZF protein domain is fused to one or more chromatin-modulating enzymes that (1) catalyze the chemical modification of DNA or histone residues (e.g., DNA methyltransferase, histone methyltransferase, histone acetyltransferase) or (2) remove the chemical modification (e.g., DNA demethylase, DNA dioxygenase, DNA hydroxylase, histone demethylase, histone deacetylase). For example, DNA methyltransferase DNMTs (DNMT1, DNMT3) catalyze the transfer of methyl groups to cytosines, which typically results in transcriptional repression via the recruitment of repressive regulatory proteins. Another example is CBP / p300 histone acetyltransferase, which is typically associated with transcriptional activation via interaction with multiple transcription factors. Relevant epigenetic effector domains involved in the deposition of biochemical marks on DNA or histone residues include HAT1, GCN5, PCAF, MLL, SET, DOT1, SUV39H, G9a, KAT2A / B, and EZH1 / 2. Relevant epigenetic effector domains involved in the removal of biochemical marks from DNA or histone residues include TET1 / 2, the SIRT family, LSD1, and the KDM family.

[0073] DNA-binding domain Described herein are synTFs that include at least one DNA-binding domain (DBD). In some embodiments of any of the aspects, the synTF polypeptides described herein (or collectively, synTF polypeptide systems) include one, two, three, four, five, or more DBDs. In some embodiments of any of the aspects, the synTF polypeptides or systems include one DBD. In embodiments that include multiple DBDs, the multiple DBDs can be different individual DBDs or multiple copies of the same DBD, or a combination of the above.

[0074] In some embodiments of any of the aspects, at least one DBD is an engineered zinc finger (ZF) binding domain. Zinc finger (ZF) is a finger-shaped fold in a protein that allows the protein to interact with nucleic acid sequences such as DNA and RNA. Such folds are well known in the art. The fold is created by the binding of specific amino acids of the protein with zinc atoms. Zinc finger-containing proteins (also known as ZF proteins) can regulate not only gene expression but also nucleic acid recognition, reverse transcription, and virus assembly.

[0075] ZFs are relatively small polypeptide domains containing approximately 30 amino acids that fold to form adjacent α-helices of an antiparallel β-sheet (known as a ββα-fold). The fold is stabilized by the coordination of a zinc ion between four largely invariant (depending on the type of zinc finger framework) Cys and / or His residues, which are further described below. Natural zinc finger domains have been well studied and described in the literature. See, for example, Miller et al., (1985) EMBO J. 4:1609-1614; Berg (1988) Proc. Natl. Acad. Sci. USA 85: 99-102; and Lee et al., (1989) Science 245: 635-637. ZF domains recognize and bind to nucleic acid triplets or overlapping quadruplets (described below) in double-stranded DNA target sequences. However, ZFs are also known to bind RNA and proteins (Clemens, KR et al. (1993) Science 260:530-533; Bogenhagen, DF (1993) Mol. Cell. Biol. 13: 5149-5158; Searles, MA et al. (2000) J. Mol. Biol. 301: 47-60; Mackay, JP & Crossley, M. (1998) Trends Biochem. Sci. 23: 1-4).

[0076] In one aspect, the term "zinc finger" (ZF) or "zinc finger motif" (ZF motif) or "zinc finger domain" (ZF domain), as used herein, refers to an individual "finger" comprising a beta-beta-alpha (ββα)-protein fold stabilized by zinc ions, as described elsewhere herein. The Zn-coordinated ββα protein fold produces a "fingers" that are finger-like protrusions. Each ZF motif typically contains about 30 amino acids. The term "motif" as used herein refers to a structural motif. A ZF motif is a super-secondary structure with a ββα fold stabilized by zinc ions.

[0077] In one aspect, the term "ZF motif," as commonly used in the art, refers to a distinct, continuous portion of a polypeptide's amino acid sequence that can be considered to have a specific function. ZF motifs are largely structurally independent and can retain their structure and function in different environments. Because ZF motifs are structurally and functionally independent, they also qualify as domains and are therefore often referred to as ZF domains. Thus, ZF domains are protein motifs containing multiple finger-like protrusions that make tandem contacts with their target molecules. Typically, ZF domains bind to triplet or (overlapping) quadruplet nucleotide sequences. Tandemly arranged adjacent ZF domains are connected together by linker sequences to form an array. ZF peptides typically contain ZF arrays and are composed of multiple "ZF domains" that do not exist together in nature. Therefore, they are considered to be artificial or synthetic ZF peptides or proteins.

[0078] C2H2 zinc fingers (C2H2-ZFs) are the most common type of vertebrate DNA-binding domain and typically appear as tandem arrays (ZFAs) with consecutive C2H2-ZFs, each contacting three (or more) consecutive bases. C2H2-ZFs can be assembled modularly. Given a set of modules with a given triplet specificity, assembly of modules also offers a method for constructing artificial proteins with specific DNA-binding preferences.

[0079] ZF-containing proteins generally contain a string or chain of ZF motifs that form a ZF array (ZFA). Thus, a natural ZF protein may contain two or more ZFs, i.e., ZFAs consisting of two or more ZF motifs, which may be directly adjacent to each other (i.e., separated by a short (classical) linker sequence) or may be separated by a longer, flexible, or structured polypeptide sequence. Directly adjacent ZF domains are expected to bind to consecutive nucleic acid sequences, i.e., adjacent trinucleotides / triplets. In some cases, cross-linking may occur between adjacent ZFs and their respective target triplets, which helps to strengthen or enhance target sequence recognition, leading to the binding of overlapping quadruplet sequences (Isalan et al., (1997) Proc. Natl. Acad. Sci. USA, 94: 5617-5621). In comparison, separate ZF domains within the same protein may recognize (or bind to) non-contiguous nucleic acid sequences or even different molecules (eg, proteins rather than nucleic acids).

[0080] The engineered ZF-containing protein is a chimeric protein composed of a DNA-binding zinc finger protein domain (ZF protein domain) and another domain, and exerts its effect (effector domain) through this other domain. The effector domain can be a transcription activator or repressor, a methylation domain, or a nuclease. The DNA-binding ZF protein domain contains an engineered zinc finger array (ZFA). See, for example, Khalil et al., Cell Volume 150, Issue 3, 3 August 2012, Pages 647-658; U.S. Patent No. 10,138,493; U.S. Patent Application Publication No. US20200002710A1, the contents of each of which are incorporated herein by reference in their entirety.

[0081] The engineered ZF-containing proteins are non-naturally occurring and suitably contain three or more, e.g., 4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or more (e.g., up to about 30 or 32) ZF motifs arranged in tandem adjacent to each other to form an array of ZF motifs, or ZFA. In particular, the ZF-containing synTF proteins (ZF-containing synTF fusion proteins, or simply synTFs) of the disclosure contain at least three ZFs, at least four ZF motifs, at least five ZF motifs, or at least six ZF motifs, at least seven ZF motifs, at least eight ZF motifs, at least nine ZF motifs, at least ten ZF motifs, at least 11, or at least 12 ZF motifs; and optionally at least 18 ZF motifs. In other embodiments, the ZF synTF contains up to 6, 7, 8, 10, 11, 12, 16, 17, 18, 22, 23, 24, 28, 29, 30, 34, 35, 36, 40, 41, 42, 46, 47, 48, 54, 55, 56, 58, 59, or 60 ZF motifs. In some embodiments, the ZF array comprises one or more ZF motifs. The ZF-containing synTFs of the present disclosure bind to contiguous orthogonal target nucleic acid binding sites. That is, the ZF or ZFA contained in the ZF domain of the fusion protein binds to the orthogonal target nucleic acid sequence.

[0082] In one aspect, "engineered synthetic transcription factor" or "engineered synTF" or "synTF" as used herein refers to an engineered ZF-containing chimeric protein that has at least one of the following characteristics, and may have more than one: binds to a target orthogonal specific DNA sequence and has, for example, reduced or minimal functional binding ability in the host eukaryotic genome; is derived from a mammalian protein scaffold, conferring minimal immunogenicity compared to other prokaryotic-derived domains; and can be packaged in a viral delivery system, for example, a lentiviral delivery construct.

[0083] As used herein, in another embodiment, the term "engineered synthetic transcription factor" or "engineered synTF," abbreviated as "synTF" or "ZF synTF," refers to an engineered ZF containing synthetic transcription factor polypeptide, or in other words, a ZF-containing synthetic transcription factor protein. These synTFs contain a ZF array (ZFA) therein for binding to a specific target nucleic acid sequence. SynTFs are chimeric fusion proteins that include a DNA-binding ZF-containing protein domain and an effector domain through which the synTF exerts its effect on gene expression. These synTFs can modulate gene expression by increasing or decreasing expression of a gene operably linked to a promoter that is also operably linked to a specific target nucleic acid sequence to which the DNA-binding ZF-containing protein domain of the synTF binds.

[0084] As used herein, the term "ZF array," abbreviated as "ZFA," refers to an array, or string, or chain, of ZF motifs arranged in tandem. A ZFA can have six ZF motifs (six-finger ZFA), seven ZF motifs (seven-finger ZFA), or eight ZF motifs (eight-finger ZFA).

[0085] As used herein, the terms "engineered responsive / response promoter," "engineered promoter," or "engineered responsive / response promoter element" refer to a nucleic acid construct containing a promoter sequence with at least one orthogonal DNA target sequence operably linked upstream of the promoter sequence, such that the orthogonal DNA target sequence confers a responsive characteristic to the promoter when bound by its respective transcription factor. The responsive characteristic is whether the initiation of gene transcription from the promoter is enhanced or repressed when the adjacent upstream orthogonal DNA target sequence is bound by a ZF-containing synthetic transcription factor. There may be more than one orthogonal DNA target sequence operably linked upstream of a promoter sequence. When there is one orthogonal DNA target sequence, the promoter is referred to as a "1X" promoter, where "1X" refers to the number of orthogonal DNA target sequences present in the promoter construct. For example, a 4X responsive promoter is identified as having four orthogonal DNA target sequences in an engineered responsive promoter construct, and the four orthogonal DNA target sequences are upstream of the promoter sequence.

[0086] ZF protein domains are modular in design, based on zinc finger arrays (ZFAs), with each ZFA consisting of six to eight ZF motifs. ZF protein domains contain at least one ZFA and can contain as many as ten ZFAs. ZF protein domains can have from one to as many as ten ZFAs.

[0087] The design of the synTF or any engineered fusion protein described herein is also modular, meaning that the synTF is made up of a ZF domain module (ZFA) and an effector domain / protein interaction domain / ligand-binding domain / dimerization domain module, where the individual modules are covalently conjugated together as described herein and function independently of each other. The number of ZFAs can range from 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10. When there are two or more ZFAs, the ZFAs are covalently conjugated to each other in tandem, e.g., via an L1 peptide linker, in an NH2-terminal to COOH-terminal configuration, to form an array of ZFAs. The ZFAs collectively form a ZF protein domain, which is covalently linked to the N- or C-terminus of the effector domain or regulatory protein. When there are two or more ZFAs in the ZF protein domain of a synTF or ZF-containing fusion protein described herein, the ZFAs can be the same or different.

[0088] Each modular ZFA in the ZF protein domain of a synTF or ZF-containing fusion protein disclosed herein is composed of six to eight ZF motifs. See Figure 2B for an example of a single ZFA with seven ZF motifs, a seven-finger ZFA. The ZF motif is a small protein structural motif consisting of an α-helix and an antiparallel β-sheet (αββ), characterized by the coordination of a zinc ion by two histidine residues and two cysteine ​​residues in the motif to stabilize the finger-like protruding fold, the "fingers." The ZF motif in the ZF protein domain of a synTF disclosed herein is a Cys2His2 zinc finger motif. In one embodiment, the ZF motif is represented by the formula 1: [X 0-3 CX 1-5 CX 2-7 -(Helix)-HX 3-6The ZF motifs may comprise, consist essentially of, or consist of a peptide of the formula [X, C, H] (SEQ ID NO:219), where X is any amino acid, the subscript indicates the number of possible amino acid residues, C is cysteine, H is histidine, and (helix) is a peptide of 6 (or 7) consecutive amino acid residues that form a short α-helix. The helix is ​​variable. This short α-helix forms one facet of a finger formed by the coordination of a zinc ion by two histidine and two cysteine ​​residues in the ZF motif. For each ZFA, 6 to 8 ZF motifs are interconnected from their NH2- to COOH-termini by peptide linkers of approximately 4 to 6 amino acid residues to form an array of ZF motifs, or ZF. The finger-like protruding fold of each ZF motif interacts with and binds to a nucleic acid sequence. For every two ZF motifs, approximately one peptide sequence interacts with and binds to a nucleic acid sequence of approximately 6 base pairs (bp). The multiple ZF motifs in a ZFA form finger-like overhangs that contact the orthogonal target DNA sequence. Thus, for example, a ZFA having six ZF motifs or finger-like overhangs (6-finger ZFA) interacts and binds to a nucleic acid sequence of about 18-20 bp, and an 8-finger ZFA binds to a nucleic acid sequence of about 24-26 bp. Thus, in one embodiment, the ZFA in the ZF protein domain of a synTF is N'-[(Formula 1)-L2] 6-8 -C', where the subscript 6-8 indicates the number of ZF motifs, L2 is a linker peptide having 4 to 6 amino acid residues, and N'- and C'- indicate the N- and C-termini of the peptide sequence, respectively. For example, for a ZFA consisting essentially of six ZF motifs, the sequence is N'-[(formula 1)-L2]-[(formula 1)-L2]-[(formula 1)-L2]-[(formula 1)-L2]-[(formula 1)-L2]-[(formula 1)-L2]-C', and for a ZFA consisting essentially of eight ZF motifs, the sequence is N'-[(formula 1)-L2]-[(formula 1)-L2]-[(formula 1)-L2]-[(formula 1)-L2]-[(formula 1)-L2]-[(formula 1)-L2]-[(formula 1)-L2]-C'.

[0089] SEQ ID NO:219 TIFF0007663513000039.tif3128

[0090] In another embodiment of the aspects described herein, the ZF motif comprises a peptide of formula 2: [X3CX2CX5-(helix)-HX3H] (SEQ ID NO:220), where X is any amino acid, the subscripted numbers indicate the number of possible amino acid residues, C is cysteine, H is histidine, and (helix) is a 6 (or 7) consecutive amino acid residue peptide that forms a short alpha helix. Thus, in one embodiment, the ZFA in the ZF protein domain of a synTF has the sequence: N'-[(formula 2)-L2] 6-8 -C', where the subscript 6-8 indicates the number of ZF motifs, L2 is a linker peptide having 4 to 6 amino acid residues, and N'- and C'- indicate the N- and C-termini of the peptide sequence, respectively. For example, for a ZFA consisting essentially of six ZF motifs, the sequence is N'-[(formula 2)-L2]-[(formula 2)-L2]-[(formula 2)-L2]-[(formula 2)-L2]-[(formula 2)-L2]-[(formula 2)-L2]-C', and for a ZFA consisting essentially of eight ZF motifs, the sequence is N'-[(formula 2)-L2]-[(formula 2)-L2]-[(formula 2)-L2]-[(formula 2)-L2]-[(formula 2)-L2]-[(formula 2)-L2]-[(formula 2)-L2]-C'.

[0091] SEQ ID NO:220 TIFF0007663513000040.tif3128

[0092]

[0013] In one embodiment of any aspect described herein, for a single ZFA that is a ZF protein domain of a synTF disclosed herein, the ZFA in the ZF protein domain has the sequence: Comprising, consisting essentially of, or consisting of TIFF0007663513000041.tif24170, where (helix) is a 6 (or 7) consecutive amino acid residue peptide forming a short alpha helix, which may also be expressed as plain text "xxxxxxx".

[0093] TIFF0007663513000042.tif23161TIFF0007663513000043.tif18165.

[0094] SEQ ID NO:101, a zinc finger domain scaffold, wherein [helix 1], [helix 2], [helix 3], [helix 4], [helix 5], and [helix 6] may also be represented as plain text "xxxxxxx": TIFF0007663513000044.tif18170.

[0095] TIFF0007663513000045.tif23163TIFF0007663513000046.tif18165.

[0096] In some embodiments of any of the aspects, the zinc finger scaffold comprises one of SEQ ID NOs:76, 101, 377, 380, or an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs:76, 101, 377, or 380 and maintains the same function.

[0097] In one embodiment, all six of helices 1, 2, 3, 4, 5, and 6 are distinct and different from one another. In another embodiment, all six of helices 1, 2, 3, 4, 5, and 6 are identical to one another. Alternatively, at least two of the six helices are identical and the same to one another. In other embodiments, at least three of the six helices in the ZFA are identical and the same to one another, at least four of the six helices in the ZFA are identical and the same to one another, or at least five of the six helices in the ZFA are identical and the same to one another.

[0098] In some embodiments of any aspect described herein, the helices of 6 to 8 ZF motifs of an individual ZFA disclosed herein are selected from the 6 amino acid (or 7 amino acid) residue peptide sequences disclosed in one of the following groups 1-11 (e.g., SEQ ID NOs:122-180, 192). In some embodiments, at least 4 of the ZF motifs in an individual ZFA disclosed herein are selected from the 6 amino acid (or 7 amino acid) residue peptide sequences disclosed in one of the following groups 1-11. In other embodiments, all of the ZF motifs, i.e., 6, 7, or 8 ZF motifs in an individual ZFA disclosed herein, are selected from the 6 amino acid (or 7 amino acid) residue peptide sequences disclosed in one of the following groups 1-11. In any individual ZFA, the helices selected for a single ZF comprising the ZFA can be repeated two or more times in the ZFA. This means that for any given single ZFA, at least 4 or all of the helices in the ZFA are selected from the same group disclosed herein. For example, if a ZFA consists essentially of six ZF motifs, this means there are six α-helices. All six to eight helices (helix 1; helix 2; helix 3; helix 4; helix 5; helix 6; helix 7; helix 8) of the ZFs in each ZFA are selected from one of the following groups 1 to 11, e.g., all six helices are selected from group 2. That is, all helices for all ZFs comprising a single ZFA are from the same group. Alternatively, at least four of the six helices are selected from the same group, a group selected from groups 1 to 11. For example, four of the six helices are selected from group 5, and the remaining two helices of a six-ZF motif ZFA can be selected from other groups 1 to 4, 6 to 11, or any other helices that form short α-helices. The remaining helices making up a ZFA can be those known in the art.

[0099] Table 10: Helices of groups 1 to 4 TIFF0007663513000047.tif90166

[0100] Table 11: Helices of groups 5-7 TIFF0007663513000048.tif76166

[0101] Table 12: Helices of groups 8-11 TIFF0007663513000049.tif69168

[0102] Non-limiting examples of combinations and arrangements of six helices in a single ZFA where the helices are selected from Group 1 and the motif is arranged from NH2-terminus to COOH-terminus (Group 1 ZFA helix combo) are as follows: TIFF0007663513000050.tif104162.

[0103] A non-limiting example of a combination and arrangement of six helices in a single six-finger ZFA where the helices are selected from Group 2 and the motif is arranged from NH2-terminus to COOH-terminus (Group 2 ZFA helix combo) is as follows: TIFF0007663513000051.tif105163.

[0104] A non-limiting example of a combination and arrangement of six helices in a single six-finger ZFA where the helices are selected from Group 3 and the motif is arranged from NH2-terminus to COOH-terminus (Group 3 ZFA helix combo) is as follows: TIFF0007663513000052.tif105164.

[0105] In some embodiments of any of the aspects, QRNNLGR (SEQ ID NO:192) is used in place of QTNNLGR (SEQ ID NO:141). A non-limiting example of a combination and arrangement of six helices in a single six-finger ZFA where the helices are selected from Group 3 and the motif is arranged from NH2-terminus to COOH-terminus (Group 3 ZFA helix combo) is as follows: TIFF0007663513000053.tif104163.

[0106] A non-limiting example of a combination and arrangement of six helices in a single six-finger ZFA where the helices are selected from Group 4 and the motif is arranged from NH2-terminus to COOH-terminus (Group 4 ZFA helix combo) is as follows: TIFF0007663513000054.tif105163.

[0107] A non-limiting example of a combination and arrangement of six helices in a single six-finger ZFA where the helices are selected from Group 5 and the motif is arranged from NH2-terminus to COOH-terminus (Group 5 ZFA Helix Combo) is as follows: TIFF0007663513000055.tif105162.

[0108] A non-limiting example of a combination and arrangement of six helices in a single six-finger ZFA where the helices are selected from Group 6 and the motif is arranged from NH2-terminus to COOH-terminus (Group 6 ZFA Helix Combo) is as follows: TIFF0007663513000056.tif104164.

[0109] A non-limiting example of a combination and arrangement of six helices in a single six-finger ZFA where the helices are selected from Group 7 and the motif is arranged from NH2-terminus to COOH-terminus (Group 7 ZFA helix combo) is as follows: TIFF0007663513000057.tif105164.

[0110] A non-limiting example of a combination and arrangement of six helices in a single six-finger ZFA where the helices are selected from Group 8 and the motif is arranged from NH2-terminus to COOH-terminus (Group 8 ZFA Helix Combo) is as follows: TIFF0007663513000058.tif51162.

[0111] A non-limiting example of a combination and arrangement of six helices in a single six-finger ZFA where the helices are selected from Group 9 and the motif is arranged from NH2-terminus to COOH-terminus (Group 9 ZFA Helix Combo) is as follows: TIFF0007663513000059.tif51162.

[0112] A non-limiting example of a combination and arrangement of six helices in a single six-finger ZFA where the helices are selected from Group 10 and the motif is arranged from NH2-terminus to COOH-terminus (Group 10 ZFA Helix Combo) is as follows: TIFF0007663513000060.tif11158.

[0113] A non-limiting example of a combination and arrangement of six helices in a single six-finger ZFA where the helices are selected from Group 11 and the motif is arranged from NH2-terminus to COOH-terminus (Group 11 ZFA helix combo) is as follows: TIFF0007663513000061.tif11163.

[0114] Thus, in some aspects, provided herein is an engineered synTF or ZF-containing fusion protein described herein comprising a ZF protein domain, an effector domain, and a regulatory protein, wherein the ZF protein domain comprises at least one ZFA having a ZFA helix combo selected from one of ZFA helix combo groups 1-11 disclosed herein. When there are two or more ZFAs in the ZF protein domain (i.e., a ZF array), each ZFA in the domain has a ZFA helix combo selected from one of ZFA helix combo groups 1-11 disclosed herein, and the selected ZFA helix combo groups can be different or overlapping for each ZFA in the ZF protein domain of the synTF. For example, if a synTF contains a ZF protein domain consisting essentially of three ZFAs (ZFA-1-ZFA-2-ZFA-3 in a 3-ZFA array) and an effector domain, ZFA-1 has a ZFA helix combo selected from the ZFA helix combos of group 1, ZFA-2 has a ZFA helix combo selected from the ZFA helix combos of group 5, and ZFA-3 has a ZFA helix combo selected from the ZFA helix combos of group 7. In other embodiments, the selected ZFA helix combo groups can be overlapping or triplicating for the ZF array in the synTF. For example, in a 3-ZFA array-containing ZF protein domain of a synTF, two of the ZFAs contain the same ZFA helix combo group, e.g., a ZFA helix combo selected from group 2, and the third ZFA has a ZFA helix combo selected from a different ZFA helix combo group, e.g., group 4. Two ZFAs having ZFA helix combos selected from the same Group 2 ZFA helix combos can have different or the same actual combination and arrangement of helical ZFAs.For example, if a synTF contains a ZF protein domain consisting essentially of five ZFAs (ZFA-1-ZFA-2-ZFA-3-ZFA-4-ZFA-5 in a 5-ZFA array) and an effector domain, ZFA-1 has a ZFA helix combo selected from the ZFA helix combos of group 1, ZFA-2 has a ZFA helix combo selected from the ZFA helix combos of group 5, ZFA-3 has a ZFA helix combo also selected from the ZFA helix combos of group 1, ZFA-4 has a ZFA helix combo selected from the ZFA helix combos of group 4, and ZFA-5 has a ZFA helix combo selected from the ZFA helix combos of group 2. While ZFA-1 and ZFA-3 both have ZFA helix combos selected from the ZFA helix combos of group 1, the actual combination and arrangement of helices within ZFA-1 and ZFA-3 can be different or the same. For example, ZFA-1 and ZFA-3 have the ZFA helix combo ZF 1-1 and ZF 1-5, respectively, or ZFA-1 and ZFA-3 both have the ZFA helix combo ZF 1-1.

[0115] In other aspects, provided herein is an engineered synTF or ZF-containing fusion protein as described herein, comprising a ZF protein domain and an effector domain, or comprising a ZF protein domain, an effector domain, and a ligand-binding domain, or comprising a ZF protein domain and a ligand-binding domain or a dimerization domain, wherein the ZF protein domain is selected from the group consisting of 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 and ZF 11-1, ZF 12-2, ZF 13-3, ZF 14-4, ZF 15-1, ZF 16-2, ZF 17-3, ZF 18-4, ZF 19-1, ZF 20-2, ZF 21-3, ZF 22-4, ZF 23-5, ZF 24-6, ZF 25-7, ZF 26-8, ZF 27-9, ZF 28-1, ZF 29-1, ZF 30-1, ZF 31-2, ZF 32-3, ZF 33-4, ZF 34-5, ZF 35-6, ZF 36-7, ZF 37-8, ZF 38-9, ZF 39-1, ZF 40-1, ZF 41-1, ZF 42-

[0116] In some embodiments of any of the aspects, the ZF protein domain comprises at least one ZFA having a ZFA helix combo selected from the group consisting of ZF1-3, ZF2-6, ZF3-5, ZF4-8, ZF5-7, ZF6-4, ZF7-3, ZF8-1, ZF9-2, ZF10-1, and ZF11-1, also referred to herein as ZF1, ZF2, ZF3, ZF4, ZF5, ZF6, ZF7, ZF8, ZF9, ZF10, and ZF11, respectively.

[0117] In some embodiments of any aspect described herein, in a described synTF or any ZF-containing fusion protein described herein, the individual ZFAs described therein are specifically designed to bind to an orthogonal target DNA sequence (also referred to herein as a DNA-binding motif), such as: TIFF0007663513000062.tif71128.

[0118] In some embodiments of any aspect, the ZF binding domain specifically binds to a sequence comprising at least one of SEQ ID NOs:181-191, or to a nucleic acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs:181-191 and maintains the same function.

[0119] In some embodiments of any of the aspects, ZF 1-1, ZF 1-2, ZF 1-3, ZF 1-4, ZF 1-5, ZF 1-6, ZF 1-7, and ZF 1-8 bind to target 1. In some embodiments of any of the aspects, ZF 2-1, ZF 2-2, ZF 2-3, ZF 2-4, ZF 2-5, ZF 2-6, ZF 2-7, and ZF 2-8 bind to target 2. In some embodiments of any of the aspects, ZF 3-1, ZF 3-2, ZF 3-3, ZF 3-4, ZF 3-5, ZF 3-6, ZF 3-7, and ZF 3-8 bind to target 3. In some embodiments of any of the aspects, ZF 4-1, ZF 4-2, ZF 4-3, ZF 4-4, ZF 4-5, ZF 4-6, ZF 4-7, and ZF 4-8 bind to target 4. In some embodiments of any of the aspects, ZF 5-1, ZF 5-2, ZF 5-3, ZF 5-4, ZF 5-5, ZF 5-6, ZF 5-7, and ZF 5-8 bind to target 5. In some embodiments of any of the aspects, ZF 6-1, ZF 6-2, ZF 6-3, ZF 6-4, ZF 6-5, ZF 6-6, ZF 6-7, and ZF 6-8 bind to target 6. In some embodiments of any of the aspects, ZF 7-1, ZF 7-2, ZF 7-3, ZF 7-4, ZF 7-5, ZF 7-6, ZF 7-7, and ZF 7-8 bind to target 7. In some embodiments of any of the aspects, ZF 8-1, ZF 8-2, ZF 8-3, and ZF 8-4 bind to target 8. In some embodiments of any of the aspects, ZF 9-1, ZF 9-2, ZF 9-3, and ZF 9-4 bind to target 9. In some embodiments of any of the aspects, ZF10-1 binds to target 10. In some embodiments of any of the aspects, ZF11-1 binds to target 11.

[0120] In one embodiment of any aspect described herein, the sequence: N'-[(Formula 1)-L2] that targets a target DNA sequence selected from Targets 1 to 11 6-8 -C' or sequence N'-[(Formula 2)-L2] 6-8 Provided herein are ZFAs comprising, consisting of, or consisting essentially of —C′, wherein Formula 1 is [X 0-3 CX1-5 CX 2-7 -(Helix)-HX 3-6 H] (SEQ ID NO:219) and Formula 2 is [X3CX2CX5-(helix)-HX3H] (SEQ ID NO:220).

[0121] In other aspects, provided herein is an engineered synTF or ZF-containing fusion protein as described herein, comprising a ZF protein domain and an effector domain, or comprising a ZF protein domain, an effector domain, and a ligand-binding domain, or comprising a ZF protein domain and a ligand-binding domain or a dimerization domain, wherein the ZF protein domain comprises at least one ZFA, wherein at least the ZFA has the sequence: N'-[(Formula 1)-L2] 6-8 -C' or sequence N'-[(Formula 2)-L2] 6-8 -C', wherein the ZFA targets a target DNA sequence selected from targets 1-11, and formula 1 is [X 0-3 CX 1-5 CX 2-7 -(Helix)-HX 3-6 H] (SEQ ID NO:219) and Formula 2 is [X3CX2CX5-(helix)-HX3H] (SEQ ID NO:220).

[0122] In some embodiments of any of the aspects, the ZF binding domain comprises one of SEQ ID NOs:1-3 or an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs:1-3 and maintains the same function.

[0123] SEQ ID NO: 1, sequence of ZF1-3 (ZF1) TIFF0007663513000063.tif17164.

[0124] SEQ ID NO:2, sequence of ZF3-5 (ZF3) TIFF0007663513000064.tif17165.

[0125] SEQ ID NO:3, sequence of ZF10-1 (ZF10) TIFF0007663513000065.tif17165.

[0126] In some embodiments of any of the aspects, the DBD comprises a three-unit ZF protein. In some embodiments of any of the aspects, the three-unit ZF protein comprises one of SEQ ID NOs:221-228 or an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs:221-228 and maintains the same function.

[0127] SEQ ID NO:221 high affinity scaffold (bold indicates mutated residues, plain text "xxxxxxx" indicates the three ZF helices, e.g., helix 1, helix 2, and helix 3 from N-terminus to C-terminus, respectively) TIFF0007663513000066.tif10164.

[0128] SEQ ID NO:222 Low affinity scaffold (bold indicates mutated residues, bold italic text indicates helices 1, 2, and 3, respectively) TIFF0007663513000067.tif10164.

[0129] In some embodiments of any of the aspects, at least one DBD is selected from the group consisting of 13-6, 14-3, 21-16, 36-4, 37-12, 42-10, 43-8, 54-8, 55-1, 62-1, 92-1, 93-10, 97-4, 129-3, 150-4, 151-1, 158-2, 172-5, and 173-3; see, e.g., Khalil et al., Cell Volume 150, Issue 3, 3 August 2012, Pages 647-658; U.S. Patent No. 10,138,493; U.S. Patent Application Publication No. US20200002710A1; the contents of each of which are incorporated by reference herein in their entirety. In some embodiments of any of the aspects, at least one DBD is selected from one or more of: 36-4 (SEQ ID NO:223), 43-8 (SEQ ID NO:224 or 225), 42-10 (SEQ ID NO:226-227), 97-4 (SEQ ID NO:228).

[0130] SEQ ID NO: 223, 36-4 (bold italic text indicates helices 1, 2, and 3, respectively) TIFF0007663513000068.tif11164.

[0131] SEQ ID NO: 224, 43-8 low affinity (bold indicates mutated residues, bold italic text indicates helices 1, 2, and 3, respectively) TIFF0007663513000069.tif10165.

[0132] SEQ ID NO: 225, 43-8 high affinity (bold indicates mutated residues, bold italic text indicates helices 1, 2, and 3, respectively) TIFF0007663513000070.tif10165.

[0133] SEQ ID NO: 226, 42-10 low affinity (bold indicates mutated residues, bold italic text indicates helices 1, 2, and 3, respectively) TIFF0007663513000071.tif10165.

[0134] SEQ ID NO: 227, 42-10 high affinity (bold indicates mutated residues, bold italic text indicates helices 1, 2, and 3, respectively) TIFF0007663513000072.tif10165.

[0135] SEQ ID NO: 228, 97-4 (bold italic text indicates helices 1, 2, and 3, respectively) TIFF0007663513000073.tif11165.

[0136] In some embodiments of any of the aspects, the DBD binds to a DNA binding motif (DBM) comprising any of SEQ ID NOs:229-240.

[0137] SEQ ID NO:229 is an exemplary DBM (DNA binding motif) nucleic acid sequence for 36-4: c GAA GAC GCT g.

[0138] SEQ ID NO:230 to SEQ ID NO:232 are exemplary DBM affinity variant nucleic acid sequences for 43-8. Bold text indicates residues mutated from the WT sequence. SEQ ID NO:230 is 43-8 DBM1 - aGAGTGAGGAc. SEQ ID NO:231 is 43-8 DBM2 - aCAGTGAGGAc. SEQ ID NO:232 is 43-8 DBM3 - aTAGTGAGGAc.

[0139] SEQ ID NOs: 233-239 are exemplary DBM affinity variant nucleic acid sequences for 42-10. Bold text indicates residues mutated from the WT sequence. SEQ ID NO: 233 is 42-10 DBM1 - aGACGCTGCTc. SEQ ID NO: 234 is 42-10 DBM2 - tGACGCTGCTt. SEQ ID NO: 235 is 42-10 DBM3 - aGACGGTGCTc. SEQ ID NO: 236 is 42-10 DBM4 - aCACGCTGCTc. SEQ ID NO: 237 is 42-10 DBM5 - aGACGCTACTc. SEQ ID NO: 238 is 42-10 DBM6 - aGACGCTGCTa. SEQ ID NO: 239 is 42-10 DBM7 - aGACTCTGCTc.

[0140] SEQ ID NO:240 is an exemplary DBM (DNA binding motif) nucleic acid sequence for 97-4: a TTA TGG GAG a.

[0141] Inhibitory protease domain In some embodiments of any of the aspects, a synTF described herein comprises a regulatory protein, wherein the regulatory protein is an inhibitory protease domain (referred to herein as a PRO or RPD). As used herein, the term "inhibitory protease" refers to a protease that can be inactivated by the presence or absence of a specific agent (e.g., one that specifically binds to the protease). In some embodiments, the inhibitory protease is active in the absence of the specific agent (e.g., cleaves the protease cleavage site) and inactive in the presence of the specific agent (e.g., does not cleave the protease cleavage site). In some embodiments, the specific agent is a protease inhibitor. In some embodiments, the protease inhibitor specifically inhibits a given inhibitory protease described herein.

[0142] In some embodiments of any of the aspects, a synTF polypeptide (or collectively, a synTF polypeptide system) described herein comprises one, two, three, four, five, or more inhibitory proteases. In some embodiments of any of the aspects, a synTF polypeptide or system comprises one inhibitory protease. In embodiments comprising multiple inhibitory proteases, the multiple inhibitory proteases can be different individual inhibitory proteases or multiple copies of the same inhibitory protease, or a combination of the above.

[0143] Non-limiting examples of inhibitory proteases include hepatitis C virus proteases (e.g., NS3 and NS2-3); HIV1 protease; coronavirus (main) protease; tobacco etch virus (TEV) protease; signal peptidase; proprotein convertases of the subtilisin / kexin family (furin, PCI, PC2, PC4, PACE4, PC5, PC); proprotein convertases that cleave at hydrophobic residues (e.g., Leu, Phe, Val, or Met); proprotein convertases that cleave at small amino acid residues, e.g., Ala or Thr; proprotein convertases that cleave at small amino acid residues, e.g., Ala or Thr; These include pyomelanocortin convertase (PCE); chromaffin granule aspartic protease (CGAP); prohormone thiol proteases; carboxypeptidases (e.g., carboxypeptidase E / H, carboxypeptidase D, and carboxypeptidase Z); aminopeptidases (e.g., arginine aminopeptidase, lysine aminopeptidase, aminopeptidase B); prolyl endopeptidase; aminopeptidase N; insulin-degrading enzyme; calpain; high molecular weight proteases; and caspases 1, 2, 3, 4, 5, 6, 7, 8, and 9.Other proteases include aminopeptidase N, puromycin-sensitive aminopeptidase, angiotensin-converting enzyme, pyroglutamyl peptidase II, dipeptidyl peptidase IV, N-arginine dibasic convertase, endopeptidase 24.15, endopeptidase 24.16, amyloid precursor protein secretases α, β, and γ, angiotensin-converting enzyme secretase, TGFα secretase, TFα secretase, FAS ligand secretase, TNF receptor-I and -II secretases, CD30 secretase, KL1 and KL2 secretases, IL6 receptor secretase, CD43 and CD44 secretases, and CD16-1 and CD16-11 secretases. These include, but are not limited to, retase; L-selectin secretase; folate receptor secretase; MMP1, 2, 3, 7, 8, 9, 10, 11, 12, 13, 14, and 15; urokinase-type plasminogen activator; tissue plasminogen activator; plasmin; thrombin; BMP-1 (procollagen C-peptidase); ADAM1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11; and granzymes A, B, C, D, E, F, G, and H.For a description of proteases, see, for example, VYH Hook, Proteolytic and cellular mechanisms in prohormone and proprotein processing, RG Landes Company, Austin, Tex., USA (1998); NM Hooper et al., Biochem. J. 321: 265-279 (1997); Z. Werb, Cell 9 1: 439-442 (1997); TG Wolfsberg et al., J. Cell Biol. 131: 275-278 (1995); K. Murakami and JD Etlinger, Biochem. Biophys. Res. Comm. 146: 1249-1259 (1987); T. Berg et al., Biochem. J. 307: 313-326 (1995); MJ Smyth and JA Trapani, Immunology Today 16: 202-206 (1995); RV Talanian et al., J. Biol. Chem. 272: 9677-9682 (1997); and NA Thomberry et a, J. Biol. Chem. 272: 17907-1791 1 (1997); International patent application WO2019118518; al., Methods Mol Biol. 2012;903:393-405; Gao et al. Science 21 Sep 2018: Vol. 361, Issue 6408, pp. 1252-1258; Tague et al., Nat Methods. 2018 Jul;15(7):519-522; Lin et al. PNAS June 3, 2008 105 (22) 7744-7749; U.S. Patent Application No. 16 / 832,751, filed March 27, 2020, the contents of each of which are incorporated by reference herein in their entirety.

[0144] In some embodiments of any of the aspects, the inhibitory protease is hepatitis C virus (HCV) nonstructural protein 3 (NS3). NS3, also known as p-70, is a viral nonstructural protein that is a 70 kDa cleavage product of the hepatitis C virus polyprotein. The 631-residue HCV NS3 protein is a dual-function protein containing a trypsin / chymotrypsin-like serine protease in the N-terminal region and a helicase and nucleoside triphosphatase in the C-terminal region. The minimal sequence required for functional serine protease activity includes the N-terminal 180 amino acids of the NS3 protein, which may also be referred to as "NS3a." Deletion of up to 14 residues from the N-terminus of the NS3 protein is tolerated while maintaining serine protease activity. Thus, the inhibitory proteases described herein contain at least residues 14-180 of the wild-type NS3 protein.

[0145] HCV has at least seven genotypes, designated 1 through 7, which may be further designated with "a" and "b" subtypes. Thus, the inhibitory protease can be HCV genotype 1 NS3, HCV genotype 1a NS3, HCV genotype 1b NS3, HCV genotype 2 NS3, HCV genotype 2a NS3, HCV genotype 2b NS3, HCV genotype 3 NS3, HCV genotype 3a NS3, HCV genotype 3b NS3, HCV genotype 4 NS3, HCV genotype 4a NS3, HCV genotype 4b NS3, HCV genotype 5 NS3, HCV genotype 5a NS3, HCV genotype 5b NS3, HCV genotype 6 NS3, HCV genotype 6a NS3, HCV genotype 6b NS3, HCV genotype 7 NS3, HCV genotype 7a NS3, or HCV genotype 7b NS3. In some embodiments of any of the aspects, the inhibitory protease can be, for example, any known HCV NS3 genotype, variant, or mutant that maintains the same function. In some embodiments of any of the aspects, the NS3 sequence includes residues 1-180 of the NS3 protein from HCV-H, HCV-1, HCV-J1, HCV-BK, HCV-JK1, HCV-J4, HCV-J, HCV-J6, C14112, HCV-J8, D14114, HCV-Nz11, or HCV-K3a (see, e.g., Chao Lin, Chapter 6: HCV NS3-4A Serine Protease, Hepatitis C Viruses: Genomes and Molecular Biology, Editor: Tan SL, Norfolk (UK): Horizon Bioscience, 2006, the contents of which are incorporated herein by reference in their entirety). In some embodiments of any of the aspects, the inhibitory protease is a chimera of two, three, four, five, or more different NS3 genotypes, variants, or mutations described herein, such that the protease maintains its cleavage and / or binding function.

[0146] In some embodiments of any of the aspects, the inhibitory protease of a synTF polypeptide described herein comprises SEQ ID NO:82, 91, 241-255, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs:82, 91, 241-255 and maintains the same function.

[0147] In some embodiments of any of the aspects, the inhibitory proteases of the synTF polypeptides described herein do not include at most the first (i.e., N-terminal) residues of SEQ ID NOs: 82, 91, 241-255. In some embodiments of any of the aspects, the inhibitory proteases of the synTF polypeptides described herein do not include at most the first (i.e., N-terminal) residues of SEQ ID NOs: 82, 91, 241-255. and NO:82, 91, 241-255, including residues 1-180, 2-180, 3-180, 4-180, 5-180, 6-180, 7-180, 8-180, 9-180, 10-180, 11-180, 12-180, 13-180, 14-180, 15-180, 16-180, 17-180, 18-180, 19-180, 20-180, 21-180, 22-180, 23-180, 24-180, 25-180, 26-180, 27-180, 28-180, 29-180, or 30-180.

[0148] TIFF0007663513000074.tif23162TIFF0007663513000075.tif17165.

[0149] SEQ ID NO:91, NS3 protease domain (genotype 1A) TIFF0007663513000076.tif17165.

[0150] SEQ ID NO: 241, NS3 (genotype 1A), 180 aa (see, e.g., residues 1027-1206 of the Hepatitis C virus genotype 1 polyprotein, NCBI reference sequence: NP_671491.1) TIFF0007663513000077.tif17165.

[0151] SEQ ID NO:242, NS3 (genotype 1B), 180 aa (see, e.g., chain A of residues 1-180, Ns3 protease, PDB: 4K8B_A) TIFF0007663513000078.tif17164.

[0152] SEQ ID NO: 243, NS3 (genotype 2), 180 aa (see, e.g., residues 1031-1210 of the Hepatitis C virus genotype 2 polyprotein, NCBI reference sequence: YP_001469630.1) TIFF0007663513000079.tif17165.

[0153] SEQ ID NO: 244, NS3 (genotype 3), 180 aa (see, e.g., residues 1033-1212 of the Hepatitis C virus genotype 3 polyprotein, NCBI reference sequence: YP_001469631.1) TIFF0007663513000080.tif17164.

[0154] SEQ ID NO: 245, NS3 (genotype 4), 180 aa (see, e.g., residues 1027-1206 of the Hepatitis C virus genotype 4 polyprotein, NCBI reference sequence: YP_001469632.1) TIFF0007663513000081.tif17165.

[0155] SEQ ID NO: 246, NS3 (genotype 5), 180 aa (see, e.g., residues 1028-1207 of the Hepatitis C virus genotype 5 polyprotein, NCBI reference sequence: YP_001469633.1) TIFF0007663513000082.tif17165.

[0156] SEQ ID NO: 247, NS3 (genotype 6), 180 aa (see, e.g., residues 1032-1211 of the Hepatitis C virus genotype 6 polyprotein, NCBI reference sequence: YP_001469634.1) TIFF0007663513000083.tif17164.

[0157] SEQ ID NO: 248, NS3 (genotype 7), 180 aa (see, e.g., residues 1031-1210 of the Hepatitis C virus genotype 7 polyprotein, NCBI reference sequence: YP_009272536.1) TIFF0007663513000084.tif17165.

[0158] SEQ ID NO:249, NS3 genotype 1a (HCV-H), 180aa, TIFF0007663513000085.tif17165.

[0159] SEQ ID NO:250, NS3 genotype 1b (HCV-BK), 180aa, TIFF0007663513000086.tif17164.

[0160] SEQ ID NO:251, NS3 genotype 2a (HCV-J6), 180aa, TIFF0007663513000087.tif17165.

[0161] SEQ ID NO:252, NS3 genotype 2b (HCV-J8), 180aa, TIFF0007663513000088.tif17164.

[0162] SEQ ID NO:253, NS3 genotype 3a (HCV-Nz11), 180aa, TIFF0007663513000089.tif17164.

[0163] In some embodiments of any of the aspects, the inhibitory proteases described herein are resistant to one, two, three, four, five, or more different protease inhibitors described herein. Non-limiting examples of NS3 amino acid substitutions that confer resistance to HCV NS3 protease inhibitors include V36L (e.g., genotype 1b), V36M (e.g., genotype 2a), T54S (e.g., genotype 1b), Y56F (e.g., genotype 1b), Q80L (e.g., genotype 1b), Q80R (e.g., genotype 1b), Q80K (e.g., genotypes 1a, 1b, 6a), Y132I (e.g., genotype 1b), A156S (e.g., genotype 2a), A156G, A156T, A156R, A156S, A156T, A156R ... V, D168A (e.g., genotype 1b), I170V (e.g., genotype 1b), S20N, R26K, Q28R, A39T, Q41R, I71V, Q80R, Q86R, P89L, P89S, S101N, A111S, P115S, S122R, R155Q, L144F, A150V, R155W, V158L, D168A, D168G, D168H, D168N, D168V, D168E, D168Y, E176K, T178S, M179I, M179V, and M179T. See, e.g., Sun et al., Gene Expr. 2018, 18(1): 63-69; Kliemann et al., World J Gastroenterol. 2016 Oct 28, 22(40): 8910-8917; U.S. Pat. Nos. 7,208,309 and 7,494,660, the contents of each of which are incorporated by reference herein in their entirety.

[0164] In some embodiments of any of the aspects, the synTF polypeptide described herein comprises an NS3 protease comprising at least one resistance mutation described herein, or any combination thereof. In some embodiments of any of the aspects, the synTF polypeptide described herein comprises an NS3 protease that is resistant to one protease inhibitor but responsive to at least one other protease inhibitor. In some embodiments of any of the aspects, the synTF system comprises (a) a first synTF polypeptide comprising an inhibitory protease (e.g., NS3) that is resistant to a first protease inhibitor and sensitive to a second protease inhibitor; and (b) a second synTF polypeptide comprising an inhibitory protease (e.g., NS3) that is sensitive to the first protease inhibitor and resistant to the second protease inhibitor. Thus, the presence of a first protease inhibitor can modulate the activity of a second synTF polypeptide but not the first synTF polypeptide, whereas the presence of a second protease inhibitor can modulate the activity of a first synTF polypeptide but not the second synTF polypeptide.

[0165] In some embodiments of any of the aspects, the inhibitory proteases described herein are sensitive to one, two, three, four, five, or more different protease inhibitors described herein. In some embodiments of any of the aspects, the NS3 protease comprises at least one of the following mutations: V36M, T54A, S122G, F43L, Q80K, S122R, D168Y, or any combination thereof. In some embodiments of any of the aspects, the NS3 protease comprises at least one of the following mutations: V36M, T54A, S122G, or any combination thereof; such proteases are referred to herein as NS3 inhibitors. AIThe NS3 protease is also referred to as NS3A because these mutations increase its susceptibility to asunaprevir (see, e.g., SEQ ID NO: 254). In some embodiments of any of the aspects, the NS3 protease contains at least one of the following mutations: F43L, Q80K, S122R, D168Y, or any combination thereof; such proteases are referred to herein as NS3A. TI These mutations are also referred to as telaprevir-specific mutations because they increase its susceptibility to telaprevir (see, e.g., SEQ ID NO: 255). See, e.g., WO2019023164; Jacobs et al., StaPLs: versatile genetically encoded modules for engineering drug-inducible proteins, Nat Methods. 2018 Jul; 15(7): 523-526, the contents of each of which are incorporated herein by reference in their entirety.

[0166] SEQ ID NO:254, NS3 AI ;V36M, T54A, and S122G mutations are shown in bold, double-underlined text, respectively TIFF0007663513000090.tif17164.

[0167] SEQ ID NO:255, NS3 TI ;F43L, Q80K, S122R, and D168Y mutations are shown in bold, double-underlined text, respectively TIFF0007663513000091.tif18164.

[0168] In some embodiments of any of the aspects, the polypeptide further comprises a cofactor for the inhibitory protease. As used herein, the term "cofactor for an inhibitory protease" refers to a molecule that increases the activity of the inhibitory protease. In some embodiments of any of the aspects, the synTF polypeptide described herein comprises one, two, three, four, five, or more cofactors for the inhibitory protease. In some embodiments of any of the aspects, the synTF polypeptide comprises one cofactor for each inhibitory protease. In embodiments comprising multiple cofactors for the inhibitory protease, the multiple cofactors for the inhibitory protease can be different individual cofactors or multiple copies of the same cofactor, or a combination of the above.

[0169] In some embodiments of any of the aspects, the cofactor is the HSV NS4A domain and the inhibitory protease is HSV NS3. Nonstructural protein 4a (NS4A) is the smallest nonstructural HCV protein. The NS4A protein has multiple functions in the HCV life cycle, including (1) anchoring the NS3-4A complex to the outer leaflet of the endoplasmic reticulum and the outer mitochondrial membrane, (2) serving as a cofactor for the NS3A serine protease, (3) increasing NS3A helicase activity, and (4) regulating NS5A hyperphosphorylation and viral replication. The interactions between NS4A and the replication of the NS4B control genome and between NS3 and NS4A play a role in viral assembly.

[0170] In some embodiments of any of the aspects, the synTF polypeptide described herein comprises a portion of the NS4a polypeptide that serves as a cofactor for NS3. Deletion analysis has shown that the central region of the 54-residue NS4A protein (approximately residues 21-34) is essential and sufficient for cofactor function of the NS3 serine protease. Thus, in some embodiments of any of the aspects, the inhibitory protease cofactor comprises a 14-residue region of the wild-type NS4A protein.

[0171] In some embodiments of any of the aspects, the cofactor for the inhibitory protease can be HCV genotype 1 NS4A, HCV genotype 1a NS4A, HCV genotype 1b NS4A, HCV genotype 2 NS4A, HCV genotype 2a NS4A, HCV genotype 2b NS4A, HCV genotype 3 NS4A, HCV genotype 3a NS4A, HCV genotype 3b NS4A, HCV genotype 4 NS4A, HCV genotype 4a NS4A, HCV genotype 4b NS4A, HCV genotype 5 NS4A, HCV genotype 5a NS4A, HCV genotype 5b NS4A, HCV genotype 6 NS4A, HCV genotype 6a NS4A, HCV genotype 6b NS4A, HCV genotype 7 NS4A, HCV genotype 7a NS4A, or HCV genotype 7b NS4A. In some embodiments of any of the aspects, the cofactor for the inhibitory protease can be any known NS4A genotype, variant, or mutant, e.g., one that maintains the same function. In some embodiments of any of the aspects, the NS4A sequence includes residues 21-31 of the NS4A protein from HCV-H, HCV-1, HCV-J1, HCV-BK, HCV-JK1, HCV-J4, HCV-J, HCV-J6, C14112, HCV-J8, D14114, HCV-Nz11, or HCV-K3a (see, e.g., Chao Lin 2006, supra; see, e.g., Table 13).

[0172] In some embodiments of any of the aspects, a cofactor for an inhibitory protease of a synTF polypeptide described herein comprises SEQ ID NO:48, 98, 137-156, or an amino acid sequence that maintains the same function as one of SEQ ID NO:48, 98, 137-156, that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of one of SEQ ID NO:48, 98, 137-156. In some embodiments of any of the aspects, a cofactor for an inhibitory protease of a synTF polypeptide described herein comprises SEQ ID NO:81, 93, 96, 255-276, or an amino acid sequence that is at least 95% identical to one of SEQ ID NO:81, 93, 96, 255-276 and maintains the same function.

[0173] In some embodiments of any of the aspects, a cofactor for an inhibitory protease of a synTF polypeptide described herein comprises residues 1-14, 1-13, 1-12, 1-11, 1-10, 2-14, 2-13, 2-12, 2-11, 2-10, 3-14, 3-13, 3-12, 3-11, 3-10, 4-14, 4-13, 4-12, 4-11, or 4-10 of any of SEQ ID NOs:81, 93, 96, 255-276.

[0174] SEQ ID NO:81, NS4A (genotype 1A), 13aa, GCVVIVGRIVLSG.

[0175] SEQ ID NO:93, NS4A domain (genotype 1a) TIFF0007663513000092.tif3128.

[0176] SEQ ID NO:96, NS4 (with L6 linker in bold text) TIFF0007663513000093.tif3128.

[0177] SEQ ID NO:106, NS4A domain, TIFF0007663513000094.tif3159.

[0178] SEQ ID NO:256, NS4A (genotype 1B), 12 aa, GSVVIVGRIILS; see e.g., C chain, non-structural protein, PDB:4K8B_C.

[0179] SEQ ID NO: 257, NS4A (genotype 1), 14 aa (see, e.g., residues 1678-1691 of the Hepatitis C virus genotype 1 polyprotein, NCBI reference sequence: NP_671491.1): GCVVIVGRIVLSGK.

[0180] SEQ ID NO: 258, NS4A (genotype 2), 14 aa (e.g., residues 1682-1695 of the Hepatitis C virus genotype 2 polyprotein, see NCBI Reference Sequence: YP_001469630.1: GCVCIIGRLHINQR).

[0181] SEQ ID NO: 259, NS4A (genotype 3), 14 aa (see, e.g., residues 1684-1697 of the Hepatitis C virus genotype 3 polyprotein, NCBI reference sequence: YP_001469631.1): GCVVIVGHIELEGK.

[0182] SEQ ID NO: 260, NS4A (genotype 4), 14 aa (see, e.g., residues 1678-1691 of the Hepatitis C virus genotype 4 polyprotein, NCBI reference sequence: YP_001469632.1): GSVVIVGRVVLSGQ.

[0183] SEQ ID NO: 261, NS4A (genotype 5), 14 aa (see, e.g., residues 1679-1692 of the Hepatitis C virus genotype 5 polyprotein, NCBI reference sequence: YP_001469633.1): GSVAIVGRIILSGR.

[0184] SEQ ID NO: 262, NS4A (genotype 6), 14 aa (see, e.g., residues 1683-1696 of the Hepatitis C virus genotype 6 polyprotein, NCBI Reference Sequence: YP_001469634.1): GCVVICGRIVTSGK.

[0185] SEQ ID NO: 263, NS4A (genotype 7), 14 aa (see, e.g., residues 1682-1695 of the Hepatitis C virus genotype 7 polyprotein, NCBI Reference Sequence: YP_009272536.1): GSVVVVGRVVLGSN.

[0186] In some embodiments of any of the aspects, the NS4A sequence is selected from Table 13. In one embodiment, the NS4A comprises residues 21-31 of SEQ ID NOs:264-276, or a sequence at least 70% identical.

[0187] (Table 13) Exemplary NS4A sequences (see, e.g., Chao Lin 2006, supra). Residues 21-31 are in bold. TIFF0007663513000095.tif78170

[0188] In some embodiments of any of the aspects, the synTF polypeptide described herein can comprise any combination of the NS3 and NS4A genotypes, variants, or mutations described herein. In one embodiment, NS3 and NS4A are selected from the same genotype as each other. In some embodiments of any of the aspects, NS3 is genotype 1a and NS4A is genotype 1b. In some embodiments of any of the aspects, NS3 is genotype 1b and NS4A is genotype 1a.

[0189] In some embodiments of any of the aspects, the synTF polypeptide described herein comprises an HSV NS4A domain adjacent to the NS3 inhibitory protease. In some embodiments of any of the aspects, the NS4A domain is at the N-terminus of the NS3 inhibitory protease. In some embodiments of any of the aspects, the NS4A domain is at the C-terminus of the NS3 inhibitory protease. In some embodiments of any of the aspects, the synTF polypeptide comprises a peptide linker between the NS4A domain and the NS3 inhibitory protease. Non-limiting examples of linkers (e.g., between the NS4A domain and the NS3 inhibitory protease) include SGTS (SEQ ID NO:277) and GSGS (SEQ ID NO:278).

[0190] In some embodiments of any of the aspects, any two domains described herein in a synTF polypeptide can be joined into a single polypeptide by placing a peptide linker, e.g., a flexible linker, between them. As used herein, "peptide linker" refers to an oligopeptide or polypeptide region of about 2 to 100 amino acids in length that links together any of the polypeptide sequences described herein. In some embodiments, the linker can comprise or consist of flexible residues, such as glycine and serine, to allow adjacent protein domains to move freely relative to one another. Longer linkers may be used if it is desirable to ensure that two adjacent domains do not sterically interfere with one another. The linker may be cleavable or non-cleavable.

[0191] Described herein are synTF polypeptides containing protease cleavage sites. As used herein, the term "protease cleavage site" refers to a specific sequence or sequence motif recognized by an inhibitory protease and cleaved by the protease. A cleavage site for a protease comprises a specific amino acid sequence or motif recognized by the protease during proteolytic cleavage, typically comprising 1 to 6 amino acids on either side of the scissile bond, which bind to the active site of the protease and are used for recognition as a substrate. In some embodiments of any of the aspects, the protease cleavage site can be any site specifically bound by an inhibitory protease and cleaved by the protease. In some embodiments of any of the aspects, the synTF polypeptides described herein (or collectively, the synTF polypeptide system) comprise one, two, three, four, five, or more protease cleavage sites. In some embodiments of any of the aspects, the synTF polypeptide comprises two protease cleavage sites. In embodiments comprising multiple protease cleavage sites, the multiple protease cleavage sites can be different individual protease cleavage sites or multiple copies of the same protease cleavage site, or a combination of the above.

[0192] As a non-limiting example, during HCV replication, the NS3-4A serine protease is responsible for proteolytic cleavage of the HCV polyprotein precursor at four junctions: NS3 / NS4A (autocleavage), NS4A / NS4B, NS4B / NS5A, and NS5A / NS5B. Thus, the protease cleavage sites of the synTF polypeptides described herein can be an NS3 / NS4A cleavage site, an NS4A / NS4B cleavage site, an NS4B / NS5A cleavage site, or an NS5A / NS5B cleavage site. The protease cleavage site can be a protease cleavage site from HCV genotype 1, genotype 1a, genotype 1b, genotype 2, genotype 2a, genotype 2b, genotype 3, genotype 3a, genotype 3b, genotype 4, genotype 4a, genotype 4b, genotype 5, genotype 5a, genotype 5b, genotype 6, genotype 6a, genotype 6b, genotype 7, genotype 7a NS4A, or genotype 7b. In some embodiments of any of the aspects, the protease cleavage site can be any known NS3 / NS4A protease cleavage site or a variant or mutant thereof, e.g., one that maintains the same function. In some embodiments of any of the aspects, the NS4A sequence comprises residues 21-31 of the NS4A protein from HCV-H, HCV-1, HCV-J1, HCV-BK, HCV-JK1, HCV-J4, HCV-J, HCV-J6, C14112, HCV-J8, D14114, HCV-Nz11, or HCV-K3a (see, e.g., Chao Lin 2006, supra).

[0193] In some embodiments of any of the aspects, the protease cleavage site of a synTF polypeptide described herein comprises SEQ ID NO:78, 83, 87, 279-301, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of one of SEQ ID NOs:78, 83, 87, 279-301 and maintains the same function.

[0194] In some embodiments of any of the aspects, a protease cleavage site of a synTF polypeptide described herein comprises residues 1-20, 1-19, 1-18, 1-17, 1-16, 1-15, 2-20, 2-19, 2-18, 2-17, 2-16, 2-15, 3-20, 3-19, 3-18, 3-17, 3-16, 3-15, 4-20, 4-19, 4-18, 4-17, 4-16, 4-15, 5-20, 5-19, 5-18, 5-17, 5-16, or 5-15 of any of SEQ ID NOs:78, 83, 87, 279-301.

[0195] SEQ ID NO:78, NS5A / 5B cleavage site (CC), 10aa, EDVVCCHSIY.

[0196] SEQ ID NO:83, NS4A / 4B cleavage site (CS), 14aa, LYQEFDEMEECSQH.

[0197] SEQ ID NO:87, N3 cleavage site (NS4A / 4B cleavage site), DEMEECSQHL.

[0198] SEQ ID NO:279, QEFEDVVPCSMGS.

[0199] SEQ ID NO:280, NS5A / 5B cleavage site, EDVVCCHSI.

[0200] SEQ ID NO:281, NS4A / 4B cleavage site, DEMEECSQH.

[0201] Table 14: Exemplary NS3 / NS4A protease cleavage sites (see, e.g., Chao Lin 2006, supra) TIFF0007663513000096.tif115160

[0202] In some embodiments of any of the aspects, the synTF polypeptide described herein comprises two protease cleavage sites, one at the N-terminus of the NS3-NS4A complex and one at the C-terminus of the NS3-NS4A complex (see, e.g., Table 15). In some embodiments of any of the aspects, the two protease cleavage sites can be the same cleavage site or different cleavage sites.

[0203] (Table 15) Exemplary protease cleavage site combinations. "N" indicates the N-terminus of the NS3-NS4A complex. "C" indicates the C-terminus of the NS3-NS4A complex. "3 / 4A" indicates the NS3 / NS4A cleavage site. "4A / 4B" indicates the NS4A / NS4B cleavage site. "4B / 5A" indicates the NS4B / NS5A cleavage site. "5A / 5B" indicates the NS5A / NS5B cleavage site. TIFF0007663513000097.tif23128

[0204] In some embodiments of any of the aspects, the synTF polypeptides described herein comprise any known genotype, variant, or mutant of the NS3 / NS4A, NS4A / NS4B, NS4B / NS5A, and NS5A / NS5B cleavage sites. In one embodiment, the two protease cleavage sites are selected from the same genotype as each other.

[0205] In some embodiments of any of the aspects, the protease cleavage site is located or engineered so that when synTF cleaves itself using an inhibitory protease in the absence of a protease inhibitor, the N-terminal amino acid of the resulting newly cleaved polypeptide causes the polypeptide to degrade at a higher rate and have a shorter half-life compared to other cleaved polypeptides. According to the N-end rule, newly cleaved polypeptides containing the amino acids His, Tyr, Gln, Asp, Asn, Phe, Leu, Trp, Lys, or Arg at their N-terminus exhibit a higher degradation rate and a shorter half-life (e.g., 10 minutes or less in yeast; 1-5.5 hours in mammalian reticulocytes). In comparison, newly cleaved polypeptides containing the amino acids Val, Met, Gly, Pro, Ala, Ser, Thr, Cys, Ile, or Glu at their N-terminus exhibit a slower degradation rate and a longer half-life (e.g., 30 minutes or more in yeast; 1-100 hours in mammalian reticulocytes). See, e.g., Gonda et al., Universality and Structure of the N-end Rule, The Journal of Biological Chemistry, Vol. 264(28), pp. 16700-16712, 1989, the contents of which are incorporated herein by reference in their entirety. Thus, in some embodiments of any of the aspects, the resulting amino acid at the N-terminus of the newly cleaved synTF polypeptides described herein is His, Tyr, Gln, Asp, Asn, Phe, Leu, Trp, Lys, or Arg. In some embodiments of any of the aspects, the resulting amino acid at the N-terminus of the newly cleaved synTF polypeptides described herein is not Val, Met, Gly, Pro, Ala, Ser, Thr, Cys, Ile, or Glu.

[0206] In some embodiments of any of the aspects, the N-terminus of a newly cleaved synTF polypeptide described herein comprises SEQ ID NO:79, or an amino acid sequence that maintains His or another heavily degraded amino acid at the N-terminus and is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO:79. SEQ ID NO:79, N-end rule, 8 aa, HSIYGKKK.

[0207] In some embodiments of any of the aspects, the synTF polypeptide described herein comprises a catalytically active inhibitory protease. For HCV NS3, the catalytic triad comprises His-57, Asp-81, and Ser-139. With respect to an inhibitory protease, "catalytically active" refers to the ability to cleave at the protease cleavage site. In some embodiments of any of the aspects, the catalytically active inhibitory protease can be any inhibitory protease further described herein that maintains the catalytic triad, i.e., does not include non-synonymous substitutions at His-57, Asp-81, and / or Ser-139.

[0208] In some embodiments of any of the aspects, the synTF comprises an NS3 protease domain, NS4A, and / or at least one protease cleavage site. In some embodiments of any of the aspects, the synTF comprises SEQ ID NO:85 or 102, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of one of SEQ ID NO:85 or 102.

[0209] TIFF0007663513000098.tif23164TIFF0007663513000099.tif23166.

[0210] TIFF0007663513000100.tif24164TIFF0007663513000101.tif24165.

[0211] In some embodiments of any of the aspects, the synTF comprises a stabilizable polypeptide-linking (StaPL) domain. In some embodiments of any of the aspects, the StaPL domain comprises NS4A, the NS3 protease domain, and a portion of the NS3 helicase domain. In some embodiments of any of the aspects, the partial NS3 helicase domain comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:92, 105, 302, or the sequence of one of SEQ ID NO:92, 105, 302.

[0212] SEQ ID NO:92, NS3 partial helicase domain; TIFF0007663513000102.tif3128.

[0213] SEQ ID NO:105, NS3 partial helicase domain, NSSPPAVTLTHPITK.

[0214] SEQ ID NO:302, NS3 partial helicase domain, NSSPPAVTLTH.

[0215] In some embodiments of any of the aspects, the StaPL domain further comprises a protease cleavage site at its N-terminus, either directly linked or indirectly linked via a peptide linker, e.g., selected from EDVVCCHSI (SEQ ID NO:280) or DEMEECSQH (SEQ ID NO:281). In some embodiments of any of the aspects, the StaPL domain comprises SEQ ID NOs:304-306, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of one of SEQ ID NOs:304-306.

[0216] TIFF0007663513000103.tif24156TIFF0007663513000104.tif24166.

[0217] In some embodiments of any of the aspects, the StaPL domain comprises an inhibitory protease comprising at least one mutation that increases its sensitivity to at least one protease inhibitor. In some embodiments of any of the aspects, the NS3 protease (e.g., of the StaPL domain) comprises at least one of the following mutations: V36M, T54A, S122G, F43L, Q80K, S122R, D168Y, or any combination thereof. In some embodiments of any of the aspects, the NS3 protease (e.g., of the StaPL domain) comprises at least one of the following mutations: V36M, T54A, S122G, or any combination thereof; such StaPL is referred to herein as StaPL. AIThese mutations increase its susceptibility to asunaprevir (see, e.g., SEQ ID NOs: 254, 305). In some embodiments of any of the aspects, the NS3 protease (e.g., in the StaPL domain) contains at least one of the following mutations: F43L, Q80K, S122R, D168Y, or any combination thereof; such proteases are referred to herein as StaPL TI It is also referred to as telaprevir-associated HIV-1 mutation because these mutations increase its susceptibility to telaprevir (see, e.g., SEQ ID NOs: 255, 306).

[0218] TIFF0007663513000105.tif31162TIFF0007663513000106.tif24166.

[0219] TIFF0007663513000107.tif31165TIFF0007663513000108.tif24164.

[0220] In some embodiments of any of the aspects, the synTF comprises a TimeSTAMP domain (a time-specific tag for protein age measurement). In some embodiments of any of the aspects, the TimeSTAMP comprises an inhibitory protease, at least one protease cleavage site, and a detectable marker. The detectable marker is removed from the synTF immediately after translation by the activity of the inhibitory protease until such time as a protease inhibitor is added, after which newly synthesized synTF polypeptides retain their markers. TimeSTAMP enables time-specific tagging of protein age measurement, allowing sensitive, non-perturbative visualization and quantification of newly synthesized proteins of interest with exceptionally tight temporal control.

[0221] In some embodiments of any of the aspects, the inhibitory protease exhibits increased solubility compared to the wild-type protease. As a non-limiting example, the NS3 protease can include at least one of the following mutations, or any combination thereof: Leu13 is substituted with Glu; Leu14 is substituted with Glu; Ile17 is substituted with Gln; Ile18 is substituted with Glu; and / or Leu21 is substituted with Gln. In some embodiments of any of the aspects, the synTF polypeptide described herein comprises an inhibitory protease comprising SEQ ID NOs:307-315, or a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs:307-315 and maintains the same function (e.g., serine protease; increased solubility) as SEQ ID NOs:307-315. See, e.g., U.S. Patent No. 6,333,186 and U.S. Patent Application Publication No. 20020106642, the contents of each of which are incorporated by reference in their entirety.

[0222] TIFF0007663513000109.tif57165TIFF0007663513000110.tif224165.

[0223] In some embodiments of any of the aspects, the inhibitory protease comprises a mutation to increase binding affinity to a specific ligand. As a non-limiting example, NS3aH1 (e.g., SEQ ID NO: 315) binds to the ANR peptide (e.g., SEQ ID NO: 316, TIFF0007663513000111.tif3128) contains four mutations: A7S, E13L, I35V, and T42S, required for interaction with the ribosomal protein A7S. Thus, in some embodiments of any of the aspects, the inhibitory proteases described herein contain at least one of the following mutations: A7S, E13L, I35V, and T42S, or any combination thereof.

[0224] In some embodiments of any of the aspects, the synTF polypeptide described herein is combined with a protease inhibitor. As used herein, "combined" refers to two or more substances present in the same formulation, in any molecular or physical arrangement, e.g., in an admixture, solution, mixture, suspension, colloid, or emulsion. The formulation can be a homogeneous or heterogeneous mixture. In some embodiments of any of the aspects, the active compound can be contained by a superstructure, e.g., a nanoparticle, liposome, vector, cell, scaffold, or the like, which is in a solution, mixture, admixture, suspension, or the like with the synTF polypeptide or synTF polypeptide system. In some embodiments of any of the aspects, the synTF polypeptide is bound to a protease inhibitor bound to an inhibitory protease. In some embodiments of any of the aspects, the synTF polypeptide specifically binds to a protease inhibitor bound to an inhibitory protease.

[0225] In some embodiments of any of the aspects, the synTF polypeptide is combined with one, two, three, four, five, or more protease inhibitors. In some embodiments of any of the aspects, the synTF polypeptide is combined with one protease inhibitor. In embodiments including multiple protease inhibitors, the multiple protease inhibitors can be different individual protease inhibitors or multiple copies of the same protease inhibitor, or a combination of the above.

[0226] In some embodiments of any of the aspects, the protease inhibitor is grazoprevir (abbreviated as GZV or GZP; see, e.g., PubChem CID: 44603531). In some embodiments of any of the aspects, the protease inhibitor is danoprevir (DNV; see, e.g., PubChem CID: 11285588). In some embodiments of any of the aspects, the protease inhibitor is an approved NS3 protease inhibitor, such as, but not limited to, grazoprevir, danoprevir, simeprevir, asunaprevir, cilprevir, boceprevir, sovaprevir, paritaprevir, ombitasvir, paritaprevir, ritonavir, dasabuvir, and telaprevir. Additional non-limiting examples of NS3 protease inhibitors are listed in Table 16 (see, e.g., McCauley and Rudd, Hepatitis C virus NS3 / 4a protease inhibitors, Current Opinion in Pharmacology 2016, 30:84-92; the contents of which are incorporated herein by reference in their entirety).

[0227] Table 16: Exemplary NS3 / NS4A protease inhibitors TIFF0007663513000112.tif206166TIFF0007663513000113.tif190166TIFF0007663513000114.tif212166 TIFF0007663513000115.tif218166TIFF0007663513000116.tif205166TIFF0007663513000117.tif223166

[0228] Degron domain In some aspects, synTF polypeptides are described herein that contain a degron domain. As used herein, the term "degron domain" refers to a sequence that promotes degradation of an attached protein, e.g., via the proteasome or autophagy-lysosomal pathway; in some embodiments of any of the aspects, the terms "degron," "degradation domain," and "degradation domain" can be used interchangeably with "degron domain." In some embodiments, a degron domain is a polypeptide that, when fused to a protein, destabilizes the protein, thereby reducing the half-life of the protein by at least two-fold. In some embodiments of any of the aspects, the synTF polypeptides described herein (or collectively, the synTF polypeptide system) contain one, two, three, four, five, or more degron domains. In some embodiments of any of the aspects, the synTF polypeptide or system contains one degron domain. In embodiments containing multiple degron domains, the multiple degron domains can be different individual degron domains, multiple copies of the same degron domain, or a combination of the above.

[0229] Many different degron sequences / signals (e.g., those of the ubiquitin-proteasome system) have been described, any of which can be used as provided herein. A degron domain may be operably linked to a cellular receptor, but need not be contiguous or directly adjacent to it, as long as the degron domain is still functional to direct degradation of the cellular receptor. In some embodiments, the degron domain induces rapid degradation of the cellular receptor. For descriptions of degron domains and their function in protein degradation, see, for example, Kanemaki et al. (2013) Pflugers Arch. 465(3):419-425, Erales et al. (2014) Biochim Biophys Acta 1843(1):216-221, Schrader et al. (2009) Nat. Chem. Biol. 5(11): 815-822, Ravid et al. (2008) Nat. Rev. Mol. Cell. Biol. 9(9):679-690, Tasaki et al. (2007) Trends Biochem Sci. 32(1 1):520-528, Meinnel et al. (2006) Biol. Chem. 387(7):839-851, Kim et al. (2013) Autophagy 9(7): 1100-1103, Varshavsky (2012) Methods Mol. Biol. 832: 1-11, and Fayadat et al. (2003) Mol Biol Cell. 14(3): 1268-1278; Chassin et al., Nature Communications volume 10, Article number: 2013 (2019); Natsume and Kanemaki Annu Rev Genet. 2017 Nov 27,51:83-102; the contents of each of which are incorporated herein by reference in their entirety.

[0230] In some embodiments of any of the aspects, the degron domain comprises a ubiquitin tag, including but not limited to, UbR, UbP, UbW, UbH, UbI, UbK, UbQ, UbV, UbL, UbD, UbN, UbG, UbY, UbT, UbS, UbF, UbA, UbC, UbE, UbM, 3xUbVR, 3xUbVV, 2xUbVR, 2xUbVV, UbAR, UbVV, UbVR, UbAV, 2xUbAR, 2xUbAV. In some embodiments of any of the aspects, the degron domain comprises an auto-excising degron, which refers to a complex comprising an inhibitory protease, a protease cleavage site, and the degron domain. In some embodiments of any of the aspects, the degron domain is a conditional degron domain whose degradation is induced by a ligand (e.g., a degron stabilizer) or another input, such as a temperature change or a specific wavelength of light. Non-limiting examples of conditional degron domains include eDHFR degrons (e.g., TMP inducers); FKBP12 (e.g., rapamycin analog inducers); temperature-sensitive dihydrofolate reductase (R-DHFRts, or ts-DHFR); HCV These include the NS3 / NS4A degron; a modified version of R-DHFRts named low-temperature degron (lt-degron); auxin-inducible degradation (AID); HaloTag-hydrophobic tag, HaloPROTAC, and dTAG systems (e.g., HyT13 or HyT36 inducers); photosensitive degron (PSD); blue light-inducible degron (B-LID); tobacco etch virus (TEV) protease-inducible protein inactivation (TIPI)-degron system; deGradFP (degraded green fluorescent protein; e.g., induced by NSlmb-vhhGFP expression); or split ubiquitin (SURF; e.g., induced by rapamycin) for rescue of function.

[0231] In some embodiments of any of the aspects, the degron domain is an E. coli dihydrofolate reductase (eDHFR) degron. The eDHFR degron allows for large-scale depletion of exogenously expressed proteins in mammalian cells and C. elegans. The eDHFR degron is stabilized by tight binding to antibiotics and the degron stabilizer trimethoprim (TMP), shown below, which is harmless to eukaryotic cells. TIFF0007663513000118.tif28128

[0232] Proteins tagged with eDHFR are constitutively degraded unless cells are exposed to TMP. By modulating the TMP concentration in the growth medium, the levels of tagged proteins can be directly controlled. Unlike shRNA methods, this degron-based strategy is advantageous because the depletion kinetics is not limited by the half-life of the native protein, allowing for more rapid knockdown of stable proteins. TMP stabilizes the DD-target protein fusion in a dose-dependent manner by up to 100-fold, giving the system a substantial dynamic range. The TMP ligand acts by itself and does not require dimerization with a second protein. This system is highly effective and can be used to control the levels of transmembrane proteins, such as the synTF polypeptide described herein. See, e.g., Schrader et al., Chem Biol. 2010 Sep 24, 17(9): 917-918; Ryan M. Sheridan and David L. Bentley, Biotechniques. 2016, 60(2): 69-74; Iwamoto et al., Chem Biol. 2010 Sep 24;17(9):981-8.

[0233] In some embodiments of any of the aspects, the degron domain comprises an amino acid sequence derived from FK506-binding protein and rapamycin-binding protein (FKBP12) (UniProtKB - P62942 (FKB1A_HUMAN), incorporated herein by reference), or a variant thereof. In some embodiments of any of the aspects, the amino acid sequence derived from FKBP12 comprises a phenylalanine (F) to valine (V) mutation (F36V) at amino acid position 36 (counting excluding methionine) (also referred to as FKBP12* or FKBP*). In some embodiments of any of the aspects, the degron stabilizer is a rapamycin analog, such as Shield-1, shown below. See, e.g., Banaszynski et al., Cell. 2006 Sep 8;126(5):995-1004; U.S. Patent Application Publication No. 20180179522; U.S. Patent No. 10,137,180; the contents of each of which are incorporated herein by reference in their entirety. TIFF0007663513000119.tif57128

[0234] In some embodiments of any of the aspects, the degron domain of a synTF polypeptide described herein comprises SEQ ID NO:317 or 318, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO:317 or 318 and maintains the same function (e.g., degradation, binding to TMP or Shield-1).

[0235] SEQ ID NO:317, DHFR(V19A), 158 aa, TIFF0007663513000120.tif17165.

[0236] SEQ ID NO:318, FK506 and rapamycin binding protein (FKBP), 107 aa, TIFF0007663513000121.tif11164.

[0237] In some embodiments of any aspect, the destabilizing degron domain comprises at least one mutation that causes near-complete removal or degradation of the synTF polypeptide. Non-limiting examples of DHFR (e.g., SEQ ID NO:317) mutations include V19A, Y100I, G121V, H12Y, H12L, R98H, F103S, M42T, H114R, I61F, T68S, H12Y / Y100I, H12L / Y100I, R98H / F103S, M42T / H114R, and I61F / T68S, or any combination thereof; see, e.g., U.S. Patent No. 8,173,792, the contents of which are incorporated herein by reference in their entirety.

[0238] In some embodiments of any aspect, the degron domain comprises a ligand-induced degradation (LID) domain. Proteins comprising the LID domain are destabilized and degraded in the presence of a degron destabilizer. In some embodiments of any aspect, the LID domain of the degron domain can bind to a degron destabilizer, promoting degradation of the attached protein. This system is reversible; upon removal of the degron destabilizer, the protein is no longer destabilized and / or degraded. In some embodiments of any aspect, the synTF polypeptide is bound to the degron destabilizer bound to the degron domain. In some embodiments of any aspect, the synTF polypeptide is specifically bound to the degron destabilizer bound to the degron domain.

[0239] In some embodiments of any aspect, the synTF polypeptide is in combination with one, two, three, four, five, or more degron destabilizers. In some embodiments of any aspect, the synTF polypeptide is in combination with one degron destabilizer. In embodiments including multiple degron destabilizers, the multiple degron destabilizers can be different individual degron destabilizers or multiple copies of the same degron stabilizer, or combinations of the foregoing.

[0240] In some embodiments of any aspect, the LID degron domain comprises FK506 and rapamycin-binding protein (FKBP), and further comprises a degron fused to the C-terminus of FKBP with an intervening linker, e.g., a 10 amino acid linker (Gly4SerGly4Ser) or another linker as described herein. In some embodiments of any aspect, the degron fused to the C-terminus of FKBP (e.g., SEQ ID NO:318) has the 19 amino acid sequence: TIFF0007663513000122.tif3128 (SEQ ID NO:319), or a sequence at least 95% identical and maintaining the same function. In the absence of the small molecule Shield-1, the 19-aa degron binds to the FKBP fusion protein, stabilizing the protein. In its presence, Shield-1 tightly binds to FKBP, displacing the 19-aa degron and inducing rapid and progressive degradation of the LID domain and any fused partner protein. In some embodiments of any aspect, the degron destabilizer is Shield-1, as shown above, or an analog thereof; see, e.g., Bonger et al., Nat Chem Biol. 2011 Jul 3;7(8):531-7.

[0241] In some embodiments of any aspect, the degron domain comprises auxin-induced degradation (AID). Proteins fused to AID (also known as indole-3-acetic acid-inducible 17 or AUX / IAA transcription factor family proteins) are rapidly degraded. Degradation requires ectopic expression of the plant F-Box protein TIR1, which recruits AID-tagged proteins to the SKP1-CUL1-F-Box (SCF) ubiquitin E3 ligase in an auxin-dependent manner, resulting in their ubiquitination and proteasomal degradation. In some embodiments of any aspect, the degron domain comprises residues 65-133, 65-130, 70-130, or 70-120 of SEQ ID NO: 320. In some embodiments of any aspect, the degron domain of a synTF polypeptide as described herein comprises SEQ ID NO: 320 or 321, or an amino acid sequence that maintains the same function as SEQ ID NO: 320 or 321, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO: 320 or 321. See, e.g., Daniel et al., Nat Commun. 2018 Aug 17;9(1):3297, the contents of which are incorporated herein by reference in their entirety.

[0242] SEQ ID NO: 320, AUX / IAA transcriptional regulator family protein [Arabidopsis thaliana], NCBI reference sequence: NP_171921.1, 229 aa, TIFF0007663513000123.tif23165.

[0243] SEQ ID NO: 321, mAID (minimum AID), 68 aa, TIFF0007663513000124.tif10163.

[0244] In some embodiments of any aspect, the degron domain comprises a modified portion of NS3 helicase and NS4A. The arrangement of the NS3pro and NS4A sequences in the construct creates a functional degron. During HCV replication, the free NS4A N-terminus forms a hydrophobic α-helix that inserts into the endoplasmic reticulum membrane. This N-terminus is generated by cleavage of the HCV nonstructural polypeptide at the NS3 / 4A junction due to its positioning in the protease active site by the NS3 helicase domain. Because the engineered construct lacks the helicase domain, NS3 / 4A cleavage does not occur. The hydrophobic sequence of NS4A, which cannot insert into the membrane without the free N-terminus, exhibits degron-like activity. See, e.g., U.S. Patent 10,550,379; Chung et al., Nat Chem Biol. 2015 Sep; 11(9): 713-720 (the contents of which are incorporated herein by reference in their entirety).

[0245] In some embodiments of any aspect, the degron domain comprises SEQ ID NO:322 or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO:322 and maintains the same function.

[0246] SEQ ID NO:322, HCV NS3 / NS4 degron domain (42 aa) TIFF0007663513000125.tif3128.

[0247] Inducible Degradation Domain In some aspects, the present disclosure provides a synTF polypeptide comprising an inducible degradation domain, also referred to herein as a self-cleaving degron or small molecule-assisted shutoff (SMASh) domain. In some embodiments of any aspect, the SMASh domain comprises an inhibitory protease, at least one protease cleavage site, and a degron domain. In the absence of a protease inhibitor, the inhibitory protease cleaves the degron from the synTF, and the synTF is not degraded. In the presence of a protease inhibitor, the inhibitory protease does not cleave the degron from the synTF, and the degron domain directs degradation of the synTF.

[0248] In some embodiments of any aspect, a synTF polypeptide (or collectively, a synTF polypeptide system) as described herein comprises one, two, three, four, five, or more inducible degradation domains. In some embodiments of any aspect, a synTF polypeptide or system comprises one inducible degradation domain. In embodiments comprising multiple inducible degradation domains, the multiple inducible degradation domains can be different individual inducible degradation domains or multiple copies of the same inducible degradation domain, or a combination of the foregoing.

[0249] In some embodiments of any aspect, the degron domain (e.g., of a SMASh domain) may be selected from the group consisting of: a ubiquitin tag; an eDHFR degron (e.g., a TMP inducer); FKBP12 (e.g., a rapamycin analog inducer); a temperature-sensitive dihydrofolate reductase (R-DHFRts, or ts-DHFR); an HCV NS3 / NS4A degron; a modified form of R-DHFRts called a low-temperature degron (lt-degron); an auxin-inducible degradation (AID); HaloTag-hydrophobic Tag, HaloPROTAC, and dTAG systems (e.g., a HyT13 or HyT36 inducer); a light-sensitive degron (PSD); a blue-light-inducible degron (B-LID); a tobacco etch virus (TEV) protease-induced protein inactivation (TIPI)-degron system; deGradFP (degrading green fluorescent protein; e.g., induced by NSlmb-vhhGFP expression); or Split Ubiquitin Function Rescue (SURF; e.g., induced by rapamycin).

[0250] In some embodiments of any aspect, the degron domain (e.g., of the SMASh domain) comprises modified portions of NS3 helicase and NS4A. In some embodiments of any aspect, the degron domain (e.g., of the SMASh domain) comprises SEQ ID NO:322, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO:322 and maintains the same function.

[0251] In some embodiments of any aspect, the SMASh tag comprises an inhibitory protease, a partial protease helical domain, and a cofactor domain. In some embodiments of any aspect, the SMASh tag comprises an inhibitory protease, a partial protease helical domain, a cofactor domain, and at least one protease cleavage site. In some embodiments of any aspect, the SMASh tag comprises an NS3 inhibitory protease, an NS3 partial protease helical domain, an NS3 cofactor domain (i.e., NS4A), and at least one protease cleavage site of the NS3 inhibitory protease.

[0252] In some embodiments of any aspect, the SMASh tag is a C-terminal SMASh tag, for example, the tag is engineered to be attached to the C-terminus of a synTF. In some embodiments of any aspect, the C-terminal SMASh tag comprises a protease cleavage site at the N-terminus of the tag. In some embodiments of any aspect, the C-terminal SMASh tag comprises, from N-terminus to C-terminus, an NS3 cleavage site, at least one linker, an NS3 domain, an NS3 partial helicase, and an NS4A domain. In some embodiments of any aspect, the C-terminal SMASh tag is fused to the C-terminus of the transcription effector domain of a synTF. In some embodiments of any aspect, the C-terminal SMASh tag is fused to the C-terminus of the DNA binding domain of a synTF.

[0253] In some embodiments of any aspect, the C-terminal SMASh tag comprises SEQ ID NO:86, 324, 327, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NO:86, 324, 327 and maintains the same function.

[0254] TIFF0007663513000126.tif37163TIFF0007663513000127.tif31166.

[0255] In some embodiments of any aspect, the SMASh tag is an N-terminal SMASh tag, for example, the tag is engineered to be attached to the N-terminus of synTF. In some embodiments of any aspect, the N-terminal SMASh tag comprises a protease cleavage site at the C-terminus of the tag. In some embodiments of any aspect, the N-terminal SMASh tag comprises, from N-terminus to C-terminus, at least one linker, an NS3 domain, an NS3 partial helicase, an NS4 domain, and an NS3 cleavage site. In some embodiments of any aspect, the N-terminal SMASh tag is fused to the N-terminus of the transcription effector domain of synTF. In some embodiments of any aspect, the N-terminal SMASh tag is fused to the N-terminus of the DNA binding domain of synTF.

[0256] In some embodiments of any aspect, the N-terminal SMASh tag comprises SEQ ID NO:94, 95, 325, 326, 328, 329, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NO:94, 95, 325, 326, 328, or 329 and maintains the same function.

[0257] TIFF0007663513000128.tif36164TIFF0007663513000129.tif31166.

[0258] TIFF0007663513000130.tif36165TIFF0007663513000131.tif31166.

[0259] In some embodiments of any aspect, the SMASh domain comprises an inhibitory protease comprising at least one mutation that increases susceptibility to at least one protease inhibitor. In some embodiments of any aspect, the NS3 protease (e.g., of the SMASh domain) comprises at least one of the following mutations: V36M, T54A, S122G, F43L, Q80K, S122R, D168Y, or any combination thereof. In some embodiments of any aspect, the NS3 protease (e.g., of the SMASh domain) comprises at least one of the following mutations: V36M, T54A, S122G, or any combination thereof; these mutations increase susceptibility to asunaprevir, and such SMASh is referred to herein as SMASh. AI In some embodiments of any aspect, the NS3 protease (e.g., of the SMASh domain) contains at least one of the following mutations: F43L, Q80K, S122R, D168Y, or any combination thereof; because these mutations increase susceptibility to telaprevir, such proteases are referred to herein as SMASh. TI Also referred to as (see, e.g., SEQ ID NOs: 255, 327, 328, 329).

[0260] TIFF0007663513000132.tif37165TIFF0007663513000133.tif31166.

[0261] TIFF0007663513000134.tif37164TIFF0007663513000135.tif31165.

[0262] TIFF0007663513000136.tif37165TIFF0007663513000137.tif31165.

[0263] TIFF0007663513000138.tif37165TIFF0007663513000139.tif31165.

[0264] TIFF0007663513000140.tif37163TIFF0007663513000141.tif31166.

[0265] TIFF0007663513000142.tif37165TIFF0007663513000143.tif31166.

[0266] In some embodiments of any aspect, the SMASh domain of a synTF polypeptide is in combination with one, two, three, four, five, or more protease inhibitors. In some embodiments of any aspect, the SMASh domain of a synTF polypeptide is in combination with one protease inhibitor. In embodiments including multiple protease inhibitors, the multiple protease inhibitors can be different individual protease inhibitors or multiple copies of the same protease inhibitor, or combinations of the foregoing.

[0267] In some embodiments of any aspect, the protease inhibitor is grazoprevir (abbreviated as GZV or GZP; see, e.g., PubChem CID: 44603531). In some embodiments of any aspect, the protease inhibitor is danoprevir (DNV; see, e.g., PubChem CID: 11285588). In some embodiments of any aspect, the protease inhibitor is an approved NS3 protease inhibitor, such as, but not limited to, grazoprevir, danoprevir, simeprevir, asunaprevir, cilprevir, boceprevir, sovaprevir, paritaprevir, ombitasvir, paritaprevir, ritonavir, dasabuvir, and telaprevir. Additional non-limiting examples of NS3 protease inhibitors are listed in Table 16 (see, e.g., McCauley and Rudd, Hepatitis C virus NS3 / 4a protease inhibitors, Current Opinion in Pharmacology 2016, 30:84-92; the contents of which are incorporated herein by reference in their entirety).

[0268] Induced Proximal Domains In some aspects, described herein are synTF polypeptides that include at least two induced proximity domains, also referred to herein as heterodimerization domains. As used herein, the term "induced proximity domain" refers to at least two domains that are induced to dimerize or come into proximity in the presence of a stimulus (e.g., a chemical inducer, light, etc.). In some embodiments of any aspect, an induced proximity domain pair comprises a first induced proximity domain (IPD) and a second induced proximity domain (IPD). A ) and at least a second induced proximity domain (IPD B ), which in the presence of an inducer substance or an inducer signal, A and IPD B In some embodiments of any aspect, the synTF effector domain is an IPD in the first polypeptide. A (or IPD B) and the synTF DBD is linked to the IPD in the second polypeptide. B (or IPD A ) is linked to the IPD in the presence of an inducer substance or inducer signal. A and IPD B are ligated together, resulting in the ligation of the ED to the DBD of the synthetic TF. In the absence of an inducer agent or inducer signal, the ED is not coupled or ligated to the DBD of the synthetic TF.

[0269] In some embodiments of any aspect, a synTF polypeptide (or collectively a synTF polypeptide system) as described herein comprises one, two, three, four, five, or more induced proximity domains. In some embodiments of any aspect, a synTF polypeptide or system comprises one induced proximity domain. In embodiments comprising multiple induced proximity domains, the multiple induced proximity domains can be different individual induced proximity domains or multiple copies of the same induced proximity domain, or a combination of the foregoing.

[0270] In some embodiments of any aspect, the induced proximal domain pair (IPD pair) is an IPD pair selected from any one or more of the following: A and IPD B (1) an IPD comprising a GID1 domain or a fragment thereof, in which the GID1 domain and the GAI domain bind to an inducer substance, gibberellin ester (GIB); A , and IPDs containing GAI domains B (2) an IPD containing an FKBP domain or a fragment thereof, in which the FKBP domain and the FRB domain bind to an inducer substance, a rapalog (RAP); A , and IPDs containing FRB domains B (3) an IPD containing a PYL domain or a fragment thereof, in which the PYL domain and the ABI domain bind to the inducer substance abscisic acid (ABA); A , and the IPD containing the ABI domain B (4) IPD containing a light-inducible dimerization domain (LIDD)A wherein the LIDD is exposed to a light inducer signal of an appropriate wavelength to form a complementary LIDD (IPD B ) and dimerizes with IPD A .

[0271] In some embodiments of any aspect, the synTF polypeptide is in combination with one, two, three, four, five, or more inducer agents that induce dimerization or proximity of the IPD. In some embodiments of any aspect, the synTF polypeptide is in combination with one inducer agent. In embodiments including multiple inducer agents, the multiple inducer agents can be different individual inducer agents or multiple copies of the same inducer agent, or combinations of the foregoing.

[0272] In some embodiments of any aspect, the IPD pair comprises an ABI (ABA-insensitive) domain and a PYL (pyrabactin resistance-like) domain derived from components of the abscisic acid (ABA) signaling pathway from Arabidopsis thaliana. In some embodiments of any aspect, the IPD pair comprises the interacting complementarity (CS) of PYL1 (PYLcs, amino acids 33-209) and ABI1 (ABIcs, amino acids 126-423). In some embodiments of any aspect, the ABI domain (e.g., SEQ ID NO:66) comprises mutations A18D and E108G. In some embodiments of any aspect, the ABI domain further comprises a detectable marker (e.g., a FLAG tag). In some embodiments of any aspect, the PYL domain further comprises a detectable marker (e.g., an HA tag).

[0273] In some embodiments of any aspect, the ABI domain comprises SEQ ID NO:66 or 107, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NO:66 or 107 and maintains the same function.

[0274] In some embodiments of any aspect, the PYL domain comprises SEQ ID NO:71 or 108, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of the sequences of SEQ ID NO:71 or 108 and maintains the same function.

[0275] SEQ ID NO:66, ABI cs CO1(298 aa) TIFF0007663513000144.tif31165.

[0276] SEQ ID NO:71, PYL1cs domain (177 aa) TIFF0007663513000145.tif17165.

[0277] SEQ ID NO:107, an alternative ABI binding motif TIFF0007663513000146.tif31165.

[0278] SEQ ID NO:108, an alternative PYL-binding motif TIFF0007663513000147.tif17165.

[0279] In some embodiments of any aspect, the proximity inducer agent (e.g., for the ABI and PYL domains) is abscisic acid (ABA): The file is TIFF0007663513000148.tif24128.

[0280] In some embodiments, the IPD pair is FKBP (FK506 and rapamycin binding protein) and FKBP12-rapamycin binding protein (FRB) proteins, which dimerize together in the presence of a rapalog. In some embodiments of any aspect, the FKBP domain comprises SEQ ID NO: 109 or 318, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of one of SEQ ID NO: 109 or 318 and maintains the same function. In some embodiments of any aspect, the FRB domain comprises SEQ ID NO:110 or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO:110 and maintains the same function.

[0281] SEQ ID NO:109, FKBP aa binding motif TIFF0007663513000149.tif11163.

[0282] SEQ ID NO:110, FRB binding motif TIFF0007663513000150.tif11164.

[0283] In some embodiments of any aspect, the proximity inducer agent (e.g., for the FKBP and FRB domains) is rapamycin, as shown below. In some embodiments of any aspect, the proximity inducer agent (e.g., for the FKBP and FRB domains) is a rapalog, i.e., a rapamycin analog. In some embodiments of any aspect, the rapalog is Shield-1, as further described herein. TIFF0007663513000151.tif50128

[0284] In other embodiments, the IPD pair is the GAI (gibberellin-insensitive) and GID1 (gibberellin-insensitive dwarf1) proteins from Arabidopsis thaliana, which combine in the presence of gibberellin esters (GE). In some embodiments of any aspect, the GAI domain comprises SEQ ID NO:111, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO:111 and maintains the same function. In some embodiments of any aspect, the GAI domain comprises the amino-terminal DELLA domain of GAI. In some embodiments of any aspect, the GID domain comprises SEQ ID NO:112 or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO:112 and maintains the same function.

[0285] SEQ ID NO:111, GAI binding motif TIFF0007663513000152.tif11163.

[0286] SEQ ID NO:112, GID1 binding motif TIFF0007663513000153.tif37166.

[0287] In some embodiments of any aspect, the proximity inducer agent (eg, for the GAI and GID1 domains) is a bioactive gibberellin (shown below), a gibberellin ester (GE), or another gibberellin analog. TIFF0007663513000154.tif28128

[0288] In some embodiments, the IPD pair comprises a caffeine-induced dimerization system, such as a VHH camelid antibody (referred to as aCaffVHH), which homodimerizes with high affinity (Kd = 500 nM) in the presence of caffeine. In some embodiments, the IPD pair is selected from a combinatorial binder-enabled selection of chemically induced dimerization systems (COMBINES-CID) using a specific chemical ligand. As a non-limiting example, the ligand can be CBD (cannabidiol). In some embodiments, the IPD pair comprises a human antibody-based chemically induced dimerizer (AbCID), which is obtained from a known small molecule-protein complex by selecting a synthetic antibody that recognizes a chemical epitope generated by a small molecule bound to a protein (e.g., ABT-737). In some embodiments, the IPD pair comprises calcineurin and FKBP, which combine in the presence of FK506. In some embodiments, the IPD pair comprises calcineurin and prolyl isomerase CyP, which combine in the presence of cyclosporin A. In some embodiments, the IPD pair comprises CyP and FKBP, which combine in the presence of FKCsA, a fusion of FK506 and cyclosprin A. In some embodiments, the IPD pair comprises two copies of FKBP, which combine in the presence of FK2012, a fusion of two FK506 molecules.For example, Franco et al., Journal of Chromatography B, Volume 878, Issue 2, 15 January 2010, Pages 177-186; Liang et al. Sci Signal 2011 Mar 15;4(164):rs2; Laura A. Banaszynski et al. JACS 2005 Apr 6;127(13):4715-21; Miyamoto et al. Nat Chem Biol Nature Chemical Biology volume 8, pages 465-470(2012); Bojar et al. Nature Communications volume 9, Article number: 2318(2018); Kang et al. JACS 2019 Jul 17; 141(28): 10948-10952; Hill et al. Nat ChemBio 2018 Feb;14(2):112-117; Stanton et al. See Science 2018 Mar 9; 359(6380): eaao5902; Weinberg et al. Nat Biotech 2017 May; 35(5): 453-462; Matthew J Kennedy, Nature Methods volume 7, pages 973-975 (2010); U.S. patent applications US20180163195 and US20170183654; U.S. patent 8735096, the contents of each of which are incorporated herein by reference in their entirety.

[0289] In some embodiments, the IPD pair comprises a light-induced dimerization domain (LIDD) pair, non-limiting examples of which include nMag / nMag, CRY2 / CIBN, and photochromic proteins. In some embodiments, the IPD pair comprises a light-induced dimerization domain (LIDD) pair, e.g., nMag and pMag proteins, that dimerize together upon a blue light signal, e.g., a blue light pulse signal, or a pulse of light of an appropriate wavelength. In some embodiments of any aspect, the nMag domain comprises SEQ ID NO:113, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO:113 and maintains the same function. In some embodiments of any aspect, the pMag domain comprises SEQ ID NO:114 or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO:114 and maintains the same function.

[0290] SEQ ID NO:113, nMag binding motif TIFF0007663513000155.tif17164.

[0291] SEQ ID NO:114, pMag binding motif TIFF0007663513000156.tif17165.

[0292] In some embodiments, the IPD pair comprises a light-induced dimerization domain (LIDD) pair from Arabidopsis thaliana, e.g., cryptochrome 2 (CRY2) and CIBN (a truncated form of CIB1 (CRY2-interacting basic helix-loop-helix 1)) and protein, which dimerize together upon a blue light signal, e.g., a blue light pulse signal or a pulse of light of an appropriate wavelength. In some embodiments of any aspect, the CIBN domain comprises SEQ ID NO: 115, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO: 115 and maintains the same function. In some embodiments of any aspect, the CRY2 domain comprises SEQ ID NO:116 or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO:116 and maintains the same function.

[0293] TIFF0007663513000157.tif98165.

[0294] In some embodiments, the IPD pair comprises a light-induced dimerization domain (LIDD) pair, such as a photochromic protein domain including, but not limited to, fluorescent Dronpa, Padron, rsTagRFP, and mApple, or variants or polypeptide fragments thereof (e.g., Dronpa-145N, Padron-145N, or mApple-162H-164A). Such photochromic protein domains dimerize in the presence of a specific wavelength (e.g., blue light). In some embodiments of any aspect, the photochromic protein domain comprises one of SEQ ID NOs:330-333, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of one of SEQ ID NOs:330-333 and maintains the same function.

[0295] SEQ ID NO: 330, Dropna-145K (224 aa) TIFF0007663513000158.tif24165.

[0296] SEQ ID NO:331, Dropuna-145N (224 aa) TIFF0007663513000159.tif24165.

[0297] SEQ ID NO:332, Padron-145N (224 aa) TIFF0007663513000160.tif24165.

[0298] SEQ ID NO:333, mApple-162H-164A(236 aa) TIFF0007663513000161.tif24165.

[0299] Cytosolic Sequestration Domain In some aspects, described herein are synTF polypeptides that include a cytosolic sequestration domain or protein, also referred to herein as a translocation domain. As used herein, the term "cytosolic sequestration domain" refers to a domain that affects the intracellular location of a synTF to which it is linked, such as through ligand binding.

[0300] In some embodiments of any aspect, a synTF polypeptide (or collectively, a synTF polypeptide system) as described herein comprises one, two, three, four, five, or more cytosolic sequestration domains. In some embodiments of any aspect, a synTF polypeptide or system comprises one cytosolic sequestration domain. In embodiments comprising multiple cytosolic sequestration domains, the multiple cytosolic sequestration domains can be different individual cytosolic sequestration domains or multiple copies of the same cytosolic sequestration domain, or a combination of the foregoing.

[0301] In some embodiments of any aspect, the cytosolic sequestration protein comprises a ligand binding domain (LBD), and in the presence of a ligand, sequestration of the protein into the cytosol is inhibited. In some embodiments of any aspect, the cytosolic sequestration protein further comprises a nuclear localization signal (NLS), and in the absence of a ligand, the NLS is inhibited, thereby preventing translocation of the sequestration protein to the nucleus, and in the presence of a ligand, the nuclear localization signal is exposed, allowing translocation of the sequestration protein to the nucleus. Thus, in the absence of a ligand, synTF is sequestered in the cytosol. In the absence of a ligand, synTF is translocated to the nucleus.

[0302] In some embodiments of any aspect, the sequestration protein comprises at least a portion of an estrogen receptor (ER). ER naturally associates with cytoplasmic factors in cells in the absence of its cognate ligand, effectively sequestrating itself in the cytoplasm. Binding of a cognate ligand, such as estrogen or other steroid hormone derivatives, causes a conformational change in the receptor that allows it to dissociate from the cytoplasmic complex, expose a nuclear localization signal, and translocate to the nucleus.

[0303] In some embodiments of any aspect, the sequestration protein comprises SEQ ID NO: 334 or an amino acid sequence that maintains the same function that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO: 334. In some embodiments of any aspect, the sequestration protein comprises a portion of ER (e.g., SEQ ID NO: 334), e.g., the C-terminal ligand binding and nuclear localization domain of ER. In some embodiments of any aspect, the sequestered protein comprises residues 282-595 of SEQ ID NO:334, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to residues 282-595 of SEQ ID NO:334.

[0304] SEQ ID NO: 334, Estrogen receptor isoform 1 [Homo sapiens]; NCBI reference sequence: NP_000116.2 (595 aa) TIFF0007663513000162.tif64165.

[0305] In some embodiments of any aspect, the estrogen receptor comprises at least one mutation that reduces its ability to bind to its natural ligand (e.g., estradiol) but maintains its ability to bind to synthetic ligands such as tamoxifen and its analogs. In some embodiments of any aspect, the estrogen receptor comprises at least one of the following mutations: G400V, G521R, L539A, L540A, M543A, L544A, V595A, or any combination thereof. In some embodiments of any aspect, the triple G400V / MS43A / L544A ER mutant is referred to herein as ERT2. In some embodiments of any aspect, the sequestering protein further comprises a V595A mutation from ER (e.g., SEQ ID NO: 334). In some embodiments of any aspect, the sequestering protein comprises an estrogen ligand binding domain (ERT2) or a variant thereof. In some embodiments of any aspect, the sequestered protein comprises ERT, ERT2, ERT3, or a variant thereof. In some embodiments of any aspect, the sequestered protein comprises one of SEQ ID NOs:74, 335-337, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of one of SEQ ID NOs:74, 335-337 and maintains the same function. See, e.g., U.S. Patent 7,112,715; Feil et al., Biochemical and Biophysical Research Communications, Volume 237, Issue 3, 28 August 1997, Pages 752-757; Felker et al., PLoS One. 2016 Apr 14;11(4):e0152989, the contents of each of which are incorporated herein by reference in their entirety.

[0306] SEQ ID NO:74, ERT2 (314 aa), G400V, M543A, L544A, and V595A mutations from ER (e.g., SEQ ID NO:334) indicated in bold, double-underlined text TIFF0007663513000163.tif31165.

[0307] SEQ ID NO:335, ERT (314 aa), G521R mutation from ER (e.g., SEQ ID NO:334) indicated in bold, double-underlined text TIFF0007663513000164.tif31164.

[0308] SEQ ID NO:336, ERT3 (314 aa), M543A, L544A, and V595A mutations from ER (e.g., SEQ ID NO:334) indicated in bold, double-underlined text TIFF0007663513000165.tif31165.

[0309] SEQ ID NO:337, ERT (314 aa), G400V, L539A, L540A mutations from ER (e.g., SEQ ID NO:334) indicated in bold, double-underlined text TIFF0007663513000166.tif31164.

[0310] In some embodiments of any aspect, the sequestering protein of the synTF polypeptide is in combination with one, two, three, four, five, or more ligands. In some embodiments of any aspect, the sequestering protein of the synTF polypeptide is in combination with one ligand. In embodiments including multiple ligands, the multiple ligands can be different individual ligands or multiple copies of the same ligand, or combinations of the foregoing.

[0311] In some embodiments of any aspect, the ligand is estradiol (PubChem CID: 5757) or an analog thereof. In some embodiments of any aspect, the ligand is a synthetic ligand of the estrogen receptor, such as tamoxifen or a derivative thereof. The ligand is selected from tamoxifen, 4-hydroxytamoxifen (4OHT), endoxifen, and fulvestrant, and binding of the ligand to ERT (e.g., ERT2) exposes the NLS and leads to nuclear translocation of ERT. In some embodiments of any aspect, the ligand is 4-hydroxytamoxifen (4-OHT) (PubChem CID: 449459), which may also be referred to as afimoxifen, as shown below. In some embodiments of any aspect, the ligand is 4-hydroxy-N-desmethyltamoxifen (PubChem CID: 10090750), which may also be referred to as endoxifen, as shown below. In some embodiments of any aspect, the ligand is fulvestrant (PubChem CID 104741), shown below, which may also be referred to as ICI 182,780.

[0312] TIFF0007663513000167.tif82154

[0313] In some embodiments of any aspect, the sequestration protein of the synTF is a transmembrane receptor sequestration protein, and the DNA binding domain (DBD) and transcriptional effector (TE) domain of the synTF are linked to the cytosolic side of the transmembrane domain of the receptor. In the absence of a specific ligand for the transmembrane protein, the DBD and TA of the synTF are sequestered in the cell membrane. In the presence of a specific ligand for the transmembrane protein, the transmembrane protein cleaves itself, releasing the DBD and TA of the synTF into the cytosol for transport to the nucleus. Non-limiting examples of transmembrane receptor sequestration proteins include the synthetic Notch receptor or the first and second exogenous extracellular sensors further described herein.

[0314] In some embodiments of any aspect, the cytosolic sequestration protein comprises a Notch receptor or a variant of an endogenous Notch receptor, such as a synthetic Notch (synNotch) receptor. In some embodiments of any aspect, the synTF comprising synNotch comprises: (a) an extracellular domain comprising a first member of a specific binding pair heterologous to the Notch receptor; (b) a Notch receptor regulatory region; and (c) an intracellular domain comprising the DNA binding domain and transcription effector domain of synTF. In the presence of a second member of the specific binding pair, the binding of the first member of the specific binding pair to the second member of the specific binding pair induces cleavage of the binding-induced proteolytic cleavage site, activating the intracellular domain, thereby allowing synTF to translocate to the nucleus. In the absence of the second member of the specific binding pair, synTF remains sequestered in the cell membrane. In some embodiments of any aspect, the sequestered protein comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOS: 338-339 or one of SEQ ID NOS: 74, 335-337, and maintains the same function. See, e.g., U.S. Patent 10,590,182; Morsut et al., Cell. 2016 Feb 11;164(4):780-91, the contents of which are incorporated herein by reference in their entirety. In some embodiments of any aspect, the Notch receptor regulatory region comprises Lin-12 Notch repeat AC, heterodimerization domains HD-N and HD-C, a binding-induced proteolytic cleavage site, and a transmembrane domain. In some embodiments of any aspect, the Notch variant is a Notch receptor in which the Notch extracellular subunit (NEC), which includes the negative regulatory region (NRR), has been partially or completely deleted.In some embodiments of any aspect, the Notch receptor regulatory region is a truncated or modified variant of synNotch, e.g., lacking one or more of the following domains: the Lin-12 Notch repeat AC, the heterodimerization domains HD-N and HD-C, the binding-induced proteolytic cleavage site, the Notch extracellular domain (NEC), the negative regulatory region (NRR), or the transmembrane domain.

[0315] TIFF0007663513000168.tif84165.

[0316] Suitable first members of a specific binding pair (e.g., for synNotch) include, but are not limited to, antibody-based recognition scaffolds; antibodies (i.e., antibody-based recognition scaffolds, including antigen-binding antibody fragments); non-antibody-based recognition scaffolds; antigens (e.g., endogenous antigens; exogenous antigens; etc.); ligands for receptors; receptors; targets of non-antibody-based recognition scaffolds; Fc receptors (e.g., FcγRIIIa; FcγRIIIb; etc.); extracellular matrix components; and the like.

[0317] Specific binding pairs (e.g., of synNotch) include, for example, antigen-antibody specific binding pairs, where a first member is an antibody (or antibody-based recognition scaffold) that specifically binds to a second member that is an antigen, or where the first member is an antigen and the second member is an antibody (or antibody-based recognition scaffold) that specifically binds to the antigen; ligand-receptor specific binding pairs, where a first member is a ligand and the second member is a receptor to which the ligand binds, or where a first member is a receptor and the second member is a ligand that binds to the receptor; and non-antibody-based recognition scaffold-target specific binding pairs, where a first member is a non-antibody-based recognition scaffold. and the second member is a target that binds to a non-antibody-based recognition scaffold, or the first member is a target and the second member is a non-antibody-based recognition scaffold that binds to the target; adhesion molecule-extracellular matrix binding pairs; Fc receptor-Fc binding pairs, where the first member comprises an immunoglobulin Fc that binds to a second member that is an Fc receptor, or the first member is an Fc receptor that binds to a second member that comprises an immunoglobulin Fc; and receptor-co-receptor binding pairs, where the first member is a receptor that specifically binds to a second member that is a co-receptor, or the first member is a co-receptor that specifically binds to a second member that is a receptor.

[0318] In some embodiments of any aspect, the transmembrane receptor sequestering protein comprises a first and a second exogenous extracellular sensor, wherein the first exogenous extracellular sensor comprises (a) a ligand-binding domain, (b) a transmembrane domain, (c) a protease cleavage site, and (d) a DBD and a TA of a synTF; and the second exogenous extracellular sensor comprises (e) a ligand-binding domain, (f) a transmembrane domain, and (g) a protease domain. Such a system may also be referred to as a modular extracellular sensor architecture (MESA) system. In the presence of a ligand for the first and second exogenous extracellular sensors, the two receptors come into close proximity, allowing the protease to cleave the protease cleavage site and release the DBD and TA of the synTF into the cytosol for transport to the nucleus. In the absence of a ligand for the first and second exogenous extracellular sensors, the DBD and TA of the synTF remain sequestered in the plasma membrane. In some embodiments of any aspect, the protease includes any protease (e.g., NS3) as described herein, and the protease cleavage site includes an NS3 protease cleavage site as described herein. See, e.g., U.S. Patent Application No. 2014 / 0234851; Daringer et al., ACS Synth. Biol. 2014, 3, 12, 892-902.

[0319] Any type of suitable ligand-binding domain (LB) can be utilized with the transmembrane receptor sequestering protein. The ligand-binding domain can be derived, for example, from either an existing receptor ligand-binding domain or an engineered ligand-binding domain. Existing ligand-binding domains can also be derived, for example, from cytokine receptors, chemokine receptors, innate immune receptors (such as TLRs), olfactory receptors, steroid and hormone receptors, growth factor receptors, mutant receptors occurring in cancer, and neurotransmitter receptors. Engineered ligand-binding domains can be, for example, single-chain antibodies (see scFv constructs, described below), engineered fibronectin-based binding proteins, and engineered consensus-derived binding proteins (e.g., DARPins based on leucine-rich repeats or ankyrin-rich repeats). The ligand can be any cognate ligand of such a ligand-binding domain.

[0320] Linker peptide In some aspects, synTF polypeptides are described herein that include at least one linker peptide. As used herein, a "linker peptide" (used interchangeably with "peptide linker") refers to an oligo- or polypeptide region of about 2 to 100 amino acids in length that links together any of the polypeptide sequences described herein. In some embodiments, the linker can comprise or consist of flexible residues such as glycine and serine, allowing adjacent protein domains to move freely relative to one another. Longer linkers can be used when it is desired to prevent two adjacent domains from sterically interfering with one another. The linker can be cleavable or non-cleavable.

[0321] In some embodiments of any aspect, a synTF polypeptide (or collectively, a synTF polypeptide system) as described herein comprises one, two, three, four, five, or more linker peptides. In some embodiments of any aspect, a synTF polypeptide or system comprises one linker peptide. In embodiments comprising multiple linker peptides, the multiple linker peptides can be different individual linker peptides or multiple copies of the same linker peptide, or a combination of the foregoing. In some embodiments of any aspect, a linker peptide can be located anywhere between any two domains as described herein: for example, between the DBD and the regulatory protein, between the regulatory protein and the effector domain, between the DBD and the effector domain, or any combination thereof. In some embodiments of any aspect, a linker peptide can be positioned within the DBD, the effector domain, or the regulatory protein, for example, to link components of the domains.

[0322] In some embodiments of any one of the aspects described herein, a linker connects several ZFs to each other in tandem to form a ZF array. In some embodiments of any one of the aspects described herein, a linker connects a first ZFA to a second ZFA. In some embodiments of any one of the aspects described herein, a linker connects several ZFAs to each other in tandem to form a ZF-containing ZF protein domain. Non-limiting examples of peptide linker molecules useful in the polypeptides described herein include glycine-rich peptide linkers (see, e.g., US 5,908,626) in which more than half of the amino acid residues are glycine. Preferably, such glycine-rich peptide linkers consist of about 20 or fewer amino acids. Linker molecules can also include non-peptide or partial peptide molecules. For example, peptides can be linked to peptides or other molecules using well-known cross-linking molecules such as glutaraldehyde or EDC (Pierce, Rockford, Illinois). In some embodiments of the engineered synTFs described herein, the ZF arrays (ZFAs) in the ZF protein domain of the synTF are connected together by linker peptides in their respective fusion proteins.

[0323] Non-limiting examples of linker peptides include: Including, but not limited to, TIFF0007663513000169.tif77165.

[0324] for example, TIFF0007663513000170.tif4157 was used as a linker between ZFAs; TIFF0007663513000171.tif24164 is used to link the ZF domain and effector domain together; TIFF0007663513000172.tif11156 is used to link regions of the SMASh domain, StaPL domain, or NS3 / NS4a domain further described herein.

[0325] Flexible linkers are generally composed of small non-polar or polar residues such as Gly, Ser, and Thr. In one embodiment of any fusion protein described herein that includes a linker, the linker peptide includes at least one amino acid that is Gly or Ser. In one embodiment of the fusion protein described herein that includes a linker, the linker is a flexible polypeptide that is 1-25 residues long. A common example of a flexible peptide linker is (GGS)n, where n=1-8. TIFF0007663513000173.tif4128, or (Gly4Ser)n repeats (where n = 1 to 8) TIFF0007663513000174.tif4139, preferably n=3, 4, 5, or 6), i.e., (Gly-Gly-Gly-Gly-Ser)n (SEQ ID NO:353), GGGGS (where n indicates the number of repeats of the motif). For example, the flexible linker is (GGS)2 (SEQ ID NO:354, GGSGGS). Alternatively, the flexible peptide linker may be a Gly-Ser repeat (Gly-Ser) p (where p indicates the number of Gly-Ser repeats in the motif, p = 1 to 8) TIFF0007663513000175.tif4128, preferably where n=3, 4, 5, or 6. Another example of a flexible linker is TGSQK (SEQ ID NO:341).

[0326] In one embodiment of the engineered synTFs described herein, in which the ZF protein domain and the effector domain are connected together by a linker peptide, the linker peptide is about 1-20 amino acids in length. In one embodiment, the linker peptide does not contain, does not contain, or does not contain both Lys and Arg.

[0327] In some embodiments of the engineered synTFs described herein, the ZF protein domain and the effector domain are connected together with a chemical crosslinker. A bifunctional linker molecule is a linker molecule that possesses two separate reactive sites. For example, one of the reactive sites of the bifunctional linker molecule can react with a functional group on a peptide to form a covalent linkage, and the other reactive site can react with a functional group on another molecule to form a covalent linkage. General methods for crosslinking molecules have been reviewed (see, e.g., Means and Feeney, Bioconjugate Chem., 1: 2-12 (1990)).

[0328] Homobifunctional cross-linking molecules have two reactive sites that are chemically identical. Non-limiting examples of homobifunctional cross-linking molecules include, but are not limited to, glutaraldehyde; N,N'-bis(3-maleimido-propionyl-2-hydroxy-1,3-propanediol (a sulfhydryl-specific homobifunctional cross-linker); certain N-succinimide esters (e.g., disuccinimidyl suberate, dithiobis(succinimidyl propionate)), and soluble bis-sulfonic acids and their salts (see, e.g., Pierce Chemicals, Rockford, Illinois; Sigma-Aldrich Corp., St. Louis, Missouri).

[0329] The bifunctional linker molecule is a heterobifunctional linker molecule, meaning that the linker has at least two different reactive sites, each of which can be separately linked to a peptide or other molecule. The use of such a heterobifunctional linker allows for the chemically separate and stepwise addition (vectorial conjunction) of each of the reactive sites to a selected peptide sequence. Heterobifunctional linker molecules useful in the present disclosure include, but are not limited to, m-maleimidobenzoyl-N-hydroxysuccinimide ester (see Green et al., Cell, 28: 477-487 (1982); Parker et al., Proc. Natl. Acad. Sci (USA), 84: 2479-2483 (1987)); m-maleimido-benzoyl sulfosuccinimide ester; maleimidobutyric acid N-hydroxysuccinimide ester; and N-succinimidyl 3-(2-pyridyl-dithio)propionate (see, e.g., Carlos et al., Biochem. J., 173: 723-737 (1978); Sigma-Aldrich Corp., St. Louis, Missouri).

[0330] In some embodiments of any aspect described herein, in a synTF or ZF-containing fusion protein described herein, all helices within a ZFA are connected by a peptide linker (L2) having 4 to 6 amino acid residues.

[0331] In some embodiments of any of the aspects described herein, in a synTF or a ZF-containing fusion protein described herein, all of the helices within an individual ZFA are The ZF motifs are linked by a rigid peptide linker, such as TIFF0007663513000176.tif11151. The rigid linker helps confer synergistic binding of the ZF motif to its target DNA sequence.

[0332] In one embodiment of any aspect described herein, in the synTF or ZF-containing fusion protein described herein, (L1) or (L2) is a flexible linker. Non-limiting examples include: TIFF0007663513000177.tif4128. In one embodiment, the linker flexible peptide is 1-20 amino acids in length. The flexible linker assists in weakening the cooperativity between adjacent ZF motifs.

[0333] In one embodiment of any aspect described herein, in the synTF or ZF-containing fusion protein described herein, (L1) or (L2) is a rigid linker. Non-limiting examples include: Includes TIFF0007663513000178.tif11144.

[0334] In some embodiments of any aspect described herein, in a synTF or ZF-containing fusion protein described herein having two or more ZFAs, the individual ZFAs are linked by a flexible peptide linker, such as TGSQKP (SEQ ID NO:359). In another embodiment, the ZFAs are linked by a chemical crosslinker. Chemical crosslinkers are known in the art.

[0335] In some embodiments of any aspect described herein, in a synTF or ZF-containing fusion protein described herein, all helices within an individual ZFA are linked by a combination of rigid and flexible peptide linkers. In some embodiments of any aspect, rigid and flexible peptide linkers are alternately used to connect referents.

[0336] Self-cleaving peptides In some aspects, synTF polypeptides are described herein that include a self-cleaving peptide. As used herein, the term "self-cleaving peptide" refers to a short amino acid sequence (e.g., a peptide approximately 18-22 aa long) that can catalyze its own cleavage. In some embodiments of any aspect, a multi-component synTF system (e.g., an inducible proximity synTF system) as described herein includes at least two polypeptides that are physically linked to each other through a self-cleaving peptide domain. The self-cleaving peptide allows the nucleic acids of a first polypeptide and a second polypeptide (and / or a third polypeptide, etc.) to exist in the same vector, but after translation, the self-cleaving peptide cleaves the translated polypeptide into multiple separate polypeptides.

[0337] In some embodiments of any aspect, a synTF polypeptide (or collectively, a synTF polypeptide system) as described herein comprises one, two, three, four, five, or more self-cleaving peptides, e.g., between each synTF polypeptide. In some embodiments of any aspect, a synTF polypeptide or system comprises one self-cleaving peptide, e.g., between a first polypeptide and a second polypeptide of a synTF polypeptide system. In embodiments comprising multiple self-cleaving peptides, the multiple self-cleaving peptides can be different individual self-cleaving peptides or multiple copies of the same self-cleaving peptide, or a combination of the foregoing.

[0338] In some embodiments, a self-cleaving peptide is used, for example, in a heterodimerization domain synTF. As a non-limiting example, Figures 19 and 20 show a 2A self-cleaving peptide between a first polypeptide region comprising [ABI]-[ZF] and a second polypeptide region comprising [ED]-[PYL]. After translation of the polypeptide, the self-cleaving peptide 2A sequence cleaves the polypeptide into two polypeptides: [ABI]-[ZF] and [ED]-[PYL], which can form an [ED]-[PYL]·ABA·[ABI]-[ZF] complex in the presence of ABA, thus coupling the DBD (ZF) and ED (e.g., p65 or KRAB).

[0339] In some embodiments of any aspect, the self-cleaving peptide belongs to the 2A peptide family, which may also be referred to as a 2A ribosomal skipping sequence. Non-limiting examples of 2A peptides include P2A, E2A, F2A, and T2A (see, e.g., Table 18). F2A is derived from foot-and-mouth disease virus 18; E2A is derived from equine rhinitis A virus; P2A is derived from porcine teschovirus-1 2A; and T2A is derived from thosea asigna virus 2A. In some embodiments of any aspect, the N-terminus of the 2A peptide comprises the sequence "GSG" (Gly-Ser-Gly). In some embodiments of any aspect, the N-terminus of the 2A peptide does not comprise the sequence "GSG" (Gly-Ser-Gly).

[0340] Table 18. Exemplary self-cleaving peptides TIFF0007663513000179.tif27140

[0341] 2A peptide-mediated cleavage begins after protein translation. Cleavage is triggered by cleavage of the peptide bond between proline (P) and glycine (G) at the C-terminus of the 2A peptide. The molecular mechanism of 2A peptide-mediated cleavage involves ribosomal "skipping" of glycyl-prolyl peptide bond formation rather than true proteolytic cleavage. Different 2A peptides have different self-cleavage efficiencies, with P2A being the most efficient and F2A being the least efficient. Therefore, up to 50% of the F2A-linked protein can remain in the cell as a fusion protein.

[0342] In some embodiments of any aspect, the self-cleaving peptide of the synTF polypeptide system as described herein comprises an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:68, 360-362, or a sequence of one of SEQ ID NO:68, 360-362 that maintains the same function (e.g., self-cleavage).

[0343] In some embodiments of any aspect, it is advantageous to provide multiple polypeptides of a synTF system as described herein in a 1:1 (or 1:1:1, etc.) stoichiometric ratio (e.g., this stoichiometric ratio results in optimal functionality). In embodiments where a 1:1 (or 1:1:1, etc.) ratio of the first and second (and third, etc.) polypeptides of a synTF system is advantageous, the first and second polypeptides can be provided in a single vector, adjacent to a self-cleaving peptide as described herein. In embodiments where a 1:1 (or 1:1:1, etc.) ratio of the first and second (and third, etc.) polypeptides of a synTF system is not advantageous (e.g., this stoichiometric ratio results in suboptimal functionality and other ratios result in optimal functionality), the first and second polypeptides can be provided in multiple separate vectors, e.g., in the desired stoichiometric ratio.

[0344] Detectable Markers In some aspects, synTF polypeptides are described herein that include at least one detectable marker. As used herein, the term "detectable marker" refers to a moiety that, when attached to a synTF polypeptide, confers detectability to the polypeptide or another molecule to which the polypeptide binds. In some embodiments of any aspect, a synTF polypeptide (or collectively, a synTF polypeptide system) includes one, two, three, four, five, or more detectable markers. In some embodiments of any aspect, a synTF polypeptide or system includes one detectable marker. In embodiments that include multiple detectable markers, the multiple detectable markers can be different individual detectable markers or multiple copies of the same detectable marker, or a combination of the foregoing.

[0345] In some embodiments of any aspect, fluorescent moieties can be used as detectable markers, but detectable markers also include, for example, isotopes, fluorescent proteins and peptides, enzymes, components of specific binding pairs, chromophores, affinity tags as defined herein, antibodies, colloidal metals (i.e., gold), and quantum dots. Detectable markers can be either directly or indirectly detectable. Directly detectable markers do not require additional reagents or substrates to generate a detectable signal. Examples include isotopes and fluorophores. Indirectly detectable markers require the presence or action of one or more cofactors or substrates. Examples include enzymes such as β-galactosidase, which is detectable by the production of a colored reaction product upon cleavage of a substrate such as X-gal (5-bromo-4-chloro-3-indoyl-β-D-galactopyranoside) chromogen, horseradish peroxidase, which is detectable by the production of a colored reaction product in the presence of a diaminobenzidine substrate, and alkaline phosphatase, which is detectable by the production of a colored reaction product in the presence of nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate, as well as affinity tags. Non-limiting examples of affinity tags include Strep-tag, chitin-binding protein (CBP), maltose-binding protein (MBP), glutathione-S-transferase (GST), FLAG-tag, HA-tag, Myc-tag, poly(His)-tag, and derivatives thereof. In some embodiments of any aspect, the detectable marker is selected from GFP, V5, HA1, Myc, VSV-G, HSV, FLAG, HIS, mCherry, AU1, and biotin.

[0346] SEQ ID NO:65, NLS, PKKKRKV.

[0347] SEQ ID NO:77, 3X FLAG tag + nuclear localization sequence (bold text) TIFF0007663513000180.tif3128.

[0348] SEQ ID NO:80, AU1 tag, DTYRYI.

[0349] SEQ ID NO:84, HA tag, YPYDVPDYA.

[0350] SEQ ID NO:89, FLAG tag, DYKDDDDK.

[0351] SEQ ID NO:372, mCherry TIFF0007663513000181.tif24165.

[0352] SEQ ID NO:376, huEGFRt (truncated human EGFR polypeptide) TIFF0007663513000182.tif37165.

[0353] In some embodiments of any aspect, a detectable marker for a synTF polypeptide as described herein comprises SEQ ID NO:65, 77, 80, 80, 89, 372, 376, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of one of SEQ ID NO:65, 77, 80, 80, 89, 372, or 376, while maintaining identity (e.g., detection of a synTF polypeptide or a truncated fragment of a synTF polypeptide).

[0354] In some embodiments of any aspect, the detectable marker can be located anywhere within the synTF polypeptide as described herein. In one embodiment, the detectable marker is located between any domain of the synTF polypeptide as described herein, but is not found within a functional domain or disrupts the function of the domain. In some embodiments of any aspect, the detectable marker is located adjacent to and C-terminal to the extracellular binding domain. Such markers can be used to detect the expression of the synTF polypeptide, including cytosolic expression or nuclear translocation.

[0355] In some embodiments of any aspect, a detectable marker is located between the inhibitory protease and the protease cleavage site; such a marker can be used to detect cleavage and / or expression of the synTF polypeptide. In some embodiments of any aspect, the detectable marker located between the inhibitory protease and the protease cleavage site comprises an AU1 tag, an HA1 tag, or any other marker as described herein.

[0356] In some embodiments of any aspect, the detectable marker is located adjacent to and N-terminal to the inhibitory protease. In some embodiments of any aspect, the detectable marker is located adjacent to and N-terminal to the inhibitory protease and C-terminal to the first protease cleavage site. In some embodiments of any aspect, the detectable marker is located adjacent to and C-terminal to the inhibitory protease. In some embodiments of any aspect, the detectable marker is located adjacent to and C-terminal to the inhibitory protease and N-terminal to the second protease cleavage site.

[0357] In some embodiments of any aspect, the detectable marker is located at the C-terminus of the polypeptide.Such marker can be used to detect the intracellular expression of synTF polypeptide.In some embodiments of any aspect, the detectable marker located at the C-terminus of the polypeptide comprises mCherry or another marker as described herein.

[0358] In some embodiments of any aspect, a synTF polypeptide as described herein, particularly one administered to a subject or part of a pharmaceutical composition, does not include a detectable marker that is immunogenic. In some embodiments of any aspect, a synTF polypeptide as described herein does not include GFP, mCherry, HA1, or any other immunogenic marker.

[0359] II. Inducible synthetic transcription factors In several aspects, described herein are synthetic transcription factors comprising (a) at least one DNA-binding domain (DBD), (b) a transcriptional effector domain (ED), and (c) at least one regulatory protein (RP). In some embodiments of any aspect, the ED is directly coupled or linked to the DBD. In some embodiments of any aspect, the ED is indirectly coupled or linked to the DBD. In some embodiments of any aspect, the coupling of the ED to the DBD is regulated by at least one RP. In some embodiments of any aspect, the cellular localization of the ED is regulated by at least one regulatory protein. In some embodiments of any aspect, the at least one regulatory protein is selected from the group consisting of an inhibitory protease, an inducible degradation domain, an inducible proximity domain, and a cytosolic sequestration domain.

[0360] In some embodiments of any aspect, the domains of a synTF can be, for example, in the following order from N-terminus to C-terminus: DBD-ED-RP; DBD-RP-ED; ED-DBD-RP; ED-RP-DBD; RP-DBD-ED; or RP-ED-DBD. In embodiments including two regulatory proteins (i.e., RP1 and RP2), the domains of a synTF can be, for example, in the following order from N-terminus to C-terminus: DBD-ED-RP1-RP2; ED-DBD-RP1-RP2; RP1-DBD-ED-RP2; DBD-RP1-ED-RP2; ED-RP1-DBD-RP2; RP1-ED-DBD-RP2; RP1-ED-RP2-DBD; ED-RP1-RP2-DBD; RP2-RP1-ED-DBD; RP1-RP2-E D-DBD;ED-RP2-RP1-DBD;RP2-ED-RP1-DBD;RP2-DBD-RP1-ED;DBD-RP2-RP1-ED;RP1-RP2-DBD-ED;RP2-RP1-DBD-ED;DBD-RP1-RP2-E D; RP1-DBD-RP2-ED; ED-DBD-RP2-RP1; DBD-ED-RP2-RP1; RP2-ED-DBD-RP1; ED-RP2-DBD-RP1; DBD-RP2-ED-RP1;

[0361] In some embodiments, and by way of example only, as disclosed herein, an exemplary RP1 is selected from an inducible proximity domain (IPD) or a cytosolic sequestration domain (CS), and RP2 is selected from an inducible degradation domain (comprising a SMASh domain). In another embodiment, an exemplary RP1 is an inducible degradation domain (comprising a SMASh domain), and RP2 is selected from an inducible proximity domain (IPD) or a cytosolic sequestration domain (IPD).

[0362] In aspects, exemplary synTF polypeptides are described herein. In some embodiments of any aspect, the synTF polypeptide comprises one of SEQ ID NOs: 4-15, 40-51, or 378-379, or a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of one of SEQ ID NOs: 4-15, 40-51, or 378-379.

[0363] A. Inhibitory protease SynTF In some embodiments of any aspect, the regulatory protein is an inhibitory protease.Thus, in one aspect, the present invention provides a synTF, which comprises (a) DBD; (b) ED; and (c) an inhibitory protease domain (referred to herein as PRO or RPD).In some embodiments of any aspect, the domains of synTF can be, for example, in the following order from N-terminus to C-terminus: DBD-ED-PRO; DBD-PRO-ED; ED-DBD-PRO; ED-PRO-DBD; PRO-DBD-ED; or PRO-ED-DBD.

[0364] In some embodiments of any aspect, the inhibitory protease synTF further comprises at least one protease cleavage site (PC), as further described herein. In preferred embodiments, the at least one protease cleavage site is located between the DBD and the ED, such that when the protease cleaves at the protease cleavage site, the DBD and ED are not coupled. Thus, in some embodiments, the inhibitory protease synTF comprises, from N-terminus to C-terminus: PRO-ED-PC-DBD; ED-PRO-PC-DBD; ED-PC-PRO-DBD; DBD-PC-PRO-ED; DBD-PRO-PC-ED; PRO-DBD-PC-ED; ED-PC-DBD-PRO; and DBD-PC-ED-PRO.

[0365] In some embodiments of any aspect, the inhibitory protease synTF comprises two protease cleavage sites (PC). In some embodiments of any aspect, the two protease cleavage sites are located directly N- and C-terminal to the inhibitory protease domain, e.g., from N- to C-terminus: PC1-PRO-PC2. In some embodiments of any aspect, the inhibitory protease synTF comprises, from N- to C-terminus: DBD-PC1-PRO-PC2-ED, or ED-PC1-PRO-PC2-DBD.

[0366] In some embodiments of any aspect, the inhibitory protease synTF further comprises a cofactor of the inhibitory protease (CO), as further described herein. In some embodiments of any aspect, the cofactor of the inhibitory protease is directly linked to the inhibitory protease, e.g., from N-terminus to C-terminus: CO-PRO or PRO-CO. In some embodiments of any aspect, the inhibitory protease synTF comprises, from N-terminus to C-terminus: DBD-ED-PRO-CO; DBD-PRO-CO-ED; ED-DBD-PRO-CO; ED-PRO-CO-DBD; PRO-CO-DBD-ED; PRO-CO-ED-DBD; DBD-ED-CO-PRO; DBD-CO-PRO-ED; ED-DBD-CO-PRO; ED-CO-PRO-DBD; CO-PRO-DBD-ED; CO-PRO-ED-DBD; DBD-PC1-PRO-CO-PC2-ED; ED-PC1-PRO-CO-PC2-DBD; DBD-PC1-CO-PRO-PC2-ED; or ED-PC1-CO-PRO-PC2-DBD.

[0367] In some embodiments of any aspect, the DBD of the inhibitory protease synTF comprises ZF10-1. In some embodiments of any aspect, the DBD of the inhibitory protease synTF comprises SEQ ID NO:3 or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:3 and maintains the same function.

[0368] In some embodiments of any aspect, the effector domain (ED) comprises a transcriptional activator. In one aspect, described herein is an inhibitory protease synTF comprising, from N-terminus to C-terminus, (a) a DBD, (b) an inhibitory protease, and (c) a transcriptional activator domain. In some embodiments of any aspect, the inhibitory protease synTF comprises SEQ ID NO:8 or 44, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of the sequences of SEQ ID NO:8 or 44 and maintains the same function.

[0369] In some embodiments of any aspect, the inhibitory protease synTF is encoded by a vector or polynucleotide that comprises SEQ ID NO:20 or 32, or a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NO:20 or 32, maintaining the same function of the encoded polypeptide, or that comprises a codon-optimized version of SEQ ID NO:20 or 32.

[0370] In some embodiments of any aspect, the effector domain (ED) comprises a transcriptional repressor. In one aspect, described herein is an inhibitory protease synTF comprising, from N-terminus to C-terminus, (a) a transcriptional repressor domain, (b) an inhibitory protease; and (c) a DBD. In some embodiments of any aspect, the inhibitory protease synTF comprises SEQ ID NO:9 or 45, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of the sequences of SEQ ID NO:9 or 45 and maintains the same function.

[0371] In some embodiments of any aspect, the inhibitory protease synTF is encoded by a vector or polynucleotide that comprises SEQ ID NO:21 or 33, or a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of one of SEQ ID NO:21 or 33, maintaining the same function of the encoded polypeptide, or that comprises a codon-optimized version of SEQ ID NO:21 or 33.

[0372] B. Inducible Degradation Domain SynTF In some embodiments of any aspect, the regulatory protein is an inducible degradation domain.Thus, in one aspect, the present invention describes a synTF, which comprises (a) DBD; (b) ED; and (c) an inducible degradation domain (SMASh).As described herein, the SMASh domain can be a C-terminal SMASh domain or an N-terminal SMASh domain.In some embodiments of any aspect, the domains of synTF can be, for example, in the following order from N-terminus to C-terminus: DBD-ED-SMASh; ED-DBD-SMASh; SMASh-DBD-ED; or SMASh-ED-DBD.

[0373] In some embodiments of any aspect, the DBD of the SMASh synTF comprises ZF10- 1. In some embodiments of any aspect, the DBD of the SMASh synTF comprises SEQ ID NO:3, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:3 and maintains the same function.

[0374] In some embodiments of any aspect, the effector domain (ED) comprises a transcriptional activator. In one aspect, described herein is a SMASh synTF comprising, from N-terminus to C-terminus, (a) a DBD, (b) a transcriptional activator domain, and (c) an inducible degradation (e.g., SMASh) domain. In some embodiments of any aspect, the SMASh synTF comprises SEQ ID NO: 12 or 48, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of the sequences of SEQ ID NO: 12 or 48 and maintains the same function.

[0375] In some embodiments of any aspect, the SMASh synTF is encoded by a vector or polynucleotide that comprises SEQ ID NO:24 or 36, or a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NO:24 or 36, maintaining the same function of the encoded polypeptide, or a codon-optimized version of SEQ ID NO:24 or 36.

[0376] In some aspects, the inducible degradation domain synTF further comprises a second regulatory protein, e.g., a cytosolic sequestration domain (CS). Thus, in one aspect, described herein is a synTF comprising: (a) a DBD; (b) an ED; (c) an inducible degradation domain (SMASh); and (d) a cytosolic sequestration domain (CS). In some embodiments of any aspect, the synTF comprises, from N-terminus to C-terminus: DBD-ED-SMASh-CS; ED-DBD-SMASh-CS; SMASh-DBD-ED-CS; DBD-SMASh-ED-CS; ED-SMASh-DBD-CS; SMASh-ED-DBD-CS; SMASh-ED-CS-DBD; ED-SMASh-CS-DBD; CS-SMASh-ED-DBD; SMASh-CS-ED-DBD; ED-CS-SMASh-DBD. D;CS-ED-SMASh-DBD;CS-DBD-SMASh-ED;DBD-CS-SMASh-ED;SMASh-CS-DBD-ED;CS-SMASh-DBD-ED;DBD-SMASh-CS-ED;SMASh-DB D-CS-ED;ED-DBD-CS-SMASh;DBD-ED-CS-SMASh;CS-ED-DBD-SMASh;ED-CS-DBD-SMASh;DBD-CS-ED-SMASh;and CS-DBD-ED-SMASh. In preferred embodiments, the SMASh domain is at the C-terminus or N-terminus: for example, SMASh-DBD-ED-CS; SMASh-ED-DBD-CS; SMASh-ED-CS-DBD; SMASh-CS-ED-DBD; SMASh-CS-DBD-ED; SMASh-DBD-CS-ED; ED-DBD-CS-SMASh; DBD-ED-CS-SMASh; CS-ED-DBD-SMASh; ED-CS-DBD-SMASh; DBD-CS-ED-SMASh; and CS-DBD-ED-SMASh.

[0377] In some embodiments of any aspect, the DBD of the SMASh / cytosol-sequestering synTF comprises ZF3-5. In some embodiments of any aspect, the DBD of the SMASh / cytosol-sequestering synTF comprises SEQ ID NO:2 or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:2 and maintains the same function.

[0378] In some embodiments of any aspect, the effector domain (ED) comprises a transcriptional activator. In one aspect, described herein is a SMASh / cytosol-sequestering synTF comprising, from N-terminus to C-terminus, (a) a DBD, (b) a transcriptional activator domain, (c) a cytosol-sequestering domain, and (d) an inducible degradation (e.g., SMASh) domain. In another aspect, described herein is a SMASh / cytosol-sequestering synTF comprising, from N-terminus to C-terminus, (a) an inducible degradation (e.g., SMASh) domain, (b) a DBD, (c) a transcriptional activator domain, and (d) a cytosol-sequestering domain. In some embodiments of any aspect, the SMASh / cytosol-sequestering synTF comprises SEQ ID NO: 10, 11, 46, 47, or 379, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NO: 10, 11, 46, 47, or 379 and maintains the same function.

[0379] In some embodiments of any aspect, the SMASh / cytosol-sequestering synTF comprises SEQ ID NO:22, 23, 34, or 35, or a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of one of SEQ ID NO:22, 23, 34, or 35, maintaining the same function of the encoded polypeptide, or is encoded by a vector or polynucleotide that comprises a codon-optimized version of SEQ ID NO:22, 23, 34, or 35.

[0380] In some embodiments of any aspect, the effector domain (ED) comprises a transcriptional repressor. In one aspect, described herein is a SMASh / cytosol-sequestering synTF comprising, from N-terminus to C-terminus, (a) a transcriptional repressor domain, (b) a DBD, (c) a cytosol-sequestering domain, and (d) an inducible degradation (e.g., SMASh) domain. In another aspect, described herein is a SMASh / cytosol-sequestering synTF comprising, from N-terminus to C-terminus, (a) an inducible degradation (e.g., SMASh) domain, (b) a transcriptional repressor domain, (c) a DBD, and (d) a cytosol-sequestering domain. In some embodiments of any aspect, the SMASh / cytosol-sequestering synTF comprises one of SEQ ID NOs: 13-15 or 49-51, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to or maintains the same function as one of SEQ ID NOs: 13-15 or 49-51.

[0381] In some embodiments of any aspect, the SMASh / cytosol-sequestering synTF comprises one of SEQ ID NOs: 25-27 or 37-39, or comprises a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of one of SEQ ID NOs: 22, 23, 34, or 35, maintaining the same function of the encoded polypeptide, or is encoded by a vector or polynucleotide that comprises a codon-optimized version of SEQ ID NOs: 25-27 or 37-39.

[0382] In some aspects, the inducible degradation domain synTF further comprises a second regulatory protein, e.g., an inhibitory domain (PRO). Thus, in one aspect, described herein is a synTF comprising: (a) a DBD; (b) an ED; (c) an inducible degradation domain (SMASh); and (d) an inhibitory protease domain (PRO). In some embodiments of any aspect, the synTF comprises, from N-terminus to C-terminus: DBD-ED-SMASh-PRO; ED-DBD-SMASh-PRO; SMASh-DBD-ED-PRO; DBD-SMASh-ED-PRO; ED-SMASh-DBD-PRO; SMASh-ED-DBD-PRO; SMASh-ED-PRO-DBD; ED-SMASh-PRO-DBD; PRO-SMASh-ED-DBD; SMASh-PRO-ED-DBD; ED-PRO-SMASh-DBD In some embodiments, the SMASh domain is at the C-terminus or N-terminus, and the PRO domain is between the DBD and ED domains: for example, SMASh-ED-PRO-DBD; SMASh-DBD-PRO-ED; ED-PRO-DBD-SMASh; ED-PRO-DBD-SMASh; and DBD-PRO-ED-SMASh.

[0383] In some embodiments, the sequence of each domain is selected from exemplary domain sequences as described herein. In some embodiments, the ED of the PRO / SMASh synTF system is a transcriptional activator. In some embodiments, the ED of the PRO / SMASh synTF system is a transcriptional repressor. In the presence of a protease inhibitor of the PRO domain, the ED and DBD are coupled and the synTF is turned on; in the absence of a protease inhibitor of the PRO domain, the ED and DBD are uncoupled and the synTF is turned off. In the presence of an inducer of the inducible degradation pair (e.g., a protease inhibitor), the PRO / SMASh synTF system is degraded. In the absence of an inducer of the inducible degradation pair (e.g., a protease inhibitor), the SMASh tag is degraded and the PRO / SMASh synTF system is not degraded. In some embodiments of any aspect, the inhibitory protease domain and the inducible degradation domain each comprise a different protease, or each comprise an NS3 protease that is sensitive to a different NS3 protease inhibitor, such that separate protease inhibitors can be used to separately regulate the PRO domain and the SMASh domain.

[0384] In some aspects, the inducible degradation domain synTF further comprises a second regulatory protein, such as an induced proximity pair (IPD). Thus, in one aspect, a synTF system is described herein that comprises: (a) a DBD; (b) an ED; (c) an inducible degradation domain (SMASh); and (d) an induced proximity pair (IPD). The inducible degradation domain can be linked to any polypeptide of the IPD synTF system. In one aspect, a synTF system is described herein that comprises: (a) a first polypeptide comprising (i) a DBD, (ii) a first member of an induced proximity pair (IP1) and (iii) an inducible degradation domain (SMASh); and (b) a second polypeptide comprising (i) an ED and (ii) a second member of an induced proximity pair (IP2). In another aspect, a synTF system is described herein, comprising: (a) a first polypeptide comprising (i) a DBD and (ii) a first member of an induced proximity pair (IP1); and (b) a second polypeptide comprising (i) an ED, (ii) a second member of the induced proximity pair (IP2), and (iii) an inducible degradation domain (SMASh). In another aspect, a synTF system is described herein, comprising: (a) a first polypeptide comprising (i) a DBD, (ii) a first member of an induced proximity pair (IP1), and (iii) an inducible degradation domain (SMASh); and (b) a second polypeptide comprising (i) an ED, (ii) a second member of the induced proximity pair (IP2), and (iii) an inducible degradation domain (SMASh). The SMASh is linked, for example, through the use of a flexible linker peptide, so as not to interfere with the binding of IPD1 and IPD2 in the presence of an inducer substance. Non-limiting examples of first and second IPD / SMASh synTF systems are shown in Table 19.

[0385] Table 19: Exemplary pairs of first and second IPD / SMASh synTF systems, with each polypeptide shown from N-terminus to C-terminus. TIFF0007663513000183.tif71166

[0386] In some embodiments, the sequence of each domain is selected from exemplary domain sequences as described herein. In some embodiments, the ED of the IPD / SMASh synTF system is a transcriptional activator. In some embodiments, the ED of the IPD / SMASh synTF system is a transcriptional repressor. In the presence of an inducer of the inducible proximity pair, IP1 and IP2 bind to the inducer, resulting in the formation of a protein complex containing both polypeptides of the IPD / SMASh system; in the absence of the inducer, the polypeptides of the IPD / SMASh system do not form a complex. In the presence of an inducer of the inducible degradation pair (e.g., a protease inhibitor), the IPD / SMASh synTF system is degraded. In the absence of an inducer of the inducible degradation pair (e.g., a protease inhibitor), the SMASh tag is degraded and the IPD / SMASh synTF system is not degraded.

[0387] C. Inducible Proximity Domain SynTF In some embodiments of any aspect, the regulatory protein is a pair of induced proximity domains. Each of the two members of the induced proximity pair is directly linked to the DBD or ED. Thus, in one aspect, a synTF system is described herein, which includes: (a) a first polypeptide comprising (i) a DBD and (ii) a first member (IP1) of the induced proximity pair; and (b) a second polypeptide comprising (i) an ED and (ii) a second member (IP2) of the induced proximity pair. In some embodiments of any aspect, the domains of the synTF can be, for example, in the following order from N-terminus to C-terminus: DBD-IP1 and ED-IP2; DBD-IP1 and IP2-ED; IP1-DBD and ED-IP2; or IP1-DBD and IP2-ED. That is, the DBD is attached to one member of the induced proximity pair (i.e., IP1) and the ED is attached to the other or cognate member of the induced proximity pair (i.e., IP2), such that in the presence of an inducer of the induced proximity pair, IP1 and IP2 bind to the inducer, resulting in the formation of a protein complex containing DBD-IP1:IP2-ED; in the absence of the inducer, DBD-IP1 and ED-IP2 do not form a complex.

[0388] In some embodiments of any aspect, the two polypeptides of the inducible proximity synTF are linked by a self-cleaving peptide (SCP), such that the synTF system is expressed by a single vector and the two polypeptides are post-translationally cleaved from each other. Thus, the inducible proximity synTF system can comprise, from N-terminus to C-terminus: DBD-IP1-SCP-ED-IP2; DBD-IP1-SCP-IP2-ED; IP1-DBD-SCP-ED-IP2; or IP1-DBD-SCP-IP2-ED.

[0389] In some embodiments of any aspect, the DBD of the induced proximal synTF comprises ZF1-3. In some embodiments of any aspect, the DBD of the induced proximal synTF comprises SEQ ID NO:1 or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:1 and maintains the same function.

[0390] In some embodiments of any aspect, the effector domain (ED) comprises a transcriptional activator. In one aspect, an induced proximity synTF is described herein, in which (a) a first polypeptide comprises, from N-terminus to C-terminus, (i) a first member of an induced proximity pair and (ii) a DBD; and (b) a second polypeptide comprises, from N-terminus to C-terminus, (i) a transcriptional activator domain and (ii) a second member of the induced proximity pair. In some embodiments of any aspect, the first and second induced proximity synTFs are linked by a self-cleaving peptide. In some embodiments of any aspect, the induced proximal synTF comprises SEQ ID NO:4 or 40, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of the sequences of SEQ ID NO:4 or 40 and maintains the same function.

[0391] In some embodiments of any aspect, the induced proximal synTF comprises SEQ ID NO: 16 or 28, or a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of one of SEQ ID NO: 16 or 28, maintaining the same function of the encoded polypeptide, or is encoded by a vector or polynucleotide that comprises a codon-optimized version of SEQ ID NO: 16 or 28.

[0392] In some embodiments of any aspect, the effector domain (ED) comprises a transcriptional repressor. In one aspect, an induced proximity synTF is described herein, in which (a) a first polypeptide comprises, from N-terminus to C-terminus, (i) a first member of an induced proximity pair and (ii) a DBD; and (b) a second polypeptide comprises, from N-terminus to C-terminus, (i) a transcriptional repressor domain and (ii) a second member of the induced proximity pair. In some embodiments of any aspect, the first and second induced proximity synTFs are linked by a self-cleaving peptide. In some embodiments of any aspect, the induced proximal synTF comprises SEQ ID NO:5 or 41, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of the sequences of SEQ ID NO:5 or 41 and maintains the same function.

[0393] In some embodiments of any aspect, the induced proximal synTF comprises SEQ ID NO: 17 or 29, or a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of one of SEQ ID NO: 17 or 29, maintaining the same function of the encoded polypeptide, or is encoded by a vector or polynucleotide that comprises a codon-optimized version of SEQ ID NO: 17 or 29.

[0394] D. Cytosolic Sequestration Domain SynTF In some embodiments of any aspect, the regulatory protein is a cytosolic sequestration protein. Thus, in one aspect, the present invention provides a synTF that includes (a) DBD; (b) ED; and (c) cytosolic sequestration protein (CS). In some embodiments of any aspect, the domains of synTF can be, for example, in the following order from N-terminus to C-terminus: DBD-ED-CS; DBD-CS-ED; ED-DBD-CS; ED-CS-DBD; CS-DBD-ED; or CS-ED-DBD.

[0395] In some embodiments of any aspect, the DBD of the cytosol-sequestered synTF comprises ZF3-5. In some embodiments of any aspect, the DBD of the cytosol-sequestered synTF comprises SEQ ID NO:2, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:2 and maintains the same function.

[0396] In some embodiments of any aspect, the effector domain (ED) comprises a transcriptional activator. In one aspect, described herein is a cytosol-sequestering synTF comprising, from N-terminus to C-terminus, (a) a DBD, (b) a transcriptional activator domain, and (c) a cytosol-sequestering domain. In some embodiments of any aspect, the cytosol-sequestering synTF comprises SEQ ID NO:6 or 42, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of the sequences of SEQ ID NO:6 or 42 and maintains the same function.

[0397] In some embodiments of any aspect, the cytosol-sequestered synTF comprises SEQ ID NO: 18 or 30, or a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of one of SEQ ID NO: 18 or 30, maintaining the same function of the encoded polypeptide, or is encoded by a vector or polynucleotide that comprises a codon-optimized version of SEQ ID NO: 18 or 30.

[0398] In some embodiments of any aspect, the effector domain (ED) comprises a transcriptional repressor. In one aspect, described herein is a cytosol-sequestering synTF comprising, from N-terminus to C-terminus, (a) a transcriptional repressor domain, (b) a DBD, and (c) a cytosol-sequestering domain. In some embodiments of any aspect, the cytosol-sequestering synTF comprises SEQ ID NO:7, 43, or 378, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one of SEQ ID NOs:7, 43, or 378 and maintains the same function.

[0399] In some embodiments of any aspect, the cytosol-sequestering synTF comprises SEQ ID NO:19 or 31, or a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of one of SEQ ID NO:19 or 31, maintaining the same function of the encoded polypeptide, or is encoded by a vector or polynucleotide that comprises a codon-optimized version of SEQ ID NO:19 or 31.

[0400] III. Polynucleotides and Vectors In some aspects, polynucleotides encoding synTFs are described herein. In some embodiments of any aspect, the synTF polynucleotide comprises one of SEQ ID NOs:28-39 (see, e.g., Table 2), or a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of one of SEQ ID NOs:28-39, and maintains the same function (e.g., inducible transcription factor) as a polypeptide.

[0401] In some embodiments, the synTF polynucleotide is a codon-optimized version of SEQ ID NOs:28-39. In some embodiments of any aspect, the vectors or nucleic acids described herein are codon-optimized, e.g., the native or wild-type sequence of a nucleic acid sequence has been modified or engineered to include alternative codons, such that the modified or engineered nucleic acid encodes the same polypeptide expression product as the native / wild-type sequence but is transcribed and / or translated with improved efficiency in a desired expression system. In some embodiments of any aspect, the expression system is an organism (or cells derived from such an organism) other than the source of the native / wild-type sequence. In some embodiments of any aspect, the vectors and / or nucleic acid sequences described herein are codon-optimized for expression in a mammal or mammalian cell, e.g., a mouse, murine cell, or human cell. In some embodiments of any aspect, the vectors and / or nucleic acid sequences described herein are codon-optimized for expression in a human cell. In some embodiments of any aspect, the vectors and / or nucleic acid sequences described herein are codon-optimized for expression in a yeast or yeast cell. In some embodiments of any aspect, the vectors and / or nucleic acid sequences described herein are codon-optimized for expression in bacterial cells. In some embodiments of any aspect, the vectors and / or nucleic acid sequences described herein are codon-optimized for expression in Escherichia coli (E. coli) cells.

[0402] In some embodiments, one or more of the genes described herein (e.g., synTF, gene of interest) are expressed in a recombinant expression vector or plasmid. As used herein, the term "vector" refers to a polynucleotide sequence suitable for transferring a transgene to a host cell. The term "vector" includes plasmids, minichromosomes, phages, naked DNA, and the like. See, for example, U.S. Patent Nos. 4,980,285; 5,631,150; 5,707,828; 5,759,828; 5,888,783 and 5,919,670, and Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Press (1989). One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments are ligated. Another type of vector is a viral vector, in which additional DNA segments are ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors." In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. As the plasmid is the most commonly used form of vector, "plasmid" and "vector" are used interchangeably herein. However, the invention is intended to include other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[0403] A cloning vector is a vector that can replicate autonomously in a host cell or integrate into the genome and is further characterized by one or more endonuclease restriction sites at which the vector can be cut in a determinable manner and into which a desired DNA sequence can be ligated, so that the new recombinant vector retains its ability to replicate in the host cell. In the case of a plasmid, replication of the desired sequence can occur multiple times as the plasmid increases in copy number within a host cell, such as a host bacterium, or it can occur only once per host before the host reproduces by mitosis. In the case of a phage, replication can occur actively during a lytic phase or passively during a lysogenic phase.

[0404] An expression vector is a vector into which a desired DNA sequence can be inserted by restriction and ligation so that the desired DNA sequence is operably connected to a regulatory sequence containing a DNA-binding domain as described herein, and can be expressed as an RNA transcript. A vector can also contain one or more marker sequences suitable for use in identifying cells that have been transformed or transfected with the vector, or cells that have not. Markers include, for example, genes encoding proteins that increase or decrease resistance or sensitivity to antibiotics or other compounds, genes encoding enzymes whose activity can be detected by standard assays known in the art (e.g., β-galactosidase, luciferase, or alkaline phosphatase), and genes that visibly affect the phenotype of transformed or transfected cells, hosts, colonies, or plaques (e.g., green fluorescent protein). In certain embodiments, the vectors used herein are capable of autonomous replication and expression of structural gene products present in the DNA segments to which they are operably connected.

[0405] As used herein, a coding sequence and a regulatory sequence are said to be "operably" connected when they are covalently linked in such a way that the expression or transcription of the coding sequence is under the influence or control of the regulatory sequence. When it is desired that the coding sequence be translated into a functional protein, two DNA sequences are said to be operably connected if induction of a promoter in a 5' regulatory sequence results in transcription of the coding sequence, and if the nature of the linkage between the two DNA sequences (1) does not result in the introduction of a frameshift mutation, (2) does not interfere with the ability of the promoter region to direct transcription of the coding sequence, and (3) does not interfere with the ability of the corresponding RNA transcript to be translated into a protein. Thus, a promoter region can be operably connected to a coding sequence if it is capable of effecting transcription of that DNA sequence so that the resulting transcript can be translated into a desired protein or polypeptide.

[0406] When a nucleic acid molecule encoding any of the polypeptides described herein is expressed in a cell, a wide variety of transcription control sequences (e.g., promoter / enhancer sequences) can be used to direct its expression. The promoter can be the native promoter, i.e., the promoter of the gene in its endogenous environment, which provides for the normal regulation of expression of that gene. In some embodiments, the promoter can be constitutive, i.e., the promoter is unregulated, allowing continuous transcription of its associated gene. A wide variety of conditional promoters, such as promoters controlled by the presence or absence of a certain molecule, can also be used.

[0407] The exact nature of the regulatory sequences required for gene expression may vary depending on the species or cell type, but generally may include, as necessary, 5' non-transcribed and 5' non-translated sequences involved in initiation of transcription and translation, respectively, such as a TATA box, capping sequence, CAAT sequence, etc. In particular, such 5' non-transcribed regulatory sequences include a promoter region containing a promoter sequence for transcriptional control of an operably linked gene. Regulatory sequences may also include enhancer sequences or upstream activating sequences, if desired. Vectors of the present invention may optionally include a 5' leader sequence or signal sequence. The selection and design of an appropriate vector is within the skill and discretion of one of ordinary skill in the art.

[0408] In some embodiments of any aspect, the promoter is a eukaryotic or human constitutive promoter, including, but not limited to, the human elongation factor-1 alpha (EF-1α, EF1a) promoter; silencing-prone spleen focus-forming virus (SFFV); cytomegalovirus (CMV) promoter; ubiquitin C (UbiC, pUb, UbC) promoter; phosphoglycerate kinase 1 (PGK, pGK) promoter; cytomegalovirus (CMV) enhancer (CAG / CAGG) fused to the chicken beta-actin promoter; simian virus 40 (SV40) enhancer and early promoter; β-actin (ACTB) promoter; and the like. In some embodiments of any aspect, the promoter is a minimal or core promoter. A minimal or core promoter, by definition, is a sequence located between -35 and +35 relative to the transcription start site; a minimal promoter is typically shorter than a complete promoter and does not include additional elements such as enhancers or silencers. Non-limiting examples of minimal promoters include minCMV; CMV53 (minCMV with an upstream GC box); minSV40 (minimal simian virus 40 promoter); miniTK (region -33 to +32 of the herpes simplex thymidine kinase promoter); MLP (region -38 to +6 of the adenovirus major late promoter); pJB42CAT5 (minimal promoter derived from the human junB gene); ybTATA (synthetic minimal promoter), and TATA box alone. See, e.g., Ede et al., ACS Synth Biol. 2016 May 20, 5(5): 395-404; Qin et al., PLoS One. 2010, 5(5): e10611; Norman et al., PLoS One. 2010 Aug 26, 5(8): e12413 (the contents of each of which are incorporated herein by reference in their entirety).

[0409] In some embodiments of any aspect, the vector (e.g., SEQ ID NOs: 16-27, 58-60, 64) comprises an SFFV promoter (e.g., SEQ ID NO: 363). In some embodiments of any aspect, the vector (e.g., SEQ ID NOs: 55-57) comprises a full CMV promoter (e.g., SEQ ID NO: 364). In some embodiments of any aspect, the vector (e.g., SEQ ID NOs: 52-54, 62) comprises a minCMV promoter (e.g., SEQ ID NO: 365). In some embodiments of any aspect, the vector (e.g., SEQ ID NOs: 61, 63, 64) comprises a minTK promoter (e.g., SEQ ID NO: 366). In some embodiments of any aspect, the vector comprises a Kozak sequence (e.g., GCCGCCACC), which is a nucleic acid motif that functions as a protein translation initiation site in eukaryotic mRNA transcripts.

[0410] Expression vectors containing all the elements necessary for expression are commercially available and known to those skilled in the art.See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989.Cells are genetically engineered by introducing heterologous DNA (RNA) into cells.The heterologous DNA (RNA) is placed under the operable control of transcription elements that allow the expression of the heterologous DNA in host cells.

[0411] In some embodiments of any aspect, the synTF vector comprises one of SEQ ID NOs:16-27 or SEQ ID NOs:52-64 (see, e.g., Table 1 or 2), or comprises a sequence that maintains the same function as one of SEQ ID NOs:16-27 or SEQ ID NOs:52-64 (e.g., lentiviral vector, synTF polypeptide expression) that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of one of SEQ ID NOs:236-261.

[0412] In some embodiments, the vector is a pHR vector. In some embodiments, the vector is a lentiviral vector. The term "lentivirus" refers to a genus in the Retroviridae family. Lentiviruses are unique among retroviruses in that they can infect non-dividing cells; they are one of the most efficient gene delivery vectors because they can deliver significant amounts of genetic information into the DNA of host cells. HIV, SIV, and FIV are all examples of lentiviruses. The term "lentiviral vector" refers to a vector derived from at least a portion of a lentiviral genome, including, inter alia, self-inactivating lentiviral vectors such as those provided in Milone et al., Mol. Ther. 17(8): 1453-1464 (2009). Other examples of lentiviral vectors that can be used clinically include, but are not limited to, Oxford BioMedica's LENTIVECTOR® gene delivery technology, Lentigen's LENTIMAX™ vector system, and the like. Non-clinical lentiviral vectors are also available and would be known to those skilled in the art.

[0413] In some embodiments of any aspect, the lentiviral vector comprises a central polypurine tract (cPPT; e.g., SEQ ID NO: 367). The central polypurine tract / central termination sequence creates a "DNA flap" that increases nuclear import of the viral genome during target cell infection. The cPPT / CTS element improves vector integration and transduction efficiency. In some embodiments of any aspect, the lentiviral vector comprises a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE; e.g., SEQ ID NO: 368), which prevents poly(A) site readthrough, promotes RNA processing and maturation, and increases RNA nuclear export. In genomic transcripts, it enhances vector packaging and increases titer. In transduced target cells, the WPRE enhances transgene expression by facilitating mRNA transcript maturation. In some embodiments of any aspect, the lentiviral vector comprises long terminal repeats (LTR; e.g., SEQ ID NO: 369 and 370). LTRs are identical sequences of DNA repeated hundreds or thousands of times found at either end of retrotransposons or proviral DNA formed by reverse transcription of retroviral RNA; they are used by viruses to insert genetic material into the host genome. In some embodiments of any aspect, the lentiviral vector contains a Rev response element (RRE; e.g., SEQ ID NO:371), which is necessary for producing high-titer vectors.

[0414] Without limitation, the genes described herein can be included in one vector or separate vectors. For example, the polynucleotide encoding synTF and the polynucleotide encoding the gene of interest can be included in the same vector. In some embodiments of any aspect, the polynucleotide encoding synTF and the polynucleotide encoding the gene of interest can be included in different vectors. In some embodiments of any aspect, a single vector can include at least one polynucleotide encoding synTF. In some embodiments of any aspect, a single vector can include at least one polynucleotide encoding the gene of interest. In some embodiments of any aspect, a single vector can include at least one polynucleotide encoding synTF and at least one polynucleotide encoding the gene of interest.

[0415] In one aspect, described herein are synTF lentiviral expression vectors (e.g., SEQ ID NOs: 16-27). In some embodiments, the synTF lentiviral expression vector comprises a polynucleotide encoding synTF operably linked to a promoter (e.g., SFFV). In another aspect, described herein are lentiviral reporter vectors (e.g., SEQ ID NOs: 52-60). In some embodiments, the lentiviral reporter vector comprises (i) at least one (e.g., 1, 2, 3, 4, or more) DNA-binding motif (DBM) for the DBD of the synTF; (ii) a promoter sequence located 3' of at least one DBM; and (iii) a detectable marker (i.e., reporter; e.g., mCherry) operably linked to the promoter sequence. In some embodiments, the lentiviral reporter vector is an activation reporter for a synTF that includes a transcription activation domain (e.g., SEQ ID NOs:52-54), such that when the inducible synTF is on, the DBD of the synTF binds to the DBM of the reporter and the transcription activation domain activates transcription of the detectable marker; when the DBD of the inducible synTF is off and does not bind to the DBM of the reporter, the detectable marker is not produced because it is operably linked only to a core or minimal promoter (e.g., minCMV). In some embodiments, the lentiviral reporter vector is a repressible reporter for an inducible synTF that contains a transcriptional repressor domain (e.g., SEQ ID NOs:55-60), such that when the synTF is on, the DBD of the synTF binds to the DBM of the reporter and the transcriptional repressor domain represses transcription of the detectable marker; when the synTF is off, the DBD of the synTF does not bind to the DBM of the reporter and the detectable marker is produced because it is operably linked to a complete constitutive promoter (e.g., a complete CMV or SFFV promoter).

[0416] In one aspect, gene of interest (GOI) lentiviral expression vectors (e.g., 61-64) are described herein. In some embodiments, the GOI vector comprises at least one (e.g., one, two, three, four, or more) DNA-binding motif (DBM) for the DBD of a synTF and a gene of interest operably linked to a minimal promoter. In some embodiments, the GOI vector comprises a reporter vector as described herein, in which a detectable marker replaces or is fused to the gene of interest; thus, the synTF can induce expression of the gene of interest or repress expression of the gene of interest in the presence or absence of a regulatory protein inducer.

[0417] In some embodiments, the polypeptide encoded by the gene of interest is selected from the group consisting of chemokines, chemokine receptors, chimeric antigen receptors, cytokines, cytokine receptors, differentiation factors, growth factors, growth factor receptors, hormones, metabolic enzymes, pathogen-derived proteins, proliferation inducers, receptors, RNA-guided nucleases, site-specific nucleases, small second messenger synthesis enzymes, T cell receptors, toxin-derived proteins, transcription activators, transcription repressors, transcriptional activators, transcriptional repressors, translation regulators, translation activators, translation repressors, activating immunoreceptors, antibodies, apoptosis inhibitors, apoptosis inducers, engineered T cell receptors, immune activators, immune inhibitors, inhibitory immunoreceptors, and RNA-guided DNA-binding proteins. In some embodiments, the polypeptide encoded by the gene of interest is believed to benefit a subject in need of treatment, such as a subject with cancer, a subject with autoimmunity, or a subject in need of regenerative medicine.

[0418] In some embodiments, the polypeptide encoded by the gene of interest is CD19 CAR (e.g., SEQ ID NO:61, 373), linked to a detectable marker such as mCherry (e.g., SEQ ID NO:372). In some embodiments, the polypeptide encoded by the gene of interest is IL2 (e.g., SEQ ID NO:62, 374), which can be linked to a detectable marker, e.g., (huEGFRt), using a self-cleaving peptide. In some embodiments, the polypeptide encoded by the gene of interest is IL10 (e.g., SEQ ID NO:63-64, 375).

[0419] In one aspect, described herein is a lentiviral vector comprising both a synTF operably linked to a constitutive promoter and at least one (e.g., 1, 2, 3, 4, or more) DNA binding motif (DBM) for the DBD of the synTF and a gene of interest operably linked to a minimal promoter (e.g., SEQ ID NO:64). Thus, described herein is a nucleic acid construct comprising, in a 5' to 3' direction: (a) a nucleic acid sequence encoding a gene of interest (GOI) in reverse orientation; (b) a first promoter sequence in reverse orientation and operably linked to the nucleic acid encoding the GOI; (c) a nucleic acid sequence comprising at least one target DNA-binding motif (DBM) comprising a target nucleic acid for binding of at least one DBD of a synTF, wherein binding of the DBD of the synTF places an effector domain (ED) in proximity to the promoter sequence operably linked to the GOI; (d) a second promoter sequence; and (e) a nucleic acid sequence encoding a synthetic transcription factor (synTF) operably linked to the second promoter sequence, wherein the encoded synTF comprises at least one DBD that binds to at least one DBM of the nucleic acid sequence of (c).

[0420] In some embodiments of any aspect, the promoter sequence operably linked to the GOI is selected from any of a miniCMV promoter, a miniTK promoter, a ybTATA promoter, a minSV40 promoter, a CMV53 promoter, a pJB42CAT5 promoter, an MLP promoter, and a TATA promoter. In some embodiments of any aspect, the promoter sequence operably linked to the nucleic acid encoding a synTF is a pSFFV promoter, a CMV promoter, a pUb / UbC promoter, an EF1a promoter, a PGK / pGK promoter, a CAG / CAGG promoter, an SV40 promoter, and a β-actin / ACTB promoter.

[0421] In some embodiments, the polynucleotide encoding the synTF is operably linked to an inducible promoter that is active in the presence of a promoter activator or in the absence of a promoter repressor, and is inactive in the absence of a promoter inducer or in the presence of a promoter repressor. Non-limiting examples of inducible promoters include a doxycycline-inducible promoter, a lac promoter, a lacUV5 promoter, a tac promoter, a trc promoter, a T5 promoter, a T7 promoter, a T7-lac promoter, an araBAD promoter, a rha promoter, a tet promoter, an isopropyl β-D-1-thiogalactopyranoside (IPTG)-dependent promoter, an AlcA promoter, a LexA promoter, a temperature-inducible promoter (e.g., a promoter derived from Hsp70 or Hsp90), or a light-inducible promoter (e.g., a pDawn / YFI / FixK2 promoter / CI / pR promoter system).

[0422] In some embodiments, the vector includes a selectable marker, e.g., a selectable marker for selectively amplifying the vector in bacteria. Non-limiting examples of selectable marker genes for use in bacteria include antibiotic resistance genes that confer resistance to ampicillin, tetracycline, and kanamycin. Tetracycline (tet) and ampicillin (amp) resistance marker genes can be obtained from any of a number of commercially available vectors, including pBR322 (available from New England BioLabs, Beverly, Mass., cat. no. 303-3s). The tet coding sequence is contained within nucleotides 86-476; the amp gene is contained within nucleotides 3295-4155. The nucleotide sequence of the kanamycin (kan) gene is available from the vector pACYC 177 (New England BioLabs, Cat. no. 401-L, GenBank accession no. X06402).

[0423] In some embodiments, one or more of the recombinantly expressed genes may be integrated into the genome of the cell.

[0424] Nucleic acid molecules encoding the enzymes of the claimed invention can be introduced into one or more cells using methods and techniques that are standard in the art. For example, the nucleic acid molecules can be introduced by standard protocols such as transformation, including chemical transformation and electroporation, transduction, particle bombardment, etc. Expression of nucleic acid molecules encoding the enzymes of the claimed invention can also be achieved by integrating the nucleic acid molecules into the genome.

[0425] IV. synTF System Another aspect of the present technology relates to a synTF system for controlling gene expression of a gene of interest (GOI), the system comprising a nucleic acid construct comprising a synTF as described herein and an element to which the synTF binds to regulate gene expression. In one aspect, the present invention describes a system for controlling gene expression, comprising: (a) at least one synthetic transcription factor (synTF) as described herein; and (b) at least one nucleic acid construct as described herein, for example, (i) at least one target DNA-binding motif (DBM) comprising a target nucleic acid for binding at least one DBD of the synTF; (ii) a promoter sequence located 3' of the at least one DBM; and (iii) a nucleic acid construct comprising a gene of interest operably linked to the promoter sequence.

[0426] Exemplary systems are shown in Figures 1B, 2, 5C, 10B, 11A, 12A, 13A, 14A, and 15A-15B, depicting nucleic acid constructs comprising a synTF and a gene of interest (e.g., mCherry, CD19 CAR, IL4, IL10). In synTF embodiments where the synTF is a protease synTF or an inducible proximity domain synTF (i.e., where coupling of the ED to the DBD is regulated by at least one protease RP or at least one inducible proximity pair), in the presence of an RP inducer, coupling of the ED to the DBD of the synTF is maintained, such that when the DBD binds to the DNA-binding motif (DBM), the ED can be brought into proximity with the promoter sequence and promote the initiation or inhibition of gene expression of the gene of interest ("ED-on"). Alternatively, in the absence of an RP inducer, the coupling of the ED to the DBD of the synTF is cleaved, and the binding of the DBD to the DNA-binding motif (DBM) prevents the ED from approaching the promoter sequence, thereby preventing the ED from regulating the expression of the gene of interest ("ED-off"). Depending on whether the effector domain is a transcriptional activator (TA) or a transcriptional repressor (TR), ED-on and ED-off are thought to have different effects. That is, if the ED is a TA, gene expression can occur when the synTF is in the "ED-on" configuration, whereas if the ED is a transcriptional repressor (TR), the ED-on configuration is thought to result in repression of gene expression (e.g., no expression). Conversely, if the ED is a TA, gene expression is off when the synTF is in the "ED-off" configuration, whereas if the ED is a transcriptional repressor (TR), the "ED-off" configuration weakens repression of gene expression, thereby turning on gene expression. A summary of the effect of each regulator promoter inducer on final gene expression dependent on different synTF and effector domains is summarized in Table 20 herein.

[0427] Similarly, by way of example only, in synTF embodiments in which the synTF is a cytosolic sequestration domain synTF (i.e., the cellular localization of the ED-DBD fusion protein is regulated by at least one cytosolic sequestration regulatory protein), in the presence of an RP inducer, the ED-DBD is not sequestered in the cytosol, such that the DBD can bind to the DNA-binding motif (DBM) and the transcription effector domain (ED) is positioned near the promoter sequence to control expression of the gene of interest ("ED-on"). Thus, in the presence of an inducer, if the ED is a TA, the ED-on configuration can result in gene expression (referred to as "TA-on"), whereas if the ED is a TR, the ED-on configuration would result in repression of gene expression (referred to as "TR-on" and no gene expression). Alternatively, in the absence of an RP inducer, the ED coupled to the DBD of the synTF is sequestered in the cytosol, preventing binding of the DBD of the synTF to the DBM and preventing the proximity of the effector domain (ED) to the promoter sequence, thereby preventing expression of the gene of interest ("ED-off"). Thus, in the absence of an inducer, in embodiments where the ED is a transcriptional activator (TA), the ED results in abrogation of gene expression ("TA-off" (no expression)), whereas in embodiments where the ED is a transcriptional repressor protein (TR), the ED enables gene expression ("TR-off"; therefore, repression is off and gene expression can occur).

[0428] In some embodiments of any aspect described herein, the synTF is an inducible degradation domain synTF or further comprises an N-terminal or C-terminal small molecule-assisted shut-off (SMASh) domain. As described herein, the SMASh domain comprises a self-cleaving SMASh protease, a partial protease helical domain, and a cofactor domain. In some embodiments of any aspect, in the presence of an inhibitor of the SMASh protease (referred to as a "SMASh inhibitor"), SMASh protease activity is inhibited, resulting in the synTF being degraded, thereby preventing the DBD from binding to the DBM and controlling the expression or repression of the gene of interest ("synTF-degradation"; ED-off). In some embodiments of any aspect, in the absence of an inhibitor to the SMASh protease, the SMASh protease is active and does not cleave / couple itself from the synTF, such that the SMASh domain is not targeted for degradation and allows the DBD of the synTF to bind to the DBM and ED of the synTF to control expression of the gene of interest ("SMASh-degraded, TA-on (expressed), TR-on (no expression)").

[0429] Table 20A: A table summarizing the effect of the presence or absence of regulatory protein inducers on the final expression of genes of interest. TIFF0007663513000184.tif92166

[0430] Thus, expression of a GOI is dependent on three levels of control, including, but not limited to, (i) the type of regulatory protein in the synTF, (ii) the presence or absence of a regulatory protein inducer (RP inducer), and (iii) the type of effector domain. In some embodiments, if the synTF contains an inducible degradation domain or an SMASh domain, it can also provide an additional level of control over the expression of a GOI. The final expression of a GOI for synTFs containing an SMASh domain is shown in Table 20B.

[0431] (Table 20B) TIFF0007663513000185.tif169169

[0432] As shown in Table 20B, in the presence of a SMASh inhibitor, synTF is degraded in the absence or presence of a regulatory protein inducer (e.g., a protease inhibitor, an IPD inducer, or a ligand for CS-SynTF), and expression of the GOI depends on whether the ED is a TA or a TR. However, as shown in Table 20B, in the absence of a SMASh inhibitor, expression of the GOI is dependent on the presence or absence of a regulatory protein inducer.

[0433] Thus, in one aspect, a synthetic transcription factor (synTF) is provided, 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 regulatory protein (RP), wherein the ED is directly or indirectly coupled or linked to the DBD, and wherein the coupling is regulated by the at least one RP, or wherein the cellular localization of the ED linked to the DBD is regulated by the at least one RP, and the at least one RP is regulated by an RP inducer, and wherein the DBD is capable of binding to a target DNA-binding motif (DBM) located upstream of a promoter operably linked to a gene; (b) (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) at least a nucleic acid construct comprising (iii) a promoter sequence located 3' of one DBM, and a gene of interest operably linked to the promoter sequence, wherein for a synTF whose coupling of its ED to its DBD is regulated by at least one RP, in the presence of an RP inducer, coupling of the ED to the DBD of the synTF is maintained, allowing the ED to come into proximity with the promoter sequence upon binding of the DBD to the DNA-binding motif (DBM), whereby the ED controls expression of the gene of interest ("ED-on"), or in the absence of the RP inducer, coupling of the ED to the DBD of the synTF is disengaged, preventing the ED from coming into proximity with the promoter sequence upon binding of the DBD to the DNA-binding motif (DBM), thereby preventing gene expression of the gene of interest ("ED-off");and for a synTF in which the cellular localization of the ED linked to the DBD is regulated by at least one regulatory protein, a system is described herein in which, in the presence of an RP inducer, the ED coupled to the DBD of the synTF is not sequestered in the cytosol, allowing the DBD to bind to a DNA-binding motif (DBM), thereby allowing the transcriptional effector domain (ED) to be in close proximity to the promoter sequence and control expression of the gene of interest ("ED-on"), or, in the absence of an RP inducer, 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 close proximity to the promoter sequence, thereby preventing expression of the gene of interest ("ED-off");

[0434] Nucleic acid construct encoding a GOI for regulation by a synTF As described herein, in some aspects, the system includes a nucleic acid construct comprising a synTF and a GOI as described herein. In some embodiments, the nucleic acid construct includes, in a 5' to 3' direction, (i) a DBA-binding motif (DBM) that enables binding of the DBD of the synTF, (ii) a promoter sequence, and (iii) a nucleic acid encoding the GOI operably linked to the promoter sequence. Exemplary systems are shown in Figures 5C and 12A.

[0435] In some embodiments, the system further comprises a nucleic acid sequence encoding a synTF operably linked to a promoter, e.g., the promoter is a constitutive promoter, see e.g., Figures 10B, 13A, 14A, and 15A for exemplary systems. In such embodiments, the synTF can be expressed in the presence of an inducer for the promoter, and the activity of the synTF in regulating gene expression of the GOI is dependent on the presence or absence of the regulatory protein inducer and / or the SMASh inducer when the SMASh domain is attached.

[0436] In some embodiments, the nucleic acid construct comprises (i) a first nucleic acid encoding a synTF under a promoter, and (ii) a second nucleic acid construct comprising (i) a DBA-binding motif (DBM) that allows binding of the DBD of the expressed synTF, (ii) a promoter sequence, and (iii) a nucleic acid encoding a GOI operably linked to the promoter sequence. Thus, the nucleic acids encoding the inducible synTF and the GOI are present on the same nucleic acid construct; see, e.g., Figure 15B for an exemplary system.

[0437] In some embodiments, the nucleic acid construct comprises (i) a first promoter operably linked to a nucleic acid encoding a synTF, and (ii) a nucleic acid encoding a GOI operably linked to a second promoter, wherein 5' of the second promoter is a DBD of the synTF protein expressed under the control of the first promoter. In some embodiments, as shown in Figure 15B, the construct comprises, in 5' to 3' direction: (i) a nucleic acid encoding the GOI in an antisense orientation, (ii) a first promoter operably linked to the GOI in an antisense orientation, (iii) a DBD in an antisense orientation, (iv) a second promoter in a sense orientation, and (v) a nucleic acid encoding a synTF in a sense orientation operably linked to the second promoter. Such a system allows for expression of the synTF, wherein the expressed synTF can be used to regulate expression of the GOI in the presence or absence of an inducer of the synTF.

[0438] In some embodiments of any aspect, the promoter operably linked to the GOI or synTF is selected from any of a miniCMV promoter, a miniTK promoter, a ybTATA promoter, a minSV40 promoter, a CMV53 promoter, a pJB42CAT5 promoter, an MLP promoter, a TATA promoter, a pSFFV promoter, a CMV promoter, a pUb / UbC promoter, an EF1a promoter, a PGK / pGK promoter, a CAG / CAGG promoter, an SV40 promoter, and a β-actin / ACTB promoter.

[0439] In some embodiments of any aspect, at least one synTF expressed by the system is selected from any of the synTFs described herein. In some embodiments of any aspect, the synTF system can include any combination of at least two synTF polypeptides as described herein, controlling the same or different GOIs. As a non-limiting example, Figures 14A-14C show a system including two synTFs, each with a distinct regulatory protein and GOI: an inhibitory protease synTF controlling CD19 CAR expression and a cytosolic sequestration domain synTF controlling IL4 expression. Table 9 below shows non-limiting examples of combinations of such synTF systems. In some embodiments of any aspect, the examples shown in Table 9 can be in combination with a regulatory protein inducer (e.g., a small molecule drug such as grazoprevir, ABA, or 4OHT).

[0440] (Table 9) synTF systems. "PRO" refers to inhibitory protease synTF polypeptides and systems as described herein. "ID" refers to inducible degradation synTF polypeptides and systems as described herein. "IPD" refers to inducible proximity synTF polypeptides and systems as described herein. "CS" refers to cytosolic sequestration domain synTF polypeptides and systems as described herein. TIFF0007663513000186.tif55166

[0441] V. Cell In one aspect, described herein is a cell or population thereof comprising at least one synTF polypeptide, synTF system, synTF polynucleotide, or synTF vector as described herein (see, e.g., Tables 1-2). In some embodiments of any aspect, the cell or population thereof can comprise any combination of synTF polypeptides or systems (see, e.g., Table 9).

[0442] In one aspect, the present invention provides cells (e.g., T cells) engineered to express synTF, wherein the activity of the synTF cell can be controlled by a small molecule. In one aspect, the cell is transformed with synTF, and synTF is expressed on the cell surface. In some embodiments, the cell (e.g., T cell) is transduced with a viral vector encoding synTF. In some embodiments, the viral vector is a retroviral vector. In some embodiments, the viral vector is a lentiviral vector. In some such embodiments, the cell can stably express synTF. In another embodiment, the cell (e.g., T cell) is transfected with a nucleic acid, e.g., mRNA, cDNA, DNA, encoding synTF. In some such embodiments, the cell can transiently express synTF.

[0443] In one aspect, described herein is a cell comprising a nucleic acid sequence comprising at least one target DNA binding motif (DBM) comprising a target nucleic acid for binding of 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) operably linked to the promoter sequence.

[0444] In one aspect, described herein is 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 at least one DBD of a synTF, a promoter sequence located 3' of the at least one DBM, and a nucleic acid encoding ...

Claims

1. a. at least one DNA binding domain (DBD); b. a transcription effector domain (ED); c. At least a first and a second regulatory protein (RP) A synthetic transcription factor (synTF) comprising: the ED is indirectly coupled to the DBD, and The coupling is regulated by the first RP, the first RP is located between the DBD and the ED, and the first RP is an inhibitory protease or an inducible proximity domain pair (IPD pair), and The coupling is further regulated by the second RP, which is located between the DBD and the ED, and the second RP is an inhibitory protease or an inducible proximity domain pair (IPD pair), or the cellular localization of the ED is regulated by the second regulatory protein, which is a cytosolic sequestration domain. synTF.

2. The synTF of claim 1, wherein the transcription ED is a transcription activator (TA) domain.

3. The synTF of claim 2, wherein the TA is selected from the group consisting of p65; Rta; mini-VPR; complete VPR; VP16; VP64; p300; p300 HAT core; and CBP HAT domain.

4. The synTF of claim 2, wherein the TA is p65 or a variant thereof.

5. The synTF of claim 4, wherein the p65 comprises one of SEQ ID NOs: 69, 117-121, 193-197, or a protein having at least 90% sequence identity to one of SEQ ID NOs: 69, 117-121, 193-197.

6. The synTF of claim 1, wherein the transcription ED is a transcription repressor (TR) domain.

7. The synTF of claim 6, wherein the TR is selected from the group consisting of KRAB; KRAB-MeCP2; Hp1a; DNMT3B; EED; and HDAC4.

8. The synTF of claim 6, wherein the TR is KRAB or a variant thereof.

9. 9. The synTF of claim 8, wherein the KRAB comprises one of SEQ ID NOs: 72, 97, or 214-215, or a protein having at least 90% sequence identity to one of SEQ ID NOs: 72, 97, or 214-215.

10. The synTF of claim 1, wherein the at least one DBD is an engineered zinc finger (ZF) binding domain.

11. The synTF of claim 10, wherein the ZF binding domain comprises two or more ZF motifs.

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

13. The synTF of any one of claims 10 to 12, wherein the ZF binding domain comprises one of ZF 1-3 (SEQ ID NO:1), ZF 3-5 (SEQ ID NO:2), or ZF 10-1 (SEQ ID NO:3), or an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs:1 to 3.

14. The synTF of any one of claims 10 to 13, wherein the ZF binding domain is selected from any of SEQ ID NOs: 1-3, 76, 101, 377, or 380.

15. The synTF of claim 14, wherein the ZF binding domain specifically binds to a sequence comprising at least one of SEQ ID NOs: 181-191.

16. The synTF of any one of claims 1 to 15, wherein at least one DBD is selected from any one or more of SEQ ID NO: 221 or 222, SEQ ID NO: 223, SEQ ID NO: 224 or 225, SEQ ID NO: 226 or 227, SEQ ID NO: 228, or wherein the DBD binds to a DNA binding motif (DBM) comprising any of SEQ ID NOs: 229-240.

17. The synTF of claim 1, wherein the first and / or second regulatory protein is located between the DBD and the transcriptional effector domain.

18. The synTF of any one of claims 1 to 17, wherein the first and / or second regulatory protein is an inhibitory protease.

19. The synTF of any one of claims 1 to 18, wherein the first and / or second regulatory protein is an NS3 protease protein.

20. The synTF of any one of claims 1 to 19, wherein the first and / or second regulatory protein comprises amino acids of SEQ ID NO: 82, 85, 91, 102, 241-255, 304-315, or a homologue having at least 90% sequence identity to SEQ ID NO: 82, 85, 91, 102, 241-255, 304-315.

21. 20. The synTF of claim 19, wherein in the presence of a protease inhibitor or an inhibitor of the NS3 protease protein, the NS3 protease protein is inhibited, thereby maintaining the coupling of the DBD to the effector domain.

22. The synTF of claim 19, wherein in the absence of a protease inhibitor or an inhibitor to the NS3 protease protein, the NS3 protease protein is active and causes excision of the NS3 protease protein from the DBD, thereby uncoupling the DBD and the effector domain.

23. The synTF of claim 21 or 22, wherein the inhibitor of the NS3 protease protein is selected from any of grazoprevir (GRZ / GZV), danoprevir, simeprevir, asunaprevir, cilprevir, boceprevir, sovaprevir, paritaprevir, ombitasvir, paritaprevir, ritonavir, dasabuvir, and telaprevir.

24. The first and / or second RP is a pair of induced proximity domains (IPD pair), the IPD pair comprising: The first induced proximity domain (IPD A ), and at least a second complementary IPD (IPD B ) Including, wherein in the presence of an inducer agent or an inducer signal, said IPD A and I.P.D. B together provide coupling between the ED and the DBD of a synthetic TF; and in the absence of the inducer agent or inducer signal, the ED is not coupled to the DBD of the synthetic TF; The synTF of claim 1.

25. The induced proximal domain pair (IPD pair) a. An IPD containing a GID1 domain, in which the GID1 and GAI domains bind to the inducer substance gibberellin ester (GIB). A , and IPDs containing GAI domains B ; b. An IPD containing an FKBP domain, in which the FKBP and FRB domains bind to the inducer substance Rapalog (RAP). A , and IPDs containing FRB domains B ; c. A PYL domain-containing IPD in which the PYL and ABI domains bind to the inducer substance abscisic acid (ABA). A , and the IPD containing the ABI domain B ; d. IPD A and I.P.D. B A light-inducible dimerization domain (LIDD) pair comprising an IPD A contains LIDD and IIPD B a light-inducible dimerization domain (LIDD) pair, wherein the LIDD comprises a complementary LIDD, and the LIDD dimerizes with the complementary LIDD upon exposure to a light inducer signal of an appropriate wavelength. IPD selected from any one or more of A and I.P.D. B The synTF of claim 24, comprising:

26. The induced proximal domain pair (IPD pair) is an IPD pair comprising a PYL domain and an ABI domain. A and I.P.D. B 25. The synTF of claim 24, comprising: wherein the PYL domain and the ABI domain bind to the inducer substance abscisic acid (ABA).

27. the LIDD is nMag and the complementary LIDD is pMag, wherein nMag is capable of dimerizing with the complementary LIDD pMag upon exposure to a blue light inducible signal; or the LIDD is CIBN and the complementary LIDD is CRY2, and CIBN is capable of dimerizing with the complementary CRY2 upon exposure to a blue-inducer light signal; or The LIDD and the complementary LIDD are photochromic protein domains, which can dimerize upon exposure to a blue-inducing light signal; The synTF of claim 25.

28. 26. The synTF of claim 25, wherein the light inducer signal is a pulsed light signal.

29. The synTF of any one of claims 1 to 17, wherein the second RP is a cytosolic sequestration protein.

30. The synTF of claim 29, wherein the sequestration protein comprises a ligand binding domain (LBD), and in the presence of a ligand, sequestration of the protein into the cytosol is inhibited.

31. The synTF of claim 29, wherein the cytosolic sequestration protein comprises a ligand binding domain and a nuclear localization signal (NLS), wherein in the absence of a ligand, the NLS is inhibited, thereby preventing translocation of the sequestration protein to the nucleus, and in the presence of the ligand, the nuclear localization signal is exposed, allowing translocation of the sequestration protein to the nucleus.

32. The synTF of any one of claims 29 to 31, wherein the sequestering protein comprises at least a portion of an estrogen receptor (ER).

33. The synTF of any one of claims 29 to 32, wherein the sequestering protein comprises an estrogen ligand binding domain (ERT) or a variant thereof selected from the group consisting of ERT) 2, ERT, and ERT3.

34. The synTF of claim 33, wherein the ERT binds to one or more ligands selected from tamoxifen, 4-hydroxytamoxifen (4OHT), endoxifen, and fulvestrant, and the binding of the ligand to the ERT exposes the NLS and results in nuclear translocation of the ERT.

35. The synTF of claim 29, wherein the cytosolic sequestration protein comprises a transmembrane receptor sequestration protein.

36. The synTF of any one of claims 1 to 35, comprising an N-terminal DBD, said sequestration protein, and a C-terminal effector domain.

37. The synTF of claim 36, wherein the effector domain is a transcription activator (TA).

38. The synTF of any one of claims 1 to 35, comprising an N-terminal effector domain, a DBD, and a C-terminal cytosolic sequestration protein.

39. The synTF of claim 38, wherein the effector domain is a transcription repressor (TR).

40. The synTF of any one of claims 19 to 23, wherein the NS3 protease protein is part of a Small molecule-Assisted Shutoff (SMASh) domain, the SMASh domain comprising the NS3 protease protein, a partial protease helical domain, and an NS4A domain.

41. The synTF of any one of claims 1-9 and 24-40, wherein the synTF further comprises a small molecule assisted shutoff (SMASh) tag, the SMASh tag being an N-terminal or C-terminal SMASh domain comprising an inhibitory protease, a partial protease helical domain and a cofactor domain.

42. The synTF of claim 41, wherein the SMASh tag is a C-terminal SMASh domain, the C-terminal SMASh domain comprising, in order from N-terminus to C-terminus, an NS3 cleavage site, at least one linker, an NS3 domain, an NS3 partial helicase, and an NS4A domain, and the SMASh tag is fused to the C-terminus of the effector domain of the synTF.

43. The synTF of claim 41, wherein the SMASh tag is an N-terminal SMASh domain, the N-terminal SMASh domain comprising, in order from the N-terminus to the C-terminus, an NS3 domain, an NS3 partial helicase, an NS4 domain, and an NS3 cleavage site, and the SMASh tag is fused to the N-terminus of the synTF.

44. In the absence of an inhibitor to the NS3 protease protein, the NS3 protease protein is active and self-cleaves / uncouples from the synTF, thereby targeting the SMASh tag for degradation ("SMASh-degradation", synTF-on / TA-on / RP-on); and In the presence of an inhibitor to the NS3 protease protein, NS3 protease protein activity is inhibited, thereby resulting in the SMASh-tagged synTF being targeted for degradation ("synTF-degradation", synTF-off / TA-off / RP-off"); The synTF of any one of claims 40 to 43.

45. 45. The synTF of claim 44, wherein the inhibitor against the NS3 protease protein is selected from any of grazoprevir (GRZ / GZV), danoprevir, simeprevir, asunaprevir, cilprevir, boceprevir, sovaprevir, paritaprevir, ombitasvir, paritaprevir, ritonavir, dasabuvir, and telaprevir.

46. The synTF of any one of claims 24 to 45, comprising a SMASh tag and a cytosolic sequestration protein.

47. The synTF of claim 46, which is active in the presence of a ligand for the cytosol sequestration protein and in the absence of an inhibitor for the NS3 protease protein; and is inactive in the absence of a ligand for the cytosol sequestration protein and in the presence of an inhibitor for the NS3 protease protein.

48. The synTF further comprises a linker peptide, which may be located either between the DBD and the regulatory protein; between the regulatory protein and the effector domain; within the DBD, within the effector domain, or within the regulatory protein; or any combination thereof, and the linker peptide is PGER (SEQ ID NO: 340), TGSQK (SEQ ID NO: 341), TGEKP (SEQ ID NO: 342), THLR (SEQ ID NO: 343), TGGGEKP (SEQ ID NO: 344), FHYDRNNIAVGADESVVKEAHREVINSSTEGLLLNIDKDIRKILSGYIVEIEDTE (SEQ ID NO: 345); VEIEDTE (SEQ ID NO: 346), KDIRKILSGYIVEIEDTE (SEQ ID NO: 347); STEGLLLNIDKDIRKILSGYIVEIEDTE (SEQ ID NO: 348), EVKQENRLLNESES (SEQ ID NO: 349); VGADESVVKEAHREVINSSTEGLLLNIDKDIRKILSGYIVEIEDTE (SEQ ID NO: 350); GGSGG (SEQ ID NO: 67); GGGSG (SEQ ID NO: 70); CVRGS (SEQ ID NO: 73), GGGGSG (SEQ ID NO: 75), GGSGSGSAC (SEQ ID NO: 100), LEGGGGSGG (SEQ ID NO: 103), GGGGSGGT (SEQ ID NO: 104), SGGGSGGSGSS (SEQ ID NO: 345); PGAGSSGDIM (SEQ ID NO: 88) GSSGTGSGSGTS (SEQ ID NO: 90); SGTS (SEQ ID NO: 277); GSGS (SEQ ID NO: 278), GGSGGS (SEQ ID NO: 303), or GSSGSS (SEQ ID NO: 323) The synTF of any one of claims 1 to 47, wherein the synTF is selected from any one of the following:

49. a. at least one synthetic transcription factor (synTF) comprising at least one DNA binding domain (DBD), a transcription effector domain (ED), and a first and a second regulatory protein (RP); The ED is indirectly coupled to the DBD, and The coupling is regulated by the first RP, the first RP is located between the DBD and the ED, and the first RP is an inhibitory protease or an inducible proximity domain pair (IPD pair), and The coupling is further regulated by the second RP, the second RP being located between the DBD and the ED, and the second RP being an inhibitory protease or an inducible proximity domain pair (IPD pair), or the cellular localization of the ED coupled to the DBD is regulated by the second RP being a cytosolic sequestration domain; the first and second RPs are each regulated by an RP inducer; the DBD is capable of binding to a target DNA-binding motif (DBM) located upstream of a promoter operably linked to a gene; The synTF, at least one target DNA binding motif (DBM) comprising a target nucleic acid for binding of at least one DBD of the bi-synTF; and ii. a promoter sequence located 3' of at least one DBM; and iii. A gene of interest operably linked to a promoter sequence. A nucleic acid construct comprising: A system for controlling gene expression comprising: For a synTF, in which the first and / or second RP regulates coupling between the ED and the DBD, In the presence of an RP inducer, coupling between the ED and DBD of the synTF is maintained, allowing the ED to come into close proximity to the promoter sequence upon binding of the DBD to the DNA-binding motif (DBM), where the ED controls expression of the gene of interest ("ED-on"); or In the absence of an RP inducer, the coupling of the ED and DBD of the synTF is dissociated, preventing the ED from approaching the promoter sequence upon binding of the DBD to the DNA-binding motif (DBM), thereby preventing gene expression of the gene of interest ("ED-off"); and For synTFs, where a second regulatory protein regulates the cellular localization of the ED coupled to the DBD, In the presence of an RP inducer, the ED coupled to the DBD of the synTF is not sequestered in the cytosol, allowing the DBD to bind to the DNA-binding motif (DBM), and thus allowing the transcription effector domain (ED) to be in close proximity to the promoter sequence to control expression of the gene of interest ("ED-on"); or In the absence of an RP inducer, the ED coupled to the DBD of the synTF is sequestered in the cytosol, preventing binding of the DBD of the synTF to the DBM and preventing the effector domain (ED) from coming into close proximity with the promoter sequence, thereby preventing expression of the gene of interest ("ED-off"); The system.

50. The transcription effector domain (ED) is a transcription activator (TA), a. for a synTF, wherein said first and / or second RP regulates coupling between the ED and DBD, i. In the presence of an RP inducer, coupling between the ED and DBD of the synTF is maintained, allowing the TA domain to approach the promoter sequence upon binding of the DBD to the DNA-binding motif (DBM), where the TA turns on expression of the gene of interest ("TA-on"); or ii. in the absence of an RP inducer, coupling between the ED and DBD of the synTF is dissociated, preventing the TA domain from approaching the promoter sequence upon binding of the DBD to the DNA-binding motif (DBM), thereby preventing expression of the gene of interest ("TA-off"); and b. For a synTF, wherein the second RP regulates the cellular localization of an ED coupled to the DBD, i. in the presence of an RP inducer, the ED coupled to the DBD of the synTF is not sequestered in the cytosol, allowing the DBD to bind to a DNA-binding motif (DBM), and thus allowing the TA domain to come into close proximity with the promoter sequence to turn on expression of the gene of interest ("TA-on"); or ii. in the absence of RP inducer, the ED coupled to the DBD of the synTF is sequestered in the cytosol, preventing binding of the DBD to the DBM and preventing the proximity of the TA domain to promoter sequences, thereby preventing expression of the gene of interest ("TA-off"); 50. The system of claim 49.

51. ED is a transcriptional repressor (TR), a. for a synTF, wherein coupling of the ED to the DBD is regulated by the first and / or second regulatory protein, i. In the presence of an RP inducer, coupling between the ED and DBD of the synTF is maintained, allowing the binding of the DBD to the DNA-binding motif (DBM) to bring the transcriptional repressor (TR) into close proximity to the promoter sequence, where the TR prevents expression of the gene of interest ("TR-on" (no expression) or ii. In the absence of RP inducer, the coupling between the ED and DBD of the synTF is dissociated, and the binding of the DBD to the DNA-binding motif (DBM) prevents the transcriptional repressor (TR) from approaching the promoter sequence, thereby allowing expression of the gene of interest ("TR-off" (expressed)); and b. For a synTF, wherein the second regulatory protein regulates the cellular localization of the ED coupled to the DBD, i. In the presence of an RP inducer, the ED coupled to the DBD of the synTF is not sequestered in the cytosol, allowing the DBD to bind to the DNA-binding motif (DBM), and thus allowing the transcriptional repressor (TR) to come into close proximity with the promoter sequence to turn off expression of the gene of interest ("TR-on" (no expression) or ii. in the absence of RP inducer, the ED coupled to the DBD of the synTF is sequestered in the cytosol, preventing binding of the DBD to the DBM and preventing the transcriptional repressor (TR) from coming into close proximity with the promoter sequence, thereby allowing expression of the gene of interest (with "TR-off" expression); 50. The system of claim 49.

52. The system of any one of claims 49 to 51, wherein the synTF is selected from any one of the synTFs of any one of claims 1 to 48.

53. the at least one synTF further comprises a small molecule auxiliary shutoff (SMASh) domain at the N-terminus or C-terminus, the SMASh domain comprising a self-cleaving SMASh protease, a partial protease helical domain, and a cofactor domain; wherein in the presence of an inhibitor of the SMASh protease, the SMASh protease activity is inhibited, resulting in the synTF being degraded and preventing the binding of the DBD of the synTF to the DBM, and thus controlling the expression or repression of a gene of interest ("synTF-degradation"; TA-off (no expression), TR-off (expression)); In the absence of an inhibitor to the SMASh protease, the SMASh protease is active and self-cleaves / uncouples from the synTF, resulting in the SMASh domain being targeted for degradation, and thus allowing the DBD of the synTF to bind to the DBM and ED of the synTF to control expression of the gene of interest ("SMASh-degraded, TA-on (expressed), TR-on (no expression)"); 53. The system of any one of claims 49 to 52.

54. 54. The system of any one of claims 49 to 53, wherein the promoter is selected from the group consisting of mini-CMV promoter, mini-TK promoter, ybTATA promoter, minSV40 promoter, CMV53 promoter, pJB42CAT5 promoter, MLP promoter, TATA promoter, pSFFV promoter, CMV promoter, pUb / UbC promoter, EF1a promoter, PGK / pGK promoter, CAG / CAGG promoter, SV40 promoter, and beta-actin / ACTB promoter.

55. a. a first nucleic acid sequence comprising at least one target DNA binding motif (DBM) comprising a target nucleic acid for binding of 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) operably linked to the promoter sequence; and b. A second nucleic acid sequence comprising a nucleic acid encoding a synthetic transcription factor (synTF) according to any one of claims 1 to 48, operably linked to an inducible or constitutive promoter. comprising a cell.

56. a. a nucleic acid sequence encoding a gene of interest (GOI) in reverse orientation; b. a first promoter sequence in a reverse orientation and operably linked to a nucleic acid encoding the GOI; c. a nucleic acid sequence comprising at least one target DNA binding motif (DBM) comprising a target nucleic acid for binding of at least one DBD of a synTF, wherein binding of the DBD of the synTF places an effector domain (ED) in close proximity to the promoter sequence operably linked to the GOI; d. a second promoter sequence, and e. a nucleic acid sequence encoding a synthetic transcription factor (synTF) operably linked to the second promoter sequence, the encoded synTF comprising at least one DBD that binds to at least one DBM of the nucleic acid sequence of (c). The cell of claim 55, comprising a nucleic acid construct comprising in a 5' to 3' direction:

57. The cell of claim 55 or 56, wherein the promoter sequence operably linked to the GOI is selected from the group consisting of a mini-CMV promoter, a mini-TK promoter, a ybTATA promoter, a minSV40 promoter, a CMV53 promoter, a pJB42CAT5 promoter, an MLP promoter, and a TATA promoter.

58. The cell of claim 55 or 56, wherein the promoter sequence operably linked to the nucleic acid encoding the synTF is selected from the group consisting of a pSFFV promoter, a CMV promoter, a pUb / UbC promoter, an EF1a promoter, a PGK / pGK promoter, a CAG / CAGG promoter, an SV40 promoter, and a beta-actin / ACTB promoter.

59. A polynucleotide encoding the synTF of any one of claims 1 to 48 or the synTF system of any one of claims 49 to 54.

60. 60. A vector comprising the polynucleotide of claim 59.

61. A synTF according to any one of claims 1 to 48, a synTF system according to any one of claims 49 to 54, a synTF cell according to any one of claims 55 to 58, a polynucleotide according to claim 59, or a vector according to claim 60. A composition comprising:

62. A synTF according to any one of claims 1 to 48, a synTF system according to any one of claims 49 to 54, a synTF cell according to any one of claims 55 to 58, a polynucleotide according to claim 59, or a vector according to claim 60, A pharma- ceutically acceptable carrier; 23. A pharmaceutical composition comprising:

63. a. providing a population of cells according to any one of claims 55 to 58; and b. contacting the population of cells with an effective amount of a regulatory protein inducer. A method for regulating the activity of synTF in vitro, comprising:

64. a. providing a population of cells according to any one of claims 55 to 58; and b. contacting the population of cells with an effective amount of a regulatory protein inducer. A method for regulating expression of a gene of interest in vitro, comprising:

65. A composition comprising the population of cells according to any one of claims 55 to 58 for use in a method of treating a subject in need of cell therapy, the method comprising: a. administering to said subject a population of cells according to any one of claims 55-58; and b. administering to the subject an effective amount of a regulatory protein inducer. A composition comprising:

66. 66. The composition of claim 65, wherein said population of cells comprises immune cells.

67. The composition of claim 66, wherein the population of immune cells comprises CD4+ T cells, CD8+ T cells, Tregs, or NK cells.

68. The composition of any one of claims 65-67, wherein said regulatory protein inducer is administered simultaneously with said population of cells.

69. The composition of any one of claims 65-67, wherein said regulatory protein inducer is administered after said population of cells is administered.

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