Regulated synthetic gene activation systems

Abstract

The technology described herein is directed to regulated synthetic gene expression systems. In one aspect described herein are synthetic transcription factors (synTFs) comprising a DNA binding domain, a transcriptional activator domain, a transcriptional effector domain (TED), and optionally a regulator protein. In other aspects described herein are gene expression systems comprising said synTFs and methods of treating diseases and disorders using said synTFs.

Description

Attorney Docket No: 701586-000114WOPT REGULATED SYNTHETIC GENE ACTIVATION SYSTEMS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No.63 / 523,569 filed June 27, 2023, the contents of which are incorporated herein by reference in their entirety. GOVERNMENT SUPPORT

[0002] This invention was made with government support under contract No. R01EB029483 awarded by the National Institutes of Health. The government has certain rights in the invention. TECHNICAL FIELD

[0003] The technology described herein relates to regulated synthetic gene activation systems, including humanized hybrid transcription activator domains (hhTADs) and constructs comprising them. BACKGROUND

[0004] Cells function as intricate information processors, capable of sensing a wide range of environmental cues, carrying out elaborate calculations, and generating numerous outcomes, such as gene expression, signal molecule release, morphological alterations, and cell growth. Additionally, various cell types have developed unique abilities to adapt to diverse environments and execute specific functions. These characteristics position cells as prime candidates for intelligent therapeutics that offer improved safety and effectiveness. In fact, several cell types have been explored for the creation of cell therapies, encompassing bacteria and stem cell treatments.

[0005] Cell-based therapies offer numerous benefits, including maintaining persistence in patients for extended disease management, involving endogenous cells in a unified response, and applying synthetic biology to achieve precise control over their functions. The capabilities of engineered cell systems have advanced in several areas, such as identifying and targeting cancer cells, regulating the spatiotemporal activity of therapies in response to drugs or antigens, and guiding stem cells to differentiate into intricate tissues. Specifically, human immune cells have emerged as one of the most crucial cell types for developing therapeutics, especially in the context of cancer treatment.

[0006] There is however a need to simultaneously tackle safety and efficacy challenges to develop efficient immune cell therapies. This can be achieved via enhancing precision and control. Synthetic biology, which centers on the predictable reprogramming of living cells, holds great potential in this area. More specifically, mammalian synthetic biology is devoted to creating tools and innovative gene circuits to regulate and reprogram diverse functions in mammalian cells. 1 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT

[0007] A key aspect of synthetic biology is the engineering of new biological functions through the identification, analysis, and repurposing of molecular components. The components can then be used to attain the capability to predict and generate a desired output level for any given input. There is great need to manage simple inputs, such as small molecules, which in turn govern more intricate outputs, for example, to regulate immune responses to specific cancer antigens in engineered T cells. SUMMARY

[0008] The technology described herein is directed to compositions and methods to control gene expression. In general, the technology described herein relates to constructs comprising a transcriptional effector domain (TED) in combination with a transcriptional activator (TA) domain. The TED increases the transcriptional activation of the TA compared to a construct with the TA but lacking the TED. The TED can be derived from a human protein, such as the TIMs domain of human IWS1. Use of constructs comprising such human domains allows for clinical use, without concern for foreign immune-activation, such as can occur with non-human (e.g., virally derived) constructs. The TEDs described herein can function in both dual vector and single vector systems. The TEDs described herein can drive more robust expression of a large payload compared a construct not comprising the TED (see e.g., Fig.15).

[0009] In some aspects, described herein are synthetic transcription factors (synTFs) comprising the TED and TA, which further comprise a regulator protein (RP), where the regulator protein regulates the activity of the synTF. In particular, the synTFs according to the methods, systems and compositions as disclosed herein comprise (i) a DNA binding domain (DBD) which binds to a target nucleic acid sequence (or target DNA binding motif (DBM)) located 3’ of a promoter that is operatively linked to the nucleic acid of a gene of interest (GOI) to be expressed, (ii) an effector domain (ED) (e.g., TA and TED) and a regulator protein (RP), where the regulator protein controls the coupling of the DNA binding domain (DBD) with the effector domain (ED), 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 can function to recruit translation machinery to the promoter to regulate gene expression of a gene of interest. In some embodiments, the ED can be a transcriptional activator (TA) in combination with a transcriptional effector domain (TED), thereby turning on gene expression when the ED is present at the transcription start site of a gene of interest.

[0010] In some embodiments of the systems, compositions and methods as disclosed herein, the regulator protein of the SynTF is selected from a protease, a pair of inducible proximity domains (IPDs) or a translocation domain (i.e., a cytosolic sequestering protein), each of which are described herein and in more detail below.

[0011] In some embodiments of the systems, compositions and methods as disclosed herein, the synTF comprises a regulator protein that is a self-cleaving protease, for example, one exemplary 2 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT protease is NS3. SynTFs comprising self-cleaving proteases can also be referred to herein as “repressible proteases SynTF”. In such embodiments of the systems, compositions and methods disclosed herein, the DBD is directly linked or indirectly linked (or coupled) to the effector domain, and the protease regulator protein (typically located between the DBD and ED) controls the coupling of the DBD to the ED. In such an embodiment, in the presence of an agent which inhibits the regulator protein (e.g., 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). In such an embodiment, in the absence of an agent which inhibits the regulator protein (e.g., NS3 protein), the linkage between the DBD and ED is broken or cleaved, and therefore the ED is not brought into proximity of the transcription start site of the gene (or the ED dissociates from the start site), and therefore the TA can no longer initiate gene expression of the GOI.

[0012] In another embodiment of the systems, compositions and methods as disclosed herein, the synTF comprises a regulator protein that is a pair of inducer proximity domains (referred to as an “IPD pair”) which is located between the DBD and ED, where each domain of the IPD come together in the presence of an inducer agent, and therefore linking the DBD and ED and controlling gene expression. SynTFs comprising an IPD pair can also be referred to herein as a “heterodimerization domain SynTF”. For example, in such embodiments, where the regulator 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 bind to the inducer agent, thereby indirectly coupling the DBD with the ED, such that when the DBD binds to a promoter region, the ED can control gene expression from the promoter (i.e., turning on gene expression if the ED is a TA). In alternative embodiments where the RP is an IPD pair, in the absence of the inducer agent, the DBD and ED remain uncoupled, and therefore the ED is not in a position to regulate gene transcription from the transcription start site at the GOI. Exemplary IPD pairs and their inducing agents are disclosed herein.

[0013] In some embodiments of the systems, compositions and methods as disclosed herein, the synTF comprises a regulator protein that is a translocation domain. In some embodiments, a translocation domain is a cytosolic sequestering protein, for example, one exemplary cytosolic sequestering protein is ERT2 and variants thereof. SynTFs comprising a translocation domain, e.g., a cytosolic sequestering protein can also be referred to herein as “Translocation Domain SynTF”. In such embodiments of the systems, compositions and methods disclosed herein, the DBD is directly linked or indirectly linked (or coupled) to the effector domain, and the translocation domain, e.g., a cytosolic sequestering protein regulator protein (which can be attached to either the ED, or DBD or located between the DBD and ED) controls the cellular localization of the synTF comprising the DBD-ED. In such an embodiment, in the absence of a ligand that binds to the cytosolic sequestering 3 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT protein, the cytosolic sequestering protein sequesters the ED and coupled DBD in the cytosol, and therefore the ED is not brought into proximity of the transcription start site of the gene (or the ED dissociates from the start site), and therefore the TA can no longer initiate gene expression of the GOI. In contrast, in the presence of a ligand that binds to the cytosolic sequestering protein, the cytosolic sequestering 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., turning on gene expression of the gene of interest (GOI) if the ED is a TA).

[0014] Another aspect of the systems, compositions and methods as disclosed herein are synTFs comprising a small-molecule assisted shutoff (SMASh) domain, which can also be referred to herein as an “induced degradation domain.” In general, SMASh domains function to target the polypeptide that is attached to the SMASh domain for degradation. In some embodiments, the SMASh domain is attached to a synTF comprising a regulator protein that is an inducer proximity domain pair (IPD) or a cytosolic sequestering protein. In alternative embodiments, the regulator protein can be a SMASh domain (i.e., the SMASh domain replaces a self-cleaving protease regulator protein in the synTF).

[0015] In all aspects of the methods, systems and compositions disclosed herein, a SMASh domain comprises a self-cleaving protease and a degron domain. In some embodiments, the self- cleaving protease is a NS3 protease or variant thereof as disclosed herein. In some embodiments, the self-cleaving protease of the SMASh domain comprises: an NS3 protease domain, a partial NS3 helical domain, and NS4A domain, and can be fused to the N-terminal or C-terminal of a synTF described herein. Without being limited to theory and by way of explanation only, when a SMASh domain is attached to a synTF as disclosed herein, and when there is an inhibitor of the SMASh domain self-cleaving protease 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” - that is because the synTF is degraded, the synTF cannot bind to the DBM, or regulate the expression of the gene of interest, regardless of the type of effector domain present in the synTF. Conversely, when an inhibitor of the self-cleaving protease is absent, the self-cleaving protease cleaves (or uncouples) the SMASh domain from the synTF, and only the SMASh domain is targeted for degradation, and the activity of the released synTF is regulated by way of the regulator protein. As such, when a SMASh domain is attached to the synTF, in the absence of the protease inhibitor, it is referred to “SMASh-degradation” and results in the synTF being “SynTF-ON” permitting the synTF to be regulated by the regulator protein, and gene expression can occur when the ED is a transcription activator. Accordingly, the presence of a SMASh domain attached to the synTF enables a second level of control for the expression of the GOI in addition to the regulator protein. 4 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT

[0016] In some embodiments, the SMASh domain by itself serves as the regulator protein of a synTF (i.e., SMASh domain replaces a self-cleaving protease regulator protein), and is referred to as an Induced Degradation Domain SynTF. In such embodiments, where the SMASh domain serves as the regulator protein, the SMASh domain can be attached to either the ED or the DBD of the synTF, and in the absence of a NS3 protease inhibitor, the NS3 protease is active and the SMASh domain uncouples from the synTF, thereby resulting in only the SMASh domain being targeted for degradation, and the synTF comprising the DBD and the coupled ED enabling to control gene expression from the promoter (i.e., the DBD binds to the DBM, bringing the ED in close proximity to the promoter and turning on gene expression of the GOI if the ED is a TA). In such an embodiment, in the absence of an agent which inhibits the regulator protein (e.g., NS3 protein), the linkage between the DBD and ED is broken or cleaved, and therefore the ED is not brought into proximity of the transcription start site of the gene (or the ED dissociates from the start site), and therefore the TA can no longer initiate gene expression of the GOI.

[0017] In some embodiments, where the SMASh domain is attached to translocation domain synTF (e.g., where the regulator protein is a sequestering protein), or a heterodimerization domain synTF (i.e., where the regulator protein is pair of inducible proximity domains (IPD pair)), the SMASH domain can be referred to as a “SMASh tag” and can be attached to the C-terminal or N- terminal of a synTF. By way of an example only, a C-terminal SMASh tag attached to the C-terminal of an ED or regulator protein of a synTF can comprise in the following N-terminal to C-terminal order: a NS3 cleavage site, at least one linker, a NS3 domain, a NS3 partial helicase, a NS4A domain, wherein the SMASh tag is fused to the C-terminus of the effector domain of the synTF. In some embodiments and by way of an example only, where a SMASh tag is fused to the N-terminus of a synTF (referred to herein as a “N-terminal SMASh tag”), the SMASh tag comprises in a N-terminal to C-terminal order: at least one Linker, a NS3 domain, a NS3 partial helicase, a NS4 domain, and a NS3 cleavage site, wherein the SMASh tag is fused to the N-terminus of the synTF.

[0018] Another aspect of the technology disclosed herein relates to a system for controlling gene expression of a gene of interest (GOI), where the system comprises a synTF described herein and a nucleic acid construct comprising the elements that the synTF binds to regulate gene expression. In particular, in some aspects, the system comprising (i) at least one synthetic transcription factor (synTF) as disclosed herein, and (ii) at least a nucleic acid construct, where the synTF comprises at least one DNA binding domain (DBD), a transcriptional effector domain (ED) (e.g., TA and TED), and at least one regulator protein (RP), and where the ED is directly or indirectly coupled or linked to the DBD, and where 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 where the at least one RP is regulated by an RP inducer, where the DBD can bind to a target DNA binding motif (DBM) located upstream of a promoter operatively linked to a gene, and where the nucleic acid construct 5 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT 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) operatively linked to the promoter sequence. In some embodiments where the regulator protein of the synTFs regulates the coupling of the ED to the DBD (e.g., protease domain synTF or induced proximity domain synTFs), in the presence of the RP inducer, the coupling of the ED to the DBD of the synTF is maintained, permitting the ED to be in proximity to the promoter sequence when the DBD binds to the DNA binding motif (DBM), where the ED controls the expression of the gene of interest (“ED-on”). In embodiments where the ED is a transcriptional activator (TA), it results in turning on gene expression (“TA-on” (expression)). In contrast, where the RP inducer is absent, the coupling of the ED to the DBD of the synTF is severed, preventing the ED from being in proximity to the promoter sequence when the DBD binds to the DNA binding motif (DBM), preventing gene expression of the gene of interest (“ED-off”). In embodiments where the ED is a transcriptional activator (TA), it results in turning off the gene expression (“TA-off” (no expression)).

[0019] In embodiments where the regulator protein of the 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, permitting the DBD to bind to the DNA binding motif (DBM) and permitting the transcriptional effector domain (ED) to be in proximity to the promoter sequence to control the expression of the gene of interest (“ED-on”). In embodiments where the ED is a transcriptional activator (TA), it results in turning on gene expression (“TA-on” (expression)).

[0020] Moreover, when the RP inducer is absent, the ED coupled to the DBD of the synTF is sequestered in the cytosol, preventing the DBD of the synTF from binding to the DBM, and preventing the effector domain (ED) from being in proximity to the promoter sequence, preventing expression of the gene of interest (“ED-off”). In embodiments where the ED is a transcriptional activator (TA), it results in turning off the gene expression (“TA-off” (no expression)).

[0021] Table 2: table summarizing the influence of the presence or absence of regulator protein inducers on ultimate expression of the gene of interest.6 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT

[0022] Accordingly, whether gene expression of the GOI occurs is dependent on 3 levels of control, including but not limited to; (i) the type of regulator protein in the synTF, (ii) the presence or absence of a regulator protein inducer (RP inducer), and (iii) the type of effector domain (e.g., presence or absence of TED in combination with the TA).

[0023] Moreover, in some embodiments, the system for controlling gene expression can be configured for an additional level of control for the gene expression, depending on whether there is a SMASh domain attached to the synTF, as disclosed herein. For example, attachment of a SMASh domain to a synTF will result in the following outcomes: if an inhibitor to the SMASh protease is present, the SMASh protease activity is inhibited, resulting in the synTF being degraded (“Syn- degradation”) and preventing the DBD of the synTF binding to the DBM and controlling the expression of the gene of interest (“synTF-degradation”; TA-off (no expression). In alternative embodiments, if an inhibitor to the SMASh protease is absent, the SMASh protease is active and self cleaves / uncouples from the synTF, resulting the SMASh domain being targeted for degradation and allowing the DBD of the synTF to bind to the DBM and the ED of synTF to control the expression of the gene of interest (“SMASh-degradation”, TA-on (yes-expression)).

[0024] Other aspects of the technology described herein relate to a cell comprising the nucleic acid sequences as disclosed herein for binding of the synTF to regulate the expression of the gene of interest, and also a nucleic acid encoding the 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 and referred to as a “single vector”.

[0025] Other embodiments will become readily apparent from the disclosure. Aspects of the present invention teach certain benefits in construction and use which give rise to the exemplary advantages described below.

[0026] 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 invention.

[0027] Accordingly, in one aspect described herein is a synthetic transcription factor (synTF) comprising: (a) at least one DNA binding domain (DBD), (b) a transcriptional activator domain (TA), (c) a transcriptional effector domain (TED), and (d) at least one regulator protein (RP), and wherein the TED increases the transcriptional activation of the synTF compared to an otherwise identical synTF lacking the TED, wherein the TA is directly or indirectly coupled to the DBD, and wherein the coupling is regulated by the at least one RP, or wherein cellular localization of the TA is regulated by the at least one RP. 7 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT

[0028] In some embodiments of any of the aspects, the TED comprises an elongation domain, an activator domain, or a domain with pioneer ability.

[0029] In some embodiments of any of the aspects, the elongation domain is derived from a polypeptide selected from the group consisting of: Interacts with Suppressor Of Ty 6 (Spt6) Homolog (IWS1); Suppressor Of Ty 5 (Spt5) Homolog (SUPT5H); Bromodomain-containing protein 4 (BRD4); and cellular Myelocytomatosis (cMyc).

[0030] In some embodiments of any of the aspects, the elongation domain is derived from IWS1.

[0031] In some embodiments of any of the aspects, the elongation domain is derived from human IWS1.

[0032] In some embodiments of any of the aspects, the elongation domain comprises at least one TFIIS N-terminal domains (TND)-interacting motif (TIM) domain of IWS1.

[0033] In some embodiments of any of the aspects, the elongation domain comprises at least one TIM1, TIM2, and / or TIM3 domain from IWS1.

[0034] In some embodiments of any of the aspects, the elongation domain comprises one of SEQ ID NOs: 5-9 or an amino acid sequence with at least 80% sequence identity to one of SEQ ID NOs: 5- 9.

[0035] In some embodiments of any of the aspects, the activator domain is derived from a polypeptide selected from the group consisting of: Heat Shock Factor 1 (HSF1), Glucocorticoid Receptor (GR), and MLX interacting protein like (MLXIPL).

[0036] In some embodiments of any of the aspects, the domain with pioneer ability is derived from a polypeptide selected from the group consisting of Fused in Sarcoma (FUS) and Ewing Sarcoma Breakpoint Region (EWSR).

[0037] In some embodiments of any of the aspects, the TA is selected from the group consisting of: p65; Rta; miniVPR; full VPR; VP16; VP64; NFZ; 3Z; p300; p300 HAT Core; and a CBP HAT domain; or a variant thereof.

[0038] In some embodiments of any of the aspects, the TA is p65, or a variant thereof.

[0039] In some embodiments of any of the aspects, the p65 comprises one of SEQ ID NOs: 38, 58-67 or an amino acid sequence with at least 80% sequence identity to one of SEQ ID NOs: 38, 58- 67.

[0040] In some embodiments of any of the aspects, the at least one DBD is an engineered zinc finger (ZF)-binding domain.

[0041] In some embodiments of any of the aspects, the engineered ZF-binding domain comprises 2 or more ZF motifs.

[0042] In some embodiments of any of the aspects, the engineered ZF-binding domain comprises 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or more ZF motifs arranged adjacent to each other in tandem to form a ZF array (ZFA). 8 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT

[0043] In some embodiments of any of the aspects, the engineered ZF binding domain comprises a sequence 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 11-1.

[0044] In some embodiments of any of the aspects, the engineered ZF-binding domain comprises one of SEQ ID NO: 36 (ZF10-1), SEQ ID NO: 45 (ZF3-5), or SEQ ID NO: 86 (ZF1-3).

[0045] In some embodiments of any of the aspects, the engineered ZF-binding domain comprises ZF 10-1.

[0046] In some embodiments of any of the aspects, the engineered ZF-binding domain specifically binds to a nucleic acid with a sequence comprising at least one of SEQ ID NO: 100 (ZF10 binding site (BS)), SEQ ID NO: 93 (ZF3 BS), or SEQ ID NO: 91 (ZF1 BS).

[0047] In some embodiments of any of the aspects, the engineered ZF-binding domain comprises SEQ ID NO: 48 and specifically binds endogenous VEGF gene (VEGF ZF).

[0048] In some embodiments of any of the aspects, the at least one RP comprises a polypeptide selected from the group consisting of: a repressible protease; a pair of induced proximity domains (IPD pair); a cytosolic sequestering protein; and combinations thereof.

[0049] In some embodiments of any of the aspects, the at least one RP comprises a repressible protease.

[0050] In some embodiments of any of the aspects, the at least one RP comprises a NS3 protease protein.

[0051] In some embodiments of any of the aspects, the at least one RP comprises the amino acid sequence of SEQ ID NOs: 182-198, or a homologue with at least 80% sequence identity to one of SEQ ID NOs: 182-198.

[0052] In some embodiments of any of the aspects, in the presence of a protease inhibitor, or an inhibitor of NS3, the protease protein is inhibited, thereby maintaining the coupling of the DBD to the TA.

[0053] In some embodiments of any of the aspects, in the absence of a protease inhibitor, or an inhibitor of NS3, the protease protein is active, and the TA is excised from the DBD, thereby uncoupling the DBD and the TA.

[0054] In some embodiments of any of the aspects, the inhibitor of NS3 is selected from the group consisting of: grazoprevir (GRZ / GZV), danoprevir, simeprevir, asunaprevir, ciluprevir, boceprevir, sovaprevir, paritaprevir, ombitasvir, paritaprevir, ritonavir, dasabuvir, and telaprevir. 9 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT

[0055] In some embodiments of any of the aspects, the at least one RP is a pair of induced proximity domains (IPD pair), wherein the IPD pair comprises: a first induced proximity domain (IPDA) and at least a second complementary IPD (IPDB), wherein in the presence of an inducer agent or signal, the IPDAand IPDBspecifically bind together resulting in the coupling of the TA to the DBD, and wherein in the absence of an inducer agent or signal, the TA is uncoupled from the DBD.

[0056] In some embodiments of any of the aspects, the IPD pair comprises: (a) a IPDAcomprising a GID1 domain or a fragment thereof, and a IPDBcomprising a GAI domain, wherein the GID1 domain and GAI domain bind to the inducer agent Gibberellin Ester (GIB); (b) a IPDAcomprising a FKBP domain or a fragment thereof, and a IPDBcomprising a FRB domain, wherein the FKBP domain and FRB domain bind to the inducer agent Rapalog (RAP); (c) a IPDAcomprising a PYL domain or a fragment thereof, and a IPDBcomprising an ABI domain, wherein the PYL domain and ABI domain bind to the inducer agent Abscisic acid (ABA); and / or (d) a IPDAcomprising a Light-inducible dimerization domain (LIDD), wherein a LIDD dimerizes with a complementary LIDD (IPDB) upon exposure to a light inducer signal of an appropriate wavelength.

[0057] In some embodiments of any of the aspects, the LIDD is nMag, Calcium And Integrin- Binding Protein 1 truncation (CIBN), or a photochromic protein domain; wherein nMag can dimerize with a complementary LIDD pMag upon exposure to a blue light inducer signal; or wherein CIBN can dimerize with a complementary cryptochrome 2 (CRY2) upon exposure to a blue inducer light signal; or wherein the photochromic protein domains can dimerize upon exposure to a blue inducer light signal.

[0058] In some embodiments of any of the aspects, the light inducer signal is a pulse light signal.

[0059] In some embodiments of any of the aspects, the at least one RP comprises a cytosolic sequestering protein.

[0060] In some embodiments of any of the aspects, the cytosolic sequestering protein comprises a ligand binding domain (LBD), wherein in the presence of a ligand to which the LBD binds, sequestering of the synTF to the cytosol is inhibited.

[0061] In some embodiments of any of the aspects, the cytosolic sequestering protein comprises a LBD and a nuclear localization signal (NLS); wherein in the absence of a ligand to which the LBD binds, the NLS is inhibited thereby preventing translocation of the synTF to the nucleus; and wherein in the presence of the ligand, the NLS is exposed permitting translocation of the synTF to the nucleus.

[0062] In some embodiments of any of the aspects, the cytosolic sequestering protein comprises at least a portion of an estrogen receptor (ER).

[0063] In some embodiments of any of the aspects, the cytosolic sequestering protein comprises an estrogen ligand binding domain (ERT) or a variant thereof, selected from the group consisting of: SEQ ID NO: 43 (ERT2), SEQ ID NO: 304, and SEQ ID NO: 305 (ERT3). 10 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT

[0064] In some embodiments of any of the aspects, the ERT binds to one or more ligands selected from the group consisting of: tamoxifen, 4-hydroxytamoxifen (4OHT), endoxifen, and Fulvestrant; wherein binding of the ligand to the ERT exposes the NLS and results in nuclear translocation of the ERT.

[0065] In some embodiments of any of the aspects, the cytosolic sequestering protein comprises a transmembrane receptor sequestering protein.

[0066] In some embodiments of any of the aspects, the NS3 protease protein is part of a Small molecule-Assisted Shutoff (SMASh) domain, wherein the SMASh domain comprises the NS3 protease protein, a partial protease helical domain and a NS4A domain.

[0067] In some embodiments of any of the aspects, the synTF further comprises a Small molecule-Assisted Shutoff (SMASh) domain, wherein the SMASh domain is a N-terminal or C- terminal SMASh domain comprising a repressible protease, a partial protease helical domain and a cofactor domain.

[0068] In some embodiments of any of the aspects, the SMASh domain is a C-terminal SMASh domain comprising, in N-terminal to C-terminal order: a NS3 cleavage site, at least one linker, a NS3 domain, a NS3 partial helicase, and a NS4A domain, wherein the SMASh domain is fused to the C- terminus of the synTF.

[0069] In some embodiments of any of the aspects, the SMASh domain is a N-terminal SMASh domain comprising in N-terminal to C-terminal order: at least one Linker, a NS3 domain, a NS3 partial helicase, a NS4 domain, and a NS3 cleavage site, wherein the SMASh domain is fused to the N-terminus of the synTF.

[0070] In some embodiments of any of the aspects, in the absence of an inhibitor for the NS3 protease, the NS3 protease is active and self cleaves / uncouples from the synTF, thereby resulting in the SMASh domain targeted for degradation (“SMASh-degradation”, synTF-on / TA-on), and wherein in the presence of an inhibitor for the NS3 protease, NS3 protease activity is inhibited thereby resulting in the SMASh-comprising synTF targeted for degradation (“synTF-degradation”, synTF- OFF / TA-off”).

[0071] In some embodiments of any of the aspects, the inhibitor for the NS3 protease is selected from the group consisting of: grazoprevir (GRZ / GZV), danoprevir, simeprevir, asunaprevir, ciluprevir, boceprevir, sovaprevir, paritaprevir, ombitasvir, paritaprevir, ritonavir, dasabuvir, and telaprevir.

[0072] In some embodiments of any of the aspects, the synTF comprises a SMASh domain and a cytosolic sequestering protein.

[0073] In some embodiments of any of the aspects, the synTF is active in the presence of the ligand for the cytosolic sequestering protein and in the absence of the inhibitor for the NS3 protease; 11 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT and wherein the synTF is inactive in the absence of the ligand for the cytosolic sequestering protein and / or in the presence of the inhibitor for the NS3 protease.

[0074] In some embodiments of any of the aspects, the synTF further comprises a linker peptide, wherein the linker peptide can be positioned anywhere from: between the DBD and the RP; between the RP and TA; between the DBD and TA; between the TED and TA; between the TED and DBD; between the TED and RP; within the DBD, TA, TED, or regulator protein; or any combination thereof.

[0075] In some embodiments of any of the aspects, the DBD, TA, TED, and / or RP are human domains or humanized domains.

[0076] In some embodiments of any of the aspects, the DBD, TA, TED, and RP are human domains or humanized domains.

[0077] In one aspect described herein is a synthetic transcription factor (synTF) comprising: (a) at least one DNA binding domain (DBD), (b) a transcriptional activator domain (TA), and (c) a transcriptional effector domain (TED), wherein the TED increases the transcriptional activation of the synTF compared to an otherwise identical synTF lacking the TED.

[0078] In one aspect described herein is a hybrid transcription activator domain (hTAD), comprising: (a) a transcriptional activator domain (TA), and (b) a transcriptional effector domain (TED), wherein the TED increases the transcriptional activation of the TA compared to an otherwise identical hTAD lacking the TED.

[0079] In some embodiments of any of the aspects, the TA and / or TED are human domains or humanized domains.

[0080] In one aspect described herein is a synthetic transcription factor (synTF) comprising an hTAD as described herein and at least one DNA binding domain (DBD).

[0081] In some embodiments of any of the aspects, the synTF further comprises at least one regulator protein (RP).

[0082] In one aspect described herein is a humanized hybrid transcription activator domain (hhTAD), comprising: (a) a transcriptional activator domain (TA), and (b) a transcriptional effector domain (TED), wherein the TED increases the transcriptional activation of the TA compared to an otherwise identical hhTAD lacking the TED; and wherein the TA and TED are human domains or humanized domains.

[0083] In one aspect described herein is a synthetic transcription factor (synTF) comprising an hhTAD as described herein and at least one DNA binding domain (DBD).

[0084] In some embodiments of any of the aspects, the synTF further comprises at least one regulator protein (RP).

[0085] In one aspect described herein is a system for controlling gene expression, comprising: (a) at least one synthetic transcription factor (synTF) as described herein, wherein the at least one DBD of 12 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT the synTF can bind to a target DNA binding motif (DBM) located upstream of a promoter operatively linked to a gene; and (b) a nucleic acid construct comprising: (i) at least one target DNA binding motif (DBM) comprising a target nucleic acid for binding of the at least one DBD of the synTF; (ii) a promoter sequence located 3’ of the at least one DBM; and (iii) a gene of interest operatively linked to the promoter sequence.

[0086] In some embodiments of any of the aspects, the synTF comprises at least one RP regulated by an RP inducer.

[0087] In some embodiments of any of the aspects, the coupling of the TA to the DBD is regulated by the at least one RP.

[0088] In some embodiments of any of the aspects, the RP comprises a repressible protease or an IPD pair.

[0089] In some embodiments of any of the aspects, in the presence of the RP inducer, the coupling of the TA to the DBD of the synTF is maintained, permitting the TA to be in proximity to the promoter sequence when the DBD binds to the DBM, and wherein the TA turns on expression of the gene of interest (“TA-on”).

[0090] In some embodiments of any of the aspects, in the absence of the RP inducer, the coupling of the TA to the DBD of the synTF is severed, preventing the TA from being in proximity to the promoter sequence when the DBD binds to the DBM, preventing expression of the gene of interest (“TA-off”).

[0091] In some embodiments of any of the aspects, cellular localization of the TA linked to the DBD is regulated by the at least one RP.

[0092] In some embodiments of any of the aspects, the RP comprises a cytosolic sequestering protein.

[0093] In some embodiments of any of the aspects, in the presence of the RP inducer, the TA coupled to the DBD of the synTF is not sequestered in the cytosol, permitting the DBD to bind to the DNA binding motif (DBM) and permitting the TA domain to be in proximity to the promoter sequence to thereby turn on expression of the gene of interest (“TA-on”).

[0094] In some embodiments of any of the aspects, in the absence of the RP inducer, the TA coupled to the DBD of the synTF is sequestered in the cytosol, preventing the DBD from binding to the DBM, and preventing the TA domain from being in proximity to the promoter sequence, thereby preventing expression of the gene of interest (“TA-off”).

[0095] In some embodiments of any of the aspects, the at least one synTF further comprises a N- terminal or C-terminal Small molecule-Assisted Shutoff (SMASh) domain, wherein SMASh domain comprises a self-cleaving SMASh protease, a partial protease helical domain and a cofactor domain.

[0096] In some embodiments of any of the aspects, in the presence of an inhibitor to the SMASh protease, the SMASh protease activity is inhibited, resulting in the synTF being degraded and 13 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT preventing the DBD of the synTF binding to the DBM and controlling the expression of the gene of interest (“synTF-degradation”; TA-off (no expression)).

[0097] In some embodiments of any of the aspects, in the absence of an inhibitor to the SMASh protease, the SMASh protease is active and self cleaves / uncouples from the synTF, resulting the SMASh domain being targeted for degradation and allowing the DBD of the synTF to bind to the DBM and the TA of synTF to control the expression of the gene of interest (“SMASh-degradation, TA-on (yes-expression)).

[0098] In some embodiments of any of the aspects, the promoter is selected from the group consisting of: miniCMV promoter, miniTK 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.

[0099] In one aspect described herein is a system 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 a gene of interest (GOI) operatively linked to the promoter sequence; and (b) a second nucleic acid sequence comprising a nucleic acid encoding a synthetic transcription factor (synTF) according to any one of claims 1-51, 54-55, 57 and 58, operatively linked to an inducible or constitutive promoter.

[0100] In some embodiments of any of the aspects, the promoter sequence operatively linked to the GOI is selected from the group consisting of: miniCMV promoter, miniTK promoter, ybTATA promoter, minSV40 promoter, CMV53 promoter, pJB42CAT5 promoter, MLP promoter, and TATA promoter.

[0101] In some embodiments of any of the aspects, the promoter sequence operatively linked to the nucleic acid encoding the synTF is selected from the group consisting of pSFFV promoter, CMV promoter, pUb / UbC promoter, EF1a promoter, PGK / pGK promoter, CAG / CAGG promoter, SV40 promoter, and beta actin / ACTB promoter.

[0102] In one aspect described herein is a polynucleotide encoding a synTF as described herein; a hTAD as described herein; a hhTAD as described herein; or a system as described herein; or portion thereof.

[0103] In one aspect described herein is a nucleic acid construct, comprising in the 5’ to 3’ direction: (a) a nucleic acid sequence encoding a gene of interest (GOI) in the inverse orientation, (b) a first promoter sequence in the inverse orientation and operatively 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 synthetic transcription factor (synTF), wherein binding of the DBD places a TA of the synTF in the proximity of the promoter 14 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT sequence operatively linked to the GOI, (d) a second promoter sequence, and (e) a nucleic acid sequence encoding the synTF, operatively linked to the second promoter sequence, wherein the encoded synTF comprises at least one DBD that binds to the at least one DBM of the nucleic acid sequence of (c).

[0104] In some embodiments of any of the aspects, the promoter sequence operatively linked to the GOI is selected from the group consisting of: miniCMV promoter, miniTK promoter, ybTATA promoter, minSV40 promoter, CMV53 promoter, pJB42CAT5 promoter, MLP promoter, and TATA promoter.

[0105] In some embodiments of any of the aspects, the promoter sequence operatively linked to the nucleic acid encoding the synTF is selected from the group consisting of a pSFFV promoter, CMV promoter, pUb / UbC promoter, EF1a promoter, PGK / pGK promoter, CAG / CAGG promoter, SV40 promoter, and beta actin / ACTB promoter.

[0106] In one aspect described herein is a vector comprising a system as described herein; a polynucleotide as described herein; or a nucleic acid construct as described herein; or portion thereof.

[0107] In some embodiments of any of the aspects, the vector is a lentiviral vector.

[0108] In one aspect described herein is a cell comprising a synTF as described herein; a hTAD as described herein; a hhTAD as described herein; a system as described herein; a polynucleotide as described herein; a nucleic acid construct as described herein; or a vector as described herein; or portion thereof.

[0109] In some embodiments of any of the aspects, the cell is an immune cell.

[0110] In some embodiments of any of the aspects, the immune cell is selected from the group consisting of: a CD4+ T cell, a CD8+ T cell, a Treg, an NK cell, a monocyte, and a macrophage.

[0111] In one aspect described herein is a composition comprising a synTF as described herein; a hTAD as described herein; a hhTAD as described herein; a system as described herein; a polynucleotide as described herein; a nucleic acid construct as described herein; a vector as described herein; a cell as described herein; or portion thereof.

[0112] In one aspect described herein is a pharmaceutical composition comprising a synTF as described herein; a hTAD as described herein; a hhTAD as described herein; a system as described herein; a polynucleotide as described herein; a nucleic acid construct as described herein; a vector as described herein; a cell as described herein; or portion thereof; and a pharmaceutically acceptable carrier.

[0113] In one aspect described herein is a method of regulating the activity of a synTF, comprising the steps of: (a) providing a population of cells as described herein; and (b) contacting the population of cells with an effective amount of at least one RP inducer. 15 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT

[0114] In one aspect described herein is a method of regulating the expression of a gene of interest, comprising the steps of: (a) providing a population of cells as described herein; and (b) contacting the population of cells with an effective amount of at least one RP inducer.

[0115] In one aspect described herein is a method of treating a subject in need of a cell-based therapy, comprising the steps of: (a) administering to the subject a population of cells as described herein; and (b) administering to the subject an effective amount of at least one RP inducer.

[0116] In some embodiments of any of the aspects, the population of cells comprises immune cells.

[0117] In some embodiments of any of the aspects, the population of immune cells comprises CD4+ T cells, CD8+ T cells, Tregs, NK cells, monocytes, or macrophages.

[0118] In some embodiments of any of the aspects, the at least one RP inducer is administered at the same time the population of cells is administered.

[0119] In some embodiments of any of the aspects, the at least one RP inducer is administered after the population of cells is administered. BRIEF DESCRIPTION OF THE DRAWINGS

[0120] Fig.1A-1F. Screen of Transcription Effector Domains (TEDs). (Fig.1A) Construction of TED integrated-synZiFTRs. A set consisting of 10 effector domains (Fused in Sarcoma (FUS), Ewing Sarcoma Breakpoint Region (EWSR), Heat Shock Factor 1 (HSF1), Glucocorticoid Receptor (GR), MLX interacting protein like (MLXIPL), Kow5 domain of Suppressor Of Ty 5 (Spt5) Homolog (SUPT5H), Interacts with Suppressor Of Ty 6 (Spt6) Homolog (IWS1), Bromodomain-containing protein 4 (BRD4), cellular Myelocytomatosis (cMyc), enhanced green fluorescent protein (eGFP)) was inserted into a lentiviral backbone between the ZF10 DNA binding domain and the p65 human transcription domain. This set included domains that have been identified as intrinsically disordered domains with pioneer ability (“txn. (transcription) pioneer factors” FUS, EWSR), activator domains (ADs; “txn. activation factors (Weak) HSF1, GR, MLXIPL), elongation domains (EDs; “txn. Elongation factors” Kow5, IWS1, BRD4, cMyc), and EGFP as control. (Fig.1B) Overview of genetic circuits used to evaluate candidate TEDs in Jurkat cells. Responsive promoters and synZiFTRs were integrated into Jurkat T cells in an equal ratio using lentiviral co-transduction. mCherry was measured by flow cytometry 3 days following Dox-induction. (Fig.1C) 5 TED integrated-synZiFTRs activated stronger gene expression compared to synZiFTR. TEDs were inserted into a lentiviral backbone between the ZF10 DNA binding domain and the p65 human transcription domain. Bars represent median fluorescence intensity (MFI) values for three measurements ± standard deviation (SD). Selected domain yielding highest enrichment of mCherry fluorescent output is highlighted in gray. Statistics represent two-tailed Student’s t test; ****P < 0.0001. PhPGK, human phosphoglycerate kinase 1 promoter; TRE3G, tetracycline-responsive 16 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT promoter; pybTATA, synthetic YB_TATA promoter; p65, amino acids 361 to 551; FUS, amino acids 2-214; EWSR1, amino acids 47-267; HSF1, amino acids 406-529; GR, amino acids 187-244; MLXIP, amino acids 318-370; Kow5 (SUPT5H), amino acids 698-765; TIMs (IWS1), amino acids 449-492; BRD4, amino acids 1308-1362; cMyc, amino acids 1-70. (Fig.1D) Evaluation of TIMs integrated- synZiFTR order variants. TIMs were inserted into a lentiviral backbone containing the ZF10 DNA binding domain and the p65 human transcription domain at different locations. Experiments were conducted via same circuit design in Fig.1B. Bars represent median fluorescence intensity (MFI) values for three measurements ± SD. Statistics represent two-tailed Student’s t test; ****P < 0.0001. (Fig.1E) Evaluation of transcription synergy. Comparing min. synZiFTR2.0 mCherry fluorescent output with min. synZiFTR + ZF10-TIMs. Experiments were conducted via same circuit design in Fig.1B. Bars represent median fluorescence intensity (MFI) values for three measurements ± SD. Statistics represent two-tailed Student’s t test; ****P < 0.0001.

[0121] Fig.2A-2E. Small molecule regulated synZiFTR2.0. (Fig.2A) Integrating TIMs into orthogonal gene switches regulated by clinically relevant small molecules. Design of GZV- and 4OHT- distinct synZiFTR2.0s. Grazoprevir (GZV), and 4-hydroxytamoxifen / tamoxifen (4OHT / TMX). NS3, hepatitis C virus NS3 protease domain; ERT2, human estrogen receptor T2 mutant domain. (Fig.2B) synZiFTR2.0 evaluation gene circuit. Jurkat T cells were co-transduced with reporter, synZiFTR2.0 and synZiFTR expression lentiviral vectors in an equal ratio. mCherry fluorescence was measured by flow cytometry 3 days following induction by small molecules at indicated concentrations. (Fig.2C) GZV-synZiFTR2.0 switches permit stronger inducible gene expression in Jurkat T cells. TIMs was inserted into various locations of the GZV-synZiFTR. Selected design(s) designated as GZV-synZiFTR2.0 is highlighted in gray. Bars represent median values for three measurements ± SD. Statistics represent two-tailed Student’s t test; ****P < 0.0001. Histograms show absolute levels and mean fold activation for one representative measurement (insets). Points represent median values for three measurements ± SD. pSFFV, Spleen Focus-Forming Virus promoter. (Fig.2D) 4OHT-synZiFTR2.0 switches permit stronger inducible gene expression in Jurkat T cells. TIMs were inserted into various locations of the 4OHT-synZiFTR. Selected design(s) designated as 4OHT-synZiFTR2.0 is highlighted in gray. Bars represent median values for three measurements ± SD. Statistics represent two-tailed Student’s t test; ****P < 0.0001. Histograms show absolute levels and mean fold activation for one representative measurement (insets). Points represent median values for three measurements ± SD. Statistics represent two-tailed Student’s t test; ****P < 0.0001. (Fig.2E) Double TIMs integrated GZV- and 4OHT- SynZiFTR2.0 further enhanced gene expression in Jurkat T cells. Two TIMs domains were inserted into selected designs of both GZV- and 4OHT-SynZiFTR. Selected design(s) with 2 TIMs domains is highlighted in gray. Bars represent median values for three measurements ± SD. Statistics 17 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT represent two-tailed Student’s t test; ****P < 0.0001. Comparison of GZV-SynZiFTR vs. GZV- SynZiFTR2.0, for all genes.

[0122] Fig.3A-3D. Expansion of the synZiFTR toolkit for two-input AND gate. (Fig.3A) Comparing heterodimers’ circuit output. Circuit output of reconstituted of split min. synZiFTR is measured in Jurkat T cells via a mammalian-two-hybrid circuit. Responsive promoters, input 1, and input 2 were integrated into Jurkat T cells in an equal ratio using lentiviral co-transduction. mCherry was measured by flow cytometry 3 days following Dox-induction. Selected heterodimer is highlighted in gray. Bars represent median fluorescence intensity (MFI) values for three measurements ± SD. Selected heterodimer yielding highest mCherry fluorescent output is highlighted in gray. Statistics represent two-tailed Student’s t test; ****P < 0.0001. (Fig.3B) Characterizing ZF affinity variant. Min. synZiFTR with non-modified ZF (domain 1: SEQ ID NO: 1, ERPFQCRICMRNFS; SEQ ID NO: 2, TGEK; domains 2-6: SEQ ID NO: 3, PFQCRICMRNFS) or ZFs with 3R2A (see e.g., 3 asterisks, bottom row, indicating arginine (R) to alanine (A) mutations in ZF domains 1, 3, and 6), 5R2A (see e.g., 5 asterisks, middle row, indicating arginine (R) to alanine (A) mutations in ZF domains 1, 3, 4, and 6), and 7R2A mutants (see e.g., 7 asterisks, top row, indicating arginine (R) to alanine (A) mutations in ZF domains 1-6) were expressed in Jurkat T cells. Responsive promoters and synZiFTRs were integrated into Jurkat T cells in an equal ratio using lentiviral co-transduction. mCherry was measured by flow cytometry 3 days following Dox-induction. Points represent median fluorescence intensity (MFI) values for three measurements ± SD. Statistics represent two-tailed Student’s t test; ****P < 0.0001. (Fig.3C) Characterizing transcription output via scaffold mediated cooperativity. Min. synZiFTRs with ZF carrying 7R2A mutations regulated mCherry expression in the presence of a nc= 2 scaffold in Jurkat T cells. Responsive promoters, input 1, and input 2 were integrated into Jurkat T cells in an equal ratio using lentiviral co-transduction. mCherry was measured by flow cytometry 3 days following Dox-induction. Bars represent median fluorescence intensity (MFI) values for three measurements ± SD. Selected heterodimer yielding highest mCherry fluorescent output is highlighted in gray. Statistics represent two-tailed Student’s t test; ****P < 0.0001; **P < 0.01. (Fig.3D) Characterizing transcription output of split minimal synZiFTR2.0. Circuit output of reconstituted of split min. synZiFTR2.0 was measured in Jurkat T cells via a mammalian-two-hybrid circuit. Responsive promoters, input 1, and input 2 were integrated into Jurkat T cells in an equal ratio using lentiviral co-transduction. mCherry was measured by flow cytometry 3 days following Dox-induction. Bars represent median fluorescence intensity (MFI) values for three measurements ± SD. Selected heterodimer yielding highest mCherry fluorescent output is highlighted in gray. Statistics represent two-tailed Student’s t test; ****P < 0.0001.

[0123] Fig.4A-4B. IWS1 TIMs mechanism of action. (Fig.4A) IWS1 mutants affecting TIMs employed in this study. WT TIMs domain in GZV-synZiFTR2.0 were replaced by TIMs structural mutant variants (see e.g., SEQ ID NOs: 5-14). The amino acid sequence of the TIMs 18 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT domain is shown (see e.g., SEQ ID NO: 5). The boxes highlight each of the 3 TIM amino acid sequences (see e.g., SEQ ID NOs: 6-8) and the mutants respectively (see e.g., SEQ ID NOs: 12-14). Different TIM mutations caused loss of interactions with various transcription elongation factors (Transcription factor IIS (TFIIS) and Elongin A (EloA) for the TIM1 mutant; Protein Phosphatase 1 Nuclear Targeting Subunit (PP1-PNUTs) for the TIM2 mutant; lens epithelium-derived growth factor (LEDGF) and H3K36me2 / 3 binding protein HDGFL2 (HRP2) for the TIM3 mutant). (Fig.4B) Evaluating loss of function TIMs mutations. GZV-synZiFTR2.04 distinct mutant variants of TIMs were expressed in Jurkat T cells. The circuit outputs were compared to GZV-synZiFTR2.0 carrying WT TIMs. Jurkat T cells were co-transduced with reporter, synZiFTR2.0 and synZiFTR expression lentiviral vectors in an equal ratio. mCherry fluorescence was measured by flow cytometry 3 days after induction by small molecules at the indicated concentrations. Bars represent mean values for three measurements ± SD. Statistics represent two-tailed Student’s t test; ****P < 0.0001.

[0124] Fig.5A-5D shows examples of the hhTAD synTFs. Fig.5A is a schematic showing that the IWS1 TIMs domain can be incorporated into synTF constructs to increase transcriptional activation. Fig.5B is a schematic showing small-molecule-regulated synTFs. Fig.5C is a schematic showing an exemplary grazoprevir-regulated synTF comprising an NS3 domain. Fig.5D is a schematic showing an exemplary tamoxifen-regulated synTF comprising an ERT2 domain.

[0125] Fig.6A-6B shows that incorporation of the IWS1 TIMs domain into synTFs increases their transcriptional activation. Fig.6A is a schematic and bar graph showing that the IWS1 TIMs domain increases transcriptional activation in an NS3-comprising synTF in the presence of grazoprevir. Fig.6B is a schematic and bar graph showing that the IWS1 TIMs domain increases transcriptional activation in an ERT2-comprising synTF in the presence of 4OHT.

[0126] Fig.7 is a series of schematics showing one (left) or two (right) copies of IWS1 TND- interacting motifs (TIMs), a small scaffolding domain involved in assembling transcriptional elongation complexes, fused to the N terminus of the p65 transcriptional activator domain in the synZiFTR technologies to generate synZiFTR 2.0 systems. Top: 4OHT-inducible synZiFTR 2.0. Bottom: GZV-inducible synZiFTR 2.0.

[0127] Fig.8A-8B shows testing of dual lentiviral vectors for GZV inducible systems driving mCherry reporter in Jurkat cells (benchmarked against VPR and minVPR). Fig.8A: a single copy of TIMs domain was placed on N or C terminus of p65 in synZiFTR, and N terminally fused TIMs enhanced synZiFTR activity more. Fig.8B: fusing two copies of TIMs on N terminus of p65 drives higher output than fusing a single copy of TIMs at the same loci.

[0128] Fig.9A-9B shows testing of dual lentiviral vectors for 4OHT inducible systems driving mCherry reporter in Jurkat cells (benchmarked against VPR and minVPR). Fig.9A: a single copy of TIMs domain was placed on N or C terminus of p65 in synZiFTR, and N terminally fused TIMs enhanced synZiFTR activity with less increase in the basal induction (no drug added). Fig.9B: fusing 19 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT two copies of TIMs on N terminus of p65 drove higher output than fusing a single copy of TIMs at the same loci.

[0129] Fig.10 shows testing of dual lentiviral vectors for GZV inducible systems driving mCherry reporter in Primary T cells (benchmarked against VPR). synZiFTR 2.0 drove a high output level compared to the VPR domain. In addition, fusing TIMs domains on the N terminus also enhanced activation potency of the NFZ transcriptional activator.

[0130] Fig.11 shows testing of dual lentiviral vectors for 4OHT inducible systems driving mCherry reporter in Primary T cells (benchmarked against VPR and minVPR).

[0131] Fig.12 shows testing of single lentiviral vectors for 4OHT inducible systems driving mCherry reporter in HEK cells (benchmarked against the original (1.0) synZiFTR).

[0132] Fig.13 shows testing of dual lentiviral vectors for GZV inducible systems driving mCherry reporter in THP1 monocytes / macrophages.

[0133] Fig.14 shows testing of dual lentiviral vectors for GZV inducible systems driving mCherry reporter in Primary monocytes / macrophages.

[0134] Fig.15 shows testing of dual lentiviral vectors for GZV inducible systems driving GFP- STAT1 protein in THP1 monocytes.

[0135] Fig.16 shows testing of single lentiviral vectors for GZV inducible systems driving GFP- STAT1 in THP1 monocytes.

[0136] Fig.17 shows testing of VEGF synTFs. The bar graph shows the 450nm absorbance readout from the ELISA assay, indicating the relative level of VEGF. Only VEGF-targeting synZiFTR 2.0 successfully drove VEGF production upon addition of 4OHT, in a ZF dependent manner. DETAILED DESCRIPTION

[0137] Described herein are synTFs for use in the methods and compositions as disclosed herein, where the synTFs comprise (i) a DNA binding domain (DBD) which binds to a target nucleic acid sequence (or target DNA binding motif (DBM)), (ii) a transcriptional effector domain (TED), (iii) a transcriptional activation (TA) domain, and (iv) optionally a regulator protein (RP).

[0138] In one aspect, described herein is a synthetic transcription factor (synTF) comprising: (a) at least one DNA binding domain (DBD), (b) a transcriptional activator (TA) domain, (c) a transcriptional effector domain (TED), and (d) at least one regulator protein (RP). In some embodiments, the synTF comprises the DBD, TA, TED, and RP domains in a N-terminal to C- terminal order (or 5’ to 3’ order in a corresponding nucleic acid) selected from Table 3.

[0139] Table 3: Exemplary SynTF domain orders. The first number corresponds to the position of the DBD, the second number corresponds to the position of the TA, the third number corresponds to the position of the TED, and the fourth number corresponds to the position of the RP. 20 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT For examples, 1,2,3,4 indicates that the DBD in the first (N-terminal) position, the TA in the second position, the TED in the third position, and the RP in the fourth position (C-terminal).

[0140] In another aspect, described herein is a synthetic transcription factor (synTF) comprising: (a) at least one DNA binding domain (DBD), (b) a transcriptional activator (TA) domain, and (c) a transcriptional effector domain (TED). In some embodiments, the synTF comprises the DBD, TA, and TED domains in a N-terminal to C-terminal order (or 5’ to 3’ order in a corresponding nucleic acid) selected from Table 4.

[0141] Table 4: Exemplary SynTF domain orders. The first number corresponds to the position of the DBD, the second number corresponds to the position of the TA, and the third number corresponds to the position of the TED. For examples, 1,2,3 indicates that the DBD in the first (N- terminal) position, the TA in the second position, and the TED in the third position (C-terminal).

[0142] In one aspect, described herein is a hybrid transcription activator domain (hTAD), comprising: (a) a transcriptional activator (TA) domain, and (b) a transcriptional effector domain (TED). In some embodiments, the TA is N terminal to the TED. In some embodiments, the TA is C terminal to the TED.

[0143] In another aspect, described herein is a humanized hybrid transcription activator domain (hhTAD), comprising: (a) a transcriptional activator (TA) domain, and (b) a transcriptional effector domain (TED), wherein the TA and TED are human domains or humanized domains. In some embodiments, the hhTAD comprises SEQ ID NO: 16 or an amino acid sequence with 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% sequence identity to SEQ ID NO: 16, which 21 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT maintains the same function (e.g., increased transcriptional activation, compared to a construct lacking the TED).

[0144] The TED increases the transcriptional activation of the TA compared to a construct comprising the TA and lacking the TED. As a non-limiting example, the TED increases the transcriptional activation of the TA by at least about 10%, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10%-100%, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a construct comprising the TA and lacking the TED.

[0145] The TED can be derived from a human protein, such as the TIMs domain of human IWS1. In some embodiments, at least one, at least two, at least three, at least four, or more of the domains in the construct (e.g., DBD, TA, TED, and / or RP) are human or humanized. Use of constructs comprising such human or humanized domains allows for clinical use, without concern for foreign immune-activation, such as can occur with non-human (e.g., virally derived) constructs.

[0146] The TEDs described herein can function in both dual vector and single vector systems (see e.g., Fig.12, Fig.16 for exemplary single vector systems). The TEDs described herein can drive more robust expression of a large payload compared a construct not comprising the TED (see e.g., Fig.15). As a non-limiting example, a synTF comprising at least one TED as described herein can increase complete transcription of a nucleic acid that is at least 1000 nucleotides long, at least 1100 nucleotides long, at least 1200 nucleotides long, at least 1300 nucleotides long, at least 1400 nucleotides long, at least 1500 nucleotides long, at least 1600 nucleotides long, at least 1700 nucleotides long, at least 1800 nucleotides long, at least 1900 nucleotides long, at least 2000 nucleotides long, at least 2100 nucleotides long, at least 2200 nucleotides long, at least 2300 nucleotides long, at least 2400 nucleotides long, at least 2500 nucleotides long, at least 2600 nucleotides long, at least 2700 nucleotides long, at least 2800 nucleotides long, at least 2900 nucleotides long, at least 3000 nucleotides long, at least 3100 nucleotides long, at least 3200 nucleotides long, at least 3300 nucleotides long, at least 3400 nucleotides long, at least 3500 nucleotides long, at least 3600 nucleotides long, at least 3700 nucleotides long, at least 3800 nucleotides long, at least 3900 nucleotides long, at least 4000 nucleotides long, at least 4100 nucleotides long, at least 4200 nucleotides long, at least 4300 nucleotides long, at least 4400 nucleotides long, at least 4500 nucleotides long, at least 4600 nucleotides long, at least 4700 nucleotides long, at least 4800 nucleotides long, at least 4900 nucleotides long, at least 5000 nucleotides long, or more (see e.g., SEQ ID NO: 27-28, Example 7.8, Fig.15, SEQ ID NO: 35).

[0147] In embodiments comprising a regulator protein, the regulator protein can control the coupling or linkage of the DNA binding domain (DBD) with the TA (such coupling can also be 22 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT referred to as a “mediator domain”), or can control the cellular localization of the TA, such that when the TA and DBD are attached and / or located in the nucleus, the TA can function to recruit translation machinery to the promoter to increase gene expression of a gene of interest.

[0148] In some embodiments of any of the aspects, regulator proteins can be activated or inhibited by a variety of inputs, non-limiting examples of which include: inducers (e.g., small molecules), light-inducible control (e.g., dimerization, assembly, localization), temperature, pH, phosphorylation, oxygen, lipid, magnetic, electric, spatial mechanisms (e.g., intracellular and / or extracellular; e.g., synthetic receptors and / or soluble factors), endogenous ligands (e.g., biomarkers), cell-cycle state, native signaling pathways, or disease and / or pathogenic states (e.g., aggregation, infection).

[0149] In some embodiments of the systems, compositions and methods as disclosed herein, the regulator protein of the SynTF is selected from a protease, a pair of inducible proximity domains (IPDs), a translocation domain (i.e., a cytosolic sequestering protein), or an induced degradation domain, each of which are described herein and in more detail below.

[0150] Described herein are four general frameworks of inducible or drug-controllable synthetic transcription factors comprising the TA and TED: (1) a synTF comprising a repressible protease, referred to as a repressible protease synTF; (2) a synTF comprising induced proximity domains, referred to as a induced proximity domain SynTF; (3) a synTF system comprising a cytosolic sequestering domain, referred to as a cytosolic sequestering synTF; and (4) a synTF comprising an induced degradation domain referred to as an induced degradation domain synTF. Also described herein are polynucleotides and vector encoding said synTF polypeptides, cells expressing said synTF polypeptides, pharmaceutical compositions comprising said synTF polypeptides, and methods of using said synTF polypeptides.

[0151] Described herein is a class of engineered transcription factor proteins (synTFs) and corresponding responsive artificial engineered 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 potential in the host genome (i.e., "orthogonal" activity to the host genome). The synTF proteins described herein can comprise a DNA binding domain (DBD) which are based on engineered zinc finger (ZF) arrays that are designed to target and bind specific 18-20 nucleotide sequences that are distant and different from the host genome sequences, when the synTF proteins are used in the selected hosts. This strategy limits non- specific interactions of the synTF proteins with the host’s genome; such non-specific interactions are not ideal and therefore, are not desired.

[0152] The synTFs described herein are designed, in some aspects, according to the following parameters: (1) targetable DNA sequences (also known as ZF binding sites) are identified for the ZF arrays that are specifically designed to have reduced binding potential in a host genome; (2) ZF arrays 23 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT are designed and assembled; (3) synTFs are designed by coupling engineered (i.e., covalently linked) ZF arrays to the transcriptional activator (TA) domain and transcriptional effector domain (TED); (4) corresponding responsive promoters are designed by placing instances of the targetable DNA sequences (i.e., ZF binding sites) upstream of constitutive promoters. The targetable DNA sequences are operably linked to the promoters such that the occupancy of synTFs on the targetable DNA sequences regulates the activity of the promoter in gene expression. The combination of a synTF and a targetable DNA sequence-promoter forms a unique expression system that is artificial, scalable, and regulatable, for the expressions of desired genes placed within the expression systems, with no or minimal effects on the expression of endogenous genes, meaning no or minimal off-site gene regulation of endogenous genes.

[0153] The synTFs described herein have reduced or minimal functional binding potential in the host genome, which provides, in part, advantages of no or minimal off-site DNA targeting by the synTFs. In addition, the synthetic ZF-based proteins (synTFs) described herein are derived from mammalian or human protein scaffolds, conferring minimal degree of immunogenicity over other prokaryotically-derived domains. In contrast to other classes of programmable DNA-targeting domains, these zinc-finger-based regulatory proteins are considerably smaller (~4-5x) than TALE and dCas9 proteins, less repetitive than TALE repeat proteins, and are not as constrained by lentiviral packaging limits, permitting convenient packaging in lentiviral delivery constructs and affording space for other desirable control elements. I. Synthetic Transcription Factor Domains

[0154] In multiple aspects described herein are constructs, including synTF polypeptides or synTF polypeptide systems, which comprise at least one of the following domains: transcriptional activator domain; a transcriptional effector domain (TED); a DNA-binding domain; at least one (e.g., 1, 2, 3, 4, 5, or more) regulator protein(s) selected from the group consisting of: repressible protease, induced proximity domain, cytosolic sequestration domain, and / or an induced degradation domain (e.g., SMASh domain); at least one linker peptide, at least one detectable marker, and / or self-cleaving peptide, or any combination thereof.

[0155] In some embodiments of any of the aspects, a synTF polypeptide or a synTF polypeptide system collectively (i.e., the first polypeptide and / or the second polypeptide) comprises at least the following: a transcriptional activator (TA) domain, a transcriptional effector domain (TED), a DNA- binding domain, and at least one (e.g., 1, 2, 3, 4, 5, or more) regulator protein(s) selected from the group consisting of: repressible protease, induced proximity domain, cytosolic sequestration domain, and / or induced degradation domain. In some embodiments of any of the aspects, a synTF polypeptide or system further comprises at least one linker peptide, and / or at least one detectable marker, and / or at least one self-cleaving peptide, or any combination thereof. Specific synTFs described herein are not 24 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT to be construed as limitations. For example, the following combinations are contemplated herein (see e.g., Table 5) below.

[0156] Table 5: Exemplary Combinations of Domains in a synTF Polypeptide or synTF Polypeptide System. Each exemplary synTF polypeptide shown in Table 5 also comprises a transcriptional activator (TA) domain, a transcriptional effector domain (TED), and a DNA-binding domain. The domains can be in any order. “PRO” indicates repressible protease. “IPD” indicates induced proximity domain. “CS” indicates cytosolic sequestering domain. “DD” indicates induced degron domain. “LP” indicates linker peptide. “DM” indicates detectable marker. “SP” indicates self- cleaving peptide. 25 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT PRO IPD CS DD LP DM SP PRO IPD CS DD LP DM SP X X X X X X X X X X X X X X X X X X264887-3198-4841.9Attorney Docket No: 701586-000114WOPT PRO IPD CS DD LP DM SP PRO IPD CS DD LP DM SP X X X X X X X X X X X X X27 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT

[0157] In some embodiments, a synTF or synTF system as described herein comprises a DBD, a TA, a TED, at least one ligand binding domain (LBD) and at least one ligand specific for the LBD. In some embodiments, the DBD, TA, TED, LBD, and ligand domains can be comprised by two separate polypeptides. In some embodiments, the DBD and TA are in separate polypeptides, such that the system is active only when both polypeptides are expressed. In some embodiments, a synTF or synTF system as described herein comprises a DBD, a TA, a TED, at least one PDZ, and at least one ligand specific for the PDZ domain (see e.g., Example 4, Fig.3A-3D). As a non-limiting example, in one aspect described herein is a synTF system comprising: (a) a first polypeptide comprising: (i) a DBD, (ii) a TED, and (iii) an LBD; and (b) a second polypeptide comprising: (i) a ligand specific for the LBD, and (ii) a TA. Non-limiting examples of PDZs and PDZ-specific ligands are provided in US Patent 11,781,149 B2, the contents of which are incorporated herein by reference in their entirety. Transcriptional Effector Domain (TED)

[0158] In multiple aspects, described herein are constructs, including synTFs, comprising at least one Transcriptional Effector Domain (TED). As used herein, the term “Transcriptional Effector Domain (TED)” refers to a domain that increases the transcriptional activity of a transcriptional activator (TA) domain, compared to a construct comprising the TA but lacking the TED. In some embodiments, the TED comprises an elongation domain, an activator domain, and / or a domain with pioneer ability.

[0159] In some embodiments of any of the aspects, a synTF polypeptide as described herein (or a synTF polypeptide system collectively) comprises 1, 2, 3, 4, 5, or more TED(s). In some embodiments of any of the aspects, the synTF polypeptide or system comprises one TED. In some embodiments of any of the aspects, the synTF polypeptide or system comprises two TEDs. In embodiments comprising multiple TEDs, the multiple TEDs can be different individual TEDs or multiple copies of the same TED, or a combination of the foregoing.

[0160] In some embodiments, the TED comprises an elongation domain. As used herein, the term “elongation domain” refers to a polypeptide that promotes elongation of the transcript by a polymerase, e.g., by decreasing transcriptional pausing and / or polymerase backtracking, arrest, and / or termination (see e.g., Section 3.1.1.3 of Example 3). In some embodiments, the TED comprises an elongation domain derived from a polypeptide selected from the group consisting of: Interacts with Suppressor Of Ty 6 (Spt6) Homolog (IWS1); Suppressor Of Ty 5 (Spt5) Homolog (SUPT5H); Bromodomain-containing protein 4 (BRD4); and cellular Myelocytomatosis (cMyc).

[0161] In some embodiments, the TED comprises an elongation domain derived from IWS1. In some embodiments, the TED comprises an elongation domain derived from mammalian IWS1. In some embodiments, the TED comprises an elongation domain derived from human IWS1 (see e.g., SEQ ID NO: 4). In some embodiments, the TED comprises amino acids 449-492, 450-492, 451-492, 28 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT 452-492, 453-492, 454-492, 455-492, 456-492, 449-491, 450-491, 451-491, 452-491, 453-491, 454- 491, 455-491, or 456-491 of IWS1 in SEQ ID NO: 4.

[0162] SEQ ID NO: 4, IWS1, NCBI reference NP_001397852.1, 818 amino acids (aa) MDSEYYSGDQSDDGGATPVQDERDSGSDGEDDVNEQHSGSDTGSVERHSENETSDREDGLP KGHHVTDSENDEPLNLNASDSESEELHRQKDSDSESEERAEPPASDSENEDVNQHGSDSESEE TRKLPGSDSENEELLNGHASDSENEDVGKHPASDSEIEELQKSPASDSETEDALKPQISDSESE EPPRHQASDSENEEPPKPRMSDSESEELPKPQVSDSESEEPPRHQASDSENEELPKPRISDSESE DPPRHQASDSENEELPKPRISDSESEDPPRNQASDSENEELPKPRVSDSESEGPQKGPASDSETE DASRHKQKPESDDDSDRENKGEDTEMQNDSFHSDSHMDRKKFHSSDSEEEEHKKQKMDSD EDEKEGEEEKVAKRKAAVLSDSEDEEKASAKKSRVVSDADDSDSDAVSDKSGKREKTIASD SEEEAGKELSDKKNEEKDLFGSDSESGNEEENLIADIFGESGDEEEEEFTGFNQEDLEEEKGET QVKEAEDSDSDDNIKRGKHMDFLSDFEMMLQRKKSMSGKRRRNRDGGTFISDADDVVSAM IVKMNEAAEEDRQLNNQKKPALKKLTLLPAVVMHLKKQDLKETFIDSGVMSAIKEWLSPLP DRSLPALKIREELLKILQELPSVSQETLKHSGIGRAVMYLYKHPKESRSNKDMAGKLINEWSR PIFGLTSNYKGMTREEREQRDLEQMPQRRRMNSTGGQTPRRDLEKVLTGEEKALRPGDPGFC ARARVPMPSNKDYVVRPKWNVEMESSRPGILKKGLSRLEKHKRRFAEQKRLSKVHRAVKFS IEGNRMPL

[0163] In some embodiments, the elongation domain comprises at least one TFIIS N-terminal domains (TND)-interacting motif (TIM) domain of IWS1 (see e.g., SEQ ID NOs: 5-9). IWS1 is a transcription elongation factor, which directly interacts with RNA polymerase II (RNAPII) and is phosphorylated at casein kinase II (CKII) sites. The human IWS1 homolog physically interacts with protein arginine methyltransferase 5 (PRMT5). IWS1 also recruits a SET2 histone methyltransferase (Huntingtin-interacting protein HYPB, also known as SETD2) to RNAPII during transcription elongation and is involved in H3K36 trimethylation.

[0164] In some embodiments, the elongation domain comprises at least one TIM1, TIM2, and / or TIM3 domain from IWS1. In some embodiments, the elongation domain comprises at least one TIM1. In some embodiments, the elongation domain comprises at least one TIM2. In some embodiments, the elongation domain comprises at least one TIM3. In some embodiments, the elongation domain comprises at least one TIM1 and at least one TIM2. In some embodiments, the elongation domain comprises at least one TIM1 and at least one TIM3. In some embodiments, the elongation domain comprises at least one TIM2 and at least one TIM3. In some embodiments, the elongation domain comprises at least one TIM1, at least one TIM2, and at least one TIM3 domain from IWS1.

[0165] In some embodiments, the elongation domain comprises at least one of SEQ ID NOs: 5-9 or an amino acid sequence with 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% 29 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT sequence identity to one of SEQ ID NOs: 5-9, which maintains the same function (e.g., increased transcriptional activity of the synTF).

[0166] SEQ ID NO: 5, amino acids 449-492 of IWS1, IWS1 TIMs domain (see e.g., SEQ ID NO: 4), TIM1 bolded (see e.g., SEQ ID NO: 6), TIM2 italicized (see e.g., SEQ ID NO: 7), TIM3 bolded and italicized (see e.g., SEQ ID NO: 8), 44 amino acids (aa): SDKKNEEKDLFGSDSESGNEEENLIADIFGESGDEEEEEFTGFN.

[0167] SEQ ID NO: 6, TIM1: KDLFG.

[0168] SEQ ID NO: 7, TIM2: ADIFG.

[0169] SEQ ID NO: 8, TIM3: EFTGF.

[0170] In some embodiments, the elongation domain comprises at least one (e.g., 1, 2, 3) mutation(s) in a TIM domain. In some embodiments, the elongation domain comprises at least one of SEQ ID NOs: 9-14 or an amino acid sequence with 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% sequence identity to one of SEQ ID NOs: 9-14, which maintains the same function (e.g., increased transcriptional activity of the synTF).

[0171] SEQ ID NO: 9, amino acids 449-492 of IWS, mutant 1 (M1; L458A and F459A: bolded; TIM1 mutant; see e.g., SEQ ID NO: 12): SDKKNEEKDAAGSDSESGNEEENLIADIFGESGDEEEEEFTGFN.

[0172] SEQ ID NO: 10, amino acids 449-492 of IWS, mutant 2 (M2; I476A and F477A: bolded; TIM2 mutant; see e.g., SEQ ID NO: 13): SDKKNEEKDLFGSDSESGNEEENLIADAAGESGDEEEEEFTGFN.

[0173] SEQ ID NO: 11, amino acids 449-492 of IWS, mutant 3 (M3; F488A and F491A: bolded; TIM3 mutant; see e.g., SEQ ID NO: 14): SDKKNEEKDLFGSDSESGNEEENLIADIFGESGDEEEEEATGAN.

[0174] SEQ ID NO: 12, TIM1 mutant: KDAAG.

[0175] SEQ ID NO: 13, TIM2 mutant: ADAAG.

[0176] SEQ ID NO: 14, TIM3 mutant: EATGA.

[0177] In some embodiments, the elongation domain is derived from Suppressor Of Ty 5 (Spt5) Homolog (SUPT5H). In some embodiments, the elongation domain is the Kow5 domain of SUPT5H. In some embodiments, the elongation domain comprises approximately amino acids 698-765 of SUPT5H (see e.g., SEQ ID NO: 49). In some embodiments, the elongation domain is derived from Bromodomain-containing protein 4 (BRD4). In some embodiments, the elongation domain comprises approximately amino acids 1308-1362 of BRD4 (see e.g., SEQ ID NO: 50). In some embodiments, the elongation domain is derived from cellular Myelocytomatosis (cMyc). In some embodiments, the elongation domain comprises approximately amino acids 1-70 of cMyc (see e.g., SEQ ID NO: 51). 30 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT

[0178] SEQ ID NO: 49, human Suppressor Of Ty 5 (Spt5) Homolog (SUPT5H), DSIF elongation factor subunit, NCBI Reference Sequence: NP_001124297.1, 1083 aa MSDSEDSNFSEEEDSERSSDGEEAEVDEERRSAAGSEKEEEPEDEEEEEEEEEYDEEEEEEDD DRPPKKPRHGGFILDEADVDDEYEDEDQWEDGAEDILEKASNIDNVVLDEDRSGARRLQNL WRDQREEELGEYYMKKYAKSSVGETVYGGSDELSDDITQQQLLPGVKDPNLWTVKCKIGEE RATAISLMRKFIAYQFTDTPLQIKSVVAPEHVKGYIYVEAYKQTHVKQAIEGVGNLRLGYWN QQMVPIKEMTDVLKVVKEVANLKPKSWVRLKRGIYKDDIAQVDYVEPSQNTISLKMIPRIDY DRIKARMSLKDWFAKRKKFKRPPQRLFDAEKIRSLGGDVASDGDFLIFEGNRYSRKGFLFKSF AMSAVITEGVKPTLSELEKFEDQPEGIDLEVVTESTGKEREHNFQPGDNVEVCEGELINLQGK ILSVDGNKITIMPKHEDLKDMLEFPAQELRKYFKMGDHVKVIAGRFEGDTGLIVRVEENFVIL FSDLTMHELKVLPRDLQLCSETASGVDVGGQHEWGELVQLDPQTVGVIVRLERETFQVLNM YGKVVTVRHQAVTRKKDNRFAVALDSEQNNIHVKDIVKVIDGPHSGREGEIRHLFRSFAFLH CKKLVENGGMFVCKTRHLVLAGGSKPRDVTNFTVGGFAPMSPRISSPMHPSAGGQRGGFGS PGGGSGGMSRGRGRRDNELIGQTVRISQGPYKGYIGVVKDATESTARVELHSTCQTISVDRQ RLTTVGSRRPGGMTSTYGRTPMYGSQTPMYGSGSRTPMYGSQTPLQDGSRTPHYGSQTPLH DGSRTPAQSGAWDPNNPNTPSRAEEEYEYAFDDEPTPSPQAYGGTPNPQTPGYPDPSSPQVNP QYNPQTPGTPAMYNTDQFSPYAAPSPQGSYQPSPSPQSYHQVAPSPAGYQNTHSPASYHPTPS PMAYQASPSPSPVGYSPMTPGAPSPGGYNPHTPGSGIEQNSSDWVTTDIQVKVRDTYLDTQV VGQTGVIRSVTGGMCSVYLKDSEKVVSISSEHLEPITPTKNNKVKVILGEDREATGVLLSIDGE DGIVRMDLDEQLKILNLRFLGKLLEA

[0179] SEQ ID NO: 50, human Bromodomain-containing protein 4 (BRD4), NCBI Reference Sequence: NP_001366220.1, 1362 aa MSAESGPGTRLRNLPVMGDGLETSQMSTTQAQAQPQPANAASTNPPPPETSNPNKPKRQTNQ LQYLLRVVLKTLWKHQFAWPFQQPVDAVKLNLPDYYKIIKTPMDMGTIKKRLENNYYWNA QECIQDFNTMFTNCYIYNKPGDDIVLMAEALEKLFLQKINELPTEETEIMIVQAKGRGRGRKE TGTAKPGVSTVPNTTQASTPPQTQTPQPNPPPVQATPHPFPAVTPDLIVQTPVMTVVPPQPLQT PPPVPPQPQPPPAPAPQPVQSHPPIIAATPQPVKTKKGVKRKADTTTPTTIDPIHEPPSLPPEPKT TKLGQRRESSRPVKPPKKDVPDSQQHPAPEKSSKVSEQLKCCSGILKEMFAKKHAAYAWPFY KPVDVEALGLHDYCDIIKHPMDMSTIKSKLEAREYRDAQEFGADVRLMFSNCYKYNPPDHE VVAMARKLQDVFEMRFAKMPDEPEEPVVAVSSPAVPPPTKVVAPPSSSDSSSDSSSDSDSSTD DSEEERAQRLAELQEQLKAVHEQLAALSQPQQNKPKKKEKDKKEKKKEKHKRKEEVEENK KSKAKEPPPKKTKKNNSSNSNVSKKEPAPMKSKPPPTYESEEEDKCKPMSYEEKRQLSLDINK LPGEKLGRVVHIIQSREPSLKNSNPDEIEIDFETLKPSTLRELERYVTSCLRKKRKPQAEKVDVI AGSSKMKGFSSSESESSSESSSSDSEDSETEMAPKSKKKGHPGREQKKHHHHHHQQMQQAPA PVPQQPPPPPQQPPPPPPPQQQQQPPPPPPPPSMPQQAAPAMKSSPPPFIATQVPVLEPQLPGSV FDPIGHFTQPILHLPQPELPPHLPQPPEHSTPPHLNQHAVVSPPALHNALPQQPSRPSNRAAALP 31 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT PKPARPPAVSPALTQTPLLPQPPMAQPPQVLLEDEEPPAPPLTSMQMQLYLQQLQKVQPPTPL LPSVKVQSQPPPPLPPPPHPSVQQQLQQQPPPPPPPQPQPPPQQQHQPPPRPVHLQPMQFSTHIQ QPPPPQGQQPPHPPPGQQPPPPQPAKPQQVIQHHHSPRHHKSDPYSTGHLREAPSPLMIHSPQ MSQFQSLTHQSPPQQNVQPKKQELRAASVVQPQPLVVVKEEKIHSPIIRSEPFSPSLRPEPPKHP ESIKAPVHLPQRPEMKPVDVGRPVIRPPEQNAPPPGAPDKDKQKQEPKTPVAPKKDLKIKNM GSWASLVQKHPTTPSSTAKSSSDSFEQFRRAAREKEEREKALKAQAEHAEKEKERLRQERMR SREDEDALEQARRAHEEARRRQEQQQQQRQEQQQQQQQQAAAVAAAATPQAQSSQPQSML DQQRELARKREQERRRREAMAATIDMNFQSDLLSIFEENLF

[0180] SEQ ID NO: 51, cellular Myelocytomatosis (cMyc), human MYC proto-oncogene, bHLH transcription factor, UNIPROT reference number P01106-2, 454 aa MDFFRVVENQQPPATMPLNVSFTNRNYDLDYDSVQPYFYCDEEENFYQQQQQSELQPPAPS EDIWKKFELLPTPPLSPSRRSGLCSPSYVAVTPFSLRGDNDGGGGSFSTADQLEMVTELLGGD MVNQSFICDPDDETFIKNIIIQDCMWSGFSAAAKLVSEKLASYQAARKDSGSPNPARGHSVCS TSSLYLQDLSAAASECIDPSVVFPYPLNDSSSPKSCASQDSSAFSPSSDSLLSSTESSPQGSPEPL VLHEETPPTTSSDSEEEQEDEEEIDVVSVEKRQAPGKRSESGSPSAGGHSKPPHSPLVLKRCHV STHQHNYAAPPSTRKDYPAAKRVKLDSVRVLRQISNNRKCTSPRSSDTEENVKRRTHNVLER QRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSVQAEEQKLISEEDLLRKRREQLK HKLEQLRNSCA

[0181] In some embodiments, the TED comprises an activator domain. As used herein, the term “activator domain” refers to a polypeptide that is involved in positive transcription regulation (see e.g., Section 3.1.1.2 of Example 3). In some embodiments, the TED comprises an activator domain derived from a polypeptide selected from the group consisting of: Heat Shock Factor 1 (HSF1), Glucocorticoid Receptor (GR), and MLX interacting protein like (MLXIPL).

[0182] In some embodiments, the activator domain is derived from Heat Shock Factor 1 (HSF1). In some embodiments, the activator domain comprises approximately amino acids 406-529 of HSF (see e.g., SEQ ID NO: 52). In some embodiments, the activator domain is derived from Glucocorticoid Receptor (GR). In some embodiments, the activator domain comprises approximately amino acids 187-244 of GR (see e.g., SEQ ID NO: 53). In some embodiments, the activator domain is derived from MLX interacting protein like (MLXIPL). In some embodiments, the activator domain comprises approximately amino acids 318-370 of MLXIPL (see e.g., SEQ ID NO: 54).

[0183] SEQ ID NO: 52, human Heat Shock Factor 1 (HSF1), NCBI Reference Sequence: NP_005517.1, 529 aa MDLPVGPGAAGPSNVPAFLTKLWTLVSDPDTDALICWSPSGNSFHVFDQGQFAKEVLPKYFK HNNMASFVRQLNMYGFRKVVHIEQGGLVKPERDDTEFQHPCFLRGQEQLLENIKRKVTSVS TLKSEDIKIRQDSVTKLLTDVQLMKGKQECMDSKLLAMKHENEALWREVASLRQKHAQQQ KVVNKLIQFLISLVQSNRILGVKRKIPLMLNDSGSAHSMPKYSRQFSLEHVHGSGPYSAPSPA 32 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT YSSSSLYAPDAVASSGPIISDITELAPASPMASPGGSIDERPLSSSPLVRVKEEPPSPPQSPRVEE ASPGRPSSVDTLLSPTALIDSILRESEPAPASVTALTDARGHTDTEGRPPSPPPTSTPEKCLSVA CLDKNELSDHLDAMDSNLDNLQTMLSSHGFSVDTSALLDLFSPSVTVPDMSLPDLDSSLASIQ ELLSPQEPPRPPEAENSSPDSGKQLVHYTAQPLFLLDPGSVDTGSNDLPVLFELGEGSYFSEGD GFAEDPTISLLTGSEPPKAKDPTVS

[0184] SEQ ID NO: 53, human Glucocorticoid Receptor (GR), also referred to as Nuclear Receptor Subfamily 3 Group C Member 1 (NR3C1), NCBI Reference Sequence: NP_000167.1, 777 aa MDSKESLTPGREENPSSVLAQERGDVMDFYKTLRGGATVKVSASSPSLAVASQSDSKQRRLL VDFPKGSVSNAQQPDLSKAVSLSMGLYMGETETKVMGNDLGFPQQGQISLSSGETDLKLLEE SIANLNRSTSVPENPKSSASTAVSAAPTEKEFPKTHSDVSSEQQHLKGQTGTNGGNVKLYTTD QSTFDILQDLEFSSGSPGKETNESPWRSDLLIDENCLLSPLAGEDDSFLLEGNSNEDCKPLILPD TKPKIKDNGDLVLSSPSNVTLPQVKTEKEDFIELCTPGVIKQEKLGTVYCQASFPGANIIGNKM SAISVHGVSTSGGQMYHYDMNTASLSQQQDQKPIFNVIPPIPVGSENWNRCQGSGDDNLTSL GTLNFPGRTVFSNGYSSPSMRPDVSSPPSSSSTATTGPPPKLCLVCSDEASGCHYGVLTCGSCK VFFKRAVEGQHNYLCAGRNDCIIDKIRRKNCPACRYRKCLQAGMNLEARKTKKKIKGIQQA TTGVSQETSENPGNKTIVPATLPQLTPTLVSLLEVIEPEVLYAGYDSSVPDSTWRIMTTLNMLG GRQVIAAVKWAKAIPGFRNLHLDDQMTLLQYSWMFLMAFALGWRSYRQSSANLLCFAPDLI INEQRMTLPCMYDQCKHMLYVSSELHRLQVSYEEYLCMKTLLLLSSVPKDGLKSQELFDEIR MTYIKELGKAIVKREGNSSQNWQRFYQLTKLLDSMHEVVENLLNYCFQTFLDKTMSIEFPEM LAEIITNQIPKYSNGNIKKLLFHQK

[0185] SEQ ID NO: 54, human MLX interacting protein like (MLXIPL), NCBI Reference Sequence: NP_116569.1852 aa MAGALAGLAAGLQVPRVAPSPDSDSDTDSEDPSLRRSAGGLLRSQVIHSGHFMVSSPHSDSL PRRRDQEGSVGPSDFGPRSIDPTLTRLFECLSLAYSGKLVSPKWKNFKGLKLLCRDKIRLNNAI WRAWYIQYVKRRKSPVCGFVTPLQGPEADAHRKPEAVVLEGNYWKRRIEVVMREYHKWRI YYKKRLRKPSREDDLLAPKQAEGRWPPPEQWCKQLFSSVVPVLLGDPEEEPGGRQLLDLNCF LSDISDTLFTMTQSGPSPLQLPPEDAYVGNADMIQPDLTPLQPSLDDFMDISDFFTNSRLPQPP MPSNFPEPPSFSPVVDSLFSSGTLGPEVPPASSAMTHLSGHSRLQARNSCPGPLDSSAFLSSDFL LPEDPKPRLPPPPVPPPLLHYPPPAKVPGLEPCPPPPFPPMAPPTALLQEEPLFSPRFPFPTVPPAP GVSPLPAPAAFPPTPQSVPSPAPTPFPIELLPLGYSEPAFGPCFSMPRGKPPAPSPRGQKASPPTL APATASPPTTAGSNNPCLTQLLTAAKPEQALEPPLVSSTLLRSPGSPQETVPEFPCTFLPPTPAP TPPRPPPGPATLAPSRPLLVPKAERLSPPAPSGSERRLSGDLSSMPGPGTLSVRVSPPQPILSRG RPDSNKTENRRITHISAEQKRRFNIKLGFDTLHGLVSTLSAQPSLKVSKATTLQKTAEYILMLQ QERAGLQEEAQQLRDEIEELNAAINLCQQQLPATGVPITHQRFDQMRDMFDDYVRTRTLHN 33 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT WKFWVFSILIRPLFESFNGMVSTASVHTLRQTSLAWLDQYCSLPALRPTVLNSLRQLGTSTSIL TDPGRIPEQATRAVTEGTLGKPL

[0186] In some embodiments, the TED comprises a domain with pioneer ability. As used herein, the term “domain with pioneer ability” refers to a polypeptide that has the ability to access DNA in compacted heterochromatin (see e.g., Section 3.1.1.1 of Example 3). In some embodiments, the TED comprises a domain with pioneer ability derived from a polypeptide selected from the group consisting of: Fused in Sarcoma (FUS) and Ewing Sarcoma Breakpoint Region (EWSR).

[0187] In some embodiments, the domain with pioneer ability is derived from Fused in Sarcoma (FUS). In some embodiments, the domain with pioneer ability comprises approximately amino acids 2-214 of FUS (see e.g., SEQ ID NO: 55). In some embodiments, the domain with pioneer ability is derived from Ewing Sarcoma Breakpoint Region (EWSR). In some embodiments, the domain with pioneer ability comprises approximately amino acids 47-267 of EWSR (see e.g., SEQ ID NO: 56).

[0188] SEQ ID NO: 55, human Fused in Sarcoma (FUS), NP_001164105.1, 525 aa MASNDYTQQATQSYGAYPTQPGQGYSQQSSQPYGQQSYSGYSQSTDTSGYGQSSYSSYGQS QNSYGTQSTPQGYGSTGGYGSSQSSQSSYGQQSSYPGYGQQPAPSSTSGSYGSSSQSSSYGQP QSGSYSQQPSYGGQQQSYGQQQSYNPPQGYGQQNQYNSSSGGGGGGGGGGNYGQDQSSMS SGGGSGGGYGNQDQSGGGGSGGYGQQDRGGRGRGGSGGGGGGGGGGYNRSSGGYEPRGR GGGRGGRGGMGGSDRGGFNKFGGPRDQGSRHDSEQDNSDNNTIFVQGLGENVTIESVADYF KQIGIIKTNKKTGQPMINLYTDRETGKLKGEATVSFDDPPSAKAAIDWFDGKEFSGNPIKVSF ATRRADFNRGGGNGRGGRGRGGPMGRGGYGGGGSGGGGRGGFPSGGGGGGGQQRAGDW KCPNPTCENMNFSWRNECNQCKAPKPDGPGGGPGGSHMGGNYGDDRRGGRGGYDRGGYR GRGGDRGGFRGGRGGGDRGGFGPGKMDSRGEHRQDRRERPY

[0189] SEQ ID NO: 56, human Ewing Sarcoma Breakpoint Region (EWSR), NCBI Reference Sequence: NP_001156757.1, 655 aa MASTDYSTYSQAAAQQGYSAYTAQPTQGYAQTTQAYGQQSYGTYGQPTDVSYTQAQTTAT YGQTAYATSYGQPPTGYTTPTAPQAYSQPVQGYGTGAYDTTTATVTTTQASYAAQSAYGTQ PAYPAYGQQPAATAPTRPQDGNKPTETSQPQSSTGGYNQPSLGYGQSNYSYPQVPGSYPMQP VTAPPSYPPTSYSSTQPTSYDQSSYSQQNTYGQPSSYGQQSSYGQQSSYGQQPPTSYPPQTGS YSQAPSQYSQQSSSYGQQSSFRQDHPSSMGVYGQESGGFSGPGENRSMSGPDNRGRGRGGF DRGGMSRGGRGGGRGGMGAGERGGFNKPGGPMDEGPDLDLGPPVDPDEDSDNSAIYVQGL NDSVTLDDLADFFKQCGVVKMNKRTGQPMIHIYLDKETGKPKGDATVSYEDPPTAKAAVE WFDGKDFQGSKLKVSLARKKPPMNSMRGGLPPREGRGMPPPLRGGPGGPGGPGGPMGRMG GRGGDRGGFPPRGPRGSRGNPSGGGNVQHRAGDWQCPNPGCGNQNFAWRTECNQCKAPKP EGFLPPPFPPPGGDRGRGGPGGMRGGRGGLMDRGGPGGMFRGGRGGDRGGFRGGRGMDR GGFGGGRRGGPGGPPGPLMEQMGGRRGGRGGPGKMDKGEHRQERRDRPY 34 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT

[0190] In some embodiments, the TED comprises an elongation domain and an activator domain. In some embodiments, the TED comprises an elongation domain and a domain with pioneer ability. In some embodiments, the TED comprises an activator domain and a domain with pioneer ability. In some embodiments, the TED comprises an elongation domain, an activator domain, and a domain with pioneer ability. Transcriptional Activator (TA) Domain

[0191] Described herein are synTFs comprising a transcription activator (or activating) domain (TA). For example, the transcriptional 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; a p65 activation domain of NFkB or functional fragment thereof; an Epstein-Barr virus R transactivator (Rta) activation domain or functional fragment thereof; a tripartite activator consisting of the VP64, the p65, and the Rta activation domains, wherein the tripartite activator is known as a VPR activation domain; a miniVPR (or “minVPR”); NFZ; 3Z; a histone acetyltransferase (HAT) core domain of the human E1A-associated protein p300, known as a p300 HAT core activation domain; a CBP HAT domain. In some embodiments, the transcriptional activator domain is selected from VPR (VP64-p65-Rta), VP64, p65, Rta, minVPR, NFZ, and 3Z.

[0192] In some embodiments of any of the aspects, a synTF polypeptide as described herein (or a synTF polypeptide system collectively) comprises 1, 2, 3, 4, 5, or more transcriptional activator domain(s). In some embodiments of any of the aspects, the synTF polypeptide or system comprises one transcriptional activator domain. In embodiments comprising multiple transcriptional activator domains, the multiple transcriptional activator domains can be different individual transcriptional activator domains or multiple copies of the same transcriptional activator domain, or a combination of the foregoing.

[0193] 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; miniVPR; full VPR; VP16; VP64; NFZ; 3Z; p300; p300 HAT Core; and a CBP HAT domain. See e.g., US Patent 10,138,493; US Patent 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 entireties.

[0194] 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-kappa-B p65 subunit is a protein 35 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT that in humans is encoded by the RELA gene. In some embodiments of any of the aspects, p65 comprises SEQ ID NO: 38 or a protein having at least 85% sequence identity to SEQ ID NO: 38. In some embodiments of any of the aspects, p65 comprises SEQ ID NO: 58 or a protein having at least 85% sequence identity to SEQ ID NO: 58. In some embodiments of any of the aspects, p65 comprises SEQ ID NO: 59 or a portion of SEQ ID NO: 59, e.g., residues 150-261, 100-261, 200-261, 1-200, 1- 50, 1-100, or 50-100 of SEQ ID NO: 59. In some embodiments of any of the aspects, p65 comprises one of SEQ ID NOs: 38, 58-67 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: 38, 58-67 that maintains its function. In some embodiments of any of the aspects, p65 comprises SEQ ID NO: 61 (p65100-261) 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: 61 that maintains the same function.

[0195] SEQ ID NO: 38, p65 (amino acids 361-551 of NFkB) Activation Domain (191 aa) DEFPTMVFPSGQISQASALAPAPPQVLPQAPAPAPAPAMVSALAQAPAPVPVLAPGPPQAVAP PAPKPTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNSEFQQLLNQGIPVAP HTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGDEDFSSIADMDFSALLSQIS S

[0196] SEQ ID NO: 58, p65 (full sequence, 551 aa), transcription factor p65 isoform 1 [Homo sapiens], NCBI Reference Sequence: NP_068810.3 MDELFPLIFPAEPAQASGPYVEIIEQPKQRGMRFRYKCEGRSAGSIPGERSTDTTKTHPTIKING YTGPGTVRISLVTKDPPHRPHPHELVGKDCRDGFYEAELCPDRCIHSFQNLGIQCVKKRDLEQ AISQRIQTNNNPFQVPIEEQRGDYDLNAVRLCFQVTVRDPSGRPLRLPPVLSHPIFDNRAPNTA ELKICRVNRNSGSCLGGDEIFLLCDKVQKEDIEVYFTGPGWEARGSFSQADVHRQVAIVFRTP PYADPSLQAPVRVSMQLRRPSDRELSEPMEFQYLPDTDDRHRIEEKRKRTYETFKSIMKKSPF SGPTDPRPPPRRIAVPSRSSASVPKPAPQPYPFTSSLSTINYDEFPTMVFPSGQISQASALAPAPP QVLPQAPAPAPAPAMVSALAQAPAPVPVLAPGPPQAVAPPAPKPTQAGEGTLSEALLQLQFD DEDLGALLGNSTDPAVFTDLASVDNSEFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQ RPPDPAPAPLGAPGLPNGLLSGDEDFSSIADMDFSALLSQISS

[0197] SEQ ID NO: 59, p651-261 (261 aa) SQYLPDTDDRHRIEEKRKRTYETFKSIMKKSPFSGPTDPRPPPRRIAVPSRSSASVPKPAPQPYP FTSSLSTINYDEFPTMVFPSGQISQASALAPAPPQVLPQAPAPAPAPAMVSALAQAPAPVPVLA PGPPQAVAPPAPKPTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNSEFQQ LLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGDEDFSSIAD MDFSALL

[0198] SEQ ID NO: 60, p65150-261 (112 aa) 36 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT SLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNSEFQQLLNQGIPVAPHTTEPMLMEY PEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGDEDFSSIADMDFSALL

[0199] SEQ ID NO: 61, p65100-261 (162 aa) SVLPQAPAPAPAPAMVSALAQAPAPVPVLAPGPPQAVAPPAPKPTQAGEGTLSEALLQLQFD DEDLGALLGNSTDPAVFTDLASVDNSEFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQ RPPDPAPAPLGAPGLPNGLLSGDEDFSSIADMDFSALL

[0200] SEQ ID NO: 62, p65200-261 (62 aa) SPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGDEDFSSIADMDFSALL

[0201] SEQ ID NO: 63, p651-200 (200 aa) SQYLPDTDDRHRIEEKRKRTYETFKSIMKKSPFSGPTDPRPPPRRIAVPSRSSASVPKPAPQPYP FTSSLSTINYDEFPTMVFPSGQISQASALAPAPPQVLPQAPAPAPAPAMVSALAQAPAPVPVLA PGPPQAVAPPAPKPTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNSEFQQ LLNQGIPVA

[0202] SEQ ID NO: 64, p651-150 (150 aa) SQYLPDTDDRHRIEEKRKRTYETFKSIMKKSPFSGPTDPRPPPRRIAVPSRSSASVPKPAPQPYP FTSSLSTINYDEFPTMVFPSGQISQASALAPAPPQVLPQAPAPAPAPAMVSALAQAPAPVPVLA PGPPQAVAPPAPKPTQAGEGT

[0203] SEQ ID NO: 65, p651-100 (100 aa) SQYLPDTDDRHRIEEKRKRTYETFKSIMKKSPFSGPTDPRPPPRRIAVPSRSSASVPKPAPQPYP FTSSLSTINYDEFPTMVFPSGQISQASALAPAPPQ

[0204] SEQ ID NO: 66, p6550-150 (101 aa) SRSSASVPKPAPQPYPFTSSLSTINYDEFPTMVFPSGQISQASALAPAPPQVLPQAPAPAPAPAM VSALAQAPAPVPVLAPGPPQAVAPPAPKPTQAGEGT

[0205] SEQ ID NO: 67, p65143-261 (119 aa) PTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNSEFQQLLNQGIPVAPHTTE PMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGDEDFSSIADMDFSALL

[0206] In some embodiments of any of the aspects, the TA is Rta, or a functional fragment thereof. Rta is an Epstein-Barr virus R transactivator (Rta) activation domain. In some embodiments of any of the aspects, Rta comprises SEQ ID NO: 68 or a protein having at least 85% sequence identity to SEQ ID NO: 68. In some embodiments of any of the aspects, Rta comprises a portion of SEQ ID NO: 68, e.g., residues 75-190, 125-190, 50-175, 75-175, 100-175, or 125-175 of SEQ ID NO: 68. In some embodiments of any of the aspects, Rta comprises one of SEQ ID NOs: 68-74 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: 68-74 that maintains its function. In some embodiments of any of the aspects, Rta comprises SEQ ID NO: 70 (Rta 125-190) or a polypeptide comprising a sequence 37 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT 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: 70 that maintains the same function.

[0207] SEQ ID NO: 68, Rta (full sequence, 1-190; 190 aa) RDSREGMFLPKPEAGSAISDVFEGREVCQPKRIRPFHPPGSPWANRPLPASLAPTPTGPVHEPV GSLTPAPVPQPLDPAPAVTPEASHLLEDPDEETSQAVKALREMADTVIPQKEEAAICGQMDLS HPPPRGHLDELTTTLESMTEDLNLDSPLTPELNEILDTFLNDECLLHAMHISTGLSIFDTSLF

[0208] SEQ ID NO: 69, Rta (75-190; 116 aa) PLDPAPAVTPEASHLLEDPDEETSQAVKALREMADTVIPQKEEAAICGQMDLSHPPPRGHLDE LTTTLESMTEDLNLDSPLTPELNEILDTFLNDECLLHAMHISTGLSIFDTSLF

[0209] SEQ ID NO: 70, Rta (125-190; 66 aa) DLSHPPPRGHLDELTTTLESMTEDLNLDSPLTPELNEILDTFLNDECLLHAMHISTGLSIFDTSL F

[0210] SEQ ID NO: 71, Rta (50-175; 126 aa) SSLAPTPTGPVHEPVGSLTPAPVPQPLDPAPAVTPEASHLLEDPDEETSQAVKALREMADTVIP QKEEAAICGQMDLSHPPPRGHLDELTTTLESMTEDLNLDSPLTPELNEILDTFLNDECLLHA

[0211] SEQ ID NO: 72, Rta (75-175; 101 aa) PLDPAPAVTPEASHLLEDPDEETSQAVKALREMADTVIPQKEEAAICGQMDLSHPPPRGHLDE LTTTLESMTEDLNLDSPLTPELNEILDTFLNDECLLHA

[0212] SEQ ID NO: 73, Rta (100-175; 76 aa) SVKALREMADTVIPQKEEAAICGQMDLSHPPPRGHLDELTTTLESMTEDLNLDSPLTPELNEI LDTFLNDECLLHA

[0213] SEQ ID NO: 74, Rta (125-175; 51 aa) DLSHPPPRGHLDELTTTLESMTEDLNLDSPLTPELNEILDTFLNDECLLHA

[0214] In some embodiments of any of the aspects, the TA is VPR, or a functional fragment thereof. VPR is a tripartite activator consisting of the VP64, the p65, and the Rta activation domains. In some embodiments of any of the aspects, VPR comprises VP64 (e.g., SEQ ID NO: 78), p65 (e.g., any one of SEQ ID NOs: 38, 58 to 67 or a polypeptide with at least 85% sequence identity to any one of SEQ ID NOs: 38, 58 to 67 that maintains the same function), and Rta (e.g., any one of SEQ ID NOs: 68-74 or a polypeptide with at least 85% sequence identity to any one of SEQ ID NOs: 68-74 that maintains the same function). In some embodiments of any of the aspects, VPR comprises one of SEQ ID NOs: 75, 76, 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: 75 or 76, that maintains its function. 38 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT

[0215] SEQ ID NO: 75, miniVPR, comprising the p65 (100-261aa; SEQ ID NO: 61) truncation and the RTA (125-190aa; SEQ ID NO: 70) truncation; bold text indicates VP64 (SEQ ID NO: 78); italicized text indicates SV40 NLS; bold italicized text indicates p65 (100-261aa; SEQ ID NO: 61); double underlined text indicates RTA (125-190aa; SEQ ID NO: 70). GRADALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLINSRS SGSPKKKRKVGSGGGSGGSGSVLPQAPAPAPAPAMVSALAQAPAPVPVLAPGPPQAVAPPAPK PTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNSEFQQLLNQGIPVAPHTT EPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGDEDFSSIADMDFSALLSGGGS GGSGSDLSHPPPRGHLDELTTTLESMTEDLNLDSPLTPELNEILDTFLNDECLLHAMHISTGLSI FDTSLF

[0216] SEQ ID NO: 76, full VPR; bold text indicates VP64 (SEQ ID NO: 78); italicized text indicates SV40 NLS; bold italicized text indicates p65 (SEQ ID NO: 59); double underlined text indicates RTA (SEQ ID NO: 68). GRADALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLINSRS SGSPKKKRKVGSQYLPDTDDRHRIEEKRKRTYETFKSIMKKSPFSGPTDPRPPPRRIAVPSRSS ASVPKPAPQPYPFTSSLSTINYDEFPTMVFPSGQISQASALAPAPPQVLPQAPAPAPAPAMVSA LAQAPAPVPVLAPGPPQAVAPPAPKPTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFT DLASVDNSEFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNG LLSGDEDFSSIADMDFSALLGSGSGSRDSREGMFLPKPEAGSAISDVFEGREVCQPKRIRPFHP PGSPWANRPLPASLAPTPTGPVHEPVGSLTPAPVPQPLDPAPAVTPEASHLLEDPDEETSQAV KALREMADTVIPQKEEAAICGQMDLSHPPPRGHLDELTTTLESMTEDLNLDSPLTPELNEILD TFLNDECLLHAMHISTGLSIFDTSLF

[0217] In some embodiments of any of the aspects, the TA comprises the Herpes Simplex Virus Protein 16 (VP16) activation domain. In some embodiments of any of the aspects, the TA comprises the VP64 activation domain, which comprises four tandem copies of VP16. In some embodiments of any of the aspects, the TA comprises one of SEQ ID NOs: 77, 78, 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: 77 or 78, that maintains its function.

[0218] SEQ ID NO: 77, VP16 (11 aa) DALDDFDLDML

[0219] SEQ ID NO: 78, VP64 (53 aa), with the VP16 domain indicated by bold text, GRADALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDML

[0220] In some embodiments of any of the aspects, the TA comprises NFZ or a functional fragment thereof. In some embodiments of any of the aspects, NFZ comprises SEQ ID NO: 57 or a polypeptide comprising a sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at 39 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT 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: 57 that maintains the same function.

[0221] In some embodiments of any of the aspects, the TA comprises 3Z or a functional fragment thereof. In some embodiments of any of the aspects, 3Z comprises SEQ ID NO: 303 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: 303 that maintains the same function.

[0222] In some embodiments of any of the aspects, the TA comprises p300 or a functional fragment thereof. The adenovirus E1A-associated cellular p300 transcriptional co-activator protein functions as histone acetyltransferase that regulates transcription via chromatin remodeling. In some embodiments of any of the aspects, p300 comprises SEQ ID NO: 79 or a protein having at least 85% sequence identity to SEQ ID NO: 79. In some embodiments of any of the aspects, p300 comprises a portion of SEQ ID NO: 79, e.g., residues 1048-1664 of SEQ ID NO: 79. In some embodiments of any of the aspects, the TA comprises the p300 HAT Core activation domain. In some embodiments of any of the aspects, p300 comprises SEQ ID NO: 80 or a protein having at least 85% sequence identity to SEQ ID NO: 80. In some embodiments of any of the aspects, the TA comprises one of SEQ ID NOs: 79, 80, 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: 79 or 80, that maintains its function.

[0223] SEQ ID NO: 79, human acetyltransferase p300 (2414 aa), bold text indicates the core activation domain MAENVVEPGPPSAKRPKLSSPALSASASDGTDFGSLFDLEHDLPDELINSTELGLTNGGDINQL QTSLGMVQDAASKHKQLSELLRSGSSPNLNMGVGGPGQVMASQAQQSSPGLGLINSMVKSP MTQAGLTSPNMGMGTSGPNQGPTQSTGMMNSPVNQPAMGMNTGMNAGMNPGMLAAGNG QGIMPNQVMNGSIGAGRGRQNMQYPNPGMGSAGNLLTEPLQQGSPQMGGQTGLRGPQPLK MGMMNNPNPYGSPYTQNPGQQIGASGLGLQIQTKTVLSNNLSPFAMDKKAVPGGGMPNMG QQPAPQVQQPGLVTPVAQGMGSGAHTADPEKRKLIQQQLVLLLHAHKCQRREQANGEVRQ CNLPHCRTMKNVLNHMTHCQSGKSCQVAHCASSRQIISHWKNCTRHDCPVCLPLKNAGDK RNQQPILTGAPVGLGNPSSLGVGQQSAPNLSTVSQIDPSSIERAYAALGLPYQVNQMPTQPQV QAKNQQNQQPGQSPQGMRPMSNMSASPMGVNGGVGVQTPSLLSDSMLHSAINSQNPMMSE NASVPSLGPMPTAAQPSTTGIRKQWHEDITQDLRNHLVHKLVQAIFPTPDPAALKDRRMENL VAYARKVEGDMYESANNRAEYYHLLAEKIYKIQKELEEKRRTRLQKQNMLPNAAGMVPVS MNPGPNMGQPQPGMTSNGPLPDPSMIRGSVPNQMMPRITPQSGLNQFGQMSMAQPPIVPRQ TPPLQHHGQLAQPGALNPPMGYGPRMQQPSNQGQFLPQTQFPSQGMNVTNIPLAPSSGQAPV SQAQMSSSSCPVNSPIMPPGSQGSHIHCPQLPQPALHQNSPSPVPSRTPTPHHTPPSIGAQQPPA TTIPAPVPTPPAMPPGPQSQALHPPPRQTPTPPTTQLPQQVQPSLPAAPSADQPQQQPRSQQST 40 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT AASVPTPTAPLLPPQPATPLSQPAVSIEGQVSNPPSTSSTEVNSQAIAEKQPSQEVKMEAKMEV DQPEPADTQPEDISESKVEDCKMESTETEERSTELKTEIKEEEDQPSTSATQSSPAPGQSKKKIF KPEELRQALMPTLEALYRQDPESLPFRQPVDPQLLGIPDYFDIVKSPMDLSTIKRKLDTG QYQEPWQYVDDIWLMFNNAWLYNRKTSRVYKYCSKLSEVFEQEIDPVMQSLGYCCGR KLEFSPQTLCCYGKQLCTIPRDATYYSYQNRYHFCEKCFNEIQGESVSLGDDPSQPQTTI NKEQFSKRKNDTLDPELFVECTECGRKMHQICVLHHEIIWPAGFVCDGCLKKSARTRK ENKFSAKRLPSTRLGTFLENRVNDFLRRQNHPESGEVTVRVVHASDKTVEVKPGMKAR FVDSGEMAESFPYRTKALFAFEEIDGVDLCFFGMHVQEYGSDCPPPNQRRVYISYLDSV HFFRPKCLRTAVYHEILIGYLEYVKKLGYTTGHIWACPPSEGDDYIFHCHPPDQKIPKPK RLQEWYKKMLDKAVSERIVHDYKDIFKQATEDRLTSAKELPYFEGDFWPNVLEESIKE LEQEEEERKREENTSNESTDVTKGDSKNAKKKNNKKTSKNKSSLSRGNKKKPGMPNVS NDLSQKLYATMEKHKEVFFVIRLIAGPAANSLPPIVDPDPLIPCDLMDGRDAFLTLARDK HLEFSSLRRAQWSTMCMLVELHTQSQDRFVYTCNECKHHVETRWHCTVCEDYDLCITCY NTKNHDHKMEKLGLGLDDESNNQQAAATQSPGDSRRLSIQRCIQSLVHACQCRNANCSLPS CQKMKRVVQHTKGCKRKTNGGCPICKQLIALCCYHAKHCQENKCPVPFCLNIKQKLRQQQL QHRLQQAQMLRRRMASMQRTGVVGQQQGLPSPTPATPTTPTGQQPTTPQTPQPTSQPQPTPP NSMPPYLPRTQAAGPVSQGKAAGQVTPPTPPQTAQPPLPGPPPAAVEMAMQIQRAAETQRQ MAHVQIFQRPIQHQMPPMTPMAPMGMNPPPMTRGPSGHLEPGMGPTGMQQQPPWSQGGLP QPQQLQSGMPRPAMMSVAQHGQPLNMAPQPGLGQVGISPLKPGTVSQQALQNLLRTLRSPS SPLQQQQVLSILHANPQLLAAFIKQRAAKYANSNPQPIPGQPGMPQGQPGLQPPTMPGQQGV HSNPAMQNMNPMQAGVQRAGLPQQQPQQQLQPPMGGMSPQAQQMNMNHNTMPSQFRDIL RRQQMMQQQQQQGAGPGIGPGMANHNQFQQPQGVGYPPQQQQRMQHHMQQMQQGNMG QIGQLPQALGAEAGASLQAYQQRLLQQQMGSPVQPNPMSPQQHMLPNQAQSPHLQGQQIPN SLSNQVRSPQPVPSPRPQSQPPHSSPSPRMQPQPSPHHVSPQTSSPHPGLVAAQANPMEQGHF ASPDQNSMLSQLASNPGMANLHGASATDLGLSTDNSDLNSNLSQSTLDIH

[0224] SEQ ID NO: 80, p300 HAT Core activation domain (617 aa) IFKPEELRQALMPTLEALYRQDPESLPFRQPVDPQLLGIPDYFDIVKSPMDLSTIKRKLDTGQY QE PWQYVDDIWLMFNNAWLYNRKTSRVYKYCSKLSEVFEQEIDPVMQSLGYCCGRKLEFSPQT LC CYGKQLCTIPRDATYYSYQNRYHFCEKCFNEIQGESVSLGDDPSQPQTTINKEQFSKRKNDTL DP ELFVECTECGRKMHQICVLHHEIIWPAGFVCDGCLKKSARTRKENKFSAKRLPSTRLGTFLEN RV NDFLRRQNHPESGEVTVRVVHASDKTVEVKPGMKARFVDSGEMAESFPYRTKALFAFEEID GV 41 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT DLCFFGMHVQEYGSDCPPPNQRRVYISYLDSVHFFRPKCLRTAVYHEILIGYLEYVKKLGYTT G HIWACPPSEGDDYIFHCHPPDQKIPKPKRLQEWYKKMLDKAVSERIVHDYKDIFKQATEDRL TS AKELPYFEGDFWPNVLEESIKELEQEEEERKREENTSNESTDVTKGDSKNAKKKNNKKTSKN KS SLSRGNKKKPGMPNVSNDLSQKLYATMEKHKEVFFVIRLIAGPAANSLPPIVDPDPLIPCDLM DG RDAFLTLARDKHLEFSSLRRAQWSTMCMLVELHTQSQD

[0225] 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 the transcriptional coactivation of many different transcription factors and has intrinsic histone acetyltransferase activity. In some embodiments of any of the aspects, 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 the CBP HAT Core activation domain. In some embodiments of any of the aspects, the TA comprises one of SEQ ID NOs: 81-83, 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: 81-83, that maintains its function.

[0226] SEQ ID NO: 81, 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. VNKFLRRQNHPEAGEVFVRVVASSDKTVEVKPGMKSRFVDSGEMSESFPYRTKALFAFEEID GVDVCFFGMHVQEYGSDCPPPNTRRVYISYLDSIHFFRPRCLRTAVYHEILIGYLEYVKKLGY VTGHIWACPPSEGDDYIFHCHPPDQKIPKPKRLQEWYKKMLDKAFAERIIHDYKDIFKQATED RLTSAKELPYFEGDFWPNVLEESIKELEQEEEERKKEESTAASETTEGSQGDSKNAKKKNNK KTNKNKSSISRANKKKPSMPNVSNDLSQKLYATMEKHKEVFFVIHLHAGPVINTLPPI

[0227] SEQ ID NO: 82, residues 1954-2267 of nejire, isoform E [Drosophila melanogaster], NCBI Reference Sequence: NP_001259387.1, HAT_KAT11, Histone acetylation protein VNNFLKKKEAGAGEVHIRVVSSSDKCVEVKPGMRRRFVEQGEMMNEFPYRAKALFAFEEV DGIDVCFFGMHVQEYGSECPAPNTRRVYIAYLDSVHFFRPRQYRTAVYHEILLGYMDYVKQ LGYTMAHIWACPPSEGDDYIFHCHPTDQKIPKPKRLQEWYKKMLDKGMIERIIQDYKDILKQ AMEDKLGSAAELPYFEGDFWPNVLEESIKELDQEEEEKRKQAEAAEAAAAANLFSIEENEVS GDGKKKGQKKAKKSNKSKAAQRKNSKKSNEHQSGNDLSTKIYATMEKHKEVFFVIRLHSA QSAASLAPI 42 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT

[0228] SEQ ID NO: 83, aa 1696–2329 from Drosophila CBP (nejire), NCBI Reference Sequence: NP_001259387.1, including the bromodomain, PHD domain, and HAT domain NGKYSDPWEYVDDVWLMFDNAWLYNRKTSRVYRYCTKLSEVFEAEIDPVMQALGYCCGR KYTFNPQVLCCYGKQLCTIPRDAKYYSYQNRYTYCQKCFNDIQGDTVTLGDDPLQSQTQIKK DQFKEMKNDHLELEPFVNCQECGRKQHQICVLWLDSIWPGGFVCDNCLKKKNSKRKENKF NAKRLPTTKLGVYIETRVNNFLKKKEAGAGEVHIRVVSSSDKCVEVKPGMRRRFVEQGEMM NEFPYRAKALFAFEEVDGIDVCFFGMHVQEYGSECPAPNTRRVYIAYLDSVHFFRPRQYRTA VYHEILLGYMDYVKQLGYTMAHIWACPPSEGDDYIFHCHPTDQKIPKPKRLQEWYKKMLDK GMIERIIQDYKDILKQAMEDKLGSAAELPYFEGDFWPNVLEESIKELDQEEEEKRKQAEAAEA AAAANLFSIEENEVSGDGKKKGQKKAKKSNKSKAAQRKNSKKSNEHQSGNDLSTKIYATME KHKEVFFVIRLHSAQSAASLAPIQDPDPLLTCDLMDGRDAFLTLARDKHFEFSSLRRAQFSTL SMLYELHNQGQDKFVYTCNHCKTAVETRYHCTVCDDFDLCIVCKEKVGHQHKMEKLGFDI DDGSALADHKQANPQEARKQSI.

[0229] In another embodiment of any aspect described herein, in the synTF described or the ZF- containing fusion protein described herein, the transcriptional effector domain comprises an epigenetic effector domain. For example, at least one ZF protein domain is fused to one or more chromatin regulating enzymes that (1) catalyze chemical modifications of DNA or histone residues (e.g. DNA methyltransferases, histone methyltransferases, histone acetyltransferases) or (2) remove chemical modifications (e.g. DNA demethylases, DNA di-oxygenases, DNA hydroxylases, histone demethylases, histone deacetylases). One example is CBP / p300 histone acetyltransferase, which is typically associated with transcriptional activation through the interactions with multiple transcription factors. Related epigenetic effector domains associated with the deposition of biochemical marks on DNA or histone residue(s) include HAT1, GCN5, PCAF, MLL, SET, DOT1, SUV39H, G9a, KAT2A / B and EZH1 / 2. Related epigenetic effector domains associated with the removal of biochemical marks from DNA or histone residue(s) include TET1 / 2, SIRT family, LSD1, and KDM family. DNA-Binding Domain

[0230] Described herein are synTFs comprising at least one DNA-binding domain (DBD). In some embodiments of any of the aspects, a synTF polypeptide as described herein (or a synTF polypeptide system collectively) comprises 1, 2, 3, 4, 5, or more DBD(s). In some embodiments of any of the aspects, the synTF polypeptide or system comprises one DBD. In embodiments comprising multiple DBDs, the multiple DBDs can be different individual DBDs or multiple copies of the same DBDs, or a combination of the foregoing. 43 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT

[0231] In some embodiments of any of the aspects, the at least one DBD is an engineered zinc finger (ZF) binding domain. A zinc finger (ZF) is a finger-shaped fold in a protein that permits it to interact with nucleic acid sequences such as DNA and RNA. Such a fold is well known in the art. The fold is created by the binding of specific amino acids in the protein to a zinc atom. Zinc-finger containing proteins (also known as ZF proteins) can regulate the expression of genes as well as nucleic acid recognition, reverse transcription and virus assembly.

[0232] A ZF is a relatively small polypeptide domain comprising approximately 30 amino acids, which folds to form an α-helix adjacent an antiparallel β-sheet (known as a ββα-fold). The fold is stabilized by the co-ordination of a zinc ion between four largely invariant (depending on zinc finger framework type) Cys and / or His residues, as described further 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. A ZF domain recognizes and binds to a nucleic acid triplet, or an overlapping quadruplet (as explained below), in a double-stranded DNA target sequence. However, ZFs are also known to bind RNA and proteins (Clemens, K. R. et al. (1993) Science 260: 530-533; Bogenhagen, D. F. (1993) Mol. Cell. Biol.13: 5149-5158; Searles, M. A. et al. (2000) J. Mol. Biol.301: 47-60; Mackay, J. P. & Crossley, M. (1998) Trends Biochem. Sci.23: 1-4).

[0233] In one embodiment, as used herein, the term “zinc finger” (ZF) or “zinc finger motif” (ZF motif) or “zinc finger domain” (ZF domain) refers to an individual “finger”, which comprises a beta- beta-alpha (ββα)-protein fold stabilized by a zinc ion as described elsewhere herein. The Zn- coordinated ββα protein fold produces a finger-like protrusion, a “finger.” Each ZF motif typically includes approximately 30 amino acids. The term “motif” as used herein refers to a structural motif. The ZF motif is a supersecondary structure having the ββα-fold that stabilized by a zinc ion.

[0234] In one embodiment, the term “ZF motif” according to its ordinary usage in the art, refers to a discrete continuous part of the amino acid sequence of a polypeptide that can be equated with a particular function. ZF motifs are largely structurally independent and may retain their structure and function in different environments. Because the ZF motifs are structurally and functionally independent, the motifs also qualify as domains, thus are often referred as ZF domains. Therefore, ZF domains are protein motifs that contain multiple finger-like protrusions that make tandem contacts with their target molecule. Typically, a ZF domain binds a triplet or (overlapping) quadruplet nucleotide sequence. Adjacent ZF domains arranged in tandem are joined together by linker sequences to form an array. A ZF peptide typically contains a ZF array and is composed of a plurality of “ZF domains”, which in combination do not exist in nature. Therefore, they are considered to be artificial or synthetic ZF peptides or proteins.

[0235] C2H2zinc fingers (C2H2-ZFs) are the most prevalent type of vertebrate DNA-binding domain, and typically appear in tandem arrays (ZFAs), with sequential C2H2-ZFs each contacting 44 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT three (or more) sequential bases. C2H2-ZFs can be assembled in a modular fashion. Given a set of modules with defined three-base specificities, modular assembly also presents a way to construct artificial proteins with specific DNA-binding preferences.

[0236] ZF-containing proteins generally contain strings or chains of ZF motifs, forming an array of ZF (ZFA). Thus, a natural ZF protein may include 2 or more ZF, i.e., a ZFA consisting of 2 or more ZF motifs, which may be directly adjacent one another (i.e. separated by a short (canonical) linker sequence), or may be separated by longer, flexible or structured polypeptide sequences. Directly adjacent ZF domains are expected to bind to contiguous nucleic acid sequences, i.e. to adjacent trinucleotides / triplets. In some cases cross-binding may also occur between adjacent ZF and their respective target triplets, which helps to strengthen or enhance the recognition of the target sequence, and leads to the binding of overlapping quadruplet sequences (Isalan et al., (1997) Proc. Natl. Acad. Sci. USA, 94: 5617-5621). By comparison, distant ZF domains within the same protein may recognize (or bind to) non-contiguous nucleic acid sequences or even to different molecules (e.g. protein rather than nucleic acid).

[0237] Engineered ZF-containing proteins are chimeric proteins composed of a DNA-binding zinc finger protein domain (ZF protein domain) and another domain through which the protein exerts its effect (effector domain). The effector domain may be a transcriptional activator, a methylation domain or a nuclease. DNA-binding ZF protein domain would contain engineered zinc finger arrays (ZFAs). See e.g., Khalil et al., Cell Volume 150, Issue 3, 3 August 2012, Pages 647-658; US Patent 10,138,493; US Patent Application US20200002710A1; the contents of each of which are incorporated herein by reference in their entireties.

[0238] Engineered ZF-containing proteins are non-natural and suitably contain 3 or more, for example, 4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or more (e.g., up to approximately 30 or 32) ZF motifs arranged adjacent one another in tandem, forming arrays of ZF motifs or ZFA. Particularly ZF-containing synTF proteins (ZF-containing synTF fusion protein, or simply synTF) of the disclosure include at least 3 ZF, at least 4 ZF motifs, at least 5 ZF motifs, or at least 6 ZF motifs, at least 7 ZF motifs, at least 8 ZF motifs, at least 9 ZF motifs, at least 10 ZF motifs, at least 11 or at least 12 ZF motifs; and in some cases 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 1 or more ZF motif. The ZF-containing synTF of the disclosure bind to contiguous orthogonal target nucleic acid binding sites. That is, the ZFs or ZFAs comprising in the ZF domain of the fusion protein binds orthogonal target nucleic acid sequences.

[0239] In one embodiment, as used herein, an “engineered synthetic transcription factor” or “engineered synTF” or “synTF” refers to an engineered ZF-containing chimeric protein having at least one of the following characteristics and may have more than one: bind target orthogonal specific 45 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT DNA sequences and have, for example, reduced or minimal functional binding potential in a host eukaryotic genome; are derived from mammalian protein scaffolds, conferring minimal degree of immunogenicity over other prokaryotically-derived domains; and can be packaged in viral delivery systems, such as lentiviral delivery constructs.

[0240] In another embodiment, as used herein, the term “engineered synthetic transcription factor” or “engineered synTF,” abbreviated as “synTF” or “ZF synTF,” refers to an engineered ZF containing synthetic transcription factor that is a polypeptide, in other words, a ZF-containing synthetic transcription factor protein. These synTFs contain ZF arrays (ZFA) therein for binding to specific target nucleic acid sequences. The synTF is a chimeric, fusion protein that comprises 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, wherein the modulation is by increasing or decreasing the expression of a gene that is operably linked to a promoter that is also operably linked to the specific target nucleic acid sequence to which the DNA-binding, ZF-containing protein domain of the synTF binds.

[0241] As used herein, the term “ZF array,” abbreviated as “ZFA” refers to an array, or a string, or a chain of ZF motifs arranged in tandem. A ZFA can have six ZF motifs (a 6-finger ZFA), seven ZF motifs (a 7-finger ZFA), or eight ZF motifs (an 8-finger ZFA).

[0242] As used herein, the term “engineered responsive / response promoter,” “engineered promoter,” or “engineered responsive / response promoter element” refers is a nucleic acid construct containing a promoter sequence that has at least one orthogonal DNA target sequence operably linked upstream of the promoter sequence such that the orthogonal DNA target sequence confer a responsive property to the promoter when the orthogonal DNA target sequence is bound by its respective transcription factor, the responsive property being whether gene transcription initiation from that promoter is enhanced when the upstream nearby orthogonal DNA target sequences are bound by a ZF-containing synthetic transcription factor. There may be more than one orthogonal DNA target sequence operably linked upstream of the promoter sequence. When there is one orthogonal DNA target sequence, the promoter is referred to a “1X” promoter, where the “1X” refers to the number of orthogonal DNA target sequence present in the promoter construct. For example, a 4X responsive promoter would be identified as having four orthogonal DNA target sequences in the engineered response promoter construct, and the four orthogonal DNA target sequences are upstream of the promoter sequence.

[0243] The ZF protein domain is modular in design, with zinc finger arrays (ZFA) as the main components, and each ZFA is made of 6-8 ZF motifs. The ZF protein domain comprises at least one ZFA and can contain as many as up to ten ZFA. The ZF protein domain can have one and up to ten ZFA. 46 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT

[0244] The design of the synTF or any engineered fusion protein described herein is also modular, meaning the synTF is made up of modules of ZF domains (ZFA) and modules of effector domains / protein interaction domains / ligand binding domains / dimerization domains, the individual modules are covalently conjugated together as described herein, and the individual modules function independently of each other. The number of ZFA can range from one, two, three, four, five, six, seven, eight, nine, and up to ten. When there are two or more ZFA, the ZFAs are covalently conjugated to each other in tandem, e.g., by a L1 peptide linker, in an NH2- to COOH- terminus arrangement to form an array of ZFA. The ZFAs, as a whole, forms the ZF protein domain, is covalently linked to the N-terminus or the C-terminus of the effector domain or the regulator protein. When there are two or more ZFAs present in the ZF protein domain of a synTF or a ZF containing fusion protein described herein, the ZFAs can be the same, or different.

[0245] Each modular ZFA in the ZF protein domain of a synTF disclosed herein or a ZF containing fusion protein described herein is comprised of six to eight ZF motifs. For example, a single ZFA having seven ZF motifs is referred to as a seven-finger ZFA. The ZF motif is a small protein structural motif consisting of an α helix and an antiparallel β sheet (αββ) and is characterized by the coordination of one zinc ion by two histidine residues and two cysteine residues in the motif in order to stabilize the finger-like protrusion fold, the “finger”. The ZF motif in the ZF protein domain of a synTF disclosed herein is a Cys2His2zinc finger motif. In one embodiment, the ZF motif comprises, consisting essentially of, or consisting of a peptide of formula 1: [X0-3CX1-5CX2-7-(helix)- HX3-6H] (SEQ ID NO: 84) wherein X is any amino acid, the subscript numbers indicate the possible number of amino acid residues, C is cysteine, H is histidine, and (helix) is a-six (or seven) contiguous amino acid residue peptide that forms a short alpha helix. The helix is variable. This short alpha helix forms one facet of the finger formed by the coordination of the zinc ion by two histidine residues and two cysteine residues in the ZF motif. For each ZFA, the six to eight ZF motifs therein are linked to each other, NH2- to COOH- terminus by a peptide linker having about four to six amino acid residues to form an array of ZF motifs or ZF. The finger-like protrusion fold of each ZF motif interacts with and binds nucleic acid sequence. Approximately a peptide sequence for two ZF motif interacts with and binds a ~six-base pair (bp) nucleic acid sequence. The multiple ZF motifs in a ZFA form finger- like protrusions that would make contact with an orthogonal target DNA sequence. Hence, for example, a ZFA with six ZF motifs or finger-like protrusions (a six-finger ZFA) interacts with and binds a ~18-20 bp nucleic acid sequence, and an eight-finger ZFA would bind a ~24-26 bp nucleic acid sequence. Accordingly, in one embodiment, the ZFA in the ZF protein domain of a synTF comprises, consists essentially of, or consists of a sequence: N’-[(formula 1)-L2]6-8-C’, where the subscript 6-8 indicates the number of ZF motifs, the L2is a linker peptide having 4-6 amino acid residues, and the N’- and C’- indicates the N-terminus and C-terminus respectively of the peptide sequence. For example, for a ZFA consists essentially of six ZF motifs, the sequence is N’- [(formula 47 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT 1)-L2]-[(formula 1)-L2]-[(formula 1)-L2]-[(formula 1)-L2]-[(formula 1)-L2]-[(formula 1)-L2]-C’, and a ZFA consists 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]-[(formula 1)- L2]-C’.

[0246] SEQ ID NO: 84: XXXCXXXXXCXXXXXXXXXXXXXHXXXXXXH

[0247] In another embodiment of any aspect described herein, the ZF motif comprises a peptide of formula 2: [X3CX2CX5-(helix)-HX3H] (SEQ ID NO: 85) wherein X is any amino acid, the subscript numbers indicate the possible number of amino acid residues, C is cysteine, H is histidine, and (helix) is a-six (or seven) contiguous amino acid residue peptide that forms a short alpha helix. Accordingly, in one embodiment, the ZFA in the ZF protein domain of a synTF comprises, consists essentially of, or consists of a sequence: N’-[(formula 2)-L2]6-8-C’, where the subscript 6-8 indicates the number of ZF motifs, the L2is a linker peptide having 4-6 amino acid residues, and the N’- and C’- indicates the N-terminus and C-terminus respectively of the peptide sequence. For example, for a ZFA consists 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 a ZFA consists 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]-[(formula 2)-L2]-C’.

[0248] SEQ ID NO: 85: XXXCXXCXXXXXXXXXXXHXXXH

[0249] In one embodiment of any aspect described herein, for a single ZFA is the ZF protein domain of a synTF disclosed herein, the ZFA in the ZF protein domain comprises, consists essentially of, or consists of a sequence: N’-PGERPFQCRICMRNFS-(Helix 1)- HTRTHTGEKPFQCRICMRNFS-(Helix 2)- HLRTHTGSQK PFQCRICMRNFS-(Helix 3)- HTRTHTGEK PFQCRICMRNFS-(Helix 4)-HLRTHTGSQKPFQCRICMRNFS-(Helix 5)- HTRTHTGEK PFQCRICMRNFS-(Helix 6)-HLRTHLR-C’ (SEQ ID NO: 87), wherein the (Helix) is a-six (or seven) contiguous amino acid residue peptide that forms a short alpha helix and can also be represented as plain text “xxxxxxx”.

[0250] SEQ ID NO: 88, Zinc Finger Domain scaffold; italicized double underlined text indicates the restriction sites; plain text “xxxxxxx” indicates six ZF helices, e.g., from N terminus to C terminus: helix 1, helix 2, helix 3, helix 4, helix 5, and helix 6 SRPGERPFQCRICMRNFSxxxxxxxHTRTHTGEKPFQCRICMRNFSxxxxxxxHLRTHTGSQKPFQC RICMRNFSxxxxxxxHTRTHTGEKPFQCRICMRNFSxxxxxxxHLRTHTGSQKPFQCRICMRNFSxx xxxxxHTRTHTGEKPFQCRICMRNFSxxxxxxxHLRTHLRGS

[0251] SEQ ID NO: 89, Zinc Finger Domain scaffold, wherein [Helix 1], [Helix 2], [Helix 3], [Helix 4], [Helix 5], and [Helix 6] can also be represented as plain text “xxxxxxx” 48 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT PGERPFQCRICMRNFS [Helix1] HTRTHTGEKPFQCRICMRNFS [Helix 2] HLRTHTGSQKPFQCRICMRNFS [Helix 3] HLRTHTGEKPFQCRICMRNFS [Helix 4] HLKTHTGSQKPFQCRICMRNFS [Helix 5] HLRTHTGEKPFQCRICMRNFS [Helix 6] HLRTHLR

[0252] SEQ ID NO: 90, Zinc Finger Domain scaffold; italicized double underlined text indicates the restriction sites; plain text “xxxxxxx” indicates six ZF helices, e.g., from N terminus to C terminus: helix 1, helix 2, helix 3, helix 4, helix 5, and helix 6 SRPGERPFQCRICMRNFSxxxxxxxHTRTHTGEKPFQCRICMRNFSxxxxxxxHLRTHTGSQKPFQC RICMRNFSxxxxxxxHLRTHTGEKPFQCRICMRNFSxxxxxxxHLKTHTGSQKPFQCRICMRNFSxx xxxxxHLRTHTGEKPFQCRICMRNFSxxxxxxxHLRTHLRGS

[0253] In some embodiments of any of the aspects, the zinc finger scaffold comprises one of SEQ ID NOs: 84-90or 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: 84-90 that maintains the same function.

[0254] In one embodiment, all six of the helix 1, 2, 3, 4, 5 and 6 are distinct and different from each other. In another embodiment, all six of the helix 1, 2, 3, 4, 5 and 6 are identical to each other. Alternatively, at least two of the six helices are identical and the same with each other. In other embodiments, at least three of the six helices in a ZFA are identical and the same with each other, at least four of the six helices in a ZFA are identical and the same with each other, or at least five of the six helices in a ZFA are identical and the same with each other.

[0255] In some embodiments of any aspect described herein, the helices of the six to eight ZF motifs of an individual ZFA disclosed herein are selected from the six-amino acid (or seven-amino acid) residue peptide sequences disclosed in one of the following Groups 1-11 (e.g., SEQ ID NOs: 122-181). In some embodiments, at least four of the ZF motifs in an individual ZFA disclosed herein are selected from the six-amino acid (or seven-amino acid) residue peptide sequences disclosed in one of the following Groups 1-11. In other embodiments, all of the ZF motifs, i.e. the six, seven or eight ZF motifs in an individual ZFA disclosed herein, are selected from the six (or seven) amino acid residue peptide sequences disclosed in one of the following Groups 1-11. In any individual ZFA, the helix selected for a single ZF comprising the ZFA can be repeated twice or more in the ZFA. This means that for any given single ZFA, at least four or all the helices in the ZFA are selected from the same group disclosed herein. For example, wherein a ZFA consists essentially of six ZF motifs, that means there are six alpha helices. All the 6-8 helices (Helix 1; Helix 2; Helix 3; Helix 4; Helix 5; Helix 6; Helix 7; Helix 8) of the ZFs in an individual ZFA is selected from one of the following group 1-11, for example, all six helices are selected from group 2. That is, all the helices for all the ZF comprising a single ZFA come from the same group. Alternatively, at least four of the six helices are selected from the same group, a group selected from group 1-11. For example, four of the six helices are selected from group 5, and the remainder two helices of the six-ZF motif ZFA are selected from 49 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT the other groups 1-4, 6-11, or can be any other helices that would form a short alpha helix. The other remaining helices making up the ZFA can be those that are known in the art.

[0256] Table 6: Groups 1-4 helices

[0257] Table 7: Groups 5-7 helices

[0258] Table 8: Groups 8-11 helices50 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT

[0259] Non-limiting examples of the combinations and arrangements of six helices in a single ZFA where the helices are selected from Group 1 and where the motifs are in an NH2- to COOH- terminus arrangement, (Group 1 ZFA helix combo), are as follows: ZF 1-1: N’-DEANLRR, DPSVLKR, QSANLLR, DPSSLKR,QQTNLTR, DATQLVR-C’ (SEQ ID NOS 122, 123, 124, 125, 126, and 127, respectively, in order of appearance) ZF 1-2: N’-DEANLRR, DPSVLKR, QSANLLR, DPSSLKR,QQTNLTR, ERRSLAR-C’ (SEQ ID NOS 122, 123, 124, 125, 126, and 128, respectively, in order of appearance) ZF 1-3: N’-EEANLRR, DHSSLKR, QSANLLR, DPSSLKR QQTNLTR, DATQLVR-C’ (SEQ ID NOS 129, 130, 124, 125, 126, and 127, respectively, in order of appearance) ZF 1-4: N’-EEANLRR, DHSSLKR, QSANLLR, DPSSLKR QQTNLTR, ERRSLAR-C’(SEQ ID NOS 129, 130, 124, 125, 126, and 128, respectively, in order of appearance) ZF 1-5: N’-DEANLRR, DPSVLKR, QQTNLTR, ERRSLAR QQTNLTR, DATQLVR-C’ (SEQ ID NOS 122, 123, 126, 128, 126, and 127, respectively, in order of appearance) ZF 1-6: N’-DEANLRR, DPSVLKR, QQTNLTR, ERRSLAR QQTNLTR, ERRSLAR-C’ (SEQ ID NOS 122, 123, 126, 128, 126, and 128, respectively, in order of appearance) ZF 1-7: N’-EEANLRR, DHSSLKR, QQTNLTR, ERRSLAR QQTNLTR, DATQLVR-C’ (SEQ ID NOS 129, 130, 126, 128, 126, and 127, respectively, in order of appearance) ZF 1-8: N’-EEANLRR, DHSSLKR, QQTNLTR, ERRSLAR QQTNLTR, ERRSLAR-C’ (SEQ ID NOS 129, 130, 126, 128, 126, and 128, respectively, in order of appearance)

[0260] Non-limiting examples of the combinations and arrangements of six helices in a singlesix-finger ZFA where the helices are selected from Group 2 and where the motifs are in an NH2- toCOOH- terminus arrangement, (Group 2 ZFA helix combo), are as follows: ZF 2-1: N’-QRSSLVR, DMGNLGR, RSHDLTR, HKSSLTR, DSSNLRR, DQGNLIR-C’ (SEQ ID NOS 131, 132, 133, 134, 135, and 136, respectively, in order of appearance) ZF 2-2: N’-QKQALTR, DRGNLTR, RSHDLTR, HKSSLTR, DSSNLRR, DQGNLIR-C’ (SEQ ID NOS 137, 138, 133, 134, 135, and 136, respectively, in order of appearance) ZF 2-3: N’-QRSSLVR, DMGNLGR, RSHDLTV, HKSSLTR, DSSNLRR, DQGNLIR-C’ (SEQ ID NOS 131, 132, 139, 134, 135, and 136, respectively, in order of appearance) 51 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT ZF 2-4: N’-QKQALTR, DRGNLTR, RSHDLTV, HKSSLTR, DSSNLRR, DQGNLIR-C’ (SEQ ID NOS 137, 138, 139, 134, 135, and 136, respectively, in order of appearance) ZF 2-5: N’-QRSSLVR, DMGNLGR, RSHDLTR, HKSSLTR, EKQNLAR, DPSNLRR-C’ (SEQ ID NOS 131, 132, 133, 134, 148, and 149, respectively, in order of appearance) ZF 2-6: N’-QKQALTR, DRGNLTR, RSHDLTR, HKSSLTR, EKQNLAR, DPSNLRR-C’ (SEQ ID NOS 137, 138, 133, 134, 148, and 149, respectively, in order of appearance) ZF 2-7: N’-QRSSLVR, DMGNLGR, RSHDLTV, HKSSLTR, EKQNLAR, DPSNLRR-C’ (SEQ ID NOS 131, 132, 139, 134, 148, and 149, respectively, in order of appearance) ZF 2-8: N’-QKQALTR, DRGNLTR, RSHDLTV, HKSSLTR, EKQNLAR, DPSNLRR-C’ (SEQ ID NOS 137, 138, 139, 134, 148, and 149, respectively, in order of appearance)

[0261] Non-limiting examples of the combinations and arrangements of six helices in a single six-finger ZFA where the helices are selected from Group 3 and where the motifs are in an NH2- to COOH- terminus arrangement, (Group 3 ZFA helix combo), are as follows: ZF 3-1: N’-QRSSLVR, DKSVLAR, QRSSLVR, QTNNLGR, THAVLTR, DRGNLTR-C’ (SEQ ID NOS 131, 140, 131, 141, 142, and 138, respectively, in order of appearance) ZF 3-2: N’-QRSSLVR, DKSVLAR, QRSSLVR, QTNNLGR, TKSLLAR, DRGNLTR-C’ (SEQ ID NOS 131, 140, 131, 141, 143, and 138, respectively, in order of appearance) ZF 3-3: N’-QKQALDR, DTSVLNR, QRSSLVR, QTNNLGR, THAVLTR, DRGNLTR-C’ (SEQ ID NOS 144, 145, 131, 141, 142, and 138, respectively, in order of appearance) ZF 3-4: N’-QKQALDR, DTSVLNR, QRSSLVR, QTNNLGR, TKSLLAR, DRGNLTR-C’ (SEQ ID NOS 144, 145, 131, 141, 143, and 138, respectively, in order of appearance) ZF 3-5: N’-QRSSLVR, DKSVLAR, QRSSLVR, QTNNLGR, THAVLTR, DRGNLTR-C’ (SEQ ID NOS 131, 140, 131, 141, 142, and 138, respectively, in order of appearance) ZF 3-6: N’-QRSSLVR, DKSVLAR, QRSSLVR, QTNNLGR, TKSLLAR, DRGNLTR-C’ (SEQ ID NOS 131, 140, 131, 141, 143, and 138, respectively, in order of appearance) ZF 3-7: N’-QKQALDR, DTSVLNR, QRSSLVR, QTNNLGR, THAVLTR, DRGNLTR-C’ (SEQ ID NOS 144, 145, 131, 141, 142, and 138, respectively, in order of appearance) ZF 3-8: N’-QKQALDR, DTSVLNR, QRSSLVR, QTNNLGR, TKSLLAR, DRGNLTR-C’ (SEQ ID NOS 144, 145, 131, 141, 143, and 138, respectively, in order of appearance)

[0262] In some embodiments of any of the aspects, QRNNLGR (SEQ ID NO: 181) is used in place of QTNNLGR (SEQ ID NO: 141). Non-limiting examples of the combinations and arrangements of six helices in a single six-finger ZFA where the helices are selected from Group 3 and where the motifs are in an NH2- to COOH- terminus arrangement, (Group 3 ZFA helix combo), are as follows: ZF 3-1: N’-QRSSLVR, DKSVLAR, QRSSLVR, QRNNLGR, THAVLTR, DRGNLTR-C’ (SEQ ID NOS 131, 140, 131, 181, 142, and 138, respectively, in order of appearance) 52 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT ZF 3-2: N’-QRSSLVR, DKSVLAR, QRSSLVR, QRNNLGR, TKSLLAR, DRGNLTR-C’ (SEQ ID NOS 131, 140, 131, 181, 143, and 138, respectively, in order of appearance) ZF 3-3: N’-QKQALDR, DTSVLNR, QRSSLVR, QRNNLGR, THAVLTR, DRGNLTR-C’ (SEQ ID NOS 144, 145, 131, 181, 142, and 138, respectively, in order of appearance) ZF 3-4: N’-QKQALDR, DTSVLNR, QRSSLVR, QRNNLGR, TKSLLAR, DRGNLTR-C’ (SEQ ID NOS 144, 145, 131, 181, 143, and 138, respectively, in order of appearance) ZF 3-5: N’-QRSSLVR, DKSVLAR, QRSSLVR, QRNNLGR, THAVLTR, DRGNLTR-C’ (SEQ ID NOS 131, 140, 131, 181, 142, and 138, respectively, in order of appearance) ZF 3-6: N’-QRSSLVR, DKSVLAR, QRSSLVR, QRNNLGR, TKSLLAR, DRGNLTR-C’ (SEQ ID NOS 131, 140, 131, 181, 143, and 138, respectively, in order of appearance) ZF 3-7: N’-QKQALDR, DTSVLNR, QRSSLVR, QRNNLGR, THAVLTR, DRGNLTR-C’ (SEQ ID NOS 144, 145, 131, 181, 142, and 138, respectively, in order of appearance) ZF 3-8: N’-QKQALDR, DTSVLNR, QRSSLVR, QRNNLGR, TKSLLAR, DRGNLTR-C’ (SEQ ID NOS 144, 145, 131, 181, 143, and 138, respectively, in order of appearance)

[0263] Non-limiting examples of the combinations and arrangements of six helices in a single six-finger ZFA where the helices are selected from Group 4 and where the motifs are in an NH2- to COOH- terminus arrangement, (Group 4 ZFA helix combo), are as follows: ZF 4-1: N’-QQTNLTR, QGTSLAR, VRHNLTR, DKSVLAR, DSSNLRR, DQGNLIR-C’ (SEQ ID NOS 126, 146, 147, 140, 135, and 136, respectively, in order of appearance) ZF 4-2: N’-QQTNLTR, QGTSLAR, VRHNLTR, DKSVLAR, EKQNLAR, DPSNLRR-C’ (SEQ ID NOS 126, 146, 147, 140, 148, and 149, respectively, in order of appearance) ZF 4-3: N’-QQTNLTR, QGTSLAR, VRHNLTR, DHSNLSR, DSSNLRR, DQGNLIR-C’ (SEQ ID NOS 126, 146, 147, 150, 135, and 136, respectively, in order of appearance) ZF 4-4: N’-QQTNLTR, QGTSLAR, VRHNLTR, DHSNLSR, EKQNLAR, DPSNLRR-C’ (SEQ ID NOS 126, 146, 147, 150, 148, and 149, respectively, in order of appearance) ZF 4-5: N’-QQTNLTR, QSTSLQR, VRHNLTR, DKSVLAR, DSSNLRR, DQGNLIR-C’ (SEQ ID NOS 126, 151, 147, 140, 135, and 136, respectively, in order of appearance) ZF 4-6: N’-QQTNLTR, QSTSLQR, VRHNLTR, DKSVLAR, EKQNLAR, DPSNLRR-C’ (SEQ ID NOS 126, 151, 147, 140, 148, and 149, respectively, in order of appearance) ZF 4-7: N’-QQTNLTR, QSTSLQR, VRHNLTR, DHSNLSR, DSSNLRR, DQGNLIR-C’ (SEQ ID NOS 126, 151, 147, 150, 135, and 136, respectively, in order of appearance) ZF 4-8: N’-QQTNLTR, QSTSLQR, VRHNLTR, DHSNLSR, EKQNLAR, DPSNLRR-C’ (SEQ ID NOS 126, 151, 147, 150, 148, and 149, respectively, in order of appearance)

[0264] Non-limiting examples of the combinations and arrangements of six helices in a single six-finger ZFA where the helices are selected from Group 5 and where the motifs are in an NH2- to COOH- terminus arrangement, (Group 5 ZFA helix combo), are as follows: 53 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT ZF 5-1: N’-NMSNLTR, DRSVLRR, LQENLTR, DRSSLRR, QSGTLHR, QSGTLHR-C’ (SEQ ID NOS 152, 153, 154, 155, 156, and 156, respectively, in order of appearance) ZF 5-2: N’-QLANLAR, DQTTLRR, LQENLTR, DRSSLRR, QSGTLHR, QSGTLHR-C’ (SEQ ID NOS 157, 158, 154, 155, 156, and 156, respectively, in order of appearance) ZF 5-3: N’-NMSNLTR, DRSVLRR, DPSNLAR, DRSSLRR, QSGTLHR, QSGTLHR-C’ (SEQ ID NOS 152, 153, 159, 155, 156, and 156, respectively, in order of appearance) ZF 5-4: N’-QLANLAR, DQTTLRR, DPSNLAR, DRSSLRR, QSGTLHR, QSGTLHR-C’ (SEQ ID NOS 157, 158, 159, 155, 156, and 156, respectively, in order of appearance) ZF 5-5: N’-NMSNLTR, DRSVLRR, LQENLTR, DRSSLRR, QSGTLHR, QSGTLHR-C’ (SEQ ID NOS 152, 153, 154, 155, 156, and 156, respectively, in order of appearance) ZF 5-6: N’-QLANLAR, DQTTLRR, LQENLTR, DRSSLRR, QSGTLHR, QSGTLHR-C’ (SEQ ID NOS 157, 158, 154, 155, 156, and 156, respectively, in order of appearance) ZF 5-7: N’-NMSNLTR, DRSVLRR, DPSNLAR, DRSSLRR, QSGTLHR, QSGTLHR-C’ (SEQ ID NOS 152, 153, 159, 155, 156, and 156, respectively, in order of appearance) ZF 5-8: N’-QLANLAR, DQTTLRR, DPSNLAR, DRSSLRR, QSGTLHR, QSGTLHR-C’ (SEQ ID NOS 157, 158, 159, 155, 156, and 156, respectively, in order of appearance)

[0265] Non-limiting examples of the combinations and arrangements of six helices in a single six-finger ZFA where the helices are selected from Group 6 and where the motifs are in an NH2- to COOH- terminus arrangement, (Group 6 ZFA helix combo), are as follows: ZF 6-1: N’-QQTNLTR, QGGNLAL, DHSSLKR, RADMLRR, DSSNLRR, DQGNLIR-C’ (SEQ ID NOS 126, 160, 130, 161, 135, and 136, respectively, in order of appearance) ZF 6-2: N’-QQTNLTR, QGGNLAL, DHSSLKR, RADMLRR, EKQNLAR, DPSNLRR-C’ (SEQ ID NOS 126, 160, 130, 161, 148, and 149, respectively, in order of appearance) ZF 6-3: N’-QQTNLTR, QKANLGV, DHSSLKR, RADMLRR, DSSNLRR, DQGNLIR-C’ (SEQ ID NOS 126, 162, 130, 161, 135, and 136, respectively, in order of appearance) ZF 6-4: N’-QQTNLTR, QKANLGV, DHSSLKR, RADMLRR, EKQNLAR, DPSNLRR-C’ (SEQ ID NOS 126, 162, 130, 161, 148, and 149, respectively, in order of appearance) ZF 6-5: N’-QQTNLTR, QGGNLAL, DHSSLKR, RLDMLAR, DSSNLRR, DQGNLIR-C’ (SEQ ID NOS 126, 160, 130, 163, 135, and 136, respectively, in order of appearance) ZF 6-6: N’-QQTNLTR, QGGNLAL, DHSSLKR, RLDMLAR, EKQNLAR, DPSNLRR-C’ (SEQ ID NOS 126, 160, 130, 163, 148, and 149, respectively, in order of appearance) ZF 6-7: N’-QQTNLTR, QKANLGV, DHSSLKR, RLDMLAR, DSSNLRR, DQGNLIR-C’ (SEQ ID NOS 126, 162, 130, 163, 135, and 136, respectively, in order of appearance) ZF 6-8: N’-QQTNLTR, QKANLGV, DHSSLKR, RLDMLAR,EKQNLAR, DPSNLRR-C’ (SEQ ID NOS 126, 162, 130, 163, 148, and 149, respectively, in order of appearance) 54 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT

[0266] Non-limiting examples of the combinations and arrangements of six helices in a single six-finger ZFA where the helices are selected from Group 7 and where the motifs are in an NH2- to COOH- terminus arrangement, (Group 7 ZFA helix combo), are as follows: ZF 7-1: N’-QRSSLVR, QRGNLNM, RPQELRR, DHSSLKR, RQDNLGR, DGGNLGR-C’ (SEQ ID NOS 131, 164, 165, 130, 166, and 167, respectively, in order of appearance) ZF 7-2: N’-QRSSLVR, QQGNLQL, RPQELRR, DHSSLKR, RQDNLGR, DGGNLGR-C’ (SEQ ID NOS 131, 168, 165, 130, 166, and 167, respectively, in order of appearance) ZF 7-3: N’-QRSSLVR, QRGNLNM, RRQELTR, DHSSLKR, RQDNLGR, DGGNLGR-C’ (SEQ ID NOS 131, 164, 169, 130, 166, and 167, respectively, in order of appearance) ZF 7-4: N’-QRSSLVR, QQGNLQL, RRQELTR, DHSSLKR, RQDNLGR, DGGNLGR-C’ (SEQ ID NOS 131, 168, 169, 130, 166, and 167, respectively, in order of appearance) ZF 7-5: N’-QRSSLVR, QRGNLNM, RPQELRR, DHSSLKR, RQDNLGR, DPSNLRR-C’ (SEQ ID NOS 131, 164, 165, 130, 166, and 149, respectively, in order of appearance) ZF 7-6: N’-QRSSLVR, QQGNLQL, RPQELRR, DHSSLKR, RQDNLGR, DPSNLRR-C’ (SEQ ID NOS 131, 168, 165, 130, 166, and 149, respectively, in order of appearance) ZF 7-7: N’-QRSSLVR, QRGNLNM, RRQELTR, DHSSLKR, RQDNLGR, DPSNLRR-C’ (SEQ ID NOS 131, 164, 169, 130, 166, and 149, respectively, in order of appearance) ZF 7-8: N’-QRSSLVR, QQGNLQL, RRQELTR, DHSSLKR, RQDNLGR, DPSNLRR-C’ (SEQ ID NOS 131, 168, 169, 130, 166, and 149, respectively, in order of appearance)

[0267] Non-limiting examples of the combinations and arrangements of six helices in a single six-finger ZFA where the helices are selected from Group 8 and where the motifs are in an NH2- to COOH- terminus arrangement, (Group 8 ZFA helix combo), are as follows: ZF 8-1: N’-QASNLTR, DHSSLKR, RAHNLLL, QRSSLVR, QSTTLKR, DPSNLRR-C’ (SEQ ID NOS 170, 130, 171, 131, 172, and 149, respectively, in order of appearance) ZF 8-2: N’-QASNLTR, DHSSLKR, RAHNLLL, QRSSLVR, QGTTLKR, DPSNLRR-C’ (SEQ ID NOS 170, 130, 171, 131, 173, and 149, respectively, in order of appearance) ZF 8-3: N’-QRSNLAR, DHSSLKR, RAHNLLL, QRSSLVR, QSTTLKR, DPSNLRR-C’ (SEQ ID NOS 174, 130, 171, 131, 172, and 149, respectively, in order of appearance) ZF 8-4: N’-QRSNLAR, DHSSLKR, RAHNLLL, QRSSLVR, QGTTLKR, DPSNLRR-C’ (SEQ ID NOS 174, 130, 171, 131, 173, and 149, respectively, in order of appearance)

[0268] Non-limiting examples of the combinations and arrangements of six helices in a single six-finger ZFA where the helices are selected from Group 9 and where the motifs are in an NH2- to COOH- terminus arrangement, (Group 9 ZFA helix combo), are as follows: ZF 9-1: N’-DSSNLRR, DQGNLIR, RAHNLLL, QRSSLVR, QSTTLKR, DPSNLRR-C’ (SEQ ID NOS 135, 136, 171, 131, 172, and 149, respectively, in order of appearance) 55 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT ZF 9-2: N’-EKQNLAR, DPSNLRR, RAHNLLL, QRSSLVR, QSTTLKR, DPSNLRR-C’ (SEQ ID NOS 148, 149, 171, 131, 172, and 149, respectively, in order of appearance) ZF 9-3: N’-DSSNLRR, DQGNLIR, RAHNLLL, QRSSLVR, QGTTLKR, DPSNLRR-C’ (SEQ ID NOS 135, 136, 171, 131, 173, and 149, respectively, in order of appearance) ZF 9-4: N’-EKQNLAR, DPSNLRR, RAHNLLL, QRSSLVR, QGTTLKR, DPSNLRR-C’ (SEQ ID NOS 148, 149, 171, 131, 173, and 149, respectively, in order of appearance)

[0269] A non-limiting example of the combination and arrangement of six helices in a single six- finger ZFA where the helices are selected from Group 10 and where the motif are in an NH2- to COOH- terminus arrangement, (Group 10 ZFA helix combo), is as follows: ZF 10-1: N’-RRHGLDR, DHSSLKR, VRHNLTR, DHSNLSR, QRSSLVR, ESGHLKR-C’ (SEQ ID NOS 175, 130, 147, 150, 131, and 176, respectively, in order of appearance)

[0270] A non-limiting example of the combination and arrangement of six helices in a single six- finger ZFA where the helices are selected from Group 11 and where the motif are in an NH2- to COOH- terminus arrangement, (Group 11 ZFA helix combo), is as follows: ZF 11-1: N’-QLSNLTR, DRSSLKR, QRSSLVR, RLDMLAR, VRHSLTR, ESGALRR-C’ (SEQ ID NOS 177, 178, 131, 163, 179, and 180, respectively, in order of appearance)

[0271] Accordingly, provided herein, in some aspects, are engineered synTF or ZF-containing fusion proteins described herein comprising a ZF protein domain, an effector domain, and a regulator protein, wherein the ZF protein domain comprises at least one ZFA having the ZFA helix combo selected from one of the ZFA helix combo Groups 1-11 disclosed herein. Where there are two or more ZFAs, (i.e., a ZF array) in the ZF protein domain, each ZFAs in the domain has a ZFA helix combo selected from one of the ZFA helix combo Groups 1-11 disclosed herein, and the selected ZFA helix combo groups can be different or duplicated for the each ZFAs in the ZF protein domain of the synTF. For example, when a synTF comprises a ZF protein domain consisting essentially of three ZFAs (ZFA-1― ZFA-2― ZFA-3 in a three-ZFA array) and an effector domain, ZFA-1 has a ZFA helix combo selected from the Group 1 ZFA helix combo, ZFA-2 has a ZFA helix combo selected from the Group 5 ZFA helix combo, and ZFA-3 has a ZFA helix combo selected from the Group 7 ZFA helix combo. In other embodiments, the selected ZFA helix combo groups can be duplicated or triplicated for the ZF array in the synTF. For example, in a three-ZFA array-containing ZF protein domain of a synTF, two of the ZFAs comprises ZFA helix combo selected from the same ZFA helix combo group, e.g., Group 2, and the third ZFA has a ZFA helix combo selected from a different ZFA helix combo group, e.g., Group 4. The two ZFAs having ZFA helix combos selected from the same Group 2 ZFA helix combo can have different or the same actual combination and arrangement of the helices ZFAs. For example, when the synTF comprises of a ZF protein domain consisting essentially of five ZFAs (ZFA-1―ZFA-2―ZFA-3―ZFA-4―ZFA-5 in a five-ZFA array) and an effector domain, ZFA-1 has a ZFA helix combo selected from the Group 1 ZFA helix combo, ZFA-2 has a 56 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT ZFA helix combo selected from the Group 5 ZFA helix combo, ZFA-3 has a ZFA helix combo also selected from the Group 1 ZFA helix combo, ZFA-4 has a ZFA helix combo selected from the Group 4 ZFA helix combo, and ZFA-5 has a ZFA helix combo selected from the Group 2 ZFA helix combo. While ZFA-1 and ZFA-3 both have ZFA helix combo selected from the Group 1 ZFA helix combo, the actual combination and arrangement of the 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 both ZFA-1 and ZFA-3 have the ZFA helix combo ZF 1-1.

[0272] In other aspects, provided herein are engineered synTF or a ZF-containing fusion protein 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 having a ZFA helix combo 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 11-1 disclosed herein.

[0273] 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, which are also referred to herein as ZF1, ZF2, ZF3, ZF4, ZF5, ZF6, ZF7, ZF8, ZF9, ZF10, and ZF11, respectively.

[0274] In some embodiments of any aspect described herein, in the synTF described or any ZF- containing fusion protein described herein, the individual ZFA therein described are specifically designed to bind orthogonal target DNA sequences (also referred to herein as DNA binding motifs) such as the following: Target 1: 5’ C GTC GAA GTC GAA GTC GAC C 3’ (SEQ ID NO: 91) Target 2: 5’ G GAC GAC GTT ACG GAC GTA C 3’ (SEQ ID NO: 92) Target 3: 5’ A GAC GTC GAA GTA GCC GTA G 3’ (SEQ ID NO: 93) Target 4: 5’ G GAC GAC GCC GAT GTA GAA G 3’ (SEQ ID NO: 94) Target 5: 5’ T GAA GCA GTC GAC GCC GAA G 3’ (SEQ ID NO: 95) Target 6: 5’ G GAC GAC GCG GTC TAA GAA G 3’ (SEQ ID NO: 96) Target 7: 5’ C GAC GAG GTC GCA TAA GTA G 3’ (SEQ ID NO: 97) Target 8: 5’ A GAC GCA GTA TAG GTC GAA C 3’ (SEQ ID NO: 98) Target 9: 5’ A GAC GCA GTA TAG GAC GAC G 3’ (SEQ ID NO: 99) Target 10: 5’ C GGC GTA GCC GAT GTC GCG C 3’ (SEQ ID NO: 100) 57 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT Target 11: 5’ G GTC GTT GCG GTA GTC GAA G 3’ (SEQ ID NO: 101)

[0275] In some embodiments of any of the aspects, the ZF binding domain specifically binds to a sequence comprising at least one of SEQ ID NOs: 91-101 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: 91-101 that maintains the same function.

[0276] 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, 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.

[0277] In one embodiment of any aspect described herein, provided herein is a ZFA that comprises, consists of, or consist essentially of a sequence: N’- [(formula 1)-L2]6-8-C’ or a sequence N’- [(formula 2)-L2]6-8-C’ that targets a target DNA sequence selected from Target 1-11, wherein the formula 1 is[X0-3CX1-5CX2-7-(helix)-HX3-6H] (SEQ ID NO: 84) and the formula 2 is [X3CX2CX5- (helix)-HX3H] (SEQ ID NO: 85).

[0278] In other aspects, provided herein are engineered synTF or the ZF containing fusion protein 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 the an least ZFA comprises, consists of, or consist essentially of a sequence:N’- [(formula 1)-L2]6-8-C’ or a sequence N’- [(formula 2)-L2]6-8-C’, and wherein the ZFA(s) thereintargets a target DNA sequence selected from Target 1-11, wherein the formula 1 is[X0-3CX1-5CX2-7- (helix)-HX3-6H] (SEQ ID NO: 84) and the formula 2 is [X3CX2CX5-(helix)-HX3H] (SEQ ID NO: 85).

[0279] In some embodiments of any of the aspects, the ZF binding domain comprises one of SEQ ID NOs: 36, 45, 86, 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: 36, 45, 86 that maintains the same function. 58 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT

[0280] SEQ ID NO: 86, Sequence of ZF1-3 (ZF1) SRPGERPFQCRICMRNFSEEANLRRHTRTHTGEKPFQCRICMRNFSDHSSLKRHLRTHTGSQK PFQCRICMRNFSQSANLLRHTRTHTGEKPFQCRICMRNFSDPSSLKRHLRTHTGSQKPFQCRIC MRNFSQQTNLTRHTRTHTGEKPFQCRICMRNFSDATQLVRHLRTHLRGS

[0281] SEQ ID NO: 45, Sequence of ZF3-5 (ZF3) SRPGERPFQCRICMRNFSQRSSLVRHTRTHTGEKPFQCRICMRNFSDKSVLARHLRTHTGSQK PFQCRICMRNFSQRSSLVRHTRTHTGEKPFQCRICMRNFSQRNNLGRHLRTHTGSQKPFQCRI CMRNFSTHAVLTRHTRTHTGEKPFQCRICMRNFSDRGNLTRHLRTHLRGS

[0282] SEQ ID NO: 36, Sequence of ZF10-1 (ZF10) SRPGERPFQCRICMRNFSRRHGLDRHTRTHTGEKPFQCRICMRNFSDHSSLKRHLRTHTGSQK PFQCRICMRNFSVRHNLTRHLRTHTGEKPFQCRICMRNFSDHSNLSRHLKTHTGSQKPFQCRI CMRNFSQRSSLVRHLRTHTGEKPFQCRICMRNFSESGHLKRHLRTHLRGS

[0283] In some embodiments of any of the aspects, the DBD comprises a 3-unit ZF protein. In some embodiments of any of the aspects, the 3-unit ZF protein comprises one of SEQ ID NOs: 102- 109 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: 102-109 that maintains the same function.

[0284] SEQ ID NO: 102 high affinity scaffold (bolded letter shows mutated residue, and plain text “xxxxxxx” indicates three ZF helices, e.g., from N terminus to C terminus: helix 1, helix 2, helix 3, respectively) GERPFQCRICMANFSxxxxxxxHTRTHTGEKPFQCRICMANFSxxxxxxxHLRTHTGEKPFQCRIC MANFSxxxxxxxHLKTHLR

[0285] SEQ ID NO: 103 low affinity scaffold (bolded letter shows mutated residue, and bold italic text indicates helices 1, 2, and 3 respectively) GEAPFQCRICMANFSxxxxxxxHTRTHTGEKPFQCRICMANFSxxxxxxxHLRTHTGEKPFQCRIC MANFSxxxxxxxHLKTHLR

[0286] In some embodiments of any of the aspects, the 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; US Patent 10,138,493; US Patent Application US20200002710A1; the contents of each of which are incorporated herein by reference in their entireties. In some embodiments of any of the aspects, the at least one DBD is selected from one or more of any of: 36-4 (SEQ ID NO: 104), 43-8 (SEQ ID NO: 105 or 106), 42-10 (SEQ ID NO: 107-108), 97-4 (SEQ ID NO: 109).

[0287] SEQ ID NO: 104, 36-4 (bold italic text indicates helices 1, 2, and 3 respectively) 59 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT GERPFQCRICMANFSGRQALDRHTRTHTGEKPFQCRICMANFSDKANLTRHLRTHTGEKPFQ CRICMANFSQRNNLGRHLKTHLR

[0288] SEQ ID NO: 10543-8 low affinity (bolded letter shows mutated residue, and bold italic text indicates helices 1, 2, and 3 respectively) GEAPFQCRICMANFSRQDRLDRHTRTHTGEKPFQCRICMANFSQKEHLAGHLRTHTGEKPFQ CRICMANFSRRDNLNRHLKTHLR

[0289] SEQ ID NO: 10643-8 high affinity (bolded letter shows mutated residue, and bold italic text indicates helices 1, 2, and 3 respectively) GERPFQCRICMANFSRQDRLDRHTRTHTGEKPFQCRICMANFSQKEHLAGHLRTHTGEKPFQ CRICMANFSRRDNLNRHLKTHLR

[0290] SEQ ID NO: 10742-10 low affinity (bolded letter shows mutated residue, and bold italic text indicates helices 1, 2, and 3 respectively) GEAPFQCRICMANFSTGQILDRHTRTHTGEKPFQCRICMANFSVAHSLKRHLRTHTGEKPFQC RICMANFSDPSNLRRHLKTHLR

[0291] SEQ ID NO: 10842-10 high affinity (bolded letter shows mutated residue, and bold italic text indicates helices 1, 2, and 3 respectively) GERPFQCRICMANFSTGQILDRHTRTHTGEKPFQCRICMANFSVAHSLKRHLRTHTGEKPFQC RICMANFSDPSNLRRHLKTHLR

[0292] SEQ ID NO: 109, 97-4 (bold italic text indicates helices 1, 2, and 3 respectively) GERPFQCRICMRNFSRQSNLSRHTRTHTGEKPFQCRICMRNFSRNEHLVLHLRTHTGEKPFQC RICMRNFSQKTGLRVHLKTHLR

[0293] In some embodiments of any of the aspects, the DBD binds to DNA binding motifs (DBM) comprising any of: SEQ ID NOs: 110-121.

[0294] SEQ ID NO: 110 is an exemplary DBM (DNA binding motif) nucleic acid sequence for 36-4: c GAA GAC GCT g.

[0295] SEQ ID NO: 111- SEQ ID NO: 113 are exemplary DBM affinity variant nucleic acid sequences for 43-8. Bold text indicates residues mutated from the WT sequence. SEQ ID NO: 111 is 43-8 DBM1 – aGAGTGAGGAc. SEQ ID NO: 112 is 43-8 DBM2 – aCAGTGAGGAc. SEQ ID NO: 113 is 43-8 DBM3 – aTAGTGAGGAc.

[0296] SEQ ID NOS 114-120 are exemplary DBM affinity variant nucleic acid sequences for 42-10. Bold text indicates residues mutated from the WT sequence. SEQ ID NO: 114 is 42-10 DBM1 – aGACGCTGCTc. SEQ ID NO: 115 is 42-10 DBM2 – tGACGCTGCTt. SEQ ID NO: 116 is 42-10 DBM3 – aGACGGTGCTc. SEQ ID NO: 117 is 42-10 DBM4 – aCACGCTGCTc. SEQ ID NO: 118 is 42-10 DBM5 – aGACGCTACTc. SEQ ID NO: 119 is 42-10 DBM6 – aGACGCTGCTa. SEQ ID NO: 120 is 42-10 DBM7 – aGACTCTGCTc. 60 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT

[0297] SEQ ID NO: 121 is an exemplary DBM (DNA binding motif) nucleic acid sequence for 97-4: a TTA TGG GAG a.

[0298] In some embodiments, the engineered ZF-binding domain binds to an endogenous gene, such as an endogenous promoter. As a non-limiting example, the engineered ZF-binding domain binds to endogenous VEGF gene (VEGF ZF), such as the endogenous promoter of the human VEGF gene. In some embodiments, the engineered ZF-binding domain comprises SEQ ID NO: 48 or amino acid sequence with at least 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% sequence identity to SEQ ID NO: 48, which maintains its function (e.g., binding to the endogenous VEGF gene). Repressible Protease Domain

[0299] In some embodiments of any of the aspects, a synTF as described herein comprises a regulator protein, wherein the regulator protein is a repressible protease domain (referred to herein as PRO or RPD). As used herein, the term “repressible protease” refers to a protease that can be inactivated by the presence or absence of a specific agent (e.g., that specifically binds to the protease). In some embodiments, a repressible protease is active (e.g., cleaves a protease cleavage site) in the absence of the specific agent and is inactive (e.g., does not cleave a protease cleavage site) in the presence of the specific agent. In some embodiments, the specific agent is a protease inhibitor. In some embodiments, the protease inhibitor specifically inhibits a given repressible protease as described herein.

[0300] In some embodiments of any of the aspects, a synTF polypeptide as described herein (or a synTF polypeptide system collectively) comprises 1, 2, 3, 4, 5, or more repressible protease(s). In some embodiments of any of the aspects, the synTF polypeptide or system comprises one repressible protease. In embodiments comprising multiple repressible proteases, the multiple repressible proteases can be different individual repressible proteases or multiple copies of the same repressible protease, or a combination of the foregoing.

[0301] Non-limiting examples of repressible 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 cleaving at hydrophobic residues (e.g., Leu, Phe, Val, or Met); proprotein convertases cleaving at small amino acid residues such as Ala or Thr; proopiomelanocortin converting enzyme (PCE); chromaffin granule aspartic protease (CGAP); prohormone thiol protease; 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 61 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT protease; and, caspases 1, 2, 3, 4, 5, 6, 7, 8, and 9. Other proteases include, but are not limited to, 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 alpha, beta and gamma; angiotensin converting enzyme secretase; TGF alpha secretase; T F alpha secretase; FAS ligand secretase; TNF receptor-I and -II secretases; CD30 secretase; KL1 and KL2 secretases; IL6 receptor secretase; CD43, CD44 secretase; CD 16-1 and CD 16-11 secretases; L-selectin secretase; Folate receptor secretase; MMP 1, 2, 3, 7, 8, 9, 10, 11, 12, 13, 14, and 15; urokinase plasminogen activator; tissue plasminogen activator; plasmin; thrombin; BMP-l (procollagen C-peptidase); ADAM 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11; and, granzymes A, B, C, D, E, F, G, and H. For a discussion of proteases, see, e.g., V. Y. H. Hook, Proteolytic and cellular mechanisms in prohormone and proprotein processing, RG Landes Company, Austin, Tex., USA (1998); N. M. Hooper et al., Biochem. J.321: 265-279 (1997); Z. Werb, Cell 91: 439-442 (1997); T. G. Wolfsberg et al., J. Cell Biol.131: 275-278 (1995); K. Murakami and J. D. Etlinger, Biochem. Biophys. Res. Comm.146: 1249-1259 (1987); T. Berg et al., Biochem. J. 307: 313-326 (1995); M. J. Smyth and J. A. Trapani, Immunology Today 16: 202-206 (1995); R. V. Talanian et al., J. Biol. Chem.272: 9677-9682 (1997); and N. A. Thomberry et al., J. Biol. Chem. 272: 17907-17911 (1997); International Patent Application WO2019118518; Rajakuberan et 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, 2008105 (22) 7744-7749; US Patent Application 16 / 832,751 filed March 27, 2020; the contents of each of which are incorporated herein by reference in their entireties.

[0302] In some embodiments of any of the aspects, the repressible 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 the trypsin / chymotrypsin-like serine protease in the N-terminal region and a helicase and nucleoside triphosphatase in the C-terminal region. The minimal sequences required for a functional serine protease activity comprise the N-terminal 180 amino acids of the NS3 protein, which can also be referred to as “NS3a”. Deletion of up to 14 residues from the N terminus of the NS3 protein is tolerated while maintaining the serine protease activity. Accordingly, the repressible proteases described herein comprise at the least residues 14-180 of the wildtype NS3 protein.

[0303] HCV has at least seven genotypes, labeled 1 through 7, which can also be further designated with “a” and “b” subtypes. Accordingly, the repressible protease can be an HCV genotype 1 NS3, an HCV genotype 1a NS3, an HCV genotype 1b NS3, an HCV genotype 2 NS3, an HCV genotype 2a NS3, an HCV genotype 2b NS3, an HCV genotype 3 NS3, an HCV genotype 3a NS3, an HCV genotype 3b NS3, an HCV genotype 4 NS3, an HCV genotype 4a NS3, an HCV genotype 4b 62 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT NS3, an HCV genotype 5 NS3, an HCV genotype 5a NS3, an HCV genotype 5b NS3, an HCV genotype 6 NS3, an HCV genotype 6a NS3, an HCV genotype 6b NS3, an HCV genotype 7 NS3, an HCV genotype 7a NS3, or an HCV genotype 7b NS3. In some embodiments of any of the aspects, the repressible protease can be any known HCV NS3 genotype, variant, or mutant, e.g., that maintains the same function. In some embodiments of any of the aspects, the NS3 sequence comprises 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 content of which is incorporated herein by reference in its entirety). In some embodiments of any of the aspects, the repressible protease is a chimera of 2, 3, 4, 5, or more different NS3 genotypes, variants, or mutants as described herein, such that the protease maintains its cleavage and / or binding functions.

[0304] In some embodiments of any of the aspects, the repressible protease of a synTF polypeptide as described herein comprises SEQ ID NOs: 182-198 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: 182-198 that maintains the same function.

[0305] In some embodiments of any of the aspects, the repressible protease of a synTF polypeptide as described herein does not comprise at most the first (i.e., N-terminal) residues of SEQ ID NOs: 182-198. In some embodiments of any of the aspects, the repressible protease of a synTF polypeptide as described herein comprises 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 of SEQ ID NOs: 182-198.

[0306] SEQ ID NO: 182, NS3 (genotype 1A), 189 aa; bold text indicates His-57 of the catalytic triad; italicized double underlined text indicates Asp-81 of the catalytic triad; bold italicized indicates Ser-139 of the catalytic triad; double underlined text indicates Asp-168. APITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIVSTATQTFLATCINGVCWAVYHGAGTR TIASPKGPVIQMYTNVDQDLVGWPAPQGSRSLTPCTCGSSDLYLVTRHADVIPVRRRGDSRG SLLSPRPISYLKGSSGGPLLCPAGHAVGLFRAAVCTRGVAKAVDFIPVENLETTMRSPVFTDN SS

[0307] SEQ ID NO: 183, NS3 protease domain (genotype 1A) APITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIVSTATQTFLATCINGVCWAVYHGAGTRT IASPKGPVIQMYTNVDQDLVGWPAPQGSRSLTPCTCGSSDLYLVTRHADVIPVRRRGDSRGS LLSPRPISYLKGSSGGPLLCPAGHAVGLFRAAVCTRGVAKAVDFIPVENLETTMRSPVFTD 63 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT

[0308] SEQ ID NO: 184, NS3 (genotype 1A), 180 aa (see e.g., residues 1027-1206 of Hepatitis C virus genotype 1 polyprotein, NCBI Reference Sequence: NP_671491.1). APITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIVSTATQTFLATCINGVCWTVYHGAGTRT IASPKGPVIQMYTNVDQDLVGWPAPQGSRSLTPCTCGSSDLYLVTRHADVIPVRRRGDSRGS LLSPRPISYLKGSSGGPLLCPAGHAVGLFRAAVCTRGVAKAVDFIPVENLETTMR

[0309] SEQ ID NO: 185, NS3 (genotype 1B), 180 aa (see e.g., residues 1-180 Chain A, Ns3 Protease, PDB: 4K8B_A) APITAYSQQTRGLLGCIITSLTGRDKNQVEGEVQVVSTATQSFLATCVNGVCWTVYHGAGSK TLAGPKGPITQMYTNVDQDLVGWQAPPGARSLTPCTCGSSDLYLVTRHADVIPVRRRGDSR GSLLSPRPVSYLKGSSGGPLLCPSGHAVGIFRAAVCTRGVAKAVDFVPVESMETTMR

[0310] SEQ ID NO: 186, NS3 (genotype 2), 180 aa (see e.g., residues 1031-1210 of Hepatitis C virus genotype 2 polyprotein, NCBI Reference Sequence: YP_001469630.1) APITAYAQQTRGLLGTIVVSMTGRDKTEQAGEIQVLSTVTQSFLGTSISGVLWTVYHGAGNK TLAGSRGPVTQMYSSAEGDLVGWPSPPGTKSLEPCTCGAVDLYLVTRNADVIPARRRGDKR GALLSPRPLSTLKGSSGGPVLCPRGHAVGVFRAAVCSRGVAKSIDFIPVETLDIVTR

[0311] SEQ ID NO: 187, NS3 (genotype 3), 180 aa (see e.g., residues 1033-1212 of Hepatitis C virus genotype 3 polyprotein, NCBI Reference Sequence: YP_001469631.1) APITAYAQQTRGLLGTIVTSLTGRDKNVVTGEVQVLSTATQTFLGTTVGGVIWTVYHGAGSR TLAGAKHPALQMYTNVDQDLVGWPAPPGAKSLEPCACGSSDLYLVTRDADVIPARRRGDST ASLLSPRPLACLKGSSGGPVMCPSGHVAGIFRAAVCTRGVAKSLQFIPVETLSTQAR

[0312] SEQ ID NO: 188, NS3 (genotype 4), 180 aa (see e.g., residues 1027-1206 of Hepatitis C virus genotype 4 polyprotein, NCBI Reference Sequence: YP_001469632.1) APITAYAQQTRGLFSTIVTSLTGRDTNENCGEVQVLSTATQSFLGTAVNGVMWTVYHGAGA KTISGPKGPVNQMYTNVDQDLVGWPAPPGVRSLAPCTCGSADLYLVTRHADVIPVRRRGDT RGALLSPRPISILKGSSGGPLLCPMGHRAGIFRAAVCTRGVAKAVDFVPVESLETTMR

[0313] SEQ ID NO: 189, NS3 (genotype 5), 180 aa (see e.g., residues 1028-1207 of Hepatitis C virus genotype 5 polyprotein, NCBI Reference Sequence: YP_001469633.1) APITAYAQQTRGVLGAIVLSLTGRDKNEAEGEVQFLSTATQTFLGICINGVMWTLFHGAGSK TLAGPKGPVVQMYTNVDKDLVGWPSPPGKGSLTRCTCGSADLYLVTRHADVIPARRRGDTR ASLLSPRPISYLKGSSGGPIMCPSGHVVGVFRAAVCTRGVAKALEFVPVENLETTMR

[0314] SEQ ID NO: 190, NS3 (genotype 6), 180 aa (see e.g., residues 1032-1211 of Hepatitis C virus genotype 6 polyprotein, NCBI Reference Sequence: YP_001469634.1) APITAYAQQTRGLVGTIVTSLTGRDKNEAEGEVQVVSTATQSFLATTINGVLWTVYHGAGSK NLAGPKGPVCQMYTNVDQDLVGWPAPLGARSLAPCTCGSSDLYLVTRGADVIPARRRGDTR AALLSPRPISTLKGSSGGPLMCPSGHVVGLFRAAVCTRGVAKALDFIPVENMDTTMR 64 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT

[0315] SEQ ID NO: 191, NS3 (genotype 7), 180 aa (see e.g., residues 1031-1210 of Hepatitis C virus genotype 7 polyprotein, NCBI Reference Sequence: YP_009272536.1) APISAYAQQTRGLISTLVVSLTGRDKNETAGEVQVLSTSTQTFLGTNVGGVMWGPYHGAGT RTVAGRGGPVLQMYTSVSDDLVGWPAPPGSKSLEPCSCGSADLYLVTRNADVLPLRRKGDG TASLLSPRPVSSLKGSSGGPVLCPQSHCVGIFRAAVCTRGVAKAVQFVPIEKMQVAQR

[0316] SEQ ID NO: 192, NS3 genotype 1a (HCV-H), 180 aa APITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIVSTATQTFLATCINGVCWTVYHGAGTRT IASPKGPVIQMYTNVDQDLVGWPAPQGSRSLTPCTCGSSDLYLVTRHADVIPVRRRGDSRGS LLSPRPISYLKGSSGGPLLCPAGHAVGLFRAAVCTRGVTKAVDFIPVENLETTMR

[0317] SEQ ID NO: 193, NS3 genotype 1b (HCV-BK), 180 aa APITAYSQQTRGLLGCIITSLTGRDKNQVEGEVQVVSTATQSFLATCVNGVCWTVYHGAGSK TLAAPKGPITQMYTNVDQDLVGWPKPPGARSLTPCTCGSSDLYLVTRHADVIPVRRRGDSRG SLLSPRPVSYLKGSSGGPLLCPFGHAVGIFRAAVCTRGVAKAVDFVPVESMETTMR

[0318] SEQ ID NO: 194, NS3 genotype 2a (HCV-J6), 180 aa APITAYAQQTRGLLGTIVVSMTGRDKTEQAGEIQVLSTVTQSFLGTTISGVLWTVYHGAGNK TLAGSRGPVTQMYSSAEGDLVGWPSPPGTKSLEPCTCGAVDLYLVTRNADVIPARRRGDKR GALLSPRPLSTLKGSSGGPVLCPRGHAVGVFRAAVCSRGVAKSIDFIPVETLDIVTR

[0319] SEQ ID NO: 195, NS3 genotype 2b (HCV-J8), 180 aa APITAYTQQTRGLLGAIVVSLTGRDKNEQAGQVQVLSSVTQTFLGTSISGVLWTVYHGAGNK TLAGPKGPVTQMYTSAEGDLVGWPSPPGTKSLDPCTCGAVDLYLVTRNADVIPVRRKDDRR GALLSPRPLSTLKGSSGGPVLCSRGHAVGLFRAAVsynTFGVAKSIDFIPVESLDVATR

[0320] SEQ ID NO: 196, NS3 genotype 3a (HCV-Nz11), 180 aa APITAYAQQTRGLLGTIVTSLTGRDKNVVTGEVQVLSTATQTFLGTTVGGVIWTVYHGAGSR TLAGAKHPALQMYTNVDQDLVGWPAPPGAKSLEPCACGSSDLYLVTRDADVIPARRRGDST ASLLSPRPLACLKGSSGGPVMCPSGHVAGIFRAAVCTRGVAKSLQFIPVETLSTQAR

[0321] In some embodiments of any of the aspects, a repressible protease as described herein is resistant to 1, 2, 3, 4, 5, or more different protease inhibitors as described herein. Non-limiting examples of NS3 amino acid substitutions conferring 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., genotype 1a, 1b, 6a), Y132I (e.g., genotype 1b), A156S (e.g., genotype 2a), A156G, A156T, A156V, 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, 65 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT 22(40): 8910–8917; US Patents 7,208,309; 7,494,660; the contents of each of which are incorporated herein by reference in their entireties.

[0322] In some embodiments of any of the aspects, a synTF polypeptide as described herein comprises an NS3 protease comprising at least one resistance mutation as described herein or any combination thereof. In some embodiments of any of the aspects, a synTF polypeptide as 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, a synTF system comprises: (a) a first synTF polypeptide comprising a repressible protease (e.g., NS3) that is resistant to a first protease inhibitor and that is susceptible to a second protease inhibitor; and (b) a second synTF polypeptide comprising a repressible protease (e.g., NS3) that is susceptible to a first protease inhibitor and that is resistant to a second protease inhibitor. Accordingly, presence of the first protease inhibitor can modulate the activity of the second synTF polypeptide but not the first synTF polypeptide, while the presence of the second protease inhibitor can modulate the activity of the first synTF polypeptide but not the second synTF polypeptide.

[0323] In some embodiments of any of the aspects, a repressible protease as described herein is sensitive to 1, 2, 3, 4, 5, or more different protease inhibitors as 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 a protease is also referred to herein as NS3AI, as these mutations increase its sensitivity to asunaprevir (see e.g., SEQ ID NO: 197). In some embodiments of any of the aspects, the NS3 protease comprises at least one of the following mutations: F43L, Q80K, S122R, D168Y, or any combination thereof; such a protease is also referred to herein as NS3TI, as these mutations increase its sensitivity to telaprevir (see e.g., SEQ ID NO: 198). 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 each are incorporated herein by reference in their entireties.

[0324] SEQ ID NO: 197, NS3AI; the V36M, T54A, S122G mutations are shown in bold double underlined text, respectively APITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIMSTATQTFLATCINGVCWAVYHGAGTR TIASPKGPVIQMYTNVDQDLVGWPAPQGSRSLTPCTCGSSDLYLVTRHADVIPVRRRGDGRG SLLSPRPISYLKGSSGGPLLCPAGHAVGLFRAAVCTRGVAKAVDFIPVENLETTMRSPVFTD

[0325] SEQ ID NO: 198, NS3TI; the F43L, Q80K, S122R, D168Y mutations are shown in bold double underlined text, respectively 66 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT APITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIVSTATQTLLATCINGVCWAVYHGAGTR TIASPKGPVIQMYTNVDKDLVGWPAPQGSRSLTPCTCGSSDLYLVTRHADVIPVRRRGDRRG SLLSPRPISYLKGSSGGPLLCPAGHAVGLFRAAVCTRGVAKAVYFIPVENLETTMRSPVFTD

[0326] In some embodiments of any of the aspects, the polypeptide further comprising a cofactor for the repressible protease. As used herein the term “cofactor for the repressible protease” refers to a molecule that increases the activity of the repressible protease. In some embodiments of any of the aspects, a synTF polypeptide as described herein comprises 1, 2, 3, 4, 5, or more cofactors for the repressible protease. In some embodiments of any of the aspects, the synTF polypeptide comprises one cofactor for each repressible protease. In embodiments comprising multiple cofactors for the repressible protease, the multiple cofactors for the repressible protease can be different individual cofactors or multiple copies of the same cofactor, or a combination of the foregoing.

[0327] In some embodiments of any of the aspects, the cofactor is an HSV NS4A domain, and the repressible protease is HSV NS3. The nonstructural protein 4a (NS4A) is the smallest of the nonstructural HCV proteins. 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 mitochondrial outer membrane, (2) serving as a cofactor for the NS3A serine protease, (3) augmenting NS3A helicase activity, and (4) regulating NS5A hyperphosphorylation and viral replication. The interactions between NS4A and NS4B control genome replication and between NS3 and NS4A play a role in virus assembly.

[0328] In some embodiments of any of the aspects, a synTF polypeptide as described herein comprises the portion of the NS4a polypeptide that serves as a cofactor for NS3. Deletion analysis has shown that the central region (approximately residues 21 to 34) of the 54-residue NS4A protein is essential and sufficient for the cofactor function of the NS3 serine protease. Accordingly, in some embodiments of any of the aspects, the repressible protease cofactor comprises a 14-residue region of the wildtype NS4A protein.

[0329] In some embodiments of any of the aspects, the cofactor for the repressible protease can be an HCV genotype 1 NS4A, an HCV genotype 1a NS4A, an HCV genotype 1b NS4A, an HCV genotype 2 NS4A, an HCV genotype 2a NS4A, an HCV genotype 2b NS4A, an HCV genotype 3 NS4A, an HCV genotype 3a NS4A, an HCV genotype 3b NS4A, an HCV genotype 4 NS4A, an HCV genotype 4a NS4A, an HCV genotype 4b NS4A, an HCV genotype 5 NS4A, an HCV genotype 5a NS4A, an HCV genotype 5b NS4A, an HCV genotype 6 NS4A, an HCV genotype 6a NS4A, an HCV genotype 6b NS4A, an HCV genotype 7 NS4A, an HCV genotype 7a NS4A, or an HCV genotype 7b NS4A. In some embodiments of any of the aspects, the cofactor for the repressible protease can be any known NS4A genotype, variant, or mutant, e.g., that maintains the same function. In some embodiments of any of the aspects, the NS4A sequence comprises residues 21-31 of the NS4A 67 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT 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 9).

[0330] In some embodiments of any of the aspects, the cofactor for a repressible protease of a synTF polypeptide as described herein comprises SEQ ID NOs: 199-223, 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: 199-223that maintains the same functions as one of SEQ ID NOs: 48, 98, 137-156. In some embodiments of any of the aspects, the cofactor for a repressible protease of a synTF polypeptide as described herein comprises SEQ ID NOs: 199-223, or an amino acid sequence that is at least 95% identical to the sequence of one of SEQ ID NOs: 199- 223 that maintains the same function.

[0331] In some embodiments of any of the aspects, the cofactor for the repressible protease of a synTF polypeptide as 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: 199-223.

[0332] SEQ ID NO: 199, NS4A (genotype 1A), 13 aa, GCVVIVGRIVLSG

[0333] SEQ ID NO: 200, NS4A domain (genotype 1a) STWVLVGGVLAALAAYCLSTGCVVIVGRIVLSGKPAIIPDREVLY

[0334] SEQ ID NO: 201, NS4 (with L6 linker in bold text) STWVLVGGVLAALAAYCLSTGCVVIVGRIVLSGKPAGSSGSSIIPDREVLY

[0335] SEQ ID NO: 202, NS4A domain, IDTKYIMTCMSADLEVVTSTWVLVGGVLAALAAYCLSTGCVVIVGRIVLSGKPAIIPDREVLY

[0336] SEQ ID NO: 203, NS4A (genotype 1B), 12 aa, GSVVIVGRIILS; see e.g., Chain C, Nonstructural Protein, PDB: 4K8B_C.

[0337] SEQ ID NO: 204, NS4A (genotype 1), 14 aa (see e.g., residues 1678-1691 of Hepatitis C virus genotype 1 polyprotein, NCBI Reference Sequence: NP_671491.1): GCVVIVGRIVLSGK

[0338] SEQ ID NO: 205, NS4A (genotype 2), 14 aa (see e.g., residues 1682-1695 of Hepatitis C virus genotype 2 polyprotein, NCBI Reference Sequence: YP_001469630.1): GCVCIIGRLHINQR

[0339] SEQ ID NO: 206, NS4A (genotype 3), 14 aa (see e.g., residues 1684-1697 of Hepatitis C virus genotype 3 polyprotein, NCBI Reference Sequence: YP_001469631.1): GCVVIVGHIELEGK

[0340] SEQ ID NO: 207, NS4A (genotype 4), 14 aa (see e.g., residues 1678-1691 of Hepatitis C virus genotype 4 polyprotein, NCBI Reference Sequence: YP_001469632.1): GSVVIVGRVVLSGQ

[0341] SEQ ID NO: 208, NS4A (genotype 5), 14 aa (see e.g., residues 1679-1692 of Hepatitis C virus genotype 5 polyprotein, NCBI Reference Sequence: YP_001469633.1): GSVAIVGRIILSGR

[0342] SEQ ID NO: 209, NS4A (genotype 6), 14 aa (see e.g., residues 1683-1696 of Hepatitis C virus genotype 6 polyprotein, NCBI Reference Sequence: YP_001469634.1): GCVVICGRIVTSGK 68 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT

[0343] SEQ ID NO: 210, NS4A (genotype 7), 14 aa (see e.g., residues 1682-1695 of Hepatitis C virus genotype 7 polyprotein, NCBI Reference Sequence: YP_009272536.1): GSVVVVGRVVLGSN

[0344] In some embodiments of any of the aspects, the NS4A sequence is selected from Table 9. In one embodiment, the NS4A comprises residues 21-31 of SEQ ID NO: 211-223 or a sequence that is at least 70% identical.

[0345] Table 9: Exemplary NS4A sequences (see e.g., Chao Lin 2006 supra). Residues 21-31 are bolded.

[0346] In some embodiments of any of the aspects, a synTF polypeptide as described herein can comprise any combination of NS3 and NS4A genotypes, variants, or mutants as described herein. In one embodiment, the NS3 and NS4A are selected from the same genotype as each other. In some embodiments of any of the aspects, the NS3 is genotype 1a and the NS4A is genotype 1b. In some embodiments of any of the aspects, the NS3 is genotype 1b and the NS4A is genotype 1a.

[0347] In some embodiments of any of the aspects, a synTF polypeptide as described herein comprises an HSV NS4A domain adjacent to the NS3 repressible protease. In some embodiments of any of the aspects, the NS4A domain is N-terminal of the NS3 repressible protease. In some 69 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT embodiments of any of the aspects, the NS4A domain is C-terminal of the NS3 repressible protease. In some embodiments of any of the aspects, the synTF polypeptide comprises a peptide linker between the NS4A domain and the NS3 repressible protease. Non-limiting examples of linker (e.g., between the NS4A domain and the NS3 repressible protease) include: SGTS (SEQ ID NO: 224) and GSGS (SEQ ID NO: 225).

[0348] In some embodiments of any of the aspects, any two domains as described herein in a synTF polypeptide can be joined into a single polypeptide by positioning a peptide linker, e.g., a flexible linker between them. As used herein “peptide linker” refers to an oligo- or polypeptide region from about 2 to 100 amino acids in length, which links together any of the sequences of the polypeptides as described herein. In some embodiment, linkers can include or be composed of flexible residues such as glycine and serine so that the adjacent protein domains are free to move relative to one another. Longer linkers may be used when it is desirable to ensure that two adjacent domains do not sterically interfere with one another. Linkers may be cleavable or non-cleavable.

[0349] Described herein are synTF polypeptides comprising protease cleavage sites. As used herein, the term “protease cleavage site” refers to a specific sequence or sequence motif recognized by and cleaved by the repressible protease. A cleavage site for a protease includes the specific amino acid sequence or motif recognized by the protease during proteolytic cleavage and typically includes the surrounding one to six 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 and cleaved by the repressible protease. In some embodiments of any of the aspects, a synTF polypeptide as described herein (or the synTF polypeptide system collectively) comprises 1, 2, 3, 4, 5, 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 sites, or a combination of the foregoing.

[0350] As a non-limiting example, during HCV replication, the NS3-4A serine protease is responsible for the proteolytic cleavage at four junctions of the HCV polyprotein precursor: NS3 / NS4A (self-cleavage), NS4A / NS4B, NS4B / NS5A, and NS5A / NS5B. Accordingly, the protease cleavage site of a synTF polypeptide as described herein can be a NS3 / NS4A cleavage site, a NS4A / NS4B cleavage site, a NS4B / NS5A cleavage site, or a NS5A / NS5B cleavage site. The protease cleavage site can be a protease cleavage sites 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, 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 variant or mutant thereof, e.g., 70 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT 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).

[0351] In some embodiments of any of the aspects, the protease cleavage site of a synTF polypeptide as described herein comprises SEQ ID NOs: 226-251, 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: 226-251that maintains the same function.

[0352] In some embodiments of any of the aspects, the protease cleavage site of a synTF polypeptide as 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: 226-251.

[0353] SEQ ID NO: 226, NS5A / 5B cut site (CC), 10 aa, EDVVCCHSIY

[0354] SEQ ID NO: 227, NS4A / 4B cut site (CS), 14 aa, LYQEFDEMEECSQH

[0355] SEQ ID NO: 228, N3 cleavage site (NS4A / 4B cut site), DEMEECSQHL

[0356] SEQ ID NO: 229, QEFEDVVPCSMGS

[0357] SEQ ID NO: 230, NS5A / 5B cut site, EDVVCCHSI

[0358] SEQ ID NO: 231, NS4A / 4B cut site, DEMEECSQH

[0359] Table 10: Exemplary NS3 / NS4A protease cleavage sites (see e.g., Chao Lin 2006 supra).71 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT

[0360] In some embodiments of any of the aspects, a synTF polypeptide as described herein comprises two protease cleavage sites, with one N-terminal of the NS3-NS4A complex, and the other C-terminal of the NS3-NS4A complex (see e.g., Table 11). In some embodiments of any of the aspects, the two protease cleavage sites can be the same cleavage sites or different cleavage sites.

[0361] Table 11: Exemplary Protease Cleavage Site Combinations. “N” indicates N-terminal of the NS3-NS4A complex. “C” indicates C-terminal 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.

[0362] In some embodiments of any of the aspects, a synTF polypeptide as described herein comprise any known genotypes, variants, or mutants of 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.

[0363] In some embodiments of any of the aspects, the protease cleavage site is located or engineered such that, when the synTF cleaves itself using the repressible protease in the absence of a protease inhibitor, the resulting amino acid at the N-terminus of the newly cleaved polypeptide(s) causes the polypeptide(s) to degrade at a faster rate and have a shorter half-life compared to other cleaved polypeptides. According to the N-end rule, newly cleaved polypeptides comprising the amino acid His, Tyr, Gln, Asp, Asn, Phe, Leu, Trp, Lys, or Arg at the N-terminus exhibit a high degradation rate and a short half-life (e.g., 10 minutes or less in yeast; 1-5.5 hours in mammalian reticulocytes). Comparatively, newly cleaved polypeptides comprising the amino acid Val, Met, Gly, Pro, Ala, Ser, Thr, Cys, Ile, or Glu at the N-terminus exhibit a lower 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 content of which is incorporated herein by reference in its entirety. Accordingly, in some embodiments of any of the aspects, the resulting amino acid at the N-terminus of a newly cleaved synTF polypeptide as 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 72 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT the newly cleaved synTF polypeptide as described herein is not Val, Met, Gly, Pro, Ala, Ser, Thr, Cys, Ile, or Glu.

[0364] In some embodiments of any of the aspects, the N-terminus of a newly cleaved synTF polypeptide as described herein comprises SEQ ID NO: 252 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: 252 that maintains a His or another highly degraded amino acid at the N-terminus. SEQ ID NO: 252, N-end rule, 8 aa, HSIYGKKK.

[0365] In some embodiments of any of the aspects, a synTF polypeptide as described herein comprises a repressible protease that is catalytically active. For HCV NS3, the catalytic triad comprises His-57, Asp-81, and Ser-139. In regard to a repressible protease, “catalytically active” refers to the ability to cleave at a protease cleavage site. In some embodiments of any of the aspects, the catalytically active repressible protease can be any repressible protease as described further herein that maintains the catalytic triad, i.e., comprises no non-synonymous substitutions at His-57, Asp-81, and / or Ser-139.

[0366] In some embodiments of any of the aspects, the synTF comprises SEQ ID NO: 37 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: 37, which maintains its function.

[0367] In some embodiments of any of the aspects, the synTF comprises 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 NOs: 253 or 254 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: 253 or 254.

[0368] SEQ ID NO: 253, NS3 domain, comprising: a portion of the N-end rule (bold text), AU1 tag (italicized double underlined text), NS4A (bold italicized text), and NS3 protease (italicized text): GKKKGDIDTYRYIGSSGTGCVVIVGRIVLSGSGTSAPITAYAQQTRGLLGCIITSLTGRDKNQVEG EVQIVSTATQTFLATCINGVCWAVYHGAGTRTIASPKGPVIQMYTNVDQDLVGWPAPQGSRSLTPC TCGSSDLYLVTRHADVIPVRRRGDSRGSLLSPRPISYLKGSSGGPLLCPAGHAVGLFRAAVCTRGVA KAVDFIPVENLETTMRSPVFTDNSSPPAVTLTHPITKIDREV

[0369] SEQ ID NO: 254, NS3 domain, comprising: NS3 cleavage sites (double underlined text), N-end rule (bold text), AU1 tag (italicized text), NS4A (bold italicized text), and NS3 protease (italicized double underlined text): EDVVCCHSIYGKKKGDIDTYRYIGSSGTGCVVIVGRIVLSGSGTSAPITAYAQQTRGLLGCIITSL TGRDKNQVEGEVQIVSTATQTFLATCINGVCWAVYHGAGTRTIASPKGPVIQMYTNVDQDLVGWP 73 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT APQGSRSLTPCTCGSSDLYLVTRHADVIPVRRRGDSRGSLLSPRPISYLKGSSGGPLLCPAGHAVGLF RAAVCTRGVAKAVDFIPVENLETTMRSPVFTDNSSPPAVTLTHPITKIDREVLYQEFDEMEECSQH

[0370] In some embodiments of any of the aspects, the synTF comprises a stabilizable polypeptide linkage (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 SEQ ID NOs: 255- 257, 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: 255-257.

[0371] SEQ ID NO: 255, NS3 Partial Helicase Domain, NSSPPAVTLTHPITKIDTKYIMTCMSADLEVVT

[0372] SEQ ID NO: 256, NS3 Partial Helicase Domain, NSSPPAVTLTHPITK

[0373] SEQ ID NO: 257, NS3 partial helicase domain, NSSPPAVTLTH

[0374] In some embodiments of any of the aspects, the StaPL domain further comprises a protease cleavage site at the N terminus, e.g., selected from EDVVCCHSI (SEQ ID NO: 230) or DEMEECSQH (SEQ ID NO: 231), directly linked or indirectly linked through a peptide linker. In some embodiments of any of the aspects, the StaPL domain comprises one of SEQ ID NOs: 258-260, 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: 258-260.

[0375] SEQ ID NO: 258, StaPL domain, comprising: NS4A (bold italicized text), and NS3 protease (italicized text); linkers (); NS3 helicase (bold italicized double underlined text) TGCVVIVGRIVLSGSGTSAPITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIVSTATQTFLATCING VCWAVYHGAGTRTIASPKGPVIQMYTNVDQDLVGWPAPQGSRSLTPCTCGSSDLYLVTRHADVIPV RRRGDSRGSLLSPRPISYLKGSSGGPLLCPAGHAVGLFRAAVCTRGVAKAVDFIPVENLETTMRSPV FTDNSSPPAVTLTHGGSGGS

[0376] In some embodiments of any of the aspects, StaPL domain comprises a repressible protease that comprises 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 a StaPL is also referred to herein as StaPLAI, as these mutations increase its sensitivity to asunaprevir (see e.g., SEQ ID NO: 197, 259). 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: F43L, Q80K, S122R, D168Y, or any combination thereof; such a protease is also referred 74 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT to herein as StaPLTI, as these mutations increase its sensitivity to telaprevir (see e.g., SEQ ID NO: 198, 260).

[0377] SEQ ID NO: 259, StaPLAIdomain, comprising: NS4A (bold italicized text), and NS3 protease (italicized text); linkers (double underlined text); NS3 helicase (bold italicized double underlined text); the V36M, T54A, S122G mutations are shown in bold double underlined text, respectively. TGCVVIVGRIVLSGSGTSAPITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIMSTATQTFLATCIN GVCWAVYHGAGTRTIASPKGPVIQMYTNVDQDLVGWPAPQGSRSLTPCTCGSSDLYLVTRHADVI PVRRRGDGRGSLLSPRPISYLKGSSGGPLLCPAGHAVGLFRAAVCTRGVAKAVDFIPVENLETTMRS PVFTDNSSPPAVTLTHGGSGGS

[0378] SEQ ID NO: 260, StaPLTIdomain, comprising: NS4A (bold italicized text), and NS3 protease (italicized text); linkers (double underlined text); NS3 helicase (bold italicized double underlined text); the F43L, Q80K, S122R, D168Y mutations are shown in bold double underlined text, respectively. TGCVVIVGRIVLSGSGTSAPITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIVSTATQTLLATCING VCWAVYHGAGTRTIASPKGPVIQMYTNVDKDLVGWPAPQGSRSLTPCTCGSSDLYLVTRHADVIPV RRRGDRRGSLLSPRPISYLKGSSGGPLLCPAGHAVGLFRAAVCTRGVAKAVYFIPVENLETTMRSPV FTDNSSPPAVTLTHGGSGGS

[0379] In some embodiments of any of the aspects, the synTF comprises a TimeSTAMP domain (a time-specific tag for the age measurement of proteins). In some embodiments of any of the aspects, the TimeSTAMP comprises a repressible 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 repressible protease until the time a protease inhibitor is added, after which newly synthesized synTF polypeptides retain their markers. TimeSTAMP allows for time-specific tagging of the age measurement of proteins, and allows sensitive and nonperturbative visualization and quantification of newly synthesized proteins of interest with exceptionally tight temporal control.

[0380] In some embodiments of any of the aspects, the repressible protease exhibits increased solubility compared to the wild-type protease. As a non-limiting example, the NS3 protease can comprise at least one of the following mutations or any combination thereof: Leu13 is substituted to Glu; Leu14 is substituted to Glu; Ile17 is substituted to Gln; Ile18 is substituted to Glu; and / or Leu21 is substituted to Gln. In some embodiments of any of the aspects, a synTF polypeptide as described herein comprises a repressible protease comprising SEQ ID NOs: 261-269, 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: 261-269 that maintains the same functions (e.g., serine protease; increased solubility) as SEQ ID NOs: 261-269; see e.g., US Patent 6333186 and US 75 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT Patent Publication US20020106642, the contents of each are incorporated herein by reference in their entireties.

[0381] SEQ ID NO: 261, soluble NS3, 182 aa MAPITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIVSTAAQTFLATCINGVCWTVYHGAGT RTIASPKGPVIQMYTNVDKDLVGWPAPQGSRSLTPCTCGSSDLYLVTRHADVIPVRRRGDSR GSLLSPRPISYLKGSSGGPLLCPAGHAVGIFRAAVCTRGVAKAVDFIPVESLETTMRS

[0382] SEQ ID NO: 262, soluble NS3 / NS4A, 195 aa MKKKGSVVIVGRIVLNGAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIVSTAAQTFLATCING VCWTVYHGAGTRTIASPKGPVIQMYTNVDKDLVGWPAPQGSRSLTPCTCGSSDLYLVTRHA DVIPVRRRGDSRGSLLSPRPISYLKGSSGGPLLCPAGHAVGIFRAAVCTRGVAKAVDFIPVESL ETTMRSP

[0383] SEQ ID NO: 263, soluble NS3 / NS4A, 195 aa MKKKGSVVIVGRIVLNGAYAQQTRGEEGCQETSQTGRDKNQVEGEVQIVSTAAQTFLATCIN GVCWTVYHGAGTRTIASPKGPVIQMYTNVDKDLVGWPAPQGSRSLTPCTCGSSDLYLVTRH ADVIPVRRRGDSRGSLLSPRPISYLKGSSGGPLLCPAGHAVGIFRAAVCTRGVAKAVDFIPVES LETTMRSP

[0384] SEQ ID NO: 264, soluble NS3 / NS4A, 197 aa MKKKGSVVIVGRINLSGDTAYAQQTRGEEGCQETSQTGRDKNQVEGEVQIVSTAAQTFLAT CINGVCWTVYHGAGTRTIASPKGPVIQMYTNVDKDLVGWPAPQGSRSLTPCTCGSSDLYLVT RHADVIPVRRRGDSRGSLLSPRPISYLKGSSGGPLLCPAGHAVGIFRAAVCTRGVAKAVDFIP VESLETTMRSP

[0385] SEQ ID NO: 265, soluble NS3 / NS4A, 197 aa MKKKGSVVIVGRINLSGDTAYAQQTRGEEGCQETSQTGRDKNQVEGEVQIVSTATQTFLATC INGVCWTVYHGAGTRTIASPKGPVTQMYTNVDKDLVGWQAPQGSRSLTPCTCGSSDLYLVT RHADVIPVRRRGDSRGSLLSPRPISYLKGSSGGPLLCPAGHAVGIFRAAVCTRGVAKAVDFIP VESLETTMRSP

[0386] SEQ ID NO: 266, soluble NS3 / NS4A, 197 aa MKKKGSVVIVGRINLSGDTAYAQQTRGEEGCQETSQTGRDKNQVEGEVQIVSTATQTFLATS INGVLWTVYHGAGTRTIASPKGPVTQMYTNVDKDLVGWQAPQGSRSLTPCTCGSSDLYLVT RHADVIPVRRRGDSRGSLLSPRPISYLKGSSGGPLLCPAGHAVGIFRAAVSTRGVAKAVDFIPV ESLETTMRSP

[0387] SEQ ID NO: 267, soluble NS3 / NS4A, 197 aa MKKKGSVVIVGRINLSGDTAYAQQTRGEQGCQKTSHTGRDKNQVEGEVQIVSTATQTFLAT SINGVLWTVYHGAGTRTIASPKGPVTQMYTNVDKDLVGWQAPQGSRSLTPCTCGSSDLYLV TRHADVIPVRRRGDSRGSLLSPRPISYLKGSSGGPLLCPAGHAVGIFRAAVSTRGVAKAVDFIP VESLETTMRSP 76 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT

[0388] SEQ ID NO: 268, soluble NS3 / NS4A, 197 aa MKKKGSVVIVGRINLSGDTAYAQQTRGEQGTQKTSHTGRDKNQVEGEVQIVSTATQTFLAT SINGVLWTVYHGAGTRTIASPKGPVTQMYTNVDKDLVGWQAPQGSRSLTPCTCGSSDLYLV TRHADVIPVRRRGDSRGSLLSPRPISYLKGSSGGPLLCPAGHAVGIFRAAVSTRGVAKAVDFIP VESLETTMRSP

[0389] SEQ ID NO: 269, NS3aH1, soluble NS3 / NS4A (S139A), 196 aa KKKGSVVIVGRINLSGDTAYAQQTRGEEGCQETSQTGRDKNQVEGEVQIVSTATQTFLATSI NGVLWTVYHGAGTRTIASPKGPVTQMYTNVDKDLVGWQAPQGSRSLTPCTCGSSDLYLVT RHADVIPVRRRGDSRGSLLSPRPISYLKGSAGGPLLCPAGHAVGIFRAAVSTRGVAKAVDFIP VESLETTMRSP

[0390] In some embodiments of any of the aspects, the repressible protease comprises mutations to increase binding affinity for a specific ligand. As a non-limiting example, NS3aH1 (e.g., SEQ ID NO: 269) comprises four mutations needed for interaction with the ANR peptide (e.g., SEQ ID NO: 270, GELDELVYLLDGPGYDPIHSD): A7S, E13L, I35V and T42S. Accordingly, in some embodiments of any of the aspects, a repressible protease as described herein comprises at least one of the following mutations: A7S, E13L, I35V and T42S, or any combination thereof.

[0391] In some embodiments of any of the aspects, a synTF polypeptide as described herein is in combination with a protease inhibitor. As used herein, “in combination with” refers to two or more substances being present in the same formulation in any molecular or physical arrangement, e.g., in an admixture, in a solution, in a mixture, in a suspension, in a colloid, in an emulsion. The formulation can be a homogeneous or heterogeneous mixture. In some embodiments of any of the aspects, the active compound(s) can be comprised by a superstructure, e.g., nanoparticles, liposomes, vectors, cells, scaffolds, or the like, said superstructure is which in solution, mixture, admixture, suspension, etc., 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 the repressible protease. In some embodiments of any of the aspects, the synTF polypeptide is bound specifically to a protease inhibitor bound to the repressible protease.

[0392] In some embodiments of any of the aspects, the synTF polypeptide is in combination with 1, 2, 3, 4, 5, or more protease inhibitors. In some embodiments of any of the aspects, the synTF polypeptide is in combination with one protease inhibitor. In embodiments comprising 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 foregoing.

[0393] 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 77 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT as but not limited to grazoprevir, danoprevir, simeprevir, asunaprevir, ciluprevir, boceprevir, sovaprevir, paritaprevir, ombitasvir, paritaprevir, ritonavir, dasabuvir, and telaprevir. Additional non- limiting examples of NS3 protease inhibitors are listed in Table 12 (see e.g., McCauley and Rudd, Hepatitis C virus NS3 / 4a protease inhibitors, Current Opinion in Pharmacology 2016, 30:84–92; the content of which is incorporated herein by reference in its entirety).

[0394] Table 12: Exemplary NS3 / NS4A protease inhibitors78 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT79 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT80 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT81 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT82 4887-3198-4841.9Attorney Docket No: 701586-000114WOPTDegron Domain 83 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT

[0395] In several aspects, described herein are synTF polypeptides comprising a degron domain. As used herein, the term “degron domain” refers to a sequence that promotes degradation of an attached protein, e.g., through the proteasome or autophagy-lysosome pathways; 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 destabilize a protein such that half-life of the protein is reduced at least two-fold, when fused to the protein. In some embodiments of any of the aspects, a synTF polypeptide as described herein (or a synTF polypeptide system collectively) comprises 1, 2, 3, 4, 5, or more degron domains. In some embodiments of any of the aspects, the synTF polypeptide or system comprises one degron domain. In embodiments comprising multiple degron domains, the multiple degron domains can be different individual degron domains or multiple copies of the same degron domain, or a combination of the foregoing.

[0396] Many different degron sequences / signals (e.g., 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 cell receptor, but need not be contiguous or immediately adjacent with it as long as the degron domain still functions to direct degradation of the cell receptor. In some embodiments, the degron domain induces rapid degradation of the cell receptor. For a discussion of degron domains and their function in protein degradation, see, e.g., Kanemaki et al. (2013) Pflugers Arch.465(3):4l9-425, Erales et al. (2014) Biochim Biophys Acta 1843(l):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(11):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 is incorporated herein by reference in its entirety.

[0397] 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 a self-excising degron, which refers to a complex comprising a repressible protease, a protease cleavage site, and a degron domain. In some embodiments of any of the aspects, the degron domain is a conditional degron domain, wherein the degradation is induced by ligands (e.g., a degron stabilizer) or another input such as temperature shift or a specific wave length of light. Non-limiting examples of conditional degron domains include the eDHFR degron (e.g., TMP inducer); FKBP12 (e.g., rapamycin analog inducer); temperature-sensitive dihydrofolate reductase (R- DHFRts, or ts-DHFR); an HCV NS3 / NS4A degron; a modified version of R-DHFRts termed the low- 84 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT temperature degron (lt-degron); auxin-inducible degradation (AID); HaloTag-Hydrophobic Tag, HaloPROTAC, and dTAG system (e.g., HyT13 or HyT36 inducer); photosensitive degron (PSD); blue-light-inducible degron (B-LID); tobacco etch virus (TEV) protease-induced protein inactivation (TIPI)-degron system; deGradFP (degrade green fluorescent protein; e.g., induced by NSlmb–vhhGFP expression); or split ubiquitin for the rescue of function (SURF; e.g., induced by rapamycin).

[0398] In some embodiments of any of the aspects, the degron domain is the E. coli dihydrofolate reductase (eDHFR) degron. The eDHFR degron permits extensive depletion of exogenously expressed proteins in mammalian cells and C. elegans. The eDHFR degron is stabilized by tight binding to the antibiotic and degron stabilizer trimethoprim (TMP), shown below, which is innocuous in eukaryotic cells.

[0399] Proteins tagged with eDHFR are constitutively degraded unless the cells are exposed to TMP. The level of tagged protein can be directly controlled by modulating the TMP concentration in the growth medium. Unlike shRNA methods this degron-based strategy is advantageous since depletion kinetics are not limited by the natural protein half-life, which allows for more rapid knockdown of stable proteins. TMP stabilizes the DD-target protein fusion in a dose-dependent manner up to 100-fold, which gives the system a substantial dynamic range. The ligand TMP works by itself and does not require dimerization with a second protein. This system is so effective that it can control the levels of transmembrane proteins, such as the synTF polypeptides 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.

[0400] In some embodiments of any of the aspects, the degron domain comprises an amino acid sequence derived from an FK506- 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 FKBP12 derived amino acid sequence comprises a mutation of the phenylalanine (F) at amino acid position 36 (as counted without the methionine) to valine (V) (F36V) (also referred to as FKBP12* or FKBP*). In some embodiments of any of the aspects, the degron stabilizer is a rapamycin analog, such as Sheild-1, shown below. See e.g., Banaszynski et al., Cell. 2006 Sep 8; 126(5): 995–1004; US Patent Application US20180179522; US Patent 10,137,180; the content of each of which is incorporated herein by reference in its entirety. 85 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT

[0401] In some embodiments of any of the aspects, the degron domain of a synTF polypeptide as described herein comprises SEQ ID NOs: 270 or 271, 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 NOs: 270 or 271 that maintains the same function (e.g., degradation, binding to TMP or Shield-1).

[0402] SEQ ID NO: 270, DHFR (V19A), 158 aa, ISLIAALAVDYVIGMENAMPWNLPADLAWFKRNTLNKPVIMGRHTWESIGRPLPGRKNIILSS QPSTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVIEQFLPKAQKLYLTHIDAEVEGDTHFP DYEPDDWESVFSEFHDADAQNSHSYCFEILERR

[0403] SEQ ID NO: 271, FK506- and rapamycin-binding protein (FKBP), 107 aa GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEE GVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE

[0404] In some embodiments of any of the aspects, the destabilizing degron domain comprises at least one mutation that causes almost complete removal or degradation of the synTF polypeptide. Non-limiting examples of DHFR (e.g., SEQ ID NO: 270) 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 combinations thereof; see e.g., US Patent 8173792, the content of which is incorporated herein by reference in its entirety.

[0405] In some embodiments of any of the aspects, the degron domain comprises a ligand- induced degradation (LID) domain. Proteins comprising a LID domain are destabilized and degraded in the presence of a degron destabilizer. In some embodiments of any of the aspects, the LID domain of a degron domain can bind to a degron destabilizer, promoting the degradation of the attached protein. The system is reversible and when the degron destabilizer is withdrawn, the protein is not destabilized and / or not degraded. In some embodiments of any of the aspects, a synTF polypeptide is bound to a degron destabilizer bound to the degron domain. In some embodiments of any of the 86 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT aspects, the synTF polypeptide is bound specifically to a degron destabilizer bound to the degron domain.

[0406] In some embodiments of any of the aspects, the synTF polypeptide is in combination with 1, 2, 3, 4, 5, or more degron destabilizers. In some embodiments of any of the aspects, the synTF polypeptide is in combination with one degron destabilizer. In embodiments comprising multiple degron destabilizers, the multiple degron destabilizers can be different individual degron destabilizers or multiple copies of the same degron stabilizer, or a combination of the foregoing.

[0407] In some embodiments of any of the aspects, the LID degron domain comprises the FK506- and rapamycin-binding protein (FKBP), further comprising a degron fused to the C terminus of FKBP, e.g., with an intervening linker such as the 10-amino acid linker (Gly4SerGly4Ser) or another linker as described herein. In some embodiments of any of the aspects, the degron fused to the C terminus of FKBP (e.g., SEQ ID NO: 271) comprises the 19 amino acid sequence: TRGVEEVAEGVVLLRRRGN (SEQ ID NO: 272), or a sequence that is at least 95% identical that maintains the same function. In the absence of the small molecule Shield-1, the 19-aa degron is bound to the FKBP fusion protein, and the protein is stable. When present, Shield-1 binds tightly to FKBP, displacing the 19-aa degron and inducing rapid and processive degradation of the LID domain and any fused partner protein. In some embodiments of any of the aspects, the degron destabilizer is Sheild-1, shown above, or an analog thereof; see e.g., Bonger et al., Nat Chem Biol.2011 Jul 3;7(8):531-7.

[0408] In some embodiments of any of the aspects, the degron domain comprises an auxin- inducible degradation (AID). Proteins fused to AID (also known as indole-3-acetic acid inducible 17 or AUX / IAA transcriptional regulator family protein) are rapidly degraded. Degradation requires the ectopic expression of the plant F-Box protein TIR1, which recruits proteins tagged with AID in an auxin-dependent manner to the SKP1-CUL1-F-Box (SCF) ubiquitin E3 ligases resulting in their ubiquitylation and proteasomal degradation. In some embodiments of any of the aspects, the degron domain comprises residues 65-133, 65-130, 70-130, or 70-120 of SEQ ID NO: 273. In some embodiments of any of the aspects, the degron domain of a synTF polypeptide as described herein comprises SEQ ID NOs: 273 or 274, 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 NOs: 273 or 274 that maintains the same function. See e.g., Daniel et al., Nat Commun. 2018 Aug 17;9(1):3297; the content of which is incorporated herein by reference in its entirety.

[0409] SEQ ID NO: 273, AUX / IAA transcriptional regulator family protein [Arabidopsis thaliana], NCBI Reference Sequence: NP_171921.1, 229 aa MMGSVELNLRETELCLGLPGGDTVAPVTGNKRGFSETVDLKLNLNNEPANKEGSTTHDVVT FDSKEKSACPKDPAKPPAKAQVVGWPPVRSYRKNVMVSCQKSSGGPEAAAFVKVSMDGAP 87 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT YLRKIDLRMYKSYDELSNALSNMFSSFTMGKHGGEEGMIDFMNERKLMDLVNSWDYVPSY EDKDGDWMLVGDVPWPMFVDTCKRLRLMKGSDAIGLAPRAMEKCKSRA

[0410] SEQ ID NO: 274, mAID (minimal AID), 68 aa KEKSACPKDPAKPPAKAQVVGWPPVRSYRKNVMVSCQKSSGGPEAAAFVKVSMDGAPYLR KIDLRMYK

[0411] In some embodiments of any of the aspects, the degron domain comprises a modified portion of the NS3 helicase and NS4A. The arrangement of 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 is inserted into the endoplasmic reticulum membrane. This N-terminus is created 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. The engineered construct lacks the helicase domain, so NS3 / 4A cleavage does not occur. The hydrophobic sequences of NS4A, unable to insert into the membrane without a free N-terminus, then exhibit degron-like activity. See e.g., US 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 entireties.

[0412] In some embodiments of any of the aspects, the degron domain comprises SEQ ID NO: 275, 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: 275 that maintains the same function.

[0413] SEQ ID NO: 275, HCV NS3 / NS4A degron domain (42 aa) PITKIDTKYIMTCMSADLEVVTSTWVLVGGVLAALAAYCLST Induced Degradation Domain

[0414] In several aspects, described herein are synTF polypeptides comprising an induced degradation domain, also referred to herein as a self-excising degron or a small molecule-assisted shutoff (SMASh) domain. In some embodiments of any of the aspects, the SMASh domain comprises a repressible protease, at least one protease cleavage site, and a degron domain. In the absence of the protease inhibitor, the repressible protease cleaves the degron from the synTF, and the synTF is not degraded. In the presence of the protease inhibitor, the repressible protease does not cleave the degron from the synTF, and the degron domain leads to the degradation of the synTF.

[0415] In some embodiments of any of the aspects, a synTF polypeptide as described herein (or a synTF polypeptide system collectively) comprises 1, 2, 3, 4, 5, or more induced degradation domain(s). In some embodiments of any of the aspects, the synTF polypeptide or system comprises one induced degradation domain. In embodiments comprising multiple induced degradation domains, 88 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT the multiple induced degradation domains can be different individual induced degradation domains or multiple copies of the same induced degradation domain, or a combination of the foregoing.

[0416] In some embodiments of any of the aspects, degron domain (e.g., of the SMASh domain) is selected from the group consisting of: a ubiquitin tag; eDHFR degron (e.g., TMP inducer); FKBP12 (e.g., rapamycin analog inducer); temperature-sensitive dihydrofolate reductase (R-DHFRts, or ts- DHFR); an HCV NS3 / NS4A degron; a modified version of R-DHFRts termed the low-temperature degron (lt-degron); auxin-inducible degradation (AID); HaloTag-Hydrophobic Tag, HaloPROTAC, and dTAG system (e.g., HyT13 or HyT36 inducer); photosensitive degron (PSD); blue-light-inducible degron (B-LID); tobacco etch virus (TEV) protease-induced protein inactivation (TIPI)-degron system; deGradFP (degrade green fluorescent protein; e.g., induced by NSlmb–vhhGFP expression); or split ubiquitin for the rescue of function (SURF; e.g., induced by rapamycin).

[0417] In some embodiments of any of the aspects, the degron domain (e.g., of the SMASh domain) comprises a modified portion of the NS3 helicase and NS4A. In some embodiments of any of the aspects, the degron domain (e.g., of the SMASh domain) comprises SEQ ID NO: 275, 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: 275 that maintains the same function.

[0418] In some embodiments of any of the aspects, the SMASh tag comprises a repressible protease, a partial protease helical domain, and a cofactor domain. In some embodiments of any of the aspects, the SMASh tag comprises a repressible protease, a partial protease helical domain, a cofactor domain, and at least one protease cleavage site. In some embodiments of any of the aspects, the SMASh tag comprises an NS3 repressible protease, an NS3 partial protease helical domain, an NS3 cofactor domain (i.e., NS4A), and at least one protease cleavage site of the NS3 repressible protease.

[0419] In some embodiments of any of the aspects, the SMASh tag is a C-terminal SMASh tag, e.g., the tag is engineered to be attached to the C-terminus of the synTF. In some embodiments of any of the aspects, the C-terminal SMASh tag comprises a protease cleavage site at the N-terminus of the tag. In some embodiments of any of the aspects, the C-terminal SMASh tag comprises in a N-terminal to C-terminal order: a NS3 cleavage site, at least one linker, a NS3 domain, a NS3 partial helicase, a NS4A domain. In some embodiments of any of the aspects, the C-terminal SMASh tag is fused to the C-terminus of the transcriptional effector domain of the synTF. In some embodiments of any of the aspects, the C-terminal SMASh tag is fused to the C-terminus of the DNA-binding domain of the synTF.

[0420] In some embodiments of any of the aspects, the C-terminal SMASh tag comprises one of SEQ ID NOs: 276-278 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 89 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence of one of SEQ ID NOs: 276-278 that maintains the same function.

[0421] SEQ ID NO: 276, C-terminal SMASh domain (304 aa); bold text indicates NS3 Cleavage Site; italicized double underlined text indicates a Linker; bold italicized text indicates the FLAG tag; italicized text indicates the NS3 Protease Domain; unformatted text indicates NS3 Partial Helicase; bold italicized double underlined text indicates NS4A Domain: DEMEECSQHLPGAGSSGDIMDYKDDDDKGSSGTGSGSGTSAPITAYAQQTRGLLGCIITSLTGRD KNQVEGEVQIVSTATQTFLATCINGVCWAVYHGAGTRTIASPKGPVIQMYTNVDQDLVGWPAPQG SRSLTPCTCGSSDLYLVTRHADVIPVRRRGDSRGSLLSPRPISYLKGSSGGPLLCPAGHAVGLFRAAV CTRGVAKAVDFIPVENLETTMRSPVFTDNSSPPAVTLTHPITKIDTKYIMTCMSADLEVVTSTW VLVGGVLAALAAYCLSTGCVVIVGRIVLSGKPAIIPDREVLY

[0422] In some embodiments of any of the aspects, the SMASh tag is a N-terminal SMASh tag, e.g., the tag is engineered to be attached to the N-terminus of the synTF. In some embodiments of any of the aspects, the N-terminal SMASh tag comprises a protease cleavage site at the C-terminus of the tag. In some embodiments of any of the aspects, the N-terminal SMASh tag comprises in a N-terminal to C-terminal order at least one Linker, a NS3 domain, a NS3 partial helicase, a NS4 domain, and a NS3 cleavage site. In some embodiments of any of the aspects, the N-terminal SMASh tag is fused to the N-terminus of the transcriptional effector domain of the synTF. In some embodiments of any of the aspects, the N-terminal SMASh tag is fused to the N-terminus of the DNA-binding domain of the synTF.

[0423] In some embodiments of any of the aspects, the N-terminal SMASh tag comprises SEQ ID NOs: 279 to 284, 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: 279 to 284, that maintains the same function.

[0424] SEQ ID NO: 279, N-terminal SMASh domain (297 aa), italicized double underlined text indicates a Linker; bold italicized text indicates the FLAG tag (; italicized text indicates the NS3 Protease Domain; unformatted text indicates NS3 Partial Helicase; bold italicized double underlined text indicates NS4A Domain; bold text indicates NS3 Cleavage Site: DYKDDDDKGSSGTGSGSGTSAPITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIVSTATQTFLATC INGVCWAVYHGAGTRTIASPKGPVIQMYTNVDQDLVGWPAPQGSRSLTPCTCGSSDLYLVTRHADV IPVRRRGDSRGSLLSPRPISYLKGSSGGPLLCPAGHAVGLFRAAVCTRGVAKAVDFIPVENLETTMR SPVFTDNSSPPAVTLTHPITKIDTKYIMTCMSADLEVVTSTWVLVGGVLAALAAYCLSTGCVVI VGRIVLSGKPAIIPDREVLYQEFEDVVPCSMGS

[0425] SEQ ID NO: 280, N-terminal SMASh domain (303 aa, with GSSGSS (SEQ ID NO: 285) L6 domain in NS4A), italicized double underlined text indicates a Linker; bold italicized text 90 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT indicates the FLAG tag; italicized text indicates the NS3 Protease Domain; unformatted text indicates NS3 Partial Helicase; bold italicized double underlined text indicates NS4A Domain; bold text indicates NS3 Cleavage Site. DYKDDDDKGSSGTGSGSGTSAPITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIVSTATQTFLATC INGVCWAVYHGAGTRTIASPKGPVIQMYTNVDQDLVGWPAPQGSRSLTPCTCGSSDLYLVTRHADV IPVRRRGDSRGSLLSPRPISYLKGSSGGPLLCPAGHAVGLFRAAVCTRGVAKAVDFIPVENLETTMR SPVFTDNSSPPAVTLTHPITKIDTKYIMTCMSADLEVVTSTWVLVGGVLAALAAYCLSTGCVVI VGRIVLSGKPAGSSGSSIIPDREVLYQEFEDVVPCSMGS

[0426] In some embodiments of any of the aspects, SMASh domain comprises a repressible protease that comprises 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 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 of the aspects, the NS3 protease (e.g., of the SMASh domain) comprises at least one of the following mutations: V36M, T54A, S122G, or any combination thereof; such a SMASh is also referred to herein as SMAShAI, as these mutations increase its sensitivity to asunaprevir (see e.g., SEQ ID NOs: 197, 277, 281, 282). In some embodiments of any of the aspects, the NS3 protease (e.g., of the SMASh domain) comprises at least one of the following mutations: F43L, Q80K, S122R, D168Y, or any combination thereof; such a protease is also referred to herein as SMAShTI, as these mutations increase its sensitivity to telaprevir (see e.g., SEQ ID NOs: 198, 278, 283, 284).

[0427] SEQ ID NO: 277, C-terminal SMAShAIdomain with (304 aa); bold text indicates NS3 Cleavage Site; italicized double underlined text indicates a Linker; bold italicized text indicates the FLAG tag; italicized text indicates the NS3 Protease Domain; unformatted text indicates NS3 Partial Helicase; bold italicized double underlined text indicates NS4A Domain; the V36M, T54A, S122G mutations are shown in bold double underlined text, respectively. DEMEECSQHLPGAGSSGDIMDYKDDDDKGSSGTGSGSGTSAPITAYAQQTRGLLGCIITSLTGRD KNQVEGEVQIMSTATQTFLATCINGVCWAVYHGAGTRTIASPKGPVIQMYTNVDQDLVGWPAPQ GSRSLTPCTCGSSDLYLVTRHADVIPVRRRGDGRGSLLSPRPISYLKGSSGGPLLCPAGHAVGLFRA AVCTRGVAKAVDFIPVENLETTMRSPVFTDNSSPPAVTLTHPITKIDTKYIMTCMSADLEVVTST WVLVGGVLAALAAYCLSTGCVVIVGRIVLSGKPAIIPDREVLY

[0428] SEQ ID NO: 281, N-terminal SMAShAIdomain (297 aa), italicized double underlined text indicates a Linker; bold italicized text indicates the FLAG tag; italicized text indicates the NS3 Protease Domain; unformatted text indicates NS3 Partial Helicase; bold italicized double underlined text indicates NS4A Domain; bold text indicates NS3 Cleavage Site; the V36M, T54A, S122G mutations are shown in bold double underlined text, respectively. 91 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT DYKDDDDKGSSGTGSGSGTSAPITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIMSTATQTFLAT CINGVCWAVYHGAGTRTIASPKGPVIQMYTNVDQDLVGWPAPQGSRSLTPCTCGSSDLYLVTRHA DVIPVRRRGDGRGSLLSPRPISYLKGSSGGPLLCPAGHAVGLFRAAVCTRGVAKAVDFIPVENLETT MRSPVFTDNSSPPAVTLTHPITKIDTKYIMTCMSADLEVVTSTWVLVGGVLAALAAYCLSTGC VVIVGRIVLSGKPAIIPDREVLYQEFEDVVPCSMGS

[0429] SEQ ID NO: 282, N-terminal SMAShAIdomain (303 aa, with L6 domain in NS4A), italicized double underlined text indicates a Linker; bold italicized text indicates the FLAG tag; italicized text indicates the NS3 Protease Domain; unformatted text indicates NS3 Partial Helicase; bold italicized double underlined text indicates NS4A Domain; bold text indicates NS3 Cleavage Site; the V36M, T54A, S122G mutations are shown in bold double underlined text, respectively. DYKDDDDKGSSGTGSGSGTSAPITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIMSTATQTFLAT CINGVCWAVYHGAGTRTIASPKGPVIQMYTNVDQDLVGWPAPQGSRSLTPCTCGSSDLYLVTRHA DVIPVRRRGDGRGSLLSPRPISYLKGSSGGPLLCPAGHAVGLFRAAVCTRGVAKAVDFIPVENLETT MRSPVFTDNSSPPAVTLTHPITKIDTKYIMTCMSADLEVVTSTWVLVGGVLAALAAYCLSTGC VVIVGRIVLSGKPAGSSGSSIIPDREVLYQEFEDVVPCSMGS

[0430] SEQ ID NO: 278, C-terminal SMAShTIdomain with (304 aa); bold text indicates NS3 Cleavage Site; italicized double underlined text indicates a Linker; bold italicized text indicates the FLAG tag; italicized text indicates the NS3 Protease Domain; unformatted text indicates NS3 Partial Helicase; bold italicized double underlined text indicates NS4A Domain; the F43L, Q80K, S122R, D168Y mutations are shown in bold double underlined text, respectively. DEMEECSQHLPGAGSSGDIMDYKDDDDKGSSGTGSGSGTSAPITAYAQQTRGLLGCIITSLTGRD KNQVEGEVQIVSTATQTLLATCINGVCWAVYHGAGTRTIASPKGPVIQMYTNVDKDLVGWPAPQG SRSLTPCTCGSSDLYLVTRHADVIPVRRRGDRRGSLLSPRPISYLKGSSGGPLLCPAGHAVGLFRAAV CTRGVAKAVYFIPVENLETTMRSPVFTDNSSPPAVTLTHPITKIDTKYIMTCMSADLEVVTSTWV LVGGVLAALAAYCLSTGCVVIVGRIVLSGKPAIIPDREVLY

[0431] SEQ ID NO: 283, N-terminal SMAShTIdomain (297 aa), italicized double underlined text indicates a Linker; bold italicized text indicates the FLAG tag; italicized text indicates the NS3 Protease Domain; unformatted text indicates NS3 Partial Helicase; bold italicized double underlined text indicates NS4A Domain; bold text indicates NS3 Cleavage Site; the F43L, Q80K, S122R, D168Y mutations are shown in bold double underlined text, respectively. DYKDDDDKGSSGTGSGSGTSAPITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIVSTATQTLLATC INGVCWAVYHGAGTRTIASPKGPVIQMYTNVDKDLVGWPAPQGSRSLTPCTCGSSDLYLVTRHADV IPVRRRGDRRGSLLSPRPISYLKGSSGGPLLCPAGHAVGLFRAAVCTRGVAKAVYFIPVENLETTMR SPVFTDNSSPPAVTLTHPITKIDTKYIMTCMSADLEVVTSTWVLVGGVLAALAAYCLSTGCVVI VGRIVLSGKPAIIPDREVLYQEFEDVVPCSMGS 92 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT

[0432] SEQ ID NO: 284, N-terminal SMAShTIdomain (303 aa, with L6 domain in NS4A), italicized double underlined text indicates a Linker; bold italicized text indicates the FLAG tag; italicized text indicates the NS3 Protease Domain; unformatted text indicates NS3 Partial Helicase; bold italicized double underlined text indicates NS4A Domain; bold text indicates NS3 Cleavage Site; the F43L, Q80K, S122R, D168Y mutations are shown in bold double underlined text, respectively. DYKDDDDKGSSGTGSGSGTSAPITAYAQQTRGLLGCIITSLTGRDKNQVEGEVQIVSTATQTLLATC INGVCWAVYHGAGTRTIASPKGPVIQMYTNVDKDLVGWPAPQGSRSLTPCTCGSSDLYLVTRHADV IPVRRRGDRRGSLLSPRPISYLKGSSGGPLLCPAGHAVGLFRAAVCTRGVAKAVYFIPVENLETTMR SPVFTDNSSPPAVTLTHPITKIDTKYIMTCMSADLEVVTSTWVLVGGVLAALAAYCLSTGCVVI VGRIVLSGKPAGSSGSSIIPDREVLYQEFEDVVPCSMGS

[0433] In some embodiments of any of the aspects, the SMASh domain of the synTF polypeptide is in combination with 1, 2, 3, 4, 5, or more protease inhibitors. In some embodiments of any of the aspects, the SMASh domain of the synTF polypeptide is in combination with one protease inhibitor. In embodiments comprising 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 foregoing.

[0434] 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, ciluprevir, boceprevir, sovaprevir, paritaprevir, ombitasvir, paritaprevir, ritonavir, dasabuvir, and telaprevir. Additional non- limiting examples of NS3 protease inhibitors are listed in Table 12 (see e.g., McCauley and Rudd, Hepatitis C virus NS3 / 4a protease inhibitors, Current Opinion in Pharmacology 2016, 30:84–92; the content of which is incorporated herein by reference in its entirety). Induced Proximity Domains

[0435] In several aspects, described herein are synTF polypeptides comprising at least two induced proximity domains, also referred to herein as heterodimerization domains. As used herein the term “induced proximity domains” refers to at least two domains that are induced to dimerize or come into close proximity in the presence of a stimulus (e.g., chemical inducer, light, etc.). In some embodiments of any of the aspects, the induced proximity domain pair comprises a first induced proximity domain (IPDA) and at least a second induced proximity domain (IPDB), wherein in the presence of an inducer agent or inducer signal, the IPDAand IPDBcome together. In some embodiments of any of the aspects, the synTF effector domain is linked to IPDA(or IPDB) in a first 93 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT polypeptide, and the synTF DBD is linked to IPDB(or IPDA) in a second polypeptide. Thus, in the presence of an inducer agent or inducer signal, the IPDAand IPDBcome together linked to resulting in the linkage of the TA to the DBD of the synthetic TF. In the absence of an inducer agent or inducer signal, the TA is uncoupled or unlinked to the DBD of the synthetic TF.

[0436] In some embodiments of any of the aspects, a synTF polypeptide as described herein (or a synTF polypeptide system collectively) comprises 1, 2, 3, 4, 5, or more induced proximity domain(s). In some embodiments of any of the aspects, the 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.

[0437] In some embodiments of any of the aspects, the induced proximity domain pair (IPD pair) comprises a IPDAand IPDBselected from any one or more of: (1) a IPDAcomprising a GID1 domain or a fragment thereof, and a IPDBcomprising a GAI domain, wherein the GID1 domain and GAI domain bind to the inducer agent Gibberellin Ester (GIB); (2) a IPDAcomprising a FKBP domain or a fragment thereof, and a IPDBcomprising a FRB domain, wherein the FKBP domain and FRB domain bind to the inducer agent Rapalog (RAP); (3) a IPDAcomprising a PYL domain or a fragment thereof, and a IPDBcomprising a ABI domain, wherein the PYL domain and ABI domain bind to the inducer agent Abscisic acid (ABA); (4) a IPDAcomprising a Light-inducible dimerization domain (LIDD), wherein a LIDD dimerizes with a complementary LIDD (IPDB) upon exposure to a light inducer signal of an appropriate wavelength.

[0438] In some embodiments of any of the aspects, the synTF polypeptide is in combination with 1, 2, 3, 4, 5, or more inducer agents, i.e., that induce dimerization or proximity of the IPDs. In some embodiments of any of the aspects, the synTF polypeptides are in combination with one inducer agent. In embodiments comprising multiple inducer agents, the multiple inducer agents can be different individual inducer agents or multiple copies of the same inducer agent, or a combination of the foregoing.

[0439] In some embodiments of any of the aspects, the IPD pair comprises a 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 of the aspects, the IPD pair comprises the interacting complementary surfaces (CSs) of PYL1 (PYLcs, amino acids 33 to 209) and ABI1 (ABIcs, amino acids 126 to 423). In some embodiments of any of the aspects, the ABI domain (e.g., SEQ ID NO: 286 or 287) comprises mutations A18D and E108G. In some embodiments of any of the aspects, the ABI domain further comprises a detectable marker (e.g., a FLAG tag). In some embodiments of any of the aspects, the PYL domain further comprises a detectable marker (e.g., an HA tag). 94 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT

[0440] In some embodiments of any of the aspects, the ABI domain comprises SEQ ID NOs: 286 or 287 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: 286 or 287, that maintains the same function.

[0441] In some embodiments of any of the aspects, the PYL domain comprises SEQ ID NOs: 288 or 289 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: 288 or 289, that maintains the same function.

[0442] SEQ ID NO: 286, ABI cs CO1 (298 aa) VPLYGFTSICGRRPEMEAAVSTIPRFLQSSSGSMLDGRFDPQSAAHFFGVYDGHGGSQVANY CRERMHLALAEEIAKEKPMLCDGDTWLEKWKKALFNSFLRVDSEIESVAPETVGSTSVVAV VFPSHIFVANCGDSRAVLCRGKTALPLSVDHKPDREDEAARIEAAGGKVIQWNGARVFGVL AMSRSIGDRYLKPSIIPDPEVTAVKRVKEDDCLILASDGVWDVMTDEEACEMARKRILLWHK KNAVAGDASLLADERRKEGKDPAAMSAAEYLSKLAIQRGSKDNISVVVVDLK

[0443] SEQ ID NO: 288, PYL1cs Domain (177 aa) TQDEFTQLSQSIAEFHTYQLGNGRCSSLLAQRIHAPPETVWSVVRRFDRPQIYKHFIKSCNVSE DFEMRVGCTRDVNVISGLPANTSRERLDLLDDDRRVTGFSITGGEHRLRNYKSVTTVHRFEK EEEEERIWTVVLESYVVDVPEGNSEEDTRLFADTVIRLNLQKLASITEAMN

[0444] SEQ ID NO: 287, Alternative ABI binding motif PLYGFTSICGRRPEMEDAVSTIPRFLQSSSGSMLDGRFDPQSAAHFFGVYDGHGGSQVANYC RERMHLALAEEIAKEKPMLCDGDTWLEKWKKALFNSFLRVDSEIGSVAPETVGSTSVVAVV FPSHIFVANCGDSRAVLCRGKTALPLSVDHKPDREDEAARIEAAGGKVIQWNGARVFGVLA MSRSIGDRYLKPSIIPDPEVTAVKRVKEDDCLILASDGVWDVMTDEEACEMARKRILLWHKK NAVAGDASLLADERRKEGKDPAAMSAAEYLSKLAIQRGSKDNISVVVVDLKDYKDDDDK

[0445] SEQ ID NO: 289, Alternative PYL binding motif APTQDEFTQLSQSIAEFHTYQLGNGRCSSLLAQRIHAPPETVWSVVRRFDRPQIYKHFIKSCNV SEDFEMRVGCTRDVNVISGLPANTSRERLDLLDDDRRVTGFSITGGEHRLRNYKSVTTVHRF EKEEEEERIWTVVLESYVVDVPEGNSEEDTRLFADTVIRLNLQKLASITEAMNYPYDVPDYA

[0446] In some embodiments of any of the aspects, the proximity inducer agent (e.g., for ABI and PYL domains) is abscisic acid (ABA):95 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT

[0447] In some embodiments, the IPD pair are FKBP (FK506- and rapamycin-binding protein) and FKBP12-rapamycin-binding protein (FRB) proteins, which come together and dimerize in the presence of a rapalog. In some embodiments of any of the aspects, the FKBP domain comprises SEQ ID NOs: 290 or 271 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: 290 or 271, that maintains the same function. In some embodiments of any of the aspects, the FRB domain comprises SEQ ID NO: 291 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: 291, that maintains the same function.

[0448] SEQ ID NO: 290, FKBP aa binding motif: SRGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGW EEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE

[0449] SEQ ID NO: 291, FRB binding motif, ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLME AQEWCRKYMKSGNVKDLLQAWDLYYHVFRRIS

[0450] In some embodiments of any of the aspects, the proximity inducer agent (e.g., for FKBP and FRB domains) is rapamycin shown below. In some embodiments of any of the aspects, the proximity inducer agent (e.g., for FKBP and FRB domains) is a rapalog, i.e., a rapamycin analog. In some embodiments of any of the aspects, the rapalog is Sheild-1, as described further herein.

[0451] In other embodiments, the IPD pair are GAI (Gibberellin insensitive) and GID1 (Gibberellin insensitive dwarf1) proteins, derived from, Arabidopsis thaliana, which come together in the presence of Gibberellin Ester (GE). In some embodiments of any of the aspects, the GAI domain comprises SEQ ID NO: 292 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: 292, that maintains the same function. In some embodiments of any of the aspects, the GAI 96 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT domain comprises the amino-terminal DELLA domain of GAI. In some embodiments of any of the aspects, the GID domain comprises SEQ ID NO: 293 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: 293, that maintains the same function.

[0452] SEQ ID NO: 292, GAI binding motif, MKRDHHHHHHQDKKTMMMNEEDDGNGMDELLAVLGYKVRSSEMADVAQKLEQLEVMM SNVQEDDLSQLATETVHYNPAELYTWLDSMLTDLN

[0453] SEQ ID NO: 293, GID1 binding motif MAASDEVNLIESRTVVPLNTWVLISNFKVAYNILRRPDGTFNRHLAEYLDRKVTANANPVDG VFSFDVLIDRRINLLSRVYRPAYADQEQPPSILDLEKPVDGDIVPVILFFHGGSFAHSSANSAIY DTLCRRLVGLCKCVVVSVNYRRAPENPYPCAYDDGWIALNWVNSRSWLKSKKDSKVHIFL AGDSSGGNIAHNVALRAGESGIDVLGNILLNPMFGGNERTESEKSLDGKYFVTVRDRDWYW KAFLPEGEDREHPACNPFSPRGKSLEGVSFPKSLVVVAGLDLIRDWQLAYAEGLKKAGQEVK LMHLEKATVGFYLLPNNNHFHNVMDEISAFVNAEC

[0454] In some embodiments of any of the aspects, the proximity inducer agent (e.g., for GAI and GID1 domains) is a bioactive gibberellin (shown below), a Gibberellin Ester (GE), or another gibberellin analog.

[0455] In some embodiments, the IPD pair comprises a caffeine-induced dimerization system, such as a VHH camelid antibody (referred to as aCaffVHH) that has high affinity (Kd = 500 nM) and homodimerizes in the presence of caffeine. In some embodiments, the IPD pair is selected from a combinatorial binders-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 human antibody-based chemically induced dimerizes (AbCIDs), which are derived from known small-molecule–protein complexes by selecting for synthetic antibodies that recognize the chemical epitope created by the small molecule bound to the protein (e.g., ABT-737). In some embodiments, the IPD pair comprises Calcineurin and FKBP, which come together in the presence of FK506. In some embodiments, the IPD pair comprises Calcineurin and prolyl isomerase CyP, which come together in the presence of Cyclosporine A. In some embodiments, the IPD pair comprises CyP and FKBP, which come together in the presence of FKCsA, a fusion of FK506 and Cyclosprin A. In some embodiments, the IPD pair comprises two 97 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT copies of FKBP, which come together in the presence of FK2012, a fusion of two FK506 molecules. See e.g., 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, pages465– 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. 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, pages973–975(2010); US Patent Applications US20180163195 and US20170183654; US Patent 8735096; the contents of each of which are incorporated herein by reference in their entireties.

[0456] In some embodiments, the IPD pair comprises a light-inducible 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-inducible dimerization domain (LIDD) pair, such as nMag and pMag proteins, which come together and dimerize in a blue light signal, e.g., after a blue light pulse signal, or pulse of a light of an appropriate wavelength. In some embodiments of any of the aspects, the nMag domain comprises SEQ ID NO: 294 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: 294, that maintains the same function. In some embodiments of any of the aspects, the pMag domain comprises SEQ ID NO: 295 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: 295, that maintains the same function.

[0457] SEQ ID NO: 294, nMag binding motif HTLYAPGGYDIMGYLDQIGNRPNPQVELGPVDTSCALILCDLKQKDTPIVYASEAFLYMTGY SNAEVLGRNCRFLQSPDGMVKPKSTRKYVDSNTINTMRKAIDRNAEVQVEVVNFKKNGQRF VNFLTMIPVRDETGEYRYSMGFQCETE

[0458] SEQ ID NO: 295, pMag binding motif HTLYAPGGYDIMGYLRQIRNRPNPQVELGPVDTSCALILCDLKQKDTPIVYASEAFLYMTGY SNAEVLGRNCRFLQSPDGMVKPKSTRKYVDSNTINTMRKAIDRNAEVQVEVVNFKKNGQRF VNFLTMIPVRDETGEYRYSMGFQCETE

[0459] In some embodiments, the IPD pair comprises a light-inducible dimerization domain (LIDD) pair, such as cryptochrome 2 (CRY2) and CIBN (a truncated version of CIB1 (Calcium And Integrin-Binding Protein 1; CRY2 interacting basic-helix-loop-helix 1)) and proteins, derived from Arabidopsis thaliana, which come together and dimerize in a blue light signal, e.g., after a blue light pulse signal, or pulse of a light of an appropriate wavelength. In some embodiments of any of the 98 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT aspects, the CIBN domain comprises SEQ ID NO: 296 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: 296, that maintains the same function. In some embodiments of any of the aspects, the CRY2 domain comprises SEQ ID NO: 297 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: 297, that maintains the same function.

[0460] SEQ ID NO: 296, CIBN LIDD MNGAIGGDLLLNFPDMSVLERQRAHLKYLNPTFDSPLAGFFADSSMITGGEMDSYLSTAGLN LPMMYGETTVEGDSRLSISPETTLGTGNFKKRKFDTETKDCNEKKKKMTMNRDDLVEEGEE EKSKITEQNNGSTKSIKKMKHKAKKEENNFSNDSSKVTKELEKTDYIH

[0461] SEQ ID NO: 297, CRY2 LIDD MKMDKKTIVWFRRDLRIEDNPALAAAAHEGSVFPVFIWCPEEEGQFYPGRASRWWMKQSL AHLSQSLKALGSDLTLIKTHNTISAILDCIRVTGATKVVFNHLYDPVSLVRDHTVKEKLVERGI SVQSYNGDLLYEPWEIYCEKGKPFTSFNSYWKKCLDMSIESVMLPPPWRLMPITAAAEAIWA CSIEELGLENEAEKPSNALLTRAWSPGWSNADKLLNEFIEKQLIDYAKNSKKVVGNSTSLLSP YLHFGEISVRHVFQCARMKQIIWARDKNSEGEESADLFLRGIGLREYSRYICFNFPFTHEQSLL SHLRFFPWDADVDKFKAWRQGRTGYPLVDAGMRELWATGWMHNRIRVIVSSFAVKFLLLP WKWGMKYFWDTLLDADLECDILGWQYISGSIPDGHELDRLDNPALQGAKYDPEGEYIRQW LPELARLPTEWIHHPWDAPLTVLKASGVELGTNYAKPIVDIDTARELLAKAISRTREAQIMIG AAPDEIVADSFEALGANTIKEPGLCPSVSSNDQQVPSAVRYNGSKRVKPEEEEERDMKKSRG FDERELFSTAESSSSSSVFFVSQSCSLASEGKNLEGIQDSSDQITTSLGKNGCK

[0462] In some embodiments, the IPD pair comprises a light-inducible dimerization domain (LIDD) pair, such as photochromic protein domains including, but not limited to Dronpa, Padron, rsTagRFP, and mApple, or a variant or polypeptide fragment thereof having fluorescence characteristics (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 of the aspects, the photochromic protein domain comprises one of SEQ ID NOs: 298-301or 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: 298- 301, that maintains the same function.

[0463] SEQ ID NO: 298, Dropna-145K (224 aa) MSVIKPDMKIKLRMEGAVNGHPFAIEGVGLGKPFEGKQSMDLKVKEGGPLPFAYDILTTVFC YGNRVFAKYPENIVDYFKQSFPEGYSWERSMNYEDGGICNATNDITLDGDCYIYEIRFDGVN 99 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT FPANGPVMQKRTVKWEPSTEKLYVRDGVLKGDVNMALSLEGGGHYRCDFKTTYKAKKVV QLPDYHFVDHHIEIKSHDKDYSNVNLHEHAEAHSELPRQAK

[0464] SEQ ID NO: 299, Dropna-145N (224 aa) MSVIKPDMKIKLRMEGAVNGHPFAIEGVGLGKPFEGKQSMDLKVKEGGPLPFAYDILTTVFC YGNRVFAKYPENIVDYFKQSFPEGYSWERSMNYEDGGICNATNDITLDGDCYIYEIRFDGVN FPANGPVMQKRTVKWEPSTENLYVRDGVLKGDVNMALSLEGGGHYRCDFKTTYKAKKVV QLPDYHFVDHHIEIKSHDKDYSNVNLHEHAEAHSELPRQAK

[0465] SEQ ID NO: 300, Padron-145N (224 aa) MSVIKPDMKIKLRMEGAVNGHPFAIEGVGLGKPFEGKQSMDLKVKEGGPLPFAYDILTMAFC YGNRVFAKYPENIVDYFKQSFPEGYSWERSMHYEDGGSCNATNDITLDGDCYIYEIRFDGVN FPANGPVMQKRTVKWERSTENLYVRDGVLKSDGNYALSLEGGGHYRCDFKTTYKAKKVV QLPDYHSVDHHIEIKSHDKDYSNVNLHEHAEAHSELPRQAN

[0466] SEQ ID NO: 301, mApple-162H-164A (236 aa) MVSKGEENNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEAFQTAKLKVTKGGPLPFA WDILSPQFMYGSKVYIKHPADIPDYFKLSFPEGFRWERVMNFEDGGIIHVNQDSSLQDGVFIY KVKLRGTNFPSDGPVMQKKTMGWEASEERMYPEDGAHKAEIKKRLKLKDGGHYAAEVKT TYKAKKPVQLPGAYIVDIKLDIVSHNEDYTIVEQYERAEGRHSTGGMDELYK Cytosolic Sequestering Domain

[0467] In several aspects, described herein are synTF polypeptides comprising a cytosolic sequestering domain or protein, also referred to herein as a translocation domain. As used herein, the term “cytosolic sequestering domain” refers to a domain that influences the subcellular location of the synTF to which it is linked, e.g., through the binding of a ligand.

[0468] In some embodiments of any of the aspects, a synTF polypeptide as described herein (or a synTF polypeptide system collectively) comprises 1, 2, 3, 4, 5, or more cytosolic sequestering domain(s). In some embodiments of any of the aspects, the synTF polypeptide or system comprises one cytosolic sequestering domain. In embodiments comprising multiple cytosolic sequestering domains, the multiple cytosolic sequestering domains can be different individual cytosolic sequestering domains or multiple copies of the same cytosolic sequestering domain, or a combination of the foregoing.

[0469] In some embodiments of any of the aspects, the cytosolic sequestering protein comprises a ligand binding domain (LBD), wherein in the presence of the ligand, the sequestering of the protein to the cytosol is inhibited. In some embodiments of any of the aspects, cytosolic sequestering protein further comprises a nuclear localization signal (NLS), wherein in the absence of the ligand the NLS is inhibited thereby preventing translocation of the sequestering protein to the nucleus, and wherein in the presence of the ligand the nuclear localization signal is exposed enabling translocation of the 100 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT sequestering protein to the nucleus. Accordingly, when the ligand is absent, the synTF is sequestered to the cytosol. When the ligand is absent, the synTF is translocated to the nucleus.

[0470] In some embodiments of any of the aspects, the sequestering protein comprises at least a portion of the estrogen receptor (ER). The ER naturally associates with cytoplasmic factors in the cell in the absence of cognate ligands, effectively sequestering itself in the cytoplasm. Binding of cognate ligands, such as estrogen or other steroid hormone derivatives, cause a conformational change to the receptor that allow dissociation from the cytoplasmic complexes and expose a nuclear localization signal, permitting translocation into the nucleus.

[0471] In some embodiments of any of the aspects, the sequestering protein comprises SEQ ID NO: 302 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: 302 that maintains the same function. In some embodiments of any of the aspects, the sequestering protein comprises a portion of the ER (e.g., SEQ ID NO: 302), e.g., the C-terminal ligand-binding and nuclear localization domains of ER. In some embodiments of any of the aspects, the sequestering protein comprises residues 282-595 of SEQ ID NO: 302 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: 302.

[0472] SEQ ID NO: 302, estrogen receptor isoform 1 [Homo sapiens]; NCBI Reference Sequence: NP_000116.2 (595 aa) MTMTLHTKASGMALLHQIQGNELEPLNRPQLKIPLERPLGEVYLDSSKPAVYNYPEGAAYEF NAAAAANAQVYGQTGLPYGPGSEAAAFGSNGLGGFPPLNSVSPSPLMLLHPPPQLSPFLQPH GQQVPYYLENEPSGYTVREAGPPAFYRPNSDNRRQGGRERLASTNDKGSMAMESAKETRYC AVCNDYASGYHYGVWSCEGCKAFFKRSIQGHNDYMCPATNQCTIDKNRRKSCQACRLRKC YEVGMMKGGIRKDRRGGRMLKHKRQRDDGEGRGEVGSAGDMRAANLWPSPLMIKRSKKN SLALSLTADQMVSALLDAEPPILYSEYDPTRPFSEASMMGLLTNLADRELVHMINWAKRVPG FVDLTLHDQVHLLECAWLEILMIGLVWRSMEHPGKLLFAPNLLLDRNQGKCVEGMVEIFDM LLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLKSLEEKDHIHRVLDKITDTLIHLMA KAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMKCKNVVPLYDLLLEMLDAHRLHAP TSRGGASVEETDQSHLATAGSTSSHSLQKYYITGEAEGFPATV

[0473] In some embodiments of any of the aspects, the estrogen receptor comprises at least one mutation that decreases its ability to bind to its natural ligands (e.g., estradiol) but maintains the ability to bind to synthetic ligands such as tamoxifen and analogs thereof. In some embodiments of any of the aspects, 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 101 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT of any of the aspects, a triple G400V / MS43A / L544A ER mutant is referred to herein as ERT2. In some embodiments of any of the aspects, the sequestering protein further comprises a V595A mutation from ER (e.g., SEQ ID NO: 302). In some embodiments of any of the aspects, the sequestering protein comprises an estrogen ligand binding domain (ERT2) or a variant thereof. In some embodiments of any of the aspects, the sequestering protein comprises ERT, ERT2, ERT3, or a variant thereof. In some embodiments of any of the aspects, the sequestering protein comprises one of SEQ ID NOs: 43, 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: 43, 304-306, that maintains the same function. See e.g., US 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 entireties.

[0474] SEQ ID NO: 43, ERT2 (314 aa), G400V, M543A, L544A, and V595A mutations from ER (e.g., SEQ ID NO: 302) shown in bold, double underlined text SAGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPFSEASMM GLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEILMIGLVWRSMEHPVKLLF APNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLK SLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMK CKNVVPLYDLLLEAADAHRLHAPTSRGGASVEETDQSHLATAGSTSSHSLQKYYITGEAEGF PATA

[0475] SEQ ID NO: 304, ERT (314 aa), G521R mutation from ER (e.g., SEQ ID NO: 302) shown in bold, double underlined text SAGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPFSEASMM GLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEILMIGLVWRSMEHPGKLLF APNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLK SLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKRMEHLYSMK CKNVVPLYDLLLEMLDAHRLHAPTSRGGASVEETDQSHLATAGSTSSHSLQKYYITGEAEGF PATV

[0476] SEQ ID NO: 305, ERT3 (314 aa), M543A, L544A, and V595A mutations from ER (e.g., SEQ ID NO: 302) shown in bold, double underlined text SAGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPFSEASMM GLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEILMIGLVWRSMEHPGKLLF APNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLK SLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMK 102 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT CKNVVPLYDLLLEAADAHRLHAPTSRGGASVEETDQSHLATAGSTSSHSLQKYYITGEAEGF PATA

[0477] SEQ ID NO: 306, ERT (314 aa), G400V, L539A, L540A mutations from ER (e.g., SEQ ID NO: 302) shown in bold, double underlined text SAGDMRAANLWPSPLMIKRSKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPFSEASMM GLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEILMIGLVWRSMEHPVKLLF APNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTFLSSTLK SLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNKGMEHLYSMK CKNVVPLYDAALEMLDAHRLHAPTSRGGASVEETDQSHLATAGSTSSHSLQKYYITGEAEGF PATV

[0478] In some embodiments of any of the aspects, the sequestering protein of the synTF polypeptide is in combination with 1, 2, 3, 4, 5, or more ligands. In some embodiments of any of the aspects, the sequestering protein of the synTF polypeptide is in combination with one ligand. In embodiments comprising multiple ligands, the multiple ligands can be different individual ligands or multiple copies of the same ligands, or a combination of the foregoing.

[0479] In some embodiments of any of the aspects, the ligand is estradiol (PubChem CID: 5757), or an analog thereof. In some embodiments of any of the aspects, 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, wherein binding of the ligand to the ERT (e.g., ERT2) exposes the NLS and results in nuclear translocation of the ERT. In some embodiments of any of the aspects, the ligand is 4-hydroxytamoxifen (4-OHT), shown below (PubChem CID: 449459), which can also be referred to as afimoxifene. In some embodiments of any of the aspects, the ligand is 4-Hydroxy-N-desmethyltamoxifen, shown below (PubChem CID: 10090750), which can also be referred to as endoxifen. In some embodiments of any of the aspects, the ligand is Fulvestrant shown below (PubChem CID 104741), which can also be referred to as ICI182,780. 103 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT

[0480] In some embodiments of any of the aspects, the sequestering protein of the synTF is a transmembrane receptor sequestering protein, and the DNA-binding domain (DBD) and transcriptional activator (TA) domain and transcriptional effector domain (TED) 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, TED, and TA of the synTF are sequestered to the cellular membrane. In the presence of a specific ligand for the transmembrane protein, the transmembrane protein cleaves itself such that the DBD, TED, and TA of the synTF are released into the cytosol to be transported to the nucleus. Non-limiting examples of transmembrane receptor sequestering protein include a synthetic notch receptor or first and second exogenous extracellular sensors, described further herein.

[0481] In some embodiments of any of the aspects, the cytosolic sequestering protein comprises a Notch receptor or a variant of endogenous Notch receptor, such as a synthetic Notch (synNotch) receptor. In some embodiments of any of the aspects, the synTF comprising a synNotch comprises: (a) an extracellular domain comprising a first member of a specific binding pair that is heterologous to the Notch receptor; (b) a Notch receptor regulatory region; and (c) an intracellular domain comprising the DNA binding domain, transcriptional activator (TA) domain, and transcriptional effector domain (TED) of the synTF. In the presence of a second member of the specific binding pair, 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 to activate the intracellular domain, thereby permitting the synTF to translocate to the nucleus. In the absence of a second member of the specific binding pair, the synTF remains sequestered at the cellular membrane. In some embodiments of any of the aspects, the sequestering protein comprises one of SEQ ID NOs: 307-308 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: 307-308, that maintains the same function. See e.g., US 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 entireties. In some embodiments of any of the aspects, the Notch receptor regulatory region comprises Lin-12 Notch repeats A-C, heterodimerization domains HD-N and HD-C, a binding-induced proteolytic cleavage site, and a transmembrane domain. In some embodiments of any of the aspects, the Notch variant is a Notch receptor where the Notch extracellular subunit (NEC) (which includes the negative regulatory region 104 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT (NRR)) is partially or completely removed. In some embodiments of any of the aspects, the Notch receptor regulatory region is a truncated or modified variant of synNotch, e.g., lacking one or more of the following domains: Lin-12 Notch repeats A-C, heterodimerization domains HD-N and HD-C, a binding-induced proteolytic cleavage site, the Notch extracellular domain (NEC), the negative regulatory region (NRR), or a transmembrane domain.

[0482] SEQ ID NO: 307, synNotch (306 aa) PPQIEEACELPECQVDAGNKVCNLQCNNHACGWDGGDCSLNFNDPWKNCTQSLQCWKYFS DGHCDSQCNSAGCLFDGFDCQLTEGQCNPLYDQYCKDHFSDGHCDQGCNSAECEWDGLDC AEHVPERLAAGTLVLVVLLPPDQLRNNSFHFLRELSHVLHTNVVFKRDAQGQQMIFPYYGHE EELRKHPIKRSTVGWATSSLLPGTSGGRQRRELDPMDIRGSIVYLEIDNRQCVQSSSQCFQSAT DVAAFLGALASLGSLNIPYKIEAVKSEPVEPPLPSQLHLMYVAAAAFVLLFFVGCGVLLS

[0483] SEQ ID NO: 308, synNotch (358 aa) PCVGSNPCYNQGTCEPTSENPFYRCLCPAKFNGLLCHILDYSFTGGAGRDIPPPQIEEACELPE CQVDAGNKVCNLQCNNHACGWDGGDCSLNFNDPWKNCTQSLQCWKYFSDGHCDSQCNSA GCLFDGFDCQLTEGQCNPLYDQYCKDHFSDGHCDQGCNSAECEWDGLDCAEHVPERLAAG TLVLVVLLPPDQLRNNSFHFLRELSHVLHTNVVFKRDAQGQQMIFPYYGHEEELRKHPIKRST VGWATSSLLPGTSGGRQRRELDPMDIRGSIVYLEIDNRQCVQSSSQCFQSATDVAAFLGALAS LGSLNIPYKIEAVKSEPVEPPLPSQLHLMYVAAAAFVLLFFVGCGVLLS

[0484] Suitable first members of a specific binding pairs (e.g., of the synNotch) include, but are not limited to, antibody-based recognition scaffolds; antibodies (i.e., an antibody-based recognition scaffold, including antigen-binding antibody fragments); non-antibody-based recognition scaffolds; antigens (e.g., endogenous antigens; exogenous antigens; etc.); a ligand for a receptor; a receptor; a target of a non-antibody-based recognition scaffold; an Fc receptor (e.g., FcγRIIIa; FcγRIIIb; etc.); an extracellular matrix component; and the like.

[0485] Specific binding pairs (e.g., of the synNotch) include, e.g., antigen-antibody specific binding pairs, where the first member is an antibody (or antibody-based recognition scaffold) that binds specifically to the second member, which is an antigen, or where the first member is an antigen and the second member is an antibody (or antibody-based recognition scaffold) that binds specifically to the antigen; ligand-receptor specific binding pairs, where the first member is a ligand and the second member is a receptor to which the ligand binds, or where the first member is a receptor, and the second member is a ligand that binds to the receptor; non-antibody-based recognition scaffold- target specific binding pairs, where the first member is a non-antibody-based recognition scaffold and the second member is a target that binds to the non-antibody-based recognition scaffold, or where 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 the second member, which is 105 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT an Fc receptor, or where the first member is an Fc receptor that binds to the second member which comprises an immunoglobulin Fc; and receptor-co-receptor binding pairs, where the first member is a receptor that binds specifically to the second member which is a co-receptor, or where the first member is a co-receptor that binds specifically to the second member which is a receptor.

[0486] In some embodiments of any of the aspects, the transmembrane receptor sequestering protein comprises first and second exogenous extracellular sensors, wherein said first exogenous extracellular sensor comprises: (a) a ligand binding domain, (b) a transmembrane domain, (c) a protease cleavage site, and (d) the DBD, TED, and TA of the synTF; and wherein said second exogenous extracellular sensor comprises: (e) a ligand binding domain, (f) a transmembrane domain, and (g) a protease domain. Such a system can 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 are brought into proximity, permitting the protease to cleave the protease cleavage site and release the DBD, TED, and TA of the synTF into the cytosol to be translocated to the nucleus. In the absence of a ligand for the first and second exogenous extracellular sensors, the DBD, TED, and TA of the synTF remains sequestered at the cell membrane. In some embodiments of any of the aspects, the protease comprises any protease as described herein (e.g., NS3), and the protease cleavage site comprises an NS3 protease cleavage site as described herein. See e.g., US Patent Application 2014 / 0234851; Daringer et al., ACS Synth. Biol.2014, 3, 12, 892-902.

[0487] Any type of suitable ligand binding domain (LB) can be employed with transmembrane receptor sequestering protein. Ligand binding domains can, for example, be derived from either an existing receptor ligand-binding domain or from an engineered ligand binding domain. Existing ligand-binding domains could come, for example, from cytokine receptors, chemokine receptors, innate immune receptors (TLRs, etc.), olfactory receptors, steroid and hormone receptors, growth factor receptors, mutant receptors that occur in cancer, neurotransmitter receptors. Engineered ligand- binding domains can be, for example, single-chain antibodies (see scFv constructs discussion below), engineered fibronectin based binding proteins, and engineered consensus-derived binding proteins (e.g., based upon leucine-rich repeats or ankyrin-rich repeats, such as DARPins). The ligand can be any cognate ligand of such ligand-binding domains. Linker Peptide

[0488] In several aspects, described herein are synTF polypeptides comprising at least one linker peptide. As used herein “linker peptide” (used interchangeably with “peptide linker”) refers to an oligo- or polypeptide region from about 2 to 100 amino acids in length, which links together any of the sequences of the polypeptides as described herein. In some embodiment, linkers can include or be composed of flexible residues such as glycine and serine so that the adjacent protein domains are free to move relative to one another. Longer linkers may be used when it is desirable to ensure that two 106 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT adjacent domains do not sterically interfere with one another. Linkers may be cleavable or non- cleavable.

[0489] In some embodiments of any of the aspects, a synTF polypeptide as described herein (or a synTF polypeptide system collectively) comprises 1, 2, 3, 4, 5, or more linker peptide(s). In some embodiments of any of the aspects, the 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 of the aspects, the linker peptide can be positioned anywhere, between any two domains as described herein: e.g., between the DBD and the RP; between the RP and TA; between the DBD and TA; between the TED and TA; between the TED and DBD; between the TED and RP; within the DBD, TA, TED, or regulator protein; or any combination thereof. In some embodiments of any of the aspects, the linker peptide can be positioned within the DBD, TA, TED, or RP, e.g., to link constituents of the domains.

[0490] In some embodiments of any one aspects described herein, the linkers connect several ZFs to each other in tandem to form a ZF array. In some embodiments of any one aspects described herein, the linker connects a first ZFA with a second ZFA. In some embodiments of any one aspects described herein, the linkers connect several ZFAs to each other to 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), wherein more than half of the amino acid residues are glycine. Preferably, such glycine-rich peptide linkers consist of about 20 or fewer amino acids. A linker molecule may also include non-peptide or partial peptide molecules. For instance, the 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 joined together in the respective fusion protein with a linker peptide.

[0491] Non-limiting examples of linker peptide include, but are not limited to: PGER (SEQ ID NO: 309), TGSQK (SEQ ID NO: 310), TGEKP (SEQ ID NO: 311), THLR (SEQ ID NO: 312), TGGGEKP (SEQ ID NO: 313), FHYDRNNIAVGADESVVKEAHREVINSSTEGLLLNIDKDIRKILSGYIVEIEDTE (SEQ ID NO: 314); VEIEDTE (SEQ ID NO: 315), KDIRKILSGYIVEIEDTE (SEQ ID NO: 316); STEGLLLNIDKDIRKILSGYIVEIEDTE (SEQ ID NO: 317), EVKQENRLLNESES (SEQ ID NO: 318); VGADESVVKEAHREVINSSTEGLLLNIDKDIRKILSGYIVEIEDTE (SEQ ID NO: 319); GGSGG (SEQ ID NO: 320); GGGSG (SEQ ID NO: 321); CVRGS (SEQ ID NO: 322), GGGGSG (SEQ ID NO: 323), GGSGSGSAC (SEQ ID NO: 324), LEGGGGSGG (SEQ ID NO: 325), GGGGSGGT (SEQ ID NO: 326), SGGGSGGSGSS (SEQ ID NO: 327); PGAGSSGDIM (SEQ ID 107 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT NO: 328) GSSGTGSGSGTS (SEQ ID NO: 329); SGTS (SEQ ID NO: 224); GSGS (SEQ ID NO: 225), GGSGGS (SEQ ID NO: 330), and GSSGSS (SEQ ID NO: 285).

[0492] As non-limiting examples, TGSQK (SEQ ID NO: 310) or TGEKP (SEQ ID NO: 311) or TGGGEKP (SEQ ID NO: 313) can be used as linker between ZFAs; VEIEDTE (SEQ ID NO: 315), GGSGGS (SEQ ID NO: 330), GGSGG (SEQ ID NO: 320), GGGSG (SEQ ID NO: 70), CVRGS (SEQ ID NO: 322), GGGGSG (SEQ ID NO: 323), GGSGSGSAC (SEQ ID NO: 324), LEGGGGSGG (SEQ ID NO: 325), GGGGSGGT (SEQ ID NO: 326), or SGGGSGGSGSS (SEQ ID NO: 327) can be used to link ZF domains and effector domains together; PGAGSSGDIM (SEQ ID NO: 328) GSSGTGSGSGTS (SEQ ID NO: 329); SGTS (SEQ ID NO: 224); GSGS (SEQ ID NO: 225), or GSSGSS (SEQ ID NO: 285) can be used to link regions of a SMASh domain, a StaPL domain, or an NS3 / NS4a domain, described further herein.

[0493] 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 comprises at least one amino acid that is Gly or Ser. In one embodiment of a fusion protein described herein that includes a linker, the linker is a flexible polypeptide between 1 and 25 residues in length. Common examples of flexible peptide linkers include (GGS)n, where n = 1 to 8 (SEQ ID NO: 331, GGSGGSGGSGGSGGSGGSGGSGGS), or (Gly4Ser)n repeat where n=1-8 (SEQ ID NO: 332, GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS), preferably, n=3, 4, 5, or 6, that is (Gly–Gly–Gly–Gly–Ser)n (SEQ ID NO: 333), GGGGS, where n indicates the number of repeats of the motif. For example, the flexible linker is (GGS)2(SEQ ID NO: 334, GGSGGS). Alternatively, flexible peptide linkers include Gly-Ser repeats (Gly-Ser)pwhere p indicates the number of Gly-Ser repeats of the motif, p=1-8 (SEQ ID NO: 335 GSGSGSGSGSGSGSGS), preferably, n=3, 4, 5, or 6. Another example of a flexible linker is TGSQK (SEQ ID NO: 310).

[0494] In one embodiment of the engineered synTFs described herein, wherein the ZF protein domains and effector domains are joined together with a linker peptide, the linker peptide is about 1- 20 amino acids long. In one embodiment, the linker peptide does not comprise Lys, or does not comprise Arg, or does not comprise both Lys and Arg.

[0495] In some embodiments of the engineered synTFs described herein, the ZF protein domains and effector domains are joined together chemical cross-linking agents. Bifunctional cross-linking molecules are linker molecules that possess two distinct reactive sites. For example, one of the reactive sites of a bifunctional linker molecule may be reacted with a functional group on a peptide to form a covalent linkage and the other reactive site may be reacted with a functional group on another molecule to form a covalent linkage. General methods for cross-linking molecules have been reviewed (see, e.g., Means and Feeney, Bioconjugate Chem., 1: 2-12 (1990)). 108 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT

[0496] Homobifunctional cross-linker molecules have two reactive sites which are chemically the same. Non-limiting examples of homobifunctional cross-linker molecules include, without limitation, glutaraldehyde; N,N'-bis(3-maleimido-propionyl)-2-hydroxy-l,3-propanediol (a sulfhydryl- specific homobifunctional cross-linker); certain N-succinimide esters (e.g., disuccinimidyl suberate, dithiobis(succinimidyl propionate), and soluble bis-sulfonic acid and salt thereof (see, e.g., Pierce Chemicals, Rockford, Illinois; SIGMA-ALDRICH CORP., St. Louis, Missouri).

[0497] A bifunctional cross-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. Use of such heterobifunctional linkers permits chemically separate and stepwise addition (vectorial conjunction) of each of the reactive sites to a selected peptide sequence. Heterobifunctional linker molecules useful in the disclosure include, without limitation, m- maleimidobenzoyl-N-hydroxysuccinimide ester (see, Green et al., Cell, 28: 477-487 (1982); Palker et al., Proc. Natl. Acad. Sci (USA), 84: 2479-2483 (1987)); m-maleimido-benzoylsulfosuccinimide 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).

[0498] In some embodiments of any aspect described herein, in the synTF described or the ZF- containing fusion protein described herein, all the helices within a ZFA are linked by peptide linkers (L2) having four to six amino acid residues.

[0499] In some embodiments of any aspect described herein, in the synTF described or the ZF- containing fusion protein described herein, all the helices within an individual ZFA are linked by rigid peptide linkers such as TGEKP (SEQ ID NO: 311) or TGSKP (SEQ ID NO: 336) or TGQKP (SEQ ID NO: 337) or TGGKP (SEQ ID NO: 338). The rigid linker aids in conferring synergistic binding of the ZF motifs to its target DNA sequence.

[0500] In one embodiment of any aspect described herein, in the synTF described or the ZF containing fusion protein described herein, the (L1) or (L2) is a flexible linker. Non-limiting examples include: TGSQKP (SEQ ID NO: 339) and TGGGEKP (SEQ ID NO: 313). In one embodiment, the linker flexible peptide is 1-20 amino acids long. The flexible linker aid in weakening cooperativity between adjacent ZF motifs.

[0501] In one embodiment of any aspect described herein, in the synTF described or the ZF containing fusion protein described herein, the (L1) or (L2) is a rigid linker. Non-limiting examples include: TGEKP (SEQ ID NO: 311), TGSKP (SEQ ID NO: 336), TGQKP (SEQ ID NO: 337) and TGGKP (SEQ ID NO: 338).

[0502] In some embodiments of any aspect described herein, in the synTF described or the ZF containing fusion protein described herein, where there are two or more ZFAs, the individual ZFAs 109 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT are linked by flexible peptide linkers, such as TGSQKP (SEQ ID NO: 339). In another embodiment, the ZFAs are linked by chemical crosslinkers. Chemical crosslinkers are known in the art.

[0503] In some embodiments of any aspect described herein, in the synTF described or the ZF containing fusion protein described herein, all the helices within an individual ZFA are linked by a combination of rigid peptide linkers and flexible peptide linkers. In some embodiments of any of the aspects, the rigid peptide linkers and flexible peptide linkers are used alternatingly to connect the fingers. Self-Cleaving Peptide

[0504] In several aspects, described herein are synTF polypeptides comprising a self-cleaving peptide. As used herein, the term “self-cleaving peptide” refers to a short amino acid sequence (e.g., approximately 18–22 aa-long peptides) that can catalyze its own cleavage. In some embodiments of any of the aspects, a multi-component synTF system as described herein (e.g., induced proximity synTF system) comprises at least two polypeptides that are physically linked to one another through a self-cleaving peptide domain. The self-cleaving peptide allows the nucleic acids of the first polypeptide and second polypeptide (and / or third polypeptide, etc.) to be present in the same vector, but after translation the self-cleaving peptide cleaves the translated polypeptide into the multiple separate polypeptides.

[0505] In some embodiments of any of the aspects, a synTF polypeptide as described herein (or a synTF polypeptide system collectively) comprises 1, 2, 3, 4, 5, or more self-cleaving peptides, e.g., in between each synTF polypeptide. In some embodiments of any of the aspects, the synTF polypeptide or system comprises one self-cleaving peptide, e.g., in 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.

[0506] In some embodiments, self-cleaving peptides are used, for example, in heterodimerization domain synTFs. As a non-limiting example, a 2A self-cleaving peptide can be in between a first polypeptide region comprising [ABI]-[ZF] and a second polypeptide region comprising [TA]-[TED]- [PYL]. Following translation of the polypeptide, the 2A sequence, which is a self-cleaving peptide, cleaves the polypeptide into two polypeptides: [ABI]-[ZF] and [TA]-[TED]-[PYL], which in the presence of ABA can form a [TA]-[TED]-[PYL]⋅ABA⋅[ABI]-[ZF] complex, thus coupling the DBD (ZF), TA (e.g., p65), and TED (e.g., IWS1 TIMs domain).

[0507] In some embodiments of any of the aspects, the self-cleaving peptide belongs to the 2A peptide family, which can also be referred to as a 2A Ribosomal Skip Sequence. Non-limiting examples of 2A peptides include P2A, E2A, F2A and T2A (see e.g., Table 13). F2A is derived from foot-and-mouth disease virus 18; E2A is derived from equine rhinitis A virus; P2A is derived from 110 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT porcine teschovirus-12A; T2A is derived from thosea asigna virus 2A. In some embodiments of any of the aspects, the N-terminal of the 2A peptide comprises the sequence “GSG” (Gly-Ser-Gly). In some embodiments of any of the aspects, the N-terminal of the 2A peptide does not comprise the sequence “GSG” (Gly-Ser-Gly).

[0508] Table 13: Exemplary Self-Cleaving Peptides

[0509] The 2A-peptide-mediated cleavage commences after protein translation. The cleavage is triggered by breaking of peptide bond between the Proline (P) and Glycine (G) in the C-terminal 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 efficiencies of self-cleaving, with P2A being the most efficient and F2A the least efficient. Therefore, up to 50% of F2A-linked proteins can remain in the cell as a fusion protein.

[0510] In some embodiments of any of the aspects, the self-cleaving peptide of a synTF polypeptide system as described herein comprises SEQ ID NOs: 340-343, 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: 340-343, that maintains the same function (e.g., self-cleavage).

[0511] In some embodiments of any of the aspects, providing the multiple polypeptides of the synTF systems as described herein in a 1:1 (or 1:1:1, etc.) stoichiometric ratio is advantageous (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, then the first and second polypeptides can be provided in a single vector, flanking a self-cleaving peptide(s) 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) then the first and second polypeptides can be provided in multiple separate vectors, e.g., at the desired stoichiometric ratios. Detectable Marker

[0512] In several aspects, described herein are synTF polypeptides comprising at least one detectable marker. As used herein, the term “detectable marker” refers to a moiety that, when attached to the synTF polypeptide, confers detectability upon that polypeptide or another molecule to which the polypeptide binds. In some embodiments of any of the aspects, the synTF polypeptide (or the 111 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT synTF polypeptide system collectively) comprises 1, 2, 3, 4, 5, or more detectable markers. In some embodiments of any of the aspects, the synTF polypeptide or system comprises one detectable marker. In embodiments comprising multiple detectable markers, the multiple detectable markers can be different individual detectable markers or multiple copies of the same detectable markers, or a combination of the foregoing.

[0513] In some embodiments of any of the aspects, fluorescent moieties can be used as detectable markers, but detectable markers also include, for example, isotopes, fluorescent proteins and peptides, enzymes, components of a specific binding pair, 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 in order to generate detectable signal. Examples include isotopes and fluorophores. Indirectly detectable markers require the presence or action of one or more co-factors or substrates. Examples include enzymes such as β-galactosidase which is detectable by generation of colored reaction products upon cleavage of substrates such as the chromogen X-gal (5-bromo-4-chloro-3- indoyl-β-D-galactopyranoside), horseradish peroxidase which is detectable by generation of a colored reaction product in the presence of the substrate diaminobenzidine and alkaline phosphatase which is detectable by generation of colored reaction product in the presence of nitroblue tetrazolium and 5- bromo-4-chloro-3-indolyl phosphate, and affinity tags. Non-limiting examples of affinity tags include Strep-tags, chitin binding proteins (CBP), maltose binding proteins (MBP), glutathione-S-transferase (GST), FLAG-tags, HA-tags, Myc- tags, poly(His)-tags as well as derivatives thereof. In some embodiments of any of the aspects, the detectable marker is selected from GFP, V5, HA1, Myc, VSV- G, HSV, FLAG, HIS, mCherry, AU1, and biotin.

[0514] SEQ ID NO: 344, NLS, PKKKRKV

[0515] SEQ ID NO: 345, 3X FLAG Tag + Nuclear Localization Sequence (in bold text) DYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPGG

[0516] SEQ ID NO: 346, AU1 tag, DTYRYI

[0517] SEQ ID NO: 347, HA tag, YPYDVPDYA

[0518] SEQ ID NO: 348, FLAG Tag, DYKDDDDK

[0519] SEQ ID NO: 349, mCherry: VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFA WDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFI YKVKLRGTNFPSDGPVMQKKTMGWQASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVK TTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYK

[0520] SEQ ID NO: 350, huEGFRt (a truncated human EGFR polypeptide) RKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTV KEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIIS 112 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT GNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSC RNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGP HCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATG MVGALLLLLVVALGIGLFM

[0521] In some embodiments of any of the aspects, the detectable marker of a synTF polypeptide as described herein comprises SEQ ID NOs: 40-41 or SEQ ID NOs: 344-350, 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: 40-41 or SEQ ID NOs: 344 to 350, that maintains the same function (e.g., detection of the synTF polypeptide or cleaved fragments of the synTF polypeptide).

[0522] In some embodiments of any of the aspects, the detectable marker can be located anywhere within a synTF polypeptide as described herein. In one embodiment, the detectable marker is located between any domain of a synTF polypeptide as described herein, but is not found within a functional domain or does not disrupt the function of a domain. In some embodiments of any of the aspects, the detectable marker is located adjacent to and C terminal of the extracellular binding domain. Such a marker can be used to detect the expression of the synTF polypeptide, including cytosolic expression or nuclear translocation.

[0523] In some embodiments of any of the aspects, the detectable marker is located between the repressible protease and a protease cleavage site; such a marker can be used to detect the cleavage and / or expression of the synTF polypeptide. In some embodiments of any of the aspects, the detectable marker that is located between the repressible protease and a protease cleavage site comprises the AU1 tag, the HA1 tag, or any other marker as described herein.

[0524] In some embodiments of any of the aspects, the detectable marker is located adjacent and N-terminal to the repressible protease. In some embodiments of any of the aspects, the detectable marker is located adjacent and N-terminal to the repressible protease and C-terminal to a first protease cleavage site. In some embodiments of any of the aspects, the detectable marker is located adjacent to and C terminal to the repressible protease. In some embodiments of any of the aspects, the detectable marker is located adjacent to and C terminal to the repressible protease and N-terminal to a second protease cleavage site.

[0525] In some embodiments of any of the aspects, the detectable marker is located at the C- terminal end of the polypeptide. Such a marker can be used to detect the intracellular expression of the synTF polypeptide. In some embodiments of any of the aspects, the detectable marker located at the C-terminal end of the polypeptide comprises mCherry or another marker as described herein.

[0526] In some embodiments of any of the aspects, synTF polypeptides as described herein, especially those that are administered to a subject or those that are part of a pharmaceutical 113 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT composition, do not comprise detectable markers that are immunogenic. In some embodiments of any of the aspects, synTF polypeptides as described herein do not comprise GFP, mCherry, HA1, or any other immunogenic markers. II. Synthetic Transcription Factors

[0527] In multiple aspects described herein are synthetic transcription factors comprising: (a) at least one DNA binding domain (DBD), (b) a transcriptional activator (TA) domain; (c) a transcriptional effector domain (TED), and (d) at least one regulator protein (RP). In some embodiments of any of the aspects, the TA is directly coupled or linked to the DBD; as used herein the terms “directly coupled” or “directly linked” indicate that there are no other domain (other than a linker) found between the TA and DBD in the synTF or other construct. In some embodiments of any of the aspects, the TA is indirectly coupled or linked to the DBD; as used herein the terms “indirectly coupled” or “indirectly linked” indicate that at least one other domain is found between the TA and DBD in the synTF or other construct. In some embodiments of any of the aspects, the coupling of the TA to the DBD is regulated by the at least one RP. In some embodiments of any of the aspects, the cellular localization of the TA is regulated by the at least one regulator protein. In some embodiments of any of the aspects, at least one regulator protein is selected from the group consisting of repressible protease, induced degradation domain, induced proximity domain, and cytosolic sequestering domain.

[0528] In some embodiments of any of the aspects, the domains of the synTF can be in order, e.g., 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; where “ED” designates the combination of the TA and TED (e.g., TA- TED or TED-TA),. See also Tables 3-4. In embodiments comprising two regulator proteins (i.e., RP1 and RP2), the domains of the synTF can be in order, e.g., 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-ED-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-ED; RP1-DBD-RP2-ED; ED-DBD-RP2-RP1; DBD-ED- RP2-RP1; RP2-ED-DBD-RP1; ED-RP2-DBD-RP1; DBD-RP2-ED-RP1; and RP2-DBD-ED-RP1, where “ED” designates the combination of the TA and TED (e.g., TA-TED or TED-TA).

[0529] In some embodiments and by way of an example only, an exemplary RP1 is selected from an induced proximity domain (IPD), or cytosolic sequestering domain (CS), and the RP2 is selected from the induced degradation domain (comprising a SMASh domain), as disclosed herein. In another embodiment, an exemplary RP1 is an induced degradation domain (comprising a SMASh domain) and the PR2 is selected from an induced proximity domain (IPD) or a cytosolic sequestering domain (IPD). A. Repressible Protease SynTF 114 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT

[0530] In some embodiments of any of the aspects, the regulator protein is a repressible protease. Accordingly, in one aspect described herein is a synTF comprising: (a) a DBD; (b) a TA; (c) a TED; and (d) a repressible protease domain (referred to herein as a PRO or RPD). In some embodiments of any of the aspects, the domains of the synTF can be in order, e.g., 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, where “ED” designates the combination of the TA and TED (e.g., TA-TED or TED-TA) (see e.g., Table 3).

[0531] In some embodiments of any of the aspects, the repressible protease synTF further comprises at least one protease cleavage site (PC), as described further herein. In a preferred embodiment, the at least one protease cleavage site is located in between the DBD and TA, such that when the protease cleaves at the protease cleavage site, the DBD and TA are uncoupled. Accordingly, in some embodiments, the repressible 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, where “ED” designates the combination of the TA and TED (e.g., TA-TED or TED-TA).

[0532] In some embodiments of any of the aspects, the repressible protease synTF comprises two protease cleavage sites (PC). In some embodiments of any of the aspects, the two protease cleavage s...

Claims

Attorney Docket No: 701586-000114WOPT CLAIMS What is claimed herein is:

1. A synthetic transcription factor (synTF) comprising: (a) at least one DNA binding domain (DBD), (b) a transcriptional activator domain (TA), (c) a transcriptional effector domain (TED), and (d) at least one regulator protein (RP), and wherein the TED increases the transcriptional activation of the synTF compared to an otherwise identical synTF lacking the TED, wherein the TA is directly or indirectly coupled to the DBD, and wherein the coupling is regulated by the at least one RP, or wherein cellular localization of the TA is regulated by the at least one RP.

2. The synTF of claim 1, wherein the TED comprises an elongation domain, an activator domain, or a domain with pioneer ability.

3. The synTF of claim 2, wherein the elongation domain is derived from a polypeptide selected from the group consisting of: Interacts with Suppressor Of Ty 6 (Spt6) Homolog (IWS1); Suppressor Of Ty 5 (Spt5) Homolog (SUPT5H); Bromodomain-containing protein 4 (BRD4); and cellular Myelocytomatosis (cMyc).

4. The synTF of claim 2, wherein the elongation domain is derived from IWS1.

5. The synTF of claim 2, wherein the elongation domain is derived from human IWS1.

6. The synTF of claim 2, wherein the elongation domain comprises at least one TFIIS N- terminal domains (TND)-interacting motif (TIM) domain of IWS1.

7. The synTF of claim 2, wherein the elongation domain comprises at least one TIM1, TIM2, and / or TIM3 domain from IWS1.

8. The synTF of claim 2, wherein the elongation domain comprises one of SEQ ID NOs: 5-9 or an amino acid sequence with at least 80% sequence identity to one of SEQ ID NOs: 5-9.

9. The synTF of claim 2, wherein the activator domain is derived from a polypeptide selected from the group consisting of: Heat Shock Factor 1 (HSF1), Glucocorticoid Receptor (GR), and MLX interacting protein like (MLXIPL).

10. The synTF of claim 2, wherein the domain with pioneer ability is derived from a polypeptide selected from the group consisting of Fused in Sarcoma (FUS) and Ewing Sarcoma Breakpoint Region (EWSR).

11. The synTF of any one of claims 1-10, wherein the TA is selected from the group consisting of: p65; Rta; miniVPR; full VPR; VP16; VP64; NFZ; 3Z; p300; p300 HAT Core; and a CBP HAT domain; or a variant thereof. 237 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT 12. The synTF of any one of claims 1-11, wherein the TA is p65, or a variant thereof.

13. The synTF of claim 12, wherein the p65 comprises one of SEQ ID NOs: 38, 58-67 or an amino acid sequence with at least 80% sequence identity to one of SEQ ID NOs: 38, 58-67.

14. The synTF of any one of claims 1-13, wherein the at least one DBD is an engineered zinc finger (ZF)-binding domain.

15. The synTF of claim 14, wherein the engineered ZF-binding domain comprises 2 or more ZF motifs.

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

17. The synTF of any one of claims 14-16, wherein the engineered ZF binding domain comprises a sequence 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 11-1.

18. The synTF of any one of claims 14-17, wherein the engineered ZF-binding domain comprises one of SEQ ID NO: 36 (ZF10-1), SEQ ID NO: 45 (ZF3-5), or SEQ ID NO: 86 (ZF1-3).

19. The synTF of any one of claims 14-18, wherein the engineered ZF-binding domain comprises ZF 10-1.

20. The synTF of any one of claims 14-19, wherein the engineered ZF-binding domain specifically binds to a nucleic acid with a sequence comprising at least one of SEQ ID NO: 100 (ZF10 binding site (BS)), SEQ ID NO: 93 (ZF3 BS), or SEQ ID NO: 91 (ZF1 BS).

21. The synTF of any one of claims 14-20, wherein the engineered ZF-binding domain comprises SEQ ID NO: 48 and specifically binds endogenous VEGF gene (VEGF ZF).

22. The synTF of any one of claims 1-21, wherein the at least one RP comprises a polypeptide selected from the group consisting of: a repressible protease; a pair of induced proximity domains (IPD pair); a cytosolic sequestering protein; and combinations thereof.

23. The synTF of any one of claims 1-22, wherein the at least one RP comprises a repressible protease.

24. The synTF of any one of claims 1-23, wherein the at least one RP comprises a NS3 protease protein. 238 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT 25. The synTF of any one of claims 1-24, wherein the at least one RP comprises the amino acid sequence of SEQ ID NOs: 182-198, or a homologue with at least 80% sequence identity to one of SEQ ID NOs: 182-198.

26. The synTF of any one of claims 24-25, wherein in the presence of a protease inhibitor, or an inhibitor of NS3, the protease protein is inhibited, thereby maintaining the coupling of the DBD to the TA.

27. The synTF of any one of claims 24-26, wherein in the absence of a protease inhibitor, or an inhibitor of NS3, the protease protein is active, and the TA is excised from the DBD, thereby uncoupling the DBD and the TA.

28. The synTF of claim 26 or 27, wherein the inhibitor of NS3 is selected from the group consisting of: grazoprevir (GRZ / GZV), danoprevir, simeprevir, asunaprevir, ciluprevir, boceprevir, sovaprevir, paritaprevir, ombitasvir, paritaprevir, ritonavir, dasabuvir, and telaprevir.

29. The synTF of any one of claims 1-28, wherein the at least one RP is a pair of induced proximity domains (IPD pair), wherein the IPD pair comprises: a first induced proximity domain (IPDA) and at least a second complementary IPD (IPDB), wherein in the presence of an inducer agent or signal, the IPDAand IPDBspecifically bind together resulting in the coupling of the TA to the DBD, and wherein in the absence of an inducer agent or signal, the TA is uncoupled from the DBD.

30. The synTF of claim 29, wherein the IPD pair comprises: (a) a IPDAcomprising a GID1 domain or a fragment thereof, and a IPDBcomprising a GAI domain, wherein the GID1 domain and GAI domain bind to the inducer agent Gibberellin Ester (GIB); (b) a IPDAcomprising a FKBP domain or a fragment thereof, and a IPDBcomprising a FRB domain, wherein the FKBP domain and FRB domain bind to the inducer agent Rapalog (RAP); (c) a IPDAcomprising a PYL domain or a fragment thereof, and a IPDBcomprising an ABI domain, wherein the PYL domain and ABI domain bind to the inducer agent Abscisic acid (ABA); and / or (d) a IPDAcomprising a Light-inducible dimerization domain (LIDD), wherein a LIDD dimerizes with a complementary LIDD (IPDB) upon exposure to a light inducer signal of an appropriate wavelength.

31. The synTF of claim 30, wherein the LIDD is nMag, Calcium And Integrin-Binding Protein 1 truncation (CIBN), or a photochromic protein domain; wherein nMag can dimerize with a 239 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT complementary LIDD pMag upon exposure to a blue light inducer signal; or wherein CIBN can dimerize with a complementary cryptochrome 2 (CRY2) upon exposure to a blue inducer light signal; or wherein the photochromic protein domains can dimerize upon exposure to a blue inducer light signal.

32. The synTF of claim 30 or 31, wherein the light inducer signal is a pulse light signal.

33. The synTF of any one of claims 1-32, wherein the at least one RP comprises a cytosolic sequestering protein.

34. The synTF of claim 33, wherein the cytosolic sequestering protein comprises a ligand binding domain (LBD), wherein in the presence of a ligand to which the LBD binds, sequestering of the synTF to the cytosol is inhibited.

35. The synTF of claim 33 or 34, wherein the cytosolic sequestering protein comprises a LBD and a nuclear localization signal (NLS); wherein in the absence of a ligand to which the LBD binds, the NLS is inhibited thereby preventing translocation of the synTF to the nucleus; and wherein in the presence of the ligand, the NLS is exposed permitting translocation of the synTF to the nucleus.

36. The synTF of any one of claims 33-35, wherein the cytosolic sequestering protein comprises at least a portion of an estrogen receptor (ER).

37. The synTF of any one of claims 33-36, wherein the cytosolic sequestering protein comprises an estrogen ligand binding domain (ERT) or a variant thereof, selected from the group consisting of: SEQ ID NO: 43 (ERT2), SEQ ID NO: 304, and SEQ ID NO: 305 (ERT3).

38. The synTF of claim 37, wherein the ERT binds to one or more ligands selected from the group consisting of: tamoxifen, 4-hydroxytamoxifen (4OHT), endoxifen, and Fulvestrant; wherein binding of the ligand to the ERT exposes the NLS and results in nuclear translocation of the ERT.

39. The synTF of any one of claims 33-38, wherein the cytosolic sequestering protein comprises a transmembrane receptor sequestering protein.

40. The synTF of any one of claims 24-39, wherein the NS3 protease protein is part of a Small molecule-Assisted Shutoff (SMASh) domain, wherein the SMASh domain comprises the NS3 protease protein, a partial protease helical domain and a NS4A domain.

41. The synTF of any one of claims 1-40, further comprising a Small molecule-Assisted Shutoff (SMASh) domain, wherein the SMASh domain is a N-terminal or C-terminal SMASh domain comprising a repressible protease, a partial protease helical domain and a cofactor domain.

42. The synTF of claim 41, wherein the SMASh domain is a C-terminal SMASh domain comprising, in N-terminal to C-terminal order: a NS3 cleavage site, at least one linker, a NS3 domain, a NS3 partial helicase, and a NS4A domain, wherein the SMASh domain is fused to the C-terminus of the synTF. 240 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT 43. The synTF of claim 41, wherein the SMASh domain is a N-terminal SMASh domain comprising in N-terminal to C-terminal order: at least one Linker, a NS3 domain, a NS3 partial helicase, a NS4 domain, and a NS3 cleavage site, wherein the SMASh domain is fused to the N-terminus of the synTF.

44. The synTF of any one of claims 40-43, wherein in the absence of an inhibitor for the NS3 protease, the NS3 protease is active and self cleaves / uncouples from the synTF, thereby resulting in the SMASh domain targeted for degradation (“SMASh-degradation”, synTF- on / TA-on), and wherein in the presence of an inhibitor for the NS3 protease, NS3 protease activity is inhibited thereby resulting in the SMASh-comprising synTF targeted for degradation (“synTF-degradation”, synTF-OFF / TA-off”).

45. The synTF of claim 44, wherein the inhibitor for the NS3 protease is selected from the group consisting of: grazoprevir (GRZ / GZV), danoprevir, simeprevir, asunaprevir, ciluprevir, boceprevir, sovaprevir, paritaprevir, ombitasvir, paritaprevir, ritonavir, dasabuvir, and telaprevir.

46. The synTF of any one of claims 1-45, wherein the synTF comprises a SMASh domain and a cytosolic sequestering protein.

47. The synTF of claim 46, wherein the synTF is active in the presence of the ligand for the cytosolic sequestering protein and in the absence of the inhibitor for the NS3 protease; and wherein the synTF is inactive in the absence of the ligand for the cytosolic sequestering protein and / or in the presence of the inhibitor for the NS3 protease.

48. The synTF of any of claims 1-47, further comprising a linker peptide, wherein the linker peptide can be positioned anywhere from: between the DBD and the RP; between the RP and TA; between the DBD and TA; between the TED and TA; between the TED and DBD; between the TED and RP; within the DBD, TA, TED, or regulator protein; or any combination thereof.

49. The synTF of any one of claims 1-48, wherein the DBD, TA, TED, and / or RP are human domains or humanized domains.

50. The synTF of any one of claims 1-49, wherein the DBD, TA, TED, and RP are human domains or humanized domains.

51. A synthetic transcription factor (synTF) comprising: (a) at least one DNA binding domain (DBD), (b) a transcriptional activator domain (TA), and (c) a transcriptional effector domain (TED), wherein the TED increases the transcriptional activation of the synTF compared to an otherwise identical synTF lacking the TED. 241 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT 52. A hybrid transcription activator domain (hTAD), comprising: (a) a transcriptional activator domain (TA), and (b) a transcriptional effector domain (TED), wherein the TED increases the transcriptional activation of the TA compared to an otherwise identical hTAD lacking the TED.

53. The hTAD of claim 52, wherein the TA and / or TED are human domains or humanized domains.

54. A synthetic transcription factor (synTF) comprising the hTAD of claim 52 or 53 and at least one DNA binding domain (DBD).

55. The synTF of claim 54, further comprising at least one regulator protein (RP).

56. A humanized hybrid transcription activator domain (hhTAD), comprising: (a) a transcriptional activator domain (TA), and (b) a transcriptional effector domain (TED), wherein the TED increases the transcriptional activation of the TA compared to an otherwise identical hhTAD lacking the TED; and wherein the TA and TED are human domains or humanized domains.

57. A synthetic transcription factor (synTF) comprising the hhTAD of claim 56 and at least one DNA binding domain (DBD).

58. The synTF of claim 57, further comprising at least one regulator protein (RP).

59. A system for controlling gene expression, comprising: (a) at least one synthetic transcription factor (synTF) of any one of claims 1-51, 54-55, 57 and 58, wherein the at least one DBD of the synTF can bind to a target DNA binding motif (DBM) located upstream of a promoter operatively linked to a gene; and (b) a nucleic acid construct comprising: (i) at least one target DNA binding motif (DBM) comprising a target nucleic acid for binding of the at least one DBD of the synTF; (ii) a promoter sequence located 3’ of the at least one DBM; and (iii) a gene of interest operatively linked to the promoter sequence.

60. The system of claim 59, wherein the synTF comprises at least one RP regulated by an RP inducer.

61. The system of claim 59 or 60, wherein the coupling of the TA to the DBD is regulated by the at least one RP.

62. The system of claim 61, wherein the RP comprises a repressible protease or an IPD pair.

63. The system of any one of claims 61-62, wherein in the presence of the RP inducer, the coupling of the TA to the DBD of the synTF is maintained, permitting the TA to be in 242 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT proximity to the promoter sequence when the DBD binds to the DBM, and wherein the TA turns on expression of the gene of interest (“TA-on”).

64. The system of claim 61-63, wherein in the absence of the RP inducer, the coupling of the TA to the DBD of the synTF is severed, preventing the TA from being in proximity to the promoter sequence when the DBD binds to the DBM, preventing expression of the gene of interest (“TA-off”).

65. The system of any one of claims 59-64, wherein cellular localization of the TA linked to the DBD is regulated by the at least one RP.

66. The system of claim 65, wherein the RP comprises a cytosolic sequestering protein.

67. The system of any one of claims 65-66, wherein in the presence of the RP inducer, the TA coupled to the DBD of the synTF is not sequestered in the cytosol, permitting the DBD to bind to the DNA binding motif (DBM) and permitting the TA domain to be in proximity to the promoter sequence to thereby turn on expression of the gene of interest (“TA-on”).

68. The system of any one of claims 65-67, wherein in the absence of the RP inducer, the TA coupled to the DBD of the synTF is sequestered in the cytosol, preventing the DBD from binding to the DBM, and preventing the TA domain from being in proximity to the promoter sequence, thereby preventing expression of the gene of interest (“TA-off”).

69. The system of any one of claims 59-68, wherein the at least one synTF further comprises a N- terminal or C-terminal Small molecule-Assisted Shutoff (SMASh) domain, wherein SMASh domain comprises a self-cleaving SMASh protease, a partial protease helical domain and a cofactor domain.

70. The system of claim 69, wherein in the presence of an inhibitor to the SMASh protease, the SMASh protease activity is inhibited, resulting in the synTF being degraded and preventing the DBD of the synTF binding to the DBM and controlling the expression of the gene of interest (“synTF-degradation”; TA-off (no expression)).

71. The system of claim 69 or 70, wherein in the absence of an inhibitor to the SMASh protease, the SMASh protease is active and self cleaves / uncouples from the synTF, resulting the SMASh domain being targeted for degradation and allowing the DBD of the synTF to bind to the DBM and the TA of synTF to control the expression of the gene of interest (“SMASh- degradation, TA-on (yes-expression)).

72. The system of any of claims 59-71, wherein the promoter is selected from the group consisting of: miniCMV promoter, miniTK 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.

73. A system comprising: 243 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT (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) operatively linked to the promoter sequence; and (b) a second nucleic acid sequence comprising a nucleic acid encoding a synthetic transcription factor (synTF) according to any one of claims 1-51, 54-55, 57 and 58, operatively linked to an inducible or constitutive promoter.

74. The system of claim 73, wherein the promoter sequence operatively linked to the GOI is selected from the group consisting of: miniCMV promoter, miniTK promoter, ybTATA promoter, minSV40 promoter, CMV53 promoter, pJB42CAT5 promoter, MLP promoter, and TATA promoter.

75. The system of claim 73 or 74, wherein the promoter sequence operatively linked to the nucleic acid encoding the synTF is selected from the group consisting of pSFFV promoter, CMV promoter, pUb / UbC promoter, EF1a promoter, PGK / pGK promoter, CAG / CAGG promoter, SV40 promoter, and beta actin / ACTB promoter.

76. A polynucleotide encoding the synTF of any one of claims 1-51, 54-55, 57, and 58; the hTAD of claim 52 or 53; the hhTAD of claim 56; or the system of any one of claims 59-75; or portion thereof.

77. A nucleic acid construct, comprising in the 5’ to 3’ direction: (a) a nucleic acid sequence encoding a gene of interest (GOI) in the inverse orientation, (b) a first promoter sequence in the inverse orientation and operatively 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 synthetic transcription factor (synTF), wherein binding of the DBD places a TA of the synTF in the proximity of the promoter sequence operatively linked to the GOI, (d) a second promoter sequence, and (e) a nucleic acid sequence encoding the synTF, operatively linked to the second promoter sequence, wherein the encoded synTF comprises at least one DBD that binds to the at least one DBM of the nucleic acid sequence of (c).

78. The nucleic acid construct of claim 77, wherein the promoter sequence operatively linked to the GOI is selected from the group consisting of: miniCMV promoter, miniTK promoter, ybTATA promoter, minSV40 promoter, CMV53 promoter, pJB42CAT5 promoter, MLP promoter, and TATA promoter. 244 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT 79. The nucleic acid construct of claim 77 or 78, wherein the promoter sequence operatively linked to the nucleic acid encoding the synTF is selected from the group consisting of a pSFFV promoter, CMV promoter, pUb / UbC promoter, EF1a promoter, PGK / pGK promoter, CAG / CAGG promoter, SV40 promoter, and beta actin / ACTB promoter.

80. A vector comprising the system of any one of claims 59-75; the polynucleotide of claim 76; or the nucleic acid construct of any one of claims 77-79; or portion thereof.

81. The vector of claim 80, wherein the vector is a lentiviral vector.

82. A cell comprising the synTF of any one of claims 1-51, 54-55, 57, and 58; the hTAD of claim 52 or 53; the hhTAD of claim 56; the system of any one of claims 59-75; the polynucleotide of claim 76; the nucleic acid construct of any one of claims 77-79; or the vector of claim 80 or 81; or portion thereof.

83. The cell of claim 82, wherein the cell is an immune cell.

84. The cell of claim 83, wherein the immune cell is selected from the group consisting of: a CD4+ T cell, a CD8+ T cell, a Treg, an NK cell, a monocyte, and a macrophage.

85. A composition comprising the synTF of any one of claims 1-51, 54-55, 57, and 58; the hTAD of claim 52 or 53; the hhTAD of claim 56; the system of any one of claims 59-75; the polynucleotide of claim 76; the nucleic acid construct of any one of claims 77-79; the vector of claim 80 or 81; or the cell of any one of claims 82-84; or portion thereof.

86. A pharmaceutical composition comprising the synTF of any one of claims 1-51, 54-55, 57, and 58; the hTAD of claim 52 or 53; the hhTAD of claim 56; the system of any one of claims 59-75; the polynucleotide of claim 76; the nucleic acid construct of any one of claims 77-79; the vector of claim 80 or 81; or the cell of any one of claims 82-84; or portion thereof; and a pharmaceutically acceptable carrier.

87. A method of regulating the activity of a synTF, comprising the steps of: (a) providing a population of cells of claim 82-84; and (b) contacting the population of cells with an effective amount of at least one RP inducer.

88. A method of regulating the expression of a gene of interest, comprising the steps of: (a) providing a population of cells of claim 82-84; and (b) contacting the population of cells with an effective amount of at least one RP inducer.

89. A method of treating a subject in need of a cell-based therapy, comprising the steps of: (a) administering to the subject a population of cells of claim 82-84; and (b) administering to the subject an effective amount of at least one RP inducer.

90. The method of any one of claims 87-89, wherein the population of cells comprises immune cells. 245 4887-3198-4841.9Attorney Docket No: 701586-000114WOPT 91. The method of claim 90, wherein the population of immune cells comprises CD4+ T cells, CD8+ T cells, Tregs, NK cells, monocytes, or macrophages.

92. The method of any one of claims 87-91, wherein the at least one RP inducer is administered at the same time the population of cells is administered.

93. The method of any one of claims 87-92, wherein the at least one RP inducer is administered after the population of cells is administered. 246 4887-3198-4841.9

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